The Effects Of CETP On Mouse Pancreatic β-cell Lipid Composition And β-cell Function

Objective：Insulin resistance and pancreatic β cell dysfunction are two mainly factors in thepathogenesis of Type2diabetes, and β cell dysfunction plays a pivotal role in thedevelopment of Type2diabetes. More cholesterol accumulation in β cell will causeisletsβ cell dysfunction. CETP, the cholesteryl ester transfer protein, plays as a keyrole in the process of reverse cholesterol transport. Many studies focuse on thecorrelation between CETP and lipid metabolism but fewer studies are about therelationship between CETP and glucose metabolism. Patients with Type2diabetesand obese often show low HDL-C and high activity of plasma CETP which suggeststhat CETP may also have an impact on glucose metabolism. In this study, differentanimal model, NFR-CETPTg mice and AP2-CETPTg mice were used to investigatethe effect of CETP on pancreatic β-cell lipid composition and β-cell function in micein order to know the further role of CETP on development of Type2diabetes.Methods:1. Three groups’ mice with different genetic backgrounds, wild-type C57BL/6Jmice, AP2-CETPTg mice and NFR-CETPTg mice were selected for the first part ofthis study. Characteristic of tissue expression and plasma CETP activitymeasurements were conducted among these animals. We also explored whether therewas expression of CETP in mice islet2. In the second part of this study, wild type C57BL/6J mice(control group),AP2-CETPTg mice and NFR-CETPTg mice were included. We determined fastingand15min plasma insulin after glucose load meanwhile we used HOMA formula tocalculate HOMA-IR and HOMA-β. Glucose stimulate insulin secretion wasconducted to evaluate the function of islet of CETPTg mice. All these were done to explore the effect of different levels of CETP on β cell function.3. In the third part of this study, Islets of mice were also isolated and lipidprofile was measured using enzymatic analysis and LS/MS/MS methods to explorethe regulation of different levels of CETP on islet lipid profile. We also extractedRNA from mice islet and determined genes expression which participated inregulating of cholesterol balance in islets of mice using real-time PCR methods inorder to explore the effect of CETP on these genes expression.4. In the fourth part of experiments, mice were sacrificed. Islets of AP2-CETPTg mice, NFR-CETPTg and WT mice were separated and collected. Pancreasbiopsies were done and islets morphological changes were observed in these mice inorder to analyse the reason for the decrease of insulin secretion in CETPTg mice.5. In the fifth part of experiments, islets of wild type C57BL/6J mice,AP2-CETPTg mice and NFR-CETPTg mice were also isolated. RNA was extractedfrom these mice islet and genes expression which participated in islet proliferationand inflammation were detected in order to find the reason for the islet massreduction in CETPTg mice. We also investigated the effect of different levels ofCETP on Glut2expression in mice islet.6. In the sixth part of experiments, Wild C57BL/6J mice, NFR-CETPTg mice,AP2-CETPTg mice were fed by high fat diet for sixteen weeks since eight weeks. Allmice were selected to accept intraperitoneal injection of glucose tolerance test(IPGTT) and intraperitoneal injection of insulin tolerance test(IPITT). We alsodetected fasting insulin and conducted glucose stimulate insulin secretion in thesemice. Thus, high fat diet feeding on body weight and glucose metabolism of differentgenetic background mice is documented.Results:1. The first part of the experiment 1.1Characteristic of tissue expression in NFR-CETPTg and AP2-CETPTg miceAP2-CETPTg mice are adipose tissue-specific CETP expression transgenic micewhile other tissues did not express CETP mRNA. The tissue distribution of CETPmRNA expression in NFR-CETPTg mice was the same as in human. We also foundthat CETP mRNA expressed in islet of NFR-CETPTg mice but it did not express inislet of WT and AP2-CETPTg mice.1.2Serum CETP activity measurementsSerum CETP activity of NFR-CETPTg mice was the same as human whileSerum CETP activity of AP2-CETPTg mice was approximately three times as human.2. The second part of experiments2.1The determination of serum insulin in miceFasting and15min serum insulin after glucose load of CETPTg mice werelower than that of WT mice while fasting plasma insulin was higher in NFR-CETPTgmice than in AP2-CETPTg mice (p<0.05), but there was no difference of15minplasma insulin after glucose load between these two types of CETP Tg mice2.2Calculation HOMA-IR using HOMA formulaCompared with WT mice, HOMA-IR of CETPTg mice were lower whichsuggested that CETPTg mice were insulin sensitivity while AP2-CETPTg mice weremore sensitive.2.3Calculation HOMA-β using HOMA formulaCompared with WT mice, HOMA-β of CETPTg mice were lower whichsuggested that CETPTg mice with β cell dysfunction. There was no difference ofHOMA-β between these two CETPTg mice.2.4Glucose stimulate insulin secretionCompared with WT mice, CETPTg mice showed dysfunction of glucosestimulating insulin secretion while it was serious in AP2-CETPTg mice. 3. The third part of experiments3.1.1The determination of lipid profile of islet using enzymatic analysisTC, FC, CE were higher in islets of CETPTg mice when compared with WTmice (p<0.05). While more cholesterol accumulated in AP2-CETPTg mice.3.1.2The determination of lipid profile of islet using LS/MS/MSCompared with WT mice, FC, FC/CE were higher in islets of AP2-CETPTgmice (p<0.05).3.2The effect of CETP on genes expression which participate in regulating ofcholesterol balance in islets of miceCompared with WT mice, the expression of LDLR, HMG-COA, SREBP-2weredecreased while the expression of ABCA1, ABCG1were up-regulation in CETPTgmice (p<0.05). There was no difference between NFR-CETPTg mice andAP2-CETPTg mice.4. The forth part of experiments4.1The number of mice isletsCompared with WT mice, the number of CETPTg mice islets were reduced(p<0.05). While there was no difference between NFR-CETPTg mice andAP2-CETPTg mice.4.2Morphological change of CETPTg mice isletsPancreatic biopsy by hematoxylin-eosin (HE) staining, Observed under themicroscope, compared with the control group, CETPTg mice islets number anddensity were significantly reduced. While there was no difference betweenNFR-CETPTg mice andAP2-CETPTg mice.5. The fifth part of experiment5.1The effect of CETP on genes expression which participated in proliferationand inflammation of islets Compared with WT mice, the expression of PDX1and BETA2were decreasedwhile the expression of IL-β、CHOP、f4/80were up-regulation in CETPTg mice(p<0.05). This phenomenon was significant in AP2-CETPTg mice.5.2The effect of CETP on Glut2expressionThe expression of Glut2in AP2-CETPTg mice was lowest in these mice whilethere was no difference between NFR-CETPTg mice and WT mice.6. The sixth part of experiments6.1IPGTT after16weeks group feedingAfter high fat diet feeding, compared with WT mice, AP2-CETPTg mice andNFR-CETPTg mice showed impaired glucose tolerance （P<0.05） whileAP2-CETPTg mice impaired more glucose tolerance. Mice feed high fat dietshowed impaired glucose tolerance than which feed the standard diet.6.2IPITT after16weeks group feedingAfter high fat diet feeding, compared with WT mice, there was higher insulinsensitivity in CETPTg mice while there was no difference between NFR-CETPTgmice and AP2-CETPTg mice.6.3The determination of plasma insulin in miceAfter high fat diet feeding, fasting plasma insulin of CETPTg mice were lowerthan WT mice while there was no difference between NFR-CETPTg mice andAP2-CETPTg mice. Fasting plasma insulin of mice feed high fat diet were higherthan which feed the standard diet.6.4Glucose stimulate insulin secretionAfter high fat diet feeding, compared with WT mice, CETPTg mice showeddysfunction of glucose stimulating insulin secretion while there was no differencebetween NFR-CETPTg mice and AP2-CETPTg mice. The dysfunction of islet wasmore serious in mice feed high fat diet were higher than which feed the standard diet. Conclusion：1. We demonstrate that CETP mRNA expressed in islet of NFR-CETPTg micebut it does not express in islets of WT and AP2-CETPTg mice. We speculate thatCETP has directly or indirectly impact on the function of islet for NFR-CETPTg micewhile this effect is indirect for AP2-CETPTg mice.2. Fasting and15min serum insulin after glucose load of AP2-CETPTg mice andNFR-CETPTg mice are lower than WT mice which suggests that CETP Tg miceshow impaired β cell dysfunction. There are more cholesterol accumulating in isletsof CETP Tg mice and pancreatic biopsy shows that there are fewer islets of CETP Tgmice which suggests that CETP has effect on β cell function by influencing the levelof cholesterol in islet. All of these explain that CETP may play an important role inthe development of type2diabetes.3. CETP influences the transports participating in cholesterol balance expressionin islet which suggests that these transportation are important for the cholesterolbalance of islets.4. High-fat diet, purchased from research diet company (D12331) could establishan obesity mice model with impaired insulin resistance.5. High-fat diet increases glucose and lipid metabolism disorders of CETPTgmice. After feed high-fat diet, islet dysfunction was more serious in CETPTg mice. Ithints that CETP and obesity could play a role in incidence and development of type2diabetes.