Markus Neumeier

Fatty acid-induced induction of Toll-like receptor-4/nuclear factor-κB 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‐κB (TLR‐4/NF‐κB) 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‐κB p65 nuclear translocation and TLR‐4 expression were investigated by Western blot. The effects mediated by NF‐κB were tested using a specific NF‐κB‐inhibitor and TLR‐4‐induced effects were analysed with a neutralizing TLR‐4 antibody. Binding of 14C‐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‐κB. 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.

Different effects of adiponectin isoforms in human monocytic cells.

Adiponectin (APM) is an adipocyte‐derived adipokine with immunosuppressive, antidiabetic, and antiatherosclerotic properties. Low molecular weight (LMW)‐ and higher molecular weight (HMW)‐APM circulate in the serum and activate different signaling pathways. We were interested to see whether LMW‐APM exerts different effects on monocytic cells compared with the HMW isoform. Therefore, the effects of recombinant LMW‐APM produced in insect cells and the APM from higher eukaryotic cells containing HMW forms on monocytic cells were investigated with respect to apoptosis and inflammation. LMW‐ and HMW‐APM induce apoptosis in nondifferentiated THP‐1 cells, reduce macrophage scavenger receptor (MSR) A mRNA expression, and stimulate phosphorylation of adenosine monophosphate‐activated protein kinase (AMPK). However, HMW‐APM induces the secretion of interleukin (IL)‐6 in human monocytes and THP‐1 cells but does not suppress lipopolysaccharide (LPS)‐induced IL‐6 secretion. In contrast, LMW‐APM reduces LPS‐mediated IL‐6 release and furthermore, stimulates IL‐10 secretion, most likely by reducing the abundance of inhibitor of nuclear factor (NF)‐κB kinase β, leading to a diminished nuclear translocation of NF‐κB p65. Our data indicate that the different APM isoforms do share common effects on monocytic cells but also induce isoform‐specific responses. Although apoptosis, the activation of AMPK, and the reduction of MSR are mediated by all APM isoforms, only LMW‐APM displays anti‐inflammatory properties.

Systemic chemerin is related to inflammation rather than obesity in type 2 diabetes

Background  The adipokine chemerin modulates the function of innate immune cells and may link obesity and inflammation, and therefore, a possible relation of chemerin to inflammatory proteins in obesity and type 2 diabetes (T2D) was analysed. As visceral fat contributes to systemic inflammation, chemerin was measured in portal venous (PVS), hepatic venous (HVS) and systemic venous (SVS) blood of patients with liver cirrhosis.

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.

Profiling adipocytokine secretion from creeping fat in Crohn's disease

Background: Adipose tissue is recognized as a compartment secreting highly active molecules. Creeping fat represents a characteristic feature of Crohns disease (CD). Proinflammatory or anti‐inflammatory adipose‐derived secretory products, now generally called adipocytokines, may play a role in the pathogenesis of CD. Materials and Methods: Adipose tissue specimens were obtained from creeping fat contiguous to the involved intestine of 10 patients with CD. Mesenteric adipose tissue specimens resected from 13 patients with colon cancer (CC) and from 7 patients with diverticulitis served as controls. Three fat tissue specimen per well and 6 to 8 wells per patient were incubated ex vivo for 24 h. The release of adiponectin, resistin, leptin, interleukin‐6, macrophage colony‐stimulating factor, monocyte chemotactic protein‐1, and migration inhibitory factor was measured by ELISA. The expression of AdipoR1 and AdipoR2 receptors was investigated by real‐time quantitative polymerase chain reaction in a subset of adipose tissues. Results: The secretion of adiponectin and macrophage colony‐stimulating factor, as well as leptin and migration inhibitory factor, was significantly upregulated in CD compared with CC and diverticulitis or CC only, respectively. Resistin, interleukin‐6, and monocyte chemotactic protein‐1 were not specifically induced in CD but were associated with unspecific inflammation. Adiponectin receptor expression was not different in CC, CD, or diverticulitis. Steroid treatment in CD patients had differential effects on the ex vivo secretion of adipocytokines. Conclusions: A specific secretion pattern of proinflammatory and anti‐inflammatory adipocytokines indicates the significance of adipose tissue in intestinal inflammation in CD. Future investigations of this intestinal compartment may provide novelinsights into the pathophysiological role of creeping fat and CD.

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 NFκB 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 NFκB. 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.

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.

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.

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 Crohns 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. (Inflamm Bowel Dis 2009;)

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.