Stimulated Insulin Secretion Research Articles

Genomic and biochemical analysis reveals fermented product of a putative novel Romboutsia species involves the glycometabolism of tilapia

Fish have a restricted capacity to utilize carbohydrates. Gut microbiota contribute to carbohydrate degradation in the host. The connection between the intestinal microbiota and carbohydrate metabolism is not well known in fish. We found that when feed was transformed from zooplankton-based to plant-based diet, the population of genus Romboutsia in tilapia gut increased. We inferred that the abundance of the genus Romboutsia was positively correlated with dietary sugar levels. Then, a strain of genus Romboutsia was obtained and designated MY01. Strain MY01 was a novel species of Romboutsia based on its genomic analysis. Many genes involved in carbohydrate metabolism were found in its genome, including Carbohydrate Active enZymes (CAZymes) genes associated with dietary fiber fermentation. An increased quantity of short-chain fatty acids (SCFAs) and decreased quantity of carbohydrates in feed fermented with MY01 were revealed by GC–MS and HPLC analysis. Fermented feed with MY01 increased the insulin content and decreased the glucose content of tilapia serum. Our in vivo and in vitro results are the first to suggest that fermented feed with Romboutsia regulates the tilapia glycometabolism by producing SCFAs to stimulate insulin secretion, and by reducing carbohydrates content of feed simultaneously to decrease blood glucose eventually. In further, the study provides a potential source of SCFAs production.

The NERP-4–SNAT2 axis regulates pancreatic β-cell maintenance and function

Insulin secretion from pancreatic β cells is regulated by multiple stimuli, including nutrients, hormones, neuronal inputs, and local signalling. Amino acids modulate insulin secretion via amino acid transporters expressed on β cells. The granin protein VGF has dual roles in β cells: regulating secretory granule formation and functioning as a multiple peptide precursor. A VGF-derived peptide, neuroendocrine regulatory peptide-4 (NERP-4), increases Ca2+ influx in the pancreata of transgenic mice expressing apoaequorin, a Ca2+-induced bioluminescent protein complex. NERP-4 enhances glucose-stimulated insulin secretion from isolated human and mouse islets and β-cell–derived MIN6-K8 cells. NERP-4 administration reverses the impairment of β-cell maintenance and function in db/db mice by enhancing mitochondrial function and reducing metabolic stress. NERP-4 acts on sodium-coupled neutral amino acid transporter 2 (SNAT2), thereby increasing glutamine, alanine, and proline uptake into β cells and stimulating insulin secretion. SNAT2 deletion and inhibition abolish the protective effects of NERP-4 on β-cell maintenance. These findings demonstrate a novel autocrine mechanism of β-cell maintenance and function that is mediated by the peptide–amino acid transporter axis.

Diabetes mellitus in acromegaly: prevalence, pathophysiological particulars and treatment strategies

The prevalence of diabetes mellitus in acromegaly is significantly higher than that in the general population. Carbohydrate metabolism abnormalities often precede other phenotypic manifestations of acromegaly. The review presents data on the prevalence of carbohydrate metabolism disorders in acromegaly and describes their pathophysiological characteristics and approaches to treatment.
 Growth hormone (GH) excess is recognized as a key factor of glucose homeostasis abnormalities due to decreased insulin sensitivity (resulting from active lipolysis) and direct stimulation of insulin secretion. Insulin-like growth factor 1 (IGF1) improves insulin sensitivity; however, the GH diabetogenic effects prevail over the insulin-sensitizing impact of IGF1. Surgical and radiation treatment for somatotropinoma may indirectly improve carbohydrate metabolism, because they decrease the GH levels. Treatment with first generation somatostatin analogues can both improve glycemic control due to decreased GH levels and worsen it due to deterioration of postprandial insulin release, especially in patients with already manifest carbohydrate metabolism disorders. The GH receptor antagonist pegvisomant blocks the effects of excess GH on the target tissues without suppressing insulin secretion, which results in better glucose control; treatment with this agent can be preferred in patients with acromegaly and diabetes mellitus. Management of carbohydrate metabolism disorders in acromegaly is done in accordance with general treatment principles for type 2 diabetes mellitus.

Microencapsulation and Peptide identification of purified bioactive fraction from spirulina protein hydrolysates with dipeptidyl peptidase IV (DPP-IV) inhibitory activity

The growing worldwide occurrence of type 2 diabetes mellitus (T2DM) necessitates the development of effective treatments. This study presents a comprehensive investigation into the inhibitory effect of trypsin-digested Spirulina protein hydrolysate (SPH) on the dipeptidyl peptidase IV (DPP-IV) enzyme, responsible for deactivating incretin hormones that stimulate insulin secretion. Following purification through hydrophobic interaction chromatography, the DPP-IV inhibition potential was enhanced, achieving a level of 74.20% ± 5.78%, with a half-maximal inhibitory concentration (IC50) value of 0.46 ± 0.16 mg/ml. By subsequently encapsulating the purified SPH within alginate and chitosan microcapsules, a controlled and sustained release profile of bioactive peptides was achieved while retaining their DPP-IV inhibitory activity. The encapsulation process exhibited an efficiency of 86.30% ± 0.16% and an efficacy of 80.65% ± 0.16% for SPH loading. Further analysis via LC-MS/MS revealed a potent peptide, GPNYASSER, derived from phycocyanin, exhibiting an IC50 value of 0.358 ± 0.188 mM. Beyond the search for specific peptides displaying strong DPP-IV inhibitory properties, the integration of SPH as function components for Type 2 Diabetes Mellitus (T2DM) management holds significant promise as a pathway for long-term preventive strategies.

Glucagon-like peptide 1 agonists: a ray of hope for the treatment of polycystic ovarian syndrome

Dear editor, The Glucagon-like-peptide receptor- 1, agonists belong to a class of drugs that have been used to treat type 2 diabetes. Liraglutide and Semaglutide are drugs of this class, commonly sold under the brand names Saxenda and Wegovy respectively. These drugs have also been approved by the FDA for weight loss in diabetic and non-diabetic obese people.1 The mechanism of action of GLP-1 agonists involve stimulating insulin secretion and delaying gastric emptying. Moreover, GLP-1 receptors are found on adipose tissue, where they promote the conversion of stored fat into energy. They also target the appetite center in the hypothalamus which increases satiety and reduces food intake to cause weight loss. A large number of females who struggle with obesity, concomitantly suffer from Polycystic Ovarian syndrome (PCOS) also which is a multifactorial endocrine disorder in women of reproductive age, affecting 4-20% women around the world.2 One of the major manifestations of this syndrome is weight gain and difficulty in losing it. In contemporary medicine, the treatment of PCOS is limited to lifestyle intervention and symptomatic management. Thus, the patients of PCOS are often told by their doctors to simply lose weight. Physicians around the world agree that weight loss is crucial for women with PCOS who are obese to attain better metabolic, reproductive, and cardiovascular outcomes, because if not treated, obesity worsens the presentation of PCOS. But it is noteworthy that the ongoing hormonal imbalances, increased appetite and insulin resistance in this syndrome, all work against the body’s natural mechanism to lose weight and make it extremely difficult for the patient. This creates a dire need for external interventions regarding weight management in PCOS. Where conventional methods like dietary modifications and exercise alone do not provide substantial results, GLP-1 agonists can be a breakthrough for the management of PCOS since recent research and clinical data also provides evidence of correlation between obesity, GLP-1 kinetic alterations, and pathophysiology of PCOS.3 Hence the use of GLP-1 agonists can be a viable option for treatment of PCOS as multiple clinical trials have shown significant weight reduction, decrease in testosterone levels, improvement in insulin sensitivity and menstrual cycles.4 As the prevalence of PCOS continues to rise in Pakistan, the major presenting complaints include; weight gain, facial hair, acne and menstrual irregularities in teenage girls and infertility in married females. ---Continue

High Doses of Exogenous Glucagon Stimulate Insulin Secretion and Reduce Insulin Clearance in Healthy Humans.

Glucagon is generally defined as a counter-regulatory hormone with a primary role to raise blood glucose concentrations by increasing endogenous glucose production (EGP) in response to hypoglycemia. However, glucagon has long been known to stimulate insulin release, and recent preclinical findings support a paracrine action of glucagon directly on islet β-cells that augments secretion. In mice, the insulinotropic effect of glucagon is glucose dependent and not present during basal euglycemia. To test the hypothesis that the relative effects of glucagon on hepatic and islet function were also varied with blood glucose, a group of healthy subjects received glucagon (100 ng/kg) during fasting glycemia or experimental hyperglycemia (~150 mg/dl) on two separate days. During fasting euglycemia, administration of glucagon caused blood glucose to rise due to increased EGP, with a delayed increase of insulin secretion. When given during experimental hyperglycemia, glucagon caused a rapid, 3-fold increase in insulin secretion, as well as a more gradual increase in EGP. Under both conditions, insulin clearance was decreased in response to glucagon infusion. The insulinotropic action of glucagon, which is proportional to elevated levels of blood glucose, suggests distinct physiologic roles in the fasting and prandial states.

Profiling of secondary metabolite and evaluation of anti-diabetic potency of Crotalaria quinquefolia (L): In-vitro, in-vivo, and in-silico approaches

Traditional medicinal plants have played a promising role in the human health system. In folklore medicine, Crotalaria quinquefolia L. is used to treat fever, pain, eczema, impetigo, lung infections, scabies. The present investigation was executed to identify secondary metabolites responsible for anti-diabetic potential of C. quinquefolia L. leaf extract along with their possible mechanistic pathways. The anti-hyperglycemic activity was assessed by in vitro α-amylase and α-glucosidase inhibitory assays and an in vivo oral glucose tolerance test and diabetogenic effect of streptozotocin in mice, followed by an integrative computational analysis. A total of 23 compounds were identified through GCMS and HPLC. The extract showed potent in-vitro α-amylase and α-glucosidase suppressive activity with IC50 values of 12.8 ± 0.1 µg/mL and 36.3 ± 0.07 µg/mL, respectively. In an in vivo oral glucose tolerance test, the extract (400 mg/kg body weight) prompted blood glucose levels to plummet by 18.9 % after 30 min, compared to the normal control and streptozotocin induced diabetes test, maximum glucose reduction was observed 11.67 % by dose of 200 mg/kg compared to the control; glibenclamide and extract (400 mg/kg) reduced blood glucose levels by 1.3 % and 16.7 %, respectively, compared to diabetic control at the end of the trial. Additionally, among the identified compounds, myricetin, quercetin, rutin, and kaempferol revealed good binding affinity as well as stability with the studied anti-diabetic proteins in docking and molecular dynamics simulation studies. Furthermore, QSAR analysis and network pharmacology studies of the identified compounds divulged enhanced insulin secretion stimulation, insulin receptor kinase activity, PPARγ expression; enzyme inhibition (α-glucosidase, α-amylase) and protection of the pancreas -mediated antidiabetic effects. Besides, they proved strong inhibitory potential against the studied antidiabetic proteins in other computational analysis. Based on the present findings, it can be affirmed that C. quinquefolia extract possesses anti-diabetic activity.

Partial Deletion of Perk Improved High-Fat Diet-Induced Glucose Intolerance in Mice.

Although pancreatic endoplasmic reticulum kinase (PERK) is indispensable to beta cells, low-dose PERK inhibitor improved glucose- stimulated insulin secretion (GSIS) and hyperglycemia in diabetic mice. Current study examined if partial deletion of Perk (Perk+/-) recapitulated the effects of PERK inhibitor, on the contrary to the complete deletion. Perk+/- mice and wild-type controls were fed with a high-fat diet (HFD) for 23 weeks. Glucose tolerance was evaluated along with serum insulin levels and islet morphology. Perk+/- mice on normal chow were comparable to wild-type mice in various metabolic features. HFD-induced obesity was not influenced by Perk reduction; however, HFD-induced glucose intolerance was significantly improved since 15-week HFD. HFD-induced compromises in GSIS were relieved by Perk reduction, accompanied by reductions in phosphorylated PERK and activating transcription factor 4 (ATF4) in the islets. Meanwhile, HFD-induced islet expansion was not significantly affected. In summary, partial deletion of Perk improved glucose tolerance and GSIS impaired by diet-induced obesity, without changes in body weights or islet mass.

Spatiotemporal regulation of GIPR signaling impacts glucose homeostasis as revealed in studies of a common GIPR variant

ObjectiveGlucose-dependent insulinotropic polypeptide (GIP) has a role in controlling postprandial metabolic tone. In humans, a GIP receptor (GIPR) variant (Q354, rs1800437) is associated with a lower body mass index (BMI) and increased risk for Type 2 Diabetes. To better understand the impacts of GIPR-Q354 on metabolism, it is necessary to study it in an isogeneic background to the predominant GIPR isoform, E354. To accomplish this objective, we used CRISPR-CAS9 editing to generate mouse models of GIPR-Q354 and GIPR-E354. Here we characterize the metabolic effects of GIPR-Q354 variant in a mouse model (GIPR-Q350). MethodsWe generated the GIPR-Q350 mice for in vivo studies of metabolic impact of the variant. We isolated pancreatic islets from GIPR-Q350 mice to study insulin secretion ex vivo. We used a β−cell cell line to understand the impact of the GIPR-Q354 variant on the receptor traffic. ResultsWe found that female GIPR-Q350 mice are leaner than littermate controls, and male GIPR-Q350 mice are resistant to diet-induced obesity, in line with the association of the variant with reduced BMI in humans. GIPR-Q350 mice of both sexes are more glucose tolerant and exhibit an increased sensitivity to GIP. Postprandial GIP levels are reduced in GIPR-Q350 mice, revealing feedback regulation that balances the increased sensitivity of GIP target tissues to secretion of GIP from intestinal endocrine cells. The increased GIP sensitivity is recapitulated ex vivo during glucose stimulated insulin secretion assays in islets. Generation of cAMP in islets downstream of GIPR activation is not affected by the Q354 substitution. However, post-activation traffic of GIPR-Q354 variant in β-cells is altered, characterized by enhanced intracellular dwell time and increased localization to the Trans-Golgi Network (TGN). ConclusionsOur data link altered intracellular traffic of the GIPR-Q354 variant with GIP control of metabolism. We propose that this change in spatiotemporal signaling underlies the physiologic effects of GIPR-Q350/4 and GIPR-E350/4 in mice and humans. These findings contribute to a more complete understanding of the impact of GIPR-Q354 variant on glucose homeostasis that could perhaps be leveraged to enhance pharmacologic targeting of GIPR for the treatment of metabolic disease.

The impact of a single dose of whey protein on glucose flux and metabolite profiles in normoglycemic males: insights into glucagon and insulin biology.

Protein ingestion concurrently stimulates euglycemic glucagon and insulin secretion, a response that is particularly robust with rapidly absorbing proteins. Previously, we have shown that ingestion of repeated doses of rapidly absorbing whey protein equally stimulated endogenous glucose production (EGP) and glucose disposal (Rd), thus explaining the preservation of euglycemia. Here, we aimed to determine if a smaller single dose of whey could elicit a large enough glucagon and insulin response to stimulate glucose flux. Therefore, in normoglycemic young adult males (N=10; Age ~26; BMI ~25), using [6,6-2H2] glucose tracing and quantitative targeted metabolite profiling, we determined the metabolic response to a single 25g 'standard' dose of whey protein. Whey protein ingestion did not alter glycemia, but increased circulating glucagon (peak 4-fold basal), insulin (peak 6-fold basal), amino acids and urea while also reducing free fatty acid and glycerol concentrations. Interestingly, the postprandial insulin response was driven by both a stimulation of insulin secretion and marked reduction in hepatic insulin clearance. Whey protein ingestion resulted in a modest stimulation of EGP and Rd, both peaking at ~20% above baseline 1 h after protein ingestion. These findings demonstrate that the ingestion of a single standard serving of whey protein can induce a euglycemic glucagon and insulin response that stimulates glucose flux. We speculate on a theory that could potentially explain how glucagon and insulin synergistically provide hardwired control of nitrogen and glucose homeostasis.

Adjudin improves beta cell maturation, hepatic glucose uptake and glucose homeostasis

Aims/hypothesisRecovering functional beta cell mass is a promising approach for future diabetes therapies. The aim of the present study is to investigate the effects of adjudin, a small molecule identified in a beta cell screen using zebrafish, on pancreatic beta cells and diabetes conditions in mice and human spheroids.MethodsIn zebrafish, insulin expression was examined by bioluminescence and quantitative real-time PCR (qPCR), glucose levels were examined by direct measurements and distribution using a fluorescent glucose analogue, and calcium activity in beta cells was analysed by in vivo live imaging. Pancreatic islets of wild-type postnatal day 0 (P0) and 3-month-old (adult) mice, as well as adult db/db mice (i.e. BKS(D)-Leprdb/JOrlRj), were cultured in vitro and analysed by qPCR, glucose stimulated insulin secretion and whole mount staining. RNA-seq was performed for islets of P0 and db/db mice. For in vivo assessment, db/db mice were treated with adjudin and subjected to analysis of metabolic variables and islet cells. Glucose consumption was examined in primary human hepatocyte spheroids.ResultsAdjudin treatment increased insulin expression and calcium response to glucose in beta cells and decreased glucose levels after beta cell ablation in zebrafish. Adjudin led to improved beta cell function, decreased beta cell proliferation and glucose responsive insulin secretion by decreasing basal insulin secretion in in vitro cultured newborn mouse islets. RNA-seq of P0 islets indicated that adjudin treatment resulted in increased glucose metabolism and mitochondrial function, as well as downstream signalling pathways involved in insulin secretion. In islets from db/db mice cultured in vitro, adjudin treatment strengthened beta cell identity and insulin secretion. RNA-seq of db/db islets indicated adjudin-upregulated genes associated with insulin secretion, membrane ion channel activity and exocytosis. Moreover, adjudin promoted glucose uptake in the liver of zebrafish in an insulin-independent manner, and similarly promoted glucose consumption in primary human hepatocyte spheroids with insulin resistance. In vivo studies using db/db mice revealed reduced nonfasting blood glucose, improved glucose tolerance and strengthened beta cell identity after adjudin treatment.Conclusions/interpretationAdjudin promoted functional maturation of immature islets, improved function of dysfunctional islets, stimulated glucose uptake in liver and improved glucose homeostasis in db/db mice. Thus, the multifunctional drug adjudin, previously studied in various contexts and conditions, also shows promise in the management of diabetic states.Data availabilityRaw and processed RNA-seq data for this study have been deposited in the Gene Expression Omnibus under accession number GSE235398 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE235398).Graphical

A metabolic role for CD47 in pancreatic β cell insulin secretion and islet transplant outcomes.

Diabetes is a global public health burden and is characterized clinically by relative or absolute insulin deficiency. Therapeutic agents that stimulate insulin secretion and improve insulin sensitivity are in high demand as treatment options. CD47 is a cell surface glycoprotein implicated in multiple cellular functions including recognition of self, angiogenesis, and nitric oxide signaling; however, its role in the regulation of insulin secretion remains unknown. Here, we demonstrate that CD47 receptor signaling inhibits insulin release from human as well as mouse pancreatic β cells and that it can be pharmacologically exploited to boost insulin secretion in both models. CD47 depletion stimulated insulin granule exocytosis via activation of the Rho GTPase Cdc42 in β cells and improved glucose clearance and insulin sensitivity in vivo. CD47 blockade enhanced syngeneic islet transplantation efficiency and expedited the return to euglycemia in streptozotocin-induced diabetic mice. Further, anti-CD47 antibody treatment delayed the onset of diabetes in nonobese diabetic (NOD) mice and protected them from overt diabetes. Our findings identify CD47 as a regulator of insulin secretion, and its manipulation in β cells offers a therapeutic opportunity for diabetes and islet transplantation by correcting insulin deficiency.

SAT050 Liver Androgen Receptor Knockout Delayed The Onset Of High Fructose Diet-induced Dysglycemia In Female Mice

Abstract Disclosure: C. Falzarano: None. T. Oliver: None. A. Osei-Ntansah: None. T. Lofton: None. S. Andrisse: None. Introduction: Previous research has indicated that a lipid-independent pathogenic mechanism plays a role in androgen-induced hepatic insulin resistance where liver androgen receptors may play a role in modulating disease. Consequently, this study aims to investigate the pathophysiology of high fructose diet (HFrD) induced dysglycemia in male and female liver androgen receptor knockout (LivARKO) mice. Andrisse et al 2021 showed that deleting the liver androgen receptor prevented female mice from developing hyperandrogenemia (HA)-induced insulin resistance. From this, we asked the question, does LivARKO prevent HFrD induced dysglycemia? Hypothesis: It was hypothesized that HFrD LivARKO female mice would display impaired whole body glucose metabolism in comparison to the Control diet fed LivARKO female mice, suggesting that AR does not play a significant role in regulating HFrD-induced dysglycemia. Methods: We performed insulin tolerance tests (ITT), glucose tolerance tests (GTT), pyruvate tolerance tests (PTT), and glucose stimulated insulin secretion (GSIS) tests on LivARKO female mice fed a high fat diet (HFD) or a control diet (CD) (from Research Diets Inc) during months 1 or 2 after starting the diet. Results: After 1-month, LivARKO female mice fed a Control diet had impaired glucose tolerance compared to LivARKO-HFrD female mice; however, after 2-months, the LivARKO-HFrD female mice displayed impaired glucose tolerance above that of the Control diet LivARKO female mice. LivARKO female mice fed a high fructose diet (HFrD) did not display increased fasting insulin levels compared to LivARKO female mice on a control diet. HFrD LivARKO mice did not display any increase in GSIS at 15-min or 30-min. Conclusion: These findings suggest that LivARKO delayed the onset of HFrD-Induced dysglycemia in female mice. Further research may consist of molecular data to elucidate the mechanistic pathways involved in these whole body phenotypes observed. Presentation: Saturday, June 17, 2023

THU237 A Case Of Primary Hypothyroidism, Dysglycemia And Epilepsy With PAX8 And SETD1A Mutation

Abstract Disclosure: S.K. Singh: None. KEYWORDS: PAX8, SETD1A, DYSGLYCEMIA INTRODUCTION: Hypothyroidism is associated with dysglycemia linked with insulin resistance and shared common genetic determinants.PAX 8 gene regulates the development of thyroid gland as well as the expression of TG,TPO and NIS genes.It impacts glucose homeostasis by favoring beta-cell survival and through thyroid hormones.Epilepsy in hypothyroid patients can be idiopathic or due to Hashimoto’s Encephalopathy (HE). Thyroid hormones can impact epileptogenesis through mitochondrial dysfunction, oxidative-stress and GABAergic transmission. THE INDEX PATIENT: A 13 year old female(weight and height age 1.5 years below her chronological age by Indian growth chart and a further lag of 18 months and 6 months, respectively by CDC chart)was diagnosed as a case of thyroprivic hypothyroidism with TSH - 100 mIU/L and positive anti-TPO antibody. She was euthyroid on 112 ug/d L-T4.She is on anticonvulsant since 12 years of age for tonic-clonic epilepsy. The MRI of brain showed focal gliosis with few small encephalomalacic streaks in right occipital lobe. On evaluation at 12.5 years of age, her fasting plasma glucose was 80 mg/dl and her HbA1c was 4.8% but 2-h post glucose was 233 mg/dl with another value of 270 mg/dl. Her father has T2DM and Hypothyroidism. Her mother is Hypothyroid. Her genetic study showed SETDIA and PAX8(-) mutations. DISCUSSION: PAX8 regulates development of thyroid gland and biosynthesis of thyroid hormones. It can impact glucose homeostasis by enhancing beta-cell survival by inhibiting apoptosis, immunomodulation, maintaining local anti-inflammatory milieu in pancreas, ensuring systemic levels of thyroid hormone to stimulate insulin secretion and protect beta-cells from environmental stressors.PAX8 mutation in a family has been shown to be associated with a spectrum of disorders encompassing Gestational Thyroid Dysfunction(GTD),Gestational Diabetes Mellitus(GDM) and T2DM.[1] Although mutation analysis was done only in the index patient, Hypothyroidism and Dysglycemia (post- glucose>200 mg/dl)in the patient and family history of Hypothyroidism and T2DM could be linked with PAX8 mutation.SETD1A mutation has been linked with early-onset epilepsy with or without developmental delay. The mutation study can aid in deciding appropriate therapy including steroids for HE. CONCLUSION: This case highlights the significance of evaluation for PAX8 mutation in hypothyroid patients, even if anti-TPO positive, particularly when there is family history of thyroid dysfunction, GDM, T2DM. It also underlies the importance of SETD1A mutation analysis in hypothyroid patients with epilepsy. Presentation: Thursday, June 15, 2023

SAT064 Establishment And Initial Characterization Of Two Parallel Targeted Human ZnT8 Knock-in Murine Lines For Studying The Type 2 Diabetes Risk Variant At SNP rs13266634 Of The ZnT8 Gene

Abstract Disclosure: J.C. Wang: None. C. Adelma: None. M.G. Hayes: None. M. El Muayed: None. Background: Studies showed an association between R variant at position 325 in the β-cell zinc transporter ZnT8 and an increased risk for developing type 2 diabetes mellitus (T2DM). However, the mechanism by which this variant confers an increased risk for type 2 diabetes remains unclear. Methods: We generated a murine model for studying the effect of SNP rs13266634 on β-cell physiology. This model consists of two parallel murine lines in which conditional (RIP-Cre induced) expression constructs for the human variants of SLC30A8 (ZnT8), namely R (hZnT8R325, T2DM risk allele) and W (hZnT8W325, non-risk allele) of SNPrs13266634 were inserted into the rosa26 locus via targeted integration. β-cell specific expression of each of ZnT8R325 and ZnT8W325 was induced by cross breeding with a RIP-Cre line, expressing Cre recombinase in β-cells. All mouse lines were backcrossed into a C57BL/6N background null for the mouse variant of ZnT8. Islet zinc concentrations was measured by ICP-MS following hydrolysis in HNO3. Ex-vivo Glucose Stimulated Insulin Secretion Ratio (GSISR) was measured by incubation of isolated islets in 2.8 mM glucose, followed by 16.7 mM glucose minutes after an initial 60 min equilibration phase and calculating the insulin secretion at 16.7 mM/2.8 mM glucose. Data are reported as averages ± SEM from 3 independent experiments (n=3), each with 4 technical replicates. Results: Expression of both variants was verified by mRNA PCR from islets. Levels of expression in both lines were identical, owing to the targeted integration approach. Expression of each of hZnT8R325 and ZnT8W325 resulted in restoration of islet zinc concentration to levels similar to those found in normal human islets and wild type mouse islets. This confirms proper function of the hZnT8R325 transgenes in this newly established model. hZnT8R expressing islets showed a trend to lower insulin response to ex-vivo glucose stimulation compared to hZnT8W325 expressing islets (GSISR- 1.37 ± 0.41 vs 1.97 ± 0.32 respectively), as well as Cre expressing (GSISR 1.53 ± 0.40) or inactive construct controls (GSISR 2.43 ± 0.57). In-vivo glucose stimulated insulin secretion was not significantly different between hZnT8R325 expressing mice and those expressing hZnT8W325. As previously reported by others, all mice with β-cell Cre expression (RIP-Cre activated hZnT8 expressing lines and RIP-Cre controls) showed a lower insulin secretory response to glucose compared to non-Cre expressing mice (inactive construct controls). Conclusion: The newly created murine mouse model expresses functional human homologues of the ZnT8 variants hZnT8R325 and hZnT8W325 that result in restored beta cell zinc uptake. A trend to lower insulin secretory capacity was observed in ex-vivo glucose stimulated insulin secretion experiments. This model will be valuable tool to elucidate the functional consequences of the T2DM risk variant at SNP rs13266634. Presentation: Saturday, June 17, 2023

Mesenchymal stromal cell secretory molecules improve the functional survival of human islets.

Human islet transplantation as a therapy for type 1 diabetes is compromised by the loss of functional beta cells in the immediate post-transplantation period. Mesenchymal stromal cells (MSCs) and MSC-derived secretory peptides improve the outcomes of islet transplantation in rodent models of diabetes. Here, we utilized a mouse model for human islet transplantation and assessed the effects of a cocktail of MSC-secreted peptides (screened by MSC-secretome for human islet GPCRs) on the functional survival of human islets. Human islets from nine donors (Age: 36-57; BMI: 20-35) were treated with a cocktail of human recombinant annexin A1 (ANXA1), stromal cell-derived factor-1 (SDF-1/CXCL12) and complement component C3 (C3a). Glucose-stimulated insulin secretion (GSIS) was assessed in static incubation, and cytokine-induced apoptosis was assessed by measuring caspase 3/7 activity. mRNA expression levels were determined by qPCR. Human islet function in vivo was assessed using a novel model for human islet transplantation into a T1D mouse model. Human islet function in vivo was assessed using islet transplantation under the kidney capsule of immunodeficient mice prior to STZ destruction of endogenous mouse beta cells to model T1DM. Pretreatment with a cocktail of MSC-secreted peptides increased GSIS in vitro and protected against cytokine-induced apoptosis in human islets isolated from nine donors. Animals transplanted with either treated or untreated human islets remained normoglycaemic for up to 28 days after STZ-administration to ablate the endogenous mouse beta cells, whereas non-transplanted animals showed significantly increased blood glucose immediately after STZ administration. Removal of the human islet graft by nephrectomy resulted in rapid increases in blood glucose to similar levels as the non-transplanted controls. Pretreating human islets with the MSC-derived cocktail significantly improved glucose tolerance in graft recipients, consistent with enhanced functional survival of the treated islets in vivo. Pretreating human islets before transplantation with a defined cocktail of MSC-derived molecules could be employed to improve the quality of human islets for transplantation therapy for type 1 diabetes.

Mechanism of the antidiabetic action of Nigella sativa and Thymoquinone: a review.

Long used in traditional medicine, Nigella sativa (NS; Ranunculaceae) has shown significant efficacy as an adjuvant therapy for diabetes mellitus (DM) management by improving glucose tolerance, decreasing hepatic gluconeogenesis, normalizing blood sugar and lipid imbalance, and stimulating insulin secretion from pancreatic cells. In this review, the pharmacological and pharmacokinetic properties of NS as a herbal diabetes medication are examined in depth, demonstrating how it counteracts oxidative stress and the onset and progression of DM. This literature review drew on databases such as Google Scholar and PubMed and various gray literature sources using search terms like the etiology of diabetes, conventional versus herbal therapy, subclinical pharmacology, pharmacokinetics, physiology, behavior, and clinical outcomes. The efficiency and safety of NS in diabetes, notably its thymoquinone (TQ) rich volatile oil, have drawn great attention from researchers in recent years; the specific therapeutic dose has eluded determination so far. TQ has anti-diabetic, anti-inflammatory, antioxidant, and immunomodulatory properties but has not proved druggable. DM's intimate link with oxidative stress, makes NS therapy relevant since it is a potent antioxidant that energizes the cell's endogenous arsenal of antioxidant enzymes. NS attenuates insulin resistance, enhances insulin signaling, suppresses cyclooxygenase-2, upregulates insulin-like growth factor-1, and prevents endothelial dysfunction in DM. The interaction of NS with mainstream drugs, gut microbiota, and probiotics opens new possibilities for innovative therapies. Despite its strong potential to treat DM, NS and TQ must be examined in more inclusive clinical studies targeting underrepresented patient populations.

Abstract P379: Acute Mineralocorticoid Receptor Antagonism Does Not Alter Insulin Sensitivity Or Secretion In Metabolic Syndrome

We previously demonstrated that maximal dietary sodium restriction impairs insulin secretion in humans, and that aldosterone impairs insulin secretion ex vivo in isolated islets. Aldosterone is inappropriately elevated in obesity and metabolic syndrome, but it is unknown if mineralocorticoid receptor antagonism with eplerenone improves insulin secretion in metabolic syndrome (MS) during moderate sodium restriction. We hypothesized that eplerenone would improve insulin secretion and insulin sensitivity in participants with the metabolic syndrome. We conducted a randomized, double-blind cross-over study in 16 volunteers with MS and elevated blood pressure in whom we provided either Eplerenone 50 mg or Amlodipine 5 mg and a standardized 50mmol Na/day diet for 7 days. Insulin sensitivity was assessed with hyperinsulinemic clamp and insulin secretion was assessed with hyperglycemic clamp at 5 and 7 days of diet, respectively. EPL increased plasma aldosterone compared to AML treatment (19.8±9.3 vs 12.2±3.9 ng/dL, in EPL and AML, respectively; mean±SD, for all descriptions; P <0.001) but did alter systolic blood pressure (127.5±15.6 vs 124.5±14.5 mmHg; P =0.51). EPL did not alter insulin sensitivity assessed by hyperinsulinemic clamp (19.3±7.8 vs 18.7±7.3 μg/kg/min per uU/mL Insulin; P =0.82).Fasting glucose (101.8±9.0 vs 100.5±6.8 mg/dL, P=0.76) and insulin (28.1±18.0 vs 23.9±12.7 μU/mL; P =0.7) were similar during EPL treatment. EPL did not alter acute glucose-stimulated insulin secretion (68.2±45.5 vs 58.7±43.7 μU/ml; P =0.7) or L-arginine stimulated insulin secretion (578±238 vs 579±228 μU/ml; P =0.8) assessed by hyperglycemic clamp. Disposition index was similarly unchanged (1.2±0.8 vs 1.1±0.9; P =0.8). We conclude that mineralocorticoid receptor antagonism with eplerenone during 7 days of moderate sodium restriction does not alter insulin sensitivity or insulin secretion in Metabolic Syndrome.

Pitfalls of Mitochondrial Redox Signaling Research.

Redox signaling from mitochondria (mt) to the cytosol and plasma membrane (PM) has been scarcely reported, such as in the case of hypoxic cell adaptation or (2-oxo-) 2-keto-isocaproate (KIC) β-like-oxidation stimulating insulin secretion in pancreatic β-cells. Mutual redox state influence between mitochondrial major compartments, the matrix and the intracristal space, and the cytosol is therefore derived theoretically in this article to predict possible conditions, when mt-to-cytosol and mt-to-PM signals may occur, as well as conditions in which the cytosolic redox signaling is not overwhelmed by the mitochondrial antioxidant capacity. Possible peroxiredoxin 3 participation in mt-to-cytosol redox signaling is discussed, as well as another specific case, whereby mitochondrial superoxide release is diminished, whereas the matrix MnSOD is activated. As a result, the enhanced conversion to H2O2 allows H2O2 diffusion into the cytosol, where it could be a predominant component of the H2O2 release. In both of these ways, mt-to-cytosol and mt-to-PM signals may be realized. Finally, the use of redox-sensitive probes is discussed, which disturb redox equilibria, and hence add a surplus redox-buffering to the compartment, where they are localized. Specifically, when attempts to quantify net H2O2 fluxes are to be made, this should be taken into account.

Effects of Dipeptidyl Peptidase 4 Inhibition on the Endothelial Control of the Vascular Tone.

Dipeptidyl peptidase 4 (DPP4) is a serine protease known to cleave incretin hormones, which stimulate insulin secretion after food intake, a fact that supported the development of its inhibitors (DPP4i, or gliptins) for the treatment of type 2 diabetes mellitus. In addition to their glucose lowering effects, DPP4i show benefits for the cardiovascular system that could be related, at least in part, to their protective action on the vascular endothelium. DPP4i has been associated with the reversal of endothelial dysfunction, an important predictor of cardiovascular events and a hallmark of diseases such as atherosclerosis, diabetes mellitus, hypertension, and heart failure. In animal models of these diseases, DPP4i increase nitric oxide bioavailability and limit oxidative stress, thereby improving the endothelium-dependent relaxation. Similar effects on flow-mediated dilation and attenuation of endothelial dysfunction have also been noted in human studies, suggesting a value for gliptins in the clinical scenario, despite the variability of the results regarding the DPP4i used, treatment duration and presence of comorbidities. In this mini review we discuss the advances in our comprehension of the DPP4i effects on endothelial regulation of vascular tone. Understanding the role of DPP4 and its involvement in the signaling mechanisms leading to endothelial dysfunction will pave the way for a broader use of DPP4i in conditions which endothelial dysfunction is an important pathophysiological player.