Protective Effects Of Sulforaphane On Type 2 Diabetes-induced Cardiomyopathy Via AMPK-mediated Activation Of Lipid Metabolic Pathways And NRF2 Function

Background:The prevalence of diabetes is increasing worldwide,and about 95% of adults diagnosed with diabetes are type 2 diabetes(T2D).According to a large number of epidemiological data,diabetes has become a very critical risk factor for the onset and development of cardiovascular diseases(CVD),independent of coronary atherosclerosis and hypertension.Among the diabetes-derived CVDs,diabetic cardiomyopathy(DCM)has arisen extensive attention because of its unique remodeling pattern.Clinically,DCM was characterized by diastolic dysfunction in the early stage,followed by systolic dysfunction and decreased left ventricular function in the late stage,and the patients will develop pulmonary edema,dyspnea and other symptoms until death.In terms of pathological mechanisms,DCM is mainly characterized by abnormal energy metabolism in cardiomyocytes.First of all,under the condition of T2 D,myocardium will develop the resistance to insulin,and this will cause cellular dysregulation of glucose utilization,T2 D can also lead to excessive fatty acids(FAs)into cardiomyocytes.However,cardiomyocytes exhibit the low capacity of fatty acid oxidation(FAO)in T2 D.As a result,the redundant FAs will store in the form of triglycerides in the heart,and it will cause cardiac lipotoxicity: oxidative stress(damage)、inflammatory responses、apoptosis and mitochondrial dysfunction,consequently resulting in cardiac remodeling and cardiac dysfunction.AMP-activated protein kinase(AMPK)plays an important role in maintaining the homeostasis of energy metabolism.AMPK is a heterotrimeric enzyme comprised of one catalytic subunit(α1 or α2)and two regulatory subunits.Studies have shown that α2 subunit mainly exists in myocardial tissue,and only α2 plays an important role in energy metabolism.AMPK regulates myocardial FA metabolism by modulating downstream genes,and the activation of AMPK plays an important role in promoting myocardial FAO and inhibiting lipogenesis.The transcription factor NRF2 is a major regulator in cellular defense system.NRF2 has a highly-conserved basic leucine zipper(bZIP)structure,and it is a member of the Cap ‘n’ Collar family of basic leucine zipper(bZip)transcription factors.Activated NRF2 translocates into the cell nucleus and then binds to the antioxidant response element in the promoters of various detoxifying and anti-oxidative genes.Sulforaphane(SFN)is a phytochemical belonging to isothiocyanates derived from cruciferous vegetables,such as broccoli,cabbage,and watercress.Recently,Sulforaphane is well-known as a potent activator of NRF2.Studies have shown that sulforaphane can activate NRF2-mediated anti-oxidant pathways to improve DCMinduced cardiac dysfunction.Recently,SFN was found to alleviate lipid accumulation both in vivo and in vitro,suggesting SFN can also become a potential breAKThrough point in therapy for dyslipidemia.In support of this notion,in our previous study,we found that 4-month sulforaphane treatment alleviated T2D-induced cardiac lipid deposition and reduced T2D-induced myocardial lipotoxicity,thereby reversing pathological ventricular remodeling and improving cardiac dysfunction.At the same time,we also found that SFN prevented type 2 diabetes(T2D)-induced cardiomyopathy accompanied by the activation of AMPK.Objects:We used global deletion of AMPKα2 gene(AMPKα2-KO)mice and their WT(Wild-type)mice,and the aims of the present study is to(1)determine whether AMPK is indispensable for SFN-induced prevention against cardiomyopathy in T2 D and the possible underlying mechanisms;(2)investigate the impact of SFN treatment in the early course and the lasting effects in the late course of T2D-cardiomyopathy.Methods and Results:T2D was established by feeding 3-month high-fat diet(HFD)to induce insulin resistance,followed by an intraperitoneal injection of streptozotocin(STZ)to induce mild hyperglycemia in both AMPKα2-KO and WT mice.Other age-matched mice were fed a normal diet as control.Then both T2 D and control mice were subsequently treated with or without SFN for 3 months while continually feeding HFD or normal diet.Upon completion of the 3-month treatment,five mice from each group were sacrificed as a 3-month time-point(3M).The rest continued normal diet or HFD until terminating study at the sixth month(6M)of diabetes.During the establishment of the 6-month animal model,we monitored the dynamic changes of body weight and blood glucose every month.Cardiac function was examined with echocardiography before sacrifice at both 3M and 6M.SFN prevented T2D-induced progression of cardiac dysfunction,remodeling(hypertrophy and fibrosis),inflammation,and oxidative damage in wild-type diabetic mice,but not in AMPKα2-KO mice.Mechanistically,SFN prevented T2D-induced cardiomyopathy not only by improving AMPKmediated lipid metabolic pathways,including upregulating the expression of PGC-1α 、 PPAR-α and CPT-1B,downregulating the expression of SCD-1,but also enhancing NRF2 activation via AMPK/AKT/GSK3β pathway.However,these improving effects of SFN were abolished in AMPKa2-KO diabetic mice.Conclusions:(1)AMPK is indispensable for the SFN-induced prevention of cardiomyopathy in T2 D,and the preventive effects induced by 3-month SFN treatment can last at least 3 more months forward;(2)The activation of NRF2 by SFN is mediated by AMPK/AKT/GSK3β signaling pathways.