Suppression of basal, but not of glucose-stimulated insulin secretion by human insulin in healthy and obese hyperinsulinemic subjects

To evaluate the suppressive effect of biosynthetic human insulin (BHI; 2.5 U/m2 . h) on basal and glucose-stimulated insulin secretion in healthy and obese hyperinsulinemic subjects, the plasma C-peptide response was measured during maintenance of euglycemia and hyperglycemia by means of the glucose clamp technique. In five healthy subjects in whom arterial insulin concentration was increased to 94 +/- 8 microU/mL, but euglycemia was maintained at the fasting level. C-peptide concentration fell from 1.3 +/- 1.0 ng/mL by 21 +/- 8% (P less than 0.05). When hyperglycemia of 7 mmol/L above basal was induced by a variable glucose infusion, the C-peptide response was similar in the control (5.0 +/- 0.6 ng/mL) and BHI experiments (4.7 +/- 0.6 ng/mL) and was paralleled by an identical increase in plasma insulin above the prevailing insulin concentration. In seven obese patients plasma C-peptide fell from 3.5 +/- 0.4 to 2.8 +/- 0.5 ng/mL (P less than 0.05) when BHI was infused at the same rate of euglycemia maintained as in the lean subjects. As in healthy subjects, however, the plasma C-peptide response to the hyperglycemic stimulus (8.7 +/- 0.9 ng/mL) was not altered by BHI (7.9 +/- 0.8 ng/mL). Glucose utilization as determined by the glucose infusion rate necessary to maintain the desired glucose level was reduced by half in the obese patients compared with that of normal subjects. From these data we conclude that in healthy as well as obese hyperinsulinemic subjects, insulin at concentrations capable of suppressing its basal secretion fails to suppress its glucose-stimulated secretion.     

Role of insulin in glucose-stimulated insulin secretion in beta cells

Diabetes is on the increase worldwide and greater than 90% are type 2. There are two features to type 2 diabetes: muscle, fat and liver tissues are insulin resistant and beta cells lose the ability to secrete insulin. Prior to developing diabetes, however, insulin resistant individuals lose the first-phase insulin secretion response. Transgenic mice lacking insulin receptors in their beta cells have no first-phase response. Primary cultures of mouse islets pre-exposed to anti-insulin do not exhibit a first-phase insulin secretion response. That is, beta cells, like muscle, fat, and liver, are an insulin sensitive tissue and in the presence of insulin resistance (type 2 diabetes), in the absence of insulin receptors (transgenic mice lacking beta cell insulin receptors), or in the absence of constitutively secreted insulin (anti-insulin treatment), beta cells are unable to respond properly to post-prandial glucose. The purpose of this report is to review our understanding of the glucose-stimulus response and of insulin signaling, and to suggest why the latter may be necessary for the former to proceed.     

gamma-tocopherol, but not alpha-tocopherol, potently inhibits neointimal formation induced by vascular injury in insulin resistant rats

Insulin resistance may enhance the neointima formation via increased oxidative stress. However, clinical trials investigating the benefit of antioxidant therapy with alpha-tocopherol showed negative results. Recent studies showed that chemical characteristics of gamma-tocopherol are distinct from those of alpha-tocopherol. We hypothesized that gamma-tocopherol is superior to alpha-tocopherol in preventing the neointima growth after arterial injury in insulin resistance. Male rats were fed with standard chow or a high fructose diet for induction of insulin resistance. Thereafter, the left carotid artery was injured with a balloon catheter. After 2 weeks, the carotid arteries were harvested and histomorphometrically analyzed. The neointima-media ratio of the injured artery was significantly greater in insulin resistance group (n=8, 1.33+/-0.12) than in normal group (n=10, 0.76+/-0.11, p<0.01). gamma-Tocopherol (100 mg/kg/day) reduced the ratio (n=5, 0.55+/-0.21, p<0.01 vs. insulin resistance group), while alpha-tocopherol was without effect (n=7, 1.08+/-0.14). The quantification of plasma phosphatidylcholine hydroperoxide, an indicator of systemic oxidative stress, and dihydroethidium fluorescence staining of the carotid artery, an indicator of the local superoxide production, showed that oxidative stress in the systemic circulation and local arterial tissue was increased in insulin resistance. Both tocopherols decreased plasma phosphatidylcholine hydroperoxide, but failed to suppress the superoxide production in the carotid arteries. Increased 3-nitrotyrosine in neointima by insulin resistance was greatly reduced only by gamma-tocopherol. In conclusion, gamma-tocopherol, but not alpha-tocopherol, reduces the neointima proliferation in insulin resistance, independently of its effects on superoxide production. The beneficial effect may be related with its inhibitory effects on nitrosative stress.     

Nocturnal growth hormone secretion does not affect diurnal variations in arginine and glucose-stimulated insulin secretion

There is a diurnal variation in insulin secretion, with higher values in the morning (AM) than in the afternoon (PM). This study tested the hypothesis that nocturnal human growth hormone (hGH) secretion might be the mechanism producing this diurnal variation in insulin secretion. Six healthy normal-weight men were studied on four occasions: twice in the early morning (AM) and twice in the afternoon (PM). Oral methscopolamine (Pamine), an anticholinergic agent that blocks hGH release, was administered at bedtime prior to the AM study or before breakfast for the PM study. An index of insulin secretion in all four tests was obtained from measurement of the acute release of insulin in response to two intravenous (IV) boluses of arginine, one given basally and the other given after raising glucose levels to approximately 150 mg/dL above the baseline. Insulin secretion was significantly greater in the morning than in the afternoon in both control and methscopolamine-pretreated subjects. The mean peak hGH was reduced in subjects pretreated with oral methscopolamine. Drug treatment reduced insulin secretion proportionally in the morning and afternoon. These results suggest that the diurnal insulin response to stimulation with arginine during a hyperglycemic clamp persists despite complete suppression of hGH by anticholinergic blockade, and that the diurnal insulin secretion is not caused by sleep- or meal-induced GH secretion.     

Sweet taste receptor signaling in beta cells mediates fructose-induced potentiation of glucose-stimulated insulin secretion

Postprandial insulin release is regulated by glucose, but other circulating nutrients may target beta cells and potentiate glucose-stimulated insulin secretion via distinct signaling pathways. We demonstrate that fructose activates sweet taste receptors (TRs) on beta cells and synergizes with glucose to amplify insulin release in human and mouse islets. Genetic ablation of the sweet TR protein T1R2 obliterates fructose-induced insulin release and its potentiating effects on glucose-stimulated insulin secretion in vitro and in vivo. TR signaling in beta cells is triggered, at least in part, in parallel with the glucose metabolic pathway and leads to increases in intracellular calcium that are dependent on the activation of phospholipase C (PLC) and transient receptor potential cation channel, subfamily M, member 5 (TRPM5). Our results unveil a pathway for the regulation of insulin release by postprandial nutrients that involves beta cell sweet TR signaling.     

Sodium channel beta1 regulatory subunit deficiency reduces pancreatic islet glucose-stimulated insulin and glucagon secretion

Glucose-stimulated insulin and glucagon release regulates glucose homeostasis by an excitation-secretion coupling pathway beginning with ATP-sensitive K(+) channel closure, membrane depolarization, and entry of calcium ions to stimulate exocytosis. The contribution of voltage-gated sodium channels to this release pathway is still being elucidated. We demonstrate that loss of Scn1b, a major regulatory subunit expressed with Na(v)1.7 protein in mouse pancreatic islets, reduces glucose-stimulated insulin and glucagon secretion in vitro and in vivo, resulting in severe fed and fasting hypoglycemia. This genetic mouse model is the first to demonstrate that sodium channelopathy impairs the physiological excitation-release coupling pathway for pancreatic insulin and glucagon release.     

Acute plasma glucose increase, but not early insulin response, regulates plasma ghrelin

OBJECTIVE: The independent role of glucose and insulin in ghrelin regulation is still controversial; this is also because in healthy subjects it is difficult to isolate the increase of glucose from that of insulin. The aim of this study was to discriminate the effect of glucose increase alone and early insulin response on plasma ghrelin, comparing ghrelin variation after i.v. glucose between healthy subjects and type 2 diabetic (T2DM) subjects, in whom the early insulin response to i.v. glucose is abolished. METHODS: Plasma glucose, insulin and ghrelin levels were measured 0, 3, 5, 10, 30, 45 and 60 min after a 5 g glucose i.v. bolus in seven healthy control subjects and eight T2DM subjects. RESULTS: There were no significant differences in body mass index, basal insulin and basal ghrelin between T2DM and healthy subjects. Basal glucose levels were higher in T2DM subjects than in controls. After i.v. glucose administration, plasma glucose increased significantly in both groups and the glucose peak was higher in T2DM subjects than in controls (9.67+/-1.25 (s.d.) vs 6.88+/-1.00 mmol/l, P<0.01). Insulin increased rapidly in controls, while in T2DM subjects, plasma insulin did not rise in the first 10 min. After the glucose bolus, plasma ghrelin showed a significant reduction both in controls and in T2DM subjects after 5 min. CONCLUSION: These findings indicate that a low-dose i.v. glucose bolus reduces ghrelin both in controls and in T2DM subjects and therefore that early insulin response does not affect plasma ghrelin.     

The nucleotide exchange factor SIL1 is required for glucose-stimulated insulin secretion from mouse pancreatic beta cells in vivo

Aims/hypothesis

Regulation of insulin secretion along the secretory pathway is incompletely understood. We addressed the expression of SIL1, a nucleotide exchange factor for the endoplasmic reticulum (ER) chaperone glucose-regulated protein 78 kD (GRP78), in pancreatic beta cells and investigated whether or not SIL1 is involved in beta cell function.

Methods

SIL1 expression was analysed by immunoblotting and immunofluorescence. Metabolic and islet variables, including glucose tolerance, beta cell mass, insulin secretion, islet ultrastructure, insulin content and levels of ER stress marker proteins, were addressed in Sil1 knockout (Sil1 ^?/?) mice. Insulin, proinsulin and C-peptide release was addressed in Sil1 ^?/? islets, and SIL1 overexpression or knockdown was explored in MIN6 cells in vitro. Models of type 1 diabetes and insulin resistance were induced in Sil1 ^?/? mice by administration of streptozotocin (STZ) and a high-fat diet (HFD), respectively.

Results

We show that SIL1 is expressed in pancreatic beta cells and is required for islet insulin content, islet sizing, glucose tolerance and glucose-stimulated insulin secretion in vivo. Levels of pancreatic ER stress markers are increased in Sil1 ^?/? mice, and Sil1 ^?/? beta cell ER is ultrastructurally compromised. Isolated Sil1 ^?/? islets show lower proinsulin and insulin content and impaired glucose-stimulated insulin secretion. Modulation of SIL1 protein levels in MIN6 cells correlates with changes in insulin content and secreted insulin. Furthermore, Sil1 ^?/? mice are more susceptible to STZ-induced type 1 diabetes with increased apoptosis. Upon HFD feeding, Sil1 ^?/? mice show markedly lower insulin secretion and exacerbated glucose intolerance compared with control mice. Surprisingly, however, HFD-fed Sil1 ^?/? mice display pronounced islet hyperplasia with low amounts of insulin in total pancreas.

Conclusions/interpretation

These results reveal a novel role for the nucleotide exchange factor SIL1 in pancreatic beta cell function under physiological and disease conditions such as diabetes and the metabolic syndrome.     

The dense core transmembrane vesicle protein IA-2 is a regulator of vesicle number and insulin secretion

IA-2 is an enzymatically inactive member of the transmembrane protein tyrosine phosphate family located in dense core secretory vesicles and a major autoantigen in type 1 diabetes. Recent studies showed that targeted disruption of the IA-2 gene in mice resulted in impairment of insulin secretion and glucose intolerance. Insulin homeostasis, however, is a complex process involving a cascade of regulatory factors, and IA-2 is widely expressed in neuroendocrine cells throughout the body. Consequently, it is uncertain whether the impairment of insulin secretion in IA-2 knockout mice is a direct result of the knockout of IA-2 in beta cells or to counter regulatory alterations resulting from IA-2 knockout in other neuroendocrine cells. To define the function of IA-2, we studied the secretion of insulin in a single cell type, MIN-6, by overexpressing and knocking down IA-2. Our experiments showed that overexpression of IA-2 resulted in a 6-fold increase in glucose- or K+-induced insulin secretion and a approximately 3-fold increase in the number of secretory vesicles and the insulin content of cells. In contrast, knockdown of endogenous IA-2 by short interfering RNA resulted in nearly a complete loss of glucose-induced insulin secretion and a 50% decrease in basal insulin release. The half-life of insulin in cells overexpressing IA-2 was nearly twice as great as that in mock-transfected cells, suggesting that IA-2 was stabilizing the insulin-containing vesicles. From these results we conclude that in beta cells, IA-2 is an important regulator of dense core vesicle number and glucose-induced and basal insulin secretion.     

Involvement of nitric oxide in arginine, but not glucose, induced insulin secretion in normal men

OBJECTIVE At present, there are no reports in the literature of studies in humans concerning a possible role of nitric oxide (NO) in the regulation of pancreatic endocrine secretions, whereas studies in the rat provided discrepant results. The aim of this study was to clarify whether NO is involved in the control of insulin and/or glucagon secretion in basal conditions and/or in response to arginine or glucose administration in normal male subjects. DESIGN We investigated whether an intravenous infusion of the NO synthase (NOS) inhibitor L-NAME, at a dose previously demonstrated not to produce blood pressure alterations or untoward side-effects, modifies insulin and/or glucagon secretory patterns. SUBJECTS Fourteen healthy male volunteers aged 24–35 years, within 10–13% of their ideal body weight and without family history of diabetes mellitus or other endocrine diseases. METHODS Seven normal men were treated intravenously with L -arginine (30g in 50ml of normal saline over 30 minutes) or glucose (0.33g/kg body weight in a bolus) with or without the concomitant infusion of L-NAME (90μg/kg in 50 ml of normal saline). L-NAME was infused for 30 minutes before and during arginine infusion and over 30 minutes before and 30 minutes after glucose injection. Another group of 7 men was infused over 60 minutes with L-NAME (90μg/kg in 50 ml of normal saline) alone or saline alone. RESULTS Basal and L -arginine or glucose induced glucagon secretions and basal and glucose stimulated insulin secretions were not altered by L-NAME administration. In contrast, the drug produced a partial but significant decrease in the insulin response to L -arginine. In fact, the mean peak insulin response to L -arginine was 5.3 times (53±5mU/l (mean±SE)) higher than basal value (10±2) in the absence of L-NAME, but only 3.33 times (40±4) higher than baseline (12±3) during the infusion of the NOS-inhibitor. CONCLUSION These data suggest that NO at least partially mediates the stimulatory action of L -arginine on insulin secretion in normal human subjects.     

Nitric oxide inhibits, and carbon monoxide activates, islet acid alpha-glucoside hydrolase activities in parallel with glucose-stimulated insulin secretion

We have studied the influence of nitric oxide (NO) and carbon monoxide (CO), putative messenger molecules in the brain as well as in the islets of Langerhans, on glucose-stimulated insulin secretion and on the activities of the acid alpha-glucoside hydrolases, enzymes which we previously have shown to be implicated in the insulin release process. We have shown here that exogenous NO gas inhibits, while CO gas amplifies glucose-stimulated insulin secretion in intact mouse islets concomitant with a marked inhibition (NO) and a marked activation (CO) of the activities of the lysosomal/vacuolar enzymes acid glucan-1,4-alpha-glucosidase and acid alpha-glucosidase (acid alpha-glucoside hydrolases). Furthermore, CO dose-dependently potentiated glucose-stimulated insulin secretion in the range 0.1-1000 microM. In intact islets, the heme oxygenase substrate hemin markedly amplified glucose-stimulated insulin release, an effect which was accompanied by an increased activity of the acid alpha-glucoside hydrolases. These effects were partially suppressed by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one. Hemin also inhibited inducible NO synthase (iNOS)-derived NO production probably through a direct effect of CO on the NOS enzyme. Further, exogenous CO raised the content of both cGMP and cAMP in parallel with a marked amplification of glucose-stimulated insulin release, while exogenous NO suppressed insulin release and cAMP, leaving cGMP unaffected. Emiglitate, a selective inhibitor of alpha-glucoside hydrolase activities, was able to markedly inhibit the stimulatory effect of exogenous CO on both glucose-stimulated insulin secretion and the activityof acid glucan-1,4-alpha-glucosidase and acid alpha-glucosidase, while no appreciable effect on the activities of other lysosomal enzyme activities measured was found. We propose that CO and NO, both produced in significant quantities in the islets of Langerhans, have interacting regulatory roles on glucose-stimulated insulin secretion. This regulation is, at least in part, transduced through the activity of cGMP and the lysosomal/vacuolar system and the associated acid alpha-glucoside hydrolases, but probably also through a direct effect on the cAMP system.     

Urocortin 3 regulates glucose-stimulated insulin secretion and energy homeostasis

Urocortin 3 (Ucn 3), a member of the corticotropin-releasing factor (CRF) family of peptides, is strongly expressed in mammalian pancreatic beta cells and has been shown to stimulate insulin secretion. Here we report the investigation of the hypothesis that endogenous Ucn 3 regulates insulin secretion, particularly in the presence of nutrient excess. Secretion of Ucn 3-like immunoreactivity from cultured beta cells was stimulated by high glucose and insulin secretagogs such as GLP-1; furthermore, 5 pancreatic Ucn 3 mRNA levels in vivo were increased during the positive energy balance caused by high-fat diet and by the absence of leptin. Immunoneutralization of Ucn 3 or pharmacologic blockade of its receptor, the type 2 CRF receptor (CRFR2), attenuated high but not low glucose-induced insulin secretion from isolated islets in vitro. Cultured islets isolated from Ucn 3-null mice also secreted less insulin in response to high glucose concentrations. Consistently, peripheral injection of a selective CRFR2 antagonist before the administration of a glucose challenge significantly attenuated glucose-induced insulin secretion in vivo. Ucn 3-null mice were relatively protected from the hyperinsulinemia, hyperglycemia, glucose intolerance, hepatic steatosis, and hypertriglyceridemia induced by high-fat diet. Additionally, we found that aged Ucn 3-null mice maintained better glucose tolerance than age-matched wild-type littermates. These results suggest that endogenous Ucn 3 in the pancreas is induced under excessive caloric conditions and acts locally to augment insulin production, which in the long-term may contribute to reduced insulin sensitivity and harmful metabolic consequences.     

Tumour necrosis factor-alpha-induced glucose-stimulated insulin secretion inhibition in INS-1 cells is ascribed to a reduction of the glucose-stimulated Ca2+ influx

The present study was undertaken to determine how tumour necrosis factor-alpha (TNF-alpha) elicits the inhibition of glucose-stimulated insulin secretion (GSIS) in rat insulinoma cells (INS)-1 beta-cells. TNF-alpha pretreatment did not change the expression levels of insulin, PDX-1, glucose transporter 2, glucokinase, K(ATP) channels, Ca(2)(+) channels, and exocytotic molecules and, furthermore, did not reduce the glucose-stimulated ATP level. On the other hand, TNF-alpha reduced the glucose-stimulated influx of Ca(2)(+). The TNF-alpha treatment was thought to activate c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and NF-kappaB inflammatory signals, since TNF-alpha increased phospho-JNK and phospho-p38 and reduced I kappaB levels. Inhibitors of these signaling pathways prevented the TNF-alpha-induced reduction of the Ca(2)(+) influx and GSIS. Overexpression of MEKK3, a possible mediator from the TNF-alpha receptor to the JNK/p38 and NK-kappaB signaling cascade, increased the levels of phospho-JNK, phospho-p38, and NF-kappaB, and reduced the glucose-stimulated Ca(2)(+) influx and GSIS. The reduction of the Ca(2)(+) influx and GSIS in MEKK3-overexpressing INS-1 cells was also prevented by inhibitors of JNK, p38, and NF-kappaB. These data demonstrate that TNF-alpha inhibits GSIS by reducing the glucose-stimulated Ca(2)(+) influx, possibly through the activation of JNK and p38 MAPK and NF-kappaB inflammatory signals. Thus, our findings suggest that the activation of stress and inflammatory signals can contribute to the inhibition of GSIS in the development of diabetes.     

Circulating insulin inhibits glucagon secretion induced by arginine in type 1 diabetes

OBJECTIVE: To evaluate if insulin has a suppressive effect on the glucagon secretion stimulated by arginine in type 1 diabetes. RESEARCH DESIGN AND METHODS: The alpha-cell response to an i.v. bolus of arginine (150mgkg(-1)) followed by an infusion of arginine (10mgkg(-1)min(-1)) was studied in random order during either low dose infusion (LDT) or high dose infusion (HDT) of insulin in ten patients with type 1 diabetes. The blood glucose level was clamped at an arterialized level of 5mmoll(-1) by a variable infusion of glucose. Venous C-peptide, glucagon, growth hormone, and insulin were analyzed. RESULTS: The mean plasma concentration of insulin was four times higher during the HDT. The C-peptide level did not differ between the LDT and the HDT. During the LDT in response to arginine the blood glucose level increased from 5.0 to 5.8mmol l(-1) although the glucose infusion was markedly reduced, while no change was seen during the HDT. A significantly smaller increase in the glucagon levels during the HDT was seen (area under the curve of 413+/-45 vs 466+/-44pgml(-1)h(-1), P=0.03) while the growth hormone levels were almost identical. CONCLUSION: This study demonstrates that a high level of circulating insulin exerts an inhibitory effect on the glucagon response to arginine in type 1 diabetes. Thus, the suppressive effect of insulin on the glucagon release from the alpha-cell seems to be general and not only dependent on stimulation by hypoglycemia.     

Twelve weeks' treatment with diazoxide without insulin supplementation in Type 2 diabetes is feasible but does not improve insulin secretion.

AIMS: Treatment with K-ATP channel openers, such as diazoxide, can have beneficial effects on insulin secretion in both Type 1 and Type 2 diabetes. However, the precise conditions for obtaining beneficial effects without untoward events have not been determined. We tested the hypothesis that intermittent administration of diazoxide at bedtime for 12 weeks could produce beneficial effects in the absence of side-effects in Type 2 diabetic patients who were not taking insulin. METHODS: After an 8-week run-in period, during which treatment with repaglinide and metformin was optimized, we randomized 26 patients to either diazoxide, 100 mg at bedtime, or placebo. RESULTS: Side-effects were absent or minimal. HbA(1c) did not change. However day-time glucose concentrations by home glucose monitoring were approximately 1.5 mmol/l higher with diazoxide vs. placebo. Stimulation tests (C-peptide-glucagon and breakfast) did not indicate improved pancreatic B-cell function, except by posthoc analysis, in a subgroup of younger age. CONCLUSION: Compared with previous results with diazoxide together with bedtime insulin, the present results are less favourable and indicate that concomitant insulin treatment is needed during intervention with K-ATP channel openers.