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  • 02 for Windows. Total RNA was prepared from liver, gastrocnemius skeletal muscle and subcutaneous white adipose tissue (WAT) of LC (n=10) and HC (n=10) mice after 12 weeks on the diets following a 6-h light-phase fast. Liver RNA was prepared using an RNeasy Plus Mini Kit (Qiagen, USA). Skeletal muscle RNA was prepared using an RNeasy fibrous Mini Kit, and WAT was prepared using an RNeasy Lipid Tissue Mini Kit (Qiagen, USA). The RNA concentration was assessed using a NanoDrop ND-1000 spectrophotometer check details (Thermo-Fischer Scientific). The ratio of absorbance at 260 and 280 nm was used to assess the purity of RNA. The extracted RNA was stored at ?80��C. cDNA was prepared using Superscript III reverse transcriptase (Invitrogen). Quantitative polymerase chain reaction (PCR) was performed using TaqMan Gene Expression Assay reagents and TaqMan FAM dye-labeled probes (Applied Biosystems Inc., USA) using an ABIPRISM 7700 Fast Real-Time PCR System (Applied Biosystems Inc., USA). Data were normalized to expression INNO-406 order of the endogenous housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and analyzed by the comparative ����CT method to determine the difference between LC and HC groups. Data are expressed as mean��S.E.M. Comparison between groups was performed with a Student's t test for independent samples using Microsoft Excel 2003 for Windows. All tests were two-tailed, and P<.05 was considered <a href="http://en.wikipedia.org/wiki/PRDX5">PRDX5 statistically significant. The HC group gained consistently more weight than LC over the 12 weeks of the study (Fig. 1). At week 12, the percent weight change from baseline in the HC group was 33% higher than in the LC group (P=.004). Body weight in LC and HC groups over the 12 weeks matched or exceeded the standard diet group (Fig. 1), confirming that the protein intake in LC and HC groups was adequate for maintenance requirements in these mature mice as recommended by Tobin et al. [20]. Subsequent analysis was restricted to comparing the LC and HC groups. Total fat mass and lean mass measured by DEXA were similar in HC and LC groups at baseline, but were significantly higher in HC at weeks 4, 8 and 12 (Fig. 2A). Total fat mass as a percent of body weight remained similar in both groups, whereas peri-intestinal fat mass dissected at the termination of the study (week 12) was ?30% higher in HC whether normalized to total fat mass or body weight (P=.003) ( Fig. 2B, C). There was no difference in the weights of epididymal or brown fat pads. The HC mice also had elevated hepatic triglycerides and free fatty acids at week 12 ( Fig. 2D). At week 12, HC mice had higher plasma low-density lipoprotein cholesterol (LDL-C) and triglycerides than LC mice. Plasma glycerol was significantly reduced in HC mice, suggesting decreased lipolysis, but there was no difference in free fatty acid concentrations (Table 2).

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