Increased insulin sensitivity and hypoglycaemia in mice lacking the p85 alpha subunit of phosphoinositide 3-kinase.

The hallmark of type 2 diabetes, the most common metabolic disorder, is a defect in insulin-stimulated glucose transport in peripheral tissues. Although a role for phosphoinositide-3-kinase (PI3K) activity in insulin-stimulated glucose transport and glucose transporter isoform 4 (Glut4) translocation has been suggested in vitro, its role in vivo and the molecular link between activation of PI3K and translocation has not yet been elucidated. To determine the role of PI3K in glucose homeostasis, we generated mice with a targeted disruption of the gene encoding the p85alpha regulatory subunit of PI3K (Pik3r1; refs 3-5). Pik3r1-/- mice showed increased insulin sensitivity and hypoglycaemia due to increased glucose transport in skeletal muscle and adipocytes. Insulin-stimulated PI3K activity associated with insulin receptor substrates (IRSs) was mediated via full-length p85 alpha in wild-type mice, but via the p50 alpha alternative splicing isoform of the same gene in Pik3r1-/- mice. This isoform switch was associated with an increase in insulin-induced generation of phosphatidylinositol(3,4,5)triphosphate (PtdIns(3,4,5)P3) in Pik3r1-/- adipocytes and facilitation of Glut4 translocation from the low-density microsome (LDM) fraction to the plasma membrane (PM). This mechanism seems to be responsible for the phenotype of Pik3r1-/- mice, namely increased glucose transport and hypoglycaemia. Our work provides the first direct evidence that PI3K and its regulatory subunit have a role in glucose homeostasis in vivo.

A novel cDNA clone encoding a prolactin-like protein that lacks the two C-terminal cysteine residues isolated from bovine placenta.

A new prolactin-like cDNA clone, bPLP-IV, was isolated from a bovine placental cDNA library and the complete nucleotide sequence was determined. The bPLP-IV encodes a protein consisting of 237 amino acids, which is related to, but different from seven other known bovine prolactin-like proteins including two placental lactogens. The predicted amino acid sequence of the bPLP-IV shows over 52% identity to other known members of bovine prolactin-like proteins, 48% to bovine prolactin, 40% to both two bovine placental lactogens and only 22% to bovine growth hormone. The bPLP-IV protein has a unique feature in its primary structure, lacking the two C-terminal cysteine residues which are completely conserved in all other known members of prolactin-growth hormone-placental lactogen gene family. The expression of bPLP-IV in developing bovine placenta was apparently stage-specific, being maximal in the full-term placenta.

Preferential expression of long form prolactin receptor mRNA in the rat brain during the oestrous cycle, pregnancy and lactation: hormones involved in its gene expression.

The mRNA species for prolactin receptor (PRL-R) isoforms, long and short form PRL-Rs, were estimated by the reverse transcription-polymerase chain reaction method in the rat brain (cerebrum) during the oestrous cycle, pregnancy and lactation. The levels of long form PRL-R mRNA increased at pro-oestrus and oestrus, at the same time as serum prolactin levels increased, whereas the mRNA level of short form PRL-R was relatively unchanged. Long form PRL-R mRNA expression was also markedly increased in the brain at mid- and late gestation, and this elevated mRNA level was maintained during the period of lactation. In contrast, basal levels of short form PRL-R mRNA were also maintained throughout these periods of gestation and lactation. Ovariectomy moderately reduced brain mRNA levels of both long and short form PRL-R from the levels of those in control dioestrous rats, and hypophysectomy further suppressed them to the lowest levels. Administration of oestradiol valerate (E2V) or 17 alpha-hydroxyprogesterone caproate (17OHPC) to ovariectomized rats resulted in dramatic increases in long form PRL-R mRNA levels in the brain, whereas no significant increase in short form PRL-R mRNA was observed. In rats which were ovariectomized and hypophysectomized, the administration of 17OHPC, rat prolactin or rat GH partially restored the brain level of long form PRL-R mRNA but not short form PRL-R mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Pup contact induces the expression of long form prolactin receptor mRNA in the brain of female rats: effects of ovariectomy and hypophysectomy on receptor gene expression.

Prolactin receptor (PRL-R) mRNA expression levels in the female rat brain (cerebrum) during pup contact stimulation were determined by the reverse transcription-PCR method. The high expression levels of long form PRL-R mRNA found in the brain of lactating rats were markedly reduced by removal of pups, and long form PRL-R mRNA levels were recovered by resumption of pup contact. Interestingly, pup contact stimuli of nulliparous virgin rats also markedly induced long form but not short form PRL-R mRNA expression in the brain in 1.3 days, together with the expression of maternal behaviour. In ovariectomized (OVX) or hypophysectomized (HYPOX) virgin rats, or in OVX plus HYPOX virgin rats, however, brain long form PRL-R mRNA was not significantly induced by pup contact stimuli for as long as 7 days, while maternal behaviour was fully expressed in these rats after 7 days of pup contact. The in situ hybridization experiments revealed that the long form PRL-R mRNA induced in virgin rats in contact with pups or in lactating rats was localized in the epithelial cells of the choroid plexus. No significant increase in mRNA was detected in other regions of the brain, such as the hypothalamus or cortex, in these maternal female rats. These results suggest that pup contact induces the expression of long form PRL-R mRNA in the choroid plexus of the brain in the presence of female sex steroid and pituitary hormones for the rapid expression of maternal behaviour. Our studies also suggested that maternal behaviour can be expressed in OVX or HYPOX rats after exposure to pups for 7 days without any significant increase in brain PRL-R mRNA expression.

Relation of rat fetal body weight to the relative expression of insulin genes I and II.

This study investigates the relation between the ratio of the abundance of mRNAs encoded by insulin genes I and II (insulin I/II mRNA ratio) and the weight of rat fetuses on gestational day 20. Total RNA was extracted from the pancreas of the fetuses with the maximum and minimum body weight in each litter on gestational day 20. The amount of insulin mRNAs I and II in each RNA preparation was determined by reverse transcription-polymerase chain reaction (RT-PCR) analysis and restriction enzyme digestion. The maximum and minimum weight of the fetuses were 4.07 +/- 0.23 and 3.23 +/- 0.34 g, respectively (N = 18, P < .01) and the corresponding insulin I/II mRNA ratios were 3.65 +/- 0.55 and 1.42 +/- 0.21 (N = 18, P < .01). Furthermore, the insulin I/II mRNA ratio correlated significantly with fetal weight (r = .526, P < .05). These results suggest that the relative expression of insulin genes I and II may affect the extent of fetal growth.

A prospective randomized comparison of routine buserelin acetate and a decreasing dosage of nafarelin acetate with a low-dose gonadotropin-releasing hormone agonist protocol for in vitro fertilization and intracytoplasmic sperm injection.

OBJECTIVE: To compare the efficacy of a draw-back nafarelin acetate protocol with routine buserelin acetate administration for in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). DESIGN: Prospective clinical study. SETTING: Mie University School of Medicine, Tsu, Mie, Japan. PATIENT(S): One hundred sixty-nine women treated with IVF and 183 women treated with ICSI. INTERVENTION(S): Nafarelin acetate and buserelin acetate in ovarian hyperstimulation in IVF and ICSI were administered. MAIN OUTCOME MEASURE(S): The concentrations of estradiol (E(2)), FSH, LH, gonadotropin dosages; the number of oocytes retrieved, oocytes fertilized, and embryos; and pregnancy rates. RESULT(S): A prospective study was conducted with 44 cycles for 34 couples with nafarelin acetate (group 1) and 47 cycles for 40 couples with buserelin acetate (group 2) with a long IVF protocol; 68 cycles for 46 couples with nafarelin acetate (group 3) and 56 cycles for 39 couples with buserelin acetate (group 4) with a short IVF protocol; 39 cycles for 32 couples with nafarelin acetate (group 5) and 50 cycles for 30 couples with buserelin acetate (group 6) with a long ICSI protocol; and 87 cycles for 60 couples with nafarelin acetate (group 7) and 81 cycles for 61 couples with buserelin acetate (group 8) with a short ICSI protocol. Patients were randomized to receive either full-dose nafarelin acetate (200 microg b.i.d.) treatment for 7 days followed by half-dose nafarelin acetate (200 microg daily) or buserelin acetate (300 microg t.i.d.). There were no statistically significant differences in baseline concentrations of E(2) and FSH, concentrations of E(2), P4, FSH, LH on hCG administration, gonadotropin dosage, the number of oocytes retrieved and embryos transferred, or pregnancy rates between groups 1 and 2, groups 3 and 4, groups 5 and 6, and groups 7 and 8. CONCLUSION(S): Full-dose nafarelin acetate treatment for 7 days followed by half-dose nafarelin acetate ("draw-back" protocol) is an effective new protocol for IVF and ICSI.

Relative expression of insulin genes I and II during pancreatic development in fetuses of rats with streptozotocin-induced diabetes.

The relative expression of insulin genes I and II in the developing pancreas of fetuses from female rats with streptozotocin-induced diabetes was investigated by reverse transcription and polymerase chain reaction analysis. Unlike the fetuses of normal rats, in which the ratio of the pancreatic abundance of insulin I mRNA to that of insulin II mRNA increases during late gestational development, peaks around birth, and then decreases to adult values, the ratio in the offspring of diabetic mothers remained relatively constant (1.6 to 2.2) throughout this period at values similar to those of normal adult animals. These results suggest that changes in the maternal environment attributable to diabetes affect the relative expression of insulin genes I and II, and that the two genes are regulated by distinct mechanisms.

Possible direct effect of gonadotropin releasing hormone on human endometrium and decidua.

Decidualization of endometrial tissues, which is essential for implantation and the continuation of pregnancy, is induced by pituitary hormones that are regulated by gonadotropin releasing hormone (GnRH). Our objective was to determine the role of a direct action of GnRH on endometrial tissues by comparing the characteristics of receptors for GnRH in human endometrial and decidual tissues. Competitive binding studies were performed with the protease-resistant GnRH analogues, buserelin and [125I] buserelin. The effects of buserelin on phosphoinositol turnover were determined by the measurement of inositol 1,4,5-triphosphate(IP3). The values for the dissociation constant (Kd) and number of binding sites (Bmax) per unit protein versus buserelin for endometrial tissues did not differ from the values for decidual tissues. However, the Bmax per unit DNA was significantly higher in endometrial tissues. Also, buserelin induced a significant increase in IP3 in decidual tissue. These results indicate that GnRH may be a potential modulator of the function in human endometrium and decidua. The signal transduction mechanism for GnRH action appeared to involve the accelerated turnover of phosphoinositol.

Expression of glucose transporter 4 mRNA in adipose tissue and skeletal muscle of ovariectomized rats treated with sex steroid hormones.

The effects of 17beta-estradiol (E) and/or progesterone (P) on glucose transporter 4 (GLUT4) expression in the adipose tissue and skeletal muscle of ovariectomized female rats were studied. The Sprague-Dawley rats received daily subcutaneous injections of various doses of E and/or P for 7 days (n=5-6 per dose). The expression of GLUT4 mRNA was assessed by performing ribonuclease protection assays. GLUT4 protein levels were assessed by Western blotting assays. The adipose tissue levels of GLUT4 mRNA were reduced by the administration of 50 microg E, which resulted in unphysiologically high serum E concentrations. Although the GLUT4 mRNA levels did not change after the administration of 10 microg E or 5 mg P, they were reduced significantly to approximately half the control group level by the administration of both hormones (p <0.01). The skeletal muscle GLUT4 mRNA levels were not changed significantly by hormone treatment. These findings suggest that E and P may be involved in the regulation of GLUT4 mRNA expression in adipose tissue.

GnRH agonist. Increasing the pregnancy rate after combined treatment with hMG/hCG and direct intraperitoneal insemination.

OBJECTIVE: To evaluate the efficacy of the GnRH agonist (GnRHa) administered in conjunction with human menopausal gonadotropin/human chorionic gonadotropin hMG/hCG and direct intraperitoneal insemination (DIPI) in women with long-standing unexplained infertility. STUDY DESIGN: A prospective, randomized, non-blind analysis. During the period May 1995-December 1996, couples with unexplained infertility who failed to conceive following superovulation combined with IUI for at least seven cycles were prospectively enrolled and followed. Pregnancy rates per cycle and per patient of DIPI were compared between groups of hMG/hCG with (GnRHa[+] controlled ovarian hyperstimulation [COH] group) or without (GnRHa[-] COH group) GnRHa. RESULTS: Thirty-four women (59 cycles) underwent COH with hMG and GnRHa, and 31 women (49 cycles) received hyperstimulation with hMG alone. The pregnancy rates for the women administered GnRHa significantly exceeded those of the patients who did not receive GnRHa both per treatment cycle (35.6% versus 14.3%) and per couple (55.9% versus 22.5%). CONCLUSION: The use of GnRHa with hMG/hCG and DIPI treatment significantly increased the pregnancy rate in women with long-standing infertility.

Dependence of the photoelectrochemical performance of sensitised ZnO on the crystalline orientation in electrodeposited ZnO thin films.

The influence of the crystal orientation in porous crystalline films of ZnO electrodeposited on the photoelectrochemical characteristics of the films is studied. For differently oriented ZnO thin films following removal of the respective structure-directing agent (SDA) and adsorption of a sensitiser, time-resolved photocurrent measurements, intensity modulated photocurrent spectroscopy (IMPS), intensity modulated photovoltage spectroscopy (IMVS) and current-voltage curves were measured in acetonitrile-based electrolytes containing I(3)(-)/I(-) as the redox electrolyte. The crystal orientation has a significant influence on the charge transport across such films and hence is reflected in the observed electrode kinetics. Films originally grown in the presence of, e.g., Coumarin 343 as a SDA, showed a significantly faster response to illumination. Increased electron diffusion coefficients and diffusion lengths were calculated from the results of IMPS and IMVS, caused by a faster electron movement in the films. Implications of these findings on further improvements of sensitised ZnO films prepared by electrochemical deposition are discussed.

Mechanism by which a novel non-thiazolidinedione peroxisome proliferator-activated receptor gamma agonist, FK614, ameliorates insulin resistance in Zucker fatty rats.

AIM: The aim of this study was to examine the mechanism by which a novel non-thiazolidinedione (TZD) peroxisome proliferator-activated receptor (PPAR) gamma agonist, FK614, ameliorates insulin resistance in Zucker fatty rats. METHODS: FK614 (1, 3.2 or 10 mg/kg) and a TZD PPARgamma agonist, pioglitazone (1, 3.2 or 10 mg/kg), were orally administered to Zucker fatty rats (genetically obese and insulin resistant) once a day for 14 days, and an oral glucose tolerance test was performed. The expression levels of various genes in the white adipose tissue (WAT) of Zucker fatty rats treated with FK614 (3.2 mg/kg), pioglitazone (10 mg/kg) and another TZD PPARgamma agonist, rosiglitazone (3.2 mg/kg), were determined using a real-time reverse transcription-polymerase chain reaction method. Morphometric analysis of the WAT of Zucker fatty rats treated with FK614 (3.2 mg/kg) and pioglitazone (10 mg/kg) was performed. Glucose transport activity in the isolated soleus muscle of FK614-treated Zucker fatty rats was also investigated. RESULTS: FK614 and pioglitazone both improved glucose tolerance in Zucker fatty rats. FK614 significantly increased the expression levels of acyl CoA oxidase, a PPAR-responsive gene, and adipocyte fatty acid-binding protein (aP2), an adipocyte differentiation marker gene, in epididymal WAT. It also significantly decreased the level of gene expression of tumour necrosis factor-alpha, an insulin resistance-inducing factor in retroperitoneal WAT, as did pioglitazone and rosiglitazone. FK614 and pioglitazone both significantly increased the total number of adipocytes and decreased their average size in WAT, mainly by increasing the number of small adipocytes. Additionally, administration of FK614 to Zucker fatty rats enhanced insulin sensitivity for glucose uptake in the soleus muscle. CONCLUSION: This study suggests the possibility that FK614 induces adipocyte differentiation in Zucker fatty rats by stimulating PPARgammain vivo, thereby changing the character of WAT and improving insulin sensitivity throughout the body.

Photoelectrochemical characterisation and optimisation of electrodeposited ZnO thin films sensitised by porphyrins and phthalocyanines.

Hybrid thin films of crystalline ZnO modified by 5,10,15,20-tetrakis(4-sulfonatophenyl) porphyrinato zinc (TSTPPZn) and 2,9,16,23-tetrasulfophthalocyaninatozinc(II) (TSPcZn) were prepared by electrochemical deposition from aqueous zinc salt solutions. A "one-step" process with the sensitisers adsorbed during ZnO deposition represented the most simple approach. ZnO was also grown independently in the presence of Eosin Y as a structure-directing agent, which was then removed and the sensitisers were chemisorbed from solutions ("readsorption" method). The photoelectrochemical characteristics of the electrodes were studied by photocurrent spectra and by time-resolved photocurrent measurements in an acetonitrile-based solution containing I3(-)/I(-) as the redox electrolyte. In films with both sensitisers present, both sensitisers worked in parallel providing panchromatic sensitisation. Recombination of electrons injected into the conduction band of ZnO with remaining holes in the HOMO of the sensitisers was indicated for the one-step films, but was considerably suppressed for the films prepared by the readsorption method. Films prepared by the readsorption method showed a significantly increased efficiency by an increased surface area and suppressed recombination.

Host immunoglobulin G titre and antibody activity in haemolymph of the tick, Ornithodoros moubata.

Immunoglobulin G (IgG) in tick haemolymph was analysed immunochemically and biochemically for its antigenicity, antibody activity and relative concentration in a soft tick, Ornithodoros moubata (Murray) sensu Walton 1962 (Acari: Argasidae). Ouchterlony immunodiffusion tests showed that haemolymph from a tick engorged on rabbit IgG (or human IgG) through an artificial membrane, reacted with anti-rabbit IgG (anti-human IgG) but not with anti-human IgG (anti-rabbit IgG). This indicates that haemolymph of the fed tick contains IgG with a similar antigen specificity to host blood IgG. IgG from tick haemolymph was demonstrated by enzyme immunoassay to have the same antibody activity as ingested IgG. The IgG concentration in tick haemolymph was measured by a quantitative single immunodiffusion test. Changes of IgG titre after a bloodmeal were correlated with IgG activity, which was low for 5 days after a bloodmeal and then suddenly increased. The IgG titre reached a maximum 7 days post-engorgement, and remained high for over 4 months during and after oviposition. 125I-labelled IgG was injected into the tick haemocoel to determine the persistence of IgG in the haemolymph. Recovery of labelled IgG was low at 1 and 3 days, and high at 5, 8 and 16 days after engorgement. The data suggest that IgG in haemolymph disappears quickly soon after engorgement possibly by degradation and/or absorption (adhesion to tissues).

Effects of epidermal growth factor on preimplantation mouse embryos.

PURPOSE: Our purpose was to clarify the effects of epidermal growth factor (EGF) on mouse preimplantation embryos. METHODS: We examined the effect of EGF on two-cell and four-cell stage mouse embryos cultured in vitro. In preimplantation embryos, we analyzed the binding of 125I-EGF by autoradiography and EGF receptor mRNA by the reverse transcription-polymerase chain reaction. RESULTS: At more than 0.005 ng/ml, EGF relieved the two-cell block and regulated the differentiation of morula-stage embryos. These effects were negated by antiserum. EGF did not exhibit any marked effect on embryos between the four-cell and the morula stages. Specific binding for EGF and EGF receptor mRNA was detected during and after the morula stage. CONCLUSIONS: The effects of EGF on preimplantation mouse embryos differed according to the stage of development, promoting cleavage before the four-cell stage and regulating differentiation after the morula stage. This regulatory action is thought to be transmitted to the cells via EGF receptors.

Ornithodoros moubata: host immunoglobulin G in tick hemolymph.

Hemolymph proteins of a soft tick, Ornithodoros moubata, were analyzed immunochemically and biochemically. The components of tick hemolymph proteins were shown to be totally different from the host (rabbit) serum proteins by polyacrylamide gel electrophoresis with sodium dodecyl sulfate and Coomassie blue or silver stain. However, in the hemolymph of ticks engorged from rabbits immunoglobulin G was detected by immunoblotting analysis with goat anti-rabbit immunoglobulin G. The concentration of rabbit Immunoglobulin G in tick hemolymph changed with the physiological stages after a blood meal. Immunoglobulin G was isolated from tick hemolymph by affinity chromatography on a Protein A-Sepharose 4B column. Analysis of the isolated immunoglobulin G from tick hemolymph with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Ouchterlony double diffusion test showed it to be composed of the same subunits as heavy and light chains of host (rabbit) immunoglobulin G. Tracer experiments showed that 125I-labeled heavy and light chains of immunoglobulin G were detected in an intact form in hemolymph from ticks that sucked 125I-labeled rabbit immunoglobulin G through an artificial membrane. These facts suggested that the host rabbit immunoglobulin G ingested in the tick midgut passed through the gut wall without digestion. By solid-phase enzyme immunoassay, immunoglobulin in the hemolymph was shown to retain its antibody activity.

Changes in ovarian expression of tissue-type plasminogen activator and plasminogen activator inhibitor type-1 messenger ribonucleic acids during ovulation in rat.

We investigated the time course and localization of ovarian tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor type-1 (PAI-1) expression during the ovulatory period in rat by RNase protection assay and in situ hybridization. Immature female Wistar rats were injected with 25 IU pregnant mare serum gonadotropin (PMSG), followed 50 h later by 25 IU human chorionic gonadotropin (hCG). Levels of tPA mRNA were low before hormone treatment and after PMSG treatment. After hCG treatment, tPA mRNA levels increased rapidly, the first peak at 4 h after hCG treatment and reached a maximum just prior to ovulation, 12 h later, before declining again. PAI-1 mRNA was barely detectable before hormone treatment but was transiently induced by hCG treatment, reaching peak levels after 4 h. Subsequently, PAI-1 mRNA levels decreased until early luteinization. The expression of tPA mRNA 4 h after hCG treatment occurred mainly in the follicular thecal-interstitial cells, but was barely detectable in the granulosa cells, whereas 12 h after hCG treatment it was maximal in the granulosa cells of the large follicles destined to ovulate. PAI-1 mRNA was expressed mainly in ovarian stromal tissue and in the thecal external interstitial cells encapsulating the follicles at 4 h after hCG treatment. These results suggest that the temporal regulation of tPA biosynthesis after hCG induction depends on the cell types and size classes in the various ovarian compartments. PAI-1 may be produced by the stormal tissue and the thecal external interstitial cells and is perhaps implicated in structural changes during follicular growth, ovulation and luteinization.

In vitro fertilization and intracytoplasmic sperm injection for couples with unexplained infertility after failed direct intraperitoneal insemination.

PURPOSE: The objective was to determine the optimal insemination technique in patients undergoing in vitro fertilization (IVF) after failed direct intraperitoneal insemination (DIPI) and the outcome of intracytoplasmic sperm injection (ICSI) in such cases. METHODS: In case-control studies, 53 couples with unexplained infertility who underwent IVF after four failed DIPI cycles were compared with 75 couples with tubal or endometriosis infertility as controls. Thirty couples with unexplained infertility after failing to conceive with DIPI and conventional IVF who underwent ICSI and 58 couples with male-factor infertility as controls also were compared. Fertilization cleavage, embryo quality, implantation, and pregnancy were compared after IVF and after ICSI. RESULTS: There was a significant difference in fertilization rates after IVF between cases of unexplained infertility after failing to conceive with DIPI (40.4%) and patients with tubal or endometriosis infertility (67.9%). There also was a significant difference in total fertilization failure rates between the two groups (30.4% and 3.9%, respectively). There was a slight but significant difference in numbers of fertilized oocytes after ICSI between patients with low fertilization rate undergoing IVF after failing to conceive DIPI (85.8%) and patients with male factor (90.4%). Total fertilization failure was not observed in these cases. CONCLUSIONS: Couples with unexplained infertility after failing to conceive with DIPI show a failed fertilization or a low fertilization rate after IVF. However, they demonstrated a good chance of becoming pregnant after subsequent ICSI, even with statistically significant difference in fertilization rate as compared with male-factor cases.