Degradation and glycemic effects of His(7)-glucitol glucagon-like peptide-1(7-36)amide in obese diabetic ob/ob mice

Glucagon-like peptide-1(7-36)amide (tGLP-1) has attracted considerable potential as a possible therapeutic agent for type 2 diabetes. However, tGLP-1 is rapidly inactivated in vivo by the exopeptidase dipeptidyl peptidase IV (DPP IV), thereby terminating its insulin releasing activity. The present study has examined the ability of a novel analogue, His(7)-glucitol tGLP-1 to resist plasma degradation and enhance the insulin-releasing and antihyperglycemic activity of the peptide in 20-25-week-old obese diabetic ob/ob mice. Degradation of native tGLP-1 by incubation at 37 degreesC with obese mouse plasma was clearly evident after 3 h (35% intact). After 6 h, more than 87% of tGLP-1 was converted to GLP-1(9-36)amide and two further N-terminal fragments, GLP-1(7-28) and GLP-1(9-28). In contrast, His7-glucitol tGLP-1 was completely resistant to N-terminal degradation. The formation of GLP-1(9-36)amide from native tGLP-1 was almost totally abolished by addition of diprotin A, a specific inhibitor of DPP IV. Effects of tGLP-1 and His7-glucitol tGLP-1 were examined in overnight fasted obese mice following i.p. injection of either peptide (30 nmol/kg) together with glucose (18 mmol/kg) or in association with feeding. Plasma glucose was significantly lower and insulin response greater following administration of His7-glucitol tGLP-1 as compared to glucose alone. Native tGLP-1 lacked antidiabetic effects under the conditions employed, and neither peptide influenced the glucose-lowering action of exogenous insulin (50 units/kg). Twice daily s.c. injection of ob/ob mice with His(7)-glucitol tGLP-1 (10 nmol/kg) for 7 days reduced fasting hyperglycemia and greatly augmented the plasma insulin response to the peptides given in association with feeding. These data demonstrate that His(7)-glucitol tGLP-1 displays resistance to plasma DPP IV degradation and exhibits antihyperglycemic activity and substantially enhanced insulin-releasing action in a commonly used animal model of type 2 diabetes. (C) 2001 Elsevier Science B.V. All rights reserved.

Improved glycaemic control in obese diabetic ob/ob mice using N-terminally modified gastric inhibitory polypeptide

Gastric inhibitory polypeptide (GIP) is an important insulin-releasing hormone of the enteroinsular axis which is rapidly inactivated by the exopeptidase dipeptidyl peptidase (DPP) IV. The present study has examined the ability of Tyr(1)-glucitol GIP to be protected from plasma degradation and to enhance insulin-releasing and antihyperglycaemic activity in 20- to 25-week-old obese diabetic ob/ob mice. Degradation of GIP by incubation at 37 degrees C with obese mouse plasma was clearly evident after 3 h (35% degraded). After 6 h, more than 61% of GIP was converted to GIP(3-42) whereas N-terminally modified Tyr(1)-glucitol GIP was resistant to degradation in plasma (>99% intact after 6 h). The formation of GIP(3-42) was almost completely abolished by inhibition of plasma DPP IV with diprotin A. Effects of GIP and Tyr(1)-glucitol GIP were examined in overnight-fasted obese mice following i.p. injection of either peptide (20 nmol/kg) together with glucose (18 mmol/kg) or in association with feeding. Most prominent effects were observed in the former group where plasma glucose values at 60 min together with the area under the curve (AUC) for glucose were significantly lower following GIP (AUC, 874 +/- 72 mmol/l.min; P

Evaluation of glycated insulin in diabetic animals using immunocytochemistry and radioimmunoassay

Glycated insulin was evaluated in plasma and biological tissues of diabetic animal models by immuno. cytochemistry (ICC) and a novel radioimmunoassay. Glycated insulin circulated at 0.10 +/-0.04 ng/ml and 2.20 +/-0.14 ng/ml in lean and diabetic obese (ob/ob) mice, corresponding to 12.5 and 9.8% total plasma insulin, respectively. The concentration of glycated insulin was elevated 22-fold in obese mice compared to controls (P10 and 83 +/-4 ng/g wt (P0.17 mug/g wt). ICC revealed fluorescent positively stained cells in pancreatic islets from hydrocortisone (HC)treated diabetic rats. Fasting of HC-treated rats, resulted in 3-fold and 15-fold reductions in plasma glycated insulin (P

Langerhans cells are critical in the development of atopic dermatitis-like inflammation and symptoms in mice

Genetic or vitamin D3-induced overexpression of thymic stromal lymphopoietin (TSLP) by keratinocytes results in an atopic dermatitis (AD)-like inflammatory phenotype in mice echoing the discovery of high TSLP expression in epidermis from AD patients. Although skin dendritic cells (DC) are suspected to be involved in AD, direct evidence of a pathogenetic role for skin DC in TSLP-induced skin inflammation has not yet been demonstrated. In a mouse model of AD, i.e. mice treated with the low-calcemic vitamin D3 analogue, MC903, we show that epidermal Langerhans cells (LC)-depleted mice treated with MC903 do neither develop AD-like inflammation nor increased serum IgE as compared to vitamin D3 analogue-treated control mice. Accordingly, we show that, in mice treated with MC903 or in K14-TSLP transgenic mice, expression of maturation markers by LC is increased whereas maturation of dermal DC is not altered. Moreover, only LC are responsible for the polarization of naive CD4+ T cells to a Th2 phenotype, i.e. decrease in interferon-gamma and increase in interleukin (IL)-13 production by CD4+ T cells. This effect of LC on T-lymphocytes does not require OX40-L/CD134 and is mediated by a concomitant down-regulation of IL-12 and CD70. Although it was previously stated that TSLP up-regulates the production of thymus and activation-regulated chemokine (TARC)/chemokine (C-C motif) ligand 17 (CCL17) and macrophage-derived chemokine (MDC)/CCL22 by human LC in vitro, our work shows that production of these Th2- cell attracting chemokines is increased only in keratinocytes in response to TSLP overexpression. These results demonstrate that LC are required for the development of AD in mouse models of AD involving epidermal TSLP overexpression.

Rod photoreceptor loss in Rho(-/-) mice reduces retinal hypoxia and hypoxia-regulated gene expression

PURPOSE. This study was conducted to evaluate whether regions of the retinal neuropile become hypoxic during periods of high oxygen consumption and whether depletion of the outer retina reduces hypoxia and related changes in gene expression.

METHODS. Retinas from rhodopsin knockout (Rho(-/-)) mice were evaluated along with those of wild-type (WT) control animals. Retinas were also examined at the end of 12-hour dark or light periods, and a separate group was treated with L-cis-diltiazem at the beginning of a 12-hour dark period. Hypoxia was assessed by deposition of hypoxyprobe (HP) and HP-protein adducts were localized by immunohistochemistry and quantified using ELISA. Also, hypoxia-regulated gene expression and transcriptional activity were assessed alongside vascular density.

RESULTS. Hypoxia was observed in the inner nuclear and ganglion cell layers in WT retina and was significantly reduced in Rho (-/-) mice (P < 0.05). Retinal hypoxia was significantly increased during dark adaptation in WT mice (P < 0.05), whereas no change was observed in Rho(-/-) or with L-cis-diltiazem-treated WT mice. Hypoxia-inducible factor (HIF)-1 alpha DNA-binding and VEGF mRNA expression in Rho(-/-) retina was significantly reduced in unison with outer retinal depletion (P < 0.05). Retina from the Rho(-/-) mice displayed an extensive intraretinal vascular network after 6 months, although there was evidence that capillary density was depleted in comparison with that in WT retinas.

CONCLUSIONS. Relative hypoxia occurs in the inner retina especially during dark adaptation. Photoreceptor loss reduces retinal oxygen usage and hypoxia which corresponds with attenuation of the retinal microvasculature. These studies suggest that in normal physiological conditions and diurnal cycles the adult retina exists in a state of borderline hypoxia, making this tissue particularly susceptible to even subtle reductions in perfusion.

Sustained intraocular VEGF neutralization results in retinal neurodegeneration in the Ins2Akita diabetic mouse

Current therapies that target vascular endothelial growth factor (VEGF) have become a mainstream therapy for the management of diabetic macular oedema. The treatment involves monthly repeated intravitreal injections of VEGF inhibitors. VEGF is an important growth factor for many retinal cells, including different types of neurons. In this study, we investigated the adverse effect of multiple intravitreal anti-VEGF injections (200 ng/μl/eye anti-mouse VEGF164, once every 2 weeks totalling 5-6 injections) to retinal neurons in Ins2(Akita) diabetic mice. Funduscopic examination revealed the development of cotton wool spot-like lesions in anti-VEGF treated Ins2(Akita) mice after 5 injections. Histological investigation showed focal swellings of retinal nerve fibres with neurofilament disruption. Furthermore, anti-VEGF-treated Ins2(Akita) mice exhibited impaired electroretinographic responses, characterized by reduced scotopic a- and b-wave and oscillatory potentials. Immunofluorescent staining revealed impairment of photoreceptors, disruptions of synaptic structures and loss of amacrine and retinal ganglion cells in anti-VEGF treated Ins2(Akita) mice. Anti-VEGF-treated WT mice also presented mild amacrine and ganglion cell death, but no overt abnormalities in photoreceptors and synaptic structures. At the vascular level, exacerbated albumin leakage was observed in anti-VEGF injected diabetic mice. Our results suggest that sustained intraocular VEGF neutralization induces retinal neurodegeneration and vascular damage in the diabetic eye.

Metabolic effects of sub-chronic ablation of the incretin receptors by daily administration of (Pro(3))GIP and exendin(9-39)amide in obese diabetic (ob/ob) mice

Effects of chemical ablation of the GIP and GLP-1 receptors on metabolic aspects of obesity-diabetes were investigated using the stable receptor antagonists (Pro(3))GIP and exendin(9-39)amide. Ob/ob mice received a daily i.p. injection of saline vehicle, (Pro(3))GIP, exendin(9-39)amide or a combination of both peptides over a 14-day period. Non-fasting plasma glucose levels were significantly (p <0.05) lower in (Pro(3))GIP-treated mice compared to control mice after just 9 days of treatment. (Pro(3))GIP-treated mice also displayed significantly lower plasma glucose concentrations in response to feeding and intraperitoneal administration of either glucose or insulin (p <0.05 to p <0.001). The (Pro(3))GIP-treated group also exhibited significantly (p <0.05) reduced pancreatic insulin content. Acute administration of exendin(9-39) amide immediately prior to re-feeding completely annulled the beneficial effects of sub-chronic (Pro(3))GIP treatment, but non-fasting concentrations of active GLP-1 were unchanged. Combined sub-chronic administration of (Pro(3)GIP) with exendin(9-39)amide revealed no beneficial effects. Similarly, daily administration of exendin(9-39)amide alone had no significant effects on any of the metabolic parameters measured. These studies highlight an important role for GIP in obesity-related forms of diabetes, suggesting the possible involvement of GLP-1 in the beneficial actions of GIP receptor antagonism.

Effects of the novel (Pro(3))GIP antagonist and exendin(9-39)amide on GIP- and GLP-1-induced cyclic AMP generation, insulin secretion and postprandial insulin release in obese diabetic (ob/ob) mice: evidence that GIP is the major physiological incretin

AIMS/HYPOTHESIS: This study examined the biological effects of the GIP receptor antagonist, (Pro3)GIP and the GLP-1 receptor antagonist, exendin(9-39)amide.

METHODS: Cyclic AMP production was assessed in Chinese hamster lung fibroblasts transfected with human GIP or GLP-1 receptors, respectively. In vitro insulin release studies were assessed in BRIN-BD11 cells while in vivo insulinotropic and glycaemic responses were measured in obese diabetic ( ob/ ob) mice.

RESULTS: In GIP receptor-transfected fibroblasts, (Pro(3))GIP or exendin(9-39)amide inhibited GIP-stimulated cyclic AMP production with maximal inhibition of 70.0+/-3.5% and 73.5+/-3.2% at 10(-6) mol/l, respectively. In GLP-1 receptor-transfected fibroblasts, exendin(9-39)amide inhibited GLP-1-stimulated cyclic AMP production with maximal inhibition of 60+/-0.7% at 10(-6) mol/l, whereas (Pro(3))GIP had no effect. (Pro(3))GIP specifically inhibited GIP-stimulated insulin release (86%; p<0.001) from clonal BRIN-BD11 cells, but had no effect on GLP-1-stimulated insulin release. In contrast, exendin(9-39)amide inhibited both GIP and GLP-1-stimulated insulin release (57% and 44%, respectively; p<0.001). Administration of (Pro(3))GIP, exendin(9-39)amide or a combination of both peptides (25 nmol/kg body weight, i.p.) to fasted (ob/ob) mice decreased the plasma insulin responses by 42%, 54% and 49%, respectively (p<0.01 to p<0.001). The hyperinsulinaemia of non-fasted (ob/ob) mice was decreased by 19%, 27% and 18% (p<0.05 to p<0.01) by injection of (Pro3)GIP, exendin(9-39)amide or combined peptides but accompanying changes of plasma glucose were small.

CONCLUSIONS/INTERPRETATION: These data show that (Pro(3))GIP is a specific GIP receptor antagonist. Furthermore, feeding studies in one commonly used animal model of obesity and diabetes, (ob/ob) mice, suggest that GIP is the major physiological component of the enteroinsular axis, contributing approximately 80% to incretin-induced insulin release.

Comparison of the anti-diabetic effects of GIP- and GLP-1-receptor activation in obese diabetic (ob/ob) mice: studies with DPP IV resistant N-AcGIP and exendin(1-39)amide

Background The two major incretin hormones, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are being actively explored as anti-diabetic agents because they lower blood glucose through multiple mechanisms. The rapid inactivation of GIP and GLP-1 by the ubiquitous enzyme, dipeptidyl peptidase IV (DPP IV) makes their biological actions short-lived, but stable agonists such as N-acetylated GIP (N-AcGIP) and exendin(1-39)amide have been advocated as stable and specific GIP and GLP-1 analogues.

Vitamin C supplementation decreases insulin glycation and improves glucose homeostasis in obese hyperglycemic (ob/ob) mice

The effects of dietary vitamin C supplementation on glucose homeostasis and insulin glycation were examined in adult lean and obese hyperglycemic (ob/ob) mice. In lean mice, supplementation of the drinking water with vitamin C (25 g/L) for 14 days did not affect food intake, fluid intake, glycated hemoglobin, plasma glucose, or plasma insulin concentrations. Total pancreatic insulin content and the percentage of glycated pancreatic insulin were also similar to control lean mice. In ob/ob mice, vitamin C supplementation caused significant reductions by 26% to 48% in food intake and fluid intake, glycated hemoglobin, plasma glucose, and insulin concentrations compared with untreated control ob/ob mice. The total insulin content and the extent of insulin glycation in the pancreas of ob/ob mice were also significantly decreased by 42% to 45% after vitamin C supplementation. This change was accompanied by a significant 80% decrease in the percentage of glycated insulin in the circulation of vitamin C- supplemented ob/ob mice. These data demonstrate that vitamin C supplementation can decrease insulin glycation and ameliorate aspects of the obesity-diabetes syndrome in ob/ob mice. Copyright 2002, Elsevier Science (USA). All rights reserved.

Detection of glycated gastric inhibitory polypeptide within the intestines of diabetic obese (ob/ob) mice

Gastric inhibitory polypeptide (GIP) is produced within endocrine cells of the small intestine and released into the circulation upon nutrient ingestion. This study has quantified the levels of this insulinotropic peptide in the intestines of lean and diabetic obese ob/ob mice and estimated the proportion that is glycated. The total intestinal GIP concentration and content of the diabetic mice were significantly greater (p

Evaluation of the antidiabetic activity of DPP IV resistant N-terminally modified versus mid-chain acylated analogues of glucose-dependent insulinotropic polypeptide

Glucose dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone with therapeutic potential for type 2 diabetes due to its insulin-releasing and antihyperglycaemic actions. However, development of GIP-based therapies is limited by N-terminal degradation by DPP IV resulting in a very short circulating half-life. Numerous GIP analogues have now been generated exhibiting DPP IV resistance and extended bioactivity profiles. In this study, we report a direct comparison of the long-term antidiabetic actions of three such GIP molecules, N-AcGIP, GIP(LyS(37)PAL) and N-AcGIP(LyS(37)PAL) in obese diabetic (ob/ob) mice. An extended duration of action of each GIP analogue was demonstrated prior to examining the effects of once daily injections (25 nmol kg(-1) body weight) over a 14-day period. Administration of either N-AcGIP, GIP(LyS(37)PAL) or N-AcGIP(LyS37PAL) significantly decreased non-fasting plasma glucose and improved glucose tolerance compared to saline treated controls. All three analogues significantly enhanced glucose and nutrient-induced insulin release, and improved insulin sensitivity. The metabolic and insulin secretory responses to native GIP were also enhanced in 14-day analogue treated mice, revealing no evidence of GIP-receptor desensitization. These effects were accompanied by significantly enhanced pancreatic insulin following N-AcGIP(Lys(37)PAL) and increased islet number and islet size in all three groups. Body weight, food intake and circulating glucagon were unchanged. These data demonstrate the therapeutic potential of once daily injection of enzyme resistant GIP analogues and indicate that N-AcGIP is equally as effective as related palmitate derivatised analogues of GIP. (c) 2006 Elsevier Inc. All rights reserved.