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NPY is an important central orexigenic hormone, but little is known about its peripheral actions in human adipose tissue (AT) or its potential paracrine effects. Our objective was to examine NPY's role in AT, specifically addressing NPY protein expression, the effect of NPY on adipokine secretion, and the influence of insulin and rosiglitazone (RSG) on adipocytederived NPY in vitro. Ex vivo human AT was obtained from women undergoing elective surgery [age: 42.7 [+ or -] 1.5 yr (mean [+ or -] SE), BMI: 26.2 [+ or -] 0.7 kg/[m.sup.2]; n = 38]. Western blot analysis was used to determine NPY protein expression in AT depots. Abdominal subcutaneous (AbSc) adipocytes were isolated and treated with recombinant (rh) NPY, insulin, and RSG. NPY and adipokine levels were measured by ELISA. Our results were that NPY was localized in human AT and adipocytes and confirmed by immunohistochemistry. Depot-specific NPY expression was noted as highest in AbSc AT (1.87 [+ or -] 0.23 ODU) compared with omental (Om; 1.03 [+ or -] 0.15 ODU, P = 0.029) or thigh AT (Th; 1.0 [+ or -] 0.29 ODU, P = 0.035). Insulin increased NPY secretion (control: 0.22 [+ or -] 0.024 ng/ml; 1 nM insulin: 0.26 [+ or -] 0.05 ng/ml; 100 nM insulin: 0.29 [+ or -] 0.04 ng/ml; 1,000 nM insulin: 0.3 [+ or -] 0.04 ng/ml; P < 0.05, n = 13), but cotreatment of RSG (10 nM) with insulin (100 nM) had no effect on NPY secretion. Furthermore, adipocyte treatment with rh-NPY downregulated leptin secretion (control: 6.99 [+ or -] 0.89 ng/ml; 1 nmol/l rh-NPY: 4.4 [+ or -] 0.64 ng/ml; 10 nmol/l rh-NPY: 4.3 [+ or -] 0.61 ng/ml, 100 nmol/l rh-NPY: 4.2 [+ or -] 0.67 ng/ml; P < 0.05, n = 10) but had no effect on adiponectin or TNF-[alpha] secretion. We conclude that NPY is expressed and secreted by human adipocytes. NPY secretion is stimulated by insulin, but this increment was limited by cotreatment with RSG. NPY's antilipolytic action may promote an increase in adipocyte size in hyperinsulinemic conditions. Adipose-derived NPY mediates reduction of leptin secretion and may have implications for central feedback of adiposity signals. adipokines; insulin; rosiglitazone; leptin

Abstract Background Emerging 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. Methods Fasted 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). Results Endotoxin 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. Conclusions Endotoxin 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.

Abstract Introduction Chronic low-grade inflammation is a significant factor in the development of obesity associated diabetes. This is supported by recent studies suggesting endotoxin, derived from gut flora, may be key to the development of inflammation by stimulating the secretion of an adverse cytokine profile from adipose tissue. Aims The study investigated the relationship between endotoxin and various metabolic parameters of diabetic patients to determine if anti-diabetic therapies exerted a significant effect on endotoxin levels and adipocytokine profiles. Methods Fasting blood samples were collected from consenting Saudi Arabian patients (BMI: 30.2 ± (SD)5.6 kg/m2, n = 413), consisting of non-diabetics (ND: n = 67) and T2DM subjects (n = 346). The diabetics were divided into 5 subgroups based on their 1 year treatment regimes: diet-controlled (n = 36), metformin (n = 141), rosiglitazone (RSG: n = 22), a combined fixed dose of metformin/rosiglitazone (met/RSG n = 100) and insulin (n = 47). Lipid profiles, fasting plasma glucose, insulin, adiponectin, resistin, TNF-α, leptin, C-reactive protein (CRP) and endotoxin concentrations were determined. Results Regression analyses revealed significant correlations between endotoxin levels and triglycerides (R2 = 0.42; p < 0.0001); total cholesterol (R2 = 0.10; p < 0.001), glucose (R2 = 0.076; p < 0.001) and insulin (R2 = 0.032; p < 0.001) in T2DM subjects. Endotoxin showed a strong inverse correlation with HDL-cholesterol (R2 = 0.055; p < 0.001). Further, endotoxin levels were elevated in all of the treated diabetic subgroups compared with ND, with the RSG treated diabetics showing significantly lower endotoxin levels than all of the other treatment groups (ND: 4.2 ± 1.7 EU/ml, RSG: 5.6 ± 2.2 EU/ml). Both the met/RSG and RSG treated groups had significantly higher adiponectin levels than all the other groups, with the RSG group expressing the highest levels overall. Conclusion We conclude that sub-clinical inflammation in T2DM may, in part, be mediated by circulating endotoxin. Furthermore, that whilst the endotoxin and adipocytokine profiles of diabetic patients treated with different therapies were comparable, the RSG group demonstrated significant differences in both adiponectin and endotoxin levels. We confirm an association between endotoxin and serum insulin and triglycerides and an inverse relationship with HDL. Lower endotoxin and higher adiponectin in the groups treated with RSG may be related and indicate another mechanism for the effect of RSG on insulin sensitivity.

Abstract Introduction Inflammation contributes to cardiovascular disease and is exacerbated with increased adiposity, particularly omental adiposity; however, the role of epicardial fat is poorly understood. Methods For these studies the expression of inflammatory markers was assessed in epicardial fat biopsies from coronary artery bypass grafting (CABG) patients using quantitative RT-PCR. Further, the effects of chronic medications, including statins, as well as peri-operative glucose, insulin and potassium infusion, on gene expression were also assessed. Circulating resistin, CRP, adiponectin and leptin levels were determined to assess inflammation. Results The expression of adiponectin, resistin and other adipocytokine mRNAs were comparable to that in omental fat. Epicardial CD45 expression was significantly higher than control depots (p < 0.01) indicating significant infiltration of macrophages. Statin treated patients showed significantly lower epicardial expression of IL-6 mRNA, in comparison with the control abdominal depots (p < 0.001). The serum profile of CABG patients showed significantly higher levels of both CRP (control: 1.28 ± 1.57 μg/mL vs CABG: 9.11 ± 15.7 μg/mL; p < 0.001) and resistin (control: 10.53 ± 0.81 ng/mL vs CABG: 16.8 ± 1.69 ng/mL; p < 0.01) and significantly lower levels of adiponectin (control: 29.1 ± 14.8 μg/mL vs CABG: 11.9 ± 6.0 μg/mL; p < 0.05) when compared to BMI matched controls. Conclusion Epicardial and omental fat exhibit a broadly comparable pathogenic mRNA profile, this may arise in part from macrophage infiltration into the epicardial fat. This study highlights that chronic inflammation occurs locally as well as systemically potentially contributing further to the pathogenesis of coronary artery disease.

A letter to the editor is presented in response to the article "High Fat Intake Leads to Acute Postprandial Exposure to Circulating Endotoxin in Type 2 Diabetic Subjects," published in a previous issue of "Diabetes Care."

Adipose tissue (AT) distribution has significant impact on disease, with central abdominal fat increasing the risk of T2DM and CVD compared with gluteo-femoral (thigh) fat, however limited studies to date have addressed the site-specific nature of human epicardial AT in relation to thigh fat. Our previous studies have implicated epicardial AT as a source of inflammatory cytokines which may influence myocardial function, due to its location. This study examined, in matched paired epicardial and thigh AT from patients undergoing coronary artery bypass grafting (CABG), the intrinsic nature of epicardial and thigh AT during inflammation, mediators of the innate immune pathway toll like receptors (TLR) 2 & 4, NFκB & JNK as potential key mediators of adipocytokine production, CD68 gene expression (macrophage marker, to investigate the role of macrophages in epicardial AT) and whether elevated systemic endotoxin levels may initiate the innate immune response. Fasted serum was taken from CABG subjects (Age:63.8±8.4yrs, BMI:27.8±3.5kg/m², n=72) and non-CAD subjects (Age:61.8±8.0yrs, BMI:27.9±3.3kg/m², n=60). A sub-cohort of paired epicardial and gluteo-femoral AT was obtained from CABG subjects (Age:66.7±6.7yrs, BMI:28.4±3.6 kg/m⊃2, n=16). Endotoxin was measured by LAL assay. In epicardial AT, gene expression data showed associations between CD68 with TLR2 (r=0.94, p<0.001), TLR4 (r=0.95, p<0.001) and TNFα (r=0.81, p<0.001). NFκB and the IKK protein expression was higher in epicardial AT compared with thigh (NF κB p=0.06, IKKβ p=0.01, IKKγp=0.001) as well as JNK expression (p<0.01). Lastly, serum endotoxin was raised in CABG patients compared with controls (p<0.05). In brief epicardial AT showed increased NF κB, IKKβ and JNK levels compared with matched thigh AT; suggesting a depot-specific inflammatory response. Thus enhanced activation of the NF κB and JNK pathways in the epicardial depot may arise through increased simultaneous activation of macrophages and adipocytes. Also such an inflammatory response in AT may arise due to gut-derived endotoxin levels which were increased in CABG patients. In conclusion, these studies show epicardial AT as a source of inflammatory cytokines through NFκB and other key inflammatory pathways highlighting the significance of macrophages in epicardial AT. [ABSTRACT FROM AUTHOR]

Chronic low-grade inflammation is a significant factor in the development of obesity associated diabetes. This is supported by recent studies suggesting that endotoxin, derived from gut flora, may be key to the development of inflammarion by stimulating the secretion of an adverse cytokine profile from adipose tissue. The study investigated the relationship of endotoxin on several metabolic parameters of diabetic patients. It also determined if various anti-diabetic therapies exerted a significant effect on endotoxin levels and the adipocytokine profiles of Type 2 diabetes mellitus (T2DM) patients. Fasting blood samples were collected from consenting Saudi Arabian subjects (BMI:30.2 Kg/M²±5.6, n=413), consisting of non-diabetics (NDs, n=67) and T2DM subjects (n=346). The diabetics were divided into 5 subgroups based on their 1 year treatment regimes: diet-control (n=36), insulin (n=47), metformin (n=141), rosiglitazone (RSG, n=22) or a combined fixed dose of metformin/rosiglitazone (Avandamet n=100). Lipid profiles, fasting plasma glucose, insulin, adiponectin, resistin, TNF-α, leptin, C-reactive protein (CRP) and endotoxin concentrations were determined. Regression analyses revealed significant correlations between endotoxin levels and triglycerides (TGs, r²=0.40; p<0.0001); total cholesterol (r²=0.10; p<0.0001), glucose (r²=0.14; p<0.0001) and insulin (r²0.12; p=0.01). Endotoxin showed a strong inverse correlation with HDL (r²=0.10; p<0.0001) and was elevated in all of the treated diabetic subgroups compared with NDs. Only circulating endotoxin levels of the RSG treated diabetics were comparable to ND controls (Control:4.2±1.7, RSG:5.6±2.2 EU/mL). Both the Avandamet and RSG treated groups had the highest adiponectin levels. In summary, sub-clinical inflammation in T2DM may be mediated by endotoxin in serum. Furthermore, whilst the endotoxin and adipocytokine profiles of diabetic patients treated with different therapies were comparable, the RSG group demonstrated significant differences in both adiponectin and endotoxin levels. In addition, a positive association between endotoxin, serum insulin and TGs was noted and an inverse relationship with HDL. Lower endotoxin and higher adiponectin in the groups treated with RSG may be related and indicate another mechanism for the effects of RSG on insulin sensitivity. [ABSTRACT FROM AUTHOR]

Central obesity is strongly associated with sub-clinical inflammation, insulin resistance (IR) and type 2 diabetes mellitus (T2DM), but the intracellular mechanisms involved in the pathogenesis of central obesity remain unclear. Recent studies have implicated NF κB and c-Jun N terminal kinase (JNK) as central molecules linking insulin action and inflammation in mice. Our study aimed to investigate (1) the central inflammatory intracellular signaling pathways involving NF κB and JNK activation in abdominal subcutaneous (AbdSc) and omental (Ore) human adipose tissue, (2) the influence of adiposity and diabetic status on the activation of the NF κB and its key intermediates and JNK within these AT depots and (3) the effects of TNFα, a known mediator of inflammation in mice, on NF κB and JNK activity in human AbdSc adipocytes. Our data showed that ex vivo IKKβ, IKKγ, I κBα and I κB α -P protein expression was increased in Om AT compared with AbdSc AT (p<0.01). The ratio of active NF κB to total NF κB expression (NF κB ratio) was upregulated in Om AT compared with AbdSc in both lean and obese subjects (p<0.05). Whilst, phosphospecific JNK1 was increased 5.8 fold in the AbdSc depot, (p<0.01), compared with Om AT. In in vitro TNF α treated AbdSc adipocytes NF κB activity increased over time (2-48 hr, p<0.05), however JNK activity remained unchanged. In conclusion, these findings suggest distinct inflammatory signaling pathways with increased NF κB in human Om AT and JNK in AbdSc AT. In addition, NF κB was influenced by obesity and diabetic status, whilst TNF α treated AbdSc lead to activation of NF κB and not JNK. Consequently, NF κB appears to play a more central role in inflammatory mediated metabolic disease than JNK, highlighting NF κB as a potential key target for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Peripheral action of irisin improves glucose homeostasis and increases energy expenditure, with no data on a central role of irisin in metabolism. These studies sought to examine 1) presence of irisin in human cerebrospinal fluid (CSF) and banked human hypothalamic tissue, 2) serum irisin in maternal subjects across varying adiposities with or without gestational diabetes (GDM), and 3) their respective neonate offspring. CSF, serum, and neonatal cord serum were collected from 91 pregnant women with and without GDM attending for an elective cesarean section [body mass index (BMI): 37.7 ± 7.6 kg/m(2); age: 32 ± 8.3 yr]. Irisin was assessed by ELISA and correlated with biochemical and anthropometric data. Irisin expression was examined in human hypothalamus by immunohistochemical staining. Serum irisin in pregnant women was significantly lower in nonobese compared with obese and GDM subjects, after adjusting for BMI, lipids, and glucose. Irisin was present in neonatal cord serum (237 ± 8 ng/ml) and maternal CSF (32 ± 1.5 ng/ml). CSF irisin correlated positively with serum irisin levels from nonobese and obese pregnant women (P < 0.01), with CSF irisin significantly raised in GDM subjects (P < 0.05). Irisin was present in human hypothalamic sections in the paraventricular neurons, colocalized with neuropeptide Y. Irisin was detectable in CSF and in paraventricular neurons. Maternal serum irisin was lower in nonobese pregnant women after adjusting for BMI and a number of metabolic parameters. These studies indicate that irisin may have a central role in metabolism in addition to the known peripheral role. Further studies investigating the central action of irisin in human metabolic disease are required.