👤 Loranne Agius

Stable isotope studies have shown that hepatic de novo lipogenesis (DNL) plays an important role in the pathogenesis of intrahepatic lipid (IHL) deposition. Furthermore, previous research has demonstrated that fructose 1-phosphate (F1P) not only serves as a substrate for DNL, but also acts as a signalling metabolite that stimulates DNL from glucose. The aim of this study was to elucidate the mediators of F1P-stimulated DNL, with special focus on two key regulators of intrahepatic glucose metabolism, i.e., glucokinase regulatory protein (GKRP) and carbohydrate response element binding protein (ChREBP). Aldolase B deficient mice (Aldob Aldob Both GKRP and ChREBP mediate F1P-stimulated DNL in aldolase B deficient mice. Further studies are needed to unravel the role of GKRP and hepatic ChREBP in regulating IHL accumulation in aldolase B deficiency. Show less

Small molecule activators of glucokinase (GKAs) have been explored extensively as potential anti-hyperglycaemic drugs for type 2 diabetes (T2D). Several GKAs were remarkably effective in lowering blood glucose during early therapy but then lost their glycaemic efficacy chronically during clinical trials. We used rat hepatocytes to test the hypothesis that GKAs raise hepatocyte glucose 6-phosphate (G6P, the glucokinase product) and down-stream metabolites with consequent repression of the liver glucokinase gene ( Gck). We compared a GKA with metformin, the most widely prescribed drug for T2D. Treatment of hepatocytes with 25 mM glucose raised cell G6P, concomitantly with Gck repression and induction of G6pc (glucose 6-phosphatase) and Pklr (pyruvate kinase). A GKA mimicked high glucose by raising G6P and fructose-2,6-bisphosphate, a regulatory metabolite, causing a left-shift in glucose responsiveness on gene regulation. Fructose, like the GKA, repressed Gck but modestly induced G6pc. 2-Deoxyglucose, which is phosphorylated by glucokinase but not further metabolized caused Gck repression but not G6pc induction, implicating the glucokinase product in Gck repression. Metformin counteracted the effect of high glucose on the elevated G6P and fructose 2,6-bisphosphate and on Gck repression, recruitment of Mlx-ChREBP to the G6pc and Pklr promoters and induction of these genes. Elevation in hepatocyte G6P and downstream metabolites, with consequent liver Gck repression, is a potential contributing mechanism to the loss of GKA efficacy during chronic therapy. Cell metformin loads within the therapeutic range attenuate the effect of high glucose on G6P and on glucose-regulated gene expression. Show less

Type 2 diabetes and non-alcoholic fatty liver disease (NAFLD) are associated with elevated hepatic glucose production and fatty acid synthesis (de novo lipogenesis (DNL)). High carbohydrate diets also increase hepatic glucose production and lipogenesis. The carbohydrate-response element-binding protein (ChREBP, encoded by MLXIPL) is a transcription factor with a major role in the hepatic response to excess dietary carbohydrate. Because its target genes include pyruvate kinase (PKLR) and enzymes of lipogenesis, it is regarded as a key regulator for conversion of dietary carbohydrate to lipid for energy storage. An alternative hypothesis for ChREBP function is to maintain hepatic ATP homeostasis by restraining the elevation of phosphate ester intermediates in response to elevated glucose. This is supported by the following evidence: (i) A key stimulus for ChREBP activation and induction of its target genes is elevation of phosphate esters; (ii) target genes of ChREBP include key negative regulators of the hexose phosphate ester pool (GCKR, G6PC, SLC37A4) and triose phosphate pool (PKLR); (iii) ChREBP knock-down models have elevated hepatic hexose phosphates and triose phosphates and compromised ATP phosphorylation potential; (iv) gene defects in G6PC and SLC37A4 and common variants of MLXIPL, GCKR and PKLR in man are associated with elevated hepatic uric acid production (a marker of ATP depletion) or raised plasma uric acid levels. It is proposed that compromised hepatic phosphate homeostasis is a contributing factor to the elevated hepatic glucose production and lipogenesis that associate with type 2 diabetes, NAFLD and excess carbohydrate in the diet. Show less

In the liver, a high glucose concentration activates transcription of genes encoding glucose 6-phosphatase and enzymes for glycolysis and lipogenesis by elevation in phosphorylated intermediates and recruitment of the transcription factor ChREBP (carbohydrate response element binding protein) and its partner, Mlx, to gene promoters. A proposed function for this mechanism is intracellular phosphate homeostasis. In extrahepatic tissues, MondoA, the paralog of ChREBP, partners with Mlx in transcriptional induction by glucose. We tested for glucose induction of regulatory proteins of the glycogenic pathway in hepatocytes and identified the glycogen-targeting proteins, G(L) and PTG (protein targeting to glycogen), as being encoded by Mlx-dependent glucose-inducible genes. PTG induction by glucose was MondoA dependent but ChREBP independent and was enhanced by forced elevation of fructose 2,6-bisphosphate and by additional xylitol-derived metabolites. It was counteracted by selective depletion of fructose 2,6-bisphosphate with a bisphosphatase-active kinase-deficient variant of phosphofructokinase 2/fructosebisphosphatase 2, which prevented translocation of MondoA to the nucleus and recruitment to the PTG promoter. We identify a novel role for MondoA in the liver and demonstrate that elevated fructose 2,6-bisphosphate is essential for recruitment of MondoA to the PTG promoter. Phosphometabolite activation of MondoA and ChREBP and their recruitment to target genes is consistent with a mechanism for gene regulation to maintain intracellular phosphate homeostasis. Show less

In population studies hepatic steatosis in subjects with Non-alcoholic fatty liver disease (NAFLD) is strongly associated with insulin resistance. This association has encouraged debate whether hepatic steatosis is the cause or the consequence of hepatic insulin resistance? Although genome-wide studies have identified several gene variants associated with either hepatic steatosis or type 2 diabetes, no variants have been identified associated with both hepatic steatosis and insulin resistance. Here, the hypothesis is proposed that high-carbohydrate diets contribute to the association between hepatic steatosis and insulin resistance through activation of the transcription factor ChREBP (Carbohydrate response element binding protein). Postprandial hyperglycaemia raises the hepatic concentrations of phosphorylated intermediates causing activation of ChREBP and induction of its target genes. These include not only enzymes of glycolysis and lipogenesis that predispose to hepatic steatosis but also glucose 6-phosphatase (G6PC) that catalyses the final reaction in glucose production and GCKR, the inhibitor of hepatic glucokinase that curtails hepatic glucose uptake. Induction of G6PC and GCKR manifests as hepatic glucose intolerance or insulin resistance. Induction of these two genes by high glucose serves to safeguard intrahepatic homeostasis of phosphorylated intermediates. The importance of GCKR in this protective mechanism is supported by "less-active" GCKR variants in association not only with hepatic steatosis and hyperuricaemia but also with lower fasting plasma glucose and decreased insulin resistance. This supports a role for GCKR in restricting hepatic glucose phosphorylation to maintain intrahepatic homeostasis. Pharmacological targeting of the glucokinase-GCKR interaction can favour either glucose clearance by the liver or intrahepatic metabolite homeostasis. Show less

Glucose metabolism in the liver activates the transcription of various genes encoding enzymes of glycolysis and lipogenesis and also G6pc (glucose-6-phosphatase). Allosteric mechanisms involving glucose 6-phosphate or xylulose 5-phosphate and covalent modification of ChREBP (carbohydrate-response element-binding protein) have been implicated in this mechanism. However, evidence supporting an essential role for a specific metabolite or pathway in hepatocytes remains equivocal. By using diverse substrates and inhibitors and a kinase-deficient bisphosphatase-active variant of the bifunctional enzyme PFK2/FBP2 (6-phosphofructo-2-kinase-fructose-2,6-bisphosphatase), we demonstrate an essential role for fructose 2,6-bisphosphate in the induction of G6pc and other ChREBP target genes by glucose. Selective depletion of fructose 2,6-bisphosphate inhibits glucose-induced recruitment of ChREBP to the G6pc promoter and also induction of G6pc by xylitol and gluconeogenic precursors. The requirement for fructose 2,6-bisphosphate for ChREBP recruitment to the promoter does not exclude the involvement of additional metabolites acting either co-ordinately or at downstream sites. Glucose raises fructose 2,6-bisphosphate levels in hepatocytes by reversing the phosphorylation of PFK2/FBP2 at Ser32, but also independently of Ser32 dephosphorylation. This supports a role for the bifunctional enzyme as the phosphometabolite sensor and for its product, fructose 2,6-bisphosphate, as the metabolic signal for substrate-regulated ChREBP-mediated expression of G6pc and other ChREBP target genes. Show less

Rapsyn, a 43-kDa postsynaptic protein, is essential for anchoring and clustering acetylcholine receptors (AChRs) at the endplate (EP). Mutations in the rapsyn gene have been found to cause a postsynaptic congenital myasthenic syndrome (CMS). We detected six patients with CMS due to mutations in the rapsyn gene (RAPSN). In vitro studies performed in the anconeus muscle biopsies of four patients showed severe reduction of miniature EP potential amplitudes. Electron microscopy revealed various degrees of impaired development of postsynaptic membrane folds. All patients carried the N88K mutation. Three patients were homozygous for N88K and had less severe phenotypes and milder histopathologic abnormalities than the three patients who were heterozygous and carried a second mutation (either L14P, 46insC, or Y269X). Surprisingly, two N88K homozygous patients had one asymptomatic relative each who carried the same genotype, suggesting that additional genetic factors to RAPSN mutations are required for disease expression. Show less