Ingmar Lundquist

Deletion of the G protein-coupled receptor 30 impairs glucose tolerance, reduces bone growth, increases blood pressure, and eliminates estradiol-stimulated insulin release in female mice.

In vitro studies suggest that the G protein-coupled receptor (GPR) 30 is a functional estrogen receptor. However, the physiological role of GPR30 in vivo is unknown, and it remains to be determined whether GPR30 is an estrogen receptor also in vivo. To this end, we studied the effects of disrupting the GPR30 gene in female and male mice. Female GPR30((-/-)) mice had hyperglycemia and impaired glucose tolerance, reduced body growth, increased blood pressure, and reduced serum IGF-I levels. The reduced growth correlated with a proportional decrease in skeletal development. The elevated blood pressure was associated with an increased vascular resistance manifested as an increased media to lumen ratio of the resistance arteries. The hyperglycemia and impaired glucose tolerance in vivo were associated with decreased insulin expression and release in vivo and in vitro in isolated pancreatic islets. GPR30 is expressed in islets, and GPR30 deletion abolished estradiol-stimulated insulin release both in vivo in ovariectomized adult mice and in vitro in isolated islets. Our findings show that GPR30 is important for several metabolic functions in female mice, including estradiol-stimulated insulin release.

SUR1 Regulates PKA-independent cAMP-induced Granule Priming in Mouse Pancreatic B-cells

Measurements of membrane capacitance were applied to dissect the cellular mechanisms underlying PKA-dependent and -independent stimulation of insulin secretion by cyclic AMP. Whereas the PKA-independent (Rp-cAMPS–insensitive) component correlated with a rapid increase in membrane capacitance of ∼80 fF that plateaued within ∼200 ms, the PKA-dependent component became prominent during depolarizations >450 ms. The PKA-dependent and -independent components of cAMP-stimulated exocytosis differed with regard to cAMP concentration dependence; the K d values were 6 and 29 μM for the PKA-dependent and -independent mechanisms, respectively. The ability of cAMP to elicit exocytosis independently of PKA activation was mimicked by the selective cAMP-GEFII agonist 8CPT-2Me-cAMP. Moreover, treatment of B-cells with antisense oligodeoxynucleotides against cAMP-GEFII resulted in partial (50%) suppression of PKA-independent exocytosis. Surprisingly, B-cells in islets isolated from SUR1-deficient mice (SUR1−/− mice) lacked the PKA-independent component of exocytosis. Measurements of insulin release in response to GLP-1 stimulation in isolated islets from SUR1−/− mice confirmed the complete loss of the PKA-independent component. This was not attributable to a reduced capacity of GLP-1 to elevate intracellular cAMP but instead associated with the inability of cAMP to stimulate influx of Cl− into the granules, a step important for granule priming. We conclude that the role of SUR1 in the B cell extends beyond being a subunit of the plasma membrane KATP-channel and that it also plays an unexpected but important role in the cAMP-dependent regulation of Ca2+-induced exocytosis.

Starch availability in vitro and in vivo after flaking, steam-cooking and popping of wheat

The availability for digestion of starch in vitro after flaking, steam cooking and popping of whole grain wheat was studied and compared with that of raw and boiled wheat. Some physical and morphological characteristics were also investigated and correlated with starch availability for amylolysis. In an in vitro assay using hog pancreatic α-amylase after preincubation with pepsin, starch in flaked wheat was less available than that in boiled, popped and steam-cooked wheat. The starch in raw wheat was digested only slightly. When pepsin was omitted, the availability to α-amylase of starch in raw and boiled wheat decreased substantially, indicating that a large fraction of the starch was encapsulated in a protein matrix. The starch in flaked wheat elicited lower plasma glucose and plasma insulin levels in rats in vivo than that in boiled wheat after a gastric load. With raw wheat, the plasma glucose peak was much delayed and of lower magnitude compared with that with the heat-treated samples. In order to obtain good agreement with the in vivo results, a pepsin step had to be included in the in vitro assay using α-amylase. Differential scanning calorimetry measurements indicated that starch in flaked wheat was not gelatinised completely and this fact probably accounted for the lower availability in flaked wheat compared with boiled, popped or steam-cooked wheat.

Impaired insulin secretion and glucose tolerance in beta cell-selective Ca(v)1.2 Ca2+ channel null mice.

Insulin is secreted from pancreatic β cells in response to an elevation of cytoplasmic Ca2+ resulting from enhanced Ca2+ influx through voltage‐gated Ca2+ channels. Mouse β cells express several types of Ca2+ channel (L‐, R‐ and possibly P/Q‐type). β cell‐selective ablation of the gene encoding the L‐type Ca2+ channel subtype Cav1.2 (βCav1.2−/− mouse) decreased the whole‐cell Ca2+ current by only ∼45%, but almost abolished first‐phase insulin secretion and resulted in systemic glucose intolerance. These effects did not correlate with any major effects on intracellular Ca2+ handling and glucose‐induced electrical activity. However, high‐resolution capacitance measurements of exocytosis in single β cells revealed that the loss of first‐phase insulin secretion in the βCav1.2−/− mouse was associated with the disappearance of a rapid component of exocytosis reflecting fusion of secretory granules physically attached to the Cav1.2 channel. Thus, the conduit of Ca2+ entry determines the ability of the cation to elicit secretion.

Proghrelin-derived peptides influence the secretion of insulin, glucagon, pancreatic polypeptide and somatostatin: a study on isolated islets from mouse and rat pancreas.

Proghrelin, the precursor of the orexigenic and adipogenic peptide hormone ghrelin, is synthetized in endocrine (A-like) cells in the gastric mucosa. During its cellular processing, proghrelin gives rise to the 28-amino acid peptide desacyl ghrelin, which after octanoylation becomes active acyl ghrelin, and to the 23-amino acid peptide obestatin, claimed to be a physiological opponent of acyl ghrelin. This study examines the effects of the proghrelin products, alone and in combinations, on the secretion of insulin, glucagon, pancreatic polypeptide (PP) and somatostatin from isolated islets of mice and rats. Surprisingly, acyl ghrelin and obestatin had almost identical effects in that they stimulated the secretion of glucagon and inhibited that of PP and somatostatin from both mouse and rat islets. Obestatin inhibited insulin secretion more effectively than acyl ghrelin. In mouse islets, acyl ghrelin inhibited insulin secretion at low doses and stimulated at high. In rat islets, acyl ghrelin inhibited insulin secretion in a dose-dependent manner but the IC(50) for the acyl ghrelin-induced inhibition of insulin release was 7.5 x 10(-8) M, while the EC(50) and IC(50) values, with respect to stimulation of glucagon release and to inhibition of PP and somatostatin release, were in the 3 x 10(-12)-15 x 10(-12) M range. The corresponding EC(50) and IC(50) values for obestatin ranged from 5 x 10(-12) to 20 x 10(-12) M. Desacyl ghrelin per se did not affect islet hormone secretion. However, at a ten times higher concentration than acyl ghrelin (corresponding to the ratio of the two peptides in circulation), desacyl ghrelin abolished the effects of acyl ghrelin but not those of obestatin. Acyl ghrelin and obestatin affected the secretion of glucagon, PP and somatostatin at physiologically relevant concentrations; with obestatin this was the case also for insulin secretion. The combination of obestatin, acyl ghrelin and desacyl ghrelin in concentrations and proportions similar to those found in plasma resulted in effects that were indistinguishable from those induced by obestatin alone. From the data it seems that the effects of endogenous, circulating acyl ghrelin may be overshadowed by obestatin or blunted by desacyl ghrelin.

Cholecystokinin-A and Cholecystokinin-B/Gastrin Receptor mRNA Expression in the Gastrointestinal Tract and Pancreas of the Rat and Man: A Polymerase Chain Reaction Study

BACKGROUND Gastrin and cholecystokinin (CCK) are thought to exert trophic effects on the gastrointestinal tract and pancreas. Two types of receptors have been cloned, CCK-A and CCK-B/ gastrin. We have examined the occurrence of CCK-A and CCK-B receptor mRNA in the brain, digestive tract, pancreas, and kidney of the rat and man by Northern blot and reverse transcribed polymerase chain reaction (RT-PCR). METHODS Total RNA was isolated from rat tissues and reverse transcribed into cDNA. cDNA from brain, kidney, and pancreas of the rat and man and from human whole stomach were commercially available. Northern blot and a PCR technique based on Taq polymerase-antibody interaction and using CCK-A and CCK-B receptor-specific primers, followed by Southern blot analysis, were the methods used. RESULTS By means of Northern blots, CCK-A receptor mRNA was detected in rat fundus mucosa and pancreas but not in the remaining GI tract or brain. By means of RT-PCR, CCK-A receptor mRNA was demonstrated in the brain and the mucosa of the fundus, antrum, duodenum, and colon, kidney, pancreas and pancreatic islets. CCK-B receptor mRNA was detected by Northern blot analysis in the brain and the fundus mucosa but not in the rest of the digestive tract and not in the pancreas, pancreatic islets, or kidney. By RT-PCR, expression of CCK-B receptor mRNA could also be detected in antrum mucosa. In man, CCK-A receptor mRNA was detected in the brain, stomach, pancreas, and kidney, whereas CCK-B receptor mRNA was found in the brain, stomach, and pancreas but not in the kidney. Cloning and DNA-sequence analysis of the PCR-amplified rat and human CCK-A and CCK-B receptor DNA fragments, which cover the protein-encoding regions of the intracellular loop C3, showed complete sequence homology as compared with published rat and human sequences. CONCLUSIONS It appears unlikely that CCK will have effects in the ileum, at least not effects mediated by CCK-A receptors. It also appears unlikely that physiologic concentrations of gastrin in the circulation will promote growth (or exert other effects) in the pancreas, duodenum, ileum, and colon, since CCK-B receptor mRNA is not expressed or is poorly expressed in these tissues.

Neuropeptide Y: intrapancreatic neuronal localization and effects on insulin secretion in the mouse

SummaryThe intrapancreatic localization and the effects on basal and stimulated insulin secretion of neuropeptide Y (NPY) were investigated in the mouse. Immunocyto-chemistry showed NPY to be confined to intrapancreatic nerve fibers mainly associated with blood vessels. Fine varicose NPY fibers were also detected in the exocrine parenchyma and occasionally also within the islets. Double-staining experiments with the use of antisera for both NPY and tyrosine hydroxylase (TH) indicated that most of the NPY fibers were nonadrenergic in nature. Only a population of the NPY fibers occurring around blood vessels showed TH immunoreactivity. Under in vivo conditions, NPY was found to elevate plasma insulin levels slightly when injected intravenously at the high dose level of 8.5 nmol/kg. At lower dose levels, NPY did not affect basal plasma insulin levels, but instead inhibited glucose-induced insulin secretion. Thus, the glucose-induced increment in plasma insulin levels, which was 120±7μU/ml in controls, was reduced to 87 ±5 μU/ml by NPY at 4.25 nmol/kg (p<0.01) and to 98±6μU/ml by NPY at 1.06 nmol/kg (p<0.05). In contrast, the insulin secretory response to the cholinergic agonist carbachol was not affected by NPY. We conclude that NPY nerve fibers occur in the mouse pancreas and that most of these NPY nerve fibers are nonadrenergic. Furthermore, in the mouse, NPY enhances basal plasma insulin levels at high dose levels and inhibits glucose-induced, but not cholinergically induced insulin secretion at lower dose levels under in vivo conditions.

Accumulation of dopamine in mouse pancreatic B-cells following injection of L-DOPA. Localization to secretory granules and inhibition of insulin secretion.

SummaryAccumulation and subcellular localization of dopamine (DA) in pancreatic B-cells and its effects on insulin secretion were investigated in mice following a single injection of L-3,4-dihydroxyphenyl-alanine (L-DOPA). Electron microscopic autoradiography showed that3H-DA formed from administered3H-DOPA was present over B-cells as well as over other types of islet cells. Pretreatment of the animals with a decarboxylase inhibitor greatly reduced the number of autoradiographic grains. In the B-cells the3H-DA-grains were associated with the secretory granules. The location of the label may suggest an incorporation in the periphery of the β-granule, rather than in the dense core, supposed to contain insulin. Accumulation of DA in the B-cells following L-DOPA administration was found to inhibit partially the insulin secretory response to different insulin secretagogues (glucose, glibenclamide and L-isopropylnoradrenaline (L-IPNA)). Treatment with monoamine oxidase inhibitor + L-DOPA induced an almost total suppression of L-IPNA-stimulated insulin secretion, whereas glucose-induced insulin release was still only partially inhibited. Pretreatment with a decarboxylase inhibitor abolished the effects of L-DOPA. It is suggested that intracellularly accumulated DA in the B-cell exerts an inhibitory action on insulin releasing mechanisms induced by different secretagogues and that this action might involve interference with a calcium translocation process at the level of the secretory granule.

Influence of nitric oxide synthase inhibition, nitric oxide and hydroperoxide on insulin release induced by various secretagogues

1 Recent studies have suggested that the generation of nitric oxide (NO) and hydrogen peroxide (H2O2) by islet NO synthase and monoamine oxidase, respectively, may have a regulatory influence on insulin secretory processes. We have investigated the pattern of insulin release from isolated islets of Langerhans in the presence of various pharmacological agents known to perturb the intracellular levels of NO and the oxidation state of SH‐groups. 2 The NO synthase inhibitor, NG‐nitro‐l‐arginine methyl ester (l‐NAME) dose‐dependently increased l‐arginine‐induced insulin release. d‐Arginine did not influence l‐arginine‐induced insulin secretion. However, d‐NAME which reportedly has no inhibitory action on NO synthase, modestly increased l‐arginine‐induced insulin release, but was less effective than l‐NAME. High concentrations (10 mm) of d‐arginine as well as l‐NAME and d‐NAME could enhance basal insulin release. 3 The intracellular NO donor, hydroxylamine, dose‐dependently inhibited insulin secretion induced by l‐arginine and l‐arginine + l‐NAME. 4 Glucose‐induced insulin release was increased by NO synthase inhibition (l‐NAME) and inhibited by the intracellular NO donor, hydroxylamine. Sydnonimine‐1 (SIN‐1), an extracellular donor of NO and superoxide, induced a modest suppression of glucose‐stimulated insulin release. SIN‐1 did not influence insulin secretion induced by l‐arginine or the adenylate cyclase activator, forskolin. 5 The intracellular ‘hydroperoxide donor’ tert‐butylhydroperoxide in the concentration range of 0.03–3mm inhibited insulin release stimulated by the nutrient secretagogues glucose and l‐arginine. Low concentrations (0.03–30 μm) of tert‐butylhydroperoxide, however enhanced insulin secretion induced by the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX). 6 Islet guanosine 3’:5′‐cyclic monophosphate (cyclic GMP) content was not influenced by 10 mm l‐arginine or tert‐butylhydroperoxide at 3 or 300 μm but was markedly increased (14 fold) by a high hydroxylamine concentration (300 μm). In contrast, islet adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) content was increased (3 fold) by l‐arginine (10 mm) and (2 fold) by tert‐butylhydroperoxide (300 μm). 7 Our results strongly suggest that NO is a negative modulator of insulin release induced by the nutrient secretagogues l‐arginine and glucose. This effect is probably not mediated to any major extent by the guanylate cyclase‐cyclic GMP system but may rather be exerted by the S‐nitrosylation of critical thiol groups involved in the secretory process. Similarly the inhibitory effect of tert‐butylhydroperoxide is likely to be elicited through affecting critical thiol groups. The mechanism underlying the secretion‐promoting action of tert‐butylhydroperoxide on IBMX‐induced insulin release is probably linked to intracellular Ca2+‐perturbations affecting exocytosis. 8 Taken together with previous data the present results suggest that islet production of low physiological levels of free radicals such as NO and H2O2 may serve as important modulators of insulin secretory processes.

Glucagon immunoreactivity in plasma from normal and dystrophic mice

SummaryThe present investigation was undertaken to determine and characterize glucagon immunoreactivity in plasma from normal NMRI mice and from dystrophic mice and their unaffected littermates of the 129/ReJ strain. Very young dystrophic mice (6 weeks old) displayed much higher basal levels of plasma glucagon immunoreactivity than normal mice. In contrast, plasma concentrations of insulin and glucose were lower in these dystrophic mice than in normal NMRI mice. The plasma glucagon levels declined with age in both strains during the time-period studied (1.5–5 months). Gel filtration of plasma from dystrophic as well as normal mice on Sephadex G-200 revealed that a large part of the total glucagon immunoreactivity was eluted in fractions containing the immunoglobulins. The amount of the ‘true’ glucagon part was lower in plasma from normal mice (about 0.2 μg/l) than in plasma from mice of the dystrophic strain (0.4–0.5 μg/l)). This finding was indirectly corroborated by the observation that a large intravenous glucose load decreased plasma glucagon by approximately 0.2 μg/l in the non-dystrophic NMRI strain and by about 0.4–0.6 μg/l in the dystrophic strain. Thus, the ability of glucose to suppress glucagon secretion appeared unaffected in the dystrophic mice. Glucose-induced insulin release, however, was considerably impaired in these animals. It is concluded that mice of the dystrophic 129/ReJ strain have higher plasma levels of ‘true’ glucagon than mice of the non-dystrophic NMRI strain. Whether the abnormally high plasma glucagon levels in the dystrophic strain, particularly in very young dystrophic mice, might contribute to the development of the muscular dystrophy remains to be elucidated.