Porcine phosphotyrosine interaction domain containing 1 modulates 3T3-L1 preadipocyte proliferation and differentiation

The phosphotyrosine interaction domain containing 1 (PID1) gene was firstly isolated from obese subjects and involved in obesity-associated insulin resistance. In the present study, Duroc×Landrace×Yorkshire (DLY) pig PID1 cDNA was cloned. The entire open reading frame of the cloned porcine PID1 is 654 bp. The predicted protein is composed of 217 amino acids residues with a molecular mass of 24,774 Da. Over-expression of porcine PID1 significantly accelerated the proliferation of 3T3-L1 preadipocyte, but inhibited preadipocyte differentiation by decreasing the numerous fat droplets appeared and down-regulating the mRNA expression levels of peroxisome proliferators-activated receptor-γ, CCAAT/enhancer binding protein α, fat acid synthase and lipoprotein lipase. Together, these results suggest that porcine PID1 plays a role in regulating adipose development.     

pGhrelin对离体猪脂肪细胞Caspase-3活性及基因表达的影响

研究新近发现的猪Ghrelin (porcine ghrelin,pGhrelin)对猪前体脂肪细胞Caspase-3活性及其基因表达的影响．采用细胞培养技术,以仔猪背部皮下前体脂肪细胞为靶细胞,经0、1、10和100 nmol/L pGhrelin处理细胞48 h后,于倒置生物显微镜下进行脂肪细胞形态学观察．利用MTT法测定pGhrelin对细胞增殖的影响．采用分光光度法检测Caspase-3活性．以实时荧光定量RT-PCR方法测定Caspase-3的基因表达．结果显示,10 nmol/L pGhrelin可以显著降低脂肪细胞Caspase-3的活性与mRNA的表达水平(P < 0.05),100 nmol/L pGhrelin对猪脂肪前体细胞增殖有极显著促进作用(P < 0.01)．上述结果表明,pGhrelin可以下调Caspase-3的活性与基因表达,促进脂肪细胞增殖,抑制脂肪细胞凋亡,其机制可能与Caspase-3依赖性凋亡调节信号通路有关．     

Biological role of MicroRNA-103 based on expression profile and target genes analysis in pigs

MicroRNAs (miRNAs) are endogenously expressed RNAs consisting of 20–24 nucleotides. These molecules are thought to repress protein translation by binding to target mRNAs. However, biological functions have not been assigned to most of the 175 porcine miRNAs registered in miRBase (release 15.0). In an effort to uncover miR-103 important in pigs, we examined the integrative tissue expression profile and gene ontology (GO) term enrichment of predicted target genes to determine the global biological functions of miR-103. Our results demonstrated that miR-103 is involved in various biological processes including brain development, lipid metabolism, adipocyte differentiation, hematopoiesis, and immunity. Moreover, we also experimentally verified effects of miR-103 in porcine preadipocytes. miR-103 levels increased in differentiating adipocytes, and inhibition of miR-103 effectively inhibited preadipocyte differentiation. In addition, mRNA levels of the putative miR-103 target RAI14 were higher in miR-103 inhibitor-treated adipocytes. These results demonstrate that miR-103 is involved in porcine preadipocyte differentiation and may act through the putative target gene RAI14. In a word, our data provide new insights into the global biological role of miR-103.     

MiR-127 attenuates adipogenesis by targeting MAPK4 and HOXC6 in porcine adipocytes

MicroRNAs (miRNAs) have critical roles during adipogenesis; however, their precise functions are not completely understood. Porcine miRNA expression profiles show that miR-127 is dramatically downregulated with age in adipose tissue. We aimed to identify the precise functions and mechanisms of miR-127 in proliferation and adipogenesis. Preadipocytes were cultured under conditions to induce proliferation or differentiation and the effect of miR-127 overexpression on these processes, and the associated bioinformatically predicted target genes, were assessed using luciferase assays, quantitative real-time PCR, western blot analysis, and cell staining techniques. miR-127 increased proliferation by promoting cell cycling, whereas it suppressed differentiation, which was accompanied by reduced lipid accumulation. miR-127 targeted mitogen-activated protein kinase 4 and homeobox C6 (HOXC6) to activate preadipocyte proliferation. During differentiation, miR-127 targeted HOXC6 to attenuate adipogenesis. These findings identify miR-127 as an inhibitor of porcine adipogenesis, which may inform future strategies to reduce porcine fat deposition and treat human obesity.     

Alteration of mitochondrial oxidative capacity during porcine preadipocyte differentiation and in response to leptin

Mitochondrial apparatus is a fundamental aspect in cell, serving for amino acid biosynthesis, fatty acid oxidation (FAO), and ATP production. In this article, we investigated the change of mitochondrial oxidative capacity during porcine adipocyte differentiation and in response to leptin. Rhodamine 123 staining analysis showed about 2-fold increase of mitochondrial membrane electric potential in differentiated adipocyte in comparison with preadipocyte. The mRNA expression of Cytochromes c (Cyt c), carnitine palmitoyltransferase 1 (CPT1), and malate dehydrogenases (MDH) increased markedly (P < 0.05), but that of UCP2 decreased (P < 0.05). Moreover PGC-1α and UCP3 was very low and showed no changes during the adipocyte differentiation. The protein expression of Cyt c and the enzyme activity of Cytochrome c oxidase (COX) increased with preadipocyte differentiation, but cellular ATP level decreased. Furthermore, at the level of 10 and 100 ng/ml leptin not only selectively increased the gene expression of PGC-1α, CPT1, Cyt c, UCP2, and UCP3 (P < 0.05), but also enhanced COX enzyme activity which related to mitochondrial FAO. There is no change of Mitochondrial membrane electric potential and ATP level in cell treated by leptin. These results suggested Mitochondrial is not only critical in FAO, but also play an important role in adipogenesis.     

瘦素(Leptin )对猪前体脂肪细胞分化及PGC-1α和UCPs mRNA表达的影响

 以体外培养的猪前体脂肪细胞为研究对象,分析瘦素(leptin )对前体脂肪细胞分化及能量代谢相关基因PGC- 1α和UCPs mRNA表达的影响.油红O染色提取结果显示,10～90nmol/L的leptin对猪前体脂肪细胞甘油三酯合成均无显著影响(P>0.05).RT-PCR检测结果表明,30　nmol/L和100nmol/L leptin可显著促进LPL mRNA表达,处理24 h较12 h 作用效果明显(P<0.05);两个浓度的leptin对脂肪细胞分化转录因子C/EBPα和PPARγ2在mRNA水平上均没有明显的影响(P>0.05).对能量代谢相关基因的RT-PCR检测结果表明,30 nmol/L 和100nmol/L leptin 可显著促进PGC-1α和UCP3转录 (P<0.05),30nmol/L leptin可明显提高前脂肪细胞中UCP2 mRNA水平(P<0.05),且作用24h促进效果明显优于12h处理(P<0.05).研究结果提示,leptin不影响猪前体脂肪细胞分化,但可能通过上调PGC-1α和UCPs转录,促进能量消耗.     

FTO Promotes Adipogenesis through Inhibition of the Wnt/β-catenin Signaling Pathway in Porcine Intramuscular Preadipocytes

Numerous studies have demonstrated that FTO plays an important role in adipogenesis. Herein, we designed a small interfering RNA targeting FTO to knock down its endogenous expression and investigated its effects on the proliferation and differentiation of porcine intramuscular preadipocytes. Its possible mechanism was also investigated. We showed that FTO silencing significantly decreased the level of phospho-Histone H3 protein and inhibited the proliferation of porcine intramuscular preadipocytes. In addition, the expressions of peroxisome proliferators-activated receptor γ (PPARγ) and CAAT/enhancer binding protein (C/EBPα) were down-regulated, but the expression of β-catenin was up-regulated, by FTO silencing. Of specific interest here was that LiCl, a Wnt/β-catenin signaling specific activator, attenuated the FTO-induced upregulation of PPARγ and downregulation of β-catenin. Collectively, our data demonstrated that FTO silence decreased the proliferation and differentiation of porcine intramuscular preadipocytes, and FTO affects the porcine intramuscular preadipocytes differentiation might be via Wnt/β-catenin signaling pathway.     

The adipokine Chemerin induces lipolysis and adipogenesis in bovine intramuscular adipocytes

The adipokine Chemerin is reported to regulate adipogenesis and glucose homeostasis in vivo and in 3T3-L1 cells. Our team is focused on the role of Chemerin in metabolism and intramuscular adipocyte differentiation because intramuscular fat is the basic material for the formation of marbling in livestock and poultry meat. In this study, bovine intramuscular mature adipocytes were cultured in medium with Chemerin, and the process of lipolysis of mature adipocytes and the adipogenesis of de-differentiated preadipocytes were investigated. The results showed that Chemerin induced significant lipolytic metabolism in intramuscular mature adipocytes, indicated by increased levels of glycerol, FFA, and up-regulated expression of the lipolysis critical factors HSL, LPL, and leptin. Meanwhile, the expressions of adipogenic key factors PPARγ, C/EBPα, and A-FABP were decreased by Chemerin during lipolysis or dedifferentiation in mature adipocytes. The de-differentiated preadipocytes could re-differentiate into mature adipocytes. Intriguingly, the formation of cells’ lipid droplets was promoted by Chemerin during preadipocyte differentiation. In addition, mRNA and protein expressions of PPARγ, C/EBPα, and A-FABP were up-regulated by Chemerin during preadipocytes differentiation. These results suggest that Chemerin promotes lipolysis in mature adipocytes and induces adipogenesis during preadipocyte re-differentiation, further indicating a dual role for Chemerin in the deposition of intramuscular fat in ruminant animals.

     

CCAAT/Enhancer-Binding Protein β is not Affected by Tetrachlorodibenzo-p-dioxin (TCDD) Inhibition of 3T3-L1 Preadipocyte Differentiation

The differentiation of 3T3-L1 preadipocytes is induced by the coordinate activation of trans-acting factors in response to inducers. Depending on the time of treatment, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was effective in inhibiting 3T3-L1 preadipocyte differentiation and the expression of differentiation-dependent trans-acting factors. Based on glycerol-3-phosphate dehydrogenase activity, the differentiation of 3T3-L1 cells was decreased by 70% in cells treated with TCDD before the induction of differentiation, 25% during induction, and not at all after induction. This time-dependent inhibition of cell differentiation by TCDD was correlated with the levels of aryl hydrocarbon receptor (AhR). TCDD treatment decreased the mRNA levels of C/EBPα and PPAR-γ2 but did not affect the mRNA levels of RXRα and RARα. Furthermore, TCDD did not change the mRNA or protein levels of C/EBPβ, which is thought to play a role in inducing C/EBPα and PPARγ2 expression. These results suggest that TCDD inhibited 3T3-L1 preadipocyte differentiation through the AhR pathway, and the change of C/EBPβ mRNA and protein was not involved in reducing mRNA expression of C/EBPα and PPARγ2.     

Decreased steady-state insulin-like growth factor binding protein-3 (IGFBP-3) mRNA level is associated with differentiation of cultured porcine myogenic cells

Insulin-like growth factor binding proteins (IGFBPs) affect the biological activity of IGF-I in several cell types, including cultured muscle cells. Additionally, at least one of the IGFBPs, IGFBP-3, has been shown to have IGF-independent effects on cell proliferation. Numerous studies have shown that immortalized muscle cell lines produce various IGFBPs, but to date no muscle cell line has been reported to produce IGFBP-3 protein or mRNA. Unlike muscle cell lines, primary cultures of porcine embryonic myogenic cells express IGFBP-3 mRNA and secrete a protein that is immunologically identifiable as IGFBP-3. Additionally, steady-state IGFBP-3 levels change significantly during differentiation. Here we report that differentiation of porcine myogenic cells in an IGFBP-3-free medium is accompanied by reduced steady-state IGFBP-3 mRNA levels. Steady-state levels of IGFBP-3 mRNA decreased approximately sevenfold (P < .05) during differentiation and then increased to predifferentiation levels once differentiation was complete. Addition of TGF-beta1 (0.5 ng/ml) to porcine myogenic cultures suppressed fusion and resulted in a sevenfold increase in steady-state IGFBP-3 mRNA and a 1.8-fold increase in IGFBP-3 protein levels as compared to untreated control cultures (P < .05). Results suggest that alterations in IGFBP-3 mRNA and protein may play a role in differentiation of porcine embryonic muscle cells.     

过表达RBP4抑制体外培养的猪前体脂肪细胞分化

为研究视黄醇结合蛋白4(retinol binding protein 4,RBP4)对猪前体脂肪细胞分化的影响,实验构建了RBP4重组腺病毒表达载体,包装并感染猪前体细胞,采用油红O染色和Real-time PCR等方法,检测了过表达RBP4对成脂分化的作用. 研究结果显示,重组腺病毒RBP4载体构建成功,转染猪前体脂肪细胞后,使RBP4的mRNA水平和蛋白水平分别增加了约400倍和20倍. 过表达RBP4能减少脂肪细胞的脂质积累,降低成脂关键基因过氧化物酶体增生物激活受体γ (peroxisome proliferator-activated receptor gamma, PPARγ)和脂肪酸结合蛋白2 (adipocyte protein 2, aP2)的表达. 结果表明,RBP4对猪前体脂肪细胞分化有抑制作用,为进一步研究RBP4对猪前体脂肪细胞分化的作用机制奠定基础.     

Txnip基因siRNA慢病毒表达质粒构建及促进猪前体脂肪细胞分化

硫氧还蛋白互作蛋白(thioredoxin interacting protein, Txnip)是一种氧化还原调节蛋白质,与硫氧还蛋白结合并抑制其活性,调节细胞氧化还原状态,影响细胞多种生理过程,然而其在猪脂肪细胞分化中的作用尚不明确。本文设计合成3对靶向猪Txnip基因的shRNA寡核苷酸,分别连接于重组慢病毒载体pGLV_3/H_1/GFP+Puro构建siRNA表达质粒。测序验证后,与包装质粒共转染293T细胞,获得滴度1×10~8 pfu/mL的慢病毒干扰质粒。以MOI值100转染原代培养猪前体脂肪细胞,转染率均达80%以上,其中Txnip-shRNA-2转染细胞Txnip基因沉默率达75%。转染Txnip-shRNA-2的猪前体脂肪细胞用成脂分化培养液诱导后,每隔1 d检测细胞成脂分化及相关基因表达。结果发现,其分化比阴性对照质粒转染或未转染细胞显著增强(P<0.05),PPARγ和FAS mRNA表达水平显著提高(P<0.05)。本文构建siRNA慢病毒表达质粒能有效干扰猪Txnip基因表达,Txnip表达沉默可通过上调PPARγ表达促进猪前体脂肪细胞分化。本研究提示,Txnip可能是猪脂肪细胞分化的抑制因子。     

Modulation of Sirt1 by resveratrol and nicotinamide alters proliferation and differentiation of pig preadipocytes

Sirt1, a NAD⁺-dependent histone deacetylase, may regulate senescence, metabolism, and apoptosis. In this study, primary pig preadipocytes were cultured in DMEM/F12 medium containing 10% fetal bovine serum (FBS) with or without reagents affecting Sirt1 activity. The adipocyte differentiation process was visualized by light microscopy after Oil red O staining. Proliferation and differentiation of preadipocytes was measured using methylthiazolyldiphenyl-tetrazolium bromide (MTT) and Oil red O extraction. Expression of Sirt1, FoxO1, and adipocyte specific genes was detected with semi-quantitive RT-PCR. The results showed that Sirt1 mRNA was widely expressed in various pig tissues from different developmental stages. Sirt1 mRNA was expressed throughout the entire differentiation process of pig preadipocytes. Resveratrol significantly increased Sirt1 mRNA expression, but decreased the expression of FoxO1 and adipocyte marker gene PPARγ2. Resveratrol significantly inhibited pig preadipocyte proliferation and differentiation. Nicotinamide decreased the expression of Sirt1 mRNA, but increased the expression of FoxO1 and adipocyte specific genes. Nicotinamide greatly stimulated the proliferation and differentiation of pig preadipocytes. In conclusion, these results indicate that Sirt1 may modulate the proliferation and differentiation of pig preadipocytes. Sirt1 may down-regulate pig preadipocytes proliferation and differentiation through repression of adipocyte genes or FoxO1.     

CTSB超表达促进体外培养的猪前体脂肪细胞分化

为研究溶酶体组织蛋白酶B(cathepsin B,CTSB)对脂肪细胞分化的影响,本实验构建了Ctsb重组腺病毒超表达载体,包装并侵染体外培养的猪前体脂肪细胞,采用油红O染色,油红O提取比色法检测猪前体脂肪细胞分化的情况,并通过real-time PCR法检测成脂关键基因mRNA水平的变化.结果显示,重组腺病毒Ctsb载体构建成功,转染猪前体脂肪细胞后,使Ctsb的mRNA和蛋白质表达量分别提高了约16倍和12倍. CTSB超表达能促进脂肪细胞的分化和脂质积累,成脂关键基因过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptor gamma, PPARγ)、脂肪酸结合蛋白2(adipocyte protein 2, aP2)的表达量均有显著升高. 研究表明,提高Ctsb的表达能促进猪前体脂肪细胞分化,揭示了Ctsb在猪前体脂肪细胞分化过程中可能发挥关键作用. 研究结果为进一步研究其作用机制奠定了基础.     

The mRNA of lipin1 and its isoforms are differently expressed in the <Emphasis Type="Italic">longissimus dorsi</Emphasis> muscle of obese and lean pigs

Lipin1 has been documented to play an important role in adipogenesis. In the present study, the mRNA expression level of lipin1 and its isoforms in longissimus dorsi muscle were determined by semi-quantification RT-PCR in lean PIC and obese Rongchang pigs. Further, we determined mRNA expression for lipin1 and its two isoforms in Rongchang obese pigs which had either a high or low intramuscular fat content. We demonstrate for the first time that porcine lipin1 has two alternative forms, lipin-α and lipin-β. Unlike mice and humans where the lipin-β has 99 more nucleotides than lipin-α, we found that in swine, lipin-β has 108 more nucleotides than lipin-α. Our results indicate that the longissimus dorsi muscle of Rongchang obese pigs have a higher level of mRNA expression for lipin1 and its isoforms than PIC lean pigs. Furthermore, Rongchang pigs with higher intramuscular fat content had a higher lipin1 and lipin-β mRNA expression in longissimus dorsisi muscle than Rongchang pigs with lower intramuscular fat content (P < 0.05), whereas no difference was seen in lipin-α mRNA expression between Rongchang pigs with high or low intramuscular fat. The ratio of lipin-β mRNA to lipin-α mRNA was also significantly different between Rongchang pigs distinguished by a high intramuscular fat content compared with those with low intramuscular fat (P < 0.05). These data suggested that the lipin1 gene may have a crucial effect on body lipid accumulation in pigs, whereas the lipin-β isoform may play an important role in intramuscular fat deposition in obese pigs.     

Adipocyte Development is Dependent Upon Stem Cell Recruitment and Proliferation of Preadipocytes

KRAS, KRYSTYNA M., DOROTHY B. HAUSMAN, GARY J. HAUSMAN, AND ROY J. MARTIN. Adipocyte development is dependent upon stem cell recruitment and proliferation of preadipocytes. Obes Res. Objectives: The ability to acquire fat cells persists over the life spans of animals. It is unknown whether adipocyte acquisition is the result of preadipocyte proliferation or stem cell recruitment to become adipocytes. The purposes of these studies were 1) to characterize early differentiation of stromal vascular (S-V) cells to preadipocytes as it is influenced by insulin, dexamethasone (DEX), and insulin-like growth factor-I (IGF-I); and 2) to determine whether new fat cells arise from stem cell recruitment or preadipocyte proliferation. Research Methods and Procedures: Freshly isolated S-V cells from rat inguinal adipose tissues were plated for 24 hours then exposed to serum-free medium. Results: Approximately 15% of freshly plated S-V cells were preadipocytes as determined by a preadipocyte specific marker, AD3. Total cell number and proportion of preadipocytes were significantly greater with 100 nM insulin treatment than with 0, 0. 1, or 1. 0 nM, but IGF-I treatment at 10 nM resulted in preadipocyte development similar to that with 100 nM insulin treatment. The addition of 5 nM DEX to the 100 nM insulin treatment resulted in a 20% increase in preadipocyte number by day 2 when compared to either treatment alone. 5-Bromo-2′-deoxyuridine treatment suppressed the increased proportion of preadipocytes from days 0–2 in non-insulin treated cells and prevented the increase typically observed with insulin. A mitosis inhibitor also significantly reduced the proportion of preadipocytes. Discussion: These results show for the first time that S-V cells are recruited as preadipocytes and that proliferation of these preadipocytes and early differentiation occur simultaneously.