Bidda Rolin

GLP-1 derivative liraglutide in rats with β-cell deficiencies: influence of metabolic state on β-cell mass dynamics

Liraglutide is a long‐acting GLP‐1 derivative, designed for once daily administration in type II diabetic patients. To investigate the effects of liraglutide on glycemic control and β‐cell mass in rat models of β‐cell deficiencies, studies were performed in male Zucker diabetic fatty (ZDF) rats and in 60% pancreatectomized rats. When liraglutide was dosed s.c. at 150 μg kg−1 b.i.d. for 6 weeks in ZDF rats 6–8 weeks of age at study start, diabetes development was markedly attenuated. Blood glucose was approximately 12 mM lower compared to vehicle (P<0.0002), and plasma insulin was 2–3‐fold higher during a normal 24‐h feeding period (P<0.001). Judged by pair feeding, approximately 53% of the antihyperglycemic effect observed on 24‐h glucose profiles was mediated by a reduction in food intake, which persisted throughout the study and averaged 16% (P<0.02). Histological analyses revealed that β‐cell mass and proliferation were significantly lower in prediabetic animals still normoglycemic after 2 weeks treatment compared to vehicle‐treated animals that had begun to develop diabetes. When the treatment period was 6 weeks, the liraglutide‐treated animals were no longer completely normoglycemic and the β‐cell mass was significantly increased compared to overtly diabetic vehicle‐treated animals, while β‐cell proliferation was unaffected. In the experiments with 60% pancreatectomized rats, 8 days treatment with liraglutide resulted in a significantly lower glucose excursion in response to oral glucose compared to vehicle treatment. Again, part of the antihyperglycemic effect was due to reduced food intake. No effect of liraglutide on β‐cell mass was observed in these virtually normoglycemic animals. In conclusion, treatment with liraglutide has marked antihyperglycemic effects in rodent models of β‐cell deficiencies, and the in vivo effect of liraglutide on β‐cell mass may in part depend on the metabolic state of the animals.

Liraglutide, a Long-Acting Glucagon-Like Peptide-1 Analog, Reduces Body Weight and Food Intake in Obese Candy-Fed Rats, Whereas a Dipeptidyl Peptidase-IV Inhibitor, Vildagliptin, Does Not

Metabolic effects of the glucagon-like peptide-1 analog liraglutide and the dipeptidyl peptidase-IV inhibitor vildagliptin were compared in rats made obese by supplementary candy feeding. Female Sprague-Dawley rats were randomized to 12-week diets of chow or chow plus candy. The latter were randomized for 12 further weeks to continue their diet while receiving 0.2 mg/kg liraglutide twice daily subcutaneously, 10 mg/kg vildagliptin twice daily orally, or vehicle or to revert to chow-only diet. Energy expenditure was measured, and oral glucose tolerance tests (OGTTs) were performed. Body composition was determined by dual-energy X-ray absorptiometry scanning, and pancreatic β-cell mass was determined by histology. Candy feeding increased weight, fat mass, and feeding-associated energy expenditure. Liraglutide or reversal to chow diet fully reversed weight and fat gains. Liraglutide was associated with decreased calorie intake and shifted food preference (increased chow/decreased candy consumption). Despite weight loss, liraglutide-treated rats did not decrease energy expenditure compared with candy-fed controls. Vildagliptin affected neither weight, food intake, nor energy expenditure. OGTTs, histology, and blood analyses indirectly suggested that both drugs increased insulin sensitivity. Liraglutide and vildagliptin inhibited obesity-associated increases in β-cell mass. This was associated with weight and fat mass normalization with liraglutide, but not vildagliptin, where the ratio of β-cell to body mass was low.

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.

Ampicillin-Improved Glucose Tolerance in Diet-Induced Obese C57BL/6NTac Mice Is Age Dependent

Ampicillin has been shown to improve glucose tolerance in mice. We hypothesized that this effect is present only if treatment is initiated prior to weaning and that it disappears when treatment is terminated. High-fat fed C57BL/6NTac mice were divided into groups that received Ampicillin at different ages or not at all. We found that both diet and Ampicillin significantly changed the gut microbiota composition in the animals. Furthermore, there was a significant improvement in glucose tolerance in Ampicillin-treated, five-week-old mice compared to nontreated mice in the control group. At study termination, expressions of mRNA coding for tumor necrosis factor, serum amyloid A, and lactase were upregulated, while the expression of tumor necrosis factor (ligand) superfamily member 15 was downregulated in the ileum of Ampicillin-treated mice. Higher dendritic cell percentages were found systemically in high-fat diet mice, and a lower tolerogenic dendritic cell percentage was found both in relation to high-fat diet and late Ampicillin treatment. The results support our hypothesis that a “window” exists early in life in which an alteration of the gut microbiota affects glucose tolerance as well as development of gut immunity and that this window may disappear after weaning.

Recombinant Adiponectin Does Not Lower Plasma Glucose in Animal Models of Type 2 Diabetes

Aims/Hypothesis Several studies have shown that adiponectin can lower blood glucose in diabetic mice. The aim of this study was to establish an effective adiponectin production process and to evaluate the anti-diabetic potential of the different adiponectin forms in diabetic mice and sand rats. Methods Human high molecular weight, mouse low molecular weight and mouse plus human globular adiponectin forms were expressed and purified from mammalian cells or yeast. The purified protein was administered at 10–30 mg/kg i.p. b.i.d. to diabetic db/db mice for 2 weeks. Furthermore, high molecular weight human and globular mouse adiponectin batches were administered at 5–15 mg/kg i.p. b.i.d. to diabetic sand rats for 12 days. Results Surprisingly, none of our batches had any effect on blood glucose, HbA1c, plasma lipids or body weight in diabetic db/db mice or sand rats. In vitro biological, biochemical and biophysical data suggest that the protein was correctly folded and biologically active. Conclusions/Interpretation Recombinant adiponectin is ineffective at lowering blood glucose in diabetic db/db mice or sand rats.

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.

Modulating the Gut Microbiota Improves Glucose Tolerance, Lipoprotein Profile and Atherosclerotic Plaque Development in ApoE-Deficient Mice

The importance of the gut microbiota (GM) in disease development has recently received increased attention, and numerous approaches have been made to better understand this important interplay. For example, metabolites derived from the GM have been shown to promote atherosclerosis, the underlying cause of cardiovascular disease (CVD), and to increase CVD risk factors. Popular interest in the role of the intestine in a variety of disease states has now resulted in a significant proportion of individuals without coeliac disease switching to gluten-free diets. The effect of gluten-free diets on atherosclerosis and cardiovascular risk factors is largely unknown. We therefore investigated the effect of a gluten-free high-fat cholesterol-rich diet, as compared to the same diet in which the gluten peptide gliadin had been added back, on atherosclerosis and several cardiovascular risk factors in apolipoprotein E-deficient (Apoe-/-) mice. The gluten-free diet transiently altered GM composition in these mice, as compared to the gliadin-supplemented diet, but did not alter body weights, glucose tolerance, insulin levels, plasma lipids, or atherosclerosis. In parallel, other Apoe-/- mice fed the same diets were treated with ampicillin, a broad-spectrum antibiotic known to affect GM composition. Ampicillin-treatment had a marked and sustained effect on GM composition, as expected. Furthermore, although ampicillin-treated mice were slightly heavier than controls, ampicillin-treatment transiently improved glucose tolerance both in the absence or presence of gliadin, reduced plasma LDL and VLDL cholesterol levels, and reduced aortic atherosclerotic lesion area. These results demonstrate that a gluten-free diet does not seem to have beneficial effects on atherosclerosis or several CVD risk factors in this mouse model, but that sustained alteration of GM composition with a broad-spectrum antibiotic has beneficial effects on CVD risk factors and atherosclerosis. These findings support the concept that altering the microbiota might provide novel treatment strategies for CVD.