Porcine Endogenous Retroviruses: Quantification of the Viral Copy Number for the Four Miniature Pig Breeds in China.

Domestic pigs has served not only as one of the most important economy livestock but also as ideal organ-source animals owing to similarity in anatomy, physiology, and organ size to humans. Howerer, the barrier of the cross-species transmission risk of porcine endogenous retrovirus (PERVs) blocked the pig-to-human xenotransplantation. PERVs are integrated into pigs' genomes and cannot be eliminated by designated or specified pathogen-free breeding. PERVs are an important biosafety issue in xenotransplantation because they can be released from normal pig cells and infect human cells in vitro under certain conditions. Screening and analyzing the presence of PERVs in pig genome will provide essential parameters for pig breed sources. In China, four miniature pig breeds, such as Guizhou miniature pig (GZ), Bama miniature pig (BM), Wuzhishan miniature pig (WZS), and Juema miniature pig (JM), were the main experimental miniature pig breeds, which were widely used. In this study, PCR was performed to amplify env-A, env-B, and env-C for all individuals from the four breeds. The results revealed that PERV env-A and env-B were detected in all individuals and the lowest ratios of PERV env-C was 17.6% (3/17) in the GZ breed. Then, PERV pol and GAPDH were detected using the droplet digital PCR (ddPCR) method. As the reference of GAPDH copy number, the copy numbers of PERVs were at the median of 12, 16, 14, and 16 in the four miniature pig breeds (GZ, BM, WZS, and JM), respectively. Furthermore, the copy number of the PERV pol gene in many organs from the GZ breed was analyzed using ddPCR. The copy numbers of PERV pol gene were at the median of 7 copies, 8 copies, 8 copies, 11 copies, 5 copies, 6 copies, and 7 copies in heart, liver, spleen, lung, kidney, muscle, and skin, and the maximum number was 11 copies in the lung. The minimum number was 5 copies in the kidney as the reference of GAPDH. These data suggest that GZ breed has the lower PERVs copy number in the genome, and may be an ideal organ-source miniature pig breed for the study of the pig-to-human xenotransplantation.

Bifenthrin Induces Fat Deposition by Improving Fatty Acid Uptake and Inhibiting Lipolysis in Mice.

Chemical residues in the environment are considered to be important factors that cause obesity. Bifenthrin is one of the pyrethroid pesticides and is widely used worldwide. However, its effect on adipose tissue is ill-defined. Here, we administered bifenthrin/corn oil to adult C57BL/6 mice by gavage. After 6 weeks, the bifenthrin treatment significantly increased their body weight (P = 0.015) and fat mass (P < 0.001). Then we identified 246 differently expressed proteins by proteomic analysis, and they were highly involved in fatty acid uptake and lipid metabolism processes. Interestingly, protein hormone-sensitive lipase and adipose triacylglyceride lipase were downregulated while lipoprotein lipase is upregulated after bifenthrin treatment. Similar effects in 3T3-L1 cells treated with bifenthrin validated the in vivo results. Thus, this study suggests that long-term exposure to low-dose bifenthrin induces fat deposition in mice by improving fatty acid uptake and inhibiting lipolysis, and it may cause obesity in humans.

MicroRNA-214-3p Targeting Ctnnb1 Promotes 3T3-L1 Preadipocyte Differentiation by Interfering with the Wnt/ß-Catenin Signaling Pathway.

Differentiation from preadipocytes into mature adipocytes is a complex biological process in which miRNAs play an important role. Previous studies showed that miR-214-3p facilitates adipocyte differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) in vitro. The detailed function and molecular mechanism of miR-214-3p in adipocyte development is unclear. In this study, the 3T3-L1 cell line was used to analyze the function of miR-214-3p in vitro. Using 5-Ethynyl-2'-deoxyuridine (EdU) staining and the CCK-8 assay, we observed that transfection with the miR-214-3p agomir visibly promoted proliferation of 3T3-L1 preadipocytes by up-regulating the expression of cell cycle-related genes. Interestingly, overexpression of miR-214-3p promoted 3T3-L1 preadipocyte differentiation and up-regulated the expression of key genes for lipogenesis: PPARγ, FABP4, and Adiponectin. Conversely, inhibition of miR-214-3p repressed 3T3-L1 preadipocyte proliferation and differentiation, and down-regulated the expression of cell cycle-related genes and adipogenic markers. Furthermore, we proved that miR-214-3p regulates 3T3-L1 preadipocyte differentiation by directly targeting the 3'-untranslated regions (3'UTR) of Ctnnb1, which is an important transcriptional regulatory factor of the Wnt/ß-Catenin pathway. Taken together, the data indicate that miR-214-3p may positively regulate preadipocyte proliferation and enhance differentiation through the Wnt/ß-Catenin signaling pathway.

An Additive Effect of Promoting Thermogenic Gene Expression in Mice Adipose-Derived Stromal Vascular Cells by Combination of Rosiglitazone and CL316,243.

It is well-documented that CL316,243 (a ß3 agonist) or rosiglitazone (a PPARγ agonist) can induce white adipocyte populations to brown-like adipocytes, thus increasing energy consumption and combating obesity. However, whether there is a combined effect remains unknown. In the present study, stromal vascular cells of inguinal white adipose tissue (iWAT-SVCs for short) from mice were cultured and induced into browning by CL316,243, rosiglitazone, or both. Results showed that a combination of CL316,243 and rosiglitazone significantly upregulated the expression of the core thermogenic gene Ucp1 as well as genes related with mitochondrial function (Cidea, Cox5b, Cox7a1, Cox8b, and Cycs), compared with the treatment of CL316,243 or rosiglitazone alone. Moreover, co-treatment with rosiglitazone could reverse the downregulation of Adiponectin resulting from CL316,243 stimuli alone. Taken together, a combination of rosiglitazone and CL316,243 can produce an additive effect of promoting thermogenic gene expression and an improvement of insulin sensitivity in mouse iWAT-SVCs.

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.

Mulberry 1-Deoxynojirimycin Inhibits Adipogenesis by Repression of the ERK/PPARγ Signaling Pathway in Porcine Intramuscular Adipocytes.

Intramuscular fat (IMF), which is modulated by adipogenensis of intramuscular adipocytes, plays a key role in pork quality associated with marbling, juiceness, and flavor. However, the regulatory mechanism of 1-deoxynojirimycin (DNJ) on adipogenesis is still unknown. Here, we found that both DNJ (2.0, 3.0, 4.0, 5.0, and 6.0 µM) and rosiglitazone (RSG; 0.1, 0.2, 0.3, 0.4, and 0.5 mM) had no effect on cell viability. Moreover, 4 µM DNJ significantly inhibited adipogenesis, whereas 0.4 mM RSG increased lipogenesis of porcine intramuscular adipocytes. Interestingly, DNJ sharply inhibited phosphorylation of extracellular regulated protein kinases 1/2 (ERK1/2), but did not change phosphorylation of AKT (protein kinase B) in intramuscular adipocytes. We further found that the inhibitory adipogenesis of DNJ was attenuated by RSG via up-regulation of PPARγ. On the basis of the above findings, we suggest that DNJ inhibited adipogenesis through the ERK/PPARγ signaling pathway in porcine intramuscular adipocytes.

3,3'-Diindolylmethane increases bone mass by suppressing osteoclastic bone resorption in mice.

3,3'-Diindolylmethane (DIM), a major acid-condensation product or metabolite of indole-3-carbinol which is found in cruciferous vegetables, has been shown to have anticancer, anti-inflammatory, and multiple immune stimulating effects. However, its function in bone metabolism is poorly understood. This study evaluated the effect of DIM on bone mass in mice under physiological and pathological conditions. Eight-week-old female mice received injections of a vehicle or 0.1mg/g of DIM, twice a week for four weeks. We found that DIM treatment significantly increased bone mass as assessed by dual-energy X-ray absorptiometry (DEXA) and micro-computed tomography (µCT). Further, Bone histomorphometric analyses showed that this treatment significantly reduced bone resorption parameters, but did not increase bone formation parameters. Furthermore, we use ovariectomized (OVX)-induced osteoporotic mouse model, and explore function of DIM in skeletal pathological processes. Bone phenotype analyses revealed that the administration of DIM in this study effectively prevented OVX-induced bone loss resulting from increased bone resorption. Our results demonstrated that DIM increased bone mass by suppressing osteoclastic bone resorption in bone metabolism under both physiological and pathological conditions. Accordingly, DIM may be of value in the treatment and the possible prevention of bone diseases characterized by bone loss, such as postmenopausal osteoporosis.

Aryl hydrocarbon receptors in osteoclast lineage cells are a negative regulator of bone mass.

Aryl hydrocarbon receptors (AhRs) play a critical role in various pathological and physiological processes. Although recent research has identified AhRs as a key contributor to bone metabolism following studies in systemic AhR knockout (KO) or transgenic mice, the cellular and molecular mechanism(s) in this process remain unclear. In this study, we explored the function of AhR in bone metabolism using AhR(RANKΔOc/ΔOc) (RANK(Cre/+);AhR(flox/flox)) mice. We observed enhanced bone mass together with decreased resorption in both male and female 12 and 24-week-old AhR(RANKΔOc/ΔOc) mice. Control mice treated with 3-methylcholanthrene (3MC), an AhR agonist, exhibited decreased bone mass and increased bone resorption, whereas AhR(CtskΔOc/ΔOc) (Ctsk(Cre/+);AhR(flox/flox)) mice injected with 3MC appeared to have a normal bone phenotype. In vitro, bone marrow-derived macrophages (BMDMs) from AhR(RANKΔOc/ΔOc) mice exhibited impaired osteoclastogenesis and repressed differentiation with downregulated expression of B lymphocyte-induced maturation protein 1 (Blimp1), and cytochrome P450 genes Cyp1b1 and Cyp1a2. Collectively, our results not only demonstrated that AhR in osteoclast lineage cells is a physiologically relevant regulator of bone resorption, but also highlighted the need for further studies on the skeletal actions of AhR inhibitors in osteoclast lineage cells commonly associated with bone diseases, especially diseases linked to environmental pollutants known to induce bone loss.

Aryl hydrocarbon receptor catabolic activity in bone metabolism is osteoclast dependent in vivo.

Bone mass is regulated by various molecules including endogenous factors as well as exogenous factors, such as nutrients and pollutants. Aryl hydrocarbon receptor (AhR) is known as a dioxin receptor and is responsible for various pathological and physiological processes. However, the role of AhR in bone homeostasis remains elusive because the cell type specific direct function of AhR has never been explored in vivo. Here, we show the cell type specific function of AhR in vivo in bone homeostasis. Systemic AhR knockout (AhRKO) mice exhibit increased bone mass with decreased resorption and decreased formation. Meanwhile, osteoclast specific AhRKO (AhR(ΔOc/ΔOc)) mice have increased bone mass with reduced bone resorption, although the mice lacking AhR in osteoblasts have a normal bone phenotype. Even under pathological conditions, AhR(ΔOc/ΔOc) mice are resistant to sex hormone deficiency-induced bone loss resulting from increased bone resorption. Furthermore, 3-methylcholanthrene, an AhR agonist, induces low bone mass with increased bone resorption in control mice, but not in AhR(ΔOc/ΔOc) mice. Taken together, cell type specific in vivo evidence for AhR functions indicates that osteoclastic AhR plays a significant role in maintenance of bone homeostasis, suggesting that inhibition of AhR in osteoclasts can be beneficial in the treatment of osteoporosis.

Tissue expression of porcine FoxO1 and its negative regulation during primary preadipocyte differentiation.

The Forkhead transcription factor O 1 (FoxO1) gene plays an important role in the integration of hormone-activated signaling pathways with the complex transcriptional cascade that promotes clonal cell line differentiation. However, tissue expression of porcine FoxO1 and its function during porcine preadipocyte differentiation remained poorly understood. In the present study, we investigated tissue expressions of FoxO1 in pig by real time quantitative RT-PCR (qRT-PCR) and western blotting, and explored its role in porcine preadipocytes differentiation by RNA interference technique and qRT-PCR. FoxO1 gene expressions were highly in subcutaneous adipose and visceral adipose tissues, and higher in piglets than those in adults (P < 0.05). We showed that expression of endogenous FoxO1 in preadipocytes transfected with pBS/U6-siFoxO1-1748 expression vector was inhibited efficiently. After reducing expression of FoxO1, glycerol-3-phosphate dehydrogenase (GPDH) activity and triglyceride (TG) content increased from day 1 to 9, and the time-course expressions of several key adipogenic genes mRNA, including peroxisome proliferator-activated receptor gamma on day 3, 5, and 7, adipocyte fatty acid binding protein on day 1, 3, and 5, and sirtuin1 on day 1, 3, and 5, were increased significantly (P < 0.05). Lipoprotein lipase was unrelevant to FoxO1. By using insulin-like growth factor-I treating, expression of FoxO1 reduced at day 3 and 5 (P < 0.05), and significant differentiation of porcine preadipocyte with increasing number of filled-lipid cell and size of lipid droplets, GPDH activity and TG content were promoted. These results suggested that porcine FoxO1 gene took part in the regulation of adipose and was a negative transcription regulation factor in preadipocyte differentiation.

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-1alpha 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-1alpha, 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.