Effect And Mechanism Of Bariatric Surgery On Diabetic Cardiomyopathy In Type 2 Diabetic Rats

Part 1 Restoration of myocardial glucose uptake with facilitated GLUT-4 translocation contributes to alleviation of diabetic cardiomyopathy in rats after duodenal-jejunal bypassBackgroudIn 2017,the number of people with diabetes worldwide has reached 425 million.It is estimated that it will exceed 629 million by 2045.Diabetes mellitus has become a serious global burden of public health,with type 2 diabetes mellitus(T2DM)being the most common.The prevalence of T2DM in China has exploded in the past 20 years.Traditional methods of treatment have poor control of blood glucose,and further exploration of more effective treatment options is needed.Bariatric surgery was originally designed to treat morbid obesity.However,besides the weight loss after surgery,the scholars also observed significant remission in both T2DM and its related metabolic complications.Since then,the basic and clinical research about the effect of bariatric surgery on T2DM has been carried out.Bariatric surgery is widely used in more and more areas as a mature treatment nowadays.It also has been proved to provide rapid,effective,and lasting remission in T2DM and associated complications.Diabetic cardiomyopathy is one of the major cardiac complications of diabetes and refers to myocardial lesions that occur in diabetic patients but cannot be explained by arterial hypertension or coronary artery disease.Diabetic cardiomyopathy usually manifests as decreased myocardial compliance and diastolic dysfunction at early stage,with both systolic and diastolic cardiac dysfunction in the advanced stage.Diabetic cardiomyopathy can significantly increase the risk of heart failure and cardiogenic shock,but there is currently no specific treatment in clinical practice.Studies have shown that bariatric surgery can effectively ameliorate diabetic cardiomyopathy,but the potential mechanism is not yet clear.Exploring the effect of bariatric surgery on diabetic cardiomyopathy independent of weight loss and the related mechanisms has become an urgent problem to be solved.Duodenal-jejunal bypass(DJB)is a commonly used experimental model to investigate the anti-diabetic effect of bariatric surgery independent of weight loss.However,there has been limited study regarding the therapeutic effects of DJB on DCM to date.ObjectivesIn the present study,we performed DJB and sham surgeries on rats with diabetic cardiomyopathy induced by a high-fat diet and a low dose of streptozotocin(STZ),with chow-diet fed rats as controls.We aim to investigate the alterations in myocardial glucose uptake after DJB and the associated mechanisms,as well as its morphological and functional effects on diabetic cardiomyopathy with the help of a series of morphological and functional tests as along as experiments of molecular biology.Methods1.In the present study,the diabetic rats were induced by a high-fat diet(for 4 weeks)in combination with low-dose STZ(35 mg/kg;ip).16 weeks later,with chow-diet fed rats as controls,the following tests were performed to evaluate the validity of the rat model of diabetic cardiomyopathy:(1)Determination of body weight,urine volume,blood pressure level and other general characteristics in Control group and Diabetic group;(2)The cardiac function of rats was assessed by transthoracic echocardiography.2.After the validiation of the above animal model,rats with diabetic cardiomyopathy were randomly allocated to undergo corresponding surgery in the DJB group or the Sham group.The following analysis were performed post-operation:(1)Dynamic monitor the body weight,food intake,and indicators of glucose metabolism such as fasting blood glucose,glucose tolerance,and fasting insulin in each group;(2)The severity of myocardial remodeling was evaluated by HE staining,Masson staining,oil red O staining,transmission electron microscopy,and other histomorphometric examinations;(3)The cardiac function was evaluated by transthoracic echocardiography combined with hemodynamic analysis;(4)The positron-emission tomography(PET)with fluorine-18 labeled fluorodeoxyglucose(18F-FDG)was carried to evaluate the myocardial glucose uptake in vivo;(5)The myocardial expression of glucose transporter(GLUT)was assessed to elucidate the reason of alteration of myocardial glucose uptake after DJB;(6)The myocardial insulin signaling and GLUT-4 translocation-related proteins were investigated to study the underlying mechanisms.Results1.After 16 weeks of diabetes(just before surgery),the water intake and urine volume of the Diabetic group were significantly higher than those of the Control group(Both P<0.01).There was no significant difference in blood pressure between the two groups(P>0.05).However,the systolic and diastolic function of the diabetic rats were significantly impaired.These results confirm the validation of the rat model of diabetic cardiomyopathy.2.Due to the effects of perioperative fasting and surgical stress,the body weight and food intake of the DJB group and the Sham group showed significant reduction during the first week after surgery,and then gradually recovered.There was no significant difference in body weight or food intake between the DJB group and the Sham group at all time points after surgery(All P>0.05).3.The glucose metabolism of rats in the DJB group was significantly improved after the surgery.At 2,4,6 and 8 weeks after operation,the fasting blood glucose of the DJB group was significantly lower than that of the Sham group(All P<0.01).There was no significant difference in fasting serum insulin between the DJB group and the Sham group(P>0.05).However,the homeostasis model assessment of basal insulin resistance(HOMA-IR)was significantly lower in the DJB group than that in the Sham group(P<0.01).Meanwhile,the area under the curve for oral glucose tolerance test(AUCOGTT)in the the DJB group was significantly lower than that in the Sham group at both 2 and 8 weeks after surgery(Both P<0.01).In addition,the level of serum GLP-1 in the DJB group was significantly higher than that in the Sham group at both 2 and 8 weeks after surgery(Both P<0.01).4.The myocardial remodeling was significantly relieved in the DJB group.Compared with the Sham group,the DJB group showed significantly relieved myocardial hypertrophy,myocardial lipid deposition,myocardial fibrosis and myocardial microstructural disorder.5.The cardiac function of the DJB group was significantly improved after surgery.At 8 weeks after operation,both the diastolic and systolic cardiac function of the DJB group were significantly improved compared with the Sham group.In addition,the left ventricular end diastolic pressure(LVEDP)was significantly lower in the DJB group than that in the Sham group(P<0.01).6.The PET scan results showed that both the fasting and glucose-stimulated myocardial standard uptake value(SUV)of the DJB group was significantly higher than that of the Sham group(All P<0.01).7.There was no significant difference in the total protein expression of GLUT-1 or GLUT-4 between the three groups(Both P>0.05).No significant difference was observed in the expression of cell membrane GLUT-1 between the three groups(P>0.05).However,the expression of cell membrane GLUT-4 in the DJB group was significantly higher than that in the Sham group(P<0.01).8.The myocairdal GLUT-4 translocation was facilitated in the DJB group.Rats in the DJB group showed significant improvement in myocardial insulin sensitivity after surgery.The phosphorylation of insulin receptor substrate 1(IRS1),phosphatidylinositol 3-kinase(PI3K)and protein kinase B(Protein Kinase B,PKB/AKT)were significantly up-regulated in the DJB group than those in the Sham group.In addition,the phosphorylation of AKT substrate of 160kDa(AS 160)and Tre-2/BUB2/cdcl domain family 1(TBC1D1)were significantly enhanced in the DJB group compared with those in the Sham group(Both P<0.01).There were no significant differences in the expression of vesicle-associated membrane protein(VAMP2),Synaptosome-associated protein of 23 kDa(SNAP23)or Syntaxin4 in the myocardium of the three groups(All P>0.05).ConclusionsThe present findings provide evidence that restoration of MGU is implicated in the alleviation of diabetic caridiomyopathy after DJB through facilitating GLUT-4 translocation.Part 2 Facilitated Ca2+ homeostasis and attenuated myocardial autophagy contribute to alleviation of diabetic cardiomyopathy after bariatric surgeryBackgroundDiabetes is a chronic metabolic disease that seriously threaten public health.The incidence of type 2 diabetes mellitus(T2DM)keeps increasing,which brings mental and physical damage to patients and also a heavy economic burden to the country and individuals.In addition to the damage caused by abnormal blood glucose itself,persistent hyperglycemia and long-term metabolic disorders can cause systemic organ dysfunction,causing serious complications.Diabetic cardiomyopathy is a serious cardiac complication that can significantly increase the risk of heart failure,cardiogenic shock,and sudden death.There is currently no specific treatment for diabetic cardiomyopathy in clinic.Bariatric surgery,also known as metabolic surgery,was originally designed to treat morbid obesity.Subsequent clinical studies have confirmed that bariatric surgery can achieve rapid,effective and lasting remission of T2DM and related complications.Recently,studies have reported that bariatric surgery can effectively alleviate diabetic myocardial dysfunction independent of weight loss or dietary changes.It provides a new direction for the treatment of diabetic cardiomyopathy.However,the potential mechanism is still unclear.The occ.urrence and development of diabetic cardiomyopathy involves a variety of pathophysiological factors.The excitatory-contraction coupling of cardiomyocytes is dependent on fluctuations in intracellular calcium.Deregulation of intracellular calcium homeostasis can lead to systolic and diastolic dysfunction.To date,the effect of bariatric surgery on myocardial calcium homeostasis has not been reported.Our previous studies have shown that bariatric surgery can significantly ameliorate myocardial sarcoplasmic reticulum stress in diabetic rats.Given that sarcoplasmic reticulum is the storage site of Ca2+ and that modulation of sarcoplasmic reticulum stress is a large benefit to normalize the perturbed Ca2+ homeostasis,it raises the hypothesis that the alleviation of diabetic cardiomyopathy after bariatric surgery may be associated with facilitated myocardial Ca2+ homeostasis.Cardiomyocyte autophagy is also closely related to the occurrence and development of diabetic cardiomyopathy.Autophagy itself acts as a protective mechanism to help cells survive.However,dysregulation or loss of control of autophagy often leads to serious consequences.Previous studies have shown great controversy about the autophagy of cardiomyocytes in T2DM state.The effect of bariatric surgery on myocardial autophagy and related mechanisms have not been reported yet.ObjectivesIn the present study,the diabetic cardiomyopathy was induced on a diabetic rat model constructed by a high-fat diet and a low dose of streptozotocin(STZ).Then we carried out bariatric surgeries on rats with diabetic cardiomyopathy.Through a series of morphological,functional tests and molecular biology experiments,we aim to determine the effects of bariatric surgery on DCM via modulation of myocardial Ca2+homeostasis and autophagy.MethodsIn this study,the diabetic cardiomyopathy was induced on a diabetic rat model constructed by high-fat diet(for 4 weeks)in combination with low-dose STZ(35 mg/kg;ip).Then rats with diabetic cardiomyopathy were randomly allocated to undergo corresponding surgery in the SHAM group,sleeve gastrectomy(SG)group,and duodenal-jejunal bypass(DJB)group.The following analysis were performed after surgery:1.Dynamic monitor the indicators of glucose metabolism such as fasting blood glucose,glucose tolerance,and fasting insulin;2.The severity of myocardial remodeling was evaluated by HE staining,Masson staining,oil red O staining,and other histomorphometric examinations;3.The cardiac function of was evaluated by transthoracic echocardiography combined with hemodynamic analysis;4.Ca2+ homeostasis was evaluated by measuring Ca2+ transients with Fura-2/AM in isolated ventricular myocytes along with detection of the abundance of Ca2+regulatory proteins in the myocardium;5.Myocardial autophagic flux was determined by expression of autophagy-related proteins in the absence and presence of chloroquine.Rusults1.There was no significant difference in heart rate and blood pressure among the three groups at 4 and 16 weeks after operation(Both P>0.05).The urine volume of the SG group and the DJB group was significantly lower than that of the SHAM group(Both P<0.01).At 16 weeks postoperatively,the heart weight of the SG group and the DJB group was significantly lower than that of the SHAM group(P<0.05 for SG;P<0.01 for DJB).2.The glucose metabolism in the SG group and the DJB group was significantly improved after operation.At 2,4,8,12,and 16 weeks after surgery,the fasting blood glucose of the SG group and the DJB group was significantly lower than that of the SHAM group(All P<0.01).There was no significant difference in fasting serum insulin levels among the three groups(P>0.05).However,the homeostasis model assessment of basal insulin resistance(HOMA-IR)was significantly lower in the SG group and the DJB group than in the SHAM group(All P<0.01).In addition,the oral glucose tolerance test(OGTT)showed that the glucose tolerance of rats in the SG group and the DJB group was also significantly improved.3.The postoperative cardiac function of the SG group and the DJB group was significantly improved.The myocardial diastolic and systolic function of the SG group and the DJB group were significantly improved compared with the SHAM group at 16 weeks after surgery.The left ventricular end diastolic pressure(LVEDP)was significantly lower in the SG group and the DJB group than that in the SHAM group(Both P<0.01).In addition,the level of serum BNP in the SG group and the DJB group were significantly lower than that in the SHAM group(Both P<0.01).4.The myocardial remodeling was significantly relieved in the SG group and the DJB group after surgery.The myocardial cell area of the SG group and the DJB group was significantly lower than that of the SHAM group(Both P<0.01).The myocardial lipid deposition and myocardial fibrosis were also alleviated significantly in the SG group and the DJB group.5.The myocardial calcium homeostasis was significantly improved in the SG group and the DJB group.Ca2+ transients exhibited a significantly higher amplitude and faster decay in the SG group and the DJB group,which could be partially explained by increased expression of ryanodine receptor 2(RyR2),sarco(endo)plasmic reticulum Ca2+-2ATPase(SERCA2a),12.6-kDa FK506-binding protein(FKBP12.6),and also hyperphosphorylation of phospholamban(PLB).6.The autophagy of cardiomyocytes in the SG group and the DJB group were inhibited.Compared with the SHAM group,the number of autophagosomes in the cardiomyocytes of the SG group and the DJB group decreased significantly(Both P<0.01).In addition,a lower level of light chain 3B-II(LC3B-II)and higher level of p62 were detected in both SG group and DJB group,which could not be reversed by chloroquine treatment and associated with activated mammalian target of rapamycin(mTOR)and attenuated AMP-activated protein kinase(AMPK)signaling pathway.ConclusionsBariatric surgery could alleviate DCM morphologically and functionally,which may result,at least in part,from facilitated Ca2+ homeostasis and attenuated autophagy.