Molecular Mechanisms Of Glucose Metabolim In The Heart:-AMPK And SNARK Pathway

Background:Recently, the incidence of diabetes is significantly increased in the world. Individual with diabetes have a significantly increased risk of developing cardiovascular disease which is the major cause of mortality associated with diabetes. However, the mechanism of diabetes increases the risk of cardiovascular diseases is still not clear. But insulin resistance and glucose metabolism disorder are the main characters and the major mechanisms. So it is necessary to investigate the molecular mechanisms of cardiac glucose metabolism.Obese diabetes patients always occur insulin resistance. And high fat diet is one of the major risk factors of insulin resistance. Previous studies showed that lipid metabolic disorder is both a cause and a result of insulin resistance. In the recent years, the incidence of obese is increased. The high-fat and low-carbohydrate diet is one of the major reasons.Previous studies showed that the high-fat and low-carbohydrate diet may result in cardiac energy disorder. However, the specific mechanisms have not elucidated. It is well known that AMP-activated protein kinase (AMPK) can regulate cardiac glucose utilization and energy balance, and that it is activated by an upstream protein kinase, LKB1. Following activation by phosphorylated LKB1, AMPK activates its downstream targets, such as peroxisome proliferatoractivated receptor gamma coactivator-1-alpha (PGC-1α) and phosphofructokinase2(PFK-2). PGC-1α is α nuclear-encoded transcriptional coactivator, and it plays an important role in glucose metabolism, adaptive thermogenesis, glucose uptake, and insulin sensitivity in muscle cells. PFK2belongs to the phosphoglycerate mutase family, and is required for the activation of cellular glycolysis. Previous studies have reported that high-fat and lowcarbohydrate diet feeding (45%calories as fat,35%carbohydrates, and20%protein; or contained35.5%fat) for13weeks caused marked effects on both glucose metabolism and insulin sensitivity in both adipocytes and skeletal muscle. Recent studies have also demonstrated that a high-fat and low-carbohydrate diet (35or45%of kcal from fat) was associated with cardiac dysfunction and aberrant energy metabolism. However, the effects of a highfat and low-carbohydrate diet on energy metabolism in cardiac muscle have not been elucidated, and the underlying molecular mechanism remains a mystery.High-fat and low-carbohydrate diet (containing17%lard) feeding for60days decreases the AMPK phosphorylation and activation in the epididymal adipose tissue, as well as PGC-1a expression in skeletal muscle. In addition, AMPK-mediated PFK-2activation is likely to be involved in the stimulation of heart glycolysis during ischemia. However, the effects of high-fat and low-carbohydrate diet feeding on cardiac function and the related molecular mechanisms have rarely been studied.Thus, we kept rats on different diets and studied the effects of high-fat and low-carbohydrate diet intake on cardiac function and the expression of AMPK, LKB1, PGC-1a, and PFK-2in rat cardiac muscle.Objective:1. To observe the effects of high-fat and low-carbohydrate diet on glucose and lipid metabolism, as well as cardiac function in rat.2. To investigate the effects of high-fat and low-carbohydrate diet on AMPK activity in cardiac muscle.3. To test the effects of high-fat and low-carbohydrate diet on the expression of LKB1,AMPK,PGC-1α and PFK-2in rat cardiac muscle.Methods:1. Animal feeding:Twenty-four male Wistar rats (4weeks old,body weight160-180g) were individually housed in cages located in a room under controlled housing conditions. They received either an experimental diet or control diet for22weeks. On a caloric basis, the experimental diet consisted of59%fat (mainly from lard, a representative food full of saturated fatty acids),24%carbohydrate, and17%protein (5.95kcal/g total), whereas the normal control diet contained10%fat,70%carbohydrate, and20%protein (4.24kcal/g total). 2. The effects of high-fat and low-carbohydrate diet on biochemical characters and cardiac function:Fasting blood glucose levels (FBG) and insulin concentrations were measured by the glucose oxidase method and RIA,respectively. Serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL) levels were measured by chemiluminescence immune assay. Transthoracic echocardiography was performed on rats at baseline and, after22weeks on the diets. Left ventricular (LV) end systole diameter (ESD), end diastole diameter (EDD), interventricular septum systole (IVSS) wall thicknesses, interventricular septum diastole (IVSD) wall thicknesses, posterior systole (PS) wall thicknesses, and posterior diastole (PD) wall thicknesses were determined for three cardiac cycles and averaged.3. Effects of high-fat and low-carbohydrate diet on AMPK activity:Briefly, the left ventricle muscles were weighed and then the AMPK activity was assessed with32P.4. To test the effects of high-fat and low-carbohydrate diet on LKB1, AMPK,PGC-1α,PFK-2in rat cardiac tissue:The gene level of AMPK a1and AMPK α2in rat cardiac muscle were verified using RT-PCR. The protein expression of total-LKB1, Phospho-LKB1(Ser431), AMPK α,Phospho-AMPK a (Thr172),PGC-1α and Phospho-PFK-2(ser466) were tested using Western blot.Results:1. The effects of high-fat and low-carbohydrate diet on biochemical characters and cardiac function:1.1The effects of high-fat and low-carbohydrate diet on biochemical characters:Before experimental diet intake, the two groups destined for experimental or normal diets were of similar body weights (P>0.05). After22weeks of feeding, the body weight in experimental diet feeding was increased by13.2%compared to that of normal diet feeding (P<0.01). The experimental diet increased the fasting glucose level by28.1(P>0.05) and the fasting insulin concentration by29.3%(P<0.05). Meanwhile, in comparison to the normal diet group, the serum levels of TC, LDL, and TG were increased by15.0%(P>0.05),38.7%(P<0.01), and33.5%(P<0.01), respectively, in the experimental diet group. 1.2The effects of high-fat and low-carbohydrate diet on cardiac function:The LV dimensions were determined at the start and at22weeks after initiation of the diet interventions. Baseline parameters were similar in the two groups. After22weeks on the diet, ESD and IVSS were increased in the experimental diet-fed group, whereas EDD, IVSD, PD and PS was unaffected by the diet. Accordingly, FS%and EF%were decreased in the experimental group compared with control group hearts.2. Effects of high-fat and low-carbohydrate diet on AMPK activity and expression:The levels of α1and α2activity were inhibited in the hearts of rats fed the experimental diet. The gene expression of AMPK a1and AMPK a2was significantly reduced, by43.33%(P<0.05) and27.52%(P<0.05), respectively, in the experimental diet group. Meanwhile, compared to the normal diet group, the protein levels of total and phosphorylated AMPK a were also decreased, by54.47%(P<0.05) and58.76%(P<0.05), respectively, in the experimental diet group. However, no statistically significant difference was found in the ratio of phospho-AMPK a to the total AMPK a between the two groups (P>0.05).3. To test the effects of high-fat and low-carbohydrate diet on the expression of LKB1,PGC-1α and PFK-2in rat cardiac tissue:We failed to find any difference in the level of total LKB1between the two groups. However, the phospho-LKB1level was significantly attenuated in experimental diet group (59.09%, P<0.05). As expected, both PGC-1α expression (P <0.05) and PFK-2phosphorylation (P<0.05) were significantly reduced in hearts from rats fed the experimental diet.Conclusions:1. High-fat and low-carbohydrate diet increases the body weight, fasting insulin level, serum levels of TC, LDL and TG. High-fat and low-carbohydrate diet impairs cardiac contractile function.2. High-fat and low-carbohydrate diet significantly decreases the AMPK activity and expression in rat cardiac muscle.3. High-fat and low-carbohydrate diet down-regulate LKB1,AMP,PGC-1α and PFK-2in rat cardiac muscle. The signaling mechanisms mediating myocardial glucose transport are not fully understood. Sucrose non-fermenting AMPK-related kinase (SNARK) is an AMPK-related protein kinase that is expressed in the heart and has been implicated in contraction-stimulated glucose transport in mouse skeletal muscle. To study SNARK in the heart, we first determined if SNARK is phosphorylated on Thr208, a site critical for SNARK activity. Mice were treated with exercise (treadmill running,0.6mph,12.5%grade for10,30, or60min), ischemia (global,3min), submaximal insulin (induced by i.p. injection of1g glucose/kg bw), or maximal insulin (1U/kg bw, i.p.), and the hearts were dissected and processed. Treadmill exercise slightly, but significantly increased SNARK Thr208phosphorylation. Ischemia also increased SNARK Thr208phosphorylation, but there was no effect of submaximal or maximal insulin. HL1cardiomyocytes were used to overexpress wild type SNARK (2-fold) and to knockdown endogenous SNARK (by shRNA,50-60%). Glucose transport and glycogen concentrations were measured in cells in the basal state and in response to ischemia (acidic N2gassed buffer,45min) and insulin (100nM,20min). Overexpression of wild type SNARK significantly increased basal glucose transport and glycogen concentrations in the cells. Overexpression of wild type SNARK had no effect on ischemia-stimulated glucose transport and glycogen; however, SNARK knockdown significantly decreased ischemia-stimulated glucose transport. SNARK overexpression or knockdown did not alter insulin-stimulated glucose transport or glycogen concentrations. To study SNARK function in vivo, SNARK heterozygous knockout mice (SNARK+/-) and wild type littermates performed treadmill exercise (0.6mph,12.5%grade,30min), and glucose transport was measured. Exercise-stimulated glucose transport was decreased by～50%in hearts from SNARK+/-mice. In summary, exercise and ischemia increase SNARK Thr208phosphorylation in the heart and SNARK regulates exercise-and ischemia-stimulated glucose transport. SNARK is a novel mediator of insulin-independent glucose transport in the heart.