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Gluconeogenesis is essential for blood glucose homeostasis during fasting and is regulated by various enzymes, which are encoded by gluconeogenic genes. Those genes are controlled by various transcription factors. Zinc finger and BTB domain-containing 7c (Zbtb7c, also called Kr-pok) is a BTB-POZ family transcription factor with proto-oncogenic activity. Previous findings have indicated that Zbtb7c is involved in the regulation of fatty acid biosynthesis, suggesting an involvement also in primary metabolism. We found here that fasting induced Zbtb7c expression in the mouse liver and in primary liver hepatocytes. We also observed that Zbtb7c-knockout mice have decreased blood glucose levels, so we investigated whether Zbtb7c plays a role in gluconeogenesis. Indeed, differential gene expression analysis of Zbtb7c-knockout versus wild type mouse livers showed downregulated transcription of gluconeogenic genes encoding the glucose 6-phosphatase catalytic subunit (G6pc) and phosphoenolpyruvate carboxykinase 1 (Pck1), while Zbtb7c expression upregulated these two genes, under fasting conditions. Mechanistically, we found that when complexed with histone deacetylase 3 (Hdac3), Zbtb7c binds insulin response elements (IREs) within the G6pc and Pck1 promoters. Moreover, complexed Zbtb7c deacetylated forkhead box O1 (Foxo1), thereby increasing Foxo1 binding to the G6pc and Pck1 IREs, resulting in their transcriptional activation. These results demonstrate Zbtb7c to be a crucial metabolic regulator of blood glucose homeostasis, during mammalian fasting.

Prolactin regulatory element-binding (PREB) protein is a transcription factor that regulates prolactin (PRL) gene expression. PRL, also known as luteotropic hormone or luteotropin, is well known for its role in producing milk. However, the role of PREB, in terms of hepatic glucose metabolism, is not well elucidated. Here, we observed expression of Preb in the mouse liver, in connection with glucose homeostasis. Morevoer, Preb was downregulated in db/db, ob/ob and high-fat diet-induced obese (DIO) mice, concurrent with upregulation of the liver genes glucose-6-phosphatase (G6pc) and phosphoenolpyruvate carboxykinase-1 (Pck). Administration of adenovirus-Preb (Ad-Preb) to db/db, ob/ob, and DIO mice diminished glucose, insulin, and pyruvate tolerance, which analogously, were impaired in normal (C57BL/6) mice knocked down for Preb, via infection with Ad-shPreb (anti-Preb RNA), indicating Preb to be a negative regulator of liver gluconeogenic genes. We further demonstrate that Preb negatively influences gluconeogenic gene expression, by directly binding to their promoters at a prolactin core-binding element (PCBE). A better understanding of Preb gene expression, during the pathogenesis of hepatic insulin resistance, could ultimately provide new avenues for therapies for metabolic syndrome, obesity, and type-2 diabetes mellitus, disorders whose worldwide incidences are increasing drastically.

Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step of triglyceride synthesis and plays a critical role in the development of fatty liver disease and insulin resistance. The expression of DGAT2 is mostly induced by dietary carbohydrate in the mammalian liver; however, the transcription factors that regulate DGAT2 expression have yet to be identified. In this study, we investigated the molecular mechanism underlying the glucose-induced transcriptional regulation of human DGAT2 in HepG2 cells. Human DGAT2 expression was increased by glucose in HepG2 cells. Transfection studies of the DGAT2 promoter-luciferase reporter construct in vitro and in silico analysis identified two glucose-responsive regions in the DGAT2 promoter. Each region contains one ChREBP/MLX (carbohydrate response element binding protein/Max-like protein) binding site (ChoRE, −539 to −551 and −6067 to −6083) and one specificity protein 1 (SP1) binding site (GC-rich motif, −556 to −572 and −6016 to −6032). Mutational an...

Insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2), one of the most abundant circulating IGFBPs, is known to attenuate the biological action of IGF-1. Although the effect of IGFBP-2 in preventing metabolic disorders is well known, its regulatory mechanism remains unclear. In the present study, we demonstrated the transcriptional regulation of the Igfbp-2 gene by peroxisome-proliferator-activated receptor (PPAR) α in the liver. During fasting, both Igfbp-2 and PPARα expression levels were increased. Wy14643, a selective PPARα agonist, significantly induced Igfbp-2 gene expression in primary cultured hepatocytes. However, Igfbp-2 gene expression in Pparα null mice was not affected by fasting or Wy14643. In addition, through transient transfection and chromatin immunoprecipitation assay in fasted livers, we determined that PPARα bound to the putative PPAR-responsive element between −511 bp and −499 bp on the Igfbp-2 gene promoter, indicating that the Igfbp-2 gene transcription is activated directly by PPARα. To explore the role of PPARα in IGF-1 signalling, we treated primary cultured hepatocytes with Wy14643 and observed a decrease in the number of IGF-1 receptors (IGF-1Rs) and in Akt phosphorylation. No inhibition was observed in the hepatocytes isolated from Pparα null mice. These results suggest that PPARα controls IGF-1 signalling through the up-regulation of hepatic Igfbp-2 transcription during fasting and Wy14643 treatment., Insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2) is known to attenuate the biological action of IGF-1, but its regulatory mechanism remains unclear. We demonstrate the transcriptional regulation of the hepatic Igfbp-2 gene by peroxisome-proliferator-activated receptor (PPAR) α during fasting. We also show how PPARα controls IGF-1 signalling through IGFBP-2.

Elongation of very long chain fatty acids protein 6 (ELOVL6), a rate-limiting enzyme for the elongation of saturated and monounsaturated fatty acids with 12, 14, and 16 carbons, plays a key role in energy metabolism and insulin sensitivity. Hepatic Elovl6 expression is upregulated in the fasting-refeeding response and in leptin-deficient ob/ob mice. Mouse Elovl6 has been shown to be a direct target of sterol regulatory element binding protein-1 (SREBP-1) in response to insulin. In the present study, we demonstrated that mouse and human Elovl6 expression is under the direct transcriptional control of carbohydrate response element binding protein (ChREBP), a mediator of glucose-induced gene expression. Serial deletion and site-directed mutagenesis studies revealed functional carbohydrate response elements (ChoREs) in the mouse and human Elovl6 promoters and gel shift assays and chromatin immunoprecipitation assays confirmed the binding of ChREBP to the Elovl6-ChoRE sites. In addition, the ectopic co-expression of ChREBP and SREBP-1c in HepG2 cells synergistically stimulated Elovl6 promoter activity and this synergistic activation was abolished by mutating the Elovl6 promoter ChoREs. Taken together, these results suggest that the synergistic action of ChREBP and SREBP-1c is necessary for the maximal induction of Elovl6 expression in the liver.

The present study examined the effects of krill-derived phosphatidylserine (Krill-PS) on the learning and memory function and the neural activity in rats with trimethyltin (TMT)-induced memory deficits. The rats were administered vehicle (medium-chain triglyceride: MCT) or Krill-PS (50, 100 mg/kg, p.o.) daily for 21 days. The cognitive improving efficacy of Krill-PS in TMT-induced amnesic rats was investigated by assessing the Morris water maze test and by performing choline acetyltransferase (ChAT), acetylcholinesterase (AChE) and cAMP responsive element binding protein (CREB) immunohistochemistry. The rats with TMT injection showed impaired learning and memory of the tasks and treatment with Krill-PS produced a significant improvement of the escape latency to find the platform in the Morris water maze at the 2(nd) and 4(th) day compared to that of the MCT group (p0.05). In the retention test, the Krill-PS+MCT groups showed increased time spent around the platform compared to that of the MCT group. Consistent with the behavioral data, Krill-PS 50+MCT group significantly alleviated the loss of acetylcholinergic neurons in the hippocampus and medial septum compared to that of the MCT group. Treatment with Krill-PS significantly increased the CREB positive neurons in the hippocampal CA1 area as compared to that of the MCT group. These results suggest that Krill-PS may be useful for improving the cognitive function via regulation of cholinergic marker enzyme activity and neural activity.

Glucose-6-phosphatase (G6Pase) is a key enzyme that is responsible for the production of glucose in the liver during fasting or in type 2 diabetes mellitus (T2DM). During fasting or in T2DM, peroxisome proliferator-activated receptor α (PPARα) is activated, which may contribute to increased hepatic glucose output. However, the mechanism by which PPARα up-regulates hepatic G6Pase gene expression in these states is not well understood. We evaluated the mechanism by which PPARα up-regulates hepatic G6Pase gene expression in fasting and T2DM states. In PPARα-null mice, both hepatic G6Pase and phosphoenolpyruvate carboxykinase levels were not increased in the fasting state. Moreover, treatment of primary cultured hepatocytes with Wy14,643 or fenofibrate increased the G6Pase mRNA level. In addition, we have localized and characterized a PPAR-responsive element in the promoter region of the G6Pase gene. Chromatin immunoprecipitation (ChIP) assay revealed that PPARα binding to the putative PPAR-responsive element of the G6Pase promoter was increased in fasted wild-type mice and db/db mice. These results indicate that PPARα is responsible for glucose production through the up-regulation of hepatic G6Pase gene expression during fasting or T2DM animal models.

During a state of fasting, the blood glucose level is maintained by hepatic gluconeogenesis. SIRT1 is an important metabolic regulator during nutrient deprivation and the liver-specific knockdown of SIRT1 resulted in decreased glucose production. We hypothesize that SIRT1 is responsible for the upregulation of insulin-suppressed gluconeogenic genes through the deacetylation of FOXO1. Treatment of primary cultured hepatocytes with resveratrol increased insulin-repressed PEPCK and G6Pase mRNA levels, which depend on SIRT1 activity. We found that the resveratrol treatment resulted in a decrease in the phosphorylation of Akt and FOXO1, which are independent of SIRT1 action. Fluorescence microscopy revealed that resveratrol caused the nuclear localization of FOXO1. In the nucleus, FOXO1 is deacetylated by SIRT1, which might make it more accessible to the IRE of the PEPCK and G6Pase promoter, causing an increase in their gene expression. Our results indicate that resveratrol upregulates the expression of gluconeogenic genes by attenuating insulin signaling and by deacetylating FOXO1, which are SIRT1-independent in the cytosol and SIRT1-dependent in the nucleus, respectively.

My professional journey to understand the glucose homeostasis began in the 1990s, starting from cloning of the promoter region of glucose transporter type 2 (GLUT2) gene that led us to establish research foundation of my group. When I was a graduate student, I simply thought that hyperglycemia, a typical clinical manifestation of type 2 diabetes mellitus (T2DM), could be caused by a defect in the glucose transport system in the body. Thus, if a molecular mechanism controlling glucose transport system could be understood, treatment of T2DM could be possible. In the early 70s, hyperglycemia was thought to develop primarily due to a defect in the muscle and adipose tissue; thus, muscle/adipose tissue type glucose transporter (GLUT4) became a major research interest in the diabetology. However, glucose utilization occurs not only in muscle/adipose tissue but also in liver and brain. Thus, I was interested in the hepatic glucose transport system, where glucose storage and release are the most actively occurring.

Expression of the HER2 oncogene is increased in approximately 30% of human breast carcinomas and is closely correlated with the expression of fatty acid synthase (FASN). In the present study, we determined the mechanism by which FASN and acetyl-CoA carboxylase alpha (ACCalpha) could be induced by HER2 overexpression. SK-BR-3 and BT-474 cells, breast cancer cells that overexpress HER2, expressed higher levels of FASN and ACCalpha compared with MCF-7 and MDA-MB-231 breast cancer cells in which HER2 expression is low. The induction of FASN and ACCalpha in BT474 cells were not mediated by the activation of SREBP-1. Exogenous HER2 expression in MDA-MB-231 cells induced the expression of FASN and ACCalpha, and the HER2-mediated increase in ACCalpha and FASN was inhibited by both LY294002, a phosphatidylinositol 3-kinase inhibitor, and rapamycin, a mammalian target of rapamycin (mTOR) inhibitor. In addition, the activation of mTOR by the overexpression of RHEB in MDA-MB-231 cells increased the synthetic rates of both FASN and ACCalpha. On the other hand, FASN and ACCalpha were reduced in BT-474 cells by a blockade of the mTOR signaling pathway. These changes observed in their protein levels were not accompanied by changes in their mRNA levels. The 5'- and 3'-untranslated regions of both FASN and ACCalpha mRNAs were involved in selective translational induction that was mediated by mTOR signal transduction. These results strongly suggest that the major mechanism of HER2-mediated induction of FASN and ACCalpha in the breast cancer cells used in this study is translational regulation primarily through the mTOR signaling pathway.

We have investigated the function and mechanisms of the CARM1-SNF5 complex in T3-dependent transcriptional activation. Using specific small interfering RNAs (siRNA) to knock down coactivators in HeLa alpha2 cells, we found that coactivator associated arginine methyltransferase 1 (CARM1) and SWI/SNF complex component 5 (SNF5) are important for T3-dependent transcriptional activation. The CARM1- SWI/SNF chromatin remodeling complex serves as a mechanism for the rapid reversal of H3-K9 methylation. Importantly, siRNA treatment against CARM1 and/or SNF5 increased the recruitment of HMTase G9a to the type 1 deiodinase (D1) promoter even with T3. Knocking-down either CARM1 or SNF5 also inhibited the down-regulation of histone macroH2A, which is correlated with transcriptional activation. Finally, knocking down CARM1 and SNF5 by siRNA impaired the association of these coactivators to the D1 promoter, suggesting functional importance of CARM1- SNF5 complex in T3-dependent transcriptional activation.

Follicle-stimulating hormone (FSH) was produced in Chinese hamster ovary (CHO) cells using a perfusion bioreactor. Perfusion culture at 37 degrees C yielded a high cell density but a low FSH production. To investigate the effect of culture temperature in the range of 26-37 degrees C on cell growth and FSH production, batch cultures were performed. Lowering culture temperature below 32 degrees C resulted in growth suppression. However, specific productivity of FSH, q (FSH), increased as culture temperature decreased, and the maximum q (FSH) of 43.4 ng/10(6) cells/h was obtained at 28 degrees C, which is 13-fold higher than that at 37 degrees C. Based on the results obtained from batch cultures, we performed perfusion cultures with two consecutive temperatures. CHO cells were grown up to 3.2 x 10(7) cells/ml at 37 degrees C and culture temperature shifted down to 28 degrees C to obtain a high FSH titer. Soon after the maximum FSH titer of 21 mug/ml was achieved, a rapid loss of not only viable cell concentration but also cell viability was observed, probably due to the low activities of enzymes related to cell growth. Thus, the extension of production period at 28 degrees C is critical for the enhancement of FSH production, and the use of antiapoptotic genes seems to be promising.

Derangement of glucose metabolism is a key feature of T2DM, with the liver and pancreatic beta-cells playing a key role in glucose homeostasis. In the postprandial state, glucose is transported into hepatocytes and either metabolized to fatty acids or CO(2), or stored as glycogen. Glucose also acts as a key signal in pancreatic beta-cells for regulating insulin secretion. Because GLUT2 and GK expressed in liver and beta-cells are responsible for sensing glucose levels in the blood, studies on the regulation of these biomolecules are important in understanding glucose homeostasis in vivo. These molecules are known to be regulated either transcriptionally or post-transcriptionally, and recent studies on the structure and function of promoters of these genes have revealed the involvement of various transcriptional factors in their regulation. Here, we review recent progress in elucidating the transcriptional regulation of glucose sensors in the liver and pancreatic beta-cells and the relevance to T2DM.

Acetyl-CoA carboxylase beta (ACCbeta) is a critical enzyme in the regulation of fatty acid oxidation and is dominantly expressed in the skeletal muscle, heart, and liver. It has been established that two promoters, P-I and P-II, control the transcription of the ACCbeta gene. However, the precise mechanism involved in controlling tissue-specific gene expression of ACCbeta is largely unknown yet. In this study we revealed that promoter P-I, active in the skeletal muscle and heart but not in the liver, could be activated by myogenic regulatory factors and retinoid X receptors in a synergistic manner. Moreover, P-I was also activated markedly by the cardiac-specific transcription factors, Csx/Nkx2.5 and GATA4. These results suggest that the proper stimulation of P-I by these tissue-specific transcription factors is important for the expression of ACCbeta according to the tissue types. In addition, CpG sites around human exon 1a transcribed by P-I are half-methylated in muscle but completely methylated in the liver, where P-I is absolutely inactive. In humans, the skeletal muscle uses P-II as well as P-I, whereas only P-I is active in rat skeletal muscle. The proximal myogenic regulatory factor-binding sites in human P-II, which are not conserved in rat P-II, might contribute to this difference in P-II usage between human and rat skeletal muscle. Hepatoma-derived cell lines primarily use another novel promoter located about 3 kilobases upstream of P-I, designated as P-O. This study is the first to explain the mechanisms underlying the differential regulation of ACCbeta gene expression between tissues in living organisms.

To achieve the liver-directed expression in sufficient amounts of therapeutic genes for successful and safe gene therapy, natural liver-specific promoters can be used to direct the expression of therapeutic genes in the liver, whereas strong viral enhancers were used to obtain sufficient amounts of expressed therapeutic gene products. However, very often use of either the former or the latter does not guarantee both potent and liver-specific therapeutic gene expression. Here we conglomerate them and thus create a potent tissue-specific promoter by characterizing and using the liver-type pyruvate kinase proximal promoter (LPKPP) harboring its TATA box and a HNF-1alpha binding site. Alone it hardly activated its reporter gene expression in non-hepatocytes or hepatocytes. However, in the presence of the SV40 viral enhancer (SV40VE), which is active in multiple cell types, it was able to potently activate its reporter gene expression specifically in hepatocytes. The tissue-specific activation of the LPKPP by the viral enhancer was attributed to HNF-1alpha binding to the LPKPP. Taken together, these results support the idea that the constitutively active SV40VE could be used to activate the LPKPP in a tissue-specific manner in the presence of HNF-1alpha. To our knowledge, this is the first study to utilize HNF-1alpha and its binding site, in the context of the LPKPP, to generate a basal promoter that is transcriptionally activated potently in a tissue-specific manner by a viral enhancer that is active in multiple cell types.

Ligand activation of peroxisome proliferator-activated receptor gamma (PPARgamma) has been reported to induce growth inhibition and apoptosis in various cancers including hepatocellular carcinoma (HCC). However, the effect of hepatitis B virus X protein (HBx) on PPARgamma activation has not been characterized in hepatitis B virus (HBV)-associated HCC. Herein, we demonstrated that HBx counteracted growth inhibition caused by PPARgamma ligand in HBx-associated HCC cells. We found that HBx bound to DNA binding domain of PPARgamma and HBx/PPARgamma interaction blocked nuclear localization and binding to recognition site of PPARgamma. HBx significantly suppressed a PPARgamma-mediated transactivation. These results suggest that HBx modulates PPARgamma function through protein-protein interaction.

Insulin is the major hormone that regulates hepatic glucose metabolism by repressing gluconeogenic enzyme-encoding genes, and the counteracting glucagon/protein kinase (PKA)–inducible coactivating peroxisome proliferator–activated receptor γ coactivator-1α (PGC-1α) signaling pathway is also well characterized in hepatic glucose metabolism (1–3). Until now, however, the regulation of the insulin/protein kinase B (PKB)/Akt–inducible corepressor signaling pathway has remained largely unknown. Previously, it was believed that insulin suppression of gluconeogenesis was largely mediated through PKB activity via direct phosphorylation and dephosphorylation mechanisms (4). However, in this issue of Diabetes , Lee et al. (5) dissect the role of the small heterodimer partner–interacting leucine zipper protein (SMILE) in insulin-mediated hepatic glucose metabolism. SMILE is a member of the CREB/ATF family of basic-region leucine zipper (bZIP) transcription factors and has been reported to function as a corepressor of nuclear receptor superfamily genes, including estrogen-related receptor γ (ERRγ), glucocorticoid receptor (GR), hepatocyte nuclear factor 4α (HNF4α), and cAMP-responsive element–binding protein H (CREBH) (6–8). In fact, ERRγ, GR, HNF4α, and CREBH have all been implicated in upregulating gluconeogenic gene expression (9–12). Lee et al. (5) show that SMILE is an insulin-inducible corepressor …

Zinc is an important trace element in the body and is involved in both the proliferation and growth arrest of many kinds of cells including colorectal epithelial cells. The aim of this study was to identify the molecular mechanism of the growth regulation of colorectal cancer cells by extracellular zinc. Zinc-stimulated activation of the mitogen-activated protein kinase (MAPK) cascade was measured by immunoblotting and Elk-1 dependent trans-reporter gene expression, and zinc-stimulated p21(Cip/WAF1) activation by immunoblotting, Northern blot analysis and immunochemistry. Cell proliferation was measured by thymidine and bromodeoxyuridine (BrdU) incorporation. By treating colorectal cancer cells with 100 microM ZnCl2, MAPKs were activated in two different phases, the initial weak activation occurred within 5 min and this was followed by a stronger and more prolonged activation. Zinc concomitantly activated Raf-1-MEK-MAPK kinases, and induced Elk-1 dependent trans-reporter gene expression. Prolonged activation of MAPKs by 100 microM of ZnCl2 resulted in the induction and nuclear localization of p21(Cip/WAF1) and was related to the inhibition of both thymidine and BrdU incorporations. These results not only suggest the presence of a mechanism for p21(Cip/WAF1) dependent negative regulation of colorectal cancer cell growth by zinc but also suggest potential usage of zinc to control the growth of colorectal cancer cells.

Thiazolidinediones, synthetic ligands of peroxisomal proliferator-activated receptor-gamma (PPAR-gamma), improve peripheral insulin sensitivity and glucose-stimulated insulin secretion in pancreatic beta-cells. To explore the role of PPAR-gamma in glucose sensing of beta-cells, we have dissected the beta-cell-specific glucokinase (betaGK) promoter, which constitutes glucose-sensing apparatus in pancreatic beta-cells, and identified a peroxisomal proliferator response element (PPRE) in the promoter. The betaGK-PPRE is located in the region between +47 and +68 bp. PPAR-gamma/retinoid X receptor-alpha heterodimer binds to the element and activates the betaGK promoter. The betaGK promoter lacking or having mutations in PPRE cannot be activated by PPAR-gamma. PPAR-gamma activates the betaGK promoter in beta-cells as well as non-beta-cells. Furthermore, troglitazone increases endogenous GK expression and its enzyme activity in beta-cell lines. These results indicate that PPAR-gamma can regulate GK expression in beta-cells. Taking these results together with our previous work, we conclude that PPAR-gamma regulates gene expression of glucose-sensing apparatus and thereby improves glucose-sensing ability of beta-cells, contributing to the restoration of beta-cell function in type 2 diabetic subjects by troglitazone.

A partial C-terminal cDNA sequence of a novel Drosophila mitogen-activated protein kinase phosphatase (MKP), designated DMKP-3, was identified from an epitope expressed sequence tag database, and the missing N-terminal cDNA fragment was cloned from a Drosophila cDNA library. DMKP-3 is a protein of 411 amino acids, with a calculated molecular mass of 45.8kDa; the deduced amino acid sequence is most similar to that of mammalian MKP-3. Recombinant DMKP-3 produced in Escherichia coli retained intrinsic tyrosine phosphatase activity. In addition, DMKP-3 specifically inhibited extracellular-signal-regulated kinase (ERK) activity, but was without a significant affect on c-Jun N-terminal kinase (JNK) and p38 activities, when it was overexpressed in Schneider cells. DMKP-3 interacted specifically with Drosophila ERK (DERK) via its N-terminal domain. In addition, DMKP-3 specifically inhibited Elk-1-dependent trans-reporter gene expression in mammalian CV1 cells, and dephosphorylated activated mammalian ERK in vitro. DMKP-3 is uniquely localized in the cytoplasm within Schneider cells, and gene expression is tightly regulated during development. Thus DMKP-3 is a Drosophila homologue of mammalian MKP-3, and may play important roles in the regulation of various developmental processes.

Bcl-2 has been reported to inhibit neurotoxicity induced by cisplatin. However, neither the mechanism of cisplatin-induced neurotoxicity nor the mechanism by which Bcl-2 confers neuroprotection is clear. In this study, the signaling pathways involved in cisplatin-induced neurotoxicity were examined using a rat neuroblastoma cell line, B104. Treatment of B104 cells with cisplatin induced apoptosis, accompanying the accumulation of p53 and Bax protein. Interestingly, extracellular signal-regulated kinase 1/2 (ERK1/2) activities of MAP kinases were markedly enhanced prior to cisplatin-induced accumulation of p53 and Bax. Inhibition of ERK1/2 activities using PD98059, a selective MEK inhibitor, blocked the apoptotic cell death preventing cisplatin-induced accumulation of p53 and Bax. These results suggest that ERK mediates cisplatin-induced p53 activation to trigger apoptosis in B104 cells. Overexpression of Bcl-2 in B104 cells resulted in the complete resistance to cisplatin-induced apoptosis blocking ERK activation and the subsequent signaling pathway of p53. Our study clearly demonstrates that the action site of Bcl-2 localizes upstream of ERK in cisplatin-induced apoptotic signaling pathway.

Upstream open-reading frames are unusual in mammalian mRNAs. The 5′ untranslated region of ADH5 mRNA contains an upstream open-reading frame (uORF) with two possible AUG start codons. Myf6 mRNA contains three tandem AUG repeats at the translation start site, a rare feature. Mutation at one or both of the upstream AUG codons in the ADH5 mRNA increased gene expression twofold in CV-1, NIH/3T3, HeLa, and SL2 cells. Mutation of these AUG codons led to 3- to 5-fold increases in activity as measured by in vitro translation assays using capped mRNAs.RNA toeprint analysis demonstrated many stalled ribosomes flanking the AUG codons and secondary structures near the AUGs. Secondary structures may increase the ability of ribosomes to recognize the two AUGs, despite their poor initiation context. The degree of repression by uAUGs varied significantly depending on the cell lines tested, which may partly explain the differential tissue expression. Myf6 is a critical myogenic transcription factor with the striking feature of three tandem AUG codons at the translation initiation site. This structure reduced expression; removing two of these AUGs led to a doubling of activity in CV-1, HeLa, and NIH/3T3 cells.

The effect of low levels of carbon dioxide (CO2) in the gas phase on the production of recombinant human erythropoietin (EPO)in CHO cells was explored. A T-flask culture in an incubator without CO2 addition showed a slow cell growth initially followed by the cessation of growth, while other cultures incubated under 0.5–5% CO2 concentrations grew normally at the same rate during the entire period of cultivation. Interestingly, the production of EPO in the culture incubated under no CO2 supply was highest among the tested cultures. The cell specific secretion rate of EPO (qEPO) of the culture under no CO2 supply was about 3 times higher than that of the culture under 5% CO2 supply. Western blot analysis and in vivo bioassay of EPO showed no apparent changes in EPO quality between the two cases of different CO2 environments (air vs. 5% CO2), suggesting robust glycosylation of EPO by CHO cells even under very reduced CO2 environment. Various combinations of the two extreme cases, with 5% CO2 supply (suitable for cell growth) and no CO2 addition (better for EPO production), were made in order to maximize the volumetric productivity of EPO secretion (PV) in CHO cells. The PV of the cultures programmed with initial incubation under 5% CO2 followed by no CO2 supply was about 2 times superior to that of the culture incubated only under no CO2 supply. The PV of the culture under no CO2 supply was slightly lower than that of culture grown under 5% CO2. However, the qEPO of the no CO2 supply case was more than 5 times higher than that of the culture under 5% CO2 supply. In conclusion, we have demonstrated that a simple programming of CO2 supply to an incubator can enhance the production of EPO in CHO cells remarkably, without any apparent change of the EPO quality.

The 280-kDa beta-isoform of acetyl-CoA carboxylase (ACCbeta) is predominantly expressed in heart and skeletal muscle, whereas the 265-kDa alpha-isoform (ACCalpha) is the major ACC in lipogenic tissues. The ACCbeta promoter showed myoblast-specific promoter activity and was strongly induced by MyoD in NIH3T3 cells. Serial deletions of the promoter revealed that MyoD acts on the E-boxes located at positions -498 to -403 and on the proximal region including the 5'-untranslated region. Destruction of the E-boxes at positions -498 to -403 by site-directed mutagenesis resulted in a significant decrease of MyoD responsiveness. The "TGAAA" at -32 to -28 and the region around the transcription start site play important roles in basal transcription, probably as a TATA box and an Inr element, respectively. Mutations of another E-box at -14 to -9 and a "GCCTGTCA" sequence at +17 to +24 drastically decreased the MyoD responsiveness. The novel cis-element GCCTGTCA was preferentially bound by MyoD homodimer in EMSA and conferred MyoD responsiveness to a luciferase reporter, which was repressed by the overexpression of E12. This finding is unique since activation via E-boxes is mediated by heterodimers of MyoD and E-proteins. We screened a human skeletal muscle cDNA library to isolate clones expressing proteins that bind to the region around the GCCTGTCA (+8 to +27) sequence, and isolated Myf4 and Myf6 cDNAs. Electrophoretic mobility shift assay showed that recombinant Myf4 and Myf6 bind to this novel cis-element. Moreover, transient expression of Myf6 induced significant activation on the ACCbeta promoter or an artificial promoter harboring this novel cis-element. These findings suggest that muscle regulatory factors, such as MyoD, Myf4, and Myf6, contribute to the muscle-specific expression of ACCbeta via E-boxes and the novel cis-element GCCTGTCA.

ATP citrate-lyase (ACL) is a key enzyme supplying acetyl-CoA for fatty acid and cholesterol synthesis. Its expression is drastically up-regulated when an animal is fed a low fat, high carbohydrate diet after prolonged fasting. In this report, we describe the role of sterol regulatory element-binding proteins (SREBPs) in the transactivation of the rat ACL promoter. ACL promoter activity was markedly stimulated by the overexpression of SREBP-1a and, to a lesser extent, by SREBP-2 in Alexander human hepatoma cells. The promoter elements responsive to SREBPs were located within the 55-base pair sequences from -114 to -60. The gel mobility shift assay revealed four SREBP-1a binding sites in this region. Of these four elements, the -102/-94 region, immediately upstream of the inverted Y-box, and the -70/-61 region, just adjacent to Sp1 binding site, played critical roles in SREBPs-mediated stimulation. The mutation in the inverted Y-box and the coexpression of dominant negative nuclear factor-Y (NF-Y) significantly attenuated the transactivation by SREBP-1a, suggesting that NF-Y binding is a prerequisite for SREBPs to activate the ACL promoter. However, the multiple Sp1 binding sites did not affect the transactivation of the ACL promoter by SREBPs. The binding affinity of SREBP-1a to SREs of the ACL promoter also was much higher than that of SREBP-2. The transactivation potencies of the chimeric SREBPs, of which the activation domains (70 amino acids of the amino terminus) were derived from the different species of their carboxyl-terminal region, were similar to those of SREBPs corresponding to their carboxyl termini. Therefore, it is suggested that the carboxyl-terminal portions of SREBPs containing DNA binding domains are important in determining their transactivation potencies to a certain promoter.

We identified the peroxisomal proliferator response element (PPRE) in the +68/+89 region of the rat GLUT2 gene. To identify whether the putative PPRE in the GLUT2 gene (GLUT2-PPRE) is functional, GLUT2 promoter-luciferase reporter constructs were transfected into CV-1 cells. Promoter activities were increased by coexpression of peroxisomal proliferator-activated receptor (PPAR)-gamma, retinoid X receptor (RXR)-alpha, and treatment of their ligands; troglitazone and 9-cis retinoic acid potentiated the transactivational effects. Introduction of mutations in GLUT2-PPRE resulted in loss of transactivational effects of the PPAR-gamma/RXR-alpha heterodimer. Electrophoretic mobility shift assay using nuclear extracts of CV-1 cells, which were transfected with various combinations of PPARs or RXR-alpha expression plasmids, revealed that heterodimers of PPAR-gamma and RXR-alpha preferentially bound to GLUT2-PPRE. In HIT-T15 cells, promoter activity of the rat GLUT2 gene was increased by troglitazone and 9-cis retinoic acid, and mutations of GLUT2-PPRE resulted in reduction of promoter activity. In addition, we observed increased GLUT2 transcription by troglitazone and 9-cis retinoic acid in isolated rat primary islets. These results suggested that the GLUT2-PPRE is functional and plays a significant role in gene expression of GLUT2 in pancreatic beta-cells. This is the first report identifying PPRE in a gene involved in glucose homeostasis, linking the effect of troglitazone on the regulation of insulin secretion.

We investigated transacting factors binding to the cis-element important in tissue-specific expression of the human glucose transporter type 2 isoform (GLUT2) gene. By transient transfection assay, we determined that the 227-base pair fragment upstream of the ATG start site contained promoter activity and that the region from +87 to +132 (site C) was responsible for tissue-specific expression. DNase I footprinting and electrophoretic mobility shift assay indicated that site C contained one binding site for hepatocyte nuclear factor 1 (HNF1) and two binding sites for HNF3. The mutations at positions +101 and +103, which are considered to be critical in binding HNF1 and HNF3, resulted in a 53% decrease in promoter activity, whereas the mutation of the proximal HNF3 binding site (+115 and +117) reduced promoter activity by 28%. The mutations of these four sites resulted in marked decrease (70%) in promoter activity as well as diminished bindings of HNF1 and HNF3. A to G mutation, which causes conversion of the HNF1 and HNF3 binding sequence to the NF-Y binding site, resulted in a 22% decrease in promoter activity. We identified that both HNF1 and HNF3 function as transcriptional activators in GLUT2 gene expression. Coexpression of the pGL+74 (+74 to +301) construct with the HNF1alpha and HNF3beta expression vectors in NIH 3T3 cells showed the synergistic effect on GLUT2 promoter activity compared with the expression of HNF1alpha, HNF3beta, or a combination of HNF1beta and HNF3beta. These data suggest that HNF1alpha and HNF3beta may be the most important players in the tissue-specific expression of the human GLUT2 gene.

The specific secretion rate of recombinant erythropoietin (EPO) from Chinese Hamster Ovary (CHO) cells was increased by reducing agents. Addition of 5 mM cysteamine to serum free medium led to almost 8-fold increase of specific EPO secretion rate although it was not effective in thiol free medium. Each reducing agent has its own optimal concentration for maximizing the EPO secretion from CHO cells.

Two phage clones, lambda hgACL21 and lambda hgACL28, harboring the 5' flanking region of human ATP-citrate lyase (ACL) gene were identified by screening about 1.5 X 10(6) recombinant plaques from the lambdaEMBL3-human placental genomic DNA library. The 5' flanking region of ACL had the CAAT box on -92 bp from the transcription initiation site (+1), however, the TATA box was not found. The primer extension and rapid amplification of cDNA end showed that mRNA is transcribed at a thymine extending 12 bp upstream of the reported cDNA end. The sequences of 5' flanking region in 1.5 kb size of human ACL showed 60% homology with those of rat; however, no homology was found in the exon 1 and intron 1 region. Several consensus sequences, including four Sp1 binding sites, were found in the 5' flanking region of this gene. The promoter activity was assayed by transfecting the 3' or 5' deletion clones of ACL-chloramphenicol acetyl transferase (CAT) plasmid into PLC/PRF5 cells. The clone that contains the part of the first intron sequences from -659 to +440 bp showed the highest CAT activity in the transient transfection assay. High promoter activities were maintained until the transcription initiation site was removed. It is suggested that the sequences from -213 to +12 which contain three Sp1-binding sequences, CAAT box, and the transcription initiation site were necessary as a mean of for exerting the basal promoter activity of ACL gene.

Peroxisome proliferator-activated receptor gamma (PPARγ) belongs to a nuclear receptor superfamily; members of which play key roles in the control of body metabolism principally by acting on adipose tissue. Ligands of PPARγ, such as thiazolidinediones, are widely used in the treatment of metabolic syndromes and type 2 diabetes mellitus (T2DM). Although these drugs have potential benefits in the treatment of T2DM, they also cause unwanted side effects. Thus, understanding the molecular mechanisms governing the transcriptional activity of PPARγ is of prime importance in the development of new selective drugs or drugs with fewer side effects. Recent advancements in molecular biology have made it possible to obtain a deeper understanding of the role of PPARγ in body homeostasis. The transcriptional activity of PPARγ is subject to regulation either by interacting proteins or by modification of the protein itself. New interacting partners of PPARγ with new functions are being unveiled. In addition, post-translational modification by various cellular signals contributes to fine-tuning of the transcriptional activities of PPARγ. In this review, we will summarize recent advancements in our understanding of the post-translational modifications of, and proteins interacting with, PPARγ, both of which affect its transcriptional activities in relation to adipogenesis.

The conventional saccharification of cassava root by enzymatic hydrolysis is improved by using a little amount of cellulase and cellobiase in addition to conventional enzyme, glucoamylase. With new saccharification[glucoamylase 0.45SGU/g cassava, cellulase 4.5NCU/g cassava, cellobiase 0.09U/g cassava, pH 4.3, temperature 60°C, total volume 465ml : 100g of cassava/400ml of water], the reaction time was reduced by about 5 hours, the concentration of reducing sugar was increased by 40%, glucose production was enhanced by 10% , and the viscosity was reduced by 30%.

The present study examined the effects of Tremella fuciformis (TF) on the learning and memory function and the neural activity in rats with trimethyltin (TMT)-induced memory deficits. The rats were administered saline or TF (TF 25, 50, 100 mg/kg, p.o.) daily for 21 days. The cognitive improving efficacy of TF on the amnesic rats, which was induced by TMT, was investigated by assessing the Morris water maze test and by performing Choline acetyltransferase (ChAT) and cAMP responsive element binding protein (CREB) immunohistochemistry. In order to confirm the underlying mechanisms of the memory enhancing effects of TF, we assessed the neurite outgrowth of PC12 cells. We also administered 18F-fluorodeoxyglucose and performed a PET scan of the frontal lobe. The rats with TMT injection showed impaired learning and memory of the tasks and treatment with TF produced a significant improvement of the escape latency to find the platform in the Morris water maze compared to that of the control group. In the retention test, the TF50 group showed increased time spent around the platform compared to that of the control group. Consistent with the behavioral data, TF50 mg/kg significantly alleviated the loss of ChAT-ir neurons in the hippocampus compared to that of the control group. Treatment with TF significantly increased the CREB positive neurons in the hippocampal CA1 area as compared to that of the control group. In addition, TF treatment (50 mg/kg) increased the glucose uptake approximately sevenfold in the frontal lobe and it significantly promoted neurite outgrowth of the PC12 cells, as compared to that of the controls. These results suggest that TF may be useful for improving the cognitive function via regulation of the CREB signaling pathway and cholinergic system in the hippocampus.

Pancreatic β-cells and the liver play a key role in glucose homeostasis. After a meal or in a state of hyperglycemia, glucose is transported into the β-cells or hepatocytes where it is metabolized. In the β-cells, glucose is metabolized to increase the ATP:ADP ratio, resulting in the secretion of insulin stored in the vesicle. In the hepatocytes, glucose is metabolized to CO(2), fatty acids or stored as glycogen. In these cells, solute carrier family 2 (SLC2A2) and glucokinase play a key role in sensing and uptaking glucose. Dysfunction of these proteins results in the hyperglycemia which is one of the characteristics of type 2 diabetes mellitus (T2DM). Thus, studies on the molecular mechanisms of their transcriptional regulations are important in understanding pathogenesis and combating T2DM. In this paper, we will review a recent update on the progress of gene regulation of glucose sensors in the liver and β-cells.

Liver glucokinase (LGK) plays an essential role in controlling blood glucose levels and maintaining cellular metabolic functions. Expression of LGK is induced mainly regulated by insulin through sterol regulatory element-binding protein-1c (SREBP-1c) as a mediator. Since LGK expression is known to be decreased in the liver of liver X receptor (LXR) knockout mice, we have investigated whether LGK might be directly activated by LXRalpha. Furthermore, we have studied interrelationship between transcription factors that control gene expression of LGK. In the current studies, we demonstrated that LXRalpha increased LGK expression in primary hepatocytes and that there is a functional LXR response element in the LGK gene promoter as shown by electrophoretic mobility shift and chromatin precipitation assay. In addition, our studies demonstrate that LXRalpha and insulin activation of the LGK gene promoter occurs through a multifaceted indirect mechanism. LXRalpha increases SREBP-1c expression and then insulin stimulates the processing of the membrane-bound precursor SREBP-1c protein, and it activates LGK expression through SREBP sites in its promoter. LXRalpha also activates the LGK promoter by increasing the transcriptional activity and induction of peroxisome proliferator-activated receptor (PPAR)-gamma, which also stimulates LGK expression through a peroxisome proliferator-responsive element. This activation is tempered through a negative mechanism, where a small heterodimer partner (SHP) decreases LGK gene expression by inhibiting the transcriptional activity of LXRalpha and PPARgamma by directly interacting with their common heterodimer partner RXRalpha. From these data, we propose a mechanism for LXRalpha in controlling the gene expression of LGK that involves activation through SREBP-1c and PPARgamma and inhibition through SHP.

Lipin1 expression was induced at a late stage of differentiation of 3T3-L1 preadipocytes and maintained at high levels in mature adipocytes. Knockdown of expression of lipin1 by small interfering RNA in 3T3-L1 preadipocytes almost completely inhibited differentiation into adipocytes, whereas overexpression of lipin1 accelerated adipocyte differentiation, demonstrating that lipin1 is required for adipocyte differentiation. In mature adipocytes, transfection of lipin1-small interfering RNA decreased the expression of adipocyte functional genes, indicating the involvement of lipin1 in the maintenance of adipocyte function. Lipin1 increases the transcription-activating function of peroxisome proliferator-activated receptor gamma(2) (PPAR gamma(2)) via direct physical interaction, whereas lipin1 did not affect the function of other adipocyte-related transcription factors such as C/EBP alpha, liver X-activated receptor alpha, or sterol regulatory element binding protein 1c. In mature adipocytes, lipin1 was specifically recruited to the PPAR gamma-response elements of the phosphoenolpyruvate carboxykinase gene, an adipocyte-specific gene. C/EBP alpha up-regulates lipin1 transcription by directly binding to the lipin1 promoter. Based on the existence of a positive feedback loop between C/EBP alpha and PPAR gamma(2), we propose that lipin1 functions as an amplifier of the network between these factors, resulting in the maintenance of high levels of the specific gene expression that are required for adipogenesis and mature adipocyte functions.

KLF5 (Krüppel-like factor 5) is a zinc-finger transcription factor that plays a critical role in the regulation of cellular signalling involved in cell proliferation, differentiation and oncogenesis. In the present study, we showed that KLF5 acts as a key regulator controlling the expression of FASN (fatty acid synthase) through an interaction with SREBP-1 (sterol-regulatory-element-binding protein-1) in the androgen-dependent LNCaP prostate cancer cell line. The mRNA level of KLF5 increased when cells were treated with a synthetic androgen, R1881. Furthermore, KLF5 bound to SREBP-1 and enhanced the SREBP-1-mediated increase in FASN promoter activity. The results also demonstrated that the expression of KLF5 in LNCaP prostate cancer cells enhanced FASN expression, whereas silencing of KLF5 by small interfering RNA down-regulated FASN expression. The proximal promoter region and the first intron of the FASN gene contain multiple CACCC elements that mediate the transcriptional regulation of the gene by KLF5. However, other lipogenic and cholesterogenic genes, such as those encoding acetyl-CoA carboxylase, ATP-citrate lyase, the LDL (low-density lipoprotein) receptor, HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) synthase and HMG-CoA reductase are irresponsive to KLF5 expression, owing to the absence of CACCC elements in their promoter regions. Taken together, these results suggest that the FASN gene is activated by the synergistic action of KLF5 and SREBP-1, which was induced by androgen in androgen-dependent prostate cancer cells.

The mechanism of how PPARgamma decrease gluconeogenic gene expressions in liver is still unclear. Since PPARgamma is a transcriptional activator, it requires a mediator to decrease the transcription of gluconeogenic genes. Recently, SHP has been shown to mediate the bile acid-dependent down regulation of gluconeogenic gene expression in liver. This led us to explore the possibility that SHP may mediate the antigluconeogenic effect of PPARgamma. In the present study, we have identified and characterized the presence of functional PPRE in human SHP promoter. We show the binding of PPARgamma/RXRalpha heterodimer to the PPRE and increased SHP expression by rosiglitazone in primary rat hepatocytes. Taken together with the previous reports about the function of SHP on gluconeogenesis, our results indicate that SHP can mediate the acute antigluconeogenic effect of PPARgamma.

The gene expression of glucose transporter type 4 isoform (GLUT4) is known to be controlled by metabolic, nutritional, or hormonal status. Understanding the molecular mechanisms governing GLUT4 gene expression is critical, because glucose disposal in the body depends on the activities of GLUT4 in the muscle and adipocytes. The GLUT4 activities are regulated by a variety of mechanisms. One of them is transcriptional regulation. GLUT4 gene expression is regulated by a variety of transcriptional factors in muscle and adipose tissue. These data are accumulating regarding the transcriptional factors regulating GLUT4 gene expression. These include MyoD, MEF2A, GEF, TNF-alpha, TR-1alpha, KLF15, SREBP-1c, C/EBP-alpha, O/E-1, free fatty acids, PAPRgamma, LXRalpha, NF-1, etc. These factors are involved in the positive or negative regulation of GLUT4 gene expression. In addition, there is a complex interplay between these factors in transactivating GLUT4 promoter activity. Understanding the mechanisms controlling GLUT4 gene transcription in these tissues will greatly promote the potential therapeutic drug development for obesity and T2DM.

Expression of the GLUT4 (glucose transporter type 4 isoform) gene in adipocytes is subject to hormonal or metabolic control. In the present study, we have characterized an adipose tissue transcription factor that is influenced by fasting/refeeding regimens and insulin. Northern blotting showed that refeeding increased GLUT4 mRNA levels for 24 h in adipose tissue. Consistent with an increased GLUT4 gene expression, the mRNA levels of SREBP (sterol-regulatory-element-binding protein)-1c in adipose tissue were also increased by refeeding. In streptozotocin-induced diabetic rats, insulin treatment increased the mRNA levels of GLUT4 in adipose tissue. Serial deletion, luciferase reporter assays and electrophoretic mobility-shift assay studies indicated that the putative sterol response element is located in the region between bases −109 and −100 of the human GLUT4 promoter. Transduction of the SREBP-1c dominant negative form to differentiated 3T3-L1 adipocytes caused a reduction in the mRNA levels of GLUT4, suggesting that SREBP-1c mediates the transcription of GLUT4. In vivo chromatin immunoprecipitation revealed that refeeding increased the binding of SREBP-1 to the putative sterol-response element in the GLUT4. Furthermore, treating streptozotocin-induced diabetic rats with insulin restored SREBP-1 binding. In addition, we have identified an Sp1 binding site adjacent to the functional sterol-response element in the GLUT4 promoter. The Sp1 site appears to play an additive role in SREBP-1c mediated GLUT4 gene upregulation. These results suggest that upregulation of GLUT4 gene transcription might be directly mediated by SREBP-1c in adipose tissue.

We investigated effects of glycine betaine on erythropoietin(EPO) production by CHO cells in hyperosmotic serum free media. It has been known that the physiological osmolality of media in animal cell culture is 320 to 340 mOsm/kg. Most of researchers usually ignore osmolality change of media in cell culture. In this report we showed that the osmolality of media might be important factor in CHO cell culture for protein production.

GLUT2 is mainly expressed in the liver, β-cells of the pancreas, and the basolateral membrane of kidney proximal tubules and plays an important role in glucose homeostasis in living organisms. The transcription of the GLUT2 gene is known to be upregulated in the liver during postprandial hyperglycemic states or in type 2 diabetes. However, a molecular mechanism by which glucose activates GLUT2 gene expression is not known. In this study, we report evidence that sterol response element–binding protein (SREBP)-1c plays a key role in glucose-stimulated GLUT2 gene expression. The GLUT2 promoter reporter is activated by SREBP-1c, and the activation is inhibited by a dominant-negative form of SREBP-1c (SREBP-1c DN). Adenoviral expression of SREBP-1c DN suppressed glucose-stimulated GLUT2 mRNA level in primary hepatocytes. An electrophoretic mobility shift assay and mutational analysis of the GLUT2 promoter revealed that SREBP-1c binds to the −84/−76 region of the GLUT2 promoter. Chromatin immunoprecipitation revealed that the binding of SREBP-1c to the −84/−76 region was increased by glucose concentration in a dose-dependent manner. These results indicate that SREBP-1c mediates glucose-stimulated GLUT2 gene expression in hepatocytes.

In the present study, we show that the expression of type 2 glucose transporter isoform (GLUT2) could be regulated by PPAR-gamma in the liver. Rosiglitazone, PPAR-gamma agonist, activated the GLUT2 mRNA level in the primary cultured hepatocytes and Alexander cells, when these cells were transfected with PPAR-gamma/RXR-alpha. We have localized the peroxisome proliferator response element in the mouse GLUT2 promoter by serial deletion studies and site-directed mutagenesis. Chromatin immunoprecipitation assay using ob/ob mice also showed that PPAR-gamma rather than PPAR-alpha binds to the -197/-184 region of GLUT2 promoter. Taken together, liver GLUT2 may be a direct target of PPAR-gamma ligand contributing to glucose transport into liver in a condition when PAPR-gamma expression is increased as in type 2 diabetes or in severe obesity.

The regulation of hepatic glucose metabolism is important in glucose homeostasis, and liver glucokinase (LGK) plays a central role in this process. Hepatic glucokinase expression is known to be regulated by insulin. Recently it has been suggested that sterol regulatory element binding protein-1c (SREBP-1c) mediates the action of insulin on LGK transcription; however, the precise mechanism is not, to date, well known. In the present study, we identified two functional SREBP-1c response elements, SREa and SREb, in the rat LGK promoter. SREBP-1c could bind to these SREs and activate the LGK promoter, and insulin activated the LGK promoter in Alexander cells. The physical interaction between the protein and SREs of the LGK promoter in vivo was also confirmed. Insulin selectively increased SREBP-1c and LGK expression in primary hepatocytes. Adenoviral expression of SREBP-1c stimulated LGK expression, and the dominant negative mutant of SREBP-1c blocked the increased gene expression of LGK by insulin and SREBP-1c. A chromatin immunoprecipitation assay using primary hepatocytes showed increased binding of SREBP-1 to SREs of the LGK promoter by insulin.

Thiazolidinediones (TZDs), synthetic ligands of peroxisome proliferator-activated receptor (PPAR)-gamma, are known to decrease hepatic glucose production and increase glycogen synthesis in diabetic animals. Recently it was reported that glucokinase (GK) expression was increased by TZDs in the liver of diabetic ZDF rats. However, the mechanism whereby TZDs increase GK expression is not yet studied. We have assumed that liver type glucokinase (LGK) induction by TZDs could be achieved by direct transcriptional activation. Thus, we have dissected the LGK promoter to explore the presence of a PPAR response element (PPRE) in the promoter. From this study, we were able to localize a PPRE in the -116/-104 region of the rat LGK gene. The PPAR-gamma/retinoid X receptor-alpha heterodimer was bound to the element and activated the LGK promoter. The LGK promoter lacking the PPRE or having mutations in the PPRE could not be activated by PPAR-gamma. Furthermore, troglitazone increased endogenous GK mRNA in primary hepatocytes. These results indicate that PPAR-gamma can directly activate GK expression in liver and may contribute to improving glucose homeostasis in type 2 diabetes.

Acetyl-CoA carboxylase (ACC) exists as two major isoforms originated from separate genes: ACCalpha (or ACC1) and ACCbeta (or ACC2). Previous data revealed that ACCbeta has two forms of mRNA with different 5'-untranslated regions derived by different usage of promoters, I and II, in human. In this study, we revealed that ACCbeta expression in liver is markedly stimulated by food intake at the transcriptional level. In the process of this induction in rat liver, promoter II plays the major role in regulating the expression of ACCbeta gene. The transient transfection with promoter II-luciferase reporters elucidated that the region from -93 to -38 nucleotides is important for the responsiveness to sterol regulatory element-binding protein-1 (SREBP-1), which is known to be the principle mediator for the stimulation of gene transcriptions by insulin and diet. The Sp1-binding site (-71 to -66) and neighboring two conserved SREs (-62 to -44) play a critical role in the stimulation of ACCbeta gene expression by SREBP-1. In vivo chromatin immunoprecipitation assay revealed that SREBP-1 directly bound to ACCbeta promoter II in liver, and its binding was regulated by the diet. This study provides evidence that ACCbeta expression in liver is regulated at the transcriptional level by the direct interaction of SREBP-1 with promoter II.

In this study, we identified a new mechanism for the anti-proliferation of HCT15 colorectal cancer cells by troglitazone (TRO). Treating HCT15 cells with 20 microM of TRO transiently increased extracellular signal regulated kinase (ERK) activity within 15 min, and this subsequently induced p21Cip/WAF1 cell cycle regulator and localized in the nucleus. Raf-1 modification and MEK activation also occurred after TRO treatment, and Elk-1-dependent trans-reporter gene expression was concomitantly induced. The induction and nuclear localization of p21Cip/WAF1 by TRO were blocked by PD98059 pre-treatment, which suggested a role for the ERK pathway in p21Cip/WAF1 activation. TRO inhibited BrdU incorporation and no BrdU incorporation was observed in most p21Cip/WAF1-activated cells. Therefore, TRO regulates the proliferation of HCT15 cells at least partly by a mechanism involving the activation of p21Cip/WAF1.

Understanding the molecular networks that regulate adipogenesis is crucial for combating obesity. However, the identity and molecular actions of negative regulators that regulate the early development of adipocytes remain poorly understood. In this study, we investigated the role of CREB3L4, a member of the CREB3-like family, in the regulation of adiposity. Constitutive overexpression of CREB3L4 resulted in the inhibition of adipocyte differentiation, whereas knockdown of Creb3l4 expression caused differentiation of preadipocytes into mature adipocytes, bypassing the mitotic clonal expansion step. In 3T3-L1 preadipocytes, Creb3l4 knockdown resulted in increased expression of peroxisome proliferator-activated receptor γ (PPARγ2) and CCAAT/enhancer binding protein (C/EBPα), either by increasing the protein stability of C/EBPβ or by decreasing the expression of GATA3, a negative regulator of PPARγ2 expression. Consequently, increased PPARγ2 and C/EBPα levels induced adipocyte differentiation, even in the presence of minimal hormonal inducer. Thus, it can be speculated that CREB3L4 has a role as gatekeeper, inhibiting adipogenesis in 3T3-L1 preadipocytes. Moreover, adipocytes of Creb3l4-knockout mice showed hyperplasia caused by increased adipogenesis, and exhibited improved glucose tolerance and insulin sensitivity, as compared with littermate wild-type mice. These results raise the possibility that Creb3l4 could be a useful therapeutic target in the fight against obesity and metabolic syndrome.

A possible role of hepatocyte nuclear factor 1 (HNF1) or HNF3, a predominant trans-acting factors of hepatic or pancreatic beta-cells, was examined on the tissue specific interdependent expression of glucokinase (GK) in liver, H4IIE, HepG2, HIT-T15 and MIN6 cell line. The tissues or cell lines known to express GK showed abundant levels of HNF1 and HNF3 mRNA as observed in liver, H4IIE, HepG2, HIT-T15 and MIN6 cells, whereas they were not detected in brain, heart, NIH 3T3, HeLa cells. The promoter of glucokinase contains several HNF3 consensus sequences and are well conserved in human, mouse and rat. Transfection of the glucokinase promotor linked with luciferase reporter to liver or pancreatic beta cell lines showed high interacting activities with HNF1 and HNF3, whereas minimal activities were detected in the cells expressing very low levels of HNFs. The binding of HNF1 or HNF3 to the GK promoter genes was confirmed by electrophoretic mobility shift assay (EMSA). From these data, we propose that the expression of HNF1 and/or HNF3 may, in part, contribute to the tissue specific expression of GK

In order to develop a serum-free (SF) medium for the production of erythropoietin (EPO) by suspension culture of recombinant Chinese hamster ovary (rCHO) cells, a statistical optimization approach based on a Plackett-Burman design was adopted. A basal medium was prepared by supplementing Iscove's modified Dulbecco's medium (IMDM) with Fe(NO3)3.9H2O, CuCl2 and ZnSO4.7H2O which are generally contained in SF medium formulations. Insulin, transferrin and ethanolamine were also supplemented to the basal medium to determine their optimal concentrations. From this statistical analysis, glutamate, serine, methionine, phosphatidylcholine, hydrocortisone and pluronic F68 were identified as positive determinants for cell growth. The SF medium was formulated by supplementing the basal medium with components showing positive effects on cell growth in suspension culture. An EPO titer in this optimized SF medium was 79% of that in IMDM supplemented with 5% dialyzed fetal bovine serum (dFBS). Furthermore, the in vitro and in vivo biological activities of EPO produced in the SF medium were comparable to those produced in the serum-supplemented medium. Taken together, the results obtained here show that a Plackett-Burman design facilitates the development of SF media for the production of EPO by suspension culture of rCHO cells.

The liver-specific expression of the GLUT2 glucose transporter gene is suppressed in cultured hepatoma cell lines as well as in hepatocytes in primary culture. To understand the underlying mechanism involved in this process, we analysed the rat GLUT2 promoter region. A DNase I footprinting assay with rat liver nuclear extract revealed eight protected regions within a -500 bp region of the GLUT2 promoter (sites A to H). Three of these sites (B, F and H) were occupied by transcription factors that are considerably enriched in liver cells compared with spleen or kidney. The proteins binding to these sites were investigated by a combination of DNase I footprinting assay and electrophoretic mobility-shift assay with the addition of specific oligonucleotide competitors and specific antibody against known transcription factors. As a result it was revealed that hepatocyte nuclear factor 3 binds to site B (-120 to -70), and CCAAT/enhancer binding protein alpha (C/EBPalpha) and C/EBPbeta bind to site F (-375 to -356) and site H (-500 to -471). The binding of C/EBP to sites F and H was markedly decreased within 4 h when liver cells were subjected to primary culture, suggesting that C/EBP might be responsible for the decreased expression of GLUT2 in this process. In contrast, Western blot analysis revealed that C/EBPalpha began to decrease after 1 h of hepatocyte culture, and C/EBPbeta was not changed significantly throughout the culture period, suggesting that C/EBP could be regulated at the transcriptional level as well as the post-translational level when hepatocytes were put in culture. To confirm the role of C/EBP in the regulation of GLUT2 promoter activity, sites F and H were ligated to a chloramphenicol acetyltransferase (CAT) reporter gene and co-transfected with a C/EBP expression vector into HepG2 cells. The co-expression of C/EBPalpha and C/EBPbeta resulted in 9.1-fold and 3. 8-fold increases of CAT activities in the site F-CAT and site H-CAT constructs respectively. These results indicate that C/EBPalpha and C/EBPbeta regulate the promoter activity of the GLUT2 gene and might be responsible for the down-regulation of the GLUT2 gene when hepatocytes are subjected to primary culture.