Polyunsaturated fatty acids of marine origin upregulate mitochondrial biogenesis and induce-beta oxidation in white fat

Aims/hypothesis Intake of n-3 polyunsaturated fatty acids reduces adipose tissue mass, preferentially in the abdomen. The more pronounced effect of marine-derived eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids on adiposity, compared with their precursor -linolenic acid, may be mediated by changes in gene expression and metabolism in white fat. Methods The effects of EPA/DHA concentrate (6% EPA, 51% DHA) admixed to form two types of high-fat diet were studied in C57BL/6J mice. Oligonucleotide microarrays, cDNA PCR subtraction and quantitative real-time RT-PCR were used to characterise gene expression. Mitochondrial proteins were quantified using immunoblots. Fatty acid oxidation and synthesis were measured in adipose tissue fragments. Results Expression screens revealed upregulation of genes for mitochondrial proteins, predominantly in epididymal fat when EPA/DHA concentrate was admixed to a semisynthetic high-fat diet rich in -linolenic acid. This was associated with a three-fold stimulation of the expression of genes encoding regulatory factors for mitochondrial biogenesis and oxidative metabolism (peroxisome proliferator-activated receptor gamma coactivator 1 alpha [Ppargc1a, also known as Pgc1] and nuclear respiratory factor-1 [Nrf1] respectively). Expression of genes for carnitine palmitoyltransferase 1A and fatty acid oxidation was increased in epididymal but not subcutaneous fat. In the former depot, lipogenesis was depressed. Similar changes in adipose gene expression were detected after replacement of as little as 15% of lipids in the composite high-fat diet with EPA/DHA concentrate, while the development of obesity was reduced. The expression of Ppargc1a and Nrf1 was also stimulated by n-3 polyunsaturated fatty acids in 3T3-L1 cells. Conclusions/interpretation The anti-adipogenic effect of EPA/DHA may involve a metabolic switch in adipocytes that includes enhancement of -oxidation and upregulation of mitochondrial biogenesi

Beta-carotene and the application of transcriptomics in risk-benefit evaluation of natural dietary components

Beta-carotene is a natural food component that is present in fruits and vegetables and is also used as a food colorant and a supplement. Beta-carotene is an anti-oxidant and a source of vitamin A. It is endowed with health beneficial properties, but a number of studies showed that with high intakes it may increase the risk for lung cancer in at risk individuals (heavy smokers, asbestos workers and alcohol users). To establish the window of benefit, it is necessary to identify early markers of effect and to obtain insight in the mechanism of action of beta-carotene, in the absence and presence of environmental risk factors. Genomics technologies are well suited to dissect the mechanisms of action and identify the markers of effect. Human cell lines can be used to analyse the effects of beta-carotene, but exposure studies with beta-carotene show that cell lines display a widely variant behaviour, which hampers translation to the in vivo situation in humans. Alternatively, animal studies can be used. Especially the ferret seems to be a good model, but little sequence information of this species is available. However, heterologous hybridization on human cDNA seems possible and provides and a new tool for molecular analysis of health effects of beta-caroten

Individual variation of adipose gene expression and identification of covariated genes by cDNA microarrays

Gene expression profiling through the application of microarrays provides comprehensive assessment of gene expression levels in a given tissue or cell population, as well as information on changes of gene expression in altered physiological or pathological situations. Microarrays are particularly suited to study interactions in the regulation of large numbers of different genes, since their expression is analyzed simultaneously. For improved understanding of the physiology of adipose tissue, and consequently obesity and diabetes, identification of covariability in gene expression was attempted by analysis of the individual variability of gene expression in subcutaneous white and brown fat of the Siberian dwarf hamster using microarrays containing 300 cDNA fragments of adipose genes. No sex-dependant variability in gene expression could be found, and overall individual variability was rather low, with more than 80% of clones showing a coefficient of variation lower than 30%. Uncoupling protein 1 (UCP1) displayed a high variability of gene expression in brown fat, which was negatively correlated with the gene expression of complement factor B (FactB), implying a possible functional relationshi

Individual variation of adipose gene expression and identification of covariated genes by cDNA microarrays

Gene expression profiling through the application of microarrays provides comprehensive assessment of gene expression levels in a given tissue or cell population, as well as information on changes of gene expression in altered physiological or pathological situations. Microarrays are particularly suited to study interactions in the regulation of large numbers of different genes, since their expression is analyzed simultaneously. For improved understanding of the physiology of adipose tissue, and consequently obesity and diabetes, identification of covariability in gene expression was attempted by analysis of the individual variability of gene expression in subcutaneous white and brown fat of the Siberian dwarf hamster using microarrays containing 300 cDNA fragments of adipose genes. No sex-dependant variability in gene expression could be found, and overall individual variability was rather low, with more than 80% of clones showing a coefficient of variation lower than 30%. Uncoupling protein 1 (UCP1) displayed a high variability of gene expression in brown fat, which was negatively correlated with the gene expression of complement factor B (FactB), implying a possible functional relationshi

The intraclass correlation coefficient applied for evaluation of data correction, labeling methods and rectal biopsy sampling in DNA microarray experiments

We show that the intraclass correlation coefficient (ICC) can be used as a relatively simple statistical measure to assess methodological and biological variation in DNA microarray analysis. The ICC is a measure that determines the reproducibility of a variable, which can easily be calculated from an ANOVA table. It is based on the assessment of both systematic deviation and random variation, and it facilitates comparison of multiple samples at once. We used the ICC first to optimize our microarray data normalization method and found that the use of median values instead of mean values improves data correction. Then the reproducibility of different labeling methods was evaluated, and labeling by indirect fluorescent dye incorporation appeared to be more reproducible than direct labeling. Finally, we determined optimal biopsy sampling by analyzing overall variation in gene expression. The variation in gene expression of rectal biopsies within persons decreased when two biopsies were taken instead of one, but it did not considerably improve when more than two biopsies were taken from one person, indicating that it is sufficient to use two biopsies per person for DNA microarray analysis under our experimental conditions. To optimize the accuracy of the microarray data, biopsies from at least six different persons should be used per grou

Factors influencing cDNA microarray hybridization on silylated glass slides

cDNA microarray technology is becoming the technique of choice for studying gene expression and gene expression patterns. Although experimental protocols are available, only limited methodological information on microarray manufacture, hybridization, and signal interpretation has been published. The aim of this paper is to provide more insight into the practical aspects of microarray construction and hybridization. The influence of the size, composition, and concentration of the spotted DNA fragments on the final hybridization signal and the effect of hybridization volume, sample concentration, and sample depletion have been tested and are discusse

Adipose gene expression patterns of weight gain suggest counteracting steroid hormone synthesis

VAN SCHOTHORST, EVERT M., NICOLE FRANSSEN-VAN HAL, MIRJAM M. SCHAAP, JEROEN PENNINGS, BARBARA HOEBEE, AND JAAP KEIJER. Adipose gene expression patterns of weight gain suggest counteracting steroid hormone synthesis. Obes Res. 2005;13:1031-1041. Objective: To identify early molecular changes in weight gain, using analysis of gene expression changes in adipose tissue of mice fed well-defined humanized (Western) high-fat and low-fat (control) diets during a short (3- to 5-week) time interval. Research Methods and Procedures: An adipose-enriched cDNA microarray was constructed and used for the expression analyses of visceral adipose tissues of wildtype young adult C57BL/6J male mice on different diets. Results: Mice on a high-fat diet had significantly higher body weight (at most, 9.6% greater) and adipose tissue weights compared with mice on a control diet. Gene expression analyses revealed 31 transcripts significantly differentially expressed in visceral adipose tissue between the diet groups. Most of these genes were expressed more on the high-fat diet. They mainly encode proteins involved in cellular structure (e.g., myosin, procollagen, vimentin) and lipid metabolism (e.g., leptin, lipoprotein lipase, carbonic anhydrase 3). This increase in gene expression was accompanied by a decrease in oxidative phosphorylation and carbohydrate metabolism (ATP citrate lyase). Importantly, genes belonging to steroid hormone biosynthesis (3 beta-hydroxysteroid dehydrogenase-1, cholesterol side-chain cleavage cytochrome P450, and steroid-11 beta-hydroxylase) were all expressed less in mice on a high-fat diet. Discussion: A short time period of 3 to 5 weeks of high-fat feeding altered gene expression patterns in visceral adipose tissue in male mice. Gene expression changes indicate initiation of adipose tissue enlargement and the down-regulation of adipose steroid hormone biosynthesis. The latter suggests a mechanism by which initial progression toward weight gain is counteracted

Differential gene expression in white and brown preadipocytes

White (WAT) and brown (BAT) adipose tissue are tissues of energy storage and energy dissipation, respectively. Experimental evidence suggests that brown and white preadipocytes are differentially determined, but so far not much is known about the genetic control of this determination process. The aim of this study was to identify differentially expressed genes involved in brown and white preadipocyte development. Using representational difference analysis (cDNA RDA) and DNA microarray screening, we identified four genes with higher expression in white preadipocytes (three different complement factors and -6 fatty acid desaturase) and seven genes with higher expression levels in brown preadipocytes, of which three are structural genes implicated in cell adhesion and cytoskeleton organization (fibronectin, -actinin-4, metargidin) and four that might function in gene transcription and protein synthesis (vigilin, necdin, snRNP polypeptide A, and a homolog to human hepatocellular carcinoma-associated protein). The expression profile of these genes was analyzed during preadipocyte differentiation, upon ß-adrenergic stimulation, and in WAT and BAT tissue in vivo compared with references genes such as peroxisome proliferator-activated receptor- (PPAR), uncoupling protein 1 (UCP1), cytochrome c oxidas

Differential gene expression in white and brown preadipocytes

White (WAT) and brown (BAT) adipose tissue are tissues of energy storage and energy dissipation, respectively. Experimental evidence suggests that brown and white preadipocytes are differentially determined, but so far not much is known about the genetic control of this determination process. The aim of this study was to identify differentially expressed genes involved in brown and white preadipocyte development. Using representational difference analysis (cDNA RDA) and DNA microarray screening, we identified four genes with higher expression in white preadipocytes (three different complement factors and -6 fatty acid desaturase) and seven genes with higher expression levels in brown preadipocytes, of which three are structural genes implicated in cell adhesion and cytoskeleton organization (fibronectin, -actinin-4, metargidin) and four that might function in gene transcription and protein synthesis (vigilin, necdin, snRNP polypeptide A, and a homolog to human hepatocellular carcinoma-associated protein). The expression profile of these genes was analyzed during preadipocyte differentiation, upon ß-adrenergic stimulation, and in WAT and BAT tissue in vivo compared with references genes such as peroxisome proliferator-activated receptor- (PPAR), uncoupling protein 1 (UCP1), cytochrome c oxidas