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.

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.

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.

The calcium-sensing receptor (CASR), a plasma membrane G-protein-coupled receptor, is expressed in parathyroid gland and kidney, and controls systemic calcium homeostasis. Inactivating CASR mutations are associated with familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism, and activating mutations cause autosomal dominant hypocalcemia (ADH). CASR mutation identification plays an important role in the clinical management of mineral metabolism disorders. We describe here a high-throughput method using screening with denaturing high performance liquid chromatography (DHPLC) to initially interrogate 12 amplicons covering translated exons and exon/intron boundaries, followed by sequencing of any amplicon with a modified melting curve relative to wild type, and direct sequencing of a 13th amplicon encoding the COOH-terminal tail to distinguish causative mutations from three common missense single nucleotide polymorphisms. A blinded analysis of 32 positive controls representing mutations throughout the CASR sequence, as well as 22 negative controls, yielded a concordance rate of 100%. We report eight novel and five recurrent FHH mutations, along with six novel and two recurrent ADH mutations. Thus, DHPLC provides a rapid and effective means to screen for CASR mutations.

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).