Although the beta-cells of the pancreatic islets of Langerhans express both prolactin (PRL) and GH receptors, we have observed that PRL is considerably more effective than GH in the up-regulation of islet function in vitro. This study examined whether differences in the activation of the Janus kinase 2/signal transducer and activator of transcription (STAT) 5 signaling pathway by these closely related receptors may be involved in this disparity. The activation of STAT5B by PRL was biphasic, with an initial peak within 30 min, a nadir between 1 and 3 h, and prolonged activation after 4 h. In contrast, the response to GH was transient for 1 h. The importance of the long-term activation of STAT5B by PRL was supported by the similar dose response curves for STAT5B activation and the PRL-induced increases in insulin secretion and islet cell proliferation. Because the pulsatile secretion of GH affects its actions in other target tissues, the ability of pretreatment with either hormone to affect subsequent stimulation was also examined. Surprisingly, the response to PRL was inhibited by prior exposure for less than 3 h to either PRL or GH and disappeared with a longer pretreatment with either hormone. Similar to other tissues, the response to GH was inhibited by any length of prior exposure to GH. However, pretreatment with PRL had no effect. These experiments are the first demonstration of the transient desensitization of the PRL receptor by either PRL or GH pretreatment in any tissue and the desensitization of GH stimulation in islet cells. These observations provide insight into the mechanisms that regulate the desensitization of these receptors and, more importantly, allow the long-term activation of STAT5B by the PRL receptor. These results may apply to other members of the cytokine superfamily of receptors. We also demonstrate that the increase in islet cell proliferation required continuous stimulation with PRL, whereas the smaller effect with GH occurred with either continuous or pulsatile stimulation. In summary, this study demonstrates that islets are sensitive to the temporal pattern of stimulation by these hormones and provides a new basis for understanding their physiological roles in the regulation of islet function.

Glucokinase activity is increased in pancreatic islets during pregnancy and in vitro by prolactin (PRL). The underlying mechanisms that lead to increased glucokinase have not been resolved. Since glucose itself regulates glucokinase activity in b-cells, it was unclear whether the lactogen effects are direct or occur through changes in glucose metabolism. To clarify the roles of glucose metabolism in this process, we examined the interactions between glucose and PRL on glucose metabolism, insulin secretion, and glucokinase expression in insulin 1 (INS-1) cells and rat islets. Although the PRLinduced changes were more pronounced after culture at higher glucose concentrations, an increase in glucose metabolism, insulin secretion, and glucokinase expression occurred even in the absence of glucose. The presence of comparable levels of insulin secretion at similar rates of glucose metabolism from both control and PRL-treated INS-1 cells suggests the PRL-induced increase in glucose metabolism is responsible for the increase in insulin secretion. Similarly, increases in other known PRL responsive genes (e.g. the PRL receptor, glucose transporter-2, and insulin) were also detected after culture without glucose. We show that the upstream glucokinase promoter contains multiple STAT5 binding sequences with increased binding in response to PRL. Corresponding increases in glucokinase mRNA and protein synthesis were also detected. This suggests the PRLinduced increase in glucokinase mRNA and its translation are sufficient to account for the elevated glucokinase activity in b-cells with lactogens. Importantly, the increase in islet glucokinase observed with PRL is in line with that observed in islets during pregnancy.

This study examined whether an immunohistochemical method examining the subcellular localization of STAT5 could be used to characterize the activation of the JAK2/STAT5 pathway by prolactin (PRL) in intact cells or tissues. In the Ins-1 beta-cell line, STAT5A and STAT5B were distributed almost equally in the cytoplasm and the nucleus in unstimulated cells. STAT5A was also detected along the border of cells and in the perinuclear region. After exposure to PRL, the redistribution from the cytoplasm to the nucleus was much higher for STAT5B compared to STAT5A. This translocation represented 12% of the STAT5A and 22% of the STAT5B originally located in the cytoplasm before stimulation. In isolated rat islets of Langerhans, PRL stimulated the nuclear translocation of both STAT5A and STAT5B only in beta-cells. The expression of the PRL receptor only by beta-cells was confirmed with a rabbit polyclonal antiserum raised against the rat PRL receptor. It was estimated that 4% of STAT5A and 9% of STAT5B originally located in the cytoplasm was translocated to the nucleus after stimulation. The presence of a functional JAK2/STAT5 signaling pathway in all islet cells was demonstrated by the nuclear translocation of STAT5B in all islet cells (i.e., alpha-, beta-, and delta-cells) after stimulation with fetal calf serum. The nuclear translocation and tyrosine phosphorylation of STAT5B was biphasic, with an initial peak within 30 min, a nadir between 1 and 3 hr, and prolonged activation after 4 hr. In contrast, the tyrosine phosphorylation of STAT5A was also biphasic but its nuclear translocation peaked within 30 min and was then reduced to a level slightly above that observed before PRL stimulation. This method is able to detect changes in STAT5 activation as small as 2% of the total cell content. These observations demonstrate the utility of this approach for studying the activation of STAT5 in a mixed population of cells within tissues or organs. In addition, the dose response for the nuclear translocation of STAT5B in normal beta-cells was similar to those for changes in proliferation and insulin secretion in isolated rat islets. Therefore, the subcellular localization can be used to monitor the activation of STAT5 and it may be a key event in the upregulation of the pancreatic islets of Langerhans during pregnancy.

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.

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.