Carbohydrates mediate their conversion to triglycerides in the liver by promoting both rapid posttranslational activation of ratelimiting glycolytic and lipogenic enzymes and transcriptional induction of the genes encoding many of these same enzymes. The mechanism by which elevated carbohydrate levels affect transcription of these genes remains unknown. Here we report the purification and identification of a transcription factor that recognizes the carbohydrate response element (ChRE) within the promoter of the L-type pyruvate kinase (LPK) gene. The DNA-binding activity of this ChRE-binding protein (ChREBP) in rat livers is specifically induced by a high carbohydrate diet. ChREBP's DNA-binding specificity in vitro precisely correlates with promoter activity in vivo. Furthermore, forced ChREBP overexpression in primary hepatocytes activates transcription from the L-type Pyruvate kinase promoter in response to high glucose levels. The DNA-binding activity of ChREBP can be modulated in vitro by means of changes in its phosphorylation state, suggesting a possible mode of glucoseresponsive regulation. ChREBP is likely critical for the optimal long-term storage of excess carbohydrates as fats, and may contribute to the imbalance between nutrient utilization and storage characteristic of obesity.
Recently we purified and identified a previously uncharacterized transcription factor from rat liver binding to the carbohydrate responsive element of the L-type pyruvate kinase (L-PK) gene. This factor was named carbohydrate responsive element binding protein (ChREBP). ChREBP, essential for L-PK gene transcription, is activated by high glucose and inhibited by cAMP. Here, we demonstrated that (i) nuclear localization signal and basic helix-loop-helix͞leucine-zipper domains of ChREBP were essential for the transcription, and (ii) these domains were the targets of regulation by cAMP and glucose. Among three cAMP-dependent protein kinase phosphorylation sites, Ser 196 and Thr 666 were the target sites. Phosphorylation of the former resulted in inactivation of nuclear import, and that of the latter resulted in loss of the DNA-binding activity and L-PK transcription. On the other hand, glucose activated the nuclear import by dephosphorylation of Ser 196 in the cytoplasm and also stimulated the DNAbinding activity by dephosphorylation of Thr 666 in the nucleus. These results thus reveal mechanisms for regulation of ChREBP and the L-PK transcription by excess carbohydrate and cAMP. T he liver is the principal organ responsible for conversion of excess dietary carbohydrate to triglycerides. A high carbohydrate diet leads to activation of several regulatory enzymes of glycolysis and lipogenesis including L-type pyruvate kinase (L-PK), acetyl CoA carboxylase, and fatty acid synthase (1). Excess carbohydrate also results in post-translational activation of several key enzymes involved in carbohydrate metabolism and lipogenesis (1). Until recently it was thought that hormones such as insulin and glucagon regulate the transcription of genes. It has only been appreciated recently that nutrients themselves play an important role in the regulation.Glucose-stimulated L-PK gene expression in liver is mediated through the glucose or carbohydrate response element (ChRE) that is located in the region Ϫ183 to Ϫ96 base pairs upstream from the cap site of the L-PK gene (2). The binding site for the ChRE contains an E-box sequence, CACGGG, separated by 5 bases that corresponds to the consensus binding site (CACGTG) for upstream stimulatory factors and their related family members. Several transcription factors binding the ChRE have been reported previously (3-5), but none of these factors vary with diet, and the mechanisms of regulation are still unclear.We recently purified, identified, and cloned a transcription factor that binds specifically to the ChRE of the L-PK gene (6). We termed this new transcription factor ChRE-binding protein (ChREBP; ref. 6). ChREBP is expressed specifically in liver and is responsive to excess carbohydrate, i.e., ChREBP is activated by high glucose diet and inhibited by high fat diet. Overexpression of ChREBP in primary cultured hepatocytes results in increased transcription activity of the L-PK gene in response to high glucose.ChREBP is a member of the basic helix-loop-helix͞leucine zipper (bHLH͞ZIP) f...
Embryonic stem cells (ESCs) of nonhuman primates are important for research into human gametogenesis because of similarities between the embryos and fetuses of nonhuman primates and those of humans. Recently, the formation of germ cells from mouse ESCs in vitro has been reported. In this study, we established cynomolgus monkey ES cell lines (cyESCs) and attempted to induce their differentiation into germ cells to obtain further information on the development of primate germ cells by observing the markers specific to germ cells. Three cyESCs were newly established and confirmed to be pluripotent. When the cells are induced to differentiate, the transcripts of Vasa and some meiotic markers were expressed. VASA protein accumulated in differentiated cell clumps and VASA-positive cells gathered in clumps as the number of differentiation days increased. In the later stages, VASA-positive clumps coexpressed OCT-4, suggesting that these cells might correspond to early gonocytes at the postmigration stage. Furthermore, meiosis-specific gene expression was also observed. These results demonstrate that cyESCs can differentiate to developing germ cells such as primordial germ cells (PGCs) or more developed gonocytes in our differentiation systems, and may be a suitable model for studying the mechanisms of primate germ cell development.
Although volatile anesthetic agents have been used clinically for many years, the mechanisms by which they act on the nervous system to produce anesthesia are not known. A possible site of action is the voltage-gated calcium-selective channel (Krnjevic and Puil, 1988). Accordingly, the action of the halogenated alkane anesthetic halothane on voltage-dependent Ca currents in neonatal rat sensory neurons was examined using whole-cell patch-clamp recordings. Halothane reversibly reduced the low-voltage-activated Ca current with an EC50 of about 100 microM. Similar effects were seen using a halogenated ether anesthetic (isoflurane) and in sensory neurons from adult rats. At higher concentrations, both halothane and isoflurane reduced the high-voltage-activated Ca current. Because low-voltage-activated Ca current. Because low-voltage-activated Ca current has been postulated to be involved in the control of neuronal excitability and bursting (Llinas, 1988), this block may explain some of the clinical actions of volatile anesthetics.
This work shows that CT can give insight into ovarian function after heterotopic transplantation, and that heterotopic autografts of vitrified ovarian cortex can give rise to long-term ovarian function and embryos in a primate model. It remains to be established how outcomes following rapid vitrification compared with outcomes following conventional slow cooling procedures.
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