Effect of pharmacological agents and fasting on pancreatic islet norepinephrine in the golden hamster

Summary Using a specific and sensitive radioenzymatic assay that utilizes the partially purified enzyme phenylethanolamine-N-methyltransferase, studies were done to determine if pharmacological agents and/or fasting alter the norepinephrine concentration of collagenase-isolated golden hamster pancreatic islets. The norepinephrine concentration (42.1±8.07 mol/kg net weight, mean±SEM) and the monoamine oxidase activity (5,407±530 pmol product /mg tissue/min) of hamster pancreatic islets was at least five times higher than acinar pancreas, kidney, heart, median eminence or cerebral cortex. The catechol-o-methyltransferase activity of hamster islets (7±2.3 pmol product/mg tissue/min) was one half or less than the other tissues. Islet norepinephrine was not increased by two days administration of the monoamine oxidase inhibitor tranylcypromine. Islet norepinephrine concentration was increased 2-fold by administration of the norepinephrine precursor DL-threo-dihydroxyphenylserine.This increase was enhanced by prior administration of tranylcypromine (3.5-fold) and prevented by prior administration of the decarboxylase inhibitor N¹-(DL-seryl)-N²-(2,3,4-trihydroxybenzyl) hydrazine (RO-4-4602). There was a good correlation between the islet norepinephrine concentration and the plasma glucose concentration after pharmacological agents. Reserpine administration markedly depleted the islet norepinephrine concentra tion. Fasting of 24, 48 and 72 h did not alter the norepinephrine concentration in islets and heart. It is concluded that the pancreatic islets of the hamster have an active noradrenergic system, but that islet norepinephrine does not appear to play an important role in the impaired insulin secretion of fasting hamsters.     

Therapy of malignant hamster insulinomas with monoamine precursors

Summary The hyperinsulinaemia and hypoglycaemia produced by malignant insulinomas pose a difficult therapeutic problem. The non-polar monoamine precursors 5-hydroxytryptophan, L-3,4-dihydroxyphenylalanine and DL-threo-dihydroxyphenylserine are readily taken up by normal hamster pancreatic islets and are converted intracellularly to serotonin, dopamine and norepinephrine. These intracellular monoamines then inhibit glucose-stimulated insulin secretion. In the present study we have determined if the monoamine oxidase inhibitor pargyline, and monoamine precursors, could modify the severe hypoglycaemia of hamsters bearing a rapidly growing transplantable insulinoma. One hour after receiving the respective precursor there was an 11, 18, and 54-fold increase in the concentration of serotonin, dopamine or norepinephrine in the insulinomas. Twenty four hours after the administration of the respective precursors there were the following increases in plasma glucose in the respective groups: 5-hydroxytryptophan, 1.9 ± 0.5 to 4.9 ± 0.3 mmol/l (mean ± SEM); L-3,4-dihydroxyphenylalanine, 1.5 ± 0.3 to 3.4 ± 0.6 mmol/l; and DL-threo-dihydroxyphenylserine, 1.5 ± 0.2 to 6.4 ± 0.7 mmol/l. The change in plasma insulin concentration was statistically significant only in the group receiving DL-threo-dihydroxyphenylserine (229 ± 59 to 81 ± 42 mU/l). To determine if this therapeutic approach could modify the rapidly fatal outcome of the tumour-bearing hamsters, they received 0.154 mol/l saline, DL-threo-dihydroxyphenylserine or pargyline plus DL-threo-dihydroxyphenylserine every two days. The survival of the groups (mean ± SEM) was: saline 8.4 ± 1.1 d; DL-threo-dihydroxyphenylserine 17.9 ± 3.5 d; and pargyline plus DL-threo-dihydroxyphenylserine 24.4 ± 5.5 d. The present study suggests that increasing the monoamine content of malignant insulinomas may favourably modify the course of these devastating tumours.     

Monoamine oxidase and catechol-o-methyltransferase activity in hamster and rat insulinomas

Hamster and rat insulinomas were assayed for norepinephrine, dopamine and serotonin concentration and for monoamine oxidase and catechol-o-ethyltransferase (COMT) activity. The concentration of norepinephrine (mean 0.55 mumol/kg, range less than 0.20 to 2.64 mumol/kg) and serotonin (mean 5.22 mucol/kg, rang less than 0.6 to 26.5 mumol/kg) in hamster insulinomas were comparable to previously reported concentrations. Dopamine conentration (mean 0.34 mumol/kg, range less than 0.20 to 0.95 mumol/kg) was only 2 to 2.5% of that reported previously. Monoamine oxidase activity of the hamster and rat insulinomas were comparable to those of normal hamster islets. In contrast, the COMT activity of both insulinomas was much greater than the COMT activity of normal pancreatic islets of both species and was greater than in several other tissues and tumours. The tumour COMT, which was predominantly in the cytosol, was Mg2+ dependent and had a comparable sensitivity to inhibition by tropolone as purified beef-liver COMT. Hamster insulinoma monoamine oxidase was more sensitive than rat insulinoma monoamine oxidase to inhibition by tranylcypromine and deprenyl, while rat insulinoma monoamine oxidase was more sensitive to inhibition by clorgyline and was more heat labile.     

Role of vesicular monoamine transporter type 2 in rodent insulin secretion and glucose metabolism revealed by its specific antagonist tetrabenazine

Despite different embryological origins, islet beta-cells and neurons share the expression of many genes and display multiple functional similarities. One shared gene product, vesicular monoamine transporter type 2 (VMAT2, also known as SLC18A2), is highly expressed in human beta-cells relative to other cells in the endocrine and exocrine pancreas. Recent reports suggest that the monoamine dopamine is an important paracrine and/or autocrine regulator of insulin release by beta-cells. Given the important role of VMAT2 in the economy of monoamines such as dopamine, we investigated the possible role of VMAT2 in insulin secretion and glucose metabolism. Using a VMAT2-specific antagonist, tetrabenazine (TBZ), we studied glucose homeostasis, insulin secretion both in vivo and ex vivo in cultures of purified rodent islets. During intraperitoneal glucose tolerance tests, control rats showed increased serum insulin concentrations and smaller glucose excursions relative to controls after a single intravenous dose of TBZ. One hour following TBZ administration we observed a significant depletion of total pancreas dopamine. Correspondingly, exogenous L-3,4-dihydroxyphenylalanine reversed the effects of TBZ on glucose clearance in vivo. In in vitro studies of rat islets, a significantly enhanced glucose-dependent insulin secretion was observed in the presence of dihydrotetrabenazine, the active metabolite of TBZ. Together, these data suggest that VMAT2 regulates in vivo glucose homeostasis and insulin production, most likely via its role in vesicular transport and storage of monoamines in beta-cells.     

Long-term infusion of norepinephrine plus serotonin into the ventromedial hypothalamus impairs pancreatic islet function.

To examine the possibility of a cause-effect relationship between enhanced monoamine content in the ventromedial hypothalamus ([VMH] a characteristic of hyperinsulinemic and insulin-resistant animals) and islet dysfunction, we infused norepinephrine ([NE] 25 nmol/h) and/or serotonin ([5-HT] 2.5 nmol/h) into the VMH of normal hamsters for 5 weeks and then examined insulin release from the isolated pancreatic islets. VMH infusion of NE + 5-HT, but not of either neurotransmitter alone, produced a marked leftward shift in the dose-response curve of glucose-induced insulin release (twofold to sixfold increase at 5 to 7.5 mmol/L glucose v vehicle-treated animals). In addition, the islet responsiveness to 1 micromol/L NE and 10 micromol/L acetylcholine was abolished in these NE + 5-HT VMH-infused hamsters. These findings indicate that an increase of NE and 5-HT content in the VMH can induce dysregulation of islet insulin release in response to glucose and neurotransmitters. Inasmuch as VMH NE and 5-HT levels are elevated in hyperinsulinemic and insulin-resistant animals, the present findings suggest that an endogenous increase in these hypothalamic monoamines may contribute to islet dysfunction, which is one of the characteristics of type 2 diabetes.     

Stimulation of the hypothalamo-pituitary-adrenocortical axis by the central serotonergic pathway: involvement of endogenous corticotropin-releasing hormone but not vasopressin.

Many reports indicate that serotonin plays a role in the regulation of the hypothalamo-pituitary-adrenocortical axis. The present study was designed to elucidate whether the activation of the central serotonergic pathway enhances adrenocorticotropin and corticosterone secretion, and if so, whether the CRH and vasopressin neuronal systems could be mediating this effect. Intraperitoneal administration of a low dose of L-5-hydroxytryptophan (an aromatic L-amino acid precursor of serotonin synthesis; 20 mg/kg bw, 30 minutes before the sacrifice) in rats pretreated with pargyline (a brain monoamine oxidase inhibitor, which enhances monoamine activity; 75 mg/Kg bw, 16 hours before the sacrifice) and carbidopa (a peripheral active inhibitor of the decarboxylation of aromatic L-amino acids, which would permit more monoamine precursor to be available to the brain; 50 mg/Kg bw, 90 minutes before the sacrifice) increased ACTH and corticosterone secretion in plasma. Such an effect was partially blocked by metergoline (a serotonin type-1 and-2 receptor blocker; 1 mg/Kg bw, 90 minutes before the sacrifice), but not by spiperone (a serotonin type-2 and dopamine receptor antagonist; 0.5 mg/Kg bw. 90 minutes before the sacrifice). The activation of the central serotonergic system enhanced the CRH content in the median eminence, whereas it decreased the content of this neuropeptide in the medial basal hypothalamus. These effects were fully abolished by metergoline, but not by spiperone pretreatment. The activation of the serotonergic pathway did not influence the vasopressinergic neuronal system. In vitro experiments using hypothalamic-median eminence fragments incubated with serotonin solutions indicate that this monoamine possesses a CRH releasing effect at concentrations of 1 microM or more.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biogenic amine regulation of glucagon secretion

Summary Norepinephrine, dopamine and serotonin directly stimulated glucagon secretion by isolated perifused hamster islet. Propranolol blocked norepinephrine, dopamine and serotonin-stimulated glucagon release, suggesting that norepinephrine, dopamine and serotonin may modulate glucagon secretion via a beta-adrenergic mechanism. Dopamine and serotonin may exert their action directly via the beta receptor, stimulate residual adrenergic nerve terminals, or the release of other islet biogenic amines. We conclude that part of the stimulating effect of norepinephrine on glucagon release is beta adrenergically mediated and that dopamine and serotonin may affect glucagon secretion either directly or through the release of neurotransmitters that lead to glucagon release. Thus the intra-islet released dopamine and serotonin may contribute to the islets paracrine system by stimulating intra-islet adrenergic neurons. This study was supported by N1H RR08167.     

Characterization of pancreatic islet monoamine oxidase

Monoamine oxidase (MAO) is present in isolated islets of Langerhans of rabbits, golden hamsters, and rats. Tryptamine, tyramine, serotonin, and dopamine can serve as substrates for this enzyme. We compared the properties of islet and liver MAO in the rabbit. The Michaelis constant (K_m) for tryptamine of islet MAO (6.5 × 10^?5M) is greater than the K_m of liver MAO (3 × 10^?5M). The K_m for tyramine of islet MAO (1.5 × 10^?4M) is similar to the K_m of liver MAO (1.8 × 10^?4M). Islet MAO appeared to be more susceptible to heat inactivation (50°C) than did liver MAO. This may be an artifact produced by the collagenase technique used in the preparation of the islets, as collagenase treatment of liver increased the thermal lability of the MAO in this tissue. Liver and islet MAO have a comparable sensitivity to MAO inhibitors such as clorgyline, deprenyl, tranylcypromine, pargyline, and harmine. The present report, along with previous reports that MAO inhibitors alter insulin secretion, suggests that islet MAO may modify insulin secretion.     

Monoamines stimulate sex reversal in the saddleback wrasse

Monoamine neurotransmitters (norepinephrine, dopamine, and serotonin) play an important role in reproduction and sexual behavior throughout the vertebrates. They are the first endogenous chemical signals in the regulation of the hypothalamo-pituitary-gonadal (HPG) axis. In teleosts with behavioral sex determination, much is known about behavioral cues that induce sex reversal. The cues are social, processed via the visual system and depend on the ratio of females to males in the population. The mechanisms by which these external behavioral cues are converted to an internal chemical regulatory process are largely unknown. The protogynous Hawaiian saddleback wrasse, Thalassoma duperrey, was used to investigate the biological pathway mediating the conversion of a social cue into neuroendocrine events regulating sex reversal. Because monoamines play an important role in the regulation of the HPG axis, they were selected as likely candidates for such a conversion. To determine if monoamines could affect sex reversal, drugs affecting monoamines were used in an attempt to either induce sex reversal under non-permissive conditions, or prevent sex reversal under permissive conditions. Increasing norepinephrine or blocking dopamine or serotonin lead to sex reversal in experimental animals under non-permissive conditions. Increasing serotonin blocked sex reversal under permissive conditions, while blocking dopamine or norepinephrine retarded the process. The results presented here demonstrate that monoamines contribute significantly to the control sex reversal. Norepinephrine stimulates initiation and completion of gonadal sex of reversal as well as color change perhaps directly via its effects on the HPG axis. Dopamine exercises inhibitory action on the initiation of sex reversal while 5-HT inhibits both initiation and completion of sex reversal. The serotonergic system appears to be an integral part of the pathway mediating the conversion of a social cue into a neuroendocrine event. The complex organization of neurochemical events controlling the psychosocial, physiological, and anatomical events that constitute reversal of sexual identity includes monoamine neurotransmitters.     

The effect of serotonin on in vitro insulin secretion and biosynthesis in mice

Summary The effect of serotonin on insulin secretion and biosynthesis was studied using isolated islets of mice. Serotonin produced a small stimulatory effect on insulin secretion when glucose was present in the incubation medium at a low concentration. On the other hand, an inhibition of insulin secretion was obtained with serotonin when glucose in the medium reached 3.0 mg/ml concentration. No significant effect of serotonin was obtained on insulin biosynthesis, neither in the presence of low nor with a high glucose concentration. These results suggest that the effect of this monoamine on insulin secretion is not mediated via its effect on insulin biosynthesis.Supported by Deutsche Forschungsgemeinschaft Bonn-Bad Godesberg, SFB 87 Ulm     

Time-dependent potentiation of insulin release induced by alpha-ketoisocaproate and leucine in rats: possible involvement of phosphoinositide hydrolysis

Summary The ability of the amino acid leucine and its keto acid, alpha-ketoisocaproate, to induce insulin release, to initiate phosphoinositide hydrolysis, and to amplify the subsequent insulin secretory response to glucose was assessed. In islets whose inositol-containing lipids were prelabelled with myo[2-³H]inositol, the addition of either compound resulted in an increase in insulin output, an increase in ³H efflux, rapid and significant increases in labelled inositol phosphate accumulation and a sustained increase in ³H efflux after removal of the stimulant. Direct measurements of labelled inositol phosphate accumulation in islets previously stimulated with alpha-ketoisocaproate demonstrate that this sustained increase in ³H efflux was the result of a persistent increase in phosphoinositide hydrolysis and was not simply a consequence of the hydrolysis of preformed inositol phosphates into more membrane permeable species. Prior exposure of islets to alpha-ketoisocaproate or leucine also resulted in an amplified secretory response to a subsequent glucose (10 mmol/l) stimulus. While peak first phase insulin release averaged 66±4 (mean±SEM, n=18) pg-islet^–1. min^–1 from control islets, this value increased to 204±14 and 246±11 pg·islet^–1· min^–1 in the leucine or alpha-ketoisocaproate pretreated islets respectively. The duration of this amplified response paralleled the duration of the persistent increase in ³H efflux. Prior alpha-ketoisocaproate exposure also amplified the subsequent insulin secretory response to tolbutamide and glyceraldehyde. While control (non-pretreated) islets in response to tolbutamide (200 mol/l) released insulin at a rate of 50±6pg·islet^–1·min^–1 (n = 3), this first phase response increased to 506±38 pg·islet^–1. min^–1 in prior alpha-ketoisocaproate treated islets. Peak first and second phase insulin responses to glyceraldehyde were increased 5-fold and 2-fold, respectively, by prior alpha-ketoisocaproate. These results suggest that events coupled to the hydrolysis of membrane inositol-containing phospholipids induced by leucine and alpha-ketoisocaproate participate not only in their acute insulin stimulatory action, but also in their ability to induce time-dependent potentiation (memory) in isolated islets.     

Effect of diabetes mellitus and chemical sympathectomy on tissue monoamine oxidase activity and norepinephrine concentration in the golden hamster

The pancreatic islets of the golden hamster have an extremely high monoamine oxidase (MAO) activity and norepinephrine (NE) concentration. To determine the cellular component responsible for these high values, we assayed MAO activity and NE concentration in collagenase-isolated islets from normal, streptozotocin-induced diabetic, and 6-hydroxydopamine (6-OHDA) induced sympathectomized golden hamsters. There was a 53% reduction in islet MAO activity in the diabetic hamsters, with no alteration in islet MAO activity in the sympathectomized ones, suggesting that the pancreatic B cells contain a large proportion of the MAO activity. There was a 98% reduction in islet NE concentration in the sympathectomized hamsters, with no alteration in islet NE concentration in the diabetic hamsters, suggesting that the adrenergic nerve endings in the islets contain the majority of the NE. There was no alteration in the MAO activity or NE concentration in the cerebral cortex, pituitary gland, liver, kidney, acinar pancreas, or heart of diabetic hamsters. Although the MAO activity of the hypothalamus was not altered, the diabetic hamsters had a 166% increase in NE and a 199% increase in dopamine in their hypothalamus, with no change in these amines in their cerebral cortex.     

Effect of L-dopa administration on islet monoamine oxidase activity and glucose-induced insulin release in the mouse

Previous studies have shown that the amine precursor L-3,4-dihydroxyphenylalanine (L-DOPA) is rapidly converted to its corresponding amine, dopamine, in islet beta-cells. In the present investigation, we studied the effect of acute L-DOPA administration on islet monoamine oxidase (MAO) activity and on glucose-induced insulin secretory response in mice. It was observed that at 2 min after intravenous L-DOPA administration, there was a marked increase (+35%) in islet MAO activity, with serotonin as substrate. At 7 min, MAO activity towards dopamine was enhanced by 32% and that towards serotonin and phenylethylamine (PEA) was decreased by 23 and 25%, respectively. The inhibitor of L-aromatic amino acid decarboxylase, benserazide, abolished L-DOPA-induced changes of MAO activity, suggesting that the formed dopamine, and not L-DOPA itself, was responsible for the observed effects. At 60 min, no effect by L-DOPA administration on islet MAO activity was noticed. L-DOPA (125 or 250 mumol/kg), given together with glucose, induced a decrease in glucose-induced insulin response. L-DOPA (125 mumol/kg), given 7 min before glucose, totally suppressed glucose-induced insulin response. This inhibition was eliminated through pretreatment with benserazide. Enhancement of glucose-stimulated insulin response, after deposition of horseradish peroxidase (HRP) in beta-cell vacuolar system, was suppressed by L-DOPA. We conclude that acute L-DOPA-induced dopamine accumulation in pancreatic islets is accompanied by rapid changes in MAO activity, concomitant with an inhibitory effect on glucose-stimulated insulin response. Increased hydrogen peroxide production, following increased MAO activity, may possibly augment the inhibitory effect of dopamine accumulation on insulin release.     

Monoamines in pancreatic islets of guinea pig, hamster, rat, and mouse determined by high performance liquid chromatography

Previous studies on the occurrence of catecholamines and serotonin in pancreatic islets using various histochemical and chemical methods have given widely different results. We therefore performed a comparative analysis of these amines in whole pancreas and islet tissue from hamster, guinea pig, rat, and mouse by the use of high performance liquid chromatography. Whole pancreas of guinea pig, hamster, and rat had a norepinephrine concentration of approximately 1.1 mumol/kg of pancreatic wet weight. The mouse pancreas had less than one-half of that concentration. Epinephrine and dopamine concentrations were on the order of 0.02 mumol/kg of pancreatic wet weight in all four species. The serotonin concentration was 2.1 mumol/kg of pancreatic wet weight in the guinea pig pancreas and approximately 0.2 mumol/kg in the other three species studied. The catecholamine concentrations were much higher in the pancreatic islets than in the exocrine pancreas. Thus, the norepinephrine concentration was approximately 35 mumol/kg of islet wet weight in hamster islets and 5-10 mumol/kg in rat, guinea pig, and mouse islets. The epinephrine concentration in islet tissue ranged between 1 and 7 mumol/kg of islet wet weight and the dopamine concentration between 0.5 and 4 mumol/kg except for guinea pig islets (12 mumol/kg). The islet tissue in the mouse, rat, and guinea pig contained disproportionately more epinephrine and dopamine relative to norepinephrine than did the exocrine pancreas. Chemical sympathectomy (6-hydroxydopamine treatment) in the mouse reduced the norepinephrine and epinephrine concentrations in islet tissue to nondetectable levels, whereas the dopamine concentration was essentially unchanged, thus suggesting an extra-neuronal source of this amine in addition to its occurrence in adrenergic nerves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of hypothalamic monoamines in the regulation of gonadotropin secretion

Experiments on rats were made to examine the time course of changes in the content of dopamine, noradrenaline and serotonin in different areas of the hypothalamus during the estrous cycle and the effects of local administration of monoamines to the nuclear formations of the hypothalamus on luteinizing hormone (LH) release in intact and ovariectomized female rats. The amplitude of circadian fluctuations in the monoamine level was demonstrated to depend on the phase of the estrous cycle, with the minimal values in the preovulatory period. The differences were detected in the pattern of the response of pituitary gonadotrophics to the administration of monoamines to the hypothalamus depending on the function of the sexual glands. It was noted that noradrenergic innervation of the preoptic area and dopaminergic innervation of the arcuate nucleus exert a stimulant effect on the preovulatory release of LH and that serotoninergic innervation of the indicated areas has an inhibitory effect on the same process. Estradial was demonstrated to have a modulatory action on the studied effects of monoamines. The data obtained indicate the involvement of noradrenaline, dopamine and serotonin in the control of cyclic secretion of LH in female rats.     

Insulin inhibits its own secretion from isolated,perifused human pancreatic islets

It is still a controversial question whether insulin suppresses its own secretion. We prepared pure human islets from three pancreases by collagenase digestion and density gradient purification. Aliquots of 200 islet equivalents (IE, 150-m sized-islets) were sequentially perifused at 37°C with 3.3 mmol/l glucose (3.3G, 40 min), 16.7 mmol/l glucose (16.7G, 30 min) and again 3.3G (30 min) after 24 h, 37°C culture in CMRL 1066 medium with or without the addition of either 200 or 400 U/ml human insulin in the incubation medium (6 replicates each). Insulin secretion was assessed by C-peptide (Cp) measurement in the perufusate. Without added insulin (C) and with 200 (Ins200) or 400 (Ins400) U/ml added insulin, basal Cp release was 0.12±0.03, 0.14±0.02 and 0.14±0.04 ng/ml, respectively. At 16.7G, the first-phase secretion peak (expressed as Cp value) was significantly lower with Ins200 (0.47±0.13 ng/ml,P<0.02) and Ins400 (0.68±0.15 ng/ml,P<0.05) than C (0.83±0.15 ng/ml). The second-phase secretion peak was also significantly (P<0.05) reduced with added insulin (Ins200: 0.47±0.08 ng/ml; Ins400: 0.45±0.07 ng/ml) than in its absence (C: 0.65±0.09 ng/ml). Accordingly, total Cp secretion was lower with Ins200 (10.6±2.3 ng/ml,P=0.03) and Ins400 (11.8±2.3 ng/ml) than with C (16.0±2.2 ng/ml). Thus, the addition for 24 h of either 200 or 400 U/ml insulin in the culture medium caused a significant decrease of insulin (as assessed by Cp measurement) secretion from perifused human islets, suggesting that feedback suppression of insulin release is at least in part due to a direct action of insulin on the islets.     

Abnormal insulin secretion and glucose metabolism in pancreatic islets from the spontaneously diabetic GK rat

Summary Insulin secretion and islet glucose metabolism were compared in pancreatic islets isolated from GK/Wistar (GK) rats with spontaneous Type 2 (non-insulin-dependent) diabetes mellitus and control Wistar rats. Islet insulin content was 24.5±3.1 U/ng islet DNA in GK rats and 28.8±2.5 U/ng islet DNA in control rats, with a mean (±SEM) islet DNA content of 17.3±1.7 and 26.5±3.4 ng (p < 0.05), respectively. Basal insulin secretion at 3.3 mmol/l glucose was 0.19±0.03 · ng islet DNA^–1· h^–1 in GK rat islets and 0.40±0.07 in control islets. Glucose (16.7 mmol/l) stimulated insulin release in GK rat islets only two-fold while in control islets five-fold. Glucose utilization at 16.7 mmol/l glucose, as measured by the formation of ³H₂O from [5-³ H]glucose, was 2.4 times higher in GK rat islets (3.1±0.7 pmol · ng islet DNA^–1 · h^–1) than in control islets (1.3±0.1 pmol · ng islet DNA^–1 · h^–1; p<0.05). In contrast, glucose oxidation, estimated as the production of ¹⁴CO₂ from [U-¹⁴C]glucose, was similar in both types of islets and corresponded to 15±2 and 30±3 % (p<0.001) of total glucose phosphorylated in GK and control islets, respectively. Glucose cycling, i. e. the rate of dephosphorylation of the total amount of glucose phosphorylated, (determined as production of labelled glucose from islets incubated with ³H₂O) was 16.4±3.4% in GK rat and 6.4±1.0% in control islets, respectively (p<0.01). We conclude that insulin secretion stimulated by glucose is markedly impaired in GK rat islets. Glucose metabolism is also altered in GK rat islets, with diminished ratio between oxidation and utilization of glucose, and increased glucose cycling, suggesting links between impaired glucose-induced insulin release and abnormal glucose metabolism.     

Properties of isolated human islets of langerhans: insulin secretion,glucose oxidation and protein phosphorylation

Summary In the present study, human islets were isolated by collagenase digestion from the pancreases of three kidney donors. Maintainance of the islets in tissue culture enabled insulin release, glucose oxidation and Ca²⁺-calmodulin-dependent protein phosphorylation to be determined using the same islets. Increasing glucose over a range 0–20 mmol/l resulted in a sigmoidal stimulation of insulin release (28.8±5.2 to 118.4±25.8 U-islet^–-h^–, n=10; threshold <4 mmol/l). There was a marked correlation between the insulin secretory response of the islets to glucose and their rate of glucose oxidation (5.9±0.3 at glucose 2 mmol/l up to 25.8±1.8 pmol-islet^–.h^– at 20 mmol/l, r = 0.98). N-acetylglucosamine (20 mmol/l) failed to elicit a secretory response from the islets. Stimulation of insulin secretion by glucose was dependent upon the presence of extracellular Ca²⁺. Extracts of the islets contained a Ca²⁺-calmodulin-dependent protein kinase which phosphorylated a 48-kdalton endogenous polypeptide. Myosin light-chain kinase activity was demonstrated in the presence of exogenous myosin light chains. This report demonstrates for the first time the sigmoidal nature of glucose-stimulated insulin release from isolated human islets, and its correlation with enhanced glucose oxidation. Furthermore, this is the first report of the presence of Ca²⁺-dependent protein kinases in human islets.     

Insulin secretion profiles are modified by overexpression of glutamate dehydrogenase in pancreatic islets

Aims/hypothesis Glutamate dehydrogenase (GDH) is a mitochondrial enzyme playing a key role in the control of insulin secretion. However, it is not known whether GDH expression levels in beta cells are rate-limiting for the secretory response to glucose. GDH also controls glutamine and glutamate oxidative metabolism, which is only weak in islets if GDH is not allosterically activated by L-leucine or (+/–)-2-aminobicyclo-[2,2,1]heptane-2-carboxylic acid (BCH).Methods We constructed an adenovirus encoding for GDH to overexpress the enzyme in the beta-cell line INS-1E, as well as in isolated rat and mouse pancreatic islets. The secretory responses to glucose and glutamine were studied in static and perifusion experiments. Amino acid concentrations and metabolic parameters were measured in parallel.Results GDH overexpression in rat islets did not change insulin release at basal or intermediate glucose (2.8 and 8.3 mmol/l respectively), but potentiated the secretory response at high glucose concentrations (16.7 mmol/l) compared to controls (+35%). Control islets exposed to 5 mmol/l glutamine at basal glucose did not increase insulin release, unless BCH was added with a resulting 2.5-fold response. In islets overexpressing GDH glutamine alone stimulated insulin secretion (2.7-fold), which was potentiated 2.2-fold by adding BCH. The secretory responses evoked by glutamine under these conditions correlated with enhanced cellular metabolism.Conclusions/interpretation GDH could be rate-limiting in glucose-induced insulin secretion, as GDH overexpression enhanced secretory responses. Moreover, GDH overexpression made islets responsive to glutamine, indicating that under physiological conditions this enzyme acts as a gatekeeper to prevent amino acids from being inappropriate efficient secretagogues.Abbreviations AUC Area under the curve - BCH (+/–)-2-aminobicyclo-[2,2,1]heptane-2-carboxylic acid - _m mitochondrial membrane potential - FCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone - GABA -aminobutyric acid - GDH glutamate dehydrogenase - HPLC high-pressure liquid chromatography - KRBH Krebs-Ringer bicarbonate HEPES buffer - MTT 3-(4,5-dimethylthiazol-2-yl)-2,5,-diphenyl tetrazolium bromide - RPMI Roswell Park Memorial Institute - TCA tricarboxylic acid