Diet-induced obesity in stem cell antigen-1 KO mice

Stem Cell Antigen-1 (Sca-1) is a member of the lymphocyte-activated protein 6 family and has served as a marker for the identification of stem cells in various tissues, including fat depots. In vitro and in vivo studies suggest the possible involvement of Sca-1 in adipogenic differentiation and link Sca-1 antigenicity with adipocyte progenitors. Previously, we showed that Sca-1-enriched populations of ear mesenchymal stem cells possess enhanced capacity to differentiate into adipocytes. Additionally, we determined the natural frequency and localization of Sca-1-positive progenitor/stem cells in brown and white fat in situ. The present study addressed the question whether Sca-1 deficiency alters the white adipose tissue response to a high-saturated-fat diet. Our results show that Sca-1 null mice (Sca-1(-/-)) fed high-fat diet developed obesity equally well as wild-type mice, suggesting either an indirect in vivo effect of Sca-1 or a compensatory response to Sca-1 deficiency. However, contrary to wild-type mice, high fat diet-fed Sca-1(-/-) mice showed no alterations in serum adipocytokines. The data lead to the conclusion that Sca-1 is either redundant or a nonessential marker of adipose progenitor/stem cells. Nevertheless, since Sca-1-deficient mice displayed elevated blood glucose at fasting and exhibited glucose intolerance and insulin resistance, Sca-1 has subtle effects on adipose function. Thus, the Sca-1-deficient mice may provide a useful model for metabolic studies.     

Leukemia inhibitory factor influences the fate choice of mesenchymal progenitor cells

Osteoblasts and adipocytes derive from a common mesenchymal precursor, and in at least some circumstances, differentiation along these two lineages is inversely related. For example, we have recently observed that concomitant with inhibition of osteoblast differentiation and bone nodule formation, leukemia inhibitory factor (LIF) induces genes regulating lipid metabolism in fetal rat calvaria (RC) cell cultures. In this study, we further investigated the adipogenic capacity of LIF-treated RC cells. Quantitative analyses revealed that LIF increased the adipocyte differentiation induced by the peroxisome proliferator-activated receptor gamma agonist BRL49653 (BRL) in RC cell populations. Gene expression profiling of individual RC cell colonies in untreated cells or cells treated with LIF, BRL, or combined LIF-BRL suggested that some adipocytes arose from bipotential or other primitive precursors, including osteoprogenitors, since many colonies co-expressed osteoblast and adipocyte differentiation markers, whereas some arose from other cell pools, most likely committed preadipocytes present in the population. These analyses further suggested that LIF and BRL do not act at the same stages of the mesenchymal hierarchy, but rather that LIF modifies differentiation of precursor cells, whereas BRL acts later to favor adipocyte differentiation. Taken together, our data suggest that LIF increased adipocyte differentiation at least in part by altering the fate of osteoblastic cells and their precursors.     

快速诱导大鼠骨髓间充质干细胞成脂分化的培养体系

背景：目前常用的成脂刺激剂主要由胰岛素、地塞米松、IBMX和吲哚美辛组成,该体系涉及处理因素多、诱导周期长且对细胞毒性大,不利于脂肪形成抑制效应的研究。 目的：建立快速诱导大鼠骨髓间充质干细胞成脂分化的培养体系。 方法：大鼠全骨髓细胞原代培养,胰酶消化传代,富集形态均质的间充质干细胞,利用过氧化物酶体增殖物活化受体γ激动剂噻唑烷二酮类药物罗格列酮和吡咯列酮体外单独诱导大鼠骨髓间充质干细胞成脂分化,设立无糖、低糖和高糖DMEM三种基础培养体系,以经典的成脂刺激剂作为阳性对照,在诱导的不同时间点对分化细胞进行形态学观察和油红O染色。 结果与结论：与经典成脂刺激剂相比,罗格列酮或吡咯列酮单独均能诱导大鼠骨髓间充质干细胞向脂肪细胞分化,吡咯列酮诱导48 h和罗格列酮诱导72 h均可观察到富含脂滴或脂泡以及油红O染色的阳性细胞,吡咯列酮与罗格列酮的最佳诱导浓度分别为0.125 mmol/L和10 μmol/L,而高糖环境利于大鼠骨髓间充质干细胞成脂分化。说明基于高糖培养环境的以吡咯列酮或罗格列酮做为成脂刺激剂的培养体系可快速诱导大鼠骨髓间充质干细胞向脂肪细胞分化。     

Fucoxanthin and its metabolite, fucoxanthinol, suppress adipocyte differentiation in 3T3-L1 cells

Fucoxanthin is a major carotenoid found in edible seaweed such as Undaria pinnatifida and Hijikia fusiformis. We investigated the suppressive effects of fucoxanthin and its metabolite, fucoxanthinol, on the differentiation of 3T3-L1 preadipocytes to adipocytes. Fucoxanthin inhibited intercellular lipid accumulation during adipocyte differentiation of 3T3-L1 cells. Furthermore, fucoxanthin was converted to fucoxanthinol in 3T3-L1 cells. Fucoxanthinol also exhibited suppressive effects on lipid accumulation and decreased glycerol-3-phosphate dehydrogenase activity, an indicator of adipocyte differentiation. The suppressive effect of fucoxanthinol was stronger than that of fucoxanthin. In addition, in 3T3-L1 cells treated with fucoxanthin and fucoxanthinol, peroxisome proliferator-activated receptor gamma (PPARgamma), which regulates adipogenic gene expression, was down-regulated in a dose-dependent manner. These results suggest that fucoxanthin and fucoxanthinol inhibit the adipocyte differentiation of 3T3-L1 cells through down-regulation of PPARgamma. Fucoxanthinol had stronger suppressive effects than fucoxanthin on adipocyte differentiation in 3T3-L1 cells.     

猪脂肪源间充质干细胞诱导成脂分化及Krüppel样因子2的表达

目的旨在阐明猪脂肪源间充质干细胞(AMSCs)在诱导成脂分化过程中Krüppel样因子2(KLF2)的表达模式。方法采用I型胶原酶消化法处理猪脂肪组织,经原代和传代培养分离、纯化和扩增AMSCs,用流式细胞仪检测AMSCs纯度,在倒置显微镜下用形态学和油红O染色法检测AMSCs的成脂分化,用实时定量PCR检测KLF2的表达模式,并与过氧化物酶体增殖物激活受体2(PPARγ2)的表达进行了比较。结果分离、纯化的AMSCs,其间充质干细胞表面抗原CD29、CD44和CD105表达量分别为91.7%、95.1%和95.6%,而造血干细胞表面抗原CD34表达量仅为3.39%;在诱导分化2d后,个别细胞质中出现小脂滴,且含脂滴的细胞数量和脂滴量随诱导时间的延长而增加,在诱导分化第18天成脂分化率达48.2%;在诱导分化第2天、第8天和第16天,KLF2的表达量分别为1.84±0.206、1.07±0.072和0.83±0.095,而PPARγ2 mRNA的表达量分别为3.06±0.542、13.22±0.5736和15.11±1.073。结论获得纯度较高的AMSCs,具有良好的成脂分化潜能,KLF2的表达量在成脂分化早期的前脂肪细胞阶段增加,成脂分化开始后逐步减少,KLF2作为脂肪分化负调控转录因子而发挥作用。     

Interferon gamma inhibits adipogenesis in vitro and prevents marrow fat infiltration in oophorectomized mice

Interferon gamma (IFNγ) has been reported to induce osteoblastogenesis from mesenchymal stem cells (MSCs) both in vitro and in vivo. With ageing, adipocytes outnumber osteoblasts within the bone microenvironment leading to a decrease in bone formation. Since both osteoblasts and adipocytes are of mesenchymal origin, we hypothesized that IFNγ treatment might negatively affect adipogenesis while stimulating osteoblastogenesis in human MSC. To test this hypothesis, human MSCs were induced to differentiate into adipocytes in the presence or absence of osteogenic doses of IFNγ (1, 10, and 100 ng/ml). IFNγ-treated MSC showed a decrease in adipocyte differentiation and lipid deposition when compared with vehicle-treated controls. Additionally, adipogenic markers were significantly decreased by IFNγ treatment at the same doses that have been reported to have a strong osteogenic effect in vitro. Furthermore, DNA binding of peroxisome proliferator-activated receptor gamma was significantly lower in IFNγ-treated differentiating MSC. Subsequently, ovariectomized C57BL6 mice were treated with osteogenic doses of IFNγ three times a week for 6 weeks. In distal femur, treated mice showed significantly higher hematopoiesis concomitant with lower levels of fat volume/total volume, adipocyte number, and expression of adipogenic markers when compared with the vehicle-treated mice. Together, these findings demonstrate that, at osteogenic doses, IFNγ also acts as an inhibitor of adipogenesis in vitro and prevents marrow fat infiltration while favors hematopoiesis in ovariectomized mice.     

骨形态发生蛋白2抑制骨髓间充质干细胞成脂分化的优选浓度

BACKGROUND:Bone morphogenetic protein 2 cannot only promote osteogenesis, but also at different concentration ranges regulate the differentiation of bone marrow mesenchymal stem cells into adipocytes. Therefore, it is very important for the prevention and treatment of osteoporosis, if the range of concentrations of bone morphogenetic protein 2 can be found to regulate the differentiation of bone marrow mesenchymal stem cells into adipocytes, after the induction of bone marrow mesenchymal stem cells. OBJECTIVE:To determine the optimal concentration of bone morphogenetic protein 2 to inhibit the differentiation of bone marrow mesenchymal stem cells into adipocytes. METHODS:Bone marrow samples were obtained from the femur and tibia of rats. Primary cultured bone marrow mesenchymal stem cells were obtained by adherent culture method in vitro. Flow cytometry was used to identify the surface antigens of passage 2 bone marrow mesenchymal stem cells. Cells at passages 2 and 3 were taken and cultured in adipogenic induction medium for 3 days followed by the addition of 0.5, 5, 50, 100, 200 μg/L bone morphogenetic protein 2, or culture medium of bone marrow mesenchymal stem cells (control group), respectively. RESULTS AND CONCLUSION: Bone marrow mesenchymal stem cells cultured were identified by the flow cytometry. Oil red O staining results showed that the increasing number of lipid droplets was shown in bone marrow mesenchymal stem cells induced by bone morphogenetic protein 2 under the increasing concentration of 0.5 to 50 μg/L; while the number of lipid droplets in bone marrow mesenchymal stem cells decreased after 17 days of induction with bone morphogenetic protein 2 under the concentration of 100 to 200 μg/L. These findings indicate that 100 μg/L bone morphogenetic protein 2 can inhibit adipogenic differentiation of bone marrow mesenchymal stem cell, so as to reduce the formation of adipocytes in rats. Therefore, 100 μg/L bone morphogenetic protein 2 is better to inhibit adipogenic differentiation of bone marrow mesenchymal stem cells.     

Embryonic stem cell-derived adipogenesis

Key events leading to terminal differentiation of preadipocytes into adipocytes have been characterized in the recent years. However, master genes that commit progression from multipotent mesenchymal stem cell to the adipoblast stage of development have not yet been identified. The use of embryonic stem (ES) cells as a route to study early events in adipogenesis and to characterize factors involved in the decision of stem cells to follow the adipogenic pathway is described in this paper. The capacity of lif-/- and lifr-/- ES cells to undergo adipocyte differentiation is reported as an application of mutant ES cells to study gene function during the development of adipose cells.     

In vivo differentiation of brown adipocytes in adult mice: An electron microscopic study

The differentiation of brown adipocyte precursor cells was studied in interscapular brown adipose tissue of adult mice by electron microscopy. Different stages of cell differentiation were characterized in situ. Previous autoradiographic studies suggested that interstitial cells represent the precursor cells of fully differentiated brown adipocytes. The present observations provide morphological evidence for a progressive differentiation of interstitial stem cells into mature brown adipocytes. Four typical stages of development were identified: (1) interstitial cells, (2) protoadipocytes, (3) preadipocytes, and (4) mature brown adipocytes. Interstitial stem cells were small spindle shaped cells, situated between brown adipocytes and characterized by a high nuclear-cytoplasmic ratio, the scarcity of organelles, and the absence of lipid inclusions. Protoadipocytes resembled interstitial cells except that they contained a few tiny lipid droplets in their cytoplasm. Preadipocytes had a larger cytoplasm enclosing many mitochondria and lipid droplets; the smooth endoplasmic reticulum was well developed surrounding the lipid droplets, and was closely associated with the mitochondria. Preadipocytes had the typical structure of growing cells, developing long cytoplasmic processes between and around blood capillaries. Mature brown adipocytes represented the final stage of differentiation. Almost all their cellular volume was occupied by lipid droplets and numerous mitochondria with very dense cristae. Brown adipocytes were also characterized by a tight association with blood capillaries, as expected from metabolically active cells requiring oxygen and substrates. These observations provide direct ultrastructural evidence for a progressive differentiation of interstitial cells into brown adipocytes with a continuum of intermediate cellular types.     

土鳖虫含药血清干预骨髓间充质干细胞的成脂分化

背景：实验拟通过土鳖虫对激素诱导骨髓间充质干细胞向脂肪细胞分化的影响,解释中药土鳖虫治疗股骨头缺血性坏死的作用机制。 目的：观察土鳖虫对地塞米松诱导骨髓间充质干细胞成脂分化的干预作用。 方法：通过大剂量地塞米松诱导体外培养的骨髓间充质干细胞成脂分化,在诱导成脂的同时给予土鳖虫含药血清干预。检测干预6 d后细胞内成脂标志物三酰甘油、氧化物酶增殖物激活受体mRNA和脂肪酸相关蛋白mRNA的表达。 结果与结论：土鳖虫可抑制骨髓间充质干细胞分化过程中氧化物酶增殖物激活受体基因和脂肪酸相关蛋白基因的表达,降低三酰甘油水平,说明土鳖虫药物可以逆转因大量激素导致的骨髓间充质干细胞成脂分化增加。     

芥子碱对H_2O_2诱导BMSCs成脂分化的影响

目的:探讨中药有效单体芥子碱对过氧化氢(H_2O_2)诱导骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)成脂分化的影响。方法 :先通过全骨髓培养法和流式细胞术鉴定并筛选未分化的大鼠BMSCs;一方面利用H_2O_2诱导BMSCs成脂分化建立模型,一方面采用CCK-8实验检测芥子碱对BMSCs的毒性作用;模型建立成功与芥子碱药用浓度确定后进行油红O半定量实验检测细胞中脂滴含量,筛选出最佳的药物浓度;随后采用油红O染色拍片直接观察药物干预24 h后对BMSCs成脂分化的影响,并且采用qPCR和Western blot实验检测BMSCs中成脂分化相关蛋白——脂肪细胞蛋白2(aP2)、过氧化物酶体增殖物激活受体γ(PPARγ)和葡萄糖转运蛋白4(Glut4)的变化情况。结果:浓度为200μmol/L的H_2O_2作用1 h可诱导BMSCs成脂分化;CCK-8实验结果显示在芥子碱浓度40μmol/L的条件下对BMSCs的活力没有显著影响,同时结合油红O半定量实验结果筛选出芥子碱抑制其成脂分化的最佳浓度为15μmol/L;油红O染色实验观察到芥子碱组(15μmol/L)组脂滴较模型组数量明显减少,qPCR和Western blot实验结果亦显示正常对照组和芥子碱组aP2、PPARγ和Glut4表达均低于模型组(P0.01)。结论:芥子碱能够抑制H_2O_2诱导BMSCs向脂肪细胞分化,其机制可能与PPARγ/AMPK信号通路有关。     

Derivation of adipocytes from human embryonic stem cells

Human embryonic stem (hES) cells are undifferentiated and pluripotent cells that hold great therapeutic potential, but are hampered by our limited knowledge to promote specific cell differentiation. Here we provide the first report of the directed differentiation of hES cells into adipocytes. Embryoid bodies (EBs) derived from hES cells are shown to respond to factors that promote adipogenesis. Differentiated cells were observed that displayed the key features of adipocytes, i.e., expression of specific molecular markers, such as peroxisome proliferator-activated receptor gamma2 (PPARgamma2), adipocyte fatty acid binding protein (aP2) and adiponectin, the secretion of leptin, and the accumulation of lipid droplets in cytoplasm. Taken together, our results demonstrate that adipocytes derived from hES cells in vitro can provide a novel model system to study human adipogenesis and obesity.     

Activation of Sirt1 decreases adipocyte formation during osteoblast differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSC) can differentiate into osteoblasts, adipocytes, chondrocytes and myoblasts. It has been suggested that a reciprocal relationship exists between the differentiation of MSC into osteoblasts and adipocytes. Peroxisome proliferator-activated receptor gamma2 (PPARgamma2) is a key element for the differentiation into adipocytes. Activation of the nuclear protein deacetylase Sirt1 has recently been shown to decrease adipocyte development from preadipocytes via inhibition of PPARgamma2. In vitro, MSC differentiate to osteoblasts when exposed to bone-inducing medium. However, adipocytes are also developed. In the present study we have targeted Sirt1 to control adipocyte development during differentiation of MSC into osteoblasts. The finding that resveratrol and isonicotinamide markedly inhibited adipocyte and promoted osteoblast differentiation demonstrates an interesting alternative to PPARgamma antagonists. These results are important for the evolving field of cell-based tissue engineering, but may also be relevant in the search for new treatments of osteoporosis.     

Commitment of mouse embryonic stem cells to the adipocyte lineage requires retinoic acid receptor beta and active GSK3

Key events leading to terminal differentiation of preadipocytes into adipocytes have been identified in recent years. However, signaling pathways involved in the decision of stem cells to follow the adipogenic lineage have not yet been characterized. We have previously shown that differentiating mouse embryonic stem (mES) cells give rise to functional adipocytes upon an early treatment with retinoic acid (RA). The goal of this work was to identify regulators of RA-induced commitment of mES cells to the adipocyte lineage. First, we investigated the role of RA receptor (RAR) isotypes in the induction of mES cell adipogenesis. Using synthetic retinoids selective of RAR isotypes, we show that RARbeta activation is both sufficient and necessary to trigger commitment of mES cells to adipocytes. Then, we performed a small-scale drug screening to find signaling pathways involved in RARbeta-induced mES cell adipogenesis. We show that pharmacological inhibitors of glycogen synthase kinase (GSK) 3, completely inhibit RARbeta-induced adipogenesis in mES cells. This finding uncovers the requirement of active GSK3 in RARbeta-induced commitment of mES cells toward the adipocyte lineage. Finally, we investigated the role of the Wnt pathway, in which GSK3 is a critical negative regulator, in adipocyte commitment by analyzing Wnt pathway activity in RA- and RARbeta-induced mES cell adipogenesis. Our results suggest that although RARbeta and active GSK3 are required for RA-induced adipogenesis, they might be acting through a Wnt pathway-independent mechanism.     

Gene Expression Pattern of Human Chorion-Derived Mesenchymal Stem Cells during Adipogenic Differentiation

Purpose: The aim of this study was to identify the adipocyte-specific gene expression patterns in chorion-derived mesenchymal stem cells during adipogenic differentiation. Materials and Methods: Chorionic cells were isolated from the third trimester chorions from human placenta at birth and identified morphologically and by fluorescence-activated cell sorting analysis. After inducing adipogenic differentiation for 28 days, cells at days 3, 10, 21 and 28 were analyzed by Oil red O staining and RNA extraction in order to assess the expression levels of adipocyte marker genes, including CCAAT-enhancer binding protein α (C/EBPα), peroxisome proliferator- activated receptor γ (PPARγ), fatty acid binding protein 4 (FABP4) and Glycerol-3-phosphate dehydrogenase (GPD2). Cells not induced for differentiation were compared with the induced cells as a control group. Results: Chorion-derived cells showed the same pattern as fibroblasts, and expressed CD73, CD105, and CD166 antigens, but not CD45, CD34, and HLA-DR antigens. On day 3 after differentiation, cells began to stain positively upon Oil red O staining, and continuously increased in lipid granules for 4 weeks. The expression level of C/EBPα increased 4.6 fold on day 3 after induction, and continued to increase for 4 weeks. PPARγ was expressed at a maximum of 2.9 fold on day 21. FABP4 and GPD2 were significantly expressed at 4.7- and 3.0-fold, respectively, on day 21, compared to controls, and further increased thereafter. Conclusion: Human chorion-derived mesenchymal stem cells exhibited the sequential expression pattern of adipocyte marker genes during differentiation, corresponding to adipogenesis.     

Nuclear lamin A inhibits adipocyte differentiation: implications for Dunnigan-type familial partial lipodystrophy

Mutations in the LMNA gene encoding A-type lamins cause several diseases, including Emery-Dreifuss muscular dystrophy and Dunnigan-type familial partial lipodystrophy (FPLD). We analyzed differentiation of 3T3-L1 preadipocytes to adipocytes in cells overexpressing wild-type lamin A as well as lamin A with amino acid substitutions at position 482 that cause FPLD. We also examined adipogenic conversion of mouse embryonic fibroblasts lacking A-type lamins. Overexpression of both wild-type and mutant lamin A inhibited lipid accumulation, triglyceride synthesis and expression of adipogenic markers. This was associated with inhibition of expression of peroxisome-proliferator-activated receptor gamma 2 (PPARgamma2) and Glut4. In contrast, embryonic fibroblasts lacking A-type lamins accumulated more intracellular lipid and exhibited elevated de novo triglyceride synthesis compared with wild-type fibroblasts. They also had increased basal phosphorylation of AKT1, a mediator of insulin signaling. We conclude that A-type lamins act as inhibitors of adipocyte differentiation, possibly by affecting PPARgamma2 and insulin signaling.     

Curcumin represses adipogenic differentiation of human bone marrow mesenchymal stem cells via inhibiting kruppel-like factor 15 expression

Understanding the mechanisms of adipogenic differentiation may lead to the discovery of novel therapeutic targets for obesity. The natural plant polyphenol compound curcumin can improve obesity-associated inflammation and diabetes in obese mice. The role of curcumin in adipogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs) is still unclear. We used hMSCs to investigate the details of the mechanism underlying the adipogenic effects of curcumin. At different time points (i.e., 5 days and 10 days) of hMSC adipocyte differentiation, an accumulation of large lipid droplets was analyzed in Oil Red O-stained cultured cells in two curcumin (5?μM and 10?μM) groups and the control group. The cells were also harvested for the detection of mRNA and protein expressions by quantitative real-time polymerase chain reaction and Western blot analysis. The results showed that curcumin can suppresses adipocyte differentiation in a dose-dependent manner and inhibited the expression of PPARγ, C/EBPα, and FABP4. Importantly, curcumin can also suppress the expression of Kruppel-like factor 15, which may bind to the PPARγ promoter, resulting in downregulation of PPARγ expression to inhibit the adipogenic differentiation of hMSCs.     

Inhibition of Lysine-Specific Demethylase-1 (LSD1/KDM1A) Promotes the Adipogenic Differentiation of hESCs Through H3K4 Methylation

Given their totipotency, human embryonic stem cells (hESCs) can differentiate into all types of cells, including adipocytes, and provide an excellent research model for studying diseases associated with the metabolism of adipocytes, such as obesity and diabetes mellitus. Epigenetic regulation, including DNA methylation and histone modification, plays an essential role in the development and differentiation of hESCs. Lysine-specific demethylase 1 (LSD1), a well-characterized histone-modifying enzyme, demethylates dimethylated histone H3 lysine 4 (H3K4) through a flavin adenine dinucleotide (FAD)-dependent oxidative reaction. LSD1 affects the growth and differentiation of human and mouse ES cells, and the deletion of this gene in mice leads to embryonic lethality. Here, we investigated the functional role of LSD1 during the adipogenic differentiation of hESCs involving the demethylation of H3K4. We also found that treating hESCs with the LSD1 inhibitor CBB1007 promotes the adipogenic differentiation of hESCs.