Polyclonal Akt antibody (Upstate Biotechnology), coupled to protein A-sepharose beads. The immune complicated was washed, and Akt activity was determined as described (21). Lipid metabolites. Tissue triglycerides have been extracted using the method of Bligh and Dyer (22), and content was measured applying a DCL Triglyceride Reagent (Diagnostic Chemical substances, Oxford, CT). Fatty acyl-CoA, diacylglycerol, and ceramide extraction and measurement by liquid chromatography/tandem mass spectrometry have been described previously (23). Gene expression. RNA was isolated from skeletal muscle, WAT, and liver, and cDNAs were synthesized with SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA) and oligo dT. Gene expression was assessed by real-time quantitative PCR making use of distinct primers and TaqMan probes for fatty acid transport protein-1, -2, -4, and -5 and CD36 (Applied Biosystems). Quantification was performed by the CT threshold cycle process, and relative gene expression was normalized to GAPDH levels. Statistical analysis. Results are expressed as indicates SE. Statistical significance of variations amongst experimental Ubiquitin Conjugating Enzyme E2 L3 Proteins web groups was assessed utilizing the unpaired Student’s t test.RESULTSPref-1 overexpressing mice are resistant to dietinduced obesity. We recently reported that overexpression of a Pref-1/hFc fusion protein in mice impaired adipocyte differentiation (19). Interestingly, these mice exhibited a mild degree of glucose intolerance and insulin resistance at young age ( ten weeks old). To further investigate the effects of Pref-1 overexpression on tissuespecific insulin sensitivity, we carried out comparative studies on Pref-1 Tg mice and Wt littermates fed a high-fat diet program for 17 weeks. High-fat diets are recognized to induce obesity and to market insulin resistance and diabetes in mice and humans, particularly if individuals are subject to such diets for a extended time frame. At weaning, Pref-1 transgenic male mice weighed slightly significantly less than Wt littermates (Wt 9.five 0.five g, n 17, vs. Tg eight.4 0.5 g, n 15; P 0.074) (Fig. 1A). Following 17 weeks of high-fat diet feeding, Wt mice became evidently obese, PAC1-R Proteins Source exhibiting an typical of eight g in physique weight above Pref-1 transgenic mice, which remained considerably leaner (Wt 43.1 1.1 g, n 17, vs. Tg 34.8 1.3 g, n 15; P 0.01). Similarly, female transgenic mice had been also resistant to diet-induced obesity (Wt 34.2 1.0 g, n 10, vs. Tg 28.5 1.5 g, n ten; P 0.01) (Fig. 1B). The resistance toHIGH-FAT Diet plan AND INSULIN RESISTANCEAWild typeB40 30 20 ten 0 three 5 7 9 11 13 15 17 19 21 3 five 7 9 11 13 15 17 19 21 Males FemalesPref-1 TgBody Weight (g)Age (Weeks)Age (Weeks)157/group). B: Females (nFIG. 1. Body weight of wild-type (f) and Pref-1 transgenic (E) mice fed a high-fat diet program for 17 weeks. A: Males (n 10/group).high-fat diet nduced obesity occurred regardless of related food intake (Wt 0.420 0.04 kcal g 1 day 1, n six, vs. Tg 0.417 0.03 kcal g 1 day 1, n 7). Compared with Wt, Pref-1 Tg mice exhibited a significant reduction in the mass in the major WAT depots (Fig. 2A), for example gonadal, inguinal, and renal fat. The interscapular brown adipose tissue was also significantly decreased. The reduction in adipose tissue mass wasaccountable for most on the reduce observed in physique weight, since 1H magnetic resonance spectroscopy revealed no differences in lean mass among Wt and Pref-1 Tg mice (Fig. 2B). Fat depots of Pref-1 transgenic mice appeared regular by gross morphological examination, despite the fact that adipocytes were significantly smaller sized than these.