Dexamethasone counteracts the effect of prolactin on islet function: implications for islet regulation in late pregnancy.

Islets undergo a number of up-regulatory changes to meet the increased demand for insulin during pregnancy, including increased insulin secretion and beta-cell proliferation. It has been shown that elevated lactogenic hormone is directly responsible for these changes, which occur in a phasic pattern, peaking on day 15 of pregnancy and returning to control levels by day 20 (term). As placental lactogen levels remain elevated through late gestation, it was of interest to determine whether glucocorticoids (which increase during late gestation) could counteract the effects of lactogens on insulin secretion, beta-cell proliferation, and apoptosis. We found that insulin secretion measured over 24 h in culture and acute secretion measured over 1 h in response to high glucose were increased at least 2-fold by PRL treatment after 6 days in culture. Dexamethasone (DEX) treatment had a significant inhibitory effect on secretion in a dose-dependent manner at concentrations greater than 1 nM. At 100 nM, a concentration equivalent to the plasma corticosteroid level during late pregnancy, DEX inhibited secretion to below control levels. The addition of DEX (>1 nM) inhibited secretion from PRL-treated islets to levels similar to those produced by DEX treatment alone. Bromodeoxyuridine (10 microM) staining for the final 24 h of a 6-day culture showed that PRL treatment increased cell proliferation 6-fold over the control level. DEX treatment alone (1-1000 nM) did not reduce cell division below the control level, but significantly inhibited the rate of division in PRL-treated islets. YoYo-1, an ultrasensitive fluorescent nucleic acid stain, was added (1 microM; 8 h) to the medium after 1-3 days of culture to examine cell death. Islets examined under confocal microscopy showed that DEX treatment (100 nM) increased the number of cells with apoptotic nuclear morphologies. This was quantified by counting the number of YoYo-labeled nuclei per islet under conventional epifluorescence microscopy. The numbers of YoYo-1-positive nuclei per islet in control and PRL-treated islets were not different after 3 days of culture. However, DEX treatment increased YoYo-1 labeling 7-fold over that in controls. DEX also increased YoYo-1 labeling in PRL-treated islets 3-fold over the control level. These data show that the increased plasma glucocorticoid levels found during the late stages of pregnancy could effectively reverse PRL-induced up-regulation of islet function by inhibiting insulin secretion and cell proliferation while increasing apoptosis.

Role of cAMP in upregulation of insulin secretion during the adaptation of islets of Langerhans to pregnancy.

Islets undergo a number of upregulatory changes to meet the increased demand for insulin during pregnancy, including an increase in glucose-stimulated insulin secretion with a reduction in the stimulation threshold. Treatment with the lactogenic hormone prolactin (PRL) in vitro has been shown to induce changes in islets similar to those observed during pregnancy. We examined cAMP production in islets treated with PRL to determine if changes in cAMP are involved in the upregulation of insulin secretion. Insulin secretion and cAMP concentrations were measured from islets in response to a suprathreshold (6.8 mmol/l) or high (16.8 mmol/l) glucose concentration in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. Insulin secretion increased by 2.1-, 5.0-, and 5.9-fold at the suprathreshold glucose concentration and by 1.6-, 2.3-, and 2.9-fold at the higher glucose concentration after 1, 3, and 5 days of PRL treatment, respectively. After a similar pattern, cAMP metabolism increased by 1.2-, 1.6-, and 2.1-fold at the suprathreshold glucose concentration and by 1.2-, 1.7-, and 2.2-fold at the high glucose concentration after 1, 3, and 5 days of PRL treatment, respectively. The similar increases in insulin secretion and cAMP concentration suggest that changes in cAMP metabolism are involved in lactogen-induced upregulation of insulin secretion. To gain additional insight into the role of cAMP in the upregulation of islet function after lactogen treatment, we examined the relationship between changes in cAMP concentration and insulin secretion. Under all conditions (differing glucose concentrations and time periods), the increase in insulin release was directly proportional to the increase in cAMP. Thus increased glucose-stimulated insulin secretion from lactogen-treated islets could be accounted for by increased generation of cAMP and did not appear to require any further specific changes in intracellular processes mediated by cAMP. Because the PRL receptor is not directly involved in cAMP metabolism, the lactogen-induced increase in cAMP was most likely due to the increase in glucose metabolism that we have previously demonstrated in PRL-treated islets and in islets during pregnancy.

Mechanisms of arginine-induced increase in cytosolic calcium concentration in the beta-cell line NIT-1.

The effects of L-arginine and its analogues NG-nitro-L-arginine, NG-methyl-L-arginine, L-homoarginine and D-arginine on cytosolic calcium concentration were investigated to characterise the mechanisms of arginine-induced stimulation and to determine if nitric oxide production played a role in this stimulation. NIT-1 cells, a transgenic beta-cell line, were used for this purpose since they release insulin in response to typical beta-cell stimuli. Our data demonstrate that the arginine-induced increase in cytosolic calcium concentration was completely dependent on the influx of extracellular Ca2+ via verapamil-sensitive voltage-activated Ca2+ channels and that arginine stimulation requires the presence of a nutrient in order to cause an increase in cytosolic calcium concentration. The nutrient likely acted by closing the K+ ATP channels, since its effect could be inhibited by activation of these channels with diazoxide. L-arginine, as well as nitro-arginine and methyl-arginine which are not substrates for the production of nitric oxide, caused similar increases in cytosolic calcium concentration. Non-metabolisable arginine analogues homoarginine and D-arginine also caused increases in the cytosolic calcium concentration although not to the same extent. Insulin secretion was enhanced to the same extent by all analogues of arginine. It can be concluded that the arginine-induced increase in cytosolic calcium concentration in NIT-1 cells is attributable to an electrogenic effect following the transport of arginine leading to depolarisation of the plasma membrane potential, although metabolism of the amino acid itself may also partially contribute to the response.

Glucose- and GTP-dependent stimulation of the carboxyl methylation of CDC42 in rodent and human pancreatic islets and pure beta cells. Evidence for an essential role of GTP-binding proteins in nutrient-induced insulin secretion.

Several GTP-binding proteins (G-proteins) undergo post-translational modifications (isoprenylation and carboxyl methylation) in pancreatic beta cells. Herein, two of these were identified as CDC42 and rap 1, using Western blotting and immunoprecipitation. Confocal microscopic data indicated that CDC42 is localized only in islet endocrine cells but not in acinar cells of the pancreas. CDC42 undergoes a guanine nucleotide-specific membrane association and carboxyl methylation in normal rat islets, human islets, and pure beta (HIT or INS-1) cells. GTPgammaS-dependent carboxyl methylation of a 23-kD protein was also demonstrable in secretory granule fractions from normal islets or beta cells. AFC (a specific inhibitor of prenyl-cysteine carboxyl methyl transferases) blocked the carboxyl methylation of CDC42 in five types of insulin-secreting cells, without blocking GTPgammaS-induced translocation, implying that methylation is a consequence (not a cause) of transfer to membrane sites. High glucose (but not a depolarizing concentration of K+) induced the carboxyl methylation of CDC42 in intact cells, as assessed after specific immunoprecipitation. This effect was abrogated by GTP depletion using mycophenolic acid and was restored upon GTP repletion by coprovision of guanosine. In contrast, although rap 1 was also carboxyl methylated, it was not translocated to the particulate fraction by GTPgammaS; furthermore, its methylation was also stimulated by 40 mM K+ (suggesting a role which is not specific to nutrient stimulation). AFC also impeded nutrient-induced (but not K+-induced) insulin secretion from islets and beta cells under static or perifusion conditions, whereas an inactive structural analogue of AFC failed to inhibit insulin release. These effects were reproduced not only by S-adenosylhomocysteine (another methylation inhibitor), but also by GTP depletion. Thus, the glucose- and GTP-dependent carboxyl methylation of G-proteins such as CDC42 is an obligate step in the stimulus-secretion coupling of nutrient-induced insulin secretion, but not in the exocytotic event itself. Furthermore, AFC blocked glucose-activated phosphoinositide turnover, which may provide a partial biochemical explanation for its effect on secretion, and implies that certain G-proteins must be carboxyl methylated for their interaction with signaling effector molecules, a step which can be regulated by intracellular availability of GTP.

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.

Insulin secretagogues, but not glucose, stimulate an increase in [Ca2+]i in the fetal rat beta-cell.

Fetal pancreatic islets release insulin poorly in response to glucose; however, the cellular mechanism for this is controversial. By using fura 2 to measure changes in the cytoplasmic free Ca2+ concentration ([Ca2+]i) in beta-cells, we have examined islets from fetal, neonatal, and adult rats to determine the ability of glucose and other secretagogues to cause an increase in [Ca2+]i. The effects of glucose (20 mmol/l), glyceraldehyde (20 mmol/l), leucine (20 mmol/l), arginine (20 mmol/l), and the channel effectors glipizide (50 mumol/l), BAY K8644 (2 mumol/l), diazoxide (300 mumol/l), and verapamil (20 mumol/l) on changes in [Ca2+]i were studied. In both the fetal and the mature islet, glyceraldehyde, leucine, arginine, glipizide, and BAY K8644 caused an increase in [Ca2+]i. In mature islets, glucose also increased [Ca2+]i; however, in the fetal islet, glucose had no effect on [Ca2+]i. The stimulus-induced increases in [Ca2+]i in fetal and adult islets were both significantly inhibited by the addition of either diazoxide or verapamil. Similar results were obtained when insulin secretion was measured. Our data show that various secretagogues are able to stimulate fetal islets and cause an increase in [Ca2+]i. Glucose, however, fails to cause an increase in [Ca2+]i in the fetal islet. Hence, the immature insulin secretory response to glucose by the fetal islet is due to the inability of the fetal beta-cell to translate glucose stimulation into the increase in [Ca2+]i required for exocytosis of the insulin granule.

Cultured neonatal islet transplants in alloxan-treated and spontaneously diabetic rats.

Neonatal Fischer-344 (FSH) islets isolated by a nonenzymatic method have been shown to survive indefinitely in Wistar/Furth (WF) recipients. We have applied this islet isolation method to six different donor strains and transplanted the resulting islets across 20 different strain combinations. We report the variable results obtained, with FSH being the most consistently successful donor strain and ACI being the best recipient strain. Based on the complete success of culture-derived FSH (Rt1lv1) and BN (Rt1n) transplants to WF (Rt1u) recipients, we used this system to test the MHC-restriction of autoimmune beta cell destruction in the BB/Wor rat (Rt1u). Culture-derived FSH and/or WF islets were transplanted to pre-diabetic BB/Wor recipients. Animals were sacrificed at the onset of disease or up to 33 days after disease onset. No immune response developed in FSH and WF grafts in non-diabetic animals. In diabetic animals, all FSH grafts and 12/14 WF grafts survived intact, although some grafts exhibited mild-moderate infiltration which was more pronounced in MHC-matched grafts. In two animals which developed severe hyperglycemia and ketosis, the FSH grafts were found to be intact while the WF grafts were destroyed by disease recurrence. In addition, FSH and BN islets were transplanted to diabetic BB/Wor recipients. Three of four FSH islet recipients and two of four BN islet recipients were reversed of their disease. Neither these nor the non-reversed animals showed signs of disease recurrence in the MHC-mismatched grafts. Therefore, in these studies, MHC-mismatched cultured grafts survived well and were capable of reversing the diabetic syndrome in spontaneously diabetic BB/Wor recipients.

Growth of neonatal islet graft following transplantation to the BB/Wor rat.

Culture-derived neonatal Fischer-344 (Rtllvl) rat islets have reduced immunogenicity and have been shown to be fully transplantable into Wistar Furth (Rtlu) recipients. In studies designed to test the MHC-restriction of the autoimmune disease process in the BB/Wor rat, MHC-matched (Wistar Furth) and MHC-mismatched (Fischer-344) neonatal islets were isolated by a nonenzymatic tissue culture procedure and transplanted to the renal subcapsular site of BB/Wor rats before the onset of disease. All MHC-mismatched and most MHC-matched grafts survived intact. In this study we report the growth of MHC-matched and mismatched islet tissue at the graft site. Grafts in diabetes resistant animals averaged 60 +/- 26 micrograms, a comparable mass to that which was transplanted. Grafts in diabetic animals were significantly increased in size, averaging 176 +/- 156 micrograms (P = 0.01). Analysis of nuclear to cytoplasmic volume ratios indicates that the increase in mass was primarily due to islet hyperplasia rather than hypertrophy. Factors contributing to the growth of islet tissue in situ are discussed.

Islet transplantation in spontaneously diabetic BB/Wor rats.

We investigated the effectiveness of islet transplantation as therapy in an animal model of spontaneous type I (insulin-dependent) diabetes mellitus. Grafting MHC-matched and -mismatched islets with the spontaneously diabetic BB rat as a model has been previously reported to result in recurrence of the disease in the grafted tissue. When transplanted with nonimmunogenic islets isolated by nonenzymatic culture, we found that MHC-matched grafts proved to be susceptible to disease recurrence when allowed to remain in situ until ketosis developed in the host. Conversely, the MHC-mismatched grafts did not succumb to the disease process despite the destruction of the beta-cell population of the endogenous pancreas. Four manifestly hyperglycemic BB/Wor rats received sufficient islet mass by allotransplantation to reverse this state. All four animals had ameliorated conditions, and three of the four were restored to a normoglycemic state. Recurrence of diabetes in the BB rat was not observed.

The successful allotransplantation of neonatal rat islets across multiple combined major and minor histocompatibility barriers.

Cultured neonatal rat islets were transplanted across six strain combinations into nonimmunosuppressed allogeneic recipients. Islets were isolated nonenzymatically by an in vitro method and were cultured at 37 degrees C in 5% CO2 in air for 10 days prior to transplant. Transplants to nondiabetic recipients across four allogeneic barriers resulted in morphologically intact and well-granulated islet tissue present at the graft site in 54 of 55 cases for periods lasting as long as 445 days (mean day of sacrifice was 163). In trials using diabetic recipients, ACIs receiving WF islets (n = 3) and outbred Holtzmans receiving Holtzman islets (n = 3) were reversed and did not return to the hyperglycemic state for experimental periods of up to 430 days.