Project description:Caloric Restriction in Leptin Deficiency Worsens Myocardial Steatosis: Failure to Upregulate PPAR gamma and Thermogenic Glyecrolipid/Fatty Acid Cycling Growing evidence supports an anti-lipotoxic role for leptin in preventing inappropriate peripheral tissue lipid deposition. Obese, leptin deficient ob/ob mice develop left ventricular (LV) hypertrophy and myocardial steatosis with increased apoptosis and decreased longevity. Here we investigated the cardiac effects of caloric restriction in leptin deficiency. Echocardiography was performed on C57Bl/6 wild-type mice (WT) and 7-month-old ob/ob mice fed ad lib, leptin-repleted (LR-ob/ob), or calorie-restricted (CR-ob/ob) for four weeks. Ventricular tissue was examined by electron microscopy (EM), mitochondrial coupling assay, and microarray expression profiling. LR and CR-ob/ob mice showed decreased body weight, heart weight, and LV wall thickness compared to ad lib ob/ob mice. LV fractional shortening was decreased in ad lib ob/ob mice, but restored to WT levels in LR and CR groups. However, EM revealed severe cardiac steatosis in the CR-ob/ob group compared to only moderate steatosis in ad lib ob/ob . Despite marked cardiac steatosis, CR (like LR) restored mitochondrial coupling to WT levels. CR up-regulated genes associated with oxidative stress and cell death, changes suggestive of cardiac lipotoxicity. LR, but not CR was shown to induce core genes involved in glycerolipid/free fatty acid cycling, a highly thermogenic pathway that can reduce intracellular lipid stores. LR, but not CR up-regulated and restored PGC1 and PPARto wild type levels; CR paradoxically further suppressed cardiac PPAR. Thus, leptin is essential in protecting the heart from lipotoxicity, and the inability to up-regulate the thermogenic glycerolipid/free fatty acid cycling pathway may impair the response of leptin deficient animals to the lipotoxic stress of calorie restriction. 6 month aged ob/ob mice were either leptin repleted with osmotic mini-pumps, calorie restricted to match the caloric intake of the leptin repleted mice, or fed ad lib for one month. 6-8 month C57Bl/6J mice were aged to serve as controls.

Project description:Gallstone disease is a major contributor to health care costs in the United States. Approximately 12 % of the U.S. population has gallstones. As a result, more than 700,000 cholecystectomies are performed in this country each year. Many of these patients are obese and have a positive family history; but surprisingly, little is known about the link between obesity, genetics and gallstone formation. Obese individuals have been shown to have supersaturated bile, larger gallbladder fasting volumes and impaired gallbladder emptying. We have recently demonstrated that leptin plays a role in gallbladder motility. In an effort to understand the genetic basis for these observations, we tested the hypothesis that leptin would alter gallbladder gene expression. Methods: Affymetrix oligonucleotide microarrays 430 2.0 were used to compare gallbladder gene expression profiles from 12 week old control saline-treated leptin-deficient (Lep ob) and from leptin-treated Lep ob female mice. Analyses were performed on pooled RNA (n=4) from the gallbladders of 12 saline-treated Lep ob mice and from 12 Lep ob mice which were administered daily IP 5 ug/g of recombinant murine leptin for 4 weeks. Resulting data were analyzed utilizing Gene Chip Operating Software or MAS 5.0. Results: Of the genes analyzed 314 were upregulated and 108 were downregulated by leptin administration. Numerous genes related to gallstone pathogenesis, gallbladder absorption/secretion, inflammatory cytokines, and insulin resistance were altered by leptin. Experiment Overall Design: Female B6.V-lepob obese mice (n=24) aged 7 weeks were obtained from The Jackson Laboratory (Bar Harbor, ME). Upon arrival, mice were placed on a standard chow diet and were allowed to acclimate for 1 week before starting the experiment. For leptin treatments, mice received daily intraperitoneal injections of either recombinant mouse leptin (R&D Systems, Minneapolis, MN) (n=12) at a dose of 5 µg/g body weight or with saline as a control (n=12) for 4 weeks. At 12 weeks of age, mice were fasted overnight with free access to water. The following morning the mice underwent cholecystectomy. Three gallbladders were pooled to create 4 pools in each treatment and total RNA was isolated. Affymetrix murine 430 2.0 arrays were utilized to examine altered gallbladder gene expression as a result of leptin administration. The resulting data was analyzed utlizing Gene Chip Operating Software or MAS 5.0.

Project description:Exposure to environmental contaminants can disrupt normal development of the early vertebrate skeleton. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) impairs craniofacial skeletal development across many vertebrate species and its effects are especially prominent in early life stages of fish. TCDD activates the aryl hydrocarbon receptor (AHR), a transcription factor that mediates most if not all TCDD responses. We investigated the transcriptional response in the developing zebrafish jaw following TCDD exposure using DNA microarrays. Zebrafish larvae were exposed to TCDD at 96 h postfertilization (hpf) and jaw cartilage tissue was harvested for microarray analysis at 1, 2, 4 and 12 h postexposure (hpe). Numerous chondrogenic transcripts were misregulated by TCDD in the jaw. Comparison of transcripts altered by TCDD in jaw with transcripts altered in embryonic heart showed that the transcriptional responses in the jaw and the heart were strikingly different. Sox9b, a critical chondrogenic transcription factor, was the most significantly reduced transcript in the jaw. We hypothesized that the TCDD reduction of sox9b expression plays an integral role in affecting formation of the embryonic jaw. Morpholino knock down of sox9b expression demonstrated that partial reduction of sox9b expression alone was sufficient to produce a TCDD-like jaw phenotype. Heterozygous sox9b deletion mutant embryos were sensitized to TCDD. Lastly, embryos injected with sox9b mRNA and then exposed to TCDD blocked TCDD-induced jaw toxicity in approximately 14% of sox9b-injected embryos. These results suggest that reduced sox9b expression in TCDD-exposed zebrafish embryos contributes to jaw malformation. Experiment Overall Design: Three independent replicate microarray time course experiments were performed comparing transcript levels between TCDD-exposed and control zebrafish. For each experiment, zebrafish were exposed to TCDD for 1 h starting at 96 hpf as described above. For each time point (97, 98, 100 and 108 hpf) and treatment jaw samples were pooled from 10 dissections for RNA isolation and hybridization with Affymetrix zebrafish arrays (Affymetrix, Santa Clara, CA). Each microarray contains roughly 14,900 probes corresponding to approximately 30% of the zebrafish genome. For each array, total RNA (1 Âµg) was isolated from 10 jaw microdissections with the QIAGEN RNeasy Mini kit following the manufacturerâs protocol (QIAGEN, Valencia, CA). The One-Cycle Target Labeling and Control Reagents kit was used to synthesize cDNA and biotinylated cRNA following the manufacturerâs protocol (Affymetrix, Santa Clara, CA). Biotin-labeled cRNA (15 Âµg) was fragmented and hybridized onto Affymetrix Zebrafish Genechip Arrays following the protocol in the Affymetrix Genechip Expression Analysis Technical Manual. Following hybdrization, the arrays were washed and stained with streptavidin-phycoerythrin on an Affymetrix Fluidics Station 400 using the protocol EukGE WS2v4. Arrays were scanned with an Agilent Gene Array Scanner.

Project description:Ambient temperature affects organisms comprehensively, however cold responses are different among tissues. Here, we adopt a transcript screening approach to explore and compare the cold responses in zebrafish gills and brain. Zebrafish were exposed to cold and the oligonucleotide-based microarray was used to identify cold-induced genes. Principle component analysis (PCA) of the gene expression profiles indicated that gills develop different strategies for the increasing of exposure period while brain relatively remained stable. Combining statistic and clustering methods, we found that gills showed higher protein metabolism and cell activity while brain showed higher stress responses and detoxification during cold acclimation. According to the microarray data sets, we extended the study on ionocyte- and isotocin neuron-related genes in gills and brain, respectively, and found these genes were broadly stimulated by cold. These data suggest that cold activates specific physiological functions in different tissues. Taken together, our results provide molecular evidences to elucidate the cold acclimation in zebrafish gills and brain. Keywords: Time course, Tissue types After the low-temperature treatment, brain and gill tissues dissected from 10 individuals were pooled as a sample and then homogenized in 0.8 ml Trizol reagent (Invitrogen, Carlsbad, CA). 120 individuals (60 for low-temperature treatment and 60 for control) were sacrificed for each microarray hybridization experiment, and another 200 individuals were used for quantitative reverse-transcription polymerase chain reaction. After chloroform extraction, RNA precipitation and ethanol washing, the RNA samples were purified and treated with DNase1 to remove the genomic DNA by using RNeasy Mini Kit (Qiagen, Huntsville, Alabama). The quantity and quality of total RNA were assessed by spectrophotometry and Bioanalyzer (Aglient Technologies, Foster City, CA). The commercial zebrafish 14K oligonucleotides set (MWG Biotech AG, Ebersbach, Germany) were obtained and were printed on UltraGAPS Coated slide (Corning, New York, NY ) with use of the OmniGrid 100 microarrayer (Genomic Solutions, Ann Arbor, MI) according to the manufactureâ??s instructions. The 14,067 oligonucleotides represent 9666 genes (7009 singlet genes and 2657 redundant genes), and the redundancy of this chip is 31 %. The detailed description of the oligonucleotides information can be obtained on the Ocimun Biosolution website. cDNA probes were synthesized and purified by reverse transcription of 20 Î¼g total RNA using a SuperScript indirect cDNA labeling system (Invitrogen) and MinElute PCR purification kit (Qiagen) and were labeled with Alexa 647 dye (cold treatment groups) and Alexa 555 dye (control groups)(Invitrogen), respectively. The zebrafish 14K OciChip array chip was pretreated with 1% bovine serum albumin (BSA) (fraction V), 4x saline-sodium citrate (SSC), and 1% sodium dodecylsulfate (SDS) at 42 Â°C for 45 min, and then hybridized overnight in a cocktail containing 5x Denhardt's solution, 6x SSC, 0.5% SDS, 50% formamide, 50 mM sodium phosphate, and 2 Âµg/Âµl yeast tRNA. Slides were washed with 2x SSC and 0.1% SDS (5 min), 1x SSC and 0.1% SDS (5 min), 0.5x SSC (5 min), and twice with 0.1x SSC (2 min each). Scanning was performed with a Genepix scanner (Molecular Devices, Sunnyvale, CA). The acquired images were analyzed using Genepix and Genespring software (Aglient Technologies, Foster City, CA). The measurements of spots were filtered by flags, and the Lowess normalization was performed after subtraction of the median background. Each experiment contained 3 biological replicates (including 1 dye swap) with different samples. The differentially expressed genes were selected from those with at least 2 of 3 significant signals (ratio > 2 or < 0.5), and then the Significant Analysis of Microarray method (SAM 3.02) was used to determine statistical significances.

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Microarray transcriptional analysis of leptin signaling knockdown and rescue in 72-hour post fertilization zebrafish

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