Pharmacokinetic,insulinotropic, and glucagonostatic properties of GLP-1 [7–36 amide] after subcutaneous injection in healthy volunteers. Dose-response-relationships

Summary Intravenous infusions of glucagon-like peptide 1 (GLP-1) [7–36 amide] are glucose-dependently insulinotropic and glucagonostatic and normalize plasma glucose concentrations in non-insulin-dependent diabetic patients. It was the aim of this study to investigate whether subcutaneous GLP-1 [7–36 amide] also has an influence on insulin and glucagon secretion, and which doses are required for significant effects. Therefore, eight healthy volunteers (24±2 years, body mass index [BMI] 21.9±2.3 kg/ m²) were studied in the fasting state on five occasions in randomized order. Placebo (0.9% NaCl with 1% human serum albumin) or GLP-1 [7–36 amide] in doses of 0.15, 0.5, 1.5 or 4.5 nmol/kg body weight (volume 1 ml or, at the highest dose, 2 ml) was administered subcutaneously. An intravenous glucose bolus (0.33 g/kg body weight) was injected 30 min later. Blood was drawn for the measurement of glucose, insulin, C-peptide, GLP-1 [7–36 amide], and glucagon using specific radioimmunoassays. There were dose-related increments in GLP-1 [7–36 amide] concentrations (p<0.0001). However, basal values were reached again after 90–120 min. Before glucose administration, insulin (p<0.0001) and C-peptide (p<0.0004) increased, whereas glucagon (p = 0.0018) and glucose (p<0.0001) decreased in a dose-dependent manner. After glucose stimulation, integrated increments in insulin (p=0.0007) and C-peptide (p=0.02) were augmented and k_G-values increased (p<0.0001) in a dose-related fashion. The extent of reactive hypoglycaemia was related to the GLP-1 [7–36 amide] dose. With the highest GLP-1 [7–36 amide] dose, at the time of peak plasma concentrations, most volunteers felt unwell, and nausea and vomiting were observed in four subjects. In conclusion, subcutaneous GLP-1 [7–36 amide] is also able to stimulate insulin and inhibit glucagon secretion, thereby altering glucose assimilation. However, with unmodified GLP-1 [7–36 amide], the duration of action is short, and with high doses side effects are common.Abbreviations BMI Body mass index - GLP-1 glucagon-like peptide-1 - RIA radioimmunoassay - RM-ANOVA repeated-measures analysis of variance - GIP gastric inhibitory polypeptide     

A comparison of the artificial pancreas (glucose controlled insulin infusion system) and a manual technique for assessing insulin sensitivity during euglycaemic clamping

Summary Two main methods are available for assessing insulin sensitivity with the hyperinsulinaemic euglycaemic clamp technique: one employs a glucose-controlled insulin infusion system (the Biostator) with automatic feedback control; the second depends on frequent glucose measurement and the use of an algorithm and a pocket calculator (manual) to determine the glucose infusion rate. The amount of glucose infused is a measure of insulin sensitivity. The efficiency of the two methods was compared in nine normal subjects (seven lean, two obese). After an overnight fast subjects were infused with insulin at 50 mU · kg^-1 · h^-1 for 2 h; this rate was doubled during the first 10 min for the manual technique. Blood glucose averaged 4.7 ± 0.1 and 4.8 ± 0.1 mmol/l from 0 to 120 min for Biostator and manual techniques and did not deviate significantly from the desired level. Variability of the clamp was also similar over the same period (coefficient of variation 5.1 ±0.6% and 6.4 ±0.7%, Biostator and manual). Glucose infused to maintain steady state from 60 to 120 min was higher, however, with the manual than the Biostator method (5.7±0.6 versus 4.4 ± 0.6 mg·kg^-1·min^-1, p< 0.01) even when the loading dose was omitted, although the two methods correlated closely (p< 0.05). Glucose infusion rate varied more from minute to minute with the Biostator (coefficient of variation 28.8 ± 3% versus 12.2 ± 2.1%). Steady-state serum insulin levels (30–120 min) were the same during both methods. Thus both methods give effective clamping but the manual method is simpler and shows less variability in glu cose insulin infusion rate.     

Impaired intestinal proglucagon processing in mice lacking prohormone convertase 1

The neuroendocrine prohormone convertases 1 and 2 (PC1 and PC2) are expressed in endocrine intestinal L cells and pancreatic A cells, respectively, and colocalize with proglucagon in secretory granules. Mice lacking PC2 have multiple endocrinopathies and cannot process proglucagon to mature glucagon in the pancreas. Disruption of PC1 results in dwarfism and also multiple neuroendocrine peptide processing defects. This study compares the pancreatic and intestinal processing of proglucagon in mice lacking PC1 expression with that in age-matched wild-type controls. Because proglucagon was found to precipitate in acidic extracts, the intestinal processing profile was analyzed in both acidic and neutral extracts by gel filtration, HPLC, and RIA. Supporting a central role for PC2 in glucagon biosynthesis, we found normal processing of proglucagon to glucagon in the pancreas, whereas the intestinal proglucagon processing showed marked defects. Tissue proglucagon levels in null mice were elevated, and proglucagon processing to glicentin, oxyntomodulin, and glucagon-like peptide-1 and -2 (GLP-1 and GLP-2) was markedly decreased, indicating that PC1 is essential for the processing of all the intestinal proglucagon cleavage sites. This includes the monobasic site R(77) and, thereby, production of mature, biologically active GLP-1. We also found elevated glucagon levels, suggesting that factors other than PC1 that are capable of processing to mature glucagon are present in the secretory granules of the L cell. These findings strongly suggest that PC1 is essential for intestinal proglucagon processing in vivo and, thereby, plays an important role in production of the incretin hormone GLP-1 and the intestinotrophic hormone GLP-2.     

Effects of glipizide on glucose metabolism and muscle content of the insulin-regulatable glucose transporter (GLUT 4) and glycogen synthase activity during hyperglycaemia in type 2 diabetic patients

To examine whether sulphonylureas influence hyperglycaemia-induced glucose disposal and suppression of hepatic glucose production (HGP) in type 2 diabetes mellitus, a 150-min hyperglycaemic (plasma glucose 14 mmol/l) clamp with concomitant somatostatin infusion was used in eight type 2 diabetic patients before and after 6 weeks of glipizide (GZ) therapy. During the clamp a small replacement dose of insulin was given (0.15 mU/kg per min). Isotopically determined glucose-induced glucose uptake was similar before and after GZ administration which led to improved glycaemic control (basal plasma glucose 12.2±1.3 vs 8.9±0.7 mmol/l;P<0.01). Glucose-induced suppression of HGP was, however, more pronounced during GZ treatment (0.96±0.14 vs 1.44±0.20 mg/kg per min;P<0.02). Following GZ treatment hyperglycaemia failed to stimulate glycogen synthase activity. Moreover, GZ resulted in a significant increase in the immunoreactive abundance of the insulin-regulatable glucose transport protein (GLUT 4) (P<0.02). In conclusion, these results suggest that GZ therapy in type 2 diabetic patients enhances hepatic sensitivity to hyperglycaemia, while glucose-induced glucose uptake remains unaffected. In addition, GZ tends to normalize the activity of glycogen synthase and increases the content of GLUT 4 protein in skeletal muscle.     

Effects of subcutaneous glucagon-like peptide 1 (GLP-1 [7–36 amide]) in patients with NIDDM

Summary Intravenous glucagon-like peptide (GLP)-1 [7–36 amide] can normalize plasma glucose in non-insulin-dependent diabetic (NIDDM) patients. Since this is no form for routine therapeutic administration, effects of subcutaneous GLP-1 at a high dose (1.5 nmol/kg body weight) were examined. Three groups of 8, 9 and 7 patients (61 ± 7, 61 ± 9, 50 ± 11 years; BMI 29.5 ± 2.5, 26.1 ± 2.3, 28.0 ± 4.2 kg/m²; HbA_{1 c} 11.3 ± 1.5, 9.9 ± 1.0, 10.6 ± 0.7 %) were examined: after a single subcutaneous injection of 1.5 nmol/kg GLP [7–36 amide]; after repeated subcutaneous injections (0 and 120 min) in fasting patients; after a single, subcutaneous injection 30 min before a liquid test meal (amino acids 8 %, and sucrose 50 g in 400 ml), all compared with a placebo. Glucose (glucose oxidase), insulin, C-peptide, GLP-1 and glucagon (specific immunoassays) were measured. Gastric emptying was assessed with the indicator-dilution method and phenol red. Repeated measures ANOVA was used for statistical analysis. GLP-1 injection led to a short-lived increment in GLP-1 concentrations (peak at 30–60 min, then return to basal levels after 90–120 min). Each GLP-1 injection stimulated insulin (insulin, C-peptide, p < 0.0001, respectively) and inhibited glucagon secretion (p < 0.0001). In fasting patients the repeated administration of GLP-1 normalized plasma glucose (5.8 ± 0.4 mmol/l after 240 min vs 8.2 ± 0.7 mmol/l after a single dose, p = 0.0065). With the meal, subcutaneous GLP-1 led to a complete cessation of gastric emptying for 30–45 min (p < 0.0001 statistically different from placebo) followed by emptying at a normal rate. As a consequence, integrated incremental glucose responses were reduced by 40 % (p = 0.051). In conclusion, subcutaneous GLP-1 [7–36 amide] has similar effects in NIDDM patients as an intravenous infusion. Preparations with retarded release of GLP-1 would appear more suitable for therapeutic purposes because elevation of GLP-1 concentrations for 4 rather than 2 h (repeated doses) normalized fasting plasma glucose better. In the short term, there appears to be no tachyphylaxis, since insulin stimulation and glucagon suppression were similar upon repeated administrations of GLP-1 [7–36 amide]. It may be easier to influence fasting hyperglycaemia by GLP-1 than to reduce meal-related increments in glycaemia. [Diabetologia (1996) 39: 1546–1553]     

GLP-1 (glucagon-like peptide 1) and truncated GLP-1, fragments of human proglucagon,inhibit gastric acid secretion in humans

Glucagon-like peptide 1 amide (GLP-1 amide), a predicted product of the glucagon gene (proglucagon 72-107-amide), and truncated GLP-1 (proglucagon 78-107-amide), recently isolated from porcine small intestine, were infused in doses of 100 and 400 ng/kg/hr and 12.5 and 50 ng/kg/hr, respectively, into eight volunteers to study pharmacokinetics and effects on pentagastrin- stimulated gastric acid secretion (plateau stimulation with pentagastrin at D ₅₀:100ng/kg/hr). The concentration of GLP-1 in plasma increased from 64±12 to 189±23 and 631±76 pmol/liter, respectively. The concentration of truncated GLP increased from approximately 7 pmol/liter to 28±3 pmol/liter during the high rate of infusion. A similar increase was seen in response to a mixed meal in eight normal volunteers. The metabolic clearance rate (MCR) of GLP-1 was 2.2±0.3 and 2.6±0.3 ml/kg/min, respectively, and the half- life in plasma was 17±2 min. The MCR of truncated GLP-1 was 13±2.8 ml/kg/min and the half- life 11.4±2.1 min. GLP-1 reduced the pentagastrin- stimulated acid secretion 16±9% during the low-rate infusion and 23±12% during the high rate (P<0.05). Truncated GLP-1 caused a 36±3% inhibition during the high infusion rate. Thus truncated GLP-1, a naturally occurring peptide, is a potent inhibitor of acid secretion in man and more so than GLP-1.This study was supported by the Danish Medical Research Council, Novo's Fond and Owesen's Fond.     

Inhibition of muscle glycogen synthase activity and non-oxidative glucose disposal during hypoglycaemia in normal man

Summary The purpose of the present study was to evaluate the role of muscle glycogen synthase activity in the reduction of glucose uptake during hypoglycaemia. Six healthy young men were examined twice; during 120 min of hyperinsulinaemic (1.5 mU · kg^–1 · min^–1) euglycaemia followed by: 1) 240 min of graded hypoglycaemia (plasma glucose nadir 2.8 mmol/l) or 2) 240 min of euglycaemia. At 350–360 min a muscle biopsy was taken and indirect calorimetry was performed at 210–240 and 330–350 min. Hypoglycaemia was associated with markedly increased levels of adrenaline, growth hormone and glucagon and also with less hyperinsulinaemia. During hypoglycaemia the fractional velocity for glycogen synthase was markedly reduced; from 29.8±2.3 to 6.4±0.9%, p<0.05. Total glucose disposal was decreased during hypoglycaemia (5.58±0.55 vs 11.01±0.75 mg · kg^–1 · min^–1 (euglycaemia); p<0.05); this was primarily due to a reduction of non-oxidative glucose disposal (2.43±0.41 vs 7.15±0.7 mg · kg^–1 · min^–1 (euglycaemia); p<0.05), whereas oxidative glucose disposal was only suppressed to a minor degree. In conclusion hypoglycaemia virtually abolishes the effect of insulin on muscle glycogen synthase activity. This is in keeping with the finding of a marked reduction of non-oxidative glucose metabolism.Abbreviations HGP Hepatic glucose production - Rd glucose disposal - GH growth hormone - 3-OHB 3-hydroxybutyrate - G 6-P glucose 6-phosphate - NEFA non-esterified fatty acids - PP-1 phosphatase-1 - Ra rate of appearance - Rd-nonox non-oxidative glucose disposal - Rd-ox oxidative glucose disposal - A0.5 half-maximal activity     

The relationship between the circulating IGF system and the presence of retinopathy in Type 1 diabetic patients.

BACKGROUND: Patients with proliferative diabetic retinopathy (PDR) have increased vitreous levels of insulin-like growth factor (IGF)-I, IGF-II and IGF binding proteins (IGFBPs). This accumulation is probably caused by increased leakiness of the blood-retina barrier and influx of circulating IGFs and IGFBPs. To date, interest has focused on the role of circulating total IGF-I in the development of PDR, and there are only sparse data on circulating levels of free IGF-I and IGFBPs. METHODS: We compared fasting serum samples from matched groups of Type 1 diabetic patients with no retinopathy (n = 29), non-PDR (n = 13) and PDR (n = 16). We also included matched controls (n = 26). Serum was analysed for free and total IGF-I and -II, free plus dissociable IGF-I, IGFBP-1, -2 and -3, IGFBP-1-bound IGF-I as well as IGFBP-3 proteolysis. RESULTS: When compared with controls, diabetic patients (n = 58) showed reduced (P < 0.0005) levels of free and total IGFs, free plus dissociable IGF-I and IGFBP-3, whereas levels of IGFBP-1, IGFBP-1-bound IGF-I and IGFBP-2 were elevated. IGFBP-3 proteolysis remained unaltered. When comparing diabetic patients with different degrees of retinopathy, IGFBP-2 and IGFBP-1-bound IGF-I were the only parameters that differed significantly. Patients with retinopathy (non-PDR as well as PDR) had elevated IGFBP-2 (P < 0.03) and reduced IGFBP-1-bound IGF-I (P < 0.03), when compared with patients without retinopathy. Noteworthy, both parameters correlated (0.11 < r2 < 0.14, P < 0.02) with the severity of retinopathy. CONCLUSION: Our study gives no evidence of a direct role of circulating free IGF-I in the development of PDR, whereas IGFBP-2 and IGFBP-1-bound IGF-I showed a relationship with the degree of retinopathy. However, further investigations are needed in order to clarify the basis and clinical relevance of this finding.     

Acute hyperinsulinemia restrains endotoxin-induced systemic inflammatory response: an experimental study in a porcine model

BACKGROUND: Intensive insulin therapy in critically ill patients reduces morbidity and mortality. The current study elucidates whether acute hyperinsulinemia per se could attenuate the systemic cytokine response and improve neutrophil function during endotoxin (lipopolysaccharide)-induced systemic inflammation in a porcine model. METHODS: Pigs were anesthetized, mechanically ventilated, randomized into four groups, and followed for 570 min: group 1 (anesthesia solely, n = 10), group 2 (hyperinsulinemic euglycemic clamp [HEC], n = 9), group 3 (lipopolysaccharide, n = 10), group 4 (lipopolysaccharide-HEC, n = 9). Groups 3 and 4 were given a 180-min infusion of lipopolysaccharide (total, 10 microg/kg). Groups 2 and 4 were clamped (p-glucose: 5 mM/l, insulin 0.6 mU.kg(-1).min(-1)) throughout the study period. Changes in pulmonary and hemodynamic function, circulating cytokines, free fatty acids, glucagon, and neutrophil chemotaxis were monitored. RESULTS: Tumor necrosis factor alpha and interleukin 6 were significantly reduced in the lipopolysaccharide-HEC group compared with the lipopolysaccharide group (both P = 0.04). In the lipopolysaccharide-HEC group, the glucagon response was diminished compared with the lipopolysaccharide group (P < 0.05). Serum free fatty acid concentrations were decreased in animals exposed to HEC. Animals receiving lipopolysaccharide showed an increase in pulmonary pressure (P < 0.001), but otherwise, there were no major changes in pulmonary or hemodynamic function. Neutrophil function was impaired after lipopolysaccharide administration. CONCLUSION: Hyperinsulinemia concomitant with normoglycemia reduces plasma concentrations of tumor necrosis factor alpha and the catabolic hormone glucagon in lipopolysaccharide-induced systemic inflammation in pigs. The finding strongly supports the role of insulin as an antiinflammatory hormone. Whether the effect to some extent operates via a reduced free fatty acid concentration is unsettled.     

The metabolic effects of dopamine in man

Summary The metabolic effects of dopamine have been investigated by its infusion in normal man with and without simultaneous somatostatin administration. Dopamine was infused into overnight fasted men at 1.5 µg/kg/min (n=6) and 3.0 µg/kg/min (n=5) for 120 min. Plasma dopamine concentrations at 120 min were 78±9 nmol/l and 117±17 nmol/l respectively, associated with a marginal rise in plasma noradrenaline. Dopamine (1.5 µg/kg/min) induced an early and sustained rise in plasma glucagon (48±9 pg/ml versus 19±6 pg/ml in saline controls at 10 min, p<0.01)and a transient elevation in serum growth hormone which peaked to 17.7 (range 4.5–71.8)mU/l at 60 min (7.2 (range 0.6–37.7) mU/l with saline, p<0.05), but did not alter serum insulin, blood glucose or other metabolite levels. At 3.0 µg/kg/min, dopamine in addition provoked mild and transient elevations in blood glucose and serum insulin. Somatostatin (250 µg/h) suppressed circulating insulin, glucagon, and growth hormone levels and abolished the small hyperglycaemic effect seen with the higher dopamine dose. Somatostatin alone induced a progressive rise in circulating non-esterified fatty acid and 3-hydroxybutyrate levels reflecting insulin deficiency. This rise in NEFA and 3-hydroxybutyrate was increased by dopamine particularly at the higher dosage (plasma NEFA; somatostatin alone, 1.08±0.13 mmol/l; somatostatin plus dopamine 3 µg/kg/min, 1.44±0.17 mmol/l at 120 min, p<0.01: blood 3-hydroxybutyrate; somatostatin alone, 0.32±0.04 mmol/l; somatostatin plus dopamine 3 µg/kg/min, 0.56±0.12 mmol/l at 120 min, p<0.05). Thus: 1) dopamine at pharmacological dosage has minor effects when other endocrine mechanisms are intact, 2) it enhances lipolysis and ketogenesis during somatostatin-induced insulin deficiency, 3) the hyperglycaemic effect of the higher dopamine dose is probably mediated through stimulated glucagon secretion.