Leif Hunter

Zinc-α2-glycoprotein, a lipid mobilizing factor, is expressed and secreted by human (SGBS) adipocytes

Zinc‐α2‐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 PPARγ agonist rosiglitazone induced a 3‐fold increase in ZAG mRNA level, while TNF‐α led to a 4‐fold decrease. Human adipocytes express and secrete ZAG, with ZAG expression being regulated particularly through TNF‐α and the PPARγ nuclear receptor. ZAG is a novel adipokine, which may be involved in the local regulation of adipose tissue function.

Improvement in insulin resistance and reduction in plasma inflammatory adipokines after weight loss in obese dogs

Obesity is now a major disease of dogs, predisposing to numerous disorders including diabetes mellitus. Adipocytes are active endocrine cells, and human obesity is characterized by derangements in inflammatory adipokine production. However, it is unclear as to whether similar changes occur in dogs. The purpose of the current study was to assess insulin sensitivity and inflammatory adipokine profiles in dogs with naturally occurring obesity and to investigate the effect of subsequent weight loss. Twenty-six overweight dogs were studied, representing a range of breeds and both sexes. All dogs underwent a weight loss program involving diet and exercise. Body fat mass was measured by dual-energy x-ray absorptiometry; plasma concentrations of insulin, glucose, and a panel of inflammatory adipokines (including acute-phase proteins, cytokines, and chemokines) were also analyzed. Body fat mass before weight loss was positively correlated with both plasma insulin concentrations (Kendall tau=0.30, P=0.044) and insulin:glucose ratio (Kendall tau=0.36, P=0.022), and both decreased after weight loss (P=0.0037 and 0.0063, respectively). Weight loss also led to notable decreases in plasma tumor necrosis factor-alpha (TNF-alpha), haptoglobin, and C-reactive protein concentrations (P<0.05 for all), suggesting improvement of a subclinical inflammatory state associated with obesity. This study has demonstrated that in obese dogs, insulin resistance correlates with degree of adiposity, and weight loss improves insulin sensitivity. Concurrent decreases in TNF-alpha and adipose tissue mass suggest that in dogs, as in humans, this adipokine may be implicated in the insulin resistance of obesity.

Interleukin-1β mediates macrophage-induced impairment of insulin signaling in human primary adipocytes.

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.

Expression of class III facilitative glucose transporter genes (GLUT-10 and GLUT-12) in mouse and human adipose tissues

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.

NGF Gene Expression and Secretion by Canine Adipocytes in Primary Culture: Upregulation by the Inflammatory Mediators LPS and TNFα

Obesity is the commonest nutritional disorder of companion animals. In rodents and humans, white adipose tissue is a major endocrine and secretory organ, releasing adipokines linked to inflammation. In this study, we examined whether nerve growth factor (NGF), a target-derived neurotrophin central to the development/maintenance of sympathetic innervation and an inflammatory response protein, is synthesized and secreted by canine adipocytes. NGF mRNA was detected in each of the major fat depots (the subcutaneous, inguinal, gonadal, perirenal, and falciform ligaments) of dogs at similar levels. Canine adipocytes, differentiated from preadipocytes (inguinal depot) in primary culture, expressed the NGF gene and secreted NGF both pre- and post-differentiation. Treatment of the differentiated adipocytes with LPS resulted in a dramatic increase in NGF mRNA levels (20-fold at 24 h) and in NGF protein in the medium (60-fold at 24 h). The proinflammatory cytokine TNFalpha also led to a substantial increase in NGF mRNA levels (11-fold) and protein secretion (16-fold), while IL-6 had little effect. In contrast, dexamethasone decreased both NGF mRNA levels (80%) and protein release (60%). The PPARgamma agonist rosiglitazone also reduced NGF secretion. These results demonstrate that canine white adipocytes synthesize and secrete NGF, the powerful upregulation by LPS and TNFalpha indicating that the neurotrophin is strongly linked to the inflammatory response in canine WAT. Canine adipocytes appear highly sensitive to inflammatory stimuli.

Inflammatory Gene Expression Patterns Revealed by DNA Microarray Analysis in TNF-alpha-treated SGBS Human Adipocytes.

We report here the use of human inflammation arrays to study the inflammatory gene expression profile of TNF-α-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-α, and RNA from differentiated adipocytes with or without TNF-α treatment was hybridized to MWG human inflammation arrays to compare expression profiles. Eleven genes were up- or down-regulated in TNF-α-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-α. 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-α-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.

Adipose tissue-derived adiponectin expression is significantly associated with increased post operative mortality in horses undergoing emergency abdominal surgery

REASONS FOR PERFORMING STUDY Adipose tissue is an important source of inflammatory cytokines (adipokines) and adiposity has been identified as having a significant effect on human morbidity and mortality. Obesity is also an emerging welfare problem in the UK horse population, but the role that it plays in secondary diseases is unclear. OBJECTIVES To examine the expression of inflammation-related adipokine genes in retroperitoneal adipose tissue of horses undergoing emergency abdominal surgery and to explore associations with adiposity and post operative survival. METHODS Retroperitoneal adipose tissue samples were obtained from 76 horses undergoing emergency abdominal surgery. Real-time PCR was used to measure gene expression for leptin, adiponectin, tumour necrosis factor-alpha, macrophage chemoattractant protein-1, macrophage inhibitory factor, serum amyloid A, haptoglobin and interleukin-1. Multivariate patterns of adipokine expression were explored with principal component analysis (PCA), whilst univariable associations with post operative survival were tested in a Cox proportional hazards model. RESULTS Leptin gene expression was higher in overweight and obese horses than in lean animals. Expression of mRNA encoding adiponectin mRNA in visceral adipose tissue was positively associated with increased post operative mortality (hazard ratio 1.31, 95% CI 1.05-1.65). However, PCA did not demonstrate multivariable patterns of adipokine gene expression from visceral adipose tissue associated with body mass index or with survival. CONCLUSIONS In horses presented with acute intestinal disease, increased adiponectin gene expression from retroperitoneal adipose tissue is associated with an increased risk of mortality. Obesity assessed by BMI had no association with increased post operative mortality in horses with primary gastrointestinal disease. POTENTIAL RELEVANCE Further study is warranted on the expression and effects of adipokines, particularly adiponectin, and correlation with postoperative outcome.

11-Hydroxy-β-steroid dehydrogenase gene expression in canine adipose tissue and adipocytes: Stimulation by lipopolysaccharide and tumor necrosis factor α

The enzyme 11β-hydroxysteroid dehydrogenase 1 (11β-HSD-1) is expressed in a number of tissues in rodents and humans and is responsible for the reactivation of inert cortisone into cortisol. Its gene expression and activity are increased in white adipose tissue (WAT) from obese humans and may contribute to the adverse metabolic consequences of obesity and the metabolic syndrome. The extent to which 11β-HSD-1 contributes to adipose tissue function in dogs is unknown; the aim of the present study was to examine 11β-HSD-1 gene expression and its regulation by proinflammatory and anti-inflammatory agents in canine adipocytes. Real-time PCR was used to examine the expression of 11β-HSD-1 in canine adipose tissue and canine adipocytes differentiated in culture. The mRNA encoding 11β-HSD-1 was identified in all the major WAT depots in dogs and also in liver, kidney, and spleen. Quantification by real-time PCR showed that 11β-HSD-1 mRNA was least in perirenal and falciform depots and greatest in subcutaneous, omental, and gonadal depots. Greater expression was seen in the omental depot in female than in male dogs (P=0.05). Gene expression for 11β-HSD-1 was also seen in adipocytes, from both subcutaneous and visceral depots, differentiated in culture; expression was evident throughout differentiation but was generally greatest in preadipocytes and during early differentiation, declining as cells progressed to maturity. The inflammatory mediators lipopolysaccharide and tumor necrosis factor α had a main stimulatory effect on 11β-HSD-1 gene expression in canine subcutaneous adipocytes, but IL-6 had no significant effect. Treatment with dexamethasone resulted in a significant time- and dose-dependent increase in 11β-HSD-1 gene expression, with greatest effects seen at 24 h (2 nM: approximately 4-fold; 20 nM: approximately 14-fold; P=0.010 for both). When subcutaneous adipocytes were treated with the peroxisome proliferator activated receptor γ agonist rosiglitazone, similar dose- and time-dependent effects were noted. However, no effects were seen when adipocytes from the gonadal WAT depot were treated with rosiglitazone. The induction of 11β-HSD-1 expression, by the pro-inflammatory cytokine tumor necrosis factor α and by lipopolysaccharide may have implications for the pathogenesis of obesity and its associated diseases in the dog.