Transcription profiling by array of epididymal adipose tissue from male hemizygous fat-1(+/-) transgenic mice and their wild-type littermates, all fed with a high-fat diet
ABSTRACT: Dietary administration of n-3 polyunsaturated fatty acids (PUFA) is often associated with altered adipose tissue (AT) morphology and/or function in obese mice. However, it is unclear whether these observations are an indirect consequence of reduced weight gain or result from direct actions of n-3 PUFA. Here we studied the AT of fat-1 transgenic mice that convert endogenous n-6 to n-3 PUFA. These mice display equivalent weight gain and fat accretion to their wild-type (WT) counterparts. We performed Affymetrix microarray in epididymal AT of male hemizygous fat-1(+/-) transgenic mice and their wild type littermates that had been fed high fat diets from 6 weeks of age (diet-93075, 55% Kcal from fat, Harlan Teklad). Mice were sacrificed after 8 weeks on the HF diet. At sacrifice epididymal adipose tissues excised for the microarray study (n=3 per group) were rapidly homogenized in QIAZOL (QIAGEN) and snap frozen in liquid nitrogen.
Project description:Gene transcription in epididymal fat pads was investigated in an F2 cross of 129S6 x Balb/c mice using Illumina gene expression arrays. Expression data was determined in 5 months old male mice fed a high fat diet (40% fat) for 15 weeks.
Project description:Age-related and/or high caloric intake driven changes in visceral adipose tissue contribute to the development of diabetes and cardiovascular disease. The lack of reliable, high-throughput methods to analyze the adipose tissue protein composition has limited our understanding of the protein networks responsible for metabolic regulation of various adipose depots. We have developed a proteomic approach using multiple-dimension liquid chromatography tandem mass spectrometry and multiplexed TMT labeling to analyze protein composition of epididymal adipose tissues isolated from mice fed either low or high fat diet for a short (8 weeks) or a long (18 weeks) term, and from mice that aged (26 weeks old) on low vs. high fat diets. Advancing age and high-fat diet feeding led to graded deterioration, with long-term high fat diet exposure being the worst of all measures of peripheral metabolic health such as body weight, adiposity, plasma fasting glucose, insulin, triglycerides, total cholesterol levels, and glucose and insulin tolerance tests. Epididymal adipose depot proteomic analysis identified >3300 proteins per sample. In response to short-term high fat diet, 43 proteins representing lipid metabolism (e.g., AACS, ACOX1, ACLY) and red-ox pathways (e.g., CPD2, CYP2E, SOD3) were significantly altered (FDR < 10%). Long-term high fat diet significantly altered 55 proteins associated with immune response (e.g., IGTB2, IFIT3, LGALS1) and rennin angiotensin system (e.g. ENPEP, CMA1, CPA3, ANPEP). Age-related changes on low fat diet significantly altered only 18 proteins representing mainly urea cycle (e.g., OTC, ARG1, CPS1), and amino acid biosynthesis (e.g., GMT, AKR1C6). Surprisingly, high fat diet driven age-related changes culminated with alterations in 155 proteins involving primarily the urea cycle (e.g., ARG1, CPS1), immune response/complement activation (e.g., C3, C4b, C8, C9, CFB, CFH, FGA), extracellular remodeling (e.g., EFEMP1, FBN1, FBN2, LTBP4, FERMT2, ECM1, EMILIN2, ITIH3) and apoptosis (e.g., YAP1, HIP1, NDRG1, PRKCD, MUL1) pathways. Our unbiased mass spectrometry method, tailored to adipose tissue, identified both age-related and high fat diet specific proteomic signatures. In addition to well-described immune and extracellular remodeling response to high fat feeding, we have uncovered the pronounced involvement of arginine metabolism in response to advancing age, and branched chain amino acid metabolism in early response to high fat feeding. We were able to uncouple age-related metabolic responses from those associated with caloric intake, a task otherwise difficult to achieve.
Project description:Prolonged intervention studies investigating molecular metabolism are necessary for a deeper understanding of dietary effects on health. Here we provide mechanistic information about metabolic adaptation to fat-rich diets. Healthy men ingested saturated (SFA) or poly unsaturated (PUFA) fat-rich diets for six weeks during weight maintenance. Hyperinsulinemic clamps combined with leg balance technique revealed unchanged peripheral insulin sensitivity, independent of fatty acid type. Both diets increased fat oxidation potential in muscle. Hepatic insulin clearance increased, while glucose production, de novo lipogenesis and plasma triacylglycerol decreased. High fat intake changed the plasma proteome in immune-supporting direction and the gut microbiome displayed changes at taxonomical and functional level with PUFA. In mice, eucaloric feeding of human PUFA and SFA diets lowered hepatic triacylglycerol content compared to low-fat fed control mice, and induced adaptations in the liver supportive of decreased gluconeogenesis and lipogenesis. Intake of fat-rich diets thus induces extensive metabolic adaptations enabling disposition of dietary fat without metabolic complications.
Project description:Diet-induced obesity is reported to induce a phenotypic switch in adipose tissue macrophages from an antiinflammatory M2 state to a proinflammatory M1 state. Telmisartan, an angiotensin II type 1 receptor antagonist and a peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist, reportedly has beneficial effects on insulin sensitivity. We studied the effects of telmisartan on the adipose tissue macrophage phenotype in high fat-fed mice. Telmisartan was administered for 5 weeks to high fat-fed C57BL/6 mice. Insulin sensitivity, macrophage infiltration, and the gene expressions of M1 and M2 markers in epididymal fat tissues were examined. Insulin- or a glucose-tolerance test showed that telmisartan treatment improved insulin resistance, decreasing the body weight gain, visceral fat weight and adipocyte size without affecting the amount of food intake. Telmisartan treatment reduced the number of CD11c-positive cells and crown-like structures. Telmisartan reduced the mRNA expressions of M1 macrophage markers, such as TNF-alpha and IL-6, and increased the expression of M2 markers, such as IL-10 and Mgl2. The reduction of M1 macrophage markers, as well as the increased gene expression of M2 markers especially IL-10, is a possible mechanism for the improvement of insulin sensitivity by telmisartan. Six-week-old male C57BL/6J mice were purchased from CLEA Japan. The mice were fed a chow that contained 10% of its calories from fat (control) or a high-fat diet (HFD) that contained 30% of its calories from fat for 24 weeks. The high fat-fed mice were randomized to 3 groups. Either telmisartan (~3 mg/kg/day) in drinking water (HFD+Tel), candesartan (~3 mg/kg/day) in drinking water (HFD+Can), or a HFD without any drugs (HFD) was administered for the next 5 weeks. Two mice were treated per group. Epididymal adipose tissues were rapidly removed from each mouse. Gene expression in epididymal fat tissue was analyzed using a GeneChip® system with the Mouse Genome 430 2.0 Array, which was spotted with 45,101 probe sets (Affymetrix, Santa Clara, CA, USA). Sample preparation for the array hybridization was performed according to the manufacturer’s instructions. In short, 5 μg of total RNA was used to synthesize double-stranded cDNA using the GeneChip® Expression 3′-Amplification Reagents One-Cycle cDNA Synthesis Kit (Affymetrix). Biotin-labeled cRNA was then synthesized from the cDNA using GeneChip® Expression 3′-Amplification Reagents for IVT Labeling (Affymetrix). After fragmentation, the biotinylated cRNA was hybridized to arrays at 45 °C for 16 h. The arrays were washed, stained with streptavidin-phycoerythrin, and scanned using a probe array scanner. The scanned chip was analyzed using the GeneChip Analysis Suite software (Affymetrix). Hybridization intensity data were converted into a presence/absence call for each gene, and changes in gene expression between experiments were detected by a comparison analysis. Data was shown as the fold change relative to the expression level of normal chow-fed mice.
Project description:We identified differentially expressed genes in epididymal white adipose tissue of high fat diet(HFD)-fed mice compared to low fat diet-fed mice using microarray analysis. Microarray analysis revealed that genes related to lipolysis, fatty acid metabolism, mitochondrial energy transduction, oxidation-reduction, insulin sensitivity, and skeletal system development were downregulated in HFD-fed mice, and genes associated with extracellular matrix (ECM) components, ECM remodeling, and inflammation were upregulated. The top 10 up- or downregulated genes include Acsm3, mt-Nd6, Fam13a, Cyp2e1, Rgs1, and Gpnmb, whose roles in obesity-associated adipose tissue deterioration are poorly understood. Total RNA of epididymal white adipose tissue was obtained from low fat diet (10 kcal% fat)- and high fat diet(45 kcal% fat)-fed mice and mRNA expression was measured using microarray analysis.
Project description:We performed RNA-Seq for brwon fat, epididymal white fat and soleus muscle of mice to identify brown fat-selective, white fat-selective and common fat genes. RNA-Seq for brown fat, white fat and soleus muscle of wild type C56BL6 mice.
Project description:The beneficial effects of dietary long-chain (LC) n-3 polyunsaturated fatty acids (PUFA) in the prevention and/or treatment of some metabolic disorders result largely from their capacity to regulate the transcription level of many genes involved in metabolic and physiological homeostasis, especially in the liver. In this respect, they are known to bind and activate the Peroxisome Proliferator-Activated Receptor alpha (PPARalpha). The precursor of LC-PUFA, a-linoleic acid (ALA, C18:3 n-3) share some beneficial metabolic effects with its LC derivatives, however its role in gene regulation is poorly documented. Here, we analysed the hepatic transcriptome of mice fed for 5 weeks diets rich in either saturated FA from palm oil (PALM group) or ALA from linseed oil (LIN group). This modification of dietary fatty acid composition in a context of a high fat diet had a subtle but significant effect on the hepatic transcriptome. We identified mainly a group of genes that were upregulated in the LIN vs the PALM group and that include several well-known PPARalpha target genes involved in lipid and xenobiotic metabolism. Liver gene expression was measured in male C57BL/6J mice fed during 5 weeks a high fat diet (51% energy from fat) containing palm oil, rich in saturated fatty acids (n=10) or linseed oil, rich in 18:3 n-3 (n=8)
Project description:Transcriptome analysis of total RNA from C57BL/6 epididymal adipose tissue samples Exogenously administered glucocorticoids (GC) are used for the treatment of numerous pathologies, however their chronic usage is associated with alterations in lipid metabolism and adipose tissue function. The combination of chronic GC usage with consumption of a high fat diet poses an even greater risk for the development of adverse metabolic outcomes. We evaluated the effect of chronic GC usage in combination with a 45% high fat diet rich in n-6 polyunsaturated fatty acids (PUFA) on visceral adipose tissue gene expression in C57BL/6 male mice. Overall design: We analyzed epididymal adipose tissue from 8 male C57BL/6 mice using the Affymetrix GeneChip Mouse Gene 2.0 ST Array. Array data was processed by Affymetrix GeneChip Command Console (AGCG) Software and Affymetrix Expression Console Software. No techinical replicates were performed.
Project description:Single nucleotide polymorphisms in intron 1 of the fat mass and obesity-associated (FTO) gene were found to be associated with an increased risk of adult obesity. Enhanced FTO expression in mice leads to hyperphagia, increased fat mass, and higher body weight. Neuronal-specific FTO–deleted mice have an identical lean body weight phenotype to global FTO-deleted mice. The physiological role of adipose FTO in the homeostasis of energy regulation remains to be elucidated. We used microarrays to elucidate the metabolic pathways that are regulated by FTO in the white fat. FTO flox/flox and Adiponectin-cre FTO flox/flox (AFO) mice were fed with chow diet. White fat tissues from epididymal adipose pad were harvested under ad lib condition for RNA isolation. Three independent pools of FTO flox/flox and AFO mouse white fat RNA were included in the study.