Adipocyte Hypertrophy and Improved Postprandial Lipid Response in Beta 2 Syntrophin Deficient Mice.

BACKGROUND/AIMS: Adipocyte hypertrophy in obesity is associated with inflammation and adipose tissue fibrosis which both contribute to metabolic diseases. Mechanisms regulating lipid droplet expansion are poorly understood. Knock down of the scaffold protein beta 2 syntrophin (SNTB2) increases lipid droplet size of 3T3-L1 adipocytes and the physiological relevance of SNTB2 in adipose tissue morphology and metabolic health was analyzed herein. METHODS: Wild type and SNTB2-/- mice were challenged with 24 weeks high fat diet. Adipose tissue morphology and expression of various genes / proteins including collagens and caveolin-1 was examined. Glucose, insulin, fasting and fed free fatty acids were measured in serum. SNTB2 expression was determined in adipose tissues of patients. RESULTS: Upon high fat diet SNTB2-/- mice displayed reduced adiposity and adipocyte hypertrophy. Expression of various proteins was normal in the different white fat depots of SNTB2-/- mice while caveolin-1 protein and collagen mRNA levels were diminished. Null mice had reduced systemic glucose while fasting and postprandial insulin and insulin response were normal. Fatty acid clearance in the fed state and after insulin injection was enhanced. SNTB2 and caveolin-1 were increased in fat of ob/ob mice. However, no correlation between body mass index and SNTB2 protein in adipose tissues of seven patients was found. In subcutaneous but not in visceral fat the ratio of SNTB2 to alpha syntrophin protein, which affects lipid droplet size in the opposite manner, was associated with BMI. In subcutaneous fat of extremely obese patients SNTB2 mRNA levels were not correlated with weight loss after bariatric surgery. CONCLUSION: Current study shows that high SNTB2 in obese adipose tissues restricts adipocyte growth and thereby may contribute to metabolic diseases.

Lipid abnormalities in alpha/beta2-syntrophin null mice are independent from ABCA1.

The syntrophins alpha (SNTA) and beta 2 (SNTB2) are molecular adaptor proteins shown to stabilize ABCA1, an essential regulator of HDL cholesterol. Furthermore, SNTB2 is involved in glucose stimulated insulin release. Hyperglycemia and dyslipidemia are characteristic features of the metabolic syndrome, a serious public health problem with rising prevalence. Therefore, it is important to understand the role of the syntrophins herein. Mice deficient for both syntrophins (SNTA/B2-/-) have normal insulin and glucose tolerance, hepatic ABCA1 protein and cholesterol. When challenged with a HFD, wild type and SNTA/B2-/- mice have similar weight gain, adiposity, serum and liver triglycerides. Hepatic ABCA1, serum insulin and insulin sensitivity are normal while glucose tolerance is impaired. Liver cholesterol is reduced, and expression of SREBP2 and HMG-CoA-R is increased in the knockout mice. Scavenger receptor-BI (SR-BI) protein is strongly diminished in the liver of SNTA/B2-/- mice while SR-BI binding protein NHERF1 is not changed and PDZK1 is even induced. Knock-down of SNTA, SNTB2 or both has no effect on hepatocyte SR-BI and PDZK1 proteins. Further, SR-BI levels are not reduced in brown adipose tissue of SNTA/B2-/- mice excluding that syntrophins directly stabilize SR-BI. SR-BI stability is regulated by MAPK and phosphorylated ERK2 is induced in the liver of the knock-out mice. Blockage of ERK activity upregulates hepatocyte SR-BI showing that increased MAPK activity contributes to low SR-BI. Sphingomyelin which is well described to regulate cholesterol metabolism is reduced in the liver and serum of the knock-out mice while the size of serum lipoproteins is not affected. Current data exclude a major function of these syntrophins in ABCA1 activity and insulin release but suggest a role in regulating glucose uptake, ERK and SR-BI levels, and sphingomyelin metabolism in obesity.

Manganese superoxide dismutase knock-down in 3T3-L1 preadipocytes impairs subsequent adipogenesis.

Adipogenesis is associated with the upregulation of the antioxidative enzyme manganese superoxide dismutase (MnSOD) suggesting a vital function of this enzyme in adipocyte maturation. In the current work, MnSOD was knocked-down with small-interference RNA in preadipocytes to study its role in adipocyte differentiation. In mature adipocytes differentiated from these cells, proteins characteristic for mature adipocytes, which are strongly induced in late adipogenesis like adiponectin and fatty acid-binding protein 4, are markedly reduced. Triglycerides begin to accumulate after about 6 days of the induction of adipogenesis, and are strongly diminished in cells with low MnSOD. Proteins upregulated early during differentiation, like fatty acid synthase and cytochrome C oxidase-4, are not altered. Cell viability, insulin-mediated phosphorylation of Akt, antioxidative capacity (AOC), superoxide levels, and heme oxygenase 1 with the latter being induced upon oxidative stress are not affected. L-Buthionine-(S,R)-sulfoximine (BSO) depletes glutathione and modestly lowers AOC of mature adipocytes. Addition of BSO to 3T3-L1 cells 3 days after the initiation of differentiation impairs triglyceride accumulation and expression of proteins induced in late adipogenesis. Of note, proteins that increased early during adipogenesis are also diminished, suggesting that BSO causes de-differentiation of these cells. Preadipocyte proliferation is not considerably affected by low MnSOD and BSO. These data suggest that glutathione and MnSOD are essential for adipogenesis.

Free fatty acids, lipopolysaccharide and IL-1α induce adipocyte manganese superoxide dismutase which is increased in visceral adipose tissues of obese rodents.

Excess fat storage in adipocytes is associated with increased generation of reactive oxygen species (ROS) and impaired activity of antioxidant mechanisms. Manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme involved in detoxification of ROS, and objective of the current study is to analyze expression and regulation of MnSOD in obesity. MnSOD is increased in visceral but not subcutaneous fat depots of rodents kept on high fat diets (HFD) and ob/ob mice. MnSOD is elevated in visceral adipocytes of fat fed mice and exposure of differentiating 3T3-L1 cells to lipopolysaccharide, IL-1α, saturated, monounsaturated and polyunsaturated free fatty acids (FFA) upregulates its level. FFA do not alter cytochrome oxidase 4 arguing against overall induction of mitochondrial enzymes. Upregulation of MnSOD in fat loaded cells is not mediated by IL-6, TNF or sterol regulatory element binding protein 2 which are induced in these cells. MnSOD is similarly abundant in perirenal fat of Zucker diabetic rats and non-diabetic animals with similar body weight and glucose has no effect on MnSOD in 3T3-L1 cells. To evaluate whether MnSOD affects adipocyte fat storage, MnSOD was knocked-down in adipocytes for the last three days of differentiation and in mature adipocytes. Knock-down of MnSOD does neither alter lipid storage nor viability of these cells. Heme oxygenase-1 which is induced upon oxidative stress is not altered while antioxidative capacity of the cells is modestly reduced. Current data show that inflammation and excess triglyceride storage raise adipocyte MnSOD which is induced in epididymal adipocytes in obesity.

Systemic resistin is increased in type 2 diabetic patients treated with loop diuretics.

Increased serum resistin was found in rodent models of obesity and insulin resistance, whereas contradictory results have been obtained in human studies. In humans, resistin is primarily released by monocytes/macrophages, suggesting that soluble levels may be associated with macrophage activation. Here, systemic and monocyte-released resistin levels were found to be similar in type 2 diabetic (T2D) patients, overweight controls and normal-weight controls. When adjusted for body mass index and age, serum resistin modestly correlated with gamma-glutamyltransferase levels, fasting glucose and interleukin-6. Systemic resistin was marginally increased in T2D patients treated with beta-blockers or urate-lowering drugs and was considerably higher in patients treated with loop diuretics. Monocyte-released resistin was even reduced by the loop diuretic furosemide, excluding the possibility that this drug may directly stimulate resistin synthesis. In summary, the current data indicate that changes accompanying renal dysfunction but not obesity or type 2 diabetes are associated with increased serum resistin.

Adiponectin, a key adipokine in obesity related liver diseases.

Non-alcoholic fatty liver disease (NAFLD) comprising hepatic steatosis, non-alcoholic steatohepatitis (NASH), and progressive liver fibrosis is considered the most common liver disease in western countries. Fatty liver is more prevalent in overweight than normal-weight people and liver fat positively correlates with hepatic insulin resistance. Hepatic steatosis is regarded as a benign stage of NAFLD but may progress to NASH in a subgroup of patients. Besides liver biopsy no diagnostic tools to identify patients with NASH are available, and no effective treatment has been established. Visceral obesity is a main risk factor for NAFLD and inappropriate storage of triglycerides in adipocytes and higher concentrations of free fatty acids may add to increased hepatic lipid storage, insulin resistance, and progressive liver damage. Most of the adipose tissue-derived proteins are elevated in obesity and may contribute to systemic inflammation and liver damage. Adiponectin is highly abundant in human serum but its levels are reduced in obesity and are even lower in patients with hepatic steatosis or NASH. Adiponectin antagonizes excess lipid storage in the liver and protects from inflammation and fibrosis. This review aims to give a short survey on NAFLD and the hepatoprotective effects of adiponectin.

Adiponectin induces the transforming growth factor decoy receptor BAMBI in human hepatocytes.

Transforming growth factor (TGF) ß is the central cytokine in fibrotic liver diseases. We analyzed whether hepatoprotective adiponectin directly interferes with TGFß1 signaling in primary human hepatocytes (PHH). Adiponectin induces the TGFß decoy receptor BMP-and activin-membrane-bound inhibitor (BAMBI) in PHH. Overexpression of BAMBI in hepatoma cells impairs TGFß-mediated phosphorylation of SMAD2 and induction of connective tissue growth factor. BAMBI is lower in human fatty liver with a higher susceptibility to liver fibrosis and negatively correlates with BMI of the donors. Hepatic BAMBI is reduced in rodent models of liver inflammation and fibrosis. In summary, the current data show that hepatoprotective effects of adiponectin include induction of BAMBI which is reduced in human fatty liver and rodent models of metabolic liver injury.

Portal levels of latent transforming growth factor-ß are related to liver function in patients with liver cirrhosis.

OBJECTIVE: Transforming growth factor-ß1 (TGFß1) is a short-lived immune suppressive and profibrotic protein. Its latent precursor is relatively stable and may even protect from fibrosis. Latent TGFß1 is synthesized by various tissues including the liver and portal, hepatic, and systemic concentrations of latent TGFß1 were determined in patients with liver cirrhosis and patients with normal liver function to find out whether circulating levels are affected by liver disease. METHODS: Latent TGFß1 was measured in portal venous serum (PVS), hepatic venous serum (HVS), and systemic venous serum (SVS) of 26 patients with liver cirrhosis and nine patients with normal liver function. RESULTS: Latent TGFß1 was similarly abundant in HVS, PVS,and SVS of patients with liver cirrhosis and controls. There was a strong positive correlation of HVS, PVS, and SVS TGFß1 to each other. PVS levels of latent TGFß1 were significantly lower in patients with CHILD-PUGH stage C compared with CHILD-PUGH stage A, SVS levels were modestly and HVS levels tended to be reduced. PVS and SVS TGFß1 concentrations were also lower in patients with a higher model for end-stage liver disease score. Only PVS concentrations were reduced in patients with massive ascites compared with the patients without ascites. Creatinine clearance as a marker of renal function and parameters of coagulation did not correlate with this cytokine indicating that latent TGFß1 levels are not linked to kidney function and coagulation. Interleukin-6, which is elevated in patients with liver cirrhosis negatively correlated with latent TGFß1 in PVS and SVS. CONCLUSION: In patients with liver cirrhosis splanchnic organ-derived latent TGFß1 is negatively associated with the liver function.

Sterol regulatory element-binding protein 2 (SREBP2) activation after excess triglyceride storage induces chemerin in hypertrophic adipocytes.

Chemerin is an adipokine whose systemic concentration and adipose tissue expression is increased in obesity. Chemerin is highly abundant in adipocytes, yet the molecular mechanisms mediating its further induction in obesity have not been clarified. Adipocyte hypertrophy contributes to dysregulated adipokine synthesis, and we hypothesized that excess loading with free fatty acids (FFA) stimulates chemerin synthesis. Chemerin was expressed in mature adipocytes, and differentiation of 3T3-L1 cells in the presence of FFA further increased its level. TNF and IL-6 were induced by FFA, but concentrations were too low to up-regulate chemerin. Sterol regulatory element-binding protein 2 (SREBP2) was activated in these cells, indicative for cholesterol shortage. Suppression of cholesterol synthesis by lovastatin led to activation of SREBP2 and increased chemerin, and supplementation with mevalonate reversed this effect. Knockdown of SREBP2 reduced basal and FFA-induced chemerin. EMSA confirmed binding of 3T3-L1 adipocyte nuclear proteins to a SREBP site in the chemerin promotor. SREBP2 was activated and chemerin was induced in adipose tissue of mice fed a high-fat diet, and higher systemic levels seem to be derived from adipocytes. Lipopolysaccharide-mediated elevation of chemerin was similarly effective as induction by FFA, indicating that both mechanisms are equally important. Chemokine-like receptor 1 was not altered by the incubations mentioned above, and higher expression in fat of mice fed a high-fat diet may reflect increased number of adipose tissue-resident macrophages in obesity. In conclusion, the current data show that adipocyte hypertrophy and chronic inflammation are equally important in inducing chemerin synthesis.

Impaired hepatic removal of interleukin-6 in patients with liver cirrhosis.

Systemic concentrations of interleukin-6 (IL-6) are elevated in patients with liver cirrhosis, and impaired hepatic uptake of IL-6 was suggested to contribute to higher levels in these patients. To test this hypothesis IL-6 was measured in portal venous serum (PVS), hepatic venous serum (HVS) and systemic venous serum (SVS) of 41 patients with liver cirrhosis and four patients with normal liver function. IL-6 was higher in PVS than HVS of all blood donors and about 43% of portal vein derived IL-6 was extracted by the healthy liver, and 6.3% by the cirrhotic liver demonstrating markedly impaired removal of IL-6 by the latter. Whereas in patients with CHILD-PUGH stage A IL-6 in HVS was almost 25% lower than in PVS, in patients with CHILD-PUGH stage C IL-6 was similarly abundant in the two blood compartments. Ascites is a common complication in cirrhotic patients and was associated with higher IL-6 levels in all blood compartments without significant differences in hepatic excretion. Hepatic venous pressure gradient did not correlate with the degree of hepatic IL-6 removal excluding hepatic shunting as the principal cause of impaired IL-6 uptake. Furthermore, patients with alcoholic liver cirrhosis had higher IL-6 in all blood compartments than patients with cryptogenic liver cirrhosis. Aetiology of liver cirrhosis did not affect hepatic removal rate indicating higher IL-6 synthesis in patients with alcoholic liver cirrhosis. In summary, the current data provide evidence that impaired hepatic removal of IL-6 is explained by hepatic shunting and liver dysfunction in patients with liver cirrhosis partly explaining higher systemic levels.

C1q/TNF-related protein-3 represents a novel and endogenous lipopolysaccharide antagonist of the adipose tissue.

Proteins secreted by adipocytes (adipokines) play an important role in the pathophysiology of type 2 diabetes mellitus and the associated chronic and low-grade state of inflammation. It was the aim to characterize the antiinflammatory potential of the new adipocytokine, C1q/TNF-related protein-3 (CTRP-3), which shows structural homologies to the pleiotropic adipocytokine adiponectin. mRNA and protein expression of CTRP-3 was analyzed by RT-PCR and Western blot. Recombinant CTRP-3 and small interfering RNA-based strategies were used to investigate the effect of CTRP-3 on toll-like receptor (TLR) ligand, lipopolysaccharide (LPS)-, and lauric acid-induced chemokine release of monocytes and adipocytes. Together with complex ELISA-based techniques, a designed TLR4/myeloid differentiation protein-2 fusion molecule shown to bind LPS was used to prove the ability of CTRP-3 to act as endogenous LPS antagonist. CTRP-3 is synthesized in monocytes and adipocytes. The recombinant protein dose-dependently inhibits the release of chemokines in monocytes and adipocytes that were induced by lauric acid, LPS, and other TLR ligands in vitro and ex vivo. CTRP-3 inhibits monocyte chemoattractant protein-1 release in adipocytes, whereas small interfering RNA-mediated knockdown of CTRP-3 up-regulates monocyte chemoattractant protein-1 release, reduces lipid droplet size, and decreases intracellular triglyceride concentration in adipocytes, causing a dedifferentiation into a more proinflammatory and immature phenotype. By using a designed TLR4/MD-2 fusion molecule, it is shown by different techniques that CTRP-3 specifically and effectively inhibits the binding of LPS to its receptor, TLR4/MD-2. CTRP-3 inhibits three basic and common proinflammatory pathways involved in obesity and type 2 diabetes mellitus (adipo-inflammation) by acting as an endogenous LPS antagonist of the adipose tissue.

Toll-like receptor ligands cause proinflammatory and prodiabetic activation of adipocytes via phosphorylation of extracellular signal-regulated kinase and c-Jun N-terminal kinase but not interferon regulatory factor-3.

Here, we aim to investigate the mechanisms of Toll-like receptor (TLR)-induced prodiabetic and proinflammatory activation of adipocytes and to detect differences in the responsiveness of TLRs to their respective ligands between adipocytes isolated from inflamed vs. noninflamed adipose tissue. Experiments using specific ligands for all known TLRs were performed in murine 3T3-L1 adipocytes and in human adipocytes isolated from noninflamed and inflamed adipose tissue. IL-6 and monocyte chemoattractant protein-1 (MCP-1) release were measured by ELISA. The expression of the signal transduction proteins phospho-extracellular signal-regulated kinase (P-Erk), P-c-Jun N-terminal kinase (JNK), and P-interferon regulatory factor-3 was investigated by Western blot analysis. Additionally, functional inhibitors of MAPK kinase-1/-2 and JNK-1/-2 were used in the stimulation experiments. Activation of TRL4 by lipopolysaccharide (LPS) and TLR1/2 by Pam(3)Cys up-regulates IL-6 and MCP-1 release in adipocytes via specific activation of Erk. Stimulation of adipocytes by macrophage activating lipopeptide-2 (MALP-2) induces MCP-1 but has no effect on IL-6 release. This stimulatory effect on MCP-1 release is antagonized by inhibition of both mitogen-activated protein kinase-1/-2 and JNK-1/-2. Phosphorylation of Erk and JNK is up-regulated after stimulation by MALP-2. In human adipocytes isolated from noninflamed adipose tissue, LPS and Pam(3)Cys, but not MALP-2, are potent inducers of IL-6 and MCP-1. MALP-2 is able to induce IL-6 and MCP-1 release in adipocytes isolated from inflamed adipose tissue, whereas these adipocytes lost their ability to respond to LPS. The present results point to a role of the adipose tissue in innate immunity. TLR-ligand-induced proinflammatory and prodiabetic activation of adipocytes might couple visceral adipose tissue dysfunction with insulin resistance and type 2 diabetes mellitus.

Serum galectin-3 is elevated in obesity and negatively correlates with glycosylated hemoglobin in type 2 diabetes.

CONTEXT: Adipocytes synthesize galectin-3 whose deficiency protects from inflammation associated with metabolic diseases. We aimed to study circulating galectin-3 in obesity and type 2 diabetes (T2D). STUDY DESIGN: Galectin-3 was measured by ELISA in the serum of male normal-weight and overweight controls and T2D patients and in T2D patients of both sexes. Because visceral fat contributes to systemic inflammation, galectin-3 was analyzed in paired samples of human and rodent sc and visceral adipose tissue. Visceral adipose tissue adipokines are released to the portal vein, and galectin-3 was analyzed in portal, hepatic, and systemic venous serum (PVS, HVS, and SVS, respectively) of patients with liver cirrhosis and in patients who underwent surgery for nonhepatic diseases. The effect of metformin on adipocyte galectin-3 was analyzed by immunoblot. RESULTS: Circulating galectin-3 was similarly elevated in T2D and obesity compared with normal-weight individuals and revealed a body mass index-dependent positive correlation with leptin, resistin, IL-6, and age. In T2D patients, galectin-3 was increased in serum of patients with elevated C-reactive protein and negatively correlated with glycated hemoglobin. Metformin treatment was associated with lower systemic galectin-3. Reduced galectin-3 in metformin-incubated human adipocytes indicated that low galectin-3 may be a direct effect of this drug. Galectin-3 was higher in PVS compared with HVS and SVS, suggesting that the splanchnic region is a major site of galectin-3 synthesis. Low galectin-3 in HVS compared with PVS demonstrated hepatic removal. CONCLUSIONS: Systemic galectin-3 is elevated in obesity and negatively correlates with glycated hemoglobin in T2D patients, pointing to a modifying function of galectin-3 in human metabolic diseases.

Effects of the new adiponectin paralogous protein CTRP-3 and of LPS on cytokine release from monocytes of patients with type 2 diabetes mellitus.

AIMS/HYPOTHESIS: It was the aim to investigate the hypothesis that the new C1q/TNF-family member CTRP-3 (C1q/TNF-related protein-3) acts anti-inflammatory in human monocytes from healthy controls and patients with type 2 diabetes mellitus (T2D). METHODS: Monocytes were isolated from 20 healthy controls and 30 patients with T2D. IL-6 and TNF concentrations were measured by ELISA. CTRP-3 was expressed in insect cells and used for stimulation experiments. RESULTS: Basal IL-6 and TNF were not different in control and in T2D monocytes. LPS-stimulation (1 microg/ml) significantly (p<0.001) increased IL-6 and TNF in the supernatants of control and in T2D monocytes to a similar extent. CTRP-3 (1 microg/ml) significantly (p=0.03) inhibited LPS-induced IL-6 in control monocytes but not in T2D monocytes. TNF upon co-stimulation with LPS and CTRP-3 was significantly (p=0.012) lower in control than in T2D monocytes. LPS-induced TNF concentration was significantly and positively correlated with serum total cholesterol and LDL cholesterol in T2D patients. CONCLUSIONS: CTRP-3 inhibits LPS-induced IL-6 and TNF release. This anti-inflammatory effect is lost in T2D. Serum cholesterol concentration affects the pro-inflammatory potential of LPS to induce TNF release from T2D monocytes in the presence or absence of CTRP-3. CTRP-3 might partly account for the pro-inflammatory state in T2D.

Circulating levels of chemerin and adiponectin are higher in ulcerative colitis and chemerin is elevated in Crohn's disease.

BACKGROUND: Chemerin is an adipokine that stimulates chemotaxis of cells of the innate immune system. Inflammatory bowel disease (IBD) is linked to an impaired immune response and, therefore, we hypothesized that systemic chemerin may be altered in IBD patients. METHODS: Serum was collected from patients with Crohn's disease (CD, 230 patients), ulcerative colitis (UC, 80 patients), and healthy controls (HC, 80 probands). Chemerin and adiponectin, which has already been measured in the serum of similar cohorts by others, were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Chemerin was elevated in IBD compared to HC and was higher in male CD than UC patients. Female and male CD patients had lower adiponectin levels compared to UC, and adiponectin was lower in female CD patients compared to female HC. Adiponectin tended to be higher in female and male UC patients compared to HC and this difference became significant in the whole study group. Correlations with disease activity were only found in males. Here, chemerin was higher in CD patients on remission but was reduced in UC with nonactive disease. Adiponectin was higher in UC with inactive disease. Treatment with corticosteroids was linked to elevated adiponectin in male CD patients and higher chemerin in female UC patients. Unlike adiponectin, which was elevated in female serum in all cohorts, chemerin was only higher in female UC patients. CONCLUSIONS: These findings further indicate potential regulatory functions of adipokines in intestinal inflammation that are partly gender-dependent and that may even be associated with the distinct immunopathogenesis of UC and CD.

Adiponectin-stimulated CXCL8 release in primary human hepatocytes is regulated by ERK1/ERK2, p38 MAPK, NF-kappaB, and STAT3 signaling pathways.

Adiponectin is believed to exert hepatoprotective effects and induces CXCL8, a chemokine that functions as a survival factor, in vascular cells. In the current study, it is demonstrated that adiponectin also induces CXCL8 expression in primary human hepatocytes but not in hepatocellular carcinoma cell lines. Knock down of the adiponectin receptor (AdipoR) 1 or AdipoR2 by small-interfering RNA indicates that AdipoR1 is involved in adiponectin-stimulated CXCL8 release. Adiponectin activates nuclear factor (NF)-kappaB in primary hepatocytes and pharmacological inhibition of NF-kappaB, the p38 mitogen-activated protein kinase, and extracellular signal-regulated kinase (ERK) 1/ERK2 reduces adiponectin-mediated CXCL8 secretion. Furthermore, adiponectin also activates STAT3 involved in interleukin (IL)-6 and leptin-mediated CXCL8 induction in primary hepatocytes. Inhibition of JAK2 by AG-490 does not abolish adiponectin-stimulated CXCL8, indicating that this kinase is not involved. Pretreatment of primary cells with "STAT3 Inhibitor VI," however, elevates hepatocytic CXCL8 secretion, demonstrating that STAT3 is a negative regulator of CXCL8 in these cells. In accordance with this assumption, IL-6, a well-characterized activator of STAT3, reduces hepatocytic CXCL8. Therefore, adiponectin-stimulated induction of CXCL8 seems to be tightly controlled in primary human hepatocytes, whereas neither NF-kappaB, STAT3, nor CXCL8 are influenced in hepatocytic cell lines. CXCL8 is a survival factor, and its upregulation by adiponectin may contribute to the hepatoprotective effects of this adipokine.

Innate immunity and adipocyte function: ligand-specific activation of multiple Toll-like receptors modulates cytokine, adipokine, and chemokine secretion in adipocytes.

The aim of this study was to analyze Toll-like receptor (TLR) expression in preadipocytes and mature adipocytes and to investigate whether TLR ligands influence the release of cytokines, chemokines, and adipokines. Murine 3T3-L1 preadipocytes and mature adipocytes were used for stimulation experiments. The effects of lipopolysaccharide (LPS), flagellin, Poly (U), Poly (I:C), macrophage-activating lipopeptide-2 (MALP2), Pam3Cys, and CpG on the release of interleukin-6 (IL-6), resistin, and monocyte chemoattractant protein-1 (MCP-1) were determined by enzyme-linked immunosorbent assay (ELISA). Nuclear translocation and promoter binding of NFkappaB were analyzed by electrophoretic mobility shift assays. TLR expression was investigated by reverse-transcriptase (RT-PCR). All TLRs except TLR5 and TRL7 are expressed in the stromal vascular cell (SVC) fraction and in mature adipocytes of different fat stores. Whereas basal and LPS-induced IL-6 release is higher in preadipocytes, basal and LPS-induced MCP-1 release is higher in mature adipocytes. Mature adipocytes respond to corticosterone regarding MCP-1 and resistin release. The ligands for TLRs influence IL-6, MCP-1, and resistin release differentially. Some of these ligands induce nuclear translocation and promoter binding of NFkappaB. Besides TLR5, that is not expressed in mature adipocytes, all TLR family members are involved. There exists a functional TRL pathway in adipocytes that connects innate immunity with adipocyte function. As a consequence, the role of the adipose tissue in both immunity and metabolism has to be investigated in future studies. The results of this approach will help to explain the metabolic changes such as insulin resistance observed during infection and the immunological phenomena such as macrophage infiltration of adipose tissue seen in obesity.

Fatty acid-induced induction of Toll-like receptor-4/nuclear factor-kappaB pathway in adipocytes links nutritional signalling with innate immunity.

To study the effects of fatty acids and the involvement of the Toll-like receptor-4/nuclear factor-kappaB (TLR-4/NF-kappaB) pathway with respect to the secretion of adipokines from adipocytes 3T3-L1 adipocytes were stimulated with increasing doses of fatty acids. The secretion of adiponectin, resistin and monocyte chemoattractant protein-1 (MCP-1) was measured by enzyme-linked immunosorbent assay. The NF-kappaB p65 nuclear translocation and TLR-4 expression were investigated by Western blot. The effects mediated by NF-kappaB were tested using a specific NF-kappaB-inhibitor and TLR-4-induced effects were analysed with a neutralizing TLR-4 antibody. Binding of (14)C-labelled fatty acids to TLR-4/MD-2 was investigated using a FLAG-tagged extracellular part of TLR-4 fused to full-length MD-2 via a linker (lipopolysaccharide-Trap). The messenger RNA (mRNA) expression of adipokines in abdominal adipose tissue of rats fed a standard chow or a high-fat diet was investigated by reverse transcription-polymerase chain reaction. The TLR-4 is induced during adipocyte differentiation and its expression is enhanced following fatty acid stimulation. The stimulatory effects of stearic and palmitic acids on MCP-1 secretion and of palmitoleic acid on resistin secretion are mediated via NF-kappaB. The stimulatory effects of stearic, palmitic and palmitoleic acids on resistin secretion and the stimulatory effect of stearic acid on MCP-1 secretion are mediated via TLR-4. Fatty acid-mediated effects are caused by an endogenous ligand because fatty acids were shown not to bind directly to TLR-4/MD-2. Adipose tissue mRNA expression and serum levels of adipokines did not differ in rats fed a high-fat diet. These data provide a new molecular mechanism by which fatty acids can link nutrition with innate immunity.

Reduced response to adiponectin and lower abundance of adiponectin receptor proteins in type 2 diabetic monocytes.

The abundance of the adiponectin receptors, AdipoR1 and AdipoR2, and the effects of the antidiabetic adipokine adiponectin in monocytes of normal-weight and overweight controls and type 2 diabetic patients (T2D) were analyzed. AdipoR1 and AdipoR2 mRNAs were increased in monocytes of obese controls and T2D patients when compared to normal-weight controls, and AdipoR1 mRNA positively correlated to AdipoR2 mRNA, the waist to hip ratio and systemic adiponectin. However, AdipoR1 and AdipoR2 proteins were lower in monocytes of T2D compared to normal-weight donors. Induction of IL-6 and IL-8 by adiponectin, an effect involving p38 MAPK, was also reduced in T2D monocytes.

Effects of the new C1q/TNF-related protein (CTRP-3) "cartonectin" on the adipocytic secretion of adipokines.

BACKGROUND: Cartonectin (collagenous repeat-containing sequence of 26-kDa protein; CORS-26) was described as a new adipokine of the C1q/TNF molecular superfamily C1q/TNF-related protein-3 (CTRP-3), secreted by the adipocytes of mice and humans. The receptor and function of cartonectin are unknown and the recombinant protein is not commercially available. OBJECTIVE: To investigate the effects of recombinant cartonectin on the secretion of adipokines such as adiponectin, leptin, and resistin from adipocytes of human and murine origin. The effect of the BMI of the adipocyte donor was also investigated. METHODS AND PROCEDURES: Human adipocytes from pooled lean and preobese healthy individuals and murine 3T3-L1 adipocytes were used for stimulation experiments. Recombinant cartonectin was expressed in insect H5 cells. Adipokine secretion was measured using enzyme-linked immunosorbent assay. In addition, western blot analysis and luciferase reporter gene assays were employed. RESULTS: Cartonectin (1, 10, 50, and 250 ng/ml) in higher doses stimulates the secretion of adiponectin and resistin from murine adipocytes. This effect is not caused by an induction of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) protein expression, as confirmed by western blot analysis. Also, luciferase reporter gene assay revealed that cartonectin failed to induce luciferase activity at the peroxisome proliferator-activated receptor responsive element site containing the adiponectin/luciferase promoter fragment. Human adipocytes from lean individuals secrete higher amounts of adiponectin and leptin when compared with adipocytes of individuals with a preobesity BMI (25-30 kg/m(2)). Cartonectin failed to stimulate adiponectin or leptin secretion from human adipocytes, irrespective of the BMI value. DISCUSSION: Cartonectin is a new adipokine that differentially regulates the secretion of classical adipokines, with marked differences between the human and the murine systems. These effects are species-dependent, while basal adipokine secretion is influenced by the BMI.