Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) provide a consistent ex vivo cellular system that are functionally like that of in vivo adipocytes. Use of ADSCs in biomedical research allows for cellular investigation of adipose tissue metabolic regulation and function. ADSC differentiation is necessary for adequate adipocyte expansion, and suboptimal differentiation is a major mechanism of adipose dysfunction. Understanding changes in ADSC differentiation is crucial to understanding the development of metabolic dysfunction and disease. The protocols described in this manuscript, when followed, will yield mature adipocytes that can be used for several in vitro functional tests to assess ADSC metabolic function, including but not limited to assays measuring glucose uptake, lipolysis, lipogenesis, and secretion. Rhesus macaques (Macaca mulatta) are physiologically, anatomically, and evolutionarily similar to humans and as such, their tissues and cells have been used extensively in biomedical research and for development of treatments. Here, we describe ADSC isolation using fresh subcutaneous and omental adipose tissue obtained from 4–9-year old rhesus macaques. Adipose tissue samples are enzymatically digested in collagenase followed by filtration and centrifugation to isolate ADSCs from the stromal vascular fraction. Isolated ADSCs are proliferated in stromal media followed by approximately 14–21 days of differentiation using a cocktail of 0.5 μg/mL dexamethasone, 0.5 mM isobutyl methylxanthine, and 50 μM indomethacin in stromal media. Mature adipocytes are observed at approximately 14 days of differentiation. In this manuscript, we describe protocols for ADSC isolation, proliferation, and differentiation in vitro. Although, we have focused on ADSCs from rhesus macaque adipose tissue, these protocols can be utilized for adipose tissue obtained from other animals with minimal adjustments.
Adipose tissue maladaptations including cell death, inflammation, adipokine disruption, dysfunction of adipocytes and adipose tissue metabolism are central to metabolic alterations in HIV, at-risk alcohol use, and aging. Though adipose tissue dysregulation is a well-accepted mechanism meditating metabolic comorbidities in HIV, the effects of alcohol use and estrogen loss on adipose tissue cell death are not fully understood. Using a relevant preclinical model of HIV-infection, the aim of our study was to investigate the differential effects of chronic binge alcohol (CBA) administration and ovarian hormone loss, simulated by ovariectomy (OVX), on alterations in omental adipose tissue (OmAT) expression of genes involved in cell death. CBA or isovolumetric water (VEH) was administered through an intragastric catheter at a concentration of 30% (w/v) for 30 minutes, 5 days a week, with blood alcohol concentrations reaching 50-60 mM. Three months after initiation of CBA/VEH administration, macaques were infected with SIVMac251 and 2.5 months later initiated on ART. After 1 month of ART, macaques underwent sham surgery or ovariectomy. Unbiased quantitative proteomic analysis of OmAT revealed that proteins implicated in the apoptotic pathway were differentially regulated by CBA, OVX and the combination of both. Specifically, CBA and OVX independently led to proteome changes consistent with activation of apoptosis and necrosis pathways while the combination of CBA and OVX led to proteome changes consistent with inhibition of both apoptosis and necrosis. To confirm these results, OmAT expression of genes involved in apoptosis was determined using quantitative PCR. B-cell lymphoma 2 (BCL2), an antiapoptotic gene was increased by CBA but decreased in the CBA/OVX group. CBA increased the gene expression of BCL-2 homologous antagonist killer (BAK), a proapoptotic gene and increased the ratio of BCL2 associated X protein (BAX) to BCL2, an indicator of cell susceptibility to apoptosis. Our quantitative PCR results confirm and support the results of the OmAT proteomic analysis, suggesting that CBA and OVX differentially regulate the apoptotic pathway. However, in contrast to the results from the proteomic analysis, the preliminary gene expression data suggest that CBA is a major driver of apoptosis in the OmAT of female rhesus macaques. Our ongoing studies using TUNEL staining and analysis of crown-like structures will determine the effects of CBA and OVX on OmAT cell death. Additionally, the functional effects of CBA and OVX on adipose-derived stem cell death will be determined using flow cytometry.
The incidence of at-risk alcohol use among people living with HIV (PLWH) is two times greater than in the general population. Though antiretroviral therapy (ART) has significantly reduced HIV-associated mortality, increased survival of PLWH on ART is associated with increased incidence of metabolic comorbidities. Adipose tissue maladaptation is central to metabolic alterations in PLWH and at-risk alcohol use. This involves adipokine disruption, adipocyte dysfunction, inflammation, and dysregulation of the extracellular milieu. Using a relevant preclinical model of HIV-infection, the objective of our study was to investigate the effects of chronic binge alcohol (CBA) administration on omental adipose tissue (OmAT) and subcutaneous adipose tissue (SAT) extracellular matrix (ECM) phenotype. Rhesus macaques were administered CBA or isovolumetric water (VEH) through an intragastric catheter for 30 min, 5 days a week, with blood alcohol concentrations reaching 50-60 mM. Three months after initiation of CBA or VEH administration, macaques were inoculated with SIVMac251, and 2.5 months later animals were initiated on ART. CBA or VEH administration was continued throughout the duration of the study (14.5 mo). Animals were euthanized at 11.5 mo of SIV infection, and adipose (OmAT and SAT) samples excised. Collagen content, adipocyte diameter and adipocyte numbers were measured by Picrosirius Red Staining (PSR). A custom PCR Array was used to assess differential expression of 41 ECM-related genes. CBA administration significantly increased collagen expression and decreased adipocyte diameter in the OmAT without altering adipocyte numbers. There was a strong negative correlation of OmAT adipocyte diameter and collagen expression. CBA administration did not result in significant differences in collagen expression, adipocyte diameter or number in SAT. CBA administration significantly upregulated expression of transforming growth factor beta-1 (TGFB1), platelet-derived growth factor receptor alpha (PDGFRA) and secreted protein acidic and rich in cysteine (SPARC), mediators of a pro-fibrotic milieu in OmAT. In contrast, CBA did not significantly alter expression of ECM-related genes in the SAT. These data suggest that the maladaptive adipose changes associated with CBA in SIV infection are depot specific. We speculate that changes in the ECM contribute to altered gene expression and impaired adipocyte metabolic capacity.
Effective antiretroviral therapy (ART) has significantly reduced mortality of people living with HIV (PLWH), and the prevalence of at-risk alcohol use is higher among PLWH. Increased survival and aging of PLWH is associated with increased prevalence of metabolic comorbidities especially among menopausal women, and adipose tissue metabolic dysregulation may be a significant contributing factor. We examined the differential effects of chronic binge alcohol (CBA) administration and ovariectomy (OVX) on the omental adipose tissue (OmAT) proteome in a subset of simian immunodeficiency virus (SIV)-infected macaques of a longitudinal parent study. Quantitative discovery-based proteomics identified 1,429 differentially expressed proteins. Ingenuity Pathway Analysis (IPA) was used to calculate z-scores, or activation predictions, for functional pathways and diseases. Results revealed that protein changes associated with functional pathways centered around the "OmAT metaboproteome profile." Based on z-scores, CBA did not affect functional pathways of metabolic disease but dysregulated proteins involved in adenosine monophosphate-activated protein kinase (AMPK) signaling and lipid metabolism. OVX-mediated proteome changes were predicted to promote pathways involved in glucose- and lipid-associated metabolic disease. Proteins involved in apoptosis, necrosis, and reactive oxygen species (ROS) pathways were also predicted to be activated by OVX and these were predicted to be inhibited by CBA. These results provide evidence for the role of ovarian hormone loss in mediating OmAT metaboproteome dysregulation in SIV and suggest that CBA modifies OVX-associated changes. In the context of OVX, CBA administration produced larger metabolic and cellular effects, which we speculate may reflect a protective role of estrogen against CBA-mediated adipose tissue injury in female SIV-infected macaques.
People with HIV (PWH) are at increased risk for cardiometabolic comorbidities. We have reported that lifetime alcohol use among people with HIV (PWH) is associated with increased risk for metabolic syndrome. Dysfunctional adipose tissue and altered circulating adipokines mediate metabolic dysregulation. The objective of this study was to determine the associations of circulating adipokine concentration with metabolic measures, and the moderating effects of lifetime and recent alcohol use in PWH.
Abstract Background Increased survival, prolonged antiretroviral treatment (ART), and lifestyle choices, including alcohol misuse, increase the risk for comorbid conditions, including cardiometabolic comorbidities among people with HIV (PWH). Published studies indicate that dysregulated adipose tissue phenotype, particularly of the visceral adipose depot, contributes to metabolic dysregulation. Using a nonhuman primate model of simian immunodeficiency virus (SIV) infection, we previously demonstrated that chronic binge alcohol (CBA) administration to ART‐treated rhesus macaques decreases whole‐body glucose‐insulin dynamics, increases omental adipose tissue (OmAT) collagen content, decreases OmAT adipocyte size, and alters pancreatic endocrine function. The objective of this study was to delineate the depot‐specific effects of CBA on visceral (VAT) and subcutaneous adipose tissue (SAT) extracellular matrix (ECM) phenotype, the potential mechanisms involved in AT ECM remodeling, and the implications of increased tissue stiffness on AT metabolic alterations in female SIV‐infected macaques. Methods Omental and subcutaneous adipose samples were obtained from female SIV‐infected, ART‐treated macaques that received intragastric administration of CBA (12–15 g/kg/week, CBA/SIV) or water (VEH/SIV) for 14.5 months. Results CBA preferentially altered the ECM phenotype in OmAT, a VAT depot. The CBA‐associated changes included increased ECM accumulation, increased collagen I–III ratio, a profibrotic milieu, and decreased matrix metalloproteinase 13 activity. These changes were associated with smaller adipocyte size, decreased triglyceride content, decreased gene expression of perilipins, and a potential dysregulation of peroxisome proliferator‐activated receptor gamma signaling. Conclusions Collectively, these findings suggest that CBA‐mediated ECM remodeling “traps” adipocytes within a stiff environment that we propose disrupts adipocyte metabolic programming and may increase the risk for metabolic comorbidities.
