Glucose-dependent arginine stimulation test for characterization of islet function: studies on reproducibility and priming effect of arginine

Summary Quantitative determination of insulin secretion is of importance both clinically and in research. The optimal method has not been established, although several different methods have been used. We determined the reproducibility of islet function parameters obtained by the glucose-dependent arginine stimulation test, and also studied the priming effect of arginine on subsequent acute insulin responses. The test measures the acute insulin (AIR) and glucagon (AGR) responses to i. v. arginine (5 g injected over 45 s) at fasting glucose and glucose concentrations clamped at 14 and above 25 mmol/l, as well as the glucose potentiation of insulin secretion (slope_AIR) and the glucose inhibition of glucagon secretion (slope_AGR). When the test was performed twice in seven healthy women (mean ± SD age 58.7 ± 0.5 years, BMI 27.6 ± 5.5 kg/m²), the AIRs to arginine had a within-subject coefficient of variation (CV) of 18.6 % at fasting glucose, 18.7 % at 14 mmol/l glucose and 16.3 % at above 25 mmol/l glucose. The CVs for AGR were 11.6, 14.9 and 8.9 %, respectively. The CV of the slope_AIR was 24 % and of the slope_AGR 17.2 %. The arginine priming study was performed in six healthy women (age 63.7 ± 0.3 years, BMI 28.0 ± 6.9 kg/m²). Saline or arginine (5 g) was injected at fasting glucose, followed by arginine (5 g) at 14 mmol/l glucose. There was no difference between the acute insulin or glucagon responses to arginine at 14 mmol/l glucose in the two conditions, suggesting that there is no priming effect of arginine on the subsequent acute insulin or glucagon responses. Therefore, this method is a good tool to determine insulin secretion as, apart from its good reproducibility, it also provides several important parameters of islet function. [Diabetologia (1998) 41: 772–777] Received: 4 December 1997 and in final revised form: 13 February 1998     

Glucagon receptor antagonism improves islet function in mice with insulin resistance induced by a high-fat diet

Aims/hypothesis Increased glucagon secretion predicts deterioration of glucose tolerance, and high glucagon levels contribute to hyperglycaemia in type 2 diabetes. Inhibition of glucagon action may therefore be a potential novel target to reduce hyperglycaemia. Here, we investigated whether chronic treatment with a glucagon receptor antagonist (GRA) improves islet dysfunction in female mice on a high-fat diet (HFD). Materials and methods After 8 weeks of HFD, mice were treated with a small molecule GRA (300 mg/kg, gavage once daily) for up to 30 days. Insulin secretion was studied after oral and intravenous administration of glucose and glucagon secretion after intravenous arginine. Islet morphology was examined and insulin secretion and glucose oxidation were measured in isolated islets. Results Fasting plasma glucose levels were reduced by GRA (6.0 ± 0.2 vs 7.4 ± 0.5 mmol/l; p = 0.017). The acute insulin response to intravenous glucose was augmented (1,300 ± 110 vs 790 ± 64 pmol/l; p < 0.001). The early insulin response to oral glucose was reduced in mice on HFD + GRA (1,890 ± 160 vs 3,040 ± 420 pmol/l; p = 0.012), but glucose excursions were improved. Intravenous arginine significantly increased the acute glucagon response (129 ± 12 vs 36 ± 6 ng/l in controls; p < 0.01), notably without affecting plasma glucose. GRA caused a modest increase in alpha cell mass, while beta cell mass was similar to that in mice on HFD + vehicle. Isolated islets displayed improved glucose-stimulated insulin secretion after GRA treatment (0.061 ± 0.007 vs 0.030 ± 0.004 pmol islet⁻¹ h⁻¹ at 16.7 mmol/l glucose; p < 0.001), without affecting islet glucose oxidation. Conclusions/interpretation Chronic glucagon receptor antagonism in HFD-fed mice improves islet sensitivity to glucose and increases insulin secretion, suggesting improvement of key defects underlying impaired glucose tolerance and type 2 diabetes.     

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     

Evidence that autonomic mechanisms contribute to the adaptive increase in insulin secretion during dexamethasone-induced insulin resistance in humans

Aims/hypothesis This study examined whether autonomic mechanisms contribute to adaptively increased insulin secretion in insulin-resistant humans, as has been proposed from studies in animals. Methods Insulin secretion was evaluated before and after induction of insulin resistance with or without interruption of neural transmission. Insulin resistance was induced by dexamethasone (15 mg given over 3 days) in nine healthy women (age 67 years, BMI 25.2 ± 3.4 kg/m², fasting glucose 5.1 ± 0.4 mmol/l, fasting insulin 46 ± 6 pmol/l). Insulin secretion was evaluated as the insulin response to intravenous arginine (5 g) injected at fasting glucose and after raising glucose to 13 to15 mmol/l or to >28 mmol/l. Neural transmission across the ganglia was interrupted by infusion of trimethaphan (0.3–0.6 mg kg⁻¹ min⁻¹). Results As an indication of insulin resistance, dexamethasone increased fasting insulin (to 75 ± 8 pmol/l, p < 0.001) without significantly affecting fasting glucose. Arginine-induced insulin secretion was increased by dexamethasone at all glucose levels (by 64 ± 12% at fasting glucose, by 80 ± 19% at 13–15 mmol glucose and by 43 ± 12% at >28 mmol glucose; p <0.001 for all). During dexamethasone-induced insulin resistance, trimethaphan reduced the insulin response to arginine at all three glucose levels. The augmentation of the arginine-induced insulin responses by dexamethasone-induced insulin resistance was reduced by trimethaphan by 48 ± 6% at fasting glucose, 61 ± 8% at 13–15 mmol/l glucose and 62 ± 8% at >28 mmol/l glucose (p < 0.001 for all). In contrast, trimethaphan did not affect insulin secretion before dexamethasone was given. Conclusions/interpretations Autonomic mechanisms contribute to the adaptative increase in insulin secretion in dexamethasone-induced insulin resistance in healthy participants.     

Failure to adequately adapt reduced insulin sensitivity with increased insulin secretion in women with impaired glucose tolerance

Summary To study the islet adaptation to reduced insulin sensitivity in normal and glucose intolerant post-menopausal women, we performed a euglycaemic, hyperinsulinaemic clamp in 108 randomly selected women, aged 58–59 years. Of the 20 women with the lowest insulin sensitivity, 11 had impaired glucose tolerance (IGT) whereas 9 had normal glucose tolerance (NGT). These women together with 15 women with medium insulin sensitivity and 16 women with high insulin sensitivity and NGT were further examined with arginine stimulation at three glucose levels (fasting, 14 and >25 mmol/l). In NGT, the acute insulin response (AIR) to 5 g i. v. arginine at all three glucose levels and the slope_AIR, i. e. the glucose potentiation of insulin secretion, were markedly increased in the women with the lowest insulin sensitivity and NGT compared to those with medium or high insulin sensitivity. In contrast, in low insulin sensitivity, AIR was significantly lower in IGT than in NGT (at glucose 14 mmol/l p=0.015, and at >25 mmol/l p=0.048). The potentiation of AIR induced by low insulin sensitivity in women with NGT was reduced by 74% (AIR at 14 mmol/l glucose) and 57% (AIR at >25 mmol/l glucose), respectively, in women with IGT. Also the slope_AIR was lower in IGT than in NGT (p=0.025); the increase in slope_AIR due to low insulin sensitivity was abolished in IGT. In contrast, glucagon secretion was not different between women with IGT as opposed to NGT. We conclude that as long as there is an adequate beta-cell adaptation to low insulin sensitivity with increased insulin secretory capacity and glucose potentiation of insulin secretion, NGT persists.Abbreviations NIDDM Non-insulin-dependent diabetes mellitus - AIR acute insulin response - AGR acute glucagon response     

Near normalisation of blood glucose improves the potentiating effect of GLP-1 on glucose-induced insulin secretion in patients with type 2 diabetes

Aims/hypothesis The ability of glucagon-like peptide-1 (GLP-1) to enhance beta cell responsiveness to i.v. glucose is impaired in patients with type 2 diabetes mellitus compared with healthy individuals. We investigated whether 4 weeks of near normalisation of blood glucose (BG) improves the potentiation of glucose-stimulated insulin secretion by GLP-1. Methods Nine obese patients with type 2 diabetes and inadequate glycaemic control (HbA_1c 8.0 ± 0.4%) were investigated before and after 4 weeks of near normalisation of BG using insulin treatment (mean diurnal blood glucose 6.4 ± 0.3 mmol/l, HbA_1c 6.6 ± 0.3%). Nine matched healthy participants were also studied. Beta cell function was investigated before and after insulin treatment using stepwise glucose infusions and infusion of saline or GLP-1 (1.0 pmol kg⁻¹ min⁻¹), resulting in supraphysiological total GLP-1 concentrations of approximately 200 pmol/l. The responsiveness to glucose or glucose+GLP-1 was expressed as the slope of the linear regression line relating insulin secretion rate (ISR) and plasma glucose concentration (pmol kg⁻¹ min⁻¹ [mmol/l]⁻¹). Results In the diabetic participants, the slopes during glucose+saline infusion did not differ before and after insulin treatment (0.33 ± 0.07 and 0.39 ± 0.04, respectively; p = NS). In contrast, near normalisation of blood glucose improved beta cell sensitivity to glucose during glucose+GLP-1 infusion (1.27 ± 0.2 before vs 1.73 ± 0.31 after; p < 0.01). In the healthy participants, the slopes during the glucose+saline and glucose+GLP-1 infusions were 1.01 ± 0.14 and 4.79 ± 0.53, respectively. Conclusions/interpretation A supraphysiological dose of GLP-1 enhances beta cell responses to glucose in patients with type 2 diabetes, and 4 weeks of near normalisation of blood glucose further improves this effect. ClinicalTrials.gov ID no.: NCT00612625     

Inappropriate suppression of glucagon during OGTT but not during isoglycaemic i.v. glucose infusion contributes to the reduced incretin effect in type 2 diabetes mellitus

Aims/hypothesis We investigated glucagon responses during OGTT and isoglycaemic i.v. glucose infusion, respectively, to further elucidate the mechanisms behind the glucose intolerance in patients with type 2 diabetes. Materials and methods Ten patients (eight men) with type 2 diabetes (age: 64 [51–80] years; BMI: 23 [21–26] kg/m²; HbA_1c: 6.9 [6.2–8.7]%, values mean [range]) and ten control subjects matched for sex, age and BMI were studied. Blood was sampled on two separate days following a 4-h 50-g OGTT and an isoglycaemic i.v. glucose infusion, respectively. Results Isoglycaemia during the 2 days was obtained in both groups. In the control subjects no difference in glucagon suppression during the first 45 min of OGTT and isoglycaemic i.v. glucose infusion (−36 ± 12 vs −64 ± 23 mmol/l × 45 min; p = NS) was observed, whereas in the group of patients with type 2 diabetes significant glucagon suppression only occurred following isoglycaemic i.v. glucose infusion (−63 ± 21 vs 10 ± 16 mmol/l × 45 min; p = 0.002). The incretin effect was significantly reduced in patients with type 2 diabetes compared with control subjects, but no significant differences in the secretion of glucagon-like peptide-1 or glucose-dependent insulinotropic polypeptide between the two groups during OGTT or isoglycaemic i.v. glucose infusion, respectively, could explain this. Conclusions/interpretation Attenuated and delayed glucagon suppression in patients with type 2 diabetes occurs after oral ingestion of glucose, while isoglycaemic i.v. administration of glucose results in normal suppression of glucagon. We suggest that this phenomenon contributes both to the glucose intolerance and to the reduced incretin effect observed in patients with type 2 diabetes.     

The effects of epinephrine on islet hormone secretion in the dog

Summary We investigated the direct effects of physiological levels of epinephrine on the basal and arginine-stimulated secretion of insulin, glucagon, and somatostatin from the in situ pancreas in halothane-anaesthetized dogs. An IV infusion of 20 ng/kg/min of epinephrine increased plasma epinephrine levels to 918±103 pg/ml (P<0.001), and increased the baseline pancreatic output of insulin (P<0.05), glucagon (P<0.05) and somatostatin (P<0.05). The acute insulin response (AIR) to 2.5 g of arginine during this infusion of epinephrine was significantly higher (P<0.05) than in controls as were the acute glucagon response (AGR) (P<0.05) and the acute somatostatin response (ASLIR) (P<0.05). Plasma glucose levels increased slightly and transiently during infusion of epinephrine from 99±2 mg/dl to a maximum of 110±3 mg/dl (P<0.05). An IV infusion of 80 ng/kg/min of epinephrine produced plasma epinephrine levels of 2948±281 pg/ml, and increased the baseline pancreatic output of insulin (P<0.05) and glucagon (P<0.05). In contrast, baseline somatostatin output decreased transiently during this high dose infusion of epinephrine. The AIR and ASLIR to arginine were both significantly lower (P<0.05) than those during the infusion of epinephrine at the low dose. The AGR to arginine remained potentiated (P<0.05). Plasma glucose levels increased from 99±3 mg/dl to 119±4 mg/dl (P<0.01). We conclude that the effect of epinephrine on islet hormone secretion is dependent on the plasma level of epinephrine. At stress levels of 900–1000 pg/ml, both insulin and somatostatin secretion are stimulated; only at near pharmacologic, or extreme stress levels, does epinephrine produce net inhibition.     

Impaired glucose tolerance (IGT) is associated with reduced insulin-induced suppression of glucagon concentrations

Aims/hypothesis: We aimed to examine whether impaired glucose tolerance is associated with reduced suppression of glucagon concentrations. Methods: Eighty-four non-diabetic women of Caucasian origin and 61 years of age, of whom 48 had normal glucose tolerance (NGT) and 36 had IGT, underwent a 75 g OGTT and a hyperinsulinaemic, euglycaemic clamp with measurement of glucagon, insulin and glucose concentrations. Results: At 2 h after 75 g oral glucose, glucagon concentrations were reduced by 7.1 ± 1.1 ng/l in NGT vs 8.0 ± 1.4 ng/l in IGT, (NS). However, the 2 h reductions in glucagon per mmol/l increase in 2 h glucose or per pmol/l increase in 2 h insulin were both impaired in IGT (p = 0.002 and p = 0.043, respectively) because the 2 h increases in glucose and insulin were higher in IGT than in NGT. Furthermore, suppression of glucagon concentrations during a euglycaemic clamp at hyperinsulinaemic concentrations (NGT: 607 ± 19 pmol/l, IGT: 561 ± 21 pmol/l) was lower in IGT (13.6 ± 1.6 ng/l) than in NGT (23.1 ± 1.2 ng/l; p < 0.001). The suppression of glucagon concentrations during the hyperinsulinaemic, euglycaemic clamp correlated with insulin sensitivity (r = 0.24, p = 0.027) and with the 2 h glucose value during the OGTT (r = –0.52, p < 0.001). Conclusion/interpretation: Impaired glucose tolerance is associated with reduced insulin-induced suppression of glucagon secretion, which could be caused by A-cell insulin resistance. Inappropriately high glucagon secretion could therefore contribute to the metabolic perturbations in IGT. [Diabetologia (2001) 44: 1998–2003] Received: 15 May 2001 and in revised form: 13 July 2001     

Influence of gestational diabetes on the long-term control of glucose tolerance

Aims/hypothesis Gestational diabetes (GDM) carries a high risk of subsequent diabetes. We asked what impact prior GDM has on beta cell function and insulin action in women who maintain normal glucose tolerance (NGT) for a long time. Methods Ninety-one women with NGT (aged 41 ± 8 years, mean±SD) were studied (by mathematical modelling of the C-peptide response to an OGTT) 7 [6] years (median [interquartile range]) after the index pregnancy, during which 52 had GDM (pGDM) and 39 had NGT (pNGT). In all women an OGTT had also been performed at 29 ± 3 weeks of the index pregnancy. Results Women with pGDM were matched with women with pNGT for age, familial diabetes, time and weight gain since index pregnancy, parity, BMI (25.4 ± 3.9 vs 26.8 ± 6.4 kg/m²), and fasting (4.64 ± 0.56 vs 4.97 ± 0.46 mmol/l) and 2 h plasma glucose levels (5.91 ± 1.14 vs 5.91 ± 1.21 mmol/l). Nonetheless, fasting (49 [29] vs 70 [45] pmol min⁻¹ m⁻², p < 0.001) and total insulin secretion (32 [17] vs 48 [21] nmol m⁻², p < 0.0001) and beta cell glucose sensitivity (slope of the insulin secretion/plasma glucose concentration–response function) (95 [71] vs 115 [79] pmol min⁻¹ m⁻² (mmol/l)⁻¹, p = 0.025) were reduced in the pGDM group compared with the pNGT group, while insulin sensitivity was preserved (424 [98] vs 398 [77] ml min⁻¹ m⁻²). At index pregnancy, women with pGDM and those with pNGT had similar age and BMI. However, both insulin sensitivity (359 [93] vs 417 [92] ml min⁻¹ m⁻², p = 0.0012) and the insulin/glucose incremental area ratio (an empirical index of beta cell function; 98 [74] vs 138 [122] pmol/mmol, p = 0.028) were reduced in women with pGDM. Conclusions Even in women who maintain normal insulin sensitivity, impaired beta cell function is carried over into the NGT status several years after a GDM pregnancy.     

Reduced intrahepatic fat content is associated with increased whole-body lipid oxidation in patients with type 1 diabetes

Aims/hypothesis Insulin resistance may be associated with ectopic fat accumulation potentially determined by reduced lipid oxidation. In patients with type 1 diabetes peripheral insulin resistance is associated with higher intramyocellular lipid content. We assessed whether these patients are also characterised by intrahepatic fat accumulation and abnormal fat oxidation. Methods Nineteen patients with type 1 diabetes (6 women, 13 men, age 35±7 years, BMI 23±3 kg/m², HbA₁c 8.7±1.4%) and 19 healthy matched individuals were studied by (1) euglycaemic–hyperinsulinaemic clamp combined with [6,6−²H₂]glucose infusion to assess whole–body glucose metabolism; (2) indirect calorimetry to assess glucose and lipid oxidation; and (3) localised ¹H−magnetic resonance spectroscopy of the liver to assess intrahepatic fat content. Results Patients with type 1 diabetes showed a reduced insulin−stimulated metabolic clearance rate of glucose (4.3±1.3 ml kg⁻¹ min⁻¹) in comparison with normal subjects (6.0±1.6 ml kg⁻¹ min⁻¹; p<0.001). Endogenous glucose production was higher in diabetic patients (p=0.001) and its suppression was impaired during insulin administration (66±30 vs 92±8%; p=0.047) in comparison with normal subjects. Plasma glucagon concentrations were not different between groups. The estimated hepatic insulin concentration was lower in diabetic patients than in normal subjects (p<0.05), as was the intrahepatic fat content (1.5±0.7% and 2.2±1.0% respectively; p<0.03), the latter in association with a reduced respiratory quotient (0.74±0.05 vs 0.84±0.06; p=0.01) and increased fasting lipid oxidation (1.5±0.5 vs 0.8±0.4 mg kg⁻¹ min⁻¹; p<0.01). Conclusions/interpretation In patients with type 1 diabetes, insulin resistance was not associated with increased intrahepatic fat accumulation. In fact, diabetic patients had reduced intrahepatic fat content, which was associated with increased fasting lipid oxidation. The unbalanced hepatic glucagon and insulin concentrations affecting patients with type 1 diabetes may be involved in this abnormality of intrahepatic lipid metabolism.     

Copper addition prevents the inhibitory effects of interleukin 1-β on rat pancreatic islets

Summary Since copper [Cu(II)] is a necessary cofactor for both intra-mitochondrial enzymes involved in energy production and hydroxyl scavenger enzymes, two hypothesised mechanisms for action of interleukin-Iβ (IL-1β), we studied whether CU(II) addition could prevent the inhibitory effect of IL-1β on insulin release and glucose oxidation in rat pancreatic islets. Islets were incubated with or without 50 U/ml IL-1β, in the presence or absence of various concentrations of Cu(II)-GHL (Cu(II) complexed with glycyl-l-histidyl-l-lysine, a tripeptide known to enhance copper uptake into cultured cells). CuSO₄ (1–1000 ng/ml) was used as a control for Cu(II) effect when present as an inorganic salt. At the end of the incubation period, insulin secretion was evaluated in the presence of either 2.8 mmol/l (basal insulin secretion) or 16.7 mmol/l glucose (glucose-induced release). In control islets basal insulin secretion was 92.0±11.4 pg · islet⁻¹ h⁻¹ (mean ± SEM,n=7) and glucose-induced release was 2824.0±249.0 pg · islet⁻¹ h⁻¹. In islets pre-exposed to 50 U/ml IL-1β, basal insulin release was not significantly affected but glucose-induced insulin release was greatly reduced (841.2±76.9,n=7,p<0.005). In islets incubated with IL-1β and Cu-GHL (0.4 μmol/l, maximal effect) basal secretion was 119.0±13.1 pg · islet⁻¹ h⁻¹ and glucose-induced release was 2797.2±242.2, (n=7,p<0.01 in respect to islets exposed to IL-1β alone). In contrast to data obtained with Cu(II)-GHL, increasing concentrations of CuSO4 (up to 10 μmol/l) did not influence the inhibitory effect of IL-1β on glucose-stimulated insulin release. Glucose oxidation (in the presence of 16.7 mmol/l glucose) was 31.5±2.4 pmol · islet⁻¹·90min⁻¹ in control islets and 7.0±0.9 (p<0.01) in IL-1β-exposed islets. In islets exposed to IL-1β and Cu-GHL glucose oxidation was similar to control islets (31.9±1.9). In contrast, Cu-GHL did not prevent the IL-1β-induced increase in nitric oxide production. Nitrite levels were 5±1.7, 26±5 and to 29±4 pmol · islet⁻¹·48 h⁻¹ (mean ± SEM,n=5) in the culture medium from control IL-1β and IL-1β+Cu-GHL exposed islets, respectively. These data indicate that the Cu(II) complexed to GHL is able to prevent the inhibitory effects of IL-1β on insulin secretion and glucose oxidation, but not on NO production. The mechanism of action of Cu-GHL is still unclear, but it might restore the activity of the enzymatic systems inhibited by IL-1β. [Diabetologia (1995) 38∶39–45]     

Evidence for direct action of alloxan to induce insulin resistance at the cellular level

Summary To determine whether long-term insulin deficiency alters insulin movement across the endothelium, plasma and lymph dynamics were assessed in dogs after alloxan (50 mg/kg; n = 8) or saline injection (n = 6). Glucose tolerance (K_G) and acute insulin response were assessed by glucose injection before and 18 days after treatment. Two days later, hyperglycaemic (16.7 mmol/l) hyperinsulinaemic (60 pmol · min⁻¹· kg⁻¹) glucose clamps were carried out in a subset of dogs (n = 5 for each group), with simultaneous sampling of arterial blood and hindlimb lymph. Alloxan induced fasting hyperglycaemia (12.9 ± 2.3 vs 5.7 ± 0.2 mmol/l; p = 0.018 vs pre-treatment) and variable insulinopenia (62 ± 14 vs 107 ± 19 pmol/l; p = 0.079). The acute insulin response, however, was suppressed by alloxan (integrated insulin from 0–10 min: 155 ± 113 vs 2745 ± 541 pmol · l⁻¹· 10 min⁻¹; p = 0.0027), resulting in pronounced glucose intolerance (K_G: 0.99 ± 0.19 vs 3.14 ± 0.38 min⁻¹; p = 0.0002 vs dogs treated with saline). During clamps, steady state arterial insulin was higher in dogs treated with alloxan (688 ± 60 vs 502 ± 38 pmol/l; p = 0.023) due to a 25 % reduction in insulin clearance (p = 0.045). Lymph insulin concentrations were also raised (361 ± 15 vs 266 ± 27 pmol/l; p = 0.023), such that the lymph to arterial ratio was unchanged by alloxan (0.539 ± 0.022 vs 0.533 ± 0.033; p = 0.87). Despite higher lymph insulin, glucose uptake (R_d) was significantly diminished after injection of alloxan (45.4 ± 2.5 vs 64.3 ± 6.5 μmol · min⁻¹· kg⁻¹; p = 0.042). This was reflected in resistance of target tissues to the lymph insulin signal (ΔR_d/Δlymph insulin: 3.389 ± 1.093 vs 11.635 ± 2.057 · 10⁻⁶· l · min⁻¹· kg^–1· pmol⁻¹· l⁻¹; p = 0.012) which correlated strongly with the K_G (r = 0.86; p = 0.0001). In conclusion, alloxan induces insulinopenic diabetes, with glucose intolerance and insulin resistance at the target tissue level. Alloxan treatment, however, does not alter lymph insulin kinetics, indicating that insulin resistance of Type 1 (insulin-dependent) diabetes mellitus reflects direct impairment at the cellular level. [Diabetologia (1998) 41: 1327–1336] Received: 3 November 1997 and in final revised form: 2 June 1998     

Assessment of insulin sensitivity and secretion with the labelled intravenous glucose tolerance test: improved modelling analysis

Summary A new modelling analysis was developed to assess insulin sensitivity with a tracer-modified intravenous glucose tolerance test (IVGTT). IVGTTs were performed in 5 normal (NGT) and 7 non-insulin-dependent diabetic (NIDDM) subjects. A 300 mg/kg glucose bolus containing [6,6-²H₂]glucose was given at time 0. After 20 min, insulin was infused for 5 min (NGT, 0.03; NIDDM, 0.05 U/kg). Concentrations of tracer, glucose, insulin and C-peptide were measured for 240 min. A circulatory model for glucose kinetics was used. Glucose clearance was assumed to depend linearly on plasma insulin concentration delayed. Model parameters were: basal glucose clearance (Cl_b), glucose clearance at 600 pmol/l insulin concentration (Cl₆₀₀), basal glucose production (P_b), basal insulin sensitivity index (BSI = Cl_b/basal insulin concentration); incremental insulin sensitivity index (ISI = slope of the relationship between insulin concentration and glucose clearance). Insulin secretion was calculated by deconvolution of C-peptide data. Indices of basal pancreatic sensitivity (PSI_b) and first (PSI₁) and second-phase (PSI₂) sensitivity were calculated by normalizing insulin secretion to the prevailing glucose levels. Diabetic subjects were found to be insulin resistant (BSI: 2.3 ± 0.6 vs 0.76 ± 0.18 ml · min^–1· m^–2· pmol/l^–1, p < 0.02; ISI: 0.40 ± 0.06 vs 0.13 ± 0.05 ml · min^–1· m^–2· pmol/l^–1, p < 0.02; Cl₆₀₀: 333 ± 47 vs 137 ± 26 ml · min^–1· m^–2, p < 0.01; NGT vs NIDDM). P_b was not elevated in NIDDM (588 ± 169 vs 606 ± 123 μmol · min^–1· m^–2, NGT vs NIDDM). Hepatic insulin resistance was however present as basal glucose and insulin were higher. PSI₁ was impaired in NIDDM (67 ± 15 vs 12 ± 7 pmol · min^–1· m^–2· mmol/l^–1, p < 0.02; NGT vs NIDDM). In NGT and in a subset of NIDDM subjects (n = 4), PSI_b was inversely correlated with BSI (r = 0.95, p < 0.0001, log transformation). This suggests the existence of a compensatory mechanism that increases pancreatic sensitivity in the presence of insulin resistance, which is normal in some NIDDM subjects and impaired in others. In conclusion, using a simple test the present analysis provides a rich set of parameters characterizing glucose metabolism and insulin secretion, agrees with the literature, and provides some new information on the relationship between insulin sensitivity and secretion. [Diabetologia (1998) 41: 1029–1039] Received: 17 September 1997 and in final revised form: 28 April 1998     

Effect of the amino acid alanine on glucagon secretion in non-diabetic and type 1 diabetic subjects during hyperinsulinaemic euglycaemia,hypoglycaemia and post-hypoglycaemic hyperglycaemia

Aims/hypothesis The aim of our study was to establish whether the well-known defective or absent secretion of glucagon in type 1 diabetes in response to hypoglycaemia is selective or includes lack of responses to other stimuli, such as amino acids. Materials and methods Responses of glucagon to hypoglycaemia were measured in eight patients with type 1 diabetes and six non-diabetic subjects during hyperinsulinaemic (insulin infusion 0.5 mU kg⁻¹ min⁻¹) and eu-, hypo- and hyperglycaemic clamp studies (sequential steps of plasma glucose 5.0, 2.9, 5.0, 10 mmol/l). Subjects were studied on three randomised occasions with infusion of low- or high-dose alanine, or saline. Results With saline, glucagon increased in hypoglycaemia in non-diabetic subjects but not in diabetic subjects. Glucagon increased further with low-dose (181 ± 16 ng l⁻¹ min⁻¹) and high-dose alanine (238 ± 20 ng l⁻¹ min⁻¹) in non-diabetic subjects, but only with high-dose alanine in diabetic subjects (area under curve 112 ± 5 ng l⁻¹ min⁻¹). The alanine-induced glucagon increase in diabetic subjects paralleled the spontaneous glucagon response to hypoglycaemia in non-diabetic subjects not receiving alanine. The greater responses of glucagon to hypoglycaemia with alanine infusion were offset by recovery of eu- or hyperglycaemia. Conclusions/interpretation In type 1 diabetes, the usually deficient responses of glucagon to hypoglycaemia may improve after increasing the concentration of plasma amino acids. Amino acid-enhanced secretion of glucagon in response to hypoglycaemia remains under physiological control since it is regulated primarily by the ambient plasma glucose concentration. These findings might be relevant to improving counter-regulatory defences against insulin-induced hypoglycaemia in type 1 diabetes.     

Niflumic acid-sensitive ion channels play an important role in the induction of glucose-stimulated insulin secretion by cyclic AMP in mice

Aims/hypothesis  We have previously reported that glucose-stimulated insulin secretion (GSIS) is induced by glucagon-like peptide-1 (GLP-1) in mice lacking ATP-sensitive K⁺ (K_ATP) channels (Kir6.2 ^−/− mice [up-to-date symbol for Kir6.2 gene is Kcnj11]), in which glucose alone does not trigger insulin secretion. This study aimed to clarify the mechanism involved in the induction of GSIS by GLP-1. Methods  Pancreas perfusion experiments were performed using wild-type (Kir6.2 ^+/+ ) or Kir6.2 ^−/− mice. Glucose concentrations were either changed abruptly from 2.8 to 16.7 mmol/l or increased stepwise (1.4 mmol/l per step) from 2.8 to 12.5 mmol/l. Electrophysiological experiments were performed using pancreatic beta cells isolated from Kir6.2 ^−/− mice or clonal pancreatic beta cells (MIN6 cells) after pharmacologically inhibiting their K_ATP channels with glibenclamide. Results  The combination of cyclic AMP plus 16.7 mmol/l glucose evoked insulin secretion in Kir6.2 ^−/− pancreases where glucose alone was ineffective as a secretagogue. The secretion was blocked by the application of niflumic acid. In K_ATP channel-inactivated MIN6 cells, niflumic acid similarly inhibited the membrane depolarisation caused by cAMP plus glucose. Surprisingly, stepwise increases of glucose concentration triggered insulin secretion only in the presence of cAMP or GLP-1 in Kir6.2 ^+/+ , as in Kir6.2 ^−/− pancreases. Conclusions/interpretation  Niflumic acid-sensitive ion channels participate in the induction of GSIS by cyclic AMP in Kir6.2 ^−/− beta cells. Cyclic AMP thus not only acts as a potentiator of insulin secretion, but appears to be permissive for GSIS via novel, niflumic acid-sensitive ion channels. This mechanism may be physiologically important for triggering insulin secretion when the plasma glucose concentration increases gradually rather than abruptly.     

Vildagliptin therapy reduces postprandial intestinal triglyceride-rich lipoprotein particles in patients with type 2 diabetes

Aims/hypothesis We assessed the effects of vildagliptin, a novel dipeptidyl peptidase IV inhibitor, on postprandial lipid and lipoprotein metabolism in patients with type 2 diabetes.Subjects, materials and methods This was a single-centre, randomised, double-blind study in drug-naive patients with type 2 diabetes. Patients received vildagliptin (50 mg twice daily, n=15) or placebo (n=16) for 4 weeks. Triglyceride, cholesterol, lipoprotein, glucose, insulin, glucagon and glucagon-like peptide-1 (GLP-1) responses to a fat-rich mixed meal were determined for 8 h postprandially before and after 4 weeks of treatment.Results Relative to placebo, 4 weeks of treatment with vildagliptin decreased the AUC_0–8h for total trigyceride by 22±11% (p=0.037), the incremental AUC_0–8h (IAUC_0–8h) for total triglyceride by 85±47% (p=0.065), the AUC_0–8h for chylomicron triglyceride by 65±19% (p=0.001) and the IAUC_0–8h for chylomicron triglyceride by 91±28% (p=0.002). This was associated with a decrease in chylomicron apolipoprotein B-48 (AUC_0–8h, −1.0±0.5 mg l⁻¹ h, p=0.037) and chylomicron cholesterol (AUC_0–8h, −0.14±0.07 mmol l⁻¹ h, p=0.046). Consistent with previous studies, 4 weeks of treatment with vildagliptin also increased intact GLP-1, suppressed inappropriate glucagon secretion, decreased fasting and postprandial glucose, and decreased HbA_1c from a baseline of 6.7% (change, −0.4±0.1%, p<0.001), all relative to placebo.Conclusions/interpretation Treatment with vildagliptin for 4 weeks improves postprandial plasma triglyceride and apolipoprotein B-48-containing triglyceride-rich lipoprotein particle metabolism after a fat-rich meal. The mechanisms underlying the effects of this dipeptidyl peptidase IV inhibitor on postprandial lipid metabolism remain to be explored.     

Glucoregulatory function of glucagon in hypo-, eu- and hyperthyroid miniature pigs

Summary The glucoregulatory function of glucagon was investigated in hypo-, eu- and hyperthyroid miniature pigs. Infusion glucagon, (3 ng x kg body weight⁻¹ · min⁻¹) transiently increased blood glucose (p<0.01) and hepatic glucose production (p<0.01) in euthyroidism, but was without effect in hyperthyroidism. Infusing glucagon plus somatostatin (2 ng x kg body weight⁻¹ · min⁻¹ and 0.2 μg x kg body weight⁻¹ · min⁻¹) transiently increased blood glucose (Δ 3.0 to 4.3 mmol/l) and hepatic glucose production (Δ 3.3 to 7.7 umol x kg body weight⁻¹ · min⁻¹) in all thyroid states, the effect was less pronounced in hyperthyroid pigs. By contrast, hypoglucagonaemia (74 to 107 pg/ml) at basal insulin (28 to 35 μU/ml) provoked hypoglycaemia (1.4 to 2.2 mmol/l) and a fall in glucose production (Δ 4.7 to 8.3 umol x kg body weight⁻¹ · min⁻¹), which was independent of the thyroid state; the effect was most pronounced in hyperthyroidism (p<0.01). Hepatic glycogen content, arterial gluconeogenic precursor concentrations as well as the glycaemic response (Δ 0.60 mmol/l) to alanine infusion (23 umol x kg body weight⁻¹ · min⁻¹) were all unaffected by hyperthyroidism. We conclude that moderate experimental hyperthyroidism reduces glucagon action due to reduced glycogen mobilisation. This may in part result from increased insulin sensitivity.     

Glucose intolerance is predicted by low insulin secretion and high glucagon secretion: outcome of a prospective study in postmenopausal Caucasian women

Aims/hypothesis. To study the pathophysiological importance of changes in insulin sensitivity and islet function over time for alterations in glucose tolerance in a randomly selected large group of non-diabetic women aged 57–59 years over a 3-year period.¶Methods. At baseline and at the 3-year follow-up, glucose tolerance (WHO 75 g oral glucose), insulin sensitivity (euglycaemic, hyperinsulinaemic clamp) and insulin and glucagon secretion (2 to 5-min responses to 5 g i. v. arginine at fasting, 14 and > 25 mmol/l glucose) were measured.¶Results. At baseline, women with impaired glucose tolerance (IGT, n = 28) had lower insulin sensitivity (p = 0.048) than normal women (NGT, n = 58). The arginine-induced insulin responses (AIR) were inversely associated with insulin sensitivity (r≥– 0.55, p < 0.001). When related to the 3-year follow-up, the baseline product of AIR at 14 mmol/l glucose times insulin sensitivity, insulin effect index (IE) (r = – 0.40, p < 0.001) and the arginine-induced glucagon response at 14 mmol/l glucose (AGR, r = 0.28, p = 0.009) both correlated with follow-up 2-h glucose. In a multiple regression model, baseline 2-h glucose, insulin effect index and arginine-induced glucagon response independently predicted 2-h glucose at follow-up (total r = 0.668, p < 0.001). Furthermore, Δinsulin sensitivity (i. e. follow-up minus baseline) correlated with Δinsulin secretion (r = – 0.30, p = 0.006), whereas Δglucagon secretion correlated with Δ2-h glucose (r = 0.30, p = 0.006) over the 3 years. In a multiple regression, alterations in 2-h glucose over the 3 years were independently determined by changes in fasting insulin and glucagon secretion (r = 0.424, p < 0.001).¶Conclusion/interpretation. Low insulin secretion, when judged in relation to insulin sensitivity, and high glucagon secretion, determine glucose tolerance over time in the individual subject. These processes are therefore potential targets for prevention of deterioration in glucose tolerance. [Diabetologia (2000) 43: 194–202]     

Evidence that insulin can directly inhibit hepatic glucose production

Summary In order to evaluate the role of portal insulin in the modulation of hepatic glucose production (HGP), measurements of plasma glucose and insulin concentrations and both HGP and peripheral glucose disappearance rates were made following an infusion of a dose of tolbutamide (0.74 mg · m⁻²· min⁻¹) in healthy volunteers that does not result in an increase in peripheral vein insulin concentrations or metabolic clearance rate of glucose. The results showed that the infusion of such a dose of tolbutamide was associated with a significant and rapid decline in both HGP (from 9.0 ± 0.5 to 7.7 ± 0.5 μmol · kg⁻¹· min⁻¹ or Δ = − 13.8 ± 4.5 %; p < 0.001 compared to saline) and plasma glucose concentration (from 5.1 ± 0.2 to 4.4 ± 0.1 mmol/l or Δ = − 13.0 ± 2.1 %; p < 0.01 compared to saline). Since neither HGP nor fasting glucose fell when tolbutamide-stimulated insulin secretion was inhibited by the concurrent administration of somatostatin, it indicated that tolbutamide by itself, does not directly inhibit HGP. Finally, HGP fell by 26.3 ± 6.0 % at 10 min after a dose of tolbutamide that elevated both peripheral and portal insulin concentrations, at a time when HGP had barely increased (Δ = + 6.9 ± 5.3 %). The difference in the magnitude of the two responses was statistically significant (p < 0.03), providing further support for the view that insulin can directly inhibit HGP, independent of any change in flow of substrates from periphery to liver. [Diabetologia (1997) 40: 1300–1306] Received: 8 April 1997 and in revised form: 20 June 1997