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Novel aspects of adipocyte-induced skeletal muscle insulin resistanceKristin Eckardt a; Henrike Sell a; Juergen Eckel a

a Institute of Clinical Biochemistry and Pathobiochemistry, German Diabetes Center, Düsseldorf, Germany

Online Publication Date: 01 October 2008

To cite this Article Eckardt, Kristin, Sell, Henrike and Eckel, Juergen(2008)'Novel aspects of adipocyte-induced skeletal muscle insulinresistance',Archives Of Physiology And Biochemistry,114:4,287 — 298

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REVIEW ARTICLE

Novel aspects of adipocyte-induced skeletal muscle insulin resistance

KRISTIN ECKARDT, HENRIKE SELL, & JUERGEN ECKEL

Institute of Clinical Biochemistry and Pathobiochemistry, German Diabetes Center, Dusseldorf, Germany

AbstractInsulin resistance in skeletal muscle is an early event in the development of diabetes with obesity being one of the majorcontributing factors. Conditioned medium (CM) from differentiated human adipocytes impairs insulin signalling in humanskeletal muscle cells. Recent data on adipocyte-induced insulin resistance in skeletal muscle cells describes underlyingmechanisms of this process. Skeletal muscle insulin resistance involves multiple pathways and irreversible changes in theexpression level of critical proteins. Furthermore, the reversibility of insulin resistance could be demonstrated. Severalstrategies to combat insulin resistance have been developed. One recent approach to treat obesity and the metabolicsyndrome is the use of endocannabinoid receptor antagonists such as rimonabant. These compounds might also reduceinsulin resistance in type 2 diabetes with effects on adipose tissue and liver and possibly skeletal muscle.

Key words: Insulin resistance, obesity, adipokine, cannabinoid receptor antagonists.

Obesity and skeletal muscle insulin resistance

Insulin resistance and beta-cell dysfunction are core

defects in the development of type 2 diabetes. The

development of insulin resistance in peripheral

organs such as skeletal muscle and adipose tissue is

an early defect in the pathogenesis of type 2 diabetes.

Especially skeletal muscle is relevant for insulin

resistance as it constitutes one of the major insulin-

sensitive organs and contributes for about 80% of

insulin-stimulated glucose disposal (DeFronzo et al.,

1981). Consequentially, disturbed insulin signalling

in skeletal muscle as well as mechanisms leading to

diminished insulin action in diabetic patients

or animals represents a key area of research in

diabetes.

Obesity is one of the major components of the

metabolic syndrome and a strong risk factor for the

development of type 2 diabetes. It is associated with

increased circulating plasma levels of free fatty acids

and triglycerides which contribute to insulin resis-

tance in peripheral tissues like skeletal muscle (Kahn

& Flier, 2000). Adipose tissue is the major organ

where mammals store triglycerides and traditionally

it was thought that the function of this tissue is

restricted to energy storage, but it has become

evident over the last decade that adipocytes, in

addition to releasing free fatty acids (FFA) by

lipolysis, are also active secretory cells capable of

producing a variety of cytokines, the so-called

adipokines (Rajala & Scherer, 2003; Trayhurn &

Beattie, 2001). Adipose tissue is the source of key

hormones in the control of body weight and secretes

a range of adipokines, some of which may directly be

involved metabolic disturbances associated with

obesity (Trayhurn, 2005). In obesity, various fat

depots as well as single adipocytes themselves are

enlarged and characterized by an altered secretion

profile of adipokines compared to lean conditions.

Adiponectin levels are reduced and leptin levels

increased in the obese state (Arita et al., 1999;

Considine et al., 1996; Hu et al., 1996; Ostlund et al.,

1996; Trayhurn & Beattie, 2001).

The link between obesity and insulin resistance is

now well recognized and increased adiposity, espe-

cially in the visceral region, was proven to lead to

metabolic disorders such as insulin resistance and to

a higher risk factor for cardiovascular diseases

(Bosello & Zamboni, 2000). Many studies in humans

and in various animal models have shown that

obesity is strongly related to the development of

diabetes (Bloomgarden, 2000; Felber & Golay, 2002;

Finegood, 2003). The obvious association between

the epidemics of obesity and diabetes has promoted

Correspondence: Kristin Eckardt, German Diabetes Center, Auf’m Hennekamp 65, D-40225 Dusseldorf, Germany. Tel: þ49 211 3382583.

Fax: þ49 211 3382697. E-mail: kristin.eckardt@ddz.uni-duesseldorf.de. Internet: www.ddz.uni-duesseldorf.de

Received for publication 24 April 2008. Accepted 8 August 2008.

Archives of Physiology and Biochemistry, October 2008; 114(4): 287–298

ISSN 1381-3455 print/ISSN 1744-4160 online ª 2008 Informa UK Ltd.

DOI: 10.1080/13813450802404761

Downloaded By: [Eckardt, Kristin] At: 12:25 24 October 2008

research especially on the endocrine link between

lipid and glucose homeostasis. It is well-known that

elevated circulating plasma levels of triglycerides and

FFA due to obesity highly contribute to insulin

resistance in peripheral tissues such as skeletal

muscle (Kahn & Flier, 2000). In addition to lipids,

several adipokines were identified and shown to

influence insulin action in the muscle (Dietze et al.,

2002; Pittas et al., 2004) and are therefore emerging

regulators of insulin sensitivity. Tumor necrosis

factor (TNF)-a, IL-6, adiponectin, and other still

unknown factors might constitute the missing link

between adipose tissue and insulin resistance

(Greenberg & McDaniel, 2002). These adipokines

are found to be involved in the deregulation of

glucose and lipid homeostasis but also in inflamma-

tion by representing pro-inflammatory factors. With

over 50 adipokines and frequent identification of new

adipose derived factors, adipose tissue can be

described as ‘‘champion’’ when it comes to its

endocrine function (Gong et al., 2003). Adipocyte-

derived factors like IL-6 and IL-8 are significantly

increased in obesity and are good predictors of the

development of type 2 diabetes (Festa et al., 2002;

Pradhan et al., 2001).

The model of adipocyte-induced insulin

resistance

Many clinical studies and the use of various animal

models have provided good evidence that a negative

crosstalk between excess body fat and skeletal muscle

leads to disturbances in skeletal muscle insulin

signalling and finally to insulin resistance. The co-

culture model of skeletal muscle cells and adipocytes

was established in our laboratory to study the direct

interaction between these two cell types (Dietze et al.,

2002). As co-culture is a generally accepted approach

for studies on the paracrine interaction between

different cell types, the co-culture of skeletal muscle

cells and adipocytes provided the proof of a direct

negative crosstalk between adipocytes and myocytes.

Co-culture clearly leads to insulin resistance in

skeletal muscle cells similar to the defects observed

in skeletal muscle of diabetic patients (Cozzone et al.,

2008; Krook et al., 2000; Krook et al., 1998; Zierath

et al., 2000). Skeletal muscle cells undergoing co-

culture display decreased insulin-stimulated insulin

receptor substrate (IRS)- 1, Akt, glycogen synthase

kinase (GSK) 3 phosphorylation and a markedly

reduced glucose transporter (GLUT) 4-translocation

to the plasma membrane (Dietze et al., 2002; Dietze-

Schroeder et al., 2005). An alternative approach to

co-culture is the use of adipocyte-conditioned

medium (CM) containing a complex mixture of

adipokines. CM mimics co-culture with reduced

insulin-stimulated IRS-1 and Akt phosphorylation

(Dietze et al., 2004; Dietze-Schroeder et al., 2005;

Sell et al., 2006) (Figure 1).

The following review summarizes novel aspects of

adipocyte-induced insulin resistance in skeletal mus-

cle with special emphasis to the reversibility of insulin

resistance. Furthermore, the endocannabinoid sys-

tem is reviewed as a promising target for the

treatment of obesity and obesity-related disorders

such as insulin resistance.

Defects in skeletal muscle cells in parallel to

insulin resistance

Release of myokines

While the release of adipokines from adipose tissue is

characterized in many studies, the secretory function

Figure 1. Adipocyte-induced insulin resistance and concomitant defects in skeletal muscle cells. Skeletal muscle cells treated with adipocyte-

conditioned medium (CM) develop insulin resistance on the level of insulin-stimulated Akt- and GSK3-phosphorylation with a parallel

decrease in GLUT4-translocation and glucose uptake. Insulin resistant skeletal muscle cells are also characterized by other defects such as a

decreased expression of myogenic marker, disturbed secretory function and increased oxidative stress. While insulin resistance is reversible

upon CM-withdrawal, some defects such as decreased IL-8 secretion and the downregulation of myogenin are irreversibly perturbed.

288 K. Eckardt et al.

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of skeletal muscle has been studied to a much lesser

extent. In the context of crosstalk between adipose

tissue and skeletal muscle, the secretory output of

skeletal muscle might also be interesting in the

context of insulin resistance. Secretory products of

muscle, also referred to as myokines, are part of a

muscle-to-fat signalling possibly modulating the ratio

of lean-to-fat mass as well as insulin sensitivity. IL-6,

IL-8 and monocyte chemotactic protein (MCP)- 1

are all adipokines but also known myokines with

different roles in myogenesis, exercise, inflammation

and insulin sensitivity.

IL-6 is certainly the most prominent and best

studied myokine. IL-6 is strongly induced in skeletal

muscle during and after exercise (Steensberg et al.,

2002). Short-term stimulation of myocytes with IL-6

at concentrations similar to those reached during

exercise has a positive effect on skeletal muscle cell

insulin sensitivity (Weigert et al., 2005). As IL-6 also

activates lipolysis in adipose tissue it might play a role

in energy supply during exercise (van Hall et al.,

2003). While chronically increased IL-6 levels are

associated with insulin resistance in vivo (Kern et al.,

2001), short-term treatment of skeletal muscle cells

with IL-6 was shown to increase insulin sensitivity

(Weigert et al., 2005). The seeming contradiction

between increased levels of IL-6 during exercise and

increased IL-6 in obese and diabetic patients

illustrates that IL-6 might have completely different

effects in acute and chronic ways. As for myogenesis,

IL-6 is a promyogenic factor (Baeza-Raja & Munoz-

Canoves, 2004). When skeletal muscle cells are

treated with CM to induce insulin resistance, we

observe a marked decrease in IL-6 secretion (Sell

et al., 2008) (Figure 1). Whether this effect is an

adaptation to high IL-6 levels present in CM or

effects myotube physiology is not clear.

IL-8 and MCP-1 are both pro-inflammatory

chemokines being increased in serum of obese and

diabetic patients (Kim et al., 2006; Sartipy &

Loskutoff, 2003). We could already demonstrate

that MCP-1 is able to induce insulin resistance in

skeletal muscle cells (Sell et al., 2006). Furthermore,

MCP-1 is induced in skeletal muscle during myo-

pathies and TNF-a has been described to induce

MCP-1 transcription in myoblasts (De Rossi et al.,

2000). While IL-8 secretion is almost completely

inhibited in CM-treated skeletal muscle cells MCP-1

release increases pointing to an inflammatory effect

of CM (Sell et al., 2008).

Mitochondrial dysfunction and oxidative stress

Mitochondrial dysfunction in skeletal muscle leading

to fat accumulation has been proposed to be involved

in the development of type 2 diabetes (Schrauwen-

Hinderling et al., 2007). It could be shown that

diabetic patients possess less and smaller mitochon-

dria compared to control subjects (Kelley et al.,

2002). In addition, mitochondrial dysfunction has

been reported in pre-diabetic and diabetic subjects

(Petersen et al., 2003; 2004). Mitochondrial dysfunc-

tion could contribute to massive lipid accumulation

in skeletal muscle causally related to the development

of insulin resistance. Succinate dehydrogenase

(SDH) activity as a marker for mitochondrial

dysfunction is significantly reduced in skeletal

muscle of diabetic patients (Oberbach et al., 2006).

We also observe decreased SDH activity in insulin-

resistant skeletal muscle cells indicating a possible

role of decreased oxidative capacity in the initiation

of adipocyte-derived muscle insulin resistance (Sell

et al., 2008) (Figure 1). One possible explanation for

the loss of mitochondria in insulin resistance is the

occurrence of oxidative stress. As a result of

increased reactive oxygen species (ROS) formation

oxidative stress can lead to oxidation and damage of

DNA, protein and lipids. We could demonstrate

increasing ROS production in insulin-resistant ske-

letal muscle cells also explaining decreased SDH

activity in our model (Figure 1). In addition to CM,

L6 muscle cells were shown to display higher levels of

ROS in palmitate-induced insulin resistance (Rachek

et al., 2007). As fatty acids are very low in CM,

adipocyte-derived factors could cause oxidative stress

similarly to fatty acids.

Myogenic marker and fibre type

Depending on the location within the body skeletal

muscle in vivo contains a unique composition of

different muscle fibres referred to as ‘‘slow-twitch’’

and ‘‘fast-twitch’’. These fibre types express a typical

set of fast and slow protein isoforms and different sets

of metabolic enzymes and exhibit different contrac-

tile properties and endurance capacity (Bottinelli &

Reggiani, 2000). In vivo, conversion of fibre type

happens, for example, depending on the firing

patterns of motor neurons (Salmons and Sreter,

1976). In vitro, one study could also provide evidence

of conversion of fibre type in C2C12 skeletal muscle

cells (Zebedin et al., 2004). Differentiation of skeletal

muscle involves a group of transcription factors

including myogenin and myoD which activate

muscle-specific gene expression and have each a

distinct function during myogenesis (Perry &

Rudnick, 2000). In rat skeletal muscle, myoD is

prevalent in fast type II muscle fibres while myogenin

could mainly be found in type I fibres thus

associating myogenin expression with slow-twitch

and most insulin-sensitive muscle fibres (Hughes

et al., 1993). In CM-treated skeletal muscle cells, we

observed a parallel decrease in myogenin and myoD

expression in insulin resistance (Figure 1). The

particularly prominent down-regulation of myogenin

might be indicative of the formation of less oxidative

myotubes more similar to type II fibres. In fact, fibre

types are difficult to describe in vitro but the

observation of decreased SDH activity in cell lysates

might be an additional hint for in vitro fibre type

Novel aspects of adipocyte-induced skeletal muscle insulin resistance 289

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conversion and might have some relevance for the

process of insulin resistance. Furthermore, IL-6 is

mainly expressed in type I fibres (Plomgaard et al.,

2005) and its reduced secretion in CM-treated

myotubes may also point to a loss of type I fibre-

like properties. Whether IL-8 and MCP-1 are fibre

type-specific transcripts is not known.

In summary, we could demonstrate that adipocyte-

derived insulin resistance in skeletal muscle cells

effects various aspects of skeletal muscle cell physiol-

ogy such as myokine release, expression of myogenic

markers, mitochondrial function and oxidative capa-

city. The analysis of mechanisms involved in skeletal

muscle insulin resistance will eventually lead to a

better understanding of this process and a possible

discovery of muscular targets for the treatment of

type 2 diabetes.

Reversibility of insulin resistance

The development of insulin resistance and type 2

diabetes is a reversible process, at least to a certain

extend. Reduction of adipose tissue mass by weight

loss is a validated approach to reverse insulin

resistance (Fukuda et al., 1989; Petersen et al.,

2005). In parallel to improved insulin sensitivity,

weight reduction also reduces adipokine blood level

which has been demonstrated for IL-6 (Corpeleijn

et al., 2005), MCP-1 (Christiansen et al., 2005) and

TNF-a (Marfella et al., 2004). Furthermore, it could

be shown that high molecular weight adiponectin is

increased after weight loss (Bobbert et al., 2005). In

vitro, it could be shown that insulin resistance

disappears in cultured skeletal muscle biopsies from

obese, insulin-resistant patients (Brozinick et al.,

2003; Pender et al., 2005) demonstrating that insulin

resistance might be a reversible feature that can be

acquired with obesity. However, other studies provide

evidence for retained defects in culture of muscle cells

from obese and diabetic patients (Cozzone et al.,

2008; Henry et al., 1995). In our model of acquired

insulin resistance by treatment with CM, insulin

resistance can be reversed by simple withdrawal of

CM (Sell et al., 2008) (Figure 1). Together with

normalized insulin signalling, IL-6 secretion and the

expression of myoD is restored. However, several

defects present in insulin-resistant skeletal muscle

cells do not reverse upon CM withdrawal. Despite

normal insulin-stimulated Akt phosphorylation, myo-

tubes display decreased myogenin levels and abnor-

mal secretion of IL-8 and MCP-1 after withdrawal of

CM. These novel observations are hints for insulin

resistance in skeletal muscle to be a defect that can be

acquired with obesity and that is at least partially

reversible upon reduction of adipose tissue mass.

The endocannabinoid system – an overview

The endocannabinoid system is a complex network

involved in various physiological processes. Its

discovery was prompted by studies with tetrahydro-

cannabinol (THC), the psychoactive compound of

Cannabis sativa, which led to the characterization

(Devane et al., 1988; Herkenham et al., 1991) and

molecular cloning of the G protein-coupled canna-

binoid receptors type 1 (CB1R) and 2 (CB2R)

(Matsuda et al., 1990). Further research demon-

strated the existence of endogenous ligands, the so-

called endocannabinoids, as well as a complex

enzymatic machinery for their synthesis, release,

transport and degradation. The endocannabinoids

are derivatives of arachidonic acid which are

conjugated with ethanolamine or glycerol.

Arachidonoyl-ethanolamide (AEA, anandamide)

and 2-arachidonoylglycerol (2-AG) are the most

intensively studied compounds among the endocan-

nabinoids, and different pathways are involved in

their synthesis and release.

The formation of anandamide is carried out by a

specific phospholipase D (PLD) which cleaves N-

arachidonoyl-phosphatidylethanolamine (NAPE),

the precursor of anandamide (Figure 2a). The Ca2þ

dependent biosynthesis of NAPE is processed by the

enzyme N-acyltransferase, its activity being enhanced

by phosphorylation through the cAMP-dependent

activity of protein kinase A (Cadas et al., 1996;

Piomelli, 2003). The activity of PLD is regulated by

depolarization or by activation of G-protein coupled

receptors like ionotropic glutamate N-methyl-D-

aspartate (NMDA) receptors or metabotropic recep-

tors of major neurotransmitters including dopamine,

glutamate and acetylcholine. 2-AG formation is also

Ca2þ dependent but it is synthesized via a different

pathway. The activation of phosphatidylinositol-

specific phospholipase C (PLC) generates diacylgly-

cerol by cleavage of membrane phospholipids, which

is further processed to 2-AG by diacylglycerol lipase

(DAGL) (Figure 2b). The activation of metabotropic

P2Y purinergic receptors coupled to the PLC and

DAGL pathway systematically increases the produc-

tion of 2-AG (Piomelli, 2003; Stella et al., 1997),

whereby the contribution of ionotropic purinergic

receptors like P2XT boosts 2-AG formation. These

receptors, when activated, allow large quantities of

extracellular calcium to enter the cells and thus

enhancing the activity of DAGL while inhibiting the

2-AG degrading enzyme monoacylglycerol lipase

(MAGL) (Witting et al., 2004).

After synthesis, the endocannabinoids are released

in the extracellular space and act as retrograde

messengers at presynaptic CB1R (e.g. regulation of

neurotransmitter release) or target the receptors

in an utocrine/paracrine manner (Rodriguez de

Fonseca et al., 2005). The signals induced by the

endocannabinoids are terminated very rapidly by a

transporter-mediated uptake (Beltramo et al., 1997)

and subsequent degradation. The intracellular hy-

drolysis of anandamide to arachidonic acid and

ethanolamine is catalyzed by the integral membrane

enzyme fatty acid amide hydrolase (FAAH)

290 K. Eckardt et al.

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(Cravatt et al., 1996). FAAH expression has been

found in various tissues with high concentrations in

the brain and liver, and beside anandamide, it

degrades a variety of fatty acid amides such as

oleamide. 2-AG can also be inactivated by FAAH but

it is mainly hydrolyzed by MAGL (Dinh et al., 2002).

This enzyme is also widely distributed throughout

the body. For example, it has been shown that

MAGL is expressed in pre-synaptic terminals sug-

gesting a role in terminating retrograde signalling at

pre-synaptic neuron (Basavarajappa, 2007; Dinh

et al., 2002; Gulyas et al., 2004).

The endocannabinoids exert their action by bind-

ing to specific receptors. Beside the two major

cannabinoid receptors, CB1R and CB2R, pharma-

cological studies have revealed the existence of other

endocannabinoid targets including the transient

receptor potential vanilloid type 1 (TRPV1)

(Zygmunt et al., 1999) and at least two non-CB1,

non-CB2 cannabinoid receptors (Howlett et al.,

2002; Rodriguez de Fonseca et al., 2005). The

molecular characterization of CB1R and CB2R has

shown that they are members of the superfamily of

seven-transmembrane-spanning (7-TM) receptors

that can associate with G proteins. Cannabinoid

receptors are associated with G proteins of the Gi/o

family and signal transduction via Gi inhibits adenylyl

cyclase in most tissues and cells (Howlett, 2005).

Therefore, activation of CB1R and CB2R leads to

inhibition of cAMP production, resulting in a

decrease of protein kinase A-dependent phosphoryla-

tion processes as well. Furthermore, these receptors

regulate activity of ion channels, resulting in inhibi-

tion of calcium influx through N, P/Q and L type

calcium channels, and activation of inwardly rectify-

ing potassium channels (Howlett et al., 2002).

Stimulation of CB1R and CB2R also leads to

phosphorylation and activation of p42/p44

mitogen-activated protein kinase (MAPK), p38

MAPK and Jun N-terminal kinase (JNK) as signaling

pathways to regulate nuclear transcription factors

(Derkinderen et al., 2003; Howlett, 2005). Addition-

ally, they have been shown to be coupled to the

phosphatidylinositol 3-kinase pathway, to the focal

adhesion kinase, to ceramide signaling and to nitric

oxide production (Rodriguez de Fonseca et al., 2005).

The expression of CB1R mRNA has been demon-

strated in central and peripheral nerve tissue

(mainly located in terminals of neurons and glial

cells) (Devane et al., 1988; Hohmann & Herkenham,

1999; Piomelli, 2003; Tsou et al., 1998) as well as in

various peripheral tissues like fat (Bensaid et al.,

2003; Cota et al., 2003; Gasperi et al., 2007;

Pagano et al., 2007; Roche et al., 2006), liver

(Osei-Hyiaman et al., 2005), endocrine pancreas

(Bermudez-Silva et al., 2008; Juan-Pico et al., 2006;

Starowicz et al., 2008), skeletal muscle (Cavuoto

et al., 2007b), in the reproductive system (Gye et al.,

2005), some glandular systems and in the micro-

circulation (Pacher et al., 2005; Rodriguez de Fonseca

et al., 2005). The CB2R was initially described in

immune cells and multiple lymphoid organs. Mean-

while it is known, that CB2 receptors are also present

in non-immune cells such as skeletal muscle cells

(Cavuoto et al., 2007b), endocrine pancreas

(Juan-Pico et al., 2006), adipose tissue (Roche et al.,

2006), and skeleton (Bab and Zimmer, 2008).

The endocannabinoids exhibit different binding

properties and intrinsic activity at CB1R and CB2R.

Anandamide is a partial agonist at both receptors, but

has a higher affinity for the CB1R. Its intrinsic

activity at CB1R is 3-40 fold higher than at CB2 R

(Howlett et al., 2002; Rodriguez de Fonseca et al.,

2005). 2-AG behaves as a complete agonist at CB1R

Figure 2. Main biosynthesis and degradation pathways of anandamide and 2-Arachidonoylglycerol (2-AG). (a) The precursor of anandamide

is synthesized through activity of N-acyltransferase (NAT), which is Ca2þ dependent and enhanced by phosphorylation of cAMP-stimulated

proteinkinase A (PKA). The activity of the specific phospholipase D (PLD) is regulated by depolarization or by activation of G-protein

coupled receptors (GPCR). The degradation of anandamide is processed by fatty acid amide hydrolase (FAAH). (b) The production of 2-

AG is increased by activation of metabotropic purinergic P2Y receptors, which stimulate the phosphatidylinositol-specific phospholipase C

(PLC). Activation of ionotropic purinergic P2X7 raises the intracellular Ca2þ concentration thereby stimulating activity of diacylglycerol

lipase (DAGL) and inhibiting activity of monoacylglycerol lipase (MAGL).

Novel aspects of adipocyte-induced skeletal muscle insulin resistance 291

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and CB2R, and exhibits higher relative intrinsic

activity than anandamide at both receptors. Like

anandamide, 2-AG has marginally higher affinity for

CB1R than CB2R (Howlett et al., 2002).

Dysregulation of the endocannabinoid system

in obesity and hyperglycaemia

In recent time it has become clear that the

endocannabinoid system plays an important role for

the control of energy homeostasis and body weight at

both the level of food intake as well as peripheral

control of metabolism. This is based on studies in

CB1R knockout mice and/or in models of pharma-

cological blockade of CB1R resulting in reduced

food intake. However, the decrease in body weight

was to a higher extent than was predicted from the

effect of reduced energy intake (Cota et al., 2003;

Ravinet Trillou et al., 2003). Several studies have

reported evidences in animal models as well as in

humans, that in conditions of obesity and hypergly-

caemia the endocannabinoid system is over-acti-

vated. This over-activity occurs in the hypothalamus

as well as in peripheral tissues (liver, pancreas,

adipose tissue) and involves altered levels of en-

docannabinoids and/or CB1R expression.

In obese or hyperglycaemic patients due to type 2

diabetes, circulating levels of anandamide and 2-AG

are increased and elevated levels of 2-AG are found

in visceral adipose tissue (Bluher et al., 2006; Engeli

et al., 2005; Matias et al., 2006). Genetically and diet-

induced obese animal models show elevated levels of

endocannabinoids in hypothalamus, adipose tissue,

liver and endocrine pancreas (Di Marzo et al., 2001;

Matias et al., 2006; Osei-Hyiaman et al., 2005).

Additionally, the endocannabinoid level might be

influenced by the fatty acid composition of the food

due to availability of biosynthetic precursors

(Artmann et al., 2008; Berger et al., 2001; Matias

et al., 2008a; Watanabe et al., 2003). Very recently,

Matias et al. (2008b) showed that the level of

endocannabinoids during high-fat diet changes tissue

specific and depending on the composition and

duration of the diet. Interestingly, they also found

effects in the thyroid and adrenal glands, which are

indirectly involved in control of metabolism, as well

as in heart and kidney, which are affected in the long

term by the metabolic consequences of a dysregu-

lated endocannabinoid system.

Several studies revealed, that the expression of

CB1R mRNA is elevated in adipose tissue (Bensaid

et al., 2003), skeletal muscle (Pagotto et al., 2006) and

liver (Osei-Hyiaman et al., 2005) of diet-induced

obese rodents. In humans, controversial data regard-

ing CB1R expression in obese vs. lean patients exist.

While Pagano et al. (2007) found an increased

expression of CB1R mRNA in visceral fat tissue of

obese patients, other working groups (Bluher et al.,

2006; Engeli et al., 2005; Kempf et al., 2007) reported

decreased levels in visceral and subcutaneous fat

tissue compared to lean patients. Another study

carried out by Lofgren et al. (2007) did not find any

association between subcutaneous or visceral adipose

CB1R mRNA levels and body weight.

Additionally, the mRNA expression of FAAH, the

enzyme responsible for degradation of anandamide,

was shown to be decreased in adipose tissue of obese

humans (Bluher et al., 2006; Engeli et al., 2005;

Kempf et al., 2007). In mice on high-fat diet the

hepatic FAAH activity was reduced by more than

80% with apparently unchanged anandamide synth-

esis (Osei-Hyiaman et al., 2005).

The endocannabinoid system in peripheral

tissues

Adipose tissue

Analysis revealed the presence of a functional

endocannabinoid system in the adipose tissue and

data from several studies suggest that in state of

imbalanced energy supply the level of endocannabi-

noids and CB1R expression level are changed, as

outlined above.

The presence of CB1R was shown in human

(Pagano et al., 2007; Roche et al., 2006; Spoto et al.,

2006), mouse (Bensaid et al., 2003; Cota et al., 2003;

Di Marzo et al., 2008; Gasperi et al., 2007) and rat

adipoctyes (Bensaid et al., 2003; Yan et al., 2007).

Beside CB1R, the expression of CB2R could also be

demonstrated in human adipocytes but its functional

implication are currently not known (Pagano et al.,

2007; Roche et al., 2006). In human adipose tissue,

the expression of NAPE-PLD, DAGL, FAAH, and

MAGL and the presence of endocannabinoids were

proven (Pagano et al., 2007; Roche et al., 2006) as

well as altered expression level of these enzymes in

obese humans compared to lean controls (Pagano

et al., 2007). Furthermore, Gonthier et al. (2007)

used isolated mature adipocytes from human sub-

cutaneous fat to verify that adipocytes are able to

synthesize 2-AG and anandamide.

Studies in cultured mouse adipocytes revealed that

the level of CB1R mRNA as well as the CB1R protein

raised 3–4 fold during the differentiation (Bensaid

et al., 2003). Also, the amount of 2-AG increased

during differentiation, which is accompanied by an

elevated expression of DAGL but unchanged expres-

sion of MAGL. For anandamide, a peak at day 4 was

observed which goes along with a peak of NAPE-PLD

at day 4 and its decrease thereafter, while FAAH

expression increased from day 0 to day 12 (Matias

et al., 2006). Treatment of pre-adipocytes with CB1R

agonist HU-210 accelerated the differentiation as

shown by stimulation of peroxisome proliferator-

activated receptor (PPAR)-s expression and accumu-

lation of lipid droplets at day 8 (Matias et al., 2006).

In differentiated adipocytes the stimulation of

CB1R increases glucose uptake and GLUT4

translocation (Pagano et al., 2007) as well as

292 K. Eckardt et al.

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insulin-stimulated glucose uptake (Gasperi et al.,

2007), lipoprotein lipase activity (Cota et al., 2003),

and expression of fatty acid synthase mRNA (Osei-

Hyiaman et al., 2005). Additionally, endocannabi-

noids inhibit the AMP-activated protein kinase

(AMPK) (Kola et al., 2005). The described effects of

CB1R stimulation were prevented by treatment with

specific CB1R antagonists like rimonabant. Adminis-

tration of rimonabant reduced body weight and

stimulated the expression of adiponectin in adipose

tissue of obese Zucker (fa/fa) rats. In mouse adipo-

cytes rimonabant caused overexpression of adiponec-

tin mRNA and protein (Bensaid et al., 2003).

Taken together, an over-activated endocannabi-

noid system results in routeing excess energy to the

adipocytes and enhancing the storage of fat, thereby

increasing the fat depots (Figure 3). The lipogenic

action of activated CB1R in adipocytes can explain in

part why CB1 knockout mice fed with the same

amount of food as wild-type mice still develop less fat

mass (Cota et al., 2003).

Liver

The liver is the central metabolic organ and involved

in the regulation of glucose, fat and amino acid

metabolism. It plays an important role in de novo

lipogenesis and gluconeogenesis as well as in storage

of glycogen. In fact, the liver plays an even greater

role in lipogenesis than does adipose tissue (Diraison

et al., 2003).

Osei-Hyiaman and co-workers investigated the

impact of CB1R stimulation in the liver using

CB1R7/7 and diet-induced obese mouse model

(Osei-Hyiaman et al., 2005). The presence of CB1R

was confirmed by multiple methods, and after 3

weeks of high-fat diet its expression was increased

compared to controls. Also, the hepatic level of

anandamide was increased while no change was

observed for 2-AG level. This observation is accom-

panied by a decreased activity of FAAH while the

activity of N-acyltransferase and the expression of

FAAH remained unaltered. These changes were

observed before obesity was detectable.

The stimulation of CB1R inhibits the activity of

AMPK (Kola et al., 2005), induces expression of the

lipogenic transcription factor SREBP-1c and its

target enzymes acetyl co-enzyme-A carboxylase-1

(ACC1) and fatty acid synthase, and also increases de

novo fatty acid synthesis. Pre-treatment of mice on

high-fat diet with rimonabant reduced the rate of

fatty acid synthesis. In CB1R7/7 mice which are

resistant to diet-induced obesity, the high-fat diet did

not result in altered basal rate of fatty acid synthesis

(Osei-Hyiaman et al., 2005).

The results of this study suggest that the hepatic

endocannabinoid system is activated during early

stages of high-fat diet-induced obesity and that this

may be required for the development of obesity,

primarily due to an increase in de novo lipogenesis

(Kunos, 2007) (Figure 3).

Endocrine pancreas

The endocrine pancreas is an important part of the

regulatory network to maintain glucose homeostasis

Figure 3. Consequences of obesity-related overactivation of the endocannabinoid system in peripheral tissues. In general, obesity is

associated with increased endocannabinoid (EC) level and CB1R expression, and decreased FAAH activity or expression. In pancreas,

regulation of CB1R and FAAH activity or expression in obese state are not yet characterized, as well as altered EC level and regulation of

FAAH activity or expression in obese-derived skeletal muscle. Elevated EC level may take part in induction of insulin resistance. ACC1,

acteyl coenzym-a carboxylase-1; AMPK, AMP activated protein kinase; FAS, fatty acid synthase.

Novel aspects of adipocyte-induced skeletal muscle insulin resistance 293

Downloaded By: [Eckardt, Kristin] At: 12:25 24 October 2008

by secreting insulin in response to increasing blood

glucose level. Expression of the endocannabinoid

system components in the pancreas suggests that a

connection exists between the endocrine function

and the endocannabinoid system.

The expression of cannabinoid receptors in the

pancreas were shown by several working groups

(Bermudez-Silva et al., 2008; Juan-Pico et al., 2006;

Matias et al., 2006; Starowicz et al., 2008), whereby

the distribution of cannabinoid receptors appears to

be species-specific. A detailed study by Bermudez-

Silva et al. (2007) revealed a distinct distribution of

cannabinoid receptors in human pancreas by im-

munofluorescence staining. CB1R expression was

detected in alpha cells and in a small portion of beta

cells, but not in delta cells or exocrine pancreas. On

the other hand, CB2R expression was found in delta

cells and exocrine pancreas, but absent in alpha and

beta cells. In mice, CB1R is not expressed in beta

cells but in non-beta cells, while CB2R is expressed

in beta and non-beta cells. In rats, both cannabinoid

receptors are expressed in beta and non-beta cells

(Bermudez-Silva et al., 2007; Juan-Pico et al., 2006).

Also, the enzymes for synthesis and degradation of

endocannabinoids are expressed in a specific pattern.

Within the islet the, DAGL and MAGL expression

were found, and FAAH expression was detected in

beta cells but not in alpha cells. NAPE-PLD was

almost absent in islet but detected in acinar

surrounding tissue. Beside in human islets, the

enzymes of biosynthesis and degradation were also

demonstrated in rat insulinoma RIN-m5F beta cells

(Matias et al., 2006) and mouse pancreas (Starowicz

et al., 2008).

Analysis of pancreatic endocannabinoid level in

diet-induced obese mice revealed an increase of

anandamide and 2-AG level compared to lean

controls (Matias et al., 2006; Starowicz et al.,

2008). Stimulating RIN-m5F beta cells with

33 mM glucose elevated both anandamide and

2-AG level. If these cells were cultured under low

glucose condition (13 mM), costimulation of

33 mM glucose and insulin prevented glucose-

induced increase of endocannabinoid level. On the

other hand, culture in high glucose condition

(33 mM) mimicking hyperglycaemia not only

prevented the inhibitory effect of insulin on

glucose-induced increase of endocannabinoid

level, but also enhanced the level of anandamide

and 2-AG per se.

A study in isolated mouse pancreatic islets showed,

that glucose-induced Ca2þ oscillation and insulin

secretion was prevented by stimulation of CB1R

(Nakata & Yada, 2008) and CB2R (Juan-Pico et al.,

2006). Administration of anandamide to rats resulted

in glucose intolerance which could be improved by

treatment with CB1R antagonist (Bermudez-Siva

et al., 2006). Interestingly, administration of CB2R

agonists resulted in improved glucose tolerance

which is opposite to the results in mouse islets

(Bermudez-Silva et al., 2007). The action of CB2R

on insulin release may be species-specific as also

suggested by the species-specific distribution of the

cannabinoid receptors in the various pancreatic cell

types. In the insulinoma model, stimulation of CB1R

increased insulin secretion when the cells were kept

in high glucose condition but not in low glucose

condition (Matias et al., 2006). In isolated human

islets the stimulation of pancreatic CB1R resulted in

secretion of insulin, despite the relative low abundant

of CB1R in beta cells (Bermudez-Silva et al., 2008).

It seems that the result of CB1R stimulation depends

on the glycaemic status, and more studies are needed

to understand the role of the endocannabinoid

system in the pancreas.

The available data suggest, that under hypergly-

caemic conditions the endocannabinoid level in the

endocrine pancreas raises and is no longer depressed

by insulin (Figure 3). The endocannabinoid system

becomes dysregulated and the subsequent over-

stimulation of CB1R might reinforce insulin release

resulting in hyperinsulinemia which in turn start a

vicious circle by further increasing endocannabinoid

level. These findings are mirrored in the pre- and

postprandial blood endocannabinoid level of lean

normoglycaemic and obese hyperglycaemic subjects.

While in the normoglycaemic group the anandamide

level decreased postprandial, in the hyperglycaemic

group the level of both anandamide and 2-AG

increased significantly, suggesting that normal reg-

ulation of blood endocannabinoid level are disrupted

(Matias et al., 2006).

Skeletal muscle

Currently, only few data are published regarding the

impact of the endocannabinoid system in skeletal

muscle. Liu et al. reported in the genetically obese

Lepob/Lepob mouse model an increase in glucose

uptake in isolated soleus muscle preparations after

treating the mice with rimonabant. This effect may

contribute to the improved glycaemia seen in

previous studies with rimonabant (Liu et al., 2005).

Cavuoto et al. (2007b) reported the expression of

CB1R, CB2R, and FAAH mRNA in human and

rodent skeletal muscle. In our lab, we detected CB1

and CB2R protein in differentiated human primary

muscle cells (unpublished data). Furthermore,

CB1R mRNA was shown to be elevated in skeletal

muscle of diet-induced obese mice compared with

lean controls (Pagotto et al., 2006), while no

difference in mRNA level was detected between

human myotubes derived from lean or obese subjects

(Cavuoto et al., 2007a) (Figure 3).

One study investigated the effects of CB1R

agonism or antagonism on the expression of genes

involved in regulating the energy metabolism

(Cavuoto et al., 2007a). For this purpose, human

skeletal muscle cells derived from lean or obese

subjects were treated with anandamide and AM251,

294 K. Eckardt et al.

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separately or in combination. Stimulation with

anandamide alone did not alter the gene expression

of AMPKa1, AMPKa2, PPAR-g co-activator

(PGC)- 1a or PDK4. The antagonist AM251

increased mRNA level of AMPKa1 and decreased

PDK4, while no effects were seen in gene expression

of AMPKa2 and PGC-1a. Differences between

myotubes derived from lean or obese subjects were

observed when antagonist and agonist were com-

bined. In myotubes from obese subject a decrease of

AMPKa1, AMPKa2 and PGC-1a mRNA levels was

observed. In myotubes from lean subjects an increase

of AMPKa2 gene expression was found but no effect

on AMPKa1 and PGC-1a mRNA levels. Taken

together, these data are consistent with an overall

increase in metabolic capacity within skeletal muscle

following antagonism of CB1R.

The anti-diabetic effects of rimonabant are

mediated by its action on different organs and tissue

such as adipose tissue and liver, and the skeletal

muscle as the primary tissue of glucose uptake

(DeFronzo et al., 1981) is supposed as target as well.

Further studies are needed to understand the role of

the endocannabinoid system in the skeletal muscle

with special regard to the development of insulin

resistance, which is associated with obesity and type 2

diabetes.

Conclusion

The mechanisms behind the development of insulin

resistance in skeletal muscle are very complex and far

from being completely understand. A complicated

network is involved in this process and many players,

adipokines, myokines and other still unknown factors

might be involved – a long list that is regularly

extended. The endocannabinoid system, which has

been shown to be over-activated in the state of

obesity, has drawn much attention due to results

from studies with specific CB1R antagonists like

rimonabant, which are able not only to reduce food

intake and body weight but also to improve several

metabolic parameters. The implication of endocan-

nabinoids like anandamide or 2-AG, their level being

elevated in obesity, in the development of skeletal

muscle insulin resistance is currently under investi-

gation. Another important question is the reversi-

bility of insulin resistance. As our data and those of

other working groups indicate, the process of insulin

resistance is reversible only to a certain extent. Even

short periods of insulin resistance seem to leave

prolonged disturbance of skeletal muscle metabo-

lism. In summary, the feature of tissue insulin

resistance needs to be further explored to allow the

development of effective strategies against this

prominent characteristic of type 2 diabetes.

Declaration of interest: The authors report no

conflicts of interest. The authors alone are respon-

sible for the content and writing of the paper.

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