Project description:Skeletal muscle insulin resistance, an early metabolic defect in the pathogenesis of type 2 diabetes, may be a cause or consequence of altered protein expressions profiles. Proteomics technology offers enormous promise to investigate molecular mechanisms underlying pathologies, however, the analysis of skeletal muscle is challenging. Using a state-of-the-art mass spectrometry (MS) based workflow, we performed a global proteomics analysis of skeletal muscle from leptin-deficient, obese, type 2 diabetic (ob/ob) and lean mice, identifying more than 6,000 proteins with 118 proteins differentially regulated in obesity. This included protein kinases, phosphatases, and secreted and fiber type associated proteins. Enzymes involved in lipid metabolism in skeletal muscle from ob/ob mice were increased, providing evidence against reduced fatty acid oxidation in lipid-induced insulin resistance. Mitochondrial and peroxisomal proteins, as well as components of pyruvate and lactate metabolism were likewise increased. Finally, the skeletal muscle proteome from ob/ob mice displayed a shift towards the ‘slow fiber type’. This detailed characterization of obese rodent models of type 2 diabetes demonstrates an efficient workflow for skeletal muscle proteomics, which may easily be adapted to other complex tissues.
Project description:To identify novel transcriptional factors involved in dysfunctional hepatic lipids homeostasis in obesity, mRNA microarray analysis were performed to of livers of ob/ob mice, a widely used obese model, and C57BL/6J control mice. Chow-fed ob/ob and C57BL/6J mice were housed in a 12 h of light and 12 h of dark cycle and fed ad libitum a regular chow diet. Mice were sacrificed at 16:00 (Zeitgeber Time 8 during light phase). Total RNA was prepared from each liver using TRIzol (Invitrogen). Equal aliquots of total RNA from each of four mouse livers in each group were pooled and used for biotin labeling as described in the Affymetrix technical bulletin. Then the transcriptional profiles of samples were probed using the Gene-Chip Mouse Genome 430 2.0 arrays.
Project description:Prodynorphine-expressing neurons were targeted by eGFP-RPL10a, a transgene that is expressed under the control of the Pdyn promoter in a BAC containing the Pdyn backbone. Trangenic mice were made with FvB embryos via pro-nuclear injection. Transgene-positive progenies were bred to C57BL/6J for more than five generations before being used in the current experiment. Triplicates of 6-8 hypothalami were collected per sample from wild-type and ob/ob adult mice. Polysomal IP RNA and total Input RNA were purified from each sample. Affymetrix GeneChip Mouse 430 2.0 arrays (900495) were used to identify transcripts that are specifically expressed in Pdyn neurons with a fold change (IP vs. Input) of >=2 in wild-type mice. The same microarrays were also used to identify Pdyn-specific transcripts that are differentially regulated transcriptionally by leptin deficiency with a fold change (ob IP vs. wt IP) of >=1.5.
Project description:ob/ob mice is an obese mice. CIDE family proteins including Cidea, Cideb and Cidec play important role in lipid metabolism. Cidea is mainly expressed in the brown adipose tissue (BAT). Cidec is mainly expressed in the BAT and white adipose tissue (WAT). We generated ob/ob/Cidea-/-/Cidec-/- mice to investigate the phenotype of fat tissue. ob/ob/Cidea-/-/Cidec-/- mice are lean when compared with ob/ob mice. The tissue weight and TAG content of BAT and WAT was extreamly decreased in ob/ob/Cidea-/-/Cidec-/- mice compared with that in ob/ob mice. We next extract the total RNA from the BAT and WAT of ob/ob and ob/ob/Cidea-/-/Cidec-/- mice, to perform microarray analysis using Mouse Gene 1.0 ST array system, Affymetrix. We then analysised the up-regulated and down regulated pathways.
Project description:Liver gene expression was compared in wild type, SHP knockout, ob/ob, and ob/ob SHP double mutant mice. A total of eight samples were analyzed using Affymetrix arrays. Each of the four genotypes studied were assayed in duplicate.
Project description:We hypothesize that gene expression in the lungs of these differentially-treated mice are divergent thus contributing to the disparity in their phenotypes. More specifically, (1) Effects of Leptin-treatment of ob/ob postnatal mice lungs are known to be volume-dependent from 2 to 10 wks of age, and are independent of the hypometabolism associated with leptin deficiency. ; (2) Leptin is critical to postnatal lung remodeling, particularly related to enlarged alveolar surface area. In order to test these hypotheses at the gene expression level, we utilized microarray analysis to examine transcriptional differences between lungs of leptin or saline-treated ob/ob postnatal mice. Keywords: leptin, ob, lung
Project description:Prodynorphine-expressing neurons were targeted by eGFP-RPL10a, a transgene that is expressed under the control of the Pdyn promoter in a BAC containing the Pdyn backbone. Trangenic mice were made with FvB embryos via pro-nuclear injection. Transgene-positive progenies were bred to C57BL/6J for more than five generations before being used in the current experiment. Triplicates of 6-8 hypothalami were collected per sample from wild-type and ob/ob adult mice. Polysomal IP RNA and total Input RNA were purified from each sample.