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RNA-Seq. Liver tissues from WT or adiponectin KO male mice (

RNA-Seq. Liver tissues from WT or adiponectin KO male mice (n = 9 or 10) at 12 wk of age were harves… Show more RNA-Seq. Liver tissues from WT or adiponectin KO male mice (n = 9 or 10) at 12 wk of age were harvested and subjected to total RNA extraction. Total RNA from 9 or 10 mice of the same strain was pooled together as one biological sample. The mRNA sequencing samples were prepared by using the Illumina sample preparation protocol (RS-930-1001; Illumina). Briefly, mRNA was isolated from total RNA (10 μg) and then was used to synthesize cDNA. The resulting cDNA (fragments of 200–250 bp) was purified and enriched by PCR. The cDNA libraries were sequenced by an Illumina Genome analyzer II at the Whitehead Genome Technology Core. Read Mapping and Expression-Level Normalization. A total of 21.8 million and 22.7 million 36-bp single reads were obtained from the RNA of adiponectin KO and WT mice, respectively. TopHat (27) was used for mapping the reads to mouse mm9 genome, guided with Ensembl (NCBIM37 62) gtf file. The segment size for TopHat was reduced to 18, and the maximum mismatch within each segment was set to 1. More than 82% of the reads from both samples were mapped. Gene fpkms were calculated and compared with Cuffdiff (8) with –b and –u options, and rRNAs downloaded from Ensembl were excluded with –M option. Only genes with “Test Status” as OK and with at least 2 fpkm were kept for further analysis. RT-PCR. Total RNA from liver tissues of WT or adiponectin KO mice (n = 9 or 10) was isolated, and qRT-PCR was performed on an ABI Prism 7900HT Sequence Detector (Applied Biosystems) from 2 ng of total RNA after reverse transcription. 18S was used as the internal control. Data were analyzed by the relative quantification (ΔΔCt) method. Primer sequences are available upon request. HFD Study. Eighteen- to 20-wk-old male WT or adiponectin KO mice were divided randomly into four groups. Three groups were fed a HFD D12492 (60% kcal% fat) (Research Diets) for 10, 20, and 30 d, and one group was fed a chow diet for 30 d (11.8% kcal% fat) (LabDiet; 5P00 ProLab RMH 3000). Hepatic Triglyceride Contents. Liver tissues from mice under HFD studies were homogenized and extracted with chloroform/methanol (2:1; vol/vol) three times. The organic layer was collected after centrifugation and dried. The resulting samples were resuspended in 3 M KOH at 70 °C. Triglyceride level was measured by using a serum triglyceride determination kit (TR0100; Sigma). Histology. Liver, white adipose, and skeletal muscle tissues harvested from WT or adiponectin KO mice were stained with H&E or Oil Red O staining. All histology procedures were performed by the Histology Core Facility at the Koch Institute, Massachusetts Institute of Technology. ChIP Experiment. ChIP was performed as described (28, 29) with modifications. Five micrograms of specific or control antibody (HNF4-α, sc-8971; rabbit IgG, sc-2027) was incubated with preblocked Dyanbead Protein A (100.02D; Invitrogen) overnight. The specificity of the antibodies was described (30). Livers from WT or adiponectin KO mice were cross-linked, lysed, and then sonicated (28). The resulting chromatin was incubated with the antibody-coupled beads overnight. The beads were then washed, and the chromatin was eluted in ChIP elution buffer, reverse-cross-linked at 65 °C overnight, and treated with RNase and Proteinase K. The DNA was extracted, and 2 μL of DNA was used for each ChIP-PCR experiment. Primer sequences are available upon request. Statistical Analysis. Results were presented as mean ± SEM. Differences between groups were examined for statistical significance by unpaired onetailed Student’s t test or two-way ANOVA. P < 0.05 was regarded as statistically significant. Results Rate-limiting enzymes catalyze the slowest or irreversible steps in metabolic pathways, which makes them the targets of transcriptional regulation and/or posttranslational modifications such as phosphorylation in the regulation of metabolism (7). Because adiponectin plays an important role in energy metabolism, our analysis on hepatic gene expression in adiponectin KO and WT mice was specifically focused on the expression levels of rate-limiting enzymes [retrieved from the Rate-Limiting Enzymes database (RLEdb); ref. 7] in glucose and lipid metabolic pathways. In addition, we examined the mRNA expression levels of important lipid binding proteins and transporters as well as components of the mitochondrial electron transport chain and oxidative phosphorylation. question What are the major conclusions reached by the author(s)?
 How do these results tie in with the current understanding of obesity and type II diabetes? What additional experiments do these results suggest to you? (This should be well thought out and at least 3 sentences) • Show less

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