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Accumulation of dysfunctional white adipose tissues increases risks for cardiometabolic diseases in obesity. In addition to white, brown or brite adipose tissues are also present in adult humans and increasing their amount may be protective. Therefore, understanding factors regulating the amount and function of each adipose depot is crucial for developing therapeutic targets for obesity and its associated metabolic diseases. The transforming growth factor beta (TGFβ) superfamily, which consists of TGFβ, BMPs, GDFs, and activins, controls multiple aspects of adipose biology. This review focuses on the recent development in understanding the role of TGFβ superfamily in the regulation of white, brite and brown adipocyte differentiation, adipose tissue fibrosis, and adipocyte metabolic and endocrine functions. TGFβ family and their antagonists are produced locally within adipose tissues and their expression levels are altered in obesity. We also discuss their potential contribution to adipose tissue dysfunction in obesity.

Objective Regulation of fatty acid (FA) metabolism is central to adipocyte dysfunction during diet-induced obesity (DIO). Long-chain acyl-CoA synthetase-4 (ACSL4) has been hypothesized to modulate the metabolic fates of polyunsaturated FA (PUFA), including arachidonic acid (AA), but the in vivo actions of ACSL4 are unknown. The purpose of our studies was to determine the in vivo role of adipocyte ACSL4 in regulating obesity-associated adipocyte dysfunction. Methods We developed a novel mouse model with adipocyte-specific ablation of ACSL4 (Ad-KO) using loxP Cre recombinase technology. Metabolic phenotyping of Ad-KO mice relative to their floxed littermates (ACSL4floxed) was performed, including body weight and body composition over time; insulin and glucose tolerance tests; and energy expenditure, activity, and food intake in metabolic cages. Adipocytes were isolated for ex vivo adipocyte oxygen consumption by Clark electrode and lipidomics analysis. In vitro adipocyte analysis including oxygen consumption by Seahorse and real-time PCR analysis were performed to confirm our in vivo findings. Results Ad-KO mice were protected against DIO, adipocyte death, and metabolic dysfunction. Adipocytes from Ad-KO mice fed high-fat diet (HFD) had reduced incorporation of AA into phospholipids (PL), free AA, and levels of the AA lipid peroxidation product 4-hydroxynonenal (4-HNE). Additionally, adipocytes from Ad-KO mice fed HFD had reduced p53 activation and increased adipocyte oxygen consumption (OCR), which we demonstrated are direct effects of 4-HNE on adipocytes in vitro. Conclusion These studies are the first to elucidate ACSL4's in vivo actions to regulate the incorporation of AA into PL and downstream effects on DIO-associated adipocyte dysfunction. By reducing the incorporation of AA into PL and free fatty acid pools in adipocytes, Ad-KO mice were significantly protected against HFD-induced increases in adipose and liver fat accumulation, adipocyte death, gonadal white adipose tissue (gWAT) inflammation, and insulin resistance (IR). Additionally, deficiency of adipocyte ACSL4 expression in mice fed a HFD resulted in increased gWAT adipocyte OCR and whole body energy expenditure (EE)., Highlights • ACSL4 expression is upregulated in murine white adipocytes during diet-induced obesity. • Mice with adipocyte-specific ablation of ACSL4 (Ad-KO) are protected against diet-induced obesity, adipocyte death and metabolic dysfunction. • Lipidomics profiling of isolated adipocytes from Ad-KO mice fed a high-fat diet (HFD) had reduced arachidonic acid (AA) in phospholipids. • Adipocytes from Ad-KO mice fed HFD had reduced free AA and levels of the AA lipid peroxidation product 4-hydroxynonenal (4-HNE). • Adipocytes from Ad-KO mice fed HFD had reduced p53 activation and increased adipocyte oxygen consumption (OCR). • P53 activation and inhibited adipocyte OCR are direct effects of 4-HNE on adipocytes in vitro.

Vitamin D insufficiency is associated with obesity and its related metabolic diseases. Adipose tissues store and metabolize vitamin D and expression levels of vitamin D metabolizing enzymes are known to be altered in obesity. Sequestration of vitamin D in large amount of adipose tissues and low vitamin D metabolism may contribute to the vitamin D inadequacy in obesity. Vitamin D receptor is expressed in adipose tissues and vitamin D regulates multiple aspects of adipose biology including adipogenesis as well as metabolic and endocrine function of adipose tissues that can contribute to the high risk of metabolic diseases in vitamin D insufficiency. We will review current understanding of vitamin D regulation of adipose biology focusing on vitamin D modulation of adiposity and adipose tissue functions as well as the molecular mechanisms through which vitamin D regulates adipose biology. The effects of supplementation or maintenance of vitamin D on obesity and metabolic diseases are also discussed.

Objective To review recent advances in understanding the cellular mechanisms that regulate fat distribution.

Objective The study goal was to determine the effect of weight loss (WL) alone and with aerobic exercise (WL + AEX) on serum amyloid A (SAA) levels and adipose SAA secretion from gluteal and abdominal depots. Methods Ninety-six overweight or obese postmenopausal women undertook a 6-month WL alone (n = 47) or with AEX training (n = 49) (6 months WL and WL + AEX are considered WL when groups were combined). Their serum SAA levels, body weight, and adipose SAA secretion ex vivo from gluteal and abdominal depot were measured before and after WL interventions. Results The participants lost an average of 8% body weight with a 10% decrease of serum SAA. Serum SAA levels remained significantly correlated with body weight before and after WL. However, the changes of serum SAA level did not correlate with changes of body weight. The gluteal adipose tissue secreted ∼50% more SAA than the abdominal tissue, but the changes of abdominal, but not gluteal, SAA secretion correlated (R2 = 0.19, p < 0.01) with those of serum SAA levels during WL. Conclusions No linear correlation between the decrease in systemic SAA and WL was found. There is a depot-dependent difference in adipose SAA secretion and abdominal SAA secretion, which may partially account for the systemic SAA reduction during WL.

Obesity, defined as excess fat mass, increases risks for multiple metabolic diseases, such as type 2 diabetes, cardiovascular disease and several types of cancer. Over and above fat mass per se, the pattern of fat distribution, android or truncal as compared to gynoid or peripheral, has a profound influence on systemic metabolism and hence risk for metabolic diseases. Increases in upper body adipose tissue (visceral and abdominal subcutaneous) confer an independent risk, while the quantity of gluteofemoral adipose tissue is protective. Variations in the capacity of different depots to store and release fatty acids and to produce adipokines are important determinants of fat distribution and its metabolic consequences. Depot differences in cellular composition and physiology, including innervation and blood flow, likely influence their phenotypic properties. A number of lines of evidence also support the idea that adipocytes from different anatomical depots are intrinsically different as a result of genetic or developmental events. In this chapter, we will review the phenotypic characteristics of different adipose depots and mechanisms that link their depot-specific biology to metabolic complications in men and women.

Glucocorticoids promote fat accumulation in visceral compared to subcutaneous depots, but the molecular mechanisms involved remain poorly understood. To identify long-term changes in gene expression that are differentially sensitive or responsive to glucocorticoids in these depots, paired samples of human omental (Om) and abdominal subcutaneous (Abdsc) adipose tissues obtained from obese women during elective surgery were cultured with the glucocorticoid receptor agonist dexamethasone (Dex, 0, 1, 10, 25 and 1000 nM) for 7 days. Dex regulated 32% of the 19,741 genes on the array, while 53% differed by Depot and 2.5% exhibited a Depot*Dex concentration interaction. Gene set enrichment analysis showed Dex regulation of the expected metabolic and inflammatory pathways in both depots. Cluster analysis of the 460 transcripts that exhibited an interaction of Depot and Dex concentration revealed sets of mRNAs for which the responses to Dex differed in magnitude, sensitivity or direction between the two depots as well as mRNAs that responded to Dex only in one depot. These transcripts were also clearly depot different in fresh adipose tissue and are implicated in processes that could affect adipose tissue distribution or functions (e.g. adipogenesis, triacylglycerol synthesis and storage, insulin action). Elucidation of the mechanisms underlying the depot differences in the effect of Dex on the expression of specific genes and pathways that regulate adipose function may offer novel insights into understanding the biology of visceral adipose tissues and their links to metabolic health.

Increased adipocyte size is hypothesized to signal the recruitment of adipose progenitor cells (APCs) to expand tissue storage capacity. To investigate depot and sex differences in adipose growth, male and female C57BL/6J mice (10 wk-old) were challenged with high-fat (HF) or low-fat (LF) diets (D) for 14 wk. The HFD increased gonadal (GON) depot weight by adipocyte hypertrophy and hyperplasia in females but hypertrophy alone in males. In both sexes, inguinal (ING) adipocytes were smaller than GON, and depot expansion was due to hypertrophy. Matrix metalloproteinase 3 (Mmp3), an antiadipogenic factor, and its inhibitor Timps modulate the extracellular matrix remodeling needed for depot expansion. Mmp3 mRNA was depot different (ING > GON), higher in females than males and mainly expressed in APCs. In males, HFD-induced obesity increased tissue and APC Mmp3 mRNA levels and MMP3 protein and enzymatic activity. In females however, HFD significantly decreased MMP3 protein without affecting its mRNA levels. MMP3 activity also decreased (significant in ING). Timp4 mRNA was expressed mainly in adipocytes, and HFD-induced obesity tended to increase the ratio of TIMP4 to MMP3 protein in females, whereas it decreased it in males. Overexpression of Mmp3 in 3T3-L1 preadipocytes or rhMMP3 protein added to primary human preadipocytes inhibited differentiation, whereas rhTIMP4 improved adipogenesis and attenuated the inhibitory effect of rhMMP3. These data suggest that HFD-induced obesity downregulates APC MMP3 expression to trigger adipogenesis, and adipocyte TIMP4 may modulate this process to regulate hyperplastic vs. hypertrophic adipose tissue expansion, fat distribution, and metabolic health in a sex- and depot-dependent manner.

The glucocorticoid-induced leucine zipper (GILZ), a primary target of glucocorticoids, is expressed in human adipocytes, but its importance in adipocyte function is unknown. Because TNFα is increased in obese adipose tissue and antagonizes a number of glucocorticoid actions, we investigated the interplay of these pathways. GILZ knockdown increased and GILZ overexpression decreased interleukin-6 (IL-6) and leptin mRNA and protein secretion. GILZ knockdown increased the magnitude of the glucocorticoid effect on leptin secretion, but did not affect the glucocorticoid suppression of IL-6. Although GILZ silencing decreased adiponectin mRNA levels, it did not affect the amount of adiponectin secreted. GILZ negatively modulated pro-inflammatory signaling pathways, blocking basal and TNFα-stimulated (1 h) p65 nuclear factor κB nuclear translocation and transcriptional activity by binding to p65 in the cytoplasm. GILZ silencing increased basal ERK1/2 and JNK phosphorylation, and decreased MAPK phosphatase-1 protein levels. Longer term TNFα (4 h or 24 h) treatment decreased GILZ expression in human adipocytes. Furthermore, adipose tissue GILZ mRNA levels were reduced in proportion to the degree of obesity and expression of inflammatory markers. Overall, these results suggest that GILZ antagonizes the pro-inflammatory effects of TNFα in human adipocytes, and its downregulation in obesity may contribute to adipose inflammation and dysregulated adipokine production, and thereby systemic metabolism.

Objective Insulin signaling plays a unique role in the regulation of energy homeostasis and the impairment of insulin action is associated with altered lipid metabolism, obesity, and Type 2 Diabetes. The main aim of this study was to provide further insight into the regulatory mechanisms governing the insulin signaling pathway by investigating the role of non-proteolytic ubiquitination in insulin-mediated activation of AKT. Methods The molecular mechanism of AKT regulation through ubiquitination is first dissected in vitro in 3T3-L1 preadipocytes and then validated in vivo using mice with adipo-specific deletion of GPS2, an endogenous inhibitor of Ubc13 activity (GPS2-AKO mice). Results Our results indicate that K63 ubiquitination is a critical component of AKT activation in the insulin signaling pathway and that counter-regulation of this step is provided by GPS2 preventing AKT ubiquitination through inhibition of Ubc13 enzymatic activity. Removal of this negative checkpoint, through GPS2 downregulation or genetic deletion, results in sustained activation of insulin signaling both in vitro and in vivo. As a result, the balance between lipid accumulation and utilization is shifted toward storage in the adipose tissue and GPS2-AKO mice become obese under normal laboratory chow diet. However, the adipose tissue of GPS2-AKO mice is not inflamed, the levels of circulating adiponectin are elevated, and systemic insulin sensitivity is overall improved. Conclusions Our findings characterize a novel layer of regulation of the insulin signaling pathway based on non-proteolytic ubiquitination of AKT and define GPS2 as a previously unrecognized component of the insulin signaling cascade. In accordance with this role, we have shown that GPS2 presence in adipocytes modulates systemic metabolism by restricting the activation of insulin signaling during the fasted state, whereas in absence of GPS2, the adipose tissue is more efficient at lipid storage, and obesity becomes uncoupled from inflammation and insulin resistance., Graphical abstract, Highlights • Ubc13-mediated ubiquitination of AKT is required for activation of the insulin signaling pathway. • GPS2 regulates insulin signaling by inhibiting AKT ubiquitination and activation. • Adipo-specific deletion of GPS2 results in increased adiposity and altered lipid flux in the adipocytes. • GPS2-AKO mice have higher levels of circulating adiponectin and are insulin sensitive despite being obese.

Recent studies demonstrate that adipose tissue undergoes a continuous process of remodeling that is pathologically accelerated in the obese state. Contrary to earlier dogma, adipocytes die and are replaced by newly differentiated ones. This review will summarize recent advances of our knowledge of the mechanisms that regulate adipose tissue remodeling and highlight the influences of obesity, depot, and sex, as well as the relevance of rodent models to humans.A substantial literature now points to the importance of dynamic changes in adipocyte and immune cell turnover, angiogenesis, and extracellular matrix remodeling in regulating the expandability and functional integrity of this tissue. In obesity, the macrophages are recruited, surrounding dead adipocytes and polarized toward an inflammatory phenotype. The number of dead adipocytes is closely associated with the pathophysiological consequences of obesity, including insulin resistance and hepatic steatosis. Further, there are substantial depot, sex and species differences in the extent of remodeling.Adipose tissue undergoes a continuous remodeling process that normally maintains tissue health, but may spin out of control and lead to adipocyte death in association with the recruitment and activation of macrophages, and systemic insulin resistance.

This review summarizes recent advances in our understanding of the pre- and posttranscriptional mechanisms that regulate leptin production and secretion in adipocytes. Basal leptin production is proportional to the status of energy stores, i.e., fat cell size, and this is mainly regulated by alterations in leptin mRNA levels. Leptin mRNA levels are regulated by hormones, including glucocorticoids and catecholamines, but little is known about the transcriptional mechanisms involved. Leptin synthesis and secretion is also acutely modulated in response to hormones such as insulin and the availability of metabolic fuels. Acute variations in leptin production over a time course of minutes to hours are mediated at the levels of both translation and secretion. Increases in amino acids and insulin after a meal activate the mammalian target of rapamycin (mTOR) pathway, leading to an increase in specific rates of leptin biosynthesis. Cross-talk among mTOR, PKA, and AMP-activated protein kinase pathways appears to integrate hormonal and nutrient signals that regulate leptin mRNA translation, at least in part through mechanisms involving its 5′- and 3′-untranslated regions. In addition, the rate of leptin secretion from preformed stores in response to hormonal cues is also regulated. Insulin stimulates, and adrenergic agonists inhibit, leptin secretion, and this likely contributes to variations in the magnitude of nutrition-related leptin excursions and oscillations. Overall, the study of leptin production has contributed to a deepening understanding of leptin biology and, more broadly, to our understanding of the cellular and molecular mechanisms by which the adipocyte integrates hormonal and nutrient signals to regulate adipokine production.

OBJECTIVE—We examined the relationship between the expression of thrombospondin (TSP)1, an antiangiogenic factor and regulator of transforming growth factor-β activity, obesity, adipose inflammation, and insulin resistance. RESEARCH DESIGN AND METHODS—TSP1 gene expression was quantified in subcutaneous adipose tissue (SAT) of 86 nondiabetic subjects covering a wide range of BMI and insulin sensitivity, from visceral adipose (VAT) and SAT from 14 surgical patients and from 38 subjects with impaired glucose tolerance randomized to receive either pioglitazone or metformin for 10 weeks. An adipocyte culture system was also used to assess the effects of pioglitazone and coculture with macrophages on TSP1 gene expression. RESULTS—TSP1 mRNA was significantly associated with obesity (BMI) and insulin resistance (low insulin sensitivity index). Relatively strong positive associations were seen with markers of inflammation, including CD68, macrophage chemoattractant protein-1, and plasminogen activator inhibitor (PAI)-1 mRNA (r ≥ 0.46, P = 0.001 for each), that remained significant after controlling for BMI and Si. However, TSP1 mRNA was preferentially expressed in adipocyte fraction, whereas inflammatory markers predominated in stromal vascular fraction. Coculture of adipocytes and macrophages augmented TSP1 gene expression and secretion from both cell types. Pioglitazone (not metformin) treatment resulted in a 54% decrease (P < 0.04) in adipose TSP gene expression, as did in vitro pioglitazone treatment of adipocytes. CONCLUSIONS—TSP1 is a true adipokine that is highly expressed in obese, insulin-resistant subjects; is highly correlated with adipose inflammation; and is decreased by pioglitazone. TSP1 is an important link between adipocytes and macrophage-driven adipose tissue inflammation and may mediate the elevation of PAI-1 that promotes a prothrombotic state.

Retinol binding protein 4 (RBP4) was recently found to be expressed and secreted by adipose tissue, and was strongly associated with insulin resistance.The aim was to determine the relationship between RBP4 and obesity, insulin resistance, and other markers of insulin resistance in humans.RBP4 mRNA levels in adipose tissue and muscle of nondiabetic human subjects with either normal or impaired glucose tolerance (IGT) were studied, along with plasma RBP4. RBP4 gene expression was also measured in adipose tissue fractions, and from visceral and sc adipose tissue (SAT) from surgical patients.The study was conducted at University Hospital and General Clinical Research Center.Insulin sensitivity (S(I)) was measured, and fat and muscle biopsies were performed. In IGT subjects, these procedures were performed before and after treatment with metformin or pioglitazone.The relationship between RBP4 expression and obesity, S(I), adipose tissue inflammation, and intramyocellular lipid level, and response to insulin sensitizers was measured.RBP4 was expressed predominantly from the adipocyte fraction of SAT. Although SAT RBP4 expression and the plasma RBP4 level demonstrated no significant relationship with body mass index or S(I), there was a strong positive correlation between RBP4 mRNA and adipose inflammation (monocyte chemoattractant protein-1 and CD68), and glucose transporter 4 mRNA. Treatment of IGT subjects with pioglitazone resulted in an increase in S(I) and an increase in RBP4 gene expression in both adipose tissue and muscle, but not in plasma RBP4 level, and the in vitro treatment of cultured adipocytes with pioglitazone yielded a similar increase in RBP4 mRNA.RBP4 gene expression in humans is associated with inflammatory markers, but not with insulin resistance. The increase in RBP4 mRNA after pioglitazone treatment is unusual, suggesting a complex regulation of this novel adipokine.

Growth hormone (GH) administration reduces abdominal, but not lower body, fat mass. To gain insight into the underlying mechanisms, this study examined the expression of the GH receptor (GHR) and some of its targets in abdominal and gluteal adipose tissue.GHR and GH targets in the lipolytic pathway were assessed (quantitative PCR/Western blotting) in adipose aspirates from premenopausal women [n = 15, age 26.9 ± 6.1 years, body mass index (BMI) 28.0 ± 6.8 kg/m(2) ] and men (n = 28, age 29.2 ± 7.0 years, BMI 26.9 ± 3.7 kg/m(2) ).GHR mRNA expression was lower in the gluteal depot when compared with the abdominal depot (P = 0.01). Abdominal GHR correlated negatively with age and BMI, whereas gluteal GHR was associated with lower waist-to-hip ratio (WHR), that is, pear shape. In both sites, GHR mRNA correlated strongly with genes important for the regulation of lipolysis: adipose tissue triglyceride lipase (ATGL), hormone-sensitive lipase, perilipin, and CIDEA (all P 0.001), independently of BMI, WHR, age, and sex. GHR protein was lower in the gluteal fat when compared with the abdominal fat (P = 0.03) and correlated with ATGL protein in the gluteal depot (P 0.001).GHR levels correlate with levels of lipases and lipid droplet-associated proteins crucial for lipolysis. Thus, higher GHR expression in the abdominal depot when compared with the gluteal depot may underlie the in vivo effect of GH to specifically reduce abdominal adipose tissue mass.

Obesity-associated alterations in adipocyte metabolism are important to the development of metabolic disorders, including type 2 diabetes, a leading cause of chronic disease and death in the United...

In vivo and in vitro studies indicate that β-adrenergic receptor agonists decrease leptin release from fat cells in as little as 30 min. Our objective was to determine whether alterations in leptin biosynthesis or secretion were involved in the short-term adrenergic regulation of leptin in human and rat adipose tissue. Isoproterenol (Iso) decreased leptin release from incubated adipose tissue of both nonobese and obese subjects to similar extent (−28 vs. −21% after 3 h). Inhibition of protein synthesis with cycloheximide did not block the effect of Iso on leptin release from human adipose tissue, suggesting that the Iso effect is independent of leptin synthesis. Iso also tended to increase tissue leptin content at the end of the 3-h incubation, as expected from the observed inhibition of release. Consistent with a posttranslational mechanism, Iso treatment did not affect leptin mRNA levels or relative rate of leptin biosynthesis as directly assessed by [35S]methionine incorporation into immunoprecipitable leptin. In contrast to these results in human adipose tissues, Iso did not decrease basal leptin release from rat adipose tissue. However, Iso did decrease insulin-stimulated leptin release by inhibiting the ability of insulin to increase leptin biosynthesis without detectably affecting leptin mRNA levels. Thus, in both human and rat, adrenergic regulation of posttranscriptional events (secretion in humans, translation in rats) may contribute to the rapid decline in circulating leptin that occurs when the sympathetic nervous system is activated, such as during fasting and cold exposure. Furthermore, the rat does not provide an ideal model to study mechanisms of cellular leptin regulation in humans.

Obesity is reaching epidemic proportions so there is growing interest in the mechanisms that regulates adipose tissue development and function. Although murine adipose cell lines are useful for many mechanistic studies, primary human adipose stromal cells (ASCs), which can be isolated from distinct adipose depots and cultured in vitro, have clear translational relevance. We describe the methods to isolate, culture, and differentiate human ASCs to adipocytes that respond to physiologically relevant hormones, such as insulin and β-adrenergic agonists. We also describe methods for assaying hormonal effects on glucose transport and lipolysis.

Fat distribution differs in men and women, but in both sexes, a predominantly gluteal-femoral compared with abdominal (central) fat distribution is associated with lower metabolic risk. Differences in cellular characteristics and metabolic functions of these depots have been described, but the molecular mechanisms involved are not understood.Our objective was to identify depot- and sex-dependent differences in gene expression in human abdominal and gluteal sc adipose tissues.Abdominal and gluteal adipose tissue aspirates were obtained from 14 premenopausal women [age 27.5 ± 7.0 yr, body mass index (BMI) 27.3 ± 6.2 kg/m(2), and waist-to-hip ratio 0.82 ± 0.04] and 21 men (age 29.7±7.4 yr, BMI 27.2 ± 4.5 kg/m(2), and waist-to-hip ratio 0.91 ± 0.07) and transcriptomes were analyzed using Illumina microarrays. Expression of selected genes was determined in isolated adipocytes and stromal vascular fractions from each depot, and in in vitro cultures before and after adipogenic differentiation.A total of 284 genes were differentially expressed between the abdominal and gluteal depot, either specifically in males (n = 66) or females (n = 159) or in both sexes (n = 59). Most notably, gene ontology and pathway analysis identified homeobox genes (HOXA2, HOXA3, HOXA4, HOXA5, HOXA9, HOXB7, HOXB8, HOXC8, and IRX2) that were down-regulated in the gluteal depot in both sexes (P = 2 × 10(-10)). Conversely, HOXA10 was up-regulated in gluteal tissue and HOXC13 was detected exclusively in this depot. These differences were independent of BMI, were present in both adipocytes and stromal vascular fractions of adipose tissue, and were retained throughout in vitro differentiation.We conclude that developmentally programmed differences may contribute to the distinct phenotypic characteristics of peripheral fat.

1,25(OH)(2)D(3) inhibits adipogenesis in mouse 3T3-L1 adipocytes, but little is known about its effects or local metabolism in human adipose tissue. We showed that vitamin D receptor (VDR) and 1α-hydroxylase (CYP27B1), the enzyme that activates 25(OH)D(3) to 1,25(OH)(2)D(3), were expressed in human adipose tissues, primary preadipocytes and newly-differentiated adipocytes. Preadipocytes and newly-differentiated adipocytes were responsive to 1,25(OH)(2)D(3), as indicated by a markedly increased expression of CYP24A1, a primary VDR target. 1,25(OH)(2)D(3) enhanced adipogenesis as determined by increased expression of adipogenic markers and triglyceride accumulation (50% to 150%). The magnitude of the effect was greater in the absence of thiazolidinediones. 1,25(OH)(2)D(3) was equally effective when added after the removal of differentiation cocktail on day 3, but it had no effect when added only during the induction period (day 0-3), suggesting that 1,25(OH)(2)D(3) promoted maturation. 25(OH)D(3) also stimulated CYP24A1 expression and adipogenesis, most likely through its conversion to 1,25(OH)(2)D(3). Consistent with this possibility, incubation of preadipocytes with 25(OH)D(3) led to 1,25(OH)(2)D(3) accumulation in the media. 1,25(OH)(2)D(3) also enhanced adipogenesis in primary mouse preadipocytes. We conclude that vitamin D status may regulate human adipose tissue growth and remodeling.

Previous studies showed that dietary L-arginine supplementation decreased white fat mass in genetically obese rats. This study tested the effectiveness of L-arginine in diet-induced obesity. Male Sprague-Dawley rats were fed for 15 wk a high-fat (HF) (40% energy) or low-fat (LF) (10% energy) diet beginning at 4 wk of age, resulting in 18% higher body weight gains and 74% higher weights of major white fat pads (retroperitoneal, epididymal, subcutaneous, and mesenteric adipose tissues) in HF than in LF fed rats. Starting at 19 wk of age, rats in each dietary group were supplemented for 12 wk with 1.51% L-arginine-HCl or 2.55% L-alanine (isonitrogenous control) (n = 8 per treatment) in drinking water and arginine groups were individually pair-fed to alanine controls. Despite similar energy intake, absolute weights of white fat pads increased by 98% in control rats over a 12-wk period but only by 35% in arginine-supplemented rats. The arginine treatment reduced the relative weights of white fat pads by 30% and enhanced those of soleus muscle by 13%, extensor digitorum longus muscle by 11%, and brown fat by 34% compared with control rats. Serum concentrations of insulin, adiponectin, growth hormone, corticosterone, triiodothyronine, and thyroxine did not differ between control and arginine-supplemented rats. However, arginine treatment resulted in lower serum concentrations of leptin, glucose, triglycerides, urea, glutamine, and branched-chain amino acids, higher serum concentrations of nitric-oxide metabolites, and improvement in glucose tolerance. Thus, dietary arginine supplementation shifts nutrient partitioning to promote muscle over fat gain and may provide a useful treatment for improving the metabolic profile and reducing body white fat in diet-induced obese rats.

Central obesity and the accumulation of visceral fat are risk factors for the development of type 2 diabetes and cardiovascular disease. Omentin is a protein expressed and secreted from visceral but not subcutaneous adipose tissue that increases insulin sensitivity in human adipocytes. To determine the impact of obesity-dependent insulin resistance on the regulation of two omentin isoforms, gene expression and plasma levels were measured in lean, overweight, and obese subjects. Omentin 1 was shown to be the major circulating isoform in human plasma. Lean subjects had significantly higher plasma omentin 1 levels than obese and overweight subjects. In addition, higher plasma omentin 1 levels were detected in women compared with men. Plasma omentin 1 levels were inversely correlated with BMI, waist circumference, leptin levels, and insulin resistance as measured by homeostasis model assessment and positively correlated with adiponectin and HDL levels. Both omentin 1 and omentin 2 gene expression were decreased with obesity and were highly correlated with each other in visceral adipose tissue. In summary, decreased omentin levels are associated with increasing obesity and insulin resistance. Therefore, omentin levels may be predictive of the metabolic consequences or co-morbidities associated with obesity.

Serum leptin levels are upregulated in proportion to body fat and also increase over the short term in response to meals or insulin. To understand the mechanisms involved, we assessed leptin synthesis and secretion in samples of adipose tissue from subjects with a wide range of BMI. Tissue leptin content and relative rates of leptin biosynthesis, as determined by metabolic labeling, were highly correlated with each other and with BMI and fat cell size. To understand mechanisms regulating leptin synthesis in obesity, we used biosynthetic labeling to directly assess the effects of insulin and glucocorticoids (dexamethasone) on leptin synthesis and secretion in human adipose tissue. Chronic treatment (1–2 days in organ culture) with insulin increased relative rates of leptin biosynthesis without affecting leptin mRNA levels. In contrast, dexamethasone increased leptin mRNA and biosynthesis in parallel. Acute treatment with insulin or dexamethasone (added during 1-h preincubation and 45-min pulse labeling) did not affect relative rates of leptin biosynthesis, but pulse-chase studies showed that addition of insulin nearly doubled the release of [35S]leptin after a 1-h chase. We conclude that the higher leptin stores in adipose tissue of obese humans are maintained by chronic effects of insulin and glucocorticoids acting at pre- and posttranslational levels and that the ability of insulin to increase the release of preformed leptin may contribute to short-term variations in circulating leptin levels.

Visfatin (VF) is a recently described adipokine preferentially secreted by visceral adipose tissue (VAT) with insulin mimetic properties.The aim of this study was to examine the association of VF with insulin sensitivity, intramyocellular lipids (IMCL), and inflammation in humans.VF mRNA was examined in paired samples of VAT and abdominal sc adipose tissue (SAT) obtained from subjects undergoing surgery. Plasma VF and VF mRNA was also examined in SAT and muscle tissue, obtained by biopsy from well-characterized subjects with normal or impaired glucose tolerance, with a wide range in body mass index (BMI) and insulin sensitivity (S(I)).The study was conducted at a University Hospital and General Clinical Research Center.S(I) was measured, and fat and muscle biopsies were performed. In impaired glucose tolerance subjects, these procedures were performed before and after treatment with pioglitazone or metformin.We measured the relationship between VF and obesity, S(I), adipose tissue inflammation, IMCL, and response to insulin sensitizers.No significant difference in VF mRNA was seen between SAT and VAT depots. VAT VF mRNA associated positively with BMI, whereas SAT VF mRNA decreased with BMI. SAT VF correlated positively with S(I), and the association of SAT VF mRNA with S(I) was independent of BMI. IMCL and markers of inflammation (adipose CD68 and plasma TNFalpha) were negatively associated with SAT VF. Impaired glucose tolerance subjects treated with pioglitazone showed no change in SAT VF mRNA despite a significant increase in S(I). Plasma VF and muscle VF mRNA did not correlate with BMI or S(I) or IMCL, and there was no change in muscle VF with either pioglitazone or metformin treatments.SAT VF is highly expressed in lean, more insulin-sensitive subjects and is attenuated in subjects with high IMCL, low S(I), and high levels of inflammatory markers. VAT VF and SAT VF are regulated oppositely with BMI.

Background Obesity is associated with low-grade chronic inflammation, and serum markers of inflammation are independent risk factors for cardiovascular disease (CVD). However, the molecular and cellular mechanisms that link obesity to chronic inflammation and CVD are poorly understood. Methods and Findings Acute-phase serum amyloid A (A-SAA) mRNA levels, and A-SAA adipose secretion and serum levels were measured in obese and nonobese individuals, obese participants who underwent weight-loss, and persons treated with the insulin sensitizer rosiglitazone. Inflammation-eliciting activity of A-SAA was investigated in human adipose stromal vascular cells, coronary vascular endothelial cells and a murine monocyte cell line. We demonstrate that A-SAA was highly and selectively expressed in human adipocytes. Moreover, A-SAA mRNA levels and A-SAA secretion from adipose tissue were significantly correlated with body mass index ( r = 0.47; p = 0.028 and r = 0.80; p = 0.0002, respectively). Serum A-SAA levels decreased significantly after weight loss in obese participants ( p = 0.006), as well as in those treated with rosiglitazone ( p = 0.033). The magnitude of the improvement in insulin sensitivity after weight loss was significantly correlated with decreases in serum A-SAA ( r = −0.74; p = 0.034). SAA treatment of vascular endothelial cells and monocytes markedly increased the production of inflammatory cytokines, e.g., interleukin (IL)-6, IL-8, tumor necrosis factor alpha, and monocyte chemoattractant protein-1. In addition, SAA increased basal lipolysis in adipose tissue culture by 47%. Conclusions A-SAA is a proinflammatory and lipolytic adipokine in humans. The increased expression of A-SAA by adipocytes in obesity suggests that it may play a critical role in local and systemic inflammation and free fatty acid production and could be a direct link between obesity and its comorbidities, such as insulin resistance and atherosclerosis. Accordingly, improvements in systemic inflammation and insulin resistance with weight loss and rosiglitazone therapy may in part be mediated by decreases in adipocyte A-SAA production., Editors' Summary Background. Obesity often alters an individual's overall metabolism, which in turn leads to complications like diabetes, high blood pressure, and an increased risk of cardiovascular disease (disease of the heart and blood vessels, such as stroke or heart attacks). Having established a strong link between inflammation and cardiovascular disease, scientists now think that obesity might cause persistent low-level inflammation, and that this is the reason for the cardiovascular problems seen in many obese people. By better understanding the links between obesity, inflammation, and cardiovascular disease, the hope is that scientists may be able to find medications that can be given to obese people to reduce their risk of heart attacks and strokes. Why Was This Study Done? Previous research had suggested that a substance in the blood called A-SAA, which is raised by inflammation, might be a “missing link” between inflammation and cardiovascular disease, since an individual's baseline level of A-SAA is associated with the risk for cardiovascular disease (in other words, the higher the A-SAA, the higher the risk of cardiovascular disease). In the new study, researchers wanted to know whether the reason that obese people have a higher risk of cardiovascular disease is because they have higher blood levels of A-SAA. What Did the Researchers Do and Find? They found that obese people had higher levels of A-SAA in their blood. A-SAA appears to be produced in fat cells (or adipocytes) and then released into the blood. Obese people have higher numbers of fat cells, which could by itself account for the higher blood levels of A-SAA, but the researchers also found that the average fat cell from an obese individual produces and secretes higher levels of A-SAA than fat cells from lean individuals. When the researchers studied people who underwent weight loss, they found that A-SAA levels fell in response to weight loss, and this was associated with improvements in their metabolism. They then studied obese individuals who received the diabetes drug rosiglitazone (which is known to reduce inflammation). They found that even though these individuals did not lose weight, their A-SAA levels dropped as their metabolism improved. Trying to get at the mechanisms by which A-SAA might cause inflammation and diabetes, the researchers found that exposure to A-SAA can stimulate the activation of proinflammation molecules in a number of different cells, including blood vessel cells. It can also stimulate cells to break down fat stores and release fats, which could lead to metabolic complications and ultimately contribute to diabetes. What Do These Findings Mean? Together with similar results from other studies, the findings here suggest that A-SAA could promote inflammation, and that elevated levels of A-SAA in obese individuals could contribute to the chronic low-level inflammatory state that puts them at higher risk for cardiovascular complications. The authors speculate that drugs that reduce the blood levels of A-SAA might be useful as treatments for obese patients (to lower their risk of heart attacks and strokes). However, as they acknowledge, additional studies are needed to establish that A-SAA is indeed a causal link between obesity and inflammation and whether it plays a major role before it could be considered a promising drug target. Additional Information. Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030287. • MedlinePlus pages on obesity and cardiovascular disease • US Centers for Disease Control and Prevention pages on obesity and cardiovascular disease • Wikipedia pages on obesity and cardiovascular disease (note: Wikipedia is a free Internet encyclopedia that anyone can edit), Higher levels of Acute-phase serum amyloid A (A-SAA), a proinflammatory adipokine, in obese individuals may contribute to the chronic low-level inflammatory state that puts them at higher risk for cardiovascular complications.

Central (visceral) obesity is more closely associated with insulin resistance, type 2 diabetes, and cardiovascular disease than is peripheral [subcutaneous (sc)] obesity, but the underlying mechanism for this pathophysiological difference is largely unknown. To understand the molecular basis of this difference, we sequenced 10,437 expressed sequence tags (ESTs) from a human omental fat cDNA library and discovered a novel visceral fat depot-specific secretory protein, which we have named omentin. Omentin ESTs were more abundant than many known adipose genes, such as perilipin, adiponectin, and leptin in the cDNA library. Protein sequence analysis indicated that omentin mRNA encodes a peptide of 313 amino acids, containing a secretory signal sequence and a fibrinogen-related domain. Northern analysis demonstrated that omentin mRNA was predominantly expressed in visceral adipose tissue and was barely detectable in sc fat depots in humans and rhesus monkeys. Quantative real-time PCR showed that omentin mRNA was expressed in stromal vascular cells, but not fat cells, isolated from omental adipose tissue, with >150-fold less in sc cell fractions. Accordingly, omentin protein was secreted into the culture medium of omental, but not sc, fat explants. Omentin was detectable in human serum by Western blot analysis. Addition of recombinant omentin in vitro did not affect basal but enhanced insulin-stimulated glucose uptake in both sc (47%, n = 9, P = 0.003) and omental (∼30%, n = 3, P < 0.05) human adipocytes. Omentin increased Akt phosphorylation in the absence and presence of insulin. In conclusion, omentin is a new adipokine that is expressed in omental adipose tissue in humans and may regulate insulin action.

TNF increases plasma leptin in humans in vivo, but previous studies showed it decreases leptin in vitro.The objective of this study was to determine the effect of TNF on leptin release from human adipose tissue (AT) from healthy subjects undergoing elective surgery or needle aspirations of AT at a university hospital.Human omental and abdominal sc AT fragments from non- obese and obese subjects were placed in organ culture without or with TNF added in the presence or absence of insulin and/or dexamethasone (dex; a synthetic glucocorticoid) for up to 2 d.In the absence of hormones, culture with TNF decreased leptin release. In contrast, when added in the presence of dex, TNF increased secreted leptin and leptin mRNA abundance in AT from nonobese and obese subjects. The TNF + dex-stimulated increase in leptin was associated with an increase in p38 MAPK activity and was totally blocked by p38 MAPK inhibitors. In contrast, inhibition of p38 MAPK only partially blocked the effect of TNF on IL-6 production. Culture of obese AT with either p38 or p44/42 MAPK inhibitors also blunted the spontaneous increase in media leptin that occurred from d 1-2 of culture in omental AT of obese subjects.Synergistic effects of increased local or systemic TNF in combination with glucocorticoids may contribute to increased leptin expression in response to stress, including infection and obesity.

Objective: Perilipins are phosphoproteins that are localized to the surface of triacylglycerol droplets within adipocytes where they regulate the rate of lipolysis. We sought to determine the effects of severe obesity and depot [omental (Om) vs. subcutaneous (Sc)] on perilipin expression in the adipose tissue of individuals. Research Methods and Procedures: Samples of Om and Sc adipose tissues obtained at surgery from severely obese subjects and fat aspirations from nonobese subjects were analyzed for perilipin protein and mRNA levels by Northern and Western analysis. Results: Perilipin A (periA) was the major perilipin expressed in adipose tissues. periA mRNA relative abundance was significantly lower in Sc adipose tissue from severely obese compared to that from nonobese subjects. Western blotting of adipose tissue extracts showed that periA protein levels expressed relative to tissue protein or fat cell surface area were significantly lower (∼ −40%) in abdominal Sc adipose tissue from severely obese compared to that from nonobese subjects. However, the calculated mass of perilipin per fat cell did not differ between the two groups. Perilipin mRNA levels were higher in Sc compared to Om adipose tissue from obese individuals (p < 0.025; n = 26; 17 women, 9 men); however, periA protein levels did not differ. In addition, perilipin protein, but not mRNA, levels were higher in Sc adipose tissue from obese men than from women (p < 0.025). Discussion: Variations in perilipin expression may contribute to the higher basal lipolytic rates observed in obese compared to nonobese individuals and in obese women compared to obese men.