Prolactin regulation of islet-derived INS-1 cells: characteristics and immunocytochemical analysis of STAT5 translocation.

The major changes in pancreatic islet function during pregnancy and after exposure to lactogens are an increase in beta-cell proliferation and enhanced insulin secretion. In this study we examined INS-1 cells as a potential model for further inquiry into PRL signaling in beta-cells. Proliferation of beta-cells, insulin secretion, and quantitative immunocytochemical analysis of STAT5 translocation were studied. PRL treatment of INS-1 cells resulted in a 2- to 4-fold increase in cell proliferation compared to that in the control group. In contrast, there was no effect of PRL treatment on HIT cell proliferation and only a very small effect on RIN cell proliferation. A significant effect on INS-1 cell proliferation was observed at 10 ng/ml and reached a maximum at 200 ng/ml. PRL treatment resulted in enhanced insulin secretion from INS-1 cells. There was a time-dependent increase in insulin secretion, which when corrected for cell number was 1.5-fold greater in the PRL-treated cells. The effects of PRL on cell division and insulin secretion were glucose dependent. The presence of the JAK family of tyrosine kinases and the transcription factor STAT5 in INS-1 cells was examined by immunocytochemical techniques. Although all members of the JAK family of kinases were detected, the staining intensity of JAK-2 was noticeably more intense. Initial studies of STAT5 translocation were performed using PRL-dependent Nb2 lymphoma cells, in which PRL treatment resulted in a nearly complete translocation of cytoplasmic STAT5 to the nucleus. Under control conditions there was a near-equal fluorescence intensity of STAT5 staining in the nucleus and cytoplasm of INS-1 cells. PRL treatment resulted in a time-dependent increase in STAT5 staining in the nucleus, with a corresponding decrease in the cytoplasm. The STAT5 staining intensity in the nucleus remained elevated for the duration of PRL treatment. This effect was reversible upon removal of PRL from the medium. Besides PRL, both GH and FBS induced a similar translocation of STAT5 to the nucleus. Although present in RIN cells, no detectable changes in STAT5 were observed in RIN cells after exposure to PRL, GH, or FBS. INS-1 cells should provide a good model for further inquiry into the intracellular signaling pathways used by PRL and how these events alter islet function.

Immunohistochemical localization of insulin-degrading enzyme along the rat intestine, in the human colon adenocarcinoma cell line (Caco-2), and in human ileum.

Insulin-degrading enzyme (IDE) has been implicated in the intracellular degradation of insulin in insulin target cells. Knowledge of the existence of this enzyme in the intestine will be beneficial to the achievement of clinical oral efficacy of insulin. A comparative study was conducted with rat intestine, human colon adenocarcinoma (Caco-2) cells, and human ileum. Confocal microscopy analysis using the anti-IDE antibody showed that IDE was localized in the mucosal cells of rat and human intestines, as well as in Caco-2 cells. Immunostaining of this enzyme was homogeneous throughout the cell excluding nucleus, indicating a typical cytosolic distribution in rat and human enterocytes and in Caco-2 cells.

Glucokinase, hexokinase, glucose transporter 2, and glucose metabolism in islets during pregnancy and prolactin-treated islets in vitro: mechanisms for long term up-regulation of islets.

During pregnancy, islets undergo a number of up-regulatory changes to meet the increased need for insulin. One of the most important changes is an increase in glucose-stimulated insulin secretion with a reduction in the glucose-stimulated threshold. Similarly, placental lactogen and PRL induce the same changes in islets as pregnancy. In this study, we examined the effects of pregnancy and PRL treatment of islets in vitro on insulin secretion; glucokinase and hexokinase activities; glucokinase, hexokinase, and glucose transporter 2 protein levels; and rates of glucose utilization and oxidation. Glucokinase activity was 4.9 +/- 0.4 pmol glucose/ng DNA.h in control islets and was significantly increased by 50% in islets on day 15 of pregnancy and by 60% on day 20 of pregnancy. Hexokinase activity was 11.7 +/- 0.9 pmol glucose/ng DNA.h in control islets and was increased by 20% in islets on day 15 of pregnancy and by 90% on day 20 of pregnancy. In the in vitro studies, glucokinase activity was 7.4 +/- 0.89 pmol glucose/ng DNA.h in control islets. PRL treatment of islets in vitro increased glucokinase activity by 60%, an effect similar to that observed in the pregnancy islets. In contrast, hexokinase activity was nearly undetectable in cultured islets, whether control or PRL treated. Quantitative Western blot analysis of glucokinase and hexokinase was performed using equivalent number of protein per lane for all experimental groups. On a protein equivalency basis, glucokinase expression levels were the same in control islets on days 15 and 20 of pregnancy. Likewise, hexokinase levels were not different between control islets and islets on days 15 and 20 of pregnancy. Similarly, Western blot analysis of cultured islets indicated that there were not effect of PRL on glucokinase or hexokinase levels. However, when enzyme levels were normalized on the basis of DNA, the levels of expression appeared to be commensurate with their activities. In cultured islets, the very low level of hexokinase activity corresponded to the low level of hexokinase detected by Western blots. Glucose transporter 2, as determined by Western blot quantification, was increased 2-fold in pregnancy islets on day 15 and increased by 45% in pregnancy islets on day 20. Similar results were observed in cultured islets where glucose transporter 2 was increased 2-fold in PRL-treated islets. Islet glucose utilization and oxidation rates on day 15 of pregnancy were significantly greater than those in control islets at all glucose concentrations examined. This enhanced glucose sensitivity resulted in a shift of the glucose utilization and oxidation response curves to the left. Comparable results were obtained from islets on day 20 of pregnancy. PRL treatment of islets in vitro resulted in the same changes in glucose utilization and oxidation rates that were observed during pregnancy. These results demonstrate changes in glucokinase, hexokinase, and glucose transporter 2 levels and glucose metabolism that occur as islets adapt to an increased need for insulin secretion during pregnancy. The results also indicate that these same changes can be induced by PRL treatment of islets in vitro. This provides further evidence that the long term adaptive changes that occur under the normoglycemic conditions of pregnancy are mediated by lactogen-regulated events.

Prolactin receptors and JAK2 in islets of Langerhans: an immunohistochemical analysis.

Lactogenic hormones, PRL and placental lactogen, are important regulators of insulin secretion and islet beta-cell proliferation. In this study we examined the presence of PRL receptor immunoreactivity in pancreatic islets of Langerhans using PRL receptor monoclonal antibodies provided by Dr. Paul Kelly. Studies were performed using islets isolated from neonatal, adult, and day 14 pregnant rats. The islets were examined by immunohistochemistry and laser scanning confocal microscopy. In neonatal rat islets, PRL receptors were observed in beta- and alpha-cells, but not in delta-cells. Among islet beta- and alpha-cells there was heterogeneity of cellular staining for PRL receptors. A small portion of the cells was intensely stained for PRL receptors. However, the majority of the cells had a much lower level of staining intensity, suggesting that most islet cells have a low level of PRL receptors. In general, alpha-cells were more uniformly stained than beta-cells. Similar results were obtained with adult rat islets, in which, again, there was a large range of staining intensity and many cells with low levels of PRL receptor. Rats on day 14 of pregnancy had an increased level of islet PRL receptor expression compared with age-matched control animals. There was also a decrease in cellular heterogeneity for PRL receptors, with nearly all cells having a uniformly high level of PRL receptor expression. JAK2, the tyrosine kinase associated with PRL receptors, was examined in Nb2 cells and islets. JAK2 immunoreactivity was detected at the cell membrane in very low levels in Nb2 cells. It was also found in numerous vesicular structures in the cytoplasm, where it colocalized with PRL receptors. A prominent feature of all cells was the presence of JAK2 in the nucleus, but not the nucleolus. In islets, JAK2 immunoreactivity was similarly observed in the nucleus of nearly all cells. However, the vesicular cytoplasmic location of JAK2 was less frequently observed and did not colocalize with PRL receptors. For comparison, JAK2 immunoreactivity was examined in several other tissues where it was detected in fibroblasts (endomysial and endoneurial cells), smooth muscle cells, and ganglion cells in the pancreas. JAK2 was notably absent from pancreas acinar cells, hepatocytes, skeletal muscle cells, and Schwann cells. This study demonstrates the presence of PRL receptors in islet beta- and alpha-cells, but not delta-cells. There was an increase in PRL receptor expression in islets during pregnancy, which is commensurate with the up-regulation of islet function. In addition, JAK2 immunoreactivity was detected in most islet cells and Nb2 node cells.

Evidence for the role of pancreatic acinar cells in the production of ornithine and guanidinoacetic acid by L-arginine:glycine amidinotransferase.

L-Arginine:glycine amidinotransferase (transamidinase) occurs at high concentrations in the kidney and the pancreas of rats. The cellular localization of transamidinase was investigated in fetal, neonatal, and adult rat pancreatic tissue using three indicators of the presence of transamidinase: (1) immunofluorescence microscopy, (2) in vitro enzymatic activity measurements on homogenates of whole pancreas and on isolated acinar and islet tissue from adult rats, and (3) ornithine production from perfused adult rat pancreas. The cellular localization of transamidinase was determined in fetal, neonatal, and adult rat pancreas, using a polyclonal guinea pig antibody made against a highly purified preparation of kidney transamidinase. Immunoreactive transamidinase was detected only in the pancreatic acinar cells. The cellular distribution of the immunostaining was compatible with the presence of transamidinase in mitochondria. The transamidinase enzymatic activity of whole pancreatic homogenates was 13.4 +/- 0.7 U/g wet weight (n = 11). In pancreata where islets had been isolated away from the acinar tissue, the transamidinase activity was similar to that of the whole pancreatic homogenates (16.8 +/- 2 U/g wet weight). Any transamidinase activity present in isolated islets was below the sensitivity of the assay. Transamidinase activity in the isolated perfused pancreas was determined by measuring the amount of ornithine released into the perfusate. The transamidinase activity of the perfused pancreas was 16.4 +/- 1.8 U/g pancreas and is an estimate of the physiological production capacity of the enzyme (270 +/- 29 nmol ornithine/min/g pancreas). These results indicate that transamidinase is present at high concentrations in the pancreas.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphological and functional characterization of beta TC-6 cells--an insulin-secreting cell line derived from transgenic mice.

Morphological analysis of hormone content and functional assessment of hormone secretion were conducted in beta TC-6 cells, an insulin-secreting cell line derived from transgenic mice expressing the large T-antigen of simian virus 40 (SV40) in pancreatic beta-cells. We observed by immunohistochemistry and confocal microscopy that beta TC-6 cells contain abundant insulin and small amounts of glucagon and somatostatin (SRIF). Glucagon usually co-localized with insulin, whereas cells containing SRIF did not contain insulin or glucagon. Static incubation and perifusion experiments demonstrated that beta TC-6 cells at passage 30-45 secrete insulin in response to glucose. In static incubations, maximal stimulation was achieved for glucose concentrations > 2.8 mmol/l glucose, and the half-maximal effect was observed at 0.5 mmol/l. Maximal stimulation was four times greater than HIT-T15 cells at passage 72-81, although HIT cells had a greater response over their basal levels. The magnitude of the insulin response to glucose in perifusion was 1,734 +/- 384 pmol.l-1. min and was 4.6-fold greater in the presence of 3-isobutyl-1-methylxanthine. Low amounts of glucagon were released in response to amino acids. Epinephrine (EPI), and to a lesser extent SRIF, inhibited phasic glucose-induced insulin secretion. A major portion of these inhibitory effects was mediated by pertussis toxin-sensitive substrates. Immunoblots detected the presence of the G-proteins Gi alpha 2, Gi alpha 3, and Go alpha 2. These results indicate that beta TC-6 cells are a glucose-responsive cell line in which insulin exocytosis is physiologically regulated by EPI and SRIF through Gi/Go-mediated mechanisms.

Absence of transforming growth factor-beta responsiveness in the tamoxifen growth-inhibited human breast cancer cell line CAMA-1.

Tamoxifen has been an effective antiestrogen in suppressing breast cancer growth which is estrogen-responsive or dependent. Early studies have provided circumstantial evidence that transforming growth factor-beta (TGF-beta) may be an autocrine mediator of tamoxifen action. Therefore, it is both fundamentally important and clinically relevant to investigate the relationship between tamoxifen and TGF-beta. In this study, we demonstrated that CAMA-1 cells, which are sensitive to tamoxifen inhibition, did not respond to TGF-beta growth inhibition. The type I and II TGF-beta receptors were undetectable by the radio-ligand affinity labeling technique. Despite the presence of a normal TGF-beta type II receptor gene, the mRNA transcript of the gene was undetectable by the extremely sensitive Intron-differential RNA/PCR method. The possibility that the lack of TGF-beta receptors might be intimately linked to the absence of normal retinoblastoma (Rb) gene products, as suggested by previous studies of retinoblastoma cells, was further investigated. The lack of TGF-beta receptor expression was found due to reasons other than the absence, deletion or abnormality of the Rb gene because a normal Rb gene and its hyper- and hypo-phosphorylated protein products were detected in CAMA-1 cells. In conclusion, our results suggest that the TGF-beta system is not obligatory for antiestrogen growth inhibition of CAMA-1 cells.

Differential induction of indoleamine-2,3-dioxygenase (IDO) by interferon-gamma in human gynecologic cancer cells.

Induction of indoleamine-2,3-dioxygenase (IDO) by interferon-gamma (IFN-gamma) is thought to be one mechanism underlying IFN-gamma's antineoplastic properties. Since clinical trials with IFN-gamma have yielded variable efficacy in treating cancers of gynecological origin, we tested the effects of IFN-gamma on cell growth and IDO activity in cell lines from seven gynecologic and five breast cancers. At a dose of 250 IU/ml, IFN-gamma suppressed cell growth and induced IDO activity in one cervical (C41), one vulva (A431), one breast (HS578T) and two ovarian (OVCAR-3, CAOV-3) cancer cell lines. Differing inhibition of cell growth, but with no induction of IDO activity, was found with IFN-gamma treatment of the other cell lines.

Direct positive effect of epidermal growth factor on the cytoplasmic maturation of mouse and human oocytes.

OBJECTIVE: Immature mammalian oocytes cultured in vitro undergo inadequate cytoplasmic maturation and hence have a limited potential for fertilization. Our primary objective was to determine if the addition of epidermal growth factor (EGF) to the in vitro culture system would have a positive effect on oocyte cytoplasmic maturation. DESIGN: We studied the effect of different EGF concentrations on both denuded and cumulus-enclosed mouse oocytes cultured in vitro. MAIN OUTCOME MEASURES: The percentage of oocytes undergoing germinal vesicle breakdown (GVBD) and polar body one formation over time as a function of EGF concentration was determined. RESULTS: A dose-related positive effect of EGF on both GVBD and polar body one formation over time was observed for mouse oocytes. As well, a similar effect of EGF was seen on immature human oocytes that had not been stimulated with exogenous gonadotropins. CONCLUSIONS: The use of EGF may allow for the performance of successful in vitro fertilization procedures using immature human oocytes retrieved during unstimulated cycles.

Characterization of molybdate-stabilized estrogen receptors by hydrophobic interaction HPLC: resolution of two 8S subunits.

This report describes the separation and characterization of estrogen receptors (ER) according to their degree of hydrophobicity and surface charges. Molybdate-stabilized [3H]ER from rabbit uterine cytosol was sequentially purified by passage through a size-exclusion pre-column, an anion-exchange column, and a hydrophobic interaction column. With fresh cytosol, a major radioactive peak was eluted from the DEAE columns; a major peak and a minor, less hydrophobic, peak were eluted from the hydrophobic column. In contrast, ER from frozen cytosol showed one peak in the DEAE-column and exhibited four radioactive peaks in the hydrophobic column. By sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, [3H] tamoxifen aziridine(TA)-labelled ER showed radioactive bands at 62 and 48 kd. The subunits which were characterized by these radioactive bands were successfully separated by the hydrophobic column; the more hydrophobic subunit corresponded to the 62 kd band. The HPLC-purified [3H]TA-labelled ER subunits sediment at a 7.4-8.5S region in a low-salt sucrose gradient. These results show that differential negative surface charge and hydrophobic areas exist in the holo-receptor and its subunits, and the hydrophobic interaction HPLC column separates the two major 8S steroid binding subunits of ER.