Rovided by fat. The remaining 3 groups received HF chow (Purina Mills International) from which 45 of calories had been supplied by carbohydrate, 22 were offered by protein, and 33 were provided by fat). As a result, we studied four groups of mice: group 1 consisted of SC-fed mice treated with control ASO, group two consisted of HF-fed mice treated with handle ASO, group three consisted of HF-fed mice treated with resistin ASO, and group four consisted of HF-fed mice treated with resistin ASO and acutely infused with recombinant mouse resistin. All mice received two i.p. injections (25 mg/kg) of either control ASO (groups 1 and 2) or resistin ASO (groups three and 4) throughout the week preceding the clamp study (Figure 1A). For insulin tolerance testing, basal plasma values and hepatic kinase phosphorylation studies, adult male C57BL6J mice had been fed SC and HF diets and treated with handle and resistin ASO as described above. Following an overnight fast, tail blood was sampled for serum glucose and hormone evaluation, and animals have been injected i.p. with 100 mU insulin (human recombinant; Sigma-Aldrich, St. Louis, Missouri, USA) in a remedy of 5 glucose (Sigma-Aldrich) in regular saline. Soon after 15 minutes, animals had been sacrificed and livers and intracardial blood have been sampled. Cell culture. Primary rat hepatocytes were obtained from the Cell Culture and Genetic Engineering Core Facility on the Marion Bessin Liver Analysis Center of the Albert Einstein College of Medicine (37). Immediately after cell attachment towards the culture plate development media was Contactin-3 Proteins Biological Activity changed to DMEM (PTPN22 Proteins medchemexpress Invitrogen, Carlsbad, California, USA) + 10 FBS (Invitrogen) with either insulin (10 ng/ml; SigmaAldrich) or insulin plus recombinant resistin (1 /ml). Cell lysates have been ready immediately after an overnight incubation and analyzed by Western blot as described below.The Journal of Clinical InvestigationDesign of oligodeoxynucleotide antisense against resistin mRNA. The antisense oligodeoxynucleotide (ODN), Res-AS (ISIS Inc., Carlsbad, California, USA), was designed to hybridize to the sequence-spanning mouse resistin mRNA. All nucleotides were synthesized as uniform phosphothiorate chimeric ODNs, with 2-O-methoxyethyl (MOE) groups on bases 1 to 5 and 16 to 20. The ODN were synthesized on an Applied Biosystems 380B automated DNA synthesizer (PerkinElmer-Applied Biosystems, Boston, Massachusetts, USA) and purified as described (38). Mouse resistin ASO (ISIS 167308) is usually a 20-base, 5-10-5 MOE chimeric ASO with the following sequence: TTCACGAATGTCCCACGAGC. It hybridizes to position 331 around the mouse resistin sequence (GenBank accession number AF323080.1). The handle ASO (ISIS 29848) is a chemistry control ASO that has the same length and chemical makeup because the resistin ASO but is composed of all 419 possible ASO combinations when each and every base position is randomly synthesized with any in the four possible nucleotides (A, G, T, or C). Hence, it really is not expected to hybridize to any mRNA sequence. Primers and real-time PCR. Liver G6Pase and PEPCK mRNAs had been measured by quantitative PCR with all the following mouse primers: forward primer 5-TCCTGGGACAGACACACAAG-3 and reverse primer 5-CAACTTTAATATACGCTATTGG-3 for G6Pase; forward primer 5-CTTCTCTGCCAAGGTCATCC-3 and reverse primer 5-TTTTGGGGATGGGCAC-3 for PEPCK. The mRNA levels for G6Pase and PEPCK were normalized to 18S expression (forward primer 5-AGGGTTCGATTCCGGAGAGG-3, reverse primer 5-CAACTTTAATATACGCTATTGG-3). Total RNA was isolated with Trizol (Invitrogen) and single-strand cDNA was sy.
