Optimisation of an in vitro model for

anti-diabetic screening

by

Gayle Pamela Wilson

Submitted in partial fulfillment of the requirements for the

degree of

MAGISTER SCIENTIAE

in the

Faculty of Science

at the

Nelson Mandela Metropolitan University

2006

Supervisor: Dr S Roux

Co-Supervisor: Dr M van de Venter

brought to you by COREView metadata, citation and similar papers at core.ac.uk

provided by South East Academic Libraries System (SEALS)

II

Contents Summary..........................................................................................................................IV

Acknowledgements .........................................................................................................VI

List of Abbreviations .................................................................................................... VII

List of Figures................................................................................................................... X

List of Tables ................................................................................................................XIII

Chapter 1 Literature Review ........................................................................................... 1

1.1 Introduction..........................................................................................................1 1.2 Insulin ..................................................................................................................3

1.2.1 Insulin Secretion ...........................................................................................4 1.2.2 Insulin Receptor ............................................................................................6

1.3 Glucose Absorption By Cells...............................................................................9 1.4 Diabetes Mellitus ...............................................................................................13

1.4.1 Pharmacological Treatment ........................................................................15 1.4.1.1) Biguanides ..........................................................................................17 1.4.1.2) Sulfonylurea Drugs.............................................................................18 1.4.1.3) Thiazolidinediones..............................................................................19 1.4.1.4) Insulin .................................................................................................24

1.5 Plants and the treatment of Diabetes Mellitus ...................................................24 1.5.1 Sutherlandia frutescens (Fabaceae) ...........................................................25 1.5.2 Toxicity of Hypoglycaemic Plants..............................................................26

Chapter 2 Introduction to the present study................................................................ 28

Chapter 3 Models for glucose utilization ...................................................................... 30

3.1 General methods ................................................................................................33 3.1.1 Maintenance of cell lines ............................................................................33 3.1.2 Cytotoxicity Assay......................................................................................34 3.1.3 Sulforhodamine B Assay ............................................................................34 3.1.4 Glucose oxidase assay.................................................................................35 3.1.5 Cell viability assay......................................................................................35

3.2 3T3-L1 cell line..................................................................................................35 3.2.1 Induction of 3T3-L1 preadipocyte differentiation ......................................35 3.2.2 Adipocyte differentiation assays.................................................................36

3.2.2.1 Glycerol-3-phosphate dehydrogenase assay ........................................36 3.2.3 Protein concentration determination ...........................................................37

3.2.3.1 Adapted Folin method for microtiter plates.........................................37 3.2.4 Glucose Uptake...........................................................................................37

3.3 C2C12 cell line ..................................................................................................37 3.3.1 Glucose uptake............................................................................................37

3.4 Chang cell line ...................................................................................................38 3.4.1 Glucose uptake............................................................................................38

III

3.5 Statistical analysis..............................................................................................39 3.6 Results and Discussion ......................................................................................39

3.6.1 3T3-L1 cell line...........................................................................................39 3.6.1.1 Cytotoxicity..........................................................................................39 3.6.1.2 Glycerol-3-phosphate dehydrogenase activity.....................................41 3.6.1.3 Glucose uptake.....................................................................................43

3.6.2 C2C12 skeletal muscle cell line..................................................................51 3.6.2.1 Cytotoxicity..........................................................................................51 3.6.2.2 Glucose uptake.....................................................................................52

3.6.3 Chang liver cell line ....................................................................................56 3.6.3.1 Cytotoxicity..........................................................................................56 3.6.3.2 Glucose uptake.....................................................................................57

Chapter 4 Models for insulin secretion......................................................................... 64

4.1 Materials and methods .......................................................................................65 4.1.1 INS cells maintenance.................................................................................65

4.1.1.1 Mercaptoethanol ..................................................................................65 4.1.1.2 Complete Medium ...............................................................................65 4.1.1.3 Poly-L-Lysine (PLL) ...........................................................................66 4.1.1.4 Pretreatment of culture dishes..............................................................66

4.1.2 Insulin Secretion by INS-1 cells .................................................................66 4.1.3 Plasma insulin determination from rat model.............................................67

4.1.3.1 Preparation of medication ....................................................................67 1) Sutherlandia frutescens (kankerbos) .......................................................67 2) Amitriptyline and Metformin ..................................................................67

4.1.3.2 Experimental Procedure.......................................................................68 4.1.4 Binding studies with INS-1 rat pancreatic cells..........................................69

4.1.4.1 Binding time study...............................................................................69 4.1.4.2 Saturation binding study ......................................................................70 4.1.4.3 Displacement study..............................................................................70

4.1.5 Statistical analysis.......................................................................................70 4.2 Results and Discussion ......................................................................................71

4.2.1 Cytotoxicity.................................................................................................71 4.2.2 Insulin Secretion by INS-1 cells .................................................................72 4.2.3 Serum insulin determination in an animal model .......................................82 4.2.4 Binding studies using INS-1 rat pancreatic cells ........................................83

4.2.4.1 Choice of incubation time....................................................................83 4.2.4.2 Saturation binding................................................................................84 4.2.4.3 Displacement studies ...........................................................................85

Chapter 5 Conclusion ..................................................................................................... 90

References........................................................................................................................ 99

Appendix A.................................................................................................................... 105

IV

Summary The need for alternative strategies for the prevention and treatment of diabetes is

growing rapidly as type II diabetes is reaching epidemic status in our society. This

need was the basis for the creation of this study, as it was necessary to start looking

towards medicinal plants as potential antidiabetic treatment and no comprehensive in

vitro model existed.

In creating a model for determining the effects of alternative traditional medicines as

antidiabetic potentiates, it was necessary that two metabolic pathways, namely

glucose uptake and insulin secretion, which play a significant role in glucose

homeostasis, be at the centre of our investigations. The objective of this project was to

optimize the methodology required to screen and ultimately determine the

effectiveness of the plant extracts Kankerbos and MRC2003, as antidiabetic

potentiates, through observing their effects on glucose utilisation and insulin

secretion. If these medicinal plants are going to make a positive contribution to the

health of type II diabetic South Africans, then the determination of their efficacy is

essential. The cell lines used in this study included 3T3-L1 preadipocytes, Chang

liver, C2C12 muscle and INS-1 rat pancreatic cells. Each cell line represents a

different in vivo organ that is known to have an influence on glucose homeostasis in

our bodies, each with its own unique metabolic pathways and mechanisms of activity,

thereby making each one a vital component in the study.

The positive controls for the two models were insulin and metformin (glucose

utilisation) and glibenclamide (insulin secretion). Insulin was shown to provide a

significant increase in the amount of glucose taken up in C2C12 muscle and Chang

liver cells for acute conditions. Chronic treatments with metformin provided a

significant increase in glucose utilised by Chang liver cells. Glibenclamide was an

effective positive control for stimulating insulin secretion by INS-1 cells under acute

conditions as there was a significant increase in the amount of insulin secreted.

MRC2003 did not show any significant antidiabetic activity. Sutherlandia frutescens

(Kankerbos) showed biological activities comparable to some of the more recognized

V

antidiabetic compounds throughout the study. With regards to the glucose utilisation

model, Kankerbos was seen to have both acute and chronic effects in different cell

lines. In the C2C12 muscle cell line, Kankerbos significantly increased glucose uptake

when they were exposed to acute conditions. Kankerbos also had a significant effect

on the Chang liver cells as it was observed that under both acute and chronic

conditions, this plant extract induced the uptake of glucose into these cells. With

respect to the insulin secretion model involving INS-1 cells, no significant effect was

seen during acute exposure with Kankerbos treatment. However during chronic

exposure, an increase in insulin secretion was initiated by this plant extract. Overall,

the results of this study suggest that Kankerbos has a twofold mechanism of action for

its glucose-lowering effects. Given that Kankerbos is widely available in South

Africa, this study was valuable as it provided an indication that Kankerbos has

antidiabetic activities and could possibly be used as an alternative antidiabetic

medication.

VI

Acknowledgements I would like to extend my sincere thanks and appreciation to everyone who made this

study possible, especially to:

God, for His strength and faithfulness

Dr Roux and Dr van de Venter for their unwavering guidance and support as well as

their willing assistance in the preparation of this manuscript

All staff and postgraduate students in the Department of Biochemistry and

Microbiology, Nelson Mandela Metropolitan University, for their continual support

and encouragement

The National Research Foundation and University of Port Elizabeth for their financial

support

My Mom and Dad, Greg, family and friends for their unconditional love and support

VII

List of Abbreviations

ADD1/SREBP1 Adipocyte determination and differentiation factor 1/ sterol regulatory element binding protein-1

ADP Adenosine diphosphate

α Alpha

AMP Adenosine monophosphate

Arg Arginine

ATP Adenosine triphosphate

β Beta

BSA Bovine serum albumin

Ca2+ Calcium ion

CaCl2 Calcium chloride

C/EBP CCAAT/enhancer-binding proteins

δ Delta

DEX Dexamethazone

DHAP Dihydroxyacetone phosphate

DMEM Dulbecco’s Modification of Eagle’s Medium

DMSO Dimethyl sulfoxide

EDTA Ethylenediaminetetraacetic acid tetrasodium salt

FCS Fetal Calf Serum

FFA Free fatty acid

γ Gamma

GABA Gamma-aminobutyric acid

GLUT Glucose transporter

G3PDH Glycerol-3-phosphate dehydrogenase

g Gram

HDL High density lipoprotein

HCl Hydrochloric acid

HEPES N-[2-Hydroxyethyl]piperazine-N- [2- ethanesulfonic acid]

IRS Insulin receptor substrate

IBMX Isobutylmethylxanthine

KD Dissociation constant

kg Kilogram

VIII

Kir 6.x Potassium channel subunit

K+ Potassium ion

K+ATP ATP- sensitive potassium channel

KRBH Krebs-Ringer-bicarbonate-HEPES buffer

l Litre

LXR Lipid-modulated nuclear receptor

Lys Lysine

m Milli

MAPK Ras/mitogen-activated protein kinase

MgSO4 Magnesium sulphate

2 - ME 2 - Mercaptoethanol

μ Micro

M Molar

n Nano

NaCl Sodium chloride

NaHCO3 Sodium bicarbonate

PBS Complete phosphate buffered saline

PBSA Phosphate buffered saline (without Ca2+ and Mg2+)

PC-1 Plasma cell antigen-1

PI3-kinase Phosphatidylinositol 3-kinase

PLL Poly-L-lysine

PPARγ Peroxisome proliferator-activated receptor γ

P Probability

r2 Correlation coefficient

RIA Radioimmunoassay

rpm Revolutions per minute

RPMI 1640 Roswell Park Memorial Institute culture medium

RXR Retinoid X receptor

SD Standard deviation

SEM Standard error of mean

SRB Sulforhodamine B

SUR Sulfonylurea receptor

TCA Trichloroacetic acid

TEA Triethylamine

IX

Tris Tris(hydroxymethyl)aminomethane

TNFα Tumor necrosis factor α

TZD Thiazolidinediones

WHO World Health Organisation

X

List of Figures Figure 1.1: Insulin is the principal regulator of energy metabolism…………………..3

Figure 1.2: Schematic diagram of the probable structure of the insulin receptor

tetramer in the activated state……………………………………………..7

Figure 1.3: Pathways of insulin control……………………………………………… 7

Figure 1.4: Central role of Akt in the actions of insulin………………………………8

Figure 1.5: Hypothetical model of the action of insulin on glucose transport

specifically for GLUT 4…………………………………………………12

Figure 1.6: Diabetes mellitus type II: the tip of the iceberg………………………….16

Figure 1.7: Common mechanism of insulin release in response to hyperglycaemia

or sulfonylurea administration…………………………………………...18

Figure 1.8: The transcriptional control of adipogenesis involves the activation

of several families of transcription factors………………………………20

Figure 1.9: Sutherlandia frutescens…………………………………………………..26

Figure 3.1: Cytotoxicity results of compounds (BDM1, BDH1 BDP1 and metformin)

and plant extracts (Kankerbos and MRC2003) on 3T3-L1 adipocyte cells

after a 48 hour exposure period………………………………………….40

Figure 3.2: The effect of plant extracts (Kankerbos and MRC; 12.5μg/well)

(with and without inducers) on glycerol-3-phosphate dehydrogenase

(G3PDH) specific activity……………………………………………….42

Figure 3.3: The percentage glucose taken up by undifferentiated (a) and differentiated

(b) 3T3-L1 cells when exposed to treatments under acute conditions…..45

Figure 3.4: Acute effect of treatments on glucose uptake in 3T3-L1 cells after

10 minute incubation time………………………………………………..45

Figure 3.5: Acute effect of treatments on glucose uptake in 3T3-L1 cells after

120 minute incubation time………………………………………………46

Figure 3.6: Comparison of glucose standard curve with and without 10% serum in

8mM RPMI 1640………………………………………………………..47

Figure 3.7: Glucose uptake as a percent of control in 3T3-L1 cells with wells lined

with PLL (a) and FCS (b)………………………………………………..48

Figure 3.8: Acute effects of treatments (1μM BDM1, BDH1 and BDP1) on glucose

uptake in 3T3-L1 cells after 60 minute incubation……………………...49

XI

Figure 3.9: Cytotoxicity results of compounds (BDM1, BDH1, BDP1 and metformin)

and plant extracts (Kankerbos and MRC2003) on C2C12 muscle cells

after a 48 hour exposure period………………………………………….51

Figure 3.10: The acute effect of positive controls, metformin and insulin (1μM)

on glucose uptake in C2C12 cells after 90 minute exposure…………..53

Figure 3.11: Acute effects of treatments on glucose uptake by C2C12 cells after

90 minutes……………………………………………………………...54

Figure 3.12: Effect of insulin (1μM) on glucose uptake in C2C12 muscle cells

compared to control after chronic exposure……………………………55

Figure 3.13: Cytotoxicity results of compounds (BDM1, BDH1 and BDP1) and

plant extract (MRC2003) on Chang liver cells after a 48 hour

exposure period………………………………………………………...56

Figure 3.14: Acute effect of two positive controls (1μM metformin and insulin)

on glucose uptake in Chang liver cells…………………………………57

Figure 3.15: Acute effect of treatments on glucose uptake in Chang liver cells……..58

Figure 3.16: Acute effect of treatments on glucose uptake in Chang liver cells……..59

Figure 3.17: Chronic effects of treatments exposed to Chang liver cells for 48 hours

prior to glucose uptake experimentation ………………………………60

Figure 3.18: Effect of different treatments (BDM1, BDH1, BDP1; 1μM and

Kankerbos and MRC2003; 12.5μg/well) on glucose uptake in Chang

liver cells under chronic conditions……………………………………61

Figure 3.19: CellTiter-BlueTM results of Chang liver cells exposed to

different treatments for chronic experimentation………………………62

Figure 4.1: Cytotoxicity results of compounds (Glibenclamide and Amitriptyline)

and plant extracts (Kankerbos and MRC2003) on INS-1 cell line………72

Figure 4.2: Effect of 1μM glibenclamide (30 minute exposure) on insulin secretion

in INS-1 including 2mM glucose in the medium………………………..74

Figure 4.3: Effect of extracellular glucose concentration (glucose-free, 2mM and

10mM glucose) on insulin secretion in INS-1 cells……………………...74

Figure 4.4: Insulin secretion after 30 minute treatment without glucose and with

effect of extracellular glucose concentration (glucose-free, 2mM and

10mM glucose) (10 and 30 minute exposure) on insulin secretion in

INS-1 cells including glibenclamide in the test medium………………..75

XII

Figure 4.5: Effect of extracellular glucose concentration (glucose-free, 2mM

and 10mM glucose) (10 and 30 minute exposure) on insulin secretion

in INS-1 cells including glibenclamide in the test medium……………..76

Figure 4.6: Effect of different glucose concentrations (basal, 2mM and 10mM) on

insulin stimulation in INS-1 cells under acute conditions……………….77

Figure 4.7: Effect of compounds (glibenclamide and amitriptyline, 1μM) and

plant extracts (Kankerbos and MRC2003, 0.5ng/well) on insulin secretion

in INS-1 cells under acute conditions (10 minute exposure)……………78

Figure 4.8: Effect of extracellular glucose as well as the test compounds

(Glibenclamide and Amitriptyline, 1μM) and plant extracts

(Kankerbos and MRC2003, 0.5ng/well) on insulin secretion in

INS-1 cells under chronic conditions……………………………………80

Figure 4.9: Fasting serum insulin concentrations as a result of a 19 week treatment

with the medication……………………………………………………...82

Figure 4.10: Specific binding of [3H]glibenclamide by INS-1 cells as a function

of incubation time………………………………………………………84

Figure 4.11: Saturation binding curve………………………………………………..84

Figure 4.12: Saturation binding curve………………………………………………..85

Figure 4.13: Displacement of [3H]glibenclamide by unlabelled glibenclamide

(1.34nM – 1.34µM) and amitriptyline (0.67µM and 6.67µM)…………87

Figure 4.14: Displacement of [3H]glibenclamide by unlabelled glibenclamide

(1.34nM – 1.34µM), amitriptyline and trimipramine

(0.67µM and 6.67µM)…………………………………………………..88

Figure 4.15: Displacement of [3H]glibenclamide by unlabelled glibenclamide

(1.34nM – 1.34µM) and varying concentrations of unlabelled

amitriptyline (0.67nM – 6.67μM).……………………………………..88

Figure 5.1: Summary of carbohydrate metabolism ………………………………….92

Figure 5.2: Mechanisms of insulin action……………………………………………93

Figure 5.3: Mechanisms of metformin action on hepatic glucose production and

muscle consumption……………………………………………………...94

XIII

List of Tables Table 4.1: Experimental plan for medicinal exposure .................................................68 Table 4.2: Probability (P) of binding to SUR-1 receptors ...........................................86 Table A1: Conversion table for plant extract concentrations ....................................105

1

Chapter 1

Literature Review 1.1 Introduction The identification of substances that mediate or mimic the action of insulin could lead to

the development of novel structures which may be of clinical use in the treatment of

persons having disorders of glucose metabolism, such as impaired glucose tolerance,

elevated blood glucose associated with type II diabetes and insulin resistance (Larner et

al., 1997).

Modern drug discovery requires a systematic approach to optimize time and resource use

in order to test the maximum number of samples in systems which hopefully predict

therapeutic efficacy (Wagner and Farnsworth, 1994).

Techniques for the study of hypoglycaemic activity in vivo employ animals with

normoglycaemia or induced hyperglycaemia, as well as diabetic humans. In vivo

bioassays are essential to prove the value of new hypoglycaemic agents, however, animal

tests reveal relatively little about the specific mechanisms of action of the compound, and

it is evident that there are a great many mechanisms by which blood glucose levels may

be reduced. Some of these mechanisms, such as those producing hypoglycaemia as a

side-effect of their toxicity, are obviously not useful in diabetes treatment. The lack of

perfect models for type II diabetes, coupled with financial restrictions on obtaining and

maintaining animals, and social restrictions on extensive use of animals in

experimentation, indicate that a more practical approach would involve a series of in vitro

prescreens before testing a potential new hypoglycaemic agent in animals. Many in vitro

techniques have been developed to elucidate the varied mechanisms of action of

hypoglycaemic agents discovered by in vivo bioassays. Three aspects of the

hypoglycaemic response are commonly studied in vitro: insulin release from the

pancreatic islets, peripheral insulin binding and glucose uptake, and effects on hepatic

enzymes (Wagner and Farnsworth, 1994).

2

During the last 20 years, the considerable and significant advances in tissue culture

methodology, the use of chemically-defined cell and tissue culture media, and the

availability of mammalian cells have transformed in vitro methods from a new

technology to a valuable research tool. In the past, in vitro methodology was used as the

last approach in product development. Today, the use of in vitro tests in product

development, drug discovery and safety evaluation has become commonplace, resulting

almost exclusively from the evolution of science rather than any fundamental change in

philosophy. Yet all in vitro methods are alternatives to animal testing. It is not known

how well any in vitro system would recapitulate the in vivo system. Thus, it would be

difficult to design an in vitro test battery to replace in vivo test systems.

In vitro systems are well suited to the study of biological processes in a more isolated

context, therefore, in vitro tests have their greatest potential in providing information on

basic mechanistic processes in order to refine specific experimental questions to be

addressed in the animal.

Advantages of in vitro models include the ability to directly manipulate cultivation

conditions and isolate the target tissue from the physiological effects of other organs and

tissues. Additional unique competitive advantages of in vitro models include: 1) Rapid

large-scale screening of drug candidates allows promising substances to be identified at

an early stage of the drug development process; 2) The time and costs involved in

developing active agents are significantly reduced and 3) Cost-intensive and ethically

controversial animal experiments can be reduced to a minimum.

In order to determine the best model for antidiabetic screening, it is essential to elucidate

and understand the molecular machinery involved in the regulation of blood glucose

levels.

3

1.2 Insulin

Insulin is the most potent anabolic hormone known and it is essential for appropriate

tissue development, growth, and maintenance of whole-body glucose homeostasis. It

regulates glucose homeostasis at many sites, reducing hepatic glucose output (via

decreased gluconeogenesis and glycogenolysis) and increasing the rate of glucose uptake,

primarily into striated muscle and adipose tissue (Figure 1.1). It also profoundly affects

lipid metabolism, increasing lipid synthesis in liver and fat cells, and attenuating fatty

acid release from triglycerides in fat and muscle (Pessin and Saltiel, 2000).

Figure 1.1: Insulin is the principal regulator of energy metabolism. When glucose or other nutrients are absorbed from the gastrointestinal tract, this elicits insulin secretion. Insulin regulates the metabolism of multiple fuels. Selected actions of insulin are indicated (+, activation; -, inhibition). Insulin activates transport of glucose into muscle and adipose tissue, and promotes synthesis of glycogen and triglycerides. Insulin also inhibits hepatic glucose production by inhibiting both glycogenolysis and glucogenesis. Insulin does not directly regulate the metabolism of red blood cells, which uses glycolysis to provide energy. Although the brain uses glucose in the fed state, it can also use ketone bodies when levels rise high enough (eg. during fasting) (Taylor, 1999)

4

Despite significant advances in past years on the chemistry and biology of insulin and its

receptor, the signaling mechanisms involved in the various biologic responses to insulin

remain somewhat elusive (Pessin and Saltiel, 2000). Progress in this area has been

complicated by the pleiotropic nature of the actions of insulin. The relative activation and

coordination of these distinct cellular processes by insulin varies with cell type, state of

differentiation of the cell, presence of other hormones, and insulin dose response and

time course, suggesting that insulin action involves a network of inter-related and

independent pathways with differing levels of divergence regarding mechanisms of

regulation (Saltiel, 1990).

1.2.1 Insulin Secretion

Insulin is released from the pancreatic β-cell directly from the granules by exocytosis and

the movement of the granules to the cell membrane in response to stimulation probably

involves microfilaments and microtubules. Insulin is released from pancreatic β -cells at a

low basal rate and at a much higher stimulated rate in response to a stimulus. The most

important stimulus for insulin secretion is an increase in the extracellular concentration of

glucose. Within one minute of the addition of glucose to the tissue an increased rate of

secretion occurs. The response of insulin secretion to the change in glucose concentration

is sigmoid so that there is little response below 5mM, and a 50% response at about 8mM

(Newsholme and Leech, 1992).

Pancreatic β cells secrete insulin in a pulsatile fashion and, in response to a square-wave

increase in interstitial glucose concentration, release insulin in a biphasic manner,

characterized by a “spike” lasting approximately 10 minutes (first-phase release) and

followed by gradually increasing release (second-phase release). It has been suggested

that these different phases of insulin released represent two different intra-islet pools: one

– a rapidly releasable pool accounting for about 5% of islet insulin – represents granules

close to the cell membrane and is thought to be responsible for first-phase insulin release.

The second is a reserve pool, the release of which requires adenosine triphosphate-

dependent mobilization of insulin-containing granules into the rapidly releasable pool for

5

subsequent exocytosis. Both phases of insulin release are important for maintaining

normal glucose homeostasis. However, considerably more emphasis has been placed on

the importance of first-phase insulin, assuming that this is the major determinant of

“early” insulin release, that is, the increase in plasma insulin levels observed during the

initial 30 minutes following glucose or meal ingestion (Gerich, 2000).

The proposed hypothesis of insulin secretion: An increase in the extracellular glucose

concentration above 5mM increases proportionally the rate of glycolysis (through

operation of the glucose/glucose 6-phosphate cycle) and this raises the concentration of

phosphoenol pyruvate which, increases the rate of uptake of calcium ions and probably

increases the rate of release from intracellular calcium stores. An increased cytosolic

concentration of calcium ions, via calmodulin, causes contraction of the microfilaments

or microtubules and hence results in an increased rate of exocytosis and insulin secretion.

The hypothesis attempts to link the rate of glycolysis, calcium ions and rate of insulin

secretion (Newsholme and Leech, 1992).

Potassium channels are sensitive to ATP and function in coupling cell metabolism to

membrane potential in many tissues (Hernandez-Sanchez et al., 1999). K+ATP channels

have been found in a variety of tissues including heart, pancreatic β-cells, skeletal

muscle, smooth muscle and the central nervous system (Isomoto et al., 1996). K+ATP

channels comprise an octameric complex of pore-forming Kir6.x subunits and regulatory

sulfonylurea receptors (SURs) (Gribble and Ashcroft, 2000). There are three isoforms of

the sulfonylurea receptor, SUR1 and two spliced variants of SUR 2, SUR 2A and SUR

2B. The SUR1-Kir 6.2 and SUR2B-Kir 6.2 or Kir 6.1, constitute the cardiac and vascular

smooth muscle-type channels, respectively (Hernandez-Sanchez et al., 1999). SUR is a

member of the family of ATP-binding cassette (ABC) transporter proteins and appears to

be the major determinant of the pharmacological properties of K+ATP channels (Gribble

and Ashcroft, 2000; Isomoto et al., 1996). K+ATP channels which consist of SUR1 and

Kir 6.2 do not only occur in β cells but are also present in the alpha, delta and pancreatic

polypeptide cells of the pancreatic islets (Fujikura et al., 1999). The K+ATP (SUR1-Kir

6.2) channels mediate glucose-induced insulin secretion in pancreatic cells. K+ATP

6

channels are modulated by intracellular ATP/ADP ratios: ATP closes the K+ATP

dependent channels, while ADP opens them (Raab-Graham et al., 1999). When the level

of blood glucose increases, glucose enters β-cells via GLUT 1, 2 and 3 transporters,

which are insulin independent (Walker, 2000). Following glucose metabolism, the

intracellular ATP/ADP ratio increases thereby inactivating the K+ATP channels. This

increase in the ATP concentration causes closure of the K+ATP sensitive channels and

the efflux of K+ through the channel is decreased causing depolarization of the

membrane. This in turn results in the opening of the voltage-dependent calcium channels

with a subsequent increase in the intracellular Ca2+, which initiates insulin secretion

(Hernandez-Sanchez et al., 1999).

1.2.2 Insulin Receptor

Once insulin has entered the circulation it reacts with target cells that have insulin

receptors on their plasma membranes (Katzung, 1995). The most important target cells,

which have insulin receptors are liver, muscle and fat. The number of insulin receptors on

individual cells and the affinity of the receptor to insulin varies. The receptors bind

insulin with high specificity and affinity in the picomole range. Insulin action is initiated

through the binding to and activation of its cell-surface receptor, which consists of two α

subunits and two β subunits that are disulfide linked into an α2β2 heterotetrameric

complex, as illustrated in figure 1.2. Insulin binds to the extracellular α subunits,

transmitting a signal across the plasma membrane that activates the intracellular tyrosine

kinase domain of the β subunit. Although PI 3-kinase activity is clearly necessary for

insulin-stimulated glucose uptake, additional signals are also required for the stimulation

of GLUT4 translocation (Pessin and Saltiel, 2000).

7

Upon binding insulin, the protein tyrosine kinase phosphorylates itself as well as target

substances (Figure 1.3), such as the insulin receptor proteins (IRS-1 and IRS-2), Cbl and

p52Sho (Galic et al., 2005). IRS-1 plays a more prominent role in stimulating glucose

uptake by muscle and fat, whereas IRS-2 functions mainly in the liver. It has been

discovered that IRS-2 boosts insulin production by the pancreas (Alper, 2000).

Figure 1.3: Pathways of insulin control (Alper, 2000)

Figure 1.2: Schematic diagram of the probable structure of the insulin receptor tetramer in the activated state (Katzung, 1995)

8

These phosphorylation events allow for recruitment and activation of signaling pathways,

including Ras/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-

kinase (PI3-kinase)/Akt pathways that mediate the metabolic, transcriptional and

mitogenic actions of insulin (Figure 1.4) (Galic et al., 2005).

Figure 1.4: Central role of Akt in the actions of insulin (Galic et al., 2005)

9

1.3 Glucose Absorption By Cells

The entry of glucose into cells is a crucial step in life-supporting processes since glucose

is the main monosaccharide in nature that provides carbon and energy for almost all cells.

The passage of glucose into cells depends on different parameters, including expression

of the appropriate glucose transporters in the target tissues and hormonal regulation of

their function (Gorovits and Charron, 2003). Glucose is a hydrophilic compound; it

cannot pass through the lipid bilayer by simple diffusion, and therefore requires specific

carrier proteins to mediate its specific transport into the cytosol. Cells take up glucose by

facilitated diffusion, via glucose transporters (GLUTs) associated with the plasma

membrane (Medina and Owen, 2002).

Until now the search for the mammalian facilitative glucose transporters has yielded 12

carriers including GLUT1–5 and the recently discovered GLUT6–12. The kinetic

properties and substrate specificities of the different isoforms are specifically suited to the

energy requirements of the particular cell types (Medina and Owen, 2002). The

transporters form a family whose members are broadly alike in structure and function.

Each transporter consists of a polypeptide chain consisting of approximately 500 amino

acids (Lienhard et al., 1992). All GLUTs have been predicted to have 12 membrane-

spanning domains (helices) connected by hydrophilic loops, the first of which is exofacial

and contains an N-glycosylation site in GLUT1–5. Both the amino and carboxyl termini

of GLUTs reside on the cytoplasmic side of the cell membrane. Models of GLUTs

suggest that five of the transmembrane helices form an aqueous pore providing a channel

for substrate passage. Lack of a crystal structure leaves the precise structure of GLUTs

hypothetical (Gorovits and Charron, 2003).

Glucose passage into the cell is an intricate one. In the process, the transporter takes on

two shapes, one which binds glucose on the extracellular side and the other which binds it

on the intracellular side. It is suggested that a glucose molecule enters the cell through a

four step process. First, it occupies the outward-facing binding site. Second, the complex

of transporter and glucose changes conformation. The glucose occupies the binding site

10

facing into the cell. Third, the transporter releases the glucose into the cytoplasm. Fourth,

the unoccupied transporter changes back into the conformation in which the binding site

is on the outward side, thereby enabling the binding of another glucose molecule. The

transporter is envisioned as a conformational oscillator which shifts the binding pocket

for glucose between opposite sides of the cell membrane (Lienhard et al., 1992).

GLUTs expression is cell-specific and subject to hormonal and environmental control.

(Medina and Owen, 2002). GLUT1 is a high affinity glucose transporter that is

ubiquitously expressed in most mammalian tissues. It provides basal glucose transport

and, most importantly, transport of glucose through the blood-brain barrier, erythrocytes,

and neuronal cell membranes. GLUT2 is a low affinity glucose transporter with a high

turnover rate expressed in adult liver, kidney, intestinal epithelium, and pancreatic β-cells

(Gorovits and Charron, 2003). These kinetic properties allow GLUT2 to function in the

liver where glucose transport must not be rate limiting for influx or efflux. When

circulating glucose levels are high there needs to be net hepatic uptake as the intracellular

glucose is metabolized or converted into glycogen. Conversely, when glucose levels are

low, the liver needs to export glucose to the plasma. This is achieved by GLUT2 coupled

with the regulated phosphorylating activity of hexokinase IV. GLUT2 therefore functions

in the regulation of insulin levels because changes in the blood glucose level are

effectively transmitted to the liver and the pancreatic β cells by GLUT2. In order to

regulate insulin secretion, pancreatic cells need to be highly sensitive to changes in

plasma glucose concentrations. Therefore, a low-affinity transporter, such as GLUT2 will

not be saturated at physiological levels and glucose flux will be proportional to plasma

glucose concentration. As in the liver, hexokinase regulates the entry of glucose into the

glycolytic pathway and, along with GLUT2, plays a role in glucose sensing by β-cells

(Medina and Owen, 2002). GLUT3 is found in the neuronal cells of the brain and

functions to ensure a constant movement of the glucose into these cells. This transporter

takes up glucose and converts it into energy-yielding compounds in an insulin-mediated

step (Katzung, 1995; Lienhard et al., 1992). The insulin-stimulated, high affinity glucose

transporter protein GLUT4 is expressed predominantly, but not exclusively, in all insulin-

sensitive tissues: skeletal muscle, adipose tissue, and heart. GLUT4 is unique in that it is

11

the only insulin-responsive GLUT that has been characterized so far. Stimuli leading to

the increase in translocation of GLUT4 to the plasma membrane include insulin,

contraction, and hypoxia (Gorovits and Charron, 2003). One of the primary functions of

insulin is to facilitate the disposal of blood glucose into the peripheral tissues during the

post-absorptive state. Insulin increases cellular uptake and metabolism of glucose by

accelerating its transmembrane transport (Johnson, 1998). Undoubtedly GLUT4

translocation to the plasma membrane from the intracellular pool leads to an increase in

glucose uptake. Insulin-stimulated translocation of GLUT4 to the plasma membranes

requires phosphatidylinositol 3-kinase (PI3K) activity. However, the activity of PI3K

alone is not sufficient to cause a GLUT4-mediated increase in glucose transport,

suggesting that additional stimuli are important for GLUT4 translocation (Gorovits and

Charron, 2003). GLUT5 is found in the small intestine and kidney, where it functions in

the absorption of fructose (Katzung, 1995; Lienhard et al., 1992).

GLUT1 and GLUT4 are the main GLUTs functioning in adipose and muscle tissue.

GLUT1 is thought to play a constitutive role, and is responsible for basal glucose uptake

while GLUT4 is the inducible transporter. One of the most important, and well

established, models of GLUT regulation is the stimulation of GLUT expression and

translocation in adipose and muscle tissue by insulin. It is this process that provides the

regulation of whole-body glucose homeostasis and, when dysfunctional, plays a vital role

in diabetes mellitus. GLUT4 is almost completely responsible for insulin-stimulated

glucose transport. In rat adipocytes, the most studied cell system for insulin action on

glucose transport, more than 95% of GLUT4 and 30-40% of GLUT1 is associated with

intracellular membranes, and are therefore non-functional. These GLUTs are translocated

to the plasma membrane in response to insulin, where they are then able to facilitate the

transport of substrate (Medina and Owen, 2002).

It is well established that the mechanism by which glucose transport (Figure 1.5, step 6)

is upregulated in fat and skeletal muscle, in response to insulin, involves recruitment of

the insulin-responsive GLUT4 transporter from an intracellular compartment to the

plasma membrane (steps 3-5). Insulin induces intracellular vesicles containing GLUT4 to

12

move to the inner surface of the cell membrane and fuse with it. This occurs after

intracellular signaling apparatus turns on a multistep signaling pathway that activates the

glucose transporter, due to binding of and activation by insulin to its receptor (steps 1-2).

The transporters are retrieved to the interior when small vesicles formed through

membrane invagination and fission fuse with larger endosomes, where the transporter

segregates into the tubular extentions that pinch off to form new vesicles (step 8). As long

as insulin remains, the vesicles continue to fuse with the cell membrane, but a lowered

level of insulin breaks the cycle (step 7) and the glucose transporter accumulates in

intracellular vesicles (Lienhard et al., 1992).

Figure 1.5: Hypothetical model of the action of insulin on glucose transport specifically for GLUT 4 (Johnson, 1995)

13

1.4 Diabetes Mellitus

Type II diabetes mellitus is a heterogeneous disorder due to a combination of inherited

and acquired factors that adversely affect glucose metabolism. It is thought that these

factors lead to diabetes mainly by affecting β-cell function and tissue insulin sensitivity.

If the amount of insulin produced is too little to allow for glucose to be used or stored, or

if the insulin being produced does not work effectively, glucose accumulates in the blood.

Hyperglycaemia develops when rates of glucose release into the circulation exceed rates

of tissue glucose uptake. This may occur because release is increased, because uptake is

reduced, or due to a combination of factors such as increased release with a lesser

increase in uptake (Gerich, 2000). In the normal individual, the concentration of glucose

in blood is maintained at about 90 mg/dL of plasma. However, fasting blood glucose in

diabetics may be 300-400 mg/dL and may even reach 1000 mg/dL (Johnson, 1998).

Type II diabetes is associated with insulin resistance initially and later, as the function of

the β-cell decreases, insulin deficiency (Cerasi, 2000). Type II diabetes is characterized

both by abnormalities of insulin secretion progressively leading to secretion failure as

well as insulin resistance of all major target tissues (Haring, 1999). Although insulin

resistance is important in the early stages of type II diabetes, the failure in adequate β-cell

compensation leads to the progression to the diabetic state. Compensation for insulin

resistance is through increased secretion per β-cell or by an increase in β-cell mass

through neogenesis or replication of the existing β-cells (Withers et al., 1998). Beta-cell

mass is normally tightly maintained through a balance of β-cell birth (β-cell replication

and islet neogenesis) and β-cell death through apoptosis. Most of the increase in β-cell

mass with insulin resistance is probably due to increased β-cell number, but β-cell

hypertrophy may also contribute (Weir and Bonner-Weir, 2004).

It is suggested that the disease is triggered when the delicate balance between insulin

production and insulin responsiveness goes awry. First, cells in muscle, fat and liver lose

some of their ability to respond fully to insulin. In response to growing insulin resistance,

pancreatic cells temporarily over produce insulin causing hyperinsulinemia (Alper,

2000). Much of the increase in insulin secretion undoubtedly results from the increase in

14

β-cell mass. At some point, β-cells are no longer able to keep glucose levels in the

prediabetic range. This failure presumably occurring because of a critical decline of β-cell

mass and/or increase in insulin resistance (Weir and Bonner-Weir, 2004). But the insulin-

producing cells eventually die, leading to full-blown diabetes. Therefore type II diabetes

results when the body loses the fine-tuned balance between insulin action and insulin

secretion. For years up until recently, it was believed that a malfunction in the insulin

receptor leads to insulin resistance, however researchers are converging on a new

hypothesis to explain this metabolic disorder. The shift in thinking occurred as a result of

failure in linking insulin receptor malfunction and the disease. It is now believed by some

researchers that two related pathways that normally respond to insulin by signaling cells

in the tissues to remove glucose, lie at the heart of insulin resistance. They are however

unsure as to why the biochemical pathways believed to be involved are not functioning

properly. It is believed that defects in the insulin signaling pathway leading to the disease

are subtle, as no mutations in the insulin receptor substrate (IRS) genes in diabetics have

been uncovered. Due to insulin’s regulation of glucose metabolism being so finely tuned,

it is believed that one or two subtle mutations will upset the entire system when

combined with the proper environmental insults. Another new insight into type II

diabetes is that in order for this disease to develop, insulin resistance must occur in both

muscle and liver, leading researchers to conclude that the insulin regulating system must

fail at multiple points (Alper, 2000).

The new insights into type II diabetes are mainly from studies in knockout mice,

however, human genetics does not necessarily correlate with animal data, therefore not

everyone is convinced and the old dogma is still being followed. The major causes of

insulin resistance of the skeletal muscle in the prediabetic state may be discussed as

genetic background, obesity related insulin resistance and of physical inactivity. Type II

diabetes is known to have both genetic and environmental determinants and is strongly

associated with age and obesity (Sheard and Clark, 2000). Among the environmental

factors causing insulin resistance, obesity is of predominant importance (Haring, 1999).

Obesity is defined as an excess of body weight that is mainly attributable to an increased

body fat accumulation (Tremblay and Doucet, 2000). Obesity is the most important

15

modifiable risk factor for type II diabetes (Sakurai et al., 1999). About 90 percent of

people with this form of the disease are overweight (Sheard and Clark, 2000). An excess

of body fat is associated with a deterioration of glucose utilisation and promotes

development of type II diabetes, particularly in those with a genetic predisposition for the

disease (www.geocities.com/jqjacobs).

There are a number of different hypotheses explaining the mechanism of insulin

resistance in obesity. It was proposed that tumor necrosis factor alpha (TNF-α) is released

by adipose tissue and is able to impair insulin signaling through serine kinase and

tyrosine phosphatase dependent modulation of the insulin signaling chain at the level of

the insulin receptor and substrates (IRS) (Haring, 1999). Another proposed mechanism

of insulin resistance in obesity is related to a protein molecule which interferes with

insulin action. Membrane glycoprotein PC-1 (plasma cell antigen-1) has been shown to

reduce the uptake of glucose by cells through inhibiting insulin receptor tyrosine kinase

activity. Obesity’s contribution to the onset of diabetes may be by increasing the levels of

PC-1 (www.pslgroup.com). The increase in obesity prevalence has led the World Health

Organization (WHO) to refer to a ‘global epidemic’ to describe the obesity issue. Body

weight and fat losses are essential if the health burden which obesity imposes on a great

proportion of individuals throughout the world is to be alleviated (Tremblay and Doucet,

2000).

1.4.1 Pharmacological Treatment

In figure 1.6, the simplified schematic presentation illustrates the evolution of type II

diabetes mellitus. Type II diabetes is a progressive disease. In these patients the blood

glucose level often does not rise high enough to produce many symptoms and therefore

goes undiagnosed. Because insulin is being produced they can often be treated without

insulin and may respond to dietary measures with or without addition of medication

(Vardaxis, 1994). Diabetes mellitus type II represents the end stage of long lasting

metabolic disturbances caused by insulin resistance associated with hyperinsulinemia,

obesity, dyslipoproteinemia, arterial hypertension and consequently premature

atherosclerosis. Since this detrimental metabolic milieu is present for many years before

16

plasma glucose levels are elevated, it is not surprising that type II diabetic patients have

already micro- and/or macrovascular complications at the time of the initial diagnosis.

Subjects in stage I have normal glucose tolerance due to the ability of their β-cells to

compensate for the insulin-resistant state. At this stage elevated triglyceride levels and

reduced HDL levels as well as an increased waist to hip ratio may indicate insulin

resistance and should lead to therapeutic action. In stage II, glucose tolerance after an oral

glucose load (75g) is impaired due to developing insulin-secretory deficiency. To avoid

progression to clinically overt type II diabetes (stage III), these IGT subjects must receive

treatment options to reduce insulin resistance, such as dietary advice and increase of

physical activity (Matthaei et al., 2000).

Until the importance of screening for, as well as treating, the early stage of the metabolic

syndrome is appreciated, the optimal treatment of patients with type II diabetes mellitus

to avoid diabetic complications and to preserve quality of life is a major focus in today’s

medical world. Although nonpharmacological treatment modalities such as reduced

caloric intake and increased physical activity represent the basis of the treatment of

insulin resistance and their efficacy have been demonstrated in numerous studies, the

Figure 1.6: Diabetes mellitus type II: the tip of the iceberg (Matthaei et al., 2000)

17

actual number of patients sufficiently treated without pharmacological agents is

comparatively low. Therefore, pharmacological treatment is required in the vast majority

of type II diabetic patients (Matthaei et al., 2000). The choice of an oral antidiabetic

agent may be influenced by a large number of factors, but often comes down as much to

personal preference or experience as to detailed knowledge of the differential actions of

each molecule.

The following are the available antihyperglycaemic agents which are known to

ameliorate insulin resistance, namely, biguanides, sulfonylureas, thiazolidinediones and

insulin.

1.4.1.1) Biguanides

Metformin is a hypoglycaemic drug effective in the treatment of type II diabetes mellitus

(Klip and Leiter, 1990). Despite almost 40 years of research, the precise mechanism of

metformin action is still not entirely understood. Several cellular mechanisms have been

described but a single unifying site of action has yet to be identified (Matthaei et al.,

2000). Biguanides have no direct effect on insulin release and in fact actually reduce the

serum insulin level in both the basal and stimulated states. This decrease is believed to be

secondary to the biguanide-induced decrease in blood glucose concentration and is

usually attributed to one or more influences, working individually or in combination, that

include: decreased lumen-to-plasma glucose transport, suppression of hepatic

gluconeogenesis coupled with a net decrease in hepatic glucose output, and increased

anaerobic glucose utilization in tissues. At the subcellular level, metformin has been

shown to increase insulin binding to its receptor both in vitro and in vivo. Therefore,

metformin promotes glucose uptake at tissue level and insulin secretion is not enhanced

by its action (Foye et al., 1995; Klip and Leiter, 1990).

Metformin appears to be the drug of choice to start pharmacological treatment in insulin

resistant and overweight/obese diabetic subjects. Unlike other pharmacological therapies

for type II diabetes, metformin is not associated with weight gain (Matthaei et al., 2000).

18

1.4.1.2) Sulfonylurea Drugs

Sulfonylureas are a class of compounds available for treating hyperglycemia in non-

insulin-dependant diabetics. Sulfonylurea drugs are oral hypoglycaemic agents, which

increase the release of endogenous insulin as well as improve its peripheral effectiveness

(Katzung, 1995).

Sulfonylurea drugs have three mechanisms of action including the release of insulin from

pancreatic β cells, the reduction of serum glucagon levels and directly increasing tissue

responsiveness to insulin (Clark et al., 1997). Adenosine triphosphate-sensitive potassium

(K+ATP) channels are the targets for the sulfonylurea drugs used in the treatment of type

II diabetes mellitus (Figure 1.7). They are found in a wide range of tissues, however, in β

cells, K+ATP channels provide a link between the glucose concentration and the rate of

insulin secretion (Gribble and Ashcroft, 2000).

Figure 1.7: Common mechanism of insulin release in response to hyperglycaemia or sulfonylurea administration (Harrower, 2000).

19

In type II diabetes, once insulin secretion becomes defective, the drug, sulfonylurea, is

administered to increase insulin release. Sulfonylureas promote insulin secretion by

favouring the closure of K+ATP channels. Sulfonylureas block K+ATP (SUR1-Kir 6.2)

channels by binding to the receptor (SUR1). Binding of the sulfonylurea inhibits the

efflux of K+ through the channel and results in depolarization. Depolarization of the

membrane provokes calcium influx by opening a voltage-gated Ca2+ channel. The

increased intracellular Ca2+ concentration triggers the secretion of insulin (Katzung,

1995; Quesada et al., 1999).

The use of sulfonylureas in the treatment of diabetes mellitus is controversial.

Hypoglycaemia is a danger with sulfonylureas and may be caused by drug overdose, drug

interactions, altered drug metabolism or the patient failing to eat (Clark et al., 1997)

1.4.1.3) Thiazolidinediones

Adipose tissue has been the subject of intense scrutiny, and one important reason for this

is that this tissue provides a critical link in maintaining systemic energy balance. The

ongoing explosion in the incidence of obesity and its ugly stepsister, type II diabetes, has

focused attention on all aspects of adipocyte biology, including adipogenesis (Rosen et

al., 2000).

Functionally, cellular differentiation can be thought of as a shift in gene expression

patterns, such that transcripts that determine the primitive, multipotent state give way to

those that define the final phenotype. Morphological changes result from the actions of

the genes that are induced as the cells differentiate, including alterations in the cell shape

and the accumulation of lipid that accompany adipogenesis. A transcriptional cascade has

been found to drive adipogenesis. Three classes of transcription factors have been

identified that directly influence fat cell development. These include peroxisome

proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding proteins (C/EBPs)

and the basic helix-loop-helix family, adipocyte determination and differentiation factor

1/sterol regulatory element binding protein-1 (ADD1/SREBP1c) (Rosen et al., 2000)

20

After the onset of differentiation, a cascade of gene expression begins with the rapid

induction of C/EBP β and δ (Figure 1.8).

Concomitantly, synchronous re-entry into the cell cycle occurs and cells proceed through

a mitotic clonal expansion phase that consists of approximately two rounds of mitosis.

Near to or upon completion of mitotic clonal expansion, expression of C/EBPα and

PPARγ is induced, and expression of C/EBP β and δ begins to decline (Dowell et al.,

2000). This induction is likely to be a direct transcriptional effect through C/EBP binding

sites in the PPARγ promoter. PPARγ, in the current model for a transcriptional network

in adipogenesis, is then responsible for inducing C/EBPα. C/EBPα exerts positive

feedback on PPARγ to maintain the differentiated state (Rosen et al., 2000). Both

C/EBPα and PPARγ, the latter in combination with the obligate heterodimeric partner

and nuclear hormone receptor family member, retenoid X receptor (RXR)α, have been

shown to bind regulatory elements within the promoter of the 422/aP2 gene.

Figure 1.8: The transcriptional control of adipogenesis involves the activation of several families of transcription factors (Rosen et al., 2000)

21

In addition ADD1/SREBP1c, a member of the basic helix-loop-helix leucine zipper

transcription factor class, promotes lipogenic gene expression and stimulates production

of an unidentified PPARγ ligand (Dowell et al., 2000). ADD1/SREBP1 can activate

PPARγ by inducing its expression as well as by promoting the production of an

endogenous PPARγ ligand. Thus, C/EBPα, PPARγ and ADD1/SREBP1 cooperatively

promote adipogenesis and subsequent maintenance of the adipocyte phenotype (Rosen et

al., 2000).

Thiazolidinediones (TZDs) are structurally related agents used for the treatment of type II

diabetes. In addition to improving glucose metabolic insulin sensitivity and reducing the

requirement for insulin, these agents impressively reduce hypertriglyceridemia and have

been reported to lower plasma free fatty acid (FFA) levels in various animal models of

insulin resistance. Despite the fact that these compounds have been studied for almost 20

years, the in vivo mechanisms of insulin-sensitization remain unclear. Improvements in

glucoregulation may occur via their action on lipid metabolism; a body of evidence

suggests that an oversupply of fatty acids to insulin-sensitive target tissues, particularly

liver and skeletal muscle, contributes to the development of insulin resistance. Treatment-

induced reductions in fatty acid availability may thus contribute to the insulin-sensitizing

effects of these compounds (Oakes et al., 2001).

TZDs may exert their effects via ligand activation of PPARγ, which is primarily

expressed in adipose tissue. PPARγ activation contributes to the triggering of

differentiation of preadipocytes and induces expression of genes involved in the transport

and sequestration of FFA. These cellular and molecular effects of PPARγ agonism could

influence FFA exchange between adipose and other tissues, but direct evidence based on

measurements of in vivo fluxes and the metabolic fate of FFA are lacking (Oakes et al.,

2001).

Compounds that bind to and activate the PPARγ subunit of the PPARγ - RXR nuclear

receptor heterodimer alter transcription of genes involved in glucose and lipid

metabolism. Included in these target genes are lipid transporters (CD36, aquaporin), key

22

metabolic enzymes (lipoprotein lipase, phosphoenolpyruvate carboxykinase, uncoupling

protein-1), adipocyte-enriched signaling molecules (leptin, resistin, ACRP30,

FIAF/PGAR), lipid-modulated nuclear receptors (LXRα), and an intermediate in the

insulin signaling pathway (c-Cbl-associating protein) (Forman, 2002). TZD treatment

therefore results in expression of a number of adipocyte-specific genes so that PPARγ

activation and/or overexpression is essentially associated with adipose-cell differentiation

and adipogenesis. Therefore, the clinical observations that treatment with TZDs improves

insulin-stimulated glucose uptake (muscle) and endogenous (essentially hepatic) glucose

production while PPARγ is mainly expressed in fat cells makes it difficult to link cellular

and metabolic mechanisms of action. In addition, considering the well known connection

between obesity and insulin resistance, it seems paradoxical that an agent that promotes

adipogenesis should improve insulin sensitivity (Matthaei et al., 2000).

A number of hypothetical schemas to reconcile these apparent quandaries and explain the

overall mode of action of TZDs have been put forward. First, the minute quantities of

PPARγ expressed in muscle may be sufficient or alternatively might be induced during

treatment with TZDs, leading to a direct PPARγ-mediated response. Second, the effect of

TZDs may also be mediated by FFA, which have been shown to interfere with muscle

glucose metabolism and contribute to the impaired insulin-stimulated glucose. Since

TZDs have been shown to selectively stimulate lipogenic activities in fat cells, a

TZD/PPARγ-mediated “fatty-acid-steal phenomenon” has been proposed leaving less

FFAs available for muscle. Third, TZDs have been shown to reduce expression levels in

fat cells of TNFα and leptin, both of which have been implicated in obesity-related

insulin resistance. Although the definite role of the cytokine TNFα for human insulin

resistance remains to be determined, TNFα has been shown to interfere with proximal

insulin signaling events. In addition, leptin has been shown to impair insulin signaling in

isolated rat adipocytes. Since TZDs have been shown to reduce expression levels of both

TNFα and leptin, in fat cells, they could contribute to alleviating obesity-related insulin

resistance. Which of these mechanisms plays the most important role in vivo is unclear at

23

present, but since they are not mutually exclusive all of them may be involved (Matthaei

et al., 2000)

It is evident that there are a number of critical gaps in our understanding of the type II-

PPARγ connection. For example, PPARγ is required for adipogenesis and its synthetic

agonists increase adipose mass in vivo. This is unexpected, since insulin resistance

worsens in most patients as fat mass increases. Therefore the question raised is how an

adipogenic agent can also act as an antidiabetic agent. The PPARγ ligand deficiency

hypothesis claims to provide the answer to this particular unresolved matter. LG754

defines a new class of nuclear receptor agonist that has minimal coactivator recruitment

activity and therefore minimal inherent transcriptional activity. Instead, this compound

activates transcription by allosterically enhancing the ligand binding activity of its partner

receptor, PPARγ. LG754 therefore represents the first example of a nuclear receptor-

sensitizing agent. As well as being a PPARγ sensitizer, it has been found that LG754

relieves insulin resistance in vivo. These findings have important implications, since the

molecular events that result in insulin resistance remain obscure. PPARγ agonists have

the interesting property of lowering blood glucose in diabetic animals but not in non-

diabetic animals. This implies that PPARγ ligands reverse or replace a deficiency that is

unique to the diabetic state. It is speculated that insulin resistance arises from a relative

deficiency in endogenous PPARγ and that PPARγ agonists are antidiabetic agents,

because they correct this deficiency (Forman, 2002).

Although the identity of the endogenous PPARγ ligand is unknown, it is known that the

transcription factor ADD1/SREBP1c is required to produce an endogenous ligand in

adipocytes. It has been shown that SREBP-1c levels are lower in obesity, suggesting that

the obese state may be associated with a corresponding decrease in endogenous PPARγ

ligands. While this response may provide short term benefits, a chronic deficiency in

PPARγ ligands eventually lead to the development of insulin resistance (Forman, 2002).

24

1.4.1.4) Insulin

The primary goal in the treatment of diabetes mellitus is to prevent the development of

the metabolic abnormalities experienced by diabetics. For many diabetics, the goal can be

realized only by supplemental or replacement insulin therapy. Insulin is required by type

II diabetics who cannot be maintained adequately on oral hypoglycaemic agents or

dietary regulation. No single insulin preparation or combination of preparations can

successfully meet the demands of such a diverse group. Consequently, a large number of

insulin preparations have been developed, each of which has certain advantages and

disadvantages. Although all of these preparations consist primarily of insulin and exhibit

the biologic effects of insulin, they do differ in their onset and duration of action.

Accordingly, they are classified on the basis of their duration of action into short (usual

onset 0.5-2 hours; usual duration 3-6 hours), intermediate (usual onset 3-6 hours; usual

duration 12-20 hours) and long-acting categories (usual onset 6-12 hours; usual duration

18-36 hours). The time course of action of any insulin may vary considerably in different

individuals, or at different times of day in the same individual. Consequently, these time

courses should be considered only as general guidelines (Foye et al., 1995).

1.5 Plants and the treatment of Diabetes Mellitus Medicinal plants have been part of the great healing traditions around the world going

back thousands of years. The World Health Organisation (WHO) defines traditional

medicine as health practices, approaches, knowledge and beliefs incorporating plant,

animal and mineral based medicines, spiritual therapies, manual techniques applied

singularly or in combination to treat, diagnose and prevent illness or maintain well-being.

In 2002 WHO launched its first comprehensive traditional medicine strategy to assist

efforts to promote affordable, effective and safe use of traditional medicine and

complimentary alternative medicine. In Africa, traditional medicine is used by up to 80%

of the population to meet primary healthcare needs and is crucial in the fight against

diseases. The ratio of a conventional, or western-trained general practitioner to patients is

1:20 000, whereas the availability of traditional medicine practitioners is 1:200 to 1:400.

25

This highlights the need for reliable and affordable herbal medicines that are locally

available (www.i-sis.org.uk/). More than 1123 species of plants have been used

enthnopharmacologically or experimentally to treat symptoms of diabetes mellitus. These

are very large and widely distributed families and the phylogenetic distance between even

this select group is a good indication of the varied nature of the active constituents. It is

therefore necessary to learn more about particular groups of hypoglycaemic natural

products and their mechanism of action before this method of drug discovery can be

successfully employed (Wagner and Farnsworth, 1994). The high percentage of active

plants found probably reflects, at least in part, the great variety of possible active

constituents and mechanism of action.

1.5.1 Sutherlandia frutescens (Fabaceae)

There is no distinction made between the use of S. frutescens and its subspecies

microphylla for medicinal purposes. These plants are among the most multi-purpose and

useful of the medicinal plants in southern Africa. Conditions that have been treated with

these plants include fever, poor appetite, indigestion, gastritis, oesophagitis, peptic ulcer,

dysentery, cancer (prevention and treatment), diabetes, colds and flu, cough, asthma,

chronic bronchitis, kidney and liver conditions, rheumatism, heart failure, urinary tract

infections and stress and anxiety (Wagner and Farnsworth, 1994). Sutherlandia

frutescens (subspecies microphylla) genus Fabaceae (pea and bean/leguminosae) is a

perennial shrub that grows wild in the arid regions of Botswana, Namibia, Zululand,

Western and Eastern Cape regions of Africa. Sutherlandia can grow up to 1.5 metres in

height in optimum conditions of stony grasslands exposed to constant sunshine in

daylight hours. A display of blood red flowers bloom from June to December and its

seeds are carried in greenish- red papery pods, which are almost transparent, as can be

seen in figure 1.9. The pinnate and compound shaped leaves have a green-grey colour

giving the bush a silvery appearance (www.i-sis.org.uk/). Although this plant has been

renamed to Lessertia, it is still referred by its more favoured name of Sutherlandia

(Muller, 2002). In this study, Sutherlandia frutescens subspecies microphylla was

referred to by its more common name, Kankerbos.

26

A number of highly active compounds, including canavanine, pinitol and the amino acid

GABA (gamma-aminobutyric acid), occur in high quantities in Sutherlandia species,

suggesting that there is indeed a scientific basis for some of the folk uses for serious

medical conditions. L-canavanine is a potent L-arginine antagonist with documented

antiviral, anti-bacterial, antifungal and anticancer activities. Pinitol (2-deoxy-2-amino-

galactopyranosyl) is a known anti-diabetic agent (www.sutherlandia.org). Narayanan et

al, (1987) showed that pinitol isolated from Bougainvillea spectabilis had hypoglycaemic

and anti-diabetic action. GABA is an inhibitory neurotransmitter that could account for

the plant being used for stress and anxiety, and for the improvement in mood and well-

being experienced by many patients. In addition a novel triterpenoid glucoside, SU 1, has

been isolated and characterized, and has promising biological activities

(www.sutherlandia.org).

1.5.2 Toxicity of Hypoglycaemic Plants

An in-depth literature review and properly controlled experimental bioassays should be

carried out in order to confirm the non-toxicity of a specific antidiabetic plant. Toxicity is

influenced by the plant part, method of preparation, route of administration and test

organism. Diabetes mellitus is a chronic condition with no known cure and antidiabetic

drugs must be administered for a patients entire lifetime. Therefore it is important that

Figure 1.9: Sutherlandia frutescens (Seier et al., 2002)

27

chronic toxicity studies be performed before recommending a plant-derived drug for

antidiabetic therapy (Wagner and Farnsworth, 1994).

An investigation into the possible toxicity of consumption of Sutherlandia leaf powder

(Sutherlandia frutescens subspecies microphylla) in vervet monkeys (Chlorocebus

aethiops), was carried out by determining a variety of biochemical, haematological,

physiological and physical variables. These variables reflected liver, kidney, muscle,

respiratory, intestinal, bone and general biological function. The conclusions referred to

Sutherlandia leaf powder consumption in adult male vervet monkeys for three months.

At the recommended dose, 3x the recommended dose and 9x the recommended dose,

Sutherlandia leaf powder consumption was found not to be associated with toxic or other

side-effects (Seier et al., 2002).

28

Chapter 2

Introduction to the present study

Insulin resistance and non-insulin-dependent diabetes mellitus (type II) have reached

epidemic status in industrialized societies. Over 125 million people worldwide suffer

from type II diabetes, and these individuals face a dramatically increased risk for

developing atherosclerotic heart disease, stroke, renal disease, blindness and limb

amputations. It is thus alarming that the number of type II cases have increased 5-fold in

the past decade, a trend that is predicted to continue. Equally worrisome is that type II,

initially defined as a disease of adult onset, is now appearing in adolescents (Forman,

2002). Type II diabetes is more common than type I and accounts for 90% of diabetic

cases in South Africa. It has been estimated that of the four million diabetics in South

Africa, half remain undiagnosed.

The enormous costs of modern treatment and the evidence that current methods of

treatment fail to achieve the ideals of normoglycaemia and the prevention of diabetic

complications, indicate that alternative strategies for the prevention and treatment of

diabetes must be developed. Due to the fact that almost 90% of people in developing

countries still rely on traditional medicines for their primary health care and the fact that

scientific investigations of traditional medicines have led to the discovery of drugs now

in professional use worldwide, a synthesis of local traditional and modern knowledge and

techniques for the management of diabetes should be feasible (Wagner and Farnsworth,

1994). Therefore indigenous, renewable, medicinal plant resources could prove to be the

practical and cost-efficient alternative that is clearly and desperately needed.

The present study was planned keeping the above issues in mind. The objective of this

project was to optimize the methodology required to screen and determine the

effectiveness of the specific plants as antidiabetic potentiates, through observing if

increases in glucose utilisation and insulin secretion occurred under treatment. Once

optimal methodology is achieved through this specific project, the parameters used to

29

validate the models will in the future be able to be applied to scientifically establish the

antidiabetic effect(s) and mechanism(s) of the plants under investigation.

The cell lines used in this study, namely 3T3-L1 preadipocytes, Chang liver cells, C2C12

muscle cells and INS-1 rat pancreatic cells, were investigated as potential in vitro models

for type II diabetes. Indigenous antidiabetic plants were used as test samples in order to

determine optimal conditions for screening and clearing mechanisms.

30

Chapter 3

Models for glucose utilization

The raison d’etre of the adipocyte is to store energy (in the form of triacylglycerol) for

use during periods of caloric insufficiency. Adipocytes first appear late in fetal

development preparatory to postnatal life when substantial energy reserve is needed to

survive periods of fasting (Mandrup and Lane, 1997). Adipogenesis has been one of the

most intensely studied models of cellular differentiation. In part this has been because of

the availability of in vitro models that faithfully recapitulate most of the critical aspects of

fat cell formation in vivo. This includes morphological changes, cessation of cell growth,

expression of many lipogenic enzymes, extensive lipid accumulation, and the

establishment of sensitivity to most or all of the key hormones that impact on this cell

type, including insulin (Rosen and Spiegelman, 2000).

3T3-L1 is a continuous mouse strain of 3T3 developed through clonal isolation, which

has a fibroblast-like morphology. Cells undergo a pre-adipose to adipose-like conversion

as they progress from rapidly dividing to a confluent and contact inhibited state

(www.biotech.ist.unige.it/cldb/c173.html). Considerable progress has been made in

understanding the molecular mechanisms of adipocyte biology using the 3T3-L1 cell line

as a model. A large body of evidence shows that differentiation of 3T3 preadipocytes

faithfully mimics the in vivo process giving rise to cells that possess virtually all the

biochemical and morphological characteristics of adipocytes. 3T3-L1 cells propagated

under normal conditions have a fibroblastic phenotype. However, when treated with a

combination of dexamethasone (DEX), isobutylmethylxanthine (IBMX) and insulin,

3T3-L1 cells adopt a rounded phenotype and within 5 days begin to accumulate lipids

intracellularly in the form of lipid droplets. Treatment of cells with DEX activates the

transcription factor CCAAT/enhancer-binding protein b (C/EBPb). IBMX inhibits

soluble cyclic nucleotide phosphodiesterases and results in increased intracellular cAMP

levels (Elks and Manganiello, 1985). At the nuclear level, treatment with IBMX results in

activation of the related transcription factor C/EBPd. C/EBPb and d in turn induce

31

transcription of C/EBPα and PPARγ. Within 3 days of exposure to inducers, the cells

undergo two rounds of mitosis, termed mitotic clonal expansion, which are required for

differentiation. Insulin or insulin-like growth factor-1 promote adipocyte differentiation

by activating PI3-kinase and Akt activity. Modulation of the activity of the forkhead

transcription factor Foxo1 appears to be necessary for insulin to promote adipocyte

differentiation. C/EBPα and PPARγ direct the final phase of adipogenesis by activating

expression of adipocyte-specific genes, such as fatty acid synthetase, fatty acid binding

protein, leptin and adiponectin. Endogenous negative regulators of adipocyte

differentiation, such as Pref-1 and Wnt-10b, are highly expressed on undifferentiated

3T3-L1 cells, and are down-regulated upon addition of adipogenesis inducers

(www.chemicon.com).

The evolution of glycerol-3-phosphate dehydrogenase (G3PDH) activity as well as of

G3PDH protein and mRNA is used as an indicator of adipocyte differentiation. The

enzyme accumulates to a low extent during culture in the absence of insulin. When

insulin is present, the enzyme level is dramatically increased. DEX accelerates the

insulin-dependent adipose conversion but alone does not ensure the complete

differentiation process (Gaben-Cogneville et al.,1990). IBMX also functions in

accelerating the differentiation of 3T3-L1 preadipocytes. This effect seems to be

mediated by increased cyclic AMP levels (Wiederer and Loffler, 1987). Following

hormonal induction, confluent preadipocytes undergo mitotic clonal expansion, become

growth arrested, and then coordinately express adipocyte gene products (Mandrup and

Lane, 1997).

The C2C12 muscle cell line is a subclone from a myoblast line established from normal

adult C3H mouse leg muscle. The C2C12 cell line differentiates rapidly, forming

contractile myotubes and producing characteristic muscle proteins. Cultures must not be

allowed to become confluent, as this will deplete the myoblastic population in the culture

(Tortorella and Pilch, 2002).

32

Insulin promotes the postprandial clearance of glucose from the blood primarily into

skeletal muscle in both humans and rodents. Glucose transport into skeletal muscle is

regulated by translocation of the glucose transporter GLUT4 from intracellular vesicles to

the plasma membrane (Aslesen et al., 2001). Several lines of independent

experimentation support the notion that insulin-dependent regulation of GLUT4

movement is similar or identical in skeletal muscle and adipocytes. However, GLUT4

translocates in response to exercise and hypoxia in the former and not in the latter tissue.

Insulin-dependent GLUT4 translocation is PI3-kinase dependent in both tissues. Overall,

it is the inability of insulin to promote glucose uptake in skeletal muscle that causes

insulin resistance, and, eventually type II diabetes (Tortorella and Pilch, 2002).

The development of muscle and fat cell lines has facilitated our understanding of the

translocation process, specifically C2C12 and 3T3-L1 cell lines, which are unique in their

expression of the GLUT4 protein. Both of these cell lines undergo differentiation in

culture from myoblasts into myotubes in the case of C2C12 and from fibroblasts into

adipocytes in the case of 3T3-L1. In both models, GLUT4 expression occurs on and after

differentiation into myotubes or adipocytes (Ueyama et al., 1999).

The liver plays a central role in glucose homeostasis. During the postabsorptive and

fasting period, >90% of glucose production is derived from the liver. When glucose

enters the blood following feeding, the liver switches from net glucose production to net

glucose uptake to lessen the rise in blood glucose. Such switching is regulated by raised

blood glucose per se and plasma insulin. One third of an oral glucose load is taken up by

the liver. Glucose uptake by the liver is dependent on the amount of glucose reaching it,

the insulin level within the hepatic sinusoids and a signal generated by portal glucose

delivery. Impaired suppression of hepatic glucose production and a defect in hepatic

glucose uptake in response to the raise in plasma glucose and insulin are major

pathogenesis in fasting and excessive postprandial hyperglycemia, that are common

features in obesity and diabetes (Rutter et al., 2003). Liver cell membranes have non-

insulin-dependent glucose transporters and therefore do not require insulin for glucose

entry. However insulin, acting through intracellular signaling systems, stimulates the

33

liver cell enzymes that promote utilization of glucose for the synthesis of glycogen,

amino acids, proteins and fats, particularly fatty acids. The glucose transporter, GLUT2,

is primarily expressed in hepatocytes. GLUT2 is a low-affinity receptor with a high

turnover rate. These kinetic properties allow GLUT2 to function in the liver where

glucose transport must not be rate limiting for influx or efflux (Medina and Owen, 2002).

The Chang liver cell line is derived from non-malignant human tissue. These cells are

epithelial in morphology and tend to pile up in cultures with high population density

(www.biotech.ist.unige.it).

Plant extracts were tested for potential antidiabetic activity by exposing these three cell

lines (3T3-L1 adipocyte cells, C2C12 muscle cells and Chang liver cells) to the samples

and determining the amount of glucose taken up. Each cell line represents a different in

vivo organ that is known to have an involvement in glucose homeostasis, each with its

own unique metabolic pathways and mechanism of glucose uptake. Therefore in order to

develop a model for determining glucose utilization, all three cell lines were required as

each represents a small part of the complete entity.

3.1 General methods

The methods described in the following sections are the final optimised ones used in the

glucose utilization models.

3.1.1 Maintenance of cell lines

The cells were grown in a humidified atmosphere containing 5% CO2 at 37ºC (Asfari et

al., 1992). The cells were routinely maintained in antibiotic free growth medium which

consisted of RPMI 1640 (Sigma) supplemented with 10% heat-inactivated fetal calf

serum (FCS) (Highveld Biological, SA). Proliferating preadipocytes were refed fresh

growth medium every 2-3 days. For routine maintainance, trypsin in phosphate-buffered

saline without Ca2+ and Mg2+ (PBSA) was used as the treatment to detach the cells from

the culture plate and after 5-10 minute exposure at 37ºC the cells were seeded in growth

34

medium using a split ratio of 1:5. Routine cell counting was carried out using an

improved Neubauer haemocytometer (Superior).

3.1.2 Cytotoxicity Assay A cell suspension was prepared in growth medium to give 4 000 cells per well using a

96-well plate (Nunc). Cells were inoculated in a volume of 200μl per well and 200μl

aliquots of growth medium was added to cell-free wells. The cells were preincubated for

24 hours after which they were fed fresh culture medium containing test compounds and

exposed for 48 hours after which cell survival percentage was determined using the

sulforhodamine B (SRB) assay.

3.1.3 Sulforhodamine B Assay Determination of cell growth and cell viability was performed by in situ fixation of cells,

followed by staining with a protein-binding dye, sulforhodamine B (SRB) (Sigma). The

SRB binds to the basic amino acids of cellular macromolecules; the solubilized stain is

measured spectrophotometrically to determine relative cell growth (Monks et al., 1991).

After 48 hours incubation with plant extract/medicine, 50μl cold 50% TCA (4ºC) was

added on top of the growth medium in each well (final concentration 10%) and an

incubation period of 1 hour at 4ºC followed. After removal of the contents of all the

wells, the plate was washed five times with tap water and air-dried. 100μl of 0.4% SRB

(w/v in 1% acetic acid) was added to all the wells and incubated for 10 minutes at room

temperature. The plate was then washed four times with 1% acetic acid to remove

unbound dye and air-dried until no moisture was visible. The plates could then be stored

for a period of up to 7 days or processed immediately as follows: 100μl 10mM Tris base

(pH 10.5) was added to solubilise the bound stain and the 96-well plate was placed on the

shaker for 5 minutes. Optical densities were read using the Multiscan MS microtiter plate

reader (Labsystem Multiscan MS, 665 Dosimat) at 540nm.

35

3.1.4 Glucose oxidase assay

The amount of glucose taken up by the cells in each experiment was indirectly measured

by assaying the amount of glucose remaining in the incubation medium using a glucose

oxidase kit (Glu-cinet®, Bayer), after a limited incubation time.

3.1.5 Cell viability assay

Due to the variability of cell numbers in different wells, the viability of the cells in each

well in each experiment should be determined so that any significant difference between

the cells exposed to the treatments compared to the control wells under chronic

conditions can be determined. The CellTiter-BlueTM viability assay kit (Promega, USA)

was used in accordance with the instruction manual protocol. This assay uses resazurin

(indicator dye) to measure the metabolic capacity of cells, thereby indicating cell

viability.

3.2 3T3-L1 cell line The 3T3-L1 preadipocyte cell line, used in the present study, was obtained from the

American Type Culture Collection (ATCC) through Highveld Biological (Johannesburg,

South Africa).

3.2.1 Induction of 3T3-L1 preadipocyte differentiation

Cells were plated at a density of 12 000 cells/well in 24-well plates, which were coated

with poly-L-lysine (PLL) (procedure described in section 4.1.1.3) and incubated for 24

hours in the above growth medium (section 3.1.1). On day 1, the growth medium was

replaced by supplemented medium, which consisted of RPMI 1640, supplemented with

10% FCS, 10 µg insulin/ml, 10-8 M DEX, 0.1mM IBMX and the appropriate plant extract

(Kankerbos or MRC2003). Cells were refed 48 hours later with the same supplemented

medium and after another 24 hours (day 4), this medium was removed and replaced with

growth medium including the plant extracts. After a further 48 hour incubation (day 6),

the cells were assayed in their appropriate experiments.

36

3.2.2 Adipocyte differentiation assays

This assay was used as the method of measuring the degree of fat cell differentiation.

3.2.2.1 Glycerol-3-phosphate dehydrogenase assay

In this assay glycerol-3-phosphate dehydrogenase (G3PDH) acts by reducing

dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate and at the same time there

is oxidation of the cofactor NADH to NAD+, which can be followed at 340nm (Wise and

Green, 1979). 3T3-L1 cells were grown and subcultured in 24-well plates, after which

they were harvested at 4ºC by the addition of 250μl Tris buffer (pH 7.5, containing 1mM

EDTA) and scraping the bottom of each well. Once the cells were suspended and

homogenized using a Dounce homogeniser with a Teflon pestle, the homogenate of each

separate well was transferred to individual eppendorfs and microfuged at 4ºC for 5min.

The pellet formed contained cell debris and the supernatant was collected for further

assaying.

The substrate was prepared by adding 0.5g wet weight of a Dowex-50 (H+) cation

exchange resin to 25mg cyclohexylammonium dihydroxyacetone phosphate dimethyl

ketal dissolved in 2ml water. This mixture was swirled for 30s and then the resin was

allowed to settle. The supernatant was removed and incubated at 40ºC for 4 hours at

which time the ketal was hydrolysed. The pH was then increased to 4.5 with a saturated

sodium bicarbonate solution.

To assay the supernatant for G3PDH activity, the following substances were sequentially

introduced into a microcuvette: 325µl water (37ºC); 50μl TEA buffer (pH 7.5, containing

6.66ml triethylamine, 3.6µl 2-mercaptoethanol and 420mg EDTA in 50ml water) (37ºC);

100µl sample supernatant (4ºC). The reaction was initiated by addition of 20µl DHAP

(4ºC). After rapid but gentle mixing, the cuvette was immediately placed in the 37ºC

heating mantle of a spectrophotometer and the decrease in absorption at 340nm followed

against air.

37

3.2.3 Protein concentration determination

Protein concentration was determined for calculation of the specific activity of the

enzyme, G3PDH. The protein concentration of the samples was determined using an

adapted Folin-Lowry method for microtiter plates.

3.2.3.1 Adapted Folin method for microtiter plates

A 2mg/ml BSA stock solution was prepared and diluted 10x on the day of use to prepare

the dilutions for the protein standard curve. The 0.2mg/ml sample was serially diluted to

give 6 points on the standard curve.

The procedure involved addition of 100μl serially diluted standard or sample (controls

contained 100µl of 25mM Tris buffer, pH 7.4, containing 1mM EDTA) and 25µl copper-

tartrate-alkaline solution. This was mixed and incubated for 10min at room temperature.

10µl diluted Folin-Ciocalteau phenol reagent (1:1 with 0.1 N NaOH) was added, mixed

and a further incubation period of 20min at room temperature followed. The absorbance

was read at 600nm on a microplate reader.

3.2.4 Glucose Uptake 3T3-L1 cells were grown and differentiated into adipocytes in medium which consisted

of RPMI 1640, supplemented with 10% FCS, 10 µg insulin/ml, 10-8 M DEX, 0.1mM

IBMX and the appropriate test compound or plant extract. The final method was not

achieved, however the reasons and explanations are discussed in section 3.6.1.3.

3.3 C2C12 cell line 3.3.1 Glucose uptake

The C2C12 skeletal muscle cell line was purchased from Highveld Biological

(Johannesburg, South Africa) and passage 8 to 11 was used in this study. Prior to this, a

previous study had been carried out and the growth and differentiation conditions of this

cell line had been optimised (Askew, 2003). It was determined that after being cultured in

38

RPMI 1640 (including 10% FCS) for three days, the cells were differentiated and ready

to be used for experimentation. After this time period, the cells could not be left

indefinitely otherwise they died suddenly. For the glucose uptake experiment, cells were

seeded at 4 500 cells/well into 96-well microtiter plates and cultured for 3 days in RPMI

1640 (including 10% FCS). For the glucose uptake experiment, all procedures were

completed at 37°C. The incubation medium (8mM glucose RPMI 1640 + 0.1% BSA +

specific treatment) was added to the appropriate wells (50μl/well) for an incubation time

of 1.5 hours. Only 8mM glucose RPMI 1640 (0.1% BSA) was added to the control wells.

Once incubation was complete, 10μl of the medium in the well was removed and placed

into a new 96-well microtiter plate after which 200μl glucose oxidase reagent (SERA-

PAK Plus, Bayer) was added to the sample. This was incubated at 37°C for 15 minutes

and absorbance read at 492nm using a Multiscan MS microtiter plate reader

(Labsystems).

Treatments tested for their efficacy on glucose uptake by C2C12 muscle cells included

the compounds BDM1, BDH1, pinitol (BDPl), metformin and insulin, all at the chosen

concentration of 1μM. Kankerbos and MRC2003 were tested using a concentration of

2.5μg/well. The acute effect of these treatments was observed by exposing the cells to

them for 90 minutes on the day of experimentation, whereas chronic effect was seen by

pre-exposing the cells to treatment for 48 hours prior to the glucose uptake experiment

and then again for 90 minutes during the experiment.

3.4 Chang cell line 3.4.1 Glucose uptake

Cells of the Chang liver cell line (Passage 22 to 24) were seeded at 6 000 cells/well into

96-well microtiter plates and cultured for 5 days in growth medium. On the day of the

experiment, all procedures were carried out at 37°C. 25μl of incubation medium (8mM

glucose RPMI 1640 + 0.1% BSA + the specific treatment) was added to the appropriate

wells for 3 hours. Control wells contained only 8mM glucose RPMI 1640 (0.1% BSA).

After incubation, 20μl was removed and placed into a new 96-well plate into which 80μl

double distilled water was added to give a 5x dilution. 50μl of this dilution was removed

39

and 200μl glucose oxidase reagent (Glu-cinet®, Bayer) was added to the 50μl medium

left in the well. After an incubation of 15 minutes at 37°C, the absorbance was measured

at 492nm using a Multiscan MS microtiter plate reader (Labsystems).

The treatments tested for their effect on glucose uptake in Chang liver cells included the

following: BDM1, BDH1, pinitol (BDP1), metformin and insulin, all used at 1μM

concentration. The plant extracts, Kankerbos and MRC2003, were used at 12.5μg/well.

Acute and chronic effects were both investigated. Acute effect was observed by exposing

the cells to the treatments only on the day of the experiment for the 3 hour incubation

time. However, chronic effect included adding the treatments to the cells for 48 hours

prior to the day of experiment and then again for the 3 hours.

3.5 Statistical analysis

All results were analyzed using the data analysis program in Microsoft Excel. The

statistical tests were carried out on original data obtained from the experiments

performed. The relationship between two variables was determined by the use of the

t-test, two-sample assuming equal variances.

3.6 Results and Discussion 3.6.1 3T3-L1 cell line 3.6.1.1 Cytotoxicity

In order to determine the effect of treatments on the viability of 3T3-L1 cells over time,

cytotoxicity tests were carried out whereby cells where exposed to treatments for 48

hours. These tests allow one to determine the concentrations of each treatment that are

not cytotoxic to the specific cell line and thereby use one that is non-cytotoxic in further

experimentation. Basically, any concentration that shows less than 80% of control is too

cytotoxic for the specific cell line it is being tested on and is not eligible for further use.

Ideally the concentration of the treatment chosen should not be cytotoxic to the cell line

however it should elicit an effect. Concentrations tested for the compounds (BDM1,

40

BDH1, pinitol (BDP1) and metformin) included 0.1, 1, 10, 50 and 250μM. The

concentrations tested for the plant extracts, namely Kankerbos and MRC(2003), ranged

from (0.0025 – 25μg/well) (Figure 3.1).

0

20

40

60

80

100

120

-2 -1 0 1 2 3

log [BDM1]μM

% o

f con

trol

0

20

40

60

80

100

120

-2 -1 0 1 2 3

log [BDH1]μM

% o

f con

trol

0

20

40

60

80

100

-2 -1 0 1 2 3

log[BDP1]μM

% o

f con

trol

0

20

40

60

80

100

-2 -1 0 1 2 3

log[Metformin]μM

% o

f con

trol

0

20

40

60

80

100

120

0 1 2 3 4 5

log[MRC2003]ng/well

% o

f con

trol

Figure 3.1: Cytotoxicity results of compounds (BDM1, BDH1 BDP1 and metformin) and plant extracts (Kankerbos and MRC2003) on 3T3-L1 adipocyte cells after a 48 hour exposure period. Data points represent the mean ±SD (n=6 wells) from a single experiment.

0

20

40

60

80

100

120

0 1 2 3 4 5

log[Kankerbos]ng/well

% o

f con

trol

41

In figure 3.1, the compounds (BDM1 and BDH1) showed no cytotoxic effect on the 3T3-

L1 cell line. The concentrations of BDP1 showed varied results for percentage control and

metformin was only cytotoxic to the cells at 250μM. For these compounds, no significant

cytotoxicity was seen and it was decided that all further experimentation carried out with

3T3-L1 cells would be done so at a concentration of 1μM. For the plant extracts

(Kankerbos and MRC2003) only the cells exposed to the highest concentration of

25μg/well showed signs of cytotoxicity. All results shown for the plant extracts are

μg/well, however a conversion table showing the μg/ml values is shown in Appendix 1

3.6.1.2 Glycerol-3-phosphate dehydrogenase activity

As the 3T3-L1 cells differentiated, observations were an alteration in cell shape and the

accumulation of lipid droplets in the cytoplasm. Samples tested included Kankerbos

(12.5μg/well/1ml) and MRC (12.5μg/well/1ml) plant extracts and the control included

cells not exposed to any form of treatment other than inducers where applicable. Once the

cells had reached confluency, the inducers of differentiation (insulin, DEX and IBMX)

were added for 3 days to the appropriate cells. After this they were removed and the cells

fed without inducers for another 7 days.

The glycerol-3-phosphate dehydrogenase assay functions in measuring fat cell

differentiation. The reason for using this enzymatic assay is because G3PDH is an

intermediatory enzyme in the pathway by which triacylglycerols are formed, therefore

before fat droplets form the presence of G3PDH is detectable. G3PDH reduces DHAP to

G-3-P and corresponding to this reduction, NADH is oxidized to NAD+. With the

oxidation is a decrease in absorbance at 340nm and by using the slope of this decrease,

one can calculate the enzyme activity. Determination of protein concentration then

enables one to finally calculate the specific activity of G3PDH.

42

Figure 3.2 shows the G3PDH specific activity in the 3T3-L1 cells after exposure to the

plant extracts with and without induction of differentiation by inducers. The control

sample gave results that were expected, that is an increase in G3PDH specific activity

when cells were exposed to the inducers of differentiation compared to without inducers

added. The plant extracts showed increased G3PDH specific activity with and without

inducers compared to the control, although not all samples were significantly different to

the control. This indicates that both Kankerbos and MRC extracts stimulate fat cell

differentiation. These findings would lead us to believe that the plant extracts act on

PPARγ, which is a transcription factor that directly influences fat cell development

(Rosen et al, 2000). The reason why the plant extract samples with inducers gave lower

values compared to without, was due to a large variation in sample results and therefore

greater error bars. This experiment was planned to be repeated; however problems with

the 3T3-L1 cell line pursued, this will be discussed in the following section. However it

was felt based on these results, that a positive control for PPARγ involvement should be

included in further investigations. An example of a positive control would have been the

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

Control Kankerbos MRC

G3P

DH

spec

ific

activ

ity

(mU

/mg

prot

ein)

Without Inducers

With Inducers

Figure 3.2: The effect of plant extracts (Kankerbos and MRC; 12.5μg/well) (with and without inducers) on glycerol-3-phosphate dehydrogenase (G3PDH) specific activity. Values shown are the mean ± SEM (n=4 wells) from an individual experiment. *P < 0.001 when compared to the control.

*

43

use of a thiazolidinedione, such as pioglitazone, which is a PPARγ ligand/activator,

which has been found to increase insulin sensitivity and decrease blood glucose.

3.6.1.3 Glucose uptake

In the introduction to this project, it was mentioned that one of the objectives would be to

optimize the methods required to screen and determine the effectiveness of specific plant

extracts as antidiabetic potentiates. One of the ways to do this was through creating a

model for observing glucose uptake in cells.

The idea of stream-lining a method entails increasing the amount of samples to be tested

but at the same time using materials sparingly. Based on this it was initially decided to

seed the 3T3-L1 cells (4 000 cells/well) into 96-well plates and test the amount of

glucose taken up by undifferentiated and differentiated cells. However this method was

found not to be sensitive enough due to an inadequate amount of cells and it was decided

following experiments would be carried out in 24-well plates (24 000 cells/well).

The positive controls chosen for glucose uptake in 3T3-L1 cells due to their antidiabetic

activity were metformin and insulin. Insulin specifically for its positive effect on the

translocation of GLUT4 to the cell surface thereby promoting glucose uptake. In addition

to these, BDP1 was included as a test substance as it is suspected to have antidiabetic

effects (Narayanan et al., 1987). The first experiment making use of the 24-well plates

involved testing the positive controls (1μM), as well as BDP1 (1μM) and the plant

extract, Kankerbos (12.5μg/well) on differentiated 3T3-L1 cells. The effects of the

treatments were tested by exposing the cells to the specific treatment only during the

glucose uptake experiment (acute conditions).

Glucose transport in 3T3-L1 cells is facilitated due to the presence of the glucose

transporters. The intracellular GLUTs are both GLUT1 and GLUT4, and both are

recruited to the cell membrane (Zierler, 1999). Under basal conditions, GLUT4 is

efficiently sequestered in intracellular membrane compartments. When cells are treated

44

with insulin, the sequestered pool of GLUT4 is mobilized and translocates to the plasma

membrane (Perrini et al., 2004). Insulin increases the rate of GLUT4 exocytosis, with

little or no decrease in its rate of endocytosis, so that in adipocytes the proportion of

GLUT4 at the cell surface increases from less than 10% in the absence of insulin to 35 to

50% in its presence (Bogan et al., 2001). Therefore one expects to see a marked increase

in glucose uptake in these cells in response to insulin.

Although GLUT4 expression only occurs on and after differentiation into adipocytes

(Ueyama et al., 1999), it was decided that a comparison of glucose uptake in

undifferentiated and differentiated 3T3-L1 cells would be beneficial in the first phases of

method optimization.

In figure 3.3, the results of the comparison of glucose uptake in undifferentiated (a) and

differentiated (b) 3T3-L1 cells are shown. It can be seen that over time there is an

increase in the amount of glucose taken up in the basal samples as well as the treated

samples in both scenarios. However in the differentiated 3T3-L1 cells there was greater

amount taken up overall, thus confirming the presence of GLUT4 transporters due to

their expression in these cells. Due to this confirmation, it was obvious for streamlining

purposes, to only use differentiated 3T3-L1 cells for further glucose uptake experiments.

For reproducibility purposes, the experiments were repeated and the following graphs (10

minute and 120 minute incubation times) show the combined results of two individual

glucose uptake experiments under acute conditions. Figure 3.4 shows the results of

glucose uptake as a percent of the control cells (basal-state) after 10 minute incubation

time. The positive controls, metformin and insulin both at a 1μM concentration, caused a

significant increase in glucose utilization compared to the control within 10 minutes. This

immediate effect by insulin was expected as it is known to initiate the translocation of

GLUT4 to the cell surface plasma membrane. Also seen within 10 minutes of treatment

exposure was that the plant extracts, Kankerbos (12.5μg/well) and MRC2003

(2.5μg/well), significantly enhanced glucose uptake in 3T3-L1 adipocytes.

45

0

50

100

150

200

250

300

350

400

Control Kankerbos MRC2003 Metformin Insulin

% o

f con

trol

Figure 3.4: Acute effect of treatments on glucose uptake in 3T3-L1 cells after 10 minute incubation time. Values shown are the mean ± SEM (n=6 or 8) from two experiments combined. *P < 0.05; **P < 0.01; ***P<0.001 when compared to the control.

*

Figure 3.3: The percentage glucose taken up by undifferentiated (a) and differentiated (b) 3T3-L1 cells when exposed to treatments under acute conditions. Positive controls: 1μM metformin and 1μM insulin. Treatments tested included: 1μM BDP1 and 12.5μg/well Kankerbos. Values shown are the averages from two experiments tested in quadruplicate.

05

1015202530354045

Control Metformin Insulin

% G

luco

se u

ptak

e

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120 minutes

05

1015202530354045

Control BDP1 Kankerbos Metformin Insulin

% G

luco

se u

ptak

e

10 minutes

120 minutes

a)

b)

** *** **

46

The results of glucose utilization as a percent of control after 120 minutes (Figure 3.5),

showed once again an increase in glucose uptake in the cells exposed to the treatments. In

other words, the treatments stimulated glucose utilization compared to basal glucose

uptake. Although there was an increase, it can be seen that the difference between the

control and treatments is not as large at 120 minutes compared to 10 minute incubation.

This is expected for a number of reasons, including saturation of the cells with glucose

after 120 minutes of uptake, a decrease in the amount of glucose available in the media as

well as possible decrease in the activity of the treatments over such a long exposure time.

Due to these results as well as the need to streamline the method, it was decided to

choose an incubation time between 10 and 120 minutes, where a significant difference

between control and treated cells would still be seen. For all further glucose uptake

experiments with 3T3-L1 cells it was decided to sample at 60 minutes. During further

experimentation, it was observed that the viability of the cells during the glucose uptake

experiments were questionable, so it was thought that the addition of 10% serum might

be beneficial and glucose standard curves with and without serum were carried out.

0

50

100

150

200

Control Kankerbos MRC2003 Metformin Insulin

% o

f con

trol

Figure 3.5: Acute effect of treatments on glucose uptake in 3T3-L1 cells after 120 minute incubation time. Values shown are the mean ± SEM (n=6 or 8) from two experiments combined. ***P<0.001 when compared to the control.

***

47

Figure 3.6 shows that adding 10% serum to the incubation media (8mM glucose RPMI

1640) during experimentation would not affect the amount of glucose taken up by the

cells as the glucose standard curves (with or without 10% serum) gave similar results.

Based on these findings, it was decided to include 10% serum in the media in future

glucose uptake experiments involving 3T3-L1. This would hopefully increase their

viability during the experiment.

The results obtained up until this stage in the project allowed some decisions to be made

regarding the optimization of the experiment. As mentioned 10% serum would be

included in the incubation media during the glucose uptake experiment. Also, as

mentioned, it was decided to take samples at 60 minutes only in order to streamline the

Figure 3.6: Comparison of glucose standard curve with and without 10% serum in 8mM RPMI 1640. Data points are the mean ±SD (n=4) from an individual experiment.

Glucose standard curve without 10% serum

y = 15.866xR2 = 0.9957

0.0

0.5

1.0

1.5

2.0

0.00 0.02 0.04 0.06 0.08 0.10 0.12

[Glucose] μg/well

A49

2nm

Glucose standard curve with 10% serum

y = 15.802xR2 = 0.9816

0.0

0.5

1.0

1.5

2.0

0 0.02 0.04 0.06 0.08 0.1 0.12

[Glucose] μg/well

A492

nm

48

procedure. However due to the cells beginning to peel during the experiment, it was

thought that lining the wells with PLL when seeding, would possibly be beneficial and

aid in cell attachment. Originally during the G3PDH experiments, the wells used were

treated with PLL and no problems were seen.

Following these changes in the methodology, seeding and differentiation induction of

3T3-L1 cells was continued on numerous occasions with glucose uptake experiments

being the final step. However despite the above attempts to overcome the problems, the

peeling of the cells from the edges into the centers of the wells persisted. This hampered

obtaining accurate results, as it was not known what the effect of the peeling was on the

ability of the cells to take up glucose. Although the wells were being plated with PLL

before seeding this did not seem to be improving the situation, so another possibility was

tested, that of plating the wells with FCS (Figure 3.7).

050

100150200250300350400450

Control BDP1 MRC(methanol) MRC(2002) MRC(2003)

% o

f con

trol

050

100150200250300350400450

Control BDP1 Kankerbos MRC(2003)

% o

f con

trol

a)

b)

Figure 3.7: Glucose uptake as a percent of control in 3T3-L1 cells with wells lined with PLL (a) and FCS (b). Values shown are the averages from a single experiment tested in quadruplicate.

49

Changing certain criteria continued, with the aim of finding the right method that allowed

glucose uptake experiments to proceed without the cells lifting and peeling. Pretreating

the wells with PLL and FCS was one change that was abandoned as they were not

improving the situation. Another change was that of cell number. Up until this stage, 24

000 cells/well were used, however it was thought that by decreasing this, peeling might

be prevented. Seeding at 12 000, 8 000 and 6 000 cells/well was tried, however no

improvement was observed and with low seeding numbers it prolonged induction of

differentiation due to waiting for the cells to get to confluency.

The samples, BDM1 and BDH1, were obtained from the Medical Research Council

(MRC). These compounds were expected to be within the plant extracts being tested on

3T3-L1 cells, therefore they were included in the following experiment.

The graph in figure 3.8 indicates glucose uptake in 3T3-L1 cells, however the results

confirmed the problems with the cells. When the incubation time for a glucose uptake

experiment is completed, the amount of glucose left in each well is determined with a

glucose oxidase assay and absorption values are determined (492nm). In this specific

0200400600800

100012001400160018002000

Control BDM1 BDH1 BDP1 Metformin Insulin

% o

f con

trol

Figure 3.8: Acute effects of treatments (1μM BDM1, BDH1 and BDP1) on glucose uptake in 3T3-L1 cells after 60 minute incubation. Positive controls: 1μM metformin and insulin. Values represent the averages from a single experiment tested in quadruplicate.

50

experiment, the differences in absorption between the 0 time and 60 minute samples for

the control cells were minimal. Due to these nominal values, the subsequent increases in

glucose uptake calculated for treated cells compared to control cells were invalid.

Contamination also plagued the 3T3-L1 cell line and this was also thought to be adding

to their inability to grow and behave normally. Micoplasmas were suspected and so on

two separate occasions the cells were run through treatments with ciprobay. However this

did not seem to affect their well-being. Different types of preincubation media were also

tested, including Krebs Ringer bicarbonate buffer and RPMI 1640 instead of DMEM,

however neither of these made any significant difference in preventing the cells from

peeling into the center of the wells and forming clumps. Also on numerous occasions, the

number of days the cells were being cultured was decreased, however once again this

made no positive impact on the cells.

Over time, the 3T3-L1 behaviour had changed and their morphology had changed to

more spindly cells, so overall they were not behaving as expected. Finally it was

confirmed by the European Collection of Cell Culture (ECACC) that the 3T3-L1 cell line

had transformed and was not exhibiting contact inhibition, so this aided in explaining all

the problems discussed.

51

3.6.2 C2C12 skeletal muscle cell line 3.6.2.1 Cytotoxicity

Cytotoxicity studies on C2C12 skeletal muscle cells were conducted to ascertain which

concentrations of the compounds and plant extracts would be noncytotoxic to these cells

and thereby be used for further glucose uptake experiments (Figure 3.9).

020406080

100120

-1.5 -1 -0.5 0 0.5 1 1.5

log[Metformin]μM

% o

f con

trol

020406080

100120

-1.5 -1 -0.5 0 0.5 1 1.5

log[BDP1]μM

% o

f con

trol

020406080

100120

-1.5 -1 -0.5 0 0.5 1 1.5

log[BDM1]μM

% o

f con

trol

020406080

100120

-1.5 -1 -0.5 0 0.5 1 1.5

log[BDH1]μM

% o

f con

trol

020406080

100120

0 1 2 3 4 5

log[Kankerbos]ng/well

% o

f con

trol

0

20

40

60

80

100

0 1 2 3 4 5

log[MRC2003]ng/well

% o

f con

trol

Figure 3.9: Cytotoxicity results of compounds (BDM1, BDH1, BDP1 and metformin) and plant extracts (Kankerbos and MRC2003) on C2C12 muscle cells after a 48 hour exposure period. Data points represent the mean ±SD (n=4 wells) from a single experiment.

52

The results of the range of concentrations (0.1 to 10μM) tested for the compounds,

BDM1, BDH1, BDP1 and metformin on C2C12 cells can be seen in figure 3.9. No

cytotoxic effect was observed at either of these concentrations of the compounds on this

cell line. For streamlining purposes as well as the benefit of being able to compare results

of these compounds between cell lines, it was decided to continue using 1μM as the

concentration for further use. Figure 3.9 reveals that Kankerbos was not cytotoxic at any

of the concentrations tested (0.25, 2.5 and 25μg/well). The MRC2003 sample however

gave less than 80% of control for the highest concentration of 25μg/well. In order to

maintain comparability between the plant extracts within the C2C12 cell line, a

concentration of 2.5μg/well for Kankerbos and MRC2003 was chosen to be used for

further glucose uptake experiments. All results shown for the plant extracts are μg/well,

however a conversion table showing the μg/ml values is shown in Appendix 1.

3.6.2.2 Glucose uptake

Muscle is the major site of glucose disposal, in which insulin increases glucose uptake by

promoting the translocation of GLUT4 from an intracellular compartment to the plasma

membrane via a PI 3-kinase pathway. Skeletal muscle accounts for more than 80% of the

total insulin mediated glucose uptake (Kumar and Dey, 2003). Although the C2C12

muscle cell line expresses far less GLUT4 than muscle tissue and its insulin

responsiveness is minimal, it has been used extensively for in vitro cell culture studies of

glucose transport and signaling mechanisms. Therefore it would seem as if insulin

stimulation of glucose transport is not solely dependent on the presence of the insulin

receptor and the GLUT4 protein (Purintrapiban and Ratanachaiyavang, 2003; Tortorella

and Pilch, 2002).

As we were trying to create a model for glucose uptake using C2C12 muscle cells as one

of the cell lines, it had to be deciphered which positive control would best suit this

specific cell line.

53

Figure 3.10 reveals the results of the two positive controls tested for their effect on the

amount of glucose taken up by C2C12 skeletal muscle cells under acute conditions. One

can see that the obvious choice of positive control for further experiments of this nature is

insulin (1μM). Metformin is known to increase glucose uptake in skeletal muscle,

however its effect is somewhat more chronic due to its slow permeation across the inner

membrane (Kirpichnikov et al., 2002). Therefore with regards to the C2C12 cell line, it

would seem not to have an effect within acute conditions.

The acute effects of treatments on glucose uptake by C2C12 cells can be seen in figure

3.11. The C2C12 cells were exposed to the specific treatments for 90 minutes and this

graph shows the typical glucose uptake experiment results found in this cell line. Insulin,

our positive control, significantly increased the amount of glucose taken up by these cells

compared to the control cells (*P<0.05). Under acute conditions, BDM1 had no effect on

glucose uptake by C2C12 cells, however cells exposed to BDH1 took up glucose

significantly more than the control cells (*P<0.05). BDP1 is a compound that has been

reported to possess insulin-like properties (Davis et al., 2000). It has also been said to

have a direct effect on insulin sensitization, which thereby facilitates glucose uptake.

Greenwood et al. (2001) reported BDP1 to stimulate glucose uptake into L6 skeletal

muscle cells and as can be seen in figure 3.11 it would seem to stimulate glucose uptake

by C2C12 skeletal muscle cells, as there was an increase in glucose uptake compared to

020406080

100120140160180

Control Metformin Insulin

% o

f con

trol

Figure 3.10: The acute effect of positive controls, metformin and insulin (1μM) on glucose uptake in C2C12 cells after 90 minute exposure. Values shown are the mean ±SEM (n=8 wells) of two experiments.

54

the control (**P<0.01). The two main plant extracts under investigation, namely

Kankerbos and MRC2003, gave differing results in their effect on glucose uptake into

C2C12 cells. Kankerbos significantly increased the glucose entering the cells (**P<0.01),

whereas the cells exposed to the MRC2003 extract showed no significant glucose uptake.

C2C12 cells were exposed to treatments over a longer time period, by adding them 48

hours prior to experimentation, to investigate the chronic effect of the treatments.

Glucose uptake experiments were carried out under chronic conditions a number of

times, however the results were unexplainably inconsistent every time. It was therefore

decided that for the C2C12 cell line model, acute exposure experiments would only be

carried out in future investigations. The one finding that did remain consistent time after

time, was for the cells exposed to insulin (1μM) under chronic conditions (figure 3.12).

0

50

100

150

200

250

300

Control BDM1 BDH1 BDP1 Kankerbos MRC2003 Insulin

% o

f con

trol

Figure 3.11: Acute effects of treatments on glucose uptake by C2C12 cells after 90 minutes. Treatments included: 1μM BDM1, BDH1 and BDP1 as well as plant extracts, Kankerbos and MRC2003 (2.5μg/well). Positive control: 1μM insulin. Values shown are the mean ± SEM (n=8 wells) from two experiments. *P < 0.05; **P < 0.01 when compared to the control.

* *

**

**

55

Figure 3.12 revealed that the chronic presence of insulin inhibited the uptake of glucose

by C2C12 cells. It would seem that resistance to insulin was induced in the C2C12 cells

under these conditions. Insulin resistance in skeletal muscle is characteristic in type II

diabetic patients (Dela et al., 1994). The results obtained in this experiment are not

unknown, because insulin resistance has been developed in skeletal muscle cell lines such

as C2C12 muscle cells (Kumar and Dey, 2003). This part of the glucose utilisation model

could possibly play an important role, as it could be used to screen the potential

antidiabetic compounds and plant extracts for their effects on insulin resistance in skeletal

muscle.

0

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60

80

100

120

140

160

Control Insulin

% o

f con

trol

Figure 3.12: Effect of insulin (1μM) on glucose uptake in C2C12 muscle cells compared to control after chronic exposure. Data points are the mean ±SEM (n=6 wells) from two experiments.

56

3.6.3 Chang liver cell line 3.6.3.1 Cytotoxicity

Assays to determine the effect of different concentrations of specific treatments on Chang

liver cells were carried out. The compounds tested included BDM1, BDH1 and BDP1,

where the cells were exposed to these using a range of concentrations (0.1 to 50μM). The

effect of different concentrations (0.025, 0.25, 2.5 and 25μg/well) of the plant extract,

MRC2003, on these cells was also determined.

Based on the theory of cytotoxicity testing previously discussed, figure 3.13 indicates that

none of the compounds tested (BDM1, BDH1 and BDP1) showed any cytotoxicity toward

the Chang liver cell line they were tested on. Therefore to maintain some consistency and

020406080

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log[BDH1]μM

% o

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-2 -1 0 1 2

log[BDM1]μM

% o

f con

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-2 -1 0 1 2

log[BDP1]μM

% o

f con

trol

0

50

100

150

200

250

0 1 2 3 4 5

log[MRC2003]ng/well

% o

f con

trol

Figure 3.13: Cytotoxicity results of compounds (BDM1, BDH1 and BDP1) and plant extract (MRC2003) on Chang liver cells after a 48 hour exposure period. Data points represent the mean ±SD (n=8 wells) from a single experiment.

57

thereby be able to compare the effect of these compounds on different cell lines, it was

decided to continue with the 1μM concentration for BDM1, BDH1 and BDP1. The plant

extract sample, MRC2003, appeared to have no cytotoxic effect in the Chang liver cells.

It was decided to use the MRC2003 sample at a concentration of 12.5μg/well (acute) and

10μg/well (chronic) for further experimentation with the Chang liver cell line. Although

the plant extract, Kankerbos, was not tested specifically for cytotoxicity in this cell line, it

was decided, based on previous findings and on the results obtained for MRC2003, that

its concentration for glucose uptake experiments in Chang liver cells under acute

conditions would also be 12.5μg/well and for chronic conditions, 10μg/well. All results

shown for the plant extracts are μg/well, however a conversion table showing the μg/ml

values is shown in Appendix 1.

3.6.3.2 Glucose uptake

For any experiment carried out one needs a positive control in order to substantiate the

results obtained. The two possible candidates for their positive effect on glucose uptake

in Chang liver cells were metformin and insulin (Figure 3.14).

0

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140

160

Control Metformin Insulin

% o

f con

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Figure 3.14: Acute effect of two positive controls (1μM metformin and insulin) on glucose uptake in Chang liver cells. Values shown are the mean ± SEM (n=8 or 15 wells) from an individual experiment. *P < 0.01 when compared to the control.

*

58

Shown in figure 3.14 is the glucose uptake results found when Chang liver cells were

exposed to metformin and insulin under acute conditions. It has been found for

metformin that there is an effect if the exposure to the drug is over a longer time period.

This is probably due to the relatively slow permeation of the drug across cell membranes

and its subsequent accumulation within the mitochondrial matrix, where it exerts a dose-

dependent inhibitory effect on complex I of the respiratory chain (Rutter et al., 2003).

Therefore these results correlate to this as it is evident that under acute conditions,

metformin has no effect and there is no significant uptake of glucose by the cells. Insulin,

with regards to exposure time, appears to work oppositely as can be seen that it has acute

effect allowing a significant increase in glucose uptake. Therefore based on these

findings, it was decided that for the glucose uptake experiment under acute conditions

that insulin would act as a positive control and seeing that metformin is known to have a

chronic effect on glucose uptake that it would be used as the positive control for chronic

conditions.

The graph in figure 3.15 indicates the results obtained in a typical glucose uptake

experiment in Chang liver cells, where cells were exposed to treatments for the acute

exposure time of three hours.

0

20

40

60

80

100

120

140

160

Control BDM1 BDH1 BDP1 Kankerbos MRC2003 Insulin

% o

f con

trol

Figure 3.15: Acute effect of treatments on glucose uptake in Chang liver cells. Treatments included: 1μM BDM1, BDH1 and BDP1 as well as plant extracts, Kankerbos and MRC2003 (12.5μg/well). Positive control: 1μM insulin. Values shown are the mean ± SEM (n=8 or 15 wells) from an individual experiment. *P < 0.05; **P < 0.01 when compared to the control.

** *

59

Our positive control, namely insulin (1μM), showed a significant increase in glucose

uptake in Chang liver cells compared to the control (**P<0.01). The two samples

supplied by the MRC, namely BDM1 and BDH1, showed no effect on glucose uptake in

Chang cells under acute conditions. BDP1, a compound found in Kankerbos, is alleged to

have anti-diabetic properties (Davis et al., 2000). BDP1 supposedly enhances the action

of insulin and therefore the uptake of glucose. However it was found that under acute

conditions, BDP1 (1μM) had no significant impact on glucose uptake in Chang liver cells.

The plant extracts had differing effects. The MRC2003 sample (12.5μg) did not show any

effect on glucose uptake however Kankerbos (12.5μg) had an effect, as it increased the

uptake of glucose by the Chang cells significantly compared to the control (*P<0.05).

Figure 3.16 shows the combined results of three individual glucose uptake experiments

under acute conditions, to give an indication of reproducibility. As one can see, all the

effects of the different treatments are maintained and are comparable to those shown in

figure 3.15.

0

20

40

60

80

100

120

140

160

Control BDM1 BDH1 BDP1 Kankerbos MRC2003 Insulin

% o

f con

trol

Figure 3.16: Acute effect of treatments on glucose uptake in Chang liver cells. Treatments included: 1μM BDM1, BDH1 and BDP1 as well as plant extracts, Kankerbos and MRC2003 (12.5μg/well). Positive control: 1μM insulin. Data points represent the mean ±SEM (n=8 or 15) of three experiments.

60

The chronic effect of the treatments on glucose uptake in Chang liver cells are shown in

the figure 3.17.

Metformin decreases blood glucose levels by reducing hepatic glucose output

(gluconeogenesis) (Moriaka et al., 2005). Therefore a bigger difference between glucose

levels before and after incubation was expected. Also prolonged exposure to metformin is

more effective, so the effect of metformin seen in figure 3.17 was to be expected.

Metformin as our positive control for glucose uptake under chronic conditions gave a

significant increase in the amount of glucose taken up by the cells compared to the

control (**P<0.001). BDH1 (1μM) showed an effect under chronic conditions, whereas it

had no effect when it was exposed to the cells for only three hours. A significant increase

in glucose uptake was observed compared to the control (*P<0.05), as seen in figure 3.17.

However, BDM1 (1μM) had no effect again, therefore this sample seems to have no

effect on glucose uptake in Chang liver cells under acute or chronic conditions. Once

again BDP1 showed no significant effect on glucose uptake and so under chronic

conditions no effect was observed. An effect was observed when Kankerbos (10μg/well)

was exposed to the cells for a long time, a significantly increased amount of glucose was

Figure 3.17: Chronic effects of treatments exposed to Chang liver cells for 48 hours prior to glucose uptake experimentation. Treatments included: 1μM BDM1, BDH1 and BDP1 as well as plant extracts, Kankerbos and MRC2003 (10μg/well). Positive control: 1μM metformin. Data points represent the mean ±SEM (n=15 wells) from a single experiment. *P < 0.05; **P < 0.001 when compared to the control.

0

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40

60

80

100

120

140

Control BDM1 BDH1 BDP1 Kankerbos MRC2003 Metformin

% o

f con

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** *

**

61

taken up by the Chang cells compared to the control (**P<0.001). Therefore it would

seem that the plant extract, Kankerbos, has an impact on glucose uptake under acute and

chronic conditions. The other plant extract tested, namely MRC2003 (10μg/well), showed

no effect on glucose uptake under chronic conditions, as under acute conditions.

Therefore the MRC2003 sample seems to have no effect on this specific cell line when it

comes to glucose uptake experiments. Through combining three different glucose uptake

experiments, one can see the reproducibility of the results in figure 3.18.

After exposing the Chang liver cells to the different treatments for 48 hours prior to the

glucose uptake experiment, it was very important to determine the viability of the cells.

This was in order to determine if any specific treatment had a positive or negative impact

on cell growth and viability and to make sure that at the time of experimentation the cells

in each well were all at a similar density. By accomplishing this, one could cancel out any

doubt as to the difference cell numbers play in the role of differing results.

0

20

40

60

80

100

120

140

160

Control BDM1 BDH1 BDP1 Kankerbos MRC2003 Metformin

% o

f con

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Figure 3.18: Effect of different treatments (BDM1, BDH1, BDP1; 1μM and Kankerbos and MRC2003; 10μg/well) on glucose uptake in Chang liver cells under chronic conditions. Positive control: 1μM metformin. Data points represent the mean ±SEM (n=8 or 15 wells) of three experiments

62

Figure 3.19 shows the CellTiter-BlueTM results and it was found that there was no

significant difference between the cells exposed to the treatments compared to the control

well under chronic conditions. These results were not used in the calculations of the

previous figures (figures 3.17 and 3.18) because there was no significant difference

found, however it is advisable that CellTiter-BlueTM treatment be carried out for all

experiments to confirm cell viability.

In conclusion, the two successful cell lines used for the glucose utilisation model in this

study were the C2C12 skeletal muscle and Chang liver cell lines. Unfortunately the

problems experienced with the 3T3-L1 cell line prevented any conclusions being made

with regards to the best methods for glucose uptake in these cells.

The C2C12 skeletal muscle cell line was an important cell line to include in this model,

as it is known that skeletal muscle accounts for a large amount of the total insulin

mediated glucose uptake and is therefore one of the major insulin target tissues. Although

the chronic exposure experiments with C2C12 cells were not achieved, the acute

0

10

20

30

40

50

60

70

80

90

BDM1 BDH1 BDP1 Kankerbos MRC2003 Metformin

Alam

ar b

lue

Figure 3.19: CellTiter-BlueTM results of Chang liver cells exposed to different treatments for chronic experimentation. Treatments included: 1μM BDM1, BDH1 and BDP1 as well as plant extracts, Kankerbos and MRC2003 (10μg/well). Positive control: 1μM metformin. Values represent the mean ±SEM (n=8 or 9 wells) from a single experiment.

63

condition (1.5 hours) experiments were successful using insulin as the positive control for

glucose uptake. As a result of these findings, it was decided that in the future, all glucose

uptake experiments with C2C12 muscle cells would only include acute exposure

conditions.

For the Chang liver cell line, both the acute (3 hours) and chronic (48 hour pretreatment +

3 hours) condition experiments were successful. When observing the acute effects of the

treatments on glucose uptake in these cells, it was found that insulin was the best positive

control. However due to metformin’s mechanisms of action, it showed the required effect

when exposed to the Chang liver cells chronically and was therefore chosen as the

positive control under these conditions.

All together, these two cell lines provided a good basis for the creation of a model

whereby the effects on glucose utilisation could be investigated for treatments that were

possible antidiabetic potentiates.

64

Chapter 4

Models for insulin secretion

The insulin-secreting cell lines (INS-1 and INS-2) were established from the cells

isolated from an x-ray-induced rat transplantable insulinoma. INS-1 cell morphology,

insulin biosynthesis and secretion are remarkably similar to those of the parent tumour

propagated in vivo (Asfari et al., 1992).

The continuous growth of INS cells depends on the presence of 2-mercaptoethanol (2-

ME) in the culture medium. It was found that the presence of 2-ME (optimal

concentration 50µM) promoted cell proliferation. In the absence of 2-ME, these cells do

not only stop proliferating, but actually do not survive after trypsinization for culture

transfer. The high and stable insulin content of INS-1 cells (averaging 8µg/106 cells) as

compared to normal islets (40µg/106 cells), is probably due to the continuous presence of

2-ME. Several mechanisms have been proposed to explain the effect of 2-ME, however

whether 2-ME participates in sustaining the differentiated state of INS-1 cells remains

hypothetical. 2-ME may exert its effects through participation in cellular metabolism,

which leads to the enhancement of growth factor receptor activation, or on the expression

of oncogenes (Asfari et al., 1992).

Therefore, compared to normal β-cells, INS-1 cells retain a differentiated β-cell

phenotype with respect to glucose transport and metabolism, which are important

functions for normal metabolism-secretion coupling (Janjic et al., 1999).

Unwanted weight gain has been associated with the use of tricyclic antidepressants and it

has been postulated that the mechanism is due to antidepressants increasing blood insulin

levels (Garland et al., 1988 ; Hurr, 1996). Obesity, which is a condition caused by an

excessive amount of adipose tissue, is strongly associated with the development of type II

diabetes (Sheard and Clark, 2000). Due to type II diabetes complications and chronicity,

reducing risk factors such as obesity through lifestyle modification is crucial to the long-

term health of patients. Therefore weight gain, which is an undesired effect of some

65

antidepressants, is a major problem, which needs to be solved and further investigation is

required.

One of the main aims of the project was to create an in vitro model using an insulin-

secreting cell line in order to determine the effect of different drugs (antidepressants and

traditional medicine) on insulin secretion. Through setting up a model, one would be able

to determine the positive or negative effects of the drugs on insulin secretion.

4.1 Materials and methods

Methods described in the following sections are the final optimised methods used in the

model, decided upon when the initial set of experiments were completed.

4.1.1 INS cells maintenance

INS–1 cells were kindly donated by Professor Guy Rutter from the University of Bristol,

England.

4.1.1.1 Mercaptoethanol

Stock mercaptoethanol (3.48µl) (0.5mM) was made up to 10ml with RPMI 1640 and this

solution was filtered through a 0.22µm filter. Aliquots (0.6ml) were frozen in cryo vials

at –20ºC.

4.1.1.2 Complete Medium

The INS–I cells are maintained in complete medium composed of RPMI 1640

supplemented with 10% heat-inactivated FCS and 50µM 2-ME. The complete medium

was made up by adding 0.5ml mercaptoethanol from frozen aliquots to 5ml heat

inactivated serum (10%) and then adding 44.5ml RPMI to make 50ml, which could be

kept for 10 days at 4ºC.

66

4.1.1.3 Poly-L-Lysine (PLL)

Twenty five millilitres of sterile water was added to 5mg PLL (Sigma) in the bottle and

1ml aliquots were placed into vials. For use, 19ml sterile water was added to the 1ml PLL

in the vial.

4.1.1.4 Pretreatment of culture dishes

Prior to use of culture dishes, 2ml PLL was placed into the culture dish, coating the

surface. This was left for 10 minutes at room temperature, removed through aspiration

and the surface washed once with PBSA.

4.1.2 Insulin Secretion by INS-1 cells

Optimal seeding density was determined as 30 000 cells/well. Cells were seeded into 96-

well microtiter plates and cultured for 2 days in complete medium. For the experiments,

each well was washed once with 50μl Krebs-Ringer-bicarbonate-HEPES buffer (KRBH)

composed of 118.4mM NaCl, 4.75mM KCl, 1.192mM MgSO4, 2.54mM CaCl2, 10mM

HEPES, 2mM NaHCO3, 0.1% BSA (Janjic et al., 1999). The cells were then exposed to

50μl preincubation medium (KRBH) for 30 minutes and after removal, treatment

incubation medium (KRBH including applicable medication) (100μl/well) was added for

10 minutes. After this incubation was complete, 50μl incubation medium was removed

and a 50x dilution was made for insulin determination using the LINCO Research rat

insulin radioimmunoassay (RIA) kit (Cat no. RI-13K), which makes use of rat insulin as

a standard. All incubations were carried out at 37ºC. Samples were stored at -20°C until

required for insulin determination. Assay buffer from the LINCO kit was used as the

diluant for all the dilutions required in these experiments.

INS-I cells were exposed to an antidepressant, namely amitriptyline (1μM), plant extracts

of S frutenscens (Kankerbos) (0.5μg/well) and MRC2003 (0.5μg/well) as well as a

positive control: glibenclamide (a sulfonylurea) (1μM), at different exposure times.

67

4.1.3 Plasma insulin determination from rat model To determine whether results obtained with cell culture experiments correlate with results

in an animal model, a collaborative project between Wayne Chadwick (NMMU

Biochemistry PhD Student) and myself was done where I determined the concentration of

insulin in plasma samples from rats. The care of the animals as well as the preparation of

the medication was the responsibility of Wayne. All procedures were first tested on rats

that were not part of the experimental setup.

4.1.3.1 Preparation of medication

1) Sutherlandia frutescens (kankerbos) Dried kankerbos leaves (2.5g) were weighed and placed in 100 ml boiling water to form

an infusion, which was then brewed overnight. The extract was filtered and the rats

received a dose concentration of 0.01ml/g body weight in 100ml tea extract in water. The

drinking water with extract was replaced daily and the volume remaining each day was

recorded.

2) Amitriptyline and Metformin Amitriptyline, the best documented antidepressant that is still used by some patients and

is known to cause weight gain was used in this study. Metformin, a biguanide drug, was

given as a positive control for glucose uptake in Wayne’s project. To be consistent in the

project the pancreases of all the rats were tested for insulin secretion. An amitriptyline

tablet (0.11g) containing 25 mg amitriptyline (highest dose available) or a metformin

tablet (0.55g) containing 500 mg metformin (highest dose available) was dissolved in 1

ml of a 0.1 M HCl solution, ensuring that the drugs would dissolve. The relevant drug

and water was then added to the water bottles (200ml) and these were placed in the

respective cages. Due to the fact that the stability of these drugs in solution is unknown,

the medication was changed twice daily and the volume remaining was monitored.

Rats were weighed weekly. The doses of the different treatments were relevant to the

body weights of the rats in the specific cage. Rats were administered a daily dosage of

68

0.0044mg amitriptyline tablet/g body weight/day and 0.012mg metformin tablet/g body

weight/day.

4.1.3.2 Experimental Procedure

Table 4.1: Experimental plan for medicinal exposure. Each group consisted of 4 rats. Time 0 1.5 weeks 1.5 months 3 months

Control

Amitriptyline

Sutherlandia frutescans

Metformin

Each group to be sacrificed consisted of 4 rats (Table 4.1) therefore the 48 rats were

divided into 12 cages. The cages were cleaned weekly and the rats were fed dog pellets

and received their various medications in their drinking water on a daily basis. To give an

indication on whether any of the drugs played a role on weight gain and to what degree,

the rats were weighed on a weekly basis throughout the experiment.

On the day of sacrifice (after a 12 hour fast), blood was taken from the heart so as to

determine the blood glucose level and the blood insulin level prior to death. An

intramuscular injection (3μl ketamine/g body weight) was administered and as soon as

there was no response from the rat, blood was taken from the heart and then placed into a

tube (2ml) containing sodium fluoride oxalate, which acts as an anticoagulant. The tube

was then immediately placed on ice and once the rat sacrifice was complete, the blood

was centrifuged for 20 minutes at 3 000 rpm. Two equal volume aliquots of plasma were

taken and frozen at -80ºC.

Insulin quantification was carried out with the use of the LINCO Research rat insulin

RIA kit. Assay buffer from the LINCO kit was used as the diluant for the dilution of the

samples.

69

4.1.4 Binding studies with INS-1 rat pancreatic cells INS-1 cells were seeded into 24-well plates (maintained in complete medium) and once

confluency was reached, experimentation was carried out in quadruplicate. For total

binding wells, 100µl RPMI 1640 was added, whereas 100µl RPMI 1640/unlabelled

glibenclamide solution was added to each non-specific binding well (see sections

4.1.4.1/2 for concentrations). The incubation, carried out at 37ºC, was initiated through

the addition of 50µl of [3H]Glibenclamide (50 Ci/mmol) in RPMI 1640 to each well.

Once incubation with [3H]Glibenclamide was complete, the medium was removed and

each well rapidly washed three times with 1ml ice cold PBS. 50µl of 1mM NaOH was

added to each well to aid in detachment of the cells from the well surface. Each aliquot

was then added to a scintillation vial containing 3ml scintillation cocktail (Packard

BioScience). Each well was rinsed with 50μl PBS and that was also transferred to the

scintillation vials. The vials were shaken and radioactivity was determined using a

Packard Liquid Scintillation Analyzer, Tri-carb 2300TR (Packard Instrument Company).

Non-specific binding was determined by incubations in the additional presence of

unlabelled glibenclamide in excess (10 -100x more than labeled). Specific binding was

determined by subtracting non-specific from total binding.

4.1.4.1 Binding time study

Initially, an incubation time for [3H]Glibenclamide binding was determined. Time

intervals investigated included: 2, 6, 12, 30, 60 and 90 minutes at an incubation

temperature of 37°C. The final concentration of [3H]Glibenclamide in the incubation

medium was 0.67nM, which was in accordance with the KD value reported for

glibenclamide in the Amersham catalogue. Non-specific binding was defined by binding

of [3H]Glibenclanide in the presence of 67nM glibenclamide (100x excess).

70

4.1.4.2 Saturation binding study

For saturation experiments, the concentration of [3H]Glibenclamide was varied in order

to determine the actual KD value in the experimentation. This was performed using

concentrations of [3H]Glibenclamide between 0.25µM and 67nM. Non-specific binding

was determined by incubations in the presence of 1.34µM unlabelled glibenclamide.

Saturation binding studies were performed using a 30 minute incubation time at 37°C.

4.1.4.3 Displacement study

Inhibition of [3H]Glibenclamide binding to INS-1 cells was studied in the presence of

[3H]Glibenclamide (8nM) and unlabelled glibenclamide (1.34nM - 1.34µM) or the two

antidepressants, amitriptyline and trimipramine (0.67µM - 6.67µM). The concentration of

the two antidepressants was in accordance with reported plasma levels. Displacement

studies were carried out at a temperature of 37°C for 30 minutes.

4.1.5 Statistical analysis

Statistics were carried out on results using the data analysis program in Microsoft Excel

as described in section 3.5. The actual KD value for [3H]Glibenclamide was calculated

using GraphPad Prism Version 4.

71

4.2 Results and Discussion 4.2.1 Cytotoxicity

The assays to determine the cytotoxicity of treatments to INS-1 cells were carried out in

the same way seen in sections 3.1.1 and 3.1.2. Similarily the concentrations tested for the

plant extracts, namely Kankerbos and MRC(2003), ranged from (0.0025 – 25μg/well).

The antidepressant, amitriptyline, was tested using the same concentrations used for other

treatments tested, ranging from 0.1 to 250μM. Glibenclamide was used as the positive

control and the concentrations tested included: 0.001, 0.01, 0.1, 1 and 10μM. The reason

that the highest concentration tested was only 10μM compared to the usual 250μM, was

due to solubility problems experienced as well as information obtained from the binding

studies performed.

The cytotoxicity results shown in figure 4.1 reveal at which concentration each treatment

should be tested on the INS-1 cells. The basic rule of cytotoxicity tests is that any

concentration that shows less than 80% of control is too cytotoxic for the specific cell

line it is being tested on. The graph of amitriptyline indicates that at high concentrations

(50 and 250 μM) it is very cytotoxic to INS-1 cells. One does not want too low a dosage

of a treatment for experimentation because one requires an effect of some sort to be seen.

Therefore the 1μM concentration was chosen to be used on all the tests on INS-1 cells.

All the concentrations of glibenclamide tested on INS-1 showed no cytotoxic effect. 1μM

glibenclamide was chosen for further studies on the INS-1 cell line, as it was decided to

keep the treatment concentrations the same. The plant extract Kankerbos was found to be

cytotoxic at the two highest concentrations tested, namely 2.5 and 25 μg/well. A

concentration of 0.5 μg/well was decided upon for further experimentation and the same

concentration would be used for the MRC2003 plant extract as well. All results shown

for the plant extracts are μg/well, however a conversion table showing the μg/ml values is

shown in Appendix 1.

72

4.2.2 Insulin Secretion by INS-1 cells

In order to optimize the specific method to determine the amount of insulin secreted, an

initial series of experiments was performed to evaluate the incubation times and

combinations of treatments required before the final procedure was chosen. Due to the

fact that these initial experiments were carried out just to get an indication of the most

suitable protocol to use in the final insulin secretion experiments, cell number or viability

was not determined. For each individual experiment it was visually observed that the

surface area of INS-1 cells of each well tested was similar. However this could not be

determined between different experiments and therefore comparison of these initial

0

20

40

60

80

100

0 1 2 3 4 5

log[Kankerbos]ng/well

% o

f con

trol

0

20

40

60

80

100

0 1 2 3 4 5

log[MRC2003]ng/well

% o

f con

trol

0

20

40

60

80

100

120

140

-4 -3 -2 -1 0 1 2

log[Glibenclamide]μM

% o

f con

trol

0

20

40

60

80

100

120

-2 -1 0 1 2 3

log[Amitriptyline]μM

% o

f con

trol

Figure 4.1: Cytotoxicity results of compounds (Glibenclamide and Amitriptyline) and plant extracts (Kankerbos and MRC2003) on INS-1 cell line. Data points represent the mean ±SD (n=6 or 12 wells) from an individual experiment

73

experiments is not possible. Each specific experiment carried out was done so in order to

determine some specific feature for the final model. Glibenclamide, the sulphonylurea

used by type II diabetic patients, was used as the positive control.

Firstly, the concentration of glucose to be used in the medium had to be tested. Three

options were tested: glucose-free (basal), 2mM glucose and 10mM glucose. For the first

experiment these options were all carried out at two time intervals that needed

investigating, namely 10 and 30 minutes. These concentrations and incubation times were

chosen due to results described in literature (Salt et al., 1998). In addition, each glucose

concentration was tested with and without glibenclamide (1μM). All of the above

combinations were tested in triplicate and using a 5x dilution of the 20μl sample,

however due to the cost of the LINCO kit and budget constraints thereby leading to only

limited numbers of samples being able to be tested, it was decided to only carry out the

kit on the 30 minute samples collected. This was also in order to get an indication of the

kit’s sensitivity, which according to the results obtained was found to be too sensitive and

therefore further dilutions would have to be done in future experiments. As previously

mentioned glibenclamide was used as the positive control in the insulin secretion

experiments. Sulphonylureas inhibit KATP-channels in pancreatic β-cell plasma

membranes and thereby initiate insulin release. The released insulin caused by the

interaction of sulphonylureas with their receptors is largely preformed insulin because

these agents have little immediate effect on insulin synthesis. In general, the secretory

pattern obtained with the sulphonylureas is similar to, but distinct from that obtained with

glucose stimulation. Because sulphonylureas apparently stimulate insulin secretion by a

mechanism that differs from that of glucose, they can be used in the treatment of

diabetics who have lost their ability to respond to a glycaemic stimulus, but who have

retained residual pancreatic β-cell function, for example type II diabetics (Foye et al.,

1995). As shown in figure 4.2, the amount of insulin secreted was increased when

glibenclamide was added to the test medium. Insulin secretion is promoted synergistically

by glucose and sulphonylurea sensitization of the pancreas. It has been seen that if

glucose levels are reduced, that despite sulphonylurea concentrations being maintained,

insulin release decreases rapidly. Therefore based on these facts and the need for a

74

positive control (glibenclamide) for insulin stimulation, a glucose-free medium would not

be feasible.

An observation made that can be seen in figures 4.3 and 4.5 was that of a decrease in

insulin stimulation when cells were exposed for 30 minutes to 10mM glucose with the

addition of glibenclamide. This decrease was in comparison to the effect of 2mM

glucose. It would seem that the higher glucose concentration inhibits the full effect of

glibenclamide.

0

200

400

600

800

1000

1200

1400

1600

1800

2mM glucose (-glibenclamide) 2mM glucose (+glibenclamide)

[Insu

lin] n

g/m

l

Figure 4.2: Effect of 1μM glibenclamide (30 minute exposure) on insulin secretion in INS-1 including 2mM glucose in the medium. Data points represent the mean (±SD) of an individual experiment assayed in triplicate.

0200400600800

10001200140016001800

Glucose-free(+glibenclamide)

2mM glucose(+glibenclamide)

10mM glucose(+glibenclamide)

[Insu

lin] n

g/m

l

Figure 4.3: Effect of extracellular glucose concentration (glucose-free, 2mM and 10mM glucose) on insulin secretion in INS-1 cells. Cells exposed for 30 minutes and included treatment with glibenclamide. Data points represent the mean ±SD (n = 3 wells) from one experiment.

75

As previously mentioned, dilutions of the 20μl sample was decided upon because there

was too much insulin secreted by the INS-1 cells under the specific treatments and

therefore the concentrations of insulin were off-scale and did not fall on the standard

curve. A 5x dilution had already been tested, so further dilutions including 10x, 50x and

100x were decided upon. For this experiment only two of the treatments were tested to

get an indication of where the sample concentrations fell on the standard curve. The 30

minute glucose-free and 10mM glucose samples without glibenclamide were tested in

this experiment and results are shown in Figure 4.4.

Based on the results seen in figure 4.4, it was decided that for all 30 minute samples to be

tested in the future, a 100x dilution would be made. At this dilution the values obtained

fell in the middle of the standard curve, thereby assuring accurate determination of the

amount of insulin secreted. Another decision made at this point was that the incubation

medium volume would be increased from 20μl to 100μl. The 10 minute samples also

needed to be tested and a dilution factor of 50x was decided upon as less insulin would

have been secreted after 10 minutes as compared to 30 minutes. These results can be seen

in figure 4.5.

0

100

200

300

400

500

600

10x 50x 100x 10x 50x 100x

Glucose-free 10mM glucose

[Insu

lin] n

g/m

l

Figure 4.4: Insulin secretion after 30 minute treatment without glucose and with 10mM glucose, including dilutions of the glucose concentrations. All results shown are without glibenclamide. Values shown are the means of one experiment assayed in duplicate.

Figure 4.4: Insulin secretion after 30 minute treatment without glucose and with 10mM glucose, including dilutions of the glucose concentrations. All results shown are without glibenclamide. Values shown are the means of one experiment assayed in duplicate.

76

At this point the initial set of experiments were complete and based on the results seen in

figure 4.5 and previous findings, it was decided that the best protocol to use for optimal

determination of insulin secretion would be 2mM glucose in the incubation medium and

a 10 minute incubation time. Remembering that the effect of the samples to be tested was

unknown, the reasoning for choosing 2mM instead of 10mM glucose was that the

purpose of this experiment was to determine the effect of the plant extracts or treatments

on insulin secretion. Therefore knowing that the lower glucose concentration would not

saturate the glucose transporter and/or the expressed isoform of hexokinase, preventing

any effect of the treatments, it was chosen. It is known that glucose-provoked release of

insulin is biphasic, including early and late phases. The acute phase occurs almost

immediately and peaks within 5 to 10 minutes, whereas the second phase of insulin

release occurs 15 to 20 minutes later. This second phase only persists if the glucose level

remains elevated (Foye et al., 1995). Based on this theory, the choice of the 10 minute

samples was decided upon. One of the main purposes of this project was to finalize

0

50

100

150

200

250

300

350

400

450

Glucose-free(+glibenclamide)

2mM glucose(+glibenclamide)

10mM glucose(+glibenclamide)

[Insu

lin] n

g/m

l

10 minutes

30 minutes

Figure 4.5: Effect of extracellular glucose concentration (glucose-free, 2mM and 10mM glucose) (10 and 30 minute exposure) on insulin secretion in INS-1 cells including glibenclamide in the test medium. Data points represent the mean ±SD (n = 4 wells) of an individual experiment.

77

methods for the screening of plant extracts. To facilitate the screening of many plant

extracts in the future for possible stimulation of insulin release, the aim was to streamline

the method including practical and financial consideration. Therefore in finalizing this

model for insulin secretion, many decisions were made keeping this in mind.

At this stage all the initial experiments were completed and the model for determining

insulin secretion was complete. Therefore our aim to create an in vitro model using the

INS-1 cell line had been achieved and the determination of the effect of different drugs

(antidepressants and traditional medicine) on insulin secretion was now possible.

Although 2mM glucose had been chosen to be included in the incubation medium for the

chronic exposure experiments, a control for the acute exposure experiments was to

include basal (glucose-free), 2mM and 10mM glucose and see their effect on the

secretion of insulin after 10 minutes exposure. Figure 4.6 provides proof that as the

concentration of glucose increases, so the amount of insulin secreted into the medium is

increased, both the 2mM and 10mM glucose treatments showed significantly increased

0

5

10

15

20

25

30

35

40

45

50

55

Basal 2mM glucose 10mM glucose

[Insu

lin] n

g/m

l

Figure 4.6: Effect of different glucose concentrations (basal, 2mM and 10mM) on insulin stimulation in INS-1 cells under acute conditions. Data points represent the mean ±SEM (n = 4 wells) from an individual experiment. *P <0.05 when compared to basal.

*

*

78

levels compared to the glucose-free treatment (*P<0.05). This confirmed that the final

model and insulin determination method chosen for insulin secretion was functioning

correctly. The findings found in figures 4.7 and 4.8 represent an individual experiment,

however the experiment was repeated in triplicate and the results were confirmed.

For the acute exposure experiments where treatments were only added for the duration of

the experiment (10 minutes), the control used was basal wells where glucose-free

medium was added.

Figure 4.7 shows the stimulation of insulin secretion by exposure to the different

medicines tested. Firstly, it can be seen that the 1μM concentration of glibenclamide

produced a significant difference compared to the basal control (*P<0.05), under acute

exposure as expected.

0

5

10

15

20

25

30

35

40

45

50

Basal Glibenclamide(1uM)

Amitriptyline(1uM)

Kankerbos(0.5μg/w ell)

MRC2003(0.5μg/w ell)

[Insu

lin] n

g/m

l

Figure 4.7: Effect of compounds (Glibenclamide and Amitriptyline, 1μM) and plant extracts (Kankerbos and MRC2003, 0.5μg/well) on insulin secretion in INS-1 cells under acute conditions (10 minute exposure). Positive control: 1μM glibenclamide. The values shown are the mean values (±SEM) from an individual experiment assayed in quadruplicate. The experiment was performed three times with similar results. *P <0.05 when compared to basal.

*

*

79

In the experiment, amitriptyline (1μM) also stimulated the INS-1 cells to produce

significantly more insulin than the basal control (*P<0.05). Unwanted weight has been

associated with the use of tricyclic antidepressants. It has been postulated that the

mechanism is due to antidepressants increasing blood insulin levels thereby lowering

blood glucose levels which leads to carbohydrate craving as described in literature

(Flava, 2000). It is interesting that these results correlate with this hypothesis. The graph

indicates that the plant extracts, Kankerbos and MRC2003 (0.5μg/well), do not seem to

have any significant effect on the stimulation of insulin secretion when INS-1 cells are

exposed to these under acute conditions.

Chronic exposure is defined as such because the cells are exposed to the treatments for 48

hours prior to the experiment and then are re-exposed for the duration of the experiment

(10 minutes) to the applicable treatments. As previously mentioned, the wells were pre-

exposed to the specific treatment before the day of experimentation. On the day of the

experiment, basal wells were exposed to glucose-free medium for 10 minutes, this was to

determine the effect of the treatment under chronic conditions only, as there was no

further stimulation. Two millimolar glucose added for 10 minutes gave an indication of

results when further insulin stimulation occurred. The final query was to see the effect of

the treatment when the cells were re-exposed for an additional acute period. Therefore

this was the effect of pre-exposure (chronic) and re-exposure (acute). This test included

2mM glucose, so when comparing to the 2mM glucose only treatment, one could get an

indication of insulin stimulation from the treatment only.

80

The hyperinsulinemia realized with sulphonylureas (glibenclamide) is transient. When

the sulphonylureas are administered chronically, the plasma insulin levels usually, but not

always, decline to pretreatment levels or below. The decline has been shown to be

associated with decreases in proinsulin synthesis, pancreatic insulin content and insulin

secretion (Foye et al., 1995). Based on the theory discussed, glibenclamide (1μM) was

not expected to have a chronic effect, as its primary effect is seen with acute exposure

conditions. This was confirmed with the results obtained (figure 4.8), as no significant

difference was noted. As discussed previously, amitriptyline increased the amount of

insulin secreted by the INS-1 cells under acute conditions (figure 4.7), however one can

see in figure 4.8 that there was no significant difference between the basal, 2mM glucose,

2mM glucose including amitriptyline, thereby indicating that amitriptyline does not affect

insulin secretion on the chronic exposure level. There was no significant difference seen

between basal and 2mM for the plant extract, Kankerbos (0.5μg/well), indicating that

0

10

20

30

40

50

60

Glibenclamide(1μM)

Amitriptyline(1μM)

Kankerbos(0.5μg/w ell)

MRC2003(0.5μg/w ell)

[Insu

lin] n

g/m

l

Basal

2mM glucose

2mM glucose + treatment

Figure 4.8: Effect of extracellular glucose as well as the test compounds (Glibenclamide and Amitriptyline, 1μM) and plant extracts (Kankerbos and MRC2003, 0.5μg/well) on insulin secretion in INS-1 cells under chronic conditions. The values shown are the mean values (±SEM) from an individual experiment assayed in quadruplicate. The experiment was performed three times with similar results. *P <0.05 when compared to 2mM glucose.

*

81

chronic exposure alone does not seem to have an impact on insulin secretion. However,

of important significance to this project were the results found when Kankerbos

(0.5μg/well) was added in the experiment for an additional 10 minutes, so representing

the accumulative effect of chronic and acute exposure. A significant increase in the

amount of insulin secreted was seen for 2mM glucose including Kankerbos (*P<0.05)

compared to the 2mM glucose only results. MRC2003 (0.5μg/well), the other plant

extract of interest in this project, was shown to have no significant effect on insulin

secretion.

82

4.2.3 Serum insulin determination in an animal model

The hypoglycaemic effect of certain treatments was investigated using male Wistar rats,

the findings of which can be seen in figure 4.9. The rats were starved for approximately

12 hours prior to sacrifice.

Fasting blood glucose concentration of rats receiving amitriptyline was significantly

lower than the control group, indicating an increase in the transfer of glucose from the

plasma into the cytoplasm of the cells. Also, an increase in the stimulation of insulin

secretion by amitriptyline treated INS-1 cells was seen (figure 4.7). Experiments done by

Wayne Chadwick showed that the percentage insulin degraded in the liver, kidney and

muscle for the amitriptyline treated group was significantly higher than the control group.

Due to these results, increased serum insulin levels were expected for the amitriptyline

treated rats. However, it was found that there was no significant difference between the

control and amitriptyline for the concentration of insulin in the serum (Figure 4.9).

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Control Amitriptyline Metformin Kankerbos

[Insu

lin] n

g/m

l

Figure 4.9: Fasting serum insulin concentrations as a result of a 19 week treatment with the medication. Data are the mean ± SD (n=6 or 7 wells) from one experiment.

83

A possible explanation could be that the concentration of amitriptyline was a threshold

dose as the lowest dose (75 mg/kg/day) was given to the rats. There could have been an

increase in insulin secreted into the blood under amitriptyline treatment, but it was being

degraded before detection could be made. Metformin enhances glucose uptake at tissue

level and therefore insulin secretion is not enhanced. So the results found were to be

expected and metformin acted more as a negative control for the experiment of insulin

secretion into the blood. The rats receiving the traditional anti-diabetic medicine,

Sutherlandia frutescens (Kankerbos), did not have any significant difference in serum

insulin levels compared to the control group.

4.2.4 Binding studies using INS-1 rat pancreatic cells

Binding experiments were performed in order to measure the binding of

[3H]Glibenclamide (50 Ci/mmol, Amersham) to SUR-1 receptors present in INS-1 rat

pancreatic cells. Glibenclamide is a sulphonylurea drug that is used by diabetics and has

been reported to bind with high affinity to SUR-1. To enhance the model being created, it

was thought that it would be beneficial to have experiments in place, whereby one could

determine if a test compound or plant extract interacted with SUR-1 specifically. This

would then enable us to determine if its mechanism of action with regards to it

stimulating insulin secretion was through binding to these receptors or not.

4.2.4.1 Choice of incubation time

The radioligand associates with the SUR-1 receptor in a time dependant manner. In order

to determine the time needed for maximum saturation, [3H]Glibenclamide was incubated

for different time intervals with the INS-1 cells at a constant concentration (0.67nM).

The results are shown in figure 4.10. Non-specific binding was determined in the

presence of 67nM unlabelled glibenclamide. For all further binding experiments, the

duration of incubation required to reach binding equilibrium was chosen as 30 minutes.

84

4.2.4.2 Saturation binding

Non-specific binding was measured at different radioligand concentrations in

combination with an excess (saturating) of unlabelled competing ligand. Through the

subtraction of these values from the total binding at each radioligand concentration, the

specific binding was calculated (figure 4.11). Non-specific binding increased linearly as

expected, whereas specific binding was saturable indicating that all the receptors were

saturated with glibenclamide.

Figure 4.10: Specific binding of [3H]glibenclamide by INS-1 cells as a function of incubation time. Data points represent the mean ±SEM (n = 3 or 4 wells) from a single experiment.

0

200

400

600

800

1000

1200

0 10 20 30 40 50 60 70 80

[Glibenclamide] (nM)

Bou

nd li

gand

(cpm

/wel

l)

Total

Non-specific

Specific

Figure 4.11: Saturation binding curve. Data points for total, non-specific and specific binding are mean ±SD (n=3 or 4 wells) from a single experiment.

020406080

100120140160

0 20 40 60 80 100

Time (min)

Spe

cific

bin

ding

(C

PM

/wel

l)

85

The actual dissociation constant (KD) for [3H]Glibenclamide was then calculated at half-

maximal bound and as in figure 4.12, was shown to be 14.23 ± 2.04nM (R2=0.9682; 99%

confidence values: 8.5 to 19.97nM). The actual KD value calculated was found to be

higher than what was theoretically stated (0.4nM according to Amersham catalogue) and

this was probably due to variations between cell types and experimental conditions.

Based on the calculated KD value in the range of 8.5 to 19.97nM, displacement studies

were performed using a [3H] Glibenclamide concentration of 8nM.

4.2.4.3 Displacement studies

The purpose of carrying out displacement studies is to determine if there is a compound

in a plant extract that binds to SUR-1 and thereby potentially stimulating insulin

secretion. As there were no plant extracts in this study that were expected to do this, the

antidepressants amitiptyline and trimipramine were used to test the above hypothesis.

0 20 40 60 8014.230

50

100

150

200

250

300

350

400

450

500

550

600

650

Kd Glibenclamide(nM)

Bou

nd li

gand

(cpm

/wel

l)

Figure 4.12: Saturation binding curve. Data points for specific binding are mean ±SD (n=3 or 4 wells) from a single experiment.

86

The hypothesis to be tested followed a computational overlay study (Wilson, 2002). By

determining the similarity of the sulphonylurea and antidepressants 3-D structures, the

possibility of the antidepressant binding to SUR-1 receptors, blocking the K+ATP

channel and resulting in increased insulin secretion was explored. Through the overlay

study the probability (P) of a specific antidepressant agent binding to SUR-1 and thereby

initiating insulin secretion was determined. For the purpose of the current investigation,

the two tricyclic antidepressants amitriptyline and trimipramine were focused on. Table

4.2 shows the P values for these two antidepressants as found in the previous study.

Table 4.2: Probability (P) of binding to SUR-1 receptors Tricyclic Antidepressant P

Amitriptyline 0.023711

Trimipramine 0.48985

P is the probabilty of a specific antidepressant agent binding to SUR1 and thereby

initiating insulin secretion. If P<0.05 this indicates that the Root Mean Square (RMS) of

a particular antidepressant is significantly different to the training set’s selected RMS

range and there is therefore no possibility of binding. However, if P>0.05 there is a 40%

chance that binding will occur. The P value calculated for amitriptyline revealed that

there would be no possibility of binding to SUR-1, whereas trimipramine has a 40%

chance of binding.

For these studies, a constant amount of [3H]Glibenclamide was added to each of the wells

together with varying concentrations of unlabelled competitors of interest. In all the

displacement studies carried out, unlabelled glibenclamide was shown to be a significant

competitor to [3H]Glibenclamide. Figure 4.13 shows as the concentration of unlabelled

glibenclamide increases, so the percent bound [3H]Glibenclamide decreases indicating

displacement of the radioligand.

87

The initial displacement assay (figure 4.13) revealed that amitriptyline did not

significantly displace [3H]Glibenclamide and therefore did not significantly interact with

the SUR-1 receptors of INS-1 cells. This finding correlates with results found through

computational structural overlay studies done by Wilson, 2002 as seen in Table 4.2.

Although the binding study confirmed the computational overlay results with regard to

amitriptyline, it was decided to carry out another displacement study with trimipramine in

order to confirm the previous findings. It is evident in figure 4.14 that there is a large

difference between amitriptyline and trimipramine, which could explain the results found

in the computational overlaying experiments which identified a 40% chance of binding

for trimipramine compared to a 0% chance for amitriptyline. However figure 4.14 reveals

that there was no significant displacement of [3H]Glibenclamide by trimipramine.

020406080

100120140160180

1.34 13.4 134 1340 670 6670

[Competitor]nM

% B

ound

[3 H]G

liben

clam

ide

Amitriptyline

UnlabelledGlibenclamide

Figure 4.13: Displacement of [3H]glibenclamide by unlabelled glibenclamide (1.34nM – 1.34µM) and amitriptyline (0.67µM and 6.67µM). The values shown are the mean values (±SEM) from one experiment assayed in triplicate or quadruplicate. The experiment was repeated with similar results. *P <0.05 compared to the total bound in absence of competitor.

*

88

Figure 4.14 shows a continual finding during the binding studies was that amitriptyline

competition produced an increase in bound [3H]Glibenclamide greater than 100%. This

was investigated by implementing the use of a range of lower concentrations of

amitriptyline in the displacement studies, as seen in figure 4.15.

0

25

50

75

100

125

150

175

1.34 13.4 134 1340 670 6670

[Competitor]nM

% B

ound

[3 H]G

liben

clam

ide

Amitriptyline

Trimipramine

UnlabelledGlibenclamide

Figure 4.14: Displacement of [3H]glibenclamide by unlabelled glibenclamide (1.34nM – 1.34µM), amitriptyline and trimipramine (0.67µM and 6.67µM). The values shown are the mean values (±SEM) from one experiment assayed in triplicate or quadruplicate. *P <0.05 compared to the total bound in absence of competitor.

Figure 4.15: Displacement of [3H]glibenclamide by unlabelled glibenclamide (1.34nM – 1.34µM) and varying concentrations of unlabelled amitriptyline (0.67nM – 6.67μM). Mean ±SEM (n=3 or 4 wells) from an individual experiment.

*

0

20

40

60

80

100

120

140

1.34 13.4 134 1340 0.67 67 670 6670

[Competitor]nM

% B

ound

[3 H]G

liben

clam

ide

0

20

40

60

80

100

120

140

Amitriptyline

UnlabelledGlibenclamide

89

Figure 4.15 confirms that amitriptyline does not bind to the receptor, as there is no

displacement of [3H]Glibenclamide. Even at the lower concentrations of amitriptyline

tested, results produced binding of [3H]Glibenclamide that was greater than 100%.

Previous experiments (see figure 4.7), however show that amitriptyline does stimulate

insulin secretion by INS-1 cells under acute conditions. The mechanism is therefore not

clear at this stage, though we know it is not via the SUR-1 receptors, like sulphonylureas.

This confirms the results obtained previously ie. theoretical binding possibilities of

tricyclic antidepressants to SUR-1 (Wilson, 2002) in the computational overlaying

studies.

This series of experiments successfully determined the conditions needed to screen the

effects of drugs (compounds) or plant extracts on pancreatic cells.

90

Chapter 5

Conclusion

The present study investigated the in vitro antidiabetic properties of two indigenous

plants, with the aim to optimise the processes required for their screening and ultimately

the determination of their effectiveness. The study was based on the fact that these plant

extracts are being widely used as medicinal plants, however there is a lack of scientific

evidence for their efficacy. Determination of their efficacy is vital as these medicinal

plants may serve as important participants in the management of type II diabetes in the

future and thereby make a positive contribution to South Africa’s health care services.

Type II diabetes is characterised by abnormal glucose homeostasis leading to

hyperglycaemia. The mechanism of action of many hypoglycaemic drugs used for type II

diabetes is based on their ability to positively affect glucose utilisation and insulin

secretion. Therefore in creating a model for determining the effects of alternative

traditional medicines as antidiabetic potentiates, it was necessary that these two metabolic

pathways (glucose uptake and insulin secretion) play the role of centre pivot in our

investigations.

In chapter 1 the pharmacological treatments available for type II diabetic patients were

discussed. The hypoglycaemic drugs which base their mechanisms on glucose utilisation

are namely the biguanides (metformin), thiazolidinediones and insulin. Sulfonylurea

drugs such as glibenclamide in this study, employ insulin secretion as their mechanism of

action. This study encompassed these two vital processes in creating a model for

antidiabetic screening, therefore a model for glucose utilisation (Chapter 3) and a model

for insulin secretion (Chapter 4) was developed. The biguanides function as antidiabetic

agents by stimulating hepatic glucose metabolism, reducing hepatic glucose output and

reducing intestinal glucose absorption (Moriaka et al., 2005). The thiazolidinediones

increase peripheral glucose uptake and metabolism as well as increase insulin sensitivity

(Oakes et al., 2001). Insulin functions by stimulating hepatic glucose metabolism as well

as increasing peripheral glucose uptake and metabolism (Levinthal and Tavill, 1999). The

91

sulfonylureas stimulate insulin secretion from pancreatic β-cells (Foye et al., 1995). So

overall one can see that drugs used for the treatment of diabetes have a variety of tissues

that they act on, therefore it was necessary to use the relevant cell lines to represent all

the possible scenarios. Due to this the 3T3-L1 adipocyte, Chang liver, C2C12 skeletal

muscle and INS-1 rat pancreatic cell lines were involved in the glucose utilisation and

insulin secretion models of this study.

The entry of glucose into cells is a crucial step in life-supporting processes since glucose

is the main monosaccharide in nature that provides carbon and energy for almost all cells.

The passage of glucose into cells depends on different parameters, including expression

of the appropriate glucose transporters in the target tissues and hormonal regulation of

their function. In mammalian cells a tight regulation of blood glucose levels is needed to

meet the energetic demands of the brain, a tissue that uses glucose as its primary energy

source. Adequate glucose flux into tissues provides maintenance of glucose homeostasis

that is critical in well being (Gorovits and Charron, 2003).

Glucose that is taken up by a cell may be oxidized to form energy (glycolysis). In type II

diabetes, excessive hepatic glucose output contributes to the fasting hyperglycaemia.

Increased gluconeogenesis is the predominant mechanism responsible for this increased

glucose output, while glycogenolysis has not been shown to be increased in patients with

type II diabetes. Hyperglucagonemia has been shown to augment increased rates of

hepatic glucose output, probably through enhanced gluconeogenesis (Figure 5.1)

(Levinthal and Tavill, 1999).

92

In this study, the two contenders for use as positive controls in the glucose utilisation

model were insulin and metformin.

Insulin's actions are summarised in figure 5.2. Insulin promotes glycogen synthesis

(glycogenesis) in the liver and inhibits its breakdown (glycogenolysis). It promotes

protein, cholesterol, and triglyceride synthesis and stimulates formation of very-low-

density lipoprotein cholesterol. It also inhibits hepatic gluconeogenesis, stimulates

glycolysis, and inhibits ketogenesis (Levinthal and Tavill, 1999).

Figure 5.1: Summary of carbohydrate metabolism (www.np.edu.sg)

93

Exposure to insulin, which dominates in the prandial and immediate postprandial period,

encourages storage of carbohydrate and fat and reduces or prevents hepatic glucose

production. There is a general agreement that the major factor in insulin's indirect

suppression of endogenous glucose production is decreased plasma FFA concentration,

due to the antilipolytic effect of insulin on adipose tissue. When hyperinsulinaemia

becomes sufficiently high, the insulin-receptor complex in adipocytes and in skeletal

muscle initiates a transduction chain that results in translocation of intracellular vesicles

to fuse with the plasma membrane. Laced in the walls of these vesicles are GLUTs,

GLUT1 and GLUT4 in adipocytes, but almost exclusively GLUT4 in skeletal muscle

(Zierler, 1999). Insulin does not directly stimulate hepatic glucose uptake through

translocation of glucose transporters to the plasma membrane as it does in skeletal

muscle. Instead, insulin promotes glucose uptake in the liver cell, at least in part, through

increased expression of hepatic glucokinase (Iozza et al., 2003).

Figure 5.2: Mechanisms of insulin action. Key: stimulated by insulin; inhibited by insulin. (Saltiel and Kahn, 2001)

94

Metformin is an insulin-sensitising agent with potent antihyperglycaemic properties

(Tankova, 2003). Its glucose-lowering effects are mainly a consequence of reduced

hepatic glucose output (primarily through inhibition of gluconeogenesis and, to a lesser

extent, glycogenolysis) and increased insulin-stimulated glucose-uptake in skeletal

muscle and adipocytes. Its major mode of action is to reduce hepatic glucose production,

which is increased at least twofold in patients with type II diabetes. Metformin facilitates

insulin-induced suppression of gluconeogenesis from several substances (lactate,

pyruvate, glycerol and amino acids) and opposes the gluconeogenic actions of glucagon.

In addition, it increases intramitochondrial levels of calcium, a modulator of

mitochondrial respiration (Figure 5.3) (Kirpichnikov et al., 2002).

The primary site of metformin’s action on reduction of hepatic glucose production

appears to be hepatocyte mitochondria, where it disrupts respiratory chain oxidation of

complex I substrates. Inhibition of cellular respiration decreases gluconeogenesis and

may induce expression of glucose transporters, thereby facilitating glucose utilisation. In

several tissues, including skeletal muscle and adipocytes, metformin facilitates glucose

Figure 5.3: Mechanisms of metformin action on hepatic glucose production and muscle consumption (Kirpichnikov et al., 2002)

95

transport by increasing tyrosine kinase activity in insulin receptors and enhancing the

trafficking of glucose transporters 1 and 4 to the cell membrane (Kirpichnikov et al.,

2002).

Insulin and metformin were chosen as positive controls for glucose uptake in 3T3-L1

cells. In the C2C12 cell line, the results for the determination of the ideal positive control

correlated to theory, as it is known that insulin increases peripheral glucose uptake and

metabolism in skeletal muscle. Insulin was shown to provide a significant increase in the

amount of glucose taken up by these cells (Figure 3.11), however this only qualified for

acute conditions as no viable results were obtained with these cells when they were

exposed to chronic circumstances. Considering the role of muscle in glucose clearance in

the body, and the mechanism of glucose uptake in this tissue, it would be more

appropriate for an antidiabetic agent to act immediately upon administration rather than

over a long period of time.

Knowing that both insulin and metformin stimulate hepatic glucose metabolism, the

results obtained for Chang liver cells in this study were expected. For acute exposure,

insulin showed a significant effect on glucose utilisation by Chang liver cells (Figures

3.14 and 3.15) and because of this significant effect was chosen as the positive control for

this condition. Due to the results found in figure 3.14 as well as the fact that it is known

that metformin has an effect with chronic exposure, it was decided to use it as the

positive control for glucose utilisation in the cells under chronic conditions. Chronic

treatments with metformin provided a significant increase in glucose utilised by Chang

liver cells, as was seen in figure 3.17.

The positive control used for the insulin secretion model in this study was the

sulfonylurea, glibenclamide. Glibenclamide is used by type II diabetics and it functions

through its ability to bind to SUR-1 receptors of pancreatic β-cells, thereby initiating the

release of insulin. Glibenclamide was an effective positive control for stimulating insulin

secretion by INS-1 cells under acute conditions as there was a significant increase in the

amount of insulin secreted when these cells were exposed to this drug (Figure 4.7).

96

However no significant effect was seen with regards to the chronic conditions tested

when glibenclamide was included. This was not unexpected as it is known that

glibenclamide’s activity does not favour the chronic case but rather the acute one.

Therefore the results from this study correlated with previous information of

glibenclamide’s mechanism of action.

BDM1 and BDH1 are compounds expected to be present in the plant extracts tested in this

study. Although these compounds were included as test substances for their effect on

glucose utilisation in 3T3-L1 cells, results were inconclusive due to the unforeseen

problems experienced with the cell line. BDM1 was shown to have no effect on glucose

uptake in C2C12 muscle cells for either acute or chronic conditions. However, BDH1

significantly increased the amount of glucose taken up by the C2C12 cells being exposed

to acute conditions (Figure 3.11) but not in chronic exposure conditions. When tested on

the Chang liver cell line, BDM1 once again seemed to have no significant effect on

glucose utilisation in these cells. When the Chang cells were exposed to BDH1 under

acute conditions, no significant effect was seen either. On the other hand, when these

cells were treated to BDH1 chronically, a significant increase in glucose uptake was seen

(Figure 3.17). BDP1, as mentioned, is a compound that in previous studies has been found

to display similar characteristics to insulin and has been said to be an antidiabetic

compound. In this study, the only positive results that correlate to these previous findings

were observed in C2C12 muscle cells. It was found that when these cells were exposed to

BDP1 acutely, that a significant increase in glucose utilisation was seen (Figure 3.11).

Other than BDP1’s significant effect on C2C12 cell (acute exposure), no other significant

results were found for this compound on any of the other cell lines.

Throughout the course of this study it was seen that Kankerbos has biological activities

which are comparable to some of the more well-known antidiabetic compounds. The

three successful cell lines that Kankerbos was tested on included: C2C12 muscle, Chang

liver and INS-1 pancreatic cells. With regards to the glucose utilisation model,

Kankerbos was seen to have both acute and chronic effects in different cell lines. In the

C2C12 muscle cells, Kankerbos significantly increased glucose uptake when they were

97

exposed to acute conditions (Figure 3.11). Kankerbos also had a significant effect on the

Chang liver cell line as it was observed that under both acute (Figure 3.15) and chronic

(Figures 3.17) conditions, this plant extract induced the uptake of glucose into these cells.

With respect to the insulin secretion model involving INS-1 cells, no significant effect

was seen during acute exposure when they were treated with Kankerbos. However during

chronic exposure, this plant extract initiated an increased insulin secretion from INS-1

cells (Figure 4.8). Taken together, the results of this study suggest that Kankerbos has a

dual mechanism of action for its glucose-lowering effects: It stimulates glucose usage by

C2C12 muscle and Chang liver cells as well as enhances insulin release from INS-1 cells.

Since Kankerbos is widely distributed in South Africa and therefore accessible to many

communities, this study was useful as it provides an indication that Kankerbos has

antidiabetic activities and could therefore be used as a possible alternative once further

analysis has been completed.

The plant extract, MRC2003, proved not to be as effective as Kankerbos in this study.

Although it was seen in two experiments that the MRC2003 sample significantly

enhanced glucose utilisation in 3T3-L1 adipocytes (Figure 3.4), no conclusions could be

drawn due to the fact that no feasible results were obtained from the experiments carried

out on the 3T3-L1 cell line. Further experimentation involving the MRC2003 sample

included it being tested on the C2C12 skeletal muscle, Chang liver and INS-1 pancreatic

cell lines. For all these cell lines within both models, no significant effect was seen when

the cells were exposed to MRC2003 under acute and chronic conditions. Therefore no

conclusions could be made about this plant extract’s potential as an antidiabetic

compound.

Seeing that glibenclamide is known to bind with high affinity to SUR-1 and thereby

initiate insulin secretion, it was decided to use this theory for further investigations. This

was used as the basis and reasoning for carrying out binding studies. It was thought that

by carrying out these studies it would allow one to determine if the mechanism of a

compound or plant extract in stimulating insulin secretion was subsequent to its binding

to the SUR-1 receptor or whether it was accomplished by another mechanism. Owing to

98

the fact that there was no plant extract in this specific study that was expected to do this,

amitriptyline and trimipramine were used.

Due to amitriptyline being included as a test substance for the binding studies in this

study as well as the computational overlay study previously done, it was included in the

insulin secretion model of this study. The amitriptyline-treated INS-1 pancreatic cells

showed a significant increase in insulin stimulation and secretion when exposure was

acute (Figure 4.7). These results correlated with a previous hypothesis in literature:

Unwanted weight gain associated with a few antidepressants could be due to their

increasing blood insulin levels causing blood glucose to drop and thereby leading to the

food cravings experienced by the patients. This significant effect was however only

observed when the INS-1 cells were exposed to amitriptyline under acute conditions and

not chronic. Although amitriptyline had this effect on insulin secretion by INS-1 cells, the

binding studies revealed that amitriptyline does not bind to the SUR-1 receptor of

pancreatic β cells (Figures 4.13, 4.14, 4.15). Amitriptyline’s mechanism of action on

insulin secretion is still elusive at this stage, however it seems that amitriptyline does not

exert its effect on insulin secretion in the same manner as the sulphonylureas, that is via

the SUR -1 receptors.

In conclusion, the objective of this study, to optimize the techniques required for the

screening and determination of the effectiveness of specific compounds and plants as

antidiabetic potentiates, through observing if they had effects on glucose utilisation and

insulin secretion, was accomplished. Since optimal methodology was achieved through

this specific project, the criteria used to validate the models will now be able to be

applied to scientifically ascertain the antidiabetic effect(s) and mechanism(s) of plants

being researched in the future.

99

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Appendix A Table A1: Conversion table for plant extract concentrations

Cell line Plant extract Condition μg/well μl/well μg/ml

• C2C12 Kankerbos Acute 2.5 50 50 MRC2003 Acute 2.5 50 50

• Chang Kankerbos Acute 12.5 25 500

Chronic (48hr exposure) 10 200 50

MRC2003 Acute 12.5 25 500

Chronic (48hr exposure) 10 200 50

• INS-1 Kankerbos Acute 0.5 100 5

Chronic (48hr exposure) 0.5 200 2.5

MRC2003 Acute 0.5 100 5

Chronic (48hr exposure) 0.5 200 2.5