Robust anti-obesity and metabolic effects of a dual GLP-1/glucagon receptor peptide agonist in rodents and non-human primates.

AIMS: To characterize the pharmacology of MEDI0382, a peptide dual agonist of glucagon-like peptide-1 (GLP-1) and glucagon receptors. MATERIALS AND METHODS: MEDI0382 was evaluated in vitro for its ability to stimulate cAMP accumulation in cell lines expressing transfected recombinant or endogenous GLP-1 or glucagon receptors, to potentiate glucose-stimulated insulin secretion (GSIS) in pancreatic ß-cell lines and stimulate hepatic glucose output (HGO) by primary hepatocytes. The ability of MEDI0382 to reduce body weight and improve energy balance (i.e. food intake and energy expenditure), as well as control blood glucose, was evaluated in mouse models of obesity and healthy cynomolgus monkeys following single and repeated daily subcutaneous administration for up to 2 months. RESULTS: MEDI0382 potently activated rodent, cynomolgus and human GLP-1 and glucagon receptors and exhibited a fivefold bias for activation of GLP-1 receptor versus the glucagon receptor. MEDI0382 produced superior weight loss and comparable glucose lowering to the GLP-1 peptide analogue liraglutide when administered daily at comparable doses in DIO mice. The additional fat mass reduction elicited by MEDI0382 probably results from a glucagon receptor-mediated increase in energy expenditure, whereas food intake suppression results from activation of the GLP-1 receptor. Notably, the significant weight loss elicited by MEDI0382 in DIO mice was recapitulated in cynomolgus monkeys. CONCLUSIONS: Repeated administration of MEDI0382 elicits profound weight loss in DIO mice and non-human primates, produces robust glucose control and reduces hepatic fat content and fasting insulin and glucose levels. The balance of activities at the GLP-1 and glucagon receptors is considered to be optimal for achieving weight and glucose control in overweight or obese Type 2 diabetic patients.

Atypical protein kinases Clambda and -zeta associate with the GTP-binding protein Cdc42 and mediate stress fiber loss.

Both the Rho family of low-molecular-weight GTP-binding proteins and protein kinases C (PKCs) mediate responses to a variety of extracellular and intracellular signals. They share many downstream targets, including remodeling of the actin cytoskeleton, activation of p70(S6) kinase and c-jun N-terminal kinase (JNK), and regulation of transcription and cell proliferation. We therefore investigated whether Rho family GTP-binding proteins bind to PKCs. We found that Cdc42 associates with atypical PKCs (aPKCs) PKCzeta and -lambda in a GTP-dependent manner. The regulatory domain of the aPKCs mediates the interaction. Expression of activated Cdc42 results in the translocation of PKClambda from the nucleus into the cytosol, and Cdc42 and PKClambda colocalize at the plasma membrane and in the cytoplasm. Expression of activated Cdc42 leads to a loss of stress fibers, as does overexpression of either the wild type or an activated form of PKClambda. Kinase-dead PKClambda and -zeta constructs acted as dominant negatives and restored stress fibers in cells expressing the activated V12 Cdc42 mutant, indicating that Cdc42-dependent loss of stress fibers requires aPKCs. Kinase-dead PKClambda and -zeta and dominant-negative N17 Cdc42 also blocked Ras-induced loss of stress fibers, suggesting that this pathway may also be important for Ras-dependent cytoskeletal changes. N17 Rac did not block Ras-induced loss of stress fibers, nor did kinase-dead PKClambda block V12 Rac-stimulated loss of stress fibers. These results indicate that Cdc42 and Rac use different pathways to regulate stress fibers.

Predictive blood glucose lowering efficacy by Glucokinase activators in high fat fed female Zucker rats.

BACKGROUND AND PURPOSE: Glucokinase (GK) is the rate-limiting enzyme of hepatic glucose metabolism and acts as a sensor for glucose-stimulated insulin release in beta-cells. Here we examine whether the lowering of blood glucose levels in the rat by small molecule glucokinase activators (GKAs) can be predicted from in vitro enzyme potencies and plasma compound exposure. EXPERIMENTAL APPROACH: We developed an insulin resistant and hyperinsulinemic animal model, the high fat fed female Zucker (fa/fa) rat (HFFZ), and measured the acute in vivo glucose-lowering efficacy of a number of GKAs in an oral glucose tolerance test. KEY RESULTS: Four GKAs (at 1 to 30 mg kg(-1)), with different in vitro enzyme potencies, dose-dependently improved oral glucose tolerance in HFFZ rats (10-40% decrease glucose area under the curve (AUC) from vehicle control). The extent of glucose lowering, or the pharmacodynamic (PD) effect, of a GKA was directly related to the total compound concentration in the plasma; the pharmacokinetic (PK) measurement. This PK-PD relationship was extended across a series of GKAs by accounting for differences in protein binding and in the in vitro potency. CONCLUSIONS AND IMPLICATIONS: When the unbound GKA compound level is greater than the in vitro enzyme potency there was significant blood glucose lowering in vivo. This latter relationship was upheld in non-diabetic Wistar rats orally dosed with a GKA. The robust and predictive nature of the PK-PD relationship for GKAs may prove of value in testing these agents in early human clinical studies.

Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription.

BACKGROUND: Glycogen synthase kinase-3 (GSK-3) is a serine/threonine protein kinase, the activity of which is inhibited by a variety of extracellular stimuli including insulin, growth factors, cell specification factors and cell adhesion. Consequently, inhibition of GSK-3 activity has been proposed to play a role in the regulation of numerous signalling pathways that elicit pleiotropic cellular responses. This report describes the identification and characterisation of potent and selective small molecule inhibitors of GSK-3. RESULTS: SB-216763 and SB-415286 are structurally distinct maleimides that inhibit GSK-3alpha in vitro, with K(i)s of 9 nM and 31 nM respectively, in an ATP competitive manner. These compounds inhibited GSK-3beta with similar potency. However, neither compound significantly inhibited any member of a panel of 24 other protein kinases. Furthermore, treatment of cells with either compound stimulated responses characteristic of extracellular stimuli that are known to inhibit GSK-3 activity. Thus, SB-216763 and SB-415286 stimulated glycogen synthesis in human liver cells and induced expression of a beta-catenin-LEF/TCF regulated reporter gene in HEK293 cells. In both cases, compound treatment was demonstrated to inhibit cellular GSK-3 activity as assessed by activation of glycogen synthase, which is a direct target of this kinase. CONCLUSIONS: SB-216763 and SB-415286 are novel, potent and selective cell permeable inhibitors of GSK-3. Therefore, these compounds represent valuable pharmacological tools with which the role of GSK-3 in cellular signalling can be further elucidated. Furthermore, development of similar compounds may be of use therapeutically in disease states associated with elevated GSK-3 activity such as non-insulin dependent diabetes mellitus and neurodegenerative disease.

Introduction to the Kinases in Diabetes Biochemical Society Focused Meeting: are protein kinases good targets for antidiabetic drugs?

Insulin regulates whole-body glucose homoeostasis by modulating the activities of protein kinases in its target tissues: muscle, liver and fat. Defects in insulin's ability to modulate protein kinase activity lead to 'insulin resistance' or impaired insulin action. Insulin resistance in combination with defective insulin secretion from the pancreas results in the elevated blood glucose levels that are characteristic of diabetes mellitus. Pharmacological agents that selectively modulate protein kinase activities in insulin-resistant tissues may act either as insulin-sensitizing or insulin-mimetic drugs. Consistent with this, small molecule modulators of a number of protein kinases have demonstrated efficacy in animal models of insulin resistance and diabetes. Moreover, emerging data in humans suggest that marketed anti-diabetic agents may also act in part through modulating protein kinase activities. This meeting was convened to consider the potential to treat insulin resistance and Type II diabetes by modulating protein kinase activity.

Small molecule glucokinase activators as novel anti-diabetic agents.

The monomeric enzyme GK (glucokinase) has a low affinity for glucose and, quantitatively, is largely expressed in the liver and pancreatic beta-cells, playing a key 'glucose sensing' role to regulate hepatic glucose balance and insulin secretion. Mutations of GK in man can be inactivating, to cause a form of diabetes mellitus, or activating, to lower blood glucose levels. Recently, models of GK protein structure have helped to elucidate the role of inactivating and activating mutations, with the latter revealing an allosteric binding site, possibly for an unknown physiological activator. However, this discovery was pre-dated by Drug Discovery projects that have identified small organic molecules that activate pancreatic and liver GK enzyme activity. These compounds stimulate insulin secretion in islets and glucose metabolism in hepatocytes. The profile of these GK activators, both in vitro and in vivo and the potential role that GK activators play in lowering blood glucose levels in Type II diabetes mellitus will be discussed.

Phorbol esters induce insulin receptor phosphorylation in transfected fibroblasts without affecting tyrosine kinase activity.

The effects of phorbol ester induced activation of protein kinase C on insulin receptor phosphorylation and tyrosine kinase activity have been investigated in transfected fibroblasts expressing high levels of the human insulin receptor. Receptor phosphorylation was stimulated more than two-fold over basal levels upon treating CHO.T cells with PMA. This phosphorylation was additive with, rather than antagonistic to, that induced by insulin. Furthermore, PMA treatment was completely without effect on insulin-stimulated receptor tyrosine kinase activity. Similar results were obtained in NIH3T3 HIR3.5 and Rat 1 HIRc-B cells. It is concluded that the previously reported inhibitory effect of PMA on receptor kinase activity is not of general regulatory significance in all cell types.

Site-specific anti-phosphopeptide antibodies: use in assessing insulin receptor serine/threonine phosphorylation state and identification of serine-1327 as a novel site of phorbol ester-induced phosphorylation.

Rabbit antisera were raised against synthetic phosphopeptides corresponding to defined or putative sites of insulin receptor serine/threonine phosphorylation (Ser-1305, Ser-1327, Thr-1348). All of these antibodies bound specifically to the immunogenic phosphopeptide but not to the non-phosphorylated form of the peptide or to other phosphopeptides, in a microtitre plate competition enzyme-linked immunosorbent assay. Anti-PS1327 antibody reacted well with native insulin receptor prepared from phorbol ester-treated transfected CHO.T cells, but showed little reaction with receptor from untreated cells. Anti-PT1348 antibody in crude form reacted substantially with receptor from both phorbol 12-myristate 13-acetate-treated and untreated cells, but displayed specificity for phosphoreceptor after adsorption to remove antibodies reactive with dephosphopeptide. The ability to discriminate between receptor from cells treated with or without phorbol ester was retained when these antibodies were used to probe denatured receptor on Western blots. Thus anti-PS1327 and anti-PT1348 react with insulin receptor in a site-specific and phosphorylation-state-dependent manner. Anti-PT1348, but not anti-PS1327, also showed increased reactivity with receptor prepared from insulin-treated cells. The third antibody, anti-PS1305, did not react with intact insulin receptor under any conditions. It is concluded that serine 1327 is a major, previously unrecognized, site of phorbol ester-induced receptor phosphorylation, and that anti-phosphopeptide antibodies will be valuable reagents with which to examine the serine/threonine phosphorylation state of receptor extracted from tissues.

A monoclonal anti-peptide antibody reacting with the insulin receptor beta-subunit. Characterization of the antibody and its epitope and use in immunoaffinity purification of intact receptors.

A mouse monoclonal antibody (CT-1) was prepared against the C-terminal peptide sequence of the human insulin receptor beta-subunit (KKNGRILTLPRSNPS). The antibody reacted with native human and rat insulin receptors in solution, whether or not insulin was bound and whether or not the receptor had undergone prior tyrosine autophosphorylation. The antibody also reacted specifically with the receptor beta-subunit on blots of SDS/polyacrylamide gels. Preincubation of soluble receptors with antibody increased the binding of 125I-insulin approx. 2-fold. The antibody did not affect insulin-stimulated autophosphorylation, but increased the basal autophosphorylation rate approx. 2-fold. The amino acid residues contributing to the epitope for CT-1 were defined by construction and screening of an epitope library. Oligonucleotides containing 23 random bases were synthesized and ligated into the vector pCL627, and the corresponding peptide sequences expressed as fusion proteins in Escherichia coli were screened by colony blotting. Reactive peptides were identified by sequencing the oligonucleotide inserts in plasmids purified from positive colonies. Six different positive sequences were found after 900,000 colonies had been screened, and the consensus epitope was identified as GRVLTLPRS. Phosphorylation of the threonine residue within this sequence (corresponding to the known phosphorylation site Thr-1348 in the insulin receptor) decreased the affinity of antibody binding approx. 100-fold, as measured by competition in an e.l.i.s.a. Antibody CT-1 was used for immunoaffinity isolation of insulin receptor from detergent-solubilized human placental or rat liver microsomal membranes. Highly purified receptor was obtained in 60% yield by binding to CT-1-Sepharose immunoadsorbent and specific elution with a solution of peptide corresponding to the known epitope. This approach to purification under very mild conditions may in principle be used with any protein for which an antibody is available and for which a peptide epitope or 'mimotope' can be identified.

c-Jun is a JNK-independent coactivator of the PU.1 transcription factor.

The ETS domain transcription factor PU.1 is necessary for the development of monocytes and regulates, in particular, the expression of the monocyte-specific macrophage colony-stimulating factor (M-CSF) receptor, which is critical for monocytic cell survival, proliferation, and differentiation. The bZIP transcription factor c-Jun, which is part of the AP-1 transcription factor complex, is also important for monocytic differentiation, but the monocyte-specific M-CSF receptor promoter has no AP-1 consensus binding sites. We asked the question of whether c-Jun could promote the induction of the M-CSF receptor by collaborating with PU.1. We demonstrate that c-Jun enhances the ability of PU.1 to transactivate the M-CSF receptor promoter as well as a minimal thymidine kinase promoter containing only PU.1 DNA binding sites. c-Jun does not directly bind to the M-CSF receptor promoter but associates via its basic domain with the ETS domain of PU.1. Consistent with our observation that AP-1 binding does not contribute to c-Jun coactivation is the observation that the activation of PU.1 by c-Jun is blocked by overexpression of c-Fos. Phosphorylation of c-Jun by c-Jun NH2-terminal kinase on Ser-63 and -73 does not alter the ability of c-Jun to enhance PU.1 transactivation. Activated Ras enhances the transcriptional activity of PU.1 by up-regulating c-Jun expression without changing the phosphorylation pattern of PU.1. The activation of PU.1 by Ras is blocked by a mutant c-Jun protein lacking the basic domain. The expression of this mutant form of c-Jun also completely blocks 12-O-tetradecanoylphorbol-13-acetate-induced M-CSF receptor promoter activity during monocytic differentiation. We propose therefore that c-Jun acts as a c-Jun NH2-terminal kinase-independent coactivator of PU.1, resulting in M-CSF receptor expression and development of the monocytic lineage.