Neurotransmitters partially restore glucose sensitivity of insulin and glucagon secretion from perfused streptozotocin-induced diabetic rat pancreas

Summary To elucidate the mechanisms of insensitivity of hormone secretion to glucose in streptozotocin-induced diabetic rat islets, we investigated the effects of acetylcholine (ACh) and norepinephrine on insulin and glucagon secretion in response to changes in glucose concentration, using perfused pancreas preparations. Basal insulin secretion at a blood glucose level of 5.6 mmol/l was significantly higher and basal glucagon secretion significantly lower in streptozotocin-induced diabetic rats than in controls, and neither high (16.7 mmol/l) nor low (1.4 mmol/l) blood glucose concentrations influenced insulin or glucagon secretion. Addition of 10^–6 mol/l ACh to the perfusate increased glucose-stimulated insulin secretion. Also, 10^–6 mol/l ACh, 10^–7 mol/l norepinephrine, as well as a combination of both, induced marked glucagon secretion, this was suppressed by high blood glucose level. Although simultaneous addition of 10^–6 mol/l ACh and 10^–7 mol/l norepinephrine induced only a slight increase in glucagon secretion in response to glucopenia, there was a significant increase in glucagon secretion in conjunction with an ambient decrease in insulin. Histopathological examination revealed a marked decline in acetylcholinesterase and monoamine-oxidase activities in the islets of streptozotocin-induced diabetic rats. We speculate that reduction of the potentiating effects of ACh and norepinephrine lessens glucose sensitivity of islet beta and alpha cells in this rat model of diabetes.Abbreviations STZ Streptozotocin - STZD streptozotocin-induce diabetic - ACh acetylcholine - AChE acetylcholinesterase - NE norepinephrine - MAO monoamine-oxidase     

Contribution of neural intrapancreatic non-cholinergic non-adrenergic mechanisms to glucose-induced insulin release in the isolated rat pancreas

Summary In the isolated rat pancreas the effect of intrapancreatic non-adrenergic non-cholinergic nerves was examined upon insulin, glucagon and somatostatin release during perturbations of perfusate glucose. Elevation of glucose from 1.6 to 8.3 mmol/l increased insulin and somatostatin secretion and inhibited glucagon release. The first phase of insulin secretion was significantly reduced by the neurotoxin tetrodotoxin to 55% of the controls (p<0.05). The somatostatin response was attenuated by tetrodotoxin while the change of glucagon remained unaffected. In contrast the combined adrenergic and cholinergic blockade with atropine, phentolamine and propranolol (10^–5mol/l) did not modify the insulin, glucagon and somatostatin response. When glucose was changed from 8.3 to 1.6 mmol/l, the reduction of insulin and somatostatin release was not modified by tetrodotoxin, but stimulation of glucagon was significantly attenuated by 60–70% (p<0.03), which was similar to the effect of combined adrenergic and cholinergic blockade. Subsequently, the effect of neural blockade was examined during more physiological perturbations of perfusate glucose levels. When glucose was changed from 3.9 to 7.2 mmol/l, tetrodotoxin also attenuated first phase insulin response by 40% while cholinergic and adrenergic blockade had no effect. The nitric oxide synthase inhibitor N^G-Nitro-l-arginine-methylester (l-NAME) did not alter the glucose-induced insulin response indicating that nitric oxide is not involved in this mechanism. It is concluded that neural non-adrenergic noncholinergic mechanisms contribute to the first, but not second phase of glucose-induced insulin release. Non-adrenergic non-cholinergic effects do not participate in regulation of glucagon and somatostatin secretion under the conditions employed. The non-adrenergic non-cholinergic effect is most likely of peptidergic nature and remains to be examined in greater detail.     

Direct effect of insulin and insulin-like growth factor-I on the secretory activity of rat pancreatic beta cells

Summary Purified pancreatic Beta cells were labelled with ³H-tyrosine before studying their secretory activity in perifusion. At 1.4 mmol/l glucose, the cells released similar fractions (0.01% per min) of their contents in preformed and in newly formed insulin. At 20 mmol/l glucose plus 10^–8 mol/l glucagon, these fractional release rates increased by 16 and 40-fold respectively. The preferential release of newly synthesized as compared to stored insulin is attributable to a heterogeneity in individual cell responses. The secretory responsiveness to glucose plus glucagon was completely suppressed by 10^–7 mol/l clonidine. Insulin induced a 20% reduction at 10^–6 mol/l, but remained without effect at 10^–7 mol/l. Insulin-like growth factor-I provoked a 30% decrease at 5.10^–9 mol/l. It is concluded that the type-I insulin-like growth factor receptors on pancreatic Beta cells mediate a suppressive action on the insulin release process. Their high affinity for insulin-like growth factor-I allows physiologic levels of this peptide to participate in the regulation of insulin release. Their low affinity for insulin provides the basis for a minor feedback action by this hormone at concentrations exceeding the normal circulating levels.Part of this study has been presented at the 25th Annual Meeting of the European Association for the Study of Diabetes, Lisbon, Portugal, 1989     

Inhibition of glucagon release by serotonin in mouse pancreatic islets

Summary In this work we have investigated the effect of serotonin on glucagon release in mouse pancreatic islets isolated by the collagenase technique.Incubation of the islets with serotonin (4 ×10^–3mol/l) was associated with an inhibition of glucagon output both in the basal medium (3.3 mmol/l glucose) and in the presence of arginine (10 mmol/l). The inhibitory effect of serotonin on basal glucagon release was also apparent at concentrations of 2×10^–3 mol/l, 10^–3 mol/l and 5×10^–4 mol/l. Addition of 5-hydroxytryptophan (4 ×10^–3 mol/l) to the incubation medium was without effect on basal glucagon output while it significantly reduced arginine-induced glucagon release. In contrast, tryptophan (4×10^–3 mol/l) provoked glucagon secretion. As inferred from our previous human studies, the present data indicate that serotonin is able to inhibit glucagon secretion. These findings provide further support for the participation of a serotoninergic mechanism in the control of A-cell function.     

Effect of enkephalins and morphine on insulin secretion from isolated rat islets

Summary The direct effects of an enkephalin analogue, (D-Ala²/MePhe⁴/Met/(O)-ol) enkephalin (DAMME), on insulin release from isolated islets of Langerhans of the rat have been investigated. DAMME had a dose-dependent effect on insulin secretion: low concentrations (10^–10 to 10^–8 mol/l) were stimulatory while high concentrations (10^–5mol/l) were inhibitory in the presence of 8 mmol/l glucose. Similar effects were found with met-enkephalin, and with the longer acting alanine substituted metenkephalin. Morphine sulphate (5 sx 10^–7 mol/l) also stimulated insulin release. The effects of enkephalin and morphine were blocked by the specific opiate antagonist naloxone hydrochloride (1.2 × 10^–6 mol/l). The insulin secretory response of perifused islets to enkephalins and morphine was rapid, corresponding to the first phase of glucose induced insulin release. These observations suggest that there may be opiate receptors in islets, and that opioid peptides could modulate insulin release.     

Studies on glucagon secretion using isolated islets of langerhans of the rat

Summary Glucagon secretion and its control have been studied in perifused isolated islets of Langerhans of the rat. It was shown that a low concentration of glucose per se does not cause increased glucagon secretion, but that at low glucose concentrations the amino acid arginine stimulates a biphasic secretory response. Such amino acid stimulated glucagon secretion can be suppressed by increasing the glucose content of the perifused media from 1.67 to 5.5 or 16.7 mM; insulin secretion is also then increased. Since high concentrations of added porcine insulin (10 mU/ml) did not affect amino acid stimulated glucagon secretion at low glucose concentration, it was concluded that high concentrations of glucose and not insulin secreted in response to that glucose are probably responsible for suppression of glucagon secretion. At low concentrations of glucose, epinephrine (2.5 × 10^–7 M) also stimulated glucagon secretion. It is concluded that isolated rat islets of Langerhans can be used for the study of glucagon secretion in vitro, and that substances appearing in the blood in vivo at low glucose concentrations are probably responsible for increased glucagon secretion under conditions associated with hypoglycemia.     

Relative roles of insulin and hypoglycaemia on induction of neuroendocrine responses to,symptoms of,and deterioration of cognitive function in hypoglycaemia in male and female humans

Summary To assess the relative roles of insulin and hypoglycaemia on induction of neuroendocrine responses, symptoms and deterioration of cognitive function (12 cognitive tests) during progressive decreases in plasma glucose, and to quantitate glycaemic thresholds, 22 normal, non-diabetic subjects (11 males, 11 females) were studied on four occasions: prolonged fast (n=8, saline euglycaemia study, SA-EU), stepped hypoglycaemia (plasma glucose plateaus of 4.3, 3.7, 3 and 2.3 mmol/l) or euglycaemia during insulin infusion at 1 and 2 mU·kg^–1·min^–1 (n=22, high-insulin hypoglycaemia and euglycaemia studies, HI-INS-HYPO and HI-INS-EU, respectively), and stepped hypoglycaemia during infusion of insulin at 0.35 mU· kg^–1·min^–1 (n=9, low-insulin hypoglycaemia study, LO-INS-HYPO). Insulin per se (SA-EU vs HI-INS-EU), suppressed plasma glucagon (20%) and pancreatic polypeptide (30%), whereas it increased plasma noradrenaline (R10%, p<0.05). Hypoglycaemia per se (HI-INS-HYPO vs HI-INS-EU) induced responses of counterregulatory hormones (CR-HORM), symptoms and deteriorated cognitive function. With the exception of suppression of endogenous insulin secretion, which had the lowest glycaemic threshold of 4.44±0.06 mmol/l, pancreatic polypeptide, glucagon, growth hormone, adrenaline and cortisol had similar glycaemic thresholds (3.8-3.6 mmol/l); noradrenaline (3.1±0.0 mmol/l), autonomic (3.05±0.06 mmol/l) and neuroglycopenic (3.05±0.05 mmol/l) symptoms had higher thresholds. All 12 tests of cognitive function deteriorated at a glycaemic threshold of 2.45±0.06 mmol/l, but 7 out of 12 tests were already abnormal at a glycaemic threshold of 2.89±0.06 mmol/l. Although all CR-HORM had a similar glycaemic threshold, the lag time of response (the time required for a given parameter to increase) of glucagon (15±1 min) and growth hormone (14±3 min) was shorter than adrenaline (19±3 min) and cortisol (39±4 min) (p<0.05). With the exception of glucagon (which was suppressed) and noradrenaline (which was stimulated), insulin per se (HI-INS-HYPO vs LO-INS-HYPO) did not affect the responses of CR-HORM, and did not influence the symptoms or the cognitve function during hypoglycaemia. Despite lower responses of glucagon, adrenaline and growth hormone (but not thresholds) in females than males, females were less insulin sensitive than males during stepped hypoglycaemia.     

Differential mechanisms of glucose and palmitate in augmentation of insulin secretion in mouse pancreatic islets

Aims/hypothesis. To assess the possible importance of saturated fatty acids in glucose amplification of K⁺ _ATP channel-independent insulin secretion. Methods. Insulin release from perifused pancreatic islets of NMRI mice was determined by radioimmunoassay. Results. In the presence of K⁺ (20 mmol/l) and diazoxide (250 μmol/l), which stimulates Ca²⁺ influx and opens K⁺ _ATP channels, palmitate (165 μmol/l total; 1.2 μmol/l free) increased insulin secretion at 3.3, 10 and 16.7 mmol/l glucose while glucose (10; 16.7 mmol/l) did not increase insulin secretion. In the presence of K⁺ (60 mmol/l) and diazoxide (250 μmol/l), glucose (10; 16.7 mmol/l) stimulation of K⁺ _ATP channel-independent insulin secretion increased, whereas the effectiveness of palmitate (165 μmol/l total; 1.2 μmol/l free) on insulin secretion at both 3.3, 10 or 16.7 mmol/l glucose was reduced. Palmitate thereby mimicked the stimulatory pattern of the protein kinase C activator, 12-O-tetradecanoylphorbol 13-acetate (0.16 μmol/l), which also failed to increase insulin secretion at maximum depolarising concentrations of K⁺ (60 mmol/l). Furthermore, the protein kinase C inhibitor calphostin C (1 μmol/l), led to a complete suppression of the effects of both palmitate (165 μmol/l total; 1.2 μmol/l free) and myristate (165 μmol/l total; 2.4 μmol/l free) stimulation of glucose (16.7 mmol/l)-induced insulin secretion. Calphostin C (1 μmol/l), however, failed to affect insulin secretion induced by glucose (16.7 mmol/l). Conclusion/interpretation. These data suggest that glucose could increase insulin secretion independently of saturated fatty acids like palmitate and myristate, which amplify glucose-induced insulin secretion by activation of protein kinase C. [Diabetologia (2001) 44: 738–746] Received: 30 October 2001 and in revised form: 31 January 2001     

Possible role of endogenous prostaglandins in glucagon secretion by isolated guinea-pig islets

Summary Previous studies have demonstrated that prostaglandins stimulate glucagon secretionin vitro andin vivo. The present work was aimed at investigating the influence of two inhibitors of prostaglandin synthesis, isopropyl-2 nicotinoyl-3 indole (L8027) and indomethacin, on basal and arginine- or noradrenaline-stimulated glucagon release from isolated guinea-pig islets incubated in the absence of glucose. L8027 (10^–4 and 10^–5 mol/l) did not alter basal glucagon release, blocked almost completely the glucagon response to arginine (10^–2 mol/l), had no effect on the glucagon release induced by noradrenaline (10^–4H mol/l), but reduced the stimulatory effect of a lower concentration of noradrenaline (5.10^–7 mol/l). The kinetic study of this inhibitory effect demonstrated that (1) it necessitates preincubation of the islets with L8027 for 30 minutes before the addition of arginine, (2) after a short preincubation period (30 minutes) in the presence of L8027, removal of the inhibitor at the time of arginine stimulation resulted in enhanced glucagon response, (3) on the contrary, after a prolonged incubation period (75 min) with arginine and L8027, the inhibitory effect remained transiently detectable after removal of L8027. Indomethacin similarly blocked arginine- and noradrenaline-induced glucagon secretion. These results suggest that an intra-insular synthesis of prostaglandins is involved in the A cell response to arginine and noradrenaline.     

Effects of islet amyloid polypeptide on hepatic insulin resistance and glucose production in the isolated perfused rat liver

Summary The impact of (pancreatic) islet amyloid polypeptide on glucose metabolism and insulin sensitivity was examined in isolated rat livers perfused in a non-recirculating system. Continuous infusion of 10^–7mol/l islet amyloid polypeptide affected neither basal nor glucagon (10^–9 mol/l)-stimulated glucose output by livers from fed rats, but it did increase the hepatic cyclic AMP release within 44 min (7.91±12.07 vs control: 0.07±0.03 pmol·100 g body weight^–1). The effect of the peptide on the ability of insulin to inhibit glucagon-induced hepatic glycogenolysis was measured in three experimental groups (n = 6). As expected glucagon (7×10^–11 mol/l) increased integral hepatic glucose release within 84 min (763.4±161.7 vs –25.7±73.2 mol · 100 g body weight^–1 in the control group, p<0.001), while insulin (100 mU/l) decreased the glucagon-stimulated glucose production (395.2±180.0 mol·100 g body weight^–1, p<0.01). Simultaneous infusion of 10^–7 mol/l islet amyloid polypeptide however, was not able to reverse insulin-dependent inhibition of glucagon-stimulated hepatic glucose output (370.0±102.5 mol·100 g body weight^–1, NS) or to enhance lactate-induced gluconeogenesis of livers from 24 h fasted rats (n = 8). The glucose production stimulated by 10^–9 mol/l glucagon was slightly greater in islet amyloid polypeptide-pre-treated livers than in a control group without addition of islet amyloid polypeptide (5 min: 3.60±3.36 vs 1.67±1.28 mol·min^–1·100 g body weight^–1). These results suggest that islet amyloid polypeptide neither directly affects hepatic glycogenolysis nor causes insulin resistance to hormone-sensitive glucose production, but may increase the size of the hepatic glycogen pool by enhancing gluconeogenesis.     

Sulphonylurea stimulates glucose uptake in rats through an ATP-sensitive K+ channel dependent mechanism

Summary We studied the effect of gliclazide, a second-generation sulphonylurea, on rat skeletal muscle glucose uptake using perfused hindquarter muscle preparations. Gliclazide at concentrations of 10 to 1000 g/ml increased (p<0.05) the basal glucose uptake. The effect of gliclazide on glucose uptake was immediate and dose-dependent, reaching a plateau at a concentration of 300 g/ml; the half-maximal effect was obtained between 25 and 50 g/ml. The glucose uptake stimulated by gliclazide (300–1000 g/ ml) did not differ from that achieved by 10^–9 mol/l insulin, and was lower (p<0.05) than that obtained with 10^–7 mol/l insulin. The combination of gliclazide (300 g/ml) and 10^–9 mol/l insulin produced an increase in glucose uptake (7.7±0.6 mol · g^–1 · h^–1, n=8, mean±SEM) which was higher (p<0.05) than that achieved with 10^–9 mol/l insulin (5.6±0.7 mol · g^–1 · h^–1, n=11) and not different from that obtained with 10^–7 mol/l insulin (9.8±1.0 mol · g^–1 · h^–1, n=11). Diazoxide (100 mol/l), an ATP-sensitive K⁺ channel opener, reversed the stimulatory effect of gliclazide (100 g/ml) on muscle glucose uptake from 3.1±0.4 to 0.5±0.2 mol · g^–1 · h^–1, (n=7, p<0.001). The addition of diazoxide prior to gliclazide into the perfusion medium blocked the gliclazide-induced glucose uptake by the hindquarter muscle preparations. In conclusion, gliclazide alone has an immediate stimulatory effect on glucose uptake by skeletal muscle and together with insulin has an additive effect on muscle glucose uptake. The effect of gliclazide on muscle glucose uptake seems to be due to the inhibition of ATP-sensitive K⁺ channels.Abbreviations NIDDM Non-insulin-dependent diabetes mellitus - GLUT glucose transporter     

Impaired tyrosine-kinase activity of muscle insulin receptors from hypomagnesaemic rats

Summary The effect of magnesium deficiency on glucose disposal, glucose-stimulated insulin secretion and insulin action on skeletal muscle was investigated in rats which were fed a low magnesium-containing diet for 4 days. Control rats were fed a standard diet. Compared to the control rats, the rats fed with low magnesium diet presented: 1) lower serum magnesium levels (0.45±0.02 vs 0.78±0.01 mmol/l, p<0.001), 2) higher basal serum glucose (6.8±0.2 vs 5.5±0.2 mmol/l, p<0.05) and similar basal serum insulin, 3) 40% reduction (p<0.001) in the glucose disappearance rate after its i.v. administration, and 4) 45% reduction (p<0.05) in the glucose-stimulated insulin secretion. The insulin action upon the glucose uptake by skeletal muscle was determined by means of hindquarter perfusions. Compared with control rats, magnesium-deficient rats presented: 1) normal basal glucose uptake, 2) lower stimulatory effect on the glucose uptake by insulin at the concentrations of 5×10^–10 mol/l (3.0±0.9 vs 5.4±0.6, p<0.05) and 5×10^–9mol/l (6.3±0.5 vs 8.0±0.5, p<0.05), 3) normal glucose uptake at a maximal insulin concentration of 1×10^–7 mol/l, and 4) 50% reduction in the insulin sensitivity (ED₅₀: 1.3±0.3 vs 0.55±0.1 mol/l, p<0.05). In partially purified insulin receptors prepared from gastrocnemius muscle, ¹²⁵I-insulin binding was similar in both groups of rats. However, the autophosphorylation of the -subunit of the insulin receptor was significantly reduced by 50% in magnesium-deficient rats and the tyrosine kinase activity of insulin receptors toward the exogenous substrate Poly Glu4: Tyr 1 was also reduced (p<0.05) by hypomagnesaemia. The abundance of the insulin-sensitive glucose transporter protein (muscle/fat GLUT4), measured by Western blot analysis using polyclonal antisera, was similar in muscles of control and hypomagnesaemic rats. These findings indicate that hypomagnesaemia has a deleterious effect on glucose metabolism due to an impairment of both insulin secretion and action. The insulin resistance observed in skeletal muscle of magnesium-deficient rats may be attributed, at least in part, to a defective tyrosine kinase activity of insulin receptors.Abbreviations WGA Wheat germ agglutinin agarose - Hepes N-2-hydroxyethylpiperazine-N-2-ethane sulphonic acid - PMSF phenylmethylsulphonyl fluoride - TBS Tris buffered saline - IVGTT intravenous glucose tolerance test     

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.     

Reinterpretation of the effect of haloperidol and ethanol on insulin secretion

Summary We were unable to confirm the report of haloperidol induced dose-dependent inhibition of insulin and glucagon release from the isolated canine pancreas. The possibility that the inhibition was caused by ethanol, previously used as the solvent for haloperidol, was tested. Infusion of ethanol at increasing concentrations (15.8 to 252 mmol/l) caused a progressive inhibition of insulin (-17±1 to -69 ±2%) and glucagon (-13±3 to -67±3%) secretion, using a perfusate containing 200 mg/dl glucose and 2.65 mmol/l calcium. Haloperidol (5 to 20 mol/l) dissolved in ethanol (252 mmol/l) did not augment the inhibitory effects of ethanol. At a low calcium concentration (1.3 mmol/l) ethanol further inhibited insulin secretion (-83 ± 2%) with no additional inhibition by 20 mol/l haloperidol (-80±3%). At a high calcium concentration (8.8 mmol/l) the inhibitory effect of ethanol on insulin or glucagon secretions was diminished and variable. This strongly suggests that the inhibition of insulin and glucagon secretion previously attributed to haloperidol was caused by the ethanol solvent.     

Partial recovery of insulin secretion and action after combined insulin-sulfonylurea treatment in Type 2 (non-insulin-dependent) diabetic patients with secondary failure to oral agents

Summary Metabolic control, insulin secretion and insulin action were evaluated in seven Type 2 (non-insulin-dependent) diabetic patients with secondary failure to oral antidiabetic agents before and after two months of combined therapy with supper-time insulin (Ultratard: 0.4 U/kg body weight/day) plus premeal glibenclamide (15 mg/day). Metabolic control was assessed by 24 h plasma glucose, NEFA, and substrate (lactate, alanine, glycerol, ketone bodies) profile. Insulin secretion was evaluated by glucagon stimulation of C-peptide secretion, hyperglycaemic clamp (+7 mmol/l) and 24 h free-insulin and C-peptide profiles. The repeat studies, after two months of combined therapy, were performed at least 72 h after supper-time insulin withdrawal. Combining insulin and sulfonylurea agents resulted in a reduction in fasting plasma glucose (12.9±7 vs 10.4±1.2 mmol/l; p<0.05) and hepaic glucose production (13.9±1.1 vs 11.1±1.1 mol·kgc-min^–1; p<0.05). Mean 24 h plasma glucose was also lower (13.7±1.2 vs 11.1±1.4 mmol/l; p<0.05). Decrements in fasting plasma glucose and mean 24 h profile were correlated (r=0.90; p<0.01). HbA_1c also improved (11.8±0.8 vs 8.9±0.5%; p<0.05). Twenty-four hour profile for NEFA, glycerol, and ketone bodies was lower after teatment, while no difference occurred in the blood lactate and alanine profile. Insulin secretion in response to glucagon (C-peptide =+0.53±0.07 vs +0.43±0.07 pmol/ml) and hyperglycaemia (freeinsulin = 13.1±2.0 vs 12.3±2.2 mU/l) did not change. On the contrary, mean 24 h plasma freeinsulin (13.2±2.6 vs 17.5±2.2 mU/l; p<0.01) and C-peptide (0.76±0.10 vs 0.98±0.13 pmol/l; p<0.02) as well as the area under the curve (19.1±4.1 vs 23.6±3.1 U/24 h;p<0.01 and 1.16±0.14 vs 1.38±0.18 mol/24 h; p<0.02 respectively) were significantly increased. The ratio between glucose infusion (M) and plasma insulin concentration (I) during the hyperglycaemic clamp studies (M/I, an index of insulin sensitivity), was not statistically different (1.40±0.25 vs 1.81±0.40 mol·kg^–1· min^–1/mU·l^–1). These data suggest that, in Type 2 diabetic patients with secondary failure to oral antidiabetic agents, the combination of supper-time longacting insulin and premeal sulfonylurea agents can improve metabolic control. This positive effect is possibly mediated through an increased secretion of insulin in response to physiologic stimuli.     

Inhibition of the high-affinity glucose transporter GLUT 1 affects the sensitivity to glucose in a hamster-derived pancreatic beta cell line (HIT)

Summary HIT is a hamster-derived beta-cell line which in contrast to normal beta cells that only express the high K_m GLUT-2 glucose transporter, also expresses the low K_m glucose transporter GLUT 1. In HIT cells the abnormal glucose transport mechanism is associated with a marked shift to the left of the glucose-induced insulin release dose-response curve. We have used this cell model to investigate whether changes in glucose transport affect the glucose-induced insulin release. HIT cells were first incubated with a concentration of cytochalasin B (0.4 mol/l) that selectively inhibits the GLUT-1 but not the GLUT-2 transporter. The consequences of blocking glucose phosphorylation and insulin release were studied. Exposure to 0.4 mol/l cytochalasin B for 1 h caused a selective loss of the low K_m transport: the calculated V_max of GLUT 1 was reduced from 1726±98 to 184±14 pmol · mg protein^–1 5 min^–1 (mean±SEM, n=6, p<0.005), while no major difference in the high K_m (GLUT-2) transport was observed. In cytochalasin B exposed HIT cells the glucose phosphorylating activity (due to hexokinase and glucokinase) was unaffected. In these cells, however, the dose-response curve of glucose-induced insulin release was significantly shifted to the right: the 50% of maximal response (increment over baseline) was reached at an average glucose concentration of 2.9±0.2 mmol/l (vs 0.6±0.01 mmol/l in control HIT cells mean±SE, n=5, p<0.05) and the maximal effect was reached at 11.0 mmol/l glucose (vs 2.8 mmol/l in control HIT cells p<0.005). These results are consistent with the hypothesis that the affinity of the glucose transport system may contribute to determination of the glucose threshold concentration that triggers insulin secretion.     

Enhancement of glucose transport by insulin at 37 °C in rat adipocytes is accounted for by increased Vmax

Summary The kinetics of 3-O-methyl-D-glucose and D-glucose transport was reinvestigated at 37 °C in rat adipocytes exhibiting very slow transport rates in the absence of insulin and approximately 30-fold insulin stimulation. The apparent maximal velocity for 3-O-methylglucose was increased at least 15-fold from 0.04±0.01 to 0.73±0.17 mmol ·1^–1·s^–1 (SD, n=7). The apparent half saturation constant was decreased by insulin from 8.1±1.6 mmol/l to 3.3±0.8 mmol/l. However, approximately 10% of the 3-O-methylglucose at a very low concentration was transferred by routes other than the glucose transporter, e. g. via nonmediated diffusion or via the fructose transporter, and the estimated value for the half saturation constant was therefore too high in the nonstimulated cells. After appropriate correction this value was estimated as 5.0±1.8 mmol/l. The inhibition constant of 3-O-methylglucose on the conversion of 12 mol/l ¹⁴C-labelled glucose to cell-associated products was about 4 mmol/l and was not changed significantly by insulin. Transport experiments with labelled glucose gave results similar to those obtained for transport of 3-O-methylglucose. It is concluded that insulin can cause a 15- to 30-fold increase in the maximal velocity for transport of 3-O-methylglucose and glucose, and that a concomitant decrease in the half saturation constant, if present, is insignificant.     

Immunoneutralization of endogenous glucagon with monoclonal glucagon antibody normalizes hyperglycaemia in moderately streptozotocin-diabetic rats

Summary The role of glucagon in diabetic hyperglycaemia has been a matter of controversy because of difficulties in the production of selective glucagon deficiency. We developed a high-capacity (40 nmol/ ml), high-affinity (0.6·10¹¹ l/mol) monoclonal glucagon antibody (Glu-mAb) and gave i.v. injections (4 ml/kg) to rats in order to study the effect of selective glucagon deficiency on blood glucose. Controls received a mAb against trinitrophenyl. Glu-mAb completely abolished the hyperglycaemic effect of 2.86 nmol/kg glucagon in normal rats (p<0.05, n=6). In moderately hyperglycaemic rats injected with streptozotocin as neonates (N-STZ), Glu-mAb abolished a postprandial increase in blood glucose (from 11.2±0.7 mmol/l to 17.3±1.8 mmol/l in controls vs 10.5±0.9 mmol/l to 9.3±1.0 mmol/l; cross-over: n=6, p<0.05). No significant effect of Glu-mAb treatment was observed in more hyperglycaemic N-STZ rats (cross-over, n=4) and in severely hyperglycaemic rats injected with STZ as adults (n=6), but after insulin treatment of the latter, at doses partially restoring blood glucose levels (12.7±4.3 mmol/l), Glu-mAb administration almost normalized blood glucose (maximal difference: 6.0±3.8 mmol/l; cross-over: n=5, p<0.05). In conclusion, our results provide strong additional evidence for the hypothesis that glucagon is involved in the pathogenesis of diabetes. The hormone plays an important role in the development of STZ-diabetic hyperglycaemia, but glucagon neutralization only leads to normoglycaemia in the presence of insulin.Abbreviations A-STZ rats Adult streptozotocin injected rats - AUC area under the curve - BW body weight - Con-mAb monoclonal control antibody - Glu-mAb monoclonal anti-glucagon antibody - K_a constant of association - NIDDM non-insulin-dependent diabetes mellitus - N-STZ rats neonatal streptozotocin-injected rats - OGTT oral glucose tolerance test - STZ streptozotocin     

Insulin resistant subjects lack islet adaptation to short-term dexamethasone-induced reduction in insulin sensitivity

Aims/hypothesis. To establish whether islet compensation to deterioration of insulin action depends on inherent insulin sensitivity. Methods. We examined insulin and glucagon secretion after iv arginine (5 g) at fasting, 14 and greater than 25 mmol/l glucose concentrations before and after lowering of insulin sensitivity by oral dexamethasone (3 mg twice daily for 2 1/2 days) in 10 women with normal glucose tolerance, aged 58 or 59 years. Five women had high insulin sensitivity as shown by euglycaemic, hyperinsulinaemic clamp (99 ± 12 nmol glucose · kg body weight^–1· min^–1/pmol insulin · l^–1; means ± SD) whereas five women had low insulin sensitivity (34 ± 15 nmol glucose · kg body weight^–1· min^–1/pmol insulin · l^–1). Results. Dexamethasone reduced insulin sensitivity in both groups. Fasting insulin concentration increased by dexamethasone in high insulin sensitivity (72 ± 10 vs 49 ± 9 pmol/l, p = 0.043) but not in low insulin sensitivity (148 ± 63 vs 145 ± 78 pmol/l) whereas the fasting glucose concentration increased in low insulin sensitivity (6.5 ± 0.8 vs 5.8 ± 0.6 mmol/l, p = 0.043) but not in high insulin sensitivity (5.3 ± 0.8 vs 5.3 ± 0.6 mmol/l). Fasting glucagon concentration was not changed. Plasma insulin concentrations after raising glucose to 14 and more than 25 mmol/l and the insulin response to arginine at more than 25 mmol/l glucose were increased by dexamethasone in high insulin sensitivity (p < 0.05) but not changed by dexamethasone in low insulin sensitivity. Furthermore, in high but not in low insulin sensitivity, dexamethasone reduced the glucagon response to arginine (p = 0.043). Conclusion/interpretation. The results show that adaptation in islets function to dexamethasone-induced short-term reduction in insulin sensitivity is lacking in subjects with low inherent insulin sensitivity. [Diabetologia (1999) 42: 936–943] Received: 26 January 1999 and in revised form: 1 March 1999     

Glucagon secretion in relation to insulin sensitivity in healthy subjects

Aims/hypothesis The study evaluated whether glucagon secretion is regulated by changes in insulin sensitivity under normal conditions. Materials and methods A total of 155 healthy women with NGT (aged 53–70 years) underwent a glucose-dependent arginine-stimulation test for evaluation of glucagon secretion. Arginine (5 g) was injected i.v. under fasting conditions (plasma glucose 4.8±0.1 mmol/l) and after raising blood glucose concentrations to 14.8±0.1 and 29.8±0.2 mmol/l. The acute glucagon response (AGR) to arginine during the three glucose levels (AGR₁, AGR₂, AGR₃) was estimated, as was the suppression of baseline glucagon by the increased glucose. All women also underwent a 2-h euglycaemic–hyperinsulinaemic clamp study for estimation of insulin sensitivity. Results Insulin sensitivity was normally distributed, with a mean of 73.2±29.3 (SD) nmol glucose kg⁻¹ min⁻¹/pmol insulin l⁻¹. When relating the variables obtained from the arginine test to insulin sensitivity, insulin resistance was associated with increased AGR and with increased suppression of glucagon levels by glucose. For example, the regression between insulin sensitivity and AGR₂ was r=−0.38 (p<0.001) and between insulin sensitivity and suppression of glucagon levels by 14.8 mmol/l glucose r=0.36 (p<0.001). Insulin sensitivity also correlated negatively with insulin secretion; multivariate analysis revealed that changes in insulin sensitivity and insulin secretion were independently related to changes in glucagon secretion. Conclusions/interpretation The body adapts to insulin resistance by increasing the glucagon response to arginine and by increasing the suppression of glucagon levels by glucose. Hence, not only the islet beta cells but also the alpha cells seem to undergo compensatory changes during the development of insulin resistance.