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.

PYY (3-36) protects against high fat feeding induced changes of pancreatic islet and intestinal hormone content and morphometry.

BACKGROUND: Prolonged high fat feeding negatively impacts pancreatic and intestinal morphology. In this regard, direct effects of PYY(3-36) on intestinal cell and pancreatic islet morphometry are yet to be fully explored in the setting of obesity. METHODS: We examined the influence of 21-days twice daily treatment with PYY(3-36) on these parameters in mice fed a high fat diet (HFD). RESULTS: PYY(3-36) treatment decreased food intake, body weight and circulating glucose in HFD mice. In terms of intestinal morphology, crypt depth was restored to control levels by PYY(3-36), with an additional enlargement of villi length. PYY(3-36) also reversed HFD-induced decreases of ileal PYY, and especially GLP-1, content. HFD increased numbers of PYY and GIP positive ileal cells, with PYY(3-36) fully reversing the effect on PYY cell detection. There were no obvious differences in the overall number of GLP-1 positive ileal cells in all mice, barring PYY(3-36) marginally decreasing GLP-1 villi cell immunoreactivity. Within pancreatic islets, PYY(3-36) significantly decreased alpha-cell area, whilst islet, beta-, PYY- and delta-cell areas remained unchanged. However, PYY(3-36) increased the percentage of beta-cells while also reducing percentage alpha-cell area. This was related to PYY(3-36)-induced reductions of beta-cell proliferation and apoptosis frequencies. Co-localisation of islet PYY with glucagon or somatostatin was elevated by PYY(3-36), with GLP-1/glucagon co-visualisation increased when compared to lean controls. CONCLUSION: PYY(3-36) exerts protective effects on pancreatic and intestinal morphology in HFD mice linked to elevated ileal GLP-1 content. GENERAL SIGNIFICANCE: These observations highlight mechanisms linked to the metabolic and weight reducing benefits of PYY(3-36).

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.

CRISPR/Cas9 gene editing demonstrates metabolic importance of GPR55 in the modulation of GIP release and pancreatic beta cell function.

G-protein coupled receptor-55 (GPR55), an endocannabinoid receptor, is a novel anti-diabetic target. This study aimed to assess the metabolic functionality of GPR55 ligands using CRISPR/Cas9 gene editing to determine their regulatory role in beta cell function and incretin-secreting enteroendocrine cells. A clonal Gpr55 knockout beta cell line was generated by CRISPR/Cas9 gene editing to investigate insulin secretion and Gpr55 signalling. Acute effects of GPR55 agonists were investigated in high fat fed (HFD) diabetic HsdOla:TO (Swiss TO) mice. Atypical and endogenous endocannabinoid ligands (10-7-10-4M) stimulated insulin secretion (p < 0.05-0.001) in rodent (BRIN-BD11) and human (1.1B4) beta cells, with 2-2.7-fold (p < 0.001) increase demonstrated in BRIN-BD11 cells (10-4M). The insulinotropic effect of Abn-CBD (42 %), AM251 (30 %) and PEA (53 %) were impaired (p < 0.05) in Gpr55 knockout BRIN-BD11 cells, with the secretory effect of O-1602 completely abolished (p < 0.001). Gpr55 ablation abolished the release of intracellular Ca2+ upon treatment with O-1602, Abn-CBD and PEA. Upregulation of insulin mRNA by Abn-CBD and AM251 (1.7-3-fold; p < 0.01) was greatly diminished (p < 0.001) in Gpr55 null cells. Orally administered Abn-CBD and AM251 (0.1 µmol/kgBW) improved GIP (p < 0.05-p < 0.01), GLP-1 (p < 0.05-p < 0.001), glucose tolerance (p < 0.001) and circulating insulin (p < 0.05-p < 0.001) in HFD diabetic mice. Abn-CBD in combination therapy with DPP-IV inhibitor (Sitagliptin) resulted in greater improvement in glucose tolerance (p < 0.05) and insulin release (p < 0.05). Antagonism of Gpr55 in-vivo attenuated the glucoregulatory effects of Abn-CBD (p < 0.05). Conclusively, GPR55 agonists enhance insulin, GIP and GLP-1 release, thereby promoting GPR55 agonist monotherapy and combinational therapy as a novel approach for the treatment of type-2-diabetes.

A GIP/xenin hybrid in combination with exendin-4 improves metabolic status in db/db diabetic mice and promotes enduring antidiabetic benefits in high fat fed mice.

The current study has determined the ability of exendin-4 to augment the antidiabetic benefits of the recently characterised GIP/xenin hybrid, (DAla2)GIP/xenin-8-Gln. As such, combined activation of metabolic pathways linked to various gut derived hormones has been shown to exert complementary beneficial metabolic effects in diabetes. (DAla2)GIP/xenin-8-Gln and exendin-4 were administered twice daily to high fat fed (HFF) or db/db mice for 28 days and antidiabetic benefits assessed. Persistence of beneficial metabolic effects in HFF mice was also examined. Twice-daily injection of (DAla2)GIP/xenin-8-Gln for 28 days in HFF mice significantly reduced energy intake, body weight, circulating glucose, HbA1c and improved glucose tolerance and insulin sensitivity. Overall pancreatic islet, alpha- and beta-cell areas were reduced, with concurrent reduction in alpha- and beta-cell proliferation that was more apparent in the combined treatment group. Addition of exendin-4 to (DAla2)GIP/xenin-8-Gln therapy did not significantly improve metabolic control. Remarkably, beneficial effects were still evident 14 days following complete cessation of peptide administration. Thus, circulating glucose and insulin, HbA1c concentrations and glucose tolerance were still significantly improved when compared to control HFF mice on day 42, with minimal changes to pancreatic islet architecture. In contrast to HFF mice, combined treatment of db/db mice with (DAla2)GIP/xenin-8-Gln plus exendin-4 was required to induce beneficial effects on key metabolic parameters, which were not observed with either treatment alone. This included improvements in glucose tolerance and insulin sensitivity, but no effect on pancreatic architecture. These studies highlight the clear, and persistent, metabolic advantages of sustained activation of GLP-1 receptors, alongside concurrent activation of related GIP and xenin cell signalling pathways, in diabetes.

Pharmacological potential of novel agonists for FFAR4 on islet and enteroendocrine cell function and glucose homeostasis.

BACKGROUND: To investigate the metabolic effects of FFAR4-selective agonists on islet and enteroendocrine cell hormone release and the combined therapeutic effectiveness with DPP-IV inhibitors. METHODS: Insulinotropic activity and specificity of FFAR4 agonists were determined in clonal pancreatic BRIN-BD11 cells. Expression of FFAR4 was assessed by qPCR and western blotting following agonist treatment in BRIN-BD11 cells and by immunohistochemistry in mouse islets. Acute in-vivo effects of agonists was investigated after intraperitoneal (i.p.) or oral administration in lean and HFF-obese diabetic mice. RESULTS: GSK137647 (10-11-10-4 M) and Compound-A (10-10-10-4 M) stimulated insulin secretion at 5.6 mM (p < 0.05-p < 0.001) and 16.7 mM (p < 0.05-p < 0.001) glucose in BRIN-BD11 cells, with no cytotoxicity effects as assessed by MTT. FFAR4 antagonist (AH-7614) abolished the insulintropic effect of GSK137647 (p < 0.05-p < 0.001), whilst FFAR1 antagonist (GW1100) had no effect. Incubation of BRIN-BD11 cells with GSK137647 and Compound-A increased FFAR4 (p < 0.01) gene expression at 16.7 mM glucose, with a corresponding increase in FFAR4 (p < 0.01) protein concentrations. FFAR4 upregulation was attenuated under normoglycaemic conditions. Immunohistochemistry demonstrated co-localisation of FFAR4 and insulin in mouse islets. Orally administered GSK137647 or Compound-A (0.1 µmol/kgBW) improved glucose tolerance (p < 0.001), increased plasma insulin (p < 0.001), GLP-1 (p < 0.05), GIP (p < 0.05) and induced satiety (p < 0.001) in HFF mice, with glucose-lowering effects enhanced in combination with DPP-IV inhibitor (Sitagliptin) (p < 0.05). CONCLUSIONS: Specific FFAR4 agonism improves glucose tolerance through insulin and incretin secretion, with enhanced DPP-IV inhibition in combination with Sitagliptin. GENERAL SIGNIFICANCE: These findings have for the first time demonstrated that selective FFAR4 activation regulates both islet and enteroendocrine hormone function with agonist combinational therapy, presenting a promising strategy for the treatment of type-2-diabetes.

Glucagon-like peptide 1 (GLP-1).

BACKGROUND: The glucagon-like peptide-1 (GLP-1) is a multifaceted hormone with broad pharmacological potential. Among the numerous metabolic effects of GLP-1 are the glucose-dependent stimulation of insulin secretion, decrease of gastric emptying, inhibition of food intake, increase of natriuresis and diuresis, and modulation of rodent ß-cell proliferation. GLP-1 also has cardio- and neuroprotective effects, decreases inflammation and apoptosis, and has implications for learning and memory, reward behavior, and palatability. Biochemically modified for enhanced potency and sustained action, GLP-1 receptor agonists are successfully in clinical use for the treatment of type-2 diabetes, and several GLP-1-based pharmacotherapies are in clinical evaluation for the treatment of obesity. SCOPE OF REVIEW: In this review, we provide a detailed overview on the multifaceted nature of GLP-1 and its pharmacology and discuss its therapeutic implications on various diseases. MAJOR CONCLUSIONS: Since its discovery, GLP-1 has emerged as a pleiotropic hormone with a myriad of metabolic functions that go well beyond its classical identification as an incretin hormone. The numerous beneficial effects of GLP-1 render this hormone an interesting candidate for the development of pharmacotherapies to treat obesity, diabetes, and neurodegenerative disorders.

Antidiabetic effects and sustained metabolic benefits of sub-chronic co-administration of exendin-4/gastrin and xenin-8-Gln in high fat fed mice.

The present study has examined the antidiabetic effects of 21 days co-administration of xenin-8-Gln with the dual-acting fusion peptide, exendin-4/gastrin, as well as persistence of beneficial metabolic benefits, in high fat fed (HFF) mice. Xenin-8-Gln, exendin-4 and gastrin represent compounds that activate receptors of the gut-derived hormones, xenin, glucagon-like peptide-1 (GLP-1) and gastrin, respectively. Twice-daily administration of exendin-4/gastrin, xenin-8-Gln or a combination of both peptides significantly reduced circulating glucose, HbA1c and cumulative energy intake. Combination therapy with xenin-8-Gln and exendin-4/gastrin increased circulating insulin. All HFF mice treated with exendin-4/gastrin presented with body weight similar to lean control mice on day 21. Each treatment improved glucose tolerance and the glucose-lowering actions of glucose dependent insulinotropic polypeptide (GIP), as well as augmenting glucose- and GIP-induced insulin secretion, with benefits being most prominent in the combination group. Administration of exendin-4/gastrin alone, and in combination with xenin-8-Gln, increased pancreatic insulin content and improved the insulin sensitivity index. Pancreatic beta-cell area was significantly increased, and alpha cell area decreased, by all treatments, with the combination group also displaying enhanced overall islet area. Notably, metabolic benefits were generally retained in all groups of HFF mice, and especially in the combination group, following discontinuation of the treatment regimens for 21 days. This was associated with maintenance of increased islet and beta-cell areas. Together, these data confirm the antidiabetic effects of co-activation of GLP-1, gastrin and xenin cell signalling pathways, and highlight the sustainable benefits this type of treatment paradigm can offer in T2DM.

Characterisation of Glucose-Dependent Insulinotropic Polypeptide Receptor Antagonists in Rodent Pancreatic Beta Cells and Mice.

Hypersecretion and alterations in the biological activity of the incretin hormone, glucose-dependent insulinotropic polypeptide (GIP), have been postulated as contributing factors in the development of obesity-related diabetes. However, recent studies also point to weight-reducing effects of GIP receptor activation. Therefore, generating precise experimental tools, such as specific and effective GIP receptor (GIPR) antagonists, is of key significance to better understand GIP physiology. Thus, the primary aim of the current study was to uncover improved GIPR antagonists for use in rodent studies, using human and mouse GIP sequences with N- and C-terminal deletions. Initial in vitro studies revealed that the GIPR agonists, human (h) GIP(1-42), hGIP(1-30) and mouse (m) GIP(1-30), stimulated (P < 0.01 to P < 0.001) insulin secretion from rat BRIN-BD11 cells. Analysis of insulin secretory effects of the N- and C-terminally cleaved GIP peptides, including hGIP(3-30), mGIP(3-30), h(Pro3)GIP(3-30), hGIP(5-30), hGIP(3-42) and hGIP(5-42), revealed that these peptides did not modulate insulin secretion. More pertinently, only hGIP(3-30), mGIP(3-30) and h(Pro3)GIP(3-30) were able to significantly (P < 0.01 to P < 0.001) inhibit hGIP(1-42)-stimulated insulin secretion. The human-derived GIPR agonist sequences, hGIP(1-42) and hGIP(1-30), reduced (P < 0.05) glucose levels in mice following conjoint injection with glucose, but mGIP(1-30) was ineffective. None of the N- and C-terminally cleaved GIP peptides affected glucose homeostasis when injected alone with glucose. However, hGIP(5-30) and mGIP(3-30) significantly (P < 0.05 to P < 0.01) impaired the glucose-lowering action of hGIP(1-42). Further evaluation of these most effective sequences demonstrated that mGIP(3-30), but not hGIP(5-30), effectively prevented GIP-induced elevations of plasma insulin concentrations. These data highlight, for the first time, that mGIP(3-30) represents an effective molecule to inhibit GIPR activity in mice.

C-terminal degradation of PYY peptides in plasma abolishes effects on satiety and beta-cell function.

The importance of dipeptidyl peptidase-4 mediated N-terminal metabolism of the enteroendocrine-derived hormone, peptide YY (PYY), for receptor binding and subsequent biological action profile is well established. However, an intact C-terminus may be fundamental also for bioactivity of PYY peptides. The current study has demonstrated C-terminal degradation of the major recognised circulating forms of PYY, PYY(1-36) and PYY(3-36), in plasma, resulting in production of PYY(1-34) and PYY(3-34). Interestingly, the angiotensin-converting-enzyme (ACE) inhibitor, captopril, blocked formation of PYY(3-34) from PYY(3-36) in plasma, but did result in the appearance of PYY(3-35). In addition, we were able to evidence C-terminal truncation of PYY(1-35) and PYY(3-35) to PYY(1-34) and PYY(3-34), respectively. As expected, PYY(1-36) and PYY(3-36) inhibited (P < 0.05-P < 0.001) glucose- and alanine-stimulated insulin secretion from BRIN-BD11 beta-cells. In contrast, PYY(1-34), PYY(3-34), PYY(1-35) and PYY(3-35) were devoid of insulinostatic actions. Both PYY(1-36) and PYY(3-36), but not related PYY metabolites, significantly (P < 0.05-P < 0.001) enhanced proliferation of BRIN BD11 and 1.1B4 beta-cell lines, and protected (P < 0.01-P < 0.001) these cell lines against cytokine-induced apoptosis. As expected, PYY(3-36) induced clear (P < 0.05-P < 0.01) appetite suppressive effects in mice, but this action was eliminated by mono- or di-peptide C-terminal truncation. Interestingly, captopril significantly (P < 0.05) augmented the anorexigenic effects of PYY(3-36) in mice. PYY(1-36), PYY(3-36), PYY(1-34) and PYY(3-34) lacked effects on in vivo glucose tolerance or glucose-induced insulin release. Taken together, these data highlight the unrecognised importance of C-terminal integrity of PYY peptides for biological activity and therapeutic usefulness in obesity-diabetes.

Novel dual incretin agonist peptide with antidiabetic and neuroprotective potential.

Glucose-dependent insulinotropic hormone (GIP) and glucagon-like peptide-1 (GLP-1) are incretin hormones that exert an array of beneficial actions on metabolism and cognitive function. GLP-1-based therapeutics have been highly successful in terms of obesity and diabetes management, however GIP therapies have found no clinical utility to date. In the present study we describe, for the first time, the therapeutic effectiveness of a novel GIP/GLP-1 hybrid peptide based on the amino acid sequences of GIP, GLP-1 and the clinically approved GLP-1 mimetic, exendin-4. The hybrid peptide, N-ac(d-Ala2)GIP/GLP-1-exe, was enzymatically stable for up to 12 h when incubated with DPP-4. N-ac(d-Ala2)GIP/GLP-1-exe significantly (P < 0.001) stimulated insulin secretion from BRIN-BD11 cells and isolated mouse islets, and evoked dose-dependent increases (P < 0.001) in cAMP production in both GIP-R and GLP-1-R transfected cells. In mice, injection of the hybrid in combination with glucose significantly (P < 0.001) reduced glucose and increased insulin concentrations, with metabolic actions evident (P < 0.05) 8 h post-injection. Twice-daily injection of N-ac(d-Ala2)GIP/GLP-1-exe to high fat fed (HFF) mice for 28 days significantly (P < 0.05-P < 0.001) reduced body weight, HbA1c, circulating glucose and insulin concentrations. Furthermore, both oral and i.p. glucose tolerance were improved (P < 0.001) and insulin sensitivity enhanced. The hybrid peptide also increased (P < 0.05-P < 0.001) beta cell number, islet area, pancreatic insulin content and islet insulin secretory responsiveness in HFF mice. Finally, N-ac(d-Ala2)GIP/GLP-1-exe treated mice exhibited improved (P < 0.01) recognition memory which was accompanied by enhanced (P < 0.05-P < 0.001) hippocampal neurogenesis, synapse formation and reduced neuronal oxidative stress. These data demonstrate for the first time the beneficial actions of the novel GIP/GLP-1 hybrid, N-ac(d-Ala2)GIP/GLP-1-exe, on glucose homeostasis and memory function in diabetes.

Apelin-13 analogues show potent in vitro and in vivo insulinotropic and glucose lowering actions.

Nine structurally modified apelin-13 analogues were assessed for their in vitro and acute in vivo antidiabetic potential. Stability was assessed in mouse plasma and insulinotropic efficacy tested in cultured pancreatic BRIN-BD11 cells and isolated mouse pancreatic islets. Intracellular Ca2+ and cAMP production in BRIN-BD11 cells was determined, as was glucose uptake in 3T3-L1 adipocytes. Acute antihyperglycemic effects of apelin analogues were assessed following i.p. glucose tolerance tests (ipGGT, 18 mmol/kg) in normal and diet-induced-obese (DIO) mice and on food intake in normal mice. Apelin analogues all showed enhanced in vitro stability (up to 5.8-fold, t½â€¯= 12.8 h) in mouse plasma compared to native apelin-13 (t½â€¯= 2.1 h). Compared to glucose controls, stable analogues exhibited enhanced insulinotropic responses from BRIN-BD11 cells (up to 4.7-fold, p < 0.001) and isolated mouse islets (up to 5.3-fold) for 10-7 M apelin-13 amide (versus 7.6-fold for 10-7 M GLP-1). Activation of APJ receptors on BRIN-BD11 cells increased intracellular Ca2+ (up to 3.0-fold, p < 0.001) and cAMP (up to 1.7-fold, p < 0.01). Acute ipGTT showed improved insulinotropic and glucose disposal responses in normal and DIO mice (p < 0.05 and p < 0.01, respectively). Apelin-13 amide and (pGlu)apelin-13 amide were the most effective analogues exhibiting acute, dose-dependent and persistent biological actions. Both analogues stimulated insulin-independent glucose uptake by differentiated adipocytes (2.9-3.3-fold, p < 0.05) and inhibited food intake (26-33%, p < 0.001), up to 180 min in mice, versus saline. In contrast, (Ala13)apelin-13 and (Val13)apelin-13 inhibited insulin secretion, suppressed beta-cell signal transduction and stimulated food intake in mice. Thus, stable analogues of apelin-13 have potential for diabetes/obesity therapy.

Cellular models for beta-cell function and diabetes gene therapy.

Diabetes is characterized by the destruction and/or relative dysfunction of insulin-secreting beta-cells in the pancreatic islets of Langerhans. Consequently, considerable effort has been made to understand the physiological processes governing insulin production and secretion in these cells and to elucidate the mechanisms involved in their deterioration in the pathogenesis of diabetes. To date, considerable research has exploited clonal beta-cell lines derived from rodent insulinomas. Such cell lines have proven to be a great asset in diabetes research, in vitro drug testing, and studies of beta-cell physiology and provide a sustainable, and in many cases, more practical alternative to the use of animals or primary tissue. However, selection of the most appropriate rodent beta cell line is often challenging and no single cell line entirely recapitulates the properties of human beta-cells. The generation of stable human beta-cell lines would provide a much more suitable model for studies of human beta-cell physiology and pathology and could potentially be used as a readily available source of implantable insulin-releasing tissue for cell-based therapies of diabetes. In this review, we discuss the history, development, functional characteristics and use of available clonal rodent beta-cell lines, as well as reflecting on recent advances in the generation of human-derived beta-cell lines, their use in research studies and their potential for cell therapy of 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.

Novel dual agonist peptide analogues derived from dogfish glucagon show promising in vitro insulin releasing actions and antihyperglycaemic activity in mice.

The antidiabetic potential of thirteen novel dogfish glucagon derived analogues were assessed in vitro and in acute in vivo studies. Stable peptide analogues enhanced insulin secretion from BRIN-BD11 ß-cells (p < 0.001) and reduced acute glycaemic responses following intraperitoneal glucose (25 nmol/kg) in healthy NIH Swiss mice (p < 0.05-p<0.001). The in vitro insulinotropic actions of [S2a]dogfish glucagon, [S2a]dogfish glucagon-exendin-4(31-39) and [S2a]dogfish glucagon-Lys(30)-γ-glutamyl-PAL, were blocked (p < 0.05-p<0.001) by the specific GLP-1 and glucagon receptor antagonists, exendin-4(9-39) and (desHis(1)Pro(4)Glu(9))glucagon amide but not by (Pro(3))GIP, indicating lack of GIP receptor involvement. These analogues dose-dependently stimulated cAMP production in GLP-1 and glucagon (p < 0.05-p<0.001) but not GIP-receptor transfected cells. They improved acute glycaemic and insulinotropic responses in high-fat fed diabetic mice and in wild-type C57BL/6J and GIPR-KO mice (p < 0.05-p<0.001), but not GLP-1R-KO mice, confirming action on GLP-1 but not GIP receptors. Overall, dogfish glucagon analogues have potential for diabetes therapy, exerting beneficial metabolic effects via GLP-1 and glucagon receptors.

Beneficial metabolic actions of a stable GIP agonist following pre-treatment with a SGLT2 inhibitor in high fat fed diabetic mice.

The purpose of the present study was to examine if a stable glucose-dependent insulinotropic polypeptide (GIP) agonist could exert beneficial metabolic control in diabetic mice which had been pre-treated with sodium-glucose-cotransporter-2 (SGLT2) inhibitor dapagliflozin (DAPA). High fat fed mice administered low dose streptozotocin (STZ) received vehicle, DAPA once-daily over 28 days, or DAPA once-daily for 14 days followed by (DAla(2))GIP once-daily for 14 days. Energy intake, body weight, glucose and insulin concentrations were measured at regular intervals. Glucose tolerance, insulin tolerance test, dual-energy X-ray absorptiometry (DEXA) and pancreatic histology were examined. Once-daily administration of (DAla(2))GIP for 14 days in high fat fed diabetic mice pre-treated with DAPA demonstrated significant decrease in body weight, blood glucose and increased insulin concentrations which were independent of changes in energy intake. Similarly, glucose tolerance, glucose-stimulated insulin secretion, insulin sensitivity and HOMA-ß were significantly enhanced in (DAla(2))GIP-treated mice. DEXA analysis revealed sustained percentage body fat loss with no changes in lean mass, bone mineral content and density. Pancreatic immunohistochemical analysis revealed decreased islet number and increases in islet area, beta cell area and pancreatic insulin content. The DAPA-induced increase in alpha cell area was also reversed. Additional acute in vitro and in vivo experiments confirmed that the impaired action of (DAla(2))GIP under hyperglycaemic-induced conditions was significantly reversed by DAPA treatment. These data demonstrate that (DAla(2))GIP can exert beneficial metabolic control in high fat fed diabetic mice pre-treated with DAPA. The results highlight possibility of a targeted and personalized approach using a GIP agonist and SGLT2 inhibitor for the treatment of type 2 diabetes.

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.

Stable oxyntomodulin analogues exert positive effects on hippocampal neurogenesis and gene expression as well as improving glucose homeostasis in high fat fed mice.

The weight-lowering and gluco-regulatory actions of oxyntomodulin (Oxm) have been well-documented however potential actions of this peptide in brain regions associated with learning and memory have not yet been evaluated. The present study examined the long-term actions of a stable acylated analogue of Oxm, (dS(2))Oxm(K-γ-glu-Pal), together with parent (dS(2))Oxm peptide, on hippocampal neurogenesis, gene expression and metabolic control in high fat (HF) mice. Groups of HF mice (n = 12) received twice-daily injections of Oxm analogues (both at 25 nmol/kg body weight) or saline vehicle (0.9% wt/vol) over 28 days. Hippocampal gene expression and histology were assessed together with evaluation of energy intake, body weight, non-fasting glucose and insulin, glucose tolerance, insulin sensitivity and lipids. Oxm analogues significantly reduced body weight, improved glucose tolerance, glucose-mediated insulin secretion, insulin sensitivity, islet architecture and lipid profile. Analysis of brain histology revealed significant reduction in hippocampal oxidative damage (8-oxoguanine), enhanced hippocampal neurogenesis (doublecortin) and improved hippocampal and cortical synaptogenesis (synaptophysin) following treatment. Furthermore, Oxm analogues up-regulated hippocampal mRNA expression of MASH1, Synaptophysin, SIRT1, GLUT4 and IRS1, and down-regulated expression of LDL-R and GSK3ß. These data demonstrate potential of stable Oxm analogues, and particularly (dS(2))Oxm(K-γ-glu-Pal) to improve metabolic function and enhance neurogenesis, synaptic plasticity, insulin signalling and exert protective effects against oxidative damage in hippocampus and cortex brain regions in HF mice.

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.