[show abstract][hide abstract] ABSTRACT: Weight control diets favorably affect parameters of the metabolic syndrome and delay the onset of diabetic complications. The adaptations occurring in adipose tissue (AT) are likely to have a profound impact on the whole body response as AT is a key target of dietary intervention. Identification of environmental and individual factors controlling AT adaptation is therefore essential. Here, expression of 271 transcripts, selected for regulation according to obesity and weight changes, was determined in 515 individuals before, after 8-week low-calorie diet-induced weight loss, and after 26-week ad libitum weight maintenance diets. For 175 genes, opposite regulation was observed during calorie restriction and weight maintenance phases, independently of variations in body weight. Metabolism and immunity genes showed inverse profiles. During the dietary intervention, network-based analyses revealed strong interconnection between expression of genes involved in de novo lipogenesis and components of the metabolic syndrome. Sex had a marked influence on AT expression of 88 transcripts, which persisted during the entire dietary intervention and after control for fat mass. In women, the influence of body mass index on expression of a subset of genes persisted during the dietary intervention. Twenty-two genes revealed a metabolic syndrome signature common to men and women. Genetic control of AT gene expression by cis signals was observed for 46 genes. Dietary intervention, sex, and cis genetic variants independently controlled AT gene expression. These analyses help understanding the relative importance of environmental and individual factors that control the expression of human AT genes and therefore may foster strategies aimed at improving AT function in metabolic diseases.
[show abstract][hide abstract] ABSTRACT: Genomic DNA (gDNA) contamination is an inherent problem during RNA purification that can lead to non-specific amplification and aberrant results in reverse transcription quantitative PCR (RT-qPCR). Currently, there is no alternative to RT(-) controls to evaluate the impact of the gDNA background on RT-PCR data. We propose a novel method (ValidPrime) that is more accurate than traditional RT(-) controls to test qPCR assays with respect to their sensitivity toward gDNA. ValidPrime measures the gDNA contribution using an optimized gDNA-specific ValidPrime assay (VPA) and gDNA reference sample(s). The VPA, targeting a non-transcribed locus, is used to measure the gDNA contents in RT(+) samples and the gDNA reference is used to normalize for GOI-specific differences in gDNA sensitivity. We demonstrate that the RNA-derived component of the signal can be accurately estimated and deduced from the total signal. ValidPrime corrects with high precision for both exogenous (spiked) and endogenous gDNA, contributing ∼60% of the total signal, whereas substantially reducing the number of required qPCR control reactions. In conclusion, ValidPrime offers a cost-efficient alternative to RT(-) controls and accurately corrects for signals derived from gDNA in RT-qPCR.
Nucleic Acids Research 01/2012; 40(7):e51. · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: Glucocorticoids are frequently prescribed drugs with important side-effects such as glucose intolerance and tissue remodeling. The goal was to explore the molecular basis of the response of skeletal muscle and adipose tissue during a short-term dexamethasone treatment to better understand the induction of side-effects of glucocorticoids on these metabolic tissues. Fifteen healthy male subjects were assigned to a 4-day treatment with dexamethasone at 4 mg/day. The primary outcome measures were changes in gene expression profiling of subcutaneous skeletal muscle and adipose tissue. Urinary cortisol, plasma, and metabolic biochemistry were also assessed. In both tissues the prominent observation was a response to stress and increased inflammatory responses. An upregulation of the serum amyloid A was detected in skeletal muscle, adipose tissue, and plasma, whereas circulating levels of C reactive protein, another acute phase protein, decreased along with a worsened insulin sensitivity index. As tissue-specific features, tissue remodeling was shown in skeletal muscle while the adipose tissue exhibited a decreased energy metabolism. Several limitations might be raised due to the small number of subjects investigated: a possible cross talk with the mineralocorticoid receptor, and a single time point may not identify regulations occurring during longitudinal treatment. In line with the known physiological effect of glucocorticoids the early modulation of stress response genes was observed. An unexpected feature was the upregulation of the inflammatory and immune pathways. The identification of novel impact on two glucocorticoid target tissues provides a molecular basis for the design of more specific glucocorticoids devoid of adverse effects.