The transition from net glucose production by the liver to net glucose uptake after a meal is regulated by glucose, insulin and glucagon and by neurotransmitters via mechanisms that are as yet uncharacterised. This study investigated the direct effects of serotonin on glucose metabolism in freshly isolated hepatocytes. Serotonin (5-hydroxytryptamine, 5-HT) is both a neurotransmitter and a hormone produced by the gut in the postprandial state that exerts its target actions through multiple families of 5-HT receptors (5-HTl-7). 5-HT caused a concentration-dependent stimulation of glycogen synthesis in hepatocytes maintained in short-term culture that was counteracted by antagonists of 5-HTl receptors and enhanced by antagonists of 5-HT2BC receptors. Selective agonists of 5-HTl receptors (5-HTIA, 5-HTIB and 5-HTIF), m-5-HT and 5-HT2 receptors provided evidence for a stimulatory pathway mediated by 5-HTl receptors and an inhibitory pathway mediated by 5-HT2 receptors. The activation of the stimulatory pathway was counteracted by the antipsychotic drug olanzapine. M-5-HT (a-Methyl 5-hydroxytryptamine) and selective agonists for 5-HTl receptors were used to study the stimulatory signalling pathway. The stimulation of glycogen synthesis by m-5-HT (and 5-HTl receptor agonists) was greater than the stimulation induced by insulin and was associated with inactivation of glycogen phosphorylase (conversion of phosphorylase-a to phosphorylase-b) and activation of glycogen synthase without stimulation of glucose phosphorylation or glycolysis. M-5-HT counteracted the activation of glycogen phosphorylase caused by forskolin and dibutyryl-cAMP but it did not counteract the inhibition of glycolysis or the phosphorylation of the protein kinase A (PKA) substrate phosphofructokinase-2/fructose bisphosphatase-2 (Ser32) suggesting a selective effect on the glycogenic pathway as opposed to a signalling pathway upstream of PKA. Unlike insulin action the glycogenic stimulation by m-5-HT was not blocked by wortmannin, a PI 3-kinase inhibitor, and it was not associated with activation of PKB. However, it was partially counteracted by inhibitors of cdk5 including roscovitine, kenpaullone and purvanalol. Overexpression of cdk5 and its eo-regulator p35 mimicked the stimulation of glycogen synthesis by m-5-HT supporting a role for cdk5 in control of glycogen synthesis. M-5-HT caused increased phosphorylation of both protein phosphatase-l (Thr320) and its regulatory protein, inhibitor-2 (Thr72), which are substrates of cdk5. This supports a possible role for cdk5 in the glycogenic stimulation bym-5-HT. This study provides evidence that agonists of 5-HTl receptors stimulate hepatic glycogen synthesis by a novel signalling pathway that is totally distinct from the insulin signalling pathway and involves inactivation of glycogen phosphorylase and possibly activation of cdk5. The counteraction of this pathway by olanzapine provides a possible explanation for the impaired glucose tolerance caused by this currently used drug. 11