Effects of first-line diabetes therapy with biguanides, sulphonylurea and thiazolidinediones on the differentiation, proliferation and apoptosis of islet cell populations.

AIMS: Metformin, rosiglitazone and sulfonylureas enhance either insulin action or secretion and thus have been used extensively as early stage anti-diabetic medication, independently of the aetiology of the disease. When administered to newly diagnosed diabetes patients, these drugs produce variable results. Here, we examined the effects of the three early stage oral hypoglycaemic agents in mice with diabetes induced by multiple low doses of streptozotocin, focusing specifically on the developmental biology of pancreatic islets. METHODS: Streptozotocin-treated diabetic mice expressing a fluorescent reporter specifically in pancreatic islet α-cells were administered the biguanide metformin (100 mg/kg), thiazolidinedione rosiglitazone (10 mg/kg), or sulfonylurea tolbutamide (20 mg/kg) for 10 days. We assessed the impact of the treatment on metabolic status of the animals as well as on the morphology, proliferative potential and transdifferentiation of pancreatic islet cells, using immunofluorescence. RESULTS: The effect of the therapy on the islet cells varied depending on the drug and included enhanced pancreatic islet ß-cell proliferation, in case of metformin and rosiglitazone; de-differentiation of α-cells and ß-cell apoptosis with tolbutamide; increased relative number of ß-cells and bi-hormonal insulin + glucagon + cells with metformin. These effects were accompanied by normalisation of food and fluid intake with only minor effects on glycaemia at the low doses of the agents employed. CONCLUSIONS: Our data suggest that metformin and rosiglitazone attenuate the depletion of the ß-cell pool in the streptozotocin-induced diabetes, whereas tolbutamide exacerbates the ß-cell apoptosis, but is likely to protect ß-cells from chronic hyperglycaemia by directly elevating insulin secretion.

Magainin-AM2 improves glucose homeostasis and beta cell function in high-fat fed mice.

BACKGROUND: Magainin-AM2, a previously described amphibian host-defense peptide, stimulates insulin- and glucagon-like peptide-1-release in vitro. This study investigated anti-diabetic effects of the peptide in mice with diet-induced obesity and glucose intolerance. METHODS: Male National Institute of Health Swiss mice were maintained on a high-fat diet for 12-weeks prior to the daily treatment with magainin-AM2. Various indices of glucose tolerance were monitored together with insulin secretory responsiveness of islets at conclusion of study. RESULTS: Following twice daily treatment with magainin-AM2 for 15 days, no significant difference in body weight and food intake was observed compared with saline-treated high fat control animals. However, non-fasting blood glucose was significantly (P<0.05) decreased while plasma insulin concentrations were significantly (P<0.05) increased. Oral and intraperitoneal glucose tolerance and insulin secretion following glucose administration via both routes were significantly (P<0.05) enhanced. The peptide significantly (P<0.001) improved insulin sensitivity as well as the beta cell responses of islets isolated from treated mice to a range of insulin secretagogues. Oxygen consumption, CO2production, respiratory exchange ratio and energy expenditure were not significantly altered by sub-chronic administration of magainin-AM2 but a significant (P<0.05) reduction in fat deposition was observed. CONCLUSION: These results indicate that magainin-AM2 improves glucose tolerance, insulin sensitivity and islet beta cells secretory responsiveness in mice with obesity-diabetes. GENERAL SIGNIFICANCE: The activity of magainin-AM2 suggests the possibility of exploiting this peptide for treatment of type 2 diabetes.

Dogfish glucagon analogues counter hyperglycaemia and enhance both insulin secretion and action in diet-induced obese diabetic mice.

AIMS: To investigate the antidiabetic actions of three dogfish glucagon peptide analogues [known glucagon-like peptide-1 and glucagon receptor co-agonists] after chronic administration in diet-induced high-fat-diet-fed diabetic mice. MATERIALS AND METHODS: National Institutes of Health Swiss mice were pre-conditioned to a high-fat diet (45% fat) for 100 days, and control mice were fed a normal diet (10% fat). Normal diet control and high-fat-fed control mice received twice-daily intraperitoneal (i.p.) saline injections, while the high-fat-fed treatment groups (n = 8) received twice-daily injections of exendin-4(1-39), [S2a]dogfish glucagon, [S2a]dogfish glucagon exendin-4(31-39) or [S2a]dogfish glucagon-Lys(30) -γ-glutamyl-PAL (25 nmol/kg body weight) for 51 days. RESULTS: After dogfish glucagon analogue treatment, there was a rapid and sustained decrease in non-fasting blood glucose and an associated insulinotropic effect (analysis of variance, p < .05 to <.001) compared with saline-treated high-fat-fed controls. All peptide treatments significantly improved i.p. and oral glucose tolerance with concomitant increased insulin secretion compared with saline-treated high-fat-fed controls (p <.05 to <.001). After chronic treatment, no receptor desensitization was observed but insulin sensitivity was enhanced for all peptide-treated groups (p < .01 to <.001) except [S2a]dogfish glucagon. Both exendin-4 and [S2a]dogfish glucagon exendin-4(31-39) significantly reduced plasma triglyceride concentrations compared with those found in lean controls (p = .0105 and p = .0048, respectively). Pancreatic insulin content was not affected by peptide treatments but [S2a]dogfish glucagon and [S2a]dogfish glucagon exendin-4(31-39) decreased pancreatic glucagon by 28%-34% (p = .0221 and p = .0075, respectively). The percentage of ß-cell area within islets was increased by exendin-4 and peptide analogue treatment groups compared with high-fat-fed controls and the ß-cell area decreased (p < .05 to <.01). CONCLUSIONS: Overall, dogfish glucagon co-agonist analogues had several beneficial metabolic effects, showing therapeutic potential for type 2 diabetes.

Double incretin receptor knock-out (DIRKO) mice present with alterations of trabecular and cortical micromorphology and bone strength.

UNLABELLED: A role for gut hormone in bone physiology has been suspected. We evidenced alterations of microstructural morphology (trabecular and cortical) and bone strength (both at the whole-bone--and tissue-level) in double incretin receptor knock-out (DIRKO) mice as compared to wild-type littermates. These results support a role for gut hormones in bone physiology. INTRODUCTION: The two incretins, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), have been shown to control bone remodeling and strength. However, lessons from single incretin receptor knock-out mice highlighted a compensatory mechanism induced by elevated sensitivity to the other gut hormone. As such, it is unclear whether the bone alterations observed in GIP or GLP-1 receptor deficient animals resulted from the lack of a functional gut hormone receptor, or by higher sensitivity for the other gut hormone. The aims of the present study were to investigate the bone microstructural morphology, as well as bone tissue properties, in double incretin receptor knock-out (DIRKO) mice. METHODS: Twenty-six-week-old DIRKO mice were age- and sex-matched with wild-type (WT) littermates. Bone microstructural morphology was assessed at the femur by microCT and quantitative X-ray imaging, while tissue properties were investigated by quantitative backscattered electron imaging and Fourier-transformed infrared microscopy. Bone mechanical response was assessed at the whole-bone- and tissue-level by 3-point bending and nanoindentation, respectively. RESULTS: As compared to WT animals, DIRKO mice presented significant augmentations in trabecular bone mass and trabecular number whereas bone outer diameter, cortical thickness, and cortical area were reduced. At the whole-bone-level, yield stress, ultimate stress, and post-yield work to fracture were significantly reduced in DIRKO animals. At the tissue-level, only collagen maturity was reduced by 9 % in DIRKO mice leading to reductions in maximum load, hardness, and dissipated energy. CONCLUSIONS: This study demonstrated the critical role of gut hormones in controlling bone microstructural morphology and tissue properties.

Sitagliptin, a dipeptidyl peptidase-4 inhibitor, improves recognition memory, oxidative stress and hippocampal neurogenesis and upregulates key genes involved in cognitive decline.

AIM: To examine whether prolonged dipeptidyl peptidase-4 (DPP-4) inhibition can reverse learning and memory impairment in high-fat-fed mice. METHODS: High-fat-fed mice received oral sitagliptin (50 mg/kg body weight) once daily or saline vehicle over 21 days. An additional group of mice on standard chow received saline vehicle. Energy intake, body weight, glucose and insulin concentrations were measured at regular intervals. Glucose tolerance, insulin sensitivity, novel object recognition, DPP-4 activity, hormone analysis, hippocampal gene expression and histology were performed. RESULTS: Sitagliptin decreased circulating DPP-4 activity and improved glucose tolerance, glucose-stimulated insulin secretion and insulin sensitivity, and reduced plasma triglycerides and cholesterol levels. DPP-4 inhibition improved recognition memory (1.2-fold increase) without affecting hypermoteric activity or anxiety levels. Improvement in memory and learning was linked to reduced immunostaining for 8-oxoguanine and increased doublecortin staining in the hippocampus, which were indicative of reduced brain oxidative stress and increased hippocampal neurogenesis, respectively. These effects were associated with significant upregulation of hippocampal gene expression of glucagon-like peptide-1 (GLP-1) receptor, glucose-dependent insulinotropic polypeptide receptor, synaptophysin, sirtuin 1, glycogen synthase kinase 3ß, superdioxide mutase 2, nuclear factor (erythroid-derived 2)-like 2 and vascular endothelial growth factor. Total plasma and brain GLP-1 concentrations were significantly increased after sitagliptin therapy, whereas DPP-4 activity in brain tissue was not altered. CONCLUSION: These studies show that sitagliptin can reverse memory impairment in high-fat-fed mice and is also associated with improved insulin sensitivity, enhanced hippocampal neurogenesis and reduced oxidative stress. DPP-4 inhibitors may therefore exhibit dual benefits by improving metabolic control and reducing the decline in cognitive function.

Sustained treatment with a stable long-acting oxyntomodulin analogue improves metabolic control and islet morphology in an experimental model of type 1 diabetes.

AIM: To assess the therapeutic benefits of regulatory peptides other than insulin, which have to date received limited consideration in the context of type 1 diabetes. METHODS: We assessed the effects of subchronic administration of the stable, oxyntomodulin (Oxm) analogue, (d-Ser(2) )Oxm[Lys(38) -γ-glu-PAL], for 28 days in streptozotocin (STZ)-induced insulin-deficient diabetic mice. RESULTS: Twice-daily injection with (d-Ser(2) )Oxm[Lys(38) -γ-glu-PAL] significantly countered the excessive food and fluid intake in STZ-induced diabetic mice, and maintained normal body weight. Lean body mass was normalized, whilst fat mass was significantly increased compared with control STZ-induced diabetic mice. In addition, circulating glucose was significantly reduced by the Oxm analogue, whilst plasma and pancreatic insulin concentrations were increased and glucagon decreased by day 28. Plasma lipid profile was normalized by (d-Ser(2) )Oxm[Lys(38) -γ-glu-PAL] administration and circulating amylase was not significantly altered by induction of diabetes or Oxm analogue therapy. This was associated with significantly improved glucose tolerance and insulin secretion. Peripheral insulin sensitivity was also significantly improved by Oxm analogue treatment. Histological examination of pancreata showed beneficial elevations of total islet and ß-cell area, associated with an increase in the number of smaller-sized islets. Further analysis revealed enhanced islet cell proliferation relative to apoptosis in Oxm analogue-treated mice. CONCLUSION: These studies emphasize the potential of stable Oxm-based peptides, such as (d-Ser(2) )Oxm[Lys(38) -γ-glu-PAL], as therapeutic agents for insulin-deficient type 1 diabetes.

Pharmacological characterization and antidiabetic activity of a long-acting glucagon-like peptide-1 analogue conjugated to an antithrombin III-binding pentasaccharide.

AIMS: To examine the biological characteristics of a novel glucagon-like peptide-1 (GLP-1) conjugate, in which an antithrombin III (ATIII)-binding pentasaccharide is conjugated to d-Ala(8) GLP-1 using a tetraethylene glycol linker. METHODS: We assessed GLP-1 receptor binding, cAMP generation and insulin secretory activity of the GLP-1 conjugate in vitro. Circulating half-life, glucose homeostatic and subchronic therapeutic effectiveness were then examined in vivo. RESULTS: The half-life of the GLP-1 conjugate in mice was ∼11 h. In vitro insulin secretion from clonal ß cells and islets was increased (p < 0.001) by the conjugate. The conjugate had half maximum effective concentration values of 1.3 × 10(-7) and 9.9 × 10(-8) M for displacement of (125) I-GLP-1 in competitive GLP-1 receptor binding and cAMP generation, respectively. Glucose tolerance in normal mice, immediately and 4 h after conjugate injection, resulted in significant (p < 0.001) improvements in blood glucose. These effects persisted for >48 h after administration. Daily treatment (21 days) of high-fat-fed and ob/ob mice with 25 nmol/kg conjugate resulted in significant improvement in glucose tolerance (p < 0.001) and reductions in glycated haemoglobin (HbA1c; p < 0.01) equivalent to or better than with exenatide or liraglutide. Treatment of C57BL/KsJ db/db mice for 15 days with 100 nmol/kg conjugate significantly (p < 0.001) reduced glucose and raised plasma insulin. Oral glucose tolerance was significantly (p < 0.001) improved and both 24-h glucose profile (p < 0.001) and HbA1c levels (p < 0.001) were reduced. Islet size (p < 0.001) and pancreatic insulin content were increased without change of islet cell proliferation or apoptosis. CONCLUSION: These data show that d-Ala(8) GLP-1(Lys(37) ) pentasaccharide exerts significant antidiabetic actions and has a projected pharmacokinetic/pharmacodynamic profile that merits further evaluation in humans for a possible once-weekly dosing regimen.

Effects of chronic exposure of clonal ß-cells to elevated glucose and free fatty acids on incretin receptor gene expression and secretory responses to GIP and GLP-1.

AIM: The incretin effect, mediated by glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), is impaired in type 2 diabetes. METHODS: This study examines the effects of prolonged exposure to elevated glucose and free fatty acids in clonal BRIN BD11 cells on GIP and GLP-1 action. RESULTS: Glucotoxic conditions (18 h) had no effect on GIP- or GLP-1-mediated insulinotropic responses. In contrast, 48 h glucotoxic culture impaired (p < 0.05 to p < 0.001) insulin release in response to GLP-1, and particularly GIP. Culture under lipotoxic conditions (18 h) impaired (p < 0.05 to p < 0.001) the insulin-releasing effect of GIP, but was without effect on GLP-1. However, 48 h lipotoxic culture compromised both GIP (p < 0.05 to p < 0.001) and GLP-1 (p < 0.05 to p < 0.01) insulin-releasing actions. Glucolipotoxic culture (18 h) completely annulled the insulinotropic action of GIP, whereas GLP-1 effects were similar to control. However, when glucolipotoxic culture was extended to 48 h, both GIP- and GLP-1-mediated effects were (p < 0.05 to p < 0.001) impaired. Assessment of cell viability, number and insulin content revealed detrimental (p < 0.05 to p < 0.001) effects under all culture conditions, barring 18 h glucotoxic and lipotoxic culture. Finally, GIP-R gene and protein expression was increased (p < 0.05 to p < 0.01) under glucotoxic culture, with decreased (p < 0.05 to p < 0.001) expression following glucolipotoxic culture. GLP-1-R gene expression followed a similar trend, but protein levels were generally reduced under all culture conditions. CONCLUSION: The results indicate that impaired insulinotropic response to GIP and GLP-1 under diabetic milieu involves mechanisms beyond simple expression of respective receptors.

Evaluation of the insulin-releasing and glucose-lowering effects of GPR120 activation in pancreatic ß-cells.

AIMS: To assess the potency and selectivity of various GPR120 agonists and to determine the cellular localization of GPR120 in clonal ß-cells and pancreatic islets. METHODS: Insulin secretion and alterations in intracellular Ca(2+) and cAMP response to glucose and GPR120 agonists, including endogenous agonists α-linolenic acid (ALA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and a synthetic analogue (GW-9508), were examined using clonal pancreatic BRIN-BD11 cells, mouse pancreatic islets and in vivo studies using NIH Swiss mice. Cytotoxicity was assessed by lactate dehydrogenase release. Cellular localization of GPR120 was explored by double-staining immunohistochemistry. RESULTS: The most potent and selective GPR120 agonist tested was ALA (half maximum effective concentration 1.2 × 10(-8) mol/l) with a maximum stimulation of insulin secretion of 53% at 10(-4) mol/l (p < 0.001) in BRIN-BD11 cells. Stimulation of insulin secretion was also observed with GW-9508 (6.4 × 10(-8) mol/l; 47%), EPA (7.9 × 10(-8) mol/l; 36%) and DHA (1.0 × 10(-7) mol/l; 50%). Results were corroborated by islet studies, with no evidence of cytotoxic effects. Dose-dependent insulin secretion by GPR120 agonists was glucose-sensitive and accompanied by significant elevations of intracellular Ca(2+) and cAMP. Immunocytochemistry showed GPR120 expression on BRIN-BD11 cells and was confined to islet ß-cells with no distribution on α-cells. Administration of GPR120 agonists (0.1 µmol/kg body weight) in glucose tolerance studies significantly reduced plasma glucose and augmented insulin release in mice. CONCLUSIONS: These results indicate that GPR120 is expressed on pancreatic ß-cells and that agonists at this receptor are potent insulin secretagogues with therapeutic potential for type 2 diabetes.

Evidence for inhibitory autocrine effects of proinsulin C-peptide on pancreatic ß-cell function and insulin secretion.

AIMS: Autocrine and paracrine regulatory mechanisms ensure integrated secretion of islet hormones that respond efficiently to changes in metabolic need. As proinsulin C-peptide exerts various biological effects and binds to cell membranes including insulin-secreting ß cells, its physiological role in insulin release was examined. METHODS: Insulin releasing activity of human and rat C-peptides were studied in the clonal pancreatic cell line, BRIN-BD11, with findings substantiated using isolated islets and in vivo studies employing SWISS TO mice. RESULTS: Acute exposure of clonal ß cells to human C-peptide resulted in concentration-dependent inhibitory effects on insulin secretion at 5.6 mM (p < 0.05-p < 0.001) and 16.7 mM (p < 0.01-p < 0.001) glucose. At physiologically relevant intra-islet concentrations (10(-9) -10(-6) M), C-peptide suppressed the insulin-secretory responses to a range of secretagogues acting at different points in the ß cell stimulus-secretion coupling pathway including alanine (p < 0.05), Ca(2+) (p < 0.001), arginine (p < 0.05), tolbutamide (p < 0.001), glucagon-like peptide 1 (GLP-1) (p < 0.001), isobutylmethylxanthine (IBMX) (p < 0.01) and KCl (p < 0.05). Similar results were obtained using isolated mouse pancreatic islets. Human C-peptide (3 × 10(-7) M, p < 0.001), somatostatin-14 (3 × 10(-7) M, p < 0.01) and diazoxide (300 µM, p < 0.001) reduced both alanine and glucose-stimulated insulin release by 43, 25 and 48%, respectively. The effects of human C-peptide were reproduced using rat C-peptide I and II. C-peptide also inhibited in vivo glucose-stimulated insulin release and impaired glucose tolerance in mice. CONCLUSIONS: C-peptide is a biologically active endogenous peptide hormone that exerts inhibitory autocrine effects on pancreatic ß-cell function. Mechanisms involving the activation of K(+) channels and a distal effect downstream of increased cytoplasmic Ca(2+) appear to be implicated in the inhibition of insulin secretion.

Positive interplay between FFAR4/GPR120, DPP-IV inhibition and GLP-1 in beta cell proliferation and glucose homeostasis in obese high fat fed mice.

G-protein coupled receptor-120 (GPR120; FFAR4) is a free fatty acid receptor, widely researched for its glucoregulatory and insulin release activities. This study aimed to investigate the metabolic advantage of FFAR4/GPR120 activation using combination therapy. C57BL/6 mice, fed a High Fat Diet (HFD) for 120 days to induce obesity-diabetes, were subsequently treated with a single daily oral dose of FFAR4/GPR120 agonist Compound A (CpdA) (0.1µmol/kg) alone or in combination with sitagliptin (50 mg/kg) for 21 days. After 21-days, glucose homeostasis, islet morphology, plasma hormones and lipids, tissue genes (qPCR) and protein expression (immunocytochemistry) were assessed. Oral administration of CpdA improved glucose tolerance (34% p<0.001) and increased circulating insulin (38% p<0.001). Addition of CpdA with the dipeptidyl peptidase-IV (DPP-IV) inhibitor, sitagliptin, further improved insulin release (44%) compared to sitagliptin alone and reduced fat mass (p<0.05). CpdA alone (50%) and in combination with sitagliptin (89%) induced marked reductions in LDL-cholesterol, with greater effects in combination (p<0.05). All treatment regimens restored pancreatic islet and beta-cell area and mass, complemented with significantly elevated beta-cell proliferation rates. A marked increase in circulating GLP-1 (53%) was observed, with further increases in combination (38%). With treatment, mice presented with increased Gcg (proglucagon) gene expression in the jejunum (130% increase) and ileum (120% increase), indicative of GLP-1 synthesis and secretion. These data highlight the therapeutic promise of FFAR4/GPR120 activation and the potential for combined benefit with incretin enhancing DPP-IV inhibitors in the regulation of beta cell proliferation and diabetes.

MicroRNA regulation of islet and enteroendocrine peptides: Physiology and therapeutic implications for type 2 diabetes.

The pathogenesis of type 2 diabetes (T2D) is associated with dysregulation of glucoregulatory hormones, including both islet and enteroendocrine peptides. Microribonucleic acids (miRNAs) are short noncoding RNA sequences which post transcriptionally inhibit protein synthesis by binding to complementary messenger RNA (mRNA). Essential for normal cell activities, including proliferation and apoptosis, dysregulation of these noncoding RNA molecules have been linked to several diseases, including diabetes, where alterations in miRNA expression within pancreatic islets have been observed. This may occur as a compensatory mechanism to maintain beta-cell mass/function (e.g., downregulation of miR-7), or conversely, lead to further beta-cell demise and disease progression (e.g., upregulation of miR-187). Thus, targeting miRNAs has potential for novel diagnostic and therapeutic applications in T2D. This is reinforced by the success seen to date with miRNA-based therapeutics for other conditions currently in clinical trials. In this review, differential expression of miRNAs in human islets associated with T2D will be discussed along with further consideration of their effects on the production and secretion of islet and incretin hormones. This analysis further unravels the therapeutic potential of miRNAs and offers insights into novel strategies for T2D management.

Comparison of the metabolic effects of sustained CCK1 receptor activation alone and in combination with upregulated leptin signalling in high-fat-fed mice.

AIMS/HYPOTHESIS: Cholecystokinin (CCK) and leptin are important hormones with effects on energy balance. The present study assessed the biological effects of (pGlu-Gln)-CCK-8 and [D-Leu-4]-OB3, smaller isoforms of CCK and leptin, respectively. METHODS: The actions and overall therapeutic use of (pGlu-Gln)-CCK-8 and [D-Leu-4]-OB3, alone and in combination, were evaluated in normal and high-fat-fed mice. RESULTS: (pGlu-Gln)-CCK-8 had prominent (p < 0.01 to p < 0.001), acute feeding-suppressive effects, which were significantly augmented (p < 0.05 to p < 0.01) by [D-Leu-4]-OB3. In agreement, the acute dose-dependent glucose-lowering and insulinotropic actions of (pGlu-Gln)-CCK-8 were significantly enhanced by concurrent administration of [D-Leu-4]-OB3. Twice daily injection of (pGlu-Gln)-CCK-8 alone and in combination with [D-Leu-4]-OB3 in high-fat-fed mice for 18 days decreased body weight (p < 0.05 to p < 0.001), energy intake (p < 0.01), circulating triacylglycerol (p < 0.01), non-fasting glucose (p < 0.05 to p < 0.001) and triacylglycerol deposition in liver and adipose tissue (p < 0.001). All treatment regimens improved glucose tolerance (p < 0.05 to p < 0.001) and insulin sensitivity (p < 0.001). Combined treatment with (pGlu-Gln)-CCK-8 and [D-Leu-4]-OB3 resulted in significantly lowered plasma insulin levels, normalisation of circulating LDL-cholesterol and decreased triacylglycerol deposition in muscle. These effects were superior to either treatment regimen alone. There were no changes in overall locomotor activity or respiratory exchange ratio, but treatment with (pGlu-Gln)-CCK-8 significantly reduced (p < 0.001) energy expenditure. CONCLUSIONS/INTERPRETATION: These studies highlight the potential of (pGlu-Gln)-CCK-8 alone and in combination with [D-Leu-4]-OB3 in the treatment of obesity and diabetes.

A DPP-IV-resistant triple-acting agonist of GIP, GLP-1 and glucagon receptors with potent glucose-lowering and insulinotropic actions in high-fat-fed mice.

AIMS/HYPOTHESIS: We designed a chemically modified, enzyme-resistant peptide with triple-acting properties based on human glucagon with amino acid substitutions aligned to strategic positions in the sequence of glucose-dependent insulinotropic polypeptide (GIP). METHODS: Y(1)-dA(2)-I(12)-N(17)-V(18)-I(27)-G(28,29)-glucagon (termed YAG-glucagon) was incubated with dipeptidylpeptidase IV (DPP-IV) to assess stability, BRIN-BD11 cells to evaluate insulin secretion, and receptor-transfected cells to examine cAMP production. Acute glucose-lowering and insulinotropic properties of YAG-glucagon were assessed in National Institutes of Health (NIH) Swiss mice, while longer-term actions on glucose homeostasis, insulin secretion, food intake and body weight were examined in high-fat-fed mice. RESULTS: YAG-glucagon was resistant to DPP-IV, increased in vitro insulin secretion (1.5-3-fold; p < 0.001) and stimulated cAMP production in GIP receptor-, glucagon-like peptide-1 (GLP-1) receptor- and glucagon receptor-transfected cells. Plasma glucose levels were significantly reduced (by 51%; p < 0.01) and insulin concentrations increased (1.2-fold; p < 0.01) after acute injection of YAG-glucagon in NIH Swiss mice. Acute actions were countered by established GIP, GLP-1 and glucagon antagonists. In high-fat-fed mice, twice-daily administration of YAG-glucagon for 14 days reduced plasma glucose (40% reduction; p < 0.01) and increased plasma insulin concentrations (1.8-fold; p < 0.05). Glycaemic responses were markedly improved (19-48% reduction; p < 0.05) and insulin secretion enhanced (1.5-fold; p < 0.05) after a glucose load, which were independent of changes in insulin sensitivity, food intake and body weight. CONCLUSIONS/INTERPRETATION: YAG-glucagon is a DPP-IV-resistant triple agonist of GIP, GLP-1 and glucagon receptors and exhibits beneficial biological properties suggesting that it may hold promise for treatment of type 2 diabetes.

Beneficial effects of parenteral GLP-1 delivery by cell therapy in insulin-deficient streptozotocin diabetic mice.

Parenteral delivery of long-acting glucagon-like peptide-1 (GLP-1) mimetics has received much attention as a therapeutic option for diabetes. However, cell therapy-based GLP-1 treatments may provide a more physiological regulation of blood glucose. The present study assessed the effects of chronic GLP-1 delivery by cell therapy, using the GLP-1-secreting GLUTag cell line, in normoglycemic and streptozotocin-induced diabetic mice. GLUTag cell aggregates were transplanted into the subscapular region of mice. Over 30 days, cellular transplantation gave rise to encapsulated and well-vascularized growths, which contained immunoreactive GLP-1. Cell implantation was well tolerated and had no appreciable metabolic effects in normal mice. However, transplantation significantly (P<0.001) countered excessive food and fluid intake in diabetic mice and maintained normal body weight. Circulating glucose (P<0.01) and glucagon (P<0.05) were significantly reduced and plasma insulin and GLP-1 dramatically increased. This was associated with significantly (P<0.01) improved glucose tolerance in diabetic mice. Histological examination of the pancreata of these mice revealed elevations (P<0.001) in islet and ß-cell area, with reduced (P<0.001) α-cell area. Increased ß-cell mass reflected the enhanced proliferation relative to apoptosis. These studies emphasize the potential of chronic GLP-1 delivery by cell therapy as a potential therapeutic option for diabetes.

Frog skin peptides (tigerinin-1R, magainin-AM1, -AM2, CPF-AM1, and PGla-AM1) stimulate secretion of glucagon-like peptide 1 (GLP-1) by GLUTag cells.

Skin secretions of several frog species contain host-defense peptides with multiple biological activities including in vitro and in vivo insulin-releasing actions. This study investigates the effects of tigerinin-1R from Hoplobatrachus rugulosus (Dicroglossidae) and magainin-AM1, magainin-AM2, caerulein precursor fragment (CPF-AM1) and peptide glycine leucine amide (PGLa-AM1) from Xenopus amieti (Pipidae) on GLP-1 secretion from GLUTag cells. Tigerinin-1R showed the highest potency producing a significant (P<0.05) increase in GLP-1 release at a concentration of 0.1nM for the cyclic peptide and 0.3nM for the reduced form. All peptides from X. amieti significantly (P<0.05) stimulated GLP-1 release at concentrations ⩾300nM with magainin-AM2 exhibiting the greatest potency (minimum concentration producing a significant stimulation=1nM). The maximum stimulatory response (3.2-fold of basal rate, P<0.001) was produced by CPF-AM1 at a concentration of 3µM. No peptide stimulated release of the cytosolic enzyme, lactate dehydrogenase from GLUTag cells at concentrations up to 3µM indicating that the integrity of the plasma membrane had been preserved. The data indicate that frog skin peptides, by stimulating GLP-1 release as well as direct effects on insulin secretion, show therapeutic potential as agents for the treatment of type 2 diabetes.

Liraglutide improves hippocampal synaptic plasticity associated with increased expression of Mash1 in ob/ob mice.

OBJECTIVE: Consumption of high-fat diet exerts adverse effects on learning and memory formation, which is linked to impaired hippocampal function. Activation of glucagon-like peptide-1 (GLP-1) signalling ameliorates detrimental effects of obesity-diabetes on cognitive function; however, mechanisms underlying these beneficial actions remain unclear. This study examined effects of daily subcutaneous treatment with GLP-1 mimetic, Liraglutide, on synaptic plasticity, hippocampal gene expression and metabolic control in adult obese diabetic (ob/ob) mice. RESULTS: Long-term potentiation (LTP) induced by area CA1 was completely abolished in ob/ob mice compared with lean controls. Deleterious effects on LTP were rescued (P<0.001) with Liraglutide. Indeed, Liraglutide-treated mice exhibited superior LTP profile compared with lean controls (P<0.01). Expression of hippocampal brain-derived neurotropic factor and neurotrophic tyrosine kinase receptor-type 2 were not significantly different, but synaptophysin and Mash1 were decreased in ob/ob mice. Treatment with Liraglutide over 21 days increased expression of Mash1 in ob/ob mice (2.0-fold; P<0.01). These changes were associated with significantly reduced plasma glucose (21% reduction; P<0.05) and markedly improved plasma insulin concentrations (2.1- to 3.3-fold; P<0.05 to P<0.01). Liraglutide also significantly reduced the glycaemic excursion following an intraperitonal glucose load (area under curve (AUC) values: 22%; P<0.05) and markedly enhanced the insulin response to glucose (AUC values: 1.6-fold; P<0.05). O2 consumption, CO2 production, respiratory exchange ratio and energy expenditure were not altered by Liraglutide therapy. On day 21, accumulated food intake (32% reduction; P<0.05) and number of feeding bouts (32% reduction; P<0.05) were significantly reduced but simple energy restriction was not responsible for the beneficial actions of Liraglutide. CONCLUSION: Liraglutide elicits beneficial effects on metabolic control and synaptic plasticity in mice with severe obesity and insulin resistance mediated in part through increased expression of Mash1 believed to improve hippocampal neurogenesis and cell survival.

Comparison of the independent and combined metabolic effects of subchronic modulation of CCK and GIP receptor action in obesity-related diabetes.

OBJECTIVE: Compromise of gastric inhibitory polypeptide (GIP) receptor action and activation of cholecystokinin (CCK) receptors represent mechanistically different approaches to the possible treatment of obesity-related diabetes. In the present study, we have compared the individual and combined effects of (Pro(3))GIP[mPEG] and (pGlu-Gln)-CCK-8 as an enzymatically stable GIP receptor antagonist and CCK receptor agonist molecule, respectively. RESULTS: Twice-daily injections of (pGlu-Gln)-CCK-8 alone and in combination with (Pro(3))GIP[mPEG] in high-fat-fed mice for 34 days significantly decreased the energy intake throughout the entire study (P<0.05 to P<0.01). Body weights were significantly depressed (P<0.05 to P<0.01) in all treatment groups from day 18 onwards. Administration of (pGlu-Gln)-CCK-8, (Pro(3))GIP[mPEG] or a combination of both peptides significantly (P<0.01 to P<0.001) decreased the overall glycaemic excursion in response to both oral and intraperitoneal glucose challenge when compared with the controls. Furthermore, oral glucose tolerance returned to lean control levels in all treatment groups. The beneficial effects on glucose homeostasis were not associated with altered insulin levels in any of the treatment groups. In keeping with this, the estimated insulin sensitivity was restored to control levels by twice-daily treatment with (pGlu-Gln)-CCK-8, (Pro(3))GIP[mPEG] or a combination of both peptides. The blood lipid profile on day 34 was not significantly different between the high-fat controls and all treated mice. CONCLUSION: These studies highlight the potential of (pGlu-Gln)-CCK-8 and (Pro(3))GIP[mPEG] in the treatment of obesity-related diabetes, but there was no evidence of a synergistic effect of the combined treatment.

Beneficial effects of (pGlu-Gln)-CCK-8 on energy intake and metabolism in high fat fed mice are associated with alterations of hypothalamic gene expression.

Cholecystokinin (CCK) is a gastrointestinal hormone with potential therapeutic promise for obesity-diabetes. The present study examined the effects of twice daily administration of the N-terminally modified stable CCK-8 analogue, (pGlu-Gln)-CCK-8, on metabolic control and hypothalamic gene expression in high fat fed mice. Sub-chronic twice daily injection of (pGlu-Gln)-CCK-8 for 16 days significantly decreased body weight (p<0.05), energy intake (p<0.01), circulating blood glucose (p<0.001), and plasma insulin (p<0.001) compared to high fat controls. Furthermore, (pGlu-Gln)-CCK-8 markedly improved glucose tolerance (p<0.05) and insulin sensitivity (p<0.05). Assessment of hypothalamic gene expression on day 16 revealed significantly elevated NPY (p<0.05) and reduced POMC (p<0.05) and MC4R (p<0.05) mRNA expression in (pGlu-Gln)-CCK-8 treated mice. High fat feeding or (pGlu-Gln)-CCK-8 treatment had no significant effects on hypothalamic gene expression of receptors for leptin, CCK1 and GLP-1. These studies underscore the potential of (pGlu-Gln)-CCK-8 for the treatment of obesity-diabetes and suggest modulation of NPY and melanocortin related pathways may be involved in the observed beneficial effects.

Comparison of independent and combined metabolic effects of chronic treatment with (pGlu-Gln)-CCK-8 and long-acting GLP-1 and GIP mimetics in high fat-fed mice.

AIM: The incretin hormones, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) and cholecystokinin (CCK) are gastrointestinal peptides with important physiological effects. However, rapid enzymatic degradation results in short-lived biological actions. METHODS: This study has examined metabolic actions of exendin-4, GIP[mPEG] and a novel CCK-8 analogue, (pGlu-Gln)-CCK-8 as enzymatically stable forms of GLP-1, GIP and CCK, respectively. RESULTS: All peptides significantly (p < 0.01-p < 0.001) stimulated insulin secretion from BRIN BD11 cells, and acute in vivo experiments confirmed prominent antihyperglycaemic and insulinotropic responses to GLP-1 or GIP receptor activation in normal mice. Twice daily injection of (pGlu-Gln)-CCK-8 alone and in combination with exendin-4 or GIP[mPEG] in high fat-fed mice significantly decreased accumulated food intake (p < 0.05-p < 0.01), body weight gain (p < 0.05-p < 0.01) and improved (p < 0.05) insulin sensitivity in high fat-fed mice. However, there was no evidence for superior effects compared to (pGlu-Gln)-CCK-8 alone. Combined treatment of (pGlu-Gln)-CCK-8 and exendin-4 resulted in significantly (p < 0.05) lowered circulating glucose levels and improved (p < 0.05) intraperitoneal glucose tolerance. These effects were superior to either treatment regime alone but not associated with altered insulin concentrations. A single injection of (pGlu-Gln)-CCK-8, or combined with exendin-4, significantly (p < 0.05) lowered blood glucose levels 24 h post injection in untreated high fat-fed mice. CONCLUSION: This study highlights the potential of (pGlu-Gln)-CCK-8 alone and in combination with incretin hormones for the treatment of type 2 diabetes.