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GE Rice, MH Wong, W Farrugia and KF Scott

Although phospholipase A2 (PLA2) enzymatic activities have been implicated in the regulation of phospholipid metabolism and eicosanoid formation in human gestational tissues, the role and contribution made by individual PLA2 isozymes has not been established. The aim of this study, therefore, was to determine the contribution made by Type II PLA2 to PLA2 enzymatic activity present in human term placenta. The experimental paradigm used to establish the contribution made by Type II PLA2 to total in vitro PLA2 enzymatic activity present in placental extracts was to remove Type II PLA2 by immunoaffinity extraction and then to quantify residual PLA2 enzymatic activity. Before immunoaffinity extraction, Type II PLA2 immunoactivity and total PLA2 enzymatic activity present in placental extracts averaged 28.0 +/- 10.0 ng/mg protein and 1040 +/- 367 pmol/h per mg protein (n = 3) respectively. After solid-phase immunoaffinity batch extraction of placental extracts, immunoreactive Type II PLA2 was not detectable by ELISA, and PLA2 enzymatic activity was decreased by 82 +/- 1% (P < 0.001). Residual (i.e. non-Type II) PLA2 enzymatic activity was further characterised by Western blot analysis and enzyme activity assay. The data obtained are consistent with a contribution by both cytosolic PLA2 and other secretory PLA2 isozymes (i.e. non-Type II) to residual PLA2 enzymatic activity. The results obtained in this study support the conclusion that Type II PLA2 is quantitatively the primary PLA2 isozyme that contributes to in vitro PLA2 enzymatic activity present in extracts of human term placenta, accounting for at least 80% of total activity. These data further support the involvement of this extracellularly active isozyme in the regulation of placental phospholipid metabolism and eicosanoid formation during late gestation.

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S. J. Winder, S. D. Wheatley and I. A. Forsyth

ABSTRACT

Sucrose density centrifugation was used to prepare a partially purified membrane fraction from the mammary glands of non-pregnant, pregnant and lactating sheep. The binding of125 I-labelled insulin-like growth factor-I (IGF-I) was dependent on membrane protein concentration, pH, time and temperature. The binding showed the characteristics of a type-1 IGF receptor, being displaced by IGF-I (median effective dose (ED50) 0·55 nmol/l), less effectively by IGF-II (ED50 8·8 nmol/l) and least effectively by insulin. Glucagon, ovine prolactin and ovine placental lactogen could not displace binding. A molecular weight of 135 000 was determined by affinity cross-linking using disuccinimidyl suberate; this was consistent with the reported size of the type-1 receptor α-subunit. Scatchard analysis was used to determine binding affinity and numbers of IGF-I-binding sites. A single class of high-affinity binding sites was found in all physiological states. In non-pregnant sheep and sheep at days 40, 75 and 110–120 of pregnancy and at term, the binding affinity was similar (apparent dissociation constant (K d) 2·73 ±0·31 nmol/l, n = 22). In lactating sheep (weeks 1, 4 and 10), the binding affinity was significantly (P = 0·02) higher (K d 0·77± 0·06 nmol/l n = 9). Binding capacity was similar in non-pregnant and pregnant sheep (1005 ± 113 fmol/mg, n = 19), but fell by parturition and remained low in lactation (570±52 fmol/mg membrane protein, n = 12). The results suggest that the mammary growth of pregnancy is not regulated at the level of the type-1 IGF receptor.

Journal of Endocrinology (1993) 136, 297–304

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R. C. Bonney, S. T. Qizilbash and S. Franks

ABSTRACT

The inhibition of endometrial phospholipase A2 activity by the non-steroidal anti-inflammatory agents mefenamic acid and indomethacin was studied over the concentration range 1 mmol/l–0·1 μmol/l. Both phospholipase A2 type 1 (a calcium-dependent enzyme) and phospholipase A2 type 2 (a calciumindependent enzyme) were inhibited by mefenamic acid, but the magnitude of the inhibition was dependent on calcium concentration. Phospholipase A2 type 1 was inhibited 50% by 10 μmol mefenamic acid/1 in the presence of 1·25–5 mmol calcium/l, but a concentration of 2·2 mmol mefenamic acid/l was required for 50% inhibition in the absence of calcium. On the other hand, phospholipase A2 type 2 was inhibited 50% by 22 μmol mefenamic acid/1 in the absence of calcium and by 100 μmol mefenamic acid/l in the presence of calcium (2·5 mmol/l). Although indomethacin was a less effective inhibitor of phospholipase A2 activity, a similar relationship with calcium was demonstrated. However, indomethacin also had a stimulatory effect on phospholipase A2 type 1 activity in the absence of calcium. Our findings suggest that the two endometrial enzymes may be inhibited by different mechanisms and that the dependence of the enzyme on calcium for activation may be a contributing factor.

J. Endocr. (1988) 119, 141–145

Free access

H Del Zotto, L Massa, R Rafaeloff, GL Pittenger, A Vinik, G Gold, A Reifel-Miller and JJ Gagliardino

The possible relationship between changes in islet cell mass and in islet neogenesis-associated protein (INGAP)-cell mass induced by sucrose administration to normal hamsters was investigated. Normal hamsters were given sucrose (10% in drinking water) for 5 (S8) or 21 (S24) weeks and compared with control (C) fed hamsters. Serum glucose and insulin levels were measured and quantitative immunocytochemistry of the endocrine pancreas was performed. Serum glucose levels were comparable among the groups, while insulin levels were higher in S hamsters. There was a significant increase in beta-cell mass (P<0.02) and in beta-cell 5-bromo-2'-deoxyuridine index (P<0.01), and a significant decrease in islet volume (P<0.01) only in S8 vs C8 hamsters. Cytokeratin (CK)-labelled cells were detected only in S8 hamsters. INGAP-positive cell mass was significantly larger only in S8 vs C8 hamsters. Endocrine INGAP-positive cells were located at the islet periphery ( approximately 96%), spread within the exocrine pancreas ( approximately 3%), and in ductal cells (<1%) in all groups. INGAP positivity and glucagon co-localization varied according to topographic location and type of treatment. In C8 hamsters, 49.1+/-6. 9% cells were INGAP- and glucagon-positive in the islets, while this percentage decreased by almost half in endocrine extra-insular and ductal cells. In S8 animals, co-expression increased in endocrine extra-insular cells to 36.3+/-9.5%, with similar figures in the islets, decreasing to 19.7+/-6.9% in ductal cells. INGAP-positive cells located at the islet periphery also co-expressed CK. In conclusion, a significant increase of INGAP-positive cell mass was only observed at 8 weeks when neogenesis was present, suggesting that this peptide might participate in the control of islet neogenesis. Thus, INGAP could be a potentially useful tool to treat conditions in which there is a decrease in beta-cell mass.

Free access

A Alidibbiat, C E Marriott, K T Scougall, S C Campbell, G C Huang, W M Macfarlane and J A M Shaw

Generation of new β-cells from the adult pancreas or the embryonic stem cells is being pursued by research groups worldwide. Success will be dependent on confirmation of true β-cell phenotype evidenced by capacity to process and store proinsulin. The aim of these studies was to robustly determine endocrine characteristics of the AR42J rat pancreatic acinar cell line before and after in vitro transdifferentiation. β-cell phenotypic marker expression was characterised by RT-PCR, immunostaining, western blotting, ELISA and in human preproinsulin transgene over-expression studies in wild-type AR42J cells and after culture on Matrigel basement membrane matrix with and without growth/differentiation factor supplementation. Pancreatic duodenal homeobox 1 (PDX1), forkhead box transcription factor a2 (Foxa2), glucokinase, pancreatic polypeptide and low-level insulin gene transcription in wild-type AR42J cells were confirmed by RT-PCR. Culture on Matrigel-coated plates and supplementation of medium with glucagon-like peptide 1 induced expression of the β-cell Glut 2 with maintained expression of insulin and PDX1. Increased biosynthesis and secretion of proinsulin were confirmed by immunocytochemical staining and sensitive ELISA. Absence of the regulated secretory pathway was demonstrated by undetectable prohormone convertase expression. In addition, inability to process and store endogenous proinsulin or human proinsulin translated from a constitutively over-expressed preproinsulin transgene was confirmed. The importance of robust phenotypic characterisation at the protein level in attempted β-cell transdifferentiation studies has been confirmed. Rodent and human sensitive/specific differential proinsulin/insulin ELISA in combination with human preproinsulin over-expression enables detailed elucidatation of core endocrine functions of proinsulin processing and storage in putative new β-cells.

Free access

Alison J Forhead, Juanita K Jellyman, Katherine Gillham, Janelle W Ward, Dominique Blache and Abigail L Fowden

The actions of angiotensin II on type 1 (AT1) and type 2 (AT2) receptor subtypes are important for normal kidney development before birth. This study investigated the effect of AT1 receptor antagonism on renal growth and growth regulators in fetal sheep during late gestation. From 125 days of gestation (term 145±2 days), chronically catheterised sheep fetuses were infused intravenously for 5 days with either an AT1-specific receptor antagonist (GR138950, 2–4 mg/kg per day, n=5) or saline (0.9% NaCl, n=5). Blockade of the AT1 receptor decreased arterial blood oxygenation and pH and increased blood pCO2, haemoglobin and lactate, and plasma cortisol and IGF-II. Blood glucose and plasma thyroid hormones and IGF-I were unchanged between the treatment groups. On the 5th day of infusion, the kidneys of the GR-treated fetuses were lighter than those of the control fetuses, both in absolute and relative terms, and were smaller in transverse cross-sectional width and cortical thickness. In the GR-infused fetuses, renal AT2 receptor protein concentration and glomerular density were significantly greater than in the saline-infused fetuses. Blockade of the AT1 receptor had no effect on relative cortical thickness, fractional or mean glomerular volumes, or renal protein levels of the AT1 receptor, IGF type 1 receptor, insulin receptor or protein kinase C ζ. Therefore, in the ovine fetus, AT1 receptor antagonism causes increased renal protein expression of the AT2 receptor subtype, which, combined with inhibition of AT1 receptor activity, may be partly responsible for growth retardation of the developing kidney.

Free access

Suwattanee Kooptiwut, Wanthanee Hanchang, Namoiy Semprasert, Mutita Junking, Thawornchai Limjindaporn and Pa-thai Yenchitsomanus

Hypogonadism in men is associated with an increased incidence of type 2 diabetes. Supplementation with testosterone has been shown to protect pancreatic β-cell against apoptosis due to toxic substances including streptozotocin and high glucose. One of the pathological mechanisms of glucose-induced pancreatic β-cell apoptosis is the induction of the local rennin–angiotensin–aldosterone system (RAAS). The role of testosterone in regulation of the pancreatic RAAS is still unknown. This study aims to investigate the protective action of testosterone against glucotoxicity-induced pancreatic β-cell apoptosis via alteration of the pancreatic RAAS pathway. Rat insulinoma cell line (INS-1) cells or isolated male mouse islets were cultured in basal and high-glucose media in the presence or absence of testosterone, losartan, and angiotensin II (Ang II), then cell apoptosis, cleaved caspase 3 expression, oxidative stress, and expression of angiotensin II type 1 receptor (AGTR1) and p47phox mRNA and protein were measured. Testosterone and losartan showed similar effects in reducing pancreatic β-cell apoptosis. Testosterone significantly reduced expression of AGTR1 protein in INS-1 cells cultured in high-glucose medium or high-glucose medium with Ang II. Testosterone decreased the expression of AGTR1 and p47phox mRNA and protein in comparison with levels in cells cultured in high-glucose medium alone. Furthermore, testosterone attenuated superoxide production when co-cultured with high-glucose medium. In contrast, when cultured in basal glucose, supplementation of testosterone did not have any effect on cell apoptosis, oxidative stress, and expression of AGT1R and p47phox. In addition, high-glucose medium did not increase cleaved caspase 3 in AGTR1 knockdown experiments. Thus, our results indicated that testosterone prevents pancreatic β-cell apoptosis due to glucotoxicity through reduction of the expression of ATGR1 and its signaling pathway.

Free access

LM Thurston, E Chin, KC Jonas, IJ Bujalska, PM Stewart, DR Abayasekara and AE Michael

In a range of tIssues, cortisol is inter-converted with cortisone by 11beta-hydroxysteroid dehydrogenase (11betaHSD). To date, two isoforms of 11betaHSD have been cloned. Previous studies have shown that human granulosa cells express type 2 11betaHSD mRNA during the follicular phase of the ovarian cycle, switching to type 1 11betaHSD mRNA expression as luteinization occurs. However, it is not known whether protein expression, and 11betaHSD enzyme activities reflect this reported pattern of mRNA expression. Hence, the aims of the current study were to investigate the expression and activities of 11betaHSD proteins in luteinizing human granulosa-lutein (hGL) cells. Luteinizing hGL cells were cultured for up to 3 days with enzyme activities (11beta-dehydrogenase (11betaDH) and 11-ketosteroid reductase (11 KSR)) and protein expression (type 1 and type 2 11betaHSD) assessed on each day of culture. In Western blots, an immunopurified type 1 11betaHSD antibody recognized a band of 38 kDa in hGL cells and in human embryonic kidney (HEK) cells stably transfected with human type 1 11betaHSD. The type 2 11betaHSD antibody recognized a band of 48 kDa in HEK cells transfected with human type 2 11betaHSD cDNA but the type 2 protein was not expressed in hGL cells throughout the 3 days of culture. While the expression of type 1 11betaHSD protein increased progressively by 2.7-fold over 3 days as hGL cells luteinized, both 11betaDH and reductase activities declined (by 52.9% and 34.2%; P<0.05) over this same period. Changes in enzyme expression and activity were unaffected by the suppression of ovarian steroid synthesis.

Free access

SG Derman, S Kol, I Ben-Shlomo, CE Resnick, RM Rohan and EY Adashi

Transforming growth factor beta1 (TGFbeta1) acts as an inhibitor of the actions of interleukin-1beta (IL-1beta) in various organ systems. In order better to understand the inter|P-actions between these polypeptides in the ovary, we evaluated the effect of TGFbeta1 co-treatment on various IL-1beta-mediated actions in cultures of whole ovarian dispersates. Treatment with IL-1beta enhanced media accumulation of nitrites (4.8-fold), prostaglandin E2 (PGE2, 3. 9-fold) and lactate (2.0-fold), and enhanced glucose consumption (2. 1-fold). Treatment with TGFbeta1 alone did not significantly affect any of these parameters. However, the addition of TGFbeta1 inhibited IL-1beta-stimulated nitrite (100%), PGE2 (44%) and lactate (78%) accumulation and inhibited IL-1beta-stimulated glucose consumption (74%) in a dose-dependent manner. The addition of TGFbeta1 also suppressed the steady-state levels of IL-1beta-stimulated IL-1beta, type I IL-1 receptor and IL-1 receptor antagonist transcripts (98, 67 and 83% inhibition respectively). These data suggest that TGFbeta1 is capable of inhibiting several IL-1beta-stimulated endpoints. Since IL-1 has been identified as a possible proinflammatory mediator of ovulation and TGFbeta has been implicated as a promotor of fibrosis and healing, we speculate that IL-1 and TGFbeta might play antagonistic roles in the normal ovulatory sequence.

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Akhilesh K Pandey, Wei Li, Xiangling Yin, Douglas M Stocco, Paula Grammas and XingJia Wang

Previous studies have reported the roles of Ca2+ in steroidogenesis. The present study has investigated an inhibitory effect of Ca2+ influx through L-type Ca2+ channels on gene expression of steroidogenic acute regulatory (STAR) protein that regulates the transfer of substrate cholesterol to the inner mitochondrial membrane for steroidogenesis. Blocking Ca2+ influx through L-type Ca2+ channels using the selective Ca2+ channel blocker, nifedipine, markedly enhanced cAMP-induced STAR protein expression and progesterone production in MA-10 mouse Leydig cells. This was confirmed by utilization of different L-type Ca2+ channel blockers. Reverse transcription-PCR analyses of Star mRNA and luciferase assays of Star promoter activity indicated that blocking Ca2+ influx through L-type Ca2+ channels acted at the level of Star gene transcription. Further studies showed that blocking the Ca2+ channel enhanced Star gene transcription by depressing the expression of DAX-1 (NR0B1 as listed in the MGI Database) protein, a transcriptional repressor of Star gene expression. It was also observed that there is a synergistic interaction between nifedipine and cAMP. Normally, sub-threshold levels of cAMP are unable to induce steroidogenesis, but in the presence of the L-type Ca2+ channel blocker, they increased STAR protein and steroid hormone to the maximal levels. However, in the absence of minimal levels of cAMP, none of the L-type Ca2+ channel blockers are able to induce Star gene expression. These observations indicate that Ca2+ influx through L-type Ca2+ channels is involved in an inhibitory effect on Star gene expression. Blocking L-type Ca2+ channel attenuated the inhibition and reduced the threshold of cAMP-induced Star gene expression in Leydig cells.