Project description:There is increased interest in the potential protective role of dietary Ca in the development of metabolic disorders related to the metabolic syndrome. Ca-induced intestinal precipitation of fatty acids and bile acids as well as systemic metabolic effects of Ca on adipose tissue is proposed to play a causal role. In this experiment, we have studied all these aspects to validate the suggested protective effect of Ca supplementation, independent of other dietary changes, on the development of diet-induced obesity and insulin resistance. In our diet intervention study, C57BL/6J mice were fed high-fat diets differing in Ca concentrations (50 v. 150 mmol/kg). Faecal excretion analyses showed an elevated precipitation of intestinal fatty acids (2·3-fold; P < 0·01) and bile acids (2-fold; P < 0·01) on the high-Ca diet. However, this only led to a slight reduction in fat absorption (from 98 to 95 %; P < 0·01), mainly in the distal small intestine as indicated by gene expression changes. We found no effect on body-weight gain. Lipolysis and lipogenesis-related parameters in adipose tissue also showed no significant changes on the high-Ca diet, indicating no systemic effects of dietary Ca on adiposity. Furthermore, early gene expression changes of intestinal signaling molecules predicted no protective effect of dietary Ca on the development of insulin resistance, which was confirmed by equal values for insulin sensitivity on both diets. Taken together, our data do not support the proposed protective effect of dietary Ca on the development of obesity and/or insulin resistance, despite a significant increase in fecal excretion of fatty acids and bile acids. Keywords: Diet intervention study Nine-week-old mice were fed a high fat purified diet with a low calcium concentration of 50mmol/kg (LCa diet) or a high calcium concentration of 150mmol/kg (HCa diet) for 8 weeks. Body weight was recorded weekly and after 7 weeks of diet intervention an oral glucose tolerance test was performed. For microarray analysis, after 2 weeks of diet intervention, 6 mice per diet group were anaesthetized with a mixture of isofluorane (1.5%), nitrous oxide (70%) and oxygen (30%) and the small intestines were excised. Adhering fat and pancreatic tissue were carefully removed. The small intestines were divided in three equal parts along the proximal to distal axis (SI 1, SI 2 and SI 3) and microarray analysis was performed on pooled mucosal scrapings.

Project description:<p>The GOLDN study was initiated to assess how genetic factors interact with environmental (diet and drug) interventions to influence blood levels of triglycerides and other atherogenic lipid species and inflammation markers (registered at <a href="http://clinicaltrials.gov/ct2/show/NCT00083369">clinicaltrails.gov</a>, number NCT00083369). The study recruited Caucasian participants primarily from three-generational pedigrees from two NHLBI Family Heart Study (FHS) field centers (Minneapolis, MN and Salt Lake City, UT). Only families with at least two siblings were recruited and only participants who did not take lipid-lowering agents (pharmaceuticals or nutraceuticals) for at least 4 weeks prior to the initial visit were included. A total of 1048 GOLDN participants were included in the diet intervention. The diet intervention followed the protocol of Patsch et al. (<a href="http://www.ncbi.nlm.nih.gov/pubmed/1420093">1992</a>). The whipping cream (83% fat) meal had 700 Calories/m2 body surface area (2.93 MJ/m2 body surface area): 3% of calories were derived from protein (instant nonfat dry milk) and 14% from carbohydrate (sugar). The ratio of polyunsaturated to saturated fat was 0.06 and the cholesterol content of the average meal was 240 mg. The mixture was blended with ice and flavorings. Blood samples were drawn immediately before (fasting) and at 3.5 and 6 hours after consuming the high-fat meal. For the GOLDN lipidomics study, sterols and fatty acids were measured from stored plasma (-80 degrees Celsius) collected at fasting and 3.5 hours after the diet intervention using TrueMass Panels from Lipomics (West Sacramento, CA). A total of 11 sterols were quantified in nmols/gram of sample including total cholesterol, 7-dehydrocholesterol, desmosterol, lanosterol, lathasterol, cholestanol, coprostanol, beta-sitosterol, campesterol, stigmasterol, and 7alpha-hydroxycholesterol. A total of 35 fatty acids were quantified in nmols/gram of sample inlcuding myristic acid (14:0); pentadecanoic acid (15:0); palmitic acid (16:0); stearic acid (18:0); arachidic acid (20:0); behenic acid (22:0); lignoceric acid (24:0); myristoleic acid (14:1n5); palmitoleic acid (16:1n7); palmitelaidic acid (t16:1n7); oleic acid (18:1n9); elaidic acid (t18:1n9); vaccenic acid (18:1n7); linoleic acid (18:2n6); gamma-linolenic acid (18:3n6); alpha-linolenic acid (18:3n3); stearidonic acid (18:4n3); eicosenoic acid (20:1n9); eicosadienoic acid (20:2n6); mead acid (20:3n9); di-homo-gamma-linolenic acid (20:3n6); arachidonic acid (20:4n6); eicsoatetraenoic acid (20:4n3); eicosapentaenoic acid (20:5n3); erucic acid (22:1n9); docosadienoic acid (22:2n6); adrenic acid (22:4n6); docosapentaenoic acid (22:5n6); docosapentaenoic acid (22:5n3); docosahexaenoic acid (22:6n3); nervonic acid (24:1n9); and plasmalogen derivatives of 16:0, 18:0, 18:1n9, and 18:1n7.</p>

Project description:Headline: application of the oligo-microarray to the analysis of genes modulated by SDF1α chemokine in the human circulating hematopoietic CD34+ progenitors. Summary: 1/ Introduction: The microarray analysis simultaneously explores the expression of thousands messager ribonucleic acids in the cell. The traditional method by reverse transcription requires 50 to 100 micrograms of total initial ribonucleic acids. The human circulating hematopoietic CD34+ progenitors are rare and expressed very few messager ribonucleic acids. SDF1α is a chemokine which binds a specific transmembrane receptor, the CXCR4, expressed on the human circulating hematopoietic CD34+ progenitors. A chemical gradient of SDF1α in the medullary micro-environment is implied in the regulation of the trans-endothelial migration of the hematopoietic CD34+ progenitors who leave bone marrow and then migrate towards peripheral blood. Apart from its role in the cellular mobilization, SDF1α also takes part in survival maintenance, cell cycle promotion and proliferation of hematopoietic progenitors. 2/ objectives: The objective of this work was to apply this technology to the differential analysis of genes modulated by SDF1α chemokine in the human circulating hematopoietic CD34+ progenitors. 3/ Material and methods: With regard to the biological application, CD34+ cells were incubated during sixteen hours in the presence or absence of SDF1α at the concentration of 0,5 ng/ml. For this, four experiments were carried out with CD34+ cells purified from the mononuclear cells obtained from blood of six to eight unmobilized healthy subjects. The total ribonucleic acids of untreated cells from the four experiments were pooled to synthesize a “control” microarray probe. In the same way, the total ribonucleic acids of SDF1α treated cells were used to synthesize a “SDF1α treated” microarray probe. The differential hybridization of an oligo-microarray slide (10 000 genes) was carried out simultaneously with the “control” and “SDF1α treated” probes. The bioinformatics studies and the use of classification algorithms on the results of this microarray analysis made it possible to classify the differential transcripts in intracellular functional groups. 4/ Results an discussion: Our results showed that a double linear amplification with T7 RNA polymerase of complementary retro-transcript desoxyribonucleic acids from total ribonucleic acids of human circulating hematopoietic CD34+ progenitor was necessary to obtain the probe quantity for hybridize an oligo-microarray. Indirect cyanin incorporation of the aminoallyl type during the last stage of in vitro transcription optimized the probe labelling and reduced the experimental skew during the microarray analysis. We showed that under our experimental conditions, SDF1α acted on the expression of genes mainly implied in cell cycle (mitosis induction and proliferation) and cell metabolism (energy and translation). Our microarray results, which were confirmed by quantitative RT-PCR validate the biological ones previously obtained and help us to a better understanding of the intimate regulation mechanism of gene expression modulated by SDF1α. Key words: oligo-microarray, optimization, hematopoietic CD34+ progenitors, chemokine, SDF1α, genomic, transcriptome, cell cycle, quantitative RT-PCR, bioinformatics, principal component analysis, clustering. Keywords: oligo-microarray, optimization, hematopoietic CD34+ progenitors, chemokine, SDF1α, genomic, transcriptome, cell cycle, quantitative RT-PCR, bioinformatics, principal component analysis, clustering.

Dataset Information

E-GEOD-62994

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