Marianne O. Larsen

Incretin and islet hormonal responses to fat and protein ingestion in healthy men

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) regulate islet function after carbohydrate ingestion. Whether incretin hormones are of importance for islet function after ingestion of noncarbohydrate macronutrients is not known. This study therefore examined integrated incretin and islet hormone responses to ingestion of pure fat (oleic acid; 0.88 g/kg) or protein (milk and egg protein; 2 g/kg) over 5 h in healthy men, aged 20-25 yr (n=12); plain water ingestion served as control. Both intact (active) and total GLP-1 and GIP levels were determined as was plasma activity of dipeptidyl peptidase-4 (DPP-4). Following water ingestion, glucose, insulin, glucagon, GLP-1, and GIP levels and DPP-4 activity were stable during the 5-h study period. Both fat and protein ingestion increased insulin, glucagon, GIP, and GLP-1 levels without affecting glucose levels or DPP-4 activity. The GLP-1 responses were similar after protein and fat, whereas the early (30 min) GIP response was higher after protein than after fat ingestion (P<0.001). This was associated with sevenfold higher insulin and glucagon responses compared with fat ingestion (both P<0.001). After protein, the early GIP, but not GLP-1, responses correlated to insulin (r(2)=0.86; P=0.0001) but not glucagon responses. In contrast, after fat ingestion, GLP-1 and GIP did not correlate to islet hormones. We conclude that, whereas protein and fat release both incretin and islet hormones, the early GIP secretion after protein ingestion may be of primary importance to islet hormone secretion.

Secretion and Dipeptidyl Peptidase-4-Mediated Metabolism of Incretin Hormones after a Mixed Meal or Glucose Ingestion in Obese Compared to Lean, Nondiabetic Men.

CONTEXT Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are cleaved by dipeptidyl peptidase-4 (DPP-4); plasma activity of DPP-4 may be increased in obesity. The impact of this increase on incretin hormone secretion and metabolism is not known. OBJECTIVE The aim of the study was to assess incretin hormone secretion and degradation in lean and obese nondiabetic subjects. DESIGN, SETTINGS, AND PARTICIPANTS We studied the ingestion of a mixed meal (560 kcal) or oral glucose (2 g/kg) in healthy lean (n = 12; body mass index, 20-25 kg/m(2)) or obese (n = 13; body mass index, 30-35 kg/m(2)) males at a University Clinical Research Unit. MAIN OUTCOME MEASURES We measured the area under the curve of plasma intact (i) and total (t) GIP and GLP-1 after meal ingestion and oral glucose. RESULTS Plasma DPP-4 activity was higher in the obese subjects (38.5 +/- 3.0 vs. 26.7 +/- 1.6 mmol/min . microl; P = 0.002). Although GIP secretion (AUC(tGIP)) was not reduced in obese subjects after meal ingestion or oral glucose, AUC(iGIP) was lower in obese subjects (8.5 +/- 0.6 vs. 12.7 +/- 0.9 nmol/liter x 300 min; P < 0.001) after meal ingestion. GLP-1 secretion (AUC(tGLP-1)) was reduced in obese subjects after both meal ingestion (7.3 +/- 0.9 vs. 10.0 +/- 0.6 nmol/liter x 300 min; P = 0.022) and oral glucose (6.6 +/- 0.8 vs. 9.6 +/- 1.1 nmol/liter x 180 min; P = 0.035). iGLP-1 was reduced in parallel to tGLP-1. CONCLUSIONS 1) Release and degradation of the two incretin hormones show dissociated changes in obesity: GLP-1 but not GIP secretion is lower after meal ingestion and oral glucose, whereas GIP but not GLP-1 metabolism is increased after meal ingestion. 2) Increased plasma DPP-4 activity in obesity is not associated with a generalized augmented incretin hormone metabolism.

NN2211: a long-acting glucagon-like peptide-1 derivative with anti-diabetic effects in glucose-intolerant pigs.

Glucagon-like peptide-1 (GLP-1) is an effective anti-diabetic agent, but its metabolic instability makes it therapeutically unsuitable. This study investigated the pharmacodynamics of a long-acting GLP-1 derivative (NN2211: (Arg(34)Lys(26)-(N- epsilon -(gamma-Glu(N-alpha-hexadecanoyl)))-GLP-1(7-37)), after acute and chronic treatment in hyperglycaemic minipigs. During hyperglycaemic glucose clamps, NN2211 (2 micrograms kg(-1) i.v.) treated pigs required more (P < 0.005) glucose than control animals (5.8 +/- 2.1 vs. 2.9 +/- 1.8 mg kg(-1) min(-1)). Insulin excursions were higher (P < 0.01) after NN2211 (15,367 +/- 5,438 vs. 9,014 +/- 2,952 pmol l(-1) min), and glucagon levels were suppressed (P < 0.05). Once-daily injections of NN2211 (3.3 micrograms kg(-1) s.c.) reduced the glucose excursion during an oral glucose tolerance test, to 59 +/- 15% of pre-treatment values by 4 weeks (P < 0.05), without measurable changes in insulin responses. Fructosamine concentrations were unaltered by vehicle, but decreased (from 366 +/- 187 to 302 +/- 114 micromol l(-1), P = 0.14) after 4 weeks of NN2211. Gastric emptying was reduced (P < 0.05) by NN2211. NN2211 acutely increases glucose utilization during a hyperglycaemic glucose clamp and chronic treatment results in better daily metabolic control. Therefore, NN2211, a GLP-1 derivative that can be administered once daily, holds promise as a new anti-diabetic drug with a minimal risk of hypoglycaemia.

Chamber-Dependent Expression of Brain Natriuretic Peptide and Its mRNA in Normal and Diabetic Pig Heart

Brain natriuretic peptide (BNP) is produced in cardiac myocytes, and increased secretion is closely associated with cardiac dysfunction. However, several fundamental aspects of BNP expression in the myocardium have not yet been resolved. In the present study, we report the presence of a precursor BNP mRNA transcript and a mature BNP mRNA transcript in normal porcine hearts. In normal pigs, the amount of precursor BNP mRNA was similar in atrial and ventricular myocardium, whereas the mature BNP transcript was 10- to 50-fold more abundant in atrial than in ventricular myocardium. Quantitation of proBNP in normal porcine hearts by radioimmunoassay disclosed abundant proBNP in the atria, whereas proBNP was undetectable in the ventricles. Laser confocal microscopy revealed proBNP in secretory granules of atrial but not in the ventricular myocardium of normal pigs. Mild streptozotocin-induced diabetes doubled the expression of BNP mRNA in porcine atrial myocardium (P =0.03), but was without effect on BNP mRNA in the ventricular myocardium. The data suggest that BNP mRNA processing and proBNP storage differ between the atrial and ventricular myocardium. The results also imply that diabetes increases cardiac BNP expression in a chamber-dependent manner.

High-fat high-energy feeding impairs fasting glucose and increases fasting insulin levels in the Göttingen minipig: results from a pilot study.

Abstract: High‐fat diet and obesity are known to be of major importance for development of type 2 diabetes in humans. High‐fat feeding can induce syndromes of glucose intolerance and/or insulin resistance in several species, and the Göttingen minipig might be a useful model for studying the effect of dietary high‐fat intake and obesity on glucose homeostasis and the susceptibility to diabetes. The present study was designed as a pilot study to investigate the effects of obesity caused by high‐fat high‐energy feeding on oral and intravenous glucose tolerance. Male Göttingen minipigs were fed a control diet (CD) or a high‐fat high‐energy diet (HFD) for 3 months. Body weight (32.6 ± 2.4 kg vs. 24.9 ± 0.5 kg, p < 0.001) and total (13.2 ± 3.2% vs. 6.1 ± 0.5%, p= 0.002) and truncal (11.0 ± 3.9% vs. 1.8 ± 1.1%, p= 0.001) fat percent were increased significantly, whereas relative lean body mass was decreased (84.8 ± 3.3% vs. 91.9 ± 0.5%, p= 0.002) in the HFD group compared to CD. Fasting plasma glucose (4.3 ± 0.4 mM vs. 3.6 ± 0.3 mM, p= 0.023) and insulin (80 ± 23 pM vs. 23 ± 21 pM, p= 0.012) were increased in the HFD group compared to CD, but oral glucose tolerance was not significantly changed. Insulin responses to intravenous glucose were increased (6741 ± 2538 vs. 3938 ± 771 pM 3 min, p= 0.050), while glucose clearance was not changed by HFD vs. CD, thus indicating insulin resistance. In conclusion, changes in body weight and composition, resulting in minor abnormalities in glucose tolerance and insulin sensitivity, characterized by slight hyperglycemia and compensatory hyperinsulinemia, can be induced in the male Göttingen minipig by high‐fat high‐energy feeding for 3 months. This approach seems to be an interesting and promising method for establishment of a nonrodent model of insulin resistance or type 2 diabetes.

The human GLP-1 analogs liraglutide and semaglutide: Absence of histopathological effects on the pancreas in nonhuman primates.

Increased pancreas mass and glucagon-positive adenomas have been suggested to be a risk associated with sitagliptin or exenatide therapy in humans. Novo Nordisk has conducted extensive toxicology studies, including data on pancreas weight and histology, in Cynomolgus monkeys dosed with two different human glucagon-like peptide-1 (GLP-1) receptor agonists. In a 52-week study with liraglutide, a dose-related increase in absolute pancreas weight was observed in female monkeys only. Such dose-related increase was not found in studies of 4, 13, or 87 weeks’ duration. No treatment-related histopathological abnormalities were observed in any of the studies. Quantitative histology of the pancreas from the 52-week study showed an increase in the exocrine cell mass in liraglutide-dosed animals, with normal composition of endocrine and exocrine cellular compartments. Proliferation rate of the exocrine tissue was low and comparable between groups. Endocrine cell mass and proliferation rates were unaltered by liraglutide treatment. Semaglutide showed no increase in pancreas weight and no treatment-related histopathological findings in the pancreas after 13 or 52 weeks’ dosing. Overall, results in 138 nonhuman primates showed no histopathological changes in the pancreas associated with liraglutide or semaglutide, two structurally different GLP-1 receptor agonists.

Valine Pyrrolidide Preserves Intact Glucose-Dependent Insulinotropic Peptide and Improves Abnormal Glucose Tolerance in Minipigs With Reduced β-Cell Mass

The incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are important in blood glucose regulation.However, both incretin hormones are rapidly degraded by the enzyme dipeptidyl peptidase IV (DPPIV). The concept of DPPIV inhibition as a treatment for type 2 diabetes was evaluated in a new large animal model of insulin-deficient diabetes and reduced β-cell mass, the nicotinamide (NIA) (67 mg/kg) and streptozotocin (STZ) (125 mg/kg)–treated minipig, using the DPPIV inhibitor, valine pyrrolidide (VP) (50 mg/kg).VP did not significantly affect levels of intact GLP-1 but increased levels of intact GIP (from 4543 ± 1880 to 9208 ± 3267 pM × min; P<.01), thus improving glucose tolerance (area under the curve [AUC] for glucose reduced from 1904 ± 480 to 1582 ± 353 mM × min;P = .05).VP did not increase insulin levels during the oral glucose tolerance test (OGTT) but increased the insulinogenic index in normal animals (from 83 ± 42 to 192 ± 108; P < .05), but not after NIA + STZ, possibly because of less residual insulin secretory capacity in these animals. GIP seems to contribute to the antihyperglycemic effect of VP in this model; however, additional mechanisms for the effect of DPPIV inhibition cannot be excluded. The authors conclude that DPPIV inhibitors may be useful to treat type 2 diabetes, even when this is due to reduced β-cell mass.

The conscious Göttingen minipig as a model for studying rapid pulsatile insulin secretion in vivo

Abstract Aims/hypothesis. Pulsatile secretion is important for insulin action and suitable animal models are important tools for examining the role of impaired pulsatile insulin secretion as a possible link between beta-cell mass, function and morphology and insulin resistance. This study examines the vascular sampling site, insulin kinetics, pulsatility and the response to glucose pulse entrainment to evaluate the Göttingen minipig as a model for studying pulsatile insulin secretion. Methods. Basal and glucose entrained insulin secretion was examined in normal minipigs and evaluated by autocorrelation, cross correlation and deconvolution. Results. Cross correlation showed a relation between oscillations in insulin concentrations in the portal and jugular vein in anaesthetised animals (p<0.001 in all animals), confirming the usefulness of jugular vein sampling for pulse detection. Jugular vein sampling in conscious animals showed obvious oscillations allowing estimates of burst shape and insulin kinetics. Glucose entrainment improved the pulsatile pattern (autocorrelation: 0.555±0.148 entrained vs 0.350±0.197 basal, p=0.054). Deconvolution analysis resolved almost all insulin release as secretory bursts (69±20 basal vs 99.5±1.2% entrained, p<0.01) with a pulse interval (min) of 6.6±2.2 (basal) and 9.4±1.5 (entrained) (p<0.05) and a pulse mass (pmol/l per pulse) which was higher after entrainment (228±117 vs 41.2±18.6 basal, p<0.001). Conclusion/interpretation. The ability to fit kinetic parameters directly by deconvolution of peripheral endogenous insulin concentration time series in combination with the suitability of jugular vein sampling, rapid kinetics and entrainability makes the Göttingen minipig ideal for mechanistic studies of insulin pulsatility and its effects on insulin action.

Evaluation of β-cell mass and function in the Göttingen minipig

Increased knowledge about β‐cell mass and function is important for our understanding of the pathophysiology of type 2 diabetes (T2DM). The relationship between the two is difficult to study in humans, whereas animal models allow studies of consequences of, for example, reduction of β‐cell mass and induction of obesity and procurement of the pancreas for histological examination. An overview of results obtained in the Göttingen minipig in relation to β‐cell function, and mass is provided here. Effects of a primary reduction of β‐cell mass have indicated that not all of the defects of pulsatile insulin secretion in human T2DM can be explained by reduced β‐cell mass. Furthermore, induction of obesity has shown deterioration of β‐cell function and morphological changes in the pancreas. As in humans, obesity leads to an increased β‐cell volume in the minipig, and based on the increased number of islets, neogenesis of islets is an important factor in expansion of β‐cell mass in this species. Measurement of β‐cell function as an estimate of β‐cell mass is, at present, the only method possible in humans, and this approach has been validated using lean and obese minipigs with a range of β‐cell mass. The effects on β‐cell function and mass of obesity of longer duration and/or more pronounced hyperglycaemia remains to be determined, but the models developed so far represent a valuable tool for such investigations.