SUN-080 Diminished Akt Activation and Interaction with 14-3-3ζ is Associated with Insulin Resistance in Cardiomyocytes of Metabolic Syndrome Rats

Metabolic syndrome (MetS) is a cluster of physiological and biochemical disorders that increases the risk of cardiovascular disease, being insulin resistance (IR) a pivotal condition for its development. Alterations such as metabolic inflexibility, changes in calcium dynamics, oxidative stress and impaired insulin signaling have been reported in the state of IR in the heart. Akt kinase activation plays a central role in insulin actions, leading to glucose uptake and its use as the primary energy source in the heart. Akt activity and subcellular localization are highly regulated by phosphorylation and by the interaction with other proteins. It has been reported that 14-3-3 proteins regulate insulin response and in particular the ζ isoform exerts a positive regulation on Akt activation. Therefore, we studied the changes in Akt activation and interaction with 14-3-3ζ, in response to insulin in cardiomyocytes from a MetS rat model, induced by the consumption of 30% sucrose in the drinking water during 4 months. MetS rats developed obesity (body weight in g: MetS: 592±12 vs 415±5 in control rats (C), P<0.001; epididymal fat in g: MetS: 17.5±1 vs C: 7.3±0.4, P<0.001), hypertriglyceridemia (in mg/dL MetS: 137±10 vs C: 60±3, P<0.001), low HDL-cholesterol levels (in mg/dL MetS: 31±2 vs C: 44±2, P<0.01), and systemic insulin resistance (intraperitoneal glucose tolerance test, area under the curve: MetS: 34199±2555 vs C: 17908±1844, P<0.001), without significant modifications in fasting blood glucose levels (in mg/dL MetS: 76±6 vs C: 65±5, P=0.17). Isolated cardiomyocytes from MetS rats showed insulin resistance, characterized by the absence of glucose uptake after stimulation with this hormone (3(H)-2deoxyglucose uptake in fmol/mg of protein/min: MetS basal: 84±17 vs MetS+insulin: 94±20, P=0.63; C basal: 74±19 vs C+insulin: 190±47, P<0.05). Insulin-induced Akt phosphorylation (determined by Western Blot, after 10 min of stimulation with 100 nM) in the key activation residues Ser473/Thr308 was diminished by 51% and 66%, respectively. Insulin-induced Akt translocation towards the plasma membrane (determined by immunostaining/confocal microscopy) a process involved in its activation, was also decreased in MetS cardiomyocytes. On the other hand, we found a diminished insulin-induced Akt-14-3-3ζ interaction evaluated by Akt co-immunoprecipitation/Western blot and by Akt co-immunoprecipitation/label-free mass spectrometry, since 14-3-3 was exclusively found in a pool of 3 C rats cardiomyocytes samples, but not in the MetS pool; this impairment suggest a reduced activation of AS160 protein and glucose uptake. These findings unveil a key participation of Akt regulatory mechanisms in the establishment of IR in cardiomyocytes under the MetS condition. Sources of Research Support: PRODEP research grant to the Academic Group Cinvestav-CA-10 to AR and JAO-R. CONACYT scholarship 278067 to HVL-G.