Autophagy mediates calcium-sensing receptor-induced TNFα production in human preadipocytes

Obesity is a major current public health problem worldwide due to the severe co-morbid conditions that this disease entails. The development of obesity-related cardiometabolic disorders is in direct association with adipose tissue inflammation that leads to its functional impairment. Activation of the Calcium-Sensing Receptor (CaSR) in adipose tissue contributes to inflammation and adipose dysfunction. Autophagy, a process of cell component degradation, is closely related to inflammation in many diseases, however, whether autophagy is associated with CaSR-induced inflammation remains unknown. Using LS14 and SW872 preadipose cell lines as well as primary human preadipocytes, we show that CaSR activation with the allosteric activator cinacalcet induces autophagosome formation. Cinacalcet-induced LC3II content elevation was precluded by knockdown of the CaSR and enhanced by CaSR overexpression, indicating a specific effect. Autophagy inhibition using 3-methyladenine prevented CaSR-induced TNFα production, indicating that autophagy contributes to CaSR-induced inflammation in human preadipocytes. Our results suggest that modulation of CaSR-induced autophagy is an attractive target in obese inflamed adipose tissue, to prevent the development of diseases triggered by adipose dysfunction. We describe a novel mechanism and possible new target to modulate and prevent adipose inflammation and hence the resulting disease-generating adipose tissue dysfunction.     

Adipogenic effect of calcium sensing receptor activation

We established that human adipose cells and the human adipose cell line LS14 express the calcium-sensing receptor (CaSR) and that its activation induces inflammatory cytokine production. Also, its expression is enhanced upon exposure to obesity-associated proinflammatory cytokines. We have thus proposed that CaSR activation may be associated with adipose dysfunction. Here, we evaluated a possible effect on adipogenesis. We induced adipose differentiation of primary and LS14 human preadipocytes with or without the simultaneous activation of CaSR, by the exposure to the calcimimetic cinacalcet. Activation of the receptor for 24 h decreased by 40 % the early differentiation marker CCAAT/enhancer-binding protein β. However, upon longer-term (10 day) exposure to the adipogenic cocktail, cinacalcet exerted the opposite effect, causing a dose–response increase in the expression of the mature adipose markers adipocyte protein 2, adiponectin, peroxisome proliferator-activated receptor γ, fatty acid synthase, and glycerol-3-phosphate dehydrogenase. To assess whether there was a time-sensitive effect of CaSR activation on adipogenesis, we evaluated the 10 day effect of cinacalcet exposure for the first 6, 24, 48 h, 6, and 10 days. Our observations suggest that regardless of the period of exposure, 10 day adipogenesis is elevated by cinacalcet. CaSR activation may interfere with the initial stages of adipocyte differentiation; however, these events do not seem to preclude adipogenesis from continuing. Even though adipogenesis (particularly in subcutaneous depots) is associated with insulin sensitivity and adequate adipose function, the implications of our findings in visceral adipocytes, especially in the context of inflamed AT and overnutrition, remain to be established.     

Amyloid precursor protein expression is upregulated in adipocytes in obesity

The aim of this study was to determine whether amyloid precursor protein (APP) is expressed in human adipose tissue, dysregulated in obesity, and related to insulin resistance and inflammation. APP expression was examined by microarray expression profiling of subcutaneous abdominal adipocytes (SAC) and cultured preadipocytes from obese and nonobese subjects. Quantitative real-time PCR (QPCR) was performed to confirm differences in APP expression in SAC and to compare APP expression levels in adipose tissue, adipocytes, and stromal vascular cells (SVCs) from subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) specimens. Adipose tissue samples were also examined by western blot and immunofluorescence confocal microscopy. Microarray studies demonstrated that APP mRNA expression levels were higher in SAC (approximately 2.5-fold) and preadipocytes (approximately 1.4) from obese subjects. Real-time PCR confirmed increased APP expression in SAC in a separate group of obese compared with nonobese subjects (P=0.02). APP expression correlated to in vivo indices of insulin resistance independently of BMI and with the expression of proinflammatory genes, such as monocyte chemoattractant protein-1 (MCP-1) (R=0.62, P=0.004), macrophage inflammatory protein-1alpha (MIP-1alpha) (R=0.60, P=0.005), and interleukin-6 (IL-6) (R=0.71, P=0.0005). Full-length APP protein was detected in adipocytes by western blotting and APP and its cleavage peptides, Abeta40 and Abeta42, were observed in SAT and VAT by immunofluorescence confocal microscopy. In summary, APP is highly expressed in adipose tissue, upregulated in obesity, and expression levels correlate with insulin resistance and adipocyte cytokine expression levels. These data suggest a possible role for APP and/or Abeta in the development of obesity-related insulin resistance and adipose tissue inflammation.     

Influence of secreted factors from human adipose tissue on glucose utilization and proinflammatory reaction

The objective of the present study was to characterize the nature of the autocrine/paracrine signal within human adipose tissue that may alter glucose metabolism and the inflammatory status in adipocytes. We prepared a conditioned medium from abdominal dermolipectomies in the absence (CM) or the presence (CMBSA) of bovine serum albumin (BSA), and we tested the influence of CM and CMBSA on glucose transport, maximal insulin response, and the expression of inflammation marker genes in differentiated human SGBS adipocytes. We found that CMBSA increased basal and reduced insulin-stimulated glucose incorporation along with a reduced mRNA level of the glucose transport GLUT4, and an increased expression of GLUT1. These effects were associated with a potent upregulation in the mRNA level of the proinflammatory cytokines IL-6 and MCP-1. These regulations were strongly attenuated in the absence of BSA during the preparation of CM, or after BSA depletion of CM, and were attributed to water-soluble molecules rather than lipids. Finally, fractionation of CMBSA by isoelectric focusing showed that part of its bioactivity could be reproduced with proteins with pHi ranging from 6.6 to 7.6. In conclusion, our results demonstrate that the production by human adipose tissue of autocrine/paracrine neutral proteins is able to increase the inflammatory status of the adipocytes and to deteriorate their glucose metabolism and maximal insulin response, and their release is greatly amplified by the presence of albumin.     

Evidence for activation of inflammatory lipoxygenase pathways in visceral adipose tissue of obese Zucker rats

Central obesity is associated with low-grade inflammation that promotes type 2 diabetes and cardiovascular disease in obese individuals. The 12- and 5-lipoxygenase (12-LO and 5-LO) enzymes have been linked to inflammatory changes, leading to the development of atherosclerosis. 12-LO has also been linked recently to inflammation and insulin resistance in adipocytes. We analyzed the expression of LO and proinflammatory cytokines in adipose tissue and adipocytes in obese Zucker rats, a widely studied genetic model of obesity, insulin resistance, and the metabolic syndrome. mRNA expression of 12-LO, 5-LO, and 5-LO-activating protein (FLAP) was upregulated in adipocytes and adipose tissue from obese Zucker rats compared with those from lean rats. Concomitant with increased LO gene expression, the 12-LO product 12-HETE and the 5-LO products 5-HETE and leukotriene B4 (LTB4) were also increased in adipocytes. Furthermore, upregulation of key proinflammatory markers interleukin (IL)-6, TNFα, and monocyte chemoattractant protein-1 were observed in adipocytes isolated from obese Zucker rats. Immunohistochemistry indicated that the positive 12-LO staining in adipose tissue represents cells in addition to adipocytes. This was confirmed by Western blotting in stromal vascular fractions. These changes were in part reversed by the novel anti-inflammatory drug lisofylline (LSF). LSF also reduced p-STAT4 in visceral adipose tissue from obese Zucker rats and improved the metabolic profile, reducing fasting plasma glucose and increasing insulin sensitivity in obese Zucker rats. In 3T3-L1 adipocytes, LSF abrogated the inflammatory response induced by LO products. Thus, therapeutic agents reducing LO or STAT4 activation may provide novel tools to reduce obesity-induced inflammation.     

Impact of serine protease inhibitor alpha1-antitrypsin on expression of endoplasmic reticulum stress-induced proinflammatory factors in adipocytes

Obesity-induced endoplasmic reticulum (ER) stress contributes to low-grade chronic inflammation in adipose tissue and may cause metabolic disorders such as diabetes mellitus and dyslipidemia. Identification of high serpina A1 (alpha-1 antitrypsin, A1AT) expression in mouse adipose tissue and adipocytes prompted us to explore the role of A1AT in the inflammatory response of adipocytes under ER stress. We aimed to determine the role of A1AT expression in adipocytes with ER stress during regulation of adipocyte homeostasis and inflammation. To this end, we chemically induced ER stress in A1AT small interfering RNA-transfected differentiating adipocytes using thapsigargin. Induction of CCAAT-enhancer-binding protein homologous protein (CHOP), an ER stress marker, by thapsigargin was lower in A1AT-deficient SW872 adipocytes. Thapsigargin or the proinflammatory cytokine tumor necrosis factor (TNF)α increased basal expression of cytokines such as interleukin (IL)-1β and IL-8 in both SW872 and primary omental adipocytes. This thapsigargin- or TNFα-induced expression of proinflammatory genes was increased by A1AT deficiency. These findings indicate that adipose A1AT may suppress the ER stress response to block excessive expression of proinflammatory factors, which suggests that A1AT protects against adipose tissue dysfunction associated with ER stress activation.     

Caffeine reduces TNFα up-regulation in human adipose tissue primary culture

Adipose tissue secretions play an important role in the development of obesity-related pathologies such as diabetes. Through inflammatory cytokines production, adipose tissue stromavascular fraction cells (SVF), and essentially macrophages, promote adipocyte insulin resistance by a paracrine way. Since xanthine family compounds such as caffeine were shown to decrease inflammatory production by human blood cells, we investigated the possible effect of caffeine on Tumor Necrosis Factor alpha (TNFalpha) and Interleukin-6 (IL-6) expression by human adipose tissue primary culture. For that purpose, human subcutaneous adipose tissue obtained from healthy non-obese women (BMI: 26.7 +/- 2.2 kg/m2) after abdominal dermolipectomy, was split into explants and cultured for 6 hours with or without caffeine. Three different concentrations of caffeine were tested (0.5 microg/mL, 5 microg/mL and 50 microg/mL). After 6 hours of treatment, explants were subjected to collagenase digestion in order to isolate adipocytes and SVF cells. Then, TNFalpha and IL-6 mRNA were analysed by real-time PCR alternatively in adipocytes and SVF cells. In parallel, we checked gene expression of markers involved in adipocyte differenciation and in SVF cells inflammation and proliferation. Our findings show a strong and dose dependent down-regulation of TNF-alpha gene expression in both adipocyte and SVF cells whereas IL-6 was only down regulated in SVF cells. No effect of caffeine was noticed on the other genes studied. Thus, caffeine, by decreasing TNFalpha expression, could improve adipose tissue inflammation during obesity.     

TLR-3 is Present in Human Adipocytes,but Its Signalling is Not Required for Obesity-Induced Inflammation in Adipose Tissue In Vivo

Innate immunity plays a pivotal role in obesity-induced low-grade inflammation originating from adipose tissue. Key receptors of the innate immune system including Toll-like receptors-2 and -4 (TLRs) are triggered by nutrient excess to promote inflammation. The role of other TLRs in this process is largely unknown. In addition to double-stranded viral mRNA, TLR-3 can also recognize mRNA from dying endogenous cells, a process that is frequently observed within obese adipose tissue. Here, we identified profound expression of TLR-3 in adipocytes and investigated its role during diet-induced obesity. Human adipose tissue biopsies (n=80) and an adipocyte cell-line were used to study TLR-3 expression and function. TLR-3-/- and WT animals were exposed to a high-fat diet (HFD) for 16 weeks to induce obesity. Expression of TLR-3 was significantly higher in human adipocytes compared to the non-adipocyte cells part of the adipose tissue. In vitro, TLR-3 expression was induced during differentiation of adipocytes and stimulation of the receptor led to elevated expression of pro-inflammatory cytokines. In vivo, TLR-3 deficiency did not significantly influence HFD-induced obesity, insulin sensitivity or inflammation. In humans, TLR-3 expression in adipose tissue did not correlate with BMI or insulin sensitivity (HOMA-IR). Together, our results demonstrate that TLR-3 is highly expressed in adipocytes and functionally active. However, TLR-3 appears to play a redundant role in obesity-induced inflammation and insulin resistance.     

Tomato extract suppresses the production of proinflammatory mediators induced by interaction between adipocytes and macrophages

Obese adipose tissue is characterized by enhanced macrophage infiltration. A loop involving monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNFα) between adipocytes and macrophages establishes a vicious cycle that augments inflammatory changes and insulin resistance in obese adipose tissue. Tomatoes, one of the most popular crops worldwide, contain many beneficial phytochemicals that improve obesity-related diseases such as diabetes. Some of them have also been reported to have anti-inflammatory properties. In this study, we focused on the potential protective effects of phytochemicals in tomatoes on inflammation. We screened fractions of tomato extract using nitric oxide (NO) assay in lipopolysaccharide (LPS)-stimulated RAW264 macrophages. One fraction, RF52, significantly inhibited NO production in LPS-stimulated RAW264 macrophages. Furthermore, RF52 significantly decreased MCP-1 and TNFα productions. The coculture of 3T3-L1 adipocytes and RAW264 macrophages markedly enhanced MCP-1, TNFα, and NO productions compared with the control cultures; however, the treatment with RF52 inhibited the production of these proinflammatory mediators. These results suggest that RF52 from tomatoes may have the potential to suppress inflammation by inhibiting the production of NO or proinflammatory cytokines during the interaction between adipocytes and macrophages.     

Adipose tissue tumor necrosis factor and interleukin-6 expression in human obesity and insulin resistance

Adipose tissue expresses tumor necrosis factor (TNF) and interleukin (IL)-6, which may cause obesity-related insulin resistance. We measured TNF and IL-6 expression in the adipose tissue of 50 lean and obese subjects without diabetes. Insulin sensitivity (S(I)) was determined by an intravenous glucose tolerance test with minimal-model analysis. When lean [body mass index (BMI) <25 kg/m(2)] and obese (BMI 30-40 kg/m(2)) subjects were compared, there was a 7.5-fold increase in TNF secretion (P < 0.05) from adipose tissue, and the TNF secretion was inversely related to S(I) (r = -0.42, P < 0.02). IL-6 was abundantly expressed by adipose tissue. In contrast to TNF, plasma (rather than adipose) IL-6 demonstrated the strongest relationship with obesity and insulin resistance. Plasma IL-6 was significantly higher in obese subjects and demonstrated a highly significant inverse relationship with S(I) (r = -0.71, P < 0.001). To separate the effects of BMI from S(I), subjects who were discordant for S(I) were matched for BMI, age, and gender. By use of this approach, subjects with low S(I) demonstrated a 3.0-fold increased level of TNF secretion from adipose tissue and a 2.3-fold higher plasma IL-6 level (P < 0.05) compared with matched subjects with a high S(I). Plasma IL-6 was significantly associated with plasma nonesterified fatty acid levels (r = 0.49, P < 0.002). Thus the local expression of TNF and plasma IL-6 are higher in subjects with obesity-related insulin resistance.     

Inducible Toll-like receptor and NF-kappaB regulatory pathway expression in human adipose tissue

Objective: Inflammatory activity in fat tissue has recently been implicated in mechanisms of insulin resistance and obesity‐related metabolic dysfunction. Toll‐like receptors (TLRs) play a key role in innate immune responses and recent studies implicate the TLR pathway in mechanisms of inflammation and atherosclerosis. The aim of this study was to examine differential TLR expression and function in human adipose tissue. Methods and Procedures: We biopsied subcutaneous abdominal fat from 16 obese subjects (age 39 ± 11 years, BMI 49 ± 14 kg/m²) and characterized TLR expression using quantitative real‐time PCR and confocal immunofluorescence imaging. In tissue culture, we stimulated isolated human adipocytes with Pam3CSK4 and lipopolysaccharide (LPS) (TLR2 and TLR4 agonists, respectively) and quantified TLR activity, interleukin‐6 (IL‐6) and tumor necrosis factor‐α (TNF‐α) production, and nuclear factor‐κB (NF‐κB) p65 nuclear activation using real‐time PCR, enzyme‐linked immunosorbent assay (ELISA), and immunofluorescence. Results: TLR1, 2, and 4 protein colocalized with adiponectin in human adipocytes with TLR4 exhibiting the highest immunohistochemical expression. Using real‐time PCR, we confirmed higher level of gene expression for TLR4 as compared to other members of the TLR family (TLR1, 2, 7, 8) in human adipose depots (P < 0.001). In tissue culture, adipocyte TLR2/TLR4 mRNA expression and protein increased significantly following Pam3CSK4 and LPS (P < 0.001). TLR2/TLR4 stimulation was associated with NF‐κB p65 nuclear translocation and proinflammatory cytokine production. Discussion: The findings demonstrate that TLRs are inducible in adipose tissue and linked with downstream NF‐κB activation and cytokine release. Adipose stores may play a dynamic role in the regulation of inflammation and innate immunity in human subjects via modulation of the TLR/NF‐κB regulatory pathway.     

Interleukin‐15 Contributes to the Regulation of Murine Adipose Tissue and Human Adipocytes

An alarming global rise in the prevalence of obesity and its contribution to the development of chronic diseases is a serious health concern. Recently, obesity has been described as a chronic low‐grade inflammatory condition, influenced by both adipose tissue and immune cells suggesting proinflammatory cytokines may play a role in its etiology. Here we examined the effects of interleukin‐15 (IL‐15) on adipose tissue and its association with obesity. Over expression of IL‐15 (IL‐15tg) was associated with lean body condition whereas lack of IL‐15 (IL‐15^?/?) results in significant increase in weight gain without altering appetite. Interestingly, there were no differences in proinflammatory cytokines such as IL‐6 and tumor necrosis factor‐α (TNF‐α) in serum between the three strains of mice. In addition, there were significant numbers of natural killer (NK) cells in fat tissues from IL‐15tg and B6 compared to IL‐15^?/? mice. IL‐15 treatment results in significant weight loss in IL‐15^?/? knockout and diet‐induced obese mice independent of food intake. Fat pad cross‐sections show decreased pad size with over expression of IL‐15 is due to adipocyte shrinkage. IL‐15 induces weight loss without altering food consumption by affecting lipid deposition in adipocytes. Treatment of differentiated human adipocytes with recombinant human IL‐15 protein resulted in decreased lipid deposition. In addition, obese patients had significantly lower serum IL‐15 levels when compared to normal weight individuals. These results clearly suggest that IL‐15 may be involved in adipose tissue regulation and linked to obesity.     

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.     

Naringenin suppresses macrophage infiltration into adipose tissue in an early phase of high-fat diet-induced obesity

Obese adipose tissue is characterized by increased macrophage infiltration, which results in chronic inflammation in adipose tissue and leads to obesity-related diseases such as type 2 diabetes mellitus and atherosclerosis. The regulation of macrophage infiltration into adipose tissue is an important strategy for preventing and treating obesity-related diseases. In this study, we report that naringenin, a citrus flavonoid, suppressed macrophage infiltration into adipose tissue induced by short-term (14 days) feeding of a high-fat diet in mice; although naringenin did not show any differences in high-fat diet-induced changes of serum biochemical parameters in this short administration period. Naringenin suppressed monocyte chemoattractant protein-1 (MCP-1) in adipose tissue, and this effect was mediated in part through inhibition of c-Jun NH₂-terminal kinase pathway. Naringenin also inhibited MCP-1 expression in adipocytes, macrophages, and a co-culture of adipocytes and macrophages. Our results suggest a mechanism by which daily consumption of naringenin may exhibit preventive effects on obesity-related diseases.     

Pathophysiological roles of chronic low‐grade inflammation mediators in polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is the most common hormonal imbalance disease in reproductive‐aged women. Its basic characteristics are ovulatory dysfunction and ovarian overproduction of androgens that lead to severe symptoms such as insulin resistance, hirsutism, infertility, and acne. Notwithstanding the disease burden, its underlying mechanisms remain unknown, and no causal therapeutic exists. In recent years, further studies showed that inflammation processes are involved in ovulation and play a key role in ovarian follicular dynamics. Visceral adipose tissue can cause inflammatory response and maintenance of the inflammation state in adipocytes by augmented production of inflammatory cytokines, monocyte chemoattractant proteins, and recruitment of the immune cell. Therefore, the PCOS can be related to a low‐grade inflammation state and inflammatory markers. Investigating the inflammatory processes and mediators that contribute to the commencement and development of PCOS can be a critical step for better understanding the pathophysiology of the disease and its treatment through inhibition or control of related pathways. In the present review, we discuss the pathophysiological roles of chronic low‐grade inflammation mediators including inflammasome‐related cytokines, interleukin‐1β (IL‐1β), and IL‐18 in PCOS development.     

Characterization of Herpes Virus Entry Mediator as a Factor Linked to Obesity

Herpes virus entry mediator (HVEM) is a member of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF14), which serves as a receptor for herpes viruses and cytokines such as lymphotoxin‐α (LT‐α) and LIGHT (lymphotoxin‐like inducible protein that competes with glycoprotein D for herpes virus entry on T cells). We aimed to explore the associations of HVEM with human obesity. HVEM gene expression and protein levels were studied in total adipose tissue and in their fractions (isolated adipocytes and stromovascular cells (SVCs)) obtained from 81 subjects during elective surgical procedures. HVEM ?241GA and ?14AG gene polymorphisms were also studied and associated with obesity measures in 840 subjects. Visceral adipose tissue had significantly higher expression of HVEM than subcutaneous adipose tissue (P < 0.0001). Obese patients had significantly higher subcutaneous HVEM gene expression (P = 0.03) and protein levels (P = 0.01) than lean subjects. HVEM gene expression and protein levels were found in both isolated adipocytes and SVCs. These findings were confirmed in primary cultures from human preadipocytes, in which a significant increase in HVEM was observed during the differentiation process. HVEM ?241GA and ?14AG gene polymorphisms were associated with obesity, diastolic pressure, several inflammatory parameters (C‐reactive protein and interleukin 18 (IL‐18)), and circulating LIGHT concentrations. A sample of men with the G241A gene polymorphism also showed an increased serum titer of IgG antiherpes virus 1. These results provide evidences of an existing relationship between HVEM and obesity, which suggest that this TNF superfamily receptor could be involved in the pathogenesis of obesity and inflammation‐related activity.     

Macrophages block insulin action in adipocytes by altering expression of signaling and glucose transport proteins

Obesity leads to a proinflammatory state with immune responses that include infiltration of adipose tissue with macrophages. These macrophages are believed to alter insulin sensitivity in adipocytes, but the mechanisms that underlie this effect have not been characterized. We have explored the interaction between macrophages and adipocytes in the context of both indirect and direct coculture. Macrophage-secreted factors blocked insulin action in adipocytes via downregulation of GLUT4 and IRS-1, leading to a decrease in Akt phosphorylation and impaired insulin-stimulated GLUT4 translocation to the plasma membrane. GLUT1 was upregulated with a concomitant increase in basal glucose uptake. These changes recapitulate those seen in adipose tissue from insulin-resistant humans and animal models. TNF-alpha-neutralizing antibodies partially reversed the insulin resistance produced by macrophage-conditioned media. Peritoneal macrophages and macrophage-enriched stromal vascular cells from adipose tissue also attenuated responsiveness to insulin in a manner correlating with inflammatory cytokine secretion. Adipose tissue macrophages from obese mice have an F4/80(+)CD11b(+)CD68(+)CD14(-) phenotype and form long cellular extensions in culture. Peritoneal macrophages take on similar characteristics in direct coculture with adipocytes and induce proinflammatory cytokines, suggesting that macrophage activation state is influenced by contact with adipocytes. Thus both indirect/secreted and direct/cell contact-mediated factors derived from macrophages influence insulin sensitivity in adipocytes.     

Dysregulation of adipose glutathione peroxidase 3 in obesity contributes to local and systemic oxidative stress

Glutathione peroxidase 3 (GPx3) accounts for the major antioxidant activity in the plasma. Here, we demonstrate that down-regulation of GPx3 in the plasma of obese subjects is associated with adipose GPx3 dysregulation, resulting from the increase of inflammatory signals and oxidative stress. Although GPx3 was abundantly expressed in kidney, lung, and adipose tissue, we observed that GPx3 expression was reduced selectively in the adipose tissue of several obese animal models as decreasing plasma GPx3 level. Adipose GPx3 expression was greatly suppressed by prooxidative conditions such as high levels of TNFalpha and hypoxia. In contrast, the antioxidant N-acetyl cysteine and the antidiabetic drug rosiglitazone increased adipose GPx3 expression in obese and diabetic db/db mice. Moreover, GPx3 overexpression in adipocytes improved high glucose-induced insulin resistance and attenuated inflammatory gene expression whereas GPx3 neutralization in adipocytes promoted expression of proinflammatory genes. Taken together, these data suggest that suppression of GPx3 expression in the adipose tissue of obese subjects might constitute a vicious cycle to expand local reactive oxygen species accumulation in adipose tissue potentially into systemic oxidative stress and obesity-related metabolic complications.     

No Evidence for a Role of Adipose Tissue-Derived Serum Amyloid A in the Development of Insulin Resistance or Obesity-Related Inflammation in hSAA1+/? Transgenic Mice

Obesity is associated with a low-grade inflammation including moderately increased serum levels of the acute phase protein serum amyloid A (SAA). In obesity, SAA is mainly produced from adipose tissue and serum levels of SAA are associated with insulin resistance. SAA has been described as a chemoattractant for inflammatory cells and adipose tissue from obese individuals contains increased numbers of macrophages. However, whether adipose tissue-derived SAA can have a direct impact on macrophage infiltration in adipose tissue or the development of insulin resistance is unknown. The aim of this study was to investigate the effects of adipose tissue-derived SAA1 on the development of insulin resistance and obesity-related inflammation. We have previously established a transgenic mouse model expressing human SAA1 in the adipose tissue. For this report, hSAA1^+/− transgenic mice and wild type mice were fed with a high fat diet or normal chow. Effects of hSAA1 on glucose metabolism were assessed using an oral glucose tolerance test. Real-time PCR was used to measure the mRNA levels of macrophage markers and genes related to insulin sensitivity in adipose tissue. Cytokines during inflammation were analyzed using a Proinflammatory 7-plex Assay. We found similar insulin and glucose levels in hSAA1 mice and wt controls during an oral glucose tolerance test and no decrease in mRNA levels of genes related to insulin sensitivity in adipose tissue in neither male nor female hSAA1 animals. Furthermore, serum levels of proinflammatory cytokines and mRNA levels of macrophage markers in adipose tissue were not increased in hSAA1 mice. Hence, in this model we find no evidence that adipose tissue-derived hSAA1 influences the development of insulin resistance or obesity-related inflammation.     

Expression of TWEAK and its receptor Fn14 in human subcutaneous adipose tissue. Relationship with other inflammatory cytokines in obesity

TWEAK, a cytokine of the TNF family, has been found to be expressed under different inflammatory conditions but no data is available concerning the expression of this cytokine and its receptor (Fn14) in human obesity. In the present work we have evaluated the expression of many pro-inflammatory TNF system cytokines (TNF-alpha, TWEAK and their respective receptors, TNFR1, TNFR2 and Fn14) in human adipose tissue of 84 subjects some with different degree of obesity and type 2 diabetes, and its relation with inflammation by also measuring the expression of macrophage marker CD68. We detected expression of TWEAK and Fn14 in isolated mature adipocytes and in the stromovascular fraction. Additionally, we found that LPS upregulates the expression of both genes on THP-1 human monocytic cell line. TWEAK was expressed in adipose tissue of all studied subjects with no differences between obesity group, and was associated with Fn14 expression in morbid obese, mainly in women with type 2 diabetes. The data obtained here also showed that TNF-alpha and TNFR2 mRNAs were significantly more expressed in subcutaneous adipose tissue of subjects with morbid obesity compared to obese and non-obese subjects. In contrast, TNFR1 gene expression was negatively associated with BMI. Our results suggest that the expression of TNF-derived pro-inflammatory cytokines are increased in severe obesity, where macrophage infiltrate could modulate the inflammatory environment through activation of its receptors.