OBJECTIVEThe metabolic syndrome (MetS) is highly prevalent and confers an increased risk for diabetes and cardiovascular disease (CVD). While MetS is a proinflammatory state, there is a paucity of data on cellular inflammation in MetS. Toll-like receptors (TLRs) are classical pattern recognition receptors of the innate immune response.RESEARCH DESIGN AND METHODSThe aim of this study was to examine monocyte TLR2 and TLR4 in MetS patients without diabetes or CVD and control subjects since both of the receptors have been implicated in atherosclerosis and insulin resistance. Fasting blood was obtained for TLR expression and activity.RESULTSCirculating levels of high-sensitivity C-reactive protein, interleukin (IL)-1β, IL-6, IL-8, and soluble tumor necrosis factor receptor 1 (sTNFR1) were significantly increased in MetS versus control subjects following adjustment for waist circumference. There was a significant increase in both TLR2 and TLR4 surface expression and mRNA on monocytes after adjustment for waist circumference. In addition to increased nuclear factor-κB nuclear binding, there was significantly increased release of IL-1β, IL-6, and IL-8 in MetS versus control subjects following priming of the monocytes with lipopolysaccharides. While both plasma free fatty acids and endotoxin were increased in MetS, they correlated significantly with TLR4 only.CONCLUSIONSIn conclusion, we make the novel observation that both TLR2 and TLR4 expression and activity are increased in the monocytes of patients with MetS and could contribute to increased risk for diabetes and CVD.

Several lines of evidence support a role for oxidative stress in diabetic complications Diabetic patients have increased O2− production in monocytes. Loss of SIRT1 activity may be associated with metabolic diseases such as diabetes. Several studies have shown that SIRT1 can regulate mammalian FOXO transcription factors through direct binding and/or deacetylation. However, interactions between SIRT1 and FOXO under diabetic conditions are unclear. The phytochemical resveratrol, has recently gained attention for its protection against metabolic disease. Resveratrol has been shown to increase mitochondrial function by activating SIRT1. In this study, we tested the protective effect of resveratrol on cellular oxidative stress through the SIRT1-FOXO pathway under high-glucose conditions. Human monocytic (THP-1) cells were cultured in presence of mannitol (osmolar control) or normoglycemic (NG, 5.5 mmol/L glucose) or hyperglycemic (HG, 25 mmol/L glucose) conditions in absence or presence of resveratrol (3 and 6 µmol/L) for 48 h. We first examined SIRT1 activity and oxidative stress in monocytes of T1DM patients compared to healthy controls. In T1DM patients, monocytic SIRT1 expression was significantly decreased and p47phox expression was increased compared to controls. Under HG in vitro, SIRT1 and FOXO3a were significantly decreased compared to NG, this was reversed by resveratrol treatment, concomitant with reduction in HG-induced superoxide production and p47phox. Under HG, SIRT1 small interfering RNA (siRNA), inhibited FOXO3a and there was no beneficial effect of resveratrol in siRNA treated HG-induced cells. Thus, resveratrol decreases HG-induced superoxide production via upregulation of SIRT1, induction of FOXO3a and inhibition of p47phox in monocytes.

Diabetes is a proinflammatory state. The pattern recognition receptors, Toll-like receptors (TLRs), are increased in diabetic patients and have been suggested to play a role in diabetic nephropathy (DN). Progression of DN involves altered mesangial cell (MC) function with an expansion of the mesangial matrix. There is a paucity of data examining the role of TLR and its expression in MC. We hypothesize the expression of TLRs in the mesangium might be an important factor contributing to mesangium expansion and nephropathy. Thus we evaluated the effect of high glucose on TLR2 and TLR4 expression in mouse mesangial cells (MMC) in vitro. Exposure of MMC to 25 mM glucose for 24 h resulted in increased TLR4 mRNA and cell surface receptor expression compared with 5.5 mM glucose (P < 0.05). Interestingly, we were not able to detect expression of TLR2 in MMC. Furthermore, expression of a TLR4 downstream signaling cascade including myeloid differentiation factor 88 (MyD88), interferon regulatory factor 3 (IRF3), and Toll interleukin receptor domain containing adaptor inducing interferon-β (TRIF)-related adaptor molecule (TRAM) were significantly increased in cells exposed to 25 mM glucose (P < 0.05). There was also a significant increase in NF-κB activation along with increased secretion of inflammatory cytokines IL-6 and monocyte chemotactic protein-1. Levels of transforming growth factor-β were also significantly increased in the presence of 25 mM glucose (P < 0.05). Collectively, these data suggest that hyperglycemia activates TLR4 expression and activity in MC and could contribute to DN.

The Klotho gene has been identified as an aging suppressor gene that encodes a transmembrane protein, which is expressed primarily in renal tubules. There are 2 forms of Klotho, membrane and secreted. However, there is a paucity of data on levels of soluble Klotho in diseases like diabetes and kidney disease. We validated an enzyme-linked immunosorbent assay for Klotho and quantitated Klotho levels separately in patients with diabetes and also in patients with chronic kidney disease (CKD). The Klotho assay showed good precision and was linear down to 19 ng/mL. There were no significant effects on Klotho levels with the addition of common interferents such as ascorbate, triglycerides, or hemolysis; only bilirubin (250 mg/L) significantly reduced Klotho levels (P < .05). There was a significant reduction in Klotho levels in samples with glycated hemoglobin (HbA(1c)) levels of 6.5% or more compared with control samples (HbA(1c) < 6.5%; P < .001). We also documented significantly higher levels of Klotho with CKD. Thus, we validated an assay for Klotho and made the novel observation that levels are decreased in diabetes and increased in CKD.

No abstract