IL6/adiponectin/HMGB1 feedback loop mediates adipocyte and macrophage crosstalk and M2 polarization after myocardial infarction.

Background: Differences in border zone contribute to different outcomes post-infarction, such as left ventricular aneurysm (LVA) and myocardial infarction (MI). LVA usually forms within 24 h of the onset of MI and may cause heart rupture; however, LVA surgery is best performed 3 months after MI. Few studies have investigated the LVA model, the differences in border zones between LVA and MI, and the mechanism in the border zone. Methods: The LVA, MI, and SHAM mouse models were used. Echocardiography, Masson's trichrome staining, and immunofluorescence staining were performed, and RNA sequencing of the border zone was conducted. The adipocyte-conditioned medium-treated hypoxic macrophage cell line and LVA and MI mouse models were employed to determine the effects of the hub gene, adiponectin (ADPN), on macrophages. Quantitative polymerase chain reaction (qPCR), Western blot analysis, transmission electron microscopy, and chromatin immunoprecipitation (ChIP) assays were conducted to elucidate the mechanism in the border zone. Human subepicardial adipose tissue and blood samples were collected to validate the effects of ADPN. Results: A novel, simple, consistent, and low-cost LVA mouse model was constructed. LVA caused a greater reduction in contractile functions than MI owing to reduced wall thickness and edema in the border zone. ADPN impeded cardiac edema and promoted lymphangiogenesis by increasing macrophage infiltration post-infarction. Adipocyte-derived ADPN promoted M2 polarization and sustained mitochondrial quality via the ADPN/AdipoR2/HMGB1 axis. Mechanistically, ADPN impeded macrophage HMGB1 inflammation and decreased interleukin-6 (IL6) and HMGB1 secretion. The secretion of IL6 and HMGB1 increased ADPN expression via STAT3 and the co-transcription factor, YAP, in adipocytes. Based on ChIP and Dual-Glo luciferase experiments, STAT3 promoted ADPN transcription by binding to its promoter in adipocytes. In vivo, ADPN promoted lymphangiogenesis and decreased myocardial injury after MI. These phenotypes were rescued by macrophage depletion or HMGB1 knockdown in macrophages. Supplying adipocytes overexpressing STAT3 decreased collagen disposition, increased lymphangiogenesis, and impaired myocardial injury. However, these effects were rescued after HMGB1 knockdown in macrophages. Overall, the IL6/ADPN/HMGB1 axis was validated using human subepicardial tissue and blood samples. This axis could serve as an independent factor in overweight MI patients who need coronary artery bypass grafting (CABG) treatment. Conclusion: The IL6/ADPN/HMGB1 loop between adipocytes and macrophages in the border zone contributes to different clinical outcomes post-infarction. Thus, targeting the IL6/ADPN/HMGB1 loop may be a novel therapeutic approach for cardiac lymphatic regulation and reduction of cell senescence post-infarction.

Cytoskeletal rearrangement precedes nucleolar remodeling during adipogenesis.

Differentiation of adipose progenitor cells into mature adipocytes entails a dramatic reorganization of the cellular architecture to accommodate lipid storage into cytoplasmic lipid droplets. Lipid droplets occupy most of the adipocyte volume, compressing the nucleus beneath the plasma membrane. How this cellular remodeling affects sub-nuclear structure, including size and number of nucleoli, remains unclear. We describe the morphological remodeling of the nucleus and the nucleolus during in vitro adipogenic differentiation of primary human adipose stem cells. We find that cell cycle arrest elicits a remodeling of nucleolar structure which correlates with a decrease in protein synthesis. Strikingly, triggering cytoskeletal rearrangements mimics the nucleolar remodeling observed during adipogenesis. Our results point to nucleolar remodeling as an active, mechano-regulated mechanism during adipogenic differentiation and demonstrate a key role of the actin cytoskeleton in defining nuclear and nucleolar architecture in differentiating human adipose stem cells.

HOXA9 gene inhibits proliferation and differentiation and promotes apoptosis of bovine preadipocytes.

BACKGROUND: Hox gene family is an important transcription factor that regulates cell process, and plays a role in the process of adipocytes differentiation and fat deposition. Previous transcriptome sequencing studies have indicated that the Homeobox A9 gene (HOXA9) is a candidate gene for regulating the process of bovine lipid metabolism, but the function and specific mechanism of action remain unclear. Therefore, this study aims to explore the role of HOXA9 in the proliferation, differentiation and apoptosis of bovine preadipocytes through gain-of-function and lose-of-function. RESULT: It found HOXA9 highly expressed in bovine adipose tissue, and its expression level changed significantly during adipocytes differentiation process. It gave a hint that HOXA9 may be involved in the process of bovine lipid metabolism. The results of HOXA9 gain-of-function experiments indicated that HOXA9 appeared to act as a negative regulator not only in the differentiation but also in the proliferation of bovine preadipocytes, which is mainly reflected that overexpression of HOXA9 down-regulate the mRNA and protein expression level of PPARγ, CEBPα and FABP4 (P < 0.05). The mRNA expression level of CDK1, CDK2, PCNA, CCNA2, CCNB1, CCND1 and CCNE2, as well as the protein expression of CDK2 also significantly decreased. The decrease of lipid droplets content was the main characteristic of the phenotype (P < 0.01), which further supported the evidence that HOXA9 was a negative regulator of preadipocytes differentiation. The decrease of cell proliferation rate and EdU positive rate, as well as the limitation of transition of preadipocytes from G0/G1 phase to S phase also provided evidence for the inhibition of proliferation. Apart from this above, we noted an interesting phenomenon that overexpression of HOXA9 showed in a significant upregulation of both mRNA and protein level of apoptosis markers, accompanied by a significant increase in cell apoptosis rate. These data led us not to refute the fact that HOXA9 played an active regulatory role in apoptosis. HOXA9 loss-of-function experiments, however, yielded the opposite results. Considering that HOXA9 acts as a transcription factor, we predicted its target genes. Dual luciferase reporter assay system indicated that overexpression of HOXA9 inhibits activity of PCNA promoter. CONCLUSION: Taken together, we demonstrated for the first time that HOXA9 played a role as a negative regulatory factor in the differentiation and proliferation of preadipocytes, but played a positive regulatory role in apoptosis, and it may play a regulatory role by targeting PCNA. This study provides basic data for further exploring the regulatory network of intramuscular fat deposition in bovine.

A negative feedback loop between TET2 and leptin in adipocyte regulates body weight.

Ten-eleven translocation (TET) 2 is an enzyme that catalyzes DNA demethylation to regulate gene expression by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine, functioning as an essential epigenetic regulator in various biological processes. However, the regulation and function of TET2 in adipocytes during obesity are poorly understood. In this study, we demonstrate that leptin, a key adipokine in mammalian energy homeostasis regulation, suppresses adipocyte TET2 levels via JAK2-STAT3 signaling. Adipocyte Tet2 deficiency protects against high-fat diet-induced weight gain by reducing leptin levels and further improving leptin sensitivity in obese male mice. By interacting with C/EBPα, adipocyte TET2 increases the hydroxymethylcytosine levels of the leptin gene promoter, thereby promoting leptin gene expression. A decrease in adipose TET2 is associated with obesity-related hyperleptinemia in humans. Inhibition of TET2 suppresses the production of leptin in mature human adipocytes. Our findings support the existence of a negative feedback loop between TET2 and leptin in adipocytes and reveal a compensatory mechanism for the body to counteract the metabolic dysfunction caused by obesity.

Can irisin be developed as the molecular evolutionary clock based on the origin and functions?

Irisin, a myokine identified in 2012, has garnered research interest for its capacity to induce browning of adipocytes and improve metabolic parameters. As such, the potential therapeutic applications of this exercise-induced peptide continue to be explored. Though present across diverse animal species, sequence analysis has revealed subtle variation in the irisin protein. In this review, we consider the effects of irisin on disease states in light of its molecular evolution. We summarize current evidence for irisin's influence on pathologies and discuss how sequence changes may inform development of irisin-based therapies. Furthermore, we propose that the phylogenetic variations in irisin could potentially be leveraged as a molecular clock to elucidate evolutionary relationships.

IL-33 regulates adipogenesis via Wnt/ß-catenin/PPAR-γ signaling pathway in preadipocytes.

Interleukin-33 (IL-33), an emerging cytokine within the IL-1 family, assumes a pivotal function in the control of obesity. However, the specific mechanism of its regulation of obesity formation remains unclear. In this study, we found that the expression level of IL-33 increased in visceral adipose tissue in mice fed with a high-fat diet (HFD) compared with that in mice fed with a normal diet (ND). In vitro, we also found the expression level of IL-33 was upregulated during the adipogenesis of 3T3-L1 cells. Functional test results showed that knockdown of IL-33 in 3T3-L1 cells differentiation could promote the accumulation of lipid droplets, the content of triglyceride and the expression of adipogenic-related genes (i.e. PPAR-γ, C/EBPα, FABP4, LPL, Adipoq and CD36). In contrast, overexpression of IL-33 inhibits adipogenic differentiation. Meanwhile, the above tests were repeated after over-differentiation of 3T3-L1 cells induced by oleic acid, and the results showed that IL-33 played a more significant role in the regulation of adipogenesis. To explore the mechanism, transcriptome sequencing was performed and results showed that IL-33 regulated the PPAR signaling pathway in 3T3-L1 cells. Further, Western blot and confocal microscopy showed that the inhibition of IL-33 could promote PPAR-γ expression by inhibiting the Wnt/ß-catenin signal in 3T3-L1 cells. This study demonstrated that IL-33 was an important regulator of preadipocyte differentiation and inhibited adipogenesis by regulating the Wnt/ß-catenin/PPAR-γ signaling pathway, which provided a new insight for further research on IL-33 as a new intervention target for metabolic disorders.

Diverse effects of a Cyperus rotundus extract on glucose uptake in myotubes and adipocytes and its suppression on adipocyte maturation.

Cyperus rotundus rhizomes have been used in longevity remedies in Thailand for nourishing good health, which led us to investigate the effect on energy homeostasis, especially glucose utilization in myotubes and adipocytes, and on inhibition of lipogenesis in adipocytes. The results showed that an ethyl acetate extract of C. rotundus rhizomes (ECR) containing 1.61%w/w piceatannol, with a half-maximal concentration of 17.76 ± 0.03 µg/mL in 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, caused upregulation and cell-membrane translocation of glucose transporters GLUT4 and 1 in L6 myotubes but downregulation and cytoplasmic localization of GLUT4 expression in 3T3-L1 adipocytes and was related to the p-Akt/Akt ratio in both cells, especially at 100 µg/mL. Moreover, ECR (25-100 µg/mL) significantly inhibited lipid accumulation via Adenosine Monophosphate-Activated Protein Kinase (AMPK), Acetyl CoA Carboxylase (ACC), and Glycogen Synthase Kinase (GSK) pathways. Its immunoblot showed increased expression of p-AMPKα/AMPKα and p-ACC/ACC but decreased expression of p-Akt/Akt and p-GSK3ß/GSK3ß in 3T3-L1 adipocytes. Moreover, the decreased expression of the adipogenic effectors, perilipin1 and lipoprotein lipase, in ECR-incubated adipocytes (50 and 100 µg/mL) indicated reduced de novo lipogenesis. Our study elucidated mechanisms of C. rotundus that help attenuate glucose tolerance in skeletal muscle and inhibit lipid droplet accumulation in adipose tissue.

Distinct subpopulations of human subcutaneous adipose tissue precursor cells revealed by single-cell RNA sequencing.

Adipose-derived stem cells (ADSCs) play an important role in the differential capacity for excess energy storage between upper body abdominal (ABD) adipose tissue (AT) and lower body gluteofemoral (GF) AT. We cultured ADSCs from subcutaneous ABD AT and GF AT isolated from eight women with differential body fat distribution and performed single-cell RNA sequencing. Six populations of ADSCs were identified and segregated according to their anatomical origin. The three ADSC subpopulations in GF AT were characterized by strong cholesterol/fatty acid (FA) storage and proliferation signatures. The two ABD subpopulations, differentiated by higher expression of committed preadipocyte marker genes, were set apart by differential expression of extracellular matrix and ribosomal genes. The last population, identified in both depots, was similar to smooth muscle cells and when individually isolated and cultured in vitro they differentiated less than the other subpopulations. This work provides important insight into the use of ADSC as an in vitro model of adipogenesis and suggests that specific subpopulations of GF-ADSCs contribute to the more robust capacity for GF-AT to expand and grow compared with ABD-AT in women.NEW & NOTEWORTHY Identification of distinct subpopulations of adipose-derived stem cells (ADSCs) in upper body abdominal subcutaneous (ABD) and lower body gluteofemoral subcutaneous (GF) adipose tissue depots. In ABD-ADSCs, subpopulations are more committed to adipocyte lineage. GF-ADSC subpopulations are enriched for genes involved in lipids and cholesterol metabolism. Similar depot differences were found in stem cell population identified in freshly isolated stoma vascular fraction. The repertoire of ADSCs subpopulations was different in apple-shaped versus pear-shaped women.

Adipocyte­rich microenvironment promotes chemoresistance via upregulation of peroxisome proliferator­activated receptor gamma/ABCG2 in epithelial ovarian cancer.

The effects of adipocyte­rich microenvironment (ARM) on chemoresistance have garnered increasing interest. Ovarian cancer (OVCA) is a representative adipocyte­rich associated cancer. In the present study, epithelial OVCA (EOC) was used to investigate the influence of ARM on chemoresistance with the aim of identifying novel targets and developing novel strategies to reduce chemoresistance. Bioinformatics analysis was used to explore the effects of ARM­associated mechanisms contributing to chemoresistance and treated EOC cells, primarily OVCAR3 cells, with human adipose tissue extracts (HATES) from the peritumoral adipose tissue of patients were used to mimic ARM in vitro. Specifically, the peroxisome proliferator­activated receptor Î³ (PPARγ) antagonist GW9662 and the ABC transporter G family member 2 (ABCG2) inhibitor KO143, were used to determine the underlying mechanisms. Next, the effect of HATES on the expression of PPARγ and ABCG2 in OVCAR3 cells treated with cisplatin (DDP) and paclitaxel (PTX) was determined. Additionally, the association between PPARγ, ABCG2 and chemoresistance in EOC specimens was assessed. To evaluate the effect of inhibiting PPARγ, using DDP, a nude mouse model injected with OVCAR3­shPPARγ cells and a C57BL/6 model injected with ID8 cells treated with GW9662 were established. Finally, the factors within ARM that contributed to the mechanism were determined. It was found that HATES promoted chemoresistance by increasing ABCG2 expression via PPARγ. Expression of PPARγ/ABCG2 was related to chemoresistance in EOC clinical specimens. GW9662 or knockdown of PPARγ improved the efficacy of chemotherapy in mice. Finally, angiogenin and oleic acid played key roles in HATES in the upregulation of PPARγ. The present study showed that the introduction of ARM­educated PPARγ attenuated chemoresistance in EOC, highlighting a potentially novel therapeutic adjuvant to chemotherapy and shedding light on a means of improving the efficacy of chemotherapy from the perspective of ARM.

Stimulation of estrogen receptor-alpha by hydroxyanilide fungicide, fenhexamid promotes lipid accumulation in 3 T3-L1 adipocyte.

Fenhexamid are fungicides that act against plant pathogens by inhibiting sterol biosynthesis. Nonetheless, it can trigger endocrine disruption and promote breast cancer cell growth. In a recent study, we investigated the mechanism underlying the lipid accumulation induced by fenhexamid hydroxyanilide fungicides in 3 T3-L1 adipocytes. To examine the estrogen receptor alpha (ERα)-agonistic effect, ER transactivation assay using the ERα-HeLa-9903 cell line was applied, and fenhexamid-induced ERα agonist effect was confirmed. Further confirmation that ERα-dependent lipid accumulation occurred was provided by treating 3 T3-L1 adipocytes with Methyl-piperidino-pyrazole hydrate (MPP), an ERα-selective antagonist. Fenhexamid mimicked the actions of ERα agonists and impacted lipid metabolism, and its mechanism involves upregulation of the expression of transcription factors that facilitate adipogenesis and lipogenesis. Additionally, it stimulated the expression of peroxisome proliferator-activated receptor (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), fatty acid synthase (FAS), and sterol regulatory element-binding protein 1 (SREBP1) and significantly elevated the expression of fatty acid-binding protein 4 (FABP4). In contrast, in combination with an ERα-selective antagonist, fenhexamid suppressed the expression of adipogenic/lipogenic transcription factors. These results suggest that fenhexamid affects the endocrine system and leads to lipid accumulation by interfering with processes influenced by ERα activation.

Prmt6 represses the pro-adipogenic Ppar-gamma-C/ebp-alpha transcription factor loop.

The feed-forward loop between the transcription factors Ppar-gamma and C/ebp-alpha is critical for lineage commitment during adipocytic differentiation. Ppar-gamma interacts with epigenetic cofactors to activate C/ebp-alpha and the downstream adipocytic gene expression program. Therefore, knowledge of the epigenetic cofactors associated with Ppar-gamma, is central to understanding adipocyte differentiation in normal differentiation and disease. We found that Prmt6 is present with Ppar-gamma on the Ppar-gamma and C/ebp-alpha promoter. It contributes to the repression of C/ebp-alpha expression, in part through its ability to induce H3R2me2a. During adipocyte differentiation, Prmt6 expression is reduced and the methyltransferase leaves the promoters. As a result, the expression of Ppar-gamma and C/ebp-alpha is upregulated and the adipocytic gene expression program is established. Inhibition of Prmt6 by a small molecule enhances adipogenesis, opening up the possibility of epigenetic manipulation of differentiation. Our data provide detailed information on the molecular mechanism controlling the Ppar-gamma-C/ebp-alpha feed-forward loop. Thus, they advance our understanding of adipogenesis in normal and aberrant adipogenesis.

Influence of epicardial adipose tissue inflammation and adipocyte size on postoperative atrial fibrillation in patients after cardiovascular surgery.

Epicardial adipose tissue (EAT) is an active endocrine organ that is closely associated with occurrence of atrial fibrillation (AF). However, the role of EAT in the development of postoperative AF (POAF) remains unclear. We aimed to investigate the association between EAT profile and POAF occurrence in patients who underwent cardiovascular surgery. We obtained EAT samples from 53 patients to evaluate gene expression, histological changes, mitochondrial oxidative phosphorylation (OXPHOS) capacity in the EAT, and protein secretion in EAT-conditioned medium. EAT volume was measured using computed tomography scan. Eighteen patients (34%) experienced POAF within 7 days after surgery. Although no significant difference was observed in EAT profile between patients with and without POAF, logistic regression analysis identified that the mRNA expression levels of tumor necrosis factor-alpha (TNF-α) were positively correlated and adipocyte size in the EAT was inversely correlated with onset of POAF, respectively. Mitochondrial OXPHOS capacity in the EAT was not associated with POAF occurrence; however, it showed an inverse correlation with adipocyte size and a positive correlation with adiponectin secretion. In conclusion, changes in the secretory profile and adipocyte morphology of the EAT, which represent qualitative aspects of the adipose tissue, were present before the onset of AF.

Quercetin declines LPS induced inflammation and augments adiponectin expression in 3T3-L1 differentiated adipocytes SIRT-1 dependently.

BACKGROUND: Inflammation is an important factor contributing to obesity-induced metabolic disorders. Different investigations confirm that local inflammation in adipose issues is the primary reason for such disorder, resulting in low-grade systemic inflammation. Anti-inflammatory, antioxidant, and epigenetic modification are among the varied properties of Quercetin (QCT) as a natural flavonoid. OBJECTIVE: The precise molecular mechanism followed by QCT to alleviate inflammation has been unclear. This study explores whether the anti-inflammatory effects of QCT in 3T3-L1 differentiated adipocytes may rely on SIRT-1. METHODS: The authors isolated 3T3-L1 pre-adipocyte cells and exposed them to varying concentrations of QCT, lipopolysaccharide (LPS), and a selective inhibitor of silent mating type information regulation 2 homolog 1 (SIRT-1) called EX-527. After determining the optimal dosages of QCT, LPS, and EX-527, they assessed the mRNA expression levels of IL-18, IL-1, IL-6, TNF-α, SIRT-1, and adiponectin using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). RESULTS: The study showed considerable cytotoxic effects of LPS (200 ng/mL) + QCT (100 µM) + EX-527 (10 µM) on 3T3-L1 differentiated adipocytes after 48 h of incubation. QCT significantly upregulated the expression levels of adiponectin and SIRT-1 (p < 0.0001). However, introducing SIRT-1 inhibitor (p < 0.0001) reversed the impact of QCT on adiponectin expression. Additionally, QCT reduced SIRT-1-dependent pro-inflammatory cytokines in 3T3-L1 differentiated adipocytes (p < 0.0001). CONCLUSION: This study revealed that QCT treatment reduced crucial pro-inflammatory cytokines levels and increased adiponectin levels following LPS treatment. This finding implies that SIRT-1 may be a crucial factor for the anti-inflammatory activity of QCT.

Anti-inflammatory Effects of Food Ingredients on Mice Adipose Tissues and Adipocytes.

In 2021, we published three papers related to the anti-inflammatory effects of food ingredients. The present paper reports the effects of vitamin E homologs and sweet basil powder. In these papers, we investigated whether inflammation occurs in the adipose tissue of mice fed a high-fat and high-sucrose diet for 16 weeks. Inflammatory cytokine gene expression was significantly higher in the epididymal fat of the high-fat and high-sucrose diet group than in that of the control diet group. However, the addition of α-tocopherol or δ-tocopherol to the diet could not restrain the inflammation of mice epididymal fats. Thereafter, we investigated the anti-inflammatory effects of α- and δ-tocopherols using the co-cultured cells. Consequently, we clarified that δ-tocopherol inhibited the increase in the gene expressions of inflammatory cytokines. We also examined the effect of sweet basil powder on a similar obese mice model. The final body weight in the high-fat and high-sucrose group that received sweet basil powder was significantly lower than that in the high-fat and high-sucrose diet group. Liver weights were also significantly lower in the high-fat and high-sucrose diet group that received sweet basil powder than in the high-fat and high-sucrose diet group, although adipose tissue weights were unchanged in both groups. Furthermore, sweet basil powder tended to inhibit in lipid synthesis in the mice livers. Therefore, we suggested that sweet basil powder inhibited fatty acid synthesis in mice livers, thereby suppressing liver enlargement, and resulting in body weight loss. Moreover, the gene expression of MCP-1 in the adipose tissue of mice fed a high-fat and high-sucrose diet added with sweet basil powder was significantly lower than that of mice fed a high-fat and high-sucrose diet for 12 weeks. Therefore, sweet basil powder inhibited inflammation onset in the adipose tissue of mice. Taken together, the results suggested that food ingredients, especially vitamin E homologs and sweet basil powder, have anti-inflammatory effects on mice adipose tissue and mice adipocyte-induced inflammation.

Gene and lncRNA Profiling of ω3/ω6 Polyunsaturated Fatty Acid-Exposed Human Visceral Adipocytes Uncovers Different Responses in Healthy Lean, Obese and Colorectal Cancer-Affected Individuals.

Colorectal cancer (CRC) is a major life-threatening disease, being the third most common cancer and a leading cause of death worldwide. Enhanced adiposity, particularly visceral fat, is a major risk factor for CRC, and obesity-associated alterations in metabolic, inflammatory and immune profiles in visceral adipose tissue (VAT) strongly contribute to promoting or sustaining intestinal carcinogenesis. The role of diet and nutrition in obesity and CRC has been extensively demonstrated, and AT represents the main place where diet-induced signals are integrated. Among the factors introduced with diet and processed or enriched in AT, ω3/ω6 polyunsaturated fatty acids (PUFAs) are endowed with pro- or anti-inflammatory properties and have been shown to exert either promoting or protective roles in CRC. In this study, we investigated the impact of ex vivo exposure to the ω3 and ω6 PUFAs docosahexaenoic and arachidonic acids on VAT adipocyte whole transcription in healthy lean, obese and CRC-affected individuals. High-throughput sequencing of protein-coding and long non-coding RNAs allowed us to identify specific pathways and regulatory circuits controlled by PUFAs and highlighted an impaired responsiveness of obese and CRC-affected individuals as compared to the strong response observed in healthy lean subjects. This further supports the role of healthy diets and balanced ω3/ω6 PUFA intake in the primary prevention of obesity and cancer.

Effects of mifepristone on adipocyte differentiation in mouse 3T3-L1 cells.

BACKGROUND: Both glucocorticoid receptor and peroxisome proliferator-activated receptor-γ (PPARγ) play a critical role in adipocyte differentiation. Mifepristone is not only an antagonist of the glucocorticoid receptor but also an agonist of PPARγ. Therefore, the present study investigated the effect of mifepristone on adipocyte differentiation. METHODS: Mouse 3T3-L1 cells were used as a model for adipocyte differentiation. The lipid droplet formation was evaluated with Bodipy493/503 staining and the expression of adipocyte markers [adiponectin and adipocyte fatty acid binding protein-4 (Fabp4)] was evaluated with quantitative PCR and immunoblot analyses for indication of adipocyte differentiation. siRNA and neutralizing antibodies were used to elucidate the molecular mechanism of mifepristone-induced adipocyte differentiation. Luciferase reporter assay was used to examine the effect of mifepristone on the promoter activity of PPAR-response element (PPRE). The DNA microarray analysis was used to characterize the transcriptome of the mifepristone-induced adipocytes. In vivo adipogenic effect of mifepristone was examined in mice. RESULTS: Mifepristone not only enhanced adipocyte differentiation induced by the conventional protocol consisting of insulin, dexamethasone and 3-isobutyl-1-methylxanthine but also induced adipocyte differentiation alone, as evidenced by lipid droplets formation and induction of the expression of adiponectin and Fabp4. These effects were inhibited by an adiponectin-neutralizing antibody and a PPARγ antagonist. Mifepristone activated the promoter activity of PPRE in a manner sensitive to PPARγ antagonist. A principal component analysis (PCA) of DNA microarray data revealed that the mifepristone-induced adipocytes represent some characteristics of the in situ adipocytes in normal adipose tissues to a greater extent than those induced by the conventional protocol. Mifepristone administration induced an increase in the weight of epididymal, perirenal and gluteofemoral adipose tissues. CONCLUSIONS: Mifepristone alone is capable of inducing adipocyte differentiation in 3T3-L1 cells and adipogenesis in vivo. PPARγ plays a critical role in the mifepristone-induced adipocyte differentiation. Mifepristone-induced adipocytes are closer to the in situ adipocytes than those induced by the conventional protocol. The present study proposes a single treatment with mifepristone as a novel protocol to induce more physiologically relevant adipocytes in 3T3-L1 cells than the conventional protocol.

MST1/2 regulates fibro/adipogenic progenitor fate decisions in skeletal muscle regeneration.

Defective skeletal muscle regeneration is often accompanied by fibrosis. Fibroblast/adipose progenitors (FAPs) are important in these processes, however, the regulation of FAP fate decisions is unclear. Here, using inducible conditional knockout mice, we show that blocking mammalian Ste20-like kinases 1/2 (MST1/2) of FAPs prevented apoptosis and reduced interleukin-6 secretion in vivo and in vitro, which impaired myoblast proliferation and differentiation, as well as impaired muscle regeneration. Deletion of Mst1/2 increased co-localization of Yes-associated protein (YAP) with Smad2/3 in nuclei and promoted differentiation of FAPs toward myofibroblasts, resulting in excessive collagen deposition and skeletal muscle fibrosis. Meanwhile, inhibition of MST1/2 increased YAP/Transcriptional co-activator with PDZ-binding motif activation, which promoted activation of the WNT/ß-catenin pathway and impaired the differentiation of FAPs toward adipocytes. These results reveal a new mechanism for MST1/2 action in disrupted skeletal muscle regeneration and fibrosis via regulation of FAP apoptosis and differentiation. MST1/2 is a potential therapeutic target for the treatment of some myopathies.

Crosstalk of human coronary perivascular adipose-derived stem cells with vascular cells: role of tissue factor.

The coronary perivascular adipose tissue (cPVAT) has been associated to the burden of cardiovascular risk factors and to the underlying vessel atherosclerotic plaque severity. Although the "outside to inside" hypothesis of PVAT-derived-adipokine regulation of vessel function is currently accepted, whether the resident mesenchymal stem cells (ASCs) in PVAT have a regulatory role on the underlying vascular arterial smooth muscle cells (VSMCs) is not known. Here, we investigated the interactions between resident PVAT-ASCs and VSMCs. ASCs were obtained from PVAT overlying the left anterior descending (LAD) coronary artery of hearts removed at heart transplant operations. PVAT was obtained both from patients with non-ischemic and ischemic heart disease as the cause of heart transplant. ASCs were isolated from PVAT, phenotypically characterized by flow cytometry, functionally tested for proliferation, and differentiation. Crosstalk between ASCs and VSMCs was investigated by co-culture studies. ASCs were detected in the adventitia of the LAD-PVAT showing differentiation capacity and angiogenic potential. ASCs obtained from PVAT of non-ischemic and ischemic hearts showed different tissue factor (TF) expression levels, different VSMCs recruitment capacity through the axis ERK1/2-ETS1 signaling and different angiogenic potential. Induced upregulation of TF in ASCs isolated from ischemic PVAT rescued their angiogenic capacity in subcutaneously implanted plugs in mice, whereas silencing TF in ASCs decreased the proangiogenic capacity of non-ischemic ASCs. The results indicate for the first time a novel mechanism of regulation of VSMCs by PVAT-ASCs in angiogenesis, mediated by TF expression in ASCs. Regulation of TF in ASCs may become a therapeutic intervention to increase cardiac protection.

Overactivation of the Endocannabinoid System in Adolescence Disrupts Adult Adipose Organ Function in Mice.

Cannabis use stimulates calorie intake, but epidemiological studies show that people who regularly use it are leaner than those who don't. Two explanations have been proposed for this paradoxical finding. One posits that Δ9-tetrahydrocannabinol (THC) in cannabis desensitizes adipose CB1 cannabinoid receptors, stopping their stimulating effects on lipogenesis and adipogenesis. Another explanation is that THC exposure in adolescence, when habitual cannabis use typically starts, produces lasting changes in the developing adipose organ, which impacts adult systemic energy use. Here, we consider these possibilities in the light of a study which showed that daily THC administration in adolescent mice produces an adult metabolic phenotype characterized by reduced fat mass, partial resistance to obesity and dyslipidemia, and impaired thermogenesis and lipolysis. The phenotype, whose development requires activation of CB1 receptors in differentiated adipocytes, is associated with overexpression of myocyte proteins in the adipose organ with unchanged CB1 expression. We propose that adolescent exposure to THC causes lasting adipocyte dysfunction and the consequent emergence of a metabolic state that only superficially resembles healthy leanness. A corollary of this hypothesis, which should be addressed in future studies, is that CB1 receptors and their endocannabinoid ligands may contribute to the maintenance of adipocyte differentiation during adolescence.

Generating and Characterizing Adipose Spheroids from Adipose-Derived Stromal/Stem Cells.

Advances in technology and automation over the past several decades have made it feasible to perform high-throughput compound screening with cell spheroids, a valuable approach for drug discovery. It is entirely feasible to generate multiple 384-well plates containing adipose spheroids from cryopreserved, single-donor, adipose stem cells, thus incorporating genetic diversity into the discovery stages of research. In this protocol, we describe our method for isolating primary human adipose stem cells and synthesizing cell spheroids comprised of mature adipocytes and stromal cells. Also included are representative outcome measurements useful for characterizing adipocyte metabolism and health. Wherever possible, we describe technologies that can be used to automate characterization and increase throughput.