molecular mechanisms involved in obesity-associated

Archives of Physiology and Biochemistry, 2009; 115(4): 227–239

R E V I E W A R T I C L E

Molecular mechanisms involved in obesity-associated insulin resistance: Therapeutical approach

Sonia Fernández-Veledo, Iria Nieto-Vazquez, Rocio Vila-Bedmar, Lucia Garcia-Guerra, Maria Alonso-Chamorro, and Margarita Lorenzo

Departamento de Bioquimica y Biologia Molecular II, Facultad de Farmacia, Universidad Complutense, 28040-Madrid, Spain. CIBER de Diabetes y Enfermedades Metabolicas asociadas (CIBERDEM)

Address for Correspondence: Sonia Fernández-Veledo, Departamento de Bioquimica y Biologia Molecular II, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain. Tel: 34-913941852. Fax: 34-913941779. E-mail: [email protected]

(Received 03 April 2009; revised 23 June 2009; accepted 06 July 2009)

Introduction

Insulin resistance is an important contributor to the pathogenesis of type 2 diabetes (T2D) and obesity is a risk factor for its development. In the obese state an altered secretion pattern, with increase in pro- inflammatory and decrease in anti-inflammatory factors is found (Trayhurn and Wood, 2005). Insulin resistance cor-relates with impaired insulin signalling in peripheral tissues. From the intracellular pathways activated by insulin, the tyrosine phosphorylation of insulin receptor substrate (IRS) proteins is a crucial event in mediating

insulin action. This step is one of the key molecu-lar events in insulin resistance associated with both inflammation and hyperinsulinemia. The mechanisms affecting IRSs involve proteasome-mediated degrada-tion, phosphatase-mediated dephosphorylation and serine phosphorylation of IRSs, which reduces insulin receptor (IR) tyrosine kinase activity, as previously reviewed (Pirola et al., 2004). This review is focused on examining alterations in insulin- signalling pathways in insulin resistance states associated with obesity. In this regard, stress and pro-inflamatory kinases, as well as phosphatases, seem to be involved in the molecular

ISSN 1381-3455 print/ISSN 1744-4160 online © 2009 Informa UK LtdDOI: 10.1080/13813450903164330

AbstractInsulin resistance is an important contributor to the pathogenesis of T2D and obesity is a risk factor for its development. It has been demonstrated that these obesity-related metabolic disorders are associated with a state of chronic low-intensity inflammation. Several mediators released from adipocytes and macro-phages, such as the pro-inflammatory cytokines TNF-alpha and IL-6, have been suggested to impair insulin action in peripheral tissues, including fat and skeletal muscle. Such insulin resistance can initially be com-pensated by increased insulin secretion, but the prolonged presence of the hormone is detrimental for insulin sensitivity. Stress and pro-inflamatory kinases as well as more recent players, phosphatases, seem to be involved in the molecular mechanisms by which pro-inflammatory cytokines and hyperinsulinemia disrupt insulin signalling at the level of IRSs. Pharmacological approaches, such as treatment with PPAR and LXR agonists, overcome such insulin resistance, exerting anti-inflamatory properties as well as controlling the expression of cytokines with tissular specificity.

Keywords: GLUT4; cytokines; hyperinsulinemia; PTP1B; stress and proinflammatory kinases; nuclear receptor agonists; insulin resistance; obesity

Abbreviations: ACC, acetyl-CoA carboxilase; AMPK, AMP-activated protein kinase; AS160, AKT substrate of 160 kDa; BAT, brown adipose tissue; ERK, extracellular-signal regulated kinase; FAS, fatty acid synthase; FFA, free fatty acids; GLUT4, insulin-regulated glucose transporter; HSL, hormone sensitive lipase; IL, Interleukin; IKK, inhibitor kB kinase; IR, insulin receptor; IRS, insulin receptor substrate; JNK, c-Jun N-terminal kinase; LPL, lipoprotein lipase; LXR, liver X receptor; MAPK, mitogen-activated protein kinase; MCP, monocyte chemoattractan protein; PDE, phosphodiesterase; PI3K, phosphatidylinositol 3-kinase; PK, protein kinase; PET, positron-emission tomography; PPAR, peroxisome proliferator activated receptor; PTP, protein-tyrosine phosphatase; SOCS, supressor of cytokine signalling; T2D, type 2 diabetes; TNF, tumour necrosis factor; TGA, tryglicerides; TZD, tiazolidindione; UCP, uncoupling protein; WAT, white adipose tissue.

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228 Sonia Fernández-Veledo et al.

mechanisms by which IRSs signalling could be affected in these states. Pharmacological and genetic approaches to overcoming insulin resistance will also be discussed in this review.

The impact of adipose tissue in energy metabolism and insulin sensitivity

The adipose organ consists of several depots located at various anatomical sites that have differ-ent physiological functions and pathophysiological roles. Advances over the last two decades in our understanding of the adipocyte biology have clarified its role as a key regulator of both energy balance and intermediary metabolism. White adipose tissue (WAT) has long been recognized as the main site of storage of energy excess derived from food intake. White adipocytes store dietary energy in a highly concen-trated form as triglycerides (TGA), mostly in a single large lipid droplet. In times of caloric need, these triglycerides can be rapidly hydrolysed by lipases (a process known as lipolysis) and the resulting fatty acids are transported to other tissues (mainly liver and skeletal muscle) to be oxidized in mitochondria as an energy source. In contrast, brown adipose tissue (BAT) is specialized primarily for cold-induced non-shivering thermogenesis. Brown adipocytes are char-acterized by multiple, smaller droplets of triglycerides, which are accessible to a rapid hydrolysis and oxida-tion of the fatty acids (FA). The unique thermogenic capacity of BAT results from the expression of the uncoupling protein (UCP)1, located in the mito-chondrial inner membrane. This protein allows the consumption of the energy derived from FA oxidation for the generation of heat (Nedergaard et al., 2001). Most fat depots can be characterized as either brown or white but some brown fat cells can also be found dispersed through white fat depots (Guerra et al., 1998). Until quite recently, BAT was thought to be of metabolic importance only in small mammals and infant humans. However, recent studies using posi-tron-emission tomography (PET) scanning, suggest that adult humans have several discrete areas of met-abolically active BAT (Nedergaard et al., 2007; Cypess et al., 2009). In this regard, BAT may have much more relevance in human metabolism than was previously appreciated and loss of BAT function is linked to obes-ity and metabolic disease (Lowell et al., 1993).

Adipose tissue as an endocrine organ

Currently, there is strong evidence that adipose tissue is not only an inert energy-storage depot, but

it is also an endocrine organ. This tissue secretes a large number of peptide hormones and cytokines, known as adipokines, as well as non-peptide biologically active molecules such as active lipids, which act at both the local (autocrine/paracrine) and systemic (endocrine) level (Kershaw and Flier, 2004). The adipose tissue is required for normal secretion of adipokines such as leptin and adi-ponectin, which control glucose homeosthasis, insulin sensitivity and eating patterns through effects on neuroendocrine pathways (Guilherme et al., 2008). In fact, human and mice lipodystro-phies, which are abnormalities of the adipose tissue associated with total or partial loss of body fat, are related to impaired adipokine secretion and insulin resistance (Chehab, 2008). Thus, owing to its endocrine function and its classical role as lipids storage, the presence of functional adipose tissue in proper proportion to body size is essential to control whole-body metabolism.

Insulin action in adipocytes

Insulin exerts a dominant role in regulating glucose homeostasis through orchestrated effects on the promotion of glucose uptake in peripheral tissues, such as muscle (skeletal muscle and heart) and fat (white and brown) and in suppressing hepatic glucose production. The clearance of circulating glucose in these organs depends on insulin-stimulated trans-location of glucose transporter (GLUT)4 to the cell surface, which is accomplished by the activation of the insulin intracellular signalling cascade which includes binding to specific IR, tyrosine phospho-rylation of IRS proteins, activation of phosphatidyl inositol (PI)3K, AKT and protein kinase C isoforms , , and (Huang and Czech, 2007). Skeletal muscle is responsible for the highest glucose disposal in the body whereas adipose tissue accounts for only a small fraction of insulin-dependent glucose disposal. Nevertheless, fat-selective knock-out glut4 gene mice show impaired glucose tolerance, suggesting that the functional integrity of the adipose tissue is crucial in regulating intermediate metabolism (Abel et al., 2001). Insulin action on lipid metabolism is similar to its role in glucose metabolism since it promotes anabolism as well as inhibits catabolism. Specifically, insulin upregulates lipoprotein lipase (LPL) and stimulates gene expression of intracellular lipogenic enzymes, such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), promoting trig-lyceride storage in the adipose tissue (Kersten, 2001). However, in human adipocytes, de novo lipogenesis seems to be of minor importance, as compared to

Molecular mechanisms involved in obesity-associated insulin resistance 229

the uptake and esterification of free FA (FFA) derived from plasma lipoproteins (Diraison et al., 2003). In addition, insulin inhibits lipolysis by a mechanism involving the cAMP hydrolysing enzyme phos-phodiesterase (PDE)-3B, resulting in decreased a ctivation of protein kinase A (PKA) and hormone sensitive lipase (HSL) (Belfrage et al., 1981; Smith and Manganiello, 1989).

Insulin resistance, which can be defined as a diminished ability of the cell to respond to insulin, is the most important pathophysiological feature in many pre-diabetic states and is the first detectable defect in T2D. The pathogenesis of T2D involves abnormalities in both insulin action and secretion. Insulin resistance is initially compensated by hyper-insulinemia. Although moderate hyperinsulinemia might be tolerated in the short term, chronic hyperinsulinemia exacerbates insulin resistance in peripheral tissues, including adipose tissue and contributes directly to -cell failure and diabetes (White, 2003). Resistance to insulin-stimulated glucose uptake in skeletal muscle is one of the earliest defects detected in insulin-resistant states contributing to the hyperglycemia characteristic of these states. On the other hand, the inadequate insulin action on the adipose tissue also induces alterations in glucose as well as in lipid metabolism. Thus, in the insulin-resistant state an inefficient trapping of dietary energy occurs both because of decreased LPL-mediated lipolysis and ineffective inhibition of HSL-mediated lipolysis (Coppack et al., 1992). Postpandrial lipemia and elevated plasma FA levels are well-recognized abnormalities in T2D (Axelsen et al., 1999). Moreover, reduced adipose tissue uptake and storage of TGA results in greater partitioning of dietary lipids to nonadipose tissues, including muscle and liver (Frayn, 2002). It is widely accepted that increased availability and utilization of FFA contributes to the development of skeletal muscle insulin resistance, as well as to increase hepatic glucose production (White, 2003). In fact, the progression of insulin resistance in rats on a high-fat diet is closely related to plasma FFA levels (Jiao et al., 2008). Both genetic and environmental factors can contribute to the development of insulin resistance and, in the latest group, obesity has been proposed as an important contributor.

The inflammation of adipose tissue during obesity contributes to insulin resistance

Obesity is a risk factor for developing metabolic disorders, such as insulin resistance and T2D, partly due to endocrine function of adipose tissue.

Several factors derived not only from adipocytes, but also from infiltrated macrophages, contribute to the pathogenesis of insulin resistance. Most of them are overproduced during obesity, including leptin, tumour necrosis factor (TNF)-, monocyte chemoattractant protein (MCP)-1 and resistin. Conversely, the expression and plasma levels of adipokines with anti-inflammatory properties, such as adiponectin, are down-regulated during obesity. However, body fat distribution appears to be even more important than the total amount of fat. In this regard, central (visceral) obesity is more closely related to insulin resistance and T2D than peripheral (subcutaneous) obesity (Montague and O’Rahilly, 2000). Site-depot differences in human adipocyte physiology have been demonstrated in numerous studies. Thus, visceral adipocytes present higher catecholamine- stimulated lipolysis and lower insu-lin antilipolytic effects and leptin secretion as com-pared to subcutaneous adipocytes (van Hamerlen et al., 2002). Several factors secreted from adipose tissue, including pro-inflammatory cytokines and FFA, can impair insulin signalling altering insulin-mediated processes such as glucose homeostasis and lipid metabolism (Arner, 2003). Accordingly, obesity is now considered to be a chronic state of low-intensity inflammation. In this regard, recent studies reveal that obesity is also associated with an increase in infiltration of adipose tissue with macrophages, which contributes to the inflamma-tory process through the additional secretion of cytokines (Lumeng et al., 2007). The mechanisms by which adipose tissue recruits and maintains mac-rophages could involve expression of MCP-1 and intercellular adhesion molecule-1. Recent studies revealed that those subjects with the highest tran-scription rates of genes encoding TNF- and IL-6 seemed prone to developing obesity, insulin resist-ance and T2D (Fernandez-Real and Pickup, 2008). In this regard, here we review the impact of these cytokines in modulating insulin action on glucose transport in adipose tissues and skeletal muscle.

-cell dysfunction affects metabolism of adipose tissue

Obesity is associated with both insulin resistance and hyperinsulinemia. Initially, hyperinsulinemia compensates for the insulin resistance and thereby maintains normal glucose homeostasis. Actually, fasting hyperinsulinemia is a widely used surrogate measure of insulin resistance and predicts T2D. Although this hyperinsulinemia is necessary for glycemic control, it can have harmful consequences


on many tissues including pancreatic -cells, contributing to the development of T2D.

Hyperinsulinemia induces insulin resistance in human adipocytes

A recent study from our laboratory demonstrated that long-term treatment with insulin impaired GLUT4 translocation to the plasma membrane and insulin signalling at IRS-1/AKT level in the human visceral adipocyte cell line Lisa-2 (Fernandez-Veledo et al., 2008). This in vitro situation may imitate the chronic elevation of insulin during insulin-resistant states observed in humans (Bergman and Mittelman, 1998). In addition, hyperinsulinemia also induces an increase in basal lipolysis, a decrease in isoprot-erenol responsiveness and an insulin-resistant state, not only on glucose uptake, also on the antilipolytic effect, as summarized in Figure 1 (Fernandez-Veledo et al., 2008). In fact, an alteration of the lipolytic pathway has also been one of the major hypothesis linking insulin resistance to hyperlipidemia in obes-ity and T2D (Bergman and Mittelman, 1998). The mechanism involved seems to be dependent on per-ilipin A, an essential lipid droplet-associated protein, which functions as both a suppressor of basal lipoly-sis and a necessary enhancer of PKA-stimulated lipolysis (Tansey et al., 2004). Hyperinsulinemia induced a greater movement of perilipin A and B in basal conditions, producing an increase in basal glycerol release and a decrease in isoproterenol-induced lipolysis. In this regard, chronically high insulin levels inhibit -adrenergic receptors from activating PKA (Zhang et al., 2005) and a significant positive relationship between perilipin expression and obesity has been described (Kern et al., 2004).

It is well documented that serine-phosphorylation of IRSs impairs the normal response to insulin and this situation has been associated with several insu-lin resistant states including hyperinsulinemia (Gual et al., 2005). An increase in IRS-1 phosphorylation at the Ser312 residue and a decrease in insulin-induced IRS-1 tyrosine-phosphorylation, without changes in IRS1 expression, have been observed in human adipocytes under hyperinsulinemic conditions (Fernandez-Veledo et al., 2008; Danielsson et al., 2006). This mechanism differs from those described in murine adipocytes where a decrease in IRS-1 levels was reported (Ricort et al., 1995). On the other hand, the lipid phosphatase PTEN seems to be involved in the molecular mechanism disrupting insulin signalling at

the level of IRSs found in hyperinsulinemia-induced insulin resistance (Figure 1). In this regard, hyperin-sulinemia increased PTEN protein levels in human adipocytes (Fernandez-Veledo et al., 2008). Moreover, compensatory hyperinsulinemia was not produced in muscle-specific PTEN-deficient mice (Wijesekara et al., 2005), whereas inhibition of PTEN expression in ob/ob mice reduced insulin concentrations (Butler et al., 2002).

The desregulation of the endocrine function of adipose tissue under hyperinsulinemia

Secretion of pro-inflammatory cytokines, such as MCP-1 and IL-6, as well as FFA release was markedly stimulated in human adipocytes cultured with insulin for a long-term, in accordance to elevated plasma concentrations of these factors detected in obese and diabetic patients (Takahashi et al., 2003; Kern et al., 2001). In this regard, an increase in MCP-1 expression has been described in murine adipocytes treated with insulin (Fasshauer et al., 2004). In contrast, adiponectin secretion as well as its receptors were decreased in human and murine adipocytes under hyperinsulinemic conditions (Fernandez-Veledo et al., 2008; Tsuchida et al., 2004). This desregula-tion in adipokine secretion may play a crucial role in the development of insulin resistance not only in adipocytes but also in other tissues. In fact, signals coming from undifferentiated or poorly differenti-ated human adipocytes (i.e. adiponectin) enhanced insulin-induced glucose uptake and AKT phos-phorylation in muscle cells; whereas signals from more differentiated adipocytes (i.e IL-6 or MCP-1) induced an insulin resistant-state, detected earlier than in adipocytes (Fernandez-Veledo et al., 2008). Although these results appear to represent a paradox it cannot rule out the altered secretion profile of adi-pose tissue as a primary event in obesity-associated insulin resistance. Thus, hyperinsulinemia induced a desregulation of adipokines and FFA secretion by human adipocytes, inducing insulin resistance on adipocytes as well as on myocytes in which this insulin-resistant state is detected earlier (Figure 1).

Role of TNF- in obesity-associated insulin resistance

Markers of inflammation such as TNF- have been proposed as a link between adiposity and the development of insulin resistance because adipose tissue-derived TNF- is higher in obese diabetics


than in healthy lean subjects and rodents (Kern et al., 2001). Obese mice lacking either TNF- or its recep-tors show protection from developing insulin resist-ance (Hotamisligil, 2003). Moreover, TNF- blocks skeletal muscle differentiation and produces insulin resistance in skeletal muscle in healthy humans (Plomgaard et al., 2005). Rather than acting systemi-cally, TNF- seems to act locally at the site of adipose tissue through autocrine or paracrine mechanisms, affecting insulin sensitivity and inducing IL-6 expre-sion (Arner, 2003). Circulating levels of soluble TNF- receptors seem well- correlated with BMI and impair-ment in TNF- processing can improve systemic insulin sensitivity (Serino et al., 2007). On the other hand, TNF- has lipolytic and antiadipogenic effects on WAT and BAT (Arner, 2003; Valverde et al., 2005). This paradox could be due to proliferative and anti- apoptotic effects of this cytokine in the obese adi-pocyte and may be mediated by differential expression of its soluble and membrane-anchored receptors.

Desregulation of lipid metabolism by TNF- in adipocytes

This cytokine may cause diabetogenic effects in obesity indirectly through desregulating lipid

metabolism in adipose tissue. Both FFA and ceramides were reported to induce insulin resistance in peripheral tissues. TNF- induced lipolysis in adipocytes increasing FFA release to the circulation and ceramide production as a consequence of sphin-gomyelinase activation (Green et al., 1994; Ryden et al., 2002; White, 2003; Arner, 2003). TNF- could potentially contribute to induction of systemic insu-lin resistance by causing a decrease in FA oxidation in muscle and thus an increase in plasma FFA levels (Steinberg et al., 2006). Thus, TNF- deficient mice exhibit lower circulating FFA and TGA than wild-type animals (Uysal et al., 1997). Our group has extensively studied TNF- effects on BAT and skeletal muscle. In this regard, ceramide production is activated by TNF- in brown adipocytes and exogenously added C2-ceramide inhibits AKT activity throughout ceramide-activated protein-phosphatase (PP)2A (Figure 2) (Teruel et al., 2001). In addition, TNF- also induces long-term effects of gene expression of many proteins involved in glucose and FFA uptake and storage take. For example, TNF- has been shown to downregulate the genes for adiponectin, GLUT4, IRS-1, C/EBP, PPAR and perilipin in adipocytes, involving the transcription factor NF-kB (Ruan et al., 2002). Moreover, this cytokine repressed GLUT4 gene expression in brown adipocytes by

HYPERINSULINEMIA

FFApro-Inflammatory adlpokines (MCP-1, IL-6)anti-Inflammatory adlpokines (adiponectin)

Insulin Resistance

PTENP-Ser-IRS1

P-Tyr-IRS1P-AKT

GLUT4 translocation Anti-lipolysis

WHITE ADIPOCYTES

LXR agonists PPARγ agonists

MYOCYTES

Insulin resistanceon glucose uptake

Figure 1. Hyperinsulinemia desregulates endocrine function and induces insulin resistance on glucose and lipid metabolism in human adipocytes. Long-term treatment with insulin deregulates adipocyte secretion pattern (with an increased of pro-inflammatory and a decreased of anti-inflammatory factors) inducing insulin resistance on GLUT4 translocation and on antilipolytic effect of insulin. The mechanism that involves serine-phosphorylation of IRS1 and modulation of PTEN expression. Moreover, adipocyte secreted factors modulate insulin sensitivity in skeletal muscle where insulin resistance is detected earlier than in adipocytes. Pharmacological treat-ments with LXR agonist ameliorate insulin resistance on glucose metabolism whereas PPAR agonist such as rosiglitazone presents beneficial effects on lipid metabolism.


interfering with C/EBP acumulation (Figure 2) (Fernandez-Veledo et al., 2006a). Infusion of TNF- in rodents leads to impairment of insulin-stimulated skeletal muscle glucose uptake (Nieto-Vazquez et al., 2007). Accordingly, neutralisation of TNF- with specific antibodies has the opposite effect and improves insulin resistance in rats (Hotamisligil and Spiegelman, 1994). However, in contrast to the findings in rodent models, TNF- neutralisation has no beneficial effect in terms of insulin sensitivity in humans. Variations in TNF- genotypes in the mediation of the TNF- action could explain these different effects ( Fontaine-Bisson et al., 2007).

Inflammatory pathways involved in TNF––induced insulin resistance

The interaction between TNF- and insulin signal-ling is most important for local insulin resistance in obesity. When cells are directly exposed to TNF-, this adipokine inhibits insulin signalling by affecting IRS proteins (Hotamisligil, 2003). Stress kinases and inflammatory pathways that are activated in response to TNF-, such as extracellular-signal regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), have been proposed as mediators of TNF- serine phosphoryla-tion of IRS-1 in human adipocytes and skeletal muscle cells (Bouzakri and Zierath, 2007). In this regard, ablation of jnk1 blunted insulin resistance associ-ated with dietary obesity. Furthermore, activation of ERK1/2 and p38MAPKs by TNF- could inhibit insu-lin signalling 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). In brown adipocytes, activation of ERK1/2 and

p38MAPK by TNF- is involved in this impairment of normal tyrosine phosphorylation by insulin of IRS-2 (Figure 2) (Teruel et al., 2001; Hernandez et al., 2004).

Chronic exposure to TNF- induces a state of insulin resistance on GLUT4 translocation to the plasma membrane in murine primary myotubes (de Alvaro et al., 2004), in accordance with the effect produced in muscle in vivo, systemically (Nieto-Vazquez et al., 2007). The Ser307 residue of IRS-1 seems to be one of the residues phosphor-ylated by TNF- via activation of the beta isoform of p38MAPK. Moreover, activation of inhibitor kappa B kinase (IKK), dependent on the functionality of p38MAPK, was observed during chronic treatment with TNF- in murine myotubes (de Alvaro et al., 2004). Then, IKK could act either downstream of p38MAPK or directly and mediate TNF––induced serine phosphorylation of IRS-1. Accordingly, IKK inhibition with salicylate or targeted disruption of ikk reversed obesity and diet-induced insulin resistance (Gao et al., 2003; de Alvaro et al., 2004). In this regard, the glucose- lowering effects of the anti-inflamatory compounds, salicylate and its derivative aspirin, were identified more than 100 years ago. The positive effects of high-dose aspirin are, however, limited by its toxicity profile on the gastrointestinal tract, as reviewed (de Luca and Olefsky, 2008).

Contribution of phosphatases to TNF––induced insulin resistance

The insulin-signalling cascade could also be nega-tively regulated by protein tyrosine-phosphatases such as (PTP)1B, which dephosphorylates the phos-photyrosine residues of the IR and IRS-1. The expres-sion and activity of PTP1B has been found to be

P-Tyr-IRS2 P-AKT

PPARγagonists

TNF-α

Insulin-induced GLUT4 transiocation

ERK1/2p38MAPK

PP2A PTP1B LXRagonists

Figure 2. TNF- induces insulin resistance in brown adipocytes. TNF- blocked insulin-induced GLUT4 translocation by a mechanism that involves serine phosphorylation of the IRS-2 by ERK1/2 and p38MAPK; production of ceramides and activation of PP2A, and increased activity and expression of PTP1B. Inhibition of ERK1/2 and p38MAPK activation by rosiglitazone and down-regulation of PTP1B with LXR agonist ameliorates TNF--induced insulin resistance.


increased in the muscle of diabetic and obese humans and rodents (Klaman et al., 2000; Delibegovic et al., 2007). Moreover, it has been described in various populations noncoding polymorphisms in the PTP1B gene which are associated with an increase in phos-phatase muscle expression and insulin resistance (Bento et al., 2004). In this regard, transgenic overex-pression 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 (either in total body or in skeletal muscle) exhibit increased insulin sensitivity, resistance to weight gain on a high-fat diet and an increased basal metabolic rate (Klaman et al., 2000; Delibegovic et al., 2007). Furthermore, PTP1B-deficiency also reduces the diabetic phenotype in mice with polygenic insulin resistance (Xue et al., 2007) and treatment with PTP1B anti-sense oligo-nucleotide improves insulin sensitivity in db/db mice (Gum et al., 2003). Accordingly, modulation of genes such as PTP1B might also contribute to the pathogenesis of TNF––induced insulin resist-ance. In this regard, brown adipocytes treated with TNF- showed significant enhancement of PTP1B expression and activity and the lack of ptp1b in these cells confered protection against TNF––induced insulin resistance on glucose uptake and insulin sig-nalling (Figure 2) ( Fernandez-Veledo et al., 2006b). Additionally, the expression of PTP1B was also found to be up- regulated by TNF- in murine myoblasts and in WAT and muscle (Nieto-Vazquez et al., 2007; Zabolotny et al., 2008). More importantly, chronic exposure to TNF- does not induce insulin resistance in PTP1B-deficient myocytes. Moreover, PTP1B−/− mice showed complete protection against TNF––induced systemic insulin resistance and glucose intolerance. Therefore, the lack of PTP1B expression confers protection against TNF––induced insulin resistance both in brown adipocytes and myocytes as well as in vivo (Nieto-Vazquez et al., 2007).

Thus, the different mechanisms contributing to TNF––induced insulin resistance show tis-sue specificity. TNF- impairs insulin-stimulated glucose uptake in peripheral tissues at the level of IRSs proteins by a mechanism that involves serine phosphorylation by stress and pro-inflammatory kinases and tyrosine dephosphorylation by phos-phatases, weakening the tyrosine phosphorylation induced by insulin.

Dual role of IL-6 in insulin action

IL-6 has a positive role in insulin sensitivity under physiological conditions since it is strongly induced

in skeletal muscle during and after exercise and this results in enhanced substrate metabolism and whole body glucose homeostasis (Penkowa et al., 2003; Steensberg et al., 2002; Febbraio et al., 2004). In fact, as IL-6 also activates lipolysis in WAT it might play a role in energy supply during exercise (van Hall et al., 2003). In this regard, IL-6 knockout mice showed an impaired ability to exercise and to oxidize FA and developed mature-onset obesity (Ruderman et al., 2006; Wallenius et al., 2002). Thus, IL-6 role seems to be rather anti-inflammatory in such physiological situations. Moreover, other studies reported lack of effect or a positive effect of IL-6 on whole body glucose disposal in rats and humans, respectively (Rotter et al., 2004; Carey et al., 2006) and some evidences also suggest anti-obesity effects of IL-6 through central regulation on appetite suppression and weight loss (Wernstedt et al., 2004; Wallenius et al., 2002).

Contribution of IL-6 to insulin resistance

The role of IL-6 in the etiology of insulin resistance in pathological conditions is not fully understood and has been a matter of controversy (Kristiansen and Mandrup-Poulsen, 2005; Carey and Febbraio, 2004). It is known that adipose tissue contributes to a significant proportion of total circulaling IL-6 ( Mohamed-Ali et al., 1997). Pretreatment with IL-6 in vivo blunted insulin’s ability to suppress hepatic glucose production and has been reported to be an important contributor to the chronic inflammatory state and hepatic insulin resistance of obesity (Kim et al., 2004; Klover et al., 2005). In addition, IL-6 induced insulin resistance in hepatocytes, adi-pocytes and myocytes (Senn et al., 2002; Rotter et al., 2003; Tzeng et al., 2005) and seems to be involved in palmitate-induced insulin resistance in myocytes (Senn, 2006). Alternatively, the IL-6 protein content in adipose tissue has been negatively correlated with insulin-stimulated glucose disposal and a chronic elevation of IL-6 is not desirable since it may com-promise insulin sensitivity (Bastard et al., 2002; Kern et al., 2001).

A recent study from our laboratory demonstrated a dual effect of IL-6 on insulin action: additive at short-term and negative after chronic-treatment. IL-6 per se activated glucose uptake due to the sequential phosphorylation of LKB1/AMP kinase/AKT substrate of 160 kDa (AS160) pathway in murine myotubes (Nieto-Vazquez et al., 2008). Accordingly, diminished AMPK activity was found in muscle from the IL-6 knockout mice (Ruderman et al., 2006) and improvement in glucose and insulin tolerance tests


was observed in mice treated with IL-6 for short-term (Nieto-Vazquez et al., 2008), in a similar way as after excercise. By contrast, chronic-exposure to IL-6 impaired insulin-stimulated GLUT4 transloca-tion and insulin signalling in both myotubes and skeletal muscle and caused systemic insulin resist-ance as observed from glucose and insulin tolerance tests. This situation imitates the chronic elevation of IL-6 that causes insulin resistance when is secreted by adipose tissue in obesity (Kern et al., 2001). This dual behavior of IL-6 has also been observed in human skeletal muscle cells (Al-Khalili et al., 2006). Three mechanisms seem to operate in IL-6-induced insulin resistance in myocytes: activation of JNK1/2, accumulation of suppressor of cytokine signalling (SOCS) 3 mRNA and increase in PTP1B activity, which converge at the IRS-1 level. In this regard, deficiency in PTP1B confers protection against IL-6-induced insulin resistance in skeletal muscle either in vitro or in vivo (Nieto-Vazquez et al., 2008). Accordingly, a recent study showed that JNK1-dependent secretion of IL-6 by adipose tissue caused increased expression of liver SOCS3, a protein that induces hepatic insulin resistance (Sabio et al., 2008). Overall, IL-6 remains likely to be an important contributor in obesity-associated disorders such as insulin resistance and T2D.

Agonists of nuclear receptors as therapeutic tools in ameliorating insulin resistance

Nuclear receptors, such as peroxisome prolifera-tor activated receptor (PPAR) and liver X receptor (LXR) comprise a superfamily of related proteins that act as transcription factors for target genes involved in glucose and lipid metabolism. These proteins are activated by naturally produced lipids as well as by synthetic compounds; some of them display insulin sensitizing effects and anti-inflam-matory properties (Lopez-Soriano et al., 2006). Thus, the effectiveness of different nuclear recep-tor agonists to overcome hyperinsulinemia- and cytokine-induced insulin resistance has also been evaluated in this review.

Beneficial effects of PPAR agonists in glucose and lipid metabolism

Thiazolidinediones (TZDs) have been used for the treatment of hyperglycaemia in T2D for the past 10 years. These compounds are agonists for PPAR, primarily in adipose tissue, which display insulin-sensitizing actions across a wide spectrum of

insulin-resistant states (Olefsky, 2000). Troglitazone was the first TZD to be introduced into clinical practice, but was withdrawn due to liver toxicity. Currently, pioglitazone and rosiglitazone are the only PPAR agonists licensed for patients with T2D. PPAR receptor activation by TZDs improves insu-lin sensitivity by promoting FFA uptake into the adipose tissue, increasing adiponectin production and reducing levels of inflammatory mediators such as TNF- and IL-6 (Quinn et al., 2008). The effec-tiveness of the rosiglitazone to treat TNF––induced insulin resistance in murine brown adipocytes is due to the fact that rosiglitazone impairs the activation of p38MAPK and ERK1/2 produced by TNF- and restores the insulin signalling cascade leading to normalization of insulin-induced glu-cose uptake (Figure 2) (Hernandez et al., 2004). Moreover, rosiglitazone decreases PTP1B activity, improves insulin sensitivity and is also related to an increase in thermogenic differentiation (Teruel et al., 2005) contributing globally to an accelerated glucose disposal in BAT. Likewise, rosiglitazone treatment decreases PTP1B enlargement in muscle but not in liver of diabetic rats (Wu et al., 2005). In addition, it has been shown that rosiglitazone completely restores the antilipolytic effect of insu-lin under hyperinsulinemia conditions in human adipocytes (Figure 1) (Fernandez-Veledo et al., 2008). In accordance with improvement in lipid metabolism in insulin-resistant human adipocytes, a decrease postprandial NEFA concentration in T2D has been reported in vivo. The insulin-sensitizing effects of PPAR on other tissues such as skeletal muscle have been shown to be indirect. Muscle-specific deletion of PPAR induced an increase in adiposity and whole-body insulin resistance. However, treatment with TZDs ameliorated these effects and altered expression of several lipid metabolism genes in the muscle of these mice (Norris et al., 2003). These results suggest that although PPAR is not required for the antidiabetic effects of TZDs in muscle, it is needed to mantain whole-body insulin sensitivity via altered lipid metabolism. On the other hand, PPAR appears to be the more predominant isoform in skeletal muscle. In this regard, PPAR activation using a spe-cific ligand (currently under scrutiny in a clinical trial) in human skeletal muscle cells enhances FA trans-port and oxidation (Kramer et al., 2007). However, a number of side effects are well recognized with the use of TZDs in clinical practice, such as weight gain due to expansion of the subcutaneous fat, secondary insulin resistance in adipose tissue, fluid retention, hepatotoxicity, detrimental effects on bone, as well as pro-atherogenic effects.


LXR as targets for drug treatment of insulin resistanceLXRs have recently been proposed as important regu-lators of glucose metabolism. In this regard, synthetic LXR agonists, such as T0901317 and GW3965, have been reported to improve glucose tolerance in genetic and dietary models of T2D (Cao et al., 2003; Laffitte et al., 2003) and to increase glucose-induced insulin secretion by islets (Efanov et al., 2004). In addition, LXR agonist treatment suppresses hepatic gluconeogeneis (Cao et al., 2003) and induces desir-able changes in cholesterol metabolism (Bruemmer and Law, 2005), favourable features for a potential drug against T2D. Furthermore, LXR regulates the expression of GLUT4 in vivo as well as in murine and human adipocytes, through direct interaction with a conserved LXR response element in the GLUT4 pro-moter (Laffitte et al., 2003). In addition, the ability of LXR ligands to regulate GLUT4 expression was abolished in mice lacking LXRs (Laffitte et al., 2003; Dalen et al., 2003). On the other hand, recent studies in vitro demonstrate that LXR agonists overcome insulin resistance both in adipocytes and skeletal muscle. Thus, T0901317 and GW3965 ameliorate TNF––induced insulin resistance in brown adi-pocytes, completely restoring insulin-stimulated GLUT4 translocation to the plasma membrane. This effect is parallel to the recovery of the insulin signalling cascade and could be due to the fact that these compounds preclude the enlargement in PTP1B expression produced by TNF- (Figure 2) (Fernandez-Veledo et al., 2006b). Furthermore, insulin sensitivity on glucose uptake in human adipocytes under hyperinsulinemic conditions was completely restored by T0901317 treatment (Figure 1) (Fernandez-Veledo et al., 2008). Furthermore, it has been also shown that LXR activation may regulate inflammatory response. Thus, it has been shown that LXR agonists inhibit synthesis of proinflamma-tory cytokines in lymphocytes (Walcher et al., 2006) and astrocytes (Zhang-Gandhi and Drew, 2007). More recently, beneficial effects on the endocrine function of adipose tissue have been reported since LXR agonists inhibit MCP-1 and IL-6 secretion in insulin-resistant human adipocytes (Figure 1) (Fernandez-Veledo et al., 2008).

On the other hand, LXR agonists may lead to increased utilization of lipids and glucose in human skeletal muscle cells (Cozzone et al., 2006; Kase et al., 2005). However, T0901317-induced lipogen-esis and lipid formation was more pronounced in myotubes from T2D patients than from lean individuals suggesting that increased intramyocel-lular lipid content in these patients may involve an altered response to activation of components in the

LXR pathway (Kase et al., 2007). Furthermore, when a pharmacological approach was used to ameliorate IL-6-induced insulin resistance, only LXR agonists completely restored insulin-stimulated glucose uptake (Nieto-Vazquez et al., 2008), an effect that was not produced by the PPAR agonist. Accordingly, a decrease in ptp1b gene expression by treatment with LXR agonists confers protection against insulin resistance by IL-6 (Nieto-Vazquez 2008) in a similar fashion as the mechanism described in brown adipocytes treated with TNF- (Fernandez-Veledo et al., 2006b).

In conclusion, LXR and PPAR agonists overcome cytokine-induced insulin resistance, exerting anti-inflamatory properties such as controlling the expression of cytokines with tissular specificity. Thus, nuclear receptors are interesting targets for drug treatment of insulin-resistant conditions.

Conclusion

The close association between obesity and insulin resistance and their progression to T2D is a severe health problem. The identification of the factors contributing to the development of insulin resistance and the level at which insulin signalling is impaired, is the first step for establishing the molecular basis of insulin resistance in peripheral tissues. In this regard, pro-inflammatory cytokines such as TNF-α and IL-6, have been suggested to impair insulin action. Although insulin resistance can initially be compensated by increased insulin secretion, chronic hyperinsulinemia is detrimental for insulin sensitivity. Stress and pro-inflamatory kinases as well as phosphatases, seem to be key factors in the molecular mechanisms by which insulin signalling is disrupted; however, the mechanisms involved in obesity-related insulin resistance show tissue specificity. Pharmacological approaches based on nuclear receptor agonists overcome such insulin resistance, exerting anti-inflamatory properties as well as controlling the expression of cytokines. Nevertheless, several limitations with this therapy have now emerged and new generations of selective nuclear receptors are currently being developed to improve insulin sensitivity minimizing secondary effects.

Acknowledgements

This work was supported by grants BFU2008-04043 from Ministerio de Ciencia e Innovacion, Spain, S-SAL-0159-2006 from Comunidad de Madrid, Spain.


CIBER de Diabetes y Enfermedades Metabolicas Asociadas is an ISCIII project. We also acknowl-edge the support of COST Action BM0602 from the European Commission. We thank L. Muñoz and E. Gonzalez from Universidad Complutense for their experimental and administrative support.

Decleration of Interest: The authors report no conflict of interest.

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