Identification of the signals for glucose-induced insulin secretion in INS1 (832/13) -cells using metformin-induced metabolic deceleration as a model

Metabolic deceleration in pancreatic -cells is associated with inhibition of glucose-induced insulin secretion (GIIS), but only in the presence of intermediate/submaximal glucose concentrations. Here, we used acute metformin treatment as a tool to induce metabolic deceleration in INS1 (832/13) -cells, with the goal of identifying key pathways and metabolites involved in GIIS. Metabolites and pathways previously implicated as signals for GIIS were measured in the cells at 2-25 mm glucose, with or without 5 mm metformin. We defined three criteria to identify candidate signals: 1) glucose-responsiveness, 2) sensitivity to metformin-induced inhibition of the glucose effect at intermediate glucose concentrations, and 3) alleviation of metformin inhibition by elevated glucose concentrations. Despite the lack of recovery from metformin-induced impairment of mitochondrial energy metabolism (glucose oxidation, O-2 consumption, and ATP production), insulin secretion was almost completely restored at elevated glucose concentrations. Meeting the criteria for candidates involved in promoting GIIS were the following metabolic indicators and metabolites: cytosolic NAD(+)/NADH ratio (inferred from the dihydroxyacetone phosphate:glycerol-3-phosphate ratio), mitochondrial membrane potential, ADP, Ca2+, 1-monoacylglycerol, diacylglycerol, malonyl-CoA, and HMG-CoA. On the contrary, most of the purine and nicotinamide nucleotides, acetoacetyl-CoA, H2O2, reduced glutathione, and 2-monoacylglycerol were not glucose-responsive. Overall these results underscore the significance of mitochondrial energy metabolism-independent signals in GIIS regulation; in particular, the candidate lipid signaling molecules 1-monoacylglycerol, diacylglycerol, and malonyl-CoA; the predominance of K-ATP/Ca2+ signaling control by low ADPMg(2+) rather than by high ATP levels; and a role for a more oxidized state (NAD(+)/NADH) in the cytosol during GIIS that favors high glycolysis rates.This study was supported by grants from Canadian Institutes of Health Research (to MP and SRMM) and a scholarship from Kuwait University to AA. MP holds the Canada Research Chair in Diabetes and Metabolis

Elevated expression of the toll like receptors 2 and 4 in obese individuals: its significance for obesity-induced inflammation

Abstract Background Expression profile of the toll like receptors (TLRs) on PBMCs is central to the regulation of proinflammatory markers. An imbalance in the TLRs expression may lead to several types of inflammatory disorders. Furthermore, the dynamic regulation of inflammatory activity and associated impaired production of cytokines by peripheral blood mononuclear cells (PBMCs) in obese individulas remain poorly understood. Therefore, we determined the perturbation in TLRs (TLR2 and TLR4), their adaptor proteins (MyD88, IRAK1 and TRAF6) expression in PBMCs/subcutaneous adipose tissue (AT) as well as inflammatory cytokines changes in obese individuals. Methods mRNA expression levels of TLR2, TLR4, IL-6, TNF-α and adaptor proteins were determined by RT-PCR. TLR2, TLR4 and adaptor proteins expression in AT was determined by immunohistochemistry. Results Obese and overweight individuals showed significantly increased expression of TLR2, TLR4 and MyD88 in both PBMCs and AT as compared with lean individuals (P  Conclusions TLRs and adapter proteins were overexpressed in PBMCs from obese subjects, which correlated with increased expression of TNF-α and IL-6. This association may explain a potential pathophysiological link between obesity and inflammation leading to insulin resistance.</p

Interaction of osteopontin with IL-18 in obese individuals: implications for insulin resistance.

BACKGROUND/OBJECTIVE: Osteopontin (OPN) and IL-18 are known inflammatory mediators and both participate in a wide range of biological processes linked to immunological disorders. Since an interaction between OPN and IL-18 has not been studied in obesity, we investigated whether: (i) their levels were simultaneously elevated in obese individuals; (ii) OPN was associated with IL-18 in obese individuals and (iii) their levels associated with fasting blood glucose (FBG) and BMI. SUBJECTS AND METHODS: PBMCs and plasma samples were isolated from 60 individuals including lean as well as overweight and obese individuals. Subcutaneous adipose tissue samples were obtained. OPN and IL-18 were measured by ELISA. OPN and IL-18 mRNA expression was quantified by real time quantitative RT-PCR. RESULTS: Obese individuals exhibited significantly increased circulating OPN levels as compared with lean individuals (obese 2865±101; lean 1681±116 pg/ml; P<0.0001). IL-18 levels were also high in obese individuals (obese 491±39, lean 301±26 pg/ml; P = 0.0009). OPN and IL-18 expression were simultaneously up-regulated (OPN: 5.4-Fold; IL-18: 8.9-Fold; P<0.05) in PBMCs from obese individuals compared to lean group. Adipose tissue from obese individuals had high expression of OPN (7.3-Fold) and IL-18 (9.6-Fold). Plasma OPN levels correlated positively with FBG levels (r = 0.32, P = 0.02). Similarly, IL-18 correlated positively with FBG levels (r = 0.406, P = 0.0042). Stepwise multiple regression analysis showed an independent association of BMI with OPN and IL-18. Interestingly, OPN levels increased progressively with an increase in IL-18 levels (r = 0.52, P = 0.0004). We also examined the regulatory role of IL-18 in OPN secretion from PBMCs. Neutralizing anti-IL-18Rα mAb reduced OPN secretion. CONCLUSION: These findings represent the first observation that plasma, PBMC and adipose tissue OPN and IL-18 are simultaneously increased and correlate with each other in overweight/obese individuals which may trigger the development of obesity-associated insulin resistance. Moreover, these results provide the direct evidence that IL-18 regulates OPN production in PBMCs

Expression of OPN and IL-18 mRNA in adipose tissue.

<p>Adipose tissue samples from 9 (lean: 3, overweight: 3 and obese: 3) individuals with different BMI were subjected to total RNA isolation. OPN and IL-18 mRNA expression was determined by real time quantitative RT-PCR. Relative mRNA expression was expressed as fold expression over average of gene expression of lean group. The expression level in lean group was assumed to be 1. Values are presented as mean ± SE (A and B).</p

Elevated Expression of Glycerol-3-Phosphate Phosphatase as a Biomarker of Poor Prognosis and Aggressive Prostate Cancer

The limitations of the biomarker prostate-specific antigen (PSA) necessitate the pursuit of biomarkers capable of better identifying high-risk prostate cancer (PC) patients in order to improve their therapeutic management and outcomes. Aggressive prostate tumors characteristically exhibit high rates of glycolysis and lipogenesis. Glycerol 3-phosphate phosphatase (G3PP), also known as phosphoglycolate phosphatase (PGP), is a recently identified mammalian enzyme, shown to play a role in the regulation of glucose metabolism, lipogenesis, lipolysis, and cellular nutrient-excess detoxification. We hypothesized that G3PP may relieve metabolic stress in cancer cells and assessed the association of its expression with PC patient prognosis. Using immunohistochemical staining, we assessed the epithelial expression of G3PP in two different radical prostatectomy (RP) cohorts with a total of 1797 patients, for whom information on biochemical recurrence (BCR), metastasis, and mortality was available. The association between biomarker expression, biochemical recurrence (BCR), bone metastasis, and prostate cancer-specific survival was established using log-rank and multivariable Cox regression analyses. High expression of G3PP in PC epithelial cells is associated with an increased risk of BCR, bone metastasis, and PC-specific mortality. Multivariate analysis revealed high G3PP expression in tumors as an independent predictor of BCR and bone metastasis development. High G3PP expression in tumors from patients eligible for prostatectomies is a new and independent prognostic biomarker of poor prognosis and aggressive PC for recurrence, bone metastasis, and mortality

Characteristics of the study participants.

<p>Characteristics of the study participants.</p

Correlation of plasma and PBMC gene expression levels of OPN and IL-18 with metabolic markers.

<p>Correlation of plasma and PBMC gene expression levels of OPN and IL-18 with metabolic markers.</p

Linear regression between OPN and IL-18.

<p>Plasma OPN and IL-18 levels were determined by ELISA There was a statistical significant linear regression between OPN and IL-18 (r = 0.52, P = 0.0004, n = 44) in obese and overweight individuals which were free from overweight/obesity induced major complications (A). A strong association of OPN mRNA (Fold change) with IL-18 mRNA was observed in adipose tissue (r = 0.91, P = 0.01: (B). Fold change expression of OPN and IL-18 mRNA in PBMCs of 44 overweight and obese individuals was correlated with each other (r = 0.45, P = 0.0018) (C).</p

Expression of OPN and IL-18 by PBMCs.

<p>Total RNA was extracted from PBMCs of 60 individuals with different BMI (lean: 16; overweight: 20; obese 24). OPN and IL-18 mRNA were measured by real time quantitative PCR. Relative mRNA expression was expressed as fold expression over average of gene expression of lean group. The expression level in lean group was assumed to be 1. Values are presented as mean ± SE (A and B). PBMCs from three donors of each group were cultured in RPMI supplemented with 2% FBS for 16 hours and then culture supernatants were collected. Secreted OPN protein was determined in the culture supernatants by ELISA (C). Data presented were the means of three independent experiments performed with PBMCs of three donors of each group with ± SE.</p