examined the innate immune pathway in AbdSc AT from lean, obese, and T2DM subjects, and 4) examined the association of circulating LPS in T2DM subjects. The findings showed that LPS increased TLR-2 protein expression twofold (P Ͻ 0.05). Treatment of AbdSc adipocytes with LPS caused a significant increase in TNF-␣ and IL-6 secretion (IL-6, Control: 2.7 Ϯ 0.5 vs. LPS: 4.8 Ϯ 0.3 ng/ml; P Ͻ 0.001; TNF-␣, Control: 1.0 Ϯ 0.83 vs. LPS: 32.8 Ϯ 6.23 pg/ml; P Ͻ 0.001). NF-B inhibitor reduced IL-6 in AbdSc adipocytes (Control: 2.7 Ϯ 0.5 vs. NF-B inhibitor: 2.1 Ϯ 0.4 ng/ml; P Ͻ 0.001). AbdSc AT protein expression for TLR-2, MyD88, TRAF6, and NF-B was increased in T2DM patients (P Ͻ 0.05), and TLR-2, TRAF-6, and NF-B were increased in LPStreated adipocytes (P Ͻ 0.05). Circulating LPS was 76% higher in T2DM subjects compared with matched controls. LPS correlated with insulin in controls (r ϭ 0.678, P Ͻ 0.0001). Rosiglitazone (RSG) significantly reduced both fasting serum insulin levels (reduced by 51%, P ϭ 0.0395) and serum LPS (reduced by 35%, P ϭ 0.0139) in a subgroup of previously untreated T2DM patients. In summary, our results suggest that T2DM is associated with increased endotoxemia, with AT able to initiate an innate immune response. Thus, increased adiposity may increase proinflammatory cytokines and therefore contribute to the pathogenic risk of T2DM.toll-like receptors; adipocytes; nuclear factor-B; inflammation; insulin OBESITY IS KNOWN TO REPRESENT one of the single most important risk factors for the increased risk of type 2 diabetes mellitus (T2DM) and cardiovascular disease. In addition, an increase in central (visceral) adiposity confers higher metabolic risk. This increased metabolic risk is associated with subclinical inflammation, with several studies demonstrating increased levels of proinflammatory adipocytokines, such as IL-6 and TNF-␣ (32, 33), in patients with obesity and T2DM. Activation of proinflammatory adipocytokines in adipose tissue (AT) is coordinated through NF-B, a key transcription factor in the inflammatory cascade (2,10, 11,18,21,22,33,35,37,38). Adipocytes also secrete adiponectin (29,30,36,41,42), which has been shown to possess anti-inflammatory properties through its action on NF-B and is inversely correlated with obesity and diabetes (29,30,36,41,42). Evidence for the role of NF-B in AT has been shown in studies overexpressing the NF-B activator IKK␤ in mice, which resulted in increased inflammatory cytokine production and the onset of diabetes (7). In contrast, hepatocyte IKK␤ knockout (KO) mice demonstrated a decrease in circulating proinflammatory cytokines (3). This indicates that IKK␤ KO mice do not develop hepatic insulin resistance and glucose intolerance compared with their high-fat diet-fed counterparts. Further studies also illustrate that an inflammatory reaction, induced by the bacterial endotoxin lipopolysaccharide (LPS), is markedly attenuated in the IKK␤ KO mice (3

BackgroundEmerging data indicate that gut-derived endotoxin may contribute to low-grade systemic inflammation in insulin resistant states. This study aimed to examine the importance of serum endotoxin and inflammatory markers in non-alcoholic fatty liver disease (NAFLD) patients, with and without type 2 diabetes mellitus (T2DM), and to explore the effect of treatment with a lipase inhibitor, Orlistat, on their inflammatory status.MethodsFasted serum from 155 patients with biopsy proven NAFLD and 23 control subjects were analysed for endotoxin, soluble CD14 (sCD14), soluble tumour necrosis factor receptor II (sTNFRII) and various metabolic parameters. A subgroup of NAFLD patients were re-assessed 6 and 12 months after treatment with diet alone (n = 6) or diet plus Orlistat (n = 8).ResultsEndotoxin levels were significantly higher in patients with NAFLD compared with controls (NAFLD: 10.6(7.8, 14.8) EU/mL; controls: 3.9(3.2, 5.2) EU/mL, p < 0.001); NAFLD alone produced comparable endotoxin levels to T2DM (NAFLD: T2DM: 10.6(5.6, 14.2) EU/mL; non-diabetic: 10.6(8.5, 15.2) EU/mL), whilst a significant correlation between insulin resistance and serum endotoxin was observed (r = 0.27, p = 0.008). Both sCD14 (p < 0.01) and sTNFRII (p < 0.001) increased with severity of fibrosis. A positive correlation was also noted between sTNFRII and sCD14 in the NAFLD subjects (r = 0.29, p = 0.004).Sub-cohort treatment with Orlistat in patients with NAFLD showed significant decreases in ALT (p = 0.006), weight (p = 0.005) and endotoxin (p = 0.004) compared with the NAFLD, non-Orlistat treated control cohort at 6 and 12 months post therapy, respectively.ConclusionsEndotoxin levels were considerably increased in NAFLD patients, with marked increases noted in early stage fibrosis compared with controls. These results suggest elevated endotoxin may serve as an early indicator of potential liver damage, perhaps negating the need for invasive liver biopsy. As endotoxin may promote insulin resistance and inflammation, interventions aimed at reducing endotoxin levels in NAFLD patients may prove beneficial in reducing inflammatory burden.

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Resistin, an adipocyte secreted factor, has been suggested to link obesity with type 2 diabetes in rodent models, but its relevance to human diabetes remains uncertain. Although previous studies have suggested a role for this adipocytokine as a pathogenic factor, its functional effects, regulation by insulin, and alteration of serum resistin concentration by diabetes status remain to be elucidated. Therefore, the aims of this study were to analyze serum resistin concentrations in type 2 diabetic subjects; to determine the in vitro effects of insulin and rosiglitazone (RSG) on the regulation of resistin, and to examine the functional effects of recombinant human resistin on glucose and lipid metabolism in vitro. Serum concentrations of resistin were analyzed in 45 type 2 diabetic subjects and 34 nondiabetic subjects. Subcutaneous human adipocytes were incubated in vitro with insulin, RSG, and insulin in combination with RSG to examine effects on resistin secretion. Serum resistin was increased by approximately 20% in type 2 diabetic subjects compared with nondiabetic subjects (P = 0.004) correlating with C-reactive protein. No other parameters, including adiposity and fasting insulin levels, correlated with serum resistin in this cohort. However, in vitro, insulin stimulated resistin protein secretion in a concentration-dependent manner in adipocytes [control, 1215 +/- 87 pg/ml (mean +/- SEM); 1 nM insulin, 1414.0 +/- 89 pg/ml; 1 microM insulin, 1797 +/- 107 pg/ml (P < 0.001)]. RSG (10 nM) reduced the insulin-mediated rise in resistin protein secretion (1 nM insulin plus RSG, 971 +/- 35 pg/ml; insulin, 1 microM insulin plus RSG, 1019 +/- 28 pg/ml; P < 0.01 vs. insulin alone). Glucose uptake was reduced after treatment with 10 ng/ml recombinant resistin and higher concentrations (P < 0.05). Our in vitro studies demonstrated a small, but significant, reduction in glucose uptake with human recombinant resistin in differentiated preadipocytes. In human abdominal sc adipocytes, RSG blocks the insulin-mediated release of resistin secretion in vitro. In conclusion, elevated serum resistin in human diabetes reflects the subclinical inflammation prevalent in type 2 diabetes. Our in vitro studies suggest a modest effect of resistin in reducing glucose uptake, and suppression of resistin expression may contribute to the insulin-sensitizing and glucose-lowering actions of the thiazolidinediones.