BACKGROUND AND PURPOSESmall-molecule glucokinase activators (GKAs) are currently being investigated as therapeutic options for the treatment of type 2 diabetes (T2D). Because liver overexpression of glucokinase is thought to be associated with altered lipid profiles, this study aimed at assessing the potential lipogenic risks linked to oral GKA administration. EXPERIMENTAL APPROACHNine GKA candidates were qualified for their ability to activate recombinant glucokinase and to stimulate glycogen synthesis in rat hepatocytes and insulin secretion in rat INS-1E cells. In vivo activity was monitored by plasma glucose and HbA1c measurements after oral administration in rodents. Risk-associated effects were assessed by measuring hepatic and plasma triglycerides and free fatty acids, as well as plasma aminotransferases, and alkaline phosphatase. KEY RESULTSGKAs, while efficiently decreasing glycaemia in acute conditions and HbA1c levels after chronic administration in hyperglycemic db/db mice, were potent inducers of hepatic steatosis. This adverse outcome appeared as soon as 4 days after daily oral administration at pharmacological doses and was not transient. GKA treatment similarly increased hepatic triglycerides in diabetic and normoglycaemic rats, together with a pattern of metabolic phenotypes including different combinations of increased plasma triglycerides, free fatty acids, alanine and aspartyl aminotransferases, and alkaline phosphatase. GKAs belonging to three distinct structural families induced hepatic steatosis in db/db mice, arguing in favour of a target-mediated, rather than a chemical class-mediated, effect. CONCLUSION AND IMPLICATIONSGiven the risks associated with fatty liver disease in the general population and furthermore in patients with T2D, these findings represent a serious warning for the use of GKAs in humans.

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The effect of a 8-adrenergic agonist (isoproterenol) on the tyrosine kinase activity of the insulin receptor was studied in intact adipocytes. Isoproterenol treatment rapidly ( 5 min) inhibited the insulininduced autophosphorylation of the insulin receptor on tyrosine residues in intact adipocytes. The effect of insulin on the phosphorylation of cellular proteins on tyrosine residues was also inhibited by isoproterenol. In order to understand the mechanism responsible for this inhibition, two-dimensional phosphopeptide mapping of the insulin receptor was performed. The pattern of phosphorylation of the insulin receptor in freshly isolated adipocytes showed marked differences from that previously observed in cultured cells overexpressing insulin receptors. These differences include a larger proportion of receptors being phosphorylated on the three tyrosines from the kinase domain and no apparent phosphorylation of the two tyrosines close to the C-terminus after insulin stimulation. Isoproterenol markedly inhibited the effect of insulin on the phosphorylation of the three tyrosines from the kinase domain. However, this inhibition was not associated with an increase in the phosphorylation of serinehhreonine peptides. Thus, this direct analysis of insulin receptor phosphorylation sites in intact adipocytes does no support the idea that padrenegic agents inhibit the tyrosine kinase activity of the receptor through a serinehhreonine phosphorylation-dependent mechanism.Keywords: adipocyte; insulin receptor; tyrosine kinase ; /j-adrenergic agonist ; serinekhreonine phosphorylation.It is generally agreed that the earliest event that can be detected after exposure of intact cells to insulin is the autophosphorylation of the 8-subunit of the insulin receptor on several tyrosine residues. This autophosphorylation stimulates the tyrosine kinase activity of the receptor, which is now recognized as playing a crucial role in insulin signaling, by phosphorylating cellular proteins on tyrosine residues [ I , 21. Alterations in the tyrosine kinase activity of the insulin receptor have been observed in many physiological and pathological situations [3, 41, although the underlying mechanisms remain poorly understood.It is well known that agents that increase cAMP can induce insulin resistance in fat cells. Several lines of evidence indicate that increases in cAMP may inhibit the tyrosine kinase activity of the receptor in white adipocytes 15-91, although contradictory results have been obtained by others [lo]. These contradictions may be due to the methodologies used for measuring the effect of these agents on the tyrosine kinase activity of the receptors. Indeed, this activity was generally measured in v i m , using partially purified receptors and artificial peptides as substrates t5-8, lo]. It is now becoming apparent that results obtained in vifro may not reflect the situation in intact cells 11 1 -161.Moreover, the potential mechanisms responsible for the inhibition of the tyrosine kinase activity of the receptor remain un- kno...

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