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Abstract
Previous studies showed that CCAAT/enhancer binding protein alpha (C/EBP alpha) is required for differentiation of 3T3-L1 preadipocytes induced by exogenous hormonal agents. It was not possible to ascertain, however, whether C/EBP alpha alone is sufficient to induce differentiation because its antimitogenic activity precluded propagating 3T3-L1 cell lines that constitutively express C/EBP alpha at high levels. This problem was circumvented by using 3T3-L1 preadipocytes stably transfected with an isopropyl beta-D-thiogalactoside (IPTG)-inducible p42 C/EBP alpha expression vector system. IPTG-induced expression of the 42-kDa isoform of C/EBP alpha in preadipocytes caused expression of several endogenous adipocyte-specific genes (genes encoding the 422 adipose P2 protein, glucose transporter 4, and C/EBP alpha) and the accumulation of cytoplasmic triglyceride. Thus, C/EBP alpha is not only necessary but also is sufficient to trigger differentiation of growth-arrested 3T3-L1 preadipocytes without use of exogenous hormonal agents.
... It has been well established that C/EBPα plays an essential role in adipogenesis. Overexpression of C/EBPα in 3T3-L1 preadipocytes induces cell differentiation (24), and knockdown of this gene by antisense RNA blocks this process (24). By working together with PPARγ, C/EBPα functions as a pleiotropic transcriptional activator of a large group of genes associated with adipocyte differentiation and maturation (5,7). ...
... It has been well established that C/EBPα plays an essential role in adipogenesis. Overexpression of C/EBPα in 3T3-L1 preadipocytes induces cell differentiation (24), and knockdown of this gene by antisense RNA blocks this process (24). By working together with PPARγ, C/EBPα functions as a pleiotropic transcriptional activator of a large group of genes associated with adipocyte differentiation and maturation (5,7). ...
Objective
Recent studies have shown that microRNAs (miRNAs/miRs) play key roles in adipogenesis. This study aimed to investigate the role and underlying mechanism of miR‐182 in adipogenesis.
Methods
This study used the 3T3‐L1 cell line and human visceral adipose tissue (VAT)‐derived adipocytes to determine the role of miR‐182 in adipogenesis. Adipose tissues from mice with high‐fat diet–induced obesity, ob/ob mice, or human individuals with obesity were used to determine the association of miR‐182 levels with obesity. A luciferase reporter assay was used to determine the target of miR‐182.
Results
The expression level of miR‐182 was greatly downregulated during white adipogenesis and markedly lower in the VAT of mice and humans with obesity. Ectopic expression of miR‐182 in 3T3‐L1 cells and human adipocytes suppressed the formation of lipid droplets and the expression of adipogenic genes. The luciferase reporter assay showed that miR‐182 targeted the 3′‐untranslated sequence of CCAAT/enhancer‐binding protein α (C/EBPα) directly. In addition, glucocorticoids negatively regulated miR‐182 expression, which, in turn, suppressed the glucocorticoid‐induced expression of C/EBPα.
Conclusions
Taken together, our studies identified miR‐182 as a novel negative regulator of adipogenesis and a potential therapeutic target for obesity.
... When proliferating 3T3-L1 preadipocytes become confluent in culture dishes, they enter G 1 growtharrest phase (Tang et al., 2003b). Upon a treatment of hormonal cocktail (a mixture of 3isobutyl-1-methylxanthine, dexamethasone, and insulin, hereafter called MDI) and fetal bovine serum (FBS), growth-arrested preadipocytes reenter the cell cycle and initiate differentiation via serial expression of adipogenic genes such as those encoding CCAAT/enhancer binding protein (C/EBP) β, C/EBPα, and peroxisome proliferator-activated receptor γ (PPARγ) (Lin and Lane, 1994;Tang et al., 2003a). This resumed cell cycle is called mitotic clonal expansion and the process sustains during first 2~3 days of differentiation, and is essential for 3T3-L1 differentiation because an inhibition of this step completely blocks adipogenesis (Tang et al., 2003b). ...
... The mechanisms of 3T3-L1 differentiation have been investigated since these cells were first cloned by Howard Green (Green and Meuth, 1974). These efforts have identified the roles of transcription factors C/EBPβ, C/EBPα, and PPARγ in adipogenesis (Gregoire et al., 1998;Lin and Lane, 1994), but the complex molecular processes regulating adipocyte differentiation have yet to be fully defined. In the present study, we found that Serpina3c is a regulator of adipogenesis and was responsible for the accelerated differentiation of adipocytes induced by CM (Choi et al., 2014). ...
Preadipocyte differentiation can be induced upon a hormonal treatment, and various factors secreted by the cells may contribute to adipogenesis. In this study, RNA-seq revealed Serpina3c as a critical factor regulating the signaling network during adipogenesis. Serpina3c is a secretory protein and is highly expressed in fat tissues. Knockdown of Serpina3c decreased adipogenesis by attenuating the mitotic clonal expansion of 3T3-L1 cells. These cells exhibited decreases in integrin α5, which abolished the phosphorylation of integrin β3. We found that Serpina3c inhibits a serine protease that regulates integrin α5 degradation. Knockdown of Serpina3c disrupted integrin-mediated insulin growth factor 1 (IGF-1) signaling and ERK activation. Serpina3c-mediated regulation of integrin-IGF-1 signaling is also associated with AKT activation, which affects the nuclear translocation of GSK3β. Altogether, our results indicate that Serpina3c secreted from differentiating adipocytes inhibits serine proteases to modulate integrin/IGF-1-mediated ERK and AKT signaling and thus is a critical factor contributing to adipogenesis.
... Overexpression of NKX1-2 also increased the protein expression of PPARγ, FABP4 and the p42 isoform of C/EBPα, but not the p30 isoform ( Figure 4D). The p42 isoform of C/EBPα has been shown to be sufficient to trigger the differentiation program of 3T3-L1 preadipocytes (57,58). Overall, these data support the hypothesis that NKX1-2 is a novel proadipogenic TF that upregulates the expression of the adipocyte master TFs PPARγ and C/EBPα and their downstream targets FABP4, adiponectin, resistin and HSL. ...
... These isoforms derive from alternative translation start sites (58,68), suggesting that NKX1-2 may regulate alternative translation to favor the p42 isoform (in addition to inducing expression of the C/EBPα mRNA). Previous reports showed that p42 C/EBPα but not p30 has antimitotic activity and that p42 is sufficient to trigger the differentiation program of 3T3-L1 preadipocytes (57,58). Therefore, our data suggest that selective induction of the p42 C/EBPα isoform by NKX1-2 may be an important component of its proadipogenic action. ...
Although adipogenesis is mainly controlled by a small number of master transcription factors including CCAAT/enhancer-binding protein family members and peroxisome proliferator-activated receptor γ (PPARγ), other transcription factors also are involved in this process. Thyroid cancer cells expressing a paired box 8 (PAX8)–PPARγ fusion oncogene trans-differentiate into adipocyte-like cells in the presence of the PPARγ ligand pioglitazone, but this trans-differentiation is inhibited by the transcription factor NK2 homeobox 1 (NKX2-1). Here, we tested whether NKX family members may play a role also in normal adipogenesis. Using quantitative RT-PCR (RT-qPCR), we examined the expression of all 14 NKX family members during 3T3-L1 adipocyte differentiation. We found that most NKX members, including NKX2-1, are expressed at very low levels throughout differentiation. However, mRNA and protein expression of a related family member, NKX1-2, was induced during adipocyte differentiation. NKX1-2 also was up-regulated in cultured murine ear mesenchymal stem cells (EMSCs) during adipogenesis. Importantly, shRNA-mediated NKX1-2 knockdown in 3T3-L1 preadipocytes or EMSCs almost completely blocked adipocyte differentiation. Furthermore, NKX1-2 overexpression promoted differentiation of the ST2 bone marrow–derived mesenchymal precursor cell line into adipocytes. Additional findings suggested that NKX1-2 promotes adipogenesis by inhibiting expression of the antiadipogenic protein COUP transcription factor II. Bone marrow mesenchymal precursor cells can differentiate into adipocytes or osteoblasts, and we found that NKX1- 2 both promotes ST2 cell adipogenesis and inhibits their osteoblastogenic differentiation. These results support a role for NKX1-2 in promoting adipogenesis and possibly in regulating the balance between adipocyte and osteoblast differentiation of bone marrow mesenchymal precursor cells.
... It was reported that the heritability of abdominal fat (0.82) was significantly higher than that of chest muscle (0.55) and body weight (0.55) [6], and that chicken abdominal preadipocytes have higher adipogenic differentiation ability than intramuscular preadipocytes [7]. Lipogenesis is regulated by a series of key transcription factors, such as peroxisome proliferator-activated receptor γ (PPARγ), the CCAAT/enhancer-binding protein α (C/EBPα), and fatty acid binding protein 4 (FABP4) [8,9]. Although there are currently many research reports on the deposition of abdominal fat in chickens, adipocyte differentiation is a complex and delicate process, and there are still many potential targets that have not been discovered. ...
The excessive deposition of abdominal adipocytes in chickens is detrimental to poultry production. However, the regulatory factors that affect abdominal adipogenesis in chickens are still poorly understood. SLC22A16 is differentially expressed in abdominal preadipocytes and 10-day differentiated adipocytes in chickens, but its role in regulating chicken adipogenesis has not been reported. In this study, the function of SLC22A16 in chicken abdominal preadipocytes was investigated. SLC22A16 is significantly upregulated during abdominal adipocyte differentiation. The overexpression of SLC2A16 upregulated the expression of adipogenic marker genes and proliferation-related genes, and promoted the proliferation of adipocytes and the accumulation of triglycerides. The knockdown of SLC22A16 downregulated the expression of adipogenic marker genes and proliferation-related genes, inhibited the proliferation of adipocytes, and impaired the accumulation of triglycerides in adipocytes. In addition, LNC6302 was differentially expressed in abdominal preadipocytes and mature adipocytes, and was significantly positively correlated with the expression of SLC22A16. Interference with LNC6302 inhibits the expression of adipogenic marker genes and proliferation-related genes. The data supported the notion that LNC6302 promotes the differentiation of chicken abdominal adipocytes by cis-regulating the expression of SLC22A16. This study identified the role of SLC22A16 in the differentiation and proliferation of chicken adipocytes, providing a potential target for improving abdominal adipogenesis in chickens.
... Teir expression indicates a high adipogenic potential [63]. Consequently, CEBPA, a transcriptional factor in preadipocytes, is characterized as a critical regulator of adipogenic diferentiation [64,65]. Fatty acid synthase (FASN) is a delayed adipogenic marker. ...
In this experiment, effects of Citrus sunki peel extract (CPE) on proliferation and differentiation of 3T3-L1 cells were analyzed. Citrus sunki peel was extracted with ethanol to obtain CPE. Results of measuring DPPH and hydrogen peroxide radical scavenging activity revealed that CPE had an antioxidant ability. The 3T3-L1 cells were cultured in the basal medium (C) or with 0.05% dimethyl-sulfoxide (CDMSO) or with 50, 100, 200, 300, and 400 μg/mL CPE (CPE50, CPE100, CPE200, CPE300, and CPE400, respectively). As a result of cell counting and MTS assay, CPE significantly enhanced proliferation capacity of 3T3-L1 cells. To analyze the effect of CPE on the differentiation capacity of 3T3-L1 cells, relative gene and protein expression levels of Cebpb, Cebpa, and FASN related to adipogenesis were measured by RT-qPCR and Western blot. CPE increased adipogenesis-related gene and protein expression in 3T3-L1 cells, with CPE300 being especially effective. In Oil Red O staining, adipogenesis was significantly greater in CPE200, CPE300, and CPE400 than in C. This was confirmed image wise through Nile Red staining. Through HPLC analysis, it was confirmed that nobiletin and tangeretin were most abundant in CPE. To analyze effects of nobiletin and tangeretin on 3T3-L1 cells, the cells were cultured with various concentrations of nobiletin, tangeretin, and both. As a result of cell counting and MTS assay, nobiletin and tangeretin significantly decreased the proliferation capacity of 3T3-L1 cells. In RT-qPCR, western blot, Oil Red O staining, and Nile Red staining to analyze differentiation capacity, nobiletin and tangeretin significantly enhanced the expression of genes and proteins related to adipogenic differentiation compared to the control group. In conclusion, CPE, which had high antioxidant capacity, enhanced proliferation and differentiation in 3T3-L1 adipocytes. This improvement in differentiation is due to the influence of nobiletin and tangeretin contained in CPE.
... C/EBPa and PPARg transactivate each other in a positive feedback loop 20,21 and function together to activate the expression of adipocyte-specific genes. [22][23][24][25][26] 3T3-L1 preadipocytes overexpressing Cebpb, 13 Cebpa, 27 or Pparg are able to differentiate into mature adipocytes even in the absence of hormone inducers. In addition, mouse embryonic fibroblasts (MEFs) from Cebpb À/À mice 28 fail to differentiate into iScience Article adipocytes, and adipose tissues in Cebpa À/À29 mice fail to accumulate lipid droplets. ...
Ten-eleven translocation proteins (TETs) are dioxygenases that convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), an important epigenetic mark that regulates gene expression during development and differentiation. Here, we found that the TET2 expression was positively associated with adipogenesis. Further, in vitro and in vivo experiments showed that TET2 deficiency blocked adipogenesis by inhibiting the expression of the key transcription factors CCAAT/enhancer-binding protein beta (C/EBPβ), C/EBPα and peroxisome proliferator-activated receptor gamma (PPARγ). In addition, TET2 promoted 5hmC on the CpG islands (CGIs) of Cebpb, Cebpa and Pparg at the initial time point of their transcription, which requires the cAMP-responsive element-binding protein (CREB). At last, specific knockout of Tet2 in preadipocytes enabled mice to resist obesity and attenuated the obesity-associated insulin resistance. Together, TET2 is recruited by CREB to promote the expression of Cebpb, Cebpa and Pparg via 5hmC during adipogenesis and may be a potential therapeutic target for obesity and insulin resistance.
... CEBPA, prioritized in another locus, encodes for the transcription factor CCAAT/enhancer binding protein alpha (C/EBPα), which shares binding sites with Peroxisome proliferatoractivated receptor gamma (PPARγ) and acts as a co-stimulator of adipogenesis and adipocyte differentiation 28,29 . In a previously identified PAT locus, we also prioritize TRIB2, which is a . ...
Background: While previous studies have reported associations of pericardial adipose tissue (PAT) with cardiovascular diseases such as atrial fibrillation and coronary artery disease, they have been limited in sample size or drawn from selected populations. Additionally, the genetic determinants of PAT remain largely unknown. We aimed to evaluate the association of PAT with prevalent and incident cardiovascular disease and to elucidate the genetic basis of PAT in a large population cohort.
Methods: A deep learning model was trained to quantify PAT area from four-chamber magnetic resonance images in the UK Biobank using semantic segmentation. Cross-sectional and prospective cardiovascular disease associations were evaluated, controlling for sex and age. A genome-wide association study was performed, and a polygenic score (PGS) for PAT was examined in 453,733 independent FinnGen study participants.
Results: A total of 44,725 UK Biobank participants (51.7% female, mean [SD] age 64.1 [7.7] years) were included. PAT was positively associated with male sex (β = +0.76 SD in PAT), age (r = 0.15), body mass index (BMI; r = 0.47) and waist-to-hip ratio (r = 0.55) (P < 1x10-230). PAT was more elevated in prevalent heart failure (β = +0.46 SD units) and type 2 diabetes (β = +0.56) than in coronary artery disease (β = +0.22) or AF (β = +0.18). PAT was associated with incident heart failure (HR = 1.29 per +1 SD in PAT [95% CI 1.17-1.43]) and type 2 diabetes (HR = 1.63 [1.51-1.76]) during a mean 3.2 (±1.5) years of follow-up; the associations remained significant when controlling for BMI. We identified 5 novel genetic loci for PAT and implicated transcriptional regulators of adipocyte morphology and brown adipogenesis (EBF1, EBF2 and CEBPA) and regulators of visceral adiposity (WARS2 and TRIB2). The PAT PGS was associated with T2D, heart failure, coronary artery disease and atrial fibrillation in FinnGen (ORs 1.03-1.06 per +1 SD in PGS, P < 2x10-10).
Conclusions: PAT shares genetic determinants with abdominal adiposity and is an independent predictor of incident type 2 diabetes and heart failure.
... Inactive C/EBPβ is phosphorylated by MAP kinase and GSK3β to possess DNA binding activity, and then the active C/EBPβ induces the transcription of PPARγ and C/EBPα [23]. Once expressed, PPARγ and C/EBPα coordinately trigger the transcription of adipocyte genes to produce the adipocyte phenotype [9,24]. ...
Adipogenesis is regarded as an intricate network in which multiple transcription factors and signal pathways are involved. Recently, big efforts have focused on understanding the epigenetic mechanisms and their involvement in the regulation of adipocyte development. Multiple studies investigating the regulatory role of non-coding RNAs (ncRNAs) in adipogenesis have been reported so far, especially lncRNA, miRNA, and circRNA. They regulate gene expression at multiple levels through interactions with proteins, DNA, and RNA. Exploring the mechanism of adipogenesis and developments in the field of non-coding RNA may provide a new insight to identify therapeutic targets for obesity and related diseases. Therefore, this article outlines the process of adipogenesis, and discusses updated roles and mechanisms of ncRNAs in the development of adipocytes.
... In this study, we showed that knockdown of HSPB7 increases adipogenic competency by promoting PPARγ and C/EBPα, which are master regulators of the adipocyte phenotype. Consistent with our findings, the expression of PPARγ and C/EBPα are maintained at high levels throughout the entire differentiation process and cooperate to regulate a number of adipogenic proteins such as FABP4 and Perilipin-1 [18,35]. In addition, PPARγ is involved in de novo lipogenesis by mediating Acetyl-CoA carboxylase [36], which acts as a rate-limiting enzyme and catalyzes the production of malonyl-CoA used as an essential substrate for Fatty acid synthase. ...
Background
Recent evidence suggests that accumulation of marrow adipose tissue induced by aberrant lineage allocation of bone marrow-derived mesenchymal stromal cells (BMSCs) contributes to the pathophysiologic processes of osteoporosis. Although master regulators of lineage commitment have been well documented, molecular switches between osteogenesis and adipogenesis are largely unknown.
Methods
HSPB7 gene expression during osteogenic and adipogenic differentiation of BMSCs was evaluated by qPCR and Western blot analyses. Lentiviral-mediated knockdown or overexpression of HSPB7 and its deletion constructs were used to assess its function. The organization of cytoskeleton was examined by immunofluorescent staining. ALP activity, calcium assay, Alizarin Red S staining and Oil Red O staining were performed in vitro during osteoblast or adipocyte differentiation. SB431542 and Activin A antibody were used to identify the mechanism of Activin A in the regulation of osteogenic differentiation in BMSCs.
Results
In this study, we identified HSPB7 capable of oppositely regulating osteogenic and adipogenic differentiation of BMSCs. HSPB7 silencing promoted adipogenesis while reducing osteogenic differentiation and mineralization. Conversely, overexpression of HSPB7 strongly enhanced osteogenesis, but no effect was observed on adipogenic differentiation. Deletion of the N-terminal or C-terminal domain of HSPB7 led to decreased osteoblastic potency and mineralization. Mechanistically, our data showed that Activin A is a downstream target participating in HSPB7 knockdown-mediated osteogenic inhibition.
Conclusions
Our findings suggest that HSPB7 plays a positive role in driving osteoblastic differentiation, and with the capability in maintaining the osteo-adipogenesis balance. It holds great promise as a potential therapeutic target in the treatment of bone metabolic diseases.
... In response to adipocytic inducers, the transcription factors C/EBPβ, C/EBPδ and PPARγ1 are rapidly activated in BMSCs and initiate the adipogenic cascade. This process includes elevated expression of two critical transcription factors responsible for adipogenesis, C/EBPα and PPARγ2, followed by an increase in downstream genes characterized as mature adipocytes, including fatty acid binding protein 4 (FABP4) and adiponectin [8][9][10][11][12][13]. ...
Background
Bone is a rigid organ that provides physical protection and support to vital organs of the body. Bone loss disorders are commonly associated with increased bone marrow adipose tissue. Bone marrow mesenchymal stromal/stem cells (BMSCs) are multipotent progenitors that can differentiate into osteoblasts, adipocytes, and chondrocytes. Cell division cycle 20 (CDC20) is a co-activator of anaphase promoting complex/cyclosome (APC/C), and is required for ubiquitin ligase activity. Our previous study showed that CDC20 promoted the osteogenic commitment of BMSCs and Cdc20 conditional knockout mice suggested a decline in bone mass. In this study, we found that knockdown of CDC20 promoted adipogenic differentiation of BMSCs by modulating β -catenin, which suggested a link between adipogenesis and osteogenesis.
Methods
Lentivirus containing a CDC20 shRNA was used for CDC20 knockdown in human BMSCs (hBMSCs). Primary mouse BMSCs (mBMSCs) were isolated from Cdc20 f/f and Sp7-Cre;Cdc20 f/f mice. Adipogenesis was examined using quantitative real-time reverse transcription PCR (qRT-PCR) and western blotting analysis of adipogenic regulators, Oil Red O staining, and transplantation into nude mice. CDC20 knockout efficiency was determined through immunochemistry, qRT-PCR, and western blotting of bone marrow. Accumulation of adiposity was measured through histology and staining of bone sections. Exploration of the molecular mechanism was determined through western blotting, Oil Red O staining, and qRT-PCR.
Results
CDC20 expression in hBMSCs was significantly decreased during adipogenic differentiation. CDC20 knockdown enhanced hBMSC adipogenic differentiation in vitro. CDC20 -knockdown hBMSCs showed more adipose tissue-like constructs upon hematoxylin and eosin (H&E) and Oil Red O staining. Sp7-Cre;Cdc20 f/f mice presented increased adipocytes in their bone marrow compared with the control mice. mBMSCs from Sp7-Cre;Cdc20 f/f mice showed upregulated adipogenic differentiation. Knockdown of CDC20 led to decreased β -catenin levels, and a β -catenin pathway activator (lithium chloride) abolished the role of CDC20 in BMSC adipogenic differentiation.
Conclusions
Our findings showed that CDC20 knockdown enhanced adipogenesis of hBMSC and mBMSCs adipogenesis in vitro and in vivo. CDC20 regulates both adipogenesis and osteogenesis of BMSCs, and might lead to the development of new therapeutic targets for “fatty bone” and osteoporosis.
... CCAAT/enhancer binding protein alpha (C/EBPα), adipojenez regulasyonunda anahtar role sahip bir başka transkripsiyon faktörüdür. C/EBPα'nın yüksek miktarda ekspresyonu fibroblastlarda adipojenezi indüklerken C/EBPα olmadan adipojenezin inhibe olduğu gösterilmiştir (53,54). C/EBP ekspresyonu, PPARɣ ekspresyonunu da kapsayan bir pozitif geri besleme döngüsü ile düzenlenir. ...
Mesenchymal stem cells are a subgroup of heterogeneous, non-hematopoietic fibroblast like cells that can be isolated from various tissues including bone marrow, adipose tissue and umblical cord; and characterized by their multipotent differentiation capacities in addition to being adherent under standard cell culture applications. As these multipotent cells can contribute to tissue regeneration, can modulate immune responses and promote immune tolerance and immune supression by interacting with immune system cells; these cells attracted researchers’ attention since the beginning of late 1960s when they have been first identified. Moreover, mesenchymal stem cells have become one of the most widely used cell types in the field of regenerative medicine today, as they are easily accessible, can be propagated quickly in vitro, and there are no ethical concern against their isolation as they are derived from adult tissues. However, there are various obstacles against their usage in the clinical practice, especially due the variations as a consequence of their heterogeneity. This review focuses on the history and general characteristics of mesenchymal stem cells, their impact on the immune system and their therapeutic use, while discussing the potential dangers regarding their use in clinical practice; in addition, it also offers a new nomenclature (medicinal signalling cells) based on the cellular mechanisms by which mesenchymal stem cells exert their therapeutic efficacy.
... In fact, the remaining CCNC-deficient brown adipocytes are essentially indistinguishable from control in terms of morphology, UCP1 protein levels and key thermogenic and adipogenic marker gene expression (Figure 2EeH). Coincidently, a similar disparity between in vitro and in vivo adipogenesis was also observed in the case of C/EBPa deletion [70,71]. In our previous study, we found that CCNC deficiency enhanced PPARgdependent transcription in the presence of ligands [43]. ...
Objective:
Cyclin C (CCNC) is the most conserved subunit of the Mediator complex, which is an important transcription cofactor. Recently, we have found that CCNC facilitates brown adipogenesis in vitro by activating C/EBPα-dependent transcription. However, the role of CCNC in brown adipose tissue (BAT) in vivo remains unclear.
Methods:
We generated conditional knock-out mice by crossing Ccncflox/flox mice with Myf5Cre, Ucp1Cre or AdipoqCre transgenic mice to investigate the role of CCNC in BAT development and function. We applied glucose and insulin tolerance test, cold exposure and indirect calorimetry to capture the physiological phenotypes and used immunostaining, immunoblotting, qRT-PCR, RNA-seq and cell culture to elucidate the underlying mechanisms.
Results:
Here, we show that deletion of CCNC in Myf5+ progenitor cells caused BAT paucity, despite the fact that there was significant neonatal lethality. Mechanistically different from in vitro, CCNC deficiency impaired the proliferation of embryonic brown fat progenitor cells without affecting brown adipogenesis or cell death. Interestingly, CCNC deficiency robustly reduced age-dependent lipid accumulation in differentiated brown adipocytes in all three mouse models. Mechanistically, CCNC in brown adipocytes is required for lipogenic gene expression through the activation of the C/EBPα/GLUT4/ChREBP axis. Consistent with the importance of de novo lipogenesis under carbohydrate-rich diets, high-fat diet (HFD) feeding abolished CCNC deficiency -caused defects of lipid accumulation in BAT. Although insulin sensitivity and response to acute cold exposure were not affected, CCNC deficiency in Ucp1+ cells enhanced the browning of white adipose tissue (beiging) upon prolonged cold exposure.
Conclusions:
Together, these data indicate an important role of CCNC-Mediator in the regulation of BAT development and lipid accumulation in brown adipocytes.
... As mentioned above, high concentrations of BMP2 accelerated osteoblastogenesis, whereas low concentrations of BMP2 promoted differentiation of the C3H10T1/2 mesenchymal cell line to adipocytes [35]. In addition, CCAAT/enhancer binding protein α (C/EBPα), platelet growth factor receptor β and zinc finger proteins 423 and 521 also promote lipogenic differentiation of MSCs [36][37][38][39]. ...
Bone defects repair and regeneration by various causes such as tumor resection, trauma, degeneration, etc. have always been a key issue in the clinics. As one of the few organs that can regenerate after adulthood, bone itself has a strong regenerative ability. In recent decades, bone tissue engineering technology provides various types of functional scaffold materials and seed cells for bone regeneration and repair, which significantly accelerates the speed and quality of bone regeneration, and many clinical problems are gradually solved. However, the bone metabolism mechanism is complicated, the research duration is long and difficult, which significantly restricts the progress of bone regeneration and repair research. Organoids as a new concept, which is built in vitro with the help of tissue engineering technology based on biological theory, can simulate the complex biological functions of organs in vivo. Once proposed, it shows broad application prospects in the research of organ development, drug screening, mechanism study, and so on. As a complex and special organ, bone organoid construction itself is quite challenging. This review will introduce the characteristics of bone microenvironment, the concept of organoids, focus on the research progress of bone organoids, and propose the strategies for bone organoid construction, study direction, and application prospects.
... As mentioned above, a high concentration of BMP2 accelerates osteoblast differentiation, while a low concentration of BMP2 promotes adipocyte formation in the C3H10T1/2 mesenchymal cell line (Tang et al., 2004). Additionally, CCAAT/enhancer binding protein α (C/EBPα), platelet-derived growth factor receptor β (PDGFβ) and zinc finger proteins 423 and 521 also take part in adipogenesis (Lin and Lane, 1994;Gupta et al., 2012;Huang et al., 2012;Dang et al., 2021). FIGURE 3 | Regulation of differentiation fate. ...
Bone marrow mesenchymal stromal cells (BMSCs), identified as pericytes comprising the hematopoietic niche, are a group of heterogeneous cells composed of multipotent stem cells, including osteochondral and adipocyte progenitors. Nevertheless, the identification and classification are still controversial, which limits their application. In recent years, by lineage tracing and single-cell sequencing, several new subgroups of BMSCs and their roles in normal physiological and pathological conditions have been clarified. Key regulators and mechanisms controlling the fate of BMSCs are being revealed. Cross-talk among subgroups of bone marrow mesenchymal cells has been demonstrated. In this review, we focus on recent advances in the identification and classification of BMSCs, which provides important implications for clinical applications.
... The critical role of C/EBPα in adipogenic differentiation in vitro has been extensively demonstrated, previously [22,[47][48][49][50][51][52][53][54]. Thus far, several mouse models aiming to elucidate the role of C/EBPα in adipose tissue in vivo have been used. ...
Obesity and type 2 diabetes are both significant contributors to the contemporary pandemic of non-communicable diseases. Both disorders are interconnected and associated with the disruption of normal homeostasis in adipose tissue. Consequently, exploring adipose tissue differentiation and homeostasis is important for the treatment and prevention of metabolic disorders. The aim of this work is to review the consecutive steps in the postnatal development of adipocytes, with a special emphasis on in vivo studies. We gave particular attention to well-known transcription factors that had been thoroughly described in vitro, and showed that the in vivo research of adipogenic differentiation can lead to surprising findings.
... The differentiation of BMSCs into adipocytes is driven by different transcription factors and regulated by various signaling pathways [6]. Notably, peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) can promote adipocyte differentiation [8,9]. Extensive analyses have established the genetic cascade of adipogenesis. ...
Background: Bone is a rigid organ that provides support and physical protection to vital organs of the body. Several bone loss disorders are commonly associated with increased bone marrow adipose tissue. Bone marrow mesenchymal stromal/stem cells (BMSCs) are multipotent progenitors differentiating into osteoblasts, adipocytes, and chondrocytes. CDC20 is a co-activator of APC/C, required for full ubiquitin ligase activity. In our previous study, CDC20 promoted the osteogenic commitment of BMSCs and Cdc20 conditional knockout mice suggested a decline in bone mass. In this study, we investigated the function of CDC20 in the adipogenic differentiation of BMSCs and provided a new clue between adipogenesis and osteogenesis.
Methods: Lentivirus containing CDC20 shRNA was used for CDC20 knockdown in hBMSCs. Primary mBMSCs were isolated from Cdc20f/f and Sp7-Cre;Cdc20f/f mice. Adipogenesis was examined by qRT-PCR and western blot analysis of adipogenic regulators, Oil Red O staining and transplantation into nude mice. The CDC20 knockout efficiency was determined through immunochemistry, qRT-PCR and western blot of bone marrow. Accumulation of adiposity was measured through histology and staining of bone sections.
Results: CDC20 expression in hBMSCs was significantly decreased during adipogenic differentiation. Knockdown of CDC20 enhanced adipogenic differentiation of hBMSCs in vitro. CDC20-knockdown hBMSCs showed more adipose tissue–like constructs in H&E staining and Oil Red O staining. Sp7-Cre;Cdc20f/f mice presented increased adipocytes in bone marrow compared with control mice. mBMSCs from Sp7-Cre;Cdc20f/f mice exerted upregulated adipogenic differentiation.
Conclusions: Our findings showed that knockdown of CDC20 enhanced adipogenesis of h(m)BMSCs in vitro and in vivo. Overall, CDC20 regulated both adipogenesis and osteogenesis of BMSCs, and may lead to the development of new therapeutic target for “fatty bone” and osteoporosis.
... L'activation transcriptionnelle par CEBPα induit par exemple la transcription de FABP4, du transporteur GLUT4, du récepteur à l'insuline et de plusieurs autres gènes (Christy et al., 1989;Kaestner et al., 1990;McKeon et Pham, 1991). Un haut niveau d'expression de CEBPα active la transcription spécifique de gènes adipocytaires et l'accumulation de lipides en absence d'agents inducteurs de la différenciation (Lin et Lane, 1994). En effet, l'utilisation d'ARNi (ARN interférent) contre CEBPα empêche l'accumulation de lipides et la conversion des progéniteurs en adipocytes (Lin et Lane, 1992). ...
Il existe deux types de tissus adipeux (TA). Le tissu adipeux blanc stocke les lipides sous forme de triglycérides. Le tissu adipeux brun possède une signature thermogénique via la protéine UCP1 utilisant les lipides pour former de la chaleur. Il existe aussi des adipocytes qui ont des caractéristiques similaires aux adipocytes bruns (adipocytes beiges) au sein du TA blanc. Le TA sécrète également des hormones lui conférant une fonction endocrinienne. Il maintient l’homéostasie énergétique et peut être altéré de différentes façons, ce qui conduit à des dysfonctionnements métaboliques : Une perte importante du TA dans les lipoatrophies est observée lors d’un traitement antirétroviral hautement actif contre le VIH (thérapie HAART). Ceci amène à des modifications métaboliques graves, dues à des niveaux élevés de lipides circulants et à une résistance à l’insuline systémique. Cette thérapie HAART est composée d’inhibiteurs de la protéase du VIH (IPs) ou de la transcriptase inverse (INTI). Les effets inhibiteurs des IPs sur le processus de différenciation adipocytaire blanche sont bien connus. Cependant, les mécanismes spécifiques qui affectent les différents dépôts adipeux humains distinctement ainsi que la différenciation adipocytaire brune le sont moins. Le cancer est une pathologie caractérisée par la prolifération dérégulée de cellules capables de former des métastases. Les cellules tumorales interagissent activement avec leur microenvironnement, notamment avec le TA qui est présent autour de nombreux organes et qui peut favoriser la progression tumorale (tissu adipeux associé au cancer). Le TA promeut la prolifération des cellules cancéreuses par la sécrétion d’adipocytokines. De plus, les cellules tumorales modifient le TA pour tirer leur énergie des lipides ce qui favorise leur expansion et leur dissémination. Nous avons étudié les interactions entre adipocytes et cellules tumorales de sein puisque le TA fait partie intégrante de la glande mammaire. Mon travail de thèse a consisté à identifier de nouveaux mécanismes moléculaires importants pour le développement physiopathologique et/ou l’altération du TA. Nous avons d’abord étudié les effets des IPs sur la perte de l’auto-renouvellement des progéniteurs adipeux (PAs) (1) et sur les modifications métaboliques des adipocytes (2). Nous étudions aussi les interactions entre les cellules de cancer du sein et le microenvironnement adipeux (3). Tout d’abord, les IPs inhibent l’auto-renouvellement des PAs en diminuant IER3 ce qui déstabilise en aval la boucle autocrine de l’Activine A. Les IPs bloquent la différenciation des PAs en adipocytes. La perte de ces deux processus indique que les IPs induisent des lipoatrophies retrouvées au cours de la thérapie HAART. Par la suite, nous observons que les IPs réduisent l’expression des marqueurs thermogéniques dans les adipocytes beiges et bruns par l’inhibition de la transcription d’UCP1. Ils altèrent aussi l’expression des sirtuines, enzymes antivieillissement. L’utilisation d’un activateur de la sirtuine 1 permet de renverser partiellement les effets des IPs sur l’expression d’UCP1. Enfin, nos résultats démontrent que des mammosphères de cancer de sein induisent la protéine UCP1 dans les adipocytes adjacents. L’adrénomedulline produite par les mammosphères participe à ce processus et nous avons pu caractériser son mécanisme d’action. En conclusion, les travaux réalisés pendant ma thèse ont permis de mieux comprendre les mécanismes par lesquels les IPs inhibent l’auto-renouvellement des progéniteurs adipeux ainsi que l’altération de la signature thermogénique via la perte d’UCP1 dans les adipocytes bruns. Les cellules tumorales, quant à elles, induisent l’expression d’UCP1 résultant en une conversion métabolique des adipocytes blancs en adipocytes bruns.
... Finally, our analysis suggested that expression levels of the transcription factor CEBPα were significantly lower in SIRT1-depleted adipocytes (Supplementary figure S2b). CEBPα plays a central role in adipogenesis and energy metabolism, and its overexpression is sufficient to induce terminal differentiation of 3T3-L1 preadipocytes 12,51,52 . Importantly, the adipokine leptin is a direct transcriptional target of CEBPα 53,54 and reduced levels of leptin in SIRT1-depleted adipocytes suggest that both CEBPα expression and function may be compromised when SIRT1 levels are downregulated. ...
The NAD+-dependent deacetylase SIRT1 controls key metabolic functions by deacetylating target proteins and strategies that promote SIRT1 function such as SIRT1 overexpression or NAD+ boosters alleviate metabolic complications. We previously reported that SIRT1-depletion in 3T3-L1 preadipocytes led to C-Myc activation, adipocyte hyperplasia, and dysregulated adipocyte metabolism. Here, we characterized SIRT1-depleted adipocytes by quantitative mass spectrometry-based proteomics, gene-expression and biochemical analyses, and mitochondrial studies. We found that SIRT1 promoted mitochondrial biogenesis and respiration in adipocytes and expression of molecules like leptin, adiponectin, matrix metalloproteinases, lipocalin 2, and thyroid responsive protein was SIRT1-dependent. Independent validation of the proteomics dataset uncovered SIRT1-dependence of SREBF1c and PPARα signaling in adipocytes. SIRT1 promoted nicotinamide mononucleotide acetyltransferase 2 (NMNAT2) expression during 3T3-L1 differentiation and constitutively repressed NMNAT1 and 3 levels. Supplementing preadipocytes with the NAD+ booster nicotinamide mononucleotide (NMN) during differentiation increased expression levels of leptin, SIRT1, and PGC-1α and its transcriptional targets, and reduced levels of pro-fibrotic collagens (Col6A1 and Col6A3) in a SIRT1-dependent manner. Investigating the metabolic impact of the functional interaction of SIRT1 with SREBF1c and PPARα and insights into how NAD+ metabolism modulates adipocyte function could potentially lead to new avenues in developing therapeutics for obesity complications.
... Differentiation of MSCs into adipocytes or osteoblasts is orchestrated by many signaling pathways and driven by different transcriptional factors [4]. Previous reports have shown that CCAAT/enhancer binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ or PPARG) can promote adipocyte differentiation [4,9,10], while Runx2 and Dlx5 promote osteoblast differentiation [11]. Runt-related transcription factor 1 (Runx1) is a DNA-binding partner of Core binding factor β (Cbfβ), which forms a Runx1/Cbfβ heterodimeric complex [12][13][14]. ...
Runx1 is highly expressed in osteoblasts, however, its function in osteogenesis is unclear. We generated mesenchymal progenitor-specific ( Runx1 f/f Twist2-Cre) and osteoblast-specific ( Runx1 f/f Col1α1-Cre ) conditional knockout (Runx1 CKO) mice. The mutant CKO mice with normal skeletal development displayed a severe osteoporosis phenotype at postnatal and adult stages. Runx1 CKO resulted in decreased osteogenesis and increased adipogenesis. RNA-sequencing analysis, Western blot, and qPCR validation of Runx1 CKO samples showed that Runx1 regulates BMP signaling pathway and Wnt/β-catenin signaling pathway. ChIP assay revealed direct binding of Runx1 to the promoter regions of Bmp7 , Alk3 , and Atf4 , and promoter mapping demonstrated that Runx1 upregulates their promoter activity through the binding regions. Bmp7 overexpression rescued Alk3, Runx2, and Atf4 expression in Runx1 -deficient BMSCs. Runx2 expression was decreased while Runx1 was not changed in Alk3 deficient osteoblasts. Atf4 overexpression in Runx1 -deficient BMSCs did not rescue expression of Runx1, Bmp7, and Alk3. Smad1/5/8 activity was vitally reduced in Runx1 CKO cells, indicating Runx1 positively regulates the Bmp7/Alk3/Smad1/5/8/Runx2/ATF4 signaling pathway. Notably, Runx1 overexpression in Runx2 -/- osteoblasts rescued expression of Atf4, OCN, and ALP to compensate Runx2 function. Runx1 CKO mice at various osteoblast differentiation stages reduced Wnt signaling and caused high expression of C/ebpα and Pparγ and largely increased adipogenesis. Co-culture of Runx1 -deficient and wild-type cells demonstrated that Runx1 regulates osteoblast−adipocyte lineage commitment both cell-autonomously and non-autonomously. Notably, Runx1 overexpression rescued bone loss in OVX-induced osteoporosis. This study focused on the role of Runx1 in different cell populations with regards to BMP and Wnt signaling pathways and in the interacting network underlying bone homeostasis as well as adipogenesis, and has provided new insight and advancement of knowledge in skeletal development. Collectively, Runx1 maintains adult bone homeostasis from bone loss though up-regulating Bmp7/Alk3/Smad1/5/8/Runx2/ATF4 and WNT/β-Catenin signaling pathways, and targeting Runx1 potentially leads to novel therapeutics for osteoporosis.
... The development of biotechnology has greatly promoted the ability to screen and study key genes involved in muscle and IMF development. Several critical genes have been demonstrated to mediate muscle development and adipogenesis, including peroxisome proliferative activated receptor gamma (PPARγ ) and CCAAT/enhancer-binding protein alpha (C/EBPα), which have been characterized as adipogenesis regulators (Lin and Lane, 1994;Tontonoz et al., 1994). The myogenic regulatory factors (MRFs), myocyte enhancer factor (MEF2), PAX3/PAX7, and myostatin (MSTN) have been reported to be effective for the induction of myoblasts proliferation and differentiation (Sassoon et al., 1989;McPherron and Lee, 1997;Soumillion et al., 1997;Relaix et al., 2005;Potthoff and Olson, 2007). ...
The level of muscle development in livestock directly affects the production efficiency of livestock, and the contents of intramuscular fat (IMF) is an important factor that affects meat quality. However, the molecular mechanisms through which circular RNA (circRNA) affects muscle and IMF development remains largely unknown. In this study, we isolated myoblasts and intramuscular preadipocytes from fetal bovine skeletal muscle. Oil Red O and BODIPY staining were used to identify lipid droplets in preadipocytes, and anti-myosin heavy chain (MyHC) immunofluorescence was used to identify myotubes differentiated from myoblasts. Bioinformatics, a dual-fluorescence reporter system, RNA pull-down, and RNA-binding protein immunoprecipitation were used to determine the interactions between circINSR and the micro RNA (miR)-15/16 family. Molecular and biochemical assays were used to confirm the roles played by circINSR in myoblasts and intramuscular preadipocytes. We found that isolated myoblasts and preadipocytes were able to differentiate normally. CircINSR was found to serve as a sponge for the miR-15/16 family, which targets CCND1 and Bcl-2. CircINSR overexpression significantly promoted myoblast and preadipocyte proliferation and inhibited cell apoptosis. In addition, circINSR inhibited preadipocyte adipogenesis by alleviating the inhibition of miR-15/16 against the target genes FOXO1 and EPT1. Taken together, our study demonstrated that circINSR serves as a regulator of embryonic muscle and IMF development.
... Several critical genes have been demonstrated to mediate muscle development and adipogenesis. For instance, peroxisome proliferative activated receptor (PPARγ) and CCAAT/enhancer binding protein (C/EBPα) have been characterized as the regulators of adipogenesis (6,7). The myogenic regulatory factors (MRFs), myocyte enhancer factor (MEF2), PAX3/PAX7, and myostatin (MSTN) have been reported to be effective in inducing myoblasts proliferation and differentiation (8)(9)(10)(11)(12). ...
Background: The level of muscle development directly affects the production efficiency of livestock, and the content of intramuscular fat (IMF) is an important factor affecting meat quality. Nevertheless, the molecular mechanism of embryonic circular RNA in muscle and IMF development remains largely unknown.
Methods: In this study, we isolated myoblasts and intramuscular preadipocytes from fetal bovine skeletal muscle. Oil Red O and BODIPY staining were used to identify lipid droplets of preadipocytes, and anti-MyHC immunofluorescence was used to identify myotubes differentiated from myoblasts. Bioinformatics, dual fluorescence reporter system, and RNA immunoprecipitation were used to determine the interactions between circINSR and miR-15/16 family. Molecular and biochemical assays were used to confirm the role of circINSR in myoblasts and intramuscular preadipocytes.
Results: We found that the isolated myoblasts and preadipocytes can differentiate normally. Besides, circINSR severed as a sponge of miR-15/16 family, which targeting CyclinD1 and Bcl-2. CircINSR overexpression significantly promoted myoblasts and preadipocytes proliferation, and inhibited cell apoptosis. In addition, circINSR inhibited preadipocytes adipogenesis by alleviating the inhibition of miR-15/16 on target genes FOXO1 and EPT1.
Conclusions: Taken together, our study demonstrated circINSR as a regulator of embryonic muscle and IMF development.
... This might be related to the complexity of the regulatory mechanism of cell differentiation. We have known that many proteins 45,46 and a lot of miRNAs 25,[47][48][49][50][51] have been reported to participate in adipogenic differentiation. Similarly, for angiogenesis, both proteins 52-57 and miRNAs 58-60 played a regulatory role. ...
Purpose
Small extracellular vesicles (sEV) are a heterogeneous group of vesicles that consist of proteins, lipids and miRNA molecules derived from the cell of origin. Although xenogeneic sEV have been applied for soft tissue regeneration successfully, the regeneration effect of allogeneic and xenogeneic sEV has not been compared systematically.
Methods
Our previous study has shown that sEV derived from rat adipose tissue successfully induced neoadipose regeneration. In this study, sEV were isolated from rat adipose tissue (r-sEV-AT) and porcine adipose tissue (p-sEV-AT), the morphology, size distribution and marker proteins expression of r-sEV-AT and p-sEV-AT were characterized. Besides, the sEV/AT ratio was evaluated and compared between r-sEV-AT and p-sEV-AT. Rat adipose-derived stromal/stem cells (rASCs) and rat aorta endothelial cells (rECs) were adopted to test the cellular response to allogeneic and xenogeneic sEV-AT. The effects of allogeneic and xenogeneic sEV-AT on host cells migration and neoadipose formation were evaluated in a subcutaneous custom-designed model. A full-thickness skin wound healing model was used to further compare the ability of allogeneic and xenogeneic sEV-AT in inducing complex soft tissue regeneration.
Results
p-sEV-AT showed similar morphology and size distribution to r-sEV-AT. Marker proteins of sEV were detected in both r-sEV-AT and p-sEV-AT. The sEV/AT ratio of porcine was slightly higher than that of rat. The effects of r-sEV-AT and p-sEV-AT on the differentiation of rASCs and rECs showed no significant difference. When allogeneic and xenogeneic sEV-AT were subcutaneously implanted into the back of SD rats, the host cells chemotactic infiltration was observed in 1 week and neoadipose tissue formation was induced in 8 weeks; no significant difference was observed between allogeneic and xenogeneic sEV-AT. For complex soft tissue regeneration, both allogeneic and xenogeneic sEV-AT significantly promoted wound re-epithelialization, granulation tissue formation and hair follicle regeneration and then accelerated skin wound healing.
Conclusion
Our results demonstrated that sEV derived from the same tissues of different species might be loaded with similar therapeutic substance benefitting tissue repair and regeneration, and paved the way for future research aimed at xenogeneic sEV application.
... Adipocyte differentiation is thought to be regulated by a series of transcription factors, including the C/EBPs protein family [1][2][3] and peroxisome proliferator-activated receptor g (PPARγ) [4]. On the one hand, retinoic acid (RA), the main product of aldehyde dehydrogenase 1A1 (ALDH1A1), inhibits adipogenesis through these transcription factors in mammals [5,6]. ...
ALDH1A1 (aldehyde dehydrogenase 1A1) is a crucial protein in retinoids’ metabolism, and the lack of ALDH1A1 inhibits the fat deposition in mice. However, whether ALDH1A1 has a similar effect on chickens’ fat-depot is still unknown. In this study, we investigate the role of ALDH1A1 in chickens’ adipogenesis. The immortalized chicken preadipocyte 1 (ICP1) cell line and chicken primary preadipocytes isolated from abdominal fat were used to perform a series of experiments in vitro to elucidate the effects of ALDH1A1. In addition, lentivirus was used to verify the results of cell experiments in vivo. The data showed that overexpression of ALDH1A1 significantly weakened the proliferation of preadipocytes and suppressed the differentiation of preadipocytes through the PPARγ pathway, and the knockdown experiments had the opposite results. Moreover, chickens injected with overexpression lentivirus had higher abdominal fat percentage, a bigger size of lipid droplets, and higher triglyceride content in abdominal fat, and chickens injected with interfering lentivirus had the opposite situation. We proved that ALDH1A1 not only inhibited the proliferation and differentiation of chickens’ preadipocytes in vitro, but also inhibited the fat-depot of chickens in vivo, which was completely opposite the function of ALDH1A1 in mice, indicating that ALDH1A1 may have a different mechanism that is still unknown.
... Hyperplasia occurs through the differentiation of preadipocytes into mature adipocytes that have remarkable hypertrophic potential, and increased adipocyte size is closely associated with increased adipose tissue inflammation and systemic insulin resistance (Ghaben & Scherer, 2019). Adipogenesis and lipogenesis are controlled by the sequential activation of multistep gene regulations that are mainly mediated by two primary transcription factors, proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancerbinding protein alpha (C/EBPα) (Lin & Lane, 1994;Siersbaek, Nielsen, & Mandrup, 2012;Tontonoz, Hu, & Spiegelman, 1994). As the master regulator of adipogenesis, PPARγ induces adipocyte differentiation and maturation, while C/EBPα is one of the most important downstream effectors of PPARγ (Wu et al., 1999). ...
Obesity, a major risk factor for type 2 diabetes and cardiovascular diseases, is characterized by an abnormal expansion of adipose tissue. Herein, we investigated the potential of hyaluronic acid nanoparticles (HA-NPs) as therapeutics to treat obesity-related diseases by assessing the in vitro and in vivo effects of HA-NPs on adipogenesis and lipogenesis. Treatment of 3T3-L1 preadipocytes with HA-NPs resulted in a dose-dependent suppression of adipogenesis and lipid accumulation, and decreased the expression of key adipogenic and lipogenic regulators. However, these HA-NPs mediated effects were not observed in 3T3-L1 cells transfected with siRNAs against CD44, a major HA receptor. Further, HA-NP treatment of diet-induced obese (DIO) mice reduced the epididymal fat mass and suppressed the induction of adipogenic and lipogenic regulators, while these effects were attenuated in the CD44-null mice. Thus, our study provides a better understanding of how HA-NP modulates fat accumulation and presents a potential anti-obesity strategy targeting CD44.
... Previous studies suggested that PPARγ and C/EBPα could activate the expressions of adipogenic-specific genes, such as fatty acid binding protein 4 (FABP4), sterol regulatory element-binging proteins-1c (SREBP-1c), fatty acid synthase (FASN) and adiponectin, and C1Q and collagen domain containing (ADIPOQ), thus promoting the storage of triglyceride (TG) and lipid accumulation. 6,7 Long noncoding RNAs (lncRNAs) are a class of RNAs more than 200 nt with no or weak protein-coding potential, which have been widely studied recently. 8 Increasing evidence has indicated that lncRNAs play significant roles in various biological processes, including diseases, cancers, animal development, and cell differentiation. ...
The distribution and deposition of fat tissue in different parts of the body are the key factors affecting the carcass quality and meat flavour of chickens. Adipose tissues from different locations display unique physiological, biochemical characteristics and gene expression patterns. To elucidate the transcriptomic changes of long noncoding RNAs (lncRNAs) in chicken intramuscular and abdominal adipocyte adipogenesis, we defined their transcriptome by Ribo-Zero RNA sequencing. A total of 11247 lncRNAs were observed in the adipocytes derived from IMF and AbF in chicken. Among them, we got 1624 differentiated expressed novel lncRNAs. A large amount of lncRNAs were enriched in various lipid metabolic processes and adipogenesis signaling pathways. Of these lncRNAs, lncAD is one of the most upregulated lncRNA and was co-expressed with several genes of PPAR signaling pathway. Here, we report that knockdown of lncAD inhibited its’ upstream gene TXNRD1 expression in a cis-regulation manner, thus to decrease intramuscular preadipocytes adipogenic differentiation and promoted cell proliferation. This study revealed large transcriptomic differences between IMF- and AbF-derived preadipocyte differentiation. Additionally, the microenvironment of IMF- and AbF-derived preadipocyte may play significant roles in adipogenic differentiation. Collectively, our findings provide not only valuable evidence for the identification of lncRNAs in adipocytes differentiation, but also contribute to better understanding of the molecular mechanisms underlying tissues-specific fat deposition in poultry.
... There are two main transcription factors that are involved in the differentiation of the adipocyte, CCAAT/enhancer-binding protein α (C/EBP-α), and peroxisome proliferator-activated receptor γ (PPAR-γ). PPAR-γ is the most described transcriptional factor, and its expression is regulated by the co-factor PGC1α and the production of adiponectin [76,77]. ...
... PPARγ and C/EBPα positively regulate each other to promote expression and maintain differentiation of 3T3-L1 cells [13]. PPARγ and C/EBPα have also been reported to induce adipogenesis upon ectopic expression in fibroblast cells [12,44,45]. In this study, ergosterol peroxide treatment (20 μM) also significantly reduced the protein expression and mRNA transcription of PPARγ and C/EBPα, which play an important role in differentiation of 3T3-L1 cells. ...
Ergosterol peroxide is a natural compound of the steroid family found in many fungi, and it possesses antioxidant, anti-inflammatory, anticancer and antiviral activities. The anti-obesity activity of several edible and medicinal mushrooms has been reported, but the effect of mushroom-derived ergosterol peroxide on obesity has not been studied. Therefore, we analyzed the effect of ergosterol peroxide on the inhibition of triglyceride synthesis at protein and mRNA levels and differentiation of 3T3-L1 adipocytes. Ergosterol peroxide inhibited lipid droplet synthesis of differentiated 3T3-L1 cells, expression of peroxisome proliferator-activated receptor gamma (PPARγ) and CCAT/enhancer-binding protein alpha (C/EBPα), the major transcription factors of differentiation, and also the expression of sterol regulatory element-binding protein-1c (SREBP-1c), which promotes the activity of PPARγ, resulting in inhibition of differentiation. It further inhibited the expression of fatty acid synthase (FAS), fatty acid translocase (FAT), and acetyl-coenzyme A carboxylase (ACC), which are lipogenic factors. In addition, it inhibited the phosphorylation of mitogen-activated protein kinases (MAPKs) involved in cell proliferation and activation of early differentiation transcription factors in the mitotic clonal expansion (MCE) stage. As a result, ergosterol peroxide significantly inhibited the synthesis of triglycerides and differentiation of 3T3-L1 cells, and is, therefore, a possibile prophylactic and therapeutic agent for obesity and related metabolic diseases.
... Molecules for cell shape and extracellular matrix remodeling also contribute to differentiation [1]. The major adipogenic transcriptional factors are peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT-enhancer-binding proteins (C/EBPs), which induce mature adipogenic phenotypes such as lipid storage and synthesis, lipolysis, and adipokine secretion [4][5][6]. Adipogenesis is a complicated process regulated by transcriptional activators and repressors for PPARγ and C/EBPα expression [7,8]. ...
Adipogenesis is a crucial cellular process that contributes to the expansion of adipose tissue in obesity. Shockwaves are mechanical stimuli that transmit signals to cause biological responses. The purpose of this study is to evaluate the effects of shockwaves on adipogenesis. We treated 3T3L-1 cells and human primary preadipocytes for differentiation with or without shockwaves. Western blots and quantitative real-time reverse transcriptase PCR (qRT-PCR) for adipocyte markers including peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT-enhancer-binding proteins (C/EBPα) were performed. Extracellular adenosine triphosphate (ATP) and intracellular cyclic adenosine monophosphate (cAMP) levels, which are known to affect adipocyte differentiation, were measured. Shockwave treatment decreased intracellular lipid droplet accumulation in primary human preadipocytes and 3T3-L1 cells after 11–12 days of differentiation. Levels of key adipogenic transcriptional factors PPARγ and/or C/EBPα were lower in shockwave-treated human primary preadipocytes and 3T3L-1 cells after 12–13 days of differentiation than in shockwave-untreated cells. Shockwave treatment induced release of extracellular ATP from preadipocytes and decreased intracellular cAMP levels. Shockwave-treated preadipocytes showed a higher level of β-catenin and less PPARγ expression than shockwave-untreated cells. Supplementation with 8-bromo-cAMP analog after shockwave treatment rescued adipocyte differentiation by preventing the effect of shockwaves on β-catenin, Wnt10b mRNA, and PPARγ expression. Low-energy shockwaves suppressed adipocyte differentiation by decreasing PPARγ. Our study suggests an insight into potential uses of shockwave-treatment for obesity.
... contains supplementary material, which is available to authorized users. generally accepted that preadipocyte differentiation is a highly regulated process that involves several transcription factors, including peroxisome proliferator-activated receptor gamma (PPARG) (Tontonoz et al. 1994), CCAAT/enhancer-binding protein α (CEBPA) (Lin and Lane 1994), and fatty acid binding protein 4 (FABP4) (Deng et al. 2019). ...
Emerging evidence suggests that long non-coding RNAs (lncRNAs) are critical regulators of diverse biological processes, including adipogenesis. Despite being considered an ideal animal model for studying adipogenesis, little is known about the roles of lncRNAs in the regulation of rabbit preadipocyte differentiation. In the present study, visceral preadipocytes isolated from newborn rabbits were cultured in vitro and induced for differentiation, and global lncRNA expression profiles of adipocytes collected at days 0, 3, and 9 of differentiation were analyzed by RNA-seq. A total of 2066 lncRNAs were identified from nine RNA-seq libraries. Compared to protein-coding transcripts, lncRNA transcripts exhibited characteristics of a longer length and lower expression level. Furthermore, 486 and 357 differentially expressed (DE) lncRNAs were identified when comparing day 3 vs. day 0 and day 9 vs. day 3, respectively. Target genes of DE lncRNAs were predicted by the cis-regulating approach. Prediction of functions revealed that DE lncRNAs when comparing day 3 vs. day 0 were involved in gene ontology (GO) terms of developmental growth, growth, developmental cell growth, and stem cell proliferation, and involved in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of PI3K-Akt signaling pathway, fatty acid biosynthesis, and the insulin signaling pathway. The DE lncRNAs when comparing day 9 vs. day 3 were involved in GO terms that associated with epigenetic modification and were involved in the KEGG pathway of cAMP signaling pathway. This study provides further insight into the regulatory function of lncRNAs in rabbit visceral adipose and facilitates a better understanding of different stages of preadipocyte differentiation.
... Previous literature data have shown that KCTD1 interacts with the transcription factor AP2α by the N-termini of both proteins [17]. This interaction inhibits transactivation which plays an inhibitory role of the adipogenesis by repressing the CCAAT-enhancer-binding protein alpha (C/ebpα) gene expression [29,30] that has been identified as one of the muster regulators of this differentiation program [31]. In view of the role of AP2α as repressor of the C/ebpα gene, the detection of endogenous KCTD1 in 3T3-L1 cells provided us the possibility to validate the KCTD1/AP2α partnership in this cell line and to assign a previously unrecognized function to KCTD1 in adipogenesis. ...
... To demonstrate that expression of C/ EBPα is required for adipose differentiation, Lin (Lin and Lane, 1992) inhibits expression by introducing antisense RNA in 3T3-L1 preadipocytes, and it was found that the fat-specific gene was not expressed and no aggregation of triglycerides was detected. By detecting the accumulation of lipid droplets in the cytosol and the expression of 422/aP2, GLUT4 and endogenous C/EBPα in 3T3-L1 cells, it was found that the C/EBPα conditional expression introduced by stable cloning was sufficient to induce cell adipogenic differentiation (Lin and Lane, 1994). These all indicate that the expression of C/EBPα is a sufficient and necessary condition for the adipogenic differentiation of preadipocytes. ...
The genetic regulation of lipolytic enzyme is closely related to carcass quality traits through deposition of intramuscular fat (marbling) in beef cattle breeds. The α/β hydrolase domain containing 5 (ABHD5) is an accelerating factor of adipose triglyceride lipase (ATGL), which plays a key role in triglyceride metabolism. In this study, we determined that bovine ABHD5 gene was highly expressed in adult bovine adipose tissue. To elucidate the molecular mechanisms involved in bovine ABHD5 regulation, we cloned and characterized the promoter region of ABHD5. Applying 5′-rapid amplification of cDNA end analysis (RACE), we identified transcriptional start site (TSS) found in the predicted CpG island within promoter region of ABHD5 gene. Using the recombinant dual fluorescent reporter vectors, the fragment of pGL3–109/+307 was identified as proximal minimum core promoter region in bovine intramuscular adipocytes. Site directed mutagenesis and electrophoretic mobility shift assay (EMSA) confirmed the role of two transcription factors, namely Ectopic viral integration site-1 (Evi1) and CCAAT/enhancer binding protein alpha (C/EBPα), in the regulation of ABHD5 gene. Taken together these findings we can conclude that ABHD5 gene regulated by Evi1 and C/EBPα could be used as potential marker in marker assisted selection for the improvement of Qinchuan cattle breed for carcass quality traits.
Osteoporotic vertebral compression fractures (OVCFs) significantly increase morbidity and mortality, presenting a formidable challenge in healthcare. Traditional interventions such as vertebroplasty and kyphoplasty, despite their widespread use, are limited in addressing the secondary effects of vertebral fractures in adjacent areas and do not facilitate bone regeneration. This review paper explores the emerging domain of regenerative therapies, spotlighting stem cell therapy’s transformative potential in OVCF treatment. It thoroughly describes the therapeutic possibilities and mechanisms of action of mesenchymal stem cells against OVCFs, relying on recent clinical trials and preclinical studies for efficacy assessment. Our findings reveal that stem cell therapy, particularly in combination with scaffolding materials, holds substantial promise for bone regeneration, spinal stability improvement, and pain mitigation. This integration of stem cell-based methods with conventional treatments may herald a new era in OVCF management, potentially improving patient outcomes. This review advocates for accelerated research and collaborative efforts to translate laboratory breakthroughs into clinical practice, emphasizing the revolutionary impact of regenerative therapies on OVCF management. In summary, this paper positions stem cell therapy at the forefront of innovation for OVCF treatment, stressing the importance of ongoing research and cross-disciplinary collaboration to unlock its full clinical potential.
Importance
Epicardial and pericardial adipose tissue (EPAT) has been associated with cardiovascular diseases such as atrial fibrillation or flutter (AF) and coronary artery disease (CAD), but studies have been limited in sample size or drawn from selected populations. It has been suggested that the association between EPAT and cardiovascular disease could be mediated by local or paracrine effects.
Objective
To evaluate the association of EPAT with prevalent and incident cardiovascular disease and to elucidate the genetic basis of EPAT in a large population cohort.
Design, Setting, and Participants
A deep learning model was trained to quantify EPAT area from 4-chamber magnetic resonance images using semantic segmentation. Cross-sectional and prospective cardiovascular disease associations were evaluated, controlling for sex and age. Prospective associations were additionally controlled for abdominal visceral adipose tissue (VAT) volumes. A genome-wide association study was performed, and a polygenic score (PGS) for EPAT was examined in independent FinnGen cohort study participants. Data analyses were conducted from March 2022 to December 2023.
Exposures
The primary exposures were magnetic resonance imaging–derived continuous measurements of epicardial and pericardial adipose tissue area and visceral adipose tissue volume.
Main Outcomes and Measures
Prevalent and incident CAD, AF, heart failure (HF), stroke, and type 2 diabetes (T2D).
Results
After exclusions, this study included 44 475 participants (mean [SD] age, 64.1 [7.7] years; 22 972 female [51.7%]) from the UK Biobank. Cross-sectional and prospective cardiovascular disease associations were evaluated for a mean (SD) of 3.2 (1.5) years of follow-up. Prospective associations were additionally controlled for abdominal VAT volumes for 38 527 participants. A PGS for EPAT was examined in 453 733 independent FinnGen cohort study participants. EPAT was positively associated with male sex (β = +0.78 SD in EPAT; P < 3 × 10 ⁻³²⁴ ), age (Pearson r = 0.15; P = 9.3 × 10 ⁻²²⁹ ), body mass index (Pearson r = 0.47; P < 3 × 10 ⁻³²⁴ ), and VAT (Pearson r = 0.72; P < 3 × 10 ⁻³²⁴ ). EPAT was more elevated in prevalent HF (β = +0.46 SD units) and T2D (β = +0.56) than in CAD (β = +0.23) or AF (β = +0.18). EPAT was associated with incident HF (hazard ratio [HR], 1.29 per +1 SD in EPAT; 95% CI, 1.17-1.43), T2D (HR, 1.63; 95% CI, 1.51-1.76), and CAD (HR, 1.19; 95% CI, 1.11-1.28). However, the associations were no longer significant when controlling for VAT. Seven genetic loci were identified for EPAT, implicating transcriptional regulators of adipocyte morphology and brown adipogenesis ( EBF1 , EBF2 , and CEBPA ) and regulators of visceral adiposity ( WARS2 and TRIB2 ). The EPAT PGS was associated with T2D (odds ratio [OR], 1.06; 95% CI, 1.05-1.07; P =3.6 × 10 ⁻⁴⁴ ), HF (OR, 1.05; 95% CI, 1.04-1.06; P =4.8 × 10 ⁻¹⁵ ), CAD (OR, 1.04; 95% CI, 1.03-1.05; P =1.4 × 10 ⁻¹⁷ ), AF (OR, 1.04; 95% CI, 1.03-1.06; P =7.6 × 10 ⁻¹² ), and stroke in FinnGen (OR, 1.02; 95% CI, 1.01-1.03; P =3.5 × 10 ⁻³ ) per 1 SD in PGS.
Conclusions and Relevance
Results of this cohort study suggest that epicardial and pericardial adiposity was associated with incident cardiovascular diseases, but this may largely reflect a metabolically unhealthy adiposity phenotype similar to abdominal visceral adiposity.
Bones are constantly exposed to mechanical forces from both muscles and Earth’s gravity to maintain bone homeostasis by stimulating bone formation. Mechanotransduction transforms external mechanical signals such as force, fluid flow shear, and gravity into intracellular responses to achieve force adaptation. However, the underlying molecular mechanisms on the conversion from mechanical signals into bone formation has not been completely defined yet. In the present review, we provide a comprehensive and systematic description of the mechanotransduction signaling pathways induced by mechanical stimuli during osteogenesis and address the different layers of interconnections between different signaling pathways. Further exploration of mechanotransduction would benefit patients with osteoporosis, including the aging population and postmenopausal women.
Les glucocorticoïdes (GC) font partie des médicaments les plus prescrits en raison de leurs propriétés anti-inflammatoires et immunosuppressives, cependant à fortes doses ils sont responsables du développement d’un diabète cortico-induit et d’une lipodystrophie. La contribution du récepteur adipocytaire des glucocorticoïdes (GR) dans ces effets délétères reste à ce jour peu documentée. L’objectif de ce travail de thèse était de déterminer dans un contexte d’hypercorticisme le rôle précis du GR adipocytaire dans le développement de l’insulino-résistance et des troubles métaboliques associés. Pour mener à bien ce projet, le laboratoire a généré un modèle murin d’invalidation conditionnelle du GR spécifiquement dans l’adipocyte (AdipoGR-KO), soumis à un traitement par la corticostérone. Le phénotypage métabolique montre chez les animaux AdipoGR-KO une augmentation de l’adiposité associée, paradoxalement, à une amélioration de la tolérance au glucose, de la sensibilité à l’insuline, du profil lipidique et de la stéatose hépatique. Le stockage préférentiel et bénéfique des lipides dans les dépôts adipeux nous a incité à étudier les mécanismes mis en jeu lors du développement physiopathologique du tissu adipeux, et en particulier de sa vascularisation. De façon étonnante, nos résultats indiquent un fort développement du réseau vasculaire des dépôts adipeux associé à une induction de l’expression du facteur pro-angiogénique VEGF-A et de son régulateur transcriptionnel HIF-1α. Ainsi, nous montrons pour la première fois que le GR pourrait être un facteur limitant de l’expansion du tissu adipeux via l’inhibition du processus d’angiogenèse.
Bone is a complex tissue that undergoes constant remodeling to maintain homeostasis, which requires coordinated multilineage differentiation and proper proliferation of mesenchymal stromal cells (MSCs). Mounting evidence indicates that a disturbance of bone homeostasis can trigger degenerative bone diseases, including osteoporosis and osteoarthritis. In addition to conventional genetic modifications, epigenetic modifications (i.e., DNA methylation, histone modifications, and the expression of noncoding RNAs) are considered to be contributing factors that affect bone homeostasis. Long noncoding RNAs (lncRNAs) were previously regarded as 'transcriptional noise' with no biological functions. However, substantial evidence suggests that lncRNAs have roles in the epigenetic regulation of biological processes in MSCs and related diseases. In this review, we summarized the interactions between lncRNAs and epigenetic modifiers associated with osteo-/adipogenic differentiation of MSCs and the pathogenesis of degenerative bone diseases and highlighted promising lncRNA-based diagnostic and therapeutic targets for bone diseases.
With rising global demand for food proteins and significant environmental impact associated with conventional animal agriculture, it is important to develop sustainable alternatives to supplement existing meat production. Since fat is an important contributor to meat flavor, recapitulating this component in meat alternatives such as plant based and cell cultured meats is important. Here, we discuss the topic of cell cultured or tissue engineered fat, growing adipocytes in vitro that could imbue meat alternatives with the complex flavor and aromas of animal meat. We outline potential paths for the large scale production of in vitro cultured fat, including adipogenic precursors during cell proliferation, methods to adipogenically differentiate cells at scale, as well as strategies for converting differentiated adipocytes into 3D cultured fat tissues. We showcase the maturation of knowledge and technology behind cell sourcing and scaled proliferation, while also highlighting that adipogenic differentiation and 3D adipose tissue formation at scale need further research. We also provide some potential solutions for achieving adipose cell differentiation and tissue formation at scale based on contemporary research and the state of the field.
Cytochrome P450 Family 19 (CYP19) is a crucial enzyme to catalyze the conversion of androgens to estrogens. However, the regulatory mechanism of goose CYP19 gene remains poorly understood. The present study attempted to obtain the full-length coding sequence (CDS) and 5’-flanking sequence of CYP19 gene, to investigate its expression and distribution profiles in different sized follicles, and to analyze the transcriptional regulatory mechanism of CYP19 gene in goose. Results showed that its CDS consisted of 1512 nucleotides and the encoded amino acid sequence contained a classical P450 structural domain. Homology analysis showed that there were high homologies of nucleotide and amino acid sequences between goose and other avian species. Its promoter sequence spanned from -1925 bp to the transcription start site (ATG) and several transcriptional factors were predicted in this region. Further analysis from luciferase assay showed that the luciferase activity was the highest spanning from -118 to -1 bp by constructing deletion promoter reporter vector. In addition, result from quantitative real-time polymerase chain reaction indicated that the mRNA level of CYP19 gene were highly expressed in theca layer of the fifth largest follicle, and the cellular location was in the theca externa cells by immunohistochemistry. Taken together, it could be concluded that the transcription activity of CYP19 gene was activated by transcriptional factors in its proximal region of promoter to promote the synthesis of estrogens, regulating the selection of pre-hierarchical into hierarchical follicle in goose.
While the underlying mechanism remains unknown, Rubus chingii var. suavissimus (S. K. Lee) L. T. Lu or Rubus suavissimus S. Lee (RS), a sweet plant distributed in southwest of China, has been used as beverage and folk medicine. Pharmacological studies indicated the potential of RS improving the obesity phenotype and hyperlipidemia. The mechanism is still not yet to be put forward. To verify the substantial effects of RS on lipid metabolism, a Syrian golden hamster model was adopted. The physiological and pathological evaluation of experimental animals demonstrated that RS can relieve the lipid metabolism disorder induced by high-fat diet and alleviated liver injury. RS upregulation the expressions of peroxisome proliferator-activated receptor α (PPARα), PPARγ and CCAAT/enhancer binding protein α (C/EBPα), as well as adipocyte-specific genes, glucose transporter 4 (Glut4), lipoprotein lipase (LPL) and fatty acid binding protein 4 (aP2). On the other side, RS suppressed the sterol regulatory element binding protein 1 (SREBP1) and downstream acetyl-CoA carboxylase 1 (ACC1), stearoyl-CoA desaturase-1 (SCD1) and fatty acid synthase (FAS). In conclusion, RS alleviated lipid metabolism disorder symptoms caused by high-fat diet accompanied with 8 weeks of treatment, involving enhanced β-oxidation, increased adipogenesis and decreased the metabolism of fatty acids, via modulation of the PPARs/SREBP pathway in Syrian golden hamsters.
Background and purpose:
Excessive lipid accumulation in adipose tissues and deregulation of adipogenesis-induced obesity affect millions of people worldwide. Feprazone, a nonsteroidal anti-inflammatory drug, has a wide clinical use. However, it is unknown whether Feprazone possesses an antiadipogenic ability. The aim of this study is to investigate whether Feprazone possesses an antiadipogenic ability in 3 T3-L1 cells and an antiobesity capacity in mouse models.
Methods:
An MTT assay was used to determine the optimized incubation concentrations of Feprazone in 3 T3-L1 cells. The lipid accumulation was evaluated using Oil Red O staining. The concentrations of triglyceride and glycerol release were detected to check the lipolysis during 3 T3-L1 adipogenesis. A quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the expressions of sterol regulatory element-binding protein-1C (SREBP-1C) and fatty acid binding protein 4 (FABP4) in treated cells. The expressions of peroxisome proliferator-activated receptor-γ (PPAR-γ), CCAAT/enhancer-binding protein α (C/EBP-α), adipose triglyceride lipase (ATGL), and aquaporin-7 (AQP-7) were detected using qRT-PCR and Western blot analysis. After the high-fat diet (HFD) mice were treated with Feprazone, the pathological state of adipocyte tissues was evaluated using HE staining. The adipocyte size, visceral adipocyte tissue weight, and bodyweights were recorded.
Results:
According to the proliferation result, 30 and 60 μM Feprazone were used as the optimized concentrations of Feprazone. In the in vitro study, lipid accumulation, elevated production of triglycerides, the release of glycerol, upregulated SREBP-1C, FABP4, PPAR-γ, and C/EBP-α and downregulated ATGL and AQP-7 in the 3 T3-L1 adipocytes induced by the adipocyte differentiation cocktail medium were significantly reversed by treatment with Feprazone. In the in vivo experiment, we found that the increased adipocyte size, visceral adipocyte tissue weight, and body weights induced by HFD feeding in mice were significantly suppressed by the administration of Feprazone.
Conclusion:
Feprazone might display anti-adipogenic and antiobesity capacities in in vitro 3 T3-L1 cells and in vivo mice.
Background and purpose:
obesity is defined as excessive accumulation of adipose tissues and is becoming one of the main global severe public health issues. The present study aims to investigate the anti-adipogenesis of laquinimod and the underlying mechanism.
Methods:
a differentiation cocktail was used to differentiate 3T3-L1 cells, and mice were fed with high fat food to establish the obesity animal model. Oil red O staining, glycerol production assay, and the release of triglyceride were used to evaluate the differentiation degree of 3T3-L1 cells. The expression level of sterol regulatory element binding transcription factor 1 (Srebp1), fatty acid binding protein-4 (FABP4), glucose transporter 4 (GLUT4), peroxisome proliferator-activated receptor-γ (PPAR-γ), CCAAT enhancer-binding proteins (C/EBPα), and phosphorylation of adenosine 5'-monophosphate (AMP)-activated protein kinase α (p-AMPKα) was determined by quantitative real time PCRqRT-PCR and western blot analysis. The pathological state of adipose tissues was evaluated by hematoxylin-eosin staining.
Results:
the amount and UV absorption of oil red O, glycerol production, release of triglyceride, and the expression of SREBP1, FABP4, and Glut4 in differentiated 3T3-L1 cells were decreased by the administration of laquinimod. PPAR-γ and C/EBPα were down-regulated, and p-AMPKα was up-regulated by laquinimod. The down-regulated PPAR-γ and C/EBPα, as well as the inhibited lipid accumulation functioned by laquinimod, were reversed by the coincubation with the AMPK inhibitor compound C. Decreased body weight, visceral adipocyte tissue weight, and size of adipocytes were observed in in vivo obesity mice after administration with laquinimod.
Conclusion:
laquinimod might prevent adipogenesis by down-regulating PPAR-γ and C/EBPα through activating AMPK.
Interleukin (IL)-15 is an important regulator of adipogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). This study was designed to clarify the underlying mechanism. BMSCs were obtained from Nanyang cattle and stimulated to differentiate into adipocytes using standard differentiation medium. Oil Red O staining was used to assess lipid accumulation. Western blotting and quantitative real-time polymerase chain reaction were used to assess protein and mRNA levels, respectively. Recombinant IL-15 treatment inhibited adipogenic differentiation of cattle BMSCs in vitro, as evidenced by reduced induction of the adipocyte markers, peroxisome proliferator activated receptor γ (PPARγ) and fatty acid binding protein 4 (αP2). IL-15 not only activated the signal transducers and activators of transcription (STAT) pathway, but also attenuated the activation of phosphoinositide 3-kinase (PI3K)/Akt signalling by insulin, a major inducer of adipocyte differentiation. In the presence of the STAT-specific inhibitor, 573108, the inhibitory effect of IL-15 on PPARγ and αP2 expression was abolished. Meanwhile, IL-15-attenuated PI3K/Akt signalling was also rescued. IL-15 may regulate adipogenic differentiation of BMSCs by inhibiting PI3K/Akt activation via the STAT5A pathway. Our data raise the possibility of using IL-15 in the therapy of obesity-related diseases, such as cardiovascular diseases and type 2 diabetes.
Background/aim:
The rapid increase in the number of people who are overweight or obese, which increases the risk of diseases and health problems, is becoming an important issue. Herein, we investigated whether olive leaf extract (OLE) has potent anti-obesity effects in high-fat induced mouse models.
Materials and methods:
C57BL/6 mice were randomized into normal control, high-fat diet (HFD), HFD with OLE, and HFD with garcinia groups and administered experimental diets for 12 weeks. Body weight and food intake were measured once per week and obesity-related biomarkers were evaluated in the serum and adipose tissue.
Results:
OLE significantly suppressed weight gain, food efficiency ratio, visceral fat accumulation, and serum lipid composition in HFD-induced mice. Furthermore, the expression of adipogenesis- and thermogenesis-related molecules was decreased in the OLE-treated group.
Conclusion:
OLE prevents obesity development by regulating the expression of molecules involved in adipogenesis and thermogenesis.
Numerous experimental studies have demonstrated that a series of remodeling processes occurred in the adipose tissue during the weaning, such as differentiation. Fibroblasts in the breast at weaning stage could re-differentiate into mature adipocytes. Many transcriptional factors were involved in these processes, especially the PPARγ, C/EBP, and SREBP1. There is cell apoptosis participating in the breast tissue degeneration and secretory epithelial cells loss during weaning. In addition, hormones, especially the estrogen and pituitary hormone, play a vital role in the whole reproductive processes. In this review, we mainly focus on the underlying regulated mechanisms of differentiation of adipose tissue and apoptosis of breast cell to provide a specific insight into the physiological changes during weaning.
3T3-L1 fibroblasts differentiate in culture into cells having adipocyte character. This transition is accompanied by a 40- to 50-fold rise in the incorporation of [14C]acetate into triglyceride. The increase in lipogenic rate is exactly parallel to a coordinate rise in the activities of the key enzymes of the fatty acid biosynthetic pathway (ATP-citrate lyase, acetyl-CoA carboxylase, and fatty acid synthetase). Immunological studies indicate that the elevated acetyl-CoA carboxylase activity is the product of an increased cellular enzyme level.
Previous investigations have shown that CCAAT/enhancer binding protein (C/EBP) can function as a trans-activator of the promoters of several adipocyte-specific genes--i.e., the 422 adipose P2 (422/aP2), stearoyl-CoA desaturase 1 (SCD1), and glucose transporter 4 (GLUT4) genes, in 3T3-L1 mouse preadipocytes. We now describe a cell-free system prepared from nuclei of 3T3-L1 cells that carries out transcription directed by these promoters. To measure transcript formation, we employed a polymerase chain reaction-assisted analysis. Nuclear extract from 3T3-L1 adipocytes that express C/EBP supports a higher rate of transcription of chimeric 422(aP2) promoter-chloramphenicol acetyltransferase (CAT) reporter gene constructs than nuclear extract from preadipocytes that lack C/EBP. A competitor oligonucleotide containing the C/EBP binding site sequence and antibodies raised against C/EBP inhibit transcription directed by the 422(aP2) promoter. The factor limiting transcription by nuclear extract from preadipocytes appears to be C/EBP, since recombinant C/EBP (rC/EBP) markedly activates transcription of the 422(aP2) promoter-CAT gene with preadipocyte extract but not with adipocyte extract. rC/EBP also activates cell-free transcription of SCD1 promoter-CAT and GLUT4 promoter-CAT chimeric genes. Point mutations within the C/EBP binding site in the 422(aP2) promoter markedly decrease transcription activated by rC/EBP. Consistent with activation by cAMP of the 422(aP2) promoter in intact preadipocytes, cAMP-dependent protein kinase activates transcription through this promoter with the cell-free system, this effect being independent of C/EBP. Thus, regulation of transcription directed by the 422(aP2) promoter in the cell-free system resembles that which occurs in intact 3T3-L1 cells.
During the course of differentiation of preadipocytes into adipocytes, several differentiation-linked genes are activated synchronously with morphological changes. To follow this process we have used 3T3-F442A cells, known to undergo adipocyte conversion with high frequency. Accumulation of lipid droplets in the cytoplasm constitutes an easily visualized sign of the terminally differentiated phenotype. In this report we demonstrate that expression of the CCAAT/enhancer binding protein (C/EBP) is an important factor in determining the ability to accumulate lipid droplets in terminally differentiated adipocytes. In one experiment we can suppress C/EBP expression through administration of hydrocortisone to differentiating 3T3-F442A cells, which is accompanied by an inability of the cells to accumulate lipid. In another experiment a C/EBP antisense expression vector has been stably introduced into 3T3-F442A cells and as compared with control cells, a 62% decrease of C/EBP mRNA (p less than 0.01) is demonstrated. This decrease of C/EBP mRNA is accompanied by a change in cellular morphology characterized by a reduced ability to form lipid droplets. We can also demonstrate a correlation between the degree of reduction of C/EBP mRNA and the amount of lipid present in the cells. These findings strongly support the view that C/EBP is a necessary component of terminal adipocyte differentiation.
Differentiation of 3T3-L1 preadipocytes into adipocytes is accompanied by increased expression of the nuclear protein C/EBP (CCAAT/enhancer binding protein) and by transcriptional activation of a group of adipose-specific genes. We report here the isolation of the murine C/EBP gene and the characterization of its promoter. Consistent with its proposed role in coordinating transcription during preadipocyte differentiation, an increase in the rate of transcription of the C/EBP gene precedes that of several adipose-specific genes whose promoters are transactivated by C/EBP. DNase I cleavage-inhibition patterns (footprinting) of the C/EBP gene promoter by nuclear factors from differentiated and undifferentiated 3T3-L1 cells identified two sites of differential factor binding. One site in the C/EBP gene promoter between nucleotides -252 and -239 binds a nuclear factor(s) present in preadipocytes that is lost or modified upon differentiation. Another site, between nucleotides -203 and -176, exhibits different but overlapping footprints by nuclear factors present in differentiated and undifferentiated cells. Gel retardation analysis with oligonucleotides corresponding to these sites revealed protein-oligonucleotide complexes containing these differentially expressed nuclear factors. The factor present in differentiated cells that binds at this site was identified as C/EBP (possibly in heterodimeric form with a homologous leucine-zipper protein), suggesting that C/EBP may regulate expression of its own gene.
Adipose tissue and skeletal and heart muscle, which exhibit insulin-stimulated glucose uptake, express a specific, insulin-responsive glucose transporter. Previously, a cDNA (GT2) encoding this protein was isolated from a mouse 3T3-L1 adipocyte library and was sequenced. Here we report the isolation and characterization of the corresponding mouse gene designated GLUT4. The GLUT4 gene spans 7 kilobases and consists of 11 exons and 10 introns. The start site of transcription was mapped 180 nucleotides upstream of the initial methionine codon. The GLUT4 promoter contains four potential binding sites for the nuclear transcription factor Sp1 as well as a CCAAT box. DNase I footprinting of the GLUT4 promoter with nuclear extracts from undifferentiated and differentiated 3T3-L1 cells revealed that a differentiation-specific nuclear factor binds in the region at position -258 relative to the start site of transcription. Purified CCAAT/enhancer binding protein (C/EBP) was found to bind at the same position. Transient cotransfection into 3T3-L1 preadipocytes of a GLUT4 promoter-chloramphenicol acetyltransferase gene construct that contains the C/EBP binding site, together with a C/EBP expression vector, revealed that C/EBP trans-activates the GLUT4 promoter. We suggest that C/EBP plays an important role in tissue-specific, as well as metabolic, regulation of the insulin-responsive glucose transporter gene.
Adipocyte differentiation is accompanied by the transcriptional activation of many new genes, including the gene encoding adipocyte P2 (aP2), an intracellular lipid-binding protein. Using specific deletions and point mutations, we have shown that at least two distinct sequence elements in the aP2 promoter contribute to the expression of the chloramphenicol acetyltransferase gene in chimeric constructions transfected into adipose cells. An AP-I site at -120, shown earlier to bind Jun- and Fos-like proteins, serves as a positive regulator of chloramphenicol acetyltransferase gene expression in adipocytes but is specifically silenced by adjacent upstream sequences in preadipocytes. Sequences upstream of the AP-I site at -140 (termed AE-1) can function as an enhancer in both cell types when linked to a viral promoter but can stimulate expression only in fat cells in the intact aP2 promoter. The AE-1 sequence binds an adipocyte protein identical or very closely related to an enhancer-binding protein (C/EBP) that has been previously implicated in the regulation of several liver-specific genes. A functional role for C/EBP in the regulation of the aP2 gene is indicated by the facts that C/EBP mRNA is induced during adipocyte differentiation and the aP2 promoter is transactivated by cotransfection of a C/EBP expression vector into preadipose cells. These results indicate that sequences that bind C/EBP and the Fos-Jun complex play major roles in the expression of the aP2 gene during adipocyte differentiation and demonstrate that C/EBP can directly regulate cellular gene expression.
Previous studies have shown that differentiation of 3T3-L1 preadipocytes leads to the transcriptional activation of a group of adipose-specific genes. As an approach to defining the mechanism responsible for activating the expression of these genes, we investigated the binding of nuclear factors to the promoters of two differentiation-induced genes, the 422(aP2) and stearoyl-CoA desaturase 1 (SCD1) genes. DNase I footprinting and gel retardation analysis identified two binding regions within the promoters of each gene that interact with nuclear factors present in differentiated 3T3-L1 adipocytes. One differentiation-induced nuclear factor interacts specifically with a single binding site in the promoter of each gene. Competition experiments showed that the interaction of this nuclear factor with the SCD1 promoter was prevented specifically by a synthetic oligonucleotide corresponding to the site footprinted in the 422(aP2) promoter. Several lines of evidence indicate that the differentiation-induced nuclear factor is CCAAT/enhancer binding protein (C/EBP), a DNA-binding protein first isolated from rat liver. Bacterially expressed recombinant C/EBP binds to the same site at which the differentiation-specific nuclear factor interacts within the promoter of each gene. Northern analysis with RNA from 3T3-L1 cells shows that C/EBP mRNA abundance increases markedly during differentiation. Transient cotransfection studies using a C/EBP expression vector demonstrate that C/EBP can function as a trans-activator of both the 422(aP2) and SCD1 gene promoters.
The cDNAs for two putative glucose transporters from mouse 3T3-L1 adipocytes were isolated and sequenced. One of these cDNAs encodes the murine homolog of the human hepG2/erythrocyte glucose transporter, termed GT1. GT1 mRNA is most abundant in mouse brain and is expressed in both 3T3-L1 preadipocytes and adipocytes. The other cDNA encodes a glucose transporter-like protein, termed GT2, that has a unique amino acid sequence and tissue distribution. GT2 cDNA encodes a protein with 63% amino acid sequence identity and a similar structural organization to GT1. GT2 mRNA is found at high levels in mouse skeletal muscle, heart, and adipose tissue, all of which exhibit insulin-stimulated glucose uptake. GT2 mRNA is absent from 3T3-L1 preadipocytes but is induced dramatically during differentiation into adipocytes. This increase in mRNA content correlates closely with the acquisition of insulin-stimulated glucose uptake. We propose that GT2 is an insulin-regulated glucose transporter.
We have investigated the regulation of mRNA synthesis during 3T3-adipocyte differentiation by measuring the transcription of specific genes in isolated preadipocyte and adipocyte nuclei. Transcription was assayed by hybridization of newly synthesized RNA to cDNA clones coding for glycerophosphate dehydrogenase (GPD), the induced protein of 13K which is shown here to be related to myelin protein P-2, the induced protein of 28K, actin, and two RNAs that are not developmentally regulated. Transcription of GPD and 13K was observed in adipocyte but not preadipocyte nuclei. Actin was transcribed in both types of nuclei but at a lower level in adipocytes. For most of the RNAs examined, there was a consistent relationship between amounts of nuclear transcription and the abundance of the corresponding cytoplasmic mRNA in adipocytes. However, 13K and 28K mRNAs are 10-100 times more abundant than would be predicted by their nuclear transcription alone. Preliminary mRNA turnover experiments in which 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole was used to inhibit mRNA synthesis suggest that these mRNAs are much more stable in the adipocyte cytoplasm than the other mRNAs examined. These results indicate that the transcription of specific genes is increased during adipocyte differentiation and suggest that other levels of control, particularly mRNA stability, may contribute to the relative abundance of certain developmentally-regulated mRNAs in adipocytes.
Differentiation of 3T3-L1 preadipocytes in culture is accompanied by alterations in the abundance of several mRNAs and by the appearance of many new adipocyte-specific mRNAs. To investigate the processes responsible for these alterations, the kinetics of accumulation of several specific mRNAs were compared with their respective rates of nuclear runoff transcription. The mRNAs for fructose-1,6-bisphosphate aldolase and an unidentified 4800-base mRNA increase in abundance only moderately (2-4-fold) during differentiation. Runoff transcription by nuclei isolated from 3T3-L1 cells during the course of differentiation revealed very little or no change in the rates of transcription of these mRNAs. Similar results were obtained for the beta, alpha-actin and beta-tubulin mRNAs where no difference in nuclear runoff transcription rates were observed even though a 2-fold decrease in the steady-state levels of these mRNAs accompanies differentiation. In contrast, the steady-state levels of mRNAs for 3T3-L1 P2 protein, an adipocyte homologue of myelin P2 protein, and an unidentified 5000-base mRNA increased dramatically (greater than 20-fold) during adipose conversion. These large increases in abundance were correlated with marked rises (greater than 10-fold) in nuclear runoff transcription rates for these mRNAs during differentiation of 3T3-L1 preadipocytes. No change in runoff transcription activity for these mRNAs was detected by nuclei from control nondifferentiating 3T3-C2 cells. These results strongly suggest that an increased rate of specific transcription is primarily responsible for the accumulation of these mRNAs during preadipocyte differentiation.
A density-shift method is described for analyzing insulin receptor synthesis and turnover in cultured cells labeled with "heavy" amino acids (2H, 13C, and 15N). Solubilized newly synthesized heavy and old "light" receptors are separated by isopycnic banding on CsCl gradients and then quantitated. Insulin receptor synthesis and turnover were studied by this technique in 3T3-L1 preadipocytes which undergo an increase in insulin binding capacity during differentiation. The results indicate that the increase in insulin binding capacity is a consequence of new receptor synthesis, that the insulin receptor has a relatively short half-life (6.7 hr), and that an increased rate of receptor synthesis contributes to the increase of insulin receptor level during differentiation.
3T3-L1 preadipocytes, cloned from 3T3 mouse embryo fibroblasts, differentiate in monolayer culture into cells with morphological and biochemical characteristics of adipocytes. Deposition of cytoplasmic triglyceride is associated with an increased lipogenic rate and a coordinate rise in the activities of many lipogenic enzymes (Mackall, J.C., Student, A.K., Polakis, S.E., and Lane, M.D. (1976) J. Biol. Chem. 251, 6462-6464). During differentiation induced by a 48-h treatment of postconfluent cells with methylisobutylxanthine, dexamethasone, and insulin, fatty acid synthetase activity increased to a level 19.5-fold higher than that of undifferentiated 3T3-L1 cells or nondifferentiating 3T3-C2 cells. The rate of [3H]leucine incorporation into immunoadsorbable fatty acid synthetase rose to a maximum and then declined to a new level 12.5-fold higher in differentiated than in undifferentiated 3T3-L1 cells. The kinetics of the changing [3H]leucine incorporation rate was reflected in the kinetics of the rise in fatty acid synthetase activity. The rate of degradation of fatty acid synthetase, determined by pulse-chase experiments, was unaffected by differentiation, the t1/2 remaining constant at 1.4 days. It is concluded that the higher level of fatty acid synthetase activity in differentiated 3T3-L1 cells can be attributed entirely to an increased rate of enzyme synthesis. The rate of total cellular protein synthesis also increases early early in differentiation, lending support to a model in which the synthesis of a large number of "differentiated proteins" is coordinately induced.
The CCAAT/enhancer-binding protein (C/EBP) alpha is a leucine zipper protein that is preferentially expressed in certain cell types, such as adipocytes and hepatocytes. Here we show that C/EBP alpha mRNA is translated into two major proteins, C/EBP-42 and C/EBP-30, that differ in their content of N-terminal amino acid sequences. These results are best explained by a ribosome-scanning mechanism in which a fraction of ribosomes ignore the first two AUGs and initiate translation at an AUG located 351 nt downstream of the first one. Because C/EBP-30, the translation product initiated at the third AUG, is devoid of the potent transcription-activation domain contained in C/EBP-42, the former protein stimulates transcription from the mouse albumin promoter much less efficiently than the latter. The gene encoding the liver-enriched transcriptional-activator protein LAP (C/EBP-beta) has also been shown to issue two proteins, LAP and the liver-enriched transcriptional-inhibitory protein LIP, with different transcription-activation potentials. The production of multiple proteins from a single mRNA is not only shared between different C/EBP family members but also appears to be conserved in vertebrate evolution.
The promoter region of the mouse CCAAT-Enhancer Binding Protein (C/EBP alpha) gene is capable of directing high levels of expression of reporter constructs in various cell lines, albeit even in cells that do not express their endogenous C/EBP alpha gene. To understand the molecular mechanisms underlying this ubiquitous expression, we have characterized the promoter region of the mouse C/EBP alpha gene by a variety of in vitro and in vivo methods. We show that three sites related in sequence to USF, BTE and C/EBP binding sites and present in promoter region -350/+3, are recognized by proteins from rat liver nuclear extracts. The sequence of the C/EBP alpha promoter that includes the USF binding site is also capable of forming stable complexes with purified Myc+Max heterodimers and mutation of this site drastically reduces transcription of C/EBP alpha promoter luciferase constructs both in liver and non liver cell lines. In addition, we identify three novel protein-binding sites two of which display similarity to NF-1 and a NF kappa B binding sites. The region located between nucleotides -197 and -178 forms several heat-stable complexes with liver nuclear proteins in vitro which are recognized mainly by antibodies specific for C/EBP alpha. Furthermore, transient expression of C/EBP alpha and to a lesser extent C/EBP beta expression vectors, results in transactivation of a cotransfected C/EBP alpha promoter-luciferase reporter construct. These experiments support the notion that the C/EBP alpha gene is regulated by C/EBP alpha but other C/EBP-related proteins may also be involved.
From the established mouse fibroblast line 3T3, we have isolated two clonal sublines that accumulate large amounts of triglyceride fat when the cells are in the resting state. The accumulation is reduced by lipolytic agents. Unless lipid accumulation is allowed to proceed too far, the fatty cells begin to grow again when they are transferred and in this way eliminate most of their lipid.
3T3-L1 adipoblasts that express large amounts of c-Myc cannot terminally differentiate, raising the possibility that Myc inhibits
the expression of genes that promote adipogenesis. The CCAAT/enhancer binding protein (C/EBPα) is induced during 3T3-L1 adipogenesis
when cells commit to the differentiation pathway. Transfection of 3T3-L1 adipoblasts with the gene that encodes C/EBPα caused
overt expression of the adipocyte morphology. Expression of Myc prohibited the normal induction of C/EBPα and prevented adipogenesis.
Enforced expression of C/EBPα overcame the Myc-induced block to differentiation. These results provide a molecular basis for
the regulation of adipogenesis and implicate Myc and C/EBPα as pivotal controlling elements.
This chapter discusses the mechanism of action of hormones and the regulation of cAMP -dependent protein kinases. It describes a study of3T3-L1 cell line that possesses the unusual property of differentiating from fibroblasts into adipocyte-like cells in vitro. The 3T3-L1 cell line was established and cloned from the original stock of Swiss mouse 3T3 cells. During exponential growth and the approach to confluence, 3T3-L1 cells appeared indistinguishable from other 3T3 cells with respect to growth rate, sensitivity to density-dependent inhibition, and morphological and biochemical properties. When the cells were maintained at confluence, some cells spontaneously entered a differentiation program that paralleled the development of mammalian adipose tissue. Their content of lipoprotein lipase and glutamine synthetase also changed dramatically. As mammalian fat cells responded to a wide variety of hormones, the 3T3-L1 cells also offered the potential for studying the development, regulation, and physiological coupling of hormone receptors and affecter systems. The activation of cAMP-dependent protein kinase and the phosphorylation and activation of hormone-sensitive triglyceride lipase subsequently lead to an enhanced rate of lipolysis. The chapter describes a study in which hormone sensitivity was evaluated by assessing adenylate cyclase activity in broken-cell preparations using homogenates or crude membrane fractions. Adenylate cyclase activity was measured by monitoring the conversion of [α-32P]-ATP to cyclic [32P] AMP. The accumulation of cAMP in intact cells was measured by radioimmunoassay. The incubation of cells with supraphysiological concentrations of insulin for 16 to 48 hours brought about a reversible 50% to 70% decrease in receptor number.
When cells of the established preadipose line 3T3-L1 enter a resting state, they accumulate triglyceride and convert to adipose cells. The adipose conversion is brought about by a large increase in the rate of triglyceride synthesis, as measured by the incorporation rate of labeled palmitate, acetate, and glucose. In a resting 3T3 subline which dose not undergo the adipose conversion, the rate of triglyceride synthesis from these precursors is very low, and similar to that of growing 3T3-L1 cells, before their adipose conversion begins. If 3T3-L1 cells incorporate bromodeoxyuridine during growth, triglyceride synthesis does not increase when the cells reach a stationary state, and triglycerides do not accumulate. As would be expected from their known actions on tissue adipose cells, lipogenic and lipolytic hormones and drugs affect the rate of synthesis and accumulation of triglyceride by 3T3-L1 cells, but in contrast to bromodeoxyuridine, these modulating agents do not seem to affect the proportion of cells which undergoes the adipose conversion. Insulin markedly increases the rate of synthesis and accumulation of triglyceride by fatty 3T3-L1 cells, and produces a related increase in cell protein content. Of 20 randomly selected clones isolated from the original 3T3 stock, 19 are able to convert to adipose cells. The probability of such a conversion varies greatly among the different clones, in most cases being much lower than for 3T3-L1; but once the conversion takes place, the adipose cells produced from all of the 19 clones appear similar. The adipose conversion would seem to depend on an on-off switch, which is on with a different probability in different clones. This probability is quasistably inherited by the clonal progeny.
Intact ribonucleic acid (RNA) has been prepared from tissues rich in ribonuclease such as the rat pancreas by efficient homogenization in a 4 M solution of the potent protein denaturant guanidinium thiocyanate plus 0.1 M 2-mercaptoethanol to break protein disulfide bonds. The RNA was isolated free of protein by ethanol precipitation or by sedimentation through cesium chloride. Rat pancreas RNA obtained by these means has been used as a source for the purification of alpha-amylase messenger ribonucleic acid.
When their growth is arrested in culture, susceptible 3T3 fibroblasts differentiate into adipose cells. Different clones form adipose cells with different frequency, depending upon the proportion of susceptible cells they contain. In cultures grown from small inocula, the fat cells appear in clusters formed by colonies of susceptible cells. Study of these clusters indicates the infrequent occurrence of cellular transitions from insusceptible to susceptible state. Beginning with a clone converting to adipose cells with a vary low frequency, it has been possible, by serial selection, to generate subclones which convert with a high frequency. This evolution is due to spontaneous heritable changes affecting susceptibility to the adipose conversion. Presumably, they involve the control of triglyceride synthesis. Early stages of the adipose conversion may be recognized in stained cultures. When triglyceride first begins to accumulate, the highly extended and flattened processes of the cells are probably similar to those of nonfatty cells in the same cultures. As the adipose conversion proceeds, the processes thicken and retract; the cells eventually acquire the rounded shape of the more mature adipose cells.
Classical embryology has provided a conceptual basis for our understanding of where muscle comes from. Histological and morphological studies of muscle fibre formation in the foetus and neonate have provided information on how muscle matures. More recent advances in molecular genetics have led to the characterization of muscle structural genes, and to the striking discovery of the MyoD family of myogenic regulatory factors. The question of how myogenesis takes place can now be formulated in terms of gene regulation, and molecular tools can be used to describe this process in the embryo and foetus.
Previous studies suggest that the CCAAT/enhancer-binding protein (C/EBP) functions in the coordinate expression of adipocyte genes during differentiation of 3T3-L1 preadipocytes. We sought to block expression of C/EBP selectively using a bovine papilloma virus (BPV) vector to direct transcription of a approximately 0.4-kb segment of C/EBP cDNA (in antisense orientation) containing translated sequence 5' to that encoding the basic and leucine zipper regions of the protein. Vector-directed expression of antisense C/EBP RNA in 3T3-L1 preadipocytes inhibited expression of C/EBP mRNA and protein, as well as several adipose-specific mRNAs, and also prevented cytoplasmic triglyceride accumulation. Rescue of the "adipocyte phenotype" was accomplished by transfection of cells expressing antisense RNA with a modified BPV vector that directs transcription of the complementary sense C/EBP RNA.
Significant progress has been made in defining the structural motifs that distinguish the muscle-specific from other basic helix-loop-helix proteins. Evidence is accumulating for multiple levels of regulation of the expression and action of the muscle basic helix-loop-helix factors.
The myoD gene converts many differentiated cell types into muscle. MyoD is a member of the basic-helix-loop-helix family of
proteins; this 68-amino acid domain in MyoD is necessary and sufficient for myogenesis. MyoD binds cooperatively to muscle-specific
enhancers and activates transcription. The helix-loop-helix motif is responsible for dimerization, and, depending on its dimerization
partner, MyoD activity can be controlled. MyoD senses and integrates many facets of cell state. MyoD is expressed only in
skeletal muscle and its precursors; in nonmuscle cells myoD is repressed by specific genes. MyoD activates its own transcription;
this may stabilize commitment to myogenesis.
The CCAAT-enhancer binding protein (C/EBP) has now been found to promote the terminal differentiation of adipocytes. During the normal course of adipogenesis, C/EBP expression is restricted to a terminal phase wherein proliferative growth is arrested, and specialized cell phenotype is first manifested. A conditional form of C/EBP was developed, making it feasible to test its capacity to regulate the differentiation of cultured adipocytes. Premature expression of C/EBP in adipoblasts caused a direct cessation of mitotic growth. Moreover, when abetted by the effects of three adipogenic hormones, C/EBP promoted terminal cell differentiation. Since C/EBP is expressed in a variety of tissues, it may have a fundamental role in regulating the balance between cell growth and differentiation in higher animals.
This paper presents the results of experiments that determine the chromosomal location of the mouse gene encoding CCAAT/enhancer binding protein (C/EBP) and measure its expression as a function of tissue type and temporal period of development in mice and rats. Three alleles of the C/EBP gene were identified according to restriction fragment length polymorphisms. The strain distribution pattern of the three alleles was determined in recombinant inbred mouse strains and compared to that of other mouse genes. These results mapped the gene to a position within 2.5 centimorgans (cM) of the structural gene encoding glucose phosphate isomerase on chromosome 7 of the mouse. The expression pattern of the C/EBP gene was studied by a combination of nucleic acid hybridization and antibody staining assays. High levels of C/EBP mRNA were observed in tissues known to metabolize lipid and cholesterol-related compounds at uncommonly high rates. These included liver, fat, intestine, lung, adrenal gland, and placenta. More detailed analysis of two of these tissues, liver and fat, showed that C/EBP expression was limited to fully differentiated cells. Moreover, analysis of the temporal pattern of expression of C/EBP mRNA in two tissues, liver and intestine, revealed a coordinated induction just prior to birth. These observations raise the possibility that the synthesis of C/EBP may be responsive to humoral factors and that modulation in C/EBP expression might mediate coordinated changes in gene expression that facilitate adaptive challenges met during development or during the fluctuating physiological states of adult life.
By analyzing the effects of single base substitutions around the ATG initiator codon in a cloned preproinsulin gene, I have identified ACCATGG as the optimal sequence for initiation by eukaryotic ribosomes. Mutations within that sequence modulate the yield of proinsulin over a 20-fold range. A purine in position -3 (i.e., 3 nucleotides upstream from the ATG codon) has a dominant effect; when a pyrimidine replaces the purine in position -3, translation becomes more sensitive to changes in positions -1, -2, and +4. Single base substitutions around an upstream, out-of-frame ATG codon affect the efficiency with which it acts as a barrier to initiating at the downstream start site for preproinsulin. The optimal sequence for initiation defined by mutagenesis is identical to the consensus sequence that emerged previously from surveys of translational start sites in eukaryotic mRNAs. The mechanism by which nucleotides flanking the ATG codon might exert their effect is discussed.
To identify and characterize specific mRNAs that increase in abundance during differentiation of mouse 3T3-L1 preadipocytes, a cDNA library was constructed from poly(A)+RNA isolated from differentiated 3T3-L1 adipocytes. Mixed probe isotope ratio selection and RNA blot analyses have identified several unique cDNA clones that represent mRNA species expressed either exclusively or at dramatically increased levels in differentiated cells. Further characterization of one such clone (pAL422) revealed that the corresponding mRNA, detectable only after differentiation, is approximately the same length (600 +/- 150 bases) as the cDNA insert (672 bases). The complete nucleotide sequence of the cDNA insert in pAL422 revealed a single long open reading frame that encodes a 132 amino acid polypeptide (the 422 protein) of 14.6 kDa. These and other results suggest that this cDNA may represent a nearly full-length copy of the mRNA. Computer-assisted analyses showed that the 422 protein shares 69% and 64% homology with myelin P2 proteins from rabbit and bovine peripheral nerves, respectively, as well as 23% and 30% homology with fatty-acid binding proteins from rat liver and intestine, respectively. Moreover, the mRNA hybrid selected by pAL422 DNA directs the in vitro translation of an approximately equal to 13 kDa polypeptide, and this protein is specifically immunoprecipitated by antiserum against bovine myelin P2. These observations strongly suggest that the 422 protein is a structural, and possibly functional, analog of myelin P2.
A technique for conveniently radiolabeling DNA restriction endonuclease fragments to high specific activity is described. DNA fragments are purified from agarose gels directly by ethanol precipitation and are then denatured and labeled with the large fragment of DNA polymerase I, using random oligonucleotides as primers. Over 70% of the precursor triphosphate is routinely incorporated into complementary DNA, and specific activities of over 10(9) dpm/microgram of DNA can be obtained using relatively small amounts of precursor. These "oligolabeled" DNA fragments serve as efficient probes in filter hybridization experiments.
During differentiation of 3T3-L1 preadipocytes into adipocytes, transcription of the C/EBP alpha (CCA-AT/enhancer binding protein alpha) gene is activated. The promoter of the C/EBP alpha gene contains a bipartite cis element with binding sites for C/EBP alpha undifferentiated protein (CUP) and an Sp1-like GT box binding protein. Binding of CUP to this element is markedly enhanced by its interaction with the Sp1-like protein. CUP, purified approximately 100,000-fold from HeLa cell nuclear extracts, appears to be composed of at least two types of subunit. Evidence is presented that a CUP-containing protein complex bridges between the CUP/Sp1-like GT box element and a downstream cis element, which contains a C/EBP binding site. During differentiation of 3T3-L1 preadipocytes into adipocytes, CUP activity or expression decreases as expression of C/EBP alpha increases. It is suggested that bridging by the CUP-containing protein complex may play a role in transcriptional regulation of the C/EBP alpha gene.
Full-length (42 kDa) CCAAT/enhancer binding protein alpha (C/EBP alpha) (p42) has been implicated in the transcriptional activation of adipocyte genes including the 422(aP2) and C/EBP alpha genes during differentiation of 3T3-L1 preadipocytes. We have identified a 30-kDa isoform (p30) of C/EBP alpha that is expressed by 3T3-L1 adipocytes, mouse adipose tissue, and rat liver. In vitro translation of wild-type C/EBP alpha mRNA or transient transfection with a wild-type C/EBP alpha vector gave rise to similar levels of p42 and p30. Mutational analysis revealed that p30 is an alternative translation product initiated at the third in-frame methionine codon of the C/EBP alpha message. p30C/EBP alpha binds to the C/EBP sites within and activates reporter gene expression driven by the 422(aP2) and C/EBP alpha gene promoters. Although transfection of 3T3-L1 preadipocytes with a strong p30C/EBP alpha expression vector is insufficient to induce differentiation, this vector advances the differentiation program. Unlike p42C/EBP alpha, which inhibits cell proliferation, p30C/EBP alpha is not antimitotic. Thus, the N-terminal 12-kDa segment of full-length C/EBP alpha contains an amino acid sequence necessary for antimitotic activity. During differentiation of 3T3-L1 preadipocytes and during hepatocyte development, the cellular p42C/EBP alpha/p30C/EBP alpha ratio changes, raising the possibility of a regulatory role.
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