The extreme obesity of the obese (ob/ob) mouse is attributable to mutations in the gene encoding leptin, an adipocyte-specific secreted protein which has profound effects on appetite and energy expenditure. We know of no equivalent evidence regarding leptin's role in the control of fat mass in humans. We have examined two severely obese children who are members of the same highly consanguineous pedigree. Their serum leptin levels were very low despite their markedly elevated fat mass and, in both, a homozygous frame-shift mutation involving the deletion of a single guanine nucleotide in codon 133 of the gene for leptin was found. The severe obesity found in these congenitally leptin-deficient subjects provides the first genetic evidence that leptin is an important regulator of energy balance in humans.

Recent studies in murine models suggest that resistin (also called Fizz3 [1]), a novel cysteine-rich protein secreted by adipocytes, may represent the long-sought link between obesity and insulin resistance (2). Furthermore, peroxisome proliferator-activated receptor-␥ (PPAR-␥) agonists appear to inhibit resistin expression in murine adipocytes, providing a possible explanation for the mode of action of this class of insulin sensitizers (2). Using a fluorescent real-time reverse transcriptase-polymerase chain reaction-based assay, we found that resistin mRNA levels in whole adipose tissue samples were increased in morbidly obese humans compared with lean control subjects. However, in freshly isolated human adipocytes, resistin mRNA levels were very low and showed no correlation with BMI. Resistin mRNA was undetectable in preadipocytes, endothelial cells, and vascular smooth muscle cells, but it was readily detectable in circulating mononuclear cells. Although exposure of human mononuclear cells to PPAR-␥ agonists markedly upregulated fatty acid-binding protein-4 expression, these agents had no effect on mononuclear cell resistin expression. Finally, resistin mRNA was undetectable in adipocytes from a severely insulinresistant subject with a dominant-negative mutation in PPAR-␥ (3). We conclude that the recently described relationships of murine resistin/Fizz3 expression with obesity, insulin resistance, and PPAR-␥ action may not readily translate to humans. Further studies of this novel class of proteins are needed to clarify their roles in human metabolism. Diabetes 50:2199 -2202, 2001 S teppan et al. (2) recently reported a novel cysteine-rich secreted protein, which they termed resistin, the expression of which was markedly decreased by treatment of a murine adipocyte cell line with an agonist of the nuclear hormone receptor peroxisome proliferator-activated receptor-␥ (PPAR-␥). Serum levels of resistin were elevated in obese mice, and immunoneutralization of circulating resistin in these animals improved insulin sensitivity. Administration of recombinant resistin impaired insulin action in vivo in mice and ex vivo in an adipocyte cell line. These observations led the authors to conclude that resistin might represent an adipocyte-derived mediator of the link between obesity and insulin resistance. They also suggested that the suppression of resistin expression by PPAR-␥ agonists might explain the beneficial effects of these compounds in insulin-resistant states. Contrasting conclusions were reached by Way et al. (4), who found reduced resistin mRNA levels in white adipose tissue (WAT) of several obese rodent models. Furthermore, treating these animals with PPAR-␥ agonists increased resistin mRNA levels in WAT. These discrepant observations are difficult to reconcile and indicate the need for further studies. We developed a real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR)-based assay for human resistin using primers based in exons 1 and 2 of the human gene and used it to examine ...

Activation of peroxisome proliferator-activated receptor (PPAR) gamma, a nuclear receptor highly expressed in adipocytes, induces the differentiation of murine preadipocyte cell lines. Recently, thiazolidinediones (TZDs), a novel class of insulin-sensitizing compounds effective in the treatment of non-insulin-dependent diabetes mellitus (NIDDM) have been shown to bind to PPARgamma with high affinity. We have examined the effects of these compounds on the differentiation of human preadipocytes derived from subcutaneous (SC) and omental (Om) fat. Assessed by lipid accumulation, glycerol 3-phosphate dehydrogenase activity, and mRNA levels, subcultured preadipocytes isolated from either SC or Om depots did not differentiate in defined serum-free medium. Addition of TZDs (BRL49653 or troglitazone) or 15-deoxyDelta12,14prostaglandin J2 (a natural PPARgamma ligand) enhanced markedly the differentiation of preadipocytes from SC sites, assessed by all three criteria. The rank order of potency of these agents in inducing differentiation matched their ability to activate transcription via human PPARgamma. In contrast, preadipocytes from Om sites in the same individuals were refractory to TZDs, although PPARgamma was expressed at similar levels in both depots. The mechanism of this depot-specific TZD response is unknown. However, given the association between Om adiposity and NIDDM, the site-specific responsiveness of human preadipocytes to TZDs may be involved in the beneficial effects of these compounds on in vivo insulin sensitivity.

Human omental adipocytes display a range of biochemical properties that distinguish them from adipocytes of subcutaneous origin. However, information about site-related gene expression in human fat cells is limited. We have previously demonstrated that leptin mRNA is markedly overexpressed in abdominal subcutaneous (SC) compared with omental (Om) adipocytes. To further investigate depot-specific differences in adipocyte gene expression, we have measured, in paired samples of isolated human adipocytes obtained from SC and Om fat depots, the expression of mRNAs encoding a number of proteins involved in the control of adipocyte metabolism. In contrast to the marked site-related expression of leptin, genes encoding lipoprotein lipase (LPL), hormone-sensitive lipase (HSL), peroxisome proliferator-activated receptor-gamma (PPAR-gamma), tumor necrosis factor-alpha (TNF-alpha), and adipsin were not consistently differentially expressed. Of note, a highly significant inverse correlation between adipocyte PPAR-gamma expression and BMI (r = -0.7, P = 0.0005) was found. In parallel experiments, differential display was used in an attempt to identify novel and/or unexpected adipocyte genes that were expressed in a site-related manner. No transcript that was unique to one or another depot was found, but cellular inhibitor of apoptosis protein-2 (cIAP2) mRNA, which has not previously been reported in adipocytes, was expressed at higher levels in Om than SC adipocytes (Om > SC in all eight subjects; mean Om:SC ratio 1.9 +/- 0.2, P < 0.01). Because cIAP2 may be involved in the regulation of TNF-alpha signaling, this raises the possibility that depot-specific differences may exist in the regulation of adipocyte apoptosis. Thus, of the mRNAs examined to date, only leptin and cIAP2 show consistent site-related expression, suggesting that these molecules may have important roles in determining functional properties particular to individual adipose depots. Given the importance of PPAR-gamma in adipocyte development and insulin sensitivity, the inverse correlation between adipocyte PPAR-gamma mRNA levels and adiposity may represent a local regulatory mechanism restraining fat accumulation and/or may be related to the reduction of insulin sensitivity that occurs with increasing fat mass.

Conjugated linoleic acid (CLA) is a heterogeneous group of positional and geometric isomers of linoleic acid. This study demonstrates the divergent effects of the cis-9 trans-11 (c9,t11-CLA) and trans-10 cis-12 (t10,c12-CLA) isomers of CLA on lipid metabolism and nutrient regulation of gene expression in ob/ob mice. The c9,t11-CLA diet decreased serum triacylglycerol (P ‫؍‬ 0.01) and nonesterified fatty acid (NEFA) (P ‫؍‬ 0.05) concentrations, and this was associated with reduced hepatic sterol regulatory element-binding protein-1c (SREBP-1c; P ‫؍‬ 0.0045) mRNA expression, coupled with reduced levels of both the membrane-bound precursor and the nuclear forms of the SREBP-1 protein. C9,t11-CLA significantly reduced hepatic LXR␣ (P ‫؍‬ 0.019) mRNA expression, a novel regulator of SREBP-1c. In contrast, c9,t11-CLA increased adipose tissue SREBP-1c mRNA expression (P ‫؍‬ 0.0162) proportionally to the degree of reduction of tumor necrosis factor ␣ (TNF-␣) mRNA (P ‫؍‬ 0.012). Recombinant TNF-␣ almost completely abolished adipose tissue SREBP-1c mRNA expression in vivo. The t10,c12-CLA diet promoted insulin resistance and increased serum glucose (P ‫؍‬ 0.025) and insulin (P ‫؍‬ 0.01) concentrations. T10,c12-CLA induced profound weight loss (P ‫؍‬ 0.0001) and increased brown and white adipose tissue UCP-2 (P ‫؍‬ 0.001) and skeletal muscle UCP-3 (P ‫؍‬ 0.008) mRNA expression. This study highlights the contrasting molecular and metabolic effect of two isomers of the same fatty acids. The ameliorative effect of c9,t11-CLA on lipid metabolism may be ascribed to reduced synthesis and cleavage of hepatic SREBP-1, which in turn may be regulated by hepatic LXR␣ expression.

Peroxisome proliferator-activated receptor gamma coactivator 1a (PGC1a) is an accessory protein which can potentiate the transcriptional activation function of many nuclear hormone receptors. Its tissue distribution and physiological studies suggest that its principal in vivo roles are to promote cold-induced thermogenesis, mitochondrial biogenesis, hepatic gluconeogenesis, and fatty acid b-oxidation. It is expressed in the white adipose tissue of both humans and rodents, and in rodents it has been suggested to mediate in part the leptin-induced conversion of white adipocytes from fat storing to fat oxidising cells. In this study, quantitative real-time PCR has been used in human tissue to demonstrate that (1) PGC1a mRNA levels in subcutaneous fat are three-fold lower in morbidly obese than in slim subjects; (2) there are no differences in PGC1a mRNA between omental and subcutaneous mature adipocytes; (3) there is a robust induction of PGC1a expression during subcutaneous human preadipocyte differentiation ex vivo. Whether low PGC1a expression is a prelude to the development of obesity, or a consequence of that obesity, attempts to upregulate endogenous white adipose tissue expression may prove a valuable new avenue to explore in obesity therapy. Keywords: PGC1; white adipose tissue; preadipocyte; adipocyte; obesity Peroxisome proliferator-activated receptor gamma coactivator 1a (PGC1a) was first identified as a coactivator of PPARg in vitro, 1 although subsequent studies have revealed it to be promiscuous in such systems, able to coactivate PPARa, PPARd, the oestrogen receptor, thyroid hormone receptor, and hepatic nuclear factor 4a (HNF4a) among others. It is most highly expressed in brown adipose tissue, the heart and skeletal muscle, and has been shown to be upregulated during cold exposure in rodents. Further functional studies have elucidated its central role in coordinating adaptive thermogenesis through increased mitochondrial biogenesis and uncoupled oxidative metabolism. 2 More recently, initial rodent expression data were added to by the demonstration of a strong induction of PGC1a expression in the liver during fasting, when it has been shown to act in concert with the glucocorticoid receptor, CREB, and HNF4a to stimulate gluconeogenesis, 3,4 and with CREB and HNF4a to stimulate fatty acid oxidation. 5 Thus, altering PGC1a activity has emerged as a parsimonious strategy for influencing complex programmes of gene expression in response to external stimuli in a cell-and tissue-specific manner.PGC1a expression in white adipose tissue is low in both rodents and humans, where it is around 10% of that in liver. Although the small index study found relative suppression of PGC1a mRNA in the skeletal muscle of obese humans, no difference was found in WAT expression. 6 However, recent work in rodents has shown that hyperleptinaemia results in a dramatic decrease of white adipose tissue due to increased b oxidation, attributed to increased expression of Moreover, using a range of transgenic, pharmacological, a...

Sterol regulatory element binding protein (SREBP)-1 is a transcription factor with important roles in the control of fatty acid metabolism and adipogenesis. Little information is available regarding the expression of this molecule in human health or disease. Exposure of isolated human adipocytes to insulin enhanced SREBP1 gene expression and promoted its proteolytic cleavage to the active form. Furthermore, 3 h of in vivo hyperinsulinemia also significantly increased SREBP1 gene expression in human skeletal muscle. Transcript levels of SREBP1c, the most abundant isoform in adipose tissue, were significantly decreased in the subcutaneous adipose tissue of obese normoglycemic and type 2 diabetic subjects compared with that of nonobese normoglycemic control subjects. In skeletal muscle, SREBP1 expression was significantly reduced in type 2 diabetic subjects but not in obese subjects. Within the diabetic group, the extent of SREBP1 suppression was inversely related to metabolic control and was normalized by 3 h of in vivo hyperinsulinemia. Exposure of isolated human adipocytes to tumor necrosis factor-␣ (TNF-␣) produced a marked and specific decrease in the mRNA encoding the SREBP1c isoform and completely blocked the insulin-induced cleavage of SREBP1 protein. Thus, both the expression and proteolytic maturation of human SREBP1 are positively modulated by insulin. The specific reduction in the SREBP1c isoform seen in the adipose tissue of obese and type 2 diabetic subjects can be recapitulated ex vivo by TNF-␣, suggesting a possible mechanism for this association.

Tumour necrosis factor-alpha (TNF-), secreted by cells of the macrophage-monocyte lineage, has a well established role in inflammation and host-defence. The more recent discovery that adipocytes also secrete TNF-has led to a substantial body of research implicating this molecule in the insulin resistance of obesity. However, little is known about the normal regulation of TNFrelease from human adipose tissue. In particular, it is not known whether adipocyte production of TNF-is responsive to similar or different molecular regulators than those relevant to macrophages. TNF-release from cultured human adipose tissue and isolated adipocytes was examined using an ELISA. Insulin, cortisol or the thiazolidinedione, BRL 49653, did not have a significant effect on TNF-release from adipose tissue or isolated adipocytes. In contrast, lipopolysaccaride (LPS), a major stimulus of TNF-protein production in monocytes and macrophages, resulted in a fivefold stimulation of TNFrelease from human adipose tissue. Significant stimulation of TNF-release was also seen from isolated adipocytes, indicating that the increase in TNF-release from adipose tissue in the presence of LPS is unlikely to be entirely attributable to contaminating monocytes or macrophages. Consistent with this observation was the finding that mRNA for CD14, a known cellular receptor for LPS, is expressed in human adipocytes. The increase in TNFprotein release in response to LPS was blocked by an inhibitor of the matrix metalloproteinase responsible for the cleavage of the membrane-bound proform of TNF-, indicating that this release represented regulated secretion and was not due to cell lysis.In conclusion, the regulation of TNF-protein release from human adipose tissue and isolated adipocytes appears to be similar to its regulation in cell types more traditionally implicated in host defence. The production by the adipocyte of a range of molecules involved in host defence -TNF-, factors D, B and C3, interleukin-6, and macrophage colony-stimulating factor -suggest that this cell type may make a significant contribution to innate immunity.