Coordinate Functional Regulation between Microsomal Prostaglandin E Synthase-1 (mPGES-1) and Peroxisome Proliferator-activated Receptor γ (PPARγ) in the Conversion of White-to-brown Adipocytes

Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated nuclear receptor and a master regulator of adipogenesis. Microsomal prostaglandin E (PGE) synthase-1 (mPGES-1) is an inducible enzyme that couples with cyclooxygenase-2 for the biosynthesis of PGE₂. In this study we demonstrate the existence of a coordinate functional interaction between PPARγ and mPGES-1 in controlling the process of pre-adipocyte differentiation in white adipose tissue (WAT). Adipocyte-specific PPARγ knock-out mice carrying an aP2 promoter-driven Cre recombinase transgene showed a blunted response to the adipogenic effects of a high fat diet. Pre-adipocytes from these knock-out mice showed loss of PPARγ and were resistant to rosiglitazone-induced WAT differentiation. In parallel, WAT from these mice showed increased expression of uncoupling protein 1, a mitochondrial enzyme that dissipates chemical energy as heat. Adipose tissue from mice lacking PPARγ also showed mPGES-1 up-regulation and increased PGE₂ levels. In turn, PGE₂ suppressed PPARγ expression and blocked rosiglitazone-induced pre-adipocyte differentiation toward white adipocytes while directly elevating uncoupling protein 1 expression and pre-adipocyte differentiation into mature beige/brite adipocytes. Consistently, pharmacological mPGES-1 inhibition directed pre-adipocyte differentiation toward white adipocytes while suppressing differentiation into beige/brite adipocytes. This browning effect was reproduced in knockdown experiments using a siRNA directed against mPGES-1. The effects of PGE₂ on pre-adipocyte differentiation were not seen in mice lacking PPARγ in adipose tissue and were not mirrored by other eicosanoids (i.e. leukotriene B₄). Taken together, these findings identify PGE₂ as a key regulator of white-to-brown adipogenesis and suggest the existence of a coordinate regulation of adipogenesis between PPARγ and mPGES-1.     

Up-regulated miR-145 Expression Inhibits Porcine Preadipocytes Differentiation by Targeting IRS1

Generally, most miRNAs that were up-regulated during differentiation promoted adipogenesis, but our research indicated that up-regulation of miR-145 in porcine preadipocytes did not promote but inhibit adipogenesis. In this study, miR-145 was significantly up-regulated during porcine dedifferentiated fat (DFAT) cells differentiation. In miR-145 overexpressed DFAT cells, adipogenesis was inhibited and triglycerides accumulation was decreased after hormone stimulation (P<0.05). Furthermore, up-regulation of miR-145 expression repressed induction of mRNA levels of adipogenic markers, such as CCAAT/enhancer-binding protein α (C/EBPα), and peroxisome proliferator-activated receptor γ2 (PPARγ2). These effects caused by miR-145 overexpression were mediated by Insulin receptor substrate 1 (IRS1) as a mechanism. These data suggested that induced miR-145 expression during differentiation could inhibit adipogenesis by targeting IRS1, and miR-145 may be novel agent for adipose tissue engineering.     

Hypoxia Promotes Osteogenesis but Suppresses Adipogenesis of Human Mesenchymal Stromal Cells in a Hypoxia-Inducible Factor-1 Dependent Manner

Background

Bone fracture initiates a series of cellular and molecular events including the expression of hypoxia-inducible factor (HIF)-1. HIF-1 is known to facilitate recruitment and differentiation of multipotent human mesenchymal stromal cells (hMSC). Therefore, we analyzed the impact of hypoxia and HIF-1 on the competitive differentiation potential of hMSCs towards adipogenic and osteogenic lineages.

Methodology/Principal Findings

Bone marrow derived primary hMSCs cultured for 2 weeks either under normoxic (app. 18% O₂) or hypoxic (less than 2% O₂) conditions were analyzed for the expression of MSC surface markers and for expression of the genes HIF1A, VEGFA, LDHA, PGK1, and GLUT1. Using conditioned medium, adipogenic or osteogenic differentiation as verified by Oil-Red-O or von-Kossa staining was induced in hMSCs under either normoxic or hypoxic conditions. The expression of HIF1A and VEGFA was measured by qPCR. A knockdown of HIF-1α by lentiviral transduction was performed, and the ability of the transduced hMSCs to differentiate into adipogenic and osteogenic lineages was analyzed. Hypoxia induced HIF-1α and HIF-1 target gene expression, but did not alter MSC phenotype or surface marker expression. Hypoxia (i) suppressed adipogenesis and associated HIF1A and PPARG gene expression in hMSCs and (ii) enhanced osteogenesis and associated HIF1A and RUNX2 gene expression. shRNA-mediated knockdown of HIF-1α enhanced adipogenesis under both normoxia and hypoxia, and suppressed hypoxia-induced osteogenesis.

Conclusions/Significance

Hypoxia promotes osteogenesis but suppresses adipogenesis of human MSCs in a competitive and HIF-1-dependent manner. We therefore conclude that the effects of hypoxia are crucial for effective bone healing, which may potentially lead to the development of novel therapeutic approaches.     

miR-17-5p and miR-106a are involved in the balance between osteogenic and adipogenic differentiation of adipose-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) can differentiate into several distinct cell types, including osteoblasts and adipocytes. The balance between osteogenic and adipogenic differentiation is disrupted in several osteogenic-related disorders, such as osteoporosis. So far, little is known about the molecular mechanisms that drive final lineage commitment of MSCs. In this study, we revealed that miR-17-5p and miR-106a have dual functions in the modulation of human adipose-derived mesenchymal stem cells (hADSCs) commitment by gain- and loss-of-function assays. They could promote adipogenesis and inhibit osteogenesis. Luciferase reporter assay, western blot and ELISA suggested BMP2 was a direct target of miR-17-5p and miR-106a. Downregulation of endogeneous BMP2 by RNA interference suppressed osteogenesis and increased adipogenesis, similar to the effect of miR-17-5p and miR-106a upregulation. Moreover, the inhibitory effects of miR-17-5p on osteogenic and adipogenic differentiation of hADSCs could be reversed by BMP2 RNA interference. In conclusion, miR-17-5p and miR-106a regulate osteogenic and adipogenic lineage commitment of hADSCs by directly targeting BMP2, and subsequently decreased osteogenic TAZ, MSX2 and Runx2, and increased adipogenic C/EBPα and PPARγ.     

Mechanistic target of rapamycin controls homeostasis of adipogenesis

Signaling mediated by the mechanistic target of rapamycin (mTOR) is believed to play a critical and positive role in adipogenesis, based on pharmacological evidence and genetic manipulation of mTOR regulators and targets. However, there is no direct genetic evidence for an autonomous role of mTOR itself in preadipocyte differentiation. To seek such evidence, we employed a conditional knockdown approach to deplete mTOR in preadipocytes. Surprisingly, while knockdown of S6K1, a target of mTOR, impairs 3T3-L1 preadipocyte differentiation, reduction of mTOR levels leads to increased differentiation. This enhanced adipogenesis requires the remaining mTOR activity, as mTOR inhibitors abolish differentiation in the mTOR knockdown cells. We also found that mTOR knockdown elevates the levels of CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ). Furthermore, partial reduction of mTOR levels alleviates inhibition of Akt by mTORC1 via IRS1, while at the same time maintaining its positive input through mTORC1 into the adipogenic program. The greater sensitivity of the IRS1-Akt pathway to mTOR levels provides a mechanism that explains the net outcome of enhanced adipogenesis through PPARγ upon mTOR knockdown. Our observations reveal an unexpected role of mTOR in suppressing adipogenesis and suggest that mTOR governs the homeostasis of the adipogenic process by modulating multiple signaling pathways.     

Zfp423 Promotes Adipogenic Differentiation of Bovine Stromal Vascular Cells

Intramuscular fat or marbling is critical for the palatability of beef. In mice, very recent studies show that adipocytes and fibroblasts share a common pool of progenitor cells, with Zinc finger protein 423 (Zfp423) as a key initiator of adipogenic differentiation. To evaluate the role of Zfp423 in intramuscular adipogenesis and marbling in beef cattle, we sampled beef muscle for separation of stromal vascular cells. These cells were immortalized with pCI neo-hEST2 and individual clones were selected by G418. A total of 288 clones (3×96 well plates) were isolated and induced to adipogenesis. The presence of adipocytes was assessed by Oil-Red-O staining. Three clones with high and low adipogenic potential respectively were selected for further analyses. In addition, fibro/adipogenic progenitor cells were selected using a surface marker, platelet derived growth factor receptor (PDGFR) α. The expression of Zfp423 was much higher (307.4±61.9%, P<0.05) in high adipogenic cells, while transforming growth factor (TGF)-β was higher (156.1±48.7%, P<0.05) in low adipogenic cells. Following adipogenic differentiation, the expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) were much higher (239.4±84.1% and 310.7±138.4%, respectively, P<0.05) in high adipogenic cells. Over-expression of Zfp423 in stromal vascular cells and cloned low adipogenic cells dramatically increased their adipogenic differentiation, accompanied with the inhibition of TGF-β expression. Zfp423 knockdown by shRNA in high adipogenic cells largely prevented their adipogenic differentiation. The differential regulation of Zfp423 and TGF-β between low and high adipogenic cells is associated with the DNA methylation in their promoters. In conclusion, data show that Zfp423 is a critical regulator of adipogenesis in stromal vascular cells of bovine muscle, and Zfp423 may provide a molecular target for enhancing intramuscular adipogenesis and marbling in beef cattle.     

Gentiopicroside promotes the osteogenesis of bone mesenchymal stem cells by modulation of β‐catenin‐BMP2 signalling pathway

Osteoporosis is characterized by increased bone fragility, and the drugs used at present to treat osteoporosis can cause adverse reactions. Gentiopicroside (GEN), a class of natural compounds with numerous biological activities such as anti‐resorptive properties and protective effects against bone loss. Therefore, the aim of this work was to explore the effect of GEN on bone mesenchymal stem cells (BMSCs) osteogenesis for a potential osteoporosis therapy. In vitro, BMSCs were exposed to GEN at different doses for 2 weeks, whereas in vivo, ovariectomized osteoporosis was established in mice and the therapeutic effect of GEN was evaluated for 3 months. Our results in vitro showed that GEN promoted the activity of alkaline phosphatase, increased the calcified nodules in BMSCs and up‐regulated the osteogenic factors (Runx2, OSX, OCN, OPN and BMP2). In vivo, GEN promoted the expression of Runx2, OCN and BMP2, increased the level of osteogenic parameters, and accelerated the osteogenesis of BMSCs by activating the BMP pathway and Wnt/β‐catenin pathway, effect that was inhibited using the BMP inhibitor Noggin and Wnt/β‐catenin inhibitor DKK1. Silencing the β‐catenin gene and BMP2 gene blocked the osteogenic differentiation induced by GEN in BMSCs. This block was also observed when only β‐catenin was silenced, although the knockout of BMP2 did not affect β‐catenin expression induced by GEN. Therefore, GEN promotes BMSC osteogenesis by regulating β‐catenin‐BMP signalling, providing a novel strategy in the treatment of osteoporosis.