Dietetics, Nutrition and Biological Sciences
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Item Daily administration of the GIP-R antagonist (Pro3)GIP in streptozotocin-induced diabetes suggests that insulin-dependent mechanisms are critical to anti-obesity-diabetes actions of (Pro3)GIP(Wiley, 2008-04) McClean, P. L.; Gault, V. A.; Irwin, N.; McCluskey, Jane T.; Flatt, P. R.AIM: Glucose-dependent insulinotropic polypeptide-receptor (GIP-R) antagonism using (Pro3)GIP improves glucose tolerance and ameliorates insulin resistance and abnormalities of islet structure and function in a commonly used model of obesity-diabetes, namely ob/ob mice. The effect of GIP-R antagonism in a streptozotocin (STZ)-induced model of insulin deficiency has not been evaluated. The present study has investigated the effects of daily administration of (Pro(3))GIP to STZ-treated mice. METHODS: Swiss TO mice received once-daily injection of (Pro3)GIP (25 nmol/kg body weight) or saline 4 days prior to and 16 days after injection of STZ, and effects on metabolic parameters and islet architecture were assessed. RESULTS: (Pro3)GIP treatment had no significant effect on hyperphagia or body weight loss. However, hyperglycaemia and glycated haemoglobin were worsened, glucose tolerance further decreased and insulin sensitivity was impaired by (Pro3)GIP. These effects were observed on an STZ-induced background characterized by severe reductions of circulating insulin, beta-cell mass and pancreatic insulin stores. CONCLUSIONS: These data indicate that the beneficial actions of the GIP-R antagonist, (Pro3)GIP, in obesity-diabetes appear to be largely mediated through insulin-dependent mechanisms that merit further investigation.Item A stable analogue of glucose-dependent insulinotropic polypeptide, GIP(LysPAL16), enhances functional differentiation of mouse embryonic stem cells into cells expressing islet-specific genes and hormones(De Gruyter, 2006-07) Marenah, Lamin; McCluskey, Jane T.; Abdel-Wahab, Yasser H.; O'Harte, Finbarr P. M.; McClenaghan, Neville H.; Flatt, Peter R.Embryonic stem (ES) cells can be differentiated into insulin-producing cells by conditioning the culture media. However, the number of insulin-expressing cells and amount of insulin released is very low. Glucose-dependent insulinotropic polypeptide (GIP) enhances the growth and differentiation of pancreatic beta-cells. This study examined the potential of the stable analogue GIP(LysPAL16) to enhance the differentiation of mouse ES cells into insulin-producing cells using a five-stage culturing strategy. Semi-quantitative PCR indicated mRNA expression of islet development markers (nestin, Pdx1, Nkx6.1, Oct4), mature pancreatic beta-cell markers (insulin, glucagon, Glut2, Sur1, Kir6.1) and the GIP receptor gene GIP-R in undifferentiated (stage 1) cells, with increasing levels in differentiated stages 4 and 5. IAPP and somatostatin genes were only expressed in differentiated stages. Immunohistochemical studies confirmed the presence of insulin, glucagon, somatostatin and IAPP in differentiated ES cells. After supplementation with GIP(LysPAL16), ES cells at stage 4 released insulin in response to secretagogues and glucose in a concentration-dependent manner, with 35-100% increases in insulin release. Cellular C-peptide content also increased by 45% at stages 4 and 5. We conclude that the stable GIP analogue enhanced differentiation of mouse ES cells towards a phenotype expressing specific beta-cell genes and releasing insulin.Item Prolonged exposure to homocysteine results in diminished but reversible pancreatic _-cell responsiveness to insulinotropic agents(WIley, 2007-05) Patterson, Steven; Scullion, Siobhan M. J.; McCluskey, Jane T.; Flatt, Peter R.; McClenaghan, Neville H.BACKGROUND: Plasma homocysteine levels may be elevated in poorly controlled diabetes with pre-existing vascular complications and/or nephropathy. Since homocysteine has detrimental effects on a wide diversity of cell types, the present study examined the effects of long-term homocysteine exposure on the secretory function of clonal BRIN-BD11 beta-cells. METHODS: Acute insulin secretory function, cellular insulin content and viability of BRIN-BD11 cells were assessed following long-term (18 h) exposure to homocysteine in culture. RT-PCR and Western blot analysis were used to determine the expression of key beta-cell genes and proteins. Cells were cultured for a further 18 h without homocysteine to determine any long-lasting effects. RESULTS: Homocysteine (250-1000 micromol/L) exposure reduced insulin secretion at both moderate (5.6 mmol/L) and stimulatory (16.7 mmol/L) glucose by 48-63%. Similarly, insulin secretory responsiveness to stimulatory concentrations of alanine, arginine, 2-ketoisocaproate, tolbutamide, KCl, elevated Ca2+, forskolin and PMA, GLP-1, GIP and CCK-8 were reduced by 11-62% following culture with 100-250 micromol/L homocysteine. These inhibitory effects could not simply be attributed to changes in cellular insulin content, cell viability, H2O2 generation or any obvious alterations of gene/protein expression for insulin, glucokinase, GLUT2, VDCC, or Kir6.2 and SUR1. Additional culture for 18 h in standard culture media after homocysteine exposure restored secretory responsiveness to all agents tested. CONCLUSION: These findings suggest that long-term exposure to high homocysteine levels causes a reversible impairment of pancreatic beta-cell insulinotropic pathways. The in vivo actions of hyperhomocysteinaemia on islet cell function merit investigation.Item Effects of Subchronic Treatment With the Long-Acting Glucose-Dependent Insulinotropic Polypeptide Receptor Agonist, N-AcGIP, on Glucose Homeostasis in Streptozotocin-Induced Diabetes(Wolters Kluwer, 2007-07-01) Gault, Victor A.; McClean, Paula L.; Irwin, Nigel; Power, Gavin J.; McCluskey, Jane T.; Flatt, Peter R.OBJECTIVES: N-AcGIP is a potent and dipeptidylpeptidase IV-resistant analogue of glucose-dependent insulinotropic polypeptide with significantly improved antidiabetic actions in type 2 diabetes. The present study investigated the effects of subchronic treatment with N-AcGIP on glucose homeostasis in a type 1 model, namely, streptozotocin (STZ)-induced diabetic mice. METHODS: Swiss TO mice given a single intraperitoneal injection of STZ (150 mg/kg body weight) received once-daily injection of N-AcGIP (25 nmol/kg body weight) or saline for 20 days and effects on metabolic parameters and islet architecture assessed. RESULTS: Daily injection of N-AcGIP for 20 days did not significantly alter the characteristic STZ-induced changes of pancreatic insulin content, body weight, food intake, glucose, and glycated hemoglobin levels. Glucose tolerance and insulin sensitivity were also unchanged by N-AcGIP treatment. Circulating insulin was undetectable, and the number of intact islets and insulin expression was greatly reduced in both groups. Some proliferative activity was identified by 5-bromo-2-deoxyuridine staining in the pancreas, but this and expression of glucagon and somatostatin were similar in the 2 groups. CONCLUSIONS: These data indicate that subchronic treatment with the long-acting glucose-dependent insulinotropic polypeptide receptor agonist, N-AcGIP, does not have beneficial effects in insulin-deficient STZ-diabetic mice. This supports the primary antidiabetic action of this analogue in type 2 diabetes as stimulation of beta-cell function and insulin secretion.Item Chronic exposure to tolbutamide and glibenclamide impairs insulin secretion but not transcription of KATP channel components(Elsevier, 2004-07) Ball, A. J.; McCluskey, Jane T.; Flatt, P. R.; McClenaghan, N. H.Clonal insulin-secreting BRIN-BD11 cells were used to examine effects of chronic 72-144 h exposure to the sulphonylureas tolbutamide and glibenclamide on insulin release, cellular insulin content, and mRNA levels of the Kir6.2 and SUR1 subunits of the beta-cell K(ATP) channel. Chronic exposure for 72-144 h to 5-100 microM tolbutamide and glibenclamide resulted in a time- and concentration-dependent irreversible decline in sulphonylurea-induced insulin secretion. In contrast, the decline in cellular insulin content induced by chronic exposure to high concentrations of sulphonylureas was readily reversible. Chronic exposure to tolbutamide or glibenclamide had no effect upon transcription of the Kir6.2 or SUR1 subunits of the pancreatic beta-cell K(ATP) channel. Whilst further studies are required to understand the precise nature of the chronic interactions of sulphonylurea with the insulin exocytotic mechanism, these observations may partially explain the well-known progressive failure of sulphonylurea therapy in type 2 diabetes.Item N-acetyl-GLP-1: a DPP IV-resistant analogue of glucagon-like peptide-1 (GLP-1) with improved effects on pancreatic _-cell-associated gene expression(Wiley, 2004) Liu, H. K.; Green, B. D.; Gault, V. A.; McCluskey, Jane T.; McLenaghan, N. H.; O'Harte, F. P. M.; Flatt, P. R.Glucagon-like peptide-1(7-36)amide (GLP-1) is a key insulinotropic hormone with the reported potential to differentiate non-insulin secreting cells into insulin-secreting cells. The short biological half-life of GLP-1 after cleavage by dipeptidylpeptidase IV (DPP IV) to GLP-1(9-36)amide is a major therapeutic drawback. Several GLP-1 analogues have been developed with improved stability and insulinotropic action. In this study, the N-terminally modified GLP-1 analogue, N-acetyl-GLP-1, was shown to be completely resistant to DPP IV, unlike native GLP-1, which was rapidly degraded. Furthermore, culture of pancreatic ductal ARIP cells for 72 h with N-acetyl-GLP-1 indicated a greater ability to induce pancreatic beta-cell-associated gene expression, including insulin and glucokinase. Further investigation of the effects of stable GLP-1 analogues on beta-cell differentiation is required to assess their potential in diabetic therapy.Item Function of a long-term, GLP-1-treated, insulin-secreting cell line is improved by preventing DPP IV-mediated degradation of GLP-1(Wiley, 2005-09) Green, B. D.; Liu, H. K.; McCluskey, Jane T.; Duffy, N. A.; O'Harte, F. P. M.; McClenaghan, N. H.; Flatt, P. R.Glucagon-like peptide-1 (GLP-1) is an important insulinotropic hormone with potential in the treatment of type 2 diabetes. However, the short biological half-life of the peptide after cleavage by dipeptidylpeptidase IV (DPP IV) is a major limitation. Inhibition of DPP IV activity and the development of resistant GLP-1 analogues is the subject of ongoing research. In this study, we determined cell growth, insulin content, insulin accumulation and insulin secretory function of a insulin-secreting cell line cultured for 3 days with either GLP-1, GLP-1 plus the DPP IV inhibitor diprotin A (DPA) or stable N-acetyl-GLP-1. Native GLP-1 was rapidly degraded by DPP IV during culture with accumulation of the inactive metabolite GLP-1(9-36)amide. Inclusion of DPA or use of the DPP IV-resistant analogue, N-acetyl-GLP-1, improved cellular function compared to exposure to GLP-1 alone. Most notably, basal and accumulated insulin secretion was enhanced, and glucose responsiveness was improved. However, prolonged GLP-1 treatment resulted in GLP-1 receptor desensitization regardless of DPP IV status. The results indicate that prevention of DPP IV action is necessary for beneficial effects of GLP-1 on pancreatic beta cells and that prolonged exposure to GLP-1(9-36)amide may be detrimental to insulin secretory function. These observations also support the ongoing development of DPP-IV-resistant forms of GLP-1, such as N-acetyl-GLP-1.Item Deleterious Effects of Supplementation with Dehydroepiandrosterone Sulphate or Dexamethasone on Rat Insulin-Secreting Cells Under In Vitro Culture Condition(Highwire, 2006-02) Liu, Hui-Kang; Green, Brian D.; McClenaghan, Neville H.; McCluskey, Jane T.; Flatt, Peter R.Dehydroepiandrosterone (DHEA) and glucocorticoids are steroid hormones synthesised in the adrenal cortex. Administration of DHEA, its sulphate derivative, DHEAS, and more controversially dexamethasone (DEX), a synthetic glucocorticoid, have beneficial effects in diabetic animals. Cultivating BRIN-BD11 cells for 3 days with either DHEAS (30 muM) or DEX (100 nM), reduced total cell number and reduced cell viability and cellular insulin content. DHEAS-treated cells had poor glucose responsiveness and regulated insulin release, coupled with reduced basal insulin release. In contrast, DEX-treated cells lacked responsiveness to glucose and membrane depolarisation, and both protein kinase A (PKA) and protein kinase C (PKC) secretory pathways were desensitised. Therefore, we conclude that this steroid hormone and synthetic glucocorticoid are not beneficial to pancreatic beta-cells in vitro.Item Chemical ablation of gastric inhibitory polypeptide receptor action by daily (Pro3)GIP administration improves glucose tolerance and ameliorates insulin resistance and abnormalities of islet structure in obesity-related diabetes.(American Diabetes Association, 2005-08) Gault, Victor A.; Irwin, Nigel; Green, Brian D.; McCluskey, Jane T.; Greer, Brett; Bailey, Clifford J.; Harriott, Patrick; O'harte, Finbarr P. M.; Flatt, Peter R.Glucose-dependent insulinotropic polypeptide (gastric inhibitory polypeptide [GIP]) is an important incretin hormone secreted by endocrine K-cells in response to nutrient ingestion. In this study, we investigated the effects of chemical ablation of GIP receptor (GIP-R) action on aspects of obesity-related diabetes using a stable and specific GIP-R antagonist, (Pro3)GIP. Young adult ob/ob mice received once-daily intraperitoneal injections of saline vehicle or (Pro3)GIP over an 11-day period. Nonfasting plasma glucose levels and the overall glycemic excursion (area under the curve) to a glucose load were significantly reduced (1.6-fold; P < 0.05) in (Pro3)GIP-treated mice compared with controls. GIP-R ablation also significantly lowered overall plasma glucose (1.4-fold; P < 0.05) and insulin (1.5-fold; P < 0.05) responses to feeding. These changes were associated with significantly enhanced (1.6-fold; P < 0.05) insulin sensitivity in the (Pro3)GIP-treated group. Daily injection of (Pro3)GIP reduced pancreatic insulin content (1.3-fold; P < 0.05) and partially corrected the obesity-related islet hypertrophy and beta-cell hyperplasia of ob/ob mice. These comprehensive beneficial effects of (Pro3)GIP were reversed 9 days after cessation of treatment and were independent of food intake and body weight, which were unchanged. These studies highlight a role for GIP in obesity-related glucose intolerance and emphasize the potential of specific GIP-R antagonists as a new class of drugs for the alleviation of insulin resistance and treatment of type 2 diabetes.Item Control of cell cycle gene expression in bone development and during c-Fos-induced osteosarcoma formation.(Wiley, 1998) Sunters, A.; McCluskey, Jane T.; Grigoriadis, A. E.We have used c-Fos transgenic mice which develop osteosarcomas to determine the expression patterns of cyclins, cyclin-dependent kinases (CDKs), and cyclin-dependent kinase inhibitors (CKIs) in different bone cell populations in order to define the potential mechanisms of c-Fos transformation. Immunohistochemical analysis in embryonic and early postnatal bone demonstrated that cyclin E and its kinase partner CDK2 were expressed specifically in bone-forming osteoblasts. Cyclin D1 expression was absent despite high levels of CDK4 and CDK6, and the CKI p27 was expressed in chondrocytes, osteoclasts, and at lower levels in osteoblasts. Following activation of the c-fos transgene in vivo and before overt tumor formation, cyclin D1 expression increased dramatically and was colocalized with exogenous c-Fos protein specifically in osteoblasts and chondrocytes, but not in osteoclasts. Prolonged activation of c-Fos resulted in osteosarcoma formation wherein the levels of cyclin D1, cyclin E, and CDKs 2, 4, and 6 were high in a wide spectrum of malignant cell types, especially in transformed osteoblasts. The CKI p27 was expressed at very high levels in bone-resorbing osteoclasts, and to a lesser extent in chondrocytes and osteoblasts. These in vivo observations suggest that cyclin D1 may be a target for c-Fos action and that elevation of cyclin D1 in osteoblasts which already express cyclin E/CDK2 and the cyclin D1 partners CDKs-4 and 6, may predispose cells to uncontrolled cell growth leading to osteosarcoma development. This study implicates altered cell cycle control as a potential mechanism through which c-Fos causes osteoblast transformation and bone tumor formation.
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