Central role of the adipocyte in the insulin-sensitising and cardiovascular risk modifying actions of the thiazolidinediones

Biochimie 85 (2003) 1219–1230 www.elsevier.com/locate/biochi

Central role of the adipocyte in the insulin-sensitising and cardiovascular risk modifying actions of the thiazolidinediones S.A. Smith Scientific Affairs, Diabetes, GlaxoSmithKline R&D, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK Received 12 September 2003; accepted 13 October 2003

Abstract Insulin resistance is a key metabolic defect in type 2 diabetes that is exacerbated by obesity, especially if the excess adiposity is located intra-abdominally/centrally. Insulin resistance underpins many metabolic abnormalities—collectively known as the insulin resistance syndrome—that accelerate the development of cardiovascular disease. Thiazolidinedione anti-diabetic agents improve glycaemic control by activating the nuclear receptor peroxisome proliferator activated receptor-c (PPARc). This receptor is highly expressed in adipose tissues. In insulin resistant fat depots, thiazolidinediones increase pre-adipocyte differentiation and oppose the actions of pro-inflammatory cytokines such as tumour necrosis factor-a. The metabolic consequences are enhanced insulin signalling, resulting in increased glucose uptake and lipid storage coupled with reduced release of free fatty acids (FFA) into the circulation. Metabolic effects of PPARc activation are depot specific—in people with type 2 diabetes central fat mass is reduced and subcutaneous depots are increased. Thiazolidinediones increase insulin sensitivity in liver and skeletal muscle as well as in fat, but they do not express high levels of PPARc, suggesting that improvement in insulin action is indirect. Reduced FFA availability from adipose tissues to liver and skeletal muscle is a pivotal component of the insulin-sensitising mechanism in these latter two tissues. Adipocytes secrete multiple proteins that may both regulate insulin signalling and impact on abnormalities of the insulin resistance syndrome—this may explain the link between central obesity and cardiovascular disease. Of these proteins, low plasma adiponectin is associated with insulin resistance and atherosclerosis—thiazolidinediones increase adipocyte adiponectin production. Like FFA, adiponectin is probably an important signalling molecule regulating insulin sensitivity in muscle and liver. Adipocyte production of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of fibrinolysis, and angiotensin II secretion are partially corrected by PPARc activation. The favourable modification of adipocyte-derived cardiovascular risk factors by thiazolidinediones suggests that these agents may reduce cardiovascular disease as well as provide durable glycaemic control in type 2 diabetes. © 2003 Elsevier SAS. All rights reserved. Keywords: Thiazolidinediones; Peroxisome proliferator activated receptor-c; Adipocyte; Insulin resistance; Adiponectin; Non-esterified free fatty acids; Inflammation; Plasminogen activator inhibitor-1

1. Introduction Type 2 diabetes is reaching epidemic proportions worldwide, fuelled by the increasing prevalence of obesity, as many populations adopt a western lifestyle of increased highfat food consumption and reduced physical activity [1]. Type 2 diabetes should not be considered merely to be a disorder of glucose control—many people with the disease develop se-

Abbreviations: FFA, non-esterified fatty acids; GLUT, glucose transporter; PAI-1, plasminogen activator inhibitor-1; PPARc, peroxisome proliferator activated receptor-c; TNFa, tumour necrosis factor-a. E-mail addresses: [email protected] (S.A. Smith), [email protected] (S.A. Smith). © 2003 Elsevier SAS. All rights reserved. doi:10.1016/j.biochi.2003.10.010

vere complications affecting both the microvascular (retinopathy, neuropathy and nephropathy) and macrovascular (coronary heart disease, atherosclerosis and stroke) systems. Secondary complications, particularly those affecting the heart and major blood vessels, lead to a significant reduction in quality of life and account for the excess mortality—more than 75% of people with type 2 diabetes die prematurely of cardiovascular disease [2]. The hyperglycaemia of type 2 diabetes is driven by two interacting metabolic defects: b-cell dysfunction, resulting in inadequate insulin secretion, and by reduced responsiveness of key target tissues to endogenous insulin (insulin resistance) [3]. Insulin resistance is now generally accepted to be the primary metabolic defect of type 2 diabetes and is highly correlated with obesity, especially if the excess adiposity is

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located intra-abdominally or viscerally [4]. Weight gain or weight loss are highly associated with the development of insulin resistance and increased insulin sensitivity, respectively. Whilst the severity and progression of microvascular complications correlates tightly with the level of glycaemic control, there is no such simple direct relationship between glycaemia and cardiovascular disease [5]. It is now becoming increasingly clear that insulin resistance, in non-diabetic as well as in type 2 diabetes subjects, is linked with a cluster of metabolic abnormalities—collectively known as the insulin resistance or metabolic syndrome—that together are responsible for the increased cardiovascular risk [6,7]. This cluster includes visceral obesity, dyslipidaemia, hypertension, endothelial dysfunction, reduced fibrinolysis and chronic systemic inflammation, leading to accelerated atherosclerosis. The thiazolidinediones or “glitazones” are a new class of orally active anti-hyperglycaemic agent, two of which, rosiglitazone and pioglitazone, are approved for clinical use in the management of type 2 diabetes in a large number of countries, including the US and the European Union. Thiazolidinediones exert their anti-diabetic actions by novel mechanisms, entirely distinct from those of the insulin secretagogue sulphonylurea and meglitinide drugs and the biguanide, metformin, which suppresses hepatic glucose output. Thiazolidinediones have no direct effect on insulin secretion—they improve glycaemic control by reducing insulin resistance in liver, skeletal muscle and adipose tissue. Moreover, by directly targeting insulin resistance, these agents favourably modify many of the cardiovascular risk factors present in people with type 2 diabetes thereby offering the prospect of long-term reductions in cardiovascular disease. The primary, if not the only, molecular target mediating the insulin-sensitising actions of the thiazolidinediones is the nuclear receptor peroxisome proliferator activated receptor-c (PPARc) [8]. PPARc, although expressed at low levels in multiple cell types, including those of the vasculature, is highly abundant only in adipose-derived cells [9]. This observation underscores the crucial and central role of adipose tissue in the mechanism of action of the thiazolidinediones. A corollary of this is that adverse changes in adipocyte metabolism may well be central to the development of insulin resistance not only in fat depots but also in the liver and skeletal muscle. Moreover, alterations in synthesis and secretion of adipocyte-derived molecules might also provide the driver for several of the metabolic abnormalities of the insulin resistance syndrome, particularly hypertension, hypofibrinolysis and atherosclerosis. In this review, the molecular pharmacology of the PPAR family and the influence of PPARc-targeted thiazolidinedione drugs on insulin sensitivity, glucose and lipid metabolism in adipose tissues is discussed. The contribution of PPARc-regulated changes in secretion of adipocytokines and other putative signalling molecules to the overall improvement in whole body insulin sensitivity and cardiovascular risk reduction produced by thiazolidinediones is also reviewed.

2. Peroxisome proliferator activated receptor-c is the thiazolidinedione receptor The three sub-types of PPAR, a, d, and c, are members of the ligand-activated nuclear receptor superfamily, which in man consists of 48 structurally related receptors. PPARs are only transcriptionally active after heterodimerisation with another nuclear receptor, the 9-cis retinoic acid-activated retinoid receptor, RXR [10]. Nuclear receptors directly regulate the expression of only a small subset of the total cellular pool of expressed genes. The basis of receptor selectivity is defined by the presence of receptor-specific recognition sequences in the regulatory regions of target genes. In the case of the PPAR/RXR heterodimer, the receptor binding site, known as a peroxisome proliferator activated receptor response element, or PPRE, is a direct repeat of a consensus six base (TGACCT) half site, separated by one irrelevant base (DR-1 element) [10]. In the absence of ligand, the heterodimer forms high affinity complexes with co-repressor proteins which prevent transcriptional activation by sequestration of the complex from the target gene promoter. Ligand binding produces a conformational change resulting in dissociation of repressors, allowing the heterodimer to bind to the PPRE. Co-activator proteins that facilitate assembly of the complex between the heterodimer and the basal transcriptional machinery have recently been identified. Two isoforms of PPARc protein, c1 and c2, have been identified (c2 is a splice variant having a 28 or 30 amino acid N-terminal extension in humans and mouse, respectively). Somewhat confusingly, there are three PPARc mRNA transcripts—PPARc1 and PPARc3 mRNA both encode PPARc1 protein. The c1 and c2 isoforms of PPARc are differentially expressed—in rodent tissues expression of PPARc2 mRNA is reportedly adipose specific [11]. The significantly greater abundance of PPARc in adipose tissue, compared to a wide range of peripheral tissues, has been confirmed in humans although the method used did not discriminate between receptor isoforms [9]. Limited studies on the tissue distribution of individual PPARc isoforms in man have been reported and there are some discrepancies that probably relate to small sample size and methodological differences. One study indicated that both PPARc1 and PPARc2 mRNAs are abundantly expressed in subcutaneous adipose tissue [12], whereas another showed that PPARc2 was barely detectable in adipose tissue or indeed in any of the other tissues studied [13]. More recently, a detailed investigation in obese women showed that PPARc2 mRNA is 20fold more abundant than PPARc1 mRNA in both omental and subcutaneous fat depots [14]. PPARc1 is detectable at low levels in other insulin target tissues such as the liver and heart, whereas both isoforms are expressed at low levels in skeletal muscle [12]. The role of PPARc in the control of adipocyte function as well as whole body lipid and glucose metabolism—particularly in the context of insulin resistance and type 2 diabetes has received intensive attention over the

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last decade. This stems largely from the identification, in 1995, of PPARc as the molecular target for the thiazolidinedione insulin-sensitising anti-diabetic agents, pioglitazone and rosiglitazone. Thiazolidinediones bind directly to the ligand-binding domain of recombinant PPARc, but not PPARa or PPARd, and function as receptor agonists [8]. One of the most convincing pieces of evidence implicating PPARc as the molecular target for these compounds is that the potency of the thiazolidinediones as anti-diabetic agents in vivo is highly correlated with their binding affinity and agonist potency at PPARc in vitro [15]. Although, there are only limited data published, it is apparent that the thiazolidinediones do not discriminate between PPARc1 and c2, so the cellular response is probably determined simply by total PPARc abundance rather than by the relative proportions of the individual isoforms [16]. The endogenous activating ligands are still largely unknown but, like the other two PPAR subtypes, PPARc can be activated by multiple long-chain fatty acids. Fatty acids may be metabolised first by 15-lipoxygenase to eicosanoid metabolites. PPARc is also activated by prostaglandins derived from prostaglandin D2—the terminal metabolite, 15-deoxyD12,14 PGJ2, which has been shown to bind directly to the ligand-binding domain and to activate PPARc, is a key candidate [17]. Interestingly, the tissue distribution profile of PGD2 synthase in human tissues closely matches that of PPARc itself [9].

3. PPAR responsive genes Activation of PPARc results in temporal changes in the expression of multiple genes in many tissues. The primary cellular response to a thiazolidinedione will be defined quantitatively by the abundance of PPARc and qualitatively by the cell-specific complement of genes bearing a PPRE in the promoter or associated regulatory region. The cell-type specific subset of directly PPARc-regulated genes provides the platform for subsequent, indirect changes in cellular function. Intuitively, it might be anticipated that since thiazolidinediones amplify insulin signalling and reduce hyperglycaemia, the majority of target genes would encode components of the insulin signal transduction pathway and/or glucose metabolic pathways. This has not proved to be the case. Most directly regulated genes: i.e. those bearing a PPRE, are involved in lipid metabolism, for example, lipoprotein lipase, adipose-specific fatty acid binding protein, aP2, fatty acid synthase, phosphoenolpyruvate carboxykinase and acyl CoA oxidase [18,19]. Only a limited number are directly involved in glucose metabolism (e.g. the GLUT-2 isoform of glucose transporter [20]) or insulin signalling. Recently, a functional PPRE has been identified in the promoter region of the gene encoding CAP, a protein that may be intimately involved in a newly discovered, PI3-kinaseindependent, signalling pathway for insulin stimulation of

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glucose transport in adipocytes [21]. The observation that most directly PPARc-regulated genes are associated with lipid metabolism underscores the importance of defective fatty acid metabolism in the pathogenesis of insulin resistance and type 2 diabetes. PPREs have now also been identified in two genes, encoding the adipocyte-secreted molecules adiponectin and resistin, that have been linked with regulation of whole body insulin sensitivity [22,23]. The potential role of adiponectin and resistin as circulating modulators of insulin sensitivity and as markers of cardiovascular risk will be discussed in subsequent sections of this review. Although PPREs have still only been identified in relatively few gene promoters, multiple other genes are regulated by PPARc activators. Of particular importance are the leptin and TNFa genes [24,25], mitochondrial uncoupling proteins [26,27] and certain components of the insulin signalling cascade and glucose transport, such as the insulin receptor [28], the P85aunit of PI3-kinase [29] and GLUT-4 [28]. There are also recent data to suggest that PPARc ligands can influence adipose expression of PPARc itself [30].

4. Thiazolidinediones: mechanisms involved in regulation of insulin action and improved glycaemic control Whilst it has been known for a number of years that thiazolidinedione activators of PPARc are potent insulinsensitising and anti-diabetic agents in animal models of type 2 diabetes and in humans with type 2 diabetes, the precise mechanisms linking activation of PPARc to insulin signalling have been difficult to unravel. With the exception of very rare inactivating mutations of PPARc that result in a phenotype of severe insulin resistance, hypertension and earlyonset type 2 diabetes [31], there is no evidence that gross defects in expression or function of PPARc are causal in the vast majority of people with insulin resistance or diabetes. Treatment of insulin resistant rodent models, as well as human type 2 diabetics, with thiazolidinediones increases insulin sensitivity in liver, skeletal muscle and adipose tissue, despite the fact that PPARc is abundantly expressed only in adipose tissue. Fundamental questions, therefore, that need to be resolved are whether the improvements in insulin action in liver and muscle result from direct activation of the low levels of PPARc in those tissues, or occur as an indirect consequence of activation of highly expressed PPARc in adipose tissue. If the latter mechanism is operative, then how are PPARc-mediated changes in fat cell metabolism transmitted to the liver and skeletal muscle?

5. Direct actions of PPARc activators in the liver and skeletal muscle There are a few reports that thiazolidinediones can directly influence glucose metabolism in liver and muscle cells

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in vitro, but there are insufficient data to conclude that these effects bear any relation to PPARc activation [32–34]. For example, no studies have been conducted using a number of different thiazolidinediones or structurally diverse PPARc activators to show that the potency of the compounds on glucose transport or gene expression is correlated with their agonist potencies for PPARc. Moreover, the effects described in some reports were produced only at concentrations of drug far in excess of blood levels achieved in animals at therapeutically effective doses [33]. Only after the effects of thiazolidinediones are assessed in animal models in which muscle or liver PPARc is specifically ablated, will we have definitive evidence of a direct insulin-sensitising action in either of these tissues. 6. The fat cell and insulin resistance—the central role of PPARc in adipose tissue function and the insulin-sensitising action of the thiazolidinediones In distinct contrast to the paucity of data in skeletal muscle and liver, the pharmacological effects of thiazolidinediones, via activation of PPARc, in adipose tissue have been extensively studied and are now well characterised. These studies have identified the primary mechanisms underlying thiazolidinedione-induced improvements in both adipocyte and whole body insulin action. Moreover, this emerging biological profile underscores the central role of the adipocyte in the pathogenesis of insulin resistance and type 2 diabetes.

7. The adipocyte and development of insulin resistance Adipocytes normally respond to insulin by increasing glucose uptake, triglyceride synthesis and reducing free fatty acid (FFA) release via inhibition of lipolysis. The responsiveness of adipocytes from the various fat depots of the body to insulin differs. Adipocytes from intra-abdominal or visceral fat are less sensitive to insulin, but more sensitive to lipolytic catecholamines, than subcutaneous peripheral adipocytes [35]. Thus, rates of free fatty release are greater from visceral adipocytes—these are delivered via the portal supply to the liver where they fuel elevated rates of hepatic triglyceride synthesis and hepatic glucose output. Eventually, these changes can result in insulin resistance, dyslipidaemia and type 2 diabetes. All of these metabolic changes contribute to an increase in cardiovascular risk and this explains why there is a tight correlation of type 2 diabetes and cardiovascular disease with visceral, but not subcutaneous, obesity [36]. Recent evidence points to the involvement of multiple sites in the insulin signalling pathways in the development of adipocyte insulin resistance (see Fig. 1) (reviewed in [4]). These include a reduction in IRS-1 protein and reduced activation of downstream kinases PI3-kinase and protein kinase B/Akt. Reduced activity of certain atypical PKC isoforms such as PKC f/k, reported to act as molecular switches to turn on GLUT-4 translocation/glucose transport responses, has also been identified in insulin resistant fat cells [37]. Finally, a 70–80% reduction in fat cell GLUT-4 content provides a major contribution to impaired insulin-mediated glucose transport in type 2 diabetes.

Fig. 1. Insulin signalling pathway in adipocytes, showing potential sites of thiazolidinedione interactions.

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Insulin resistance within the fat cell is both initiated and amplified by adipocyte-derived molecules. Of these, the inflammatory cytokine tumour necrosis factor-a (TNFa), may be the most important. TNFa is overexpressed in fat tissues from insulin resistant animals and humans, and induces insulin resistance by reducing insulin receptor tyrosine kinase activity and IRS-1 phosphorylation [38]. This in turn leads to a reduction in expression and activity of the GLUT-4 glucose transporter protein and consequently an impairment in insulin-stimulated glucose uptake. TNFa can not only inhibit insulin signalling directly, but also stimulate lipolysis in fat cell cultures in vitro and increase circulating FFA concentrations in vivo [4,39,40]. Chronic elevations in FFA, both in the fasting and post-prandial state are often present in type 2 diabetes and are not suppressed normally by insulin. This is probably a simple consequence of the insulin-resistancedriven enhanced FFA release from an expanded fat mass. The importance of oversupply of FFA from adipose tissue in the development of insulin resistance in liver and skeletal muscle is discussed in a subsequent section. Fat depots consist of multiple cell types. In addition to fully differentiated mature adipocytes that are able to synthesise fatty acids, metabolise glucose, and take up and store triglyceride from the blood, adipose tissues contain multipotential precursor cells that can be induced to differentiate into mature adipocytes. The adipocyte population within a fat depot ranges from small, newly differentiated highly insulin responsive adipocytes to large, lipid-filled, relatively insulin resistant adipocytes. The composition and overall insulin responsiveness of a fat depot at any one time is, therefore, in a dynamic equilibrium, reflecting the relative rates of preadipocyte differentiation, adipocyte maturation and finally, adipocyte loss by apoptosis. In insulin resistant states, such as obesity and type 2 diabetes, fat depots contain a high proportion of large-lipid filled adipocytes.

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8. Thiazolidinediones and fat cell metabolism PPARc is expressed in pre-adipocytes as well as in fully differentiated fat cells. It is now well-established that PPARc is the “master regulator” of pre-adipocyte differentiation, but the receptor also has important functions in the mature adipocyte, many of which are amplified by thiazolidinediones (reviewed in [18]). Thiazolidinediones influence adipose tissue metabolism at two distinct levels—they can influence the differentiation, size and total number of adipocytes within a particular fat depot as well as regulating insulin signalling, glucose and lipid metabolism in individual, mature adipocytes. Studies in vitro clearly show that thiazolidinedione activation of PPARc in pre-adipocytes results in enhancement of the normal insulin- and IGF-1-stimulation of the differentiation programme, with enhanced expression of adipose-specific genes such as lipoprotein lipase and the adipose-specific fatty acid binding protein aP2. Adipocyte expression of phosphoenolpyruvate carboxykinase-C is also directly stimulated by thiazolidinediones—this facilitates the enhanced rates of glyceroneogenesis required to support triacylglycerol synthesis and storage [41]. Mature adipocytes, like skeletal and cardiac myocytes, are the only cells that express high levels of the insulin-regulable GLUT, (GLUT-4) and are insulin-responsive with respect to glucose uptake. PPARc-driven pre-adipocyte differentiation, therefore, increases the population of small insulin-responsive fat cells (Fig. 2). In man, the regulation of pre-adipocyte differentiation by PPARc activators is complex and the effects may well be dependent on the anatomical source of adipocyte precursors. One study revealed that exposure of primary cultures of human pre-adipocytes to thiazolidinediones only results in differentiation of pre-adipocytes from subcutaneous, but not intra-abdominal, adipose depots [42]. In direct contrast, an-

Fig. 2. Regulation of adipose depot cell phenotype by thiazolidinedione activation of PPARc the master regulator of pre-adipocyte differentiation.

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other study found that visceral and subcutaneous preadipocytes differentiated equally well in response to thiazolidinedione stimulation [43]. Whilst these discrepancies probably result from the different methodologies used, the findings of the original study correlate perfectly with the clinical response of type 2 diabetic patients to thiazolidinedione therapy. Many studies clearly conclude that all thiazolidinediones increase peripheral adiposity but either have no effect on, or significantly reduce, the visceral fat mass [44]. The depot-specific effect on adiposity is particularly important since an increase in abdominal fat content, which is known to be associated with insulin resistance and increased cardiovascular risk, would be potentially deleterious. The depot-dependent differentiation response of pre-adipocytes is probably a reflection of a greater content of both PPARc and the heterodimeric partner RXR, combined with a greater induction of PPARc by thiazolidinediones in subcutaneous compared with visceral cells [30]. A second complementary mechanism to explain fat depot-dependent effects of thiazolidinediones lies in the ability of these agents to suppress adipocyte expression of 11b-hydroxysteroid dehydrogenase type 1 [45]. This enzyme controls intra-cellular synthesis of active cortisol from inactive cortisone and studies in humans show elevated activity in visceral compared to subcutaneous fat [46]. The importance of this is underscored by the observation that transgenic mice over-expressing 11bhydroxysteroid dehydrogenase in adipose tissue develop visceral obesity, insulin resistance and type 2 diabetes [47]. In insulin resistant animal models PPARc activation in mature adipocytes facilitates removal of large, insulinresistant, fat cells by apoptosis [48]. Thus, in animal models at least, thiazolidinediones induce a phenotypic shift in the adipose depot cellular population, away from large, lipidfilled, insulin resistant adipocytes, towards an increased number of small, metabolically active, insulin sensitive fat cells (Fig. 2). Very recent data in a small number of type 2 diabetic patients receiving either troglitazone or rosiglitazone for only 8 weeks showed a trend towards an increase in small fat cells and a decrease in large adipocytes [49]. It might be anticipated that more profound changes in fat cell populations in humans would occur after prolonged thiazolidinedione therapy. Selective alterations in adipose depot pre-adipocyte differentiation and cellular composition are clearly an important component of the insulin sensitising mechanisms of PPARc activators, but direct effects on the insulin signalling pathway itself and on other factors that influence insulin signalling and glucose transport in the mature, differentiated adipocyte are also involved. Some of these interactions are shown in Fig. 1. Thiazolidinediones have been reported to increase activity of PKB/Akt, although this might be secondary to their activation of PI3-kinase and inhibition of SHIP-2 phosphatase, which would increase intra-cellular accumulation of the PKB activator PI(3,4,5)P3. [4,50]. SHIP-2 is reportedly overexpressed in fat tissue (and muscle) of insulin resistant diabetic mice [50] and conversely, insulin sensitivity is increased in the SHIP-2 knockout mouse [51].

PPARc activation in the mature adipocyte results in enhanced expression and expression of a number of genes, including those encoding GLUTs and CAP. CAP (c-Cblassociating protein) is highly expressed in mature adipocytes, but not in pre-adipocytes. CAP serves as an adapter protein to facilitate recruitment of c-Cbl to the insulin receptor where it is tyrosine phosphorylated. The CAPphosphorylated c-Cbl complex participates in insulinmediated translocation of GLUT-4 containing vesicles to the cell surface, thus providing a second pathway, additional to the well-defined PI3-kinase-activated cascade, required for insulin stimulated glucose transport [52]. Thiazolidinediones, via activation of PPARc stimulate expression of the CAP gene both in isolated adipocytes in vitro and in the adipose tissues of intact insulin resistant rats [53]. Although CAP is expressed in muscle and liver it is not up-regulated by thiazolidinediones—providing further evidence of a lack of direct action in these two tissues. In addition to activation of the CAP/c-Cbl signalling pathway, thiazolidinediones appear to reverse the defective activation by insulin of PKC f/k—the putative molecular switch controlling the translocation of GLUT-4 from storage sites within the adipocyte up to the cell surface to facilitate increases in glucose transport [37]. These effects on CAP/cCbl and PKC f/k, combined with restoration of GLUT-4 content and translocation, probably underpin the mechanism whereby thiazolidinediones increase insulin-stimulated glucose transport in fat cells.

9. Metabolic consequences of PPARc activation in adipose tissue Activation of PPARc by thiazolidinediones in adipose tissue reduces the rate of FFA release and amplifies insulinstimulated glucose transport. Improvements in glycaemic control and insulin sensitivity in human type 2 diabetics, as well as in animal models of the disease, treated with thiazolidinediones, are invariably accompanied by a significant and sustained suppression of plasma FFA [54,55]. Reduced FFA release is probably multifactorial, arising from increased fatty acid re-esterification—secondary to enhanced adipocyte glycerol kinase expression [56], as well as from a net suppression of lipolysis. The mechanism of the anti-lipolytic action is likely to be mediated via a reduction in both TNFa levels and TNFa action, since thiazolidinediones attenuate TNFa-stimulated lipolysis in vitro and in intact rats and reduce expression of the TNFa gene [24,39,40]. An additional action of PPARc to suppress lipolysis might be via reversal of TNFa blockade of the insulin-signalling pathway to increase tyrosine phosphorylation of the insulin receptor and IRS-1 [38]. A further level of interaction between TNFa and PPARc signalling pathways comes from the recent recognition that TNFa down-regulates PPARc expression in adipocytes in vitro and, significantly, thiazolidinediones are able to reverse this action [57]. Whether the improvement in

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glycaemic control and insulin sensitivity produced by thiazolidinediones in human type 2 diabetes is accompanied by an increase in PPARc expression in any adipose tissue depot remains to be established.

10. Regulation of liver and skeletal muscle insulin sensitivity by thiazolidinediones—secondary to activation of PPARc in adipose tissue? Oversupply of FFA, a consequence of insulin resistance in an expanded adipose tissue mass, has been implicated in the generation of insulin resistance in the liver and, particularly, in skeletal muscle (Fig. 3). Elevated FFA stimulate hepatic glycogenolysis and gluconeogenesis and reduce muscle glucose uptake and oxidation. Increased systemic availability of FFA also promotes intra-hepatic and intra-myocellular triglyceride accumulation. Lipid-derived molecules can also impair efficient insulin signalling. In rats made insulin resistant by feeding on a diet containing a high percentage of saturated fat, increased muscle triglyceride is accompanied by a parallel increase in tissue diacylglycerol content [58]. Diacylglycerol selectively activates specific isoforms of novel PKC, notably e and h, which in turn can attenuate insulin signalling by reducing tyrosine phosphorylation of IRS-1 [59]. Activation of downstream targets of insulin, such as glycogen synthase, is also impaired by increased PKC activity. Elevations in muscle PKC h activity have also been reported in type 2 diabetic human skeletal muscle [60] and recent publications confirm the direct correlation of insulin resistance in muscle and liver with increased tissue triglyceride content [61,62].

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Reduced systemic lipid availability provides an attractive mechanism to explain how PPARc activators, acting primarily in adipose tissue, can regulate insulin signalling in the liver and skeletal muscle (see Fig. 4). The primacy of reduced FFA supply is reinforced by the observation that falls in plasma FFA precede the improvements in glycaemic control in diabetic subjects. Multiple factors might contribute to the response in the liver, including reduced stimulation of hepatic gluconeogenesis and glycogenolysis, secondary to reduced availability of FFA and glycerol, together with a reduction in intra-hepatic triglyceride content. The reduction in liver triglyceride content is associated with increases in whole body insulin sensitivity in human type 2 diabetic patients treated with rosiglitazone [55,63]. The major contribution to improved glycaemic control produced by thiazolidinediones in type 2 diabetes is reduced insulin resistance in skeletal muscle [55]. The mechanisms underlying the improvement in insulin sensitivity in skeletal muscle are, as in the liver, probably multifactorial. Stimulation of glucose uptake as a direct consequence of lowering of plasma FFA is clearly important, but potentiation of insulin signalling in the myocyte is also a fundamental component. As discussed above, muscle insulin resistance is directly correlated with accumulation of intra-myocellular triglyceride, activation of PKC isoforms, and reduced glucose transport and storage as glycogen. In insulin resistant rats rosiglitazone restores insulin sensitivity, promotes muscle glucose uptake and glycogen formation and these changes are accompanied by reduced tissue diacylglycerol content and decreased activation of PKC, notably e and h [64,65]. In human type 2 diabetes, rosiglitazone significantly increases insulin signalling in skeletal muscle and this is reflected by a signifi-

Fig. 3. Oversupply of FFAs from adipose tissue combined with reduced adiponectin secretion may cause insulin resistance in liver and skeletal muscle.

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Fig. 4. Proposed mechanism for reduction of hepatic and muscle insulin resistance by activation of PPARc in adipose tissue.

cant increase in glucose uptake and storage as glycogen [66]. Whether this is mediated by changes in PKC e and/or h activity is unknown but increased IRS-1 phosphorylation, which would be consistent with this hypothesis, was also reported in this study. The activity of another downstream kinase, PKC f, which is linked with insulin-stimulation of glucose transport in muscle, as well as in fat, and which is known to be defective in type 2 diabetes, is increased in type 2 diabetic subjects treated with rosiglitazone [67].

11. Other adipose secreted molecules that may regulate liver and muscle insulin sensitivity Sequestration of triglyceride in subcutaneous adipose depots, away from skeletal muscle and liver (“lipid steal”), is an important component of the insulin-sensitising actions of the thiazolidinediones. This may not, however, be the only signalling mechanism. Two adipose-derived proteins, adiponectin and resistin, have emerged as candidates that might act as signalling molecules to regulate insulin action in the liver and skeletal muscle. In humans, plasma adiponectin levels are correlated with whole body insulin sensitivity and are significantly reduced in type 2 diabetes [68]. The adiponectin knockout mouse develops insulin resistance and type 2 diabetes [69]. In animal models of insulin resistance and type 2 diabetes, administration of adiponectin targets skeletal muscle to increase fatty acid oxidation, and the liver to reduce glucose production—the net effect being a fall in plasma FFA and glucose [68]. Adiponectin production by adipocytes is directly stimulated by thiazolidinediones: a PPRE has been identified in the promoter region of the adiponectin gene [22]. There appear to be regional differ-

ences in adiponectin production in humans, with omental adipocytes secreting higher amounts than subcutaneous fat cells. Surprisingly perhaps, rosiglitazone stimulates adiponectin secretion from omental but not subcutaneous adipocytes [70]. Irrespective of the source, treatment of type 2 diabetics with rosiglitazone produces a twofold increase in circulating adiponectin concentrations [71]. If adiponectin is a key player in the insulin-sensitising mechanism, then thiazolidinediones should be inactive in the adiponection knockout mouse. The case for resistin being a PPARc-regulated signalling molecule is much weaker [72]. Adipose-expression of resistin in rodents is correlated with insulin sensitivity and administration of the protein does induce minor improvements in glycaemic control. There is no doubt that the resistin gene is a direct target for PPARc, since a PPRE has been identified in the promoter [23] and thiazolidinediones suppress resistin expression [72], but there is little support for its involvement in adipose function in man. This is because resistin is very poorly expressed in human fat cells [73]. Moreover, there are no reports of altered plasma levels of resistin in human insulin resistance and/or type 2 diabetes.

12. Modification of adipose-derived cardiovascular risk factors by thiazolidinediones The increased prevalence of cardiovascular disease in people with type 2 diabetes is known to be associated with multiple metabolic abnormalities that together comprise the insulin resistance syndrome. These include visceral obesity, dyslipidaemia, hypertension, endothelial dysfunction, reduced fibrinolysis and chronic systemic inflammation, lead-

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ing to accelerated atherosclerosis. The importance of the development of obesity, especially visceral, in the development of insulin resistance and type 2 diabetes has already been emphasised, but there is now convincing evidence that multiple adipose-derived peptides and proteins are intimately involved as cardiovascular risk factors associated with the insulin resistance syndrome. Importantly, activation of PPARc in adipose tissues by thiazolidinediones favourably modifies the profile of many biomarkers of cardiovascular risk. The renin angiotensin system (RAS) is important in the regulation of blood pressure and fat cells are a major source of RAS—this may provide the link behind the increased prevalence of hypertension in obesity. Expression of the angiotensinogen gene and secretion of angiotensin II in human fat cells is suppressed by rosiglitazone [74]. Angiotensin II not only elevates blood pressure, but it is also a powerful mitogen, increasing vascular smooth muscle proliferation, thereby promoting atherosclerosis. Thiazolidinediones, such as rosiglitazone, lower blood pressure in hypertensive nondiabetic individuals as well as in type 2 diabetic subjects [75,76]. Endothelial dysfunction is frequently present in insulin resistance and type 2 diabetes and is marked by reduced elasticity of the arterial wall and increased vascular permeability. Chronic elevations of plasma FFA have been shown to induce endothelial dysfunction [77] and thiazolidinedione therapy lowers FFA and significantly improves endothelial function in type 2 diabetes, as evidenced by direct measurements of vasodilatory responses and by reduction in microalbuminuria [78,79]. Whether improved endothelial function is mediated solely by the reduction in FFA is unlikely, since many direct actions of PPARc activators in vascular endothelial and smooth muscle cells that are consistent with a protective effect have been reported, but it may represent a significant component. Impaired fibrinolysis is a frequent abnormality contributing to increased cardiovascular risk in type 2 diabetes. Plasma concentrations of the main inhibitor of fibrinolysis, plasminogen activator inhibitor-1 (PAI-1), are increased in type 2 diabetes [80]. Adipose tissues, as well as vascular endothelial cells, are now recognised to be an important source of plasma PAI-1 and secretion of PAI-1 from human adipocytes in vitro, which is stimulated by insulin, is suppressed by rosiglitazone [81]. Again, these in vitro findings have a clinical correlate—rosiglitazone significantly reduces PAI-1 in human type 2 diabetics [82]. Finally, adipocyte-derived factors are also implicated in the development of atherosclerosis. Adiponectin levels are lower in people with coronary artery disease and, in animal models at least, adiponectin has anti-inflammatory and antiatherosclerotic actions in addition to its insulin-sensitising properties [68]. Moreover, C-reactive protein (CRP), the classic marker of systemic inflammation and strongest predictor of cardiovascular risk, is also expressed in adipose tissue [83]. It is not yet known whether adipose-expression

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and secretion of CRP is PPARc-responsive, but thiazolidinediones do produce highly significant reductions in plasma CRP levels, as well as increases in adiponectin, in people with type 2 diabetes [71,84].

13. Conclusions PPARc, a ligand-activated nuclear receptor highly expressed in adipose tissues, is the primary molecular target for the thiazolidinedione class of insulin-sensitising agents. The consequence of PPARc activation in vivo is to modify the phenotype of adipose depots by preferential stimulation preadipocyte differentiation in subcutaneous fat. Insulin sensitivity in fat is enhanced via increased populations of small fat cells, coupled with increased activity of multiple components of intra-cellular insulin signalling pathways. Metabolically, these changes result in reduced fatty acid release and increase glucose uptake. Thiazolidinediones enhance insulin action in liver and skeletal muscle, as well as adipose tissue—together, these are the three key sites of insulin resistance in type 2 diabetes. It is believed that the driving force behind thiazolidinedione-induced effects in skeletal muscle and liver is reduced availability of circulating FFAs from adipose tissue, which in turn reduces intra-cellular concentrations of triglyceride and other lipid derivatives that attenuate normal insulin signalling. In addition, increased adiponectin release from fat cells may also enhance insulin sensitivity in liver and muscle. The quantitative importance of activation of the low levels of PPARc expressed in liver and muscle to whole body glucose homeostasis remains to be established. The demonstration that multiple factors secreted from adipose tissue, such as adiponectin, PAI-1, and angiotensin II, that have a significant role in the development of some of the metabolic abnormalities associated with the insulin resistance syndrome helps explain the link between obesity and excess cardiovascular risk. The favourable modification of adipocyte-derived cardiovascular risk factors by activation of PPARc underpins the potential of thiazolidinediones not only to provide durable glycaemic control, but to reduce the severity of cardiovascular disease in people with type 2 diabetes.

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