Glucokinase activity is a major determinant of hepatic glucose metabolism and blood glucose homeostasis. Liver glucokinase activity is regulated acutely by adaptive translocation between the nucleus and the cytoplasm through binding and dissociation from its regulatory protein (GKRP) in the nucleus. Whilst the effect of glucose on this mechanism is well established, the role of hormones in regulating glucokinase location and its interaction with binding proteins remains unsettled. Here we show that treatment of rat hepatocytes with 25 mM glucose caused decreased binding of glucokinase to GKRP, translocation from the nucleus and increased binding to 6-phosphofructo 2-kinase/fructose 2,6 bisphosphatase-2 (PFK2/FBPase2) in the cytoplasm. Glucagon caused dissociation of glucokinase from PFK2/FBPase2, concomitant with phosphorylation of PFK2/FBPase2 on Ser-32, uptake of glucokinase into the nucleus and increased interaction with GKRP. Two novel glucagon receptor antagonists attenuated the action of glucagon. This establishes an unequivocal role for hormonal control of glucokinase translocation. Given that glucagon excess contributes to the pathogenesis of diabetes, glucagon may play a role in the defect in glucokinase translocation and activity evident in animal models and human diabetes.
•Hepatic glucokinase activity is acutely regulated by its cellular location.•High glucose translocates glucokinase from nuclear GKRP to cytoplasmic PFK2/FBPase2.•Here we show that glucagon counteracts glucokinase translocation by elevated glucose.•This effect of glucagon is reversed by novel glucagon receptor antagonists.•This study supports a role for glucagon in regulating glucokinase translocation.
BiFC, bimolecular fluorescence complementation; cAMP, cyclic adenosine monophosphate; EPAC, exchange protein directly activated by cAMP; F26P2, fructose 2,6-bisphosphate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GK, glucokinase; GKRP, glucokinase regulatory protein; G6pc, glucose 6-phosphatase; mRFP, red fluorescent protein; N/C ratio, nuclear-to-cytoplasmic ratio; PepO, desHis1Pro4Glu9-glucagon; PepR, desHis1Pro4Glu9Lys12(γ-glutamyl PAL)glucagon-amide; Phos-a, glycogen phosphorylase; PFK2/FBPase2, 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase-2; PKA, protein kinase A; PLA, proximity ligation assay; YFP, yellow fluorescent protein; Glucokinase; Glucokinase regulatory protein; 6-phosphofructo 2-kinase/fructose 2,6 bisphosphatase-2; Hepatocyte; Glucagon
Background & Aims
Dysregulated glucose homeostasis and lipid accumulation characterize non-alcoholic fatty liver disease (NAFLD), but underlying mechanisms are obscure. We report here that Krüppel-like factor 6 (KLF6), a ubiquitous transcription factor that promotes adipocyte differentiation, also provokes the metabolic abnormalities of NAFLD by post-transcriptionally activating PPARα-signaling.
Mice with either hepatocyte-specific depletion of KLF6 (‘DeltaHepKlf6’) or global KLF6 heterozygosity (Klf6 +/−) were fed a high fat diet (HFD) or chow for 8 or 16 weeks. Glucose and insulin tolerance tests were performed to assess insulin sensitivity. Overexpression and knockdown of KLF6 in cultured cells enabled the elucidation of underlying mechanisms. In liver samples from a cohort of 28 NAFLD patients, the expression of KLF6-related target genes was quantified.
Mice with global- or hepatocyte-depletion of KLF6 have reduced body fat content and improved glucose and insulin tolerance, and are protected from HFD-induced steatosis. In hepatocytes, KLF6 deficiency reduces PPARα-regulated genes (Trb3, Pepck) with diminished PPARα-protein but no change in Pparα-mRNA which is explained by the discovery that KLF6 represses miRNA 10b, which leads to induction of PPARα. In NAFLD-patients with advanced disease and inflammation, the expression of miRNA 10b is significantly down-regulated, while PEPCK mRNA is up-regulated; KLF6 mRNA expression also correlates with TRB3 as well as PEPCK gene expression.
KLF6 increases PPAR -activity, whereas KLF6 loss leads to PPARα repression and attenuation of lipid and glucose abnormalities associated with a high fat diet. The findings establish KLF6 as a novel regulator of hepatic glucose and lipid metabolism in fatty liver.
KLF6; PPARα; TRB3; PEPCK; CD36; miRNA-10b; NAFLD
AR42J-B-13 (B-13) cells form hepatocyte-like (B-13/H) cells in response to glucocorticoid treatment. To establish its utility in toxicity and genotoxicity screening, cytochrome P450 (CYP) induction, susceptibility to toxins, and transporter gene expression were examined. Conversion to B-13/H cells resulted in expression of male-specific CYP2C11 and sensitivity to methapyrilene. B-13/H cells constitutively expressed CYP1A, induced expression in response to an aryl hydrocarbon receptor agonist, and activated benzo[α]pyrene to a DNA-damaging species. Functional CYP1A2 was not expressed due to deletions in the Cyp1a2 gene. A B-13 cell line stably expressing the human CYP1A2 was therefore engineered (B-13−TR/h1A2) and the derived B-13/H cells expressed metabolically functional CYP1A2. Treatment with the cooked food mutagen 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine resulted in a dose-dependent increase in DNA damage. B-13/H cells expressed constitutive androstane receptor (CAR) and induced CYP2B1 mRNA levels in response to classical CAR activators. However, translation to functional CYP2B1 protein was low and increased minimally by CAR activator treatment. B-13/H cells expressed high levels of pregnane X-receptor (PXR) and induced CYP3A1 in response to classical PXR activators. CYP3A genes were inducible, functional, and activated aflatoxin B1 to a DNA-damaging species. All 23 major hepatic transporters were induced when B-13 cells were converted to B-13/H cells, although in many cases, levels remained below those present in adult rat liver. However, bile salt export pump, Abcb1b, multidrug resistance-associated protein, and breast cancer resistance protein transporters were functional in B-13/H cells. These data demonstrate that the B-13 cell generates hepatocyte-like cells with functional drug metabolism and transporter activities, which can alone—or in a humanized form—be used to screen for hepatotoxic and genotoxic endpoints in vitro.
stem cell; liver; xenobiotic metabolism; cytochrome P450; transporters.
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, GL 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.
The polymorphism, KLF6-IVS1-27A, in the Krüppel-like factor 6 (KLF6) transcription factor gene enhances its splicing into antagonistic isoforms and is associated with delayed histological progression of non-alcoholic fatty liver disease (NAFLD). To explore a potential role for KLF6 in the development of insulin resistance, central to NAFLD pathogenesis, we genotyped KLF6-IVS1-27 in healthy subjects and assayed fasting plasma glucose (FPG) and insulin sensitivities. Furthermore, we quantified mRNA expression of KLF6 and glucokinase (GCK), as an important mediator of insulin sensitivity, in human livers and in liver tissues derived from a murine Klf6 knockdown model (DeltaKlf6). Klf6 overexpression studies in a mouse hepatocyte line were utilized to mechanistically link KLF6 with Gck promoter activity.
KLF6-IVS1-27Gwt (ie., less KLF6 splicing) was associated with stepwise increases in FPG and insulin and reduced hepatic insulin sensitivity. KLF6 binds to the liver-specific Gck promoter and activates a GCK promoter-reporter, identifying GCK as a KLF6 direct transcriptional target. Accordingly, in DeltaKlf6 hepatocytes, Gck expression was reduced and stable transfection of Klf6 led to upregulation of Gck. GCK and KLF6 mRNAs correlate directly in human NAFLD tissues and immunohistochemistry studies confirm falling levels of both KLF6 and GCK in fat laden hepatocytes. In contrast to full length KLF6, splice variant KLF6-SV1 increases in NAFLD hepatocytes and inversely correlates with glucokinase regulatory protein, which negatively regulates GCK activity.
KLF6 regulation of GCK contributes to the development of hepatic insulin resistance. The KLF6-IVS1-27A polymorphism, which generates more KLF6-SV1, combats this, lowering hepatic insulin resistance and blood glucose.
Krüppel-like Factor 6; non-alcoholic fatty liver disease; hepatic insulin sensitivity; insulin resistance; glucokinase
Hepatic autonomic nerves regulate postprandial hepatic glucose uptake, but the signaling pathways remain unknown. We tested the hypothesis that serotonin (5-hydroxytryptamine [5-HT]) exerts stimulatory and inhibitory effects on hepatic glucose disposal. Ligands of diverse 5-HT receptors were used to identify signaling pathway(s) regulating glucose metabolism in hepatocytes. 5-HT had stimulatory and inhibitory effects on glycogen synthesis in hepatocytes mediated by 5-HT1/2A and 5-HT2B receptors, respectively. Agonists of 5-HT1/2A receptors lowered blood glucose and increased hepatic glycogen after oral glucose loading and also stimulated glycogen synthesis in freshly isolated hepatocytes with greater efficacy than 5-HT. This effect was blocked by olanzapine, an antagonist of 5-HT1/2A receptors. It was mediated by activation of phosphorylase phosphatase, inactivation of glycogen phosphorylase, and activation of glycogen synthase. Unlike insulin action, it was not associated with stimulation of glycolysis and was counteracted by cyclin-dependent kinase (cdk) inhibitors. A role for cdk5 was supported by adaptive changes in the coactivator protein p35 and by elevated glycogen synthesis during overexpression of p35/cdk5. These results support a novel mechanism for serotonin stimulation of hepatic glycogenesis involving cdk5. The opposing effects of serotonin, mediated by distinct 5-HT receptors, could explain why drugs targeting serotonin function can cause either diabetes or hypoglycemia in humans.
The posttranslational regulation of glucokinase (GK) differs in hepatocytes and pancreatic β-cells. We tested the hypothesis that GK mutants that cause maturity-onset diabetes of the young (GK-MODY) show compromised activity and posttranslational regulation in β-cells.
RESEARCH DESIGN AND METHODS
Activity and protein expression of GK-MODY and persistent hyperinsulinemic hypoglycemia of infancy (PHHI) mutants were studied in β-cell (MIN6) and non–β-cell (H4IIE) models. Binding of GK to phosphofructo-2-kinase, fructose-2,6-bisphosphatase (PFK2/FBPase2) was studied by bimolecular fluorescence complementation in cell-based models.
Nine of 11 GK-MODY mutants that have minimal effect on enzyme kinetics in vitro showed decreased specific activity relative to wild type when expressed in β-cells. A subset of these were stable in non–β-cells but showed increased inactivation in conditions of oxidative stress and partial reversal of inactivation by dithiothreitol. Unlike the GK-MODY mutants, four of five GK-PHHI mutants had similar specific activity to wild type and Y214C had higher activity than wild type. The GK-binding protein PFK2/FBPase2 protected wild-type GK from oxidative inactivation and the decreased stability of GK-MODY mutants correlated with decreased interaction with PFK2/FBPase2.
Several GK-MODY mutants show posttranslational defects in β-cells characterized by increased susceptibility to oxidative stress and/or protein instability. Regulation of GK activity through modulation of thiol status may be a physiological regulatory mechanism for the control of GK activity in β-cells.
The induction of hepatic glucose 6-phosphatase (G6pc) by glucose presents a paradox of glucose-induced glucose intolerance. We tested whether glucose regulation of liver gene expression is geared toward intracellular homeostasis.
RESEARCH DESIGN AND METHODS
The effect of glucose-induced accumulation of phosphorylated intermediates on expression of glucokinase (Gck) and its regulator Gckr was determined in hepatocytes. Cell ATP and uric acid production were measured as indices of cell phosphate homeostasis.
Accumulation of phosphorylated intermediates in hepatocytes incubated at elevated glucose induced rapid and inverse changes in Gck (repression) and Gckr (induction) mRNA concomitantly with induction of G6pc, but had slower effects on the Gckr-to-Gck protein ratio. Dynamic metabolic labeling in mice and liver proteome analysis confirmed that Gckr and Gck are low-turnover proteins. Involvement of Max-like protein X in glucose-mediated Gck-repression was confirmed by chromatin immunoprecipitation analysis. Elevation of the Gck-to-Gckr ratio in hepatocytes was associated with glucose-dependent ATP depletion and elevated urate production confirming compromised phosphate homeostasis.
The lowering by glucose of the Gck-to-Gckr ratio provides a potential explanation for the impaired hepatic glucose uptake in diabetes. Elevated uric acid production at an elevated Gck-to-Gckr ratio supports a role for glucose regulation of gene expression in hepatic phosphate homeostasis.
Stable isotope tracers are used to assess metabolic flux profiles in living cells. The existing methods of measurement average out the isotopic isomer distribution in metabolites throughout the cell, whereas the knowledge of compartmental organization of analyzed pathways is crucial for the evaluation of true fluxes. That is why we accepted a challenge to create a software tool that allows deciphering the compartmentation of metabolites based on the analysis of average isotopic isomer distribution.
The software Isodyn, which simulates the dynamics of isotopic isomer distribution in central metabolic pathways, was supplemented by algorithms facilitating the transition between various analyzed metabolic schemes, and by the tools for model discrimination. It simulated 13C isotope distributions in glucose, lactate, glutamate and glycogen, measured by mass spectrometry after incubation of hepatocytes in the presence of only labeled glucose or glucose and lactate together (with label either in glucose or lactate). The simulations assumed either a single intracellular hexose phosphate pool, or also channeling of hexose phosphates resulting in a different isotopic composition of glycogen. Model discrimination test was applied to check the consistency of both models with experimental data. Metabolic flux profiles, evaluated with the accepted model that assumes channeling, revealed the range of changes in metabolic fluxes in liver cells.
The analysis of compartmentation of metabolic networks based on the measured 13C distribution was included in Isodyn as a routine procedure. The advantage of this implementation is that, being a part of evaluation of metabolic fluxes, it does not require additional experiments to study metabolic compartmentation. The analysis of experimental data revealed that the distribution of measured 13C-labeled glucose metabolites is inconsistent with the idea of perfect mixing of hexose phosphates in cytosol. In contrast, the observed distribution indicates the presence of a separate pool of hexose phosphates that is channeled towards glycogen synthesis.