Human Subcutaneous Adipose Tissue Research Articles

Gene expression regional differences in human subcutaneous adipose tissue

Gene expression regional differences in human subcutaneous adipose tissue

Abstract 2960: Adipocytes sequester and metabolize daunorubicin

Abstract Obesity is associated with poorer outcome from many cancers, including childhood acute lymphoblastic leukemia (ALL). We have previously shown that adipocytes protect ALL cells from the anthracycline, daunorubicin (DNR). We therefore investigated whether adipocytes sequester and/or metabolize DNR in the ALL microenvironment. Using fluorescence and LC/MS measures, we demonstrated that adipocytes absorb DNR, reducing the intracellular DNR concentration in co-cultured BV173 ALL cells (after 48 hours, median fluorescent intensity of ALL cultured with adipocytes was 1.7±1.0 vs. 5.0±1.7 of those cultured alone, p<0.01). Mouse adipocytes convert DNR to the less active metabolite, daunorubicinol (DNR-ol); over 48 hours, media DNR decreased to 0.1±0.2 (vs. 24.8±8.76 ng/mL in control wells). At the same time, DNR-ol increased to 15.0±1.9 (vs. 0.7±0.6 in control wells; both p<0.05). Similar conversion of DNR to DNR-ol was observed in both mouse adipose explants and human adipose tissue biopsy samples ex vivo. qPCR confirmed human subcutaneous adipose tissue expresses several enzymes capable of metabolizing DNR, including AKR1A1, 1B1, 1C1, 1C2, 1C3, 7A2, and CBR1 and 3 (expression ranged between 20 and 195% β-actin). Using immunohistochemistry, we confirmed expression of AKR1C1, 1C2, and 1C3 in bone marrow adipocytes of children during the first month of treatment for ALL. Finally, two hours after an intravenous dose of DNR in mice, we found that the DNR-ol to DNR ratio was higher in subcutaneous (0.60±0.26) and omental (0.55±0.21) adipose than in white blood cells (0.16±0.11), bone marrow (undetectable DNR-ol), and spleen (undetectable DNR-ol). Together, these data demonstrate that adipocytes sequester and inactivate DNR, likely due to their expression of AKR and CBR enzymes. These findings uncover a novel and important mechanism which could promote local anthracycline resistance by cancer cells in microenvironment rich in adipocytes, such as bone marrow, omentum, and breast. Citation Format: Xia Sheng, Jean-Hugues Parmentier, Jonathan Tucci, Hua Pei, Omar Cortez-Toledo, Christina Dieli-Conwright, Matthew Oberley, Michael Neely, Etan Orgel, Stan Louie, Steven D. Mittelman. Adipocytes sequester and metabolize daunorubicin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2960. doi:10.1158/1538-7445.AM2017-2960

Physiological effects of proinsulin-connecting peptide in human subcutaneous adipose tissue.

Recent studies suggest that proinsulin-connecting peptide (C-peptide) may exhibit characteristics of a hormone and show physiological functions in various tissues. This study was aimed to determine whether C-peptide could be involved in the regulation of lipolysis, adiponectin release, and function of mesenchymal stem cells (MSCs) in adipose tissue. Human subcutaneous adipose tissue was cultured in the presence of C-peptide. The level of lipolysis was determined by glycerol measurement in the conditioned media. Effect of C-peptide on adiponectin secretion was evaluated in differentiated adipocytes. The adipogenic and osteogenic abilities of adipose MSCs were evaluated using oil red and alizarin red staining, respectively. The tetrazolium bromide test was conducted for evaluating the effect of C-peptide on MSCs proliferation. C-peptide induced a significant decrease in basal lipolysis at concentrations of 8 and 16nM (p<0.05). It had no significant effects on isoproterenol-stimulated lipolysis, adiponectin secretion, and adipogenic or osteogenic differentiation of MSCs. At a concentration of 4nM, this peptide significantly increased the proliferative capability of MSCs (p<0.05). These results suggest that C-peptide has some physiological effects in human subcutaneous adipose tissue and contributes to the regulation of basal lipolysis and pool of MSCs.

Multi-omics Integrative Investigation of Fatty Acid Metabolism in Obese and Lean Subcutaneous Tissue.

White adipose tissue (WAT) plays a central role in whole-body energy homeostasis through storage and release of fatty acids. A deeper understanding of the complex and highly integrated pathways regulating WAT fatty acid metabolism, and how they are altered with obesity, is necessary for diagnostic and therapeutic innovations in nutritional disorders. In this multi-omics study, we investigated the influence of obesity on fatty acid metabolism in human subcutaneous adipose tissue (SAT) using an approach that integrated transcriptomic, peptidomic, and fatty acid analyses. Notably, all analyses were conducted in the same adipose tissue sample from each participant, thus minimizing the chance of spurious results. In a sample of SAT from the periumbilical abdominal region of obese (n = 11, mean body mass index [BMI] = 35.0 ± 1.2 kg/m2) and lean subjects (n = 9, mean BMI = 22.1 ± 0.5 kg/m2), we found that obese SAT tended to have higher relative amounts of specific monounsaturated fatty acids and n-6 polyunsaturated fatty acids, and lower amounts of saturated fatty acids (p < 0.05). These changes were associated with differential regulation of lipogenic and lipolytic pathways in obese SAT. Fatty acid analysis showed changes in estimated fatty acid desaturase and elongase activities between lean and obese SAT (p < 0.05). Biomarkers of lipogenesis (e.g., fatty acid synthase protein) were differentially regulated between lean and obese SAT. These changes were noted in conjunction with increases in extracellular matrix remodeling proteins. Transcriptomic data revealed that the key regulators of lipolysis were reduced in obese SAT. This integrative multi-omics analysis collectively shows that obese SAT has a distinct fatty acid signature compared to lean SAT and the pathways underlying fatty acid metabolism are broadly regulated at the level of gene expression and protein abundance.

CD146 (MCAM) in human cs-DLK1−/cs-CD34+ adipose stromal/progenitor cells

To precisely characterize CD146 in adipose stromal/progenitor cells (ASCs) we sorted the stromal vascular faction (SVF) of human abdominal subcutaneous white adipose tissue (sWAT) according to cell surface (cs) expression of CD146, DLK1 and CD34. This test identified three main SVF cell populations: ~50% cs-DLK1−/cs-CD34+/cs-CD146− ASCs, ~7.5% cs-DLK1+/cs-CD34dim/+/cs-CD146+ and ~7.5% cs-DLK1+/cs-CD34dim/+/cs-CD146− cells. All cells contained intracellular CD146. Whole mount fluorescent IHC staining of small vessels detected CD146+ endothelial cells (CD31+/CD34+/CD146+) and pericytes (CD31−/CD34−/CD146+ ASCs). The cells in the outer adventitial layer showed the typical ASC morphology, were strongly CD34+ and contained low amounts of intracellular CD146 protein (CD31−/CD34+/CD146+). Additionally, we detected wavy CD34−/CD146+ and CD34dim/CD146+ cells. CD34dim/CD146+ cells were slightly more bulky than CD34−/CD146+ cells. Both CD34−/CD146+ and CD34dim/CD146+ cells were detached from the inner pericyte layer and protruded into the outer adventitial layer. Cultured early passage ASCs contained low levels of CD146 mRNA, which was expressed in two different splicing variants, at a relatively high amount of the CD146-long form and at a relatively low amount of the CD146-short form. ASCs contained low levels of CD146 protein, which consisted predominantly long form and a small amount of short form. The CD146 protein was highly stable, and the majority of the protein was localized in the Golgi apparatus. In conclusion, the present study contributes to a better understanding of the spatial localization of CD34+/CD146+ and CD34−/CD146+ cells in the adipose niche of sWAT and identifies CD146 as intracellular protein in cs-DLK1−/cs-CD34+/cs-CD146− ASCs.

Isolation and characterization of canine perivascular stem/stromal cells for bone tissue engineering.

For over 15 years, human subcutaneous adipose tissue has been recognized as a rich source of tissue resident mesenchymal stem/stromal cells (MSC). The isolation of perivascular progenitor cells from human adipose tissue by a cell sorting strategy was first published in 2008. Since this time, the interest in using pericytes and related perivascular stem/stromal cell (PSC) populations for tissue engineering has significantly increased. Here, we describe a set of experiments identifying, isolating and characterizing PSC from canine tissue (N = 12 canine adipose tissue samples). Results showed that the same antibodies used for human PSC identification and isolation are cross-reactive with canine tissue (CD45, CD146, CD34). Like their human correlate, canine PSC demonstrate characteristics of MSC including cell surface marker expression, colony forming unit-fibroblast (CFU-F) inclusion, and osteogenic differentiation potential. As well, canine PSC respond to osteoinductive signals in a similar fashion as do human PSC, such as the secreted differentiation factor NEL-Like Molecule-1 (NELL-1). Nevertheless, important differences exist between human and canine PSC, including differences in baseline osteogenic potential. In summary, canine PSC represent a multipotent mesenchymogenic cell source for future translational efforts in tissue engineering.

Apolipoprotein A5 regulates intracellular triglyceride metabolism in adipocytes.

It has previously been demonstrated that apolipoprotein A5 (apoA5) can be internalized by human adipocytes and significantly decreases intracellular triglyceride content. In the present study, endocytosis of apoA5 by adipocytes under different conditions, and the underlying mechanism by which apoA5 regulates cellular triglyceride storage, was investigated. The results revealed that the apoA5 protein was detected in human subcutaneous abdominal adipose tissues. In addition, the uptake of apoA5 was attenuated in human obese adipose tissues and in cultured adipocytes with hypertrophy or insulin resistance. Low-density lipoprotein receptor protein 1 (LRP1) knockdown in adipocytes resulted in a decrease in internalized apoA5 content, suggesting that LRP1 serves a role in apoA5 uptake. Treatment of adipocytes with apoA5 decreased the expression of the lipid droplet-associated proteins such as cidec and perilipin. ApoA5-treated adipocytes demonstrated an increase in lipolysis activity and expression of uncoupling protein 1, which is the molecular effector of thermogenesis in brown adipocytes. These results suggested that decreased triglyceride accumulation in adipocytes induced by apoA5 may be associated with enhanced lipolysis and energy expenditure, which may result from reduced expression of cidec and perilipin. In conclusion, the present study demonstrated a novel role of apoA5 in regulating the intracellular triglyceride metabolism of adipocytes. The results of the present study suggested that apoA5 may serve as a potential therapeutic target for the treatment of obesity and its related disorders.

Are Adipose-Derived Stem Cells from Liver Falciform Ligaments Another Possible Source of Mesenchymal Stem Cells?

Falciform ligaments in the liver are surrounded by adipose tissue. We investigated the capability of adipose-derived stem cells from human liver falciform ligaments (hLF-ADSCs) to differentiate into hepatic-type cells and confirmed the functional capacity of the cells. Mesenchymal stem cells (MSCs) were isolated from the liver falciform ligament and abdominal subcutaneous adipose tissue in patients undergoing partial hepatectomy for liver disease. Cells were cultivated in MSC culture medium. Properties of MSCs were confirmed by flow cytometry, RT-PCR analysis, immunocytochemistry assays, and multilineage differentiation. Hepatic induction was performed using a three-step differentiation protocol with various growth factors. Morphology, capacity for expansion, and characteristics were similar between hLF-ADSCs and adipose-derived stem cells from human abdominal subcutaneous adipose tissue (hAS-ADSCs). However, hematopoietic– and mesenchymal–epithelial transition (MET)-related surface markers (CD133, CD34, CD45, and E-cadherin) had a higher expression in hLF-ADSCs. The hepatic induction marker genes had a higher expression in hLF-ADSCs on days 7 and 10 after the hepatic induction. Albumin secretion was similar between hLF-ADSCs and hAS-ADSCs at 20 days after the hepatic induction. The hLF-ADSCs had a different pattern of surface marker expression relative to hAS-ADSCs. However, proliferation, multilineage capacity, and hepatic induction were similar between the cell types. Accordingly, it may be a useful source of MSCs for patients with liver disease.

A novel role for the Wnt inhibitor APCDD1 in adipocyte differentiation: Implications for diet-induced obesity

Impaired adipogenic differentiation during diet-induced obesity (DIO) promotes adipocyte hypertrophy and inflammation, thereby contributing to metabolic disease. Adenomatosis polyposis coli down-regulated 1 (APCDD1) has recently been identified as an inhibitor of Wnt signaling, a key regulator of adipogenic differentiation. Here we report a novel role for APCDD1 in adipogenic differentiation via repression of Wnt signaling and an epigenetic linkage between miR-130 and APCDD1 in DIO. APCDD1 expression was significantly up-regulated in mature adipocytes compared with undifferentiated preadipocytes in both human and mouse subcutaneous adipose tissues. siRNA-based silencing of APCDD1 in 3T3-L1 preadipocytes markedly increased the expression of Wnt signaling proteins (Wnt3a, Wnt5a, Wnt10b, LRP5, and β-catenin) and inhibited the expression of adipocyte differentiation markers (CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ)) and lipid droplet accumulation, whereas adenovirus-mediated overexpression of APCDD1 enhanced adipogenic differentiation. Notably, DIO mice exhibited reduced APCDD1 expression and increased Wnt expression in both subcutaneous and visceral adipose tissues and impaired adipogenic differentiation in vitro Mechanistically, we found that miR-130, whose expression is up-regulated in adipose tissues of DIO mice, could directly target the 3'-untranslated region of the APCDD1 gene. Furthermore, transfection of an miR-130 inhibitor in preadipocytes enhanced, whereas an miR-130 mimic blunted, adipogenic differentiation, suggesting that miR-130 contributes to impaired adipogenic differentiation during DIO by repressing APCDD1 expression. Finally, human subcutaneous adipose tissues isolated from obese individuals exhibited reduced expression of APCDD1, C/EBPα, and PPARγ compared with those from non-obese subjects. Taken together, these novel findings suggest that APCDD1 positively regulates adipogenic differentiation and that its down-regulation by miR-130 during DIO may contribute to impaired adipogenic differentiation and obesity-related metabolic disease.

Impact of polyunsaturated and saturated fat overfeeding on the DNA-methylation pattern in human adipose tissue: a randomized controlled trial1–3

Background: Dietary fat composition can affect ectopic lipid accumulation and, thereby, insulin resistance. Diets that are high in saturated fatty acids (SFAs) or polyunsaturated fatty acids (PUFAs) have different metabolic responses.Objective: We investigated whether the epigenome of human adipose tissue is affected differently by dietary fat composition and general overfeeding in a randomized trial.Design: We studied the effects of 7 wk of excessive SFA (n = 17) or PUFA (n = 14) intake (+750 kcal/d) on the DNA methylation of ~450,000 sites in human subcutaneous adipose tissue. Both diets resulted in similar body weight increases. We also combined the data from the 2 groups to examine the overall effect of overfeeding on the DNA methylation in adipose tissue.Results: The DNA methylation of 4875 Cytosine-phosphate-guanine (CpG) sites was affected differently between the 2 diets. Furthermore, both the SFA and PUFA diets increased the mean degree of DNA methylation in adipose tissue, particularly in promoter regions. However, although the mean methylation was changed in 1797 genes [e.g., alpha-ketoglutarate dependent dioxygenase (FTO), interleukin 6 (IL6), insulin receptor (INSR), neuronal growth regulator 1 (NEGR1), and proopiomelanocortin (POMC)] by PUFAs, only 125 genes [e.g., adiponectin, C1Q and collagen domain containing (ADIPOQ)] were changed by SFA overfeeding. In addition, the SFA diet significantly altered the expression of 28 transcripts [e.g., acyl-CoA oxidase 1 (ACOX1) and FAT atypical cadherin 1 (FAT1)], whereas the PUFA diet did not significantly affect gene expression. When the data from the 2 diet groups were combined, the mean methylation of 1444 genes, including fatty acid binding protein 1 (FABP1), fatty acid binding protein 2 (FABP2), melanocortin 2 receptor (MC2R), MC3R, PPARG coactivator 1 α (PPARGC1A), and tumor necrosis factor (TNF), was changed in adipose tissue by overfeeding. Moreover, the baseline DNA methylation of 12 CpG sites that was annotated to 9 genes [e.g., mitogen-activated protein kinase 7 (MAPK7), melanin concentrating hormone receptor 1 (MCHR1), and splicing factor SWAP homolog (SFRS8)] was associated with the degree of weight increase in response to extra energy intake.Conclusions: SFA overfeeding and PUFA overfeeding induce distinct epigenetic changes in human adipose tissue. In addition, we present data that suggest that baseline DNA methylation can predict weight increase in response to overfeeding in humans. This trial was registered at http://clinicaltrials.gov as NCT01427140.

Human adipose tissue accumulation is associated with pro-inflammatory changes in subcutaneous rather than visceral adipose tissue

The importance of the involvement of adipose tissue macrophage subpopulations in obesity-related disorders is well known from different animal models, but human data are scarcer. Subcutaneous (n=44) and visceral (n=52) adipose tissues of healthy living kidney donors were obtained during living donor nephrectomy. Stromal vascular fractions were isolated and analysed by flow cytometry using CD14, CD16, CD36 and CD163 antibodies. Total macrophage numbers in subcutaneous adipose tissue increased (P=0.02) with body mass index (BMI), with a similar increase seen in the proportion of phagocytic CD14+CD16+CD36high macrophages (P<0.01). On the other hand, there was an inverse correlation between anti-inflammatory CD14+CD16−CD163+ macrophages (P<0.05) and BMI. These correlations disappeared after excluding obese subjects (BMI ⩾30 kg m−2) from the analysis. Interestingly, none of these subpopulations were significantly related to BMI in visceral adipose tissue. Obesity per se is associated with distinct, highly phagocytic macrophage accumulation in human subcutaneous adipose tissue.

Adipose Tissue – Skeletal Muscle Crosstalk: Obese Human Subcutaneous and Visceral Adipose Tissues Both Supress Muscle Insulin Signalling in Men and Women

Adipose tissue is an important endocrine organ that communicates with other peripheral tissues via the secretion of proteins, termed adipokines. In particular, altered secretion of specific adipokines (e.g., TNF‐α and adiponectin) has previously been shown to regulate skeletal muscle insulin sensitivity. For the current study, we established a human adipose tissue – muscle crosstalk model to investigate the impact of different adipose tissue depots on skeletal myocyte insulin signaling, and if this differs between men and women. Visceral (VAT) and subcutaneous (SAT) adipose tissue samples were collected from obese men and women during laparoscopic bariatric surgery. Adipose tissue samples were subsequently cultured for 48h and secretion media was collected. Secretion media was then transferred on to human skeletal myocytes derived from healthy, non‐diabetic male and female donors for 24h. Content of insulin signaling proteins (phosphorylated and total AKT) in mature myocytes incubated with either control M199 media or secretion media from VAT or SAT was assessed using Western Blotting in basal and insulin‐stimulated conditions (100uU/well). Adipokines in secretion media were measured with a multiplex immunoassay. In men, VAT had higher interleukin‐6 gene expression compared to SAT (p=0.004), while no differences were seen in women (p=0.271). Adiponectin and TNF‐α gene expression were not different between VAT or SAT for men or women. Secretion media collected from male SAT and VAT reduced p‐AKTThr308 activation in insulin‐stimulated myocytes compared to controls (p &lt;0.01). Interestingly, myocytes incubated with VAT secretion media, compared to SAT secretion media, showed a significant reduction in p‐AKTSer473 in men after insulin stimulation (p&lt;0.01). Secretion media collected from female VAT and SAT caused significant reductions in p‐AKTThr308 and p‐AKTSer473 activation following insulin stimulation, compared to controls (p&lt;0.01). Secretion media from VAT or SAT for men or women did not affect total‐AKT levels. Independent of sex, 14 out of 18 detected adipokines (i.e., a panel consisting of cytokines, chemokines and growth factors) were more abundant in VAT secretion media compared to SAT secretion media (p&lt;0.01). Only interleukin‐21 levels showed a sex difference, with higher levels detected in secretion media from female adipose tissue (p=0.003). In conclusion, SAT and VAT secretion media from obese men and women suppress insulin signaling in myocytes to a similar extent. These results reveal the power of this human adipose tissue – muscle model to investigate crosstalk between these two tissues.Support or Funding InformationThis work was supported by the Natural Sciences and Engineering Research Council of Canada.

Silencing of the small GTPase DIRAS3 induces cellular senescence in human white adipose stromal/progenitor cells.

Inhibition of Akt-mTOR signaling protects from obesity and extends life span in animals. In the present study, we analyse the impact of the small GTPase, GTP-binding RAS-like 3 (DIRAS3), a recently identified weight-loss target gene, on cellular senescence in adipose stromal/progenitor cells (ASCs) derived from human subcutaneous white adipose tissue (sWAT). We demonstrate that DIRAS3 knock-down (KD) in ASCs induces activation of Akt-mTOR signaling and proliferation arrest. DIRAS3 KD ASCs lose the potential to form colonies and are negative for Ki-67. Moreover, silencing of DIRAS3 results in a premature senescence phenotype. This is characterized by senescence-associated β-galactosidase positive enlarged ASCs containing increased p16INK4A level and activated retinoblastoma protein. DIRAS3 KD ASCs form senescence-associated heterochromatic foci as shown by increased level of γ-H2A.X positive foci. Furthermore, these cells express a senescence-associated secretory phenotype characterized by increased interleukin-8 secretion. Human DIRAS3 KD ASCs develop also a senescence phenotype in sWAT of SCID mice. Finally, we show that DIRAS3 KD in ASCs stimulates both adipogenic differentiation and premature senescence. In conclusion, our data suggest that silencing of DIRAS3 in ASCs and subsequently hyper-activation of Akt-mTOR drives adipogenesis and premature senescence. Moreover, differentiating ASCs and/or mature adipocytes may acquire features of cellular senescence.

Mast Cells Promote Seasonal White Adipose Beiging in Humans.

Human subcutaneous (SC) white adipose tissue (WAT) increases the expression of beige adipocyte genes in the winter. Studies in rodents suggest that a number of immune mediators are important in the beiging response. We studied the seasonal beiging response in SC WAT from lean humans. We measured the gene expression of various immune cell markers and performed multivariate analysis of the gene expression data to identify genes that predict UCP1. Interleukin (IL)-4 and, unexpectedly, the mast cell marker CPA3 predicted UCP1 gene expression. Therefore, we investigated the effects of mast cells on UCP1 induction by adipocytes. TIB64 mast cells responded to cold by releasing histamine and IL-4, and this medium stimulated UCP1 expression and lipolysis by 3T3-L1 adipocytes. Pharmacological block of mast cell degranulation potently inhibited histamine release by mast cells and inhibited adipocyte UCP1 mRNA induction by conditioned medium (CM). Consistently, the histamine receptor antagonist chlorpheniramine potently inhibited adipocyte UCP1 mRNA induction by mast cell CM. Together, these data show that mast cells sense colder temperatures, release factors that promote UCP1 expression, and are an important immune cell type in the beiging response of WAT.

Gene expression regional differences in human subcutaneous adipose tissue

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.

MiRNAs in human subcutaneous adipose tissue: Effects of weight loss induced by hypocaloric diet and exercise.

Obesity is central in the development of insulin resistance. However, the underlying mechanisms still need elucidation. Dysregulated microRNAs (miRNAs; post-transcriptional regulators) in adipose tissue may present an important link. The miRNA expression in subcutaneous adipose tissue from 19 individuals with severe obesity (10 women and 9 men) before and after a 15-week weight loss intervention was studied using genome-wide microarray analysis. The microarray results were validated with RT-qPCR, and pathway enrichment analysis of in silico predicted targets was performed to elucidate the biological consequences of the miRNA dysregulation. Lastly, the messenger RNA (mRNA) and/or protein expression of multiple predicted targets as well as several proteins involved in lipolysis were investigated. The intervention led to upregulation of miR-29a-3p and miR-29a-5p and downregulation of miR-20b-5p. The mRNA and protein expression of predicted targets was not significantly affected by the intervention. However, negative correlations between miR-20b-5p and the protein levels of its predicted target, acyl-CoA synthetase long-chain family member 1, were observed. Several other miRNA-target relationships correlated negatively, indicating possible miRNA regulation, including miR-29a-3p and lipoprotein lipase mRNA levels. Proteins involved in lipolysis were not affected by the intervention. Weight loss influenced several miRNAs, some of which were negatively correlated with predicted targets. These dysregulated miRNAs may affect adipocytokine signaling and forkhead box protein O signaling.

Human Subcutaneous Adipose Tissue Adipocytes Demonstrate Two Physiological States: Insulin Responsive or Insulin Refractory

Human Subcutaneous Adipose Tissue Adipocytes Demonstrate Two Physiological States: Insulin Responsive or Insulin Refractory

Isolation, culture and identification of human adipose-derived stem cells.

The aim of the present study was to improve methods for the isolation and identification of adipose-derived stem cells (ASCs). Human subcutaneous adipose tissue was collected during liposuction surgery, without ultrasound-assisted liposuction and other assisted techniques, and digested with 0.075% collagenase I. First (P1) and second (P2) passage ASCs were applied to the subsequent experiments. ASCs were observed under a microscope, the growth curves of the cells were assessed using a cell counting kit-8 assay and the membrane expression of cell surface antigens, including cluster of differentiation (CD)44, CD105 and CD45, were detected by flow cytometry. In addition, ASCs were induced to differentiate into lipocytes and osteocytes. Oil red staining was applied to examine adipogenic induction, whereas alkaline phosphatase (ALP) staining was used to assess osteogenic induction. Primary ASCs adhered to the culture vessel wall after 72 h, were fusiform in appearance at 5 days and exhibited stable growth with active proliferation. In total, 1×105 stem cells were gained per 50 ml of lipo-aspirate. ASCs were plated in a 25 cm2 culture flask at a density of 5×104/ml; the cells underwent the first logarithmic growth period after 72 h and grew to 90% confluence within 3 days. Flow cytometry demonstrated that the cells were highly positive for CD105 and CD44, and weakly positive for CD45; 18.6% of P1 cells and 90.7% of P2 cells were CD44+CD45-CD105+. Oil red and ALP staining were positive. The results of the present study suggested that ASCs may be considered a promising cell type for tissue engineering. Furthermore, the present study established an effective method for the isolation and identification of ASCs, which reduced damage to the stem cells and simplified the identification procedure.

Glucose-dependent insulinotropic polypeptide promotes lipid deposition in subcutaneous adipocytes in obese type 2 diabetes patients: a maladaptive response.

Glucose-dependent insulinotropic polypeptide (GIP) beyond its insulinotropic effects may regulate postprandial lipid metabolism. Whereas the insulinotropic action of GIP is known to be impaired in type 2 diabetes mellitus (T2DM), its adipogenic effect is unknown. We hypothesized that GIP is anabolic in human subcutaneous adipose tissue (SAT) promoting triacylglycerol (TAG) deposition through reesterification of nonesterified fatty acids (NEFA), and this effect may differ according to obesity status or glucose tolerance. Twenty-three subjects categorized into four groups, normoglycemic lean (n = 6), normoglycemic obese (n = 6), obese with impaired glucose regulation (IGR; n = 6), and obese T2DM (n = 5), participated in a double-blind, randomized, crossover study involving a hyperglycemic clamp with a 240-min GIP infusion (2 pmol·kg-1·min-1) or normal saline. Insulin, NEFA, SAT-TAG content, and gene expression of key lipogenic enzymes were determined before and immediately after GIP/saline infusions. GIP lowered NEFA concentrations in the obese T2DM group despite diminished insulinotropic activity (mean NEFA AUC0-4 h ± SE, 41,992 ± 9,843 µmol·l-1·min-1 vs. 71,468 ± 13,605 with placebo, P = 0.039, 95% CI: 0.31-0.95). Additionally, GIP increased SAT-TAG in obese T2DM (1.78 ± 0.4 vs 0.86 ± 0.1-fold with placebo, P = 0.043, 95% CI: 0.1-1.8). Such effect with GIP was not observed in other three groups despite greater insulinotropic activity. Reduction in NEFA concentration with GIP correlated with adipose tissue insulin resistance for all subjects (Pearson, r = 0.56, P = 0.005). There were no significant gene expression changes in key SAT lipid metabolism enzymes. In conclusion, GIP appears to promote fat accretion and thus may exacerbate obesity and insulin resistance in T2DM.

Depot Dependent Effects of Dexamethasone on Gene Expression in Human Omental and Abdominal Subcutaneous Adipose Tissues from Obese Women.

Glucocorticoids promote fat accumulation in visceral compared to subcutaneous depots, but the molecular mechanisms involved remain poorly understood. To identify long-term changes in gene expression that are differentially sensitive or responsive to glucocorticoids in these depots, paired samples of human omental (Om) and abdominal subcutaneous (Abdsc) adipose tissues obtained from obese women during elective surgery were cultured with the glucocorticoid receptor agonist dexamethasone (Dex, 0, 1, 10, 25 and 1000 nM) for 7 days. Dex regulated 32% of the 19,741 genes on the array, while 53% differed by Depot and 2.5% exhibited a Depot*Dex concentration interaction. Gene set enrichment analysis showed Dex regulation of the expected metabolic and inflammatory pathways in both depots. Cluster analysis of the 460 transcripts that exhibited an interaction of Depot and Dex concentration revealed sets of mRNAs for which the responses to Dex differed in magnitude, sensitivity or direction between the two depots as well as mRNAs that responded to Dex only in one depot. These transcripts were also clearly depot different in fresh adipose tissue and are implicated in processes that could affect adipose tissue distribution or functions (e.g. adipogenesis, triacylglycerol synthesis and storage, insulin action). Elucidation of the mechanisms underlying the depot differences in the effect of Dex on the expression of specific genes and pathways that regulate adipose function may offer novel insights into understanding the biology of visceral adipose tissues and their links to metabolic health.