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Glucokinase activators (GKAs) have been developed as blood glucose lowering drugs for type 2 diabetes. Despite good short-term efficacy, several GKAs showed a decline in efficacy chronically during clinical trials. The underlying mechanisms remain incompletely understood. We tested the hypothesis that deficiency in the liver glucokinase regulatory protein (GKRP) as occurs with common human GCKR variants affects chronic GKA efficacy. We used a Gckr-P446L mouse model for the GCKR exonic rs1260326 (P446L) variant and the Gckr-del/wt mouse to model transcriptional deficiency to test for chronic efficacy of the GKA, AZD1656 in GKRP-deficient states. In the Gckr-P446L mouse, the blood glucose lowering efficacy of AZD1656 (3 mg/kg body wt) after 2 weeks was independent of genotype. However after 19 weeks, efficacy was maintained in wild-type but declined in the LL genotype, in conjunction with raised hepatic glucokinase activity and without raised liver lipids. Sustained blood glucose lowering efficacy in wild-type mice was associated with qualitatively similar but more modest changes in the liver transcriptome compared with the P446L genotype, consistent with GKA therapy representing a more modest glucokinase excess than the P446L genotype. Chronic treatment with AZD1656 in the Gckr-del/wt mouse was associated with raised liver triglyceride and hepatocyte microvesicular steatosis. The results show that in mouse models of liver GKRP deficiency in conjunction with functional liver glucokinase excess as occurs in association with common human GCKR variants, GKRP-deficiency predisposes to declining efficacy of the GKA in lowering blood glucose and to GKA induced elevation in liver lipids.

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Reversal or mitigation against an age-related decline in NAD+ has likely benefits, and this premise has driven academic and commercial endeavour to develop dietary supplements that achieve this outcome. We used a systems-based approach to improve on current supplements by targeting multiple points in the NAD+ salvage pathway. In a double-blind, randomised, crossover trial, the supplement - Nuchido TIME+® (NT) - increased NAD+ concentration in whole blood. This was associated with an increase in SIRT1 and an increase in nicotinamide phosphoribosyltransferase (NAMPT) in peripheral blood mononucleocytes, lower concentrations of pro-inflammatory cytokines in plasma, including a reduction in interleukin 2 (IL2), a reduction in glycated serum protein and a shift in the glycosylation profile of immunoglobulin G (IgG) toward a younger biological age, all of which are likely to promote a healthier ageing trajectory.

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OBJECTIVES: The Glucokinase Regulatory Protein GKRP, encoded by GCKR, enables acute regulation of liver glucokinase to support metabolic demand. The common human GCKR rs1260326:Pro446 > Leu variant within a large linkage disequilibrium region associates with pleiotropic traits including lower Type 2 diabetes risk and raised blood triglycerides and cholesterol. Whether the GCKR-P446 > L substitution is causal to the raised lipids is unknown. We determined whether mouse GKRP phenocopies the human GKRP:P446 > L substitution and studied a GKRP:P446L knockin mouse to identify physiological consequences to P446 > L. METHODS: GKRP-deficient hepatocytes were transfected with adenoviral vectors for human or mouse GKRP:446 P or 446 L for cellular comprehensive analysis including transcriptomics consequent to P446 > L. Physiological traits in the diet-challenged P446L mouse were compared with pleiotropic associations at the human rs1260326 locus. Transcriptomics was compared in P446L mouse liver with hepatocytes overexpressing glucokinase or GKRP:446 P/L. RESULTS: 1. P446 > L substitution in mouse or human GKRP similarly compromises protein expressivity of GKRP:446 L, nuclear sequestration of glucokinase and counter-regulation of gene expression. 2. The P446L knockin mouse has lower liver glucokinase and GKRP protein similar to human liver homozygous for rs1260326-446 L. 3. The diet-challenged P446L mouse has lower blood glucose, raised blood cholesterol and altered hepatic cholesterol homeostasis consistent with relative glucokinase-to-GKRP excess, but not raised blood triglycerides. CONCLUSIONS: Mouse GKRP phenocopies the human GKRP:P446 > L substitution despite the higher affinity for glucokinase of human GKRP. The diet-challenged P446L mouse replicates several traits found in association with the rs1260326 locus on chromosome 2 including raised blood cholesterol, lower blood glucose and lower liver glucokinase and GKRP protein but not raised blood triglycerides.

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The Carbohydrate response element binding protein, ChREBP encoded by the MLXIPL gene, is a transcription factor that is expressed at high levels in the liver and has a prominent function during consumption of high-carbohydrate diets. ChREBP is activated by raised cellular levels of phosphate ester intermediates of glycolysis, gluconeogenesis and the pentose phosphate pathway. Its target genes include a wide range of enzymes and regulatory proteins, including G6pc, Gckr, Pklr, Prkaa1,2, and enzymes of lipogenesis. ChREBP activation cumulatively promotes increased disposal of phosphate ester intermediates to glucose, via glucose 6-phosphatase or to pyruvate via glycolysis with further metabolism by lipogenesis. Dietary fructose is metabolized in both the intestine and the liver and is more lipogenic than glucose. It also induces greater elevation in phosphate ester intermediates than glucose, and at high concentrations causes transient depletion of inorganic phosphate, compromised ATP homeostasis and degradation of adenine nucleotides to uric acid. ChREBP deficiency predisposes to fructose intolerance and compromised cellular phosphate ester and ATP homeostasis and thereby markedly aggravates the changes in metabolite levels caused by dietary fructose. The recent evidence that high fructose intake causes more severe hepatocyte damage in ChREBP-deficient models confirms the crucial protective role for ChREBP in maintaining intracellular phosphate homeostasis. The improved ATP homeostasis in hepatocytes isolated from mice after chronic activation of ChREBP with a glucokinase activator supports the role of ChREBP in the control of intracellular homeostasis. It is hypothesized that drugs that activate ChREBP confer a protective role in the liver particularly in compromised metabolic states.

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AIM: To test the hypothesis that glucokinase activators (GKAs) induce hepatic adaptations that alter intra-hepatocyte metabolite homeostasis. METHODS: C57BL/6 mice on a standard rodent diet were treated with a GKA (AZD1656) acutely or chronically. Hepatocytes were isolated from the mice after 4 or 8 weeks of treatment for analysis of cellular metabolites and gene expression in response to substrate challenge. RESULTS: Acute exposure of mice to AZD1656 or a liver-selective GKA (PF-04991532), before a glucose tolerance test, or challenge of mouse hepatocytes with GKAs ex vivo induced various Carbohydrate response element binding protein (ChREBP) target genes, including Carbohydrate response element binding protein beta isoform (ChREBP-ß), Gckr and G6pc. Both glucokinase activation and ChREBP target gene induction by PF-04991532 were dependent on the chirality of the molecule, confirming a mechanism linked to glucokinase activation. Hepatocytes from mice treated with AZD1656 for 4 or 8 weeks had lower basal glucose 6-phosphate levels and improved ATP homeostasis during high substrate challenge. They also had raised basal ChREBP-ß mRNA and AMPK-α mRNA (Prkaa1, Prkaa2) and progressively attenuated substrate induction of some ChREBP target genes and Prkaa1 and Prkaa2. CONCLUSIONS: Chronic GKA treatment of C57BL/6 mice for 8 weeks activates liver ChREBP and improves the resilience of hepatocytes to compromised ATP homeostasis during high-substrate challenge. These changes are associated with raised mRNA levels of ChREBP-ß and both catalytic subunits of AMP-activated protein kinase.

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Metformin therapy lowers blood glucose in type 2 diabetes by targeting various pathways including hepatic gluconeogenesis. Despite widespread clinical use of metformin the molecular mechanisms by which it inhibits gluconeogenesis either acutely through allosteric and covalent mechanisms or chronically through changes in gene expression remain debated. Proposed mechanisms include: inhibition of Complex 1; activation of AMPK; and mechanisms independent of both Complex 1 inhibition and AMPK. The activation of AMPK by metformin could be consequent to Complex 1 inhibition and raised AMP through the canonical adenine nucleotide pathway or alternatively by activation of the lysosomal AMPK pool by other mechanisms involving the aldolase substrate fructose 1,6-bisphosphate or perturbations in the lysosomal membrane. Here we review current interpretations of the effects of metformin on hepatic intermediates of the gluconeogenic and glycolytic pathway and the candidate mechanistic links to regulation of gluconeogenesis. In conditions of either glucose excess or gluconeogenic substrate excess, metformin lowers hexose monophosphates by mechanisms that are independent of AMPK-activation and most likely mediated by allosteric activation of phosphofructokinase-1 and/or inhibition of fructose bisphosphatase-1. The metabolite changes caused by metformin may also have a prominent role in counteracting G6pc gene regulation in conditions of compromised intracellular homeostasis.

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The chronic effects of metformin on liver gluconeogenesis involve repression of the G6pc gene, which is regulated by the carbohydrate-response element-binding protein through raised cellular intermediates of glucose metabolism. In this study we determined the candidate mechanisms by which metformin lowers glucose 6-phosphate (G6P) in mouse and rat hepatocytes challenged with high glucose or gluconeogenic precursors. Cell metformin loads in the therapeutic range lowered cell G6P but not ATP and decreased G6pc mRNA at high glucose. The G6P lowering by metformin was mimicked by a complex 1 inhibitor (rotenone) and an uncoupler (dinitrophenol) and by overexpression of mGPDH, which lowers glycerol 3-phosphate and G6P and also mimics the G6pc repression by metformin. In contrast, direct allosteric activators of AMPK (A-769662, 991, and C-13) had opposite effects from metformin on glycolysis, gluconeogenesis, and cell G6P. The G6P lowering by metformin, which also occurs in hepatocytes from AMPK knockout mice, is best explained by allosteric regulation of phosphofructokinase-1 and/or fructose bisphosphatase-1, as supported by increased metabolism of [3-3H]glucose relative to [2-3H]glucose; by an increase in the lactate m2/m1 isotopolog ratio from [1,2-13C2]glucose; by lowering of glycerol 3-phosphate an allosteric inhibitor of phosphofructokinase-1; and by marked G6P elevation by selective inhibition of phosphofructokinase-1; but not by a more reduced cytoplasmic NADH/NAD redox state. We conclude that therapeutically relevant doses of metformin lower G6P in hepatocytes challenged with high glucose by stimulation of glycolysis by an AMP-activated protein kinase-independent mechanism through changes in allosteric effectors of phosphofructokinase-1 and fructose bisphosphatase-1, including AMP, Pi, and glycerol 3-phosphate.

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