The role of protein kinase B/Akt in insulin-induced inactivation of phosphorylase in rat hepatocytes.

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.

Hepatic glycogen synthesis is highly sensitive to phosphorylase activity: evidence from metabolic control analysis.

We used metabolic control analysis to determine the flux control coefficient of phosphorylase on glycogen synthesis in hepatocytes by titration with a specific phosphorylase inhibitor (CP-91149) or by expression of muscle phosphorylase using recombinant adenovirus. The muscle isoform was used because it is catalytically active in the b-state. CP-91149 inactivated phosphorylase with sequential activation of glycogen synthase. It increased glycogen synthesis by 7-fold at 5 mm glucose and by 2-fold at 20 mm glucose with a decrease in the concentration of glucose causing half-maximal rate (S(0.5)) from 26 to 19 mm. Muscle phosphorylase was expressed in hepatocytes mainly in the b-state. Low levels of phosphorylase expression inhibited glycogen synthesis by 50%, with little further inhibition at higher enzyme expression, and caused inactivation of glycogen synthase that was reversed by CP-91149. At endogenous activity, phosphorylase has a very high (greater than unity) negative control coefficient on glycogen synthesis, regardless of whether it is determined by enzyme inactivation or overexpression. This high control is attenuated by glucokinase overexpression, indicating dependence on other enzymes with high control. The high control coefficient of phosphorylase on glycogen synthesis affirms that phosphorylase is a strong candidate target for controlling hyperglycemia in type 2 diabetes in both the absorptive and postabsorptive states.

Subcellular localization, mobility, and kinetic activity of glucokinase in glucose-responsive insulin-secreting cells.

We investigated the subcellular localization, mobility, and activity of glucokinase in MIN6 cells, a glucose-responsive insulin-secreting beta-cell line. Glucokinase is present in the cytoplasm and a vesicular/granule compartment that is partially colocalized with insulin granules. The granular staining of glucokinase is preserved after permeabilization of the cells with digitonin. There was no evidence for changes in distribution of glucokinase between the cytoplasm and the granule compartment during incubation of the cells with glucose. The rate of release of glucokinase and of phosphoglucoisomerase from digitonin-permeabilized cells was slower when cells were incubated at an elevated glucose concentration (S0.5 approximately 15 mmol/l). This effect of glucose was counteracted by competitive inhibitors of glucokinase (5-thioglucose and mannoheptulose) but was unaffected by fructose analogs and may be due to changes in cell shape or conformation of the cytoskeleton that are secondary to glucose metabolism. Based on the similar release of glucokinase and phosphoglucoisomerase, we found no evidence for specific binding of cytoplasmic digitonin-extractable glucokinase. The affinity of beta-cells for glucose is slightly lower than that in cell extracts and, unlike that in hepatocytes, is unaffected by fructose, tagatose, or a high-K+ medium, which is consistent with the lack of change in glucokinase distribution or release. We conclude that glucokinase is present in two locations, cytoplasm and the granular compartment, and that it does not translocate between them. This conclusion is consistent with the lack of adaptive changes in the glucose phosphorylation affinity. The glucokinase activity associated with the insulin granules may have a role in either direct or indirect coupling between glucose phosphorylation and insulin secretion.

Impaired glycogen synthesis in hepatocytes from Zucker fatty fa/fa rats: the role of increased phosphorylase activity.

AIMS/HYPOTHESIS: The Zucker fatty fa/fa rat develops hyperinsulinaemia, insulin-resistance and severe obesity as a result of a homozygous mutation in the leptin receptor gene. The aim was to characterise the metabolic defect(s) in hepatocytes from fa/fa rats. METHODS: Glucose metabolism and key regulatory enzymes were investigated in hepatocytes from fa/fa and Fa/? rats after short-term culture in the absence of insulin. RESULTS: Hepatocytes from fa/fa rats have higher glucokinase activity and expression of the glucokinase regulatory protein and higher rates of glycolysis and lipogenesis, but lower rates of glycogen synthesis than hepatocytes from Fa/? controls. Insulin caused a similar stimulation of glycogen synthesis in hepatocytes from fa/fa rats as in controls ( > twofold) but did not restore the impaired glycogen synthesis in cells from fa/fa rats. Adenovirus-mediated glucokinase overexpression stimulated glycogen synthesis and glycolysis but aggravated rather than abolished the relative impairment of glycogen synthesis in cells from fa/fa rats. Inhibition of glycolysis with 2,5-anhydromannitol, an inhibitor of glycolysis and gluconeogenesis, increased glucose 6-phosphate concentrations and glycogen synthesis in hepatocytes from Fa/? and fa/fa rats but did not restore the impaired glycogen synthesis in cells from fa/fa rats. Hepatocytes from fa/fa rats had a higher activity of phosphorylase a in the basal state and after incubation with insulin or glucagon and higher total phosphorylase. CONCLUSION/INTERPRETATION: The increased activity of phosphorylase is a major contributing factor to the impaired glycogen synthesis in hepatocytes from fa/fa rats and could contribute to the lipogenic state by a glycogenolytic-glycolytic-lipogenic pathway.

Glucose-6-phosphatase overexpression lowers glucose 6-phosphate and inhibits glycogen synthesis and glycolysis in hepatocytes without affecting glucokinase translocation. Evidence against feedback inhibition of glucokinase.

In hepatocytes glucokinase (GK) and glucose-6-phosphatase (Glc-6-Pase)(1) have converse effects on glucose 6-phosphate (and fructose 6-phosphate) levels. To establish whether hexose 6-phosphate regulates GK binding to its regulatory protein, we determined the effects of Glc-6-Pase overexpression on glucose metabolism and GK compartmentation. Glc-6-Pase overexpression (4-fold) decreased glucose 6-phosphate levels by 50% and inhibited glycogen synthesis and glycolysis with a greater negative control coefficient on glycogen synthesis than on glycolysis, but it did not affect the response coefficients of glycogen synthesis or glycolysis to glucose, and it did not increase the control coefficient of GK or cause dissociation of GK from its regulatory protein, indicating that in hepatocytes fructose 6-phosphate does not regulate GK translocation by feedback inhibition. GK overexpression increases glycolysis and glycogen synthesis with a greater control coefficient on glycogen synthesis than on glycolysis. On the basis of the similar relative control coefficients of GK and Glc-6-Pase on glycogen synthesis compared with glycolysis, and the lack of effect of Glc-6-Pase overexpression on GK translocation or the control coefficient of GK, it is concluded that the main regulatory function of Glc-6-Pase is to buffer the glucose 6-phosphate concentration. This is consistent with recent findings that hyperglycemia stimulates Glc-6-Pase gene transcription.

Leptin enhances glycogen storage in hepatocytes by inhibition of phosphorylase and exerts an additive effect with insulin.

The effects of the adipocyte-derived hormone leptin on glucose metabolism in hepatocytes were investigated. Incubation of hepatocytes from Wistar rats with leptin for 16 h caused a dose-dependent increase in incorporation of [14C]glucose into glycogen, with a maximal effect at 30 nmol/l leptin. This effect of leptin was observed over a range of glucose concentrations (10-25 mmol/l) and was associated with stimulation of net glycogen deposition. It was not counteracted by mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase, indicating that it is not due to increased gluconeogenic flux. Leptin also enhanced the short-term stimulation of glycogen synthesis by insulin. These effects of leptin were associated with inhibition of phosphorylase a, which occurred after 4 h of exposure to leptin. Culture with leptin for 16 h did not affect the activities of glucose-6-phosphatase or glucokinase or the activation state of glycogen synthase. Leptin did not affect glycolysis determined from the detritiation of [3-(3)H]glucose. The inhibitory effects of leptin on phosphorylase a were counteracted by short-term incubation with glucagon but were additive with the inhibitory effects of insulin and also with the inhibition caused by resorcinol (25 pmol/l), which inhibits phosphorylase kinase by a mechanism analogous to the antidiabetic drug proglycosyn. These results show that leptin has additive effects with insulin in inhibiting phosphorylase and stimulating glycogen storage in hepatocytes, indicating that these hormones act by separate but convergent mechanisms. It is concluded that the primary action of leptin in hepatocytes is to enhance glycogen storage. This may be an important compensatory mechanism for the inhibition of insulin secretion.