Background and Design: Adiponectin is an adipokine secreted primarily from adipose tissue and can influence circulating plasma glucose and lipid levels through multiple mechanisms involving a variety of organs. In humans, reduced plasma adiponectin levels induced by obesity are associated with insulin resistance and type 2 diabetes suggesting that low adiponectin levels may contribute diet-induced diabetes.
Methods and Results: Therefore, the objective of the present study was to investigate how gene therapy designed to elevate plasma adiponectin levels altered insulin resistance in a high-fat, high-sucrose (HFHS) mouse model of diabetes. As expected, mice fed a HFHS diet for 15 weeks had a decrease in the high-molecular weight form of plasma adiponectin. Electroporation-mediated gene transfer of mouse adiponectin plasmid DNA into gastrocnemius muscle resulted in elevated serum high-molecular adiponectin compared to control mice treated with empty plasmid. Mice receiving adiponectin gene therapy gained less weight on HFHS diet, had less total fat mass and exhibited increased O2 and VO2 consumption during indirect calorimetric studies compared to mice receiving empty plasmid. Consistent with improved whole body metabolism, mice receiving adiponectin gene therapy also displayed lower plasma insulin levels, improved glucose tolerance and reduced gluconeogenesis compared to control mice. Immunoblot analysis of liver indicated increased phosphorylation of insulin signaling proteins in mice receiving adiponectin gene therapy, further supporting improved whole body insulin signaling.
Conclusions: Based on these data, we conclude that sustained expression of adiponectin ameliorated the metabolic abnormalities caused by feeding HFHS diet in mice. Together, the data suggest that long-term adiponectin gene therapy may provide a possible therapeutic opportunity to lessen the metabolic complications induced by the onset of obesity.
Matrix metalloproteinase (MMP)-2 belongs to a family of zinc-dependent proteases which are best known for their ability to proteolyse extracellular matrix proteins throughout the body, including the cardiovascular system. Increased MMP-2 activity has been demonstrated in myocardial ischaemia and reperfusion injury and the progression to congestive heart failure, with most evidence to date for its role in cardiac remodelling. Recent evidence, however, shows that MMP-2 also co-localizes with and proteolyses specific protein targets within the cardiomyocyte to cause acute, reversible contractile dysfunction, challenging the conventional wisdom on the ‘extracellular matrix only’ actions of this enzyme. In this review, we discuss the recent upsurge in MMP-2 research with regards to its activation by non-proteolytic pathways in the setting of enhanced oxidative stress in the heart. We will focus on the consequences of intracellular actions of MMP-2 within the cardiomyocyte and its regulation at several levels including its expression, post-translational modifications, and regulation by endogenous tissue inhibitors of metalloproteinases, caveolin, and small molecule MMP inhibitors. MMP-2 is emerging as an important signalling protease implicated in the proteolytic regulation of various intracellular proteins in myocardial oxidative stress injury. -- ------------------------------------------------------------------------------------------------------------------------------
AimsMatrix metalloproteinase (MMP)-2 contributes to myocardial oxidative stress injury by degrading sarcomeric and cytoskeletal proteins in cardiomyocytes. Glycogen synthase kinase (GSK)-3b is dysregulated during oxidative stress and is susceptible to proteolytic cleavage. Here we determined whether GSK-3bis a MMP-2 substrate as a result of oxidative stress. Methods and resultsMMP-2 and GSK-3bwere incubated and the cleavage fragments were identified by immunoblotting and silver stain. The intact protein and its primary cleavage fragment were subjected to trypsin digestion and the resultant peptides were analysed by LC–MS/MS. GSK-3bkinase activity was measured using a peptide substrate and [g-32 P]-ATP. Oxidative stress in H9c2 cardiomyoblasts was induced by H2O2 and the levels and activities of MMP-2 and GSK-3bwere measured. Incubation of 47 kDa GSK-3bwith MMP-2 resulted in the time- and concentration-dependant cleavage of GSK-3bas seen by appearance of an 30 kDa fragment. MS analysis and Mascot database search yielded a peptide with an amino acid sequence of GSK-3blacking the N-terminal region. GSK-3bkinase activity was significantly increased upon incubation with MMP-2 which was abrogated by the MMP inhibitor GM-6001. H2O2 challenge of H9c2 cardiomyoblasts significantly increased the activity and level of MMP-2, reduced the level of GSK-3b, and significantly increased GSK-3bkinase activity. Both the loss of intact GSK-3band increase in its kinase activity were reduced with MMP inhibitors. MMP-2 pulldown assays in H9c2 cell lysates showed the association of MMP-2 with GSK-3b. ConclusionGSK-3bmay be a target of MMP-2 and its cleavage by MMP-2 enhances its kinase activity. MMP-2 may cleave off the N-terminal of GSK-3bwhere the inhibitory phosphorylation of serine-9 occurs. MMP-2-mediated augmentation of GSK-3bkinase activity may contribute to cardiac injury resulting from enhanced oxidative stress.