CCDC69 was previously identified as a gene upregulated during adipogenic differentiation of human Simpson–Golabi–Behmel syndrome (SGBS) preadipocytes but not mouse 3T3-L1 preadipocytes. Here, we investigated the role of CCDC69 in adipogenesis using SGBS cells. CCDC69 expression during adipogenic differentiation was assessed by quantitative reverse transcription PCR and Western blotting. CCDC69 function was examined by adenovirus-mediated overexpression and knockdown, followed by analyses of lipid accumulation, adipocyte marker expression, cell morphology, and cytoskeletal organization. CCDC69 expression increased during adipogenesis. CCDC69 overexpression enhanced adipogenesis, as indicated by increased lipid accumulation, a higher proportion of cells containing lipid droplets, and increased expression of adipocyte marker genes and proteins. Unexpectedly, CCDC69 knockdown also enhanced adipogenesis. Time-course analysis showed that CCDC69 overexpression suppressed PPARG mRNA expression during early adipogenesis but increased its expression at later stages, whereas knockdown increased PPARG expression at later stages. CCDC69 overexpression induced a flattened cell morphology accompanied by reduced fibrillar α-tubulin structures and F-actin stress fibers and increased punctate F-actin structures, whereas knockdown increased the number of small, round cells. These findings indicate that CCDC69 may regulate adipogenesis in a stage-dependent manner and is associated with adipocyte morphology and cytoskeletal organization.

Adipogenesis involves intricate molecular mechanisms regulated by various transcription factors and signaling pathways. In this study, we aimed to identify factors specifically induced during adipogenesis in the human preadipocyte cell line, SGBS, but not in the mouse preadipocyte cell line, 3T3-L1. Microarray analysis revealed distinct gene expression profiles, with 1460 genes induced in SGBS cells and 1297 genes induced in 3T3-L1 cells during adipogenesis, with only 297 genes commonly induced. Among the genes uniquely induced in SGBS cells, we focused on GALNT15, which encodes polypeptide N-acetylgalactosaminyltransferase-15. Its expression increased transiently during adipogenesis in SGBS cells but remained low in 3T3-L1 cells. Overexpression of GALNT15 increased mRNA levels of CCAAT-enhancer binding protein (C/EBPα) and leptin but had no significant impact on adipogenesis in SGBS cells. Conversely, knockdown of GALNT15 suppressed mRNA expression of adipocyte marker genes, reduced lipid accumulation, and decreased the percentage of cells with oil droplets. The induction of C/EBPα and peroxisome proliferator-activated receptor γ during adipogenesis was promoted or suppressed in SGBS cells subjected to overexpression or knockdown of GALNT15, respectively. These data suggest that polypeptide N-acetylgalactosaminyltransferase-15 is a novel regulatory molecule that enhances adipogenesis in SGBS cells.

Adipogenesis involves intricate molecular mechanisms regulated by various transcription factors and signaling pathways. In this study, we aimed to identify factors specifically induced during adipogenesis in the human preadipocyte cell line, SGBS, but not in the mouse preadipocyte cell line, 3T3-L1. Microarray analysis revealed distinct gene expression profiles, with 1460 genes induced in SGBS cells and 1297 genes induced in 3T3-L1 cells during adipogenesis, with only 297 genes commonly induced. Among the genes uniquely induced in SGBS cells, we focused on GALNT15 , which encodes polypeptide N-acetylgalactosaminyl transferase-15. Its expression increased transiently during adipogenesis in SGBS cells but remained low in 3T3-L1 cells. Overexpression of GALNT15 increased mRNA levels of CCAAT-enhancer binding protein (C/EBPα) and leptin but had no significant impact on adipogenesis in SGBS cells. Conversely, knockdown of GALNT15 suppressed mRNA expression of adipocyte marker genes, reduced lipid accumulation, and decreased the percentage of cells with oil droplets. The induction of C/EBPα and peroxisome proliferator-activated receptor γ during adipogenesis was promoted or suppressed in SGBS cells subjected to overexpression or knockdown of GALNT1 5, respectively. These data suggest that GALNT15 is a novel regulatory molecule that enhances adipogenesis in SGBS cells.

The aim of this study was to identify genes that are specifically expressed in pancreatic islet β-cells (hereafter referred to as β-cells). Large-scale complementary DNA-sequencing analysis was performed for 3,429 expressed sequence tags derived from murine MIN6 β-cells, through homology comparisons using the GenBank database. Three individual ESTs were found to code for protease serine S1 family member 53 (Prss53). Prss53 mRNA is processed into both a short and long form, which encode 482 and 552 amino acids, respectively. Transient overexpression of myc-tagged Prss53 in COS-7 cells showed that Prss53 was strongly associated with the luminal surfaces of organellar membranes and that it underwent signal peptide cleavage and N-glycosylation. Immunoelectron microscopy and western blotting revealed that Prss53 localized to mitochondria in MIN6 cells. Short hairpin RNA-mediated Prss53 knockdown resulted in Ppargc1a downregulation and Ucp2 and Glut2 upregulation. JC-1 staining revealed that the mitochondria were depolarized in Prss53-knockdown MIN6 cells; however, no change was observed in glucose-stimulated insulin secretion. Our results suggest that mitochondrial Prss53 expression plays an important role in maintaining the health of β-cells.

Background/Aim: D-dopachrome tautomerase (DDT) is a macrophage migration inhibitory factor (MIF) homolog that promotes cell growth via CD74, a MIF cell surface receptor, in some types of tumors. We previously found that DDT acts as an anti-obesity adipokine independent of MIF. To understand the intrinsic properties of these two cytokines, a comparison of their actions in various tissues is necessary. In this study, we investigated the involvement of DDT in HepG2 cell (a human hepatoma cell line) proliferation, which is known to be promoted by MIF. Materials and Methods: Cell proliferation and gene expression were evaluated in HepG2 cells expressing short hairpin RNA against the DDT gene. Results: Inhibition of cell proliferation and reduced expression levels of cyclin D1 were observed in DDT-knockdown HepG2 cells. The inhibited proliferation was restored by administration of recombinant DDT. Conclusion: DDT promotes cell proliferation in HepG2 cells; therefore, its action may be similar to that of MIF.

In adipose tissue, D-dopachrome tautomerase (DDT), a cytokine with structural similarity to macrophage migration inhibitory factor, is mainly expressed in adipocytes rather than preadipocytes and acts as an anti-obesity adipokine in an autocrine manner. However, its transcriptional regulation is largely unknown. In order to explore molecules affecting DDT transcription, a chemical library screening using HEK293 cells stably expressing a DDT promoter-reporter construct was performed. Several derivatives of 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), an AMP-activated protein kinase (AMPK) activator, were identified as transcriptional activators of the DDT gene. Furthermore, DDT mRNA levels were reduced in SGBS adipocytes treated with compound C, an AMPK inhibitor, suggesting involvement of AMPK in DDT transcription. Overexpression of the FOXO1 constitutive active form reduced transcriptional activity of the DDT gene in SGBS cells, but increased it in HEK293 cells. Cell-type specific effects were also observed in the DDT gene expression of cells treated with AS1842856, a FOXO1 inhibitor. Finally, involvement of the mammalian target of rapamycin (mTOR) signaling in DDT transcription in SGBS adipocytes was investigated. Rapamycin, an inhibitor of mTOR, increased DDT mRNA levels and attenuated the inhibitory effects of compound C on DDT mRNA levels in SGBS adipocytes. In conclusion, DDT transcription may be regulated in a cell-dependent manner, and were enhanced by AMPK activation in SGBS adipocytes through inhibiting the mTOR signaling.

Growth hormone (GH)-producing pituitary adenomas (PAs) in childhood or young adulthood are rare, and the details surrounding these tumors remain enigmatic. We present the clinical, pathological and genetic features of this disease.

Mechanical stimuli regulate fundamental cell processes such as proliferation, differentiation, and morphogenesis. We attempted to identify microRNA (miRNA) whose expression is changed during compressive treatment in MC3T3-E1, a pre-osteoblastic cell line. Microarray analysis followed by reverse transcription-quantitative polymerase chain reaction revealed that compressive force at 294 Pa for 24 h in MC3T3-E1 cells increased levels of miR-494-3p, miR-146a-5p, miR-210-3p, and miR-1247-3p. Among these miRNAs, miR-494-3p was found to inhibit cell proliferation in MC3T3-E1 cells. Furthermore, cells subjected to compressive force showed slower cell growth compared with control cells. Levels of mRNA for fibroblast growth factor receptor 2 (FGFR2) and Rho-associated coiled-coil kinase 1 (ROCK1), which were predicted to be targets of miR-494-3p, were decreased by compressive force or overexpression of miR-494-3p mimics in MC3T3-E1 cells. Furthermore, binding sites of miR-494-3p within 3'-untranslated regions of Fgfr2 and Rock1 were determined using luciferase reporter assay. In conclusion, compressive force affected expressions of several miRNAs including miR-494-3p in MC3T3-E1 cells. Compressive force might inhibit cell proliferation in osteoblasts by up-regulating miR-494-3p followed by FGFR2 and ROCK1 gene repressions. (C) 2015, The Society for Biotechnology, Japan. All rights reserved.

Although the cause of familial isolated pituitary adenoma (FIPA) remains unknown in many cases, germline mutations in the aryl hydrocarbon receptor-interacting protein ( AIP ) gene were identified in approximately 20 % of families with FIPA. We investigated the AIP gene mutation by a standard sequencing method in 12 members of a Japanese two-generation FIPA family, which includes 3 patients with early-onset acromegaly. Multiplex ligation-dependent probe amplification analysis in a tumor sample was attempted to examine the loss of heterozygosity (LOH) in the locus. The effect of the detected mutation on cell proliferation was investigated. A germline mutation of c.943C > T (p.Q315X) generating an AIP protein with the C-terminal end deleted was found in the FIPA family. Biallelic inactivation of AIP by a combination of the germline mutation and LOH at 11q13 was confirmed in the tumor. The nonsense mutation disrupted the ability to inhibit cell proliferation. We conclude that p.Q315X mutation in the AIP gene is a pathogenic variant and the C-terminal region of AIP plays an important role in the predisposition to pituitary adenomas.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental toxin that induces apoptosis of neurons and a pro-inflammatory response in microglial cells. First, we found that TCDD induced proliferation of HAPI microglial cells in a dose- and time-dependent manner. Flow cytometry analysis showed that this proliferation by TCDD was due to mainly enhancing the G1 to S phase transition. Next, it was found that TCDD treatment led to up-regulation of cyclin D1, which induces cell cycle progression from G1 to S phase, in a time-dependent manner. As for molecular mechanism, we revealed that TCDD was capable of inducing Akt phosphorylation and activation, resulting in phosphorylation and inactivation of glycogen synthase kinase-3β (GSK-3β). Inactivated GSK-3β attenuated proteasomal degradation of cyclin D1 by reducing Thr(286)-phosphorylated cyclin D1 levels. Moreover, inactivated GSK-3β increased cyclin D1 gene transcription by increasing its transcription factor β-catenin in the nucleus. Further, blockage of phosphoinositide 3-kinase/Akt kinase with their specific inhibitors, LY294002 and Akt 1/2 kinase inhibitor, significantly reduced TCDD-enhanced proliferation of HAPI microglial cells. In conclusion, TCDD stimulates proliferation of HAPI microglial cells by affecting the Akt/GSK-3β/cyclin D1 signaling pathway.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent toxin that alters normal brain development, producing cognitive disability and motor dysfunction. Previous studies in rats have proved that female rats are more sensitive to TCDD lethality than male ones. Recent studies have shown that TCDD induces cell cycle arrest and apoptosis, but the regulatory proteins involved in these processes have yet to be elucidated. In this study, we constructed an acute TCDD injury female rat model, and investigated the effects of TCDD on apoptosis and expression of cell cycle regulators, forkhead box class O 3a (FoxO3a) and p27kip1, in the central nervous system (CNS). Increased levels of active caspase-3 were observed in the cerebral cortex of female rats treated with TCDD, suggesting that TCDD-induced apoptosis occurs in the CNS. The terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick-end labeling assay showed that apoptosis primarily occurred in neurons. Furthermore, Western blot analysis, reverse transcription-polymerase chain reaction, and immunohistochemistry showed a significant up-regulation of FoxO3a and p27kip1 in the cerebral cortex. Immunofluorescent labeling indicated that FoxO3a and p27kip1 were predominantly localized in apoptotic neurons, but not in astrocytes. In vitro experiments using PC12, a rat neuron-like pheochromocytoma cell line, also revealed that TCDD induced apoptosis and an increase in FoxO3a and p27kip1 expression. Furthermore, knockdown of FoxO3a expression inhibited p27kip1 transcription and TCDD-induced apoptosis. Based on our data, induction of FoxO3a may play an important role in TCDD-induced neurotoxicity.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been known to induce inflammatory signaling in a number of cell types and tissues. However, the adverse effects of TCDD on the central nervous system (CNS) have not been entirely elucidated. In this study, using reverse transcriptase PCR (RT-PCR) and ELISA, we showed that TCDD up-regulated the expression and secretion of tumor necrosis factor-alpha (TNF-alpha) in a time-dependent manner in cultured HAPI microglial cells. TCDD also caused a fast (within 30 min as judged by the increase in its mRNA level) activation of cytosolic phospholipase A2 (cPLA2). This initial action was accompanied by up-regulation of cyclooxygenase-2 (COX-2), an important inflammation marker within 1 h after TCDD treatment. These pro-inflammatory responses were inhibited by two types of Ca2+ blockers, bis-(o-aminophenoxy) ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester (BAPTA-AM) and nifedipine, thus, indicating that the effects are triggered by initial increase in the intracellular concentration of free Ca2+ ([Ca2+](i)). Further, TCDD exposure could induce phosphorylation- and ubiquitination-dependent degradation of I kappa B alpha, and the translocation of NF-kappa B p65 from the cytosol to the nucleus in this microglial cell line. Thus, the NF-kappa B signaling pathway can be activated after TCDD treatment. However, Ca2+ blockers also obviously attenuated NF-kappa B activation and transnuclear transport induced by TCDD. In concert with these results, we highlighted that the secretion of pro-inflammatory cytokine and NF-kappa B activation induced by TCDD can be mediated by elevation of [Ca2+](i) in HAPI microglial cells. (C) 2013 Elsevier Ireland Ltd. All rights reserved.

A peptide pheromone of the red-bellied male newt, sodefrin was tested for its ability to increase intracellular concentrations of Ca2+ ([Ca2+]i) in the dissociated vomeronasal (VN) cells of females by means of calcium imaging system. The pheromone elicited a marked elevation of [Ca2+]i in a small population of VN cells from sexually developed females. The population of cells exhibiting sodefrin-induced elevation of [Ca2+]i increased concentration-dependently. A pheromone of a different species was ineffective in this respect. The VN cells from non-reproductive females or from reproductive males scarcely responded to sodefrin in terms of elevating [Ca2+]i. In the cells from hypophysectomized and ovariectomized females, the sodefrin-inducible increase of [Ca2+]i never occurred. The cells from the operated newts supplemented with prolactin and estradiol exhibited [Ca2+]i responses to sodefrin with a high incidence. Thus, sex- and hormone-dependency as well as species-specificity of the responsiveness of the VN cells to sodefrin was evidenced at the cellular level. Subsequently, possibility of involvement of phospholipase C (PLC)-inositol 1,4,5-trisphosphate (IP3) and/or PLC-diacylglycerol (DAG)-protein kinase C (PKC) pathways in increasing [Ca2+]i in VN cells in response to sodefrin was explored using pharmacological approaches. The results indicated that PLC is involved in generating the Ca2+ signal in all sodefrin-responsive VN cells, whereas IP3 in approximately 50% of the cells and DAG-PKC in the remaining cells. In the latter case, the increase of [Ca2+]i was postulated to be induced by the influx of Ca2+ through the L-type channel. The significance of the finding is discussed.

Adipose tissue is a critical exchange center for complex energy transactions involving triacylglycerol storage and release. It also has an active endocrine role, releasing various adipose-derived cytokines (adipokines) that participate in complex pathways to maintain metabolic and vascular health. Here, we found D-dopachrome tautomerase (DDT) as an adipokine secreted from human adipocytes by a proteomic approach. DDT mRNA levels in human adipocytes were negatively correlated with obesity-related clinical parameters such as BMI, and visceral and subcutaneous fat areas. Experiments using SGBS cells, a human preadipocyte cell line, revealed that DDT mRNA levels were increased in an adipocyte differentiation-dependent manner and DDT was secreted from adipocytes. In DDT knockdown adipocytes differentiated from SGBS cells that were infected with the adenovirus expressing shRNA against the DDT gene, mRNA levels of genes involved in both lipolysis and lipogenesis were slightly but significantly increased. Furthermore, we investigated AMP-activated protein kinase (AMPK) signaling, which phosphorylates and inactivates enzymes involved in lipid metabolism, including hormone-sensitive lipase (HSL) and acetyl-CoA carboxylase (ACC), in DDT knockdown adipocytes. The AMPK phosphorylation of HSL Ser-565 and ACC Ser-79 was inhibited in DDT knockdown cells and recovered in the cells treated with recombinant DDT (rDDT), suggesting that down-regulated DDT in adipocytes brings about a state of active lipid metabolism. Furthermore, administration of rDDT in db/db mice improved glucose intolerance and decreased serum free fatty acids levels. In the adipose tissue from rDDT-treated db/db mice, not only increased levels of HSL phosphorylated by AMPK, but also decreased levels of HSL phosphorylated by protein kinase A (PKA), which phosphorylates HSL to promote its activity, were observed. These results suggested that DDT acts on adipocytes to regulate lipid metabolism through AMPK and/or PKA pathway(s) and improves glucose intolerance caused by obesity.

We previously identified D-dopachrome tautomerase (DDT) as a novel adipokine whose mRNA levels in adipocytes are negatively correlated with obesity-related clinical parameters, and which acts on adipocytes to regulate lipid metabolism. Here we investigated functions of DDT on preadipocytes. Recombinant DDT (rDDT) enhanced both the expression and secretion of interleukin-6 (IL-6) in SGBS cells, a human preadipocyte cell line. Treatment with rDDT increased levels of phosphorylated ERK1/2, but not p38, in SGBS cells, and rDDT-induced IL-6 mRNA expression was attenuated by pretreatment with an ERK inhibitor, U0126. Knockdown of CD74, but not CD44, inhibited rDDT-induced IL-6 mRNA expression in SGBS cells. These results suggested that the rDDT-induced IL-6 expression in preadipocytes occurred through the CD74-ERK pathway. Furthermore, in SGBS cells subjected to adipogenic induction, rDDT decreased the amount of triacylglycerol, number of cells with oil droplets, and levels of mRNA encoding adipocyte marker proteins. Increased expression of CCAAT/enhancer binding protein families and peroxisome proliferator-activated receptor γ2 during adipogenesis was inhibited in the cells treated with rDDT. These results suggested DDT to inhibit adipogenesis by suppressing the expression of genes encoding adipogenic regulators in preadipocytes.

Insulin-responsive aminopeptidase (IRAP) and GLUT4 are two major cargo proteins of GLUT4 storage vesicles (GSVs) that are translocated from a postendosomal storage compartment to the plasma membrane (PM) in response to insulin. The cytoplasmic region of IRAP is reportedly involved in retention of GSVs. In this study, vimentin was identified using the cytoplasmic domain of IRAP as bait. The validity of this interaction was confirmed by pull-down assays and immunoprecipitation in 3T3-L1 adipocytes. In addition, it was shown that GLUT4 translocation to the PM by insulin was decreased in vimentin-depleted adipocytes, presumably due to dispersing GSVs away from the cytoskeleton. These findings suggest that the IRAP binding protein, vimentin, plays an important role in retention of GSVs.

Cyclin-dependent kinase inhibitors represented by the INK4 family comprising p16(INK4A), p15(INK4B), p18(INK4C), and p19(INK4D) are regulators of the cell cycle shown to be aberrant in many types of cancer. Mice lacking p18(Ink4c) exhibit a series of phenotypes including the development of widespread organomegaly and pituitary adenomas. The objective of our study is to examine the role of p18(INK4C) in the pathogenesis of human pituitary tumors. The protein and mRNA levels of p18(INK4C) were examined by immunohistochemistry and real-time reverse transcription-polymerase chain reaction, respectively. The methylation status of the p18(INK4C) gene promoter and somatic mutations of the p18(INK4C) gene were also investigated. p18(INK4C) protein expression was lost or significantly reduced in 64% of pituitary adenomas compared with levels in normal pituitary glands. p18(INK4C) mRNA levels were low in all ACTH adenomas and non-functioning (NF)-FSH and in 42%, 70% and 66% of GH, PRL, and subtype 3 adenomas, respectively. p18(INK4C) mRNA levels were significantly associated with p18(INK4C) protein levels. Neither methylated promoters in pituitary adenomas, except in one NF-FSH adenoma, nor somatic mutations of the p18(INK4C) gene in any pituitary adenomas were detected. The down-regulation of p18(INK4C) expression may contribute to the tumorigenesis of pituitary adenomas.

Various mechanical stimuli affect differentiation of mesoderm-derived cells such as osteoblasts or myoblasts, suggesting that adipogenesis may also be influenced by mechanical stimulation. However, effects of mechanical stimuli on adipogenesis are scarcely known. Compressive force was applied to a human preadipocyte cell line, SGBS. Levels of gene expression were estimated by real-time reverse transcription-polymerase chain reaction. The accumulation of lipids was evaluated by Sudan III or Oil Red O staining. In SGBS cells subjected to a compressive force of 226 Pa for 12 h before adipogenic induction, adipogenesis was inhibited. Compressive force immediately after adipogenic induction did not affect the adipogenesis. The expression of peroxisome proliferator-activated receptor (PPAR) gamma 2 and CCAAT/enhancer binding protein (C/EBP) a mRNA during adipogenesis was inhibited by compressive force, whereas C/EBP beta and C/EBP delta mRNA levels were unaffected. In preadipocytes, compressive force increased mRNA levels of Kruppel-like factor 2, preadipocyte factor 1, WNT10b, and cyclooxygenase-2 (COX-2) which are known as negative regulators for the PPAR gamma 2 and C/EBP alpha genes. Furthermore, a COX-2 inhibitor completely reversed the inhibition of adipogenesis by compressive force. In conclusion, compressive force inhibited adipogenesis by suppressing expression of PPAR gamma 2 and C/EBP alpha in a COX-2-dependent manner. (C) 2009. The Society for Biotechnology, Japan. All rights reserved.

Obesity is considered a chronic low-grade inflammatory status and the stromal vascular fraction (SVF) cells of adipose tissue (AT) are considered a source of inflammation-related molecules. We identified YKL-40 as a major protein secreted from SVF cells in human visceral AT. YKL-40 expression levels in SVF cells from visceral AT were higher than in those from subcutaneous AT. Immunofluorescence staining revealed that YKL-40 was exclusively expressed in macrophages among SVF cells. YKL-40 purified from SVF cells inhibited the degradation of type I collagen, a major extracellular matrix of AT, by matrix metalloproteinase (MMP)-1 and increased rate of fibril formation of type I collagen. The expression of MMP-1 in preadipocytes and macrophages was enhanced by interaction between these cells. These results suggest that macrophage/preadipocyte interaction enhances degradation of type I collagen in AT, meanwhile, YKL-40 secreted from macrophages infiltrating into AT inhibits the type I collagen degradation.