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Adipose tissue expansion during obesity is associated with increased macrophage infiltration. Macrophage-derived factors significantly alter adipocyte function, inducing inflammatory responses and decreasing insulin sensitivity. Identification of the major factors that mediate detrimental effects of macrophages on adipocytes may offer potential therapeutic targets. IL-1β, a proinflammatory cytokine, is suggested to be involved in the development of insulin resistance. This study investigated the role of IL-1β in macrophage-adipocyte cross-talk, which affects insulin signaling in human adipocytes. Using macrophage-conditioned (MC) medium and human primary adipocytes, we examined the effect of IL-1β antagonism on the insulin signaling pathway. Gene expression profile and protein abundance of insulin signaling molecules were determined, as was the production of proinflammatory cytokine/chemokines. We also examined whether IL-1β mediates MC medium-induced alteration in adipocyte lipid storage. MC medium and IL-1β significantly reduced gene expression and protein abundance of insulin signaling molecules, including insulin receptor substrate-1, phosphoinositide 3-kinase p85α, and glucose transporter 4 and phosphorylation of Akt. In contrast, the expression and release of the proinflammatory markers, including IL-6, IL-8, monocyte chemotactic protein-1, and chemokine (C-C motif) ligand 5 by adipocytes were markedly increased. These changes were significantly reduced by blocking IL-1β activity, its receptor binding, or its production by macrophages. MC medium-inhibited expression of the adipogenic factors and -stimulated lipolysis was also blunted with IL-1β neutralization. We conclude that IL-1β mediates, at least in part, the effect of macrophages on insulin signaling and proinflammatory response in human adipocytes. Blocking IL-1β could be beneficial for preventing obesity-associated insulin resistance and inflammation in human adipose tissue., (Copyright © 2014 the American Physiological Society.)

Adiposity and obesity are increasing in dogs. We have examined here the endocrine function of canine adipose tissue and the regulation of production of inflammation-related adipokines by dog adipocytes. Adiponectin, leptin, IL-6, MCP-1 and TNFalpha genes were expressed in the main adipose depots of dogs, but there were no major depot differences in mRNA levels. Each adipokine was expressed in canine adipocytes differentiated in culture and secreted into the medium (leptin undetected). IL-6, MCP-1 and TNFalpha were also expressed and secreted by preadipocytes; adiponectin and leptin were only expressed after adipocyte differentiation. The inflammatory mediators LPS and TNFalpha had major stimulatory effects on the expression and secretion of IL-6, MCP-1 and TNFalpha; there was a >5,000-fold increase in IL-6 mRNA level with LPS. IL-6 release into the medium was increased >50-fold over 24 h with LPS and TNFalpha, while MCP-1 release was increased 23- and 40-fold by TNFalpha and LPS, respectively. However, there was no effect, or small reductions, in adiponectin and leptin mRNA levels with the inflammatory mediators. Dexamethasone-stimulated leptin gene expression, had no effect on adiponectin expression, but decreased the expression and secretion of IL-6 and MCP-1. The PPARgamma agonist rosiglitazone stimulated both adiponectin and leptin expression and inhibited the expression of IL-6, MCP-1 and TNFalpha; MCP-1 secretion was reduced. These results demonstrate that canine adipocytes express and secrete key adipokines and show that adipocytes of this species are highly responsive to inflammatory mediators with the induction of major increases in the production of inflammation-related adipokines.

The proposition that white adipose tissue is involved in the inflammatory response and metabolic dysregulation of endotoxaemia has been examined. Mice were injected with lipopolysaccharide (LPS; 25 mg/kg) and epididymal, perirenal and subcutaneous adipose tissue removed 4 or 24 h later. The expression of genes encoding key inflammation-related adipokines was measured by real-time polymerase chain reaction. At 24 h after the administration of LPS, there was no change in leptin mRNA level, and adiponectin mRNA fell. However, major increases in TNFalpha, MCP-1 (up to 40-fold) and IL-6 (up to 250-fold) mRNA levels were evident; a substantial elevation in these mRNAs occurred by 4 h, and adipose tissue IL-6 protein also increased (three- to eightfold). At 24 h, the responses in the subcutaneous depot were much lower than in epididymal and perirenal adipose tissue, but at 4 h, the subcutaneous tissue showed major increases in IL-6, MCP-1 and TNFalpha gene expression. In contrast to the inflammatory adipokines, the mRNA level of two macrophage markers, F4/80 and MAC-1, was unaltered in adipose tissue during endotoxaemia. Expression of the hypoxia-sensitive transcription factor, HIF-1alpha, gene was increased at both 4 and 24 h, and HIF-1alpha protein was elevated at 4 h, suggesting that the tissue was hypoxic. It is concluded that white adipose tissue may play an important role in the production of inflammatory mediators in endotoxaemia.

In subjects with obesity, diabetes and coronary artery disease, circulating levels of leptin increased while that of adiponectin is decreased. In this study we have investigated effects of physiological and pharmacological weight reduction on leptin and adiponectin mRNA expression. Wistar rats were fed either standard laboratory chow for 16 weeks (chow-fed) or given a fat-enriched, glucose-enriched diet (diet-fed) for 8 weeks. After 8 weeks, diet-fed group was subdivided into three subgroups, namely, an untreated obese, or were returned to chow diet, or treated with fenofibrate for further 8 week. After 16 weeks, compared with chow-fed group, diet-fed rats had significantly higher body weight, epididymal fat pad mass, and plasma levels of insulin, leptin, adiponectin, non-esterified fatty acids and triglycerides (P<0.001, for all). Moreover, untreated obese rats had significantly (P<0.01, for both) raised levels of Ob mRNA but reduced adiponectin mRNA levels in epididymal fat pads compared with chow-fed group. These changes were corrected by chronic removal of the high-energy diet and fenofibrate treatment. These findings indicate that physiological or pharmacological lowering of body weight together with circulating plasma lipids play a significant role in leptin and adiponectin synthesis and metabolism.

We report here the use of human inflammation arrays to study the inflammatory gene expression profile of TNF-alpha- treated human SGBS adipocytes. Human preadipocytes (SGBS) were induced to differentiate in primary culture, and adipocyte differentiation was confirmed, using Oil Red O staining. We treated the differentiated adipocytes with TNF-alpha, and RNA from differentiated adipocytes with or without TNF-alpha treatment was hybridized to MWG human inflammation arrays to compare expression profiles. Eleven genes were up- or down-regulated in TNF-alpha-treated adipocytes. As revealed by array analysis, among 6 up-regulated genes, only eotaxin-1, monocyte chemoattractant protein-1 (MCP-1), and vascular cell adhesion molecule 1 isoform a precursor (VCAM1) were confirmed by real-time polymerase chain reaction (PCR). Similarly, among 5 down-regulated genes, only IL-1 family member 5 (IL1F5), a disintegrin and metalloprotease with thrombospondin motifs-1 preproprotein (ADAMTS1), fibronectin 1 isoform 1 preprotein (FN1), and matrix metalloproteinase 15 preprotein (MMP15) were confirmed by real-time PCR. There was a substantial increase (50-fold) in eotaxin-1 in response to TNF-alpha. Taken together, we have identified several inflammatory molecules expressed in SGBS adipocytes and discovered molecular factors explaining the relationship between obesity and atherosclerosis, focusing on inflammatory cytokines expressed in the TNF-alpha-treated SGBS cells. Further investigation into the role of these up- or down-regulated cytokine genes during the pathological processes leading to the development of atherosclerosis is warranted.

Zinc-alpha2-glycoprotein (ZAG), a lipid mobilizing factor, is expressed in mouse adipose tissue and is markedly upregulated in mice with cancer cachexia. We have explored whether ZAG is expressed and secreted by human adipocytes, using SGBS cells, and examined the regulation of ZAG expression. ZAG mRNA was detected by RT-PCR in mature human adipocytes and in SGBS cells post-, but not pre-, differentiation to adipocytes. Relative ZAG mRNA levels increased rapidly after differentiation of SGBS cells, peaking at day 8 post-induction. ZAG protein was evident in differentiated adipocytes (by day 3) and also detected in the culture medium (by day 6) post-induction. The PPARgamma agonist rosiglitazone induced a 3-fold increase in ZAG mRNA level, while TNF-alpha led to a 4-fold decrease. Human adipocytes express and secrete ZAG, with ZAG expression being regulated particularly through TNF-alpha and the PPARgamma nuclear receptor. ZAG is a novel adipokine, which may be involved in the local regulation of adipose tissue function.

Objective: A great deal of attention has focused on the central role of alpha melanocyte-stimulating hormone (alpha-MSH) and its antagonism at the melanocortin-4 receptor (MC4R) by agouti related protein (AgRP) in the regulation of energy balance. However, very little is known regarding the function of circulating AgRP and alpha-MSH in humans. We aimed to determine whether circulating alpha-MSH and AgRP are responsive to long-term perturbations in energy balance, in a manner consistent with their central putative functions., Design and Measurements: Circulating alpha-MSH, AgRP and leptin were measured in both lean (n = 11) and obese (n = 18) male volunteers, some of whom (lean n = 11, obese n = 12) were then allocated one of two weight-loss dietary strategies to achieve about 5% weight loss. This was achieved by either total starvation (for 4-6 days) for rapid weight loss or a very low calorie diet (VLCD, 2.6 MJ/day) (11-12 days) for less rapid weight loss, in both the lean and obese volunteers., Results: At baseline, prior to any weight loss both plasma alpha-MSH (15.8 +/- 1.2 vs. 5.8 +/- 1.0 pmol/l +/- SEM; P < 0.001) and AgRP (49.4 +/- 2.4 vs. 10.1 +/- 0.9 pg/ml +/- SEM; P < 0.001) were elevated in obese subjects compared with lean. In both cases this correlated closely with fat mass (P < 0.001), percentage body fat (P < 0.001) and leptin (P < 0.05). Plasma AgRP increased significantly during a 6-day fast in lean individuals (11.1 +/- 1.6 vs. 21.6 +/- 3.1 pg/ml +/- SEM; P < 0.05) but not in the VLCD subjects or in the obese, while alpha-MSH was not affected by any changes in energy balance in either the lean or the obese volunteers., Conclusion: We show a difference in alpha-MSH and AgRP in lean and obese subjects that correlates closely with body fat at baseline. We demonstrate an increase in plasma AgRP during a 6-day fast in lean individuals that is coincident with a decrease in plasma leptin. This increase in AgRP was not due to weight loss per se as there was no change in AgRP as a result of the same weight loss in the VLCD intervention in lean individuals. The source of the increase in plasma AgRP and its physiological function in the periphery remains to be elucidated but we suggest that the dynamics of the change in plasma leptin may determine the elevation in fasting plasma AgRP in lean subjects.

Factors which regulate expression of the haptoglobin (acute phase reactant) gene in adipocytes have been examined using 3T3-L1 cells. Haptoglobin expression was observed by Northern blotting in each of the major white adipose tissue depots of mice (epididymal, subcutaneous, mesenteric, and perirenal) and in interscapular brown fat. Expression occurred in mature adipocytes, but not in the stromal-vascular fraction. In 3T3-L1 cells, haptoglobin mRNA was detected from day 4 after the induction of differentiation into adipocytes. Lipopolysaccharide and the cytokines, TNFalpha and interleukin-6, resulted in substantial increases in haptoglobin mRNA in 3T3-L1 adipocytes; the increase (7-fold) was highest with TNFalpha. Increases in haptoglobin mRNA level were also induced by dexamethasone, noradrenaline, isoprenaline, and a beta3-adrenoceptor agonist. In contrast, haptoglobin mRNA was reduced by nicotinic acid and the PPARgamma agonist, rosiglitazone. RT-PCR showed that the haptoglobin gene was expressed in human adipose tissue (subcutaneous, omental). It is concluded that haptoglobin gene expression in adipocytes is stimulated by inflammatory cytokines, glucocorticoids, and the sympathetic system, while activation of the PPARgamma nuclear receptor is strongly inhibitory.

We have examined whether GLUT-10 and GLUT-12, members of the Class III group of the recently expanded family of facilitative glucose transporters, are expressed in adipose tissues. The mouse GLUT-12 gene, located on chromosome 10, comprises at least five exons and encodes a 622 amino acid protein exhibiting 83% sequence identity and 91% sequence similarity to human GLUT-12. Expression of the GLUT-12 gene was evident in all the major mouse adipose tissue depots (epididymal, perirenal, mesenteric, omental, and subcutaneous white; interscapular brown). The GLUT-10 gene is also expressed in mouse adipose tissues and as with GLUT-12 expression occurred in the mature adipocytes as well as the stromal vascular cells. 3T3-L1 adipocytes express GLUT-10, but not GLUT-12, and expression of GLUT-12 was not induced by insulin or glucose. Both GLUT-10 and GLUT-12 expression was also found in human adipose tissue (subcutaneous and omental) and SGBS adipocytes. It is concluded that white fat expresses a wide range of facilitative glucose transporters.