Multiple omics analysis reveals the regulation of SIRT5 on mitochondrial function and lipid metabolism during the differentiation of bovine preadipocytes.

Preadipocyte differentiation represents a critical stage in adipogenesis, with mitochondria playing an undeniable pivotal role. Given the intricate interplay between transcription and metabolic signaling during adipogenesis, the regulation of sirtuin 5 (SIRT5) on mitochondrial function and lipid metabolism was revealed via multiple omics analysis. The findings suggest that SIRT5 plays a crucial role in promoting mitochondrial biosynthesis and maintaining mitochondrial function during preadipocyte differentiation. Moreover, SIRT5 modulates the metabolic levels of numerous bioactive substances by extensively regulating genes expression associated with differentiation, energy metabolism, lipid synthesis, and mitochondrial function. Finally, SIRT5 was found to suppress triacylglycerols (TAG) accumulation while enhancing the proportion and diversity of unsaturated fatty acids, and providing conditions for the expansion and stability of membrane structure during mitochondrial biosynthesis through numerous gene regulations. Our findings provide a foundation for the identification of crucial functional genes, signaling pathways, and metabolic substances associated with adipose tissue differentiation and metabolism.

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

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

Circular RNA- and microRNA-Mediated Post-Transcriptional Regulation of Preadipocyte Differentiation in Adipogenesis: From Expression Profiling to Signaling Pathway.

Adipogenesis is an indispensable cellular process that involves preadipocyte differentiation into mature adipocyte. Dysregulated adipogenesis contributes to obesity, diabetes, vascular conditions and cancer-associated cachexia. This review aims to elucidate the mechanistic details on how circular RNA (circRNA) and microRNA (miRNA) modulate post-transcriptional expression of targeted mRNA and the impacted downstream signaling and biochemical pathways in adipogenesis. Twelve adipocyte circRNA profiling and comparative datasets from seven species are analyzed using bioinformatics tools and interrogations of public circRNA databases. Twenty-three circRNAs are identified in the literature that are common to two or more of the adipose tissue datasets in different species; these are novel circRNAs that have not been reported in the literature in relation to adipogenesis. Four complete circRNA-miRNA-mediated modulatory pathways are constructed via integration of experimentally validated circRNA-miRNA-mRNA interactions and the downstream signaling and biochemical pathways involved in preadipocyte differentiation via the PPARγ/C/EBPα gateway. Despite the diverse mode of modulation, bioinformatics analysis shows that the circRNA-miRNA-mRNA interacting seed sequences are conserved across species, supporting mandatory regulatory functions in adipogenesis. Understanding the diverse modes of post-transcriptional regulation of adipogenesis may contribute to the development of novel diagnostic and therapeutic strategies for adipogenesis-associated diseases and in improving meat quality in the livestock industries.

A genome-wide landscape of mRNAs, lncRNAs, circRNAs and miRNAs during intramuscular adipogenesis in cattle.

BACKGROUND: Intramuscular preadipocyte differentiation plays a critical role in bovine intramuscular fat (IMF) deposition. However, the roles of different RNAs, including mRNAs, circRNAs, lncRNAs and miRNAs, in regulating the adipogenic differentiation of intramuscular preadipocytes remain largely unclear. RESULTS: In the present study, a whole transcriptome sequencing and analysis, including the analysis of mRNAs, circRNAs, lncRNAs and miRNAs, during different differentiation stages (0, 3, 6, and 9 d) of intramuscular preadipocytes from Qinchuan cattle was performed. All samples were prepared with 3 biological replicates. Here, a total of 27,153 mRNAs, 14,070 circRNAs, 7035 lncRNAs, and 427 miRNAs were annotated. Among them, we identified 4848 differentially expressed mRNAs (DEMs), 181 DE circRNAs (DECs), 501 DE lncRNAs (DELs) and 77 DE miRNAs (DEmiRs) between 0 d and other differentiation days (3, 6, and 9 d). GO and KEGG functional enrichment analyses showed that these differentially expressed genes were mainly enriched in cell differentiation, fat metabolism and adipogenesis-related pathways. Furthermore, weighted gene coexpression network analysis (WGCNA) and co-expression network analysis screened out multiple important mRNAs, circRNAs and lncRNAs related to intramuscular adipogenesis. Based on the competing endogenous RNA (ceRNA) regulatory mechanism, we finally identified 24 potential ceRNA networks and 31 potential key genes, including FOXO1/miR-330/circRNA2018/MSTRG.20301, GPAM/miR-27b/ciRNA489 and SESN3/miR-433/circRNA2627MSTRG.20342. CONCLUSIONS: This study provides new insights into the differential expression patterns of different transcript types (i.e., mRNAs, circRNAs, lncRNAs and miRNAs) in intramuscular preadipocyte differentiation. Our findings provide data support for studying the molecular mechanism of key mRNAs and noncoding RNAs in IMF deposition, and provide new candidate markers for the molecular breeding of beef cattle.

MiR-146a-5p, targeting ErbB4, promotes 3T3-L1 preadipocyte differentiation through the ERK1/2/PPAR-γ signaling pathway.

BACKGROUND: MicroRNAs (MiRNAs) are known to participate in preadipocyte differentiation, but the manner in which miR-146a-5p participates in this process remains unclear. This study was performed to examine the participation of miR-146a-5p in 3T3-L1 cell differentiation. MATERIAL AND METHODS: miR-146a-5p expression was upregulated and down-regulated to examine effects on 3T3-L1 cell differentiation. Bioinformatics analysis was performed to predict its target genes, and the signaling pathway it regulates was identified by qRT-PCR and Western blotting. The expression of miR-146a-5p in epididymal adipose tissue from obese mice and in an obese mouse adipose cell model was examined by qRT-PCR. RESULTS: 3T3-L1 cells differentiated into mature adipocytes successfully, as verified by increased areas of intracellular lipid droplets and elevated expression of mature adipocyte markers, and these cells had elevated miR-146a-5p expression. The intracellular lipid droplet and triglyceride contents and the expression of mature adipocyte markers were significantly increased in miR-146a-5p-overexpressing 3T3-L1 cells and markedly decreased in miR-146a-5p-inhibited 3T3-L1 cells. ErbB4 was a predicted target gene of miR-146a-5p. In miR-146a-5p-overexpressing 3T3-L1 cells, ErbB4 expression and ERK1/2 phosphorylation were decreased and the expression of PPAR-γ was increased; the opposite was observed in miR-146a-5p-inhibited 3T3-L1 cells. In addition, miR-146a-5p expression was significantly increased in the mouse epididymal adipose tissue and adipose cell model. CONCLUSIONS: Upregulated miR-146a-5p expression was related to 3T3-L1 cell differentiation. MiR-146a-5p promoted 3T3-L1 cell differentiation by targeting ErbB4 and via the ERK1/2/PPAR-γ signaling pathway.

Construction of miRNA-mRNA network in the differentiation of chicken preadipocytes.

1. MicroRNAs (miRNAs) play key roles in regulating lipid metabolism, adipogenesis and fat deposition in chicken. To date, there are only a few miRNAs that have been confirmed to be involved in chicken adipogenesis. The detailed mechanisms by which miRNAs regulate chicken adipogenesis remain largely unknown.2. To identify candidate miRNAs involved in chicken preadipocyte differentiation and explore potential mechanisms behind their functions, the following study analysed and identified miRNA and mRNA expression levels in undifferentiated and differentiated preadipocytes. Hub miRNA-mRNA interactions were identified, and the degree of connectivity of DE miRNAs in the network was established.3. A total of 145 DE miRNAs and 660 DE mRNAs were identified between undifferentiated and differentiated preadipocytes. An miRNA-mRNA network was constructed, including 29 DE miRNAs and 155 DE mRNAs, forming 470 miRNA-mRNA interactions. Functional enrichment analysis showed that DE mRNAs in the network were significantly enriched in 712 biological processes and 13 KEGG pathways. Based on the connectivity degree, five DE miRNAs with higher degrees miR-195-x, gga-miR-200a-3p, gga-miR-135a-5p, novel-m0067-5p and novel-m0270-5p were identified as hub miRNAs. Fifty-eight DE mRNAs interacted with these five hub miRNAs and formed 70 miRNA-mRNA interactions.4. This study constructed a miRNA-mRNA network associated with chicken preadipocyte differentiation and identified five hub miRNAs in the network. The findings identified a number of chicken adipogenic miRNAs and laid the foundation for elucidating the miRNA-mediated regulatory mechanism in chicken adipogenesis.

SIRT5 inhibits bovine preadipocyte differentiation and lipid deposition by activating AMPK and repressing MAPK signal pathways.

SIRT5 (sirtuin 5) is located in the mitochondria and plays an important role in biological processes such as maintaining the balance of lipid metabolism and promoting fatty acid oxidation mobilization. In this study, the bovine preadipocyte differentiation and obese mouse models were constructed; combined with transcriptome sequencing (RNA-seq) and morphological identification, the regulatory and molecular mechanisms underlying the effects of SIRT5 on bovine preadipocyte differentiation and lipid metabolism were studied. The results reveal that during the differentiation of preadipocytes, SIRT5 inhibited the expression of key genes that promote lipid formation and differentiation in fatty acid biosynthesis and PPAR pathways. SIRT5 significantly activated the adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathway and repressed the mitogen-activated protein kinase (MAPK) pathway. Eventually, SIRT5 significantly inhibited the differentiation of bovine preadipocytes and simultaneously inhibited lipid synthesis and lipid deposition in adipocytes. The verification experiments performed using obese mice also yielded consistent results in vivo.

SIRT6 cooperates with SIRT5 to regulate bovine preadipocyte differentiation and lipid metabolism via the AMPKα signaling pathway.

Preadipocyte differentiation and lipid synthesis are critical steps for intramuscular fat (IMF) deposition and lipid metabolism homeostasis. IMF content of beef not only determines the ratio of muscle to adipose, but also determines the beef quality, flavor, and sensory characteristics. Maintaining lipid metabolism homeostasis is the key means of preventing and treating diabetes, obesity, and other metabolic diseases. SIRT6, which is an ADP-ribosyltransferase and NAD+-dependent deacetylase of acetyl and long-chain fatty acyl groups, playing central roles in lipid and glucose metabolism, is closely related to the occurrence of diabetes and obesity caused by overnutrition and aging. This study was based on bovine preadipocyte differentiation and an obese mice model, and comprehensively used transcriptome sequencing (RNA-seq) and morphological identification methods to explore the effects of inhibition of SIRT6 on differentiation and lipid synthesis, and related molecular mechanisms. Additionally, the feedback synergistic regulation of SIRT5 and SIRT6 on differentiation and lipid deposition was analyzed. The results showed that in the differentiation process of bovine preadipocytes, inhibition of SIRT5 significantly promoted SIRT6 expression. In addition, SIRT6 inhibited bovine preadipocyte differentiation and lipid synthesis, cooperating with SIRT5 to decrease lipid deposition, and repressed cell cycle arrest of preadipocytes. Moreover, in vivo verification experiments also obtained consistent results. Furthermore, SIRT6 inhibited preadipocyte differentiation and lipid deposition by activating the adenosine monophosphate activated protein kinase alpha (AMPKα) pathway. The above results provided a novel approach for understanding the functions of SIRT6 in regulating bovine adipocyte differentiation and lipid metabolism, as well as a new target for the treatment of diabetes and obesity in a clinical setting.

Stearoyl-CoA Desaturase-2 in Murine Development, Metabolism, and Disease.

Stearoyl-CoA Desaturase-2 (SCD2) is a member of the Stearoyl-CoA Desaturase (SCD) family of enzymes that catalyze the rate-limiting step in monounsaturated fatty acid (MUFA) synthesis. The MUFAs palmitoleoyl-CoA (16:1n7) and oleoyl-CoA (18:1n9) are the major products of SCD2. Palmitoleoyl-CoA and oleoyl-CoA have various roles, from being a source of energy to signaling molecules. Under normal feeding conditions, SCD2 is ubiquitously expressed and is the predominant SCD isoform in the brain. However, obesogenic diets highly induce SCD2 in adipose tissue, lung, and kidney. Here we provide a comprehensive review of SCD2 in mouse development, metabolism, and various diseases, such as obesity, chronic kidney disease, Alzheimer's disease, multiple sclerosis, and Parkinson's disease. In addition, we show that bone mineral density is decreased in SCD2KO mice under high-fat feeding conditions and that SCD2 is not required for preadipocyte differentiation or the expression of PPARγ in vivo despite being required in vitro.

Effects of puerarin on the AKT signaling pathway in bovine preadipocyte differentiation.

OBJECTIVE: Puerarin has the potential of regulating the differentiation of preadipocytes, but its mechanism of action has not yet been elucidated. Adipocytes found in adipose tissue, the main endocrine organ, are the main sites of lipid deposition, and are widely used as a cell model in the study of in vitro fat deposition. This study aimed to investigate the effects of puerarin on adipogenesis in vitro. METHODS: Puerarin was added to the culture medium during the process of adipogenesis. The proliferation and differentiation of bovine preadipocytes was measured through cell viability and staining with Oil Red O. The content of triacylglycerol (TG) was measured using a triglyceride assay kit. The mRNA and protein expression levels of adipogenic genes, peroxisome proliferator-activated receptor-γ (PPARγ) and CCAAT/enhancer-binding protein-α (C/EBPα), were measured using quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting, respectively. RESULTS: The addition of puerarin significantly increased adipogenesis of bovine preadipocytes and enhanced the mRNA and protein level expression of PPARγ (p<0.01). The expression of P-Akt increased after adipogenic hormonal induction, whereas puerarin significantly increased PPARγ expression by promoting the Akt signaling component, P-Akt. The mechanism of adipogenesis was found to be related to the phosphorylation level of Ser473, which may activate the downstream signaling of the Akt pathway. CONCLUSION: Puerarin was able to promote the differentiation of preadipocytes and improve fat deposition in cattle. The mechanism of adipogenesis was found to be related to the phosphorylation level of Ser473.

Dynamics of transcriptome changes during subcutaneous preadipocyte differentiation in ducks.

BACKGROUND: Pekin duck is an important animal model for its ability for fat synthesis and deposition. However, transcriptional dynamic regulation of adipose differentiation driven by complex signal cascades remains largely unexplored in this model. This study aimed to explore adipogenic transcriptional dynamics before (proliferation) and after (differentiation) initial preadipocyte differentiation in ducks. RESULTS: Exogenous oleic acid alone successfully induced duck subcutaneous preadipocyte differentiation. We explored 36 mRNA-seq libraries in order to study transcriptome dynamics during proliferation and differentiation processes at 6 time points. Using robust statistical analysis, we identified 845, 652, 359, 2401 and 1933 genes differentially expressed between -48 h and 0 h, 0 h and 12 h, 12 h and 24 h, 24 h and 48 h, 48 h and 72 h, respectively (FDR < 0.05, FC > 1.5). At the proliferation stage, proliferation related pathways and basic cellular and metabolic processes were inhibited, while regulatory factors that initiate differentiation enter the ready-to-activate state, which provides a precondition for initiating adipose differentiation. According to weighted gene co-expression network analysis, pathways positively related to adipogenic differentiation are significantly activated at the differentiation stage, while WNT, FOXO and other pathways that inhibit preadipocyte differentiation are negatively regulated. Moreover, we identified and classified more than 100 transcription factors that showed significant changes during differentiation, and found novel transcription factors that were not reported to be related to preadipoctye differentiation. Finally, we manually assembled a proposed regulation network model of subcutaneous preadipocyte differentiation base on the expression data, and suggested that E2F1 may serve as an important link between the processes of duck subcutaneous preadipocyte proliferation and differentiation. CONCLUSIONS: For the first time we comprehensively analyzed the transcriptome dynamics of duck subcutaneous preadipocyte proliferation and differentiation. The current study provides a solid basis for understanding the synthesis and deposition of subcutaneous fat in ducks. Furthermore, the information generated will allow future investigations of specific genes involved in particular stages of duck adipogenesis.

Identification and screening of circular RNAs during adipogenic differentiation of ovine preadipocyte by RNA-seq.

Circular RNAs (circRNAs) are a class of non-coding RNAs that play important roles in preadipocyte differentiation and adipogenesis. However, little is known about genome-wide identification, expression profile, and function of circRNAs in sheep. To investigate the role of circRNAs during ovine adipogenic differentiation, the subcutaneous adipose tissue of Tibetan rams was collected in June 2022. Subsequently, the preadipocytes were immediately isolated from collected adipose tissue and then induced to begin differentiation. The adipocytes samples cultured on days 0, 2, and 8 of preadipocytes differentiation were used to perform RNA sequencing (RNA-seq) analysis to construct the expression profiles of circRNAs. Subsequently, the function of differentially expressed circRNAs was investigated by performing the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of their parent genes. Finally, a circRNAs-miRNAs-mRNAs network involved in adipogenic differentiation was been analyzed. As a result, a total of 6,449 candidate circRNAs were identified in ovine preadipocytes. Of these circRNAs identified, 63 candidate circRNAs were differentially expressed among the three differentiation stages and their parent genes were mainly enriched in acetyl-CoA metabolic process, positive regulation of lipid biosynthetic process, positive regulation of steroid biosynthetic process, and focal adhesion pathway (P < 0.05). Based on a circRNAs-miRNAs-mRNAs regulatory network constructed, circ_004977, circ_006132 and circ_003788 were found to function as competing endogenous RNAs (ceRNAs) to regulate ovine preadipocyte differentiation and lipid metabolism. The results provide an improved understanding of functions and molecular mechanisms of circRNAs underlying ovine adipogenesis in sheep.

The moderate fat deposition contributes to improve mutton quality, which is associated with the differentiation of preadipocytes. To investigate roles of circular RNAs (circRNAs) in preadipocyte differentiation, we identified circRNAs on days 0, 2, and 8 of preadipocytes differentiation and compared the expression profile of circRNAs at different adipogenic differentiation stages. A total of 6,449 candidate circRNAs were identified, among which 63 candidate circRNAs were differentially expressed among the three differentiation stages. The parent genes of differentially expressed circRNAs were enriched in several biological process and pathways related to lipid metabolism and synthesis. In addition, several circRNAs may regulate ovine preadipocyte differentiation by interacting with microRNAs (miRNAs). The results reveal the potential roles of circRNAs in adipogenic differentiation of sheep.

miR-26a-5p Regulates Adipocyte Differentiation via Directly Targeting ACSL3 in Adipocytes.

Preadipocytes become mature adipocytes after proliferation and differentiation, and although many genes and microRNAs have been identified in intramuscular fat, their physiological function and regulatory mechanisms remain largely unexplored. miR-26a-5p has been reported to be related to fat deposition, but its effect on porcine preadipocyte differentiation has not been explored. In this study, bioinformatics analysis and luciferase reporter assay identified that miR-26a-5p binds to the 3'UTR of Acyl-CoA synthetase long-chain family member 3 (ACSL3) mRNA. The model for porcine intramuscular preadipocyte differentiation was established to explore the function of miR-6a-5p-ACSL3 on adipocyte differentiation. ACSL3 knockdown markedly reduced the triglycerides (TG) content of cells, as well as the mRNA levels of adipogenic marker genes (PPAR-γ and SREBP-1c). The number of lipid droplets in cells transfected with a miR-26a-5p mimic is significantly reduced, consistent with ACSL3 knockdown results, while the miR-26a-5p inhibitor resulted in opposite results. Taken together, miR-26a-5p is a repressor of porcine preadipocyte differentiation and plays a vital role in ACSL3-mediated adipogenesis.

miR-33a Inhibits the Differentiation of Bovine Preadipocytes through the IRS2-Akt Pathway.

Several microRNAs (miRNAs) are known to participate in adipogenesis. However, their role in this process, especially in the differentiation of bovine preadipocytes, remains to be elucidated. This study was intended to clarify the effect of microRNA-33a (miR-33a) on the differentiation of bovine preadipocytes by cell culture, real-time fluorescent quantitative PCR (qPCR), Oil Red staining, BODIPY staining, and Western blotting. The results indicate that overexpression of miR-33a significantly inhibited lipid droplet accumulation and decreased the mRNA and protein expression of adipocyte differentiation marker genes such as peroxisome proliferator-activated receptor gamma (PPARγ), sterol regulatory element-binding protein 1 (SREBP1), and fatty acid-binding protein 4 (FABP4). In contrast, the interference expression of miR-33a promoted lipid droplet accumulation and increased the expression of marker genes. Additionally, miR-33a directly targeted insulin receptor substrate 2 (IRS2) and regulated the phosphorylation level of serine/threonine kinase (Akt). Furthermore, miR-33a inhibition could rescue defects in the differentiation of bovine preadipocytes and the Akt phosphorylation level caused by small interfering IRS2 (si-IRS2). Collectively, these results indicate that miR-33a could inhibit the differentiation of bovine preadipocytes, possibly through the IRS2-Akt pathway. These findings might help develop practical means to improve the quality of beef.

The MicroRNA-signaling-peroxisome proliferator-activated receptor gamma connection in the modulation of adipogenesis: bioinformatics projection on chicken.

Meat quality and nutritional value hinge on many factors, including fat content in the adipose tissue. In the adipogenesis process, stem cells are first committed to become preadipocytes, followed by preadipocyte differentiation. In the later event, peroxisome proliferator activated receptor gamma (PPARγ) is the gateway through which adipogenic genes are activated. This review focuses on clarifying the effects of signaling transduction on PPARγ that have been experimentally established in the adipogenesis process. Furthermore, microRNA (miRNA) are validated to target the signaling factors and impact adipogenesis are appraised to establish the miRNA-signaling-PPARγ regulatory connection in adipogenesis. As opposed to red meat, chicken is white meat, which is increasingly appreciated for health and environmental reasons. Most works reported on the miRNA-signaling-PPARγ network in adipogenesis used human and other laboratory and farm animal models. We show here that database interrogation and bioinformatics analysis may be applied to extrapolate reported findings to chicken based on evolutionary conservation. Understanding molecular modulation of adipogenesis may contribute to clinical treatment of lipid disorders and obesity in humans, and improved meat quality and commercial value in chicken farming, and possibly in the creation of artificial meat.

Differential regulatory roles of microRNAs during intramuscular adipogenesis in Chinese Guizhou Congjiang Xiang pigs.

Intramuscular fat development is regulated by a series of complicated processes, with non-coding RNA (ncRNA) such as microRNA (miRNA) having a critical role during intramuscular preadipocyte proliferation and differentiation in pigs. In the present study, the miRNA expression profiles of intramuscular preadipocytes from the longissimus dorsi muscle of Chinese Guizhou Congjiang Xiang pigs were detected by RNA-seq during various differentiation stages, namely, day 0 (D0), day 4 (D4), and day 8 (D8). A total of 67, 95, and 16 differentially expressed (DE) miRNAs were detected between D4 and D0, D8 and D0, and D8 and D4, respectively. According to gene ontology and Kyoto Encyclopedia of Genes analysis, target genes of DE miRNAs were enriched in categories and pathways related to lipid metabolic process, lipid biosynthetic process, as well as the PI3K-Akt, AMPK, and MAPK signalling pathways. Notably, miR-148a-3p was differentially expressed, with highest expressed abundance in D0, D4, and D8. Overexpression of miR-148a-3p in intramuscular preadipocytes increased cell proliferation and differentiation, and decreased apoptosis, in comparison to the knockdown of miR-148a-3p in intramuscular preadipocytes. Luciferase activity assays, quantitative polymerase-chain reaction, and western blot analysis confirmed that miR-148a-3p regulated adipogenesis by repressing PPARGC1A expression. Accordingly, the effect of miR-148a-3p mimic was attenuated by overexpression of PPARGC1A intramuscular preadipocytes. Furthermore, miR-148a-3p promoted intramuscular preadipocyte differentiation by inhibiting the AMPK/ACC/CPT1C signalling pathway. Taken together, we identified expression profiles of miRNAs in intramuscular preadipocytes and determined that miR-148a-3p acted as a promoter of adipogenesis.

Equisetin is an anti-obesity candidate through targeting 11ß-HSD1.

Obesity is increasingly prevalent globally, searching for therapeutic agents acting on adipose tissue is of great importance. Equisetin (EQST), a meroterpenoid isolated from a marine sponge-derived fungus, has been reported to display antibacterial and antiviral activities. Here, we revealed that EQST displayed anti-obesity effects acting on adipose tissue through inhibiting adipogenesis in vitro and attenuating HFD-induced obesity in mice, doing so without affecting food intake, blood pressure or heart rate. We demonstrated that EQST inhibited the enzyme activity of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), a therapeutic target of obesity in adipose tissue. Anti-obesity properties of EQST were all offset by applying excessive 11ß-HSD1's substrates and 11ß-HSD1 inhibition through knockdown in vitro or 11ß-HSD1 knockout in vivo. In the 11ß-HSD1 bypass model constructed by adding excess 11ß-HSD1 products, EQST's anti-obesity effects disappeared. Furthermore, EQST directly bond to 11ß-HSD1 protein and presented remarkable better intensity on 11ß-HSD1 inhibition and better efficacy on anti-obesity than known 11ß-HSD1 inhibitor. Therefore, EQST can be developed into anti-obesity candidate compound, and this study may provide more clues for developing higher effective 11ß-HSD1 inhibitors.

A-FABP-PTEN/AKT Regulates Insulin Resistance in Preadipocyte Cell 3T3-L1 Cells.

OBJECTIVE: The purpose of this study was to explore the regulation of A-FABP-PTEN/AKT on insulin resistance in preadipocyte 3T3-L1 cell. METHODS: siRNA interference method was used to knock-down the A-FABP expression in 3T3-L1 cells. The cell proliferation was detected by oil-O staining and MTT. The protein and mRNA expression levels of A-FABP, PTEN and AKT were detected by Western blot and qPCR. RESULTS: Inhibition of A-FABP expression increased cell proliferation activity of the 3T3-L1 cells. Moreover, siRNA3 significantly reduced A-FABP mRNA expression compared with siRNA1 and siRNA2 (P<0.05). The A-FABP mRNA level was significantly increased in the induced 3T3-L1 cells, while the PTEN mRNA expression was significantly decreased (P<0.05). Inhibition of A-FABP can significantly increase the PTEN mRNA expression in the process of induced 3T3-L1 cells (P<0.05). Overexpression of A-FABP can also increase the PTEN mRNA expression in the process of 3T3-L1 cell proliferation (P<0.05). Furthermore, the protein expression levels of PTEN and p-AKT expression were not changed in the process of 3T3-L1 cell proliferation with or without A-FABP interference (P>0.05). However, inhibition of A-FABP significantly increased the PTEN protein expression and reduced the p-AKT protein expression in the induced 3T3-L1 cells. CONCLUSION: Our finding suggested that A-FABP can directly inhibit the phosphorylation of AKT and increase the PTEN expression in the process of normal adipocyte differentiation, which speculated that A-FABP played a crucial role by adjusting the AKT activity in the process of adipocyte differentiation.

Identification of Potential Candidate Genes From Co-Expression Module Analysis During Preadipocyte Differentiation in Landrace Pig.

Preadipocyte differentiation plays an important role in lipid deposition and affects fattening efficiency in pigs. In the present study, preadipocytes isolated from the subcutaneous adipose tissue of three Landrace piglets were induced into mature adipocytes in vitro. Gene clusters associated with fat deposition were investigated using RNA sequencing data at four time points during preadipocyte differentiation. Twenty-seven co-expression modules were subsequently constructed using weighted gene co-expression network analysis. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses revealed three modules (blue, magenta, and brown) as being the most critical during preadipocyte differentiation. Based on these data and our previous differentially expressed gene analysis, angiopoietin-like 4 (ANGPTL4) was identified as a key regulator of preadipocyte differentiation and lipid metabolism. After inhibition of ANGPTL4, the expression of adipogenesis-related genes was reduced, except for that of lipoprotein lipase (LPL), which was negatively regulated by ANGPTL4 during preadipocyte differentiation. Our findings provide a new perspective to understand the mechanism of fat deposition.

KLF2 Inhibits Chicken Preadipocyte Differentiation at Least in Part via Directly Repressing PPARγ Transcript Variant 1 Expression.

Peroxisome proliferator-activated receptor gamma (PPARγ) is the master regulatory factor of preadipocyte differentiation. As a result of alternative splicing and alternative promoter usage, PPARγ gene generates multiple transcript variants encoding two protein isoforms. Krüppel-like factor 2 (KLF2) plays a negative role in preadipocyte differentiation. However, its underlying mechanism remains incompletely understood. Here, we demonstrated that KLF2 inhibited the P1 promoter activity of the chicken PPARγ gene. Bioinformatics analysis showed that the P1 promoter harbored a conserved putative KLF2 binding site, and mutation analysis showed that the KLF2 binding site was required for the KLF2-mediated transcription inhibition of the P1 promoter. ChIP, EMSA, and reporter gene assays showed that KLF2 could directly bind to the P1 promoter regardless of methylation status and reduced the P1 promoter activity. Consistently, histone modification analysis showed that H3K9me2 was enriched and H3K27ac was depleted in the P1 promoter upon KLF2 overexpression in ICP1 cells. Furthermore, gene expression analysis showed that KLF2 overexpression reduced the endogenous expression of PPARγ transcript variant 1 (PPARγ1), which is driven by the P1 promoter, in DF1 and ICP1 cells, and that the inhibition of ICP1 cell differentiation by KLF2 overexpression was accompanied by the downregulation of PPARγ1 expression. Taken together, our results demonstrated that KLF2 inhibits chicken preadipocyte differentiation at least inpart via direct downregulation of PPARγ1 expression.