Search Results

Adiponectin has been shown to regulate glucose and fatty acid uptake and metabolism in skeletal muscle. Here we investigated the role of the recently cloned adiponectin receptor (AdipoR) isoforms in mediating effects of both globular (gAd) and full-length (fAd) adiponectin, and their regulation by hyperglycemia (25 mM, 20 h) and hyperinsulinemia (100 nM, 20 h). We used L6 rat skeletal muscle cells, which were found to express both AdipoR1 and AdipoR2 mRNA in a ratio of over 6:1 respectively. Hyperglycemia and hyperinsulinemia both decreased AdipoR1 receptor expression by approximately 50%, while the latter induced an increase of approximately threefold in AdipoR2 expression. The ability of gAd to increase GLUT4 myc translocation, glucose uptake, fatty acid uptake and oxidation, as well as AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylation, was decreased by both hyperglycemia and hyperinsulinemia. Interestingly, hyperinsulinemia induced the ability of fAd to elicit fatty acid uptake and enhanced fatty acid oxidation in response to fAd. In summary, our results suggest that both hyperglycemia and hyperinsulinemia cause gAd resistance in rat skeletal muscle cells. However, hyperinsulinemia induces a switch toward increased fAd sensitivity in these cells.

In order to elucidate the roles of 17β-HSDs in fish gonadal steroidogenesis, three types of 17β-HSDs (17β-HSD1, 17β-HSD8 and putative 17β-HSD12) were cloned and characterized from the Nile tilapia, Oreochromis niloticus. The cloned cDNAs of 17β-HSD type 1, 8 and 12 were 1504, 1006 and 1930 bp long, with open reading frames encoding proteins of 289, 256 and 314 aminoacids, respectively. Tissue distribution pattern analyzed by RT-PCR and Northern blot showed that 17β-HSD1 was dominantly expressed in the ovary, while the putative 17β-HSD12, one of the two duplicates found in fish, is a male specific enzyme and expressed exclusively in testis (detected by RT-PCR only). On the other hand, 17β-HSD8 was expressed in the brain, gill, heart, liver, intestine, gonad, kidney and muscle of both male and female. Enzymatic assays of the three types of 17β-HSDs were performed using recombinant proteins expressed in E. coli or HEK 293 cells. Tilapia 17β-HSD1 expressed in E. coli had the preference for NADP(H) as cofactor and could catalyze the inter-conversion between estrone and estradiol efficiently as well as the inter-conversion between androstenedione and testosterone, but less efficiently. Tilapia 17β-HSD8 recombinant protein expressed in HEK 293 cells could catalyze the conversion of testosterone to androstenedione, as well as the inter-conversion between estrone and estradiol. However, the putative 17β-HSD12 expressed in E. coli or in HEK 293 cells showed no conversion to any of the four substrates tested in this study. Based on enzyme characterization and tissue distribution, it is plausible to attribute crucial roles to 17β-HSDs in the gonadal steroidogenesis of teleosts.

ABSTRACT Despite the intensive study of endothelin (ET) in mammals, the primary structure and biological activity of the peptide is not known for any species of non-mammalian tetrapod. Extracts of the stomach and the liver of the European green frog Rana ridibunda contained ET-like immunoreactivity measured by RIA using an antiserum raised against human ET-1. The amino acid sequence of the peptide that was isolated in pure form from the stomach extract was identical to that of human ET-1 and the peptide purified from the liver extract was identical to human ET-3 except for a single amino acid substitution (Phe(4)-->Tyr). These observations demonstrate that the amino acid sequences of ET family peptides have been very strongly conserved during evolution of tetrapods and suggest that the pathway of post-translational processing of preproendothelin in the frog is similar to that in mammals. Both frog/human ET-1, frog ET-3 and human ET-3 produced a concentration-dependent increase in the production of corticosteroids from perifused slices of the frog interrenal gland. The maximum responses produced by the peptides (approximately 2-fold increase over basal levels for both corticosterone and aldosterone production) were not significantly different. The potency of ET-1 (-log EC(50)=9.81+/-0.01 (s.e.m.) for corticosterone and 9.52+/-0.29 for aldosterone production) was significantly (P<0.01) greater than that of frog ET-3 (-log EC(50)=8.13+/-1.6 for corticosterone and 8.15+/-0.33 for aldosterone production) but the potencies of frog ET-3 and human ET-3 (-log EC(50)=8.29+/-0.34 and 7.87+/-0.18) were not significantly different.

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders; it is characterized by polycystic ovaries, hyperandrogenism and chronic anovulation. To obtain a global view of those genes that might be involved in the development of this complex clinical disorder, we used recently developed cDNA microarray technology to compare differential gene expressions between normal human ovary and ovaries from PCOS patients. A total of 9216 clones randomly selected from a commercial human ovary cDNA library were screened. Among them, 290 clones showed differential expressions, including 119 known genes and 100 known or unknown expressed sequence tags (ESTs). Among 119 known genes, 88 were upregulated and 31 downregulated in the PCOS ovary, as compared with normal human ovary. These differentially expressed genes are involved in various biologic functions, such as cell division/apoptosis, regulation of gene expression and metabolism, reflecting the complexity of clinical manifestations of PCOS. The molecular characteristics established from our study will further our understanding of the pathogenesis of PCOS and help us to identify new targets for further studies and for the development of new therapeutic interventions.

Atrial natriuretic peptide (ANP) as well as its receptors is found in mammalian ovary and follicular cells and its function in oocyte meiotic maturation has also been reported in Xenopus, hamster and rat. But the results are controversial and the physiological mechanism of ANP on oocyte maturation is not clear, especially the relationship between gonadotrophin and ANP as well as the signal transduction, and these need further study. The present study conducted experiments to examine these questions by using drug treatment and Western blot analysis and focused on pig oocyte meiotic maturation and cumulus expansion in vitro. The results revealed that ANP could inhibited FSH-induced pig oocyte maturation and cumulus expansion and prevent the full phosphorylation of mitogen-activated protein kinase in both oocytes and cumulus cells, and that these inhibitory effects could be mimicked by 8-Br-cyclic guanosine 5′-monophosphate (8-Br-cGMP), but blocked by a protein kinase G (PKG) inhibitor KT5823. Zaprinast, a cGMP-specific phosphodiesterase inhibitor, could enhance the inhibitory effect of ANP on oocyte maturation. A specific analogue of ANP, C-ANP-(4–23), which binds to the natriuretic peptide receptor-C (NPRC), had no effect in either FSH-induced or spontaneous oocyte maturation. Treatment with forskolin, a stimulator of adenylate cyclase, had a biphasic effect; 44 h treatment induced cumulus expansion but inhibited oocyte maturation while 2 h treatment induced maturation of cumulus-enclosed oocytes (CEOs). Both ANP and C-ANP-(4–23) could inhibit the effect of forskolin on CEO maturation, and these inhibitory effects of ANP/C-ANP-(4–23) could be blocked by preincubation with pertussis toxin (PT), consistent with mediation by a Gi protein(s) in the cumulus cells. All these results suggest that ANP is a multifunctional regulator of FSH and forskolin on pig CEO maturation by two signalling mechanisms: one is via a cGMP/PKG pathway, the other is via NPRC receptors in cumulus cells and the activation of the PT-sensitive Gi protein(s).

To clarify tissue-specificity of pancreatic β cells, comparison of mRNA expression in various conditions of the tissue of multiple organisms is important. Although the developed methodologies for mRNA monitoring such as microarray, rely on the growth of dbEST (database of expressed sequence tag), a large number of unknown genes in the genome, especially in the rat, have not been shown to be expressed. In this study, we have established the first database of ESTs from rat pancreatic islet and RINm5F cells. Two cDNA libraries were constructed using mRNAs from rat pancreatic islet and RINm5F cells to cover a wider spectrum of expressed genes. Over 40 000 clones were randomly selected from the two libraries and partially sequenced. The sequences obtained were subjected to BLAST database analyses. This large-scale sequencing generated 40 710 3′-ESTs. Clustering analysis and homology search of nucleotide and peptide databases using both 3′- and 5′-ESTs revealed 10 406 non-redundant transcripts representing 4078 known genes or homologs and 6328 unknown genes. To confirm actual expression, the unknown sequences were further subjected to dbEST search, resulting in the identification of 5432 significant matches to those from other sources. Interestingly, of the remaining sequences showing no match, 779 were found to be encoded by exon–intron organization in the corresponding genomic sequences, suggesting that these are newly found as actually expressed in this study. Since many genes are up- or down-regulated in differing conditions, applications of the expression profile should facilitate identification of the genes involved in cell-specific functions in normal and disease states.