Subcutaneous Adipose Tissue Samples Research Articles

Effect of genetic merit for backfat thickness and paternal breed on performance, carcass traits, and gene expression in subcutaneous adipose tissue of feedlot-finished steers

• Backfat thickness trait can be selected through genetic merit in Nellore and crossbred steers (½ Aberdeen Angus + ½ Nellore). • Animal performance is not affected by genetic merit for backfat thickness. • The expression of ACACA, ADIPOQ, FASN, PLIN 1, PPARG and SREBF1 genes in subcutaneous adipose tissue is not affected by genetic merit for backfat thickness. This study evaluated selection for backfat thickness based on genetic merit (GMB) in Nellore (N) and crossbred (½ Aberdeen Angus + ½ Nellore, AN) steers, as well as the effects of this selection on animal performance, carcass traits, and gene expression in subcutaneous adipose tissue (SAT). The animals were obtained by mating Nellore cows with Nellore or Aberdeen Angus bulls. Both paternal genetic groups were selected to have positive (+, high GMB) or negative (-, low GMB) expected progeny differences for backfat thickness. Seventy-six animals (14 N+, 20 N-, 20 AN+, and 22 AN-) were reared on pasture and finished in the feedlot. The animals were slaughtered, and SAT samples were collected to investigate the relative expression of the acetyl-CoA carboxylase alpha ( ACACA ), adiponectin, C1Q and collagen domain containing ( ADIPOQ ), fatty acid synthase ( FASN ), perilipin 1 ( PLIN1 ), peroxisome proliferator activated receptor gamma ( PPARG ), and sterol regulatory element binding protein ( SREBF1 ) genes. Carcass weight and yield, conformation, finishing score, fat distribution score, and measurements of backfat thickness and rib eye area were evaluated. There was no effect of GMB on performance or on the relative expression of the genes studied. High GMB steers had greater backfat thickness and higher finishing, conformation and fat distribution scores in the carcass than low GMB animals. The use of high GMB animals provides better carcass finishing without affecting performance. The greater fat deposition in carcasses of high GMB animals is not associated with the relative expression of the genes studied.

Uric acid is associated with morpho-functional adipose tissue markers in apparently healthy subjects

BackgroundStudies have focused on the search of novel biomarkers that allow to easily identify dysfunctional adipose tissue (AT). Uric acid (UA) could be produced and reabsorbed by AT. It has been suggested that the increases of UA concentrations participates in AT dysfunction. We investigated the association of UA with morpho-functional adipose tissue markers in apparently healthy subjects. MethodsForty apparently healthy individuals were included. Dietary habits and anthropometrical features were evaluated. Circulating concentrations of UA, adiponectin, leptin, and plasminogen activator inhibitor-1 (PAI-1) were quantified. Periumbilical subcutaneous AT samples were obtained and adipocyte number, adipocyte area, and macrophages content were assessed. ResultsThe present study included 40 healthy subjects (67% women) with an average age of 57 ± 9 y, BMI of 26 ± 4 (kg/m2). UA showed a significant association with the number and mean area of adipocytes, macrophages number, adiponectin, and PAI-1. Although UA was independently associated with the number and mean area of adipocytes, macrophages number, adiponectin into the adjusted multivariable model. ConclusionUA concentrations are associated with morpho-functional adipose tissue markers. Our results underscore the importance of UA as one earlier instigator of adipose tissue dysfunction in subjects without metabolic abnormalities.

Role of the Steroid Sulfate Uptake Transporter Soat (Slc10a6) in Adipose Tissue and 3T3-L1 Adipocytes.

In addition to the endocrine and paracrine systems, peripheral tissues such as gonads, skin, and adipose tissue are involved in the intracrine mechanisms responsible for the formation of sex steroids via the transformation of dehydroepiandrosterone and dehydroepiandrosterone sulfate (DHEA/DHEAS) into potent androgenic and estrogenic hormones. Numerous studies have examined the relationship between overweight, central obesity, and plasma levels of DHEA and DHEAS. The sodium-dependent organic anion transporter Soat (Slc10a6) is a plasma membrane uptake transporter for sulfated steroids. Significantly increased expression of Slc10a6 mRNA has been previously described in organs and tissues of lipopolysaccharide (LPS)-treated mice, including white adipose tissue. These findings suggest that Soat plays a role in the supply of steroids in peripheral target tissues. The present study aimed to investigate the expression of Soat in adipocytes and its role in adipogenesis. Soat expression was analyzed in mouse white intra-abdominal (WAT), subcutaneous (SAT), and brown (BAT) adipose tissue samples and in murine 3T3-L1 adipocytes. In addition, adipose tissue mass and size of the adipocytes were analyzed in wild-type and Slc10a6 −/− knockout mice. Soat expression was detected in mouse WAT, SAT, and BAT using immunofluorescence. The expression of Slc10a6 mRNA was significantly higher in 3T3-L1 adipocytes than that of preadipocytes and was significantly upregulated by exposure to lipopolysaccharide (LPS). Slc10a6 mRNA levels were also upregulated in the adipose tissue of LPS-treated mice. In Slc10a6 −/− knockout mice, adipocytes increased in size in the WAT and SAT of female mice and in the BAT of male mice, suggesting adipocyte hypertrophy. The serum levels of adiponectin, resistin, and leptin were comparable in wild-type and Slc10a6 −/− knockout mice. The treatment of 3T3-L1 adipocytes with DHEA significantly reduced lipid accumulation, while DHEAS did not have a significant effect. However, following LPS-induced Soat upregulation, DHEAS also significantly inhibited lipid accumulation in adipocytes. In conclusion, Soat-mediated import of DHEAS and other sulfated steroids could contribute to the complex pathways of sex steroid intracrinology in adipose tissues. Although in cell cultures the Soat-mediated uptake of DHEAS appears to reduce lipid accumulation, in Slc10a6 −/− knockout mice, the Soat deletion induced adipocyte hyperplasia through hitherto unknown mechanisms.

Macrophages in epicardial adipose tissue and serum NT-proBNP in patients with stable coronary artery disease

Coronary artery disease (CAD) is widely considered a chronic inflammatory disorder, and dysfunction of epicardial adipose tissue could be an important source of the inflammation. Amino-terminal fragment of pro-B-type natriuretic peptide (NT-proBNP) is a known marker of cardiovascular disorders of cardiac origin. Recent studies show that inflammatory stimuli may influence its secretion. Our purpose was to evaluate NT-proBNP serum concentration in relation to immune cell ratios in epicardial adipose tissue (EAT), and cytokine levels in the patients with stable CAD. Patients with stable CAD and heart failure classified into classes II–III, according to the New York Heart Association (NYHA) scale, scheduled for the coronary artery bypass graft (CABG) surgery, were recruited into the study (n = 10; 59.5 (53.0-65.0) y. o.; 50% males). The EAT and subcutaneous adipose tissue (SAT) specimens were harvested in the course of CABG surgery. Immunostaining with anti-CD68, anti-CD45, antiIL-1β and anti-TNFα monoclonal antibodies was performed to evaluate cell composition by differential counts per ten fields (400 magnification). Fasting venous blood was obtained from patients before CABG. Blood was centrifuged at 1500g, aliquots were collected and stored frozen at -40 °С until final analysis. Concentrations of NT-proBNP, IL-1β, IL-6, IL-10, TNFα were determined in serum samples by enzyme-linked immunosorbent assay (ELISA). We have found increased production of IL-1β and TNFα cytokines in EAT compared to SAT. Concentrations of NT-proBNP exceeded 125 pg/ml in 4 patients, and correlations between the CD68+macrophage counts in both EAT and SAT samples (rs= 0.762; p = 0.010 and rs= 0.835; p = 0.003, respectively). NT-proBNP levels showed positive relations with CD45+leukocyte counts (rs= 0.799; p = 0.006), and with IL-1β+cell numbers (rs= 0.705; p = 0.023) in EAT samples only. As for the serum biomarkers, NT-proBNP levels showed negative correlation with fasting glucose levels (rs= -0.684; p = 0.029), and positive correlation with serum IL-6 concentrations (rs= 0.891; p = 0.001). Increased serum concentrations of NT-proBNP in CAD patients correlate with accumulation of macrophages in EAT, which is associated with increased production of IL-1β in EAT and correlates with some metabolic parameters.

The impact of housing conditions on porcine mesenchymal stromal/stem cell populations differ between adipose tissue and skeletal muscle

Background. In pigs, the ratio between lean mass and fat mass in the carcass determines production efficiency and is strongly influenced by the number and size of cells in tissues. During growth, the increase in the number of cells results from the recruitment of different populations of multipotent mesenchymal stromal/stem cells (MSCs) residing in the tissues. We hypothesized that the impact of hygiene of housing conditions during growth on the proportions of MSCs in adipose tissue and skeletal muscle may differ between pigs with different residual feed intake (RFI), a measure of feed efficiency. Methods. At the age of 12 weeks, Large White pigs from two lines divergently selected for low and high RFI were housed in two contrasting hygiene conditions (good vs poor). After six weeks, pigs were slaughtered (n = 30; 5-9/group). Samples of subcutaneous adipose tissue and longissimus skeletal muscle were collected, and cells from the stromal vascular fraction (SVF), which includes mesenchymal stromal/stem cell populations, were isolated from each tissue. Adipose and muscle cell populations from the SVF were phenotyped by flow cytometry using antibodies that targeted different cell surface markers (CD45 to separate hematopoietic cells from MSCs; CD34, CD38, CD56 and CD140a to identify MSC populations with adipogenic and/or myogenic potential). Results. Adipose tissue and muscle shared some common MSC populations although MSC diversity was higher in muscle than in adipose tissue. In muscle, the CD45-CD56+CD34-CD140a+ and CD45-CD56+CD34+CD140a+ cell populations were abundant. Of these two cell populations, only the proportions of CD45-CD56+CD34+CD140a+ cells increased (P ≤ 0.05) in pigs housed in poor hygiene as compared with pigs in good hygiene conditions. For the CD45-CD56-CD34- cell population, present in low proportion, there was an interaction between hygiene condition and genetic line (P < 0.05) with a decrease in low RFI pigs housed in poor hygiene conditions. In adipose tissue, the two abundant MSC populations were CD45-CD56-CD34- and CD45-CD56+CD34-. The proportion of CD45-CD56-CD34- cells increased (P < 0.05) whereas the proportion of CD45-CD56+CD34- tended to decrease (P < 0.1) in pigs housed in poor conditions. This study shows that the proportions of some MSC populations were affected by hygiene of housing conditions in a tissue-dependent manner in pigs of both RFI lines. It suggests that MSCs may play a significant role in adipose tissue and skeletal muscle homeostasis and may influence later growth and body composition in growing animals.

Evaluation of Browning Markers in Subcutaneous Adipose Tissue of Newly Diagnosed Gastrointestinal Cancer Patients with and without Cachexia.

Simple SummaryCachexia occurs frequently in cancer patients with deep metabolic derangements. The browning of adipose tissue promotes thermogenesis and energy expenditure and, in cancer, has been considered a major determinant of adipose tissue atrophy. We evaluated the molecular phenotype of this phenomenon in the subcutaneous adipose tissue (SAT) of newly diagnosed gastrointestinal cancer patients compared to controls. We observed that the modulation of different markers of the browning of SAT in gastrointestinal cancer and, in particular, pancreatic cancer showed significant changes in UCP1 and PGC1α; PGC1α was highly expressed in cachectic patients. Our study highlights the relevance of browning in patients with cancer, in particular in those with pancreatic cancer. Understanding the browning phenomenon may allow us to counteract these metabolic alterations before the development of severe cachexia, which is characterized by deep adipose and muscle depletion, negatively affecting survival and quality of life.We assessed the molecular phenotype of the browning of white adipose tissue in newly diagnosed cancer patients and controls undergoing surgery for gastrointestinal tumors and for non-malignant diseases, respectively. We collected subcutaneous adipose tissue (SAT) samples and using RT-PCR, we analyzed the expression of markers of browning and using Western blot the protein levels of UCP1 and PGC1α. The Ucp1 mRNA levels were lower in cancer patients vs. controls (p = 0.01), whereas Cidea and Tmem26 mRNA levels were higher in cancer patients. We found higher PGC1α protein levels in patients vs. controls, while no differences were seen for UCP1. The Ucp1 expression was lower in cachectic and non-cachectic patients vs. controls, whereas Cidea expression was higher in cachectic and non-cachectic patients vs. controls. Pgc1α mRNA levels were higher in cachectic vs. non-cachectic patients (p = 0.03) vs. controls (p = 0.016). According to type of tumors, we did not observe differences in Cidea expression, whereas Pgc1α was higher in pancreatic cancer vs. colorectal and vs. controls. We observed the lower expression of Ucp1 in pancreatic and colorectal cancer vs. controls. We documented higher UCP1 protein levels in pancreatic cancer patients vs. colorectal (p = 0.002) and vs. controls (p = 0.031). PGC1α protein levels were higher in pancreatic cancer patients vs. controls. Different markers of the browning of SAT are modulated, and pancreatic cancer showed changes in UCP1 and PGC1α; PGC1α was highly expressed in cachectic patients, with clinical implications that should be further clarified.

NUCB2/Nesfatin-1 Reduces Obesogenic Diet Induced Inflammation in Mice Subcutaneous White Adipose Tissue.

Background: Excess adipose tissue accumulation and obesity are characterised by chronic, low-grade, systemic inflammation. Nestfatin-1 is a neuropeptide derived from the precursor protein nucleobindin-2 (NUCB2), which was initially reported to exert anorexigenic effects. The present study aimed to investigate the effects of an obesogenic diet (OD; high-fat, high-sugar) in NUCB2 knockout (KO) mice and of nesfatin-1 treatment in LPS-stimulated 3T3-L1 preadipocytes. Methods: Subcutaneous white adipose tissue (Sc-WAT) samples from wild type (WT) and NUCB2 KO mice that were fed a normal diet (ND), or the OD for 12 weeks were used for RNA and protein extraction, as well as immunohistochemistry. 3T3-L1 cells were treated with 100 nM nesfatin-1 during differentiation and stimulated with 1 µg/mL LPS for measuring the expression and secretion of pro-inflammatory mediators by qPCR, western blotting, immunofluorescence, Bioplex, and ELISA. Results: Following the OD, the mRNA, protein and cellular expression of pro-inflammatory mediators (Tnfα, Il-6, Il-1β, Adgre1, Mcp1, TLR4, Hmbgb1 and NF-kB) significantly increased in the ScWAT of NUCB2 KO mice compared to ND controls. Adiponectin and Nrf2 expression significantly decreased in the ScWAT of OD-fed NUCB2 KO, without changes in the OD-fed WT mice. Furthermore, nesfatin-1 treatment in LPS-stimulated 3T3-L1 cells significantly reduced the expression and secretion of pro-inflammatory cytokines (Tnfα, Il-6, Il-1β, Mcp1) and hmgb1. Conclusion: An obesogenic diet can induce significant inflammation in the ScWAT of NUCB2 KO mice, involving the HMGB1, NRF2 and NF-kB pathways, while nesfatin-1 reduces the pro-inflammatory response in LPS-stimulated 3T3-L1 cells. These findings provide a novel insight into the metabolic regulation of inflammation in WAT.

ABCA1 and ABCG1 DNA methylation in epicardial adipose tissue of patients with coronary artery disease

BackgroundRecent studies have focused on the potential role of epicardial adipose tissue (EAT) in the development of coronary artery disease (CAD). ABCA1 and ABCG1 transporters regulate cell cholesterol content and reverse cholesterol transport. We aimed to determine whether DNA methylation and mRNA levels of the ABCA1 and ABCG1 genes in EAT and subcutaneous adipose tissue (SAT) were associated with CAD.MethodsPaired EAT and SAT samples were collected from 82 patients undergoing elective cardiac surgery either for coronary artery bypass grafting (CAD group, N = 66) or valve surgery (NCAD group, N = 16). ABCA1 and ABCG1 mRNA levels in EAT and SAT samples were analyzed using real time polymerase chain reaction, ABCA1 protein levels in EAT samples were assessed by western blotting. ABCA1 and ABCG1 DNA methylation analysis was performed in 24 samples from the CAD group and 9 samples from the NCAD group via pyrosequencing.ResultsDNA methylation levels in the ABCA1 promoter and ABCG1 cg27243685 and cg06500161 CpG sites were higher in EAT samples from patients with CAD compared with NCAD (21.92% vs 10.81%, p = 0.003; 71.51% vs 68.42%, p = 0.024; 46.11% vs 37.79%, p = 0.016, respectively). In patients with CAD, ABCA1 and ABCG1 DNA methylation levels were higher in EAT than in SAT samples (p < 0.05). ABCA1 mRNA levels in EAT samples were reduced in the subgroup of patients with CAD and concomitant carotid artery disease or peripheral artery disease compared with the NCAD group (p = 0.024). ABCA1 protein levels in EAT samples tended to be lower in CAD patients than in the NCAD group (p = 0.053). DNA methylation levels at the ABCG1 cg27243685 site positively correlated with plasma triglyceride concentration (r = 0.510, p = 0.008), body mass index (r = 0.556, p = 0.013) and waist-to-hip ratio (r = 0.504, p = 0.012) in SAT samples.ConclusionCAD is associated with ABCA1 and ABCG1 DNA hypermethylation in EAT. CAD with concomitant carotid artery disease or peripheral artery disease is accompanied by decreased ABCA1 gene expression in EAT. DNA methylation levels at the ABCG1 cg27243685 locus in SAT are associated with hypertriglyceridemia and obesity.

CRISPR-enhanced human adipocyte browning as cell therapy for metabolic disease

Obesity and type 2 diabetes are associated with disturbances in insulin-regulated glucose and lipid fluxes and severe comorbidities including cardiovascular disease and steatohepatitis. Whole body metabolism is regulated by lipid-storing white adipocytes as well as “brown” and “brite/beige” adipocytes that express thermogenic uncoupling protein 1 (UCP1) and secrete factors favorable to metabolic health. Implantation of brown fat into obese mice improves glucose tolerance, but translation to humans has been stymied by low abundance of primary human beige adipocytes. Here we apply methods to greatly expand human adipocyte progenitors from small samples of human subcutaneous adipose tissue and then disrupt the thermogenic suppressor gene NRIP1 by CRISPR. Ribonucleoprotein consisting of Cas9 and sgRNA delivered ex vivo are fully degraded by the human cells following high efficiency NRIP1 depletion without detectable off-target editing. Implantation of such CRISPR-enhanced human or mouse brown-like adipocytes into high fat diet fed mice decreases adiposity and liver triglycerides while enhancing glucose tolerance compared to implantation with unmodified adipocytes. These findings advance a therapeutic strategy to improve metabolic homeostasis through CRISPR-based genetic enhancement of human adipocytes without exposing the recipient to immunogenic Cas9 or delivery vectors.

Changes in adipose tissue microRNA expression across the menstrual cycle in regularly menstruating females: a pilot study.

Cyclical changes in hormone profiles across the menstrual cycle are associated with alterations in metabolic control. MicroRNAs (miRNAs) contribute to regulating metabolic control, including adipose tissue metabolism. How fluctuations in hormonal profiles across the menstrual cycle affect adipose tissue miRNA expression remains unknown. Eleven healthy, regularly menstruating females underwent four sampling visits across their menstrual cycle. Subcutaneous abdominal adipose tissue and venous blood samples were collected at each sampling visit. Luteinizing hormone (LH) tests, calendar counting, and serum hormone concentrations were used to determine menstrual cycle phases: early-follicular (EF), late-follicular (LF), postovulatory (PO), and midluteal (ML). Serum follicle-stimulating hormone, LH, estrogen, progesterone, and testosterone were determined using multiplex magnetic bead panels and enzyme-linked immunosorbent assays. Global adipose tissue miRNA expression levels were determined via microarray in a subset of participants (n = 8) and 17 candidate miRNAs were validated by RT-qPCR in the whole cohort (n = 11). Global analysis of adipose tissue miRNA expression identified 33 miRNAs significantly altered across the menstrual cycle; however, no significant differences remained after correcting for multiple testing (P > 0.05). RT-qPCR analysis of candidate miRNAs revealed miR-497-5p expression was significantly altered across the menstrual cycle ([Formula: see text] = 0.18, P = 0.03); however, post hoc tests did not reveal any significant differences between menstrual cycle phases (P > 0.05). miR-30c-5p was associated with testosterone concentration (R2 = 0.13, P = 0.033). These pilot data indicate differences in adipose tissue miRNAs in healthy women across the menstrual cycle and a weak association with ovarian hormones. Further research in larger sample sizes is required to confirm regulation of miRNA expression across the menstrual cycle.

Chromatin accessibility and gene expression during adipocyte differentiation identify context-dependent effects at cardiometabolic GWAS loci.

Chromatin accessibility and gene expression in relevant cell contexts can guide identification of regulatory elements and mechanisms at genome-wide association study (GWAS) loci. To identify regulatory elements that display differential activity across adipocyte differentiation, we performed ATAC-seq and RNA-seq in a human cell model of preadipocytes and adipocytes at days 4 and 14 of differentiation. For comparison, we created a consensus map of ATAC-seq peaks in 11 human subcutaneous adipose tissue samples. We identified 58,387 context-dependent chromatin accessibility peaks and 3,090 context-dependent genes between all timepoint comparisons (log2 fold change>1, FDR<5%) with 15,919 adipocyte- and 18,244 preadipocyte-dependent peaks. Adipocyte-dependent peaks showed increased overlap (60.1%) with Roadmap Epigenomics adipocyte nuclei enhancers compared to preadipocyte-dependent peaks (11.5%). We linked context-dependent peaks to genes based on adipocyte promoter capture Hi-C data, overlap with adipose eQTL variants, and context-dependent gene expression. Of 16,167 context-dependent peaks linked to a gene, 5,145 were linked by two or more strategies to 1,670 genes. Among GWAS loci for cardiometabolic traits, adipocyte-dependent peaks, but not preadipocyte-dependent peaks, showed significant enrichment (LD score regression P<0.005) for waist-to-hip ratio and modest enrichment (P < 0.05) for HDL-cholesterol. We identified 659 peaks linked to 503 genes by two or more approaches and overlapping a GWAS signal, suggesting a regulatory mechanism at these loci. To identify variants that may alter chromatin accessibility between timepoints, we identified 582 variants in 454 context-dependent peaks that demonstrated allelic imbalance in accessibility (FDR<5%), of which 55 peaks also overlapped GWAS variants. At one GWAS locus for palmitoleic acid, rs603424 was located in an adipocyte-dependent peak linked to SCD and exhibited allelic differences in transcriptional activity in adipocytes (P = 0.003) but not preadipocytes (P = 0.09). These results demonstrate that context-dependent peaks and genes can guide discovery of regulatory variants at GWAS loci and aid identification of regulatory mechanisms.

Relationship of the degree of coronary lesion and the expression of adipocytokines of local fat depots in patients with coronary artery disease

Abstract Aims To reveal the peculiarities of the relationship between of the coronary lesion with the levels of expression of adiponectin, leptin and IL-6 by subcutaneous, epicardial and perivascular adipocytes in patients with coronary artery disease. Materials and methods The study included 90 patients with CAD who signed voluntary informed consent. During elective surgery (coronary artery bypass grafting, CABG), samples of subcutaneous adipose tissue (SAT), EAT and PVAT were obtained. Then the patients were divided, depending on the degree of CA, as determined into 3 groups by the SYNTAX Score: moderate (≤22, Group 1), severe (23–31, Group 2), and extremely severe (≥32, Group 3). The expression of adiponectin, leptin and IL-6 was determined by quantitative real-time polymerase chain reaction (qPCR) using TaqManTM Gene Expression Assays (Applied Biosystems, USA) in the ViiA 7 Real-Time PCR System (Applied Biosystems, USA). Statistical analysis was performed using Statistica 9.0. Results Evaluation of the studied parameters depending on the degree of coronary lesion according to SYNTAX Score showed that patients with moderate lesion were characterized by the highest level of adiponectin expression in all types of adipose tissue (AT). In severe coronary lesion, an increase in the level of leptin mRNA was observed in the SAT and EAT, and in extremely severe cases, in the PVAT. There was also an increase in the expression of IL-6 in the EAT and PVAT in severe and extremely severe coronary lesions. Using logistic regression analysis it was found that the most significant predictors of severe/extremely severe coronary lesion in CAD are: the expression of adiponectin in the PVAT (odds ratio (OR) 0.44, 95% confidence interval (CI 0.178–0.79, p=0.031) and age (OR=1.4, 95% CI 1.072–1.839, p=0.014) The pathogenetic significance of these markers was confirmed by discriminant analysis, which showed the significance of the expression of adiponectin and IL-6 in the PVAT, leptin in all studied local fat depots to determine the probability of developing severe/extremely severe coronary lesions. Conclusions The data obtained indicate a close relationship between the degree of coronary lesion, assessed by the SYNTAX Score, and the expression levels of adipocytokines in local fat depots in patients with CAD. Based on this, it can be concluded that the studied markers are capable of reflecting the degree of coronary artery disease in patients with CAD. The most significant predictors of severe/extremely severe coronary artery disease in CAD is the expression of adiponectin in the PVAT and age. Funding Acknowledgement Type of funding sources: None.

Beneficial Effects of High Intensity Interval Training and/or Linseed Oil Supplementation to Limit Obesity-Induced Oxidative Stress in High Fat Diet-Fed Rats.

High-intensity interval training (HIIT) and linseed oil (LO) supplementation are effective strategies to reduce obesity-induced oxidative stress. Our aim was to determine whether the HIIT + LO combination prevents obesity-induced oxidative stress in high fat diet (HFD)-fed rats. HFD-fed 8-week-old, male, Wistar rats were subdivided in four groups: HFD, LO (2% of sunflower oil replaced with 2% of LO in the HFD), HIIT (4 days/week for 12 weeks), and HIIT + LO. Wistar rats fed a low-fat diet (LFD) were used as controls. Epididymal and subcutaneous adipose tissue, gastrocnemius muscle, liver, and plasma samples were collected to measure oxidative stress markers (AOPP, oxLDL), antioxidant (SOD, CAT, and GPx activities) and pro-oxidant (NOx and XO) enzyme activities. Compared with the LFD, the HFD altered the pro/antioxidant status in different tissues (increase of AOPP, oxLDL, SOD and catalase activities in plasma, and SOD activity increase in liver and decrease in adipose tissues) but not in gastrocnemius. LO upregulated CAT activity and decreased NOx in liver. HIIT alleviated HFD negative effects in liver by reducing SOD and NOx activities. Moreover, the HIIT + LO combination potentiated SOD activity upregulation in subcutaneous tissue. HIIT and LO supplementation have independent beneficial effects on the pro/antioxidant balance. Their association promotes SOD activity in subcutaneous adipose tissue.

A novel conjunctive microenvironment derived from human subcutaneous adipose tissue contributes to physiology of its superficial layer

BackgroundIn human subcutaneous adipose tissue, the superficial fascia distinguishes superficial and deep microenvironments showing extensions called retinacula cutis. The superficial subcutaneous adipose tissue has been described as hyperplastic and the deep subcutaneous adipose tissue as inflammatory. However, few studies have described stromal-vascular fraction (SVF) content and adipose-derived stromal/stem cells (ASCs) behavior derived from superficial and deep subcutaneous adipose tissue. In this study, we analyzed a third conjunctive microenvironment: the retinacula cutis superficialis derived from superficial subcutaneous adipose tissue.MethodsThe samples of abdominal human subcutaneous adipose tissue were obtained during plastic aesthetic surgery in France (Declaration DC-2008-162) and Brazil (Protocol 145/09).ResultsThe SVF content was characterized in situ by immunofluorescence and ex vivo by flow cytometry revealing a high content of pre-adipocytes rather in superficial subcutaneous adipose tissue microenvironment. Adipogenic assays revealed higher percentage of lipid accumulation area in ASCs from superficial subcutaneous adipose tissue compared with retinacula cutis superficialis (p < 0.0001) and deep subcutaneous adipose tissue (p < 0.0001). The high adipogenic potential of superficial subcutaneous adipose tissue was corroborated by an up-regulation of adipocyte fatty acid-binding protein (FABP4) compared with retinacula cutis superficialis (p < 0.0001) and deep subcutaneous adipose tissue (p < 0.0001) and of C/EBPα (CCAAT/enhancer-binding protein alpha) compared with retinacula cutis superficialis (p < 0.0001) and deep subcutaneous adipose tissue (p < 0.0001) microenvironments. Curiously, ASCs from retinacula cutis superficialis showed a higher level of adiponectin receptor gene compared with superficial subcutaneous adipose tissue (p = 0.0409), widely known as an anti-inflammatory hormone. Non-induced ASCs from retinacula cutis superficialis showed higher secretion of human vascular endothelial growth factor (VEGF), compared with superficial (p = 0.0485) and deep (p = 0.0112) subcutaneous adipose tissue and with adipogenic-induced ASCs from superficial (p = 0.0175) and deep (p = 0.0328) subcutaneous adipose tissue. Furthermore, ASCs from retinacula cutis superficialis showed higher secretion of Chemokine (C–C motif) ligand 5 (CCL5) compared with non-induced (p = 0.0029) and induced (p = 0.0089) superficial subcutaneous adipose tissue.ConclusionsThis study highlights the contribution to ASCs from retinacula cutis superficialis in their angiogenic property previously described for the whole superficial subcutaneous adipose tissue besides supporting its adipogenic potential for superficial subcutaneous adipose tissue.

Human adipose tissue gene expression of solute carrier family 19 member 3 (SLC19A3); relation to obesity and weight-loss.

ObjectiveAdipose tissue is a specialized endocrine organ that is involved in modulating whole‐body energy homeostasis and expresses a specific subset of genes, which may play a role in adipose tissue metabolism. The aim of this study was to search for novel adipose tissue‐specific genes using a tissue panel of RNAseq expression profiles.MethodsRNAseq expression profiles from 53 human tissues were downloaded from the GTex database. SLC19A3 expression was analyzed by microarray or real‐time PCR in two sets of paired subcutaneous and omental adipose tissue samples, in two studies with adipose tissue from persons with high or low body mass index (BMI), in adipose tissue from patients who underwent weight loss with a very‐low caloric diet and during preadipocyte‐adipocyte differentiation.ResultsThe RNAseq‐based tissue distribution expression screen identified SLC19A3 (encoding the thiamine transporter 2) as adipose tissue‐specific. SLC19A3 expression was higher in subcutaneous compared with omental adipose tissue in both sample sets (p = 0.043 and p < 0.001). Preadipocyte differentiation towards adipocytes resulted in increased SLC19A3 gene expression (p = 0.018 or less at all‐time points). Subcutaneous adipose tissue expression of SLC19A3 was lower in persons with high BMI in both cohorts (p = 0.008, and p < 0.001) and increased during a weight‐loss intervention (p = 0.006).ConclusionThe specific adipose tissue expression pattern of SLC19A3, together with its regulation in obesity and during weight loss, indicate that it plays a key role in adipocyte metabolism.

COVID-19 autopsy cases: detection of virus in endocrine tissues.

PurposeThe SARS-CoV-2 genome has been detected in a variety of human samples including blood, urine, semen, and faeces. However, evidence of virus presence in tissues other than lung are limited.MethodsWe investigated whether SARS-CoV-2 could be detected in 50 autoptic specimens of endocrine organs from 29 patients who died of COVID-19.ResultsThe virus was detected in 25 specimens including ten abdominal subcutaneous adipose tissue samples (62%), six testes (67%), and nine thyroid (36%) samples. The analysis of multiple endocrine organ samples obtained from the same patients showed that, in virus-positive cases, the viral genome was consistently detected in all but two matched specimens.ConclusionOur findings show that the virus spread into endocrine organs is a common event in severe cases. Further studies should assess the rate of the phenomenon in clinically mild cases. The potential long-term effects of COVID-19 on endocrine functions should be taken into consideration.

1213-P: A Novel Relationship between Thromboxane A2 Receptor and Obesity-Related Inflammation

Animal models of obesity have shown that the expression of Thromboxane A2 Receptor (tbxa2r) and thromboxane synthase (Tbxas1) are increased in adipose tissue (AT) and are associated with insulin resistance and inflammation. Objective: To compare Tbxa2r gene expression in the AT of human subjects with different degrees of obesity and to determine the association between AT Tbxa2r with BMI and markers of AT inflammation. Methods: Subcutaneous AT samples were collected from lean (BMI 40) subjects. Gene expression of Tbxa2r was analyzed in AT and compared among different groups. Correlation analysis to determine the association between AT Tbxa2r and obesity and obesity-related inflammation. Results: The mRNA levels of Tbxa2r were significantly higher in obese compared to normal subjects (P Conclusion: Signaling of TxA2 is higher in AT in obesity and is associated with BMI as well as inflammatory genes. Tbxa2r may be a potential therapeutic target for obesity-linked diseases including type 2 DM and CV disease. Disclosure D. Alcaraz alvarez: None. S. Viswanathan: None. T. Gopal: None. N. Kumar: None. K. Kelley: None. C. Desouza: Advisory Panel; Self; Bayer AG, Novo Nordisk, Consultant; Self; AstraZeneca.

368-P: Type 2 Diabetes Mellitus Increases Epicardial Adipose Tissue Senescence in Subjects with Coronary Artery Disease during Elective Cardiac Surgery

Cellular senescence is a novel mechanism suggested to contribute to the development of type 2 diabetes mellitus (T2DM) and cardiovascular diseases. Here, we analyzed the expression of senescent markers in myocardium and adjacent adipose tissue of subjects with and without coronary artery disease (CAD) and T2DM undergoing elective cardiac surgery. Eleven subjects with CAD and T2DM (DM group), 13 subjects with CAD without T2DM (CAD group) and 8 subjects without both CAD and T2DM (C - control) group undergoing elective cardiac surgery (CABG and/or valvular surgery) were included into the study. Samples of subcutaneous (SAT) and epicardial (EAT) adipose tissue and myocardium form right atrial wall were taken along with standard biochemical and anthropometric parameters at the beginning and end of surgery. mRNA expression of senescent markers p21 and p16 was assessed using qRT PCR. All 3 groups did not differ with respect to age (65.2 ± 6.9 vs. 61.7 ± 9.8 vs. 57.9 ± 12.3 years for DM vs. CAD vs. C, n.s.) or BMI (30.8 ± 3.9 vs. 28.5 ± 3.9 vs. 30.2 ± 3.4 kg/m2, n.s.), while DM group showed higher levels of fasting glucose (8.4 ± 1.6 vs. 5.9 ± 1.1 vs. 5.9 ± 0.9 mmol/, p=0.005) and HbA1C (52.5 ± 8.9 vs. 38.9 ± 4.2 vs. 31.5 ± 8.9 mmol/mol, p&amp;lt;0.001) relative to others. At the beginning of surgery no difference in mRNA expression of either factor could be seen in any of the examined tissues except of increased expression of p16 in the myocardium of CAD group (p=0.044 vs. C group). In DM subjects, cardiac surgery significantly increased mRNA expression of p21 in myocardium (p=0.15 vs. C and CAD group) and EAT (p=0.002 vs. C group), while not such change could be seen in the nondiabetic CAD and C group. To conclude, in CAD subjects T2DM is associated with increased cellular senescence in EAT and myocardium during elective cardiac surgery. This mechanism might at least partially explain the more detrimental outcomes of CAD patients with as compared to without T2DM. Disclosure M. Mraz: None. M. Haluzik: Advisory Panel; Self; AstraZeneca, Lilly Diabetes, Novo Nordisk, Consultant; Self; Sanofi. J. Trnovska: None. B. Kasperova: None. I. Dvorakova: None. Z. Lacinova: None. D. Hlavacek: None. J. Mahrik: None. P. Ivak: None. I. Netuka: None. Funding Ministry of Health of the Czech Republic (RVO-VFN64165, NV19-0200118); IKEM (IN00023001)

Divergent immunometabolic changes in adipose tissue and skeletal muscle with ageing in healthy humans.

Ageing is associated with increased systemic inflammation and metabolic dysfunction that contributes to the development of age-associated diseases. The role of adipose tissue in immunometabolic alterations that take place with ageing is unknown in humans. We show, in healthy, active and lean older adults, that adipose tissue, but not skeletal muscle, displays considerable pro-inflammatory transcriptomic, cellular and secretory changes, as well as a reduction in insulin signalling proteins compared to younger adults. These findings indicate that adipose tissue undergoes substantial immunometabolic alterations with ageing, and that these changes are tissue-specific and more profound than those observed in skeletal muscle or in the circulation. These results identify adipose tissue as an important tissue in the biological ageing process in humans, which may exhibit signs of immunometabolic dysfunction prior to systemic manifestation. Ageing and obesity are both characterized by inflammation and a deterioration in metabolic health. It is now clear that adipose tissue plays a major role in inflammation and metabolic control in obesity, although little is known about the role of adipose tissue in human ageing. To understand how ageing impacts adipose tissue, we characterized subcutaneous adipose tissue and skeletal muscle samples from twelve younger (27±4years [Young]) and twelve older (66±5years [Old]) active/non-obese males. We performed a wide-range of whole-body and tissue measures, including RNA-sequencing and multicolour flow cytometry. We also measured a range of inflammatory and metabolic proteins in the circulation and their release by adipose tissue, exvivo. Both adipose tissue and muscle had ∼2-fold more immune cells per gram of tissue with ageing. In adipose tissue, this immune cell infiltration was driven by increased memory/effector T-cells, whereas, in muscle, the accumulation was driven by memory/effector T-cells and macrophages. Transcriptomic analysis revealed that, with ageing, adipose tissue, but not muscle, was enriched for inflammatory transcripts/pathways related to acquired and innate immunity. Ageing also increased the adipose tissue pro-inflammatory secretory profile. Insulin signalling protein content was reduced in adipose tissue, but not muscle. Our findings indicate that adipose tissue undergoes substantial immunometabolic changes with ageing in humans, and that these changes are tissue-specific and more profound than those observed in the circulation and skeletal muscle.

230-OR: Exercise Training Improved Antilipolytic Sensitivity to Insulin in Obese Adults with Low Sensitivity to Insulin before Training

Low sensitivity to the antilipolytic effects of insulin in adipose tissue has been linked with impaired insulin-mediated glucose uptake in skeletal muscle. The aims of this study were: 1) to compare adipose tissue morphology in obese adults with high- (HIGH) vs. low-sensitivity (LOW) to the antilipolytic effects of insulin, and 2) compare the effects of exercise training on adipose tissue from HIGH vs. LOW subjects. Using isotopic dilution methods before and during a hyperinsulinemic-euglycemic clamp, we measured the antilipolytic response to insulin in a total of 36 obese adults - and we identified sub-cohorts of 13 LOW (64±7% lipolytic suppression by insulin; BMI: 34±4kg/m2) and 13 HIGH (82±3% lipolytic suppression by insulin; BMI:33±3kg/m2). Abdominal subcutaneous adipose tissue (aSAT) samples were collected before the clamp (non-insulin stimulated). Measurements were made before and after a 12-week endurance exercise training program (4 days/week). Before training, capillary density (# of capillaries/mm2) in aSAT samples for LOW was significantly lower than HIGH (p=0.004). Collagen abundance in aSAT extracellular matrix (ECM) was lower in LOW (p=0.013). In response to training, the antilipolytic response to insulin was significantly improved in LOW (72±5% lipolytic suppression by insulin; p&amp;lt;0.001) but not HIGH. This was accompanied by an increased capillary density (p=0.03), and collagen abundance in ECM (p = 0.02), along with a slight reduction in fat mass (1±1kg; p=0.05). Adipocyte size was reduced in both groups (p=0.02). In summary, aSAT from obese adults with low antilipolytic sensitivity to insulin displayed lower capillarization and ECM collagen abundance compared with obese adults with high sensitivity to the antilipolytic effects of insulin. Importantly, morphological adaptations to exercise training accompanied an increased antilipolytic sensitivity to insulin in subjects with low antilipolytic sensitivity before training. Disclosure C. Ahn: None. M. W. Schleh: None. B. J. Ryan: None. A. Ludzki: None. P. Varshney: None. J. B. Gillen: None. S. Chen: None. J. F. Horowitz: None. Funding National Institutes of Health (R01DK077966, P30DK089503, T32DK007245, F32DK117522); Canadian Institutes of Health Research (DFS146190, 338735)