Adipose Hypothermia in Obesity and Its Association with Period Homolog 1, Insulin Sensitivity,and Inflammation in Fat

Visceral fat adiposity plays an important role in the development of metabolic syndrome. We reported previously the impact of human visceral fat adiposity on gene expression profile of peripheral blood cells. Genes related to circadian rhythm were highly associated with visceral fat area and period homolog 1 (PER1) showed the most significant negative correlation with visceral fat area. However, regulation of adipose Per1 remains poorly understood. The present study was designed to understand the regulation of Per1 in adipose tissues. Adipose Per1 mRNA levels of ob/ob mice were markedly low at 25 and 35 weeks of age. The levels of other core clock genes of white adipose tissues were also low in ob/ob mice at 25 and 35 weeks of age. Per1 mRNA was mainly expressed in the mature adipocyte fraction (MAF) and it was significantly low in MAF of ob/ob mice. To examine the possible mechanisms, 3T3-L1 adipocytes were treated with H₂O₂, tumor necrosis factor-α (TNF-α), S100A8, and lipopolysaccharide (LPS). However, no significant changes in Per1 mRNA level were observed by these agents. Exposure of cultured 3T3-L1 adipocytes to low temperature (33°C) decreased Per1 and catalase, and increased monocyte chemoattractant protein-1 (Mcp-1) mRNA levels. Hypothermia also worsened insulin-mediated Akt phosphorylation in 3T3-L1 adipocytes. Finally, telemetric analysis showed low temperature of adipose tissues in ob/ob mice. In obesity, adipose hypothermia seems to accelerate adipocyte dysfunction.     

Expression of aggrecan(ases) during murine preadipocyte differentiation and adipose tissue development

The expression and potential functional role of aggrecan in adipogenesis and adipose tissue development was investigated in murine models of obesity. Aggrecan, as well as the two aggrecanases ADAMTS-4 and ADAMTS-5 (A Disintegrin And Metalloproteinase with Thrombospondin motif) mRNAs, are expressed in subcutaneous (SC) and gonadal (GON) adipose tissues of mice. Their presence was confirmed by western blotting using adipose tissue extracts. In mice with nutritionally induced obesity (high fat diet) as well as in lean controls, aggrecan mRNA expression was downregulated whereas ADAMTS-4 and ADAMTS-5 were upregulated with time. In mice with genetically determined obesity (ob/ob), ADAMTS-5 mRNA was upregulated in both SC and GON adipose tissues, as compared to wild-type (WT) mice (p<0.001). Enhanced aggrecanase expression levels in these tissues were associated with significantly elevated levels of G1-NITEGE, a degradation product of aggrecan. Thus, aggrecan levels were high at the early stages of adipose tissue development in mice, whereas its production decreased and its degradation increased during development of obesity. A functional role of aggrecan in promoting early stages of adipogenesis is supported by the findings that it stimulated the in vitro differentiation of 3T3-F442A preadipocytes and the de novo in vivo accumulation of fat in Matrigel plaques injected into WT mice. Proteoglycans in the extracellular matrix of adipose tissue, such as aggrecan, may contribute to the regulation of lipid uptake and obesity in mice.     

Upregulation of uncoupling protein 2 mRNA in genetic obesity: lack of an essential role for leptin, hyperphagia, increased tissue lipid content, and TNF-alpha

Uncoupling protein 2 (UCP2) has been proposed to play a prominent role in the regulation of energy balance. UCP2 mRNA expression is upregulated in white adipose tissue (WAT) and liver, but is not altered in skeletal muscle in genetically obese ob/ob mice. The mechanisms involved in the upregulation of UCP2 in obesity have not been investigated. We have now examined the potential role of leptin, hyperphagia, increased tissue lipid content, and overexpression of tumor necrosis factor (TNF)-alpha in the upregulation of UCP2 mRNA expression in the liver and WAT in ob/ob mice. Treatment of ob/ob mice with leptin for 3 days significantly reduced their food intake but had no effect on the upregulation of UCP2 mRNA levels in the liver or WAT. To investigate the effect of feeding and higher tissue lipid content on the upregulation of UCP2 in liver and WAT, we compared UCP2 mRNA levels in ad-libitum fed and 72-h fasted control and ob/ob mice. In controls, fasting had no effect on UCP2 mRNA levels in liver, but increased UCP2 mRNA in WAT suggesting that the effects of fasting on UCP2 mRNA levels are tissue-specific. In ob/ob mice, fasting did not lower UCP2 mRNA levels in liver or WAT suggesting that the upregulation of UCP2 in ob/ob mice is not merely a direct consequence of increased food intake. 72-h fasting lowered hepatic total lipid content by 34% and 36% in control and ob/ob mice, respectively, without any corresponding decrease in hepatic UCP2 mRNA levels, suggesting that the enhanced UCP2 expression in the liver of ob/ob mice is not secondary to lipid accumulation in their livers. Although TNF-alpha has been shown to acutely increase UCP2 mRNA levels in liver and WAT, and is overexpressed in adipose tissue in obesity, deletion of the genes for both TNF receptors in ob/ob mice produces a further increase in UCP2 mRNA expression in liver and adipose tissue indicating a paradoxical inhibitory role. Taken together, these results suggest that the upregulation of UCP2 mRNA levels in the liver and WAT of ob/ob mice is not due to the lack of leptin, hyperphagia, increased tissue lipid content, or over-expression of TNF-alpha.     

Obesity-associated mouse adipose stem cell secretion of monocyte chemotactic protein-1

Studies showed that monocyte chemotactic protein-1 (MCP-1) concentrations are increased in obesity. In our current study, we demonstrate that plasma MCP-1 level in leptin-deficient ob/ob mice is significantly higher than in lean mice. Furthermore, we determined that basal adipose tissue MCP-1 mRNA levels are significantly higher in ob/ob mice compared with lean mice. To determine the mechanisms underlying obesity-associated increases in plasma and adipose tissue MCP-1 levels, we determined adipose tissue cell type sources of MCP-1 production. Our data show that adipose tissue stem cells (CD34(+)), macrophages (F4/80(+)), and stromal vascular fraction (SVF) cells express significantly higher levels of MCP-1 compared with adipocytes under both basal and lipopolysaccharide (LPS)-stimulated conditions. Furthermore, basal and LPS-induced MCP-1 secretion levels were the same for both adipose F4/80(+) and CD34(+) cells, whereas adipose CD34(+) cells have twofold higher cell numbers (30% of total SVF cells) compared with F4/80(+) macrophages (15%). Our data also show that CD34(+) cells from visceral adipose tissue depots secrete significantly higher levels of MCP-1 ex vivo when compared with CD34(+) cells from subcutaneous adipose tissue depots. Taken together, our data suggest that adipose CD34(+) stem cells may play an important role in obesity-associated increases in plasma MCP-1 levels.     

Interleukin-15 stimulates adiponectin secretion by 3T3-L1 adipocytes: evidence for a skeletal muscle-to-fat signaling pathway

Interleukin-15 (IL-15) is a cytokine which is highly expressed in skeletal muscle tissue, and which has anabolic effects on skeletal muscle protein dynamics both in vivo and in vitro. Additionally, administration of IL-15 to rats and mice inhibits white adipose tissue deposition. To determine if the action of IL-15 on adipose tissue is direct, the capacity of cultured murine 3T3-L1 preadipocytes and adipocytes to respond to IL-15 was examined. IL-15 administration inhibited lipid accumulation in differentiating 3T3-L1 preadipocytes, and stimulated secretion of the adipocyte-specific hormone adiponectin by differentiated 3T3-L1 adipocytes. The latter observation constitutes the first report of a cytokine or growth factor which stimulates adiponectin production. IL-15 mRNA expression by cultured 3T3-L1 adipogenic cells and C2C12 murine skeletal myogenic cells was also examined. Quantitative real-time PCR indicated IL-15 mRNA was expressed by C2C12 skeletal myogenic cells, and was upregulated more than 10-fold in differentiated skeletal myotubes compared to undifferentiated myoblasts. In contrast, 3T3-L1 cells expressed little or no IL-15 mRNA at either the undifferentiated preadipocyte or differentiated adipocyte stages. These findings provide support for the hypothesis that IL-15 functions in a muscle-to-fat endocrine axis which modulates fat:lean body composition and insulin sensitivity.     

Nucleoredoxin promotes adipogenic differentiation through regulation of Wnt/β-catenin signaling

Nucleoredoxin (NRX) is a member of the thioredoxin family of proteins that controls redox homeostasis in cell. Redox homeostasis is a well-known regulator of cell differentiation into various tissue types. We found that NRX expression levels were higher in white adipose tissue of obese ob/ob mice and increased in the early adipogenic stage of 3T3-L1 preadipocyte differentiation. Knockdown of NRX decreased differentiation of 3T3-L1 cells, whereas overexpression increased differentiation. Adipose tissue-specific NRX transgenic mice showed increases in adipocyte size as well as number compared with WT mice. We further confirmed that the Wingless/int-1 class (Wnt)/β-catenin pathway was also involved in NRX-promoted adipogenesis, consistent with a previous report showing NRX regulation of this pathway. Genes involved in lipid metabolism were downregulated, whereas inflammatory genes, including those encoding macrophage markers, were significantly upregulated, likely contributing to the obesity in Adipo-NRX mice. Our results therefore suggest that NRX acts as a novel proadipogenic factor and controls obesity in vivo.     

SOCS-3 inhibits insulin signaling and is up-regulated in response to tumor necrosis factor-alpha in the adipose tissue of obese mice

SOCS (suppressor of cytokine signaling) proteins are inhibitors of cytokine signaling involved in negative feedback loops. We have recently shown that insulin increases SOCS-3 mRNA expression in 3T3-L1 adipocytes. When expressed, SOCS-3 binds to phosphorylated Tyr(960) of the insulin receptor and prevents Stat 5B activation by insulin. Here we show that in COS-7 cells SOCS-3 decreases insulin-induced insulin receptor substrate 1 (IRS-1) tyrosine phosphorylation and its association with p85, a regulatory subunit of phosphatidylinositol-3 kinase. This mechanism points to a function of SOCS-3 in insulin resistance. Interestingly, SOCS-3 expression was found to be increased in the adipose tissue of obese mice, but not in the liver and muscle of these animals. Two polypeptides known to be elevated during obesity, insulin and tumor necrosis factor-alpha (TNF-alpha), induce SOCS-3 mRNA expression in mice. Insulin induces a transient expression of SOCS-3 in the liver, muscle, and the white adipose tissue (WAT). Strikingly, TNF-alpha induced a sustained SOCS-3 expression, essentially in the WAT. Moreover, transgenic ob/ob mice lacking both TNF receptors have a pronounced decrease in SOCS-3 expression in the WAT compared with ob/ob mice, providing genetic evidence for a function of this cytokine in obesity-induced SOCS-3 expression. As SOCS-3 appears as a TNF-alpha target gene that is elevated during obesity, and as SOCS-3 antagonizes insulin-induced IRS-1 tyrosine phosphorylation, we suggest that it is a player in the development of insulin resistance.     

High circulating levels of S100A8/A9 complex (calprotectin) in male Japanese with abdominal adiposity and dysregulated expression of S100A8 and S100A9 in adipose tissues of obese mice

S100A8/A9 complex, calprotectin, which serves as an endogenous ligand for immune pathways, is associated with atherosclerosis. These proteins are reported to have several functions such as activating NADPH oxidase, binding toll-like receptor 4 and associated with the receptor for advanced glycation end-products. We recently reported S100A8 mRNA was highly expressed in mouse white adipose tissues and differentiated 3T3-L1 adipocytes. However, regulation of S100A9 expression in murine adipose tissue remains to be elucidated. The results of our studies in male Japanese, obese and control mice and cultured cells showed: (1) serum levels of S100A8/A9 complex, calprotectin, correlated with visceral fat area, body mass index, subcutaneous fat area, and leukocyte count in 500 Japanese men, and (2) higher mRNA expression levels of S100A8 in mature adipocyte fraction and S100A9 in stromal vascular cell fraction of obese mice, compared with those of lean mice. Overexpression of S100A8 and S100A9 in obese adipose tissue may be involved, at least partly, in not only high circulating levels of S100A8/A9 complex in abdominal obesity but also adipose and systemic tissue inflammation.     

Expression of the adrenomedullin gene in adipose tissue

Adrenomedullin (AM) is a potent vasodilating peptide originally isolated from human pheochromocytoma cells. This report concerns the expression and secretion of AM from adipose tissue. Northern blot analysis demonstrated marked expression of AM mRNA in mouse adipose tissue. Expression levels in adipose tissues were 2.5-3.2 times higher than in the kidney. AM mRNA level in mature adipocytes was 7.3 times higher than in the stroma-vascular fraction of adipose tissue. In mature adipocyte culture, time-dependent increase of AM peptide concentration in the culture medium was detected. AM expression was also detected in human subcutaneous adipose tissue. Adipose AM expression significantly increased in obesity mouse model, high-fat diet fed mice and ob/ob mice. These results suggest that adipose tissue, especially mature adipocytes, is major source of AM in the body, and that adipocyte-derived AM plays a pathophysiological role in obesity.     

Roles of degree of fat deposition and its localization on VEGF expression in adipocytes

Vascular endothelial growth factor (VEGF) is an important angiogenic factor and is expressed in wide variety of cell types. In this study, we investigated the mechanism of VEGF production in adipocytes in three sets of experiments. First, to clarify the relation between plasma VEGF concentrations and their expressions in adipose tissues, we investigated the genetically obese db/db and KK-Ay mice. Plasma VEGF concentrations in obese mice were significantly higher than in control and were related to adiposity. VEGF expressions in visceral fat were enhanced during growth and were related to fat deposition. Next, to demonstrate the relation between VEGF production and lipid accumulation in adipocytes, we analyzed VEGF mRNA expression and its protein secretion in 3T3-L1 cells. VEGF production was enhanced during lipid accumulation in 3T3-L1 cells after adipocyte conversion. Next, to clarify the role of anatomic localization on VEGF expression in adipocytes, we implanted 3T3-L1 cells into visceral or subcutaneous fat in athymic mice. 3T3-L1 cells implanted into the mesenteric area expressed more VEGF mRNA than that into the subcutaneous area. Plasma VEGF concentration in the mice implanted in visceral fat was higher than in controls. These results suggest that both the anatomic localization and the lipid accumulation are important for the VEGF production in adipocytes.     

The role of lipocalin 2 in the regulation of inflammation in adipocytes and macrophages

Adipose tissue-derived cytokines (adipokines) are associated with the development of inflammation and insulin resistance. However, which adipokine(s) mediate this linkage and the mechanisms involved during obesity is poorly understood. Through proteomics and microarray screening, we recently identified lipocalin 2 (LCN 2) as an adipokine that potentially connects obesity and its related adipose inflammation. Herein we show that the levels of LCN2 mRNA are dramatically increased in adipose tissue and liver of ob/ob mice and primary adipose cells isolated from Zucker obese rats, and thiazolidinedione administration reduces LCN2 expression. Interestingly, addition of LCN2 induces mRNA levels of peroxisome proliferator-activated receptor-gamma (PPARgamma) and adiponectin. Reducing LCN2 gene expression causes decreased expression of PPARgamma and adiponectin, slightly reducing insulin-stimulated Akt2 phosphorylation at Serine 473 in 3T3-L1 adipocytes. LCN2 administration to 3T3-L1 cells attenuated TNFalpha-effect on glucose uptake, expression of PPARgamma, insulin receptor substrate-1, and glucose transporter 4, and secretion of adiponectin and leptin. When added to macrophages, LCN2 suppressed lipopolysaccharide-induced cytokine production. Our data suggest that LCN2, as a novel autocrine and paracrine adipokine, acts as an antagonist to the effect of inflammatory molecules on inflammation and secretion of adipokines.     

Tissue leptin and plasma insulin are associated with lipoprotein lipase activity in severely obese patients

The development of metabolic complications of obesity has been associated with the existence of depot-specific differences in the biochemical properties of adipocytes. The aim of this study was to investigate, in severely obese men and women, both gender- and depot-related differences in lipoprotein lipase (LPL) expression and activity, as well as the involvement of endocrine and biometric factors and their dependence on gender and/or fat depot. Morbidly obese, nondiabetic, subjects (9 men and 22 women) aged 41.1+/-1.9 years, with a body mass index (BMI) of 54.7+/-1.7 kg/m(2) who had undergone abdominal surgery were studied. Both expression and activity of LPL and leptin expression were determined in adipose samples from subcutaneous and visceral fat depots. In both men and women, visceral fat showed higher LPL mRNA levels as well as lower ob mRNA levels and tissue leptin content than the subcutaneous one. In both subcutaneous and visceral adipose depots, women exhibited higher protein content, decreased fat cell size and lower LPL activity than men. The gender-related differences found in abdominal fat LPL activity could contribute to the increased risk for developing obesity-associated diseases shown by men, even in morbid obesity, in which the massive fat accumulation could mask these differences. Furthermore, the leptin content of fat depots as well as plasma insulin concentrations appear in our population as the main determinants of adipose tissue LPL activity, adjusted by gender, depot and BMI.