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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
To link to this Article: DOI: 10.1080/13813450802404761
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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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