Regulation of carboxylesterase 1 (CES1) in human adipose tissue

Carboxylesterase 1 (CES1) has recently been suggested to play a role in lipolysis. Our aim was to study the regulation of CES1 expression in human adipose tissue. In the SOS Sib Pair Study, CES1 expression was higher in obese compared with lean sisters (n = 78 pairs, P = 8.7 × 10⁻¹⁸) and brothers (n = 12 pairs, P = 0.048). CES1 expression was higher in subcutaneous compared with omental adipose tissue in lean (P = 0.027) and obese subjects (P = 0.00036), and reduced during diet-induced weight loss (n = 24, weeks 8, 16, and 18 compared to baseline, P < 0.0001 for all time points). CES1 expression was higher in isolated adipocytes compared with intact adipose tissue (P = 0.0018) and higher in large compared with small adipocytes (P = 4.1 × 10⁻⁶). Basal and stimulated lipolysis was not different in individuals with high, intermediate, and low expression of CES1. Thus, CES1 expression was linked to body fat and adipocyte fat content but not to lipolytic activity.     

Resistin release by human adipose tissue explants in primary culture

Resistin, also known as Fizz3 or ADSF, is a protein found in murine adipose tissue and inflammatory lung exudates. The present studies found that resistin was released by explants of human adipose tissue but the release was quite variable ranging from 3 to 158 ng/g over 48 h. The release of resistin was 250% greater by explants of omental than by explants of human subcutaneous abdominal adipose tissue. Resistin release by adipocytes was negligible as compared to that by the non-fat cells of adipose tissue. Leptin formation by adipocytes was 8-fold greater than its formation by the non-fat cells, while the formation of PAI-1 by adipocytes was 38% of that by the non-fat cells. The conversion of glucose to lactate as well as the formation of PGE(2) and IL-8 was approximately 15% of that by the non-fat cells. In contrast the release of IL-6 and IL-1beta by adipocytes was 4-7% of that by the non-fat cells while the formation of resistin and IL-10 by adipocytes was 2% of that by non-fat cells. The release of adiponectin by explants ranged from 1000 to 5000 ng/g over 48 h but did not correlate with that of resistin. The present data suggest that resistin release by explants of human adipose tissue in primary culture is largely derived from the non-fat cells present in the explants.     

Active spice-derived components can inhibit inflammatory responses of adipose tissue in obesity by suppressing inflammatory actions of macrophages and release of monocyte chemoattractant protein-1 from adipocytes

Inflammation plays a key role in obesity-related pathologies such as cardiovascular disease, type II diabetes, and several types of cancer. Obesity-induced inflammation entails the enhancement of the recruitment of macrophages into adipose tissue and the release of various proinflammatory proteins from fat tissue. Therefore, the modulation of inflammatory responses in obesity may be useful for preventing or ameliorating obesity-related pathologies. Some spice-derived components, which are naturally occurring phytochemicals, elicit antiobesity and antiinflammatory properties. In this study, we investigated whether active spice-derived components can be applied to the suppression of obesity-induced inflammatory responses. Mesenteric adipose tissue was isolated from obese mice fed a high-fat diet and cultured to prepare an adipose tissue-conditioned medium. Raw 264.7 macrophages were treated with the adipose tissue-conditioned medium with or without active spice-derived components (i.e., diallyl disulfide, allyl isothiocyanate, piperine, zingerone and curcumin). Chemotaxis assay was performed to measure the degree of macrophage migration. Macrophage activation was estimated by measuring tumor necrosis factor-alpha (TNF-alpha), nitric oxide, and monocyte chemoattractant protein-1 (MCP-1) concentrations. The active spice-derived components markedly suppressed the migration of macrophages induced by the mesenteric adipose tissue-conditioned medium in a dose-dependent manner. Among the active spice-derived components studied, allyl isothiocyanate, zingerone, and curcumin significantly inhibited the cellular production of proinflammatory mediators such as TNF-alpha and nitric oxide, and significantly inhibited the release of MCP-1 from 3T3-L1 adipocytes. Our findings suggest that the spice-derived components can suppress obesity-induced inflammatory responses by suppressing adipose tissue macrophage accumulation or activation and inhibiting MCP-1 release from adipocytes. These spice-derived components may have a potential to improve chronic inflammatory conditions in obesity.     

Enhanced muscle by myostatin propeptide increases adipose tissue adiponectin, PPAR-alpha, and PPAR-gamma expressions

Muscle tissue utilizes a large portion of metabolic energy for its growth and maintenance. Previously, we demonstrated that transgenic over-expression of myostatin propeptide in mice fed a high-fat diet enhanced muscle mass and circulating adiponectin while the wild-type mice developed obesity and insulin resistance. To understand the effects of enhanced muscle growth on adipose tissue metabolism, we analyzed adiponectin, PPAR-α, and PPAR-γ mRNA expressions in several fat tissues. Results indicated muscled transgenic mice fed a high-fat diet displayed increased epididymal adiponectin mRNA expression by 12 times over wild-type littermates. These transgenic mice fed either a high or normal fat diet also displayed significantly high levels of PPAR-α and PPAR-γ expressions above their wild-type littermates in epididymal fat while their expressions in mesenteric fats were not significantly different between transgenic mice and their littermates. This study demonstrates that enhanced muscle growth has positive effects on fat metabolisms through increasing adiponectin expression and its regulations.     

A PPAR agonist improves TNF-alpha-induced insulin resistance of adipose tissue in mice

Thiazolidinediones (TZDs), agonists for PPARs, have been shown to block the inhibitory effects of TNF-alpha on insulin action using cultured cells. In order to clarify the in vivo effects of TZDs on the inhibition of insulin sensitivity by TNF-alpha, insulin action in muscles and adipose tissues was assessed in the TNF-alpha-overexpression mice model using transplantation of cells secreting the TNF-alpha protein. After the pioglitazone treatment for 4 weeks, glucose uptake, insulin-induced IRS-1 phosphorylation, and lipoprotein lipase mRNA levels were analyzed. Pioglitazone did not ameliorate TNF-alpha-induced hyperinsulinemia in this model, as assessed by the OGTT. Glucose uptake and lipoprotein lipase mRNA levels were decreased by TNF-alpha in adipose tissues from the TNF-alpha-overexpressing mice, and pioglitazone blocked these inhibitions by TNF-alpha. On the other hand, in muscles, pioglitazone did not reverse the effects of TNF-alpha on insulin-induced phosphorylation of IRS-1, glucose uptake, and lipoprotein lipase mRNA levels. Present study revealed the different sensitivities of pioglitazone for the recovery of decreased insulin action in a TNF-alpha-overexpressing model using cell transplantation. These results suggest that the effect of TZDs is dependent on the fat distribution and accumulation in humans.     

Nuclear factor 1 regulates adipose tissue-specific expression in the mouse GLUT4 gene

Previous studies demonstrated that an adipose tissue-specific element(s) (ASE) of the murine GLUT4 gene is located between −551 and −506 in the 5′-flanking sequence and that a high-fat responsive element(s) for down-regulation of the GLUT4 gene is located between bases −701 and −552. A binding site for nuclear factor 1 (NF1), that mediates insulin and cAMP-induced repression of GLUT4 in 3T3-L1 adipocytes is located between bases −700 and −688. To examine the role of NF1 in the regulation of GLUT4 gene expression in white adipose tissues (WAT) in vivo, we created two types of transgenic mice harboring mutated either 5′ or 3′ half-site of NF1-binding sites in GLUT4 minigene constructs. In both cases, the GLUT4 minigene was not expressed in WAT, while expression was maintained in brown adipose tissue, skeletal muscle, and heart. This was an unexpected finding, since a −551 GLUT4 minigene that did not have the NF1-binding site was expressed in WAT. We propose a model that explains the requirement for both the ASE and the NF1-binding site for expression of GLUT4 in WAT.     

Acute and chronic saturated fatty acid treatment as a key instigator of the TLR-mediated inflammatory response in human adipose tissue, in vitro

A post-prandial increase in saturated fatty acids (SFAs) and glucose (Glc) activates an inflammatory response, which may be prolonged following restoration of physiological SFAs and Glc levels — a finding referred to as ‘metabolic memory'.This study examined chronic and oscillating SFAs and Glc on the inflammatory signalling pathway in human adipose tissue (AT) and adipocytes (Ads) and determined whether Ads are subject to “metabolic memory.”Abdominal (Abd) subcutaneous (Sc) explants and Ads were treated with chronic low glucose (L-Glc): 5.6 mM and high glucose (H-Glc): 17.5 mM, with low (0.2 mM) and high (2 mM) SFA for 48 h. Abd Sc explants and Ads were also exposed to the aforementioned treatment regimen for 12-h periods, with alternating rest periods of 12 h in L-Glc.Chronic treatment with L-Glc and high SFAs, H-Glc and high SFAs up-regulated key factors of the nuclear factor-κB (NFκB) pathway in Abd Sc AT and Ads (TLR4, NFκB; P<.05), whilst down-regulating MyD88. Oscillating Glc and SFA concentrations increased TLR4, NFκB, IKKβ (P<.05) in explants and Ads and up-regulated MyD88 expression (P<.05). Both tumor necrosis factor α and interleukin 6 (P<.05) secretion were markedly increased in chronically treated Abd Sc explants and Ads whilst, with oscillating treatments, a sustained inflammatory effect was noted in absence of treatment.Therefore, SFAs may act as key instigators of the inflammatory response in human AT via NFκB activation, which suggests that short-term exposure of cells to uncontrolled levels of SFAs and Glc leads to a longer-term inflammatory insult within the Ad, which may have important implications for patients with obesity and Type 2 diabetes.     

URB is abundantly expressed in adipose tissue and dysregulated in obesity

Dysregulated production of adipocytokines in obesity is involved in the development of metabolic syndrome. URB/DRO1 contains N-terminal signal sequence and is thought to play a role in apoptosis of tumor cells. In the present study, we investigated the expression pattern of URB mRNA in adipose tissue and secretion from cultured adipocytes. In human and mouse, URB mRNA was predominantly expressed in adipose tissue and was downregulated in obese mouse models, such as ob/ob, KKAy, and diet-induced obese mice. In 3T3L1 adipocytes, insulin, TNF-α, H₂O₂ and hypoxia decreased URB mRNA level. This regulation was similar to that for adiponectin and opposite to MCP-1. URB protein was secreted in media of URB cDNA-stably transfected cells and endogenous URB was detected in media of cultured human adipocytes. In conclusion, the expression pattern of URB suggests its role in obesity and the results suggest that URB is secreted, at least in part, from adipocytes.     

Adipose-specific deletion of stearoyl-CoA desaturase 1 up-regulates the glucose transporter GLUT1 in adipose tissue

Stearoyl-CoA desaturase 1 (SCD1) deficiency protects mice from diet-induced obesity and insulin resistance. To understand the tissue-specific role of SCD1 in energy homeostasis, we have generated mice with an adipose-specific knockout of Scd1 (AKO), and report here that SCD1 deficiency increases GLUT1 expression in adipose tissue of AKO mice, but not global SCD1 knockout (GKO) mice. In 3T3-L1 adipocytes treated with an SCD inhibitor, basal glucose uptake and the cellular expression of GLUT1 were significantly increased while GLUT4 expression remained unchanged. Consistently, adipose-specific SCD1 knockout (AKO) mice had significantly elevated GLUT1 expression, but not GLUT4, in white adipose tissue compared to Lox counterparts. Concurrently, adiponectin expression was significantly diminished, whereas TNF-α expression was elevated. In contrast, in adipose tissue of GKO mice, GLUT4 and adiponectin expression were significantly elevated with lowered TNF-α expression and little change in GLUT1 expression, suggesting a differential responsiveness of adipose tissue to global- or adipose-specific SCD1 deletion. Taken together, these results indicate that adipose-specific deletion of SCD1 induces GLUT1 up-regulation in adipose tissue, associated with decreased adiponectin and increased TNF-α production, and suggest that GLUT1 may play a critical role in controlling glucose homeostasis of adipose tissue in adipose-specific SCD1-deficient conditions.     

Profiling of adipokines secreted from human subcutaneous adipose tissue in response to PPAR agonists

The role of PPARs in the regulation of human adipose tissue secretome has received little attention despite its potential importance in the therapeutic actions of PPAR agonists. Here, we have investigated the effect of selective PPARgamma, PPARalpha, and PPARbeta/delta agonists on the production of adipokines by human subcutaneous adipose tissue. Antibody arrays were used to measure secreted factors in media from cultured adipose tissue explants. Sixteen proteins were produced in significant amounts. Activation of PPARs regulated the production of five proteins. Treatments with the three PPAR agonists decreased the secretion of leptin and interleukin-6. PPARalpha and beta/delta agonists markedly enhanced hepatocyte growth factor secretion whereas PPARbeta/delta down-regulated angiogenin and up-regulated TIMP-1 release. Hepatocyte growth factor, interleukin-6, and TIMP-1 are chiefly expressed in cells from the stromal vascular fraction whereas angiogenin is expressed in both adipocytes and cells from the stromal vascular fraction. Our data show that PPAR agonists modulate secretion of bioactive molecules from the different cell types composing human adipose tissue.     

Effect of metformin on adipose tissue resistin expression in db/db mice

Resistin, a novel adipose-derived protein, has been proposed to cause insulin-resistant states in obesity. To evaluate whether an insulin-sensitizing drug, metformin, regulates adipose tissue resistin expression, murine models of obesity and diabetes, db/db mice, were treated with metformin (metformin group), insulin (insulin group), and vehicle (control group) for 4 weeks, followed by analyzing resistin protein expression in their adipose tissues. Unexpectedly, resistin protein expression was increased by 66% in the metformin group relative to the control group, while it did not differ between the insulin and control groups. Hyperinsulinemia was improved in the metformin group, while the insulin group exhibited severe hyperinsulinemia, similar to the control group. Furthermore, in comparison between obese mice (db/db mice) and age-matched lean controls, resistin protein expression was reduced by 58% in the obese mice with severe hyperinsulinemia. These data collectively suggest that resistin expression may be suppressed by hyperinsulinemia and that metformin may upregulate resistin expression via the improvement of hyperinsulinemia in obesity.     

AICAR stimulates adiponectin and inhibits cytokines in adipose tissue

5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) can be used as an experimental tool to activate 5'-AMP-activated protein kinase (AMPK) and has been shown to improve insulin sensitivity. In parallel adiponectin also seems to activate AMPK and to improve insulin sensitivity. We have investigated the effects of AICAR on the gene expression of adiponectin and on gene expression and release of cytokines in human adipose tissue in vitro. AICAR stimulated AMPK alpha1 activity 3-4-fold (p<0.001), and dose-dependently increased adiponectin mRNA levels with significant stimulation (2-4-fold) at AICAR concentrations of 0.5-2mM (p<0.05). The adipose tissue protein release of tumor necrosis factor-alpha (TNF- alpha) and interleukin-6 (IL-6) was decreased by AICAR (p<0.05). In conclusion, AICAR stimulated adipose tissue AMPK alpha1 activity and adiponectin gene expression, while attenuating the release of TNF-alpha and IL-6. Reduced concentrations of these cytokines and increased levels of adiponectin might play a role for the insulin sensitizing effects of AICAR.     

IRS-1 transgenic mice show increased epididymal fat mass and insulin resistance

Insulin receptor substrate-1 (IRS-1) is the major substrate of both the insulin receptor and the IGF-1 receptor. In this study, we created IRS-1 transgenic (IRS-1-Tg) mice which express human IRS-1 cDNA under control of the mouse IRS-1 gene promoter. In the IRS-1-Tg mice, IRS-1 mRNA expression was significantly increased in almost all tissues, but its protein expression was increased in very limited tissues (epididymal fat and skeletal muscle). IRS-1-Tg mice showed glucose intolerance and significantly enlarged epididymal fat mass, as well as elevated serum TNF-α concentrations. Importantly insulin signaling was significantly attenuated in the liver of IRS-1-Tg mice, which may contribute to the glucose intolerance. Our results suggest that excess IRS-1 expression may not provide a beneficial impact on glucose homeostasis in vivo.     

The pro-inflammatory cytokine IL-18 is expressed in human adipose tissue and strongly upregulated by TNFalpha in human adipocytes

White adipocytes have been examined as a potential source of interleukin-18 (IL-18), the circulating levels of which are increased in obesity. IL-18 gene expression was evident in human subcutaneous and visceral adipose tissue, and expression occurred in mature adipocytes and the stromal-vascular fraction. Expression of the IL-18 receptor complex (IL-18Ralpha and IL-18Rbeta) and the IL-18 binding protein (IL-18BP) genes was also observed, mirroring that of IL-18. IL-18 mRNA level increased rapidly (within 2h) and dramatically (>900-fold) in response to TNFalpha in human adipocytes differentiated in culture. IL-18 protein was detected in lysates of cultured adipocytes, though not in the medium. There was a small increase in IL-18 in lysates of adipocytes treated with TNFalpha, but the protein was again undetectable in the medium. IL-18 may be part of the inflammatory cascade within adipose tissue; however, human adipocytes do not appear to secrete significant amounts of IL-18.     

ALK7 expression is specific for adipose tissue, reduced in obesity and correlates to factors implicated in metabolic disease

Human adipose tissue is a major site of expression of inhibin beta B (INHBB) which homodimerizes to form the novel adipokine activin B. Our aim was to determine if molecules needed for a local action of activin B are expressed in adipose tissue.Microarray analysis showed that adipose tissue expressed activin type I and II receptors and that the expression of activin receptor-like kinase 7 (ALK7) was adipose tissue specific. In obesity discordant siblings from the SOS Sib Pair study, adipose tissue ALK7 expression was higher in lean (n = 90) compared to obese (n = 90) subjects (p = 4 × 10⁻³¹). Adipose tissue ALK7 expression correlated with several measures of body fat, carbohydrate metabolism and lipids. In addition, ALK7 and INHBB expression correlated but only in lean subjects and in subjects with normal glucose tolerance.We conclude that activin B may have local effects in adipose tissue and thereby influence obesity and its comorbidities.     

Attenuated metabolism is a hallmark of obesity as revealed by comparative proteomic analysis of human omental adipose tissue

Obesity is recognized as an epidemic health problem worldwide. In humans, the accumulation of omental rather than subcutaneous fat appears to be tightly linked to insulin resistance, type 2 diabetes and cardiovascular disease. Differences in gene expression profiles in the adipose tissue comparing non-obese and obese subjects have been well documented. However, to date, no comparative proteomic studies based on omental fat have investigated the influence of obesity in protein expression. In this work, we searched for proteins differentially expressed in the omental fat of non-obese and obese subjects using 2D-DIGE and MS. Forty-four proteins, several of which were further studied by immunoblotting and immunostaining analyses, showed significant differences in the expression levels in the two groups of subjects. Our findings reveal a clearly distinctive proteomic profile between obese and non-obese subjects which emphasizes: i) reduced metabolic activity in the obese fat, since most down-regulated proteins were engaged in metabolic pathways; and ii) morphological and structural cell changes in the obese fat, as revealed by the functions exerted by most up-regulated proteins. Interestingly, transketolase and aminoacylase-1 represent newly described molecules involved in the pathophysiology of obesity, thus opening up new possibilities in the study of obesity.     

Weak Functional Coupling of the Melanocortin-1 Receptor Expressed in Human Adipocytes

The melanocortin (MC) receptor type-1 (MC1-R) is the only one of the five MC receptor subtypes expressed in human adipose tissue explants, human mesenchymal stem cells (MSCs), and MSC-derived adipocytes. Following our recent expression studies (Obesity 2007, 15, 40–49), we now investigated the functional role of MC1-R in these tissues and cells to deduce the coupling state of MC1-R to intracellular output signals in human fat cells and tissue. Expression of MC1-R by undifferentiated and differentiated MSCs was quantified by real-time TaqMan PCR. Intracellular output signals (cAMP, lipolysis, secretion of IL-6, IL-10, and TNF-α), as well as effects on the metabolic rate and proliferation of human MSCs were analyzed by standard assays, exposing undifferentiated and differentiated MSCs and, in part, human adipose tissue explants to the potent MC1-R agonist, [Nle⁴, D-Phe⁷]-α -MSH (NDP-MSH). This agonist induced a weak cAMP signal in MSC-derived adipocytes. However, it did not affect lipolysis in these cells or in adipose tissue explants, nor did it modulate cytokine release and mRNA expression of IL-6, IL-8, and TNF-α upon LPS stimulation. In undifferentiated MSCs, NDP-MSH did not alter the metabolic rate, but it showed a significant antiproliferative effect. Therefore, it appears that MC1-R–effector coupling in (differentiated) human adipocytes is too weak to induce a regulatory effect on lipolysis or inflammation; by contrast, MC1-R stimulation in undifferentiated MSCs induces an inhibitory signal on cell proliferation.     

Stimulation of human breast carcinoma cell invasiveness and urokinase plasminogen activator activity by glucose deprivation

Glucose deprivation has been shown to increase the invasive and metastatic potential of tumour cells. In the present study, we determined whether the enhanced tumour cell invasiveness resulting from glucose deprivation is linked to increased activity of enzymes required for extracellular matrix degradation. Results of in vitro invasion assays revealed that the invasiveness of human MDA-MB-231 and MCF-7 breast carcinoma cells and MCF-10A1 normal breast cells was, respectively, 3.9-, 2.9-, and 2.1-fold higher when they were incubated under glucose-deprivation (0.2 mM glucose) than when incubated under physiological blood glucose levels (5 mM). This effect of glucose deprivation on invasion correlated with increased urokinase plasminogen activator (uPA) and plasmin activity. Glucose deprivation did not increase the levels of gelatinase and plasminogen activator inhibitor-1 secretion, or the expression of cell-associated uPA receptor. To determine whether the increased invasiveness resulting from glucose deprivation is causally linked to increased uPA activity, invasion assays were conducted using MDA-MB-231 cells incubated in 0.2 mM or 5 mM glucose in the presence of a neutralising anti-uPA antibody. Results revealed that the anti-uPA antibody significantly inhibited invasion in a dose-dependent manner and to a much greater extent in cells incubated in 0.2 mM glucose than in cells incubated in 5 mM glucose. These results suggest that low glucose levels in malignant cancers increase tumour cell invasiveness by stimulating uPA and plasmin activity.