REVIEW ARTICLE

Insulin resistance associated to obesity: the link TNF-alpha

IRIA NIETO-VAZQUEZ1, SONIA FERNANDEZ-VELEDO1, DAVID K. KRAMER1,2,

ROCIO VILA-BEDMAR1, LUCIA GARCIA-GUERRA1, & MARGARITA LORENZO1

1Departamento de Bioquimica y Biologia Molecular II, Facultad de Farmacia, Universidad Complutense, 28040–Madrid,

Spain, and 2Present adress: Facultad de Biologia, Universidad de Barcelona, Spain

AbstractAdipose tissue secretes proteins which may influence insulin sensitivity. Among them, tumour necrosis factor (TNF)-alphahas been proposed as a link between obesity and insulin resistance because TNF-alpha is overexpressed in adipose tissuefrom obese animals and humans, and obese mice lacking either TNF-alpha or its receptor show protection against developinginsulin resistance. The activation of proinflammatory pathways after exposure to TNF-alpha induces a state of insulinresistance in terms of glucose uptake in myocytes and adipocytes that impair insulin signalling at the level of the insulinreceptor substrate (IRS) proteins. The mechanism found in brown adipocytes involves Ser phosphorylation of IRS-2mediated by TNF-alpha activation of MAPKs. The Ser307 residue in IRS-1 has been identified as a site for the inhibitoryeffects of TNF-alpha in myotubes, with p38 mitogen-activated protein kinase (MAPK) and inhibitor kB kinase beinginvolved in the phosphorylation of this residue. Moreover, up-regulation of protein-tyrosine phosphatase (PTP)1Bexpression was recently found in cells and animals treated with TNF-alpha. PTP1B acts as a physiological negative regulatorof insulin signalling by dephosphorylating the phosphotyrosine residues of the insulin receptor and IRS-1, and PTP1Bexpression is increased in peripheral tissues from obese and diabetic humans and rodents. Accordingly, down-regulation ofPTP1B activity by treatment with pharmacological agonists of nuclear receptors restores insulin sensitivity in the presence ofTNF-alpha. Furthermore, mice and cells deficient in PTP1B are protected against insulin resistance induced by thiscytokine. In conclusion, the absence or inhibition of PTP1B in insulin-target tissues could confer protection against insulinresistance induced by cytokines.

Key words: Glucose uptake, LXR, PTP1B, p38MAPK, rosiglitazone.

Abbreviations: AMPK, AMP-activated protein kinase; AS160, AKT substrate of 160 kDa; ERK, extracelullar-signal regulated kinase; FFA, free fatty acids; FATP, fatty acid transporter protein; FAT, fatty acid translocase;GLUT4, insulin-regulated glucose transporter; HSL, hormone sensitive lipase; IKK, inhibitor kB kinase; IR, insulinreceptor; IRS, insulin receptor substrate; JNK, c-Jun N-terminal kinase; LPL, lipoprotein lipase; LXR, liver Xreceptor; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; PLC, phospholipase C; PP,protein-phosphatase; PPAR, peroxisome proliferator activated receptor; PTP, protein-tyrosine phosphatase; TNF,tumour necrosis factor; TZD, thiazolidinediones; UCP, uncoupling-protein.

Introduction

Tumour necrosis factor (TNF)-alpha was first

described as an endotoxin-induced serum factor that

causes necrosis of tumours (Carswell et al., 1975).

Later, TNF-alpha was shown to be identical to the

protein called cachectin, which is found in cultured

macrophages exposed to endotoxin. Cachectin, or

TNF-alpha, was initially found to induce cachexia in

animals (Tracey et al., 1988).

The human gene of TNF-alpha, TNFA, was

cloned in 1984 (Old, 1985). TNF-alpha is expressed

as a 26 kDa plasma membrane-bound monomer.

Proteolytic cleavage by the TNF-alpha converting

enzyme results in the formation of a 17 kD soluble

trimer of the membrane-bound precursor protein of

TNF-alpha. Two TNF-alpha receptors, denomi-

nated type I (TNFR1) and type II (TNFR2),

mediate the TNF-alpha signal by forming protein

complexes with cytoplasmic adaptor proteins. The

Correspondence: Margarita Lorenzo, Departamento de Bioquimica y Biologia Molecular II, Facultad de Farmacia, Universidad Complutense, 28040–Madrid,

Spain. Tel: 34913941858. Fax: 34913941779. E-mail: mlorenzo@farm.ucm.es

Received for publication 6 February 2008. Accepted 23 April 2008.

Archives of Physiology and Biochemistry, July 2008; 114(3): 183–194

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

DOI: 10.1080/13813450802181047

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fact that TNF-alpha signalling involves two receptors

has been complicating the dissection of its effects

(reviewed in Aggarwal, 2003). Depending on which

receptor is activated opposing intracellular pathways

with partially dramatically differential effects can be

triggered. Studies, so far, have addressed this

possibility only partially and, to date, most known

functions of TNF-alpha have been ascribed to signals

transduced by TNFR1 with TNFR2 playing primar-

ily a modulatory role in ligand passing. Interestingly,

utilizing the two different receptor types TNF-alpha

can affect cells pro-apoptotic or anti-apoptotic,

depending on conditions. This has recently been

shown to be the case in human skeletal muscle

sarcopenia, liver, and human adipocytes (for review

see Dirks and Leeuwenburgh, 2006).

Recent studies revealed that the pro-inflammatory

cytokine TNF-alpha secreted by the adipose tissue

does not only play a key role in the mediation of

immune response as a multi-functional regulator of

inflammation, cell apoptosis and survival, cytotoxi-

city, and production of other cytokines, such as

interleukin (IL)-1 and IL-6, but also in the induction

of obesity-induced insulin resistance.

Obesity, TNF-alpha, and insulin resistance

Insulin resistance, which can be defined as a

diminished ability of the cell to respond to the action

of insulin, is the most important pathophysiological

feature in many prediabetic states and is the first

detectable defect in type 2 diabetes. The pathogen-

esis of type 2 diabetes involves abnormalities in both

insulin action and secretion. Insulin resistance is

usually compensated by hyperinsulinemia. Although

moderate hyperinsulinemia might be tolerated in the

short term, chronic hyperinsulinemia exacerbates

insulin resistance and contributes directly to beta-

cell failure and diabetes (White, 2003). At the

molecular level, insulin resistance correlates with

impaired insulin signalling in peripheral tissues

(Figure 1). Insulin resistance in the adipose tissue

leads to an increase of lipolysis, with subsequent

release of glycerol and free fatty acids (FFA) into the

circulation. It is widely accepted that increased

availability and utilization of FFA contribute to the

development of skeletal muscle insulin resistance, as

well as to augmented hepatic glucose production

(White, 2003). In fact, the progression of insulin

resistance in high-fat diet rats is closely related to

plasma FFA rather than TNF-alpha and IL-6 levels

(Jiao et al., 2008). Both genetic and environmental

factors can contribute to develop insulin resistance,

and in the second group, obesity has been proposed

to be an important contributor.

Obesity is a risk factor to develop type 2 diabetes, in

part due to the fact that adipose tissue secretes proteins

named adipokines that may influence insulin sensitiv-

ity. Adipose tissue, in particular the visceral compart-

ment, is now recognized as the primary contributor to

the insulin resistance syndrome. Several factors

secreted from adipose tissue, including cytokines, che-

mokines and FFA, can impair insulin signalling

altering insulin-mediated processes, including glucose

homeostasis and lipid metabolism (Arner, 2003).

Accordingly, obesity is now being considered a chronic

state of low-intensity inflammation. In this regard,

recent studies reveal that obesity is also associated with

an increase in adipose tissue infiltration of macro-

phages, which contributes to the inflammatory process

through the additional secretion of cytokines (Lumeng

et al., 2007). The mechanisms by which adipose tissue

recruits and maintains macrophages could involve

expression of monocyte chemoattractan proteins and

intercellular adhesion molecule-1, as was described

recently (Weisberg et al., 2006).

TNF-alpha has been proposed as a link between

adiposity and the development of insulin resistance

Figure 1. The different steps in the development of type 2 diabetes.

184 I. Nieto-Vazquez et al.

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because the majority of type 2 diabetic subjects are

obese, is highly expressed in adipose tissues from

obese subjects, and obese mice lacking either TNF-

alpha or its receptors showed protection for devel-

oping insulin resistance (Hotamisligil, 2003). Rather

than acting systemically, TNF-alpha seems to act

locally at the site of adipose tissue through autocrine

or paracrine mechanisms, having effects on insulin

resistance and inducing IL-6 (Arner, 2003). Circu-

lating levels of soluble TNF-alpha receptors now

seems well correlated with BMI, and impairment in

TNF-alpha processing can improve systemic insulin

sensitivity (Zahorska-Markiewicz et al., 2000; Serino

et al., 2007). On the other hand, TNF-alpha has

lypolitic and antiadipogenic effects on adipose tissue

(Arner, 2003). This paradox could be due to

proliferative and anti-apoptotic effects of this cyto-

kine in the obese adipocyte, and could be mediated

by the differential expression of its soluble and

membrane-anchored receptors. Both ceramides and

FFA were reported to induce insulin resistance in

peripheral tissues, and the production of these

molecules could be the consequence of activation

of sphingomyelinase or lipolysis by TNF-alpha

(Arner, 2003). Several other mediators that are

activated in response to TNF-alpha such as stress

kinases and inflammatory pathways, could also

contribute to insulin resistance. In this regard,

increased phosphorylation of p38MAPK in adipo-

cytes and muscle from type 2 diabetic subjects has

been reported (Carlson et al., 2003).

Hence, TNF-alpha initiated pathways are thought

to contribute at least partially, to the metabolic

consequences of obesity in causing decreased adipo-

cyte and skeletal muscle insulin sensitivity. However,

the picture is far from being straight forward, and

rodent versus human data does not always match up.

Hence many questions remain, some of which we

will attempt to address here.

TNF-alpha deficient mice exhibit lower circulating

FFA and triglycerides than wild-type animals (Uysal

et al., 1997). Furthermore, infusion of TNF-alpha

in rodents leads to impairment of insulin stimula-

ted skeletal muscle glucose uptake. Accordingly,

neutralisation of TNF-alpha with specific antibodies

has the opposite effect and improves an insulin

resistant state in rats (Hotamisligil et al., 1994).

However, in contrast to the findings in rodent models

TNF-alpha neutralisation has been of no beneficial

effect in terms of insulin sensitivity in humans

(Rosenvinge et al., 2007). The effects of neutralisa-

tion of TNF-alpha on the expression levels of other

adipokines were differential being suggestive for why

TNF-alpha neutralisation gave no positive result. The

fact that TNF-alpha is a regulator of expression of

other adipokines, which also have effects on insulin

sensitivity, lipid metabolism and energy homoeostasis

control, complicates studies on the effects of TNF-

alpha, especially in genetically diverse human sub-

jects. While neutralisation of TNF-alpha increased

total adiponectin, the high molecular weight form of

adiponectin, which is thought to have positive effects

on insulin sensitivity was unaltered (Lo et al., 2007).

Also IL-6 expression was decreased but did not result

in any change in insulin sensitivity either (Rosenvinge

et al., 2007). Furthermore, muscle adiposity was

increased, leptin content was unaltered and resistin

levels were decreased. Additionally, in order to

induce insulin resistance using TNF-alpha infusion

requires concentrations that exceed the physiological

range of TNF-alpha usually observed in insulin

resistant rodent models (Altomonte et al., 2003).

However, TNF-alpha expression is increased in

adipose tissue from insulin-resistant obese indivi-

duals (Kern et al., 2001). Interestingly, these studies

have mostly investigated male subjects while studies

performed with females have suggested that there

might be a lack of such a correlation of TNF-alpha

with insulin resistance in women. It has been

suggested that some seemingly conflicting findings

in humans may be based on a differential expression

of the TNFR in males compared to females (Kern

et al., 2001). Further investigation will be required to

address this possibility. In order to understand the

possible mechanisms by which TNF-alpha acts upon

metabolism and insulin sensitivity, we will review the

direct and indirect effects of TNF-alpha in adipose

tissue and skeletal muscle.

The role of TNF-alpha in insulin resistance

in adipose tissue

While many secreted factors including RBP4, leptin

and IL-6 are elevated in adipose tissue from obese

humans and rodents, TNF-alpha has been most

strongly implicated in the pathogenesis of insulin

resistance, because its expression is strongly correlated

to reduced insulin-stimulated glucose disposal (Kern

et al., 2001). TNF-alpha expression is increased in

adipose tissue of humans with insulin resistance

(Hotamisligil et al., 1995). In adipose tissue TNF-

alpha is the main factor that triggers enhanced

lipolysis leading to increased secretion of FFA from

adipose tissue into the circulation (Table I) (Ruan

et al., 2003). TNF-alpha has been proposed to

stimulate lipolysis by inhibiting insulin receptor

signalling, by inhibiting signalling through the G-

protein-coupled adenosine receptor and via direct

stimulation of basal (nonhormonal) lipolysis through

interactions with the lipid-binding protein perilipin.

Lipids released from adipocytes as FFA are trans-

ported as triglycerides by VLDL and shifted to non-

adipose tissues like skeletal muscle (Sharma and

Chetty, 2005).

Concomitantly, lipoprotein lipase (LPL), the en-

zyme which hydrolyses lipids in lipoproteins into

fatty acids and glycerol and facilitates lipid FA uptake

is inhibited by TNF-alpha in 3T3-L1 adipocytes and

adipose tissue (Ramsay, 1996). Furthermore, in-

creased circulating TNF-alpha contributes also to an

Insulin resistance associated to obesity: the link TNF-alpha 185

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inhibition of adipogenesis, possibly via inhibition of

peroxisome proliferator activated receptor (PPAR)g(Zhang et al., 1996) and liponeogenesis from glucose

is reduced (Hotamisligil, 1999). Taken together, this

leads to diminished ability of adipose tissue to

accumulate lipids and thereby render clearance of

FFA from circulation impossible leading to increased

FFA in the blood stream.

Many of these described effects are a sign of an

impairment of the insulin signalling network. Insulin

initiates the biological effects in target cells by

binding to, and activating, its endogenous Tyr kinase

receptors. Insulin receptors (IR) are believed to

transduce signals by phosphorylation on Tyr residues

of several cellular substrates including insulin recep-

tor substrate (IRS) proteins 1, 2, 3 and 4 (White,

2003). A number of signalling pathways can be

activated downstream of IRS proteins organized in

two major elements, Ras/Raf/extracelullar-signal

regulated kinase (ERK) and phosphatidylinositol 3-

kinase (PI3K)/AKT/p70S6 kinase pathways. On the

other had, the insulin-signalling cascade is negatively

regulated by protein phosphatases, including Tyr,

Ser and lipid phosphatases. Most notably, protein-

tyrosine phosphatase (PTP)1B acts by dephosphor-

ylating the phosphotyrosine residues of the IR and

IRS-1 (Figure 2).

It is apparent that TNF-alpha is important for

regulating insulin action in human adipose tissue

because there is a strong negative relationship

between the release of the cytokine and insulin-

induced glucose metabolism in this tissue (Lofgren

et al., 2000). The interaction between TNF-alpha

and insulin signalling is above all important for local

insulin resistance in obesity. When cells are exposed

directly to TNF-alpha, this adipokine inhibits insulin

signalling by affecting IRS proteins (Hotamisligil,

2003). The mechanisms affecting IRSs involve

proteasome-mediated degradation, phosphatase-

mediated dephosphorylation, and Ser phosphoryla-

tion of IRS-1, which converts IRS-1 in an inhibitor of

the IR Tyr kinase activity, as reviewed previously

(White, 2003; Pirola et al., 2004). Both ERK and c-

Jun N-terminal kinase (JNK) have been proposed as

mediating TNF-alpha Ser/Thr phosphorylation of

IRS-1 in white adipocytes, with the Ser307 residue

Figure 2. A complex insulin intracellular signalling cascade controls the pleoitropic effects of this hormone.

Table I. Effect of TNF-alpha in adipose tissue and skeletal muscle.

Adipose tissue Skeletal muscle

Insulin sensitivity # (Hotamisligil, 1999) Insulin sensitivity # (Plomgaard et al., 2005)

TNF-alpha expression " (Hotamisligil et al., 1995) TNF-alpha expression " (Plomgaard et al., 2005)

Circulating FFA " (Ruan et al., 2003) Myocellular FFA storage " (Sharma and Chetty, 2005)

Lipolysis " (Ruan et al., 2003) Long term FA oxidation # (Schrauwen-Hinderling et al., 2005)

LPL activity # (Ramsay, 1996) AMPK activity # (Steinberg et al., 2006)

Liponeogenesis # (Hotamisligil, 1999) Glycogen synthesis # (Ruan et al., 2002)

GLUT4 expression # (Fernandez-Veledo et al., 2006a) GLUT4 translocation # (de Alvaro et al., 2004)

FAT/FATP expression # (Memon et al., 1998) Glucose uptake # (de Alvaro et al., 2004)

IRS-2 Ser phosphorylation " (Teruel et al., 2001) IRS-1 Ser phosphorylation " (de Alvaro et al., 2004)

PTP1B expression " (Fernandez-Veledo et al., 2006b) PTP1B expression " (Nieto-Vazquez et al., 2007)

Adipogenesis # (Zhang et al., 1996) Sarcopenia " (Dirks and Leeuwenburgh, 2006)

Ceramide levels " (Teruel et al., 2001) Diacylglycerol levels (DAG) " (Bruce and Dyck, 2004)

186 I. Nieto-Vazquez et al.

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being identified as the site for TNF-alpha phosphor-

ylation of IRS-1. In this regard, ablation of jnk1

decreases the development of insulin resistance

associated with dietary obesity. Furthermore, ERK

and p38MAPKs could inhibit insulin signalling by

TNF-alpha at the level of IRS-1 and IRS-2 in 3T3-

L1 adipocytes, whereas JNK could mediate the

feedback inhibitory effect of insulin (White, 2003;

Pirola et al., 2004). Others work has also implicated

activation of inhibitor kB kinases (IKK) by TNF-

alpha on Ser phosphorylation of IRS-1. Meanwhile,

IKK inhibition with salicylate or targeted disruption

of ikkb reversed obesity and diet-induced insulin

resistance (Gao et al., 2003).

In addition to these signalling impairments long-

term effects take place. Long term effects of TNF-

alpha are mediated via repression or stimulation of

expression of many genes responsible for glucose and

FFA uptake and storage in adipose tissue. For 3T3-

L1 adipocytes it has been shown that TNF-alpha

dependent gene up- and downregulation occurs by

regulation of the NF-kB (Ruan et al., 2002). For

example, TNF-alpha has been shown to down-

regulate the genes for adiponectin, GLUT4, IRS-1,

C/EBPa, PPARg, and perilipin in adipocytes. Inter-

estingly, in TNF-alpha injected syrian hamster fatty

acid transporter protein (FATP), and fatty acid

translocase (FAT) mRNA expression was reduced

approximately 50% in adipose tissue (Memon et al.,

1998).

TNF-alpha action on adipose tissue can alter the

production of many adipokines, and this is relevant to

the systemic effects of this cytokine on insulin

sensitivity and whole body energy homeosthasis as

review in Cawthorn and Sethi (2008). On the other

hand TNF-alpha upregulates the expression of genes

like vascular cell adhesion molecule-1, plasminogen

activator inhibitor-1, IL-6, IL-1b, angiotensinogen,

resistin and leptin (Ruan et al., 2002). Interestingly,

the drug metformin, which exerts its antihyperglyce-

mic effect by improving insulin sensitivity, which is

associated with decreased level of circulating FFA,

attenuated TNF-alpha-mediated lipolysis by suppres-

sing phosphorylation of ERK and reversing the

downregulation of perilipin protein in TNF-alpha-

stimulated adipocytes (Ren et al., 2006). Activators of

PPARg have been shown to inhibit the effects of a

number of the above mentioned effects and ultimately

the TNF-alpha induced inhibition of insulin sensitiv-

ity (Souza et al., 1998). This data indicates that TNF-

alpha action via TNFR1 might have an inhibitory

effect on PPAR-gamma mediated expression control.

Finally, the role of variations in TNF-alpha

genotypes in the mediation of the TNF-alpha action

has recently become the focus of attention. In a

recent study subjects were genotyped for the TNF-

alpha-238G 4 A, -308G 4 A, and -863C 4 A

polymorphisms. Compared with subjects who were

homozygous for the -238G allele, carriers of the

-238A allele had an altered ability to suppress

postprandial FFA. This effect was observed in

obese but not in non-obese individuals with type

2 diabetes (men, n¼ 56 and women, n¼ 67)

(Fontaine-Bisson et al., 2007). Patients with coron-

ary artery disease showed a higher frequency of the

TNF-308A allele than healthy controls, while cor-

onary artery disease patients with diabetes had a

higher frequency of the TNF-308AA genotype com-

pared with healthy controls (Sbarsi et al., 2007).

Molecular mechanisms involved in

insulin-resistance by TNF-alpha in brown

adipocytes: amelioration by agonists of

nuclear receptors

Brown adipose tissue is present and active in

mammal newborns and is responsible for their

successful defence of body temperature without

shivering. When this tissue is not adrenergically

stimulated, brown adipocytes suffer apoptosis or

transform into white adipocyte-like cells that gradu-

ally loose many brown characteristics. This phenom-

enon is particularly noticeable in adult humans, in

which brown adipose tissue is thought to be rapidly

lost postnatally, so that humans later in life do not

possess more than vestigial amounts of this tissue,

located within the white fat depots (Cannon and

Nedergaard, 2004). However, the use of fluorodeox-

yglucose positron emission tomography has revealed

the presence of symmetrical areas of increased tracer

uptake in the upper parts of the human body, which

correspond to brown adipose tissue. The human

depots are differently located from those in rodents,

mainly in the supraclavicular and the neck regions,

but no interscapular (Nedergaard et al., 2007). These

findings point out that brown adipose tissue is

present and active in a substantial fraction of adult

humans and that may thus be considered of

metabolic significance in human physiology.

Insulin exerts a dominant role in regulating glucose

homeostasis though orchestrated effects on the pro-

motion of glucose uptake in peripheral tissues, such as

muscle and fat, and suppressing hepatic glucose

production. Glucose transport in brown adipocytes is

maintained mainly by the activity of insulin-regulated

glucose transporter (GLUT)4. Insulin treatment

stimulates glucose transport by mediating GLUT4

translocation in an AKT- and PKCz-dependent

manner (Hernandez et al., 2001; Lorenzo et al.,

2002). Furthermore, inhibition of phospholipase C

(PLC)g activity precludes insulin stimulation of

glucose uptake, GLUT4 translocation and actin

reorganization, indicating that PLCg through the

production of phosphatidic acid, is a link between IR

and PKCz (Lorenzo et al., 2002). In addition, IRS-2

seems to be crucial in mediating glucose uptake in

brown adipocytes (Teruel et al., 2001).

TNF-alpha acts as a negative regulator of adipo-

genic and thermogenic differentiation and in-

duces insulin resistance in brown adipose tissue

Insulin resistance associated to obesity: the link TNF-alpha 187

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(Valverde et al., 2005), in a similar fashion as reported

in 3T3-L1 cells and in primary human adipocytes.

Moreover, TNF-alpha-induced insulin resistance on

glucose uptake in brown adipocytes seems to be due

to the hypophosphorylation of the IR and IRS-2 in

response to insulin, resulting in an impairment of

IRS-2-associated PI3K activity (Teruel et al., 2001).

As a further step, we have identified ceramide

production as one of the mediators of insulin-

resistance by TNF-alpha and exogenously added

C2-ceramide inhibited AKT activity throughout a

ceramide-activated protein-phosphatase (PP)2A

(Teruel et al., 2001). Furthermore, de novo ceramide

generation produced by chronic treatment with

TNF-alpha induces insulin resistance on GLUT4

gene expression in brown adipocytes by interfering

C/EBPa acumulation (Fernandez-Veledo et al.,

2006a). Moreover, stress kinases activated in re-

sponse to TNF-alpha, meanly ERK and p38MAPKs

also contribute to insulin resistance in brown

adipocyte primary cultures (Hernandez et al., 2004).

Modulation of genes such as PTP1B might

contribute to the pathogenesis of TNF-alpha-induced

insulin resistance. In this regard, brown adipocytes

treated with TNF-alpha showed a significant en-

hancement of PTP1B mRNA, protein and activity

(Fernandez-Veledo et al., 2006b). As expected, the

lack of PTP1B these cells confered protection against

TNF-alpha-induced insulin resistance on glucose

uptake and insulin signalling (Fernandez-Veledo

et al., 2006b). Accordingly, a complex mechanism

impairs the normal response to insulin on GLUT4

translocation in brown adipocytes in the presence of

TNF-alpha including: (1) potential Ser/Thr phos-

phorylation of IRS-2 by MAPKs, weakening the Tyr

phosphorylation induced by insulin, (2) generation of

ceramide and activation of PP2A maintaining AKT in

an inactive dephosphorylated state and (3) modula-

tion of PTP1B protein expression and activity, as

summarized in Figure 3.

Nuclear receptors, such as PPAR, and liver X

receptor (LXR), comprise a superfamily of related

proteins, which act as transcription factors to activate

expression of target genes in response to binding of

ligands. Thiazolidinediones are agonists for PPARgthat display insulin-sensitizing actions across a wide

spectrum of insulin-resistant states, and have recently

been introduced as therapeutic agents for the

treatment of type 2 diabetes (Olefsky, 2000). How-

ever, several limitations with this therapy, such as

increased adiposity, secondary insulin resistance in

adipose tissue, and pro- and anti-atherogenic effects,

are currently emerging. LXRa has been proposed

recently as important regulator of glucose metabo-

lism (Steffensen and Gustafsson, 2004). In this

regard, improved glucose tolerance and increased

glucose-induced insulin secretion by islets in genetic

and dietary models of type 2 diabetes treated with

synthetic LXR agonists was reported. Moreover,

ligand activation of LXR regulates the expression of

GLUT4 either in vivo as well as in murine and

human adipocytes, through direct interaction with a

conserved LXR response element in the GLUT4

promoter. In addition, the ability of LXR ligands to

regulate GLUT4 expression was abolished in mice

lacking LXRs.

Brown adipose tissue is a target tissue for nuclear

receptors agonists since highly expressed PPARg,LXRa, and LXRb (Steffensen and Gustafsson,

2004). In this regard, the PPARg agonist rosiglita-

zone up-regulates the expression of LPL, HSL and

uncoupling protein-1 in brown adipocytes (Teruel

et al., 2005) as well as produces insulin sensitization

by increasing the expression of IR and its tyrosine

Figure 3. Insulin resistance by TNF-a in brown adipocytes involves: (1) Ser/Thr phosphorylation of the IRS-2 by ERK and p38MAPK; (2)

generation of ceramide and activation of the phosphatase PP2A, and (3) modulation of PTP1B activity. Inhibition of ERK and p38MAPK

activation with rosiglitazone, and down-regulation of PTP1B with either rosiglitazone or T0901317 ameliorates TNF-a-induced insulin

resistance.

188 I. Nieto-Vazquez et al.

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kinase activity (Hernandez et al., 2003). The effec-

tiveness of rosiglitazone to treat TNF-a-induced

insulin resistance in these cells was due to the fact

that this TZD impaired the activation of p38MAPK

and ERK produced by TNF-alpha, and restored the

insulin signalling cascade leading to normalization of

insulin-induced glucose uptake (Hernandez et al.,

2004). Furthermore, rosiglitazone decreased the

activity of PTP1B (Hernandez et al., 2003), and

improved insulin sensitivity concomitant with an

increase in thermogenic differentiation, contributing

globally to an accelerated glucose disposal in brown

adipose tissue. Moreover, recent studies have de-

monstrated increased levels and activities of PTP1B

in skeletal muscle and liver of diabetic rats whereas

rosiglitazone treatment decreases this enlargement in

muscle but not in liver (Wu et al., 2005). On the

other hand, synthetic LXR agonists ameliorate TNF-

alpha-induced insulin resistance in fetal brown

adipocytes restoring completely insulin-stimulated

GLUT4 translocation to plasma membrane. This

effect was parallel to the recovering of insulin

signalling cascade and could be due to the fact that

T0901317 precludes the enlargement in PTP1B

expression produced by TNF-alpha (Fernandez-

Veledo et al., 2006b), supporting the hypothesis of

nuclear receptors LXR are interesting targets for

drug treatment of insulin-resistant conditions.

Therefore, inhibition of ERK and p38MAPK activa-

tion with rosiglitazone and down-regulation of

PTP1B with either rosiglitazone or LXR agonists

restores insulin sensitivity in brown adipocytes in the

presence of TNF-alpha as summarized in Figure 3.

Contribution of p38MAPK and PTP1B to

TNF-a-induced insulin resistance in skeletal

muscle

The organ in which insulin resistance is first

detectable is skeletal muscle because this tissue is

responsible for the highest glucose disposal of the

body. Acute insulin treatment stimulates glucose

transport in myocytes largely by mediating transloca-

tion of GLUT4 to the plasma membrane, which is

accomplished by activation of PI3K, AKT and

several PKC isoforms including z, l, a and d (Khan

and Pessin, 2002; Vollenweider et al., 2002). More-

over, skeletal muscle has an insulin-independent

mechanism to increase glucose transport that in-

volves the activation of AMP-activated protein kinase

(AMPK) by stimuli such as exercise, hypoxia or

ischemia (Fujii et al., 2006). The AKT substrate of

160 kDa (AS160) has emerged recently as a point of

convergence for both effectors of glucose transport

and seems to modulate GLUT4 trafficking (Fujii

et al., 2006). Whereas the GLUT4 protein content is

normal in muscle from subjects with type 2 diabetes,

the capacity of insulin to stimulate translocation of

GLUT4 to plasma membrane is impaired. In con-

trast to the effect of insulin, contraction-stimulated

glucose uptake and GLUT4 translocation in diabetic

patients is normal, providing evidence that exercise

might be able to bypass defects in insulin signalling.

TNF-alpha expression in skeletal muscle of insulin

resistant subjects and diabetic patients was fourfold

higher than in the insulin sensitive subjects (Kern

et al., 2001). TNF-alpha blocks skeletal muscle

differentiation, causes sarcopenia and produces

insulin resistance in skeletal muscle of healthy

humans and in primary cultures of mouse skeletal

muscle (Table I) (Dirks and Leeuwenburgh, 2006;

Plomgaard et al., 2005). Although ceramide and FFA

have been reported to produce insulin resistance in

skeletal muscle, a direct effect of TNF-alpha in this

tissue has been a matter of controversia. Several

reports did not detect an inhibitory action of

TNF-alpha on insulin-induced glucose uptake,

although TNF-alpha per se highly increased basal

glucose uptake. However, others observed an in-

hibitory effect of TNF-alpha on insulin action

without modifying basal glucose uptake in muscle

cells. Moreover, in most of these studies insulin

stimulation of glucose uptake was very poor because

virtually all cultured skeletal muscle cell lines,

including L6 and C2C12 myotubes, have been found

to be deficient in GLUT4 expression.

Accordingly, in our laboratory we developed

primary cultures of neonatal rat skeletal muscle that

represented a suitable system for investigating the

molecular basis of TNF-alpha-induced insulin resis-

tance. When these cells were differentiated until the

formation of myotubes in low-serum medium and

then maintained in low-glucose medium to mimick

the physiological environment, they responded to

acute insulin stimulation by increasing glucose uptake

and GLUT4 translocation to plasma membrane (de

Alvaro et al., 2004). Chronic exposure to TNF-alpha

impaired both insulin-stimulated glucose uptake and

GLUT4 translocation, without affecting the content

of GLUT4 protein or the state of differentiation of the

myotubes (de Alvaro et al., 2004), in agreement with

the effect produced in muscle in vivo. The molecular

mechanism underlying TNF-alpha -mediated insulin

resistance could involve activation of stress kinases

and proinflamatory pathways, as was observed in

neonatal myotubes (de Alvaro et al., 2004). Acute

insulin stimulation also produced a transient phos-

phorylation of p38MAPK (Conejo et al., 2002), but

insulin activated the isoform a meanwhile TNF-alpha

activates the b. When chemical inhibitors were used

to evaluate the contribution of sustained activation of

stress kinases by TNF-a to insulin resistance, only the

inhibition of p38MAPK completely restores insulin-

stimulated glucose uptake and insulin signalling

(de Alvaro et al., 2004). In this regard, adenovirus-

mediated transfections of constitutively active

MKK6/3 mutants in L6 myotubes were reported to

diminish glucose transport induced by insulin via

down-regulation of GLUT4 gene expression

(Fujishiro et al., 2003). Furthermore, the Ser307

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residue of IRS-1 seems to be one of the residues

phosphorylated by TNF-alpha via p38MAPK,

although other residues in either IRS-1 or IRS-2 or

IR cannot be excluded. The role of p38MAPK in

inflamatory diseases, including obesity and cardio-

vascular dysfunction, is well recognized because this

kinase regulated the biosynthesis of proinflamatory

cytokines, as well as is involved in the signalling

transduction pathways activated by cytokines, as

elegantly reviewed (Cuenda and Rousseau, 2007).

Several reports have also implicated IKK activa-

tion by TNF-alpha on Ser phosphorylation of IRS-1,

and aspirin rescues insulin-induced glucose uptake in

3T3-L1 adipocytes treated with TNF-alpha (Yuan

et al., 2001; Gao et al., 2003). In this regard,

activation of IKK dependent on the functionality of

p38MAPK was observed during chronic treatment

with TNF-alpha in neonatal myotubes. Moreover,

inhibition of IKK activation with salicylate comple-

tely restored insulin signalling to normal levels,

despite the presence of TNF-alpha (de Alvaro

et al., 2004), but salicylate does not affect p38MAPK

activation by the cytokine. Then, IKK could act

downstream of p38MAPK and could mediate TNF-

alpha-induced insulin resistance on skeletal muscle,

as summarized in Figure 4. Similarly to this finding

TNF-alpha also affects insulin signalling negatively

in endothelial cells via p38 MAPK (Li et al., 2007).

Interestingly, knock-down of MAP4K4 in primary

human skeletal muscle cells, the upstream kinase of

ERK1/2 and JNK inhibits the effects of TNF-alpha,

further acknowledging the involvement of these

kinases in the transmission of TNF-alpha induced

signalling (Bouzakri and Zierath, 2007).

Additionally, the phosphatase PTP1B, a negative

regulator of insulin signalling acting, has been shown

to be crucial for TNF-alpha signalling. The expres-

sion and activity of PTP1B has been found increased

in the muscle of humans and rodents in pathological

insulin-resistant states such as obesity (Ahmad et al.,

1997). Moreover, noncoding polymorphisms in the

PTP1B gene have been found in different popula-

tions, displaying increased phosphatase muscle ex-

pression and being associated with insulin resistance

(Bento et al., 2004). In this regard, transgenic

overexpression of ptp1b in muscle causes insulin

resistance, showing impaired insulin signalling and

decreased glucose uptake in this tissue (Zabolotny

et al., 2004). By contrast, mice lacking PTP1B

exhibit increased insulin sensitivity at 12 weeks of

age (attributable to enhanced phosphorylation of IR

in liver and skeletal muscle), resistance to weight gain

on a high-fat diet, and an increased basal metabolic

rate (Elchebly et al., 1999; Klaman et al., 2000). The

PTP1B-deficient mice had circulating insulin con-

centrations that were about half those of control

animals. Thus, these mice appeared to be more

insulin sensitive, because they maintained lower

glucose concentration with significantly reduced

amounts of insulin. In the fasted state, there were

no significant differences in concentrations of glu-

cose or insulin (Elchebly et al., 1999). Furthermore,

PTP1B-deficiency also reduces the diabetic pheno-

type in mice with polygenic insulin resistance (Xue

et al., 2007). Moreover, treatment with PTP1B

antisense oligonucleotide improves insulin sensitivity

in db/db mice and increases insulin signalling in

WAT and liver in ob/ob mice (Gum et al., 2003).

In our laboratory we generated myocytes lacking

PTP1B that displayed enhanced insulin sensitivity in

IR autophosphorylation and downstream signalling,

including IRS-1 and IRS-2 Tyr phosphorylation,

PI3K associated activation and AKT Ser/Thr phos-

phorylation. The phosphorylation was detected at

lower insulin doses and at shorter times in PTP1B-

deficient cells than in wild-type cells (Nieto-Vazquez

et al., 2007). Because activation of PI3K and AKT

controls glucose transport, we detected an increased

insulin-stimulated glucose uptake and GLUT4

translocation to plasma membrane in PTP1B–/– cells

Figure 4. Treatment with TNF-a impairs insulin-stimulated glucose uptake in myocytes at the level of the IRS-1 by a double mechanism that

involves: (1) Ser phosphorylation by IKK and p38MAPK and (2) Tyr dephosphorylation by the phosphatase PTP1B. Inhibition of IKK

activation with salicylate, and ablation of PTP1B restores insulin sensitivity in the presence of the cytokine.

190 I. Nieto-Vazquez et al.

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versus wild-type cells (Nieto-Vazquez et al., 2007).

This result was expected because decreased glucose

uptake in skeletal muscle was observed when PTP1B

was overexpressed selectively in muscle of transgenic

mice (Zabolotny et al., 2004). Moreover, recent data

indicate that muscle-specific PTP1B–/– mice exhibed

improved systemic insulin sensitivity and enhanced

glucose tolerance under high-fat diet (Delibegovic

et al., 2007).

As long as TNF-alpha is a strong candidate to

produce insulin resistance in skeletal muscle for the

reasons mentioned above, the lack of PTP1B might

confer protection against TNF-alpha-induced insulin

resistance. In this regard, chronic exposure to TNF-

alpha does not induce insulin resistance either on

glucose uptake or on insulin signalling in PTP1B-

deficient myocytes (Nieto-Vazquez et al., 2007).

Moreover, PTP1B–/– mice showed complete protec-

tion against TNF-alpha-induced insulin resistance

during the glucose and insulin tolerance tests (Nieto-

Vazquez et al., 2007). Accordingly, the lack of

PTP1B expression confers protection against TNF-

alpha-induced insulin resistance in skeletal muscle

either in vitro or in vivo (Nieto-Vazquez et al.,

2007).Consequently, at least part of the effects

elicited by TNF-a on pathways involving reversible

tyrosine phosphorylation may be exerted through the

dynamic modulation of PTP1B expression.

Therefore, TNF-alpha impairs insulin action in

myocytes at the level of IRS-1 by a double mechan-

ism that involves: (1) Ser phosphorylation by IKK

and p38MAPK at the Ser307 residue and (2) Tyr

dephosphorylation by PTP1B. Accordingly, inhibi-

tion of IKK activation with salicylate and ablation of

PTP1B restores insulin sensitivity in myocytes in the

presence of the cytokine, as summarized in Figure 4.

In this regard, new mono- and disalicylic acid

derivates have been used very recently as PTP1B

inhibitors and potential antiobesity drugs (Shrestha

et al., 2007).

Effect of TNF-alpha on muscle lipid

metabolism

In contrast to the numerous investigations examining

the effect of TNF-alpha glucose uptake into skeletal

muscle, only few studies have investigated the effect

of TNF-alpha on fatty acid (FA) metabolism in

muscle. However, TNF-alpha does increase FA

esterification into diacylglycerol (DAG) ultimately

leading to increased DAG content while it does not

affect FA oxidation, uptake or esterification into

triacylglycerol (TAG) in skeletal muscle and other

cell types (Bruce and Dyck, 2004). Since DAG is a

potent activator of the PKC isoforms beta and theta,

which Ser phosphorylate IRS-1, this offers further

mechanistic insight into the pathway by which TNF-

alpha potentially controls insulin sensitivity nega-

tively. PKC has been shown to be able to phosphor-

ylate PTP1B. Hence, these results would suggest a

mechanism along the axis PKC, PTP1B, MAPK to

an insulin signal inhibiting IRS-1 Ser phosphoryla-

tion. In addition to these insulin signal impairing

actions leading to reduced glucose uptake, glycogen

synthesis in muscle is inhibited by TNF-alpha which

contributes greatly to hyperglycaemia.

As mentioned above, lipids released from adipo-

cytes following TNF-alpha exposure are transported

to non-adipose tissues like skeletal muscle as FFA

(Sharma and Chetty, 2005). Excess FFA are stored

as triglyceride droplets in muscles, in turn contribut-

ing to an enhanced insulin resistance.

In addition, direct inhibition of AMPK activity via

transcriptional upregulation of PP2C by TNF-alpha

in mice has been proposed (Steinberg et al., 2006).

PP2C has only recently been indicated as a regulator

of AMPK activity in cardiomyocytes and skeletal

muscle cells (Steinberg et al., 2006). TNF-alpha

treatment of L6 myocytes for 8 h impaired AMPK

activity; thus, in turn leading to an increased activity

of acetyl CoA carboxylase leading to a reduction of

FA transport and subsequently to a diminished

utilization of lipids. This contributes even further to

the negative spiral of FA accumulation and insulin

resistance. Mice lacking the TNF-alpha receptors

were protected from this effect as one would expect

when the observations are mediated by TNF-alpha

(Uysal et al., 1997). Not surprisingly therefore, in

humans intramyocellular lipid, and TNF-alpha con-

centrations were amongst the strongest predictors of

insulin resistance.

As mentioned, it has been suggested that even

though TNF-alpha has no direct effect on beta-

oxidation in skeletal muscle (Bruce and Dyck, 2004)

the high levels of circulating FFA would enhance

beta-oxidation, which in turn would diminishes

glucose uptake and oxidation. In fact, for example

during the ‘‘first days’’ on a high-fat diet, when fat

oxidation does not equal fat intake, subjects are in

negative carbohydrate balance, leading to a decrease

in the body’s glycogen stores, a decrease in glucose

oxidation and to increased fat oxidation. However,

the consumption of a high-fat diet for ‘‘one week’’ is

accompanied by molecular changes that would

favour fat storage in muscle rather than oxidation

(Schrauwen-Hinderling et al., 2005). This circum-

stance is obviously similar to continuous exposure of

skeletal muscle to FFA due to impaired adipocyte

action as a result of high TNF-alpha levels, as

described above.

Time dependent differences, i.e., early and late

effects of TNF-alpha and lipid accumulation appear to

be of crucial importance in this context. Concluding,

the above findings rather suggest that in a long-term

perspective beta-oxidation is impaired due to TNF-

alpha exposure of skeletal muscle, ultimately enhan-

cing the problem of lipid accumulation in skeletal

muscle which causes insulin resistance to progress.

The clue to understanding impairment of

insulin action in skeletal muscle due to TNF-alpha

Insulin resistance associated to obesity: the link TNF-alpha 191

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possibility lies in the combination of two factors:

downregulation of mitochondrial FA oxidative due

to dimished AMPK activity and lipid accumulation

due to increased FFA in circulation. AMPK is

involved in the expressional control of the PPARgcoactivator (PGC)- 1alpha and even directly controls

its function in skeletal muscle (Jager et al., 2007).

PGC-1alpha in turn has been recognised to be the

crucial determining factor of mitochondrial function.

Hence, PGC-1alpha impairment ultimately leads to

impaired mitochondrial function. The combination

of lipid accumulation in skeletal muscle concomi-

tantly with impaired mitochondrial function and

reduced beta-oxidation has been described as the

crucial trigger of impaired insulin sensitivity (re-

viewed in (Schrauwen-Hinderling et al., 2005)).

As described for adipocytes, activators of PPARs,

especially PPARg, seem to hold a potential in the

treatment of TNF-alpha induced insulin resistance.

In skeletal muscle, however, PPARd appears to be

the more predominant isoform and we have pre-

viously described the potential of PPARd selective

ligands in enhancing insulin sensitivity in human

skeletal muscle cells (Kramer et al., 2005).

In cardiomyocytes a PPARd selective ligand

inhibited lipopolysaccharide (LPS)-induced TNF-

alpha production from cardiomyocytes (Ding et al.,

2006). Even more interesting was that adenoviral-

mediated overexpression of PPARd strongly inhib-

ited TNF-alpha expression; whereas overexpression

of a PPARd mutant with ablated ligand binding

domain did not show a similar effect. Hence it is

tempting to speculate that PPAR-delta may exhibit

similar effects on TNF-alpha in skeletal muscle.

Unfortunately, no data is available as yet.

However, we have demonstrated that PPARd acti-

vation using a specific ligand (which is currently under

scrutiny in clinical trial) in human skeletal muscle cells

enhances FA oxidation via expression changes, hence

addressing the problem of diminished mitochondrial

action as caused by TNF-alpha (Kramer et al., 2007).

Further investigations will be necessary to address the

question if PPARd also is a regulator of TNF-alpha

expression in skeletal muscle.

Acknowledgements

This work was supported by grants: BFU-2005-

03054 from Ministerio de Educacion y Ciencia; S-

SAL-0159-2006 from Comunidad de Madrid; San-

tander-UCM-06; REDIMET RD06-0015-0009 and

CIBERDEM CB07-08-0007 from Ministerio de

Sanidad y Consumo, Spain. We also acknowledge

the support of European Cooperation in the Field of

Scientific and Technical Reserarch (COST) Action

BM0602 from the European Commission.

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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