Overexpression of GPX2 gene regulates the development of porcine preadipocytes and skeletal muscle cells through MAPK signaling pathway.

Glutathione peroxidase 2 (GPX2) is a selenium-dependent enzyme and protects cells against oxidative damage. Recently, GPX2 has been identified as a candidate gene for backfat and feed efficiency in pigs. However, it is unclear whether GPX2 regulates the development of porcine preadipocytes and skeletal muscle cells. In this study, adenoviral gene transfer was used to overexpress GPX2. Our findings suggest that overexpression of GPX2 gene inhibited proliferation of porcine preadipocytes. And the process is accompanied by the reduction of the p-p38. GPX2 inhibited adipogenic differentiation and promoted lipid degradation, while ERK1/2 was reduced and p-p38 was increased. Proliferation of porcine skeletal muscle cells was induced after GPX2 overexpression, was accompanied by activation in JNK, ERK1/2, and p-p38. Overexpression methods confirmed that GPX2 has a promoting function in myoblastic differentiation. ERK1/2 pathway was activated and p38 was suppressed during the process. This study lays a foundation for the functional study of GPX2 and provides theoretical support for promoting subcutaneous fat reduction and muscle growth.

Elucidating the Role of circTIAM1 in Guangling Large-Tailed Sheep Adipocyte Proliferation and Differentiation via the miR-485-3p/PLCB1 Pathway.

Fat tissue-a vital energy storage organ-is intricately regulated by various factors, including circular RNA, which plays a significant role in modulating fat development and lipid metabolism. Therefore, this study aims to clarify the regulatory mechanism of sheep adipocyte proliferation and differentiation by investigating the involvement of circTIAM1, miR-485-3p, and its target gene PLCB1. Through previous sequencing data, circTIAM1 was identified in sheep adipocytes, with its circularization mechanism elucidated, confirming its cytoplasmic localization. Experimental evidence from RNase R treatment and transcription inhibitors highlighted that circTIAM1 is more stable than linear RNA. Additionally, circTIAM1 promoted sheep adipocyte proliferation and differentiation. Furthermore, bioinformatic analysis demonstrated a robust interaction between miR-485-3p and circTIAM1. Further experiments revealed that miR-485-3p inhibits fat cell proliferation and differentiation by inhibiting PLCB1, with circTIAM1 alleviating the inhibitory effect via competitive binding. In summary, our findings elucidate the mechanism through which circTIAM1 regulates Guangling Large-Tailed sheep adipocyte proliferation and differentiation via the miR-485-3p-PLCB1 pathway, offering a novel perspective for further exploring fat metabolism regulation.

The people behind the papers - Margret Bülow and Pilar Carrera.

Bez is a Class B scavenger receptor in Drosophila that is yet to be characterised. In a new study, Margret Bülow and colleagues uncover a role for Bez in mobilising lipids from Drosophila adipocytes into the ovary for oocyte maturation. To find out more about the people behind the paper, we caught up with first author, Pilar Carrera, and corresponding author, Margret Bülow, Group Leader at the University of Bonn.

The role of lncFABP4 in modulating adipogenic differentiation in buffalo intramuscular preadipocytes.

Intramuscular fat (IMF) is a crucial determinant of meat quality and is influenced by various regulatory factors. Despite the growing recognition of the important role of long noncoding RNAs (lncRNAs) in IMF deposition, the mechanisms underlying buffalo IMF deposition remain poorly understood. In this study, we identified and characterized a lncRNA, lncFABP4, which is transcribed from the antisense strand of fatty acid-binding protein 4 (FABP4). lncFABP4 inhibited cell proliferation in buffalo intramuscular preadipocytes. Moreover, lncFABP4 significantly increased intramuscular preadipocyte differentiation, as indicated by an increase in the expression of the adipogenic markers peroxisome proliferator-activated receptor gamma (PPARG), CCAAT enhancer binding protein alpha (C/EBPα), and FABP4. Mechanistically, lncFABP4 was found to have the potential to regulate downstream gene expression by participating in protein-protein interaction pathways. These findings contribute to further understanding of the intricate mechanisms through which lncRNAs modulate intramuscular adipogenesis in buffaloes.

Targeting adipocyte ESRRA promotes osteogenesis and vascular formation in adipocyte-rich bone marrow.

Excessive bone marrow adipocytes (BMAds) accumulation often occurs under diverse pathophysiological conditions associated with bone deterioration. Estrogen-related receptor α (ESRRA) is a key regulator responding to metabolic stress. Here, we show that adipocyte-specific ESRRA deficiency preserves osteogenesis and vascular formation in adipocyte-rich bone marrow upon estrogen deficiency or obesity. Mechanistically, adipocyte ESRRA interferes with E2/ESR1 signaling resulting in transcriptional repression of secreted phosphoprotein 1 (Spp1); yet positively modulates leptin expression by binding to its promoter. ESRRA abrogation results in enhanced SPP1 and decreased leptin secretion from both visceral adipocytes and BMAds, concertedly dictating bone marrow stromal stem cell fate commitment and restoring type H vessel formation, constituting a feed-forward loop for bone formation. Pharmacological inhibition of ESRRA protects obese mice against bone loss and high marrow adiposity. Thus, our findings highlight a therapeutic approach via targeting adipocyte ESRRA to preserve bone formation especially in detrimental adipocyte-rich bone milieu.

CircSSBP2 acts as a MiR-2400 sponge to promote intramuscular preadipocyte proliferation by regulating NDRG1.

Intramuscular fat (IMF) is a critical factor in beef quality. IMF is mainly distributed between muscle fibres and its accumulation can affect the marbling and meat quality of beef. IMF formation and deposition is a complex process and in recent years a group of non-coding RNAs (ncRNAs), known as circRNAs, have been discovered to play an important role in regulating intramuscular fat deposition. CircRNAs form a covalent loop structure after reverse splicing of precursor mRNAs. They can act by adsorbing miRNAs, thereby reducing their repressive effects on downstream target genes. Based on high-throughput sequencing of circRNAs in intramuscular fat of Qinchuan and Japanese black cattle, we identified a novel circSSBP2 that is differentially expressed between the two species and associated with adipogenesis. We show that circSSBP2 knockdown promotes bovine intramuscular preadipocyte proliferation, whereas overexpression inhibits bovine intramuscular preadipocyte proliferation. We also show that circSSBP2 can act as a molecular sponge for miR-2400 and that miR-2400 overexpression promotes bovine intramuscular preadipocyte proliferation. Furthermore, N-myc downstream-regulated gene 1 (NDRG1) was identified as a direct target gene of miR-2400, and NDRG1 interference promoted the proliferation of bovine intramuscular preadipocytes. In conclusion, our results suggest that circSSBP2 inhibits the proliferation of bovine intramuscular preadipocytes by regulating the miR-2400/NDRG1 axis.

Neuron-derived neurotrophic factor promotes the differentiation of intramuscular and subcutaneous adipocytes in goat.

Adipocyte play an important role in human health and meat quality by influencing the tenderness, flavor, and juiciness of mutton It has been shown that neuron-derived neurotrophic factor (NENF) is closely related to energy metabolism and adipocyte differentiation in bovine. However, the role of NENF in the goats remains unclear. The aim of this study was to detect the expression of NENF in goat subcutaneous and intramuscular adipocytes, temporal expression profiles of the NENF, and overexpressed NENF on the differentiation of different adipocytes. In this study, PCR amplification successfully cloned the goat NENF gene with a fragment length of 521 bp. In addition, the time point of highest expression of NENF differed between these two adipocytes differentiation processes. Overexpression of NENF in intramuscular and subcutaneous adipocytes promoted the expression levels of differentiation markers CEBPß and SREBP, which in turn promoted the differentiation of intramuscular and subcutaneous adipocytes. This study will provide basic data for further study of the role of goats in goat adipocyte differentiation and for the final elucidation of its molecular mechanisms in regulating goat adipocyte deposition.

TG-interacting factor 1 regulates mitotic clonal expansion during adipocyte differentiation.

Obesity is one of the significant health challenges in the world and is highly associated with abnormal adipogenesis. TG-interacting factor 1 (TGIF1) is essential for differentiating murine adipocytes and human adipose tissue-derived stem cells. However, the mode of action needs to be better elucidated. To investigate the roles of TGIF1 in differentiation in-depth, CRISPR/Cas9 knockout technology was performed to generate TGIF1-silenced preadipocytes. The absence of TGIF1 in 3 T3-F442A preadipocytes abolished lipid accumulation throughout the differentiation using Oil Red O staining. Conversely, we established 3 T3-F442A preadipocytes stably expressing TGIF1 and doxycycline-inducible TGIF1 in TGIF1-silenced 3 T3-F442A preadipocytes. Remarkably, the induction of TGIF1 by doxycycline during the initial differentiation phase successfully promoted lipid accumulation in TGIF1-silenced 3 T3-F442A cells. We further explored the mechanisms of TGIF1 in early differentiation. We demonstrated that TGIF1 promoted the mitotic clonal expansion via upregulation of CCAAT/enhancer-binding proteins ß expression, interruption with peroxisome proliferators activated receptor γ downstream regulation, and inhibition of p27kip1 expression. In conclusion, we strengthen the pivotal roles of TGIF1 in early differentiation, which might contribute to resolving obesity-associated metabolic syndromes.

LncRNA BlncAD1 Modulates Bovine Adipogenesis by Binding to MYH10, PI3K/Akt Signaling Pathway, and miR-27a-5p/CDK6 Axis.

Research on adipogenesis will help to improve the meat quality of livestock. Long noncoding RNAs (lncRNAs) are involved in mammalian adipogenesis as epigenetic modulators. In this study, we analyzed lncRNA expression during bovine adipogenesis and detected 195 differentially expressed lncRNAs, including lncRNA BlncAD1, which was significantly upregulated in mature bovine adipocytes. Gain- and loss-of-function experiments confirmed that BlncAD1 promoted the proliferation, apoptosis, and differentiation of bovine preadipocytes. RNA pull-down revealed that the nonmuscle myosin 10 (MYH10) is a potential binding protein of BlncAD1. Then, we elucidated that loss of BlncAD1 caused increased ubiquitination of MYH10, which confirmed that BlncAD1 regulates adipogenesis by enhancing the stability of the MYH10 protein. Western blotting was used to demonstrate that BlncAD1 activated the PI3K/Akt signaling pathway. Bioinformatic analysis and dual-luciferase reporter assays indicated that BlncAD1 competitively absorbed miR-27a-5p. The overexpression and interference of miR-27a-5p in bovine preadipocytes displayed that miR-27a-5p inhibited proliferation, apoptosis, and differentiation. Further results suggested that miR-27a-5p targeted the CDK6 gene and that BlncAD1 controlled the proliferation of bovine preadipocytes by modulating the miR-27a-5p/CDK6 axis. This study revealed the complex mechanisms of BlncAD1 underlying bovine adipogenesis for the first time, which would provide useful information for genetics and breeding improvement of Chinese beef cattle.

Effect of metabolically divergent pig breeds and tissues on mesenchymal stem cell expression patterns during adipogenesis.

BACKGROUND: Unraveling the intricate and tightly regulated process of adipogenesis, involving coordinated activation of transcription factors and signaling pathways, is essential for addressing obesity and related metabolic disorders. The molecular pathways recruited by mesenchymal stem cells (MSCs) during adipogenesis are also dependent on the different sources of the cells and genetic backgrounds of donors, which contribute to the functional heterogeneity of the stem cells and consequently affect the developmental features and fate of the cells. METHODS: In this study, the alteration of transcripts during differentiation of synovial mesenchymal stem cells (SMSCs) derived from fibrous synovium (FS) and adipose synovial tissue (FP) of two pig breeds differing in growth performance (German Landrace (DL)) and fat deposition (Angeln Saddleback (AS)) was investigated. SMSCs from both tissues and breeds were stimulated to differentiate into adipocytes in vitro and sampled at four time points (day 1, day 4, day 7 and day 14) to obtain transcriptomic data. RESULTS: We observed numerous signaling pathways related to the cell cycle, cell division, cell migration, or cell proliferation during early stages of adipogenesis. As the differentiation process progresses, cells begin to accumulate intracellular lipid droplets and changes in gene expression patterns in particular of adipocyte-specific markers occur. PI3K-Akt signaling and metabolic pathways changed most during adipogenesis, while p53 signaling and ferroptosis were affected late in adipogenesis. When comparing MSCs from FS and FP, only a limited number of differentially expressed genes (DEGs) and enriched signaling pathways were identified. Metabolic pathways, including fat, energy or amino acid metabolism, were highly enriched in the AS breed SMSCs compared to those of the DL breed, especially at day 7 of adipogenesis, suggesting retention of the characteristic metabolic features of their original source, demonstrating donor memory in culture. In contrast, the DL SMSCs were more enriched in immune signaling pathways. CONCLUSIONS: Our study has provided important insights into the dynamics of adipogenesis and revealed metabolic shifts in SMSCs associated with different cell sources and genetic backgrounds of donors. This emphasises the critical role of metabolic and genetic factors as important indications and criteria for donor stem cell selection.

Potential probiotic Lactobacillus delbrueckii subsp. lactis KUMS-Y33 suppresses adipogenesis and promotes osteogenesis in human adipose-derived mesenchymal stem cell.

Today, probiotics are considered to be living microorganisms whose consumption has a certain number of beneficial effects on the consumer. The present study aimed to investigate the effect of a new probiotic extract (Lactobacillus delbrueckii subsp. lactis KUMS Y33) on the differentiation process of human adipose-derived stem cells (hADSCs) into adipocytes and osteocytes and, as a result, clarify its role in the prevention and treatment of bone age disease. Several bacteria were isolated from traditional yogurt. They were evaluated to characterize the probiotic's activity. Then, the isolated hADSCs were treated with the probiotic extract, and then osteogenesis and adipogenesis were induced. To evaluate the differentiation process, oil red O and alizarin red staining, a triglyceride content assay, an alkaline phosphatase (ALP) activity assay, as well as real-time PCR and western blot analysis of osteocyte- and adipocyte-specific genes, were performed. Ultimately, the new strain was sequenced and registered on NBCI. In the probiotic-treated group, the triglyceride content and the gene expression and protein levels of C/EBP-α and PPAR-γ2 (adipocyte-specific markers) were significantly decreased compared to the control group (P < 0.05), indicating an inhibited adipogenesis process. Furthermore, the probiotic extract caused a significant increase in the ALP activity, the expression levels of RUNX2 and osteocalcin, and the protein levels of collagen I and FGF-23 (osteocyte-specific markers) in comparison to the control group (P < 0.05), indicating an enhanced osteogenesis process. According to the results of the present study, the probiotic extract inhibits adipogenesis and significantly increases osteogenesis, suggesting a positive role in the prevention and treatment of osteoporosis and opening a new aspect for future in-vivo study.

Studying Adipose Endothelial Cell/Adipocyte Cross-Talk in Human Subcutaneous Adipose Tissue.

Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. Significant heterogeneity in endothelial cell (EC) genotype and phenotype depends on their vascular bed and host disease state. The ability to isolate MVECs from tissue-specific vascular beds and individual patient groups offers the opportunity to directly compare MVEC function in different disease states. Here, using subcutaneous adipose tissue (SAT) taken at the time of insertion of cardiac implantable electronic devices (CIED), we describe a method for the isolation of a pure population of functional human subcutaneous adipose tissue MVEC (hSATMVEC) and an experimental model of hSATMVEC-adipocyte cross-talk. hSATMVEC were isolated following enzymatic digestion of SAT by incubation with anti-CD31 antibody-coated magnetic beads and passage through magnetic columns. hSATMVEC were grown and passaged on gelatin-coated plates. Experiments used cells at passages 2-4. Cells maintained classic features of EC morphology until at least passage 5. Flow cytometric assessment showed 99.5% purity of isolated hSATMVEC, defined as CD31+/CD144+/CD45-. Isolated hSATMVEC from controls had a population doubling time of approximately 57 h, and active proliferation was confirmed using a cell proliferation imaging kit. Isolated hSATMVEC function was assessed using their response to insulin stimulation and angiogenic tube-forming potential. We then established an hSATMVEC-subcutaneous adipocyte co-culture model to study cellular cross-talk and demonstrated a downstream effect of hSATMVEC on adipocyte function. hSATMVEC can be isolated from SAT taken at the time of CIED insertion and are of sufficient purity to both experimentally phenotype and study hSATMVEC-adipocyte cross-talk.

Investigating the Role of KLF6 in the Growth of Bovine Preadipocytes: Using Transcriptomic Analyses to Understand Beef Quality.

Intramuscular fat is a crucial determinant of carcass quality traits like tenderness and taste, which in turn is influenced by the proliferation of intramuscular preadipocytes. This study aimed to investigate the Krüppel-like factor 6 (KLF6)-mediated proliferation of bovine preadipocytes and identify underlying molecular mechanisms. Down-regulation of KLF6 by siKLF6 resulted in a significant (p < 0.01) suppression of cell cycle-related genes including CDK1, MCM6, ZNF4, PCNA, CDK2, CCNB1, and CDK6. Conversely, the expression level of p27 was significantly (p < 0.01) increased. Moreover, EdU (5-ethynyl-20-deoxyuridine) staining revealed a significant decrease in EdU-labeled cells due to KLF6 down-regulation. Collectively, these findings indicate that KLF6 down-regulation inhibits adipocyte proliferation. Furthermore, RNA sequencing of preadipocytes transfected with siKLF6 and NC, followed by differential gene expression analysis, identified 100 up-regulated and 70 down-regulated genes. Additionally, the differentially expressed genes also significantly influenced various Gene Ontology (GO) terms related to cell cycle, nuclear chromosomes, and catalytic activity on DNA. Furthermore, the top 20 pathways enriched in these DEGs included cell cycle, DNA replication, cellular senescence, and homologous recombination. These GO terms and KEGG pathways play key roles in bovine preadipocyte proliferation. In conclusion, the results of this study suggest that KLF6 positively regulates the proliferation of bovine preadipocytes.

Phosphorylation of TG-interacting factor 1 at carboxyl-terminal sites in response to insulin regulates adipocyte differentiation.

TG-interacting factor 1 (TGIF1) contributes to the differentiation of murine white preadipocyte and human adipose tissue-derived stem cells; however, its regulation is not well elucidated. Insulin is a component of the adipogenic cocktail that induces ERK signaling. TGIF1 phosphorylation and sustained stability in response to insulin were reduced through the use of specific MEK inhibitor U0126. Mutagenesis at T235 or T239 residue of TGIF1 in preadipocytes led to dephosphorylation of TGIF1. The reduced TGIF1 stability resulted in an increase in p27kip1 expression, a decrease in phosphorylated Rb expression and cellular proliferation, and a reduced accumulation of lipids compared to the TGIF1-overexpressed cells. These findings highlight that insulin/ERK-driven phosphorylation of the T235 or T239 residue at TGIF1 is crucial for adipocyte differentiation.

Autophagic Regulation of Adipogenesis Through TP53INP2: Insights from In Silico and In Vitro Analysis.

Obesity is an epidemic disease associated with multimorbidity resulting in higher mortality risk. The imbalance between energy storage and expenditure is the prime factor in the prognosis of the disease. Specifically, excessive lipid storage through adipogenesis leads to obesity. Adipogenesis is the process that converts preadipocytes into mature adipocytes by regulating major transcription factors like PPARγ and C/EBPα, contributes to lipid storage in adipose tissue. On the contrary, autophagy is a self-degradative process that maintains homeostasis in adipose tissue by regulating adipogenesis and lipolysis. TP53INP2 is a key player that regulates the autophagy process, and it negatively regulates adipogenesis and lipid storage. The gene expression profile GSE93637 was retrieved from the GEO database and analyzed using an integrated bioinformatics approach. The differentially expressed genes (DEGs) were analyzed using R-Bioconductor for TP53INP2 knockdown microarray dataset of 3T3L1 cells, and the DEGs were analyzed for the functional enrichment analysis. Further, the genes involved in the potential biological and molecular functions were evaluated for pathway enrichment analysis by KEGG (Kyoto Encyclopedia of Genes and Genomes). A total of 726 DEGs were found including 391 upregulated and 335 downregulated genes. Further, the functional and pathway enrichment analysis was employed to identify the highly interacting genes, and we identified a total of 56 genes that are highly interacting through a protein-protein interaction network. The DEGs mainly regulate the Peroxisome proliferator-activated receptor (PPAR) signaling pathway, lipolysis, and autophagy. Further, we investigated the associated Hub genes for enriched pathway genes and found the involvement of two autophagic genes ATG7 and sequestosome 1 (p62). In addition, in vitro studies of qRT-PCR (Quantitative real-time polymerase chain reaction) and Western blot analysis revealed that increased autophagy resulted in reduced lipid storage through down-regulation of the adipogenic gene. Moreover, increased expression of autophagic gene TP53INP2 and ATG7 facilitates the down-regulation of p62 and PPARγ gene resulting in lipolysis in mature adipocytes through autophagy. There is no specific treatment to reduce obesity other than a caloric diet and exercise. Hence, this study provides sufficient evidence to conclude that TP53INP2 negatively regulates adipogenesis and increases the degradation of lipids in mature adipocytes which is crucial for reducing obesity. Therefore, it is plausible to consider TP53INP2 as a promising therapeutic target for managing adipogenesis and obesity. However, further studies are necessary to validate their functional and molecular pathway analysis in the regulation of adipogenesis and obesity.

The P2Y2 receptor mediates terminal adipocyte differentiation and insulin resistance: Evidence for a dual G-protein coupling mode.

Several P2Y nucleotide receptors have been shown to be involved in the early stage of adipocyte differentiation in vitro and insulin resistance in obese mice; however, the exact receptor subtype(s) and its underlying molecular mechanism in relevant human cells are unclear. Here, using human primary visceral preadipocytes as a model, we found that during preadipocyte-to-mature adipocyte differentiation, the P2Y2 nucleotide receptor (P2Y2R) was the most upregulated subtype among the eight known P2Y receptors and the only one further dramatically upregulated after inflammatory TNFα treatment. Functional studies indicated that the P2Y2R induced intracellular Ca2+, ERK1/2, and JNK signaling but not the p38 pathway. In addition, stimulation of the P2Y2R suppressed basal and insulin-induced phosphorylation of AKT, accompanied by decreased GLUT4 membrane translocation and glucose uptake in mature adipocytes, suggesting a role of P2Y2R in insulin resistance. Mechanistically, we found that activation of P2Y2R did not increase lipolysis but suppressed PIP3 generation. Interestingly, activation of P2Y2R triggered Gi-protein coupling, and pertussis toxin pretreatment largely inhibited P2Y2R-mediated ERK1/2 signaling and cAMP suppression. Further, treatment of the cells with AR-C 118925XX, a selective P2Y2R antagonist, significantly inhibited adipogenesis, and P2Y2R knockout decreased mouse body weight gain with smaller eWAT mass infiltrated with fewer macrophages as compared to WT mice in response to a Western diet. Thus, we revealed that terminal adipocyte differentiation and inflammation selectively upregulate P2Y2R expression and that P2Y2R mediates insulin resistance by suppressing the AKT signaling pathway, highlighting P2Y2R as a potential new drug target to combat obesity and type-2 diabetes.

Laminin fragments conjugated with perlecan's growth factor-binding domain differentiate human induced pluripotent stem cells into skin-derived precursor cells.

Deriving stem cells to regenerate full-thickness human skin is important for treating skin disorders without invasive surgical procedures. Our previous protocol to differentiate human induced pluripotent stem cells (iPSCs) into skin-derived precursor cells (SKPs) as a source of dermal stem cells employs mouse fibroblasts as feeder cells and is therefore unsuitable for clinical use. Herein, we report a feeder-free method for differentiating iPSCs into SKPs by customising culture substrates. We immunohistochemically screened for laminins expressed in dermal papillae (DP) and explored the conditions for inducing the differentiation of iPSCs into SKPs on recombinant laminin E8 (LM-E8) fragments with or without conjugation to domain I of perlecan (PDI), which binds to growth factors through heparan sulphate chains. Several LM-E8 fragments, including those of LM111, 121, 332, 421, 511, and 521, supported iPSC differentiation into SKPs without PDI conjugation. However, the SKP yield was significantly enhanced on PDI-conjugated LM-E8 fragments. SKPs induced on PDI-conjugated LM111-E8 fragments retained the gene expression patterns characteristic of SKPs, as well as the ability to differentiate into adipocytes, osteocytes, and Schwann cells. Thus, PDI-conjugated LM-E8 fragments are promising agents for inducing iPSC differentiation into SKPs in clinical settings.

Senescent adipocytes as potential effectors of muscle cells dysfunction: An in vitro model.

Recently, there has been a growing body of evidence showing a negative effect of the white adipose tissue (WAT) dysfunction on the skeletal muscle function and quality. However, little is known about the effects of senescent adipocytes on muscle cells. Therefore, to explore potential mechanisms involved in age-related loss of muscle mass and function, we performed an in vitro experiment using conditioned medium obtained from cultures of mature and aged 3 T3-L1 adipocytes, as well as from cultures of dysfunctional adipocytes exposed to oxidative stress or high insulin doses, to treat C2C12 myocytes. The results from morphological measures indicated a significant decrease in diameter and fusion index of myotubes after treatment with medium of aged or stressed adipocytes. Aged and stressed adipocytes presented different morphological characteristics as well as a different gene expression profile of proinflammatory cytokines and ROS production. In myocytes treated with different adipocytes' conditioned media, we demonstrated a significant reduction of gene expression of myogenic differentiation markers as well as a significant increase of genes involved in atrophy. Finally, a significant reduction in protein synthesis as well as a significant increase of myostatin was found in muscle cells treated with medium of aged or stressed adipocytes compared to controls. In conclusion, these preliminary results suggest that aged adipocytes could influence negatively trophism, function and regenerative capacity of myocytes by a paracrine network of signaling.

Inhibition of nucleotide biosynthesis disrupts lipid accumulation and adipogenesis.

Energy balance and nutrient availability are key determinants of cellular decisions to remain quiescent, proliferate, or differentiate into a mature cell. After assessing its environmental state, the cell must rewire its metabolism to support distinct cellular outcomes. Mechanistically, how metabolites regulate cell fate decisions is poorly understood. We used adipogenesis as our model system to ascertain the role of metabolism in differentiation. We isolated adipose tissue stromal vascular fraction cells and profiled metabolites before and after adipogenic differentiation to identify metabolic signatures associated with these distinct cellular states. We found that differentiation alters nucleotide accumulation. Furthermore, inhibition of nucleotide biosynthesis prevented lipid storage within adipocytes and downregulated the expression of lipogenic factors. In contrast to proliferating cells, in which mechanistic target of rapamycin complex 1 is activated by purine accumulation, mechanistic target of rapamycin complex 1 signaling was unaffected by purine levels in differentiating adipocytes. Rather, our data indicated that purines regulate transcriptional activators of adipogenesis, peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α, to promote differentiation. Although de novo nucleotide biosynthesis has mainly been studied in proliferation, our study points to its requirement in adipocyte differentiation.

Kinetic networks identify TWIST2 as a key regulatory node in adipogenesis.

Adipocytes contribute to metabolic disorders such as obesity, diabetes, and atherosclerosis. Prior characterizations of the transcriptional network driving adipogenesis have overlooked transiently acting transcription factors (TFs), genes, and regulatory elements that are essential for proper differentiation. Moreover, traditional gene regulatory networks provide neither mechanistic details about individual regulatory element-gene relationships nor temporal information needed to define a regulatory hierarchy that prioritizes key regulatory factors. To address these shortcomings, we integrate kinetic chromatin accessibility (ATAC-seq) and nascent transcription (PRO-seq) data to generate temporally resolved networks that describe TF binding events and resultant effects on target gene expression. Our data indicate which TF families cooperate with and antagonize each other to regulate adipogenesis. Compartment modeling of RNA polymerase density quantifies how individual TFs mechanistically contribute to distinct steps in transcription. The glucocorticoid receptor activates transcription by inducing RNA polymerase pause release, whereas SP and AP-1 factors affect RNA polymerase initiation. We identify Twist2 as a previously unappreciated effector of adipocyte differentiation. We find that TWIST2 acts as a negative regulator of 3T3-L1 and primary preadipocyte differentiation. We confirm that Twist2 knockout mice have compromised lipid storage within subcutaneous and brown adipose tissue. Previous phenotyping of Twist2 knockout mice and Setleis syndrome Twist2 -/- patients noted deficiencies in subcutaneous adipose tissue. This network inference framework is a powerful and general approach for interpreting complex biological phenomena and can be applied to a wide range of cellular processes.