Differential Effects of Chronic Binge Alcohol and Ovariectomy on Apoptosis in Omental Adipose Tissue of SIV‐infected Macaques
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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.Adipose tissue macrophages
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The cellular growth of adipose tissue from Dorset Horn x Merino wethers was characterized by both hypertrophy and hyperplasia of adipose cells until the sheep were c. 11 months old, after which hypertrophy of existing adipose cells was solely responsible for increases in adipose tissue mass. Omental adipose tissue contained larger adipose cells than either the perirenal or subcutaneous sites. The weight of fat which had been deposited in the boneless carcass meat and the internal adipose tissue depots were linearly and positively correlated with the volume of the adipose cells. Decreases in the mass of adipose tissue, which accompanied nutritional restriction, were due to decreased adipose cell size, since no change was observed in the number of adipose cells per carcass after loss in weight. The cellularity characteristics of rehabilitated sheep were similar to those of sheep which had undergone continuous growth.
3T3-L1
Adipose tissue macrophages
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Adipose tissue can be found throughout the body but is primarily distributed in several subcutaneous and visceral locations or “depots” (1). The general distribution pattern and location of adipose tissue depots and the proportion of total body fat that each depot represents varies widely between species. In humans and swine the subcutaneous depot represents a much larger proportion of total adipose tissue than in rodents. In fact, several distinct layers of subcutaneous adipose tissue are present in humans and swine. Excessive adipose tissue accumulation in humans is often associated with the adverse health consequences of the metabolic syndrome (2). An adipose tissue distribution pattern favoring visceral adipose tissue often is implicated in this syndrome.
Subcutaneous adipose tissue
Subcutaneous fat
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GH has profound effects on the amount and distribution of adipose tissue. GHD in both children and adults is accompanied by an increased amount of adipose tissue and by the abdominal predominance of adipose tissue. In contrast, treatment with GH reduces adipose tissue, and redistributes adipose tissue from abdominal to peripheral depots.
Abdominal fat
Fat distribution
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The purpose of the present study was to correlate the type and frequency of cell death in human lymphocytes receiving variable doses of X-irradiation. Monocyte-depleted lymphocyte fractions were exposed in vitro to variable doses of X-rays of 0-20 Gy (0-2000 rads) and incubated for 4 and 16 h. An assessment of the mode of cell death (apoptosis vs. classical necrosis) was carefully evaluated using a multidisciplinary approach using light, fluorescence and electron microscopy (EM), and dye exclusion assays. Eosin Y exclusion assays indicated the absence of classical necrosis occurring in short-term cultures (4 h postirradiation). An assessment of cell counts, however, revealed a mean decrease of 4% at 0 Gy and 13% at 10-20 Gy (1000-2000 rads). The predominant mode of cell death was apoptosis, but the percent apop- totic cells (determined by EM) did not parallel this increase in cell loss with increasing radiation and actually decreased at doses above 5 Gy (500 rads). The discrepancy between percent cell loss and percent apoptosis was explained by a proposed change in overall duration of the apoptotic process. In long-term cultures (16 h postirradiation), a combination of classical necrosis, classical apoptosis, and combined apoptosis and necrosis (secondary necrosis of apoptotic cells) was apparent and was associated with a marked decrease in viability. Irradiation effects on lymphocytes showing none of the morphologic features of apoptosis or classical necrosis in short-term culture were evidenced by an increase in nuclear lobation. The results of this study indicate that the vast majority of peripheral blood lymphocytes are radioresistant. The use of irradiation in an in vitro model to study the biochemical events of the apoptotic process is also evaluated.
Viability assay
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Syngeneic adipose tissue grafts showed evidence of necrosis or lack of vascularization in intact, adult (BALB/c/Ki x Ce/Ki) F1 hybrid mice between 2 and 9 months after transplantation. One or more fat depots were surgically removed to produce a deficit in the total adipose tissue mass, and this resulted in an increased number of vascularized and viable adipose tissue grafts in similar genetic hosts during a comparable period. The anatomically dispersed fat depots appear to be under some form of self-regulation and integrated into a total adipose tissue mass.
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To assess the efficacy of cancer chemotherapy, an important index is apoptosis of the target cells, which can usually be confirmed by electron microscopy (EM). We established a new experimental technique, whereby cancer cells (MKN45) were distributed in thin collagen gel as one or two cell layers, and cultured with anti‐cancer drugs (5‐FU and CDDP). The cells were stained with fluorescent Hoechst 33258 (Ho) and photographed, then with hematoxylin and eosin (H&E) and again photographed, and processed for EM. This approach allowed us to characterize the patterns of death of single cells in detail. There were six patterns of cell damage: two patterns of apoptosis, early peripheral condensation of chromatin and late apoptotic bodies, two patterns of necrosis, cytoplasmic swelling and washed‐out images, and two further patterns, with morphological features of both apoptosis and necrosis, neither classified into necrosis nor apoptosis. The results show that cell death patterns can be mostly determined by combining observations of Ho and H&E‐stained cells without the necessity for EM observation.
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The nationally-recognized Susquehanna
Chorale will delight audiences of all
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Objective To study the changes in apoptosis and necrosis of EL\|4 cells following irradiation with different doses of X\|rays. Methods The cells were stained by PI and Hoechst 33342 six hours after irradiation,and the changes in apoptosis and necrosis were analyzed by flow cytometry. Results It was found that apoptosis and necrosis increased significantly after high dose irradiation. Conclusion The results indicate that irradiation with certain doses can not only induce apoptosis but also cause necrosis at the same time.
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To study the protective effects of ecdysterone (EDS) on the apoptosis and necrosis of cultured HUVEC induced by endotoxin.HUVEC was cultured in vitro. Apoptosis and necrosis of HUVEC were induced by LPS, and EDS was added in test specimen. Apoptosis was determined by means of flow cytometry and fluorescent microscopy.There was relatively less number of apoptosis and necrosis of in vitro cultured normal HUVEC than that after LPS stimulation, whereas apoptosis dominated over necrosis. After EDS was added to the culture of HUVEC, cell apoptosis and necrosis decreased simultaneously.EDS might exert protective effects on cultured HUVEC against apoptosis and necrosis induced by LPS.
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