Functional Studies On Carnitine Palmitoyltransferase 1A In Liver,Pancreas And Adipocyte

Carnitine palmitoyltransferase system,which includes carnitine palmitoyltransferase 1(CPT1),carnitine/acylcarnitine translocase and carnitine palmitoyltransferase 2,is localized in the mitochondrion,and its function is transferring long-chain acyl-CoA from the cytosol into the mitochondrial matrix where P-oxidation occurs.CPT1 catalyzes the rate-limiting step in fatty acid oxidation,and its main role is to maintain the balance between serum glucose and energy supply when the body lacks energy,by mobilizing fatty acids into the mitochondria for oxidation.There are three CPT1 genes,i.e.CPT1A,IB and 1C.Whereas expression of CPT1B and 1C is rather restricted in terms of their tissue distribution,CPT1A is widely expressed but mainly in liver,kidney,pancreas,adipose tissue,lymphocytes and fibroblasts.Homozygous deletion of CPT1A gene is lethal in mouse,suggesting its essential role in life process.Since liver,pancreas and adipose tissue are important peripheral tissues in glucose and lipid metabolism,to study the function of CPT1A in liver and pancreas in vivo,we constructed the CPT1A liver specific knockout(conditional knockout or CKO)mice and CPT1A pancreas CKO mice.And also we did studies of the function of CPT1A in adipose tissue in vitro by constructing different adipocyte cell lines with various levels of CPT1A.Using gene targeting technology,we had successfully constructed CPTIA-Floxed mouse model,and we have generated CPT1A liver CKO mice and CPT1A pancreas CKO mice by crossing CPTIA-Floxed mice with albumin-cre transgenic mice and Ins2-cre transgenic mice.Under different fat content diet conditions,we found that the CPT1A liver CKO mice in the high-fat conditions had more fat deposition in the liver and did not gain as much weight compared with the control mice.CPT1A liver CKO mice had lower basal serum glucose and insulin levels than control mice when fed with an ultra-high-fat diet,accompanied by higher insulin sensitivity and glucose sensitivity.To our surprise,when the mice fed with three different diets with increased fat content,the weight gain of CPT1A pancreas CKO mice appears to be the same-more-same phenomenon compared with control mice under normal diet,high fat diet and high fat/high carbohydrate diet,respectively.Unlike CPT1A liver CKO mice,CPT1A pancreas CKO mice had enhanced glucose tolerance,and the basal insulin levels were significantly increased about three times of the control mice,indicating that CPT1A deficiency in pancreas might cause insulin resistance.The observation that CPT1A CKO in different tissues caused completely different phenotypes demonstrates the important roles of CPT1A in these tissues.We adopted a PiggyBac delivery system to overexpress CPT1A gene,to decrease CPT1A expression by using shRNA and to generate a control using GFP in 3T3-L1 pre-adipocyte,and selected for stable cell lines with different CPT1A activities.Alteration in CPT1 activity,either by ectopic overexpression or pharmacological inhibition using etomoxir,did not affect adipocyte differentiation.However,when challenged with fatty acids,overexpression of CPT1A significantly reduced intracellular non-esterified fatty acids compared to the control cells.And these changes were accompanied by increased fatty acid uptake and decreased fatty acid release.CPT1A also protected the adipocyte against the insulin resistance and expression of pro-inflammatory adipokines such as TNF-a and IL-6 induced by fatty acid.CPT1A overexpression suppressed JNK activity while inhibition of CPT1A caused a significant enhancement of JNK activity.The specific inhibitor of JNK SP600125 largely abolished the changes caused by the shRNA-and etomoxir-mediated decrease in CPT1 activity.Moreover,C2C12 myocytes co-cultured with adipocytes pretreated with fatty acids decreased insulin sensitivity,and high activity of CPT1A in adipocyte appeared to alleviate the reduced insulin sensitivity.In summary,CPT1A deficiency in the liver alleviates high-fat diet induced obesity and insulin resistance,while CPT1A pancreas CKO leads to an enhanced insulin resistance.Whereas CPT1A overexpression in adipocytes attenuates fatty acid-evoked insulin resistance and inflammation via suppression of JNK.