Metformin lowers glucose 6-phosphate in hepatocytes by activation of glycolysis downstream of glucose phosphorylation.

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.

Opposite effects of a glucokinase activator and metformin on glucose-regulated gene expression in hepatocytes.

AIM: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Glucagon induces translocation of glucokinase from the cytoplasm to the nucleus of hepatocytes by transfer between 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase-2 and the glucokinase regulatory protein.

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 25mM 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.

Upregulation of Anti-Angiogenic miR-106b-3p Correlates Negatively with IGF-1 and Vascular Health Parameters in a Model of Subclinical Cardiovascular Disease: Study with Metformin Therapy.

Well-controlled type 1 diabetes mellitus (T1DM) is regarded as a model of subclinical cardiovascular disease (CVD), characterized by inflammation and adverse vascular health. However, the underlying mechanisms are not fully understood. We investigated insulin-like growth factor-1 (IGF-1) and IGF-binding protein-3 (IGFBP-3) levels, their correlation to miR-106b-3p expression in a subclinical CVD model, and the cardioprotective effect of metformin. A total of 20 controls and 29 well-controlled T1DM subjects were studied. Plasma IGF-1, IGFBP-3 levels, and miR-106b-3p expression in colony-forming unit-Hills were analyzed and compared with vascular markers. miR-106b-3p was upregulated in T1DM (p < 0.05) and negatively correlated with pro-angiogenic markers CD34+/100-lymphocytes (p < 0.05) and IGF-1 (p < 0.05). IGF-1 was downregulated in T1DM (p < 0.01), which was associated with increased inflammatory markers TNF-α, CRP, and IL-10 and reduced CD34+/100-lymphocytes. IGFBP-3 had no significant results. Metformin had no effect on IGF-1 but significantly reduced miR-106b-3p (p < 0.0001). An Ingenuity Pathway analysis predicted miR-106b-3p to inhibit PDGFA, PIK3CG, GDNF, and ADAMTS13, which activated CVD. Metformin was predicted to be cardioprotective by inhibiting miR-106b-3p. In conclusion: Subclinical CVD is characterized by a cardio-adverse profile of low IGF-1 and upregulated miR-106b-3p. We demonstrated that the cardioprotective effect of metformin may be via downregulation of upregulated miR-106b-3p and its effect on downstream targets.

Fructose 2,6-bisphosphate is essential for glucose-regulated gene transcription of glucose-6-phosphatase and other ChREBP target genes in hepatocytes.

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.

Differential routing and disposition of the long-chain saturated fatty acid palmitate in rodent vs human beta-cells.

BACKGROUND: Rodent and human ß-cells are differentially susceptible to the "lipotoxic" effects of long-chain saturated fatty acids (LC-SFA) but the factors accounting for this are unclear. Here, we have studied the intracellular disposition of the LC-SFA palmitate in human vs rodent ß-cells and present data that reveal new insights into the factors regulating ß-cell lipotoxicity. METHODS: The subcellular distribution of the LC-SFA palmitate was studied in rodent (INS-1E and INS-1 823/13 cells) and human (EndoC-ßH1) ß-cells using confocal fluorescence and electron microscopy (EM). Protein expression was assessed by Western blotting and cell viability, by vital dye staining. RESULTS: Exposure of INS-1 cells to palmitate for 24 h led to loss of viability, whereas EndoC-ßH1 cells remained viable even after 72 h of treatment with a high concentration (1 mM) of palmitate. Use of the fluorescent palmitate analogue BODIPY FL C16 revealed an early localisation of the LC-SFA to the Golgi apparatus in INS-1 cells and this correlated with distention of intracellular membranes, visualised under the EM. Despite this, the PERK-dependent ER stress pathway was not activated under these conditions. By contrast, BODIPY FL C16 did not accumulate in the Golgi apparatus in EndoC-ßH1 cells but, rather, co-localised with the lipid droplet-associated protein, PLIN2, suggesting preferential routing into lipid droplets. When INS-1 cells were treated with a combination of palmitate plus oleate, the toxic effects of palmitate were attenuated and BODIPY FL C16 localised primarily with PLIN2 but not with a Golgi marker. CONCLUSION: In rodent ß-cells, palmitate accumulates in the Golgi apparatus at early time points whereas, in EndoC- ßH1 cells, it is routed preferentially into lipid droplets. This may account for the differential sensitivity of rodent vs human ß-cells to "lipotoxicity" since manoeuvres leading to the incorporation of palmitate into lipid droplets is associated with the maintenance of cell viability in both cell types.

Pseudoislet Aggregation of Pancreatic ß-Cells Improves Glucose Stimulated Insulin Secretion by Altering Glucose Metabolism and Increasing ATP Production.

Appropriate glucose-stimulated insulin secretion (GSIS) by pancreatic ß-cells is an essential component of blood glucose homeostasis. Configuration of ß-cells as 3D pseudoislets (PI) improves the GSIS response compared to 2D monolayer (ML) culture. The aim of this study was to determine the underlying mechanisms. MIN6 ß-cells were grown as ML or PI for 5 days. Human islets were isolated from patients without diabetes. Function was assessed by GSIS and metabolic capacity using the Seahorse bioanalyser. Connexin 36 was downregulated using inducible shRNA. Culturing MIN6 as PI improved GSIS. MIN6 PI showed higher glucose-stimulated oxygen consumption (OCR) and extracellular acidification (ECAR) rates. Further analysis showed the higher ECAR was, at least in part, a consequence of increased glycolysis. Intact human islets also showed glucose-stimulated increases in both OCR and ECAR rates, although the latter was smaller in magnitude compared to MIN6 PI. The higher rates of glucose-stimulated ATP production in MIN6 PI were consistent with increased enzyme activity of key glycolytic and TCA cycle enzymes. There was no impact of connexin 36 knockdown on GSIS or ATP production. Configuration of ß-cells as PI improves GSIS by increasing the metabolic capacity of the cells, allowing higher ATP production in response to glucose.

Mitochondrial complex I subunit deficiency promotes pancreatic α-cell proliferation.

OBJECTIVE: There is strong evidence that mitochondrial DNA mutations and mitochondrial dysfunction play a role in diabetes pathogenesis. The homozygous knock-in mtDNA mutator mouse is a model of premature aging due to the accumulation of mitochondrial DNA mutations. We used this mouse model to investigate the relationship between mitochondrial subunit expression and pancreatic islet cell composition. METHODS: Quadruple immunofluorescence was used to quantify mitochondrial subunit expression (complex I and IV) and cell composition in pancreatic islets from mitochondrial DNA mutator mice (PolgAmut/mut) and control C57BL/6 mice at 12 and 44 weeks of age. RESULTS: Mitochondrial complex I subunit expression was decreased in islets from 12 week PolgAmut/mut mice. This complex I deficiency persisted with age and was associated with decreased insulin staining intensity at 44 weeks. Complex I deficiency was greater in α-cells compared with ß-cells in islets from 44 week PolgAmut/mut mice. Islet cell composition was normal in 12 week PolgAmut/mut mice, but the ß: α cell ratio was decreased in islets from 44 week PolgAmut/mut mice. This was due to an increase in α-cell number linked to an increase in α-cell proliferation. CONCLUSION: Complex I deficiency promotes α-cell proliferation and alters islet cell composition.

Exendin-4 stimulates autophagy in pancreatic ß-cells via the RAPGEF/EPAC-Ca2+-PPP3/calcineurin-TFEB axis.

Macroautophagy/autophagy is critical for the regulation of pancreatic ß-cell mass and its deregulation has been implicated in the pathogenesis of type 2 diabetes (T2D). We have previously shown that treatment of pancreatic ß-cells with the GLP1R (glucagon like peptide 1 receptor) agonist exendin-4 stimulates autophagic flux in a setting of chronic nutrient excess. The aim of this study was to identify the underlying pathways contributing to enhanced autophagic flux.Pancreatic ß-cells (INS-1E),mouse and human islets were treated with glucolipotoxic stress (0.5 mM palmitate and 25 mM glucose) in the presence of exendin-4. Consistent with our previous work, exendin-4 stimulated autophagic flux. Using chemical inhibitors and siRNA knockdown, we identified RAPGEF4/EPAC2 (Rap guanine nucleotide exchange factor 4) and downstream calcium signaling to be essential for regulation of autophagic flux by exendin-4. This pathway was independent of AMPK and MTOR signaling. Further analysis identified PPP3/calcineurin and its downstream regulator TFEB (transcription factor EB) as key proteins mediating exendin-4 induced autophagy. Importantly, inhibition of this pathway prevented exendin-4-mediated cell survival and overexpression of TFEB mimicked the cell protective effects of exendin-4 in INS-1E and human islets. Moreover, treatment of db/db mice with exendin-4 for 21 days increased the expression of lysosomal markers within the pancreatic islets. Collectively our data identify the RAPGEF4/EPAC2-calcium-PPP3/calcineurin-TFEB axis as a key mediator of autophagic flux, lysosomal function and cell survival in pancreatic ß-cells. Pharmacological modulation of this axis may offer a novel therapeutic target for the treatment of T2D.Abbreviations: AKT1/protein kinase B: AKT serine/threonine kinase 1; AMPK: 5' AMP-activated protein kinase; CAMKK: calcium/calmodulin-dependent protein kinase kinase; cAMP: cyclic adenosine monophosphate; CASP3: caspase 3; CREB: cAMP response element-binding protein; CTSD: cathepsin D; Ex4: exendin-4(1-39); GLP-1: glucagon like peptide 1; GLP1R: glucagon like peptide 1 receptor; GLT: glucolipotoxicity; INS: insulin; MTOR: mechanistic target of rapamycin kinase; NFAT: nuclear factor of activated T-cells; PPP3/calcineurin: protein phosphatase 3; PRKA/PKA: protein kinase cAMP activated; RAPGEF3/EPAC1: Rap guanine nucleotide exchange factor 3; RAPGEF4/EPAC2: Rap guanine nucleotide exchange factor 4; SQSTM1/p62: sequestosome 1; T2D: type 2 diabetes; TFEB: transcription factor EB.

A role for PFK-2/FBPase-2, as distinct from fructose 2,6-bisphosphate, in regulation of insulin secretion in pancreatic beta-cells.

PFK-2/FBPase-2 (6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase) catalyses the formation and degradation of fructose 2,6-P(2) (fructose 2,6-bisphosphate) and is also a glucokinase-binding protein. The role of fructose 2,6-P(2) in regulating glucose metabolism and insulin secretion in pancreatic beta-cells is unresolved. We down-regulated the endogenous isoforms of PFK-2/FBPase-2 with siRNA (small interfering RNA) and expressed KA (kinase active) and KD (kinase deficient) variants to distinguish between the role of PFK-2/FBPase-2 protein and the role of its product, fructose 2,6-P(2), in regulating beta-cell function. Human islets expressed the PFKFB2 (the gene encoding isoform 2 of the PFK2/FBPase2 protein) and PFKFB3 (the gene encoding isoform 3 of the PFK2/FBPase2 protein) isoforms and mouse islets expressed PFKFB2 at the mRNA level [RT-PCR (reverse transcription-PCR)]. Rat islets expressed PFKFB2 lacking the C-terminal phosphorylation sites. The glucose-responsive MIN6 and INS1E cell lines expressed PFKFB2 and PFKFB3. PFK-2 activity and the cell content of fructose 2,6-P(2) were increased by elevated glucose concentration and during pharmacological activation of AMPK (AMP-activated protein kinase), which also increased insulin secretion. Partial down-regulation of endogenous PFKFB2 and PFKFB3 in INS1E by siRNA decreased PFK-2/FBPase-2 protein, fructose 2,6-P(2) content, glucokinase activity and glucoseinduced insulin secretion. Selective down-regulation of glucose-induced fructose 2,6-P(2) in the absence of down-regulation of PFK-2/FBPase-2 protein, using a KD PFK-2/FBPase-2 variant, resulted in sustained glycolysis and elevated glucose-induced insulin secretion, indicating an over-riding role of PFK-2/FBPase-2 protein, as distinct from its product fructose 2,6-P(2), in potentiating glucose-induced insulin secretion. Whereas down-regulation of PFK-2/FBPase-2 decreased glucokinase activity, overexpression of PFK-2/FBPase-2 only affected glucokinase distribution. It is concluded that PFK-2/FBPase-2 protein rather than its product fructose 2,6-P(2) is the over-riding determinant of glucose-induced insulin secretion through regulation of glucokinase activity or subcellular targeting.

Differential effects of saturated and unsaturated fatty acids on autophagy in pancreatic ß-cells.

Long-chain saturated fatty acids are lipotoxic to pancreatic ß-cells, whereas most unsaturates are better tolerated and some may even be cytoprotective. Fatty acids alter autophagy in ß-cells and there is increasing evidence that such alterations can impact directly on the regulation of viability. Accordingly, we have compared the effects of palmitate (C16:0) and palmitoleate (C16:1) on autophagy in cultured ß-cells and human islets. Treatment of BRIN-BD11 ß-cells with palmitate led to enhanced autophagic activity, as judged by cleavage of microtubule-associated protein 1 light chain 3-I (LC3-I) and this correlated with a marked loss of cell viability in the cells. In addition, transfection of these cells with an mCherry-YFP-LC3 reporter construct revealed the accumulation of autophagosomes in palmitate-treated cells, indicating an impairment of autophagosome-lysosome fusion. This was also seen upon addition of the vacuolar ATPase inhibitor, bafilomycin A1. Exposure of BRIN-BD11 cells to palmitoleate (C16:1) did not lead directly to changes in autophagic activity or flux, but it antagonised the actions of palmitate. In parallel, palmitoleate also improved the viability of palmitate-treated BRIN-BD11 cells. Equivalent responses were observed in INS-1E cells and in isolated human islets. Taken together, these data suggest that palmitate may cause an impairment of autophagosome-lysosome fusion. These effects were not reproduced by palmitoleate which, instead, antagonised the responses mediated by palmitate suggesting that attenuation of ß-cell stress may contribute to the improvement in cell viability caused by the mono-unsaturated fatty acid.

A role for Glucagon-Like Peptide-1 in the regulation of ß-cell autophagy.

Autophagy is a highly conserved intracellular recycling pathway that serves to recycle damaged organelles/proteins or superfluous nutrients during times of nutritional stress to provide energy to maintain intracellular homeostasis and sustain core metabolic functions. Under these conditions, autophagy functions as a cell survival mechanism but impairment of this pathway can lead to pro-death stimuli. Due to their role in synthesising and secreting insulin, pancreatic ß-cells have a high requirement for robust degradation pathways. Recent research suggests that functional autophagy is required to maintain ß-cell survival and function in response to high fat diet suggesting a pro-survival role. However, a role for autophagy has also been implicated in the pathogenesis of type 2 diabetes. Thus, the pro-survival vs pro-death role of autophagy in regulating ß-cell mass requires discussion. Emerging evidence suggests that Glucagon-Like Peptide-1 (GLP-1) may exert beneficial effects on glucose homeostasis via autophagy-dependent pathways both in pancreatic ß-cells and in other cell types. The aim of the current review is to: i) summarise the literature surrounding ß-cell autophagy and its pro-death vs pro-survival role in regulating ß-cell mass; ii) review the literature describing the impact of GLP-1 on ß-cell autophagy and in other cell types; iii) discuss the potential underlying mechanisms.

Glucagon-Like Peptide 1 Protects Pancreatic ß-Cells From Death by Increasing Autophagic Flux and Restoring Lysosomal Function.

Studies in animal models of type 2 diabetes have shown that glucagon-like peptide 1 (GLP-1) receptor agonists prevent ß-cell loss. Whether GLP-1 mediates ß-cell survival via the key lysosomal-mediated process of autophagy is unknown. In this study, we report that treatment of INS-1E ß-cells and primary islets with glucolipotoxicity (0.5 mmol/L palmitate and 25 mmol/L glucose) increases LC3 II, a marker of autophagy. Further analysis indicates a blockage in autophagic flux associated with lysosomal dysfunction. Accumulation of defective lysosomes leads to lysosomal membrane permeabilization and release of cathepsin D, which contributes to cell death. Our data further demonstrated defects in autophagic flux and lysosomal staining in human samples of type 2 diabetes. Cotreatment with the GLP-1 receptor agonist exendin-4 reversed the lysosomal dysfunction, relieving the impairment in autophagic flux and further stimulated autophagy. Small interfering RNA knockdown showed the restoration of autophagic flux is also essential for the protective effects of exendin-4. Collectively, our data highlight lysosomal dysfunction as a critical mediator of ß-cell loss and shows that exendin-4 improves cell survival via restoration of lysosomal function and autophagic flux. Modulation of autophagy/lysosomal homeostasis may thus define a novel therapeutic strategy for type 2 diabetes, with the GLP-1 signaling pathway as a potential focus.

Dual role of phosphofructokinase-2/fructose bisphosphatase-2 in regulating the compartmentation and expression of glucokinase in hepatocytes.

Hepatic glucokinase is regulated by a 68-kDa regulatory protein (GKRP) that is both an inhibitor and nuclear receptor for glucokinase. We tested the role of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2) in regulating glucokinase compartmentation in hepatocytes. PFK2 catalyzes formation or degradation of the regulator of glycolysis fructose 2,6-bisphosphate (fructose 2,6-P2), depending on its phosphorylation state (ser-32), and is also a glucokinase-binding protein. Incubation of hepatocytes at 25 mmol/l glucose causes translocation of glucokinase from the nucleus to the cytoplasm and an increase in fructose 2,6-P2. Glucagon caused phosphorylation of PFK2-ser-32, lowered the fructose 2,6-P2 concentration, and inhibited glucose-induced translocation of glucokinase. These effects of glucagon were reversed by expression of a kinase-active PFK2 mutant (S32A/H258A) that overrides the suppression of fructose 2,6-P2 but not by overexpression of wild-type PFK2. Overexpression of PFK2 potentiated glucokinase expression in hepatocytes transduced with an adenoviral vector-encoding glucokinase by a mechanism that does not involve stabilization of glucokinase protein from degradation. It is concluded that PFK2 has a dual role in regulating glucokinase in hepatocytes: it potentiates glucokinase protein expression by posttranscriptional mechanisms and favors its cytoplasmic compartmentation. Thus, it acts in a complementary mechanism to GKRP, which also regulates glucokinase protein expression and compartmentation.

Glucokinase regulatory protein is associated with mitochondria in hepatocytes.

The association of glucokinase with liver mitochondria has been reported [Danial et al. (2003) BAD and glucokinase reside in a mitochondrial complex that integrates glycolysis and apoptosis. Nature 424, 952-956]. We confirmed association of glucokinase immunoreactivity with rat liver mitochondria using Percoll gradient centrifugation and demonstrated its association with the 68 kDa regulatory protein (GKRP) but not with the binding protein phosphofructokinase-2/fructose bisphosphatase-2. Substrates and glucagon induced adaptive changes in the mitochondrial glucokinase/GKRP ratio suggesting a regulatory role for GKRP. Combined with previous observations that GKRP overexpression partially inhibits glycolysis [de la Iglesia et al. (2000) The role of the regulatory protein of glucokinase in the glucose sensory mechanism of the hepatocyte. J. Biol. Chem. 275, 10597-10603] these findings suggest that there may be distinct glycolytic pools of glucokinase.

Increased sensitivity of glycogen synthesis to phosphorylase-a and impaired expression of the glycogen-targeting protein R6 in hepatocytes from insulin-resistant Zucker fa/fa rats.

Hepatic insulin resistance in the leptin-receptor defective Zucker fa/fa rat is associated with impaired glycogen synthesis and increased activity of phosphorylase-a. We investigated the coupling between phosphorylase-a and glycogen synthesis in hepatocytes from fa/fa rats by modulating the concentration of phosphorylase-a. Treatment of hepatocytes from fa/fa rats and Fa/? controls with a selective phosphorylase inhibitor caused depletion of phosphorylase-a, activation of glycogen synthase and stimulation of glycogen synthesis. The flux-control coefficient of phosphorylase on glycogen synthesis was glucose dependent and at 10 mm glucose was higher in fa/fa than Fa/? hepatocytes. There was an inverse correlation between the activities of glycogen synthase and phosphorylase-a in both fa/fa and Fa/? hepatocytes. However, fa/fa hepatocytes had a higher activity of phosphorylase-a, for a corresponding activity of glycogen synthase. This defect was, in part, normalized by expression of the glycogen-targeting protein, PTG. Hepatocytes from fa/fa rats had normal expression of the glycogen-targeting proteins G(L) and PTG but markedly reduced expression of R6. Expression of R6 protein was increased in hepatocytes from Wistar rats after incubation with leptin and insulin. Diminished hepatic R6 expression in the leptin-receptor defective fa/fa rat may be a contributing factor to the elevated phosphorylase activity and/or its high control strength on glycogen synthesis.

Glucokinase is an integral component of the insulin granules in glucose-responsive insulin secretory cells and does not translocate during glucose stimulation.

The association of glucokinase with insulin secretory granules has been shown by cell microscopy techniques. We used MIN6 insulin-secretory cells and organelle fractionation to determine the effects of glucose on the subcellular distribution of glucokinase. After permeabilization with digitonin, 50% of total glucokinase remained bound intracellularly, while 30% was associated with the 13,000g particulate fraction. After density gradient fractionation of the organelles, immunoreactive glucokinase was distributed approximately equally between dense insulin granules and low-density organelles that cofractionate with mitochondria. Although MIN6 cells show glucose-responsive insulin secretion, glucokinase association with the granules and low-density organelles was not affected by glucose. Subfractionation of the insulin granule components by hypotonic lysis followed by sucrose gradient centrifugation showed that glucokinase colocalized with the granule membrane marker phogrin and not with insulin. PFK2 (6-phosphofructo-2-kinase-2/fructose-2,6-bisphosphatase)/FDPase-2, a glucokinase-binding protein, and glyceraldehyde phosphate dehydrogenase, which has been implicated in granule fusion, also colocalized with glucokinase after hypotonic lysis or detergent extaction of the granules. The results suggest that glucokinase is an integral component of the granule and does not translocate during glucose stimulation.

Glucose induces protein targeting to glycogen in hepatocytes by fructose 2,6-bisphosphate-mediated recruitment of MondoA to the promoter.

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.

A novel mechanism for regulating hepatic glycogen synthesis involving serotonin and cyclin-dependent kinase-5.

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.