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.

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.

Acetylcholine exerts additive and permissive but not synergistic effects with insulin on glycogen synthesis in hepatocytes.

Parasympathetic (cholinergic) innervation is implicated in the stimulation of hepatic glucose uptake by portal vein hyperglycaemia. We determined the direct effects of acetylcholine on hepatocytes. Acute exposure to acetylcholine mimicked insulin action on inactivation of phosphorylase, stimulation of glycogen synthesis and suppression of phosphoenolpyruvate carboxykinase mRNA levels but with lower efficacy and without synergy. Pre-exposure to acetylcholine had a permissive effect on insulin action similar to glucocorticoids and associated with increased glucokinase activity. It is concluded that acetylcholine has a permissive effect on insulin action but cannot fully account for the rapid stimulation of glucose uptake by the portal signal.

Bioactivity of glycogen phosphorylase inhibitors that bind to the purine nucleoside site.

The bioactivity in hepatocytes of glycogen phosphorylase inhibitors that bind to the active site, the allosteric activator site and the indole carboxamide site has been described. However, the pharmacological potential of the purine nucleoside inhibitor site has remained unexplored. We report the chemical synthesis and bioactivity in hepatocytes of four new olefin derivatives of flavopiridol (1-4) that bind to the purine site. Flavopiridol and 1-4 counteracted the activation of phosphorylase in hepatocytes caused by AICAR (5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside), which is metabolised to an AMP analogue. Unlike an indole carboxamide inhibitor, the analogues 1 and 4 suppressed the basal rate of glycogenolysis in hepatocytes by allosteric inhibition rather than by inactivation of phosphorylase, and accordingly caused negligible stimulation of glycogen synthesis. However, they counteracted the stimulation of glycogenolysis by dibutyryl cAMP by both allosteric inhibition and inactivation of phosphorylase. Cumulatively, the results show key differences between purine site and indole carboxamide site inhibitors in terms of (i) relative roles of dephosphorylation of phosphorylase-a as compared with allosteric inhibition, (ii) counteraction of the efficacy of the inhibitors on glycogenolysis by dibutyryl-cAMP and (iii) stimulation of glycogen synthesis.

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.

Increased potency and efficacy of combined phosphorylase inactivation and glucokinase activation in control of hepatocyte glycogen metabolism.

Glucokinase and phosphorylase both have a high control strength over hepatocyte glycogen metabolism and are potential therapeutic targets for type 2 diabetes. We tested whether combined phosphorylase inactivation and glucokinase activation is a more effective strategy for controlling hepatic glycogen metabolism than single-site targeting. Activation of glucokinase by enzyme overexpression combined with selective dephosphorylation of phosphorylase-a by an indole carboxamide that favors the T conformation of phosphorylase caused a greater stimulation of glycogen synthesis than the sum of either treatment alone. This result is explained by the complementary roles of elevated glucose-6-phosphate (G6P; a positive modulator) and depleted phosphorylase-a (a negative modulator) in activating glycogen synthase and also by synergistic inactivation of phosphorylase-a by glucokinase activation and the indole carboxamide. Inactivation of phosphorylase-a by the indole carboxamide was counteracted by 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside, which is metabolized to an AMP analog; this effect was reversed by G6P. Our findings provide further evidence for the inverse roles of G6P and AMP in regulating the activation state of hepatic phosphorylase. It is proposed that dual targeting of glucokinase and phosphorylase-a enables improved potency and efficacy in controlling hepatic glucose metabolism.