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Intraportal infusion of serotonin (5-hydroxytryptamine, 5-HT) or inhibitors of its cellular uptake stimulate hepatic glucose uptake in vivo by either direct or indirect mechanisms. The aims of this study were to determine the direct effects of 5-HT in hepatocytes and to test the hypothesis that atypical antipsychotic drugs that predispose to type 2 diabetes counter-regulate the effects of 5-HT.Rat hepatocytes were studied in short-term primary culture.Serotonin (5-HT) stimulated glycogen synthesis at nanomolar concentrations but inhibited it at micromolar concentrations. The stimulatory effect was mimicked by alpha-methyl-5-HT, a mixed 5-HT1/5-HT2 receptor agonist, whereas the inhibition was counteracted by a 5-HT2B/2C receptor antagonist. alpha-Methyl-5-HT stimulated glycogen synthesis additively with insulin, but unlike insulin, did not stimulate glucose phosphorylation and glycolysis, nor did it cause Akt (protein kinase B) phosphorylation. Stimulation of glycogen synthesis by alpha-methyl-5-HT correlated with depletion of phosphorylase a. This effect could not be explained by elevated levels of glucose 6-phosphate, which causes inactivation of phosphorylase, but was explained, at least in part, by decreased phosphorylase kinase activity in situ. The antipsychotic drugs clozapine and olanzapine, which bind to 5-HT receptors, counteracted the effect of alpha-methyl-5-HT on phosphorylase inactivation.This study provides evidence for both stimulation and inhibition of glycogen synthesis in hepatocytes by serotonergic mechanisms. The former effects are associated with the inactivation of phosphorylase and are counteracted by atypical antipsychotic drugs that cause hepatic insulin resistance. Antagonism of hepatic serotonergic mechanisms may be a component of the hepatic dysregulation caused by antipsychotic drugs that predispose to type 2 diabetes.

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. 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. 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. 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.

A method is described for the attachment to and monolayer culture of adult rat hepatocytes on collagen-coated or fibronectin-coated microbeads or both in a chemically defined serum-free medium. Protein synthesis measured by the incorporation of [3H]leucine into protein was four-fold higher in the hepatocyte microcarrier cultures than in isolated hepatocyte suspensions. The hepatocyte microcarrier cultures showed acute responsiveness to insulin of fatty acid synthesis, glucose incorporation into glycogen, and decarboxylation of [1-14 C]pyruvate. Microcarrier-cultured hepatocytes have the combined advantages of monolayer culture and suspension systems. They are a potential tool for the study of long-term as well as acute effects of hormones.