Model of Calcium Dynamics Regulating [Formula: see text], ATP and Insulin Production in a Pancreatic [Formula: see text]-Cell.

The calcium signals regulate the production and secretion of many signaling molecules like inositol trisphosphate ([Formula: see text]) and adenosine triphosphate (ATP) in various cells including pancreatic [Formula: see text]-cells. The calcium signaling mechanisms regulating [Formula: see text], ATP and insulin responsible for various functions of [Formula: see text]-cells are still not well understood. Any disturbance in these mechanisms can alter the functions of [Formula: see text]-cells leading to diabetes and metabolic disorders. Therefore, a mathematical model is proposed by incorporating the reaction-diffusion equation for calcium dynamics and a system of first-order differential equations for [Formula: see text], ATP-production and insulin secretion with initial and boundary conditions. The model incorporates the temporal dependence of [Formula: see text]-production and degradation, ATP production and insulin secretion on calcium dynamics in a [Formula: see text]-cell. The piecewise linear finite element method has been used for the spatial dimension and the Crank-Nicolson scheme for the temporal dimension to obtain numerical results. The effect of changes in source influxes and buffers on calcium dynamics and production of [Formula: see text], ATP and insulin levels in a [Formula: see text]-cell has been analyzed. It is concluded that the dysfunction of source influx and buffers can cause significant variations in calcium levels and dysregulation of [Formula: see text], ATP and insulin production, which can lead to various metabolic disorders, diabetes, obesity, etc. The proposed model provides crucial information about the changes in mechanisms of calcium dynamics causing proportionate disturbances in [Formula: see text], ATP and insulin levels in pancreatic cells, which can be helpful for devising protocols for diagnosis and treatment of various metabolic diseases.

Insulin at the intersection of thermoregulation and glucose homeostasis.

Mammals are protected from changes in environmental temperature by altering energetic processes that modify heat production. Insulin is the dominant stimulus of glucose uptake and metabolism, which are fundamental for thermogenic processes. The purpose of this work was to determine the interaction of ambient temperature induced changes in energy expenditure (EE) on the insulin sensitivity of glucose fluxes. Short-term and adaptive responses to thermoneutral temperature (TN, ∼28 °C) and room (laboratory) temperature (RT, ∼22 °C) were studied in mice. This range of temperature does not cause detectable changes in circulating catecholamines or shivering and postabsorptive glucose homeostasis is maintained. We tested the hypothesis that a decrease in EE that occurs with TN causes insulin resistance and that this reduction in insulin action and EE is reversed upon short term (<12h) transition to RT. Insulin-stimulated glucose disposal (Rd) and tissue-specific glucose metabolic index were assessed combining isotopic tracers with hyperinsulinemic-euglycemic clamps. EE and insulin-stimulated Rd are both decreased (∼50%) in TN-adapted vs RT-adapted mice. When RT-adapted mice are switched to TN, EE rapidly decreases and Rd is reduced by ∼50%. TN-adapted mice switched to RT exhibit a rapid increase in EE, but whole-body insulin-stimulated Rd remains at the low rates of TN-adapted mice. In contrast, whole body glycolytic flux rose with EE. This higher EE occurs without increasing glucose uptake from the blood, but rather by diverting glucose from glucose storage to glycolysis. In addition to adaptations in insulin action, 'insulin-independent' glucose uptake in brown fat is exquisitely sensitive to thermoregulation. These results show that insulin action adjusts to non-stressful changes in ambient temperature to contribute to the support of body temperature homeostasis without compromising glucose homeostasis.

Insulin regulates human pancreatic endocrine cell differentiation in vitro.

OBJECTIVE: The consequences of mutations in genes associated with monogenic forms of diabetes on human pancreas development cannot be studied in a time-resolved fashion in vivo. More specifically, if recessive mutations in the insulin gene influence human pancreatic endocrine lineage formation is still an unresolved question. METHODS: To model the extremely reduced insulin levels in patients with recessive insulin gene mutations, we generated a novel knock-in H2B-Cherry reporter human induced pluripotent stem cell (iPSC) line expressing no insulin upon differentiation to stem cell-derived (SC-) ß cells in vitro. Differentiation of iPSCs into the pancreatic and endocrine lineage, combined with immunostaining, Western blotting and proteomics analysis phenotypically characterized the insulin gene deficiency in SC-islets. Furthermore, we leveraged FACS analysis and confocal microscopy to explore the impact of insulin shortage on human endocrine cell induction, composition, differentiation and proliferation. RESULTS: Interestingly, insulin-deficient SC-islets exhibited low insulin receptor (IR) signaling when stimulated with glucose but displayed increased IR sensitivity upon treatment with exogenous insulin. Furthermore, insulin shortage did not alter neurogenin-3 (NGN3)-mediated endocrine lineage induction. Nevertheless, lack of insulin skewed the SC-islet cell composition with an increased number in SC-ß cell formation at the expense of SC-α cells. Finally, insulin deficiency reduced the rate of SC-ß cell proliferation but had no impact on the expansion of SC-α cells. CONCLUSIONS: Using iPSC disease modelling, we provide first evidence of insulin function in human pancreatic endocrine lineage formation. These findings help to better understand the phenotypic impact of recessive insulin gene mutations during pancreas development and shed light on insulin gene function beside its physiological role in blood glucose regulation.

Update on the ZNT8 epitope and its role in the pathogenesis of type 1 diabetes.

Type 1 diabetes (T1D) is an organ-specific chronic autoimmune disease mediated by autoreactive T cells. ZnT8 is a pancreatic islet-specific zinc transporter that is mainly located in ß cells. It not only participates in the synthesis, storage and secretion of insulin but also maintains the structural integrity of insulin. ZnT8 is the main autoantigen recognized by autoreactive CD8+ T cells in children and adults with T1D. This article summarizes the latest research results on the T lymphocyte epitope and B lymphocyte epitope of ZnT8 in the current literature. The structure and expression of ZnT8, the role of ZnT8 in insulin synthesis and its role in autoimmunity are reviewed. ZnT8 is the primary autoantigen of T1D and is specifically expressed in pancreatic islets. Thus, it is one of biomarkers for the diagnosis of T1D. It has broad prospects for further research on immunomodulators for the treatment of T1D.

Hepatic insulin synthesis increases in rat models of diabetes mellitus type 1 and 2 differently.

Insulin-positive (+) cells (IPCs), detected in multiple organs, are of great interest as a probable alternative to ameliorate pancreatic beta-cells dysfunction and insulin deficiency in diabetes. Liver is a potential source of IPCs due to it common embryological origin with pancreas. We previously demonstrated the presence of IPCs in the liver of healthy and diabetic rats, but detailed description and analysis of the factors, which potentially can induced ectopic hepatic expression of insulin in type 1 (T1D) and type 2 diabetes (T2D), were not performed. In present study we evaluate mass of hepatic IPCs in the rat models of T1D and T2D and discuss factors, which may stimulate it generation: glycaemia, organ injury, involving of hepatic stem/progenitor cell compartment, expression of transcription factors and inflammation. Quantity of IPCs in the liver was up by 1.7-fold in rats with T1D and 10-fold in T2D compared to non-diabetic (ND) rats. We concluded that ectopic hepatic expression of insulin gene is activated by combined action of a number of factors, with inflammation playing a decision role.

Ube4A maintains metabolic homeostasis and facilitates insulin signaling in vivo.

OBJECTIVE: Defining the regulators of cell metabolism and signaling is essential to design new therapeutic strategies in obesity and NAFLD/NASH. E3 ubiquitin ligases control diverse cellular functions by ubiquitination-mediated regulation of protein targets, and thus their functional aberration is associated with many diseases. The E3 ligase Ube4A has been implicated in human obesity, inflammation, and cancer. However, its in vivo function is unknown, and no animal models are available to study this novel protein. METHODS: A whole-body Ube4A knockout (UKO) mouse model was generated, and various metabolic parameters were compared in chow- and high fat diet (HFD)-fed WT and UKO mice, and in their liver, adipose tissue, and serum. Lipidomics and RNA-Seq studies were performed in the liver samples of HFD-fed WT and UKO mice. Proteomic studies were conducted to identify Ube4A's targets in metabolism. Furthermore, a mechanism by which Ube4A regulates metabolism was identified. RESULTS: Although the body weight and composition of young, chow-fed WT and UKO mice are similar, the knockouts exhibit mild hyperinsulinemia and insulin resistance. HFD feeding substantially augments obesity, hyperinsulinemia, and insulin resistance in both sexes of UKO mice. HFD-fed white and brown adipose tissue depots of UKO mice have increased insulin resistance and inflammation and reduced energy metabolism. Moreover, Ube4A deletion exacerbates hepatic steatosis, inflammation, and liver injury in HFD-fed mice with increased lipid uptake and lipogenesis in hepatocytes. Acute insulin treatment resulted in impaired activation of the insulin effector protein kinase Akt in liver and adipose tissue of chow-fed UKO mice. We identified the Akt activator protein APPL1 as a Ube4A interactor. The K63-linked ubiquitination (K63-Ub) of Akt and APPL1, known to facilitate insulin-induced Akt activation, is impaired in UKO mice. Furthermore, Ube4A K63-ubiquitinates Akt in vitro. CONCLUSION: Ube4A is a novel regulator of obesity, insulin resistance, adipose tissue dysfunction and NAFLD, and preventing its downregulation may ameliorate these diseases.

Differentiation of stem cell-derived pancreatic progenitors into insulin-secreting islet clusters in a multiwell-based static 3D culture system.

Orbital shaker-based suspension culture systems have been in widespread use for differentiating human pluripotent stem cell (hPSC)-derived pancreatic progenitors toward islet-like clusters during endocrine induction stages. However, reproducibility between experiments is hampered by variable degrees of cell loss in shaking cultures, which contributes to variable differentiation efficiencies. Here, we describe a 96-well-based static suspension culture method for differentiation of pancreatic progenitors into hPSC-islets. Compared with shaking culture, this static 3D culture system induces similar islet gene expression profiles during differentiation processes but significantly reduces cell loss and improves cell viability of endocrine clusters. This static culture method results in more reproducible and efficient generation of glucose-responsive, insulin-secreting hPSC-islets. The successful differentiation and well-to-well consistency in 96-well plates also provides a proof of principle that the static 3D culture system can serve as a platform for small-scale compound screening experiments as well as facilitating further protocol development.

Differential expression of some termite neuropeptides and insulin/IGF-related hormones and their plausible functions in growth, reproduction and caste determination.

Background: Insulin-like growth factor (IGF) and other insulin-like peptides (ilps) are important hormones regulating growth and development in animals. Whereas most animals have a single female and male adult phenotype, in some insect species the same genome may lead to different final forms. Perhaps the best known example is the honeybee where females can either develop into queens or workers. More extreme forms of such polyphenism occur in termites, where queens, kings, workers and soldiers coexist. Both juvenile hormone and insulin-like peptides are known to regulate growth and reproduction as well as polyphenism. In termites the role of juvenile hormone in reproduction and the induction of the soldier caste is well known, but the role of IGF and other ilps in these processes remains largely unknown. Here the various termite ilps are identified and hypotheses regarding their functions suggested. Methods: Genome assemblies and transcriptome short read archives (SRAs) were used to identify insulin-like peptides and neuropeptides in termites and to determine their expression in different species, tissues and castes. Results and Discussion: Termites have seven different ilps, i.e. gonadulin, IGF and an ortholog of Drosophila insulin-like peptide 7 (dilp7), which are commonly present in insects, and four smaller peptides, that have collectively been called short IGF-related peptides (sirps) and individually atirpin, birpin, cirpin and brovirpin. Gonadulin is lost from the higher termites which have however amplified the brovirpin gene, of which they often have two or three paralogs. Based on differential expression of these genes it seems likely that IGF is a growth hormone and atirpin an autocrine tissue factor that is released when a tissue faces metabolic stress. Birpin seems to be responsible for growth and in the absence of juvenile hormone this may lead to reproductive adults or, when juvenile hormone is present, to soldiers. Brovirpin is expressed both by the brain and the ovary and likely stimulates vitellogenesis, while the function of cirpin is less clear.

Conditioning-induced expression of novel glucose transporters in canine skeletal muscle homogenate.

Athletic conditioning can increase the capacity for insulin-stimulated skeletal muscle glucose uptake through increased sarcolemmal expression of GLUT4 and potentially additional novel glucose transporters. We used a canine model that has previously demonstrated conditioning-induced increases in basal, insulin- and contraction-stimulated glucose uptake to identify whether expression of glucose transporters other than GLUT4 was upregulated by athletic conditioning. Skeletal muscle biopsies were obtained from 12 adult Alaskan Husky racing sled dogs before and after a full season of conditioning and racing, and homogenates from those biopsies were assayed for expression of GLUT1, GLUT3, GLUT4, GLUT6, GLUT8, and GLUT12 using western blots. Athletic conditioning resulted in a 1.31 ± 0.70 fold increase in GLUT1 (p <0.0001), 1.80 ± 1.99 fold increase in GLUT4 (p = 0.005), and 2.46 ± 2.39 fold increase in GLUT12 (p = 0.002). The increased expression of GLUT1 helps explain the previous findings of conditioning-induced increases in basal glucose clearance in this model, and the increase in GLUT12 provides an alternative mechanism for insulin- and contraction-mediated glucose uptake and likely contributes to the substantial conditioning-induced increases in insulin sensitivity in highly trained athletic dogs. Furthermore, these results suggest that athletic dogs can serve as a valuable resource for the study of alternative glucose transport mechanisms in higher mammals.

DYRK1A inhibitors leucettines and TGF-ß inhibitor additively stimulate insulin production in beta cells, organoids, and isolated mouse islets.

The decreased ß-cell mass and impaired ß-cell functionality are the primary causes of diabetes mellitus (DM). Nevertheless, the underlying molecular mechanisms by which ß-cell growth and function are controlled are not fully understood. In this work, we show that leucettines, known to be DYRK1A kinase inhibitors, can improve glucose-stimulated insulin secretion (GSIS) in rodent ß-cells and isolated islets, as well as in hiPSC-derived ß-cells islets. We confirm that DYRK1A is expressed in murine insulinoma cells MIN6. In addition, we found that treatment with selected leucettines stimulates proliferation of ß-cells and promotes MIN6 cell cycle progression to the G2/M phase. This effect is also confirmed by increased levels of cyclin D1, which is highly responsive to proliferative signals. Among other leucettines, leucettine L43 had a negligible impact on ß-cell proliferation, but markedly impair GSIS. However, leucettine L41, in combination with LY364947, a, a potent and selective TGF-ß type-I receptor, significantly promotes GSIS in various cellular diabetic models, including MIN6 and INS1E cells in 2D and 3D culture, iPSC-derived ß-cell islets derived from iPSC, and isolated mouse islets, by increased insulin secretion and decreased glucagon level. Our findings confirm an important role of DYRK1A inhibitors as modulators of ß-cells function and suggested a new potential target for antidiabetic therapy. Moreover, we show in detail that leucettine derivatives represent promising antidiabetic agents and are worth further evaluation, especially in vivo.

Brain insulin signaling suppresses lipolysis in the absence of peripheral insulin receptors and requires the MAPK pathway.

OBJECTIVES: Insulin's ability to counterbalance catecholamine-induced lipolysis defines insulin action in adipose tissue. Insulin suppresses lipolysis directly at the level of the adipocyte and indirectly through signaling in the brain. Here, we further characterized the role of brain insulin signaling in regulating lipolysis and defined the intracellular insulin signaling pathway required for brain insulin to suppress lipolysis. METHODS: We used hyperinsulinemic clamp studies coupled with tracer dilution techniques to assess insulin's ability to suppress lipolysis in two different mouse models with inducible insulin receptor depletion in all tissues (IRΔWB) or restricted to peripheral tissues excluding the brain (IRΔPER). To identify the underlying signaling pathway required for brain insulin to inhibit lipolysis, we continuously infused insulin +/- a PI3K or MAPK inhibitor into the mediobasal hypothalamus of male Sprague Dawley rats and assessed lipolysis during clamps. RESULTS: Genetic insulin receptor deletion induced marked hyperglycemia and insulin resistance in both IRΔPER and IRΔWB mice. However, the ability of insulin to suppress lipolysis was largely preserved in IRΔPER, but completely obliterated in IRΔWB mice indicating that insulin is still able to suppress lipolysis as long as brain insulin receptors are present. Blocking the MAPK, but not the PI3K pathway impaired the inhibition of lipolysis by brain insulin signaling. CONCLUSION: Brain insulin is required for insulin to suppress adipose tissue lipolysis and depends on intact hypothalamic MAPK signaling.

High-Throughput Quantitative Screening of Glucose-Stimulated Insulin Secretion and Insulin Content Using Automated MALDI-TOF Mass Spectrometry.

Type 2 diabetes (T2D) is a metabolic disorder characterized by loss of pancreatic ß-cell function, decreased insulin secretion and increased insulin resistance, that affects more than 537 million people worldwide. Although several treatments are proposed to patients suffering from T2D, long-term control of glycemia remains a challenge. Therefore, identifying new potential drugs and targets that positively affect ß-cell function and insulin secretion remains crucial. Here, we developed an automated approach to allow the identification of new compounds or genes potentially involved in ß-cell function in a 384-well plate format, using the murine ß-cell model Min6. By using MALDI-TOF mass spectrometry, we implemented a high-throughput screening (HTS) strategy based on the automation of a cellular assay allowing the detection of insulin secretion in response to glucose, i.e., the quantitative detection of insulin, in a miniaturized system. As a proof of concept, we screened siRNA targeting well-know ß-cell genes and 1600 chemical compounds and identified several molecules as potential regulators of insulin secretion and/or synthesis, demonstrating that our approach allows HTS of insulin secretion in vitro.

Stereochemistry and Intermolecular Interactions Influence Carrier Peptide-Mediated Insulin Delivery.

The inherent low oral bioavailability of therapeutic peptides can be enhanced by the cell-penetrating peptide penetratin and its analogues shuffle and penetramax applied as carriers for delivery of insulin. In this study, the objective was to gain mechanistic insights on the effect of the carrier peptide stereochemistry on their interactions with insulin and on insulin delivery. Insulin-carrier peptide interactions were investigated using small-angle X-ray scattering and cryogenic transmission electron microscopy, while the insulin and peptide stability and transepithelial insulin permeation were evaluated in the Caco-2 cell culture model along with the carrier peptide-induced effects on epithelial integrity and cellular metabolic activity. Interestingly, the insulin transepithelial permeation was influenced by the degree of insulin-carrier peptide complexation and depended on the stereochemistry of penetramax but not of penetratin and shuffle. The l-form of the peptides initially decreased the epithelial integrity comparable to that induced by the d-peptides, suggesting a comparable mechanism of action. The immediate decrease was reversible during exposure of the Caco-2 epithelium to the l-peptides but not during exposure to the d-peptides, likely a result of their higher stability. Overall, exploration of the stereochemistry showed to be an interesting strategy for carrier peptide-mediated insulin delivery.

Queueing theory model of mTOR complexes' impact on Akt-mediated adipocytes response to insulin.

A queueing theory based model of mTOR complexes impact on Akt-mediated cell response to insulin is presented in this paper. The model includes several aspects including the effect of insulin on the transport of glucose from the blood into the adipocytes with the participation of GLUT4, and the role of the GAPDH enzyme as a regulator of mTORC1 activity. A genetic algorithm was used to optimize the model parameters. It can be observed that mTORC1 activity is related to the amount of GLUT4 involved in glucose transport. The results show the relationship between the amount of GAPDH in the cell and mTORC1 activity. Moreover, obtained results suggest that mTORC1 inhibitors may be an effective agent in the fight against type 2 diabetes. However, these results are based on theoretical knowledge and appropriate experimental tests should be performed before making firm conclusions.

Identification of the Inner Cell Mass and the Trophectoderm Responses after an In Vitro Exposure to Glucose and Insulin during the Preimplantation Period in the Rabbit Embryo.

The prevalence of metabolic diseases is increasing, leading to more women entering pregnancy with alterations in the glucose-insulin axis. The aim of this work was to investigate the effect of a hyperglycemic and/or hyperinsulinemic environment on the development of the preimplantation embryo. In rabbit embryos developed in vitro in the presence of high insulin (HI), high glucose (HG), or both (HGI), we determined the transcriptomes of the inner cell mass (ICM) and the trophectoderm (TE). HI induced 10 differentially expressed genes (DEG) in ICM and 1 in TE. HG ICM exhibited 41 DEGs involved in oxidative phosphorylation (OXPHOS) and cell number regulation. In HG ICM, proliferation was decreased (p < 0.01) and apoptosis increased (p < 0.001). HG TE displayed 132 DEG linked to mTOR signaling and regulation of cell number. In HG TE, proliferation was increased (p < 0.001) and apoptosis decreased (p < 0.001). HGI ICM presented 39 DEG involved in OXPHOS and no differences in proliferation and apoptosis. HGI TE showed 16 DEG linked to OXPHOS and cell number regulation and exhibited increased proliferation (p < 0.001). Exposure to HG and HGI during preimplantation development results in common and specific ICM and TE responses that could compromise the development of the future individual and placenta.

Empagliflozin Induced Ketosis, Upregulated IGF-1/Insulin Receptors and the Canonical Insulin Signaling Pathway in Neurons, and Decreased the Excitatory Neurotransmitter Glutamate in the Brain of Non-Diabetics.

Sodium-glucose cotransporter-2 inhibitors (SGLT2is), such as empagliflozin, lower blood glucose in type 2 diabetes mellitus and improve cardiorenal outcomes regardless of diabetes presence. Whether SGLT2is exert any effects on the brain's metabolism has not been studied. We conducted a single-arm clinical trial to investigate the effects of once daily administration of oral empagliflozin (25 mg) for 14 days on systemic and brain metabolism in 21 non-diabetics aged 55 years old or older. Empagliflozin lowered circulating insulin and elevated ß-hydroxybutyrate over 34-h periods, both following its first administration and after 14 days of daily administration, with minor alterations in glucose homeostasis. Levels of phosphorylated insulin-like growth factor-1 receptor (pIGF-1R), phosphorylated insulin receptor (pIR), phosphorylated-in-tyrosine insulin receptor substrate-1 (pY-IRS-1), and phosphorylated protein kinase B or AKT (pAKT) were increased in extracellular vesicles enriched for neuronal origin (NEVs) following the first empagliflozin administration, but not after 14 days. Our finding of IGF-1R upregulation in NEVs is promising because several post-mortem and epidemiological studies support the idea that upregulation of IGF signaling may protect against Alzheimer's disease (AD). Moreover, our finding showing activation of insulin signaling and, in particular, the canonical pathway (pIR, pY-IRS-1, pAKT) in NEVs is important because such changes have been repeatedly associated with neuronal survival. Using brain magnetic resonance spectroscopy (MRS), we detected decreased concentrations of the excitatory neurotransmitter glutamate and its precursor glutamine after empagliflozin administration. This finding is also encouraging since glutamatergic excitotoxicity has long been implicated in AD pathology. Overall, our findings may motivate the repurposing of SGLT2is for use in AD and other, related diseases that are characterized by downregulation of IGF-1/insulin signaling in neurons and excitotoxicity.

Pregnancy induces pancreatic insulin secretion in women with long-standing type 1 diabetes.

INTRODUCTION: Pregnancy entails both pancreatic adaptations with increasing ß-cell mass and immunological alterations in healthy women. In this study, we have examined the effects of pregnancy on ß-cell function and immunological processes in long-standing type 1 diabetes (L-T1D). RESEARCH DESIGN AND METHODS: Fasting and stimulated C-peptide were measured after an oral glucose tolerance test in pregnant women with L-T1D (n=17) during the first trimester, third trimester, and 5-8 weeks post partum. Two 92-plex Olink panels were used to measure proteins in plasma. Non-pregnant women with L-T1D (n=30) were included for comparison. RESULTS: Fasting C-peptide was detected to a higher degree in women with L-T1D during gestation and after parturition (first trimester: 64.7%, third trimester: 76.5%, and post partum: 64.7% vs 26.7% in non-pregnant women). Also, total insulin secretion and peak C-peptide increased during pregnancy. The plasma protein levels in pregnant women with L-T1D was dynamic, but few analytes were functionally related. Specifically, peripheral levels of prolactin (PRL), prokineticin (PROK)-1, and glucagon (GCG) were elevated during gestation whereas levels of proteins related to leukocyte migration (CCL11), T cell activation (CD28), and antigen presentation (such as CD83) were reduced. CONCLUSIONS: In summary, we have found that some C-peptide secretion, that is, an indirect measurement of endogenous insulin production, is regained in women with L-T1D during pregnancy, which might be attributed to elevated peripheral levels of PRL, PROK-1, or GCG.

ER Redox Homeostasis Regulates Proinsulin Trafficking and Insulin Granule Formation in the Pancreatic Islet ß-Cell.

Defects in the pancreatic ß-cell's secretion system are well-described in type 2 diabetes (T2D) and include impaired proinsulin processing and a deficit in mature insulin-containing secretory granules; however, the cellular mechanisms underlying these defects remain poorly understood. To address this, we used an in situ fluorescent pulse-chase strategy to study proinsulin trafficking. We show that insulin granule formation and the appearance of nascent granules at the plasma membrane are decreased in rodent and cell culture models of prediabetes and hyperglycemia. Moreover, we link the defect in insulin granule formation to an early trafficking delay in endoplasmic reticulum (ER) export of proinsulin, which is independent of overt ER stress. Using a ratiometric redox sensor, we show that the ER becomes hyperoxidized in ß-cells from a dietary model of rodent prediabetes and that addition of reducing equivalents restores ER export of proinsulin and insulin granule formation and partially restores ß-cell function. Together, these data identify a critical role for the regulation of ER redox homeostasis in proinsulin trafficking and suggest that alterations in ER redox poise directly contribute to the decline in insulin granule production in T2D. This model highlights a critical link between alterations in ER redox and ER function with defects in proinsulin trafficking in T2D. Hyperoxidation of the ER lumen, shown as hydrogen peroxide, impairs proinsulin folding and disulfide bond formation that prevents efficient exit of proinsulin from the ER to the Golgi. This trafficking defect limits available proinsulin for the formation of insulin secretory granules during the development of T2D.

Efficacy of glucagon-like peptide-1 and estrogen dual agonist in pancreatic islets protection and pre-clinical models of insulin-deficient diabetes.

We study the efficacy of a glucagon-like peptide-1 (GLP-1) and estrogen dual agonist (GLP1-E2) in pancreatic islet protection. GLP1-E2 provides superior protection from insulin-deficient diabetes induced by multiple low-dose streptozotocin (MLD-STZ-diabetes) and by the Akita mutation in mice than a GLP-1 monoagonist. GLP1-E2 does not protect from MLD-STZ-diabetes in estrogen receptor-α (ERα)-deficient mice and fails to prevent diabetes in Akita mice following GLP-1 receptor (GLP-1R) antagonism, demonstrating the requirement of GLP-1R and ERα for GLP1-E2 antidiabetic actions. In the MIN6 ß cell model, GLP1-E2 activates estrogen action following clathrin-dependent, GLP-1R-mediated internalization and lysosomal acidification. In cultured human islet, proteomic bioinformatic analysis reveals that GLP1-E2 amplifies the antiapoptotic pathways activated by monoagonists. However, in cultured mouse islets, GLP1-E2 provides antiapoptotic protection similar to monoagonists. Thus, GLP1-E2 promotes GLP-1 and E2 antiapoptotic signals in cultured islets, but in vivo, additional GLP1-E2 actions in non-islet cells expressing GLP-1R are instrumental to prevent diabetes.

Adrenomedullin and its receptors are expressed in mouse pancreatic ß-cells and suppresses insulin synthesis and secretion.

Gestational diabetes mellitus (GDM) is associated with defective pancreatic ß-cell adaptation in pregnancy, but the underlying mechanism remains obscure. Our previous studies demonstrated that GDM women display increased plasma adrenomedullin (ADM) levels, and non-obese GDM mice show decreased serum concentrations of insulin and the number of ß-cells in pancreas islets. The aims of this study is to examine if ADM and its receptors are expressed in female mouse pancreas, and if so, whether insulin secretion is regulated by ADM in mouse ß-cell line, NIT-1 cells and isolated mouse pancreatic islets. Present study shows that ADM and its receptor components CRLR, RAMPs are present in mouse pancreatic islets and co-localized with insulin. The expressions of ADM, CRLR and RAMP2 in islets from pregnant mice are reduced compared to that of non-pregnant mice. NIT-1-ß cells express ADM and its receptor mRNA, and glucose dose-dependently stimulates expressions. Furthermore, ADM inhibits NIT-1-ß cell growth, and this inhibition is reversed by ADM antagonist, ADM22-52. The glucose-induced insulin secretion was suppressed by ADM in NIT-1-ß cells and isolated pancreatic islets from pregnant mice. These inhibitory effects are accompanied by upregulation of endoplasmic reticulum (ER) stress biomarker genes in NIT-1-ß cells. This study unveils that reduced ADM and its receptors may play a role in ß-cell adaptation during pregnancy, while increased plasma ADM in GDM may contribute to the ß-cells dysfunction, and blockade of ADM may reverse ß-cell insulin production.