Increased Release of Proinflammatory Proteins in Primary Human Adipocytes and Activation of the Inflammatory NFĸB, p38, and ERK Pathways upon Omentin Treatment.

OBJECTIVES: To investigate the impact of omentin on the release of inflammation-related biomarkers and inflammatory pathways in primary human adipocytes. METHODS: Adipocytes were treated with or without omentin (500 and 2,000 ng/mL), and the supernatants were analyzed for inflammation-related biomarkers using proximity extension assay technology. Potential upstream regulators of the omentin-stimulated proteins were identified using Ingenuity Pathway Analysis. Protein levels of components of inflammatory pathways were measured using Western blotting. RESULTS: 2,000 ng/mL omentin induced the release of 30 biomarkers 97.1 ± 31.1-fold in the supernatants (all p < 0.05). Most biomarkers were proin-flammatory chemokines and cytokines. We identified the transcription factor nuclear factor "kappa-light-chain-enhancer" of activated B cells (NFĸB) and the kinases p38 and extracellular signal-regulated kinase (ERK)1/2 as potential upstream regulators in silico. On the cellular level, treatment with 2,000 ng/mL omentin for 24 h enhanced the phosphorylation levels of NFĸB 2.1 ± 0.3-fold (p < 0.05), of p38 2.6 ± 0.4-fold (p < 0.05), and of ERK1/2 1.8 ± 0.2-fold (p < 0.05). CONCLUSIONS: These data argue that omentin exerts proinflammatory effects through the activation of the inflammatory NFĸB, p38, and ERK1/2 pathways in cultured primary adipocytes.

Sfrp5 increases glucose-stimulated insulin secretion in the rat pancreatic beta cell line INS-1E.

Previous studies reported that secreted frizzled-related protein-5 (Sfrp5) decreases beta cell proliferation and increases fasting insulin levels, but studies on direct effects of Sfrp5 on insulin secretion and its underlying mechanisms are missing. This study examined effects of Sfrp5 on (i) beta cell viability and proliferation, (ii) basal and glucose-stimulated insulin secretion and (iii) canonical and non-canonical Wnt signalling pathways. We incubated rat INS-1E cells with 0.1, 1 or 5 µg/ml recombinant Sfrp5 for 24h. We measured basal and glucose-stimulated insulin secretion at glucose concentrations of 2.5 and 20 mmol/l. Phosphorylated and total protein content as well as mRNA levels of markers of cell proliferation, canonical and non-canonical Wnt signalling pathways were examined using Western blotting and real-time PCR. Differences between treatments were analysed by repeated measurement one-way ANOVA or Friedman's test followed by correction for multiple testing using the Benjamini-Hochberg procedure. At 5 µg/ml, Sfrp5 reduced mRNA levels of cyclin-B1 by 25% (p<0.05). At 1 and 5 µg/ml, Sfrp5 increased glucose-stimulated insulin secretion by 24% and by 34% (both p<0.05), respectively, but had no impact on basal insulin secretion. Sfrp5 reduced the phosphorylation of the splicing forms p46 and p54 of JNK by 39% (p<0.01) and 49% (p<0.05), respectively. In conclusion, Sfrp5 reduced markers of cell proliferation, but increased in parallel dose-dependently glucose-stimulated insulin secretion in INS-1E cells. This effect is likely mediated by reduced JNK activity, an important component of the non-canonical Wnt signalling pathway.

Omentin-regulated proteins combine a pro-inflammatory phenotype with an anti-inflammatory counterregulation in human adipocytes: A proteomics analysis.

AIMS: Experimental and epidemiological studies reported controversial data on the role of omentin in type 2 diabetes and cardiovascular diseases. This study aimed to characterise the impact of omentin on the secretome of human adipocytes to analyse the enrichment of these proteins in metabolic and cellular signalling pathways underlying its physiological function. MATERIAL/METHODS: Differentiated primary human adipocytes were treated without or with 500 or 2000 ng/mL omentin for 24 hours. The secretome was analysed by liquid chromatography coupled tandem-mass spectrometry. Differences in protein secretion between untreated and omentin-treated adipocytes were compared using a paired t-test. Other potential upstream regulators and the overrepresentation in canonical pathways of omentin-stimulated proteins were analysed using Ingenuity Pathway Analysis. RESULTS: The supernatant of adipocytes contained 3493 proteins, of which 140 were differentially secreted by both concentrations of omentin compared with untreated adipocytes. Among the most strongly increased proteins, tumour necrosis factor-inducible gene 6 protein (TNFAIP6) was increased by 140-fold in the supernatant. Omentin-regulated proteins were overrepresented in seven canonical pathways including eukaryotic initiation factor 2 signalling, complement system, and inhibition of matrix metalloproteases. We further identified 25 other potential upstream activators of omentin-regulated proteins, mainly pro-inflammatory cytokines and transcription regulators including NFκB. CONCLUSIONS: In differentiated human adipocytes, the release of the anti-inflammatory TNFAIP6 might be part of a counterregulatory response to the pro-inflammatory action of omentin. Omentin-regulated proteins were overrepresented in pathways indicating cellular stress, a pro-inflammatory environment and a crosstalk with other organs. Other potential activators of omentin-regulated proteins point towards a central role of NFκB activation in the omentin-induced secretory process.

Effect of Sfrp5 on cytokine release and insulin action in primary human adipocytes and skeletal muscle cells.

Secreted frizzled-related protein 5 (Sfrp5) is an adipokine with anti-inflammatory and insulin-sensitizing properties in mice. However, the mechanism of Sfrp5 action, especially in humans, is largely unknown. Therefore, cytokine release and insulin signaling were analyzed to investigate the impact of Sfrp5 on inflammation and insulin signaling in primary human adipocytes and skeletal muscle cells (hSkMC). Sfrp5 neither affected interleukin (IL)-6, monocyte chemoattractant protein-1 (MCP-1) and adiponectin release from human adipocytes, nor IL-6 and IL-8 release from hSkMC. In tumor necrosis factor (TNF) α-treated adipocytes, Sfrp5 reduced IL-6 release by 49% (p<0.05), but did not affect MCP-1 and adiponectin release. In MCP-1-treated hSkMC, Sfrp5 did not affect cytokine secretion. In untreated adipocytes, Sfrp5 decreased the insulin-mediated phosphorylation of Akt-Ser473, Akt-Thr308, GSK3α-Ser21 and PRAS40-Thr246 by 34% (p<0.01), 31% (p<0.05), 37% (p<0.05) and 34% (p<0.01), respectively, and the stimulation of glucose uptake by 25% (p<0.05). Incubation with TNFα increased the phosphorylation of JNK and NFκB, and impaired insulin signaling. When Sfrp5 and TNFα were combined, there was no additional effect on insulin signaling and JNK phosphorylation, but phosphorylation of NFκB was reversed to basal levels. Sfrp5 had no effect on insulin signaling in untreated or in MCP-1 treated hSkMC. Thus, Sfrp5 lowered IL-6 release and NFκB phosphorylation in cytokine-treated human adipocytes, but not under normal conditions, and decreased insulin signaling in untreated human adipocytes. Sfrp5 did not act on hSkMC. Therefore, the cellular actions of Sfrp5 seem to depend on the type of tissue as well as its inflammatory and metabolic state.

Hypoadiponectinemia and proinflammatory state: two sides of the same coin?: results from the Cooperative Health Research in the Region of Augsburg Survey 4 (KORA S4).

OBJECTIVE: Previous studies have yielded conflicting results on the association of adiponectin levels and inflammation. Low systemic concentrations of adiponectin, as well as elevated levels of immune mediators, represent risk factors for the development of type 2 diabetes and coronary artery disease. The major aim of this cross-sectional study was to investigate the interdependence of hypoadiponectinemia and low-grade systemic inflammation. RESEARCH DESIGN AND METHODS: The study sample consisted of 606 participants aged 55-74 years (244 with normal glucose tolerance, 242 with impaired glucose tolerance, and 120 with newly diagnosed type 2 diabetes) of the population-based KORA S4 (Cooperative Health Research in the Region of Augsburg Survey 4; 1999-2001). Systemic concentrations of adiponectin and a wide range of anthropometric, metabolic, and inflammatory variables were available for analyses. The association of adiponectin with 15 immunological markers, including leukocyte count, acute-phase proteins, cytokines, cytokine receptors, and chemokines, was assessed using univariable and multivariable models. RESULTS: No evidence for a significant correlation between adiponectin and all immunological parameters except eotaxin could be found after multivariable adjustments, whereas multiple strong correlations with obesity and metabolic factors were present. CONCLUSIONS: From these data, we conclude that hypoadiponectinemia and a proinflammatory state are largely independent from each other.

The proatherogenic cytokine interleukin-18 is secreted by human adipocytes.

OBJECTIVE: Interleukin (IL)-18, an important mediator of innate immunity and strong risk factor for the development of cardiovascular disease, was shown recently to be elevated in obesity. The aim of our study was to investigate whether human adipocytes produce IL-18. METHODS: Human adipose tissue was obtained from lean women undergoing elective plastic surgery and from obese individuals undergoing laparoscopic surgery (gastric banding). Preadipocytes from mammary adipose tissue were isolated and differentiated under defined adipogenic conditions. IL-18 expression was analyzed by real-time reverse transcriptase PCR, ELISA and immunocytochemistry. RESULTS: Human preadipocytes of all differentiation stages spontaneously secreted IL-18. In parallel significant amounts of IL-18 mRNA were detected. Freshly isolated mature adipocytes from subcutaneous and omental depots also released IL-18. IL-18 release from adipocytes from obese donors was about 3-fold higher compared to adipocytes from non-obese donors. CONCLUSIONS: We conclude that human adipose tissue produces IL-18 and thereby contributes to systemic IL-18 concentrations. This finding supports the concepts that adipocytes behave as primitive immune cells and that IL-18 may mediate some of the detrimental complications of obesity such as cardiovascular disease and type 2 diabetes.

Comparison of proliferation and differentiation capacity of human adipocyte precursor cells from the omental and subcutaneous adipose tissue depot of obese subjects.

Upper body obesity is characterized by an expansion of the visceral adipose tissue and is associated with an increased susceptibility for type 2 diabetes and cardiovascular disease. In order to get a better understanding of the regulation of body fat distribution, the aim of the present study was to compare adipocyte development between the omental and subcutaneous adipose tissue region in obese subjects. Therefore, the proliferation and differentiation capacity in primary cultures of adipose tissue-derived stromal cells were compared between the 2 depots in a group of 29 obese individuals, of which 21 were women. Proliferation of the cells was stimulated using fetal calf serum (FCS) and assessed by counting the cell number in the culture dishes. Differentiation of preadipocytes was assessed in parallel by morphological criteria and determination of glycerol-3-phosphate dehydrogenase (GPDH) after stimulation by standardized adipogenic conditions. Stromal cells from the subcutaneous adipose tissue region proliferated faster (doubling time, 4 +/- 1 days) than those from the omental region (doubling time, 5 +/- 1 days), whereas there was no regional difference in adipose differentiation with any of the adipogenic media. The same findings were observed when men were excluded from the analysis. Interestingly, there were more endothelial cells in the cultures from the omental tissue as compared to those from the subcutaneous tissue, but there was no correlation between endothelial cell contamination and proliferation capacity, suggesting that the regional difference in proliferation capacity was not due to regional differences in the amount of endothelial cells. In addition, we found a negative correlation between donor age and proliferation of subcutaneous cells but not of omental cells, possibly explaining the greater capacity for adipose tissue growth in the omental as compared to the subcutaneous depot with aging. In conclusion, there may exist regional differences in adipose tissue growth with regard to proliferation capacity, whereas there are apparently no significant differences in in vitro differentiation capacity between subcutaneous and omental preadipocytes.

Impairment of insulin signaling in human skeletal muscle cells by co-culture with human adipocytes.

Adipocyte factors play a major role in the induction of insulin resistance in skeletal muscle. To analyze this cross-talk, we established a system of co-culture of human fat and skeletal muscle cells. Cells of three muscle donors were kept in co-culture with cells of various fat cell donors, and insulin signaling was subsequently analyzed in myocytes. Insulin-induced tyrosine phosphorylation of insulin receptor substrate (IRS)-1 was completely blocked, with unaltered expression of IRS-1. Troglitazone increased insulin action on IRS-1 phosphorylation, in both the absence and presence of co-culture. Insulin-regulated activation of Akt kinase in the myocytes was significantly reduced after co-culture, with troglitazone restoring insulin action. Addition of tumor necrosis factor (TNF)-alpha (2.5 nmol/l) to myocytes for 48 h reduced IRS-1 expression and inhibited IRS-1 and Akt phosphorylation comparable to the effect of co-culture. Lower doses of TNF-alpha were ineffective. After co-culture, TNF-alpha in the culture medium was below the detection limit of 0.3 pmol/l. A very low level of resistin was detected in the supernatant of myocytes, but not of adipocytes. In conclusion, the release of fat cell factors induces insulin resistance in human skeletal muscle cells; however, TNF-alpha and resistin appear not to be involved in this process.