The novel roles of liver for compensation of insulin resistance in human growth hormone transgenic rats.

Chronic excess of GH is known to cause hyperinsulinemia and insulin resistance. We developed human GH transgenic (TG) rats, which were characterized by high plasma levels of human GH and IGF-I. These TG rats showed higher levels of plasma insulin, compared with control littermates, whereas plasma glucose concentrations were normal. Insulin-dependent glucose uptake into adipocytes and muscle was impaired, suggesting that these rats developed insulin resistance. In contrast, insulin-independent glucose uptake into hepatocytes from TG rats was significantly increased, and glycogen and lipid levels in livers of TG rats were remarkably high. Because the role of liver in GH-induced insulin resistance is poorly understood, we studied insulin signaling at early stages and insulin action in liver and primary cultures of hepatocytes prepared from TG rats. There was no difference in insulin receptor kinase activity induced by insulin between TG and control rats; however, insulin-dependent insulin receptor substrate-2 tyrosine phosphorylation, glycogen synthase activation, and expression of enzymes that induce lipid synthesis were potentiated in hepatocytes of TG rats. These results suggest that impairment of insulin-dependent glucose uptake by GH excess in adipose tissue and muscle is compensated by up-regulation of glucose uptake in liver and that potentiation of insulin signaling through insulin receptor substrate-2 in liver experiencing GH excess causes an increase in glycogen and lipid synthesis from incorporated glucose, resulting in accumulation of glycogen and lipids in liver. This novel mechanism explains normalization of plasma glucose levels at least in part in a GH excess model.

Cyclopamine treatment of human embryonic stem cells followed by culture in human astrocyte medium promotes differentiation into nestin- and GFAP-expressing astrocytic lineage.

Human embryonic stem cells (hESCs) are able to differentiate into various cell types, including neuronal cells and glial cells. However, little information is available regarding astrocyte differentiation. This report describes the differentiation of hESCs into nestin- and GFAP-expressing astrocytes following treatment with cyclopamine, which is an inhibitor of Hedgehog (Hh) signaling, and culturing in human astrocyte medium (HAM). In hESCs, cyclopamine treatment suppressed the expression of Hh signaling molecules, the Hh signaling target gene, and ESC-specific markers. Clyclopamine also induced the differentiation of the cells at the edges of the hESC colonies, and these cells stained positively for the early neural marker nestin. Subsequent culturing in HAM promoted the expression of the astrocyte-specific marker GFAP, and these cells were also nestin-positive. These findings indicate that treatment with cyclopamine followed by culturing in HAM leads to the differentiation of hESCs into nestin- and GFAP-expressing astrocytic lineage.

Ligand-dependent transcriptional enhancement by DNA curvature between two half motifs of the estrogen response element in the human estrogen receptor alpha gene.

We previously reported five DNA bend sites (ERB-4 to -1, and ERB+1) in the promoter region of the human estrogen receptor alpha (ERalpha) gene [FEBS Lett. 444 (1999) 117]. One of these sites, ERB-2, was accompanied by two half motifs of the estrogen response element (ERE) and several short poly(dA)(.)poly(dT) tracts including an A(4) tract located next to a half ERE motif. This A(4) tract and the 20 bp immediate flanking sequence containing a half ERE motif (T3B) exhibited DNA curvature. Transcription assays using luciferase as a reporter gene indicated that T3B sequence conferred positive estrogen responsiveness. Mutations introduced in this sequence indicated that both bendability and estrogen responsiveness were synergistically associated with the A(4) tract located next to the half ERE motif. This motif and a mutant sequence, GGTTA, had affinity for ERalpha protein, which seems to account for ERalpha protein binding to the region without an ERE motif. These findings suggest that some DNA curvature acts as a transcriptional modulator by modifying the state of ligand effects.

The human OCT-4 isoforms differ in their ability to confer self-renewal.

OCT-4 transcription factors play an important role in maintaining the pluripotent state of embryonic stem cells and may prevent expression of genes activated during differentiation. Human OCT-4 isoform mRNAs encode proteins that have identical POU DNA binding domains and C-terminal domains but differ in their N-terminal domains. We report here the cloning and characterization of the human OCT-4B isoform. Human OCT-4B cDNA encodes a 265-amino acid protein with a predicted molecular mass of 30 kDa. Embryonic stem (ES) cell-based complementation assays using ZHBTc4 ES cells showed that unlike human OCT-4A, OCT-4B cannot sustain ES cell self-renewal. In addition, OCT-4B does not bind to a probe carrying the OCT-4 consensus binding sequence, and we demonstrate that two separate regions of its N-terminal domain are responsible for inhibiting DNA binding. We also demonstrate that OCT-4B is mainly localized to the cytoplasm. Overexpression of OCT-4B did not activate transcription from OCT-4-dependent promoters, although OCT-4A did as reported previously. Furthermore, transcriptional activation by human OCT-4A was not inhibited by co-expression of OCT-4B. Taken together, these data suggest that the DNA binding, transactivation, and abilities to confer self-renewal of the human OCT-4 isoforms differ.

Transcriptional profiling of the developmentally important signalling pathways in human embryonic stem cells.

BACKGROUND: Embryonic stem cells (ESC) maintain their 'stemness' by self-renewal. However, the molecular mechanisms underlying self-renewal of human embryonic stem cells (hESC) remain to be elucidated. In this study, expression profiles of the molecules of developmentally important signalling pathways were investigated to better understand the relationships of the signalling pathways for self-renewal in hESC. METHODS: Two human ESC lines were cultured on mouse embryonic fibroblast (MEF) feeder cells. Gene expression was analysed by RT-PCR, real-time RT-PCR and Western blotting. RESULTS: In the bone morphogenetic protein (BMP4), transforming growth factor (TGF-beta) and fibroblast growth factor (FGF4) signalling pathways, ligands and antagonists were highly expressed in hESC compared with human embryoid body (hEB). Human ESC showed abundant transcripts of intracellular molecules in the Wnt, Hh and Notch signalling pathways. No difference was detected in the expression level of the JAK/STAT signalling molecules between hESC and hEB. Western blot analysis showed that the transcriptional levels of the signalling molecules in hESC were consistent with translational levels. From the real-time PCR analysis, expression levels of some genes, such as Oct3/4, Nodal and beta-catenin, were different between two hESC lines. CONCLUSION: The self-renewal of hESC is probably maintained by coordinated regulation of signalling-specific molecules and in a signalling-specific manner.

Expressional regulation of neuronal and cancer-related genes by estrogen in adult female rats.

Adult female rats were ovariectomized and treated with or without estrogen for two weeks. mRNA was obtained from the hypothalamus, uterus, liver, kidney and skeletal muscle and analyzed by Northern blotting and/or RT-PCR. We examined two types of estrogen-responsive genes from rats, neuronal system-related genes (Amphiregulin, AR; Neuropeptide Y-Y1 receptor, NPY-Y1R; Bassoon, BSN; N-Cadherin, N-CADH) and estrogen-susceptible cancer-related genes (C-terminal binding protein interacting protein, CtIP), based on the results of a cDNA microarray analysis which was carried out to profile estrogen-responsive genes in the human breast cancer cell line MCF-7. The N-CADH gene showed identical response to that in MCF-7 cells. In the hypothalamus, all except the AR gene were down-regulated in their expression. In other tissues, the expression showed marked differences: expression of the BSN gene was not detected by either method, and the NPY-Y1R gene showed down-regulation in most tissues except for skeletal muscle. We then analyzed the time course of the estrogen-responsiveness of these genes in several tissues, finding changes in expression patterns especially in skeletal muscle but not in the hypothalamus. Our results show that the estrogen-responsive genes, which were demonstrated simply as either up- or down-regulated in their expression by estrogen in a human cell line using cDNA microarrays, exhibit tissue and temporal-specific expression patterns in adult female rats.