Porcine Preadipocytes Research Articles

Identification and Analysis of lncRNAs by Whole-Transcriptome Sequencing in Porcine Preadipocytes Induced by BMP2

Bone morphogenetic protein 2 (BMP2) can mediate the signaling of R-Smads and regulate different biological functions, including adipocyte differentiation. Long noncoding RNAs (lncRNAs) can be involved in many important biological processes, including fat metabolism, as miRNA sponges. This study aimed to investigate the molecular mechanism of fat deposition and to provide useful information for the prevention and treatment of lipid-related diseases. lncRNA sequencing was performed to compare and analyze, for the first time, the expression of lncRNAs in BMP2-induced and non-BMP2-induced preadipocytes from Junmu1 pigs. In addition, functional annotation and enrichment analysis of differentially expressed lncRNA target genes were carried out. lncRNAs and mRNAs were compared and analyzed. lncRNAs were identified that may regulate adipogenesis and lipid metabolism. The results give a theoretical basis for further studies on fat deposition mechanisms and provide potential therapeutic targets for metabolic diseases.

PRDM16 Represses the Pig White Lipogenesis through Promoting Lipolysis Activity.

The positive regulatory domain containing 16 (PRDM16) gene is a dominant transcriptional regulator that favors the “browning” of white adipocytes in rodents. Since the “browning” of white fat is important in pig in terms of producing heat fighting against cold environment, avoiding obesity, and improving meat quality, understanding the critical role that PRDM16 gene played in pig adipose “browning” and energy metabolism is of great significance. However, the constitution of pig fat differs a lot from rodents and human as they do not have brown adipose tissue (BAT) even in the newborn piglets. In this study, we isolated porcine primary preadipocytes and investigated the function of PRDM16 during preadipocytes differentiation. Our results showed that overexpression of the PR domain of PRDM16 repressed the differentiation of porcine preadipocytes, indicated by oil red O staining and the deposition of the triglyceride. Overexpression of the PR domain significantly increased the level of lipolysis and mitochondrial oxidative capacity detected by Western blotting during differentiation. Furthermore, we purified the protein coded by the PR domain and demonstrated that this protein has the H3K9me1 methyltransferase activity. In conclusion, the PR domain of the porcine PRDM16 gene repressed the mature of the porcine preadipocytes by promoting its oxidative activity.

Myostatin/SMAD4 signaling-mediated regulation of miR-124-3p represses glucocorticoid receptor expression and inhibits adipocyte differentiation.

The mechanism of adipocyte regulation specifically in muscle and the influence of muscle tissue on intramuscular fat deposition are unknown. Our previous studies have shown that myostatin, a myokine, is involved in inhibiting the differentiation of preadipocytes and may be a potential regulator that affects the deposition of intramuscular fat. Myostatin inhibited adipogenesis by downregulating the expression of glucocorticoid receptor (GR) in porcine preadipocytes. However, the mechanism of regulation is not yet clear. In this study, we demonstrate microRNA (miR-124-3p) mediates regulation of GR by myostatin. We found that miR-124-3p can target GR 3'-UTR and negatively regulate GR expression. We demonstrate that overexpression of miR-124-3p can reduce differentiation of 3T3-L1 cells by inhibiting GR, and vice versa. The expression of miR-124-3p was upregulated in 3T3-L1 cells treated with myostatin. Further study revealed that myostatin also promotes the expression of SMAD4 and its transfer and localization to the nucleus. The activated myostatin/SMAD4 signal promotes the expression of miR-124-3p by SMAD4 binding to the promoter region of miR-124-3p. When myostatin or SMAD4 activity is inhibited, the upregulation of miR-124-3p is also inhibited. All of these findings suggested that myostatin could inhibit adipogenic differentiation of 3T3-L1 cells by activating miR-124-3p to inhibit GR. These data may provide an explanation for how myostatin signaling affects intramuscular fat deposition in a tissue-specific manner.

Loss of m6 A on FAM134B promotes adipogenesis in porcine adipocytes through m6 A-YTHDF2-dependent way.

N6 -methyladenosine (m6 A) mRNA modification plays an important role in adipogenesis, but its role on single gene remains unexplored. Family with Sequence Similarity 134, Member B (FAM134B) is a cis-Golgi transmembrane protein that known to be necessary for the long-term survival of nociceptive and autonomic ganglion neurons. Recent work has shown that FAM134B plays a pivotal role in lipid homeostasis and was identified as its significant m6 A level difference between Chinese local Jinhua pigs and Landrace through RNA-sequence. Here, we construct the non-m6 A FAM134B coding sequence (CDS) plasmid (FAM134B-MUT) and found one important m6 A site on its CDS. Expression of FAM134B-MUT was more effective in promoting porcine preadipocytes adipogenic differentiation and lipid deposition than wild-type FAM134B (FAM134B-WT) both in early and ultimate differentiation stage. FAM134B-MUT functions better in promoting fat deposition by upregulating peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein (C/EBPα) level. The m6 A reader protein YTH m6 A RNA binding protein 2 (YTHDF2) interacts with FAM134B mRNA and down regulated its protein level. These results demonstrate that FAM134B was the target of YTHDF2, which may recognize and binds the m6 A site of FAM134B to reduce its mRNA lifetime and reduce its protein abundance. © 2018 IUBMB Life, 71(5):580-586, 2019.

MiR-181a regulate porcine preadipocyte differentiation by targeting TGFBR1

miRNAs have been shown to regulate a variety of biological process. It has been shown that miR-181a regulates porcine adipogenesis by targeting Tumor Necrosis Factor-α (TNF-α), but the overall functions of miR-181a in porcine preadipocyte differentiation remain unclear. This study aimed to explore the functions of miR-181a in porcine preadipocyte differentiation via the TGFβ/Smad pathway. The TargetScan program was used to predict miRNAs targeting TGFBR1, and miR-181a was selected as a candidate. To investigate the functions of miR-181a, miRNA mimics and inhibitors were used to overexpress or knockdown miR-181a, respectively. RT-qPCR and Western blotting were used to measure the expression of aP2, PPARγ, C/EBPα and TGFBR1 in porcine preadipocytes. Lipid accumulation and adipocyte apoptosis were detected using Oil Red O staining and flow cytometry, respectively. Taken together, our results indicated that miR-181a promoted porcine preadipocyte differentiation by directly targeting TGFBR1.

Grape seed procyanidin extract inhibits adipogenesis and stimulates lipolysis of porcine adipocytes in vitro.

The objective of this article was to evaluate in vitro effect of grape seed procyanidin extract (GSPE) on differentiation, proliferation, and lipolysis of porcine adipocytes, providing a molecular basis for the use of GSPE in pig fat regulation. Primary preadipocytes isolated from subcutaneous adipose tissue of pigs were used as the in vitro cell model. Treatment of GSPE repressed preadipocyte differentiation, as evidenced by reduced lipid accumulation, decreased mRNA expressions of peroxisome proliferator-activated receptor gamma (PPARγ) and fatty acid-binding protein 4 (FABP4), as well as enhanced expressions of preadipocyte factor-1. Activity of glycerol-3-phosphate dehydrogenase (GPDH), one of the most important enzymes in the pathway for triacylglycerol biosynthesis, was also decreased. Furthermore, GSPE could suppress preadipocyte proliferation by inducing G0/G1 cell cycle arrest and cell apoptosis. In porcine mature adipocytes, treatment with GSPE attenuated lipid content and GPDH activity, and the release of both free fatty acid and glycerol were enhanced; mRNA expressions of key lipolytic transcription factors, including hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), were elevated in GSPE-treated adipocytes. In summary, our results suggest GSPE inhibits porcine preadipocyte differentiation and proliferation and stimulates lipolysis of mature adipocytes, thus providing novel insights for further exploring the use of GSPE as a fat accumulation inhibitor.

MAT2A promotes porcine adipogenesis by mediating H3K27me3 at Wnt10b locus and repressing Wnt/β-catenin signaling

Methionine adenosyltransferase (MAT) is a critical biological enzyme and that can catalyze L-met and ATP to form S-adenosylmethionine (SAM), which is acted as a biological methyl donor in transmethylation reactions involving histone methylation. However, the regulatory effect of methionine adenosyltransferase2A (MAT2A) and its associated methyltransferase activity on adipogenesis is still unclear. In this study, we investigate the effect of MAT2A on adipogenesis and its potential mechanism on histone methylation during porcine preadipocyte differentiation. We demonstrated that overexpression of MAT2A promoted lipid accumulation and significantly up-regulated the levels of adipogenic marker genes including PPARγ, SREBP-1c, and aP2. Whereas, knockdown of MAT2A or inhibition MATII enzyme activity inhibited lipid accumulation and down-regulated the expression of the above-mentioned genes. Mechanistic studies revealed that MAT2A interacted with histone-lysine N-methyltransferase Ezh2 and was recruited to Wnt10b promoter to repress its expression by promoting H3K27 methylation. Additionally, MAT2A interacted with MafK protein and was recruited to MARE element at Wnt10b gene. The catalytic activity of MAT2A as well as its interacting factor-MAT2B, was required for Wnt10b repression and supplying SAM for methyltransferases. Moreover, MAT2A suppressed Wnt10b expression and further inhibited Wnt/β-catenin signaling to promote adipogenesis.

MicroRNA-130b attenuates dexamethasone-induced increase of lipid accumulation in porcine preadipocytes by suppressing PPAR-γ expression

In this study, two experiments were conducted to determine the role of miR-130b in dexamethasone (DEX)-induced lipid accumulation. Porcine preadipocytes were treated with 10−6 M DEX for 48 h to investigate effects of DEX in lipid accumulation. Next, in order to illustrate the regulatory role of miR-130b on lipid accumulation induced by DEX, miRNA scrambled control (miR-SC), miR-130b overexpression plasmid and miR-130b inhibitor were respectively transfected into porcine preadipocytes at 24 h before DEX treatment for 48 h (miR-SC-DEX, miR-130b-DEX and miR-130b-inhibitor-DEX). Results showed that 10−6 M DEX significantly increased TG concentration and expression of miR-130b as well as its target gene peroxisome proliferator-activated receptor-γ (PPAR-γ). Dual-luciferase reporter assays indicated that PPAR-γ expression was negatively regulated by miR-130b, while this effect was abolished with cotransfection of miR-130b and miR-130b inhibitor. In addition, miR-130b-DEX did not change cell proliferation but significantly decreased TG concentration and PPAR-γ expression compared to miR-SC-DEX cells, while miR-130b-inhibitor-DEX cells presented opposite results. Furthermore, miR-130b-DEX significantly reduced expression of PPAR-γ downstream factor perilipin 1 as well as adipogenesis genes fatty acid synthase, acetyl coenzyme A carboxylase, 11β hydroxysteroid dehydrogenase type 1 and fat mass and obesity-associated gene, whereas expression as well as enzyme activity of adipose triglyceride lipase and hormone-sensitive lipase were greatly increased. Overall, these results clarified the role of miR-130b in DEX-induced increase of lipid accumulation in porcine preadipocytes, suggesting that miR-130b might be deemed as a novel potential therapeutic target for DEX-induced increase of lipid accumulation, and consequently provide new insights in obesity control.

MiR-429 Inhibits Differentiation and Promotes Proliferation in Porcine Preadipocytes.

MicroRNAs (miRNAs) are crucial regulatory molecules for adipogenesis. They contribute to the controlling of proliferation and differentiation of preadipocytes. Previous studies revealed an important role of miR-429 in cell invasion, migration, and apoptosis. Our previous work has shown that the expression of miR-429 in subcutaneous fat can be observed in newly born (3-day-old) Rongchang piglets rather than their adult counterparts (180-day-old). This expression pattern suggests that miR-429 might be functionally related to postnatal adipogenesis. However, we currently lack a mechanistic understanding of miR-429 within the context of preadipocyte differentiation. In this study, we investigated the function of miR-429 in porcine subcutaneous and intramuscular preadipocyte proliferation and differentiation. In our porcine preadipocyte differentiation model, miR-429 expression decreased remarkably upon adipogenic induction. Overexpression of miR-429 notably down-regulated the expression of adipogenic marker genes: PPARγ, aP2, FAS and impaired the triglyceride accumulation, while the expression of lipolytic gene ATGL was not affected. In addition, we observed that miR-429 significantly promoted the proliferation of porcine preadipocytes. We also found that miR-429 could directly bind to the 3′-UTRs of KLF9 and p27, which have been well documented to promote preadipocyte differentiation and repress cell cycle progression. Taken together, our data support a novel role of miR-429 in regulating porcine preadipocyte differentiation and proliferation, and KLF9 and p27 are potent targets of miR-429 during these processes.

Methionine and cysteine deficiencies altered proliferation rate and time-course differentiation of porcine preadipose cells.

Methionine (Met) is an essential sulfur amino acid (AA) limiting growth and is the precursor of cysteine (Cys), the rate-limiting factor in the synthesis of glutathione, and the main intracellular non-enzymatic antioxidant. This study aimed at determining the effects of limited supplies in Met and(or) Cys in early aspects of adipose tissue development and oxidative stress in differentiated adipocytes. Incremental reductions in Met (70, 40, and 0µM) were compared with Met 100µM (control dose) in porcine preadipocytes cultured in media without or with Cys (250µM). In Cys-deprived media, both the absence (0µM) and the lowest dose of Met (40µM) reduced preadipocyte proliferation. Adding Cys in media only partly compensated for this decrease. On the opposite, mild Met deficiency (70µM) did not alter preadipocyte proliferation in media without or with Cys. Strong Met deficiency (40µM) also reduced differentiation and lipid accumulation into preadipose cells. Mild Met deficiency also reduced preadipocyte differentiation when Cys was present in the culture media, whereas in Cys-deprived media, percent of differentiated cell was similar and intracellular lipid content was slightly higher at Met 70µM than at Met 100µM. Finally, incremental reductions in Met in media with or without Cys lowered reactive oxygen species (ROS) production by differentiated cells. These results demonstrate the strong dependency of porcine adipogenesis to sulfur AA supplies. Strong Met deficiency decreases both proliferation and differentiation, whereas mild deficiency only alters differentiation.

Glucose and Insulin Stimulate Lipogenesis in Porcine Adipocytes: Dissimilar and Identical Regulation Pathway for Key Transcription Factors.

Lipogenesis is under the concerted action of ChREBP, SREBP-1c and other transcription factors in response to glucose and insulin. The isolated porcine preadipocytes were differentiated into mature adipocytes to investigate the roles and interrelation of these transcription factors in the context of glucose- and insulin-induced lipogenesis in pigs. In ChREBP-silenced adipocytes, glucose-induced lipogenesis decreased by ~70%, however insulin-induced lipogenesis was unaffected. Moreover, insulin had no effect on ChREBP expression of unperturbed adipocytes irrespective of glucose concentration, suggesting ChREBP mediate glucose-induced lipogenesis. Insulin stimulated SREBP-1c expression and when SREBP-1c activation was blocked, and the insulin-induced lipogenesis decreased by ~55%, suggesting SREBP-1c is a key transcription factor mediating insulin-induced lipogenesis. LXRα activation promoted lipogenesis and lipogenic genes expression. In ChREBP-silenced or SREBP-1c activation blocked adipocytes, LXRα activation facilitated lipogenesis and SREBP-1c expression, but had no effect on ChREBP expression. Therefore, LXRα might mediate lipogenesis via SREBP-1c rather than ChREBP. When ChREBP expression was silenced and SREBP-1c activation blocked simultaneously, glucose and insulin were still able to stimulated lipogenesis and lipogenic genes expression, and LXRα activation enhanced these effects, suggesting LXRα mediated directly glucose- and insulin-induced lipogenesis. In summary, glucose and insulin stimulated lipogenesis through both dissimilar and identical regulation pathway in porcine adipocytes.

Original Research: Orexins A and B stimulate proliferation and differentiation of porcine preadipocytes.

Orexin A (OXA) and B (OXB) are neuropeptides which regulate appetite, energy expenditure, and arousal via G-protein coupled receptors termed as OXR1 and OXR2. The aim of this study was to characterize the effects of OXA and OXB on proliferation and differentiation of porcine preadipocytes. Porcine preadipocytes express both OXRs. OXA and OXB enhance porcine preadipocyte proliferation by 54.8% or 63.2 %, respectively. OXA and OXB potentiate differentiation of porcine preadipocytes, as judged by the increased lipid accumulation and expression of proadipogenic genes. Cellular lipid content after exposure of preadipocytes for six days to 100 nM OXA or OXB increased by 82.2% or 59.2%, respectively. OXA and OXB suppressed glycerol release by 23.9% or 24.9% in preadipocytes differentiated for six days. OXA (100 nM) increased peroxisome proliferator-activated receptor gamma (PPARγ) expression in cells differentiated for 24 h by 100.5%. PPARγ expression was also stimulated in preadipocytes differentiated in the presence of 10 nM (58.3%) or 100 nM OXA (50.6%) for three days. OXB potentiated PPARγ mRNA expression at 1 nM (59%), 10 nM (53.2%), and 100 nM (73.9%) in cells differentiated for three days. OXA increased CCAAT/enhancer binding protein alpha expression in preadipocytes differentiated for six days by 65%. OXB stimulated CCAAT/enhancer binding protein beta expression in preadipocytes differentiated for three days at 10 nM (149.5%) as well as 100 nM (207.2%). Lipoprotein lipase mRNA expression increased in cells treated with 10 nM OXA by 152.6% and 100 nM OXA by 162%. Lipoprotein lipase expression increased by 134% at 100 nM OXB. Furthermore, OXA (100 nM) and OXB (100 nM) increased leptin mRNA expression in preadipocytes differentiated for three days by 49.9% or 71.3%, respectively. These data indicate that orexin receptors may be relevant in the context of white adipose tissue formation.

AMFR gene silencing inhibits the differentiation of porcine preadipocytes.

Our study clarifies the role of the autocrine motility factor receptor (AMFR) gene in porcine preadipocyte differentiation. AMFR-siRNA was transfected into porcine preadipocytes and the preadipocytes were induced to differentiation. Subsequently, qRT-PCR was conducted to examine changes in mRNA expression of a series of genes in porcine preadipocytes, including AMFR, sterol-regulatory element-binding protein-1a (SREBP1a), SREBP2, insulin-induced gene 1 (Insig1), and Insig2. Expression changes in the mRNA of genes regulating adipocyte differentiation were also analyzed using qRT-PCR, including peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein alpha (C/EBPα), and Kruppel-like factor 2 (KLF2). Western blot analysis was conducted to examine the changes in AMFR protein expression in porcine preadipocytes. Additionally, morphological changes in differentiated porcine preadipocytes were examined by oil red O staining, and changes in optical density (OD) values were measured using an ultraviolet spectrophotometer. At 24 h after transfection with AMFR-siRNA, AMFR mRNA expression significantly reduced (P < 0.01), and AMFR protein expression markedly decreased (P < 0.05). The mRNA expression of SREBP1a, SREBP2, Insig1, and C/EBPα was significantly reduced (P < 0.01), whereas the expression of KLF2 mRNA was significantly elevated (P < 0.01). After induction of preadipocyte differentiation, the number of lipid droplets decreased in the AMFR-silenced group, and the OD value markedly reduced (P < 0.05). In addition, the expression of C/EBPα mRNA significantly decreased (P < 0.05), whereas the expression of KLF2 mRNA considerably increased (P < 0.05). Taken together, silencing of the AMFR gene inhibits the differentiation of porcine preadipocytes.

PU.1 antisense lncRNA against its mRNA translation promotes adipogenesis in porcine preadipocytes.

Antisense long non-coding RNAs (AS lncRNAs) play important roles in refined regulation of animal gene expression. However, their functions and molecular mechanisms for domestic animal adipogenesis are largely unknown. Here, we found a novel AS lncRNA transcribed from the porcine PU.1 gene (also known as SPI1) by strand-specific RT-PCR. Results showed that PU.1 AS lncRNA was expressed and generally lower than the level of PU.1 mRNA in porcine subcutaneous adipose, heart, liver, spleen, lympha, skeletal muscle and kidney tissues. We further found that the levels of PU.1 mRNA and PU.1 protein were significantly lower in subcutaneous and intermuscular adipose than in mesenteric and greater omentum adipose, whereas the levels of PU.1 AS lncRNA showed no difference in porcine adipose tissues from four different parts of the body. During porcine adipogenesis, levels of PU.1 mRNA increased at day 2 and then gradually decreased. Meanwhile, PU.1 AS lncRNA exhibited an expression trend similar to PU.1 mRNA but sharply decreased after day 2. Interestingly, PU.1 protein level rose during differentiation. In addition, at day 6 after differentiation, knockdown of endogenous PU.1 promoted adipogenesis, whereas knockdown of endogenous PU.1 AS lncRNA had the opposite effect. Moreover, peroxisome proliferator-activated receptor gamma (PPARG) and fatty acid synthase (FASN) were significantly upregulated in the PU.1 shRNA treatment group (P < 0.05), whereas they were downregulated in the PU.1 AS shRNA treatment group (P < 0.05). Adipose triglyceride lipase [ATGL; also known as patatin-like phospholipase domain containing 2 (PNPLA2)] and hormone-sensitive lipase [HSL; also known as lipase, hormone-sensitive (LIPE)] contrasted with PPARG and FASN. Finally, the PU.1 mRNA/PU.1 AS lncRNA duplex was detected by an endogenous ribonuclease protection assay combined with RT-PCR. Based on the above results, we suggest that PU.1 AS lncRNA (vs. its mRNA translation) promotes adipogenesis through the formation of a sense-antisense RNA duplex with PU.1 mRNA.

DETECTION OF LEPTIN IN MUSCLE TISSUES AND ORGANS OF PIGS

The aim of this study was detection of gene leptin in muscles, liver and kidneys from pigs of breed Large White. Using Real time PRC method, we determined the Ct values of leptim gene in muscle, liver, kidney. The body weight of pigs ranged from 100 kg to 103 kg. The average body weight was 101.6 kg. The thickness of backfat ranged from 10 to 20 mm, average backfat thickness was 16 mm. The minimal Ct value of leptin gene in liver was 24.05 and the maximal value was 25.79. Average Ct value of leptin gene was 24.84. The minimal Ct value of leptin gene in muscle tissue was 25.83 and the maximal value was 27.05. Average Ct value of leptin gene was 26.41. The Ct value of leptin gene in liver ranged from 24.05 to 25.79. Average Ct value of leptin gene was 24.84. Leptin gene is expressed by porcine preadipocytes and leptin gene expression is highly dependent on dexamethasone induced preadipocyte differentiation. Hormonally driven preadipocyte recruitment and subsequent fat cell size may regulate leptin gene expression in the pig.

The proliferation and differentiation of primary pig preadipocytes is suppressed when cultures are incubated at 37°Celsius compared to euthermic conditions in pigs

Given similarities in metabolic parameters and cardiovascular physiology, the pig is well positioned as a biomedical model for metabolic disease and obesity in humans. Better understanding molecular mechanisms governing porcine adipocyte hyperplasia may provide insight into the regulation of adipose tissue development that is useful both when considering the pig as a commodity and when extrapolating porcine data to human disease. Primary cultures of pig stromal-vascular cells have served as a useful tool for investigating factors that regulate preadipocyte proliferation and differentiation. However, such cultures have generally been maintained at 37°C in vitro despite euthermia being 39°C in pigs. To address potential concerns about the physiological relevance of culturing primary pig preadipocytes under what would be hypothermic conditions in vivo, the objective of this study was to investigate the effect of culture temperature on the proliferation and differentiation of pig preadipocytes in primary culture. Culturing primary preadipocytes at 37 rather than 39°C decreases their proliferation rates based upon cleavage of the tetrazolium salt, MTT (P < 0.001), reduction of resazurin (P < 0.001), and daily cell counts (P < 0.001). Likewise, culturing primary porcine preadipocytes at 37°C suppressed their adipogenic potential based upon monitoring adipogenesis morphologically, biochemically, and via the expression of mRNA encoding adipogenic marker genes. Collectively, these data indicate the proliferation and differentiation of primary pig preadipocytes is suppressed when cultures are incubated at 37°C compared to normal body temperature of pigs. This may confound investigation of factors that impact adipocyte hyperplasia in the pig.

Myokine IL-15 regulates the crosstalk of co-cultured porcine skeletal muscle satellite cells and preadipocytes

The present study was carried out to preliminarily reveal the underlying mechanisms of the co-culture system between porcine muscle satellite cells (SCs) and stromal-vascular cells (SVs). The two cell types were co-cultured to assess both proliferation and differentiation. Desmin and Pref-1 immunofluorescence staining technique were taken to identify the two types of isolated cells. The expression of specific marker genes Myogenin was up-regulated in SCs (P<0.05) and the differentiation of SCs could be promoted when co-cultured with preadipocytes compared with the single-cultured control, while expression of c/EBPβ in SVs was down-regulated (P<0.05) and the differentiation of preadipocytes could be inhibited. Furthermore, secretion of myokine IL-15 was markedly increased, as well as its gene and protein expression levels in co-culture supernatants. However, the secretion of adipokine leptin was significantly decreased. These findings demonstrate that myokines like IL-15 could facilitate the SCs' differentiation while inhibit the SVs differentiation, and act as an important regulator of co-culture between muscle cells and adipocytes.

MiR-15a/b promote adipogenesis in porcine pre-adipocyte via repressing FoxO1.

Diabetes and many other metabolism syndromes are closely related to obesity. To reveal the underlying mechanism of fat deposition, an increasing number of studies are focusing on the functions of miRNAs during adipocytes development. Previous studies have proved that miR-15a/b play important roles in multiple physiological processes; however, their functions during adipogenesis remain unclear. To reveal this, we detected the expression profiles of miR-15a/b during adipogenesis in porcine pre-adipocyte, and found that their expression levels increased in the early stage of adipocyte differentiation and dropped after day 4. Moreover, over-expression of miR-15a/b in porcine pre-adipocytes promoted adipocyte differentiation and lipid accumulation. Target genes of miR-15a/b were predicted and examined, which revealed that Forkhead box protein O1 (FoxO1) is the target gene of miR-15a/b. The inhibition of FoxO1 expression level caused by miR-15a/b over-expression had a positive effect on adipogenesis. Thus, we conclude that miR-15a/b promote adipogenesis in porcine pre-adipocyte via repressing FoxO1.

MiR-125a inhibits porcine preadipocytes differentiation by targeting ERRα.

MicroRNAs are a family of small, non-coding RNAs that regulate gene expression in a sequence-specific manner. Estrogen-related receptor α (ERRα) is an orphan nuclear receptor which plays an important role in adipocyte differentiation. Our previous Solexa sequencing results indicated a high expression of miR-125a in adult pig backfat. In this study, we predicated and experimentally validated ERRα as a target of miR-125a. To explore the role of miR-125a in porcine preadipocytes differentiation, miRNA agomir and antagomir were used to perform miR-125a overexpression or knockdown, respectively. Our results showed that overexpression of miR-125a could dramatically reduce the mRNA expression of adipogenic markers PPARγ, LPL, and aP2, as well as its target gene ERRα. Western blotting showed the protein level of aP2 and ERRα was also significantly down-regulated. The overexpression of miR-125a also led to a notable reduction in lipid accumulation which was detected by Oil Red O staining. In contrast, we observed promoted differentiation of porcine preadipocytes upon miR-125a inhibition. In conclusion, we verified miR-125a inhibits porcine preadipocytes differentiation through targeting ERRα for the first time, which may provide new insights in pork quality improvement and obesity control.

CTSB promotes porcine preadipocytes differentiation by degrading fibronectin and attenuating the Wnt/β-catenin signaling pathway.

The process of preadipocytes differentiation plays a vital role in adipose tissue expansion and many factors are involved in this event. Cathepsin B (CTSB), secreted from lysosome, has been reported in regulating a variety of physiological processes. In this study, we demonstrated CTSB promotes lipid accumulation and adipogenic genes expression in porcine primary preadipocytes by degrading fibronectin (Fn), a key component of extracellular matrix. Lithium chloride (LiCl) is an activator of Wnt/β-catenin signaling through stabilizing β-catenin. We found that CTSB can relieve the anti-adipogenic effects of LiCl, indicating that CTSB could impact Wnt/β-catenin signaling pathway. Interestingly, Fn is an important target gene of Wnt/β-catenin. So we considered that CTSB promote preadipocytes differentiation by suppressing these two pathways.