From the Clinical to the Bench: Exploring the Insulin Modulation Effects of Tacrolimus and Belatacept.

Calcineurin inhibitors (CNIs) are critical in preventing rejection posttransplantation but pose an increased risk of post-transplant diabetes (PTD). Recent studies show that late conversion from CNIs to belatacept, a costimulation blocker, improves HbA1c in kidney transplant recipients with PTD or de novo diabetes. This study investigates whether the observed effects on PTD stem solely from CNI withdrawal or if belatacept influences PTD independently. The study assessed the impact of tacrolimus and belatacept on insulin secretion in MIN6 cells (a beta cell line) and rat islets. Tacrolimus and belatacept were administered to the cells and islets, followed by assessments of cell viability and insulin secretion. Tacrolimus impaired insulin secretion without affecting cell viability, while belatacept showed no detrimental effects on either parameter. These findings support clinical observations of improved HbA1c upon switching from tacrolimus to belatacept. Belatacept holds promise in islet or pancreas transplantation, particularly in patients with unstable diabetes. Successful cases of islet transplantation treated with belatacept without severe hypoglycemia highlight its potential in managing PTD. Further research is needed to fully understand the metabolic changes accompanying the transition from CNIs to belatacept. Preserving insulin secretion emerges as a promising avenue for investigation in this context.

Inhibition of Toll-like Receptor 4 Using Small Molecule, TAK-242, Protects Islets from Innate Immune Responses.

Islet transplantation is a therapeutic option to replace ß-cell mass lost during type 1 or type 3c diabetes. Innate immune responses, particularly the instant blood-mediated inflammatory reaction and activation of monocytes, play a major role in the loss of transplanted islet tissue. In this study, we aimed to investigate the inhibition of toll-like receptor 4 (TLR4) on innate inflammatory responses. We first demonstrate a significant loss of graft function shortly after transplant through the assessment of miR-375 and miR-200c in plasma as biomarkers. Using in vitro models, we investigate how targeting TLR4 mitigates islet damage and immune cell activation during the peritransplant period. The results of this study support the application of TAK-242 as a therapeutic agent to reduce inflammatory and innate immune responses to islets immediately following transplantation into the hepatic portal vein. Therefore, TLR4 may serve as a target to improve islet transplant outcomes in the future.

The effect of aspartame on accelerating caspase-dependent apoptosis of pancreatic islet via ZIPK/STAT3/caspase 3 signaling pathway.

Aspartame (ASP) as an important sugar substitute is widely used in pharmaceutical and food processing. Here, we compared the effects of ASP and sucrose on mice pancreatic islet cells in vivo and observed that ASP with the condition of high concentration and long-term exposure (HASP) could cause insulin secretion (500 mg/kg for 1 month). Next, we conducted iTRAQ mass spectrometry to profile the global phosphoproteome and found that phosphorylation of zipper-interacting protein kinase (ZIPK) in murine pancreatic islet tissues were induced at Thr197, Thr242, Thr282, and Ser328 by high-sucrose (HS) treatment, but only induced at Thr197 and Ser328 by HASP treatment. Simultaneously, phosphorylation of STAT3 could be induced at Tyr705 and Ser727 by HS but not by HASP. Furthermore, presence of activated STAT3 accompanied with autophagy was observed in HS treatment. In turn, the inactivation of STAT3 as well as enhanced expression of caspase 3 was observed in HASP treatment. We generated Thr242APro and Thr282Pro on ZIPK using CRISPR-Cas9 in ß-TC3 cells and found the weakened interaction with STAT3 as well as the reduced phosphorylation of STAT3 even under HS stimulation. Finally, we observed that ankyrin repeat domain containing 11 (ANKRD11) could interact with ZIPK and play an inhibitory role in the phosphorylation of Thr242APro and Thr282Pro of ZIPK. However, HASP can induce the retention of ANKRD11 in the cytoplasm by phenylpyruvic acid (the metabolite of ASP). Taken together, this study determined that ASP with high concentration and long-term exposure could lead to caspase-dependent apoptosis of pancreatic islet cells through ANKRD11/ZIPK/STAT3 inhibition. Our results give evidence of adverse effects of aspartame on islet cells in some extreme conditions, which might help people to reconsider the biosafety of non-nutritive sweeteners.

The incretin co-agonist tirzepatide requires GIPR for hormone secretion from human islets.

The incretins glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) mediate insulin responses that are proportionate to nutrient intake to facilitate glucose tolerance1. The GLP-1 receptor (GLP-1R) is an established drug target for the treatment of diabetes and obesity2, whereas the therapeutic potential of the GIP receptor (GIPR) is a subject of debate. Tirzepatide is an agonist at both the GIPR and GLP-1R and is a highly effective treatment for type 2 diabetes and obesity3,4. However, although tirzepatide activates GIPR in cell lines and mouse models, it is not clear whether or how dual agonism contributes to its therapeutic benefit. Islet beta cells express both the GLP-1R and the GIPR, and insulin secretion is an established mechanism by which incretin agonists improve glycemic control5. Here, we show that in mouse islets, tirzepatide stimulates insulin secretion predominantly through the GLP-1R, owing to reduced potency at the mouse GIPR. However, in human islets, antagonizing GIPR activity consistently decreases the insulin response to tirzepatide. Moreover, tirzepatide enhances glucagon secretion and somatostatin secretion in human islets. These data demonstrate that tirzepatide stimulates islet hormone secretion from human islets through both incretin receptors.

Prenatal exposure to a mixture of PAHs causes the dysfunction of islet cells in adult male mice: Association with type 1 diabetes mellitus.

Polycyclic aromatic hydrocarbons (PAHs) have been detected throughout the human body. Whether exposure to PAHs is associated with the incidence of type 1 diabetes mellitus should be investigated. To this end, pregnant mice were exposed to mixed PAHs (5, 50, or 500 µg/kg) once every other day during gestation. The adult male offspring displayed impaired glucose tolerance and reduced serum levels of glucagon and insulin. Immunohistochemical staining revealed increased numbers of apoptotic ß-cells and a reduced ß-cell mass in these males. The downregulated expression of pancreatic estrogen receptor α, androgen receptor, and transcription factor PDX1 was responsible for impacting ß-cell development. The relatively reduced α-cell area was associated with downregulated ARX expression. The transcription of Isn2 and Gcg in pancreatic tissue was downregulated, which indicated that the function of ß-cells and α-cells was impaired. Methylation levels in the Isn2 promotor were significantly elevated in mice prenatally exposed to 500 µg/kg PAHs, which was consistent with the change in its mRNA levels. The number of macrophages infiltrating islets was significantly increased, indicating that prenatal PAH exposure might reduce islet cell numbers in an autoimmune manner. This study shows that prenatal exposure to PAHs may promote the pathogenesis of type 1 diabetes mellitus.

Metformin attenuates high glucose-induced injury in islet microvascular endothelial cells.

As one of the most frequently prescribed antidiabetic drugs, metformin can lower glucose levels, improve insulin resistance manage body weight. However, the effect of metformin on islet microcirculation remains unclear. In the present study, to explore the effect of metformin on islet endothelial cells and investigated the underlying mechanism, we assessed the effects of metformin on islet endothelial cell survival, proliferation, oxidative stress and apoptosis. Our results suggest that metformin stimulates the proliferation of pancreatic islet endothelial cells and inhibits the apoptosis and oxidative stress caused by high glucose levels. By activating farnesoid X receptor (FXR), metformin increases the expression of vascular endothelial growth factor-A (VEGF-A) and endothelial nitric oxide synthase (eNOS), improves the production of nitric oxide (NO) and decreases the production of ROS. After the inhibition of FXR or VEGF-A, all of the effects disappeared. Thus, metformin appears to regulate islet microvascular endothelial cell (IMEC) proliferation, apoptosis and oxidative stress by activating the FXR/VEGF-A/eNOS pathway. These findings provide a new mechanism underlying the islet-protective effect of metformin.

Adult pancreatic islet endocrine cells emerge as fetal hormone-expressing cells.

The precise developmental dynamics of the pancreatic islet endocrine cell types, and their interrelation, are unknown. Some authors claim the persistence of islet cell differentiation from precursor cells after birth ("neogenesis"). Here, using four conditional cell lineage tracing ("pulse-and-chase") murine models, we describe the natural history of pancreatic islet cells, once they express a hormone gene, until late in life. Concerning the contribution of early-appearing embryonic hormone-expressing cells to the formation of islets, we report that adult islet cells emerge from embryonic hormone-expressing cells arising at different time points during development, without any evidence of postnatal neogenesis. We observe specific patterns of hormone gene activation and switching during islet morphogenesis, revealing that, within each cell type, cells have heterogeneous developmental trajectories. This likely applies to most maturating cells in the body, and explains the observed phenotypic variability within differentiated cell types. Such knowledge should help devising novel regenerative therapies.

TNF-α Antagonizes the Effect of Leptin on Insulin Secretion through FOXO1-Dependent Transcriptional Suppression of LepRb in INS-1 Cells.

Proinflammatory cytokines play a causal role in the development of hyperinsulinemia and T2MD. FOXO1, a transcription factor which is known to enhance proinflammation, was recently shown to be involved in obesity-induced ß cell dysfunction. However, molecular mechanisms for the association remained elusive. In this study, we first found that both leptin (10 nM) and TNF-α (20 ng/ml) significantly inhibited glucose-stimulated insulin secretion (GSIS) of INS-1E cells. When in combination, the GSIS function of INS-1E cells was significantly increased compared with that of the leptin alone treatment, indicating that TNF-α attenuated the inhibiting effect of leptin on GSIS of INS-1E cells. Similarly, we found that TNF-α has the same inhibitory effect on leptin in regulating insulin synthesis and secretion, and the survival and apoptosis of insulin cells. Further studies showed that TNF-α blocks leptin pathway by reducing the expression of leptin receptor (LepRb, also called OBRb) and inhibiting the activation of STAT3, a key molecule involved in the leptin signaling pathway in INS-1E cells. Besides, the downregulated expression of phosphorylated FOXO1 was found to be involved in the possible mechanism of TNF-α. Overexpression of constitutively active FOXO1 markedly aggravated the LepRb reduction by TNF-α treatment of INS-1E cells, and the endogenous FOXO1 knockdown abolished the effect of TNF-α on INS-1E cells. Furthermore, we have proved that FOXO1 could directly bind to the promoter of LepRb as a negative transcription regulator. Taken together, the results of this study reveal that TNF-α-induced LepRb downregulated in pancreatic ß cells and demonstrate that transcriptional reduction of FOXO1 might be the primary mechanism underlying TNF-α promoting INS-1E leptin resistance and ß cell dysfunction. Conclusions. Our current studies based on INS-1E cells in vitro indicate that the inflammatory factor TNF-α plays an important role in the development of INS-1E leptin resistance and glucose metabolism disorders, probably through FOXO1-induced transcription reduction of LepRb promoter in pancreatic ß cells, and FOXO1 may be a novel target for treating ß cell dysfunction in obesity-induced hyperinsulinemia and T2DM.

P2Y1 purinergic receptor identified as a diabetes target in a small-molecule screen to reverse circadian ß-cell failure.

The mammalian circadian clock drives daily oscillations in physiology and behavior through an autoregulatory transcription feedback loop present in central and peripheral cells. Ablation of the core clock within the endocrine pancreas of adult animals impairs the transcription and splicing of genes involved in hormone exocytosis and causes hypoinsulinemic diabetes. Here, we developed a genetically sensitized small-molecule screen to identify druggable proteins and mechanistic pathways involved in circadian ß-cell failure. Our approach was to generate ß-cells expressing a nanoluciferase reporter within the proinsulin polypeptide to screen 2640 pharmacologically active compounds and identify insulinotropic molecules that bypass the secretory defect in CRISPR-Cas9-targeted clock mutant ß-cells. We validated hit compounds in primary mouse islets and identified known modulators of ligand-gated ion channels and G-protein-coupled receptors, including the antihelmintic ivermectin. Single-cell electrophysiology in circadian mutant mouse and human cadaveric islets revealed ivermectin as a glucose-dependent secretagogue. Genetic, genomic, and pharmacological analyses established the P2Y1 receptor as a clock-controlled mediator of the insulinotropic activity of ivermectin. These findings identify the P2Y1 purinergic receptor as a diabetes target based upon a genetically sensitized phenotypic screen.

Circadian rhythms ­ 'inbuilt' 24-hour cycles ­ control many aspects of behaviour and physiology. In mammals, they operate in nearly all tissues, including those involved in glucose metabolism. Recent studies have shown that mice with faulty genes involved in circadian rhythms, the core clock genes, can develop diabetes. Diabetes arises when the body struggles to regulate blood sugar levels. In healthy individuals, the hormone insulin produced by beta cells in the pancreas regulates the amount of sugar in the blood. But when beta cells are faulty and do not generate sufficient insulin levels, or when insulin lacks the ability to stimulate cells to take up glucose, diabetes can develop. Marcheva, Weidemann, Taguchi et al. wanted to find out if diabetes caused by impaired clock genes could be treated by targeting pathways regulating the secretion of insulin. To do so, they tested over 2,500 potential drugs on genetically modified beta cells with faulty core clock genes. They further screened the drugs on mice with the same defect in their beta cells. Marcheva et al. identified one potential compound, the anti-parasite drug ivermectin, which was able to restore the secretion of insulin. When ivermectin was applied to both healthy mice and mice with faulty beta cells, the drug improved the control over glucose levels by activating a specific protein receptor that senses molecules important for storing and utilizing energy. The findings reveal new drug targets for treating forms of diabetes associated with deregulation of the pancreatic circadian clock. The drug screening strategy used in the study may also be applied to reveal mechanisms underlying other conditions associated with disrupted circadian clocks, including sleep loss and jetlag.

Targeted Polymeric Nanoparticles Based on Mangiferin for Enhanced Protection of Pancreatic ß-Cells and Type 1 Diabetes Mellitus Efficacy.

Mangiferin (MGF) is found in many natural plants, such as Rhizoma Anemarrhenae, and has anti-diabetes effects. However, its clinical applications and development are limited by poor solubility and low-concentration enrichment in pancreatic islets. In this paper, targeted polymeric nanoparticles were constructed for MGF delivery with the desired drug loading content (6.86 ± 0.60%), excellent blood circulation, and missile-like delivery to the pancreas. Briefly, Glucagon-like peptide 1 (GLP-1) as an active targeting agent to the pancreas was immobilized on the block copolymer polyethyleneglycol-polycaprolactone (PEG-PCL) to obtain final GLP-1-PEG-PCL amphiphiles. Spherical MGF-loaded polymeric nanoparticles were acquired from the self-assembly of the targeted GDPP nanoparticles and MGF with a homogeneous size of 158.9 ± 1.7 nm and a negative potential for a good steady state in circulation. In this drug vehicle, GLP-1 acts as the missile vanguard via the GLP-1 receptor on the surface of the pancreas for improving the accumulation and efficiency of MGF in the pancreas, the hypoglycemic effect of MGF, and the restorative effect on pancreatic islets, which were investigated. As compared to free MGF, MGF/GDPP nanoparticles appeared to be more concentrated in the pancreas, with better blood glucose and glucose tolerance, enhanced insulin levels, increased ß-cell proliferation, reduced ß-cell apoptosis, and islet repair in vivo. This targeted drug delivery system provided a novel strategy and hope for enhancing MGF delivery and anti-diabetes efficacy.

Possibility of adiponectin use to improve islet transplantation outcomes.

Although islet transplantation (ITx) is a promising therapy for severe diabetes mellitus, further advancements are necessary. Adiponectin, an adipokine that regulates lipid and glucose metabolism, exerts favorable effects on islets, such as reinforcement of the insulin-releasing function. This study evaluated the possibility of adiponectin use to improve ITx outcomes. We treated mouse islets with 10 µg/mL recombinant mouse adiponectin by overnight culture and then assessed the insulin-releasing, angiogenic, and adhesion functions of the islets. Furthermore, 80 syngeneic islet equivalents with or without adiponectin treatment were transplanted into the renal subcapsular space of diabetic mice. In in vitro assessment, released insulin at high glucose stimulation, insulin content, and expressions of vascular endothelial growth factor and integrin ß1 were improved in adiponectin-treated islets. Furthermore, adiponectin treatment improved the therapeutic effect of ITx on blood glucose levels and promoted angiogenesis of the transplanted islets. However, the therapeutic effect was not pronounced in glucose tolerance test results. In conclusion, adiponectin treatment had preferable effects in the insulin-releasing, angiogenic, and adhesion functions of islets and contributed to the improvement of ITx. The future use of adiponectin treatment in clinical settings to improve ITx outcomes should be investigated.

Glucose Regulates m6A Methylation of RNA in Pancreatic Islets.

Type 2 diabetes is characterized by chronic hyperglycemia associated with impaired insulin action and secretion. Although the heritability of type 2 diabetes is high, the environment, including blood components, could play a major role in the development of the disease. Amongst environmental effects, epitranscriptomic modifications have been recently shown to affect gene expression and glucose homeostasis. The epitranscriptome is characterized by reversible chemical changes in RNA, with one of the most prevalent being the m6A methylation of RNA. Since pancreatic ß cells fine tune glucose levels and play a major role in type 2 diabetes physiopathology, we hypothesized that the environment, through variations in blood glucose or blood free fatty acid concentrations, could induce changes in m6A methylation of RNAs in pancreatic ß cells. Here we observe a significant decrease in m6A methylation upon high glucose concentration, both in mice and human islets, associated with altered expression levels of m6A demethylases. In addition, the use of siRNA and/or specific inhibitors against selected m6A enzymes demonstrate that these enzymes modulate the expression of genes involved in pancreatic ß-cell identity and glucose-stimulated insulin secretion. Our data suggest that environmental variations, such as glucose, control m6A methylation in pancreatic ß cells, playing a key role in the control of gene expression and pancreatic ß-cell functions. Our results highlight novel causes and new mechanisms potentially involved in type 2 diabetes physiopathology and may contribute to a better understanding of the etiology of this disease.

Considerations and challenges of islet transplantation and future therapies on the horizon.

Islet transplantation is a treatment for selected adults with type 1 diabetes and severe hypoglycemia. Islets from two or more donor pancreases, a scarce resource, are usually required to impact glycemic control, but the treatment falls short of a cure. Islets are avascular when transplanted into the hypoxic liver environment and subjected to inflammatory insults, immune attack, and toxicity from systemic immunosuppression. The Collaborative Islet Transplant Registry, with outcome data on over 1,000 islet transplant recipients, has demonstrated that larger islet numbers transplanted and older age of recipients are associated with better outcomes. Induction with T-cell depleting agents and the TNF-α inhibitor etanercept and maintenance systemic immunosuppression with mTOR inhibitors in combination with calcineurin inhibitors also appear advantageous, but concerns remain over immunosuppressive toxicity. We discuss strategies and therapeutics that address specific challenges of islet transplantation, many of which are at the preclinical stage of development. On the horizon are adjuvant cell therapies with mesenchymal stromal cells and regulatory T cells that have been used in preclinical models and in humans in other contexts; such a strategy may enable reductions in immunosuppression in the early peri-transplant period when the islets are vulnerable to apoptosis. Human embryonic stem cell-derived islets are in early-phase clinical trials and hold the promise of an inexhaustible supply of insulin-producing cells; effective encapsulation of such cells or, silencing of the human leukocyte antigen (HLA) complex would eliminate the need for immunosuppression, enabling this therapy to be used in all those with type 1 diabetes.

Underappreciated roles for Rho GDP dissociation inhibitors (RhoGDIs) in cell function: Lessons learned from the pancreatic islet ß-cell.

Rho subfamily of G proteins (e.g., Rac1) have been implicated in glucose-stimulated insulin secretion from the pancreatic ß-cell. Interestingly, metabolic stress (e.g., chronic exposure to high glucose) results in sustained activation of Rac1 leading to increased oxidative stress, impaired insulin secretion and ß-cell dysfunction. Activation-deactivation of Rho G proteins is mediated by three classes of regulatory proteins, namely the guanine nucleotide exchange factors (GEFs), which facilitate the conversion of inactive G proteins to their active conformations; the GTPase-activating proteins (GAPs), which convert the active G proteins to their inactive forms); and the GDP-dissociation inhibitors (GDIs), which prevent the dissociation of GDP from G proteins. Contrary to a large number of GEFs (82 members) and GAPs (69 members), only three members of RhoGDIs (RhoGDIα, RhoGDIß and RhoGDIγ) are expressed in mammalian cells.Even though relatively smaller in number, the GDIs appear to play essential roles in G protein function (e.g., subcellular targeting) for effector activation and cell regulation. Emerging evidence also suggests that the GDIs are functionally regulated via post-translational modification (e.g., phosphorylation) and by lipid second messengers, lipid kinases and lipid phosphatases. We highlight the underappreciated regulatory roles of RhoGDI-Rho G protein signalome in islet ß-cell function in health and metabolic stress. Potential knowledge gaps in the field, and directions for future research for the identification of novel therapeutic targets to loss of functional ß-cell mass under the duress of metabolic stress are highlighted.

Phytochemicals modulate pancreatic islet ß cell function through glucagon-like peptide-1-related mechanisms.

Glucagon-like peptide-1 (GLP-1) receptor-based therapies have been developed and extensively applied in clinical practice. GLP-1 plays an important role in improving glycemic homeostasis by stimulating insulin biosynthesis and secretion, suppressing glucagon activity, delaying gastric emptying, and reducing appetite and food ingestion. Furthermore, GLP-1 has positive effects on ß-cell function by promoting ß-cell proliferation and neogenesis while simultaneously reducing apoptosis. Here, we summarize possible mechanisms of action of GLP-1 upon pancreatic islets as well as describe phytochemicals that modulate pancreatic islet ß cell function through glucagon-like peptide-1-related mechanisms. Together, this information provides potential lead compound candidates against diabetes that function as GLP-1 receptor-based pharmacotherapy.

Eugenosedin-A improves obesity-related hyperglycemia by regulating ATP-sensitive K+ channels and insulin secretion in pancreatic ß cells.

Eugenosedin-A (Eu-A) has been shown to protect against hyperglycemia- and hyperlipidemia-induced metabolic syndrome. We investigated the relationship of KATP channel activities and insulin secretion by Eu-A in vitro in pancreatic ß-cells, and examined the effect of Eu-A on streptozotocin (STZ)/nicotinamide (NA)-induced type 2 diabetes mellitus (T2DM) in vivo. We isolated pancreatic islets from adult male Wistar rats (250-350 g) and identified pancreatic ß-cells by the cell size, capacitance and membrane potential. Perforated patch-clamp and inside-out recordings were used to monitor the membrane potential (current-clamp mode) and channel activity (voltage-clamp mode) of ß-cells. The membrane potential of ß-cells was raised by Eu-A and reversed by the KATP channel activator diazoxide. Eu-A inhibited the KATP channel activity measured at - 60 mV and increased the intracellular calcium concentration ([Ca2+]i), resulting in enhanced insulin secretion. Eu-A also reduced Kir6.2 protein on the cell membrane and scattered in the cytosol under normal glucose conditions (5.6 mM). In our animal study, rats were divided into normal and STZ/NA-induced T2DM groups. Normal rats fed with regular chow were divided into control and control+Eu-A (5 mg/kg/day, i.p.) groups. The STZ/NA-induced diabetic rats fed with a high-fat diet (HFD) were divided into three groups: T2DM, T2DM+Eu-A (5 mg/kg/day, i.p.), and T2DM+glibenclamide (0.5 mg/kg/day, i.p.; a KATP channel inhibitor). Both Eu-A and glibenclamide decreased the rats' blood glucose, prevented weight gain, and enhanced insulin secretion. We found that Eu-A blocked pancreatic ß-cell KATP channels, caused membrane potential depolarization, and stimulated Ca2+ influx, thus increasing insulin secretion. Furthermore, Eu-A decreased blood glucose and increased insulin levels in T2DM rats. These results suggested that Eu-A might have clinical benefits for the control of T2DM and its complications.

1α,25-(OH)2 vitamin D3 prevents insulin resistance and regulates coordinated exocytosis and insulin secretion.

Vitamin D3 is associated with improvements in insulin resistance and glycemia. In this study, we investigated the short-term effect of 1α,25(OH)2 Vitamin D3 (1,25-D3) and cholecalciferol (vitamin D3) on the glycemia and insulin sensitivity of control and dexamethasone-induced insulin-resistance rats. 45Ca2+ influx responses to 1,25-D3 and its role in insulin secretion were investigated in isolated pancreatic islets from control rats. In vivo, 5 d treatment with 1,25-D3 (i.p.) prevented insulin resistance in dexamethasone-treated rats. Treatment with 1,25-D3 improved the activities of hepatic enzymes, serum lipids and calcium concentrations in insulin-resistant rats. 25-D3 (o.g.) does not affect insulin resistance. In pancreatic islets, 1,25-D3 increased insulin secretion and stimulated rapid response 45Ca2+ influx. The stimulatory effect of 1,25-D3 on 45Ca2+ influx was decreased by diazoxide, apamine, thapsigargin, dantrolene, 2-APB, nifedipine, TEA, PKA, PKC, and cytoskeleton inhibitor, while it was increased by glibenclamide and N-ethylmaleimide. The stimulatory effect of 1,25-D3 on 45Ca2+ influx involves the activation of L-type VDCC, K+-ATP, K+-Ca2+, and Kv channels, which augment cytosolic calcium. These ionic changes mobilize calcium from stores and downstream activation of PKC, PKA tethering vesicle traffic and fusion at the plasma membrane for insulin secretion. This is the first study highlighting the unprecedented role of 1,25-D3 (short-term effect) in the regulation of glucose homeostasis and on prevention of insulin resistance. Furthermore, this study shows the intracellular ß-cell signal transduction of 1,25-D3 through the modulation of pivotal ionic channels and proteins exhibiting a coordinated exocytosis of vesicles for insulin secretion.

Liver-Expressed Antimicrobial Peptide 2 antagonizes the insulinostatic effect of ghrelin in rat isolated pancreatic islets.

The hormone ghrelin is the endogenous agonist of the G protein-coupled receptor (GPCR) termed growth hormone secretagogue receptor (GHSR). Ghrelin inhibits glucose-stimulated insulin secretion by activating pancreatic GHSR. Recently, Liver-Expressed Antimicrobial Peptide 2 (LEAP2) was recognized as an endogenous GHSR ligand that blocks ghrelin-induced actions. Nonetheless, the effect of LEAP2 on glucose-stimulated insulin secretion from pancreatic islets is unknown. We aimed at exploring the activity of LEAP2 on glucose-stimulated insulin secretion. Islets of Langerhans isolated from rat pancreas were exposed to glucose in the presence or in the absence of LEAP2 and ghrelin and then insulin secretion was assayed. LEAP2 did not modulate glucose-stimulated insulin secretion. However, LEAP2 blocked the insulinostatic action of ghrelin. Our data show that LEAP2 behaves as an antagonist of pancreatic GHSR.