4B, E) suggesting effective GP inhibition in both conditions. of reducing glucose levels in mice under normo and hyperglycemic conditions. Moreover, these GP inhibitors induce accommodation in addition to GP inhibition – such as enhanced mitochondrial oxidation and mTORC2 signaling C to cope with the glucose influx and increased glycogen deposition in the cells, however the molecular mechanism of accommodation is usually unexplored. Introduction Glycogen content of tissues and cells depend around the concerted regulation of glycogen synthesis by glycogen synthase (GS) and glycogen breakdown by glycogen phosphorylase (GP) through an intricate network of transmission transduction pathways related to hormonal signaling [1]. These transmission transduction pathways, converging on JNJ7777120 GS and GP, exert their regulatory activity through the posttranslational modification of these enzymes to meet the energy demands of the organism [2]C[4]. GP activity is crucial in fine tuning hepatic glycogen content and hepatic glucose homeostasis [5], [6]. Glycogen breakdown by GP is usually associated with fasting responses that lead to enhanced hepatic glucose production (HGP) [7] that is reduced by GP inhibition. Moreover, GP inhibition enhances glycogen build-up in skeletal muscle mass and liver enhancing JNJ7777120 glucose uptake that contributes to glucose clearance from blood [8], [9]. Inhibition of HGP and induction of glucose uptake together reduce blood glucose that makes GP a encouraging pharmaceutical target to manage serum glucose levels. GP is usually a homodimeric enzyme existing in a phosphorylated (GPa) and an unphosphorylated form (GPb) [10]. Phosphorylase kinase phosphorylates GPb turning it to GPa, the active form [10]. Effectors influence GP activity by switching between the tense (T, less active) and relaxed (R, more active) says of both GPa and GPb. There are several effector binding sites on GP: the active site, the allosteric (AMP binding) site, the new allosteric (indole-carboxamide binding) site, the inhibitor (purin binding) site and the storage site. [11]. GP has three isoforms named after the tissues where it is dominantly expressed: liver (pygl), brain (pygb) and muscle mass (pygm). Most GP inhibitors (GPi-s) are unselective and inhibit all isoforms [10], [11]. It JNJ7777120 is important to note that glucose is considered as a physiological regulator of GP [12]. However, glucose 6-phosphate exerts a Comp similar effect on GP as glucose, although glucose and glucose 6-phosphate bind to different sites [13] and their binding converts GPa to the T conformation making it more prone to dephosphorylation [13]. Research efforts have recognized an sufficient quantity of structurally different, potent GPi-s (examined in [7], [14]). Genetic or pharmacological inhibition of GP activity ameliorates glucose tolerance supporting the possible applicability of GP inhibition in the management of glucose handling disorders in diabetes [8], [9], [15]C[18]. Indeed, a GP inhibitor, CP-316819 (Ingliforib), in clinical study was able to reduce glucagon-induced hyperglycemia [11]. Our research group has been involved in the design of glucose-derived and other GPi-s [19], [20]. In the current study we have characterized the metabolic effects of a novel glucose-based GPi N-(3,5-dimethyl-benzoyl)-N-(-D-glucopyranosyl)urea (KB228) in control, and diabetic mice and in cellular models. Materials and Methods Chemicals Unless normally stated, all chemicals were from (St. Louis, MO, USA). Glycogen phosphorylase inhibitors TH (D-glucopyranosylidene-spiro-thiohydantoin) [9], NV50 (access to water and chow (10 kcal% of excess fat) (SAFE, Augy, France) or hypercaloric high-fat diet (HFD, 60 kcal% of excess fat) (studies. KB228 was administered to C57/Bl6J mice as a single i.p. injection in a 90 mg/kg dose (lower doses were ineffective C data not shown). KB228 treatment reduced blood glucose levels 30 minutes post treatment and the reduction was managed for 6 hours (Fig. 2A) that coincided with an increment in hepatic glycogen JNJ7777120 content (Fig. 2B) without switch in the expression of GP isoforms (Fig. 2C) suggesting that KB228 treatment was effective. We induced glucose intolerance and hampered insulin sensitivity (tested in ipGTT and ipITT, data not shown) by HFD feeding (3 months feeding). Significant increase in hepatic glycogen content confirmed the efficiency.