Dataset Information

Reference Gene Optimization for Circadian Gene Expression Analysis in Human Adipose Tissue.

ABSTRACT: A hallmark of biology is the cyclical nature of organismal physiology driven by networks of biological, including circadian, rhythms. Unsurprisingly, disruptions of the circadian rhythms through sleep curtailment or shift work have been connected through numerous studies to positive associations with obesity, insulin resistance, and diabetes. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) measures oscillation in messenger RNA expression, an essential foundation for the study of the physiological circadian regulatory network. Primarily, measured oscillations have involved the use of reference gene normalization. However, the validation and identification of suitable reference genes is a significant challenge across different biological systems. This study focuses on adipose tissue of premenopausal, otherwise healthy, morbidly obese women voluntarily enrolled after being scheduled for laparoscopic sleeve gastrectomy surgery. Acquisition of tissue was accomplished by aspiratory needle biopsies of subcutaneous adipose tissue 1 to 2 weeks prior to surgery and 12 to 13 weeks following surgery and an in-surgery scalpel-assisted excision of mesenteric adipose tissue. Each biopsy was sterile cultured ex vivo and serially collected every 4 h over approximately 36 h. The candidate reference genes that were tested were 18S rRNA, GAPDH, HPRT1, RPII, RPL13?, and YWHAZ. Three analytic tools were used to test suitability, and the candidate reference genes were used to measure oscillation in expression of a known circadian clock element (Dbp). No gene was deemed suitable as an individual reference gene control, which indicated that the optimal reference gene set was the geometrically averaged 3-gene panel composed of YWHAZ, RPL13?, and GAPDH. These methods can be employed to identify optimal reference genes in other systems.

SUBMITTER: White JM

PROVIDER: S-EPMC7409766 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Reference Gene Optimization for Circadian Gene Expression Analysis in Human Adipose Tissue.

White Jeremy M JM Piron Matthew J MJ Rangaraj Vittobai R VR Hanlon Erin C EC Cohen Ronald N RN Brady Matthew J MJ

Journal of biological rhythms 20191031 1

A hallmark of biology is the cyclical nature of organismal physiology driven by networks of biological, including circadian, rhythms. Unsurprisingly, disruptions of the circadian rhythms through sleep curtailment or shift work have been connected through numerous studies to positive associations with obesity, insulin resistance, and diabetes. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) measures oscillation in messenger RNA expression, an essential foundation for the st ...[more]

PMID: 31668115

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Project description:BackgroundAccumulation of visceral adipose tissue (VAT) is clearly associated with an increased risk of obesity-related diseases and all-cause mortality, whereas gluteal subcutaneous fat accumulation (g-SAT) is associated with a lower risk. The relative contribution, in term of cardiovascular risk, of abdominal subcutaneous adipose tissue (a-SAT) is still controversial with studies showing both a detrimental effect and a protective role. Animal and in vitro studies demonstrated that adipocytes from visceral and subcutaneous depots have distinct morphological, metabolic and functional characteristics. These regional differences have a key role in the pathogenesis of obesity-related diseases. There is recent evidence that differentiation between upper-body and lower-body adipose tissues might be under control of site-specific sets of developmental genes, such as Homebox (HOX) genes, a group of related genes that control the body plan of an embryo along the anterior-posterior axis. However, the possible heterogeneity between different subcutaneous regions has not been extensively investigated. Here we studied global mRNA expression in g-SAT and a-SAT with a microarray approach. RNA was isolated from g-SAT and a-SAT biopsy, from eight healthy subjects, and hybridized on RNA microarray chips in order to detect regional differences in gene expression.ResultsA total of 131 genes are significantly and differently (>1.5 fold change, p < 0.05) expressed in a-SAT and g-SAT. Expression profiling reveals significant differences in expression of several HOX genes. Interestingly, two molecular signature of visceral adipocyte lineage, homebox genes HOXA5 and NR2F1, are up-regulated in a-SAT versus g-SAT by a 2.5 fold change.ConclusionsOur study shows that g-SAT and a-SAT have distinct expression profiles. The finding of a different expression of HOX genes, fundamental during the embryo development, suggests an early regional differentiation of subcutaneous adipose depots. Moreover, the higher expression of HOXA5 and NR2F1, two molecular signatures of visceral adipocytes, in a-SAT suggests that this subcutaneous adipose depot could be more similar to VAT than g-SAT. Our data suggest that we should look at SAT as composed of distinct depots with possibly different impact in obesity associated metabolic complications.

Project description:BackgroundProtein expression studies based on the two major intra-abdominal human fat depots, the subcutaneous and the omental fat, can shed light into the mechanisms involved in obesity and its co-morbidities. Here we address, for the first time, the identification and validation of reference proteins for data standardization, which are essential for accurate comparison of protein levels in expression studies based on fat from obese and non-obese individuals.Methodology and findingsTo uncover adipose tissue proteins equally expressed either in omental and subcutaneous fat depots (study 1) or in omental fat from non-obese and obese individuals (study 2), we have reanalyzed our previously published data based on two-dimensional fluorescence difference gel electrophoresis. Twenty-four proteins (12 in study 1 and 12 in study 2) with similar expression levels in all conditions tested were selected and identified by mass spectrometry. Immunoblotting analysis was used to confirm in adipose tissue the expression pattern of the potential reference proteins and three proteins were validated: PARK7, ENOA and FAA. Western Blot analysis was also used to test customary loading control proteins. ENOA, PARK7 and the customary loading control protein Beta-actin showed steady expression profiles in fat from non-obese and obese individuals, whilst FAA maintained steady expression levels across paired omental and subcutaneous fat samples.ConclusionsENOA, PARK7 and Beta-actin are proper reference standards in obesity studies based on omental fat, whilst FAA is the best loading control for the comparative analysis of omental and subcutaneous adipose tissues either in obese and non-obese subjects. Neither customary loading control proteins GAPDH and TBB5 nor CALX are adequate standards in differential expression studies on adipose tissue. The use of the proposed reference proteins will facilitate the adequate analysis of proteins differentially expressed in the context of obesity, an aim difficult to achieve before this study.

Project description:Two types of adipose tissues, white (WAT) and brown (BAT) are found in mammals. Increasingly novel strategies are being proposed for the treatment of obesity and its associated complications by altering amount and/or activity of BAT using mouse models.The present study was designed to: (a) investigate the differential expression of genes in LACA mice subcutaneous WAT (sWAT) and BAT using mouse DNA microarray, (b) to compare mouse differential gene expression with previously published human data; to understand any inter- species differences between the two and (c) to make a comparative assessment with C57BL/6 mouse strain. In mouse microarray studies, over 7003, 1176 and 401 probe sets showed more than two-fold, five-fold and ten-fold change respectively in differential expression between murine BAT and WAT. Microarray data was validated using quantitative RT-PCR of key genes showing high expression in BAT (Fabp3, Ucp1, Slc27a1) and sWAT (Ms4a1, H2-Ob, Bank1) or showing relatively low expression in BAT (Pgk1, Cox6b1) and sWAT (Slc20a1, Cd74). Multi-omic pathway analysis was employed to understand possible links between the organisms. When murine two fold data was compared with published human BAT and sWAT data, 90 genes showed parallel differential expression in both mouse and human. Out of these 90 genes, 46 showed same pattern of differential expression whereas the pattern was opposite for the remaining 44 genes. Based on our microarray results and its comparison with human data, we were able to identify genes (targets) (a) which can be studied in mouse model systems to extrapolate results to human (b) where caution should be exercised before extrapolation of murine data to human.Our study provides evidence for inter species (mouse vs human) differences in differential gene expression between sWAT and BAT. Critical understanding of this data may help in development of novel ways to engineer one form of adipose tissue to another using murine model with focus on human.

Dataset Information

Reference Gene Optimization for Circadian Gene Expression Analysis in Human Adipose Tissue.

Publications

Reference Gene Optimization for Circadian Gene Expression Analysis in Human Adipose Tissue.

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