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

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, RPL13α, 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, RPL13α, and GAPDH. These methods can be employed to identify optimal reference genes in other systems.

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Faculty Opinions recommendation of Human adipose-tissue derived stromal cells in combination with hypoxia effectively support ex vivo expansion of cord blood haematopoietic progenitors.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725450706.793507420 ◽

2015 ◽

Author(s):

Bjarte G Solheim

Keyword(s):

Adipose Tissue ◽

Cord Blood ◽

Stromal Cells ◽

Ex Vivo ◽

Human Adipose Tissue ◽

Ex Vivo Expansion

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Optimizations for identifying reference genes in bone and cartilage bioengineering

BMC Biotechnology ◽

10.1186/s12896-021-00685-8 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Fei Xiong ◽

Xiangyun Cheng ◽

Chao Zhang ◽

Roland Manfred Klar ◽

Tao He

Keyword(s):

Gene Expression ◽

Skeletal Muscle ◽

Adipose Tissue ◽

Real Time ◽

Reference Gene ◽

Muscle Tissue ◽

Reference Genes ◽

Target Genes ◽

The Stability ◽

And Cartilage

Abstract Background Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) remains one of the best-established techniques to assess gene expression patterns. However, appropriate reference gene(s) selection remains a critical and challenging subject in which inappropriate reference gene selction can distort results leading to false interpretations. To date, mixed opinions still exist in how to choose the most optimal reference gene sets in accodrance to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guideline. Therefore, the purpose of this study was to investigate which schemes were the most feasible for the identification of reference genes in a bone and cartilage bioengineering experimental setting. In this study, rat bone mesenchymal stem cells (rBMSCs), skeletal muscle tissue and adipose tissue were utilized, undergoing either chondrogenic or osteogenic induction, to investigate the optimal reference gene set identification scheme that would subsequently ensure stable and accurate interpretation of gene expression in bone and cartilage bioengineering. Results The stability and pairwise variance of eight candidate reference genes were analyzed using geNorm. The V0.15- vs. Vmin-based normalization scheme in rBMSCs had no significant effect on the eventual normalization of target genes. In terms of the muscle tissue, the results of the correlation of NF values between the V0.15 and Vmin schemes and the variance of target genes expression levels generated by these two schemes showed that different schemes do indeed have a significant effect on the eventual normalization of target genes. Three selection schemes were adopted in terms of the adipose tissue, including the three optimal reference genes (Opt3), V0.20 and Vmin schemes, and the analysis of NF values with eventual normalization of target genes showed that the different selection schemes also have a significant effect on the eventual normalization of target genes. Conclusions Based on these results, the proposed cut-off value of Vn/n + 1 under 0.15, according to the geNorm algorithm, should be considered with caution. For cell only experiments, at least rBMSCs, a Vn/n + 1 under 0.15 is sufficient in RT-qPCR studies. However, when using certain tissue types such as skeletal muscle and adipose tissue the minimum Vn/n + 1 should be used instead as this provides a far superior mode of generating accurate gene expression results. We thus recommended that when the stability and variation of a candidate reference genes in a specific study is unclear the minimum Vn/n + 1 should always be used as this ensures the best and most accurate gene expression value is achieved during RT-qPCR assays.

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Visceral and subcutaneous adipose tissue stearoyl-CoA desaturase-1 mRNA levels and fatty acid desaturation index positively correlate with BMI in morbidly obese women

European Journal of Lipid Science and Technology ◽

10.1002/ejlt.201400372 ◽

2015 ◽

Vol 117 (7) ◽

pp. 926-932 ◽

Cited By ~ 9

Author(s):

Adriana Mika ◽

Lukasz Kaska ◽

Justyna Korczynska ◽

Agnieszka Mirowska ◽

Piotr Stepnowski ◽

...

Keyword(s):

Adipose Tissue ◽

Fatty Acid ◽

Subcutaneous Adipose Tissue ◽

Fatty Acid Desaturation ◽

Morbidly Obese ◽

Mrna Levels ◽

Obese Women ◽

Desaturation Index ◽

Stearoyl Coa Desaturase ◽

Subcutaneous Adipose

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RANTES release by human adipose tissue in vivo and evidence for depot-specific differences

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.90511.2008 ◽

2009 ◽

Vol 296 (6) ◽

pp. E1262-E1268 ◽

Cited By ~ 42

Author(s):

Rana Madani ◽

Kalypso Karastergiou ◽

Nicola C. Ogston ◽

Nazar Miheisi ◽

Rahul Bhome ◽

...

Keyword(s):

Adipose Tissue ◽

Subcutaneous Adipose Tissue ◽

Epicardial Adipose Tissue ◽

Ex Vivo ◽

Human Adipose Tissue ◽

Fat Pad ◽

Obese Subjects ◽

Subcutaneous Adipose ◽

Circulating Levels

Obesity is associated with elevated inflammatory signals from various adipose tissue depots. This study aimed to evaluate release of regulated on activation, normal T cell expressed and secreted (RANTES) by human adipose tissue in vivo and ex vivo, in reference to monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) release. Arteriovenous differences of RANTES, MCP-1, and IL-6 were studied in vivo across the abdominal subcutaneous adipose tissue in healthy Caucasian subjects with a wide range of adiposity. Systemic levels and ex vivo RANTES release were studied in abdominal subcutaneous, gastric fat pad, and omental adipose tissue from morbidly obese bariatric surgery patients and in thoracic subcutaneous and epicardial adipose tissue from cardiac surgery patients without coronary artery disease. Arteriovenous studies confirmed in vivo RANTES and IL-6 release in adipose tissue of lean and obese subjects and release of MCP-1 in obesity. However, in vivo release of MCP-1 and RANTES, but not IL-6, was lower than circulating levels. Ex vivo release of RANTES was greater from the gastric fat pad compared with omental ( P = 0.01) and subcutaneous ( P = 0.001) tissue. Epicardial adipose tissue released less RANTES than thoracic subcutaneous adipose tissue in lean ( P = 0.04) but not obese subjects. Indexes of obesity correlated with epicardial RANTES but not with systemic RANTES or its release from other depots. In conclusion, RANTES is released by human subcutaneous adipose tissue in vivo and in varying amounts by other depots ex vivo. While it appears unlikely that the adipose organ contributes significantly to circulating levels, local implications of this chemokine deserve further investigation.

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Modulation of natriuretic peptide receptors in human adipose tissue: Molecular mechanisms behind the “natriuretic handicap” of morbidly obese patients

Nutrition Metabolism and Cardiovascular Diseases ◽

10.1016/j.numecd.2016.11.059 ◽

2017 ◽

Vol 27 (1) ◽

pp. e22

Author(s):

A. Gentili ◽

C. Orabona ◽

E. Albini ◽

M.R. Frangione ◽

M.A. Ricci ◽

...

Keyword(s):

Adipose Tissue ◽

Natriuretic Peptide ◽

Molecular Mechanisms ◽

Human Adipose Tissue ◽

Morbidly Obese ◽

Obese Patients ◽

Peptide Receptors ◽

Natriuretic Peptide Receptors

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Secretion of neuropeptide Y in human adipose tissue and its role in maintenance of adipose tissue mass

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00333.2007 ◽

2007 ◽

Vol 293 (5) ◽

pp. E1335-E1340 ◽

Cited By ~ 57

Author(s):

Katarina Kos ◽

Alison L. Harte ◽

Sean James ◽

David R. Snead ◽

Joseph P. O'Hare ◽

...

Keyword(s):

Adipose Tissue ◽

Protein Expression ◽

Elective Surgery ◽

Ex Vivo ◽

Human Adipose Tissue ◽

Tissue Mass ◽

Paracrine Effects ◽

Tnf Α ◽

Adiposity Signals

NPY is an important central orexigenic hormone, but little is known about its peripheral actions in human adipose tissue (AT) or its potential paracrine effects. Our objective was to examine NPY's role in AT, specifically addressing NPY protein expression, the effect of NPY on adipokine secretion, and the influence of insulin and rosiglitazone (RSG) on adipocyte-derived NPY in vitro. Ex vivo human AT was obtained from women undergoing elective surgery [age: 42.7 ± 1.5 yr (mean ± SE), BMI: 26.2 ± 0.7 kg/m2; n = 38]. Western blot analysis was used to determine NPY protein expression in AT depots. Abdominal subcutaneous (AbSc) adipocytes were isolated and treated with recombinant (rh) NPY, insulin, and RSG. NPY and adipokine levels were measured by ELISA. Our results were that NPY was localized in human AT and adipocytes and confirmed by immunohistochemistry. Depot-specific NPY expression was noted as highest in AbSc AT (1.87 ± 0.23 ODU) compared with omental (Om; 1.03 ± 0.15 ODU, P = 0.029) or thigh AT (Th; 1.0 ± 0.29 ODU, P = 0.035). Insulin increased NPY secretion (control: 0.22 ± 0.024 ng/ml; 1 nM insulin: 0.26 ± 0.05 ng/ml; 100 nM insulin: 0.29 ± 0.04 ng/ml; 1,000 nM insulin: 0.3 ± 0.04 ng/ml; P < 0.05, n = 13), but cotreatment of RSG (10 nM) with insulin (100 nM) had no effect on NPY secretion. Furthermore, adipocyte treatment with rh-NPY downregulated leptin secretion (control: 6.99 ± 0.89 ng/ml; 1 nmol/l rh-NPY: 4.4 ± 0.64 ng/ml; 10 nmol/l rh-NPY: 4.3 ± 0.61 ng/ml, 100 nmol/l rh-NPY: 4.2 ± 0.67 ng/ml; P < 0.05, n = 10) but had no effect on adiponectin or TNF-α secretion. We conclude that NPY is expressed and secreted by human adipocytes. NPY secretion is stimulated by insulin, but this increment was limited by cotreatment with RSG. NPY's antilipolytic action may promote an increase in adipocyte size in hyperinsulinemic conditions. Adipose-derived NPY mediates reduction of leptin secretion and may have implications for central feedback of adiposity signals.

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Early Effect of Bariatric Surgery on the Circadian Rhythms of Adipokines in Morbidly Obese Women

Metabolic Syndrome and Related Disorders ◽

10.1089/met.2015.0051 ◽

2016 ◽

Vol 14 (1) ◽

pp. 16-22 ◽

Cited By ~ 6

Author(s):

José Fabiano Costa Justus ◽

Antonio Carlos Ligocki Campos ◽

Ana Lucia C. Figueroa ◽

Ramon Gomis ◽

Marco Aurélio Santo ◽

...

Keyword(s):

Bariatric Surgery ◽

Circadian Rhythms ◽

Morbidly Obese ◽

Obese Women ◽

Early Effect

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Transduction of rat and human adipose-tissue derived mesenchymal stromal cells by adeno-associated viral vector serotype DJ

Biology Open ◽

10.1242/bio.058461 ◽

2021 ◽

Author(s):

E.S. Zubkova ◽

I.B. Beloglazova ◽

E.I. Ratner ◽

D.T. Dyikanov ◽

K.V. Dergilev ◽

...

Keyword(s):

Adipose Tissue ◽

Cellular Response ◽

Viral Vectors ◽

Viral Vector ◽

Ex Vivo ◽

Human Adipose Tissue ◽

Cell Types ◽

Specific Cell ◽

Therapeutic Benefits ◽

Rat Adipose Tissue

Ex vivo, gene therapy is a powerful approach holding great promises for the treatment of both genetic and acquired diseases. Adeno-associated virus (AAV) vectors are safe and efficient delivery system for modification of mesenchymal stem cells (MSC) that could maximize their therapeutic benefits. Assessment to MSC viability and functional activity after infection with new AAV serotypes is necessary, due to AAV tropism to specific cell types. We infected human and rat adipose-tissue MSC with hybrid AAV-DJ serotype vectors carrying GFP and SCF genes. GFP expression from AAV-DJ was about 1.5-fold superior to that observed with AAV-2 and lasted for at least 21 days as was evaluated by flow cytometry and fluorescence microscopy. AAV-DJ proves to be suitable for the infection of rat and human MSC with a similar efficiency. Infected MSC were still viable however showing 25-30%. growth rate slowdown. Moreover, we found increase of SERPINB2 mRNA expression in human MSC whereas expression of other oxidative stress markers and extracellular matrix proteins was not affected. These results suggest that there is a differential cellular response in MSC infected with AAV viral vectors, which should be taken into account as it can affect the expected outcome for the therapeutic application.

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