Fad104, a positive regulator of adipogenesis, negatively regulates osteoblast differentiation

Fad104 (factor for adipocyte differentiation 104) is a novel gene expressed temporarily in the early stages of adipocyte differentiation. Previously, we showed that fad104 promotes adipocyte differentiation in mouse 3T3-L1 cells and mouse embryonic fibroblasts (MEFs). Furthermore, we reported that implanted wild-type MEFs could develop into adipocytes, whereas fad104-deficient MEFs could not. Interestingly, bone-like tissues were only observed in the implants derived from fad104-deficient MEFs. This result implies that fad104 is involved in osteoblast differentiation. However, the functions of fad104 during osteogenesis are unknown. In this paper, we show that fad104 negatively regulates osteoblast differentiation. During the differentiation process, the level of fad104 expression decreased. Deletion of fad104 facilitated osteoblast differentiation in MEFs, and elevated the level of runx2, a master regulator of osteoblast differentiation. Disruption of fad104 suppressed BMP-2-mediated adipocyte differentiation in MEFs. In conclusion, we demonstrate that fad104 reciprocally regulates differentiation of adipocytes and osteoblast; functions as a positive regulator in adipocyte differentiation and as a negative regulator in osteoblast differentiation.     

Indispensable role of factor for adipocyte differentiation 104 (fad104) in lung maturation

Factor for adipocyte differentiation 104 (fad104) is a regulator of adipogenesis and osteogenesis. Our previous study showed that fad104-deficient mice died immediately after birth, suggesting fad104 to be essential for neonatal survival. However, the cause of this rapid death is unclear. Here, we demonstrate the role of fad104 in neonatal survival. Phenotypic and morphological analyses showed that fad104-deficient mice died due to cyanosis-associated lung dysplasia including atelectasis. Furthermore, immunohistochemistry revealed that FAD104 was strongly expressed in ATII cells in the developing lung. Most importantly, the ATII cells in lungs were immature, and impaired the expression of surfactant-associated proteins. Collectively, these results indicate that fad104 has an indispensable role in lung maturation, especially the maturation and differentiation of ATII cells.     

Targeted disruption of fad24, a regulator of adipogenesis,causes pre-implantation embryonic lethality due to the growth defect at the blastocyst stage

In previous studies, we identified a novel gene, factor for adipocyte differentiation 24 (fad24), which plays an important role during the early stages of adipogenesis in mouse 3T3-L1 cells. Moreover, overexpression of fad24 increased the number of smaller adipocytes in white adipose tissue and improved glucose metabolic activity in mice, thus indicating that fad24 functions as a regulator of adipogenesis in vivo. However, the physiological roles of fad24 in vivo are largely unknown. In this study, we attempted to generate fad24-deficient mice by gene targeting. No fad24-null mutants were recovered after embryonic day 9.5 (E9.5). Although fad24-null embryos were detected in an expected Mendelian ratio of genotypes at E3.5, none of the homozygous mutants developed into blastocysts. In vitro culture experiments revealed that fad24-null embryos develop normally to the morula stage but acquire growth defects during subsequent stages. The number of nuclei decreased in fad24-deficient morulae compared with that in wild-type ones. These results strongly suggested that fad24 is essential for pre-implantation in embryonic development, particularly for the progression to the blastocyst stage.     

CD38 deficiency suppresses adipogenesis and lipogenesis in adipose tissues through activating Sirt1/PPARγ signaling pathway

It has been recently reported that CD38 was highly expressed in adipose tissues from obese people and CD38‐deficient mice were resistant to high‐fat diet (HFD)‐induced obesity. However, the role of CD38 in the regulation of adipogenesis and lipogenesis is unknown. In this study, to explore the roles of CD38 in adipogenesis and lipogenesis in vivo and in vitro, obesity models were generated with male CD38^?/? and WT mice fed with HFD. The adipocyte differentiations were induced with MEFs from WT and CD38^?/? mice, 3T3‐L1 and C3H10T1/2 cells in vitro. The lipid accumulations and the alternations of CD38 and the genes involved in adipogenesis and lipogenesis were determined with the adipose tissues from the HFD‐fed mice or the MEFs, 3T3‐L1 and C3H10T1/2 cells during induction of adipocyte differentiation. The results showed that CD38^?/? male mice were significantly resistant to HFD‐induced obesity. CD38 expressions in adipocytes were significantly increased in WT mice fed with HFD, and the similar results were obtained from WT MEFs, 3T3‐L1 and C3H10T1/2 during induction of adipocyte differentiation. The expressions of PPARγ, AP2 and C/EBPα were markedly attenuated in adipocytes from HFD‐fed CD38^?/? mice and CD38^?/? MEFs at late stage of adipocyte differentiation. Moreover, the expressions of SREBP1 and FASN were also significantly decreased in CD38^?/? MEFs. Finally, the CD38 deficiency‐mediated activations of Sirt1 signalling were up‐regulated or down‐regulated by resveratrol and nicotinamide, respectively. These results suggest that CD38 deficiency impairs adipogenesis and lipogenesis through activating Sirt1/PPARγ‐FASN signalling pathway during the development of obesity.     

The role of microRNA-26b in human adipocyte differentiation and proliferation

Recent findings indicate that microRNAs (miRNAs) are involved in the regulatory network of adipogenesis and obesity. Thus far, only a few human miRNAs are known to function as adipogenic regulators, fanning interest in studies on the functional role of miRNAs during adipogenesis in humans. In a previous study, we used a microarray to assess miRNA expression during human preadipocyte differentiation. We found that expression of the miR-26b was increased in mature adipocytes. MiR-26b is an intronic miRNA located in the intron of CTDSP1 (carboxy terminal domain, RNA polymerase II, polypeptide A, small phosphatase 1). Target prediction and Renilla luciferase analyses revealed the phosphatase and tensin homolog gene (PTEN) as a putative target gene. In this study, we found that miR-26b was gradually upregulated during adipocyte differentiation. To understand the roles of miR-26b in adipogenesis, we adopted a loss-of-function approach to silence miR-26b stably in human preadipocytes. We found that miR-26b inhibition effectively suppressed adipocyte differentiation, as evidenced by decreased lipid droplets and the ability of miR-26b to decrease mRNA levels of adipocyte-specific molecular markers and triglyceride accumulation. Furthermore, the cell growth assay revealed that miR-26b inhibition promoted proliferation. Nevertheless, it had no effect on apoptosis. Taken together, these data indicate that miR-26b may be involved in adipogenesis and could be targeted for therapeutic intervention in obesity.     

miR-27a is a negative regulator of adipocyte differentiation via suppressing PPARγ expression

microRNAs (miRNAs) are non-coding small RNAs regulating gene expression, cell growth, and differentiation. Although several miRNAs have been implicated in cell growth and differentiation, it is barely understood their roles in adipocyte differentiation. In the present study, we reveal that miR-27a is involved in adipocyte differentiation by binding to the PPARγ 3′-UTR whose sequence motifs are highly conserved in mammals. During adipogenesis, the expression level of miR-27a was inversely correlated with that of adipogenic marker genes such as PPARγ and adiponectin. In white adipose tissue, miR-27a was more abundantly expressed in stromal vascular cell fraction than in mature adipocyte fraction. Ectopic expression of miR-27a in 3T3-L1 pre-adipocytes repressed adipocyte differentiation by reducing PPARγ expression. Interestingly, the level of miR-27a in mature adipocyte fraction of obese mice was down-regulated than that of lean mice. Together, these results suggest that miR-27a would suppress adipocyte differentiation through targeting PPARγ and thereby down-regulation of miR-27a might be associated with adipose tissue dysregulation in obesity.     

The F box protein S phase kinase-associated protein 2 regulates adipose mass and adipocyte number in vivo

Objective: The etiology of some obesity may involve adipocyte hyperplasia. However, the role of adipocyte number in establishing adipose mass is unclear. Cyclin‐dependent kinase inhibitor p27 regulates activity of cyclin/cyclin‐dependent kinase complexes responsible for cell cycle progression. This protein is critical for establishing adult adipocyte number, and p27 knockout increases adult adipocyte number. The SCF (for Skp1‐Cullin‐F‐box protein) complex targets proteins such as p27 for ubiquitin‐proteosome degradation; the F box protein S phase kinase‐associated protein 2 (Skp2), a component of the SCF complex, specifically recognizes p27 for degradation. We used Skp2 knockout (Skp2^?/?) mice to test whether Skp2 loss decreased adipose mass and adipocyte number. Research Methods and Procedures: We measured body weight, adipose mass, adipocyte diameter and number, and glucose tolerance in wild‐type (WT), Skp2^?/?, and p27^?/?Skp2^?/? mice. Mouse embryo fibroblasts (MEFs) from WT and Skp2^?/? fetuses were differentiated to determine whether Skp2 directly affected adipogenesis. Results: Skp2^?/? mice had a 50% decrease in both subcutaneous and visceral fat pad mass and adipocyte number; these decreases exceeded those in body weight, kidney, or muscle. To test the hypothesis that Skp2 effects on adipocyte number involved p27 accumulation, we used p27^?/?Skp2^?/? double knockout mice. The Skp2^?/? decrements in adipocyte number and fat pad mass were totally reversed in p27^?/?Skp2^?/? mice. Adipogenesis was inhibited in MEFs from Skp2^?/? vs. WT mice, and this inhibition was absent in MEFs from p27^?/?Skp2^?/? mice. Discussion: Our results indicate that Skp2 regulates adipogenesis and ultimate adipocyte number in vivo; thus, Skp2 may contribute to obesity involving adipocyte hyperplasia.     

Suppression of PPARγ through MKRN1-mediated ubiquitination and degradation prevents adipocyte differentiation

The central regulator of adipogenesis, PPARγ, is a nuclear receptor that is linked to obesity and metabolic diseases. Here we report that MKRN1 is an E3 ligase of PPARγ that induces its ubiquitination, followed by proteasome-dependent degradation. Furthermore, we identified two lysine sites at 184 and 185 that appear to be targeted for ubiquitination by MKRN1. Stable overexpression of MKRN1 reduced PPARγ protein levels and suppressed adipocyte differentiation in 3T3-L1 and C3H10T1/2 cells. In contrast, MKRN1 depletion stimulated adipocyte differentiation in these cells. Finally, MKRN1 knockout MEFs showed an increased capacity for adipocyte differentiation compared with wild-type MEFs, with a concomitant increase of PPARγ and adipogenic markers. Together, these data indicate that MKRN1 is an elusive PPARγ E3 ligase that targets PPARγ for proteasomal degradation by ubiquitin-dependent pathways, and further depict MKRN1 as a novel target for diseases involving PPARγ.     

PI3K/Akt is involved in brown adipogenesis mediated by growth differentiation factor-5 in association with activation of the Smad pathway

We have previously demonstrated promotion by growth differentiation factor-5 (GDF5) of brown adipogenesis for systemic energy expenditure through a mechanism relevant to activating the bone morphological protein (BMP) receptor/mothers against decapentaplegic homolog (Smad)/peroxisome proliferator-activated receptor gamma co-activator 1α (PGC-1α) pathway. Here, we show the involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in brown adipogenesis mediated by GDF5. Overexpression of GDF5 in cells expressing adipocyte protein-2 markedly accelerated the phosphorylation of Smad1/5/8 and Akt in white and brown adipose tissues. In brown adipose tissue from heterozygous GDF5^Rgsc451 mutant mice expressing a dominant-negative (DN) GDF5 under obesogenic conditions, the basal phosphorylation of Smad1/5/8 and Akt was significantly attenuated. Exposure to GDF5 not only promoted the phosphorylation of both Smad1/5/8 and Akt in cultured brown pre-adipocytes, but also up-regulated Pgc1a and uncoupling protein-1 expression in a manner sensitive to the PI3K/Akt inhibitor Ly294002 as well as retroviral infection with DN-Akt. GDF5 drastically promoted BMP-responsive luciferase reporter activity in a Ly294002-sensitive fashion. Both Ly294002 and DN-Akt markedly inhibited phosphorylation of Smad5 in the nuclei of brown pre-adipocytes. These results suggest that PI3K/Akt signals play a role in the GDF5-mediated brown adipogenesis through a mechanism related to activation of the Smad pathway.     

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.     

Rapamycin Inhibits Human Adipocyte Differentiation in Primary Culture

Objective: The immunosuppressant drug rapamycin, has been reported to inhibit 3T3‐L1 adipocyte differentiation by interfering with critical postconfluent mitoses that are required early on for successful differentiation of this cell line (clonal expansion phase). In contrast to the murine 3T3‐L1 preadipocyte cell line, human preadipocytes in primary culture do not undergo clonal expansion during differentiation. We investigated whether rapamycin could inhibit human adipocyte differentiation. Research Methods and Procedures: The effect of rapamycin on the induction of differentiation of human preadipocytes in primary culture into adipocytes was measured using Oil Red O staining and glycerol phosphate dehydrogenase activity. Results: We have observed that rapamycin severely curtails human adipocyte differentiation of both omental and abdominal subcutaneous preadipocytes (to 14% and 19% of standard differentiation, respectively). The rapamycin‐mediated inhibition of human adipocyte differentiation could be reversed in the presence of excess amounts of FK‐506, which displaces rapamycin from its intracellular receptor, FKPB12. Measurement of cytosolic protein and [³H]thymidine incorporation into DNA confirmed the absence of proliferation during differentiation of human preadipocytes in primary culture. Discussion: Our data indicate that rapamycin exerts important negative regulatory effects on adipogenesis in human preadipocytes, through a mechanism that does not depend on interruption of clonal expansion.     

Paragonimus westermani: Identification and characterization of the fasciclin I domain-containing protein

Paragonimus westermani is a trematode parasite that causes inflammatory lung disease as well as systemic infections in carnivorous mammals. The interaction of the parasite with host cells and paired worms is initiated by adhesion and plays an important role in parasite proliferation and differentiation. In this study, we isolated a cDNA encoding a P. westermani fasciclin I domain-containing protein (Pwfas-I). The fasiclin-I domain is suggested to be involved in cell adhesion, migration, and differentiation. Immunohistochemical analysis of P. westermani adult worms with polyclonal anti-Pwfas-I serum revealed immunoreactivity in the egg shells and the cells lining the sub-tegumental layer of adult worm throughout the contact regions of the cyst wall and paired worms. Using cell adhesion and spreading assays, we showed that Pwfas-I supports cell adhesion and spreading. Furthermore, we determined that the ανβ5 integrin was a functional receptor for the Pwfas-I. Taken together, these results suggest that Pwfas-I may be functional for the modulation of cell adhesion via binding with ανβ5 integrin in the extracellular matrix of Paragonimus.     

Skp2 promotes adipocyte differentiation via a p27-independent mechanism in primary mouse embryonic fibroblasts

Skp2, the substrate-binding subunit of an SCF ubiquitin ligase complex, is a key regulator of cell cycle progression that targets substrates for degradation by the 26S proteasome. We have now shown that ablation of Skp2 in primary mouse embryonic fibroblasts (MEFs) results both in impairment of adipocyte differentiation and in the accumulation of the cyclin-dependent kinase inhibitor p27^Kip1, a principal target of the SCF^Skp2 complex. Genetic ablation of p27^Kip1 in MEFs promoted both lipid accumulation and adipocyte-specific gene expression. However, depletion of p27^Kip1 by adenovirus-mediated RNA interference failed to correct the impairment of adipocyte differentiation in Skp2^-/- MEFs. In contrast, troglitazone, a high-affinity ligand for peroxisome proliferator-activated receptor γ (PPARγ), largely restored lipid accumulation and PPARγ gene expression in Skp2^−/− MEFs. Our data suggest that Skp2 plays an essential role in adipogenesis in MEFs in a manner that is at least in part independent of regulation of p27^Kip1 expression.     

Dexamethasone-induced selenoprotein S degradation is required for adipogenesis

Although adipogenesis is associated with induction of endoplasmic reticulum (ER) stress, the role of selenoprotein S (SEPS1), an ER resident selenoprotein known to regulate ER stress and ER-associated protein degradation, is unknown. We found an inverse relationship between SEPS1 level in adipose tissue and adiposity in mice. While SEPS1 expression was increased during adipogenesis, a markedly reduced SEPS1 protein level was found in the early phase of adipogenesis due to dexamethasone (DEX)-induced proteosomal degradation of SEPS1. Overexpression of SEPS1 in the early phase of cell differentiation resulted in impairment of adipogenesis with reduced levels of CCAAT/enhancer binding protein α and other adipocyte marker genes during the course of adipogenesis. Conversely, knockdown of SEPS1 resulted in the promotion of adipogenesis. Additionally, altered SEPS1 expression was associated with changes in expression of ER stress marker genes in the early phase of adipogenesis, and ubiquitin-proteasome system (UPS)-related ubiquitination and proteasome function. Our study reveals that SEPS1 is a novel anti-adipogenic selenoprotein that modulates ER stress- and UPS-dependent adipogenesis. Our results also identifies a novel function of DEX in the regulation of adipogenesis through induction of SEPS1 degradation. Taken together, DEX-dependent degradation of SEPS1 in the early phase of adipogenesis is necessary for initiating ER stress- and UPS-dependent maturation of adipocytes.