Search

Angiopoietin-related growth factor (AGF; or Angptl6) is a liver-derived, circulating factor and is considered to be a regulator of metabolic homeostasis. AGF is capable of counteracting both obesity and obesity-related insulin resistance. However, the target tissues and the molecular mechanisms underlying the antiobesity and antidiabetic actions of AGF have not been completely defined. Using rat hepatoma H4IIEc3 cells or primary hepatocytes, we demonstrate that AGF suppresses glucose production in a concentration-dependent manner through reduced expression of a key gluconeogenic enzyme, glucose-6-phosphatase (G6Pase), at both transcriptional and translational levels. The action of AGF on glucose production was inhibited by pretreatment of the cells with LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], a phosphoinositide 3-kinase (PI3K) inhibitor, and Akt (protein kinase B) inhibitors. AGF increased the phosphorylation of Akt and its substrates, glycogen synthase kinase 3beta and forkhead box class O1 (FoxO1), a key transcription factor for G6Pase expression. Furthermore, an immunohistochemical approach with anti-FoxO1 antibody demonstrated that AGF stimulation promoted translocation of FoxO1 from the nucleus to the cytoplasm in the cells. These results suggest that in hepatocytes, AGF suppresses gluconeogenesis via reduced transcriptional activity of FoxO1 resulting from the activation of PI3K/Akt signaling cascades.

Circadian rhythms govern the behavior, physiology, and metabolism of living organisms. Recent studies have revealed the role of several genes in the clock mechanism both inDrosophilaand in mammals. To study how gene expression is globally regulated by the clock mechanism, we used a high density oligonucleotide probe array (GeneChip) to profile gene expression patterns in Drosophilaunder light-dark and constant dark conditions. We found 712 genes showing a daily fluctuation in mRNA levels under light-dark conditions, and among these the expression of 115 genes was still cycling in constant darkness, i.e.under free-running conditions. Unexpectedly the expression of a large number of genes cycled exclusively under constant darkness. We found that cycling in most of these genes was lost in the arrhythmicClock(Clk) mutant under light-dark conditions. Expression of periodically regulated genes is coordinated locally on chromosomes where small clusters of genes are regulated jointly. Our findings reveal that many genes involved in diverse functions are under circadian control and reveal the complexity of circadian gene expression in Drosophila.

Pioglitazone is a thiazolidinedione drug (TZD) which potently and specifically stimulates peroxisome proliferator-activated receptor γ (PPAR γ) and sensitizes cells to insulin. Since TZDs are thought to increase energy expenditure, changes in mitochondrial thermogenesis uncoupling protein-2 and -3 mRNA levels in response to pioglitazone treatment were measured in mouse skeletal muscle. Normally hyperglycemic and hyperinsulinemic KK/Ta mice were given pioglitazone for 2 weeks to treat this non-insulin dependent diabetes-like condition. During treatment, UCP2 mRNA levels increased to 185% of normal untreated control levels in soleus muscle. In contrast, UCP3 mRNA levels significantly decreased, up to 67% of normal untreated control levels. Interestingly, UCP3 mRNA levels correlated quite strongly with blood glucose levels, with r = 0.82 for gastrocnemius tissue and r = 0.92 for soleus tissue. These results may indicate that pioglitazone increases glucose catabolism by direct upregulation of muscleUCP2gene expressionin vivo.Therefore,UCP3gene expression is controlled by a different mechanism thanUCP2expression.

The estrogen receptor (ER) regulates the expression of target genes in a ligand-dependent manner. The ligand-dependent activation function AF-2 of the ER is located in the ligand binding domain (LBD), while the N-terminal A/B domain (AF-1) functions in a ligand-independent manner when isolated from the LBD. AF-1 and AF-2 exhibit cell type and promoter context specificity. Furthermore, the AF-1 activity of the human ERα (hERα) is enhanced through phosphorylation of the Ser118residue by mitogen-activated protein kinase (MAPK). From MCF-7 cells, we purified and cloned a 68-kDa protein (p68) which interacted with the A/B domain but not with the LBD of hERα. Phosphorylation of hERα Ser118potentiated the interaction with p68. We demonstrate that p68 enhanced the activity of AF-1 but not AF-2 and the estrogen-induced as well as the anti-estrogen-induced transcriptional activity of the full-length ERα in a cell-type-specific manner. However, it did not potentiate AF-1 or AF-2 of ERβ, androgen receptor, retinoic acid receptor alpha, or mineralocorticoid receptor. We also show that the RNA helicase activity previously ascribed to p68 is dispensable for the ERα AF-1 coactivator activity and that p68 binds to CBP in vitro. Furthermore, the interaction region for p68 in the ERα A/B domain was essential for the full activity of hERα AF-1. Taken together, these findings show that p68 acts as a coactivator specific for the ERα AF-1 and strongly suggest that the interaction between p68 and the hERα A/B domain is regulated by MAPK-induced phosphorylation of Ser118.

ABSTRACTThe estrogen receptor (ER) regulates the expression of target genes in a ligand-dependent manner. The ligand-dependent activation function AF-2 of the ER is located in the ligand binding domain (LBD), while the N-terminal A/B domain (AF-1) functions in a ligand-independent manner when isolated from the LBD. AF-1 and AF-2 exhibit cell type and promoter context specificity. Furthermore, the AF-1 activity of the human ERa (hERa) is enhanced through phosphorylation of the Ser118residue by mitogen-activated protein kinase (MAPK). From MCF-7 cells, we purified and cloned a 68-kDa protein (p68) which interacted with the A/B domain but not with the LBD of hERa. Phosphorylation of hERa Ser118potentiated the interaction with p68. We demonstrate that p68 enhanced the activity of AF-1 but not AF-2 and the estrogen-induced as well as the anti-estrogen-induced transcriptional activity of the full-length ERa in a cell-type-specific manner. However, it did not potentiate AF-1 or AF-2 of ERß, androgen receptor, retinoic acid receptor alpha, or mineralocorticoid receptor. We also show that the RNA helicase activity previously ascribed to p68 is dispensable for the ERa AF-1 coactivator activity and that p68 binds to CBP in vitro. Furthermore, the interaction region for p68 in the ERa A/B domain was essential for the full activity of hERa AF-1. Taken together, these findings show that p68 acts as a coactivator specific for the ERa AF-1 and strongly suggest that the interaction between p68 and the hERa A/B domain is regulated by MAPK-induced phosphorylation of Ser118.

A rapid, highly sensitive method to determine the mRNA level of muscle-specific markers using TaqMan PCR analysis was developed and used to study sequential gene regulation of myoblasts during induced differentiation of C2C12cells. mRNA levels of muscle regulatory factor (MRF) myogenin, α-actin, thermoregulatory uncoupling protein UCP2 and glucose transporter isotype glut4 increased rapidly during early stage differentiation. In contrast, myf5, β-actin, UCP1 and glut1 mRNA levels gradually decreased during 8 days. However, the mRNA level of other genes such as MyoD, glyceraldehyde-3-phosphate dehydrogenase and hexokinase II changed only slightly in comparison. Muscle-specific uncoupling protein UCP3 mRNA was detected during differentiation and increased rapidly within 8 days. These data clearly show the sequential and the differential regulation of muscle-specific genes in C2C12cells during multinucleate myotube formation.

1.Two different molecular forms of dopamine-ß-hydroxylase were isolated from human serum; a major component (Peak I enzyme) with a molecular weight of 368000 and with a higher specific activity and a minor component (Peak II enzyme) with a molecular weight of 188000 and with a lower specific activity.2.Both forms require ascorbic acid for the activity, and are stimulated by fumarate. Addition of n-ethylmaleimide or copper also increased the activity. The optimal pH of both forms in the presence of 20mm tyramine as substrate is 5.0.3.Km values toward tyramine of Peak I enzyme and Peak II enzyme were 1.67mm and 14.2mm, respectively.4.Both Peak I enzyme and Peak II enzyme are glycoprotein.

A novel isoform of rat estrogen receptor (ER) beta, ERβ2, which is a putative alternative splicing product of the reported ERβ (ERβ1) has been identified. Rat ERβ2 cDNA contains an additional in-frame 54 base pair insertion in the ligand binding domain of ERβ1, which generates an 18 amino acid residue insertion. Northern blot and RT-PCR analyses revealed that ERβ2 coexists with ERα and ERβ1 in all tissues examined including brain, lung, liver, kidney, fat, bone, uterus, prostate, and ovary. The insertion caused loss of ligand binding activity of ERβ2, whereas the ability to bind the palindromic estrogen response element (ERE) was retained. In an ERE-containing luciferase reporter gene assay using COS-1 cells, ERβ2 failed to activate estrogen-dependent transcription. Furthermore, ERβ2 dose dependently suppressed the ERα- and ERβ1-mediated transcriptional activation. These results suggest that rat ERβ2 functions as a negative regulator of estrogen action.

Circadian rhythm is generated by genetically determined biological clock with the 24-hour light-dark cycle. In mammals, the central clock is located in the suprachiasmatic nucleus of the hypothalamus. At molecular level, the clock system is composed of interlocked transcriptional and translational feedback loops. The CLOCK and BMAL1, associated as heterodimers, bind to the E-box enhancer and positively drive the expression of Period genes, Cryptochrome genesand Rev-erbAgenes whose proteins, in turn, form multimeric complexes and feed back to repress the transactivation by CLOCK/BMAL1 in the nucleus. Circadian rhythms also exist in a variety of peripheral tissues, and are synchronized by the central pacemaker through complex interaction of neural, humoral, and behavioral cues. Ovarian granulosa cells (GCs) undergo proliferation and differentiation during folliculogenesis under the regulation of FSH and locally produced factors. The granulosa cells are finally differentiated into luteal cells (LCs) after ovulation. However, currently little is known about whether and how circadian clockwork could be affected during folliculogenesis and luteinization. In the present study, the real-time monitoring system was employed to evaluate the clockwork in the GCs and LCs obtained from transgenic rats that have been constructed with Per2promoter (chr1 (-):93289505-93293019)-destabilized luciferase reporter gene. The ovaries were obtained from DES-, and eCG and/or hCG-treated immature rats, and the GCs and LCs were prepared to culture in DMEM/F12 supplemented with 10% FBS. Cultured cells were treated for 2 h with or without dexamethasone in serum-free medium, then the medium was replaced with 2 ml of DMEM/F12 containing 5% FBS, 15 mM HEPES, and 0.1 mM luciferin. The cells were also exposed to gonadotropins (FSH or LH) without dexamethasone. Real-time monitoring of bioluminescence was performed at 37C using Kronos AB-2500 interfaced to computer for continuous data acquisition. The photon counts were integrated for 1 min at intervals of 8 min. In immature GCs from DES-treated rats, no oscillation of Per2expression was observed without dexamethasone treatment, but one oscillation was induced after dexamethasone or FSH treatment. In mature GCs from eCG-treated rats, irregular Per2oscillation was observed without dexamethasone treatment, but persistent oscillation was induced after dexamethasone or LH treatment. In LC from eCG/hCG-treated rats, persistent oscillation was induced with or without dexamethasone or LH treatment. Collectively, it is concluded that circadian clockwork system in ovarian cells is developed during folliculogenesis and luteinization, and synchronized by gonadotropins as well as dexamethasone.

Circadian rhythm is generated by genetically determined biological clock with the 24-hour light-dark cycle. In mammals, the central clock is located in the suprachiasmatic nucleus of the hypothalamus. At molecular level, the clock system is composed of interlocked transcriptional and translational feedback loops. The CLOCK and BMAL1, associated as heterodimers, bind to the E-box enhancer and positively drive the expression of Period genes, Cryptochrome genes and Rev-erbA genes whose proteins, in turn, form multimeric complexes and feed back to repress the transactivation by CLOCK/BMAL1 in the nucleus. Circadian rhythms also exist in a variety of peripheral tissues, and are synchronized by the central pacemaker through complex interaction of neural, humoral, and behavioral cues. A variety of factors including cAMP analog, forskolin, phorbol-12-myristate-13-acetate, endothelin, prostaglandin E2, fibroblast growth factor, calcium ionophors, retinoic acid, glucocorticoids, and parathyroid hormone have been unraveled having the potentials to reset circadian rhythms in peripheral cells. The uterus is regulated under both estrogen and progesterone, but very little is currently known about the molecular mechanism underlying the sex steroidal effects on circadian clockwork. In the present study, the real-time monitoring system was employed to evaluate the clockwork in the uterus stromal cells obtained from transgenic rats that have been constructed with Per2 promoter (chr1 (-):93289505-93293019)-destabilized luciferase reporter gene. The uteri were obtained from eCG and/or hCG-treated immature rats and pregnant rats (day 5 gestation) and the stromal cells prepared were cultured to be confluent in DMEM/F12 supplemented with 2% FBS. Cultured cells were treated for 2 h with or without dexamethasone or forskolin in serum-free medium, then the medium was replaced with 2 ml of DMEM/F12 containing 5% FBS, 15 mM HEPES, and 0.1 mM luciferin. Real-time monitoring of bioluminescence was performed at 37°C using Kronos AB-2500 interfaced to computer for continuous data acquisition. The photon counts were integrated for 1 min at intervals of 8 min. The cells prepared from eCG-treated immature rats (high estrogen level) showed irregular Per2 oscillation after treatment with dexamethasone or forskolin, although Per2 expression was elevated. In contrast, persistent oscillation was observed in the cells prepared from eCG/hCG-treated immature rats (high progesterone level). Interestingly, the irregular Per2 oscillation was converted to persistent oscillation with about 24-h cycle in the presence of progesterone. In pregnant rat uterus cells, also irregular Per2 oscillation was observed with estradiol, whereas the oscillation was converted to persistent oscillation with progesterone. Collectively, it is concluded that both estradiol and progesterone are involved in modification of circadian rhythm via direct regulation of the expression of clock genes.