Hyperglycemia-mediated onset of myocardial insulin resistance – unraveling molecular mechanisms and identifying therapeutic targets

[摘要] ENGLISH ABSTRACT: Background - Although acute hyperglycemic episodes are linked to lower glucose uptake, underlying mechanisms driving this process remain unclear. We hypothesized that acute hyperglycemia triggers reactive oxygen species (ROS) production and increases non-oxidative glucose pathway (NOGP) activation, i.e. stimulation of advanced glycation end products (AGE), polyol pathway (PP), hexosamine biosynthetic pathway (HBP) and protein kinase C (PKC) activation. These mechanisms attenuate cellular function, and may indeed decrease insulin-mediated cardiac glucose uptake. The role of the pentose phosphate pathway (PPP) under high glucose/diabetic conditions is a subject of contention. Activation of the PPP enzyme transketolase (TK) (by benfotiamine/BFT or thiamine) reduces flux via the other four NOGPs, and is associated with beneficial outcomes. Our aim was therefore to evaluate the effects of acute hyperglycemia on insulin-mediated glucose uptake in a cardiac-derived cell line. Specifically, we aimed to elucidate the role of ROS and NOGP induction under these conditions. Methodology - H9c2 cardiomyoblasts were exposed to 25 mM glucose for 24 hr vs. 5.5 mM glucose controls ± modulating agents during last hour of glucose exposure: a) antioxidant #1 for mitochondrial ROS (250 μM 4-OHCA), b) antioxidant #2 for NADPH oxidase-generated ROS (100 μM DPI), c) NOGP inhibitors – 100 μM aminoguanidine (AGE), 5 μM chelerythrine (PKC); 40 μM DON (HBP); and 10 μM zopolrestat (PP). We also employed BFT (50 and 100 μM) in vitro, while the effects of in vivo thiamine administration were assessed in hearts of an obese/diabetic rat model of pre-diabetes and diabetes, the OLETF strain. We evaluated insulin sensitivity by glucose uptake assay (flow cytometry), GLUT4 translocation (transfection of HA-GLUT4-GFP construct) and protein kinase B (Akt) activity assay. ROS levels (mitochondrial, intracellular) were measured by flow cytometry analysis of specific fluorescent probes. Markers of each NOGP were also assessed.Results - Acute hyperglycemia elevated ROS, activated NOGPs and blunted glucose uptake. However, TK activity (marker of PPP) did not change. Respective 4-OHCA and DPI treatment blunted ROS production, diminished NOGP activation and normalized glucose uptake. NOGP inhibitory studies identified PKCβII as a key downstream player in lowering insulin-mediated glucose uptake. When we employed BFT (known to shunt flux away from NOGPs and into the PPP), it decreased ROS generation and NOGP activation, and restored glucose uptake under acute hyperglycemic conditions. In vivo thiamine administration reduced markers of the other NOGP, while it attenuated (mainly in the pre-diabetic phase) the metabolic dysfunction observed in the OLETF rats. Conclusions - This study demonstrates that acute hyperglycemia elicits a series of maladaptive events that function in tandem to reduce glucose uptake, and that antioxidant treatment and/or attenuation of NOGP activation (PKC, polyol pathway) may limit the onset of insulin resistance.

[发布机构] Stellenbosch University