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Author(s): David J. Lloyd [sup.1] , David J. St Jean Jr [sup.2] , Robert J. M. Kurzeja [sup.2] , Robert C. Wahl [sup.2] , Klaus Michelsen [sup.2] , Rod Cupples [...], Two small-molecule disruptors of the glucokinase-glucokinase-regulatory-protein complex, AMG-1694 and AMG-3969, are identified that decrease blood glucose levels in various models of hyperglycaemic rodents. Novel glucose-lowering agents Glucokinase (GK) is involved in the regulation of glucose homeostasis and is an important target for drug discovery against diabetes, but some current GK activators are associated with an increased risk for hypoglycaemia in the clinic. This paper presents an alternative therapeutic point of intervention, focused on the liver-specific GK regulatory protein (GKRP) that binds and inhibits GK in the nucleus. David Lloyd et al. report on a small molecule, AMG-1694, that specifically disrupts the GK-GKRP complex and reduces blood glucose in hyperglycaemic rodent models. Importantly AMG-1694 has no effect in normoglycaemic animals. Disruption of the GK-GKRP complex offers a liver-specific approach of upregulating GK activity in type II diabetes while reducing the risk of hypoglycaemia. Glucose homeostasis is a vital and complex process, and its disruption can cause hyperglycaemia and type II diabetes mellitus.sup.1. Glucokinase (GK), a key enzyme that regulates glucose homeostasis, converts glucose to glucose-6-phosphate.sup.2,3 in pancreatic [beta]-cells, liver hepatocytes, specific hypothalamic neurons, and gut enterocytes.sup.4. In hepatocytes, GK regulates glucose uptake and glycogen synthesis, suppresses glucose production.sup.3,5, and is subject to the endogenous inhibitor GK regulatory protein (GKRP).sup.6,7,8. During fasting, GKRP binds, inactivates and sequesters GK in the nucleus, which removes GK from the gluconeogenic process and prevents a futile cycle of glucose phosphorylation. Compounds that directly hyperactivate GK (GK activators) lower blood glucose levels and are being evaluated clinically as potential therapeutics for the treatment of type II diabetes mellitus.sup.1,9,10. However, initial reports indicate that an increased risk of hypoglycaemia is associated with some GK activators.sup.11. To mitigate the risk of hypoglycaemia, we sought to increase GK activity by blocking GKRP. Here we describe the identification of two potent small-molecule GK-GKRP disruptors (AMG-1694 and AMG-3969) that normalized blood glucose levels in several rodent models of diabetes. These compounds potently reversed the inhibitory effect of GKRP on GK activity and promoted GK translocation both in vitro (isolated hepatocytes) and in vivo (liver). A co-crystal structure of full-length human GKRP in complex with AMG-1694 revealed a previously unknown binding pocket in GKRP distinct from that of the phosphofructose-binding site. Furthermore, with AMG-1694 and AMG-3969 (but not GK activators), blood glucose lowering was restricted to diabetic and not normoglycaemic animals. These findings exploit a new cellular mechanism for lowering blood glucose levels with reduced potential for hypoglycaemic risk in patients with type II diabetes mellitus.

Fasting hyperglycemia, a prominent finding in diabetes, is primarily due to increased gluconeogenesis. The transcription factor Foxo1 links insulin signaling to decreased transcription of PEPCK and glucose-6-phosphatase (G6Pase) and provides a possible therapeutic target in insulin-resistant states. Synthetic, optimized antisense oligonucleotides (ASOs) specifically inhibit Foxo1 expression. Here we show the effect of such therapy on insulin resistance in mice with diet-induced obesity (DIO). Reducing Foxo1 mRNA expression with ASO therapy in mouse hepatocytes decreased levels of Foxo1 protein and mRNA expression of PEPCK by 48 ± 4% and G6Pase by 64 ± 3%. In mice with DIO and insulin resistance, Foxo1 ASO therapy lowered plasma glucose concentration and the rate of basal endogenous glucose production. In addition, Foxo1 ASO therapy lowered both hepatic triglyceride and diacylglycerol content and improved hepatic insulin sensitivity. Foxo1 ASO also improved adipocyte insulin action. At a tissue-specific level, this manifested as improved insulin-mediated 2-deoxyglucose uptake and suppression of lipolysis. On a whole-body level, the result was improved glucose tolerance after an intraperitoneal glucose load and increased insulin-stimulated whole-body glucose disposal during a hyperinsulinemic-euglycemic clamp. In conclusion, Foxo1 ASO therapy improved both hepatic insulin and peripheral insulin action. Foxo1 is a potential therapeutic target for improving insulin resistance. Diabetes 55: 2042-2050, 2006 [ABSTRACT FROM AUTHOR]