Obesity, and the associated disturbed glycerolipid/fatty acid (GL/FA) cycle, contribute to insulin resistance, islet β-cell failure, and type 2 diabetes. Flux through the GL/FA cycle is regulated by the availability of glycerol-3-phosphate (Gro3P) and fatty acyl-CoA. We describe here a mammalian Gro3P phosphatase (G3PP), which was not known to exist in mammalian cells, that can directly hydrolyze Gro3P to glycerol. We identified that mammalian phosphoglycolate phosphatase, with an uncertain function, acts in fact as a G3PP. We found that G3PP, by controlling Gro3P levels, regulates glycolysis and glucose oxidation, cellular redox and ATP production, gluconeogenesis, glycerolipid synthesis, and fatty acid oxidation in pancreatic islet β-cells and hepatocytes, and that glucose stimulated insulin secretion and the response to metabolic stress, e.g., glucolipotoxicity, in β-cells. In vivo overexpression of G3PP in rat liver lowers body weight gain and hepatic glucose production from glycerol and elevates plasma HDL levels. G3PP is expressed at various levels in different tissues, and its expression varies according to the nutritional state in some tissues. As Gro3P lies at the crossroads of glucose, lipid, and energy metabolism, control of its availability by G3PP adds a key level of metabolic regulation in mammalian cells, and G3PP offers a potential target for type 2 diabetes and cardiometabolic disorders.glycerol-3-phosphate phosphatase | gluconeogenesis | glucolipotoxicity | type 2 diabetes | glucose-stimulated insulin secretion

BackgroundExpression 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.MethodsmRNA 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.ResultsObese and overweight individuals showed significantly increased expression of TLR2, TLR4 and MyD88 in both PBMCs and AT as compared with lean individuals (P < 0.05). Interestingly, we found a remarkably higher expression of TLRs in obese and overweight individuals with type 2 diabetes (P < 0.05). Increased expression of TLR2, TLR4, MyD88 and IRAK1 correlated with body mass index (BMI) (TLR2: r = 0.91; TLR4: r = 0.88, P <0.0001; MyD88: r = 0.95, P < 0.0001; IRAK1 r = 0.78, P < 0.002). TLRs’ expression was also correlated with fasting blood glucose (FBG) (TLR2: r = 0.61, P < 0.002; TLR4: r = 0.52, P < 0.01) and glycated haemoglobin (HbA1c) ( TLR2: r = 0.44, P <0.03; TLR4: r = 0.48, P < 0.03). Transcript levels of IL-6 and TNF-α were highly elevated in obese subjects compared to lean subjects. There was a strong association of TLRs’ expression in PBMCs with TNF-α (TLR2: r = 0.92; TLR4: r = 0.92; P < 0.0001) and IL-6 (TLR2: r = 0.91, P < 0.0001; TLR4: r = 0.81; P < 0.001). Similarly adaptor proteins were significantly correlated with TNF-α (MyD88: r = 0.9, P < 0.0001; IRAK1: r = 0.86; P < 0.0002) and IL-6 (MyD88: r = 0.91, P < 0.0001; IRAK1: 0.77; P < 0.002).ConclusionsTLRs 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.

Glucose metabolism promotes insulin secretion in β-cells via metabolic coupling factors that are incompletely defined. Moreover, chronically elevated glucose causes β-cell dysfunction, but little is known about how cells handle excess fuels to avoid toxicity. Here we sought to determine which among the candidate pathways and coupling factors best correlates with glucose-stimulated insulin secretion (GSIS), define the fate of glucose in the β-cell, and identify pathways possibly involved in excess-fuel detoxification. We exposed isolated rat islets for 1 h to increasing glucose concentrations and measured various pathways and metabolites. Glucose oxidation, oxygen consumption, and ATP production correlated well with GSIS and saturated at 16 mm glucose. However, glucose utilization, glycerol release, triglyceride and glycogen contents, free fatty acid (FFA) content and release, and cholesterol and cholesterol esters increased linearly up to 25 mm glucose. Besides being oxidized, glucose was mainly metabolized via glycerol production and release and lipid synthesis (particularly FFA, triglycerides, and cholesterol), whereas glycogen production was comparatively low. Using targeted metabolomics in INS-1(832/13) cells, we found that several metabolites correlated well with GSIS, in particular some Krebs cycle intermediates, malonyl-CoA, and lower ADP levels. Glucose dose-dependently increased the dihydroxyacetone phosphate/glycerol 3-phosphate ratio in INS-1(832/13) cells, indicating a more oxidized state of NAD in the cytosol upon glucose stimulation. Overall, the data support a role for accelerated oxidative mitochondrial metabolism, anaplerosis, and malonyl-CoA/lipid signaling in β-cell metabolic signaling and suggest that a decrease in ADP levels is important in GSIS. The results also suggest that excess-fuel detoxification pathways in β-cells possibly comprise glycerol and FFA formation and release extracellularly and the diversion of glucose carbons to triglycerides and cholesterol esters.

Background/ObjectiveOsteopontin (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 MethodsPBMCs 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.ResultsObese 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.ConclusionThese 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.

BackgroundMMP-9 is crucial for a normal immune response, but excessive release of this enzyme leads to severe tissue damage. Listeria monocytogenes (LM) is an opportunistic food-borne pathogen causing listerosis, meningitis and sepsis. Heat killed Listeria monocytogenes (HKLM) activates immune system and leads production of cytokines and chemokines. However, nothing is known about the involvement of HKLM in MMP-9 regulation. Therefore we investigated the role of HKLM in the regulation of MMP-9 gene expression in THP-1 cells.MethodsCommercially available heat killed Listeria monocytogenes was used in this study. HKLM-induced MMP-9 expression was assessed with quantitative real-time qPCR and ELISA. Action of HKLM in different signaling pathways were studied by using THP-1-XBlue™ cells (THP-1-cells with NF-κB/AP-1 reporter construct), THP-1-XBlue™-defMyD cells (MyD88−/− THP-1 cells), anti-TLR2 mAb and pharmacological inhibitors. Phospho and total proteins were determined by Western blotting.ResultsIncreased MMP-9 production (mRNA: 395-Fold; Protein: 8141 pg/ml; P < 0.05) was observed in HKLM stimulated THP-1 cells as compared to the un-stimulated THP-1 cells. This production of MMP-9 was completely abrogated by anti-TLR2 blocking mAb (P = 0.0024). Furthermore, THP-1-XBlue™-defMyD cells were unable to produce MMP-9 in response to HKLM. HKLM- induced activation of NF-kappaB/AP-1 was also observed in THP-1-XBlue™ Cells. In addition, inhibitors of JNK (SP600125), MEK/ERK (U0126; PD98056), p38 MAPK (SB203580) and NF-kappaB (BAY 11–7085, Triptolide and Resveratrol) significantly suppressed (P < 0.05) HKLM-stimulated MMP-9 expression.ConclusionOur results indicate that HKLM activates TLR2 and NF-κB/AP-1 signaling pathways, leading to up-regulation of MMP-9 production in THP-1 cells. Thus, MMP-9 could be an appropriate therapeutic target to stop severe tissue damage caused by infection or chronic inflammation.

Metabolic deceleration in pancreatic β-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) β-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 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, Ca, 1-monoacylglycerol, diacylglycerol, malonyl-CoA, and HMG-CoA. On the contrary, most of the purine and nicotinamide nucleotides, acetoacetyl-CoA, HO, 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/Ca signaling control by low ADP·Mg 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.

Diet induced obese (DIO) mice can be stratified according to their weight gain in response to high fat diet as low responders (LDR) and high responders (HDR). This allows the study of β-cell failure and the transitions to prediabetes (LDR) and early diabetes (HDR). C57BL/6N mice were fed for 8 weeks with a normal chow diet (ND) or a high fat diet and stratified as LDR and HDR. Freshly isolated islets from ND, LDR and HDR mice were studied ex-vivo for mitochondrial metabolism, AMPK activity and signalling, the expression and activity of key enzymes of energy metabolism, cholesterol synthesis, and mRNA profiling. Severely compromised glucose-induced insulin secretion in HDR islets, as compared to ND and LDR islets, was associated with suppressed AMP-kinase activity. HDR islets also showed reduced acetyl-CoA carboxylase activity and enhanced activity of 3-hydroxy-3-methylglutaryl-CoA reductase, which led respectively to elevated fatty acid oxidation and increased cholesterol biosynthesis. HDR islets also displayed mitochondrial membrane hyperpolarization and reduced ATP turnover in the presence of elevated glucose. Expression of protein kinase Cε, which reduces both lipolysis and production of signals for insulin secretion, was elevated in DIO islets. Genes whose expression increased or decreased by more than 1.2-fold were minor between LDR and ND islets (17 differentially expressed), but were prominent between HDR and ND islets (1508 differentially expressed). In HDR islets, particularly affected genes were related to cell cycle and proliferation, AMPK signaling, mitochondrial metabolism and cholesterol metabolism. In conclusion, chronically reduced AMPK activity, mitochondrial dysfunction, elevated cholesterol biosynthesis in islets, and substantial alterations in gene expression accompany β-cell failure in HDR islets. The β-cell compensation process in the prediabetic state (LDR) is largely independent of transcriptional adaptive changes, whereas the transition to early diabetes (HDR) is associated with major alterations in gene expression.

Metabolic stress due to nutrient excess and lipid accumulation is at the root of many age-associated disorders and the identification of therapeutic targets that mimic the beneficial effects of calorie restriction has clinical importance. Here, using C. elegans as a model organism, we study the roles of a recently discovered enzyme at the heart of metabolism in mammalian cells, glycerol-3-phosphate phosphatase (G3PP) (gene name Pgp) that hydrolyzes glucose-derived glycerol-3-phosphate to glycerol. We identify three Pgp homologues in C. elegans (pgph) and demonstrate in vivo that their protein products have G3PP activity, essential for glycerol synthesis. We demonstrate that PGPH/G3PP regulates the adaptation to various stresses, in particular hyperosmolarity and glucotoxicity. Enhanced G3PP activity reduces fat accumulation, promotes healthy aging and acts as a calorie restriction mimetic at normal food intake without altering fertility. Thus, PGP/G3PP can be considered as a target for age-related metabolic disorders.