Adipocyte Differentiation Process Research Articles

Effect Of Vitamin D3 On Lipids Metabolism And Adipose Tissue Biology

There are many different kinds of cells that make up adipose tissue, and it plays a vital part in the regulation of inflammation and the maintenance of energy balance, both locally and throughout the body. It is one of the most important organs that is not skeletal in nature and serves as the principal storage location for vitamin D. Vitamin D also has an effect on this organ. It is vital to take into consideration the specific cell type, the stage of differentiation, and the length of time that the therapy is delivered in order to determine how vitamin D can affect multiple aspects of adipose tissue function and development. This is because vitamin D can help determine how vitamin D can influence these aspects. In spite of this, vitamin D has the ability to either slow down or speed up the differentiation process of adipocytes, which are cells that are responsible for the synthesis of fat. This is dependent on the specific cell type. Vitamin D is not only responsible for this, but it also plays a role in the regulation of the energy metabolism that occurs in adipose tissue. The oxidation of fatty acids, the expression of uncoupling proteins, insulin resistance, and the synthesis of adipokines are all altered as a result of this process. Furthermore, inflammation of adipose tissue is a substantial contribution to the metabolic abnormalities that are often associated with obesity. This is because adipose tissue is a major source of fat. There is evidence that vitamin D has the potential to influence the inflammatory response of immune cells, as well as the systemic response and adipocytes that are present in adipose tissue. Vitamin D also has the ability to influence the inflammatory response of immune cells. Additionally, vitamin D has the capacity to prevent the development of cancer in some people.

Shared Mechanisms in Pparγ1sv and Pparγ2 Expression in 3T3-L1 Cells: Studies on Epigenetic and Positive Feedback Regulation of Pparγ during Adipogenesis.

We have previously reported the identification of a novel splicing variant of the mouse peroxisome proliferator-activated receptor-γ (Pparγ), referred to as Pparγ1sv. This variant, encoding the PPARγ1 protein, is abundantly and ubiquitously expressed, playing a crucial role in adipogenesis. Pparγ1sv possesses a unique promoter and 5' untranslated region (5'UTR), distinct from those of the canonical mouse Pparγ1 and Pparγ2 mRNAs. We observed a significant increase in DNA methylation at two CpG sites within the proximal promoter region (-733 to -76) of Pparγ1sv during adipocyte differentiation. Concurrently, chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) using antibodies against H3K4me3 and H3K27ac indicated marked elevations in both methylation and acetylation of histone H3, while the repressive histone mark H3K9me2 significantly decreased, at the transcription start sites of both Pparγ1sv and Pparγ2 following differentiation. Knocking down Pparγ1sv using specific siRNA also led to a decrease in Pparγ2 mRNA and PPARγ2 protein levels; conversely, knocking down Pparγ2 resulted in reduced Pparγ1sv mRNA and PPARγ1 protein levels, suggesting synergistic transcriptional regulation of Pparγ1sv and Pparγ2 during adipogenesis. Furthermore, our experiments utilizing the CRISPR-Cas9 system identified crucial PPARγ-binding sites within the Pparγ gene locus, underscoring their significance in adipogenesis. Based on these findings, we propose a model of positive feedback regulation for Pparγ1sv and Pparγ2 expression during the adipocyte differentiation process in 3T3-L1 cells.

Suramin, an antiparasitic drug, stimulates adipocyte differentiation and promotes adipogenesis

BackgroundPrevious studies demonstrated that mast cells with their degranulated component heparin are the major endogenous factors that stimulate preadipocyte differentiation and promote fascial adipogenesis, and this effect is related to the structure of heparin. Regarding the structural and physiological properties of the negatively charged polymers, hexasulfonated suramin, a centuries-old medicine that is still used for treating African trypanosomiasis and onchocerciasis, is assumed to be a heparin-related analog or heparinoid. This investigation aims to elucidate the influence of suramin on the adipogenesis.MethodsTo assess the influence exerted by suramin on adipogenic differentiation of primary white adipocytes in rats, this exploration was conducted both in vitro and in vivo. Moreover, it was attempted to explore the role played by the sulfonic acid groups present in suramin in mediating this adipogenic process.ResultsSuramin demonstrated a dose- and time-dependent propensity to stimulate the adipogenic differentiation of rat preadipocytes isolated from the superficial fascia tissue and from adult adipose tissue. This stimulation was concomitant with a notable upregulation in expression levels of pivotal adipogenic factors as the adipocyte differentiation process unfolded. Intraperitoneal injection of suramin into rats slightly increased adipogenesis in the superficial fascia and in the epididymal and inguinal fat depots. PPADS, NF023, and NF449 are suramin analogs respectively containing 2, 6, and 8 sulfonic acid groups, among which the last two moderately promoted lipid droplet formation and adipocyte differentiation. The number and position of sulfonate groups may be related to the adipogenic effect of suramin.ConclusionsSuramin emerges as a noteworthy pharmaceutical agent with the unique capability to significantly induce adipocyte differentiation, thereby fostering adipogenesis.

Combination of Panax ginseng and Diospyros kaki Leaf Inhibits White Adipocyte Differentiation and Browning Process through AMP-Activated Protein Kinase (AMPK) Activation In Vitro and In Vivo.

Activating brown adipose tissue (BAT) and stimulating white adipose tissue (WAT) browning is a prospective obesity treatment method. Dietary components derived from plants are the most effective approach to activate BAT and promote WAT browning in rodents. This study investigated the synergistic effects of Panax ginseng (PG) and Diospyros kaki leaf (DKL) extract on adipocyte differentiation and browning, as well as the molecular mechanism underlying their beneficial effects. The administration of PG and DKL to HFD-induced obese mice significantly decreased body weight and epididymal and abdominal adipose tissue mass. In in vitro, PG inhibited the adipogenesis of 3T3-L1 adipocytes by regulating the expression of key adipogenic regulators, such as peroxisome proliferator-activated receptor (PPAR)γ and CCAAT/enhancer-binding protein (C/EBP)-α. In contrast, DKL negligibly influenced the adipogenesis of 3T3-L1 adipocytes but greatly increased the protein expression of UCP-1, PGC-1α, and PPARα in BAT and/or WAT. Moreover, PG and DKL inhibited adipogenesis synergistically and activated white adipocyte browning via AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1) pathways. These results suggest that a combination of PG and DKL regulates adipogenesis in white adipocytes and browning in brown adipocytes by activating AMPK/SIRT1 axis. The potential use of PG and DKL may represent an important strategy in obesity management that will be safer and more effective.

Bone marrow adipocytes alteration in an in vitro model of Gaucher Disease

Gaucher disease (GD) is caused by biallelic pathogenic variants in GBA1 gene that encodes the lysosomal enzyme glucocerebrosidase. Up to now, specific treatment for GD cannot completely reverse bone complications. Bone is composed of different cell types; including osteoblasts, osteocytes and osteoclasts. Osteoblasts are present on bone surfaces and are derived from local mesenchymal stem cells (MSCs). Depending on environment conditions, MSCs could differentiate into osteoblasts and adipocytes. Mature adipocytes-secreted adipokines and free fatty acids affect both osteoblasts and osteoclasts formation/activity and therefore mediate skeletal homeostasis. The aim of this study was to evaluate possible alterations in GD adipocyte (GD Ad) that could contribute to bone complications. MSCs were grown in adipogenic media in order to evaluate expression of differentiation markers as PPAR-γ. PPAR-γ was observed into the nucleus of GD Ad, indicating that these cells are properly stimulated. However, these cells accumulate lesser lipid droplets (LDs) than Control Ad. In order to study lipid droplet metabolism, we evaluated the lipolysis of these structures by the measurement of free glycerol in culture supernatant. Our results indicated that GD Ad had an alteration in this process, evidenced by an increase in glycerol release. We have also evaluated two enzymes involved in LDs synthesis: fatty acid synthase (FASN) and stearoyl-coenzyme A desaturase 1 (SCD1). The transcription of these genes was decreased in GD Ad, suggesting a dysfunction in the synthesis of LDs. In conclusion, our results show an alteration in LDs metabolism of GD Ad, independent of adipocyte differentiation process. This alteration would be caused by an increase in lipolysis in early stages of differentiation and also by a reduction of lipid synthesis, which could contribute with the skeletal imbalance in GD.

Differential regulation of mRNAs and lncRNAs related to lipid metabolism in Duolang and Small Tail Han sheep

The function of long non-coding RNA (lncRNA) can be achieved through the regulation of target genes, and the deposition of fat is regulated by lncRNA. Fat has an important effect on meat quality. However, there are relatively few studies on lncRNAs in the subcutaneous adipose tissue of Duolang sheep and Small Tail Han sheep. In this study, RNA-Seq technology and bioinformatics methods were used to identify and analyze the lncRNA and mRNA in the subcutaneous adipose tissue of the two breeds of sheep. The results showed that 107 lnRNAs and 1329 mRNAs were differentially expressed. The differentially expressed genes and lncRNA target genes were significantly enriched in the biosynthesis of unsaturated fatty acids signaling pathway, fatty acid metabolism, adipocyte differentiation and other processes related to fat deposition. Among them, LOC105616076, LOC114118103, LOC105607837, LOC101116622, and LOC105603235 target FADS1, SCD, ELOVL6, HSD17B12 and HACD2, respectively. They play a key regulatory role in the biosynthesis of unsaturated fatty acids. This study lays a foundation for the study of the molecular mechanism of lncRNA on fat development, and has reference value for studying the differences in fat deposition between Duolang sheep and Small Tail Han sheep.

Screening of key miRNAs related with the differentiation of subcutaneous adipocytes and the validation of miR-133a-3p functional significance in goats.

Adipocyte differentiation is regulated by a variety of functional genes and noncoding RNAs. However, the role of miRNAs in lipid deposition of goat white adipose tissue is still unclear. Therefore, this study revealed the miRNA expression profile in goat subcutaneous adipocytes by sRNA-seq. The miRNA expressed in goat subcutaneous preadipocytes and the mature adipocytes were sequenced by sRNA-seq. The differentially expressed miRNAs (DEm) were screened and gene ontology (GO) and Kyoto encyclopedia for genes and genomes (KEGG) analyses were performed. Gain-of-function and loss-of-function combined with oil red O staining, Bodipy staining, and quantitative reverse-transcription polymerase chain reaction (qPCR) were utilized to determine the effect of miR-133a-3p on adipocyte differentiation. A total of 218 DEm were screened out. The target genes of these DEm were significantly enriched in GO items such as biological regulation and in KEGG terms such as FAK signaling pathway and MAPK signaling pathway. qPCR verified that the expression trend of miRNA was consistent with miRNA-seq. The gain-of-function or loss-of-function of miR-133a-3p showed that it promoted or inhibited the accumulation of lipid droplets, and CCAAT enhancer binding protein α (C/EBPα) and C/EBPβ were extremely significantly up-regulated or down-regulated respectively (p<0.01), the loss-of-function also led to a significant down-regulation of peroxisome proliferator activated receptor gamma (PPARγ) (p<0.01). This study successfully identified miRNAs expression patterns in goat subcutaneous adipocytes, and functional identification indicates that miR-133a-3p is a positive regulator of the differentiation process of goat subcutaneous adipocytes. Our results lay the foundation for the molecular mechanism of lipid deposition in meat-source goats from the perspective of miRNA.

Acute Myeloid Leukemia Cells Educate Mesenchymal Stromal Cells toward an Adipogenic Differentiation Propensity with Leukemia Promotion Capabilities.

Mesenchymal stromal cells (MSCs) are essential elements of the bone marrow (BM) microenvironment, which have been widely implicated in pathways that contribute to leukemia growth and resistance. Recent reports showed genotypic and phenotypic alterations in leukemia patient‐derived MSCs, indicating that MSCs might be educated/reprogrammed. However, the results have been inconclusive, possibly due to the heterogeneity of leukemia. Here, the authors report that acute myeloid leukemia (AML) induces MSCs towards an adipogenic differentiation propensity. RNAseq analysis reveal significant upregulation of gene expression enriched in the adipocyte differentiation process and reduction in osteoblast differentiation. The alteration is accompanied by a metabolic switch from glycolysis to a more oxidative phosphorylation‐dependent manner. Mechanistic studies identify that AML cell‐derived exosomes play a vital role during the AML cell‐mediated MSCs education/reprogramming process. Pre‐administration of mice BM microenvironment with AML‐derived exosomes greatly enhance leukemia engraftment in vivo. The quantitative proteomic analysis identified a list of exosomal protein components that are differently expressed in AML‐derived exosomes, which represent an opportunity for novel therapeutic strategies based on the targeting of exosome‐based AML cells‐MSCs communication. Collectively, the data show that AML‐educated MSCs tend to differentiate into adipocytes contributing to disease progression, which suggests complex interactions of leukemia with microenvironment components.

MicroRNA analysis reveals the role of miR-214 in duck adipocyte differentiation.

ObjectiveFat deposition in poultry is an important factor in production performance and meat quality research. miRNAs also play important roles in regulating adipocyte differentiation process. This study was to investigate the expression patterns of miRNAs in duck adipocytes after differentiation and explore the role of miR-214 in regulating carnitine palmitoyltransferases 2 (CPT2) gene expression during duck adipocyte differentiation.MethodsSuccessful systems for the isolation, culture, and induction of duck primary fat cells was developed in the experiment. Using Illumina next-generation sequencing, the miRNAs libraries of duck adipocytes were established. miRanda was used to predict differentially expressed (DE) miRNAs and their target genes. The expression patterns of miR-214 and CPT2 during the differentiation were verified by quantitative real-time polymerase chain reaction and western blot. Luciferase reporter assays were used to explore the specific regions of CPT2 targeted by miR-214. We used a miR-214 over-expression strategy in vitro to further investigate its effect on differentiation process and CPT2 gene transcription.ResultsThere were 481 miRNAs identified in duck adipocytes, included 57 DE miRNA candidates. And the 1,046 targets genes of DE miRNAs were mainly involved in p53 signaling, FoxO signaling, and fatty acid metabolism pathways. miR-214 and CPT2 showed contrasting expression patterns before and after differentiation, and they were selected for further research. The expression of miR-214 was decreased during the first 3 days of duck adipocytes differentiation, and then increased, while the expression of CPT2 increased both in the transcriptional and protein level. The luciferase assay suggested that miR-214 targets the 3′untranslated region of CPT2. Overexpression of miR-214 not only promoted the formation of lipid droplets but also decreased the protein abundance of CPT2.ConclusionCurrent study reports the expression profile of miRNAs in duck adipocytes differentiated for 4 days. And miR-214 has been proved to have the regulator potential for fat deposition in duck.

Serotonin 5-HT2A receptor activity mediates adipocyte differentiation through control of adipogenic gene expression

Serotonin 5-HT2 receptors are expressed in many tissues and play important roles in biological processes. Although the 5-HT2A receptor is primarily known for its role in central nervous system, it is also expressed in peripheral tissues. We have found that 5-HT2A receptor antagonists inhibit human subcutaneous primary adipocyte differentiation. We also show that siRNA knockdown of the 5-HT2A receptor blocks differentiation. Using gene expression analysis in combination with receptor antagonists we found that activity of 5-HT2A receptors is necessary very early in the differentiation process to mediate expression of adipogenic genes, including peroxisome proliferator-activated receptor gamma (ppar-γ), adipocyte protein 2 (aP2), adiponectin, and serine/threonine-protein kinase 1 (sgk1). We show here for the first time that 5-HT2A receptor activity is necessary for differentiation of human primary subcutaneous preadipocytes to adipocytes, and that 5-HT2A receptor activity mediates key genes related to adipogenesis during this process. Importantly, this work contributes to a greater understanding of the adipocyte differentiation process, as well as to the role of 5-HT2A receptors in peripheral tissues, and may be relevant to the development of novel therapeutic strategies targeting this receptor for the treatment of obesity related diseases.

Whole-Transcriptome Analysis of Preadipocyte and Adipocyte and Construction of Regulatory Networks to Investigate Lipid Metabolism in Sheep.

Many local sheep breeds in China have poor meat quality. Increasing intramuscular fat (IMF) content can significantly improve the quality of mutton. However, the molecular mechanisms of intramuscular adipocyte formation and differentiation remain unclear. This study compared differences between preadipocytes and mature adipocytes by whole-transcriptome sequencing and constructed systematically regulatory networks according to the relationship predicted among the differentially expressed RNAs (DERs). Sequencing results showed that in this process, there were 1,196, 754, 100, and 17 differentially expressed messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and circular RNAs (circRNAs), respectively. Gene Ontology analysis showed that most DERs enriched in Cell Part, Cellular Process, Biological Regulation, and Binding terms. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis found that the DERs primarily focused on Focal adhesion, phosphoinositide 3-kinase (PI3K)-Akt, mitogen-activated protein kinase (MAPK), peroxisome proliferator-activated receptor (PPAR) signaling pathways. Forty (40) DERs were randomly selected from the core regulatory network to verify the accuracy of the sequence data. The results of qPCR showed that the DER expression trend was consistent with sequence data. Four novel promising candidate miRNAs (miR-336, miR-422, miR-578, and miR-722) played crucial roles in adipocyte differentiation, and they also participated in multiple and important regulatory networks. We verified the expression pattern of the miRNAs and related pathways’ members at five time points in the adipocyte differentiation process (0, 2, 4, 6, 8, 10 days) by qPCR, including miR-336/ACSL4/LncRNA-MSTRG71379/circRNA0002331, miR-422/FOXO4/LncRNA-MSTRG54995/circRNA0000520, miR-578/IGF1/LncRNA-MSTRG102235/circRNA0002971, and miR-722/PDK4/LncRNA-MSTRG107440/circ RNA0002909. In this study, our data provided plenty of valuable candidate DERs and regulatory networks for researching the molecular mechanisms of sheep adipocyte differentiation and will assist studies in improving the IMF.

Study of the Active Components and Molecular Mechanism of Tripterygium wilfordii in the Treatment of Diabetic Nephropathy.

We aimed to explore the active ingredients and molecular mechanism of Tripterygium wilfordii (TW) in the treatment of diabetic nephropathy (DN) through network pharmacology and molecular biology. First, the active ingredients and potential targets of TW were obtained through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and related literature materials, and Cytoscape 3.7.2 software was used to construct the active ingredient-target network diagram of TW. Second, the target set of DN was obtained through the disease database, and the potential targets of TW in the treatment of DN were screened through a Venn diagram. A protein interaction network diagram (PPI) was constructed with the help of the String platform and Cytoscape 3.7.2. Third, the ClueGO plug-in tool was used to enrich the GO biological process and the KEGG metabolic pathway. Finally, molecular docking experiments and cell pathway analyses were performed. As a result, a total of 52 active ingredients of TW were screened, and 141 predicted targets and 49 target genes related to DN were identified. The biological process of GO is mediated mainly through the regulation of oxygen metabolism, endothelial cell proliferation, acute inflammation, apoptotic signal transduction pathway, fibroblast proliferation, positive regulation of cyclase activity, adipocyte differentiation and other biological processes. KEGG enrichment analysis showed that the main pathways involved were AGE-RAGE, vascular endothelial growth factor, HIF-1, IL-17, relaxin signalling pathway, TNF, Fc epsilon RI, insulin resistance and other signaling pathways. It can be concluded that TW may treat DN by reducing inflammation, reducing antioxidative stress, regulating immunity, improving vascular disease, reducing insulin resistance, delaying renal fibrosis, repairing podocytes, and reducing cell apoptosis, among others, with multicomponent, multitarget and multisystem characteristics.

TMT-based quantitative proteomics analysis reveals the key proteins related with the differentiation process of goat intramuscular adipocytes

BackgroundIntramuscular adipocytes differentiation is a complex process, which is regulated by various transcription factor, protein factor regulators and signal transduction pathways. However, the proteins and signal pathways that regulates goat intramuscular adipocytes differentiation remains unclear.ResultIn this study, based on nanoscale liquid chromatography mass spectrometry analysis (LC-MS/MS), the tandem mass tag (TMT) labeling analysis was used to investigate the differentially abundant proteins (DAPs) related with the differentiation process of goat intramuscular adipocytes. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes enrichment and protein-protein interaction network analyses were performed for the characterization of the identified DAPs. The candidate proteins were verified by parallel reaction monitoring analysis. As a result, a total of 123 proteins, 70 upregulation proteins and 53 downregulation proteins, were identified as DAPs which may be related with the differentiation process of goat intramuscular adipocytes. Furthermore, the cholesterol metabolism pathway, glucagon signaling pathway and glycolysis / gluconeogenesis pathway were noticed that may be the important signal pathways for goat Intramuscular adipocytes differentiation.ConclusionsBy proteomic comparison between goat intramuscular preadipocytes (P_IMA) and intramuscular adipocytes (IMA), we identified a series protein that might play important role in the goat intramuscular fat differentiation, such as SRSF10, CSRP3, APOH, PPP3R1, CRTC2, FOS, SERPINE1 and AIF1L, could serve as candidates for further elucidate the molecular mechanism of IMF differentiation in goats.

A Novel Role for the DNA Repair Enzyme 8-Oxoguanine DNA Glycosylase in Adipogenesis.

Cells sustain constant oxidative stress from both exogenous and endogenous sources. When unmitigated by antioxidant defenses, reactive oxygen species damage cellular macromolecules, including DNA. Oxidative lesions in both nuclear and mitochondrial DNA are repaired via the base excision repair (BER) pathway, initiated by DNA glycosylases. We have previously demonstrated that the BER glycosylase 8-oxoguanine DNA glycosylase (OGG1) plays a novel role in body weight maintenance and regulation of adiposity. Specifically, mice lacking OGG1 (Ogg1−/−) are prone to increased fat accumulation with age and consumption of hypercaloric diets. Conversely, transgenic animals with mitochondrially-targeted overexpression of OGG1 (Ogg1Tg) are resistant to age- and diet-induced obesity. Given these phenotypes of altered adiposity in the context of OGG1 genotype, we sought to determine if OGG1 plays a cell-intrinsic role in adipocyte maturation and lipid accumulation. Here, we report that preadipocytes from Ogg1−/− mice differentiate more efficiently and accumulate more lipids than those from wild-type animals. Conversely, OGG1 overexpression significantly blunts adipogenic differentiation and lipid accretion in both pre-adipocytes from Ogg1Tg mice, as well as in 3T3-L1 cells with adenovirus-mediated OGG1 overexpression. Mechanistically, changes in adipogenesis are accompanied by significant alterations in cellular PARylation, corresponding with OGG1 genotype. Specifically, deletion of OGG1 reduces protein PARylation, concomitant with increased adipogenic differentiation, while OGG1 overexpression significantly increases PARylation and blunts adipogenesis. Collectively, these data indicate a novel role for OGG1 in modulating adipocyte differentiation and lipid accretion. These findings have important implications to our knowledge of the fundamental process of adipocyte differentiation, as well as to our understanding of lipid-related diseases such as obesity.

Adipocytes and macrophages secretomes coregulate catecholamine-synthesizing enzymes.

Obesity associates with macrophage accumulation in adipose tissue where these infiltrating cells interact with adipocytes and contribute to the systemic chronic metabolic inflammation present in immunometabolic diseases. Tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) are two of the main enzymes of catecholamines (CA) synthesis. Adipocytes and macrophages produce, secrete and respond to CA, but the regulation of their synthesis in the interplay between immune and metabolic systems remains unknown. A model of indirect cell coculture with conditioned medium (CM) from RAW 264.7 macrophages with or without LPS-activation and 3T3-L1 adipocytes and preadipocytes was established to study the effect of cellular secretomes on the expression of the above enzymes. During the adipocyte differentiation process, we found a decrease of TH and PNMT expression. The secretome from LPS-activated macrophages downregulated TH and PNMT expression in preadipocytes, but not in mature adipocytes. Mature adipocytes CM induced a decrease of PNMT levels in RAW 264.7 macrophages. Pre and mature adipocytes showed a similar pattern of TH, PNMT and peroxisome proliferator-activated receptor gamma expression after exposure to pro and anti-inflammatory cytokines. We evidenced macrophages and adipocytes coregulate the expression of CA synthesis enzymes through secretome, with non-inflammatory signaling networks possibly being involved. Mediators released by macrophages seem to equally affect CA production by adipocytes, while adipocytes secretome preferentially affect AD production by macrophages. CA synthesis seems to be more determinant in early stages of adipogenic differentiation. Our results suggest that CA are key signaling molecules in the regulation of immune-metabolic crosstalk within the adipose tissue.

Perilipin2 is an Earlier Marker Than Perilipin1 for Identifying Adipocyte Regeneration in Fat Grafts.

Both perilipin1 (Plin1) and perilipin2 (Plin2) play a crucial role in regulating lipid droplet (LD) formation in fat cells. Plin2 is expressed early in the adipocyte differentiation process but is replaced by Plin1 after cell maturation. In free fat grafts, only a small number of adipocytes remain alive or are replaced by newly regenerated fat cells. It is known that Plin1-positive adipocytes participate in regeneration, but the characteristics of Plin2 expression during this process are still poorly understood. The aim of this study was to investigate whether Plin2 is a more precise early marker for detecting adipocyte regeneration in fat grafts than Plin1. Autologous fat tissue (120 mg) harvested from inguinal fat pads was injected under the scalps of C57 mice. Samples were explanted at days 3, 7, 15, and 30 after transplantation. Changes in sample size and weight were evaluated. Hematoxylin-eosin staining, real-time polymerase chain reaction, and immunostaining of Plin1 and Plin2 expression were performed. Plin1, but not Plin2, expression was detected in the freshly harvested fat, but the latter was activated after grafting. Newly regenerated Plin2-positive adipocytes increased from day 3 to day 7 and then declined, whereas the number of Plin1-positive fat cells decreased first and began to increase after day 15. The expression levels of Plin1 and Plin2 mRNA demonstrated similar changes over time. At day 30, adipocytes lost Plin2 expression and were positive for Plin1 again. Our experiments showed convincing evidence that Plin2 expression could be used to detect early adipocyte regeneration in grafted fat tissue.

A Transcriptomic Analysis Reveals Novel Patterns of Gene Expression During 3T3-L1 Adipocyte Differentiation.

BackgroundObesity is characterized by increased adipose tissue mass that results from increased fat cell size (hypertrophy) and number (hyperplasia). The molecular mechanisms that govern the regulation and differentiation of adipocytes play a critical role for better understanding of the pathological mechanism of obesity. However, the mechanism of adipocyte differentiation is still unclear.ObjectiveThe present study aims to compare the gene expression changes during adipocyte differentiation in the transcriptomic level, which may help to better understand the mechanism of adipocyte differentiation.MethodsRNA sequencing (RNA-seq) technology, GO and KEGG analysis, quantitative RT-PCR, and oil red O staining methods were used in this study.ResultsA lot of genes were up- or down-regulated between each two differentiation stages of 3T3-L1 cells. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that lipid metabolism and oxidation–reduction reaction were mainly involved in the whole process of adipocyte differentiation. Decreased immune response and cell cycle adhesion occurred in the late phase of adipocyte differentiation, which was demonstrated by divergent expression pattern analysis. Moreover, quantitative RT-PCR results showed that the mRNA expression levels of Trpv4, Trpm4, Trpm5, and Trpm7 were significantly decreased in the differentiated adipocytes. On the other hand, the mRNA expression levels of Trpv1, Trpv2, Trpv6, and Trpc1 were significantly increased in the differentiated adipocytes. Besides, the mRNA expressions of TRPV2 and TRPM7 were also significantly increased in subcutaneous white adipose tissue from diet-induced mice. In addition, the activation of TRPM7, TRPV1, and TRPV2 suppressed the differentiation of adipocytes.ConclusionThese data present the description of transcription profile changes during adipocyte differentiation and provides an in-depth analysis of the possible mechanisms of adipocyte differentiation. These data offer new insight into the understanding of the mechanisms of adipocyte differentiation.

Developing a Robust Assay to Monitor and Quantify Key Players of Metabolic Pathways during Adipogenesis by Targeted Proteomics.

Targeted data acquisition using nano liquid chromatrography (nano-LC) coupled mass spectrometry is an emerging approach when there is a need to quantify proteins with high accuracy, sensitivity, and reproducibility. Nevertheless, creating assays meeting all those criteria still remains a laborious task, especially when investigating low abundant proteins and small concentration changes. In this work a targeted data acquisition workflow is developed reducing time and effort to target and investigate key players of metabolic pathways during the process of adipocyte differentiation. This leads to accurate and sensitive quantification of proteins involved in the synthesis of fatty acids, glycerolipids, glycerophospholipids, sphingolipids, the production of energy and reduction equivalents. Additionally low abundant signaling molecules part of the peroxisome proliferator-activated receptor gamma (PPARγ) and insulin signaling pathway with ≈400 for the insulin receptor substrate and 1100 copies per cell for PPARγ are determined.

Stat5a promotes brown adipocyte differentiation and thermogenic program through binding and transactivating the Kdm6a promoter

ABSTRACT Previous studies reported that Stat5 promotes adipogenesis and white adipocyte differentiation. However, the role of Stat5 in brown adipocyte development is poorly understood. We found Stat5a was higher expressed in brown adipocytes than in white adipocytes, and its level was increased during the process of brown adipocyte differentiation. In addition, Stat5a expression was affected by cold stress and high-fat diet-feeding, suggesting a potential role in thermogenesis. Knockdown of Stat5a induced downregulation of brown fat specific genes (UCP1, PGC-1α, Acox-1 and Cidea), while overexpression of Stat5a did the opposite effect. What is more, bioinformatics analysis, ChIP assay and Luciferase activity assay all verified that Stat5a directly bind and transactivate Kdm6a promoter (Lysine-specific demethylase 6A). Further, we found that Stat5a overexpression promoted the expression of Kdm6a and inhibited the trimethylation of H3K27. While inhibiting of Kdm6a reversed the promoting effect of Stat5a overexpression on the expression of brown fat specific genes. Therefore, we conclude that Stat5a participated in brown adipocyte differentiation and thermogenic program through binding and transactivating the Kdm6a promoter. Abbreviations: Stat5: Signal transducers and activators of transcription 5; BAT: brown adipose tissue; WAT; white adipose tissue; eWAT: epididymal white adipose tissue; sWAT: subcutaneous white adipose tissue; SVFs: stromal vascular fractions; UCP1: Uncoupling protein 1; PGC-1α: Peroxisome proliferator-activated receptor gamma coactivator 1-alpha; Acox-1: Peroxisomal acyl-coenzyme A oxidase 1; Cidea: Cell death activator CIDE-A; ChIP: Chromatin Immunoprecipitation; HFD: High fat diet; FBS: Fetal bovine serum; siStat5a: Stat5a siRNA; siKdm6: Kdm6a siRNA; pcDNA-Stat5a: over expression of Stat5a pcDNA3.1 vector; IgG: mouse immunoglobulin G; Kdm6a: Lysine-specific demethylase 6A; H3K27me3: trimethylated H3K27

Obesogenic effects of chlorpyrifos and its metabolites during the differentiation of 3T3-L1 preadipocytes

Chlorpyrifos (CPF) is an organophosphorus pesticide widely and extensively used in agriculture in more than one hundred countries and found ubiquitously in the environment. The present study was aimed at providing a better understanding of the obesogenic potential of CPF and its metabolites, as well as to evaluate their effects on the adipocyte differentiation process. For it, during the initial differentiation process, 3T3-L1 mouse preadipocytes were exposed to different concentrations of CPF, CPF-oxon (CPO), or 3,5,6-trichloropyridinol (TCP), which did not affect cell survival. Results showed how CPF and, to a lesser extent, its metabolite TCP, had a positive metabolic influence over the adipogenic process by fostering an increase in the number of differentiated 3T3-L1 preadipocytes, and by enhancing the capacity to store lipid droplets. These processes seem to occur through the upregulation of the transcription factors CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ), which are related to a significant higher expression of the fatty acid-binding protein 4 (FABP4) adipokine. Based on this finding, CPF exposure could be one of the many factors that contributes to the worldwide increase in the incidence of obesity. However, additional investigations are clearly needed.