Methods for Monitoring Matrix-Induced Autophagy.

A growing body of research demonstrates modulation of autophagy by a variety of matrix constituents, including decorin, endorepellin, and endostatin. These matrix proteins are both pro-autophagic and anti-angiogenic. Here, we detail a series of methods to monitor matrix-induced autophagy and its concurrent effects on angiogenesis. We first discuss cloning and purifying proteoglycan fragment and core proteins in the laboratory and review relevant techniques spanning from cell culture to treatment with these purified proteoglycans in vitro and ex vivo. Further, we cover protocols in monitoring autophagic progression via morphological and microscopic characterization, biochemical western blot analysis, and signaling pathway investigation. Downstream angiogenic effects using in vivo approaches are then discussed using wild-type mice and the GFP-LC3 transgenic mouse model. Finally, we explore matrix-induced mitophagy via monitoring changes in mitochondrial DNA and permeability

A rationally designed monomeric peptide triagonist corrects obesity and diabetes in rodents.

We report the discovery of a new monomeric peptide that reduces body weight and diabetic complications in rodent models of obesity by acting as an agonist at three key metabolically-related peptide hormone receptors: glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and glucagon receptors. This triple agonist demonstrates supraphysiological potency and equally aligned constituent activities at each receptor, all without cross-reactivity at other related receptors. Such balanced unimolecular triple agonism proved superior to any existing dual coagonists and best-in-class monoagonists to reduce body weight, enhance glycemic control and reverse hepatic steatosis in relevant rodent models. Various loss-of-function models, including genetic knockout, pharmacological blockade and selective chemical knockout, confirmed contributions of each constituent activity in vivo. We demonstrate that these individual constituent activities harmonize to govern the overall metabolic efficacy, which predominantly results from synergistic glucagon action to increase energy expenditure, GLP-1 action to reduce caloric intake and improve glucose control, and GIP action to potentiate the incretin effect and buffer against the diabetogenic effect of inherent glucagon activity. These preclinical studies suggest that, so far, this unimolecular, polypharmaceutical strategy has potential to be the most effective pharmacological approach to reversing obesity and related metabolic disorders

Unimolecular dual incretins maximize metabolic benefits in rodents, monkeys, and humans.

We report the discovery and translational therapeutic efficacy of a peptide with potent, balanced co-agonism at both of the receptors for the incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). This unimolecular dual incretin is derived from an intermixed sequence of GLP-1 and GIP, and demonstrated enhanced antihyperglycemic and insulinotropic efficacy relative to selective GLP-1 agonists. Notably, this superior efficacy translated across rodent models of obesity and diabetes, including db/db mice and ZDF rats, to primates (cynomolgus monkeys and humans). Furthermore, this co-agonist exhibited synergism in reducing fat mass in obese rodents, whereas a selective GIP agonist demonstrated negligible weight-lowering efficacy. The unimolecular dual incretins corrected two causal mechanisms of diabesity, adiposity-induced insulin resistance and pancreatic insulin deficiency, more effectively than did selective mono-agonists. The duration of action of the unimolecular dual incretins was refined through site-specific lipidation or PEGylation to support less frequent administration. These peptides provide comparable pharmacology to the native peptides and enhanced efficacy relative to similarly modified selective GLP-1 agonists. The pharmacokinetic enhancement lessened peak drug exposure and, in combination with less dependence on GLP-1-mediated pharmacology, avoided the adverse gastrointestinal effects that typify selective GLP-1-based agonists. This discovery and validation of a balanced and high-potency dual incretin agonist enables a more physiological approach to management of diseases associated with impaired glucose tolerance

Reactive oxygen species mediate high glucose-induced heparanase-1 production and heparan sulphate proteoglycan degradation in human and rat endothelial cells: a potential role in the pathogenesis of atherosclerosis.

Item does not contain fulltextAIMS/HYPOTHESIS: The content of heparan sulphate is reduced in the endothelium under hyperglycaemic conditions and may contribute to the pathogenesis of atherosclerosis. Heparanase-1 (HPR1) specifically degrades heparan sulphate proteoglycans. We therefore sought to determine whether: (1) heparan sulphate reduction in endothelial cells is due to increased HPR1 production through increased reactive oxygen species (ROS) production; and (2) HPR1 production is increased in vivo in endothelial cells under hyperglycaemic and/or atherosclerotic conditions. METHODS: HPR1 mRNA and protein levels in endothelial cells were analysed by RT-PCR and Western blot or HPR1 enzymatic activity assay, respectively. Cell surface heparan sulphate levels were analysed by FACS. HPR1 in the artery from control rats and a rat model of diabetes, and from patients under hyperglycaemic and/or atherosclerotic conditions was immunohistochemically examined. RESULTS: High-glucose-induced HPR1 production and heparan sulphate degradation in three human endothelial cell lines, both of which were blocked by ROS scavengers, glutathione and N-acetylcysteine. Exogenous H(2)O(2) induced HPR1 production, subsequently leading to decreased cell surface heparan sulphate levels. HPR1 content was significantly increased in endothelial cells in the arterial walls of a rat model of diabetes. Clinical studies revealed that HPR1 production was increased in endothelial cells under hyperglycaemic conditions, and in endothelial cells and macrophages in atherosclerotic lesions. CONCLUSIONS/INTERPRETATION: Hyperglycaemia induces HPR1 production and heparan sulphate degradation in endothelial cells through ROS. HPR1 production is increased in endothelial cells from a rat model of diabetes, and in macrophages in the atherosclerotic lesions of diabetic and non-diabetic patients. Increased HPR1 production may contribute to the pathogenesis and progression of atherosclerosis.1 juni 201