ABSTRACT
The glucokinase (GCK) gene was one of the first candidate genes to be identified as a human "diabetes gene". Subsequently, important advances were made in understanding the impact of GCK in the regulation of glucose metabolism. Structure elucidation by crystallography provided insight into the kinetic properties of GCK. Protein interaction partners of GCK were discovered. Gene expression studies revealed new facets of the tissue distribution of GCK, including in the brain, and its regulation by insulin in the liver. Metabolic control analysis coupled to gene overexpression and knockout experiments highlighted the unique impact of GCK as a regulator of glucose metabolism. Human GCK mutants were studied biochemically to understand disease mechanisms. Drug development programs identified small molecule activators of GCK as potential antidiabetics. These advances are summarized here, with the aim of offering an integrated view of the role of GCK in the molecular physiology and medicine of glucose homeostasis.
Subject(s)
Glucose/metabolism , Hexokinase/physiology , Adaptor Proteins, Signal Transducing/metabolism , Brain/enzymology , Congenital Hyperinsulinism/enzymology , Congenital Hyperinsulinism/genetics , Diabetes Mellitus, Type 2/drug therapy , Diabetes Mellitus, Type 2/enzymology , Diabetes Mellitus, Type 2/genetics , Gene Expression Regulation , Hexokinase/chemistry , Hexokinase/genetics , Homeostasis/genetics , Homeostasis/physiology , Humans , Insulin/physiology , Islets of Langerhans/enzymology , Liver/enzymology , Mitochondrial Membranes/enzymologyABSTRACT
AIMS/HYPOTHESIS: An insulin signalling pathway leading from activation of protein kinase B (PKB, also known as Akt) to phosphorylation (inactivation) of glycogen synthase kinase-3 (GSK-3) and activation of glycogen synthase is well characterised. However, in hepatocytes, inactivation of GSK-3 is not the main mechanism by which insulin stimulates glycogen synthesis. We therefore tested whether activation of PKB causes inactivation of glycogen phosphorylase. MATERIALS AND METHODS: We used a conditionally active form of PKB, produced using recombinant adenovirus, to test the role of acute PKB activation in the control of glycogen phosphorylase and glycogen synthesis in hepatocytes. RESULTS: Conditional activation of PKB mimicked the inactivation of phosphorylase, the activation of glycogen synthase, and the stimulation of glycogen synthesis caused by insulin. In contrast, inhibition of GSK-3 caused activation of glycogen synthase but did not mimic the stimulation of glycogen synthesis by insulin. PKB activation and GSK-3 inhibition had additive effects on the activation of glycogen synthase, indicating convergent mechanisms downstream of PKB involving inactivation of either phosphorylase or GSK-3. Glycogen synthesis correlated inversely with the activity of phosphorylase-a, irrespective of whether this was modulated by insulin, by PKB activation or by a selective phosphorylase ligand, supporting an essential role for phosphorylase inactivation in the glycogenic action of insulin in hepatocytes. CONCLUSIONS/INTERPRETATION: In hepatocytes, the acute activation of PKB, but not the inhibition of GSK-3, mimics the stimulation of glycogen synthesis by insulin. This is explained by a pathway downstream of PKB leading to inactivation of phosphorylase, activation of glycogen synthase, and stimulation of glycogen synthesis, independent of the GSK-3 pathway.
Subject(s)
Hepatocytes/physiology , Insulin/pharmacology , Proto-Oncogene Proteins c-akt/physiology , Animals , Cells, Cultured , Enzyme Activation , Glycogen Synthase Kinase 3/metabolism , Hepatocytes/cytology , Hepatocytes/drug effects , Male , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Rats , Rats, WistarABSTRACT
Inhibitors of signalling pathways were used to dissect the mechanism of insulin action on expression of the gene encoding glucokinase in cultured rat hepatocytes. Wortmannin and LY 294002 completely prevented the insulin-induced increase in glucokinase mRNA seen in unhibited cells, indicating that the phosphoinositide 3-kinase module has a key role. A ligand inducible protein kinase B (PKB, also termed cAkt) fusion protein was expressed by using adenoviral transduction of hepatocytes in primary culture. The PKB activity of this protein was shown to be activated in transduced hepatocytes within 30 min of the addition of 4-hydroxytamoxifen and to stay high for 8 h, as a result of serine phosphorylation at position 473 of PKB. The increase in PKB activity was reflected in the hyperphosphorylation of phosphorylated, heat and acid stable regulated by insulin protein (PHAS-I; also termed 4E-BP1, for eukaryotic initiation factor 4E-binding protein 1), a protein involved in the regulation of translation initiation. These effects were comparable to the insulin-induced activation of endogenous PKB and phosphorylation of PHAS-I in non-transduced hepatocytes. The addition of tamoxifen to transduced hepatocytes resulted in an induction of glucokinase mRNA with kinetics and magnitude similar to those of insulin-induced mRNA accumulation. The effect of tamoxifen depended on stimulated PKB activity because it did not occur in hepatocytes that were transduced with a mutant PKB fusion protein that was refractory to activation with tamoxifen. These results establish that acute activation of PKB is sufficient to produce an insulin-like induction of glucokinase in isolated hepatocytes. Together with the inhibition by phosphoinositide 3-kinase inhibitors, they suggest that the activation of PKB might be critical in mediating the induction of glucokinase by insulin. In addition, experiments showed that PD98059 decreased by half the increase in glucokinase mRNA brought about by insulin, suggesting a contributory role of the mitogen-activated protein kinase cascade.
Subject(s)
Gene Expression Regulation, Enzymologic , Glucokinase/genetics , Hepatocytes/enzymology , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins/metabolism , Adenoviridae/genetics , Animals , Cells, Cultured , Enzyme Activation , Insulin Antagonists/pharmacology , Male , Proto-Oncogene Proteins c-akt , RNA, Messenger/genetics , Rats , Rats, Wistar , Signal Transduction/drug effects , Transduction, GeneticABSTRACT
Insulin stimulates the transcription of the sterol regulatory- element binding protein (SREBP) 1/ADD1 gene in liver. Hepatocytes in primary culture were used to delineate the insulin signalling pathway for induction of SREBP1 gene expression. The inhibitors of phosphoinositide 3-kinase (PI 3-kinase), wortmannin and LY 294002, abolished the insulin-dependent increase in SREBP1 mRNA, whereas the inhibitor of the mitogen- activated protein kinase cascade, PD 98059, was without effect. To investigate the role of protein kinase B (PKB)/cAkt downstream of PI 3-kinase, hepatocytes were transduced with an adenovirus encoding a PKB--oestrogen receptor fusion protein. The PKB activity of this recombinant protein was rapidly activated in hepatocytes challenged with 4-hydroxytamoxifen (OHT), as was endogenous PKB in hepatocytes challenged with insulin. The addition of OHT to transduced hepatocytes resulted in accumulation of SREBP1 mRNA, with a time-course and magnitude similar to the effect of insulin in non-transduced cells. The level of SREBP1 mRNA was not increased by OHT in hepatocytes expressing a mutant form of the recombinant protein whose PKB activity was not activated by OHT. Thus acute activation of PKB is sufficient to induce SREBP1 mRNA accumulation in primary hepatocytes, and might be the major signalling event by which insulin induces SREBP1 gene expression in the liver.
Subject(s)
CCAAT-Enhancer-Binding Proteins/biosynthesis , CCAAT-Enhancer-Binding Proteins/genetics , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , Gene Expression Regulation , Insulin/physiology , Liver/metabolism , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins/physiology , Tamoxifen/analogs & derivatives , Transcription Factors , Adenoviridae/genetics , Androstadienes/pharmacology , Chromones/pharmacology , Enzyme Inhibitors/pharmacology , Estrogen Antagonists/pharmacology , Flavonoids/pharmacology , Genetic Vectors , Hepatocytes/metabolism , Immunoblotting , Morpholines/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Precipitin Tests , Proto-Oncogene Proteins c-akt , RNA, Messenger/metabolism , Receptors, Estrogen/metabolism , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/metabolism , Signal Transduction , Sterol Regulatory Element Binding Protein 1 , Tamoxifen/pharmacology , Time Factors , Transduction, Genetic , WortmanninABSTRACT
Conditional expression of the glucokinase regulatory protein in insulinoma cells, under control of the reverse tetracycline-dependent transactivator, was used to investigate whether expression of this protein de novo would alter the intracellular distribution of glucokinase. The regulatory protein, which was undetectable in the basal state, could be induced by doxycycline to levels comparable to those of liver and was detected mostly in the nucleus. Concomitantly, glucokinase accumulated in the nucleus. Human embryonic kidney cells were transiently transfected to express glucokinase and the regulatory protein, either separately or together. Each protein localized predominantly to the cytoplasm when expressed alone. On co-expression, however, both proteins localized virtually entirely to the nucleus. The enzymic activity of glucokinase was not required for promoting nuclear import of the two proteins, as shown with a glucose-phosphorylation-deficient mutant. Finally, in embryonic kidney cells expressing the regulatory protein alone, treatment with leptomycin B resulted in a partial redistribution of the protein from the cytoplasm to the nucleus, suggesting that this protein can shuttle between the two compartments.
Subject(s)
Carrier Proteins , Cell Nucleus/metabolism , Glucokinase/metabolism , Proteins/metabolism , Adaptor Proteins, Signal Transducing , Animals , Antibiotics, Antineoplastic/pharmacology , Biological Transport/drug effects , Catalysis , Cell Nucleus/drug effects , Cells, Cultured , Fatty Acids, Unsaturated/pharmacology , Glucokinase/genetics , Humans , Insulinoma , Intracellular Signaling Peptides and Proteins , Nuclear Localization Signals , Proteins/genetics , Rats , Subcellular Fractions , Transfection , Tumor Cells, CulturedSubject(s)
Glucokinase/metabolism , Glucosyltransferases/metabolism , Glycolysis , Islets of Langerhans/metabolism , Saccharomyces cerevisiae/metabolism , Adenosine Triphosphate/metabolism , Glucokinase/antagonists & inhibitors , Glucosyltransferases/genetics , Liver/metabolism , Saccharomyces cerevisiae/geneticsABSTRACT
A functionally important cis-acting element termed P2 was identified in the liver promoter of the glucokinase gene. Element P2 was delineated by footprinting in vitro with nuclear proteins from rat liver and spleen. Its core sequence in the rat gene is a canonical CACGTG E-box. In the electrophoretic mobility-shift assay with nuclear proteins from rat liver, hepatocytes and hepatoma cells, an oligonucleotide with P2 in the context of the glucokinase promoter sequence gave rise to a DNA-protein complex shown to contain the upstream stimulatory factor (USF) by specific competition experiments and by reactivity with anti-USF antibodies. Transient transfection of hepatoma HepG2 cells, combined with site-directed mutagenesis, demonstrated that the P2 element was important for liver glucokinase promoter activity. Co-transfection of an expression plasmid coding for USF1 activated reporter gene expression in a manner dependent on an intact P2 element, whereas an expression plasmid for c-Myc was ineffective. Expression of a truncated form of USF1 lacking the transcription activation domain and the basic region decreased reporter activity by a dominant-negative effect. The functional significance of the P2 element was also demonstrated in transient transfection of primary hepatocytes.
Subject(s)
Glucokinase/genetics , Liver/enzymology , Liver/physiology , Promoter Regions, Genetic/physiology , Trans-Activators/physiology , Animals , Base Sequence , Binding Sites , Carcinoma, Hepatocellular/enzymology , Carcinoma, Hepatocellular/genetics , DNA/analysis , DNA/genetics , DNA/metabolism , DNA Footprinting , DNA, Neoplasm/genetics , DNA, Neoplasm/metabolism , Glucokinase/biosynthesis , Humans , Liver Neoplasms/enzymology , Liver Neoplasms/genetics , Molecular Sequence Data , Rats , Transfection , Tumor Cells, CulturedABSTRACT
The INS-r3-GK27 insulinoma cells are endowed with artificially inducible glucokinase under control of the reverse tetracycline-dependent transcriptional activator. Moderate induction of glucokinase has been shown to result in proportionate increases in glycolytic flux and in potentiation of glucose effects on insulin secretion and pyruvate kinase gene expression. In cells with 20-fold overexpression of glucokinase, however, glucose activation of secretion and gene expression was severely impaired. Measurements of the glycolytic flux in cells with 7- and 21-fold increases in glucokinase activity and determination of the flux control coefficient of this enzyme showed that control of glycolysis at the glucokinase step was lost in the cells at the higher level of overexpression. Challenging the cells with glucose above 6 mM resulted in massive accumulation of glucose 6-phosphate and caused a rapid and sustained depletion of cellular ATP, in contrast with the glucose-induced rise in ATP in cells with wild-type glucokinase levels. Loss of cell viability ensued upon prolonged culture in high glucose. In summary, in insulinoma beta cells strongly overexpressing glucokinase, an imbalance between glucose phosphorylation and turnover of glucose 6-phosphate resulted in acute glucose intolerance due to trapping of cellular orthophosphate in dead-end product and severe paralysis of energy metabolism.
Subject(s)
Glucokinase/metabolism , Glucose Intolerance/enzymology , Islets of Langerhans/enzymology , Animals , Energy Metabolism , Glucokinase/biosynthesis , Glucose-6-Phosphate/metabolism , Glycolysis , Insulin/metabolism , Insulinoma , Pyruvate Kinase/genetics , Pyruvate Kinase/metabolism , RNA, Messenger/metabolism , Rats , Tumor Cells, CulturedABSTRACT
Insulinoma beta-cells capable of overexpressing glucokinase under the control of a doxycycline-dependent transcriptional transactivator were established from parental INS-1 cells. Glucokinase could be maximally induced to a level more than 20 times the basal level after 36 h of culture with doxycycline. Intermediate levels of induction could be achieved by varying doses of, and time of culture with, the inducer. The rate of glycolysis was measured in cells with 3-, 5-, and 8-fold increment in glucokinase activity above the noninduced level. Proportionate increases in glycolytic flux occurred in cells cultured at low physiological glucose concentration. At high glucose concentration, induction of glucokinase in excess of 2-fold above basal resulted in little additional increase in glycolysis. The consequences of graded increases of glucokinase on two physiological glucose effects were investigated. Increments in glucokinase activity were accompanied by a stepwise shift to the left of the dose-response curve for the inductive effect of glucose on the L-type pyruvate kinase mRNA. Similarly, the insulin secretory response to glucose was shifted leftward in glucokinase-induced cells. The following conclusions are drawn: (i) glucokinase is the major rate-limiting enzyme for glycolysis in these cells; (ii) downstream metabolic steps become limiting at high extracellular glucose concentration with moderate increases in glucokinase over the wild-type level; (iii) within limits, glucokinase activity is a determining factor for two types of glucose responses of the beta-cell, the induction of specific gene expression, and insulin release.
Subject(s)
Glucokinase/biosynthesis , Glucose/pharmacology , Islets of Langerhans/physiology , Anti-Bacterial Agents/pharmacology , Dose-Response Relationship, Drug , Doxycycline/pharmacology , Enzyme Induction , Gene Expression Regulation , Glucokinase/genetics , Glycolysis , Insulin/metabolism , Insulin Secretion , Insulinoma , Islets of Langerhans/cytology , Islets of Langerhans/drug effects , Pancreatic Neoplasms , Pyruvate Kinase/biosynthesis , Trans-Activators , Tumor Cells, CulturedABSTRACT
The aim of our study was to investigate the relative prevalence of the different forms of diabetes in young adults and their respective clinical characteristics. Included were 51 nonobese patients (BMI < 27 kg/m2) with diabetes diagnosed before age 40, excluding typical IDDM. Each patient was subjected to screening for glucokinase gene (MODY2) and mitochondrial DNA (at nucleotide 3243) mutations, to HLA class II genotyping, and screening for the presence of islet cell antibodies (ICAs) and anti-GAD antibodies. Informative families were analyzed for linkage of diabetes to chromosome 12q (MODY3). Based on clinical criteria, patients were subdivided into MODY (n = 19) and non-MODY (n = 32). In the MODY group, we identified three patients with MODY2, one with the 3243 mitochondrial mutation, and another with autoimmune diabetes. One of the five MODY families available for linkage study was shown to have MODY3. In the non-MODY group, we found five patients with autoimmune diabetes and one with MODY2. No clinical parameter was helpful to classify patients in one of these subclasses of diabetes; however, the glucagon-stimulated C-peptide was useful to discriminate between MODY2 patients and the others. In conclusion, young and lean non-insulin-dependent diabetic patients constitute a very heterogeneous group, although they present similar clinical characteristics. The clinical distinction of MODY and non-MODY patients allows correct classification in, at most, 75% of the patients and thus is not sufficient to predict clinical course. However, immunological and genetic parameters allowed us to classify only 25% of the patients in specific diagnostic classes.
Subject(s)
Chromosomes, Human, Pair 12/genetics , Diabetes Mellitus/classification , Diabetes Mellitus/diagnosis , Glucokinase/genetics , Mutation/genetics , Adult , Autoantibodies/blood , Cohort Studies , Diabetes Mellitus/genetics , Diabetes Mellitus/immunology , Family , Female , Genetic Linkage , Genetic Markers , Glutamate Decarboxylase/immunology , Haplotypes/genetics , Humans , Islets of Langerhans/immunology , Lod Score , Male , Middle Aged , Pedigree , Phenotype , RNA, Transfer, Leu/geneticsABSTRACT
A subtype of maturity-onset diabetes of the young (MODY) is caused by mutations of the glucokinase gene, an enzyme expressed in pancreatic beta-cells and the liver. To assess the consequences of a functional alteration of glucokinase at the level of the liver, endogenous (hepatic) glucose production and glucose cycling (an indirect assessment of hepatic glucokinase activity) were measured with 2-2H glucose and 6,6-2H glucose in patients who developed MODY because of the V203A mutation of glucokinase, and in control subjects at similar levels of glycemia. Measurements were performed in the postabsorptive state and after ingestion of 13C-labeled glucose. In the postabsorptive state, MODY patients had normal glucose production (10.9 +/- 1.3 vs. 11.3 +/- 0.6 micromol x kg(-1) x min(-1)) but decreased glucose cycling (0.6 +/- 0.3 vs. 1.5 +/- 0.3 micromol x kg(-1) x min(-1); P < 0.05) when compared with control subjects. However, at plasma glucose and insulin levels similar to those observed in MODY patients, control subjects' glucose production was markedly lower (3.2 +/- 1.5 micromol x kg(-1) x min(-1). After glucose ingestion, endogenous glucose production was reduced by only 29% in MODY patients compared with 80% in control subjects at a similar level of hyperglycemia (P < 0.05). This suggests that the V203A mutation of glucokinase results in decreased activity of glucokinase in liver cells. Thus endogenous glucose production is inadequately inhibited by hyperglycemia in MODY patients, possibly as a result of impaired hepatic glucokinase activity. These alterations contribute to the pathogenesis of hyperglycemia.
Subject(s)
Diabetes Mellitus, Type 2/genetics , Glucokinase/genetics , Adult , Blood Glucose/metabolism , C-Peptide/blood , Calorimetry , Diabetes Mellitus, Type 2/enzymology , Female , Glucose/metabolism , Humans , Insulin/blood , Liver/metabolism , Male , Middle Aged , Mutation , Oxidation-ReductionABSTRACT
Glucokinase gene regions that are important for liver specific expression of the enzyme have been functionally identified using transient transfection of rat hepatocytes. Maximal luciferase activity was elicited by a reporter plasmid with 3.4 kilobase pairs of genomic DNA flanking the liver glucokinase promoter. Deletion of a gene fragment between -1000 and -600 with respect to the start of transcription resulted in a 60% decrease in luciferase activity. Further reduction, close to background level, occurred upon deletion of a 90-base pair sequence between -123 and -34. Reporter plasmids with the liver glucokinase promoter and any length of flanking sequence were minimally active in INS-1 insulinoma cells, and conversely reporters with the beta-cell-specific promoter were ineffective in primary hepatocytes. In FTO-2B hepatoma cells, a differentiated line expressing many liver-specific traits but not the endogenous glucokinase gene, the promoter proximal region between -123 and -34 markedly stimulated the expression of transfected plasmids above background. However, addition of the flanking region up to -1000 inhibited luciferase expression. The gene fragment from -1003 to -707 was shown to be a bona fide, hepatocyte-specific enhancer by the following criteria: 1) it stimulated reporter expression by more than 10- and 5-fold when inserted directly upstream of the glucokinase TATA box or complete promoter, respectively, regardless of orientation; 2) it stimulated gene expression from the heterologous SV 40 promoter 4-fold; 3) it was also effective from a downstream position; and 4) in contrast to the enhancer effect in primary hepatocytes, the sequence acted as a silencer in FTO-2B cells and was neutral in INS-1 cells. Both the promoter proximal and the enhancer regions were marked by DNase I hypersensitive sites in the chromatin of primary hepatocytes but not hepatoma or insulinoma cells. Seven footprinted elements termed A through G were mapped in the enhancer by the in vitro DNase I protection assay. Elements A-C may bind liver enriched factors, because they were not protected by spleen nuclear extract. In hepatocyte transfection, the downstream half of the enhancer containing elements A-C was about half as effective as the complete enhancer in stimulating glucokinase promoter activity. Site-directed mutagenesis of element A virtually abrogated the activity of the half-enhancer, whereas mutation of element C had a more moderate effect. The sequence between -732 and -578 upstream of the liver start of transcription in the human glucokinase gene displays 79% sequence identity with the downstream half of the rat enhancer. The human gene fragment ligated to the minimal rat liver glucokinase promoter was shown to work as an enhancer in the hepatocyte transfection system.
Subject(s)
Enhancer Elements, Genetic , Gene Expression Regulation, Enzymologic , Glucokinase/genetics , Liver/enzymology , Animals , Cells, Cultured , DNA Footprinting , Humans , Male , Mutagenesis, Site-Directed , Promoter Regions, Genetic , Rats , Rats, Wistar , Sequence Deletion , Sequence Homology, Nucleic Acid , Tumor Cells, CulturedABSTRACT
The synthesis of some class 1 acute-phase proteins (APP), including C-reactive protein (CRP) and serum amyloid A (SAA) protein is completely blocked by the IL-1 receptor antagonist (IL-1Ra), whereas the production of fibrinogen, a class 2 APP, is increased by IL-1Ra in hepatoma cells, but this has never been tested in human hepatocytes in primary culture. Since previous studies on the contributions of cytokine inhibitors in connective tissues diseases suggested that IL-1 and tumour necrosis factor-alpha (TNF-alpha) might play an important role in the regulation of CRP, we decided to examine in more detail the respective roles of IL-1 beta, IL-6, and TNF-alpha and their inhibitors in the production of APP by human primary hepatocytes versus the hepatoma cell line PLC/PRF/5. In the hepatoma cell line, IL-1 beta and/or TNF-alpha had synergistic effects with IL-6 on the production of CRP and SAA. In contrast, these cytokines were devoid of effect in normal hepatocytes. The production of fibrinogen was increased by IL-6 and decreased by IL-1 (and TNF-alpha) in both cell types. The secretion of CRP and SAA by primary hepatocytes incubated with a cytokine-rich mononuclear cell-conditioned medium was totally unaffected by IL-1Ra or anti-TNF-alpha antibodies. In contrast, the addition of IL-1Ra increased the production of fibrinogen by both hepatoma cells and primary hepatocytes incubated with the mononuclear cell-conditioned medium. We therefore conclude that IL-1 beta and TNF-alpha do not exert any significant effect on the synthesis of CRP and SAA by human primary hepatocytes.
Subject(s)
C-Reactive Protein/biosynthesis , Carcinoma, Hepatocellular/metabolism , Liver/metabolism , Serum Amyloid A Protein/biosynthesis , Sialoglycoproteins/pharmacology , Cells, Cultured , Fibrinogen/biosynthesis , Humans , In Vitro Techniques , Interleukin 1 Receptor Antagonist Protein , Interleukin-1/pharmacology , Interleukin-6/pharmacology , Receptors, Interleukin-1/antagonists & inhibitors , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
Glucokinase and phosphoenolpyruvate carboxykinase are key enzymes of glucose metabolism in the rat liver. The former is considered to be instrumental in regulating glucose hepatic release/uptake according to the glycaemia level, and cytosolic phosphoenolpyruvate carboxykinase is a major flux-generating enzyme for gluconeogenesis. The level of expression of both enzymes and the regulation of their mRNAs in the human liver cell were investigated. Surgical biopsies of liver from patients undergoing partial hepatectomies and parenchymal hepatocytes derived from the biopsies were used to assay glucokinase, hexokinase and phosphoenolpyruvate carboxykinase activities. Hepatocytes were placed in culture and the actions of insulin, glucagon and cAMP on glucokinase and phosphoenolpyruvate carboxykinase mRNAs were studied. The main results are: (a) glucokinase accounts for 95% of the glucose phosphorylation activity of human hepatocytes, although this fact is masked in assays of total liver tissue; (b) glucokinase activity is set at a lower level in human hepatocytes than in rat hepatocytes, and vice-versa for the gluconeogenic enzyme phosphoenolpyruvate carboxykinase; and (c) as previously shown in rat liver, glucokinase and phosphoenolpyruvate carboxykinase mRNAs are regulated in a reciprocal fashion in human hepatocytes, insulin inducing the first enzyme and repressing the latter, whereas glucagon has opposite effects. These data have interesting implications with respect to metabolic regulation and intracellular hormone signaling in the human liver.
Subject(s)
Gene Expression Regulation, Enzymologic , Glucokinase/biosynthesis , Liver/enzymology , Phosphoenolpyruvate Carboxykinase (GTP)/biosynthesis , Adult , Animals , Base Sequence , Biopsy , Cells, Cultured , Child , Culture Techniques/methods , Cyclic AMP/pharmacology , Cytosol/enzymology , DNA Primers , Enzyme Induction/drug effects , Female , Glucagon/pharmacology , Humans , Isoenzymes/biosynthesis , Kinetics , Liver/pathology , Male , Middle Aged , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Messenger/analysis , RNA, Messenger/biosynthesis , RatsABSTRACT
The insulinoma beta-cell line INS-1 expresses the L-type pyruvate kinase gene at high level and responds to a rise in extracellular glucose by strong induction of gene expression. Following the addition of glucose to the culture medium in the 3.5-33 mM concentration range, the cellular level of L-type pyruvate kinase mRNA increases within 2 h and reaches a maximum 15-fold above basal in 8-12 h. By run-on nuclear assay, the relative transcription rate of the pyruvate kinase gene is shown to increase 4-fold at maximal stimulation, suggesting that both transcriptional and post-transcriptional effects contribute to mRNA accumulation. The glucose effect is totally suppressed by the hexokinase inhibitor mannoheptulose, indicating a requirement for glucose phosphorylation. The mRNA induction is not inhibited in glutamine-free culture medium or by azaserine, suggesting that the hexosamine biosynthetic pathway is not involved. Moreover, metabolism along the glycolytic pathway does not appear to be an absolute requisite, since 2-deoxyglucose partly mimics the inductive effect of glucose. The glucose effect on the pyruvate kinase gene is reversibly antagonized by agents increasing intracellular cAMP. In addition, the effect is highly specific to the pyruvate kinase gene. Neither proinsulin I mRNA nor glucokinase mRNA are increased in glucose-stimulated INS-1 cells. Short term transfection with CAT plasmids driven by the pyruvate kinase L promoter reveals specific glucose-inducible reporter activity with the 183-base pair promoter region upstream of the cap site. Within this region, the previously described L4 cis-acting element is crucial for glucose responsiveness, as demonstrated by the fact that a plasmid with a mutation in this element does not elicit glucose-inducible CAT activity. Induction of L-type pyruvate kinase mRNA occurs in the islets of rats subjected to fasting and carbohydrate refeeding. In conclusion, the L-type pyruvate kinase gene provides an interesting model of glucose-regulated gene in the endocrine beta-cell type.
Subject(s)
Gene Expression Regulation, Enzymologic , Glucose/metabolism , Islets of Langerhans/enzymology , Pyruvate Kinase/genetics , Animals , Base Sequence , Cyclic AMP/analogs & derivatives , Cyclic AMP/pharmacology , DNA Primers , Glucokinase/genetics , Glucose/antagonists & inhibitors , Glucose/pharmacology , Molecular Sequence Data , Proinsulin/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Rats , Thionucleotides/pharmacology , Transcription, Genetic/drug effects , Tumor Cells, CulturedABSTRACT
Glucokinase first appears in the liver of the rat 2 weeks after birth and its activity rapidly increases after weaning on to a high-carbohydrate diet. The appearance of glucokinase is principally due to the increase of plasma insulin and to the decrease of plasma glucagon concentrations. Oral glucose administration to 1- or 10-day-old suckling rats induced an increase in plasma insulin and a fall in plasma glucagon and allowed a rapid accumulation of liver glucokinase mRNA, secondarily to a stimulation of gene transcription. When unrestrained late pregnant rats were infused with glucose during 36 h to induce an increase in fetal plasma insulin and a decrease in fetal plasma glucagon concentrations, glucokinase mRNA was detectable in fetal liver but the level was 100-fold lower than that observed in 1- or 10-day-old suckling rats. It is suggested that the hormonal environment did not allow glucokinase gene expression to be induced in fetal liver and that the absence of expression of glucokinase in suckling rat liver is due to the presence of low plasma insulin and high plasma glucagon levels. The chromatin structure of the glucokinase gene was examined during development by identification of DNase-I-hypersensitive sites from the region comprised between -8 kb upstream and +4 kb downstream of the cap site. Five hypersensitive sites were found: four liver-specific sites upstream of the cap site and one non-specific site in the first intron. These sites are already present in term fetus but the intensity of the two proximal sites located upstream of the cap site increase markedly after birth. This suggests that these sites could be implicated in the regulation of glucokinase gene expression by insulin and glucagon. Full DNase-I-hypersensitivity of these two proximal sites seems necessary for the mature response of glucokinase gene in response to changes in pancreatic hormones concentrations.
Subject(s)
Glucokinase/biosynthesis , Glucose/pharmacology , Liver/enzymology , Animals , Animals, Newborn , Chromatin , Deoxyribonuclease I/metabolism , Enzyme Induction/drug effects , Female , Fetus/metabolism , Gene Expression/drug effects , Glucagon/blood , Glucokinase/genetics , Insulin/blood , Liver/drug effects , Male , RNA, Messenger/metabolism , Rats , Rats, Wistar , Transcription, GeneticABSTRACT
In cultured rat hepatocytes, transcription of the glucokinase gene is turned on by insulin and turned off by glucagon/cAMP, the latter being the dominant effector system. It is thus possible that in the absence of hormones the gene is maintained in a repressed state by the basal level of cAMP and that insulin turns on transcription by relieving cAMP repression, for instance via activation of a cyclic-nucleotide phosphodiesterase. Three inhibitors of this class of enzymes were tested for their effect on the insulin-dependent induction of the glucokinase gene in hepatocytes. Isobutyl methylxanthine, the prototype inhibitor, abrogated the gene response to insulin, as shown by run-on transcription assay. Among the drugs investigated, Ly186126, a preferential inhibitor of type-III phosphodiesterase, proved the most potent in inhibiting insulin-induced accumulation of glucokinase mRNA. Type-III phosphodiesterase is inhibited by cGMP. Induction of glucokinase mRNA was prevented in hepatocytes challenged with insulin in presence of 8-bromoguanosine-3',5'-phosphate. These results are consistent with the involvement of type-III phosphodiesterase in transduction of the insulin signal to the glucokinase gene. However, we were unable to detect significant decreases in total cellular cAMP level or cAMP-dependent-protein-kinase ratio after the addition of insulin to hepatocytes. Many effects of glucagon are mediated via cAMP-dependent protein-kinase phosphorylation of regulatory proteins and, conversely, insulin effects are often accompanied by protein dephosphorylation. A specific inhibitor of protein phosphatases PP1 and PP2A, okadaic acid, was shown to abolish the transcriptional response of the glucokinase gene to insulin. Thus, interference of insulin with the cAMP signal transduction pathway at several steps may be a critical aspect of insulin action on hepatic glucokinase gene expression. In addition, insulin induction of glucokinase mRNA was suppressed by inhibitors of protein synthesis. The underlying mechanism was a severe inhibition of the transcriptional effect of insulin, rather than mRNA destabilization, as demonstrated by run-on transcription assays with nuclei from cycloheximide-treated or pactamycin-treated cells. Transcription of the glucokinase gene may therefore depend on de novo synthesis of the product of an early-response gene induced by insulin, or may require a short-lived trans-acting or accessory factor of transcription. Alternatively, insulin signalling may be compromised in hepatocytes by a mechanism indirectly related to the arrest of protein synthesis.
Subject(s)
Gene Expression Regulation, Enzymologic , Glucokinase/genetics , Insulin/metabolism , Liver/enzymology , Signal Transduction , 3',5'-Cyclic-AMP Phosphodiesterases/antagonists & inhibitors , Animals , Blotting, Northern , Cells, Cultured , Cyclic AMP/metabolism , Ethers, Cyclic/pharmacology , Insulin/pharmacology , Insulin Antagonists , Liver/cytology , Male , Okadaic Acid , Protein Kinases/metabolism , RNA/isolation & purification , Rats , Rats, Wistar , Transcription, GeneticABSTRACT
Amylin appears to interfere with the action of insulin in muscle and possibly in liver. We have attempted to detect a direct antagonism between amylin and insulin in cultured rat hepatocytes. The stimulation of glucokinase gene expression was used as a marker of insulin action. Amylin proved ineffective in suppressing subsequent accumulation of glucokinase mRNA in response to maximal or submaximal doses of insulin. When applied to cells already induced by prior incubation with insulin alone, amylin failed to reverse induction, in contrast to the effectiveness of glucagon under the same conditions. Thus, amylin is not a physiological antagonist of insulin in the control of hepatic glucokinase gene expression.