Apoptosis In Adipose Tissue Research Articles

Hinokiflavone resists HFD-induced obesity by promoting apoptosis in an IGF2BP2-mediated Bim m6A modification dependent manner

Obesity has emerged as a major health risk on a global scale. Hinokiflavone (HF), a natural small molecule, extracted from plants like cypress, exhibits diverse chemical structures and low synthesis costs. Using high-fat diet (HFD)-induced obese mice models, we found that HF suppresses obesity by inducing apoptosis in adipose tissue. Adipocyte apoptosis helps maintain tissue health by removing aging, damaged, or excess cells in adipose tissue, which is crucial in preventing obesity and metabolic diseases. We found that HF can specifically bind to insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) to promote the stability of N6-methyladenosine (m6A) -modified Bim, inducing mitochondrial outer membrane permeabilization (MOMP). MOMP leads to Caspase9/3-mediated adipocyte mitochondrial apoptosis, alleviating obesity induced by a high-fat diet. The pro-apoptotic effect of HF offers a controlled means for weight loss. This study reveals the potential of small molecule HF in developing new therapeutic approaches in drug development and biomedical research.

Adipocyte-specific FAK deletion promotes pancreatic β-cell apoptosis via adipose inflammatory response to exacerbate diabetes mellitus.

White adipose tissue (WAT) has a key role in maintaining energy balance throughout the body, and their dysfunction take part in the regulation of diabetes mellitus. However, the internal regulatory mechanisms underlying are still unknown. We generated adipocyte-specific FAK KO (FAK-AKO) mice and investigated their phenotype. The cascade of adipocyte, macrophage in adipocyte tissues, and pancreatic β-cells were proposed in FAK-AKO mice and validated by cell line studies using 3T3-L1, Raw264.7 and Min6. The FAK-AKO mice exhibited glucose intolerance, reduced adipose tissue mass and increased apoptosis, lipolysis and inflammatory response in adipose tissue. We further demonstrate that adipocyte FAK deletion increases β cell apoptosis and inflammatory infiltrates into islets, which is potentiated if mice were treated with STZ. In the STZ-induced diabetes model, FAK AKO mice exhibit less serum insulin content and pancreatic β cell area. Moreover, serum pro-inflammatory factors increased and insulin levels decreased after glucose stimulation in FAK AKO mice. In a parallel vitro experiment, knockdown or inhibition of FAK during differentiation also increased apoptosis, lipolysis and inflammatory in 3T3-L1 adipocytes, whereas the opposite was observed upon overexpression of FAK. Moreover, coculturing LPS-treated RAW264.7 macrophages with knockdown FAK of 3T3-L1 adipocytes increased macrophage pro-inflammatory response. Furthermore, conditioned medium from above stimulated Min6 cells apoptosis (with or without STZ), whereas the opposite was observed upon overexpression of FAK. Mechanistically, FAK protein interact with TRAF6 in adipocytes and knockdown or inhibition of FAK activated TRAF6/TAK1/NF-κB signaling, which exacerbates inflammation of adipocytes themselves. Adipocyte FAK deletion promotes both adipocyte apoptosis and adipose tissue inflammation. Pro-inflammatory factors released by the FAK-null adipose tissue further trigger apoptosis in pancreatic islets induced by the administration of STZ, thereby exacerbating the diabetes mellitus. This study reveals a link between FAK-mediated adipose inflammation and diabetes mellitus, a mechanism that has not been previously recognized.

Celastrol directly binds with VAMP7 and RAB7 to inhibit autophagy and induce apoptosis in preadipocytes.

Obesity is one of the most prevalent chronic metabolic diseases, and induction of apoptosis in preadipocytes and adipocytes is a potential strategy to treat obesity. Celastrol represents one of the most robust anti-obesity phytochemicals so far, yet its direct binding target remains elusive. Here, we determined that celastrol could induce apoptosis in preadipocytes via mitochondrial mediated pathway. Further study clarified that celastrol inhibited the fusion of autophagosome and lysosome to prohibit autophagy, leading to cell apoptosis. By conducting virtual screening and genetic manipulation, we verified that overexpression of VAMP7 and RAB7 could block the effects of celastrol on inhibiting autophagy and inducing apoptosis. The Surface Plasmon Resonance study confirmed the direct binding of celastrol with VAMP7 and RAB7. The functional study illustrated the inhibition of RAB7 GTPase activity after celastrol treatment. Moreover, celastrol induced comparable apoptosis in murine epididymal adipose tissue, human preadipocytes and adipocytes, but not in human hepatocytes. An inhibitory effect on differentiation of human primary visceral preadipocytes was also observed. In conclusion, celastrol exhibited inhibitory effect of autophagy via direct binding with VAMP7 and RAB7, leading to an increase in preadipocytes apoptosis. These results advance our understanding in the potential application of celastrol in treating obesity.

Low-dose spironolactone ameliorates adipose tissue inflammation and apoptosis in letrozole-induced PCOS rat model

Background of studyGlobally, many reproductive aged women are affected by polycystic ovarian syndrome (PCOS), which is a common endocrine and metabolic disorder that is linked with adipose dysfunction and chronic low-grade inflammation. Spironolactone (SPL), a mineralocorticoid receptor blocker has been documented as a metabolic modulator. However, its immunomodulatory effect in PCOS is unknown. Therefore, the present study hypothesized that SPL would ameliorate adipose dysfunction and inflammation in experimental PCOS animals.Materials and methodsFemale Wistar rats that were 8 weeks old were allocated into three groups. Group 1 received vehicle (distilled water; p.o.), group 2 received letrozole (1 mg/kg; p.o.) and group 3 received letrozole plus SPL (0.25 mg/kg, p.o.). The administration was performed once daily for 21 days.ResultsThe experimental PCOS animals showed insulin resistance, hyperinsulinemia and hyperandrogenism as well as oxidative stress and elevated inflammatory biomarkers (NF-kB/TNF-/IL-6) as well as a significant decrease in triglycerides, total cholesterol, free fatty acids, GSH and G6PD in the adipose tissue of PCOS animals. In addition, immunohistochemical assessment of adipose tissue showed significant expression of BAX and inflammasome, indicating apoptosis and inflammation compared to control animals. Nevertheless, administration of SPL attenuated these perturbations.ConclusionAltogether, the present study suggests that low-dose spironolactone confers protection against adipose dysfunction in experimental PCOS animals by attenuating inflammation, oxidative stress and cellular apoptosis.

The role of autophagy in high-fat diet-induced insulin resistance of adipose tissues in mice.

AimsStudies have observed changes in autophagic flux in the adipose tissue of type 2 diabetes patients with obesity. However, the role of autophagy in obesity-induced insulin resistance is unclear. We propose to confirm the effect of a high-fat diet (HFD) on autophagy and insulin signaling transduction from adipose tissue to clarify whether altered autophagy-mediated HFD induces insulin resistance, and to elucidate the possible mechanisms in autophagy-regulated adipose insulin sensitivity.MethodsEight-week-old male C57BL/6 mice were fed with HFD to confirm the effect of HFD on autophagy and insulin signaling transduction from adipose tissue. Differentiated 3T3-L1 adipocytes were treated with 1.2 mM fatty acids (FAs) and 50 nM Bafilomycin A1 to determine the autophagic flux. 2.5 mg/kg body weight dose of Chloroquine (CQ) in PBS was locally injected into mouse epididymal adipose (10 and 24 h) and 40 µM of CQ to 3T3-L1 adipocytes for 24 h to evaluate the role of autophagy in insulin signaling transduction.ResultsThe HFD treatment resulted in a significant increase in SQSTM1/p62, Rubicon expression, and C/EBP homologous protein (CHOP) expression, yet the insulin capability to induce Akt (Ser473) and GSK3β (Ser9) phosphorylation were reduced. PHLPP1 and PTEN remain unchanged after CQ injection. In differentiated 3T3-L1 adipocytes treated with CQ, although the amount of phospho-Akt stimulated by insulin in the CQ-treated group was significantly lower, CHOP expressions and cleaved caspase-3 were increased and bafilomycin A1 induced less accumulation of LC3-II protein.ConclusionLong-term high-fat diet promotes insulin resistance, late-stage autophagy inhibition, ER stress, and apoptosis in adipose tissue. Autophagy suppression may not affect insulin signaling transduction via phosphatase expression but indirectly causes insulin resistance through ER stress or apoptosis.

Modulatory Effect of Intermittent Fasting on Autophagy and Apoptosis in White Adipose Tissue of A High Fat-Fed Rat Model

Modulatory Effect of Intermittent Fasting on Autophagy and Apoptosis in White Adipose Tissue of A High Fat-Fed Rat Model

SERPINA3C ameliorates adipose tissue inflammation through the Cathepsin G/Integrin/AKT pathway

ObjectiveDue to the increasing prevalence of obesity and insulin resistance, there is an urgent need for better treatment of obesity and its related metabolic disorders. This study aimed to elucidate the role of SERPINA3C, an adipocyte secreted protein, in obesity and related metabolic disorders. MethodsMale wild type (WT) and knockout (KO) mice were fed with high-fat diet (HFD) for 16 weeks, adiposity, insulin resistance, and inflammation were assessed. AAV-mediated overexpression of SERPINA3C was injected locally in inguinal white adipose tissue (iWAT) to examine the effect of SERPINA3C. In vitro analyses were conducted in 3T3-L1 adipocytes to explore the molecular pathways underlying the function of SERPINA3C. ResultsFunctional exploration of the SERPINA3C knockout mice revealed that SERPINA3C deficiency led to an impaired metabolic phenotype (more severe obesity, lower metabolic rates, worse glucose intolerance and insulin insensitivity), which was associated with anabatic inflammation and apoptosis of white adipose tissues. Consistent with these results, overexpression of SERPINA3C in inguinal adipose tissue protected mice against diet-induced obesity and metabolic disorders with less inflammation and apoptosis in adipose tissue. Mechanistically, SERPINA3C inhibited Cathepsin G activity, acting as a serine protease inhibitor, which blocked Cathepsin G-mediated turnover of α5/β1 Integrin protein. Then, the preserved integrity (increase) of α5/β1 Integrin signaling activated AKT to decrease JNK phosphorylation, thereby inhibiting inflammation and promoting insulin sensitivity in adipocytes. Conclusions/interpretationThese findings demonstrate a previously unknown SERPINA3C/Cathepsin G/Integrin/AKT pathway in regulating adipose tissue inflammation, and suggest the therapeutic potential of targeting SERPINA3C/Cathepsin G axis in adipose tissue for the treatment of obesity and metabolic diseases.

MiR-146a-5p modulates cellular senescence and apoptosis in visceral adipose tissue of long-lived Ames dwarf mice and in cultured pre-adipocytes.

MicroRNAs (miRNAs) are potent regulators of multiple biological processes. Previous studies have demonstrated that miR-146a-5p increases in normal mice during aging, while long-living Ames dwarf (df/df) mice maintain youthful levels of this miRNA. The aim of this study was to elucidate the involvement of miR-146a-5p in modulating cellular senescence and apoptosis in visceral adipose tissue of df/df mice and cultured pre-adipocytes. To test the effects of miR-146a-5p overexpression on visceral adipose tissue, wild-type, and df/df mice, were treated with miRNA-negative control-base and df/df were transfected with 4 or 8µg/g of a miR-146a-5p mimetic, respectively. Effects of miR-146a-5p overexpression were also evaluated in 3T3-L1 cells cultured under high and normal glucose conditions. Treatment with miR-146a-5p mimetic increased cellular senescence and inflammation and decreased pro-apoptotic factors in visceral adipose tissue of df/df mice. The miR-146a-5p mimetic induced similar effects in 3T3-L1 cells cultivated at normal but not high glucose levels. Importantly, 3T3-L1 HG cells in high glucose conditions showed significantly higher expression of miR-146a-5p than 3T3-L1 grown in normal glucose conditions. These results indicate that miR-146a-5p can be a marker for cellular senescence. This miRNA represents one of the significant SASP factors that if not precisely regulated, can accentuate inflammatory responses and stimulate senescence in surrounding non-senescent cells. The role of miR-146a-5p is different in healthy versus stressed cells, suggesting potential effects of this miRNA depend on overall organismal health, aging, and metabolic state.

Pericardial adipose tissue-derived leptin promotes myocardial apoptosis in high fat diet-induced obese rats through JAK2/ROS/NKA signaling pathway

Abstract Background Pathophysiologic mechanisms underlying cardiac structural and functional changes in obesity are complex and linked to adipocytokines released from pericardial adipose tissue (PAT) and cardiomyocyte apoptosis. Although leptin is involved in various pathological conditions, its role in paracrine action of pericardial adipose tissue on myocardial apoptosis remains unknown. Purpose This study was designed to investigate the role of PAT-derived leptin on myocardial apoptosis in high fat diet-induced obese rats. Methods and results Hearts were isolated from lean or high fat diet-induced obese Wistar rats for myocardial remodeling studies. Obese rats had abnormal myocardial structure, diastolic dysfunction, greatly elevated cardiac apoptosis and increased oxidative stress level. Enzyme-linked immunosorbent assay (ELISA) detected significantly higher than circulating leptin level in PAT of obese, but not lean rats. Western blot and immunohistochemical analyses demonstrated increased leptin receptor density in obese heart. H9c2 rat cardiomyoblasts after exposed to PAT-conditioned medium of obese rats exhibited pronounced reactive oxygen species (ROS)-mediated apoptosis, which was partially reversed by leptin antagonist. Moreover, leptin derived from PAT of obese rats inhibited Na+/K+-ATPase activity of H9c2 cells through stimulating ROS, thereby activating calcium-dependent apoptosis. Pretreatment with specific inhibitors revealed that JAK2/STAT3 and PI3K/Akt signaling pathways was involved in the leptin-induced myocardial apoptosis. Conclusion PAT-derived leptin induces myocardial apoptosis in high fat diet-induced obese rats via activating JAK2/STAT3/ROS signaling pathway and inhibiting its downstream Na+/K+-ATPase activity. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Nature Science Foundation of ChinaKey Projects of Shaanxi Science and Technology Research and Development Plan

Pericardial Adipose Tissue-Derived Leptin Promotes Myocardial Apoptosis in High-Fat Diet-Induced Obese Rats Through Janus Kinase 2/Reactive Oxygen Species/Na+/K+-ATPase Signaling Pathway.

BackgroundPathophysiologic mechanisms underlying cardiac structural and functional changes in obesity are complex and linked to adipocytokines released from pericardial adipose tissue (PAT) and cardiomyocyte apoptosis. Although leptin is involved in various pathological conditions, its role in paracrine action of pericardial adipose tissue on myocardial apoptosis remains unknown. This study was designed to investigate the role of PAT‐derived leptin on myocardial apoptosis in high‐fat diet–induced obese rats.Methods and ResultsHearts were isolated from lean or high‐fat diet–induced obese Wistar rats for myocardial remodeling studies. Obese rats had abnormal myocardial structure, diastolic dysfunction, greatly elevated cardiac apoptosis, enhanced cardiac fibrosis, and increased oxidative stress level. ELISA detected significantly higher than circulating leptin level in PAT of obese, but not lean, rats. Western blot and immunohistochemical analyses demonstrated increased leptin receptor density in obese hearts. H9c2 cardiomyoblasts, after being exposed to PAT‐conditioned medium of obese rats, exhibited pronounced reactive oxygen species–mediated apoptosis, which was partially reversed by leptin antagonist. Moreover, leptin derived from PAT of obese rats inhibited Na+/K+‐ATPase activity of H9c2 cells through stimulating reactive oxygen species, thereby activating calcium‐dependent apoptosis. Pretreatment with specific inhibitors revealed that Janus kinase 2/signal transducer and activator of transcription 3 and phosphoinositide 3‐kinase/protein kinase B signaling pathways were involved in leptin‐induced myocardial apoptosis.ConclusionsPAT‐derived leptin induces myocardial apoptosis in high‐fat diet–induced obese rats via activating Janus kinase 2/signal transducer and activator of transcription 3/reactive oxygen species signaling pathway and inhibiting its downstream Na+/K+‐ATPase activity.

Differential Effects of Chronic Binge Alcohol and Ovariectomy on Apoptosis in Omental Adipose Tissue of SIV‐infected Macaques

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 anti-obesity effects of rhein on improving insulin resistance (IR) and blood lipid levels are involved in endoplasmic reticulum stress (ERs), inflammation, and oxidative stress in vivo and vitro

ABSTRACT Rhein extensive biological effects including anti-inflammatory, antioxidant stress, and improving glucose and lipid metabolism. In the present study, the effects of rhein were examined on endoplasmic reticulum stress (ERs) and inflammation in obesity-induced rats. SD rats were fed with a normal diet or a high-fat diet. Meanwhile, rats fed with high-fat diet were also administrated with different doses of rhein for 6 weeks. The pathologic changes of pathoaorta pectoralis were evaluated using hematoxyline eosin (HE) strain, and cell apoptosis levels were investigated using TUNEL staining and flow cytometry. We also performed p62 immunofluorescent staining in 3T3-L1 cells. In the present study, we found that rhein administration exerted inhibitory effects on weight, inflammatory factor levels, and oxidative stress. Meanwhile, insulin resistance (IR), blood lipid levels and pathological injury of aorta pectoralis were also improved by rhein administration. Besides, rhein also affected ERs in peripheral blood and adipose tissue in vivo. Moreover, rhein significantly reduced cell apoptosis in aorta pectoralis and adipose tissue in vivo. According to oil red staining, adipogenic differentiation was decreased by rhein treatment in vitro. Immunofluorescence staining of p62 showed that rhein contributed to a significant increase in p62 expression in vitro. In addition, rhein treatment significantly decreased peroxisome proliferators-activated receptor (PPAR)γ levels and upregulated insulin receptor (INSR) in vitro. In summary, the anti-obesity effects of rhein were considered to be related with the involvement of ERs, inflammation, oxidative stress, PPARγ, and INSR.

Preliminary exploration on the application of hydrogel from acellular porcine adipose tissue to assist lipofilling

To investigate the effect of hydrogel from acellular porcine adipose tissue (HAPA) on the survival of transplanted adipose tissue. For in vitro study, adipose tissue and HAPA-adipose tissue complex were cultured in normoxia and hypoxia atmospheres for 24 and 72 hours. TUNEL and Perilipin immunofluorescence staining were performed to observe the effect of HAPA on apoptosis and survival of adipocities. For in vivo study, 42 healthy male nude mice (4-6 weeks old) weighing 15-18 g were randomly divided into adipose group (group A), 10%HAPA group (group B), 20%HAPA group (group C), 30%HAPA group (group D), 40%HAPA group (group E), and 50%HAPA group (group F) according to different HAPA/adipose tissue volume ratio ( n=7). For each group, 1 mL adipose tissue or HAPA-adipose tissue complex was injected subcutaneously into the dorsum of the nude mice. At 4 weeks after transplantation, 7 nude mice in each group were sacrificed and grafts were harvested, gross observation, volume measurement, ultrasound examination, and histologic staining (HE staining, CD31 and Perilipin immunofluorescence stainings) were applied. Hypoxia showed a tendency of promoting adipose tissue necrosis and apoptosis, while HAPA exhibited an obvious effect of inhibiting cell apoptosis in vitro study ( P<0.05). For in vivo study, grafts of all groups had intact fibrocapsule. No obvious signs of infection and necrosis were observed at 4 weeks. Volume shrinkage was observed in all groups, however, the groups A-D had significantly higher volume retention rate than groups E and F ( P<0.05). Ultrasound examination showed that there were no significant difference in the number and volume of liquify area of the grafts in each group ( P>0.05). With the increase of HAPA's volume ratio, HE staining proved an improved fat integrity while a gradually decreased vacuoles and fibrosis. CD31 immunohistochemical staining showed that the number of neo-vascularisation in groups E and F were significantly higher than those in groups A-D ( P<0.05). Perilipin immunofluorescence staining showed that with the increase of HAPA volume ratio, the number of living adipocytes increased gradually, and more new adipocytes could be seen in the field of vision. As the volume ratio of HAPA gradually increased, the survival of transplanted adipose tissue also increased, but the volume retention rate decreased gradually. 30%HAPA was considered the relative optimal volume ratio for its superior adipose tissue survival and volume retation rate.

Downregulation of Sirt1 is correlated to upregulation of p53 and increased apoptosis in epicardial adipose tissue of patients with coronary artery disease.

The higher expression level of p53 in epithelial adipose tissue (EAT) has previously been reported in atherosclerosis. Since we hypothesized that the expression of p53 is modulated by Sirt1, the aim of this study was to determine the expression levels of Sirt1 and p53 and to investigate their correlation to apoptosis in EAT of patients with coronary artery disease (CAD). Thirty-five patients with more than 50 % stenosis in at least one of the main coronary arteries were considered as CAD group while 29 patients with no clinical signs of atherosclerosis who underwent open-heart surgery for valve replacement were classified as control group. EAT biopsy samples were collected from all participants during surgery. Sirt1, p53, Bax, and Bcl-2 gene expression levels were determined in EAT by qRT-PCR and Western blotting was carried out to assess Sirt1 and p53 protein levels. Hematoxylin and eosin staining was used for histopathological analysis. mRNA and protein levels of Sirt1 in EAT were significantly lower in patients with CAD compared with control group, whereas CAD patients showed greater p53 gene and protein expressions. In addition, inverse correlations were observed between Sirt1 and p53 at both mRNA and protein levels. The Bax and ratio of Bax/Bcl-2 gene expressions were higher in CAD group, but no difference was observed in Bcl-2 expression. Histopathological analysis showed apoptotic bodies and infiltrated immune cells in EAT of CAD group. Our results suggest that the Sirt1-p53 axis may involve in atherosclerosis by promotion of apoptosis.

The Role of SHIP1 on Apoptosis and Autophagy in the Adipose Tissue of Obese Mice.

Obesity-induced adipocyte apoptosis promotes inflammation and insulin resistance. Src homology domain-containing inositol 5′-phosphatase 1 (SHIP1) is a key factor of apoptosis and inflammation. However, the role of SHIP1 in obesity-induced adipocyte apoptosis and autophagy is unclear. We found that diet-induced obesity (DIO) mice have significantly greater crown-like structures and terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL)-positive cells than ob/ob or control mice. Using RNA sequencing (RNA-seq) analysis, we identified that the apoptosis- and inflammation-related gene Ship1 is upregulated in DIO and ob/ob mice compared with control mice. In particular, DIO mice had more SHIP1-positive macrophages and lysosomal-associated membrane protein 1 (LAMP1) as well as a higher B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)/Bcl-2 ratio compared with ob/ob or control mice. Furthermore, caloric restriction attenuated adipose tissue inflammation, apoptosis, and autophagy by reversing increases in SHIP1-associated macrophages, Bax/Bcl2-ratio, and autophagy in DIO and ob/ob mice. These results demonstrate that DIO, not ob/ob, aggravates adipocyte inflammation, apoptosis, and autophagy due to differential SHIP1 expression. The evidence of decreased SHIP1-mediated inflammation, apoptosis, and autophagy indicates new therapeutic approaches for obesity-induced chronic inflammatory diseases.

Apoptosis of hematopoietic progenitor-derived adipose tissue-resident macrophages contributes to insulin resistance after myocardial infarction.

Patients with insulin resistance have high risk of cardiovascular disease such as myocardial infarction (MI). However, it is not known whether MI can initiate or aggravate insulin resistance. We observed that patients with ST-elevation MI and mice with MI had de novo hyperglycemia and features of insulin resistance, respectively. In mouse models of both myocardial and skeletal muscle injury, we observed that the number of visceral adipose tissue (VAT)-resident macrophages decreased because of apoptosis after these distant organ injuries. Patients displayed a similar decrease in VAT-resident macrophage numbers and developed systemic insulin resistance after ST-elevation MI. Loss of VAT-resident macrophages after MI injury led to systemic insulin resistance in non-diabetic mice. Danger signaling-associated protein high mobility group box 1 was released by the dead myocardium after MI in rodents and triggered macrophage apoptosis via Toll-like receptor 4. The VAT-resident macrophage population in the steady state in mice was transcriptomically distinct from macrophages in the brain, skin, kidney, bone marrow, lungs, and liver and was derived from hematopoietic progenitor cells just after birth. Mechanistically, VAT-resident macrophage apoptosis and de novo insulin resistance in mouse models of MI were linked to diminished concentrations of macrophage colony-stimulating factor and adiponectin. Collectively, these findings demonstrate a previously unappreciated role of adipose tissue-resident macrophages in sensing remote organ injury and promoting MI pathogenesis.

1498-P: A Feedback Loop between miRNA Processing Complex and miR-3975 in Western Diet–Induced Brown Adipose Tissue Dysfunction

Brown adipose tissue (BAT) is considered as promising therapeutic targets for obesity and metabolic disorder through modulation of energy homeostasis. microRNAs (miRNAs) are established as essential modulators of biological processes in many tissues, including the differentiation and function of BAT. Here we demonstrated that high-fat/high fructose associated dysfunction of BAT coincides with impaired miRNA-processing pathway in a feedback loop with miR-3975 activation. Eight-week-old male C57BL/6 mice were fed a low-fat diet (10% fat and 17% sucrose; LFD) and high fat-high fructose (45% fat, 17% sucrose, and 17% fructose; HFHFD) for 4, 12, and 20 weeks (n=6-9 per group). We evaluated expression levels of apoptosis markers, thermogenesis markers, miRNA biogenesis regulators, and miRNAs in BAT. Mice fed an HFHFD showed a gradually significance increased body weight and brown adipose tissue weight when compared to the mice fed with LFD. BAT of HFHFD fed mice has a pale appearance versus feeding period matched LFD fed mice. The protein expression of thermogenesis markers, UCP1 (P &amp;lt;0.05), and PGC1α (P &amp;lt;0.05) were shown to decrease after 12 weeks of HFHFD diet feeding. The apoptosis marker, the Bax/Bcl-2 ratio, was upregulated at 12 weeks (P &amp;lt; 0.01). Mice fed HFHFD for 12 and 20 weeks showed a significant decrease in Dicer (P &amp;lt; 0.01 for 12 and 20 weeks) and TRBP2 (P &amp;lt; 0.01 for 12 weeks and P &amp;lt; 0.0001 for 20 weeks). miR-3975, which is predicted miRNA to regulate both Dicer and TRBP2 by IPA was activated at 12 weeks (P &amp;lt; 0.01), while inactivated at 20 weeks (P &amp;lt; 0.001). Our results suggest that miR-3975 acts as a component of a negative feedback loop that blocks miRNA biogenesis machinery through the inhibition of Dicer and TRBP2 expression. Therefore, long-term HFHFD may disturb miRNA processing and contribute to brown adipose tissue dysfunction and apoptosis by modulation of miRNA expression. We are currently investigating the novel feedback loop in brown adipocyte primary cells. Disclosure M. Park: None. S. Meruvu: None. J. Zhang: None. M. Choudhury: None.

Comparison of apoptosis between bovine subcutaneous and intramuscular adipocytes by resveratrol via SIRT1

A better understanding of the differential mechanisms regulating the deposition and release of fat between intramuscular and external adipose tissues is very important to the quality of beef. Resveratrol is a natural activator of sirtuin type 1 (SIRT1), a NAD-dependent deacetylase involved in regulating the cell cycle, energy homeostasis and apoptosis in adipose tissue. To compare the molecular mechanisms underlying differential apoptosis in bovine intramuscular and subcutaneous adipocytes, we evaluated the effect of resveratrol on differentiated adipocytes. We found that resveratrol-induced apoptosis in bovine adipocytes by regulating SIRT1 activity. In addition, we report that bovine intramuscular and subcutaneous adipocytes exhibited differential responses to resveratrol. In particular, gene and protein expression of Bcl-2 was higher, whereas that of SIRT1, AMPKα, FOXO1, Bax and caspase-3 were lower in bovine subcutaneous adipocytes than in intramuscular adipocytes. After resveratrol-treatment, the extent of up- or down-regulation was higher in subcutaneous adipocytes than in intramuscular adipocytes. These data indicate that bovine subcutaneous adipocytes are more sensitive to apoptosis than intramuscular adipocytes following treatment with resveratrol by regulating SIRT1 activity.

Abstract 604: Anti-apoptotic Role of Leptin in Adipose Tissue

Background: Leptin contributes to obesity-related cardiometabolic pathology. While the role of adipose tissue dysfunction in altering metabolic profile in obesity is recognized, the direct effects of leptin signaling in adipose tissue are not completely understood. Among factors impacting adipose tissue function in obesity, adipocyte death is associated with increased inflammation with consequent adipose tissue insulin-resistance. Furthermore, apoptosis of adipocyte progenitor cells (preadipocytes) is important as it lowers adipose tissue capacity to maintain and expand fat storage. Objective: To determine the effects of leptin on regulation of proteins important to apoptotic pathways in cultured human white preadipocytes (HWP) and differentiated HWP (dHWP). Methods: Cells were exposed to increasing concentrations of leptin to evaluate the role of leptin on regulation of anti-apoptotic Bcl2, and pro-apoptotic Bax and CD95 expression. Next, we examined if pre-exposure to leptin attenuated TNF-α and FasL induced apoptosis. Results: Leptin upregulated transcription of Bcl2 (p=0.02) and down-regulated transcription of Bax (p=0.02) and CD95 (p=0.04) in a leptin-concentration dependent manner in dHWP. Leptin dependent increases in Bcl2 (p=0.004) and decreases in Bax (p=0.003) and CD95 (P=0.03) protein expression were also observed. Furthermore, 24 hour pre-exposure to leptin protected cells from TNF-α (p=0.01; p=0.006) and FasL (p=0.03; p=0.007) induced apoptosis in dHWP and HWP respectively. Additionally, leptin attenuated TNF-mediated activation of the p53 pathway, suggesting another mechanism by which leptin mediates its inhibitory effects on TNF-induced apoptosis. Conclusion: Leptin regulates the expression of proteins involved in the apoptotic pathway such that it decreases the apoptotic potential of adipose tissue by increasing anti-apoptotic Bcl2 and decreasing pro-apoptotic Bax and CD95 protein expression. In contrast to our in-vitro findings, prior studies in adipose tissue from obese humans report decreased Bcl2 and increased Bax protein expression, along with high leptin expression. This suggests that increased adipose tissue apoptosis in obesity may at least in part be secondary to altered leptin signaling.

Slight hypercalcemia is not associated with positive responses in the Comet Assay in male rat liver

Maintenance of physiological levels of intracellular and extracellular calcium is essential for life. Increased intracellular calcium levels are involved in cell death (apoptosis and necrosis) and are associated with positive responses in the Comet assay in vitro. In addition, high calcium and vitamin D intakes were reported to induce apoptosis in adipose tissue in obese mice and to increase DNA-migration in the Comet assay. To investigate increased serum concentration of calcium as a potential confounding factor in the regulatory Comet assay in vivo, we induced mild hypercalcemia in male Wistar rats by 3-day continuous intravenous infusion of calcium gluconate and performed the Comet assay in the liver in line with regulatory guidelines.The results of the study showed that mild increases in serum calcium concentration (up to 1.4 times above the concurrent control) and increased urinary calcium concentration (up to 27.8 times above the concurrent control) results in clinical signs like mild tremor, faster respiration rate and decreased activity in a few animals. However, under the conditions of the study, no increase in the %Tail DNA in the Comet assay and no indication of liver damage as determined by histopathological means were observed. Thus, mild increases in plasma calcium did not lead to positive results in a genotoxicity assessment by the Comet assay in the rat liver. This result is important as it confirms the reliability of this assay for regulatory evaluation of safety.