The Hepatoselective Glucokinase Activator PF-04991532 Ameliorates Hyperglycemia without Causing Hepatic Steatosis in Diabetic Rats

<div><p>Hyperglycemia resulting from type 2 diabetes mellitus (T2DM) is the main cause of diabetic complications such as retinopathy and neuropathy. A reduction in hyperglycemia has been shown to prevent these associated complications supporting the importance of glucose control. Glucokinase converts glucose to glucose-6-phosphate and determines glucose flux into the β-cells and hepatocytes. Since activation of glucokinase in β-cells is associated with increased risk of hypoglycemia, we hypothesized that selectively activating hepatic glucokinase would reduce fasting and postprandial glucose with minimal risk of hypoglycemia. Previous studies have shown that hepatic glucokinase overexpression is able to restore glucose homeostasis in diabetic models; however, these overexpression experiments have also revealed that excessive increases in hepatic glucokinase activity may also cause hepatosteatosis. Herein we sought to evaluate whether liver specific pharmacological activation of hepatic glucokinase is an effective strategy to reduce hyperglycemia without causing adverse hepatic lipids changes. To test this hypothesis, we evaluated a hepatoselective glucokinase activator, PF-04991532, in Goto-Kakizaki rats. In these studies, PF-04991532 reduced plasma glucose concentrations independent of changes in insulin concentrations in a dose-dependent manner both acutely and after 28 days of sub-chronic treatment. During a hyperglycemic clamp in Goto-Kakizaki rats, the glucose infusion rate was increased approximately 5-fold with PF-04991532. This increase in glucose infusion can be partially attributed to the 60% reduction in endogenous glucose production. While PF-04991532 induced dose-dependent increases in plasma triglyceride concentrations it had no effect on hepatic triglyceride concentrations in Goto-Kakizaki rats. Interestingly, PF-04991532 decreased intracellular AMP concentrations and increased hepatic futile cycling. These data suggest that hepatoselective glucokinase activation may offer glycemic control without inducing hepatic steatosis supporting the evaluation of tissue specific activators in clinical trials.</p></div

PF-04991532 improves glucose metabolism in rats.

<p>PF-04991532 increased the rate of glucose infusion in order to maintain hyperglycemia in Goto-Kakizaki rats (n = 6/group) (<b>A</b>) which can be attributed to the increased glucose disposal and decreased glucose production (n = 6/group) during steady state (<b>B</b>). PF-04991532 decreased plasma glucose in Goto-Kakizaki rats over 28 days of dosing (n = 6–8/group) [P<0.05 for 30, 60, and 100 mg/kg compared to vehicle] (<b>C</b>) which was accompanied by an increase in plasma triglycerides at the highest dose (n = 6–8/group) [P<0.05 for 100 mg/kg] (<b>D</b>). These plasma changes were not associated with any changes in liver triglycerides compared to vehicle treated animals (<b>E</b>). **P<0.01 One-way ANOVA and Tukey’s Multiple Comparison Test were used for A,C,D, & E. Student’s t-test was used for B.</p

PF-04991532 effects on lipid metabolism and downstream hepatic metabolites.

<p>Expression of key lipid, carbohydrate, and cholesterol metabolism genes in rats treated with an acute 100/kg dose of PF-04991532 relative to vehicle (<b>A</b>). The increase in expression of the lipogenic genes can be explained by the increased nuclear ChREBP (n = 3/4/group) (<b>B</b>). Lastly, a metabolic profile of key metabolites using ³¹P-NMR and ¹H-NMR in rats treated with an acute dose of PF-04991532 (n = 5–6/group) (<b>C</b>). *P<0.05; #P<0.1 for Vehicle vs. 100 mg/kg PF-04991532. Student’s t-test was used.</p

PF-04991532 increased hepatic futile cycling.

<p>The effect of hepatic glucokinase activation on G6Pase (<b>A</b>) and the loss of positional labeling (<b>B</b>) in primary rat hepatocytes. PF-04991532 increased total hepatic substrate cycling due to increased substrate cycling between glucose and glucose-6-phosphate (n = 5–7/group) (<b>C</b>). The increased substrate cycling decreased hepatic ATP concentrations as assessed by NMR (n = 6/group) (<b>D</b>) which in turn increased hepatic pAMPK/AMPK ratio (<b>E</b>) (n = 5/group). *P<0.05, **P<0.01, ***P<0.005 for Vehicle vs. 100 mg/kg PF-04991532. Student’s t-test was used.</p

Discovery of a Selective Covalent Inhibitor of Lysophospholipase-like 1 (LYPLAL1) as a Tool to Evaluate the Role of this Serine Hydrolase in Metabolism

Lysophospholipase-like 1 (LYPLAL1) is an uncharacterized metabolic serine hydrolase. Human genome-wide association studies link variants of the gene encoding this enzyme to fat distribution, waist-to-hip ratio, and nonalcoholic fatty liver disease. We describe the discovery of potent and selective covalent small-molecule inhibitors of LYPLAL1 and their use to investigate its role in hepatic metabolism. In hepatocytes, selective inhibition of LYPLAL1 increased glucose production supporting the inference that LYPLAL1 is a significant actor in hepatic metabolism. The results provide an example of how a selective chemical tool can contribute to evaluating a hypothetical target for therapeutic intervention, even in the absence of complete biochemical characterization