An iTRAQ Proteomic Study Reveals an Association between Diet-Induced Enhanced Fatty Acid Metabolism and the Development of Glucose Intolerance in Prediabetic Mice

Abstract

High-fat diet (HFD)-induced glucose intolerance and insulin resistance increases the chances of developing type-2 diabetes and cardiovascular disease. To study the mechanism(s) by which a HFD impairs glucose tolerance, we used a quantitative proteomic platform that integrated pI-based OFFGEL fractionation and iTRAQ labeling to profile the temporal changes in adipose membrane protein expression in mice fed a HFD for up to 8 months. Within 2 months of starting the diet, the mice adipose and liver tissues accumulated fat droplets, which contributed to subsequent insulin resistance and glucose intolerance within 6 months. The membrane proteomic delineation of such phenotypic expression resulted in quantification of 1713 proteins with 266, 343, and 125 differentially expressed proteins in 2-, 6-, and 8-month HFD-fed versus control mice, respectively. Pathway analysis of these differentially expressed proteins revealed the interplay between upregulation of fatty acid metabolism and downregulation of glucose metabolism. Substantial upregulation of adipose and liver carnitine palmitoyltransferase (Cpt) 1, the rate-limiting enzyme in the transport of long-chain fatty acids into mitochondria, occurred by 2 months. The increase in hepatic Cpt 1a expression was associated with a progressive decrease in glucose uptake as evidenced by downregulation of the liver glucose transporter protein (Glut) 2. Loss of glycogen storage was found in those hepatocytes full of fat droplets. Intriguingly, skeletal muscle Cpt 1b expression was unaltered by the HFD, whereas skeletal muscle Glut 4 and tyrosine phosphoryated insulin receptor substrate 1 (p-IRS1) were substantially upregulated at the same time as abnormal glucose metabolism developed in adipose and liver tissues. This study defines some of the molecular mechanisms as well as the relationship among adipose tissue, liver and skeletal muscle during development of HFD-induced glucose intolerance in vivo and identifies Cpt 1 as a potential drug target for the control or prevention of diabetes.

Niche-Dependent Regulations of Metabolic Balance in High-Fat Diet–Induced Diabetic Mice by Mesenchymal Stromal Cells

“…We previously reported that persistent HFD feeding resulted in fatty liver, and overexpression of Cpt 1A as well as decrease in glycogen storage ability in hepatocytes accounted for liver-related glucose impairment and insulin resistance (30). Here, MSCs did not alter the morphology (Fig.…”

“…In an HFD-induced fatty liver, a massive amount of saturated fatty acids accumulated in hepatocytes results in insulin resistance via inhibiting insulin signaling through a c-Jun N-terminal kinase-dependent mechanism (9,40) and turning off glucose metabolism in hepatocytes with active b-oxidation (30). Systemic MSC transplantation restored insulin sensitivity in the liver by preventing fatty liver formation induced by an HFD (Fig.…”

“…Diabetes was induced by continuous feeding of an HFD (D12492; Research Diets, New Brunswick, NJ) (30). Thirty-six mice were used in the study of systemic MSC transplantation.…”

“…Blood glucose measurement and the GTT were performed according to the same protocol as in our previous study (30). In brief, the tail capillary blood glucose level was measured using a OneTouch Ultra meter (LifeScan, Milpitas, CA) after a mouse was starved for 8 h. For the GTT, 1 g/kg body weight of glucose in 0.9% NaCl was intraperitoneally injected, and tail capillary blood glucose levels were measured before (0 min) and 30, 60, 90, and 120 min after the glucose injection.…”

“…Protein samples were prepared according to a previously described protocol (30). Proteins (30 mg) from each sample were subjected to SDS-PAGE (10% [weight/volume] acrylamide) and transferred onto polyvinylidene difluoride membranes (Amersham Biosciences, Uppsala, Sweden).…”