Macrophage-induced preadipocyte survival depends on signaling through Akt, ERK1/2, and reactive oxygen species

Obesity is associated with adipose tissue remodeling, characterized by macrophage accumulation, adipocyte hypertrophy, and apoptosis. We previously reported that macrophage-conditioned medium (MacCM) protects preadipocytes from apoptosis, due to serum withdrawal, in a platelet-derived growth factor (PDGF)-dependent manner. We have now investigated the role of intracellular signaling pathways, activated in response to MacCM versus PDGF, in promoting preadipocyte survival. Exposure of 3T3-L1 preadipocytes to J774A.1-MacCM or PDGF strongly stimulated Akt and ERK1/2 phosphorylation from initially undetectable levels. Inhibition of the upstream regulators of Akt or ERK1/2, i.e. phosphoinositide 3-kinase (PI3K; using wortmannin or LY294002) or MEK1/2 (using UO126 or PD98509), abrogated the respective phosphorylation responses, and significantly impaired pro-survival activity. J774A.1-MacCM increased reactive oxygen species (ROS) levels by 3.4-fold, and diphenyleneiodonium (DPI) or N-acetyl cysteine (NAC) significantly inhibited pro-survival signaling and preadipocyte survival in response to J774A.1-MacCM. Serum withdrawal itself also increased ROS levels (2.1-fold), and the associated cell death was attenuated by DPI or NAC. In summary, J774A.1-MacCM-dependent 3T3-L1 preadipocyte survival requires the Akt and ERK1/2 signaling pathways. Furthermore, ROS generation by J774A.1-MacCM is required for Akt and ERK1/2 signaling to promote 3T3-L1 preadipocyte survival. These data suggest potential mechanisms by which macrophages may alter preadipocyte fate.     

Regional variation in adipogenesis and IGF regulatory proteins in the fetal baboon

Intrauterine growth rate is associated with body distribution in adulthood suggesting differential response of fetal fat depots to nutritional modifications. We hypothesize that there is regional differences in fetal adipogenesis, in part, due to depot-specific regulation of the availability of insulin growth factors. In near-term baboon fetuses (n = 3-5), the subcutaneous abdominal vs. omental preadipocytes had (1) more extensive lipid accumulation as assessed by BODIPY (lipid staining) to DAPI (nuclei) absorbance ratios (mean ± SEM; 0.51 ± 0.21, 0.35 ± 0.09, p < 0.05), (2) lower (p < 0.05) secretion of IGF-binding protein 4 (9.6 ± 1.2 vs. 17.4 ± 2.8 ng/ml) and its protease pregnancy associated plasma protein A (24.6 ± 1.9 vs. 39.1 ± 6.3 μIU/ml), (3) lower protein expression of IGF2 “clearance” receptor in cell lysate (0.28 ± 0.03 vs. 0.53 ± 0.02 OD U/mm², p < 0.05); all variables were intermediate in femoral preadipocytes. The regional variation of the adipogenesis and the IGF regulatory pathway set the stage for differential responsiveness of fat depots to external signals.     

低氧诱导大鼠内脏脂肪组织炎症状态对SePP1的影响

目的：既往研究显示SePP1具有一定的抗氧化作用,而随着年龄的增加机体逐步出现一个慢性低氧、炎症状态,我们通过4％O2浓度体外培养大鼠脂肪前体细胞模拟其体内的低氧状态,进而观察常氧（21％O2）及低氧（4％02）状态下大鼠脂肪前体细胞中炎症因子（IL-6,MCP-1,SePP1）水平的变化及不同状态下硒蛋白SePP1水平的变化。方法：取6—8周SD大鼠肾周脂肪前体细胞,分别于常氧（21％O2）及低氧（4％O2）状态下进行体外培养,诱导分化后采用油红0染色进行鉴定,至第三代后,分别采用PCR及Westem Blot技术检测两种状态下脂肪前体细胞中1L-6,MCP-1,SePPl基因及蛋白表达的不同变化,同时观察不同氧浓度对脂肪前体细胞增殖的影响。结果：4％氧浓度状态下培养的脂肪前体细胞中IL-6,MCP-1的基因及蛋白表达均明显高于正常氧浓度下的脂肪前体细胞,而SePP1的基因及蛋白表达均下降,且低氧状态下脂肪前体细胞增殖较常氧状态下加快。结论：低氧培养可进一步使机体内脏脂肪组织堆积加重,造成脂肪前体细胞的炎症状态,并且可导致SePP1的表达下降,而SePP1具有一定的抗氧化作用,与机体动脉粥样硬化等心血管疾病的发生、发展有一定的关联,本实验结论为通过干预体内SePP1的水平为靶点治疗动脉粥样硬化提供了一定的实验依据,为进一步研究SePP1在低氧状态下对动脉粥样硬化的影响及作用机制提供了一定的试验基础。     

Myotube formation is affected by adipogenic lineage cells in a cell-to-cell contact-independent manner

Intramuscular adipose tissue (IMAT) formation is observed in some pathological conditions such as Duchenne muscular dystrophy (DMD) and sarcopenia. Several studies have suggested that IMAT formation is not only negatively correlated with skeletal muscle mass but also causes decreased muscle contraction in sarcopenia. In the present study, we examined w hether adipocytes affect myogenesis. For this purpose, skeletal muscle progenitor cells were transfected with siRNA of PPARγ (siPPARγ) in an attempt to inhibit adipogenesis. Myosin heavy chain (MHC)-positive myotube formation was promoted in cells transfected with siPPARγ compared to that of cells transfected with control siRNA. To determine whether direct cell-to-cell contact between adipocytes and myoblasts is a prerequisite for adipocytes to affect myogenesis, skeletal muscle progenitor cells were cocultured with pre- or mature adipocytes in a Transwell coculture system. MHC-positive myotube formation was inhibited when skeletal muscle progenitor cells were cocultured with mature adipocytes, but was promoted when they were cocultured with preadipocytes. Similar effects were observed when pre- or mature adipocyte-conditioned medium was used. These results indicate that preadipocytes play an important role in maintaining skeletal muscle mass by promoting myogenesis; once differentiated, the resulting mature adipocytes negatively affect myogenesis, leading to the muscle deterioration observed in skeletal muscle pathologies.     

猪脂肪前体细胞分化过程中聚脂相关基因的表达模式

本实验采用胶原酶消化法分离猪皮下脂肪前体细胞,用含850 nmol/L 胰岛素和50 nmol/L地塞米松的诱导培养液进行诱导,采用油红O提取法测定了细胞中的甘油三酯含量,同时采用实时定量RT-PCR方法检测了细胞分化过程中聚脂相关基因的表达.结果显示:转录因子PPAR γ和C/EBP β在诱导后12 h即迅速表达,SREBP-1 mRNA表达水平在诱导后12 h出现显著下调,随后逐渐升高,96 h达到最高水平;脂肪合成相关酶基因GPDH、FAS、ACC和LPL呈现出与SREBP-1相似的表达模式;脂肪酸转运相关基因aP2、FAT、FATP1与VLDLR的表达量随着细胞分化过程的延长而不断增加,并且与细胞内甘油三酯的含量变化高度相关.本实验结果表明,PPAR γ、C/EBP β和SREBP-1可能是调控猪脂肪前体细胞分化的关键转录因子.猪皮下脂肪组织在聚脂过程中,在分化早期可能以脂肪细胞自身合成脂肪酸为主,而后期则主要依赖细胞外脂肪酸的跨膜转运.这些结果可能有助于揭示脂肪细胞的分化调控规律.     

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.     

Transplantation and differentiation of donor cells in the cloned pigs

The application of nuclear transfer technology is an interesting approach to investigate stem and progenitor cell transplantation therapy. If stem cells are used as a nuclear donor, donor cells can engraft into cloned animals without histocompatible problems. However, it is still uncertain whether donor cells can engraft to cloned animal and differentiate in vivo. To address this problem, we transplanted donor cells to dermal tissues of cloned pigs developed by using preadipocytes as donor cells. Preadipocytes are adipocytic progenitor which can differentiate to mature adipocytes in vitro. We showed that the donor preadipocytes were successfully transplanted into the cloned pigs without immune rejection and they differentiated into mature adipocytes in vivo 3 weeks after transplantation. In contrast, allogenic control preadipocytes, which can differentiate in vitro, did not differentiate in vivo. These results indicate that donor progenitor cells can differentiate in cloned animal.     

LMO4 modulates proliferation and differentiation of 3T3-L1 preadipocytes

Previous microarray analyses revealed that LMO4 is expressed in 3T3-L1 preadipocytes, however, its roles in adipogenesis are unknown. In the present study, using RT-PCR sequencing and quantitative real-time RT-PCR, we confirmed that LMO4 gene is expressed in 3T3-L1 preadipocytes and its expression peaks at the early stage of 3T3-L1 preadipocyte differentiation. Further analyses showed that LMO4 knockdown decreased the proliferation of 3T3-L1 preadipocytes, and attenuated the differentiation of 3T3-L1 preadipocytes, as evidenced by reduced lipid accumulation and down-regulation of PPARγ gene expression. Collectively, our findings indicate that LMO4 is a novel modulator of adipogenesis.