Effect Of Autophagy On Ectopic Lipid Deposition In Pancreas And Insulin Sensitivity

Obeject: Establish a high-fat-diet induced obese rat model, while giving theintervention of autophagy inhibitor, testing the degree of pancreas fat deposition andautophagy related protein LC3expression levels, analysis the effects of autophagyactivity to the pancreatic lipid deposition and insulin sensitivity; Observe differences ofpancreatic lipid deposition and insulin sensitivity among ones with different glucosetolerance. Investigate the relationship between pancreatic lipid deposition and insulinresistance in order to provide reference for further exploring the possible role ofautophagy in the development of type2diabetes in human body.Methods:1. Experimental research:308-week-old SD male rats were fed after1weekadaptively, who were randomly divided into3groups. Group NC: Fed with normal diet,Group HF: Fed with high fat diet, Group HFC: Fed with high fat diet and chloroquine,each group had10cases. After feeding12weeks, detect the level of triglycerides (TG),total cholesterol (TC), fasting blood glucose (FBG) and fast insulin levels. Calculatedhomeostasis model assessment of insulin resistance (HOMA-IR) index assessing insulinsensitivity; oral glucose tolerance test (OGTT) to assess the level of glucose tolerance;Take the pancreatic tissue and paraffin sections were stained with HE to observe ratpancreatic tissue morphology and the degree of lipid deposition; Detect the autophagyrelated protein LC3expression levels by Western blot. 2. Clinical research: Obeserved120cases of the subjects, including37normal glucosetolerance ones for normal control (NC) group,37impaired glucose regulation(including impaired fasting glycaemia and impaired glucose tolerance) cases for IGRgroup,46type2diabetes mellitus cases for T2DM group. collected general informationincluding height, weight, waist and hip circumference, calculate body mass index (BMI),waist-to-hip ratio (WHR), systolic blood pressure (SBP), diastolic blood pressure (DBP);After fasting10hours, fasting blood glucose (FBG),2-hour plasma glucose (2hPG), TG,TC, high-density lipoprotein cholesterol (HDL-C) and low-density lipoproteincholesterol (LDL-C) levels were detect using automatic biochemical analyzer. Fastingplasma insulin (Fins) and2-hour postprandial insulin (Ins) levels were measured byradioimmunoassay (RIA). Homeostasis model assessment HOMA-IR and HOMA-βindex; All subjects underwent upper abdominal CT scan. The CT difference betweenspleen and pancreas were measured to assess the degree of pancreatic lipid depositionand the liver and spleen CT ratio were measured to assess liver lipid deposition; Therelationships between activity level of autophagy and pancreatic lipid deposition orother variables were also analyzed by SPSS13.0software.Results:1. Experimental results:1.1Comparing the growth characters in three groupsBody weight, body length, waist circumference and other growth indicators at baselineof rats in3groups were no significant difference. After12weeks of feeding,compared with ordinary feeding group NC rats, the body weight, Lee’s index and waistcircumference (WC) in HF and HFC group which two were fed with high fat diet weresignificantly increased (P <0.05). 1.2Comparing the OGTT results in three groupsCompared with the group NC, glucose tolerance test indicates the levels of0,20,40,60,120min blood glucose and glucose area under the curve in HF and HFC group wereincreased (P <0.05). Blood glucose at20min was glycemic peak and last at a high levelfor a long time in HF and HFC group while group NC blood glucose at40min up toglycemic peak and then gradually reduced to normal levels.1.3Comparing the glucose, lipid metabolism and insulin sensitivity in three groupsCompared with the group NC, FBG, TG, TC, Fins and HOMA-IR index weresignificantly increased while HOMA-β index was significantly decreased in the HF andHFC groups (P <0.05).1.4Comparing the pancreatic morphology and lipid deposition in three groupsWe observed there were no obvious lipid droplets deposition in pancreas tissue of groupNC in HE stain. Compared with group NC, lipid droplets deposition in pancreas tissueof group HF increased significantly while a large number of lipid droplets deposition inpancreas tissue of group HFC and there were lipid infiltrating around the islet.Using image analysis software to calculate (intralobular and interlobular) thepercentage of fat cells in each high power field. We found that compared with groupNC, the degree of pancreatic lipid deposition in group HF increased significantly andcompared with group HF, the degree of pancreatic lipid deposition in group HFincreased significantly(P <0.05).1.5Correlation Analysis of pancreatic lipid deposition and metabolic indicatorsThe pancreas lipid deposition correlated positively with body weight, Lee’s index, WC,TG, TC, FBG, Fins, HOMA-IR index. In a multiple regression analysis, HOMA-IR index and TC were independently associated with pancreatic lipid deposition.1.6Comparing the expression of autophagic LC3protein in three groupsThe relative molecular weight of LC3protein was16ku. Compared with group NC,expression of LC3protein in group HF was significantly increased, in group HFC wassignificantly decreased (P <0.05).2. Clinical results:2.1Comparing the general characters in three groupsCompared with NC group, WHR, FPG, Fins,2hPG, Ins, HOMA-IR index in IGR groupand T2DM group increased significantly. Compared with NC and IGR groups, BMI,TG, TC increased significantly, while HDL-C, HOMA-β index decreased significantlyin T2DM group (P <0.05).2.2Comparing the difference of CT value between spleen and pancreas, the ratioof CT value between liver and spleen in three groupsCompared with NC group, the difference of CT value between spleen and pancreasincreased and the ratio of CT value between liver and spleen decreased in IGR group,but the difference was not statistically significant. Compared with NC and IGR groups,the difference of CT value between spleen and pancreas significantly increased and theratio of CT value between liver and spleen decreased significantly in T2DM group (P<0.05).2.3The analysis of associativity of the difference of CT value between spleen andpancreas with other parametersThe difference of CT value between spleen and pancreas correlated positively with HOMA-IR, age, BMI, WHR, FBG, Fins,2hPG, Ins, TG, TC and negatively withHOMA-β, the ratio of CT value between liver and spleen, HDL (R=0.758,0.283,0.657,0.687,0.683,0.734,0.659,0.311,0.559,0.460,-0.549,-0.732,-0.294, P <0.05).There were no significant correlation with SBP, DBP, LDL-C. In a multiple regressionanalysis, HOMA-IR, the ratio of CT value between liver and spleen, BMI, WHR, age,TC were independently associated with the difference of CT value between spleen andpancreas (P <0.05).Conclusion:1. Long term high-fat diet, it may induce obesity, lipid disorders, and pancreatic lipiddeposition, insulin resistance and β-cell dysfunction.2. Long term high-fat diet can activate the activity of autophagy in pancreas.Autophagy may play a compensatory protective role in pancreatic lipid deposition.3. Long term high-fat diet induced insulin resistance and pancreatic lipid depositionwere further aggravated when the activity of autophagy was inhibited. It may berelated to the lack of protection from autophagy.4. Autophagy may be involved in the regulation of insulin resistance and the β-cellfunction and play an important protective role in the pathogenesis of type2diabetes.5. With the degree of pancreatic lipid deposition increased, lipid metabolism disordersand IR increased significantly in different glucose tolerance ones, pancreatic lipiddeposition may be involved in insulin resistance.