2.2Heterogeneity of Adipose Tissue Composition in Relation to Adipokine and Cytokine Secretion

a short timescale, the current world epidemic of T2DM is better explained by environmental influences or possibly epigenetic modification.21 Epidemiological studies indicate that factors influencing the prevalence of T2DM include lifestyle (e.g. inactivity and dietary macronutrient composition) and obesity,22–24 age and geographical area of residence. Of these factors obesity, defined as an excess of body fat that is deemed unhealthy for the individual and a body mass index (BMI) of over 30, is one of the most important.25 Notably, both an enhanced b-cell secretory function and a compensatory increase in b-cell mass is seen in obese individuals who do not develop T2DM; these adaptations are able to counteract the increased metabolic load and obesity-associated insulin resistance.26 In the subset of obese individuals who develop T2DM, a failure in b-cell adaptation takes place.27 We hypothesize that failure of b-cell adaptation may, in part, reflect the changes in adipose composition and function that occur as it expands.

White adipose tissue comprises a range of di erent cell types: mature adipocytes (fat cells) themselves, their immediate precursors (preadipocytes) and stroma-vascular cells including fibroblastic connective tissue cells, leucocytes and macrophages.28 In adult mammals, most of the adipose tissue mass comprises lipid-filled cells (the adipocytes) in a framework of collagen fibers. An increase in body fat is caused by a combination of increased di erentiation of preadipocytes to mature adipocytes, together with hypertrophy of mature adipocytes mainly due to increased triglyceride (TAG) storage. Thus, white adipocytes vary greatly in size, and can change their diameters by 20-fold and their volumes by several-thousand-fold to accommodate lipid. T2DM, insulin resistance and the metabolic syndrome are characterized by increased circulating FA and by increased lipolysis (breakdown) of the expanded TAG stores.

The ability of adipocytes to store FA as TAG in the fed state provides animals with a fuel store for use in time of need; in addition, sequestration of excess dietary FA in adipose tissue can improve insulin action in liver and muscle by suppressing excess FA delivery to these tissues, which can cause insulin resistance and tissue dysfunction.29 In the fed state, insulin increases adipocyte TAG storage by augmenting adipocyte glucose uptake (allowing the production of glycerol 3-phosphate for FA esterification to form TAG and the formation of FA de novo), by direct e ects to stimulate enzymes in the pathway for esterification of incoming FA, mainly generated locally via adipocyte lipoprotein lipase (LPL), and by e ects to block TAG lipolysis. Adipocyte glucose uptake and its conversion to glycerol 3-phosphate is also enhanced by activation of the peroxisome proliferator-activated receptor g(PPARg).30,31 PPARg, a ‘‘lipogenic’’ transcription factor, enhances the expression of genes involved in glucose uptake and conversion to TAG32 and also stimulates glycerol transport, glyceroneogenesis and glycerol phosphorylation.30,33

In response to lipolytic stimulation, adipocytes hydrolyze stored TAG and release FA and glycerol into the circulation. FAs are used as substrates for ATP production in a range of oxidative tissues; glycerol is used for hepatic gluconeogenesis. The contribution of glycerol to glucose production depends on the nutritional state. This may vary from 5% postprandially in humans34 and

approx. 50% in the post-absorptive state in rodents35 to 20% in humans34,36 and 490% in rodents after prolonged fasting.35 The significance of glycerol as a gluconeogenic precursor is supported by the episodic hypoglycemia observed in patients with glycerol kinase deficiency.37

As well as serving as repositories for lipid, which can be mobilized when required, adipose tissue also releases adipokines involved in the regulation of normal physiological processes (e.g. the control of vascular homeostasis, blood pressure and regulation of energy balance)26 as well as contributing to the pathology of some disease states. One adipokine, leptin, is secreted from adipose tissue in proportion to adipose mass. Leptin is thought to inhibit food intake and increase energy expenditure when healthy individuals experience chronic overnourishment. To achieve the former, leptin targets hypothalamic neurons in the arcuate and in paraventricular, dorsomedial and ventromedial nuclei. Leptin has opposing actions on two populations of arcuate neurons, one that synthesizes the appetite-promoting peptides neuropeptide Y and agoutiregulated peptide (AgRP) and a second that synthesizes anorexigenic peptides, e.g. POMC (the precursor of the appetite-suppressing peptide melanocyte stimulating hormone). Leptin stimulates POMC neurons whilst inhibiting NPY and AgRP neurons. Leptin also exerts direct e ects on peripheral tissues (including liver, pancreatic islets, skeletal muscle), a ecting peripheral energy partitioning, in particular the intracellular disposition of nutrients (storage versus degradation). The ‘‘energy-sensor’’ AMP-activated protein kinase (AMPK) orchestrates critical metabolic adaptations to reduced energy availability and is activated by leptin in peripheral tissues, in particular skeletal muscle and liver.38 Obesity is characterized by leptin resistance.39 Thus, while circulating leptin levels are elevated in obesity, its e ects to limit food intake and promote energy expenditure are blunted due to the existence of leptin resistance. This may be due to leptin receptor signaling defects, for example elevated SOCS3 expression in leptin-responsive neurons of the arcuate nucleus,39 and/or defects in leptin transport across the blood-brain barrier.40

Adiponectin is another important adipokine. Adiponectin administration ameliorates insulin resistance in mice,41 whereas adiponectin-deficient mice exhibit insulin resistance42 and diabetes. This insulin-sensitizing e ect of adiponectin, like that of leptin, involves AMPK, the activation of which increases FA oxidation acutely,43 and also augments signaling via the lipo-oxidative transcription factor PPARa.44 PPARa increases the expression of genes involved in FA uptake and oxidation.45 Enhanced insulin sensitivity in response to adiponectin arises because of increased lipid clearance from the tissue via oxidation. Low circulating levels of adiponectin and increased leptin and C-reactive protein (CRP) have recently emerged as novel diabetic risk factors.46

In obesity, there is progressive adipose-tissue infiltration by macrophages that secrete proinflammatory cytokines (e.g. tumor necrosis factor (TNF) a, IL (interleukin)-1b and IL-6). TNFa over-expression is observed in adipose tissue and muscle of obese humans47 and in adipose tissue of genetically obese insulin resistant mice. Monocyte chemoattractant protein 1 (MCP-1) is mainly secreted by macrophages present in adipose tissue; however, adipocytes themselves

also release considerable amounts of MCP-148 and adipocyte MCP-1 may be the main chemokine that causes adipose tissue infiltration by macrophages within the context of obesity and insulin resistance. Mice over-expressing MCP-1 in adipose tissue using the adipocyte aP2 promoter (aP2-MCP-1 mice) are insulin resistant and have higher adipose-tissue macrophage accumulation than control mice.49 It has been suggested that a paracrine loop exists between adipocytes and macrophages that aggravates inflammatory processes.50 Coculture of di erentiated 3T3-L1 adipocytes with RAW264 macrophages results in an upregulation of macrophage TNFa secretion and a downregulation of secretion of the insulin-sensitizing adipokine adiponectin.50

The chronic inflammatory state present locally within adipose tissue itself becomes systemic when inflammatory cytokines are released into the circula-

tion. Thus obesity is characterized by increased circulating concentrations of pro-inflammatory cytokines51–53 and acute phase proteins.54,55 Circulating

leptin concentrations are increased, probably reflecting leptin resistance, whereas adiponectin concentrations are decreased in obesity.56 TNFa can impair insulin signaling and, in obese mice, lack of TNFa function improves

insulin sensitivity and glucose homeostasis, confirming the critical role of TNFa in modulating insulin action.57,58 Knowledge of the actions of the pro-

atherogenic and inflammatory mediators (cytokines and chemokines) that are derived from adipose tissue, either from the adipocytes or from the infiltrating macrophages that are associated with increased adiposity,59 may help gain a better understanding of the pathophysiology of the chronic proinflammatory state associated with obesity.

2.3 Feedback between FA and the Adipocyte

Evidence is now accumulating that FAs generated via adipocyte TAG lipolysis have pronounced direct feedback e ects on adipocytes. Within the adipocyte itself, AMPK can be activated by cAMP-inducing agents.60 This is proposed to be a consequence of stimulated lipolysis secondary to activation of cAMPdependent protein kinase (PKA) and activation by phosphorylation of key components of the lipolytic machinery. It has been suggested that AMPK activation in adipocytes in this setting is caused by an increased AMP : ATP ratio that appears to be due, at least in part, to the re-acylation of FA released by lipolysis. AMPK activation has been suggested to be part of a mechanism to restrain the energy depletion and oxidative stress caused by excessive lipolysis.

Toll-like receptors (TLR; specifically TLR-2 and -4) constitute a molecular

link between elevated circulating FA, as found in obesity, and inflammation in insulin-sensitive tissues including adipocytes.61,62 TLR-2 and -4 bind lipid-

based structures via their long-chain saturated FA moieties.63 Saturated FA, but not unsaturated FA, induce the expression of cyclooxygenase-2 via TLR-4.63,64 The adipocyte itself is capable of mounting a classical innate immune response initiated by ligand activation of TLR-4, followed by activation of nuclear factor-kB (NF-kB), increasing proinflammatory gene expression and

cytokine release.65–67 Importantly, FA (released from hypertrophic adipocytes under conditions of lipolytic stimulation) may signal to macrophages through activation of TLR-4 and stimulation of MCP-1 release by mature adipocytes in a manner dependent on dose and class of FA.68 The production of MCP-1 by adipocytes provides a potential explanation as to how macrophages are directed into the adipose tissue from the bloodstream within the context of obesity or insulin resistance (where plasma NEFA concentrations are elevated).

FAs, acting through the adipocyte TLR-4, also stimulate the release of TNFa68,69 and, somewhat surprisingly, at physiological concentrations, influence the secretion of adiponectin. Exogenous FAs enhance the nuclear translocation of NF-kB p65 and NF-kB inhibition strongly antagonizes the proinflammatory e ects of the FAs palmitic acid and stearic acid. It has therefore been argued that there might be a paracrine or autocrine role of FA in adipocyte biology, with a key involvement of NF-kB.68

2.4Autocrine E ects of Leptin and Adiponectin in Adipocytes

Leptin’s actions are initiated by its binding to a leptin receptor, encoded by the Lept gene, a member of the interleukin-6 receptor family of cytokine receptors. There are six di erent splice variants of the leptin receptor (Ob-R). Of these ObRb – the long isoform – is required for leptin’s physiological actions. Leptin links metabolic and immune responses. Leptin levels are low in infection but high in autoimmunity, both systemically and at the site of inflammation. Immune abnormalities in obesity are reversed with energy restriction (which decreases leptin levels). Leptin levels correlate with energy status and with TNFa, which is also elevated in obesity and suppresses lymphocyte function.70

Peripheral and central responses to leptin can be intimately linked. Leptin can induce changes in the brain that, in turn, a ect TAG homeostasis in adipose tissue. Leptin signaling via phosphoinositide 3-kinase (PI3K) in the hypothalamus engages the sympathetic nervous system (SNS), resulting in signaling back to the adipocytes through the endocannabinoid system.71 Thus, infusion of leptin into the basal medial hypothalamus leads to a rapid switch from lipogenesis to lipolysis.71 This change was reflected by suppression of sterol regulatory element binding protein 1 (SREBP1), together with marked downregulation of SREBP-1c targets, including the lipogenic enzymes acetylCoA carboxylase (ACC), fatty acid synthase (FAS) and stearoyl-CoA desaturase (SCD) 1.71

White adipocytes also express leptin receptors72 allowing them to respond to their own secretions. Leptin can also exert an anti-lipogenic autocrine action on adipocytes via AMPK activation (a 7-fold higher ratio of phosphorylated to total AMPK). This depletes stored adipose-tissue TAG, an e ect proposed to reflect oxidation of lipolytically generated FA in situ.73 This e ect is predicted not to be observed in adipose tissue of db/db mice. db/db mice are obese, insulin resistant and hyperleptinemic, a phenotype that develops as a result of leptin

receptor deficiency. Interestingly, OB-Rb mRNA levels in subcutaneous adipose tissue are lower in morbidly obese subjects compared with lean subjects,72 suggesting that this may exacerbate accumulation of excess adipose-tissue TAG. Furthermore, the consequences of adipocyte-selective reduction of the leptin receptors (induced by antisense mRNA) include increased adiposity, dyslipidemia, and insulin resistance.74

Adiponectin is abundant in blood plasma relative to many other hormones, approximately 0.01% of all plasma proteins, at concentrations up to 10 mg/ml. Unlike most other adipokines, adiponectin’s expression and secretion decrease significantly in obesity.75 In adults, adiponectin levels are inversely correlated with percentage body fat. Adiponectin concentrations are increased in nondiabetic subjects compared to diabetic subjects. Plasma concentrations reveal gender dimorphism with higher circulating levels of adiponectin in females.

Adiponectin exists as both a full-length protein and a proteolytic cleavage fragment (globular adiponectin). Full-length adiponectin is a trimer (low molecular weight adiponectin) that forms hexamers (medium molecular weight adiponectin). These can further oligomerize to form polymers (high molecular weight form). Two adiponectin receptors 1 and 2 (AdipoR1 and AdipoR2) have been identified by expression cloning.76 AdipoR1 activation stimulates AMPK, whereas AdipoR2 is linked to PPARa activation. AdipoR1 is highly expressed in skeletal muscle, whereas AdipoR2 is the major form in liver.76 Adiponectin decreases insulin resistance by decreasing triglyceride content in muscle and liver

in obese mice.41 Adiponectin’s action to enhance insulin sensitivity is mediated by activation of AMPK and PPARa, resulting in an increase in FA oxidation,43,76,77

together with its ability to suppress hepatic glucose production.78,79 Adiponectin increases fatty acid transport protein (FATP)-1 expression in muscle, whereas adiponectin-deficient mice are characterized by decreased FATP-1 in muscle, but not adipose tissue or liver, and impaired FA clearance.80 Adiponectin reverses insulin resistance and diabetes in db/db and KKA mice, two di erent mouse models of T2DM diabetes characterized by obesity, hyperlipidemia, insulin resistance and hyperglycemia.41 Conversely, heterozygous adiponectin-deficient (adipo(1/–)) mice exhibit mild insulin resistance, while homozygous adiponectindeficient (adipo(–/–)) mice are moderately insulin resistant and glucose intolerant even though body weight gain is una ected.42 These results confirm that adiponectin regulation is an important factor influencing insulin sensitivity. Adiponectin also enhances AMPK activity in the arcuate hypothalamus (ARH) via AdipoR1 stimulating food intake.81 ICV or intravenous administration of adiponectin increases energy expenditure and weight loss in obese (ob/ob) mice.82 Adiponectin-deficient mice exhibit decreased AMPK phosphorylation in the ARC (arcurate nucleus of the hypothalamus) together with decreased food intake, andincreased energy expenditure, as well as resistance to high-fat- diet-induced obesity.81 Administration of a high-fat/high-sucrose diet results in severe insulin resistance and increased weight gain in adiponectin-deficient mice.80 Adipose tissue TNFa expression and circulating TNFa levels are increased in adiponectin-deficient mice, e ects reversed by exogenous (adeno- virus-mediated) adiponectin.80 These data suggest that adiponectin and TNFa

act in a counter-regulatory manner. Under conditions of metabolic stress, such as diet-induced obesity, unopposed TNFa activity might lead to a shift towards insulin resistance.

Both AdipoR1 and AdipoR2 are expressed in adipocytes suggesting that adiponectin could exert an autocrine e ect in adipose tissue. In support of this, transgenic mice with moderate expression of exogenous adiponectin targeted to adipose tissue displayed reduced adiponectin mRNA levels and protein in early life, together with decreased circulating adiponectin in adult mice.83 As a result, the adult mice displayed glucose intolerance, insulin resistance and increased adiposity.83 Reduced adiponectin expression in adipose tissue in these mice was also associated with decreased AdipoR2 and UCP2 expression and increased expression of TNFa.83 Over-expression of adiponectin in 3T3-L1 fibroblasts accelerates adipogenesis, with more prolonged and robust gene expression for related transcriptional factors, CCAAT/enhancer binding protein alpha (C/ EBP2), PPARg, and adipocyte determination and di erentiation factor 1; SREBP1c (also named ADD1) together with accelerated suppression of PPARg co-activator-1a (PGC-1a).84 These changes were associated with a greater number of larger lipid droplets in di erentiated adipocytes.84 However, overexpression of human adiponectin in the livers of mice maintained on a high-fat/ high-sucrose diet impairs adipocyte di erentiation and prevents excessive fat accumulation in both visceral and subcutaneous adipose tissues.85 Macrophage infiltration in adipose tissue was also markedly suppressed in the transgenic mice.85 Importantly, adiponectin antagonizes LPS-induced NF-kB activation,66 together with production of IL-666 and TNFa.86 This autocrine e ect is observed in conjunction with increased PPARg2 expression in adipocytes.66

A recent study analyzed AdipoR1 and AdipoR2 proteins in 3T3-L1 adipocytes and subcutaneous and visceral adipocytes/adipose tissue of humans and rats.87 While di erentiation of 3T3-L1 cells in the presence of the FA palmitate did not alter AdipoR1 and AdipoR2 expression, metformin (which acts via AMPK) and fenofibrate (a PPARa agonist) upregulated AdipoR2. Furthermore, in the rat, AdipoR2 protein expression is lower in obese diabetic animals compared with obese rats with normal glucose disposal.

2.5Potential E ects of PPARa Deficiency on Autocrine Signaling in Adipose Tissue

PPARa has an established involvement in the metabolic adaptations to fasting, upregulating genes involved in FA uptake and oxidation.88,89 PPARa null mice

show an impaired response to starvation. In these mice, hypoketonemia is associated with elevated plasma FA and hepatic TAG accumulation, together with hypoglycemia.90 Although highly expressed in the liver and other oxidative tissues (e.g. heart, skeletal muscle and endocrine pancreas), PPARa is also expressed (albeit to a lower extent) in adipose tissue.91 Studies in vivo have shown altered regulation of lipogenic and cholesterogenic genes, together with increased lipogenesis de novo in adipose tissue of PPARa null mice.91

PPARa expression in white adipose tissue is enhanced in response to sustained intense hyperleptinaemia induced by adenoviral transfer of leptin cDNA.92 PPARa signaling is required for the lipopenic action of hyperleptinemia on adipose tissue as adenovirus-induced hyperleptinemia does not cause fat loss in PPARa null mice.93 The importance of autocrine e ects of leptin, in addition to e ects mediated by the hypothalamus, on the reduction of fat mass have been highlighted.73

Leptin secretion is positively correlated to adipocyte glucose handling.94,95 Adipocyte glucose uptake is enhanced by PPARg activation.30,31,33,96 PPARg

is thus predicted to facilitate leptin secretion. PPARa null mice maintained on a high-fat diet become more obese than wild-type, despite elevated blood leptin levels.97 Thus, PPARa deficiency uncouples the feedback control of fat mass by systemic leptin.

The augmented ability for storage of dietary-derived fat within the adipocyte, despite high leptin levels, protects PPARa null mice from the development of lipid-induced insulin resistance.97 However, even when maintained on low-fat diet, post-absorptive PPARa null mice show increased whole-body glucose turnover, attributed to increased glucose uptake and phosphorylation by white adipose tissue in vivo.98 Thus, in adipose tissue, PPARa deficiency induces a phenotype reminiscent of that evoked by PPARg activation.

We investigated the involvement of PPARa in the regulation of leptin physiology using isolated adipocytes from PPARa null mice, demonstrating an e ect of PPARa deficiency to increase adipocyte glucose uptake and conversion to TAG.99 We also observed (unpublished observations) that under glucose plus insulin-stimulated conditions, total leptin increased significantly with PPARa null adipocytes and a lesser proportion of this total leptin was secreted. Our data highlight e ects of PPARa, potentially important for obesity susceptibility, in the regulation of the balance between leptin synthesis and secretion. In these studies, adipose-tissue mass was not increased, but rather was lowered as a result of PPARa deficiency. This result is consistent with an increased autocrine e ect of secreted leptin to lower TAG storage. In contrast to studies indicating a requirement for PPARa for the e ects of adenovirusinduced hyperleptinemia, our study using PPARa null mice suggests that this loss of adiposity does not require the participation of PPARa. Our data instead support the concept that adipose tissue expandability may be limited by local leptin production due to increased leptin secretion. This could possibly be through a cycle of increased glucose uptake in adipocytes increasing leptin secretion, which exerts autocrine e ects on the adipocyte to reduce TAG storage. In an independent study, Yessoufou et al.100 observed a concomitant increase in both adiponectin and leptin mRNA expression in adipose tissue of PPARa null mice. While our mice, bred on an sv129 background, showed reduced adiposity, mice in this latter study, which were bred on to a di erent (C57BL/6J) background, had a higher adipose tissue mass compared to WT mice. These authors also quantified the mRNA of well-known markers of macrophages and observed that the expressions of mRNA of CD14 and CD68 (generally expressed in macrophages), but not of F4/80 (a robust macrophage