Murine GPRC6A Mediates Cellular Responses to L-Amino Acids, but Not Osteocalcin Variants.

Phenotyping of Gprc6a KO mice has shown that this promiscuous class C G protein coupled receptor is variously involved in regulation of metabolism, inflammation and endocrine function. Such effects are described as mediated by extracellular calcium, L-amino acids, the bone-derived peptide osteocalcin (OCN) and the male hormone testosterone, introducing the concept of a bone-energy-metabolism-reproduction functional crosstalk mediated by GPRC6A. However, whilst the calcium and L-amino acid-sensing properties of GPRC6A are well established, verification of activity of osteocalcin at both human and mouse GPRC6A in vitro has proven somewhat elusive. This study characterises the in vitro pharmacology of mouse GPRC6A in response to its putative ligands in both recombinant and endogenous GPRC6A-expressing cells. Using cell signalling, and glucagon-like peptide (GLP)-1 and insulin release assays, our results confirm that basic L-amino acids act as agonists of the murine GPRC6A receptor in both recombinant cells and immortalised entero-endocrine and pancreatic ß-cells. In contrast, our studies do not support a role for OCN as a direct ligand for mouse GPRC6A, suggesting that the reported in vivo effects of OCN that require GPRC6A may be indirect, rather than via direct activation of the receptor.

S 50131 and S 51434, two novel small molecule glucokinase activators, lack chronic efficacy despite potent acute antihyperglycaemic activity in diabetic mice.

BACKGROUND AND PURPOSE: Small molecule glucokinase activators (GKAs) have been associated with potent antidiabetic efficacy and hepatic steatosis in rodents. This study reports the discovery of S 50131 and S 51434, two novel GKAs with an original scaffold and an atypical pharmacological profile. EXPERIMENTAL APPROACH: Activity of the compounds was assessed in vitro by measuring activation of recombinant glucokinase, stimulation of glycogen synthesis in rat hepatocytes and increased insulin secretion from rat pancreatic islets of Langerhans. Efficacy and safety in vivo were evaluated after oral administration in db/db mice by measuring glycaemia, HbA1c and dyslipidaemia-associated events. KEY RESULTS: S 50131 and S 51434 activated GK and stimulated glycogen synthesis in hepatocytes and insulin secretion from pancreatic islets. Unexpectedly, while both compounds effectively lowered glycaemia after acute oral administration, they did not decrease HbA1c after a 4-week treatment in db/db mice. This lack of antidiabetic efficacy was associated with increased plasma free fatty acids (FFAs), contrasting with the effect of GKA50 and N00236460, two GKAs with sustained HbA1c lowering activity but neutral regarding plasma FFAs. S 50131, but not S 51434, also induced hepatic steatosis, as did GKA50 and N00236460. However, a shorter, 4-day treatment resulted in increased hepatic triglycerides without changing the plasma FFA levels, demonstrating dynamic alterations in the lipid profile over time. CONCLUSIONS AND IMPLICATIONS: In addition to confirming the occurrence of dyslipidaemia with GKAs, these findings provide new insights into understanding how such compounds may sustain or lose efficacy over time.

Small molecule glucokinase activators disturb lipid homeostasis and induce fatty liver in rodents: a warning for therapeutic applications in humans.

BACKGROUND AND PURPOSE: Small-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 APPROACH: Nine 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 RESULTS: GKAs, 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 IMPLICATIONS: Given 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. LINKED ARTICLE: This article is commented on by Rees and Gloyn, pp. 335-338 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.02201.x.

Protein tyrosine phosphatase 1B inhibitors for the treatment of type 2 diabetes and obesity: recent advances.

This review outlines the physiology of protein tyrosine phosphatase 1B (PTP1B) and its potential involvement in the states of insulin resistance that characterizes both obesity and type 2 diabetes. The primary focus of this review is upon the elucidation of the role and control of PTP1B enzyme activity in obesity and type 2 diabetes. Furthermore, since selectivity and cell permeability are the two most important requirements for the development of successful PTP1B inhibitors, recent progress in finding compounds meeting these criteria are discussed.

K+ channels in cultured bovine retinal pericytes: effects of beta-adrenergic stimulation.

Retinal pericytes are key cells involved in the regulation of retinal blood flow. The purpose of this work was to identify the K+ channel population expressed in cultured bovine retinal pericytes and to determine whether beta-adrenergic stimulation alters the activity of these channels. Isolated pericytes were obtained by homogenization and filtration of bovine retina and K+ channels were studied with the whole-cell configuration of the patch-clamp technique on 3-5 passaged pericytes. Pericytes expressed an inward current dependent on extracellular K+ concentration which was sensitive to micromolar concentrations of barium, a characteristic of an inward-rectifying K+ current. Furthermore, two voltage-dependent outward currents were also observed. Their activation and inactivation properties, as well as their respective sensitivity to 4-aminopyridine and iberiotoxin, were indicative of voltage-sensitive and large-conductance calcium-activated K+ channels (BKCa). Isoproterenol and dibutyryl cyclic adenosine monophosphate enhanced the activity of BKCa without affecting the other potassium currents. In conclusion, bovine retinal pericytes express mainly two outward potassium currents, KV and BKCa, as well as an inward rectifying K+ current, Kir. Physiologic stimuli such as an increase in extracellular potassium concentration or beta-adrenergic receptor stimulation enhance the activity of Kir and BKCa, respectively, suggesting a potential role for these channels in the control of retinal blood flow.