Pancreatic islet cell plasticity: Pathogenic or therapeutically exploitable?

The development of pancreatic islet endocrine cells is a tightly regulated process leading to the generation of distinct cell types harbouring different hormones in response to small changes in environmental stimuli. Cell differentiation is driven by transcription factors that are also critical for the maintenance of the mature islet cell phenotype. Alteration of the insulin-secreting ß-cell transcription factor set by prolonged metabolic stress, associated with the pathogenesis of diabetes, obesity or pregnancy, results in the loss of ß-cell identity through de- or transdifferentiation. Importantly, the glucose-lowering effects of approved and experimental antidiabetic agents, including glucagon-like peptide-1 mimetics, novel peptides and small molecules, have been associated with preventing or reversing ß-cell dedifferentiation or promoting the transdifferentiation of non-ß-cells towards an insulin-positive ß-cell-like phenotype. Therefore, we review the manifestations of islet cell plasticity in various experimental settings and discuss the physiological and therapeutic sides of this phenomenon, focusing on strategies for preventing ß-cell loss or generating new ß-cells in diabetes. A better understanding of the molecular mechanisms underpinning islet cell plasticity is a prerequisite for more targeted therapies to help prevent ß-cell decline in diabetes.

Sustained metabolic benefits of ΔTRTX-Ac1, a tarantula venom-derived peptide, when administered together with exenatide in high-fat fed mice.

AIM: The aim of the present study was to assess the long-term therapeutic efficacy of a recently discovered 28 amino acid peptide, Δ-theraphotoxin-Ac1 (Δ-TRTX-Ac1), originally isolated from venom of the Aphonopelma chalcodes tarantula. Δ-TRTX-Ac has previously been shown to improve pancreatic beta-cell function and suppress appetite. MATERIALS AND METHODS: Δ-TRTX-Ac1 was administered twice daily in high-fat fed (HFF) mice with streptozotocin (STZ)-induced insulin deficiency, namely HFF/STZ mice, for 28 days both alone and in combination with the venom-derived glucagon-like peptide-1 (GLP-1) mimetic, exenatide. RESULTS: Initial pharmacokinetic profiling of ΔTRTX-Ac1 revealed a plasma half-life of 2 h in mice, with ΔTRTX-Ac1 also evidenced in the pancreas 12 h post-injection. Accordingly, HFF-STZ mice received twice-daily injections of Δ-TRTX-Ac1, exenatide or a combination of both peptides for 28 days. As anticipated, HFF/STZ mice presented with hyperglycaemia, impaired glucose tolerance, decreased plasma and pancreatic insulin and disturbed pancreatic islet morphology. Administration of ΔTRTX-Ac1 reduced body weight, improved glucose tolerance and augmented pancreatic insulin content while decreasing glucagon content. Exenatide had similar benefits on body weight and pancreatic hormone content while also reducing circulating glucose. ΔTRTX-Ac1 decreased energy expenditure on day 28 whereas exenatide had no impact. All treatment regimens restored pancreatic islet and beta-cell area towards lean control levels, which was linked to significantly elevated beta-cell proliferation rates. In terms of benefits of combined ΔTRTX-Ac1 and exenatide treatment over individual agents, there was augmentation of glucose tolerance and ambulatory activity with combination therapy, and these mice presented with increased pancreatic glucagon. CONCLUSION: These data highlight the therapeutic promise of ΔTRTX-Ac1 for diabetes, with suggestion that benefits could be enhanced through combined administration with exenatide.

Beneficial metabolic effects of recurrent periods of beta-cell rest and stimulation using stable neuropeptide Y1 and glucagon-like peptide-1 receptor agonists.

AIM: To examine whether sequential administration of (d-Arg35 )-sea lamprey peptide tyrosine tyrosine (1-36) (SL-PYY) and the glucagon-like peptide-1 (GLP-1) mimetic, liraglutide, has beneficial effects in diabetes. METHODS: SL-PYY is an enzymatically stable neuropeptide Y1 receptor (NPY1R) agonist known to induce pancreatic beta-cell rest and improve overall beta-cell health. We employed SL-PYY and liraglutide to induce appropriate recurrent periods of beta-cell rest and stimulation, to assess therapeutic benefits in high fat fed (HFF) mice with streptozotocin (STZ)-induced insulin deficiency, namely HFF-STZ mice. RESULTS: Previous studies confirm that, at a dose of 0.25 nmol/kg, liraglutide exerts bioactivity over an 8-12 hour period in mice. Initial pharmacokinetic analysis revealed that 75 nmol/kg SL-PYY yielded a similar plasma drug time profile. When SL-PYY (75 nmol/kg) and liraglutide (0.25 nmol/kg) were administered sequentially at 08:00 AM and 08:00 PM, respectively, to HFF-STZ mice for 28 days, reductions in energy intake, body weight, circulating glucose, insulin and glucagon were noted. Similarly positive, but slightly less striking, effects were also apparent with twice-daily liraglutide-only therapy. The sequential SL-PYY and liraglutide treatment also improved insulin sensitivity and glucose-induced insulin secretory responses, which was not apparent with liraglutide treatment, although benefits on glucose tolerance were mild. Interestingly, combined therapy also elevated pancreatic insulin, decreased pancreatic glucagon and enhanced the plasma insulin/glucagon ratio compared with liraglutide alone. This was not associated with an enhancement of beneficial changes in islet cell areas, proliferation or apoptosis compared with liraglutide alone, but the numbers of centrally stained glucagon-positive islet cells were reduced by sequential combination therapy. CONCLUSION: These data show that NPY1R-induced intervals of beta-cell rest, combined with GLP-1R-stimulated periods of beta-cell stimulation, should be further evaluated as an effective treatment option for obesity-driven forms of diabetes.

Enzymatically stable analogue of the gut-derived peptide xenin on beta-cell transdifferentiation in high fat fed and insulin-deficient Ins1Cre/+ ;Rosa26-eYFP mice.

BACKGROUND: The antidiabetic effects of the gut hormone xenin include augmenting insulin secretion and positively affecting pancreatic islet architecture. METHODS: The current study has further probed pancreatic effects through sub-chronic administration of the long-acting xenin analogue, xenin-25[Lys13 PAL], in both high fat fed (HFF) and streptozotocin (STZ)-induced insulin-deficient Ins1Cre/+ ;Rosa26-eYFP transgenic mice. Parallel effects on metabolic control and pancreatic islet morphology, including islet beta-cell lineage tracing were also assessed. RESULTS: Xenin-25[Lys13 PAL] treatment reversed body weight loss induced by STZ, increased plasma insulin and decreased blood glucose levels. There were less obvious effects on these parameters in HFF mice, but all xenin-25[Lys13 PAL] treated mice exhibited decreased pancreatic alpha-cell areas and circulating glucagon. Xenin-25[Lys13 PAL] treatment fully, or partially, returned overall islet and beta-cell areas in STZ- and HFF mice to those of lean control animals, respectively, and was consistently associated with decreased beta-cell apoptosis. Interestingly, xenin-25[Lys13 PAL] also increased beta-cell proliferation and decreased alpha-cell apoptosis in STZ mice, with reduced alpha-cell growth noted in HFF mice. Lineage tracing studies revealed that xenin-25[Lys13 PAL] reduced the number of insulin positive pancreatic islet cells that lost their beta-cell identity, in keeping with a decreased transition of insulin positive to glucagon positive cells. These beneficial effects on islet cell differentiation were linked to maintained expression of Pdx1 within beta-cells. Xenin-25[Lys13 PAL] treatment was also associated with increased numbers of smaller sized islets in both models. CONCLUSIONS: Benefits of xenin-25[Lys13 PAL] on diabetes includes positive modulation of islet cell differentiation, in addition to promoting beta-cell growth and survival.

Amplifying the antidiabetic actions of glucagon-like peptide-1: Potential benefits of new adjunct therapies.

Clinically approved for the treatment of diabetes and obesity, glucagon-like peptide-1 (GLP-1) receptor agonists display prominent glucose- and weight-lowering effects as well as positive cardioprotective and neuroprotective actions. Despite these benefits, bariatric surgery remains superior in producing robust and sustained weight loss alongside improvements in metabolic control with possible diabetes remission. The current review considers the potential for adjunct therapies to augment the therapeutic actions of GLP-1 receptor agonists. In this regard, several gut-derived hormones also, modulated by bariatric surgery, display additive properties when combined with GLP-1 receptor agonists in both preclinical and clinical studies. In addition, glucocorticoids and oestrogen have shown promise in augmenting the biological actions of GLP-1 in animal models. Additionally, GLP-1 efficacy can also be enhanced by use of compounds that prolong GLP-1 receptor coupling to potentiate downstream receptor signalling. Taken together, therapies that activate GLP-1 receptor signalling, in combination with various other cell signalling pathways, show potential for treating type 2 diabetes and obesity with superiority over GLP-1 receptor agonist therapy alone.

Weight-reducing, lipid-lowering and antidiabetic activities of a novel arginine vasopressin analogue acting at the V1a and V1b receptors in high-fat-fed mice.

AIM: To assess the beneficial metabolic effects of the nonapeptide hormone, arginine vasopressin (AVP), on metabolism. MATERIALS AND METHODS: We exchanged amino acids at position 3 and 8 of AVP, namely phenylalanine and arginine, with those of oxytocin, to generate novel analogues with altered receptor selectivity. Secondary modification by N-terminal acetylation was used to impart stability to circulating endopeptidases. Analogues were screened for degradation, bioactivity in rodent/human clonal beta cells and primary murine islets, together with evaluation of receptor activation profile. RESULTS: Analogue Ac3IV, which lacked effects at the V2 receptors responsible for modulation of fluid balance, was selected as the lead compound for assessment of antidiabetic efficacy in high-fat-fed mice. Twice-daily administration of Ac3IV, or the gold standard control exendin-4, for 22 days, reduced energy intake as well as body weight and fat content. Both interventions decreased circulating glucose levels, enhanced insulin sensitivity, and substantially improved glucose tolerance and related insulin secretion in response to an intraperitoneal or oral glucose challenge. The peptides decreased total- and increased HDL-cholesterol, but only Ac3IV decreased LDL-cholesterol, triglyceride and non-fasting glucagon concentrations. Elevations of islet and beta-cell areas were partially reversed, accompanied by suppressed islet cell proliferation, decreased beta-cell apoptosis and, in the case of exendin-4, also decreased alpha-cell apoptosis. CONCLUSION: AVP-based therapies that exclusively target V1a and V1b receptors may have significant therapeutic potential for the treatment of obesity and related diabetes, and merit further clinical exploration.

Individual and combined effects of GIP and xenin on differentiation, glucose uptake and lipolysis in 3T3-L1 adipocytes.

The incretin hormone glucose-dependent insulinotropic polypeptide (GIP), released postprandially from K-cells, has established actions on adipocytes and lipid metabolism. In addition, xenin, a related peptide hormone also secreted from K-cells after a meal, has postulated effects on energy regulation and lipid turnover. The current study has probed direct individual and combined effects of GIP and xenin on adipocyte function in 3T3-L1 adipocytes, using enzyme-resistant peptide analogues, (d-Ala2)GIP and xenin-25-Gln, and knockdown (KD) of receptors for both peptides. (d-Ala2)GIP stimulated adipocyte differentiation and lipid accumulation in 3T3-L1 adipocytes over 96 h, with xenin-25-Gln evoking similar effects. Combined treatment significantly countered these individual adipogenic effects. Individual receptor KD impaired lipid accumulation and adipocyte differentiation, with combined receptor KD preventing differentiation. (d-Ala2)GIP and xenin-25-Gln increased glycerol release from 3T3-L1 adipocytes, but this lipolytic effect was significantly less apparent with combined treatment. Key adipogenic and lipolytic genes were upregulated by (d-Ala2)GIP or xenin-25-Gln, but not by dual peptide culture. Similarly, both (d-Ala2)GIP and xenin-25-Gln stimulated insulin-induced glucose uptake in 3T3-L1 adipocytes, but this effect was annulled by dual treatment. In conclusion, GIP and xenin possess direct, comparable, lipogenic and lipolytic actions in 3T3-L1 adipocytes. However, effects on lipid metabolism are significantly diminished by combined administration.

Beneficial actions of a long-acting apelin analogue in diabetes are related to positive effects on islet cell turnover and transdifferentiation.

AIM: The current study has tested the hypothesis that the positive effects of apelin receptor activation in diabetes are linked to benefits on islet cell apoptosis, proliferation and transdifferentiation using Ins1Cre/+ ;Rosa26-eYFP transgenic mice and induction of diabetes-like syndromes by streptozotocin (STZ) or high-fat feeding. MATERIALS AND METHODS: Groups (n = 6-8) of streptozotocin (STZ)-induced diabetic and high-fat diet (HFD)-fed mice received once-daily injection (25 nmol/kg) of the long-acting acylated apelin-13 analogue, pGlu(Lys8 Glu-PAL)apelin-13 amide, for 10 or 12 days, respectively. RESULTS: pGlu(Lys8 Glu-PAL)apelin-13 amide treatment partly reversed body weight loss induced by STZ and normalized circulating insulin. There was no effect of pGlu(Lys8 Glu-PAL)apelin-13 amide on these variables in HFD-fed mice, but an increase in pancreatic insulin content was observed. pGlu(Lys8 Glu-PAL)apelin-13 amide also fully, or partially, reversed the detrimental effects of STZ and HFD on plasma and pancreatic glucagon concentrations. In HFD-fed mice, the apelin analogue decreased dietary-induced elevations of islet, ß- and α-cell areas, whilst reducing α-cell area in STZ-induced diabetic mice. In terms of islet cell lineage, pGlu(Lys8 Glu-PAL)apelin-13 amide effectively reduced ß- to α-cell transdifferentiation and helped maintain ß-cell identity, which was linked to elevated Pdx-1 expression. These islet effects were coupled with decreased ß-cell apoptosis and α-cell proliferation in both models, and there was an accompanying increase of ß-cell proliferation in STZ-induced diabetic mice. CONCLUSION: Taken together these data demonstrate, for the first time, that pancreatic islet benefits of sustained APJ receptor activation in diabetes are linked to favourable islet cell transition events, leading to maintenance of ß-cell mass.

Peptide YY (1-36) peptides from phylogenetically ancient fish targeting mammalian neuropeptide Y1 receptors demonstrate potent effects on pancreatic ß-cell function, growth and survival.

AIM: To investigate the antidiabetic efficacy of enzymatically stable Peptide YY (PYY) peptides from phylogenetically ancient fish. MATERIALS AND METHODS: N-terminally stabilized, PYY (1-36) sequences from Amia calva (bowfin), Oncorhynchus mykiss (trout), Petromyzon marinus (sea lamprey) and Scaphirhynchus albus (sturgeon), were synthesized, and both biological actions and antidiabetic therapeutic efficacy were assessed. RESULTS: All fish PYY (1-36) peptides were resistant to dipeptidyl peptidase-4 (DPP-4) degradation and inhibited glucose- and alanine-induced (P < 0.05 to P < 0.001) insulin secretion. In addition, PYY (1-36) peptides imparted significant (P < 0.05 to P < 0.001) ß-cell proliferative and anti-apoptotic benefits. Proliferative effects were almost entirely absent in ß cells with CRISPR-Cas9-induced knockout of Npyr1. In contrast to human PYY (1-36), the piscine-derived peptides lacked appetite-suppressive actions. Twice-daily administration of sea lamprey PYY (1-36), the superior bioactive peptide, for 21 days significantly (P < 0.05 to P < 0.001) decreased fluid intake, non-fasting glucose and glucagon in streptozotocin (STZ)-induced diabetic mice. In addition, glucose tolerance, insulin sensitivity, pancreatic insulin and glucagon content were significantly improved. Metabolic benefits were linked to positive changes in pancreatic islet morphology as a result of augmented (P < 0.001) proliferation and decreased apoptosis of ß cells. Sturgeon PYY (1-36) exerted similar but less impressive effects in STZ mice. CONCLUSION: These observations reveal, for the first time, that PYY (1-36) peptide sequences from phylogenetically ancient fish replicate the pancreatic ß-cell benefits of human PYY (1-36) and have clear potential for the treatment of type 2 diabetes.

Exendin-4(Lys27 PAL)/gastrin/xenin-8-Gln: A novel acylated GLP-1/gastrin/xenin hybrid peptide that improves metabolic status in obese-diabetic (ob/ob) mice.

BACKGROUND: Therapeutic benefits of peptide-based drugs is limited by rapid renal elimination. METHODS: Therefore, to prolong the biological action profile of the recently characterized triple-acting hybrid peptide, exendin-4/gastrin/xenin-8-Gln, a fatty acid (C-16) has been covalently attached, creating exendin-4(Lys27 PAL)/gastrin/xenin-8-Gln. Exendin-4/gastrin and liraglutide/gastrin/xenin-8-Gln were also synthesized as direct comparator peptides. RESULTS: All hybrid peptides evoked significant concentration-dependent increases of insulin secretion from isolated murine islets and BRIN-BD11 cells. Following administration of peptides with glucose to mice, all hybrids significantly reduced the overall glycaemic excursion and increased insulin concentrations. In contrast to other treatments, exendin-4(Lys27 PAL)/gastrin/xenin-8-Gln displayed impressive antihyperglycaemic actions even 12 hours after administration, highlighting protracted duration of effects. Exendin-4/gastrin/xenin-8-Gln, exendin-4/gastrin, and exendin-4(Lys27 PAL)/gastrin/xenin-8-Gln were then progressed to a 31-day twice-daily treatment regimen in obese-diabetic ob/ob mice. All treatments decreased nonfasting glucose and HbA1c concentrations, as well as enhancing circulating and pancreatic insulin levels. Exendin-4/gastrin and exendin-4/gastrin/xenin-8-Gln also decreased food intake. Glucose tolerance was improved by all treatments, but only exendin-4(Lys27 PAL)/gastrin/xenin-8-Gln augmented glucose-induced insulin secretion. Interestingly, treatment regimens that included a xenin component induced clear advantages on the metabolic response to glucose-dependent insulinotropic polypeptide (GIP) and the glucose-lowering actions of insulin. CONCLUSION: This study emphasizes the therapeutic promise of long-acting, multi-targeting hybrid gut peptides for type 2 diabetes.

Sitagliptin Alters Bone Composition in High-Fat-Fed Mice.

Type 2 diabetes mellitus is recognized as a significant risk factor for fragility of bone. Among the newer anti-diabetic agents, dipeptidyl peptidase-4 inhibitors (DPP4i) have been reported to decrease the occurrence of bone fractures although the reason is unclear. The main aim of this study was to evaluate the impact of sitagliptin treatment on tissue bone strength and compositional parameters in the high-fat-fed mouse model. Male NIH swiss mice were allowed free access to high-fat diet for 150 days to induce chronic hyperglycemia and insulin resistance. Sitagliptin was administered once daily for 3 weeks. High-fat-fed mice administered with saline were used as controls. Bone strength was assessed at the organ and tissue level by three-point bending and nanoindentation, respectively. Bone microarchitecture was investigated by microcomputed tomography and bone composition was evaluated by Fourier transform infrared imaging and quantitative backscattered electron imaging. Administration of sitagliptin increased non-fasting insulin, improved glucose tolerance and increased insulin sensitivity. This was associated with clear ameliorations in bone strength at the organ and tissue level. No changes in trabecular or cortical microarchitectures were observed. On the other hand, higher values of Camean, Caturn, collagen maturity, mineral/matrix ratio, mineral maturity and crystal size index were evidenced after sitagliptin treatment. Correlation analysis significantly linked the modifications of bone strength to changes in bone compositional parameters. These results bring new light on the mode of action of sitagliptin on bone physiology and demonstrate a benefit of DPP4i.

Emerging therapeutic potential for xenin and related peptides in obesity and diabetes.

Xenin-25 is a 25-amino acid peptide hormone co-secreted from the same enteroendocrine K-cell as the incretin peptide glucose-dependent insulinotropic polypeptide. There is no known specific receptor for xenin-25, but studies suggest that at least some biological actions may be mediated through interaction with the neurotensin receptor. Original investigation into the physiological significance of xenin-25 focussed on effects related to gastrointestinal transit and satiety. However, xenin-25 has been demonstrated in pancreatic islets and recently shown to possess actions in relation to the regulation of insulin and glucagon secretion, as well as promoting beta-cell survival. Accordingly, the beneficial impact of xenin-25, and related analogues, has been assessed in animal models of diabetes-obesity. In addition, studies have demonstrated that metabolically active fragment peptides of xenin-25, particularly xenin-8, possess independent therapeutic promise for diabetes, as well as serving as bioactive components for the generation of multi-acting hybrid peptides with antidiabetic potential. This review focuses on continuing developments with xenin compounds in relation to new therapeutic approaches for diabetes-obesity.

Effect of poly(ethylene glycol) content and formulation parameters on particulate properties and intraperitoneal delivery of insulin from PLGA nanoparticles prepared using the double-emulsion evaporation procedure.

CONTEXT: Size, encapsulation efficiency and stability affect the sustained release from nanoparticles containing protein-type drugs. OBJECTIVES: Insulin was used to evaluate effects of formulation parameters on minimizing diameter, maximizing encapsulation efficiency and preserving blood glucose control following intraperitoneal (IP) administration. METHODS: Homogenization or sonication was used to incorporate insulin into poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles with increasing poly(ethylene glycol) (PEG) content. Effects of polymer type, insulin/polymer loading ratio and stabilizer in the internal aqueous phase on physicochemical characteristics of NP, in vitro release and stability of encapsulated insulin were investigated. Entrapment efficiency and release were assessed by radioimmunoassay and bicinconnic acid protein assay, and stability was evaluated using SDS-PAGE. Bioactivity of insulin was assessed in streptozotocin-induced, insulin-deficient Type I diabetic mice. RESULTS: Increasing polymeric PEG increased encapsulation efficiency, while the absence of internal stabilizer improved encapsulation and minimized burst release kinetics. Homogenization was shown to be superior to sonication, with NP fabricated from 10% PEG-PLGA having higher insulin encapsulation, lower burst release and better stability. Insulin-loaded NP maintained normoglycaemia for 24 h in diabetic mice following a single bolus, with no evidence of hypoglycemia. CONCLUSIONS: Insulin-loaded NP prepared from 10% PEG-PLGA possessed therapeutically useful encapsulation and release kinetics when delivered by the IP route.

An enzymatically stable GIP/xenin hybrid peptide restores GIP sensitivity, enhances beta cell function and improves glucose homeostasis in high-fat-fed mice.

AIMS/HYPOTHESIS: Glucose-dependent insulinotropic polypeptide (GIP) and xenin, regulatory gut hormones secreted from enteroendocrine K cells, exert important effects on metabolism. In addition, xenin potentiates the biological actions of GIP. The present study assessed the actions and therapeutic utility of a (DAla2)GIP/xenin-8-Gln hybrid peptide, in comparison with the parent peptides (DAla2)GIP and xenin-8-Gln. METHODS: Following confirmation of enzymatic stability, insulin secretory activity of (DAla2)GIP/xenin-8-Gln was assessed in BRIN-BD11 beta cells. Acute and persistent glucose-lowering and insulin-releasing effects were then examined in vivo. Finally, the metabolic benefits of twice daily injection of (DAla2)GIP/xenin-8-Gln was determined in high-fat-fed mice. RESULTS: All peptides significantly (p < 0.05 to p < 0.001) enhanced in vitro insulin secretion from pancreatic clonal BRIN-BD11 cells, with xenin (and particularly GIP)-related signalling pathways, being important for this action. Administration of (DAla2)GIP or (DAla2)GIP/xenin-8-Gln in combination with glucose significantly (p < 0.05) lowered blood glucose and increased plasma insulin in mice, with a protracted response of up to 4 h. All treatments elicited appetite-suppressive effects (p < 0.05), particularly (DAla2)GIP/xenin-8-Gln and xenin-8-Gln at elevated doses of 250 nmol/kg. Twice-daily administration of (DAla2)GIP/xenin-8-Gln or (DAla2)GIP for 21 days to high-fat-fed mice returned circulating blood glucose to lean control levels. In addition, (DAla2)GIP/xenin-8-Gln treatment significantly (p < 0.05) reduced glycaemic levels during a 24 h glucose profile assessment. Neither of the treatment regimens had an effect on body weight, energy intake or circulating insulin concentrations. However, insulin sensitivity was significantly (p < 0.001) improved by both treatments. Interestingly, GIP-mediated glucose-lowering (p < 0.05) and insulin-releasing (p < 0.05 to p < 0.01) effects were substantially improved by (DAla2)GIP and (DAla2)GIP/xenin-8-Gln treatment. Pancreatic islet and beta cell area (p < 0.001), as well as pancreatic insulin content (p < 0.05), were augmented in (DAla2)GIP/xenin-8-Gln-treated mice, related to enhanced proliferation and decreased apoptosis of beta cells, whereas (DAla2)GIP evoked increases (p < 0.05 to p < 0.01) in islet number. CONCLUSIONS/INTERPRETATION: These studies highlight the clear potential of GIP/xenin hybrids for the treatment of type 2 diabetes.

A novel chemically modified analogue of xenin-25 exhibits improved glucose-lowering and insulin-releasing properties.

BACKGROUND: Xenin-25 is a K-cell derived gut peptide with insulin-releasing activity which is rapidly degraded following release into the circulation. We hypothesized that substitution of all naturally-occurring Lys and Arg residues with Gln would lead to prolonged enzyme resistance and enhanced biological efficacy. METHODS: Peptide stability was assessed using murine plasma, in vitro insulin-releasing actions evaluated in BRIN-BD11 cells and acute glucose-lowering and insulin-releasing actions examined in high fat fed mice. For sub-chronic studies, a range of metabolic parameters and pancreatic histology were assessed in high fat fed mice which had received saline vehicle or xenin-25(gln) twice-daily for 21 days. RESULTS: In contrast to native xenin-25, xenin-25(gln) was resistant to plasma-mediated degradation and significantly stimulated insulin secretion in BRIN-BD11 cells. Acute administration of xenin-25(gln) in high fat fed mice significantly reduced blood glucose and increased plasma insulin concentrations. Twice-daily administration of xenin-25(gln) in high fat fed mice did not affect food intake, body weight or circulating insulin concentrations but significantly decreased blood glucose from day 9 onwards. Furthermore, glucose tolerance, glucose-mediated insulin secretion, insulin sensitivity and GIP-stimulated insulin-release were significantly enhanced in xenin-25(gln)-treated mice. Pancreatic immunohistochemistry revealed decreased alpha cell area with increased beta cell area and beta-to-alpha cell ratio in xenin-25(gln)-treated mice. In addition, xenin-25(gln) exerted similar beneficial actions in ob/ob mice as demonstrated by reduced blood glucose, superior glycaemic response and glucose-mediated insulin release. CONCLUSIONS: Xenin-25(gln) is resistant to plasma-mediated degradation and exerts sustained and beneficial metabolic actions in high fat fed and ob/ob mice. GENERAL SIGNIFICANCE: Glutamine (gln)-modified analogues of xenin may represent an attractive therapeutic approach for type 2 diabetes.

Locally produced xenin and the neurotensinergic system in pancreatic islet function and ß-cell survival.

Modulation of neuropeptide receptors is important for pancreatic ß-cell function. Here, islet distribution and effects of the neurotensin (NT) receptor modulators, xenin and NT, was examined. Xenin, but not NT, significantly improved glucose disposal and insulin secretion, in mice. However, both peptides stimulated insulin secretion from rodent ß-cells at 5.6 mm glucose, with xenin having similar insulinotropic actions at 16.7 mm glucose. In contrast, NT inhibited glucose-induced insulin secretion. Similar observations were made in human 1.1B4 ß-cells and isolated mouse islets. Interestingly, similar xenin levels were recorded in pancreatic and small intestinal tissue. Arginine and glucose stimulated xenin release from islets. Streptozotocin treatment decreased and hydrocortisone treatment increased ß-cell mass in mice. Xenin co-localisation with glucagon was increased by streptozotocin, but unaltered in hydrocortisone mice. This corresponded to elevated plasma xenin levels in streptozotocin mice. In addition, co-localisation of xenin with insulin was increased by hydrocortisone, and decreased by streptozotocin. Further in vitro investigations revealed that xenin and NT protected ß-cells against streptozotocin-induced cytotoxicity. Xenin augmented rodent and human ß-cell proliferation, whereas NT displayed proliferative actions only in human ß-cells. These data highlight the involvement of NT signalling pathways for the possible modulation of ß-cell function.

Influence of neuropeptide Y and pancreatic polypeptide on islet function and beta-cell survival.

BACKGROUND: In the present study we assessed the impact of neuropeptide Y receptor (NPYR) modulators, neuropeptide Y (NPY) and pancreatic polypeptide (PP), on islet function and beta-cell survival. METHODS: The effects of NPY and PP on beta-cell function were examined in BRIN BD11 and 1.1B4 beta-cells, as well as isolated mouse islets. Involvement of both peptides in pancreatic islet adaptations to streptozotocin and hydrocortisone, as well as effects on beta-cell proliferation and apoptosis was also evaluated. RESULTS: Neither NPY nor PP affected in vivo glucose disposal or insulin secretion in mice. However, both peptides inhibited (p<0.05 to p<0.001) glucose stimulated insulin secretion from rat and human beta-cells. NPY exerted similar insulinostatic effects in isolated mouse islets. NPY and PP inhibited alanine-induced changes in BRIN BD11 cell membrane potential and (Ca2+)i. Streptozotocin treatment decreased and hydrocortisone treatment increased beta-cell mass in mice. In addition, streptozotocin, but not hydrocortisone, increased PP cell area. Streptozotocin also shifted the normal co-localisation of NPY with PP, towards more pronounced co-expression with somatostatin in delta-cells. Both streptozotocin and hydrocortisone increased pancreatic exocrine expression of NPY. More detailed in vitro investigations revealed that NPY, but not PP, augmented (p<0.01) BRIN BD11 beta-cell proliferation. In addition, both peptides exerted protective effects against streptozotocin-induced DNA damage in beta-cells. CONCLUSION: These data emphasise the involvement of PP, and particularly NPY, in the regulation of beta-cell mass and function. GENERAL SIGNIFICANCE: Modulation of PP and NPY signalling is suitable for further evaluation and possible clinical development for the treatment of diabetes.

Sequential induction of beta cell rest and stimulation using stable GIP inhibitor and GLP-1 mimetic peptides improves metabolic control in C57BL/KsJ db/db mice.

AIMS/HYPOTHESIS: GIP(6-30)Cex-K(40)[Pal] has been characterised as a fatty-acid-derived gastric inhibitory polypeptide (GIP) inhibitor that can induce pancreatic beta cell rest by diminishing the incretin effect. We investigated its therapeutic efficacy with and without the glucagon-like peptide-1 (GLP-1) beta cell cytotropic agent liraglutide. METHODS: The therapeutic efficacy of GIP(6-30)Cex-K(40)[Pal] alone, and in combination with liraglutide, was determined in C57BL/KsJ db/db mice using a sequential 12 h administration schedule. RESULTS: GIP(6-30)Cex-K(40)[Pal] was devoid of cAMP-generating or insulin-secretory activity, and inhibited GIP-induced cAMP production and insulin secretion. GIP(6-30)Cex-K(40)[Pal] also inhibited GIP-induced glucose-lowering and insulin-releasing actions in mice. Dose- and time-dependent studies in mice revealed that 2.5 nmol/kg GIP(6-30)Cex-K(40)[Pal], and 0.25 nmol/kg liraglutide, imparted distinct biological effects for 8-12 h post administration. When GIP(6-30)Cex-K(40)[Pal] (2.5 nmol/kg) and liraglutide (0.25 nmol/kg) were administered sequentially at 12 h intervals (at 08:00 and 20:00 hours) to db/db mice for 28 days, mice treated with GIP(6-30)Cex-K(40)[Pal] (08:00 hours) and liraglutide (20:00 hours) displayed pronounced reductions in circulating glucose and insulin. Both oral and intraperitoneal glucose tolerance and glucose-stimulated plasma insulin concentrations were improved together with enhanced insulin sensitivity. The expression of genes involved in adipocyte lipid deposition was generally decreased. The other treatment modalities, including GIP(6-30)Cex-K(40)[Pal] (08:00 and 20:00 hours), liraglutide (08:00 and 20:00 hours) and liraglutide (08:00 hours) combined with GIP(6-30)Cex-K(40)[Pal] (20:00 hours), also imparted beneficial effects but these were not as prominent as those of GIP(6-30)Cex-K(40)[Pal] (08:00 hours) and liraglutide (20:00 hours). CONCLUSION/INTERPRETATION: These data demonstrate that periods of beta cell rest combined with intervals of beta cell stimulation benefit diabetes control and should be further evaluated as a potential treatment option for type 2 diabetes.

Stable Incretin Mimetics Counter Rapid Deterioration of Bone Quality in Type 1 Diabetes Mellitus.

Type 1 diabetes mellitus is associated with a high risk for bone fractures. Although bone mass is reduced, bone quality is also dramatically altered in this disorder. However, recent evidences suggest a beneficial effect of the glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) pathways on bone quality. The aims of the present study were to conduct a comprehensive investigation of bone strength at the organ and tissue level; and to ascertain whether enzyme resistant GIP or GLP-1 mimetic could be beneficial in preventing bone fragility in type 1 diabetes mellitus. Streptozotocin-treated mice were used as a model of type 1 diabetes mellitus. Control and streptozotocin-diabetic animals were treated for 21 days with an enzymatic-resistant GIP peptide ([D-Ala(2) ]GIP) or with liraglutide (each at 25 nmol/kg bw, ip). Bone quality was assessed at the organ and tissue level by microCT, qXRI, 3-point bending, qBEI, nanoindentation, and Fourier-transform infrared microspectroscopy. [D-Ala2]GIP and liraglutide treatment did prevent loss of whole bone strength and cortical microstructure in the STZ-injected mice. However, tissue material properties were significantly improved in STZ-injected animals following treatment with [D-Ala2]GIP or liraglutide. Treatment of STZ-diabetic mice with [D-Ala(2) ]GIP or liraglutide was capable of significantly preventing deterioration of the quality of the bone matrix. Further studies are required to further elucidate the molecular mechanisms involved and to validate whether these findings can be translated to human patients.

Positive effects of GLP-1 receptor activation with liraglutide on pancreatic islet morphology and metabolic control in C57BL/KsJ db/db mice with degenerative diabetes.

BACKGROUND: Stable glucagon-like peptide-1 (GLP-1) mimetics, such as the GLP-1 analogue liraglutide, are approved for treatment of type 2 diabetes. GLP-1 has a spectrum of anti-diabetic effects that are of possible utility in the treatment of more severe forms of diabetes. METHODS: The present study has evaluated the effect of once daily liraglutide injection (25 nmol/kg bw) for 15 days on metabolic control, islet architecture, and islet morphology in C57BL/KsJ db/db mice. RESULTS: Liraglutide had no appreciable effects on body weight, food intake, and non-fasting glucose and insulin concentrations. However, HbA1c was significantly (p < 0.001) decreased, and oral glucose tolerance improved in liraglutide treated db/db mice. Pancreatic insulin content was increased (p < 0.05) compared with saline controls, and the ratio of pancreatic insulin to glucagon in liraglutide mice was similar to lean mice. Although liraglutide did not alter islet number or area, the proportion of beta cells per islet was significantly increased (p < 0.05) and alpha cells decreased (p < 0.05), with normalization of islet architecture. In harmony with this, cell proliferation was significantly (p < 0.001) augmented and apoptosis reduced (p < 0.001) in liraglutide treated mice. Expression of pancreatic islet glucose-dependent insulinotropic polypeptide immunoreactivity was observed in lean control and, particularly, liraglutide treated db/db mice, whereas control db/db mice exhibited little glucose-dependent insulinotropic polypeptide staining. CONCLUSION: These data reveal that stable GLP-1 analogues exert important beneficial effects on pancreatic islet architecture and beta-cell turnover, indicating that they may be useful in the treatment of severe forms of diabetes with islet degeneration.