Cooperation between cAMP signalling and sulfonylurea in insulin secretion.

Although glucose is physiologically the most important regulator of insulin secretion, glucose-induced insulin secretion is modulated by hormonal and neural inputs to pancreatic ß-cells. Most of the hormones and neurotransmitters evoke intracellular signals such as cAMP, Ca²âº , and phospholipid-derived molecules by activating G protein-coupled receptors (GPCRs). In particular, cAMP is a key second messenger that amplifies insulin secretion in a glucose concentration-dependent manner. The action of cAMP on insulin secretion is mediated by both protein kinase A (PKA)-dependent and Epac2A-dependent mechanisms. Many of the proteins expressed in ß-cells are phosphorylated by PKA in vitro, but only a few proteins in which PKA phosphorylation directly affects insulin secretion have been identified. On the other hand, Epac2A activates the Ras-like small G protein Rap in a cAMP-dependent manner. Epac2A is also directly activated by various sulfonylureas, except for gliclazide. 8-pCPT-2'-O-Me-cAMP, an Epac-selective cAMP analogue, and glibenclamide, a sulfonylurea, synergistically activate Epac2A and Rap1, whereas adrenaline, which suppresses cAMP production in pancreatic ß-cells, blocks activation of Epac2A and Rap1 by glibenclamide. Thus, cAMP signalling and sulfonylurea cooperatively activate Epac2A and Rap1. This interaction could account, at least in part, for the synergistic effects of incretin-related drugs and sulfonylureas in insulin secretion. Accordingly, clarification of the mechanism of Epac2A activation may provide therapeutic strategies to improve insulin secretion in diabetes.

Treating diabetes today: a matter of selectivity of sulphonylureas.

It is well known that sulphonylureas (SUs), commonly used in the treatment of type 2 diabetes mellitus, stimulate insulin secretion by closing ATP-sensitive K(+) (K(ATP) ) channels in pancreatic ß-cells by binding to the SU receptor SUR1. SUs are now known also to activate cAMP sensor Epac2 (cAMP-GEFII) to Rap1 signalling, which promotes insulin granule exocytosis. For SUs to exert their full effects in insulin secretion, they are required to activate Epac2 as well as to inhibit the ß-cell K(ATP) channels. As Epac2 is also necessary for potentiation of glucose-induced insulin secretion by cAMP-increasing agents, such as incretin, Epac2 is a target of both cAMP and SUs. The distinct effects of various SUs appear to be because of their different actions on Epac2/Rap1 signalling as well as K(ATP) channels. Differently from other SUs, gliclazide is unique in that it is specific for ß-cell K(ATP) channel and does not activate Epac2.

cAMP-GEFII is a direct target of cAMP in regulated exocytosis.

Although cAMP is well known to regulate exocytosis in many secretory cells, its direct target in the exocytotic machinery is not known. Here we show that cAMP-GEFII, a cAMP sensor, binds to Rim (Rab3-interacting molecule, Rab3 being a small G protein) and to a new isoform, Rim2, both of which are putative regulators of fusion of vesicles to the plasma membrane. We also show that cAMP-GEFII, through its interaction with Rim2, mediates cAMP-induced, Ca2+-dependent secretion that is not blocked by an inhibitor of cAMP-dependent protein kinase (PKA). Accordingly, cAMP-GEFII is a direct target of cAMP in regulated exocytosis and is responsible for cAMP-dependent, PKA-independent exocytosis.

High-molecular-weight immunoreactive beta-endorphin in extracts of human placenta is a fragment of immunoglobulin G.

A high-molecular-weight protein with beta-endorphin- and adrenocorticotropin-immunoreactivities was isolated from extracts of human placenta after several purification steps, including immunoadsorption with a well-characterized antiserum raised to beta-endorphin. This protein was identified as the heavy chain of the human immunoglobulin class IgG1. These results have led to the recognition of homologies in the amino acid sequences of these physiologically unrelated molecules. They also suggest caution in accepting immunological competence as the sole criterion of the chemical identity of a ligand.

Roles of cAMP signalling in insulin granule exocytosis.

Insulin secretion is regulated by a series of complex events generated by various intracellular signals including Ca(2+), ATP, cAMP and phospholipid-derived signals. Glucose-stimulated insulin secretion is the principal mode of insulin secretion, and the mechanism potentiating the secretion is critical for physiological responses. Among the various intracellular signals involved, cAMP is particularly important for amplifying insulin secretion. Recently, glucagon-like peptide-1 (GLP-1) analogues and dipeptidyl peptidase-IV (DPP-IV) inhibitors have been developed as new antidiabetic drugs. These drugs all act through cAMP signalling in pancreatic beta-cells. Until recently, cAMP was generally thought to potentiate insulin secretion through protein kinase A (PKA) phosphorylation of proteins associated with the secretory process. However, it is now known that in addition to PKA, cAMP has other targets such as Epac (also referred to as cAMP-GEF). The variety of the effects mediated by cAMP signalling may be linked to cAMP compartmentation in the pancreatic beta-cells.

Age and origin of the G774A mutation in SLC22A12 causing renal hypouricemia in Japanese.

Renal hypouricemia is an inherited disorder characterized by impaired tubular uric acid transport. Impairment of the function of URAT1, the main transporter for the reabsorption of uric acid at the apical membrane of the renal tubules, causes renal hypouricemia. The G774A mutation in the SLC22A12 gene encoding URAT1 predominates in Japanese renal hypouricemia. From data on linkage disequilibrium between the G774 locus and the 13 markers flanking it (12 single nucleotide polymorphisms and 1 dinucleotide insertion/deletion locus), we here estimate the age of this mutation at approximately 6820 years [95% confidence interval (CI) 1860-11,760 years; median = 2460 years]. This indicates that the origin of the G774A mutation dates back from between the time when the Jomon people predominated in Japan and the time when the Yayoi people started to migrate to Japan from the Korean peninsula. These data are consistent with a recent finding that this G774A mutation was also predominant in Koreans with hypouricemia and indicate that the mutation originated on the Asian continent. Thus, this mutation found in Japanese patients was originally brought by immigrant(s) from the continent and thereafter expanded in the Japanese population either by founder effects or by genetic drift (or both).

Stress-induced activation of neuronal activity and corticotropin-releasing factor gene expression in the paraventricular nucleus is modulated by glucocorticoids in rats.

Intronic in situ hybridization methodology provides a means of determining the rate of gene transcription under basal and stimulated conditions. In the present study, we have used intronic in situ hybridization to the corticotropin-releasing factor (CRF) gene to measure hypothalamic CRF gene transcription after stress as well as its modulation by glucocorticoids. Using this and conventional exonic in situ hybridization we examined the time course of changes in c-fos mRNA, and CRF heteronuclear RNA (hnRNA) and mRNA concentrations in the paraventricular nucleus (PVN) of male Wistar rats after restraint stress. In addition, we determined the effects of adrenalectomy and dexamethasone administration on c-fos and CRF gene expression in the PVN. Restraint stress induced a rapid induction (within 5 min) of c-fos mRNA and CRF hnRNA expression in the PVN. Both RNA concentrations peaked at 30 min then decreased and were undetectable 2 h after stress onset. In contrast, the concentration of CRF mRNA increased gradually and a significant elevation was first detected 60 min after the beginning of stress. Adrenalectomy augmented and dexamethasone pretreatment inhibited c-fos mRNA, CRF hnRNA, and mRNA induction after stress. The data suggest that stress-induced activation of neurons, CRF gene transcription, and CRF synthesis in the PVN are modulated by glucocorticoids.

A non-acromegalic case of multiple endocrine neoplasia type 1 accompanied by a growth hormone-releasing hormone-producing pancreatic tumor.

Cases of acromegaly due to GHRHproducing pancreatic endocrine tumors have been reported. Here we present a case of a 31-yr-old nonacromegalic man with hyperparathyroidism and elevated serum IGF-I with normal serum GH levels. Serum GH was not suppressed below 1 ng/ml by the glucose tolerance test and increased in response to TR H and GHRH administration. Magnetic resonance imaging (MRI) revealed pituitary hyperplasia and an abdominal computed tomography (CT ) scan showed a tumor in the pancreatic tail. Plasma concentration of GHRH was elevated. Based on these clinical data, multiple endocrine neoplasia (MEN) type 1 was suspected. Three enlarged parathyroid glands were removed and a distal pancreatectomy was performed. Pathological examination of the parathyroid glands and pancreatic tumor showed nodular hyperplasia and a well-differentiated endocrine tumor, respectively, both compatible with MEN features. Immunohistochemistry revealed positive immunoreactivity for GHRH, SS , insulin, glucagon, chromogranin A, and pancreatic polypeptide in the pancreatic tumor. After pancreatic surgery, elevated levels of GHRH and IGF-I were normalized and pituitary hyperplasia definitely decreased in size. In cases of pituitary hyperplasia with elevated IGF-I, ectopic GHRH syndrome must be considered even if physical features of acromegaly are absent. It is also important to measure plasma GHRH concentrations in order to give a diagnosis.

Interleukin-18 mRNA expression in the rat pituitary gland.

The present study investigated the expression of IL-18 mRNA under several stimuli, and molecular structures of IL-18 mRNA of the rat pituitary. Real-time PCR demonstrated that IL-18 mRNA, highly expressed in anterior pituitary, significantly increased following stress and adrenalectomy. In situ hybridization combined with immunohistochemistry demonstrated that corticotrope cells expressed IL-18 mRNA. RACE and sequence analysis demonstrated that pituitary IL-18 mRNA possesses five new exons at the upstream of exon 1 and between exon 1 and exon 2, indicating the preferential usage of promoter 1. The present study suggests that IL-18 in the corticotrope cells may play some roles in stress responses.

A Turkish case with molybdenum cofactor deficiency.

Molybdenum cofactor deficiency (MIM 252150) is a rare progressive neurodegenerative disorder with about 100 cases reported worldwide. We have identified a male with molybdenum cofactor deficiency and analyzed the molybdenum cofactor synthesis (MOCS)1 gene, MOCS2 gene, MOCS3 gene and GEPH gene. We homozygously identified the CGA insertion after A666 of the MOCS1 gene which produces arginine insertion at codon 222 of MOCS1A. The parents, his brother and his sister who did not have any symptoms were heterozygous for the same mutation. This region was highly conserved in various species. The N-terminal part of MOCS1 a protein is suggested to form the central core of the protein and be composed of an incomplete [(alpha/beta)6] triosephosphate isomerase (TIM) barrel with a lateral opening that is covered by the C-terminal part of the protein. The insertion is located in the loop connecting the fifth beta strand to the sixth alpha helices of the TIM barrel structure. This arginine insertion would induce the conformation change and the lack of the activity.

Distribution of Corticotrophin-Releasing Factor Type 1 Receptor-Like Immunoreactivity in the Rat Pituitary.

Corticotrophin-releasing factor (CRF) regulates the hypothalamic-pituitary-adrenal axis response to stress through its type 1 receptor (CRF1 ) in the corticotrophs of the anterior pituitary. Although CRF1 mRNA expression has been confirmed in the rat pituitary, the distribution pattern of CRF1 protein in the pituitary has not been reported. Therefore, we generated an antiserum against the amino acid fragment corresponding to the 177-188 sequence of the first extracellular loop of the rat CRF1 . Using the antiserum, CRF1 -like immunoreactivity (CRF1 -LI) was detected in the anterior lobe cells of the rat pituitary where some of them expressed intense signals. CRF1 -LI also appeared in the intermediate lobe cells and on the fibre-like elements of the posterior lobe of the pituitary. Dual immunofluorescence labelling showed that corticotrophs exhibited the highest percentage of CRF1 (male: 27.1 ± 3.0%, female: 18.0 ± 3.0%), followed by lactotrophs (male: 6.7 ± 3.0%, female: 12.1 ± 1.3%), gonadotrophs (male: 2.6 ± 1.0%, female: 7.5 ± 0.5%), thyrotrophs (male: 2.9 ± 0.1%, female: 5.3 ± 1.2%) and somatotrophs (male: 1.1 ± 0.3%, female: 1.2 ± 0.5%). The percentage of CRF1 -LI-positive cells that were corticotrophs was significantly higher in male rats than in female rats, whereas CRF1 -LI-positive lactotrophs and gonadotrophs were significantly higher in female rats than in male rats. Almost all of the melanotrophs were positive for CRF1 in the intermediate lobe (98.9 ± 0.2%). CRF1 -LI and the percentage of CRF1 -LI in corticotrophs were decreased in the anterior pituitary, and the distribution patterns were altered from a diffuse to punctate one by adrenalectomy; the changes were restored by treatment with dexamethasone (100 µg/kg bw). These results suggest that CRF1 is involved in the modulation of the functions of the pituitary; moreover, protein expression and the distribution patterns of CRF1 are regulated by glucocorticoids in the rat anterior pituitary.

Ability of ubiquitin radioimmunoassay to discriminate between monoubiquitin and multi-ubiquitin chains.

Free ubiquitin (mainly monoubiquitin) and multi-ubiquitin chains coexist in eukaryote cells and serve distinct cellular roles. However, any immunoassay systems established previously have not been proved to be applicable for measuring the former without cross-reactive responses with the latter. For this purpose, we developed a radioimmunoassay specific to monoubiquitin by employing antiserum US-1 against ubiquitin. In this assay, ubiquitin-protein conjugates, prepared by a reticulocyte lysate fraction II and fractionated on Moro Q and Superdex 200 columns, exhibited practically no cross-reactivity. The cross-reactivity of fractionated ubiquitin-lysozyme conjugates was also analyzed as a function of their multi-ubiquitin chain size. As a result, the larger the conjugates were found to be, the weaker were the cross-reactive responses they showed, and the multi-ubiquitin chains (n > approx. 20) were substantially unreactive in the radioimmunoassay. By using the radioimmunoassay, heat-shock-induced decrease in the level of cellular free (mono)ubiquitin was detected. In addition, the standard preparation of multi-ubiquitin chains was not cross-reactive in all other five radioimmunoassays employing distinct antibodies to ubiquitin (four antisera and a monoclonal antibody). These data suggest that radioimmunoassays employing ubiquitin antibodies raised by the general methods can discriminate between monoubiquitin and multi-ubiquitin chains and quantitate cellular free ubiquitin.

Distribution of urocortin 2 in various tissues of the rat.

Urocortin (Ucn) 2 is a new member of the corticotrophin-releasing hormone (CRH) neuropeptide family that is expressed in the central nervous system and peripheral tissues. However, the expression levels of Ucn 2 in various tissues of the rat remains unclear. Thus, the aim of the present study was to characterise the expression of Ucn 2 in the various tissues of the rat. Reverse transcriptase-polymerase chain reaction analysis demonstrated that Ucn 2 mRNA is expressed in the hypothalamus, pituitary, adrenal, stomach, skin, ovary, uterus and skeletal muscle. Histologically, Ucn 2 mRNA and Ucn 2-like immunoreactivity (LI) were demonstrated in both the anterior and intermediate lobes of the pituitary, but not detected in the posterior lobe. Furthermore, all Ucn 2-positive cells in the anterior and intermediate lobes were also positive for beta-endorphin. Ucn 2 mRNA was detected in the adrenal cortex and medulla although Ucn 2-LI was only found in the adrenal medulla. High-performance liquid chromatography analysis of hypothalamic, pituitary, and adrenal extracts showed that the main Ucn 2-LI peak occurred at the same molecular size as that of synthetic Ucn 2. These results suggest that Ucn 2 is synthesised in various tissues, including the anterior and intermediate lobes of the pituitary and the adrenal.

Early induction of c-fos precedes increased expression of corticotropin-releasing factor messenger ribonucleic acid in the paraventricular nucleus after immobilization stress.

CRF plays a role in coordinating endocrine, physiological, and behavioral responses to stressful stimuli. Several kinds of stressors have been reported to induce an increase in CRF mRNA expression in the paraventricular nucleus of the hypothalamus (PVN). Recently, the expression of c-fos mRNA has shown promise as a useful tool for metabolic mapping at the cellular level, because various types of stimulation induce c-fos mRNA expression in specific neuron populations in various brain regions. The aim of the present study is to clarify a possible anatomical-temporal correlation between the early induction of c-fos and the enhanced expression of CRF mRNA after stress. Wistar male rats were exposed to immobilization stress for 60 min and killed before and 15, 30, 60, 90, 120, and 180 min after the beginning of immobilization. In situ hybridization was performed by hybridizing sections with 35S-labeled prepro-CRF and c-fos cRNA probes. Relative levels of CRF and c-fos mRNA were compared by estimating the number of grains over the PVN in emulsion-dipped autoradiograms. Rapid induction (within 15 min) of c-fos mRNA was noted in the parvocellular division of the PVN after immobilization stress. The level of c-fos mRNA peaked at 30 min, then gradually declined to the control level within 90 min after the beginning of stress [the number of grains over the PVN: control, 326 +/- 180; 15 min, 2091 +/- 680 (P less than 0.05 vs. control); 30 min, 3385 +/- 239 (P less than 0.05 vs. control)]. The distribution of c-fos mRNA was almost identical to that of CRF mRNA in the PVN. On the other hand, the time course of CRF mRNA induction was delayed to the c-fos mRNA expression. A significant increase in CRF mRNA levels was noted only 120 and 180 min after stress [the number of grains over PVN: control, 3868 +/- 221; 120 min, 5957 +/- 677 (P less than 0.05 vs. control); 180 min, 6600 +/- 450 (P less than 0.05 vs. control)]. The results demonstrate that increased expression of CRF mRNA is preceded by c-fos mRNA induction in the PVN after stress suggesting a role of c-fos in the activation of CRF gene expression.

Intracerebroventricular administration of the growth hormone-releasing peptide KP-102 increases food intake in free-feeding rats.

Recent evidence suggests that growth hormone-releasing peptides (GHRPs) mimic an unidentified native GH-releasing hormone (GHRH)-amplifying hormone. GHRH has been shown to stimulate food intake acting on the central nervous system. The present studies were conducted to test the hypothesis that GHRPs may also potentiate the central effect of GHRH on feeding in free-feeding rats. Intracerebroventricular (ICV) administration of picomole doses of a newly developed GHRP, KP-102, or human GHRH stimulated feeding, but the phenomenon was not reproduced by systemic injection. A prior ICV injection of a GHRH antagonist completely prevented the increase of food intake evoked by GHRH, but this pretreatment did not influence the increase in food intake induced by KP-102. When maximally effective doses of GHRH and KP-102 were co-administered ICV, the amount of food intake increased significantly compared with after ICV injection of a maximum dose of either peptide alone. These findings suggest that GHRPs stimulate food intake via a specific receptor for GHRPs in the central nervous system and amplify the central effect of GHRH on feeding.

The effect of glucose and free fatty acids on growth hormone (GH)-releasing factor-mediated GH secretion in rats.

We have examined the effect of glucose and FFA on GH-releasing factor (GHRF)-mediated GH secretion in rats under pentobarbital anesthesia. Hyperglycemia did not affect GH secretion induced by administration of 20, 100, and 200 ng GHRF/100 g body weight. In contrast, GH response to 50 ng GHRF/100 g body weight in lipid heparin-treated rats, which showed high plasma FFA levels, was significantly suppressed compared with the control group (plasma peak GH: control, 1526 +/- 263 ng/ml; lipid-heparin group, 377 +/- 69 ng/ml P less than 0.05, mean +/- SEM). This suppressive effect of FFA on GH secretion was abolished by pretreatment with antisomatostatin serum (ASS) (GH level at 4 min after GHRF administration: ASS-saline group, 1606 +/- 210 ng/ml; ASS-lipid-heparin group, 1531 +/- 174 ng/ml; mean +/- SEM). These results suggest that hyperglycemia does not change the GH response to GHRF and that elevation of plasma FFA suppresses GHRF-induced GH secretion by the stimulation of somatostatin secretion in rats.

Effects of various neuropeptides on the secretion of proopiomelanocortin-derived peptides by a cultured pituitary adenoma causing Nelson's syndrome.

To clarify whether various neuropeptides found in the hypothalamus act directly on a pituitary adenoma causing Nelson's syndrome, we examined the influence of these peptides on the secretion of immunoreactive ACTH, beta-endorphin, and melanotropins, the proopiomelanocortin (POMC)-derived peptides, by the cultured pituitary adenoma from a patient with Nelson's syndrome. Results showed that somatostatin-14 and somatostatin-28 suppressed the secretion of POMC-derived peptides by the adenoma and that somatostatin-28 was as potent as somatostatin-14. Other neuropeptides such as arginine vasopressin, vasoactive intestinal polypeptide, and oxytocin stimulate the secretion of POMC-derived peptides. Substance P, TRF, Met-enkephalin and Leu-enkephalin were also found to modulate the secretion of POMC-derived peptides. This suggests that the adenoma may have multiple receptors to various neuropeptides.

Secretion pattern of pro-opiomelanocortin-derived peptides by a pituitary adenoma from a patient with Cushing's disease.

Fragments of the pituitary adenoma of a patient with Cushing's disease were maintained in defined culture medium. Immunoreactive (IR) ACTH, IR alpha-MSH, IR beta-lipotropin (beta-LPH), IR beta-endorphin, and IR gamma-MSHs secreted from the adenoma were studied with gel permeation chromatography and the respective RIAs. The adenoma secreted roughly equimolar quantities of IR beta-LPH plus IR beta-endorphin, IR gamma 3-MSHs, and IR ACTHs. It also secreted IR alpha-MSH as well as IR gamma 1-MSH, although in a much lower concentration than the above four peptides. The secreted gamma 3-MSH-like peptides were found to be glycosylated. The secretion pattern suggests that this particular adenoma processes the pro-opiomelanocortin molecule in pathways which encompass those of both the pars distalis and the pars intermedia.

Polymorphisms of amiloride-sensitive sodium channel subunits in five sporadic cases of pseudohypoaldosteronism: do they have pathologic potential?

Pseudohypoaldosteronism (PHA) is characterized by congenital resistance of the kidney and/or other mineralocorticoid target tissues to aldosterone, resulting in excessive salt wasting. Mineralocorticoid receptor (MR) and postreceptor defects in the aldosterone-responsive amiloride-sensitive sodium channel (ENaC) subunits have been suggested as potential loci of the defect in this disease, whereas recently defects in MR and ENaC subunits were reported in familial PHA cases. Here we studied the ENaC subunit alpha, beta, and gamma complementary DNAs (cDNAs) in a series of five sporadic cases of PHA, whose MR cDNA contained nonconservative homozygous (C944-->T944, Ala241-->Val241) and/or a conservative heterozygous substitutions (A760-->G760, Ileu180-->Val180), which, however, were also present at high frequencies in a control population with apparently normal salt conservation. We found a nonconservative substitution (A2086-->G2086, Thr663-->Ala663) in the alphaENaC in all five of our patients, two of whom were homozygous and three of whom were heterozygous for this alteration, which was also present in the homozygous and heterozygous form in 31% and 64% of control subjects, respectively. We also found a nonconservative homozygous substitution (C1006-->G1006, Pro336-->Ara336) in the betaENaC and three nonconservative and conservative homozygous substitutions (T554-->A554, Trp178-->Arg178; C1526-->G1526, Pro501-->Ala501; T1862-->G1862, Ser614-->Ala614) in the gammaENaC of all five of our patients and in a substantial proportion of control subjects. Interestingly, when the patient group was compared to controls, a significantly increased concurrence of the MR and alphaENaC polymorphisms was found in the patients (P<0.025). We conclude that the changes identified in the cDNA of the three ENaC subunits in the patients with sporadic PHA are polymorphisms, which on their own have no apparent pathophysiological significance. We hypothesize, however, that these polymorphisms might influence salt conservation negatively if they are present concurrently with other genetic defects of the MR or other proteins that participate in sodium homeostasis. The latter would be compatible with a sporadic presentation and digenic or multigenic expression and heredity in PHA.

Plasma GH responses to GHRH and insulin-induced hypoglycemia in man.

Changes in plasma GH levels in response to an intravenous bolus injection of 200 micrograms GHRH-44 or 0.1 U/kg body weight regular insulin were examined in normal men who were pre-treated with 200 micrograms GHRH-44 or 0.1 U/kg body weight regular insulin 120 min in advance. The prior bolus injection of GHRH-44 inhibited the plasma GH response to the subsequent administration of GHRH-44 whereas the plasma GH response to the subsequent injection of insulin was not influenced by the prior administration of GHRH-44. The prior administration of insulin attenuated the plasma GH response to the subsequently given GHRH-44. These results suggest that desensitization of GHRH receptors in somatotrophs and/or somatostatin hypersecretion induced by increase in plasma GH levels following the prior GHRH-44 administration may be involved in the mechanism by which the prior GHRH-44 administration or insulin-induced hypoglycemia suppressed plasma GH responses to the following GHRH-44 administration. Sudden suppression of somatostatin secretion, which causes rebound of GH secretion, may occur in insulin-induced hypoglycemia.