Effects of follicle-stimulating hormone on energy balance and tissue metabolic health after loss of ovarian function.

Loss of ovarian function imparts increased susceptibility to obesity and metabolic disease. These effects are largely attributed to decreased estradiol (E2), but the role of increased follicle-stimulating hormone (FSH) in modulating energy balance has not been fully investigated. Previous work that blocked FSH binding to its receptor in mice suggested this hormone may play a part in modulating body weight and energy expenditure after ovariectomy (OVX). We used an alternate approach to isolate the individual and combined contributions of FSH and E2 in mediating energy imbalance and changes in tissue-level metabolic health. Female Wistar rats were ovariectomized and given the gonadotropin releasing hormone (GnRH) antagonist degarelix to suppress FSH production. E2 and FSH were then added back individually and in combination for a period of 3 wk. Energy balance, body mass composition, and transcriptomic profiles of individual tissues were obtained. In contrast to previous studies, suppression and replacement of FSH in our paradigm had no effect on body weight, body composition, food intake, or energy expenditure. We did, however, observe organ-specific effects of FSH that produced unique transcriptomic signatures of FSH in retroperitoneal white adipose tissue. These included reductions in biological processes related to lipogenesis and carbohydrate transport. In addition, rats administered FSH had reduced liver triglyceride concentration (P < 0.001), which correlated with FSH-induced changes at the transcriptomic level. Although not appearing to modulate energy balance after loss of ovarian function in rats, FSH may still impart tissue-specific effects in the liver and white adipose tissue that might affect the metabolic health of those organs.NEW & NOTEWORTHY We find no effect of follicle-stimulating hormone (FSH) on energy balance using a novel model in which rats are ovariectomized, subjected to gonadotropin-releasing hormone antagonism, and systematically given back FSH by osmotic pump. However, tissue-specific effects of FSH on adipose tissue and liver were observed in this study. These include unique transcriptomic signatures induced by the hormone and a stark reduction in hepatic triglyceride accumulation.

Hematopoietic Stem Cell-Derived Adipocytes Modulate Adipose Tissue Cellularity, Leptin Production and Insulin Responsiveness in Female Mice.

A subpopulation of adipocytes in the major adipose depots of mice is produced from hematopoietic stem cells rather than mesenchymal progenitors that are the source of conventional white and brown/beige adipocytes. To analyze the impact of hematopoietic stem cell-derived adipocytes (HSCDAs) in the adipose niche we transplanted HSCs in which expression of a diphtheria toxin gene was under the control of the adipocyte-specific adiponectin gene promoter into irradiated wild type recipients. Thus, only adipocytes produced from HSC would be ablated while conventional white and brown adipocytes produced from mesenchymal progenitor cells would be spared. Wild type mice transplanted with HSCs from mice containing a reporter gene, but not the diphtheria toxin gene, regulated by the adiponectin gene promoter served as controls. In mice in which HSCDA production was suppressed, adipocyte size declined while adipose depot weights were unchanged and the number of conventional adipocyte progenitors significantly increased. We also measured a paradoxical increase in circulating leptin levels while physical activity was significantly decreased in the HSCDA depleted mice. Finally, insulin sensitivity was significantly reduced in HSCDA depleted mice. In contrast, loss of HSCDA production had no effect on body weight, components of energy balance, or levels of several circulating adipokines and tissue-resident inflammatory cells. These data indicate that ablation of this low-abundance subpopulation of adipocytes is associated with changes in circulating leptin levels and leptin-regulated endpoints associated with adipose tissue function. How they do so remains a mystery, but our results highlight the need for additional studies to explore the role of HSCDAs in other physiologic contexts such as obesity, metabolic dysfunction or loss of sex hormone production.

Metabolite G-Protein Coupled Receptors in Cardio-Metabolic Diseases.

G protein-coupled receptors (GPCRs) have originally been described as a family of receptors activated by hormones, neurotransmitters, and other mediators. However, in recent years GPCRs have shown to bind endogenous metabolites, which serve functions other than as signaling mediators. These receptors respond to fatty acids, mono- and disaccharides, amino acids, or various intermediates and products of metabolism, including ketone bodies, lactate, succinate, or bile acids. Given that many of these metabolic processes are dysregulated under pathological conditions, including diabetes, dyslipidemia, and obesity, receptors of endogenous metabolites have also been recognized as potential drug targets to prevent and/or treat metabolic and cardiovascular diseases. This review describes G protein-coupled receptors activated by endogenous metabolites and summarizes their physiological, pathophysiological, and potential pharmacological roles.

Hematopoietic stem cells produce intermediate lineage adipocyte progenitors that simultaneously express both myeloid and mesenchymal lineage markers in adipose tissue.

Some adipocytes are produced from bone marrow hematopoietic stem cells. In vitro studies previously indicated that these bone marrow-derived adipocytes (BMDAs) were generated from adipose tissue macrophage (ATM) that lose their hematopoietic markers and acquire mesenchymal markers prior to terminal adipogenic differentiation. Here we interrogated whether this hematopoietic-to-mesenchymal transition drives BMDA production In vitro. We generated transgenic mice in which the lysozyme gene promoter (LysM) indelibly labeled ATM with green fluorescent protein (GFP). We discovered that adipose stroma contained a population of LysM-positive myeloid cells that simultaneously expressed hematopoietic/myeloid markers (CD45 and CD11b), and mesenchymal markers (CD29, PDGFRa and Sca-1) typically found on conventional adipocyte progenitors. These cells were capable of adipogenic differentiation In vitro and In vitro, while other stromal populations deficient in PDGFRa and Sca-1 were non-adipogenic. BMDAs and conventional adipocytes expressed common fat cell markers but exhibited little or no expression of hematopoietic and mesenchymal progenitor cell markers. The data indicate that BMDAs are produced from ATM simultaneously expressing hematopoietic and mesenchymal markers rather than via a stepwise hematopoietic-to-mesenchymal transition. Because BMDA production is stimulated by high fat feeding, their production from hematopoietic progenitors may maintain adipocyte production when conventional adipocyte precursors are diminished.

Resident mesenchymal vascular progenitors modulate adaptive angiogenesis and pulmonary remodeling via regulation of canonical Wnt signaling.

Adaptive angiogenesis is necessary for tissue repair, however, it may also be associated with the exacerbation of injury and development of chronic disease. In these studies, we demonstrate that lung mesenchymal vascular progenitor cells (MVPC) modulate adaptive angiogenesis via lineage trace, depletion of MVPC, and modulation of ß-catenin expression. Single cell sequencing confirmed MVPC as multipotential vascular progenitors, thus, genetic depletion resulted in alveolar simplification with reduced adaptive angiogenesis. Following vascular endothelial injury, Wnt activation in MVPC was sufficient to elicit an emphysema-like phenotype characterized by increased MLI, fibrosis, and MVPC driven adaptive angiogenesis. Lastly, activation of Wnt/ß-catenin signaling skewed the profile of human and murine MVPC toward an adaptive phenotype. These data suggest that lung MVPC drive angiogenesis in response to injury and regulate the microvascular niche as well as subsequent distal lung tissue architecture via Wnt signaling.

CREB depletion in smooth muscle cells promotes medial thickening, adventitial fibrosis and elicits pulmonary hypertension.

Levels of the cAMP-responsive transcription factor, CREB, are reduced in medial smooth muscle cells in remodeled pulmonary arteries from hypertensive calves and rats with chronic hypoxia-induced pulmonary hypertension. Here, we show that chronic hypoxia fails to promote CREB depletion in pulmonary artery smooth muscle cells or elicit significant remodeling of the pulmonary arteries in mice, suggesting that sustained CREB expression prevents hypoxia-induced pulmonary artery remodeling. This hypothesis was tested by generating mice, in which CREB was ablated in smooth muscle cells. Loss of CREB in smooth muscle cells stimulated pulmonary artery thickening, right ventricular hypertrophy, profound adventitial collagen deposition, recruitment of myeloid cells to the adventitia, and elevated right ventricular systolic pressure without exposure to chronic hypoxia. Isolated murine CREB-null smooth muscle cells exhibited serum-independent proliferation and hypertrophy in vitro and medium conditioned by CREB-null smooth muscle cells stimulated proliferation and expression of extracellular matrix proteins by adventitial fibroblasts. We conclude that CREB governs the pathologic switch from homeostatic, quiescent smooth muscle cells to proliferative, synthetic cells that drive arterial remodeling contributing to the development or pulmonary hypertension.

Inactivation of Tsc2 in Abcg2 lineage-derived cells drives the appearance of polycystic lesions and fibrosis in the adult kidney.

Tuberous sclerosis complex 2 (TSC2), or tuberin, is a pivotal regulator of the mechanistic target of rapamycin signaling pathway that controls cell survival, proliferation, growth, and migration. Loss of Tsc2 function manifests in organ-specific consequences, the mechanisms of which remain incompletely understood. Recent single cell analysis of the kidney has identified ATP-binding cassette G2 (Abcg2) expression in renal proximal tubules of adult mice as well as a in a novel cell population. The impact in adult kidney of Tsc2 knockdown in the Abcg2-expressing lineage has not been evaluated. We engineered an inducible system in which expression of truncated Tsc2, lacking exons 36-37 with an intact 3' region and polycystin 1, is driven by Abcg2. Here, we demonstrate that selective expression of Tsc2fl36-37 in the Abcg2pos lineage drives recombination in proximal tubule epithelial and rare perivascular mesenchymal cells, which results in progressive proximal tubule injury, impaired kidney function, formation of cystic lesions, and fibrosis in adult mice. These data illustrate the critical importance of Tsc2 function in the Abcg2-expressing proximal tubule epithelium and mesenchyme during the development of cystic lesions and remodeling of kidney parenchyma.

Modulation of Energy Expenditure by Estrogens and Exercise in Women.

Reducing estrogen in women results in decreases in energy expenditure, but the mechanism(s) remain largely unknown. We postulate that the loss of estrogens in women is associated with increased accumulation of bone marrow-derived adipocytes in white adipose tissue, decreased activity of brown adipose tissue, and reduced levels of physical activity. Regular exercise may counteract the effects of estrogen deficiency.

Ovarian Hormones Regulate the Production of Adipocytes From Bone Marrow-Derived Cells.

Sex differences in body fat distribution and menopause-associated shifts in regional adiposity suggest that sex hormones play an important role in regulating the differentiation and distribution of adipocytes, but the underlying mechanisms have not been fully explained. The aim of this study was to determine whether ovarian hormone status influences the production and distribution of adipocytes in adipose tissue arising from bone marrow-derived cells. Nine- to ten-week-old ovariectomized (OVX), surgery naïve (WT), and estrogen receptor alpha knockout (αERKO) mice underwent bone marrow transplantation from luciferase or green fluorescent protein expressing donors. A subset of OVX animals had estradiol (E2) added back. Eight-weeks posttransplant, whole body and gonadal fat BM-derived adipocyte production was highest in OVX and αERKO mice, which was attenuated in OVX mice by E2 add-back. All groups demonstrated the highest bone marrow derived adipocyte (BMDA) production in the gonadal adipose depot, a visceral fat depot in mice. Taken together, the loss of ovarian hormones increases the production of BMDAs. If translatable across species, production of BMDA may be a mechanism by which visceral adiposity increases in estrogen-deficient postmenopausal women.

Disruption of lineage specification in adult pulmonary mesenchymal progenitor cells promotes microvascular dysfunction.

Pulmonary vascular disease is characterized by remodeling and loss of microvessels and is typically attributed to pathological responses in vascular endothelium or abnormal smooth muscle cell phenotypes. We have challenged this understanding by defining an adult pulmonary mesenchymal progenitor cell (MPC) that regulates both microvascular function and angiogenesis. The current understanding of adult MPCs and their roles in homeostasis versus disease has been limited by a lack of genetic markers with which to lineage label multipotent mesenchyme and trace the differentiation of these MPCs into vascular lineages. Here, we have shown that lineage-labeled lung MPCs expressing the ATP-binding cassette protein ABCG2 (ABCG2+) are pericyte progenitors that participate in microvascular homeostasis as well as adaptive angiogenesis. Activation of Wnt/ß-catenin signaling, either autonomously or downstream of decreased BMP receptor signaling, enhanced ABCG2+ MPC proliferation but suppressed MPC differentiation into a functional pericyte lineage. Thus, enhanced Wnt/ß-catenin signaling in ABCG2+ MPCs drives a phenotype of persistent microvascular dysfunction, abnormal angiogenesis, and subsequent exacerbation of bleomycin-induced fibrosis. ABCG2+ MPCs may, therefore, account in part for the aberrant microvessel function and remodeling that are associated with chronic lung diseases.

Hematopoietic-to-mesenchymal transition of adipose tissue macrophages is regulated by integrin ß1 and fabricated fibrin matrices.

Some bona fide adult adipocytes arise de novo from a bone marrow-derived myeloid lineage. These studies further demonstrate that adipose tissue stroma contains a resident population of myeloid cells capable of adipocyte and multilineage mesenchymal differentiation. These resident myeloid cells lack hematopoietic markers and express mesenchymal and progenitor cell markers. Because bone marrow mesenchymal progenitor cells have not been shown to enter the circulation, we hypothesized that myeloid cells acquire mesenchymal differentiation capacity in adipose tissue. We fabricated a 3-dimensional fibrin matrix culture system to define the adipose differentiation potential of adipose tissue-resident myeloid subpopulations, including macrophages, granulocytes and dendritic cells. Our data show that multilineage mesenchymal potential was limited to adipose tissue macrophages, characterized by the acquisition of adipocyte, osteoblast, chondrocyte and skeletal muscle myocyte phenotypes. Fibrin hydrogel matrices stimulated macrophage loss of hematopoietic cell lineage determinants and the expression of mesenchymal and progenitor cell markers, including integrin ß1. Ablation of integrin ß1 in macrophages inhibited adipocyte specification. Therefore, some bona fide adipocytes are specifically derived from adipose tissue-resident macrophages via an integrin ß1-dependent hematopoietic-to-mesenchymal transition, whereby they become capable of multipotent mesenchymal differentiation. The requirement for integrin ß1 highlights this molecule as a potential target for controlling the production of marrow-derived adipocytes and their contribution to adipose tissue development and function.

Leukemic Stem Cells Evade Chemotherapy by Metabolic Adaptation to an Adipose Tissue Niche.

Adipose tissue (AT) has previously been identified as an extra-medullary reservoir for normal hematopoietic stem cells (HSCs) and may promote tumor development. Here, we show that a subpopulation of leukemic stem cells (LSCs) can utilize gonadal adipose tissue (GAT) as a niche to support their metabolism and evade chemotherapy. In a mouse model of blast crisis chronic myeloid leukemia (CML), adipose-resident LSCs exhibit a pro-inflammatory phenotype and induce lipolysis in GAT. GAT lipolysis fuels fatty acid oxidation in LSCs, especially within a subpopulation expressing the fatty acid transporter CD36. CD36(+) LSCs have unique metabolic properties, are strikingly enriched in AT, and are protected from chemotherapy by the GAT microenvironment. CD36 also marks a fraction of human blast crisis CML and acute myeloid leukemia (AML) cells with similar biological properties. These findings suggest striking interplay between leukemic cells and AT to create a unique microenvironment that supports the metabolic demands and survival of a distinct LSC subpopulation.

Differential Mitochondrial Adaptation in Primary Vascular Smooth Muscle Cells from a Diabetic Rat Model.

Diabetes affects more than 330 million people worldwide and causes elevated cardiovascular disease risk. Mitochondria are critical for vascular function, generate cellular reactive oxygen species (ROS), and are perturbed by diabetes, representing a novel target for therapeutics. We hypothesized that adaptive mitochondrial plasticity in response to nutrient stress would be impaired in diabetes cellular physiology via a nitric oxide synthase- (NOS-) mediated decrease in mitochondrial function. Primary smooth muscle cells (SMCs) from aorta of the nonobese, insulin resistant rat diabetes model Goto-Kakizaki (GK) and the Wistar control rat were exposed to high glucose (25 mM). At baseline, significantly greater nitric oxide evolution, ROS production, and respiratory control ratio (RCR) were observed in GK SMCs. Upon exposure to high glucose, expression of phosphorylated eNOS, uncoupled respiration, and expression of mitochondrial complexes I, II, III, and V were significantly decreased in GK SMCs (p < 0.05). Mitochondrial superoxide increased with high glucose in Wistar SMCs (p < 0.05) with no change in the GK beyond elevated baseline concentrations. Baseline comparisons show persistent metabolic perturbations in a diabetes phenotype. Overall, nutrient stress in GK SMCs caused a persistent decline in eNOS and mitochondrial function and disrupted mitochondrial plasticity, illustrating eNOS and mitochondria as potential therapeutic targets.

De novo generation of adipocytes from circulating progenitor cells in mouse and human adipose tissue.

White adipocytes in adults are typically derived from tissue resident mesenchymal progenitors. The recent identification of de novo production of adipocytes from bone marrow progenitor-derived cells in mice challenges this paradigm and indicates an alternative lineage specification that adipocytes exist. We hypothesized that alternative lineage specification of white adipocytes is also present in human adipose tissue. Bone marrow from transgenic mice in which luciferase expression is governed by the adipocyte-restricted adiponectin gene promoter was adoptively transferred to wild-type recipient mice. Light emission was quantitated in recipients by in vivo imaging and direct enzyme assay. Adipocytes were also obtained from human recipients of hematopoietic stem cell transplantation. DNA was isolated, and microsatellite polymorphisms were exploited to quantify donor/recipient chimerism. Luciferase emission was detected from major fat depots of transplanted mice. No light emission was observed from intestines, liver, or lungs. Up to 35% of adipocytes in humans were generated from donor marrow cells in the absence of cell fusion. Nontransplanted mice and stromal-vascular fraction samples were used as negative and positive controls for the mouse and human experiments, respectively. This study provides evidence for a nontissue resident origin of an adipocyte subpopulation in both mice and humans.

Shared gene expression patterns in mesenchymal progenitors derived from lung and epidermis in pulmonary arterial hypertension: identifying key pathways in pulmonary vascular disease.

Rapid access to lung-derived cells from stable subjects is a major challenge in the pulmonary hypertension field, given the relative contraindication of lung biopsy. In these studies, we sought to demonstrate the importance of evaluating a cell type that actively participates in disease processes, as well as the potential to translate these findings to vascular beds in other nonlung tissues, in this instance perivascular skin mesenchymal cells (MCs). We utilized posttransplant or autopsy lung explant-derived cells (ABCG2-expressing mesenchymal progenitor cells [MPCs], fibroblasts) and skin-derived MCs to test the hypothesis that perivascular ABCG2 MPCs derived from pulmonary arterial hypertension (PAH) patient lung and skin would express a gene profile reflective of ongoing vascular dysfunction. By analyzing the genetic signatures and pathways associated with abnormal ABCG2 lung MPC phenotypes during PAH and evaluating them in lung- and skin-derived MCs, we have identified potential predictor genes for detection of PAH as well as a targetable mechanism to restore MPCs and microvascular function. These studies are the first to explore the utility of expanding the study of ABCG2 MPC regulation of the pulmonary microvasculature to the epidermis, in order to identify potential markers for adult lung vascular disease, such as PAH.

Saxagliptin restores vascular mitochondrial exercise response in the Goto-Kakizaki rat.

Cardiovascular disease risk and all-cause mortality are largely predicted by physical fitness. Exercise stimulates vascular mitochondrial biogenesis through endothelial nitric oxide synthase (eNOS), sirtuins, and PPARγ coactivator 1α (PGC-1α), a response absent in diabetes and hypertension. We hypothesized that an agent regulating eNOS in the context of diabetes could reconstitute exercise-mediated signaling to mitochondrial biogenesis. Glucagon-like peptide 1 (GLP-1) stimulates eNOS and blood flow; we used saxagliptin, an inhibitor of GLP-1 degradation, to test whether vascular mitochondrial adaptation to exercise in diabetes could be restored. Goto-Kakizaki (GK) rats, a nonobese, type 2 diabetes model, and Wistar controls were exposed to an 8-day exercise intervention with or without saxagliptin (10 mg·kg·d). We evaluated the impact of exercise and saxagliptin on mitochondrial proteins and signaling pathways in aorta. Mitochondrial protein expression increased with exercise in the Wistar aorta and decreased or remained unchanged in the GK animals. GK rats treated with saxagliptin plus exercise showed increased expression of mitochondrial complexes, cytochrome c, eNOS, nNOS, PGC-1α, and UCP3 proteins. Notably, a 3-week saxagliptin plus exercise intervention significantly increased running time in the GK rats. These data suggest that saxagliptin restores vascular mitochondrial adaptation to exercise in a diabetic rodent model and may augment the impact of exercise on the vasculature.

ABCG2pos lung mesenchymal stem cells are a novel pericyte subpopulation that contributes to fibrotic remodeling.

Genesis of myofibroblasts is obligatory for the development of pathology in many adult lung diseases. Adult lung tissue contains a population of perivascular ABCG2(pos) mesenchymal stem cells (MSC) that are precursors of myofibroblasts and distinct from NG2 pericytes. We hypothesized that these MSC participate in deleterious remodeling associated with pulmonary fibrosis (PF) and associated hypertension (PH). To test this hypothesis, resident lung MSC were quantified in lung samples from control subjects and PF patients. ABCG2(pos) cell numbers were decreased in human PF and interstitial lung disease compared with control samples. Genetic labeling of lung MSC in mice enabled determination of terminal lineage and localization of ABCG2 cells following intratracheal administration of bleomycin to elicit fibrotic lung injury. Fourteen days following bleomycin injury enhanced green fluorescent protein (eGFP)-labeled lung MSC-derived cells were increased in number and localized to interstitial areas of fibrotic and microvessel remodeling. Finally, gene expression analysis was evaluated to define the response of MSC to bleomycin injury in vivo using ABCG2(pos) MSC isolated during the inflammatory phase postinjury and in vitro bleomycin or transforming growth factor-ß1 (TGF-ß1)-treated cells. MSC responded to bleomycin treatment in vivo with a profibrotic gene program that was not recapitulated in vitro with bleomycin treatment. However, TGF-ß1 treatment induced the appearance of a profibrotic myofibroblast phenotype in vitro. Additionally, when exposed to the profibrotic stimulus, TGF-ß1, ABCG2, and NG2 pericytes demonstrated distinct responses. Our data highlight ABCG2(pos) lung MSC as a novel cell population that contributes to detrimental myofibroblast-mediated remodeling during PF.

Identification of a common Wnt-associated genetic signature across multiple cell types in pulmonary arterial hypertension.

Understanding differences in gene expression that increase risk for pulmonary arterial hypertension (PAH) is essential to understanding the molecular basis for disease. Previous studies on patient samples were limited by end-stage disease effects or by use of nonadherent cells, which are not ideal to model vascular cells in vivo. These studies addressed the hypothesis that pathological processes associated with PAH may be identified via a genetic signature common across multiple cell types. Expression array experiments were initially conducted to analyze cell types at different stages of vascular differentiation (mesenchymal stromal and endothelial) derived from PAH patient-specific induced pluripotent stem (iPS) cells. Molecular pathways that were altered in the PAH cell lines were then compared with those in fibroblasts from 21 patients, including those with idiopathic and heritable PAH. Wnt was identified as a target pathway and was validated in vitro using primary patient mesenchymal and endothelial cells. Taken together, our data suggest that the molecular lesions that cause PAH are present in all cell types evaluated, regardless of origin, and that stimulation of the Wnt signaling pathway was a common molecular defect in both heritable and idiopathic PAH.

Targeting mitochondria to restore failed adaptation to exercise in diabetes.

Our translational research group focuses on addressing the problem of exercise defects in diabetes with basic research efforts in cell and rodent models and clinical research efforts in subjects with diabetes mellitus. CREB (cAMP-response-element-binding protein) regulates cellular differentiation of neurons, ß-cells, adipocytes and smooth muscle cells; it is also a potent survival factor and an upstream regulator of mitochondrial biogenesis. In diabetes and cardiovascular disease, CREB protein content is decreased in the vascular media, and its regulation in aberrant in ß-cells, neurons and cardiomyocytes. Loss of CREB content and function leads to decreased vascular target tissue resilience when exposed to stressors such as metabolic, oxidative or sheer stress. This basic research programme set the stage for our central hypothesis that diabetes-mediated CREB dysfunction predisposes the diabetes disease progression and cardiovascular complications. Our clinical research programme revealed that diabetes mellitus leads to defects in functional exercise capacity. Our group has determined that the defects in exercise correlate with insulin resistance, endothelial dysfunction, decreased cardiac perfusion and diastolic dysfunction, slowed muscle perfusion kinetics, decreased muscle perfusion and slowed oxidative phosphorylation. Combined basic and clinical research has defined the relationship between exercise and vascular function with particular emphasis on how the signalling to CREB and eNOS [endothelial NOS (nitric oxide synthase)] regulates tissue perfusion, mitochondrial dynamics, vascular function and exercise capacity. The present review summarizes our current working hypothesis that restoration of eNOS/NOS dysfunction will restore cellular homoeostasis and permit an optimal tissue response to an exercise training intervention.

Analysis and isolation of adipocytes by flow cytometry.

Analysis and isolation of adipocytes via flow cytometry is particularly useful to study their biology. However, the adoption of this technology has often been hampered by the presence of stromal/vascular cells in adipocyte fractions prepared from collagenase-digested adipose tissue. Here, we describe a multistep staining method and gating strategy that effectively excludes stromal contaminants. Initially, we set a gate optimized to the size and internal complexity of adipocytes. Exclusion of cell aggregates is then performed based on fluorescence of a nuclear stain followed by positive selection to collect only those cell events containing lipid droplets. Lastly, negative selection of cells expressing stromal or vascular lineage markers removes any remaining stromal contaminants. These procedures are applicable to simple analysis of adipocytes and their subcellular constituents by flow cytometry as well as isolation of adipocytes by flow sorting.