Β-cell Survival Research Articles

7765 Exploring the Synergistic Effect of GHRH Receptor Agonist MR-409 and Incretin-based Therapies in Type 1 Diabetes Using a Low-dose Streptozotocin Model

Abstract Disclosure: M. Gonzalez Medina: None. R. Louzada: None. V. Pita-Grisanti: None. M. Blandino: None. T. Cui: None. W. Sha: Owner/Co-Owner; Self; WS. is co-inventor on GHRH agonists MR-409 patent, assigned to the University of Miami and the Veterans Affairs. R. Cai: Owner/Co-Owner; Self; R.C. is co-inventor on GHRH agonists MR-409 patent, assigned to the University of Miami and the Veterans Affairs. A.V. Schally: Owner/Co-Owner; Self; A.V.S. is co-inventor on GHRH agonists MR-409 patent, assigned to the University of Miami and the Veterans Affairs.. E. Bernal-Mizrachi: None. Patients with type 1 diabetes (T1D) have decreased functional β-cell mass due to inflammatory cell infiltration and cytokine-induced β-cell death. Clinical trials using a combination of immunomodulatory agents and β-cell protecting medications such as GLP1-R agonists (GLP1-Ra) are being considered. A potential limitation for the clinical efficacy of GLP1-R agonists is the loss of GLP-1-R at the cell surface in states of hyperglycemia. Hence, novel agents that enhance β-cell survival and proliferation through parallel pathways could improve responses to GLP1-R agonists and/or synergistically potentiate β-cell responses. Previous studies have demonstrated beneficial effects of growth hormone-releasing hormone receptor agonists (GHRH-Ra), such as MR-409 on islet preconditioning in transplantation models. Our previous studies showed that GHRH-Ra protects β-cells from autoimmune injury in vitro and improves glucose levels in a model of high-dose streptozotocin-induced diabetes via stimulation of the cAMP/PKA/CREB/IRS2 axis. However, the effects of combination of incretin-based therapy with GHRHRa on β-cell survival in T1D models have not yet been explored. Using the low-dose STZ model in C57/B6 mice, we investigated how MR-409 alone or in combination with GLP1-R agonists (Exendin-4 or Ex-4) promotes β-cell survival and prevents T1D. Low-dose STZ treated mice received daily MR-409 and Ex-4 injections, either alone or in combination for six weeks. We found that, STZ-treated mice that received either MR-409, Ex-4 or combination exhibited better glucose homeostasis while the combination failed to induce any additional effect. Interestingly, glucose tolerance and insulin levels were improved only in the MR-409-treated mice. β-cell mass was similarly increased in mice that received MR-409, Ex-4 and combination groups compared to the vehicle-treated. Further, treatment of human islets with MR-409 and/or Ex-4 induced insulin receptor substrate 2 (IRS2) expression, a master regulator of survival and growth in β-cells, while the combination of both therapies did not have any additional effect. Experiments in human islets exposed to proinflammatory cytokines treatment demonstrated that MR-409 and/or Ex-4 was associated with a decrease in β-cell death and improved insulin secretory function by activation of the cAMP/PKA/CREB/IRS2 axis. These studies demonstrate that although there was no evidence for a synergistic effect between MR-409 and Ex-4, MR-409 appears to have more potent effects on improving glucose tolerance and insulin levels when compared to the long-term effect of Ex-4 in a low-dose STZ model, suggesting that MR-409 could be an alternative therapeutic agent in the treatment of β-cell death in T1D. Presentation: 6/2/2024

8030 Sexually Dimorphic Metabolic Consequences Of CCN5/WISP2 Gene Deletion Revealed A Male-Specific Pattern Of Gene Expression

Abstract Disclosure: M. Yang: None. V. Xega: None. M. Zakikhani: None. J. Liu: None. Matricellular protein CCN5/WISP2 is a novel target of IGF-1 and WNT signaling within the pancreatic islets. Our previous research suggests CCN5 promotes β-cell proliferation and survival through Akt/PKB and focal adhesion kinase signaling pathways. More recently, we have reported sexual dimorphic features of CCN5 gene deletion and overexpression. Specifically, male knockout mice demonstrated improved insulin sensitivity and glucose tolerance upon high-fat diet (HFD)-induced obesity, while females were unaffected (ENDO 2022). On the other hand, male aP2-CCN5 transgenic mice that overexpress CCN5 in adipose tissues were partially protected from HFD-streptozotocin induced diabetes, with improved glucose tolerance; females exhibited impaired glucose tolerance and exacerbated diabetes (ENDO 2023). Our findings indicate that an increased level of CCN5 protects β-cells, but only in male mice. This protection cannot be solely attributed to the pro-islet effect and suggests the involvement of extra-pancreatic and sex-specific roles. This study indirectly screened normal CCN5 gene expression by detecting β-galactosidase activity, measured in X-gal-stained tissues before fixation and sectioning. In order to create the gene deletion through homologous recombination, LacZ was inserted between exons 2 and 5 of the CCN5 gene. As a result, male knockout mice exhibited a distinct blue staining in the pseudostratified columnar epithelium with stereocilia of the epididymis and vas deferens. This type of epithelium comprises a single layer of cells but appears to have multiple layers due to the nuclei being at different levels. The epithelium possesses long projections made of actin filaments and plays a crucial role in absorbing, transporting, and maturing sperm cells. The staining was also clear in Leydig cells of the testis, which produce testosterone, which was confirmed using immunohistochemistry and authentic CCN5 antibody. In female mice, however, only a few cells in the ovary and scattered staining was observed in the endometrial glands of the uterus. Since androgens promote energy expenditure, lipolysis, and insulin sensitivity, knockout or overexpression of CCN5 may potentially affect androgen and/or sperm production, leading to metabolic consequences in males. Our results from in vitro, knockout, and overexpression models support the notion that CCN5 promotes β-cell growth and survival against diabetes and further suggest that it possesses sex-specific, metabolic effects. Presentation: 6/2/2024

9343 Protection Against Type 1 Diabetes Development In Mice With 4E-BP2 Deletion

Abstract Disclosure: V. Pita Grisanti: None. F. Pecanha: None. R. Louzada: None. M. Blandino: None. C. Jaramillo: None. N. Arenas: None. A. Bayer: None. E. Bernal-Mizrachi: None. Type 1 diabetes is an autoimmune disease characterized by β-cell destruction promoted by autoreactive T cells that acquire an effector inflammatory phenotype. 4E-BP1/2 proteins are translational repressors and downstream targets of mTORC1, a key regulator of metabolism. mTORC1 signaling disruption is implicated in human diseases including diabetes. Activation of 4E-BP2/eIF4E pathway by 4E-BP2 deletion promotes translation initiation, which induces β-cell expansion and proliferation and is crucial for the regulation of adaptive immunity. However, the involvement of 4E-BP2 in type 1 diabetes development has not been explored. Therefore, this study aimed to determine the role of 4E-BP2/eIF4E signaling in type 1 diabetes prevention by regulation of β-cell survival and immune modulation. To investigate the role of 4E-BP2/eIF4E axis in diabetes pathogenesis, we used the non-obese diabetic (NOD) mouse model as a type 1 diabetes model, and generated mice with global deletion of 4E-BP2 in the NOD background (4E-BP2KONOD). We assessed type 1 diabetes development, glucose homeostasis, pancreas morphometry and immune responses in male and female 4E-BP2KONOD and control NOD mice. We found that 4E-BP2KONOD exhibited reduced diabetes incidence in male but not female mice. Reduction in diabetes incidence in 4E-BP2KONOD male mice was associated with comparable degree of insulitis and β-cell proliferation, and with preserved β-cell mass compared to the control NOD mice. Glucose-stimulated insulin secretion of the islets, assessed in vitro by static incubation, was significantly higher in the 4E-BP2KONOD compared to the NOD control. Autoimmune responses were also evaluated by assessment of insulitis and characterization of T cell compartments, which showed a decrease in splenic CD8+ cytotoxic T cells and an increase in pancreatic regulatory T cell (Treg) infiltration mainly by increased Treg survival in 4E-BP2KONOD mice compared to control NOD. Finally, adoptive transfer studies demonstrated that lymphocytes derived from male 4E-BP2KONOD mice were less diabetogenic than those derived from control NOD mice. In conclusion, this study showed that activation of 4E-BP2/eIF4E axis in 4E-BP2KONOD male mice protects against type 1 diabetes development by dampening autoimmune responses and preserving β-cell mass. Targeting 4E-BP2 may provide a potential treatment for type 1 diabetes. Presentation: 6/1/2024

12614 Induction Of Gpt2 During Metabolic Stress Is Associated With Reduced Incretin Responsiveness And Decreased β-cell Resilience To Metabolic Stress

Abstract Disclosure: S. Sen: None. A.V. Rozo: None. M. Haemmerle: None. J. Roman: None. A.V. Scota: None. J.A. Christine: None. E.M. Morrow: None. S.A. Tersey: None. C.B. Newgard: None. N.M. Doliba: None. D.A. Stoffers: None. Elevated blood glucose and lipids compromise pancreatic β-cell survival and function, but the underlying molecular mechanisms are ill-understood. We previously found that mitochondrial glutamate-pyruvate transaminase 2 (GPT2) is induced by ER and glucolipotoxic (GLT) stress. Here, we find that islets from type 2 diabetic donors exhibited markedly elevated levels of GPT2 compared to those of non-diabetic donors. GLT stress induced GPT2 in MIN6 cells, primary murine, and human non-diabetic islets. We generated β-cell specific Gpt2-deficient (Gpt2βKO) and Gpt2 floxed control (Gpt2f/f) mice to study the in vivo role of Gpt2 in β-cells. Intraperitoneal (IP) glucose tolerance (GTT) of Gpt2βKO mice was minimally affected on chow diet; therefore, we placed Gpt2f/f and Gpt2βKO mice on 45 kcal% high-fat and matched control diets. At 1 week, HFD-fed Gpt2βKO mice exhibited reduced β-cell death, measured by droplet digital PCR of circulating unmethylated preproinsulin DNA and by TUNEL analysis. Over 37 weeks, HFD-fed Gpt2βKO mice showed lower random blood glucose similar to Gpt2f/f mice under a control diet. While IPGTT was modestly improved in HFD-fed Gpt2βKO compared to HFD-fed Gpt2f/f mice, insulin secretion was not enhanced. Remarkably, oral glucose administration markedly improved glucose tolerance and enhanced insulin secretion of control- and HFD-fed Gpt2βKO mice compared to Gpt2f/f mice, while GLP-1 and GIP levels were unaltered, suggesting enhanced incretin action on the β-cell. Indeed, Gpt2βKO mice (under both control and high-fat diets) were more responsive to administration of GLP1R agonist Exendin-4 (Ex4) than Gpt2f/f mice. Isolated Gpt2βKO islets secreted more insulin when stimulated by Ex4 and GIP but not acetylcholine. Perifusion assay revealed markedly enhanced insulin secretion of Gpt2βKO islets in response to a stepwise exposure to increasing Ex4 doses, indicating enhanced incretin sensitivity. Metabolic flux analysis of Gpt2βKO and Gpt2f/f islets exposed to uniformly labeled glucose (U-13C glucose) under low (2.8mM), high (12mM) glucose, and high glucose with Ex4 conditions revealed elevated levels of key TCA cycle intermediates in Gpt2βKO islets under high glucose and high glucose+Ex4 conditions, indicating increased TCA cycle flux that likely enhances insulin secretion. Gpt2βKO mice had higher β cell mass than Gpt2f/f littermates under control diet and HFD feeding. Bulk islet RNA-seq from Gpt2f/f and Gpt2βKO mice exposed to GLT revealed upregulation of genes responsible for cell cycle and division pathways and downregulation of apoptosis and ER stress genes, consistent with enhanced survival of Gpt2βKO islets under stress conditions. In summary, β-cell GPT2 is increased during metabolic stress and T2D. Its deficiency induces a metabolic reprogramming that improves incretin sensitivity and protects pancreatic β-cells from metabolic stress. Presentation: 6/2/2024

12331 Overcoming Limitations Of Identifying Proliferating β-cells In Dispersed Primary Islets Culture For Drug Discovery

Abstract Disclosure: H. Xu: None. S. Rebecca: None. S. Lee: None. J.P. Annes: None. Tremendous efforts have been made with screening platforms to identify islet β-cell mitogens that may be used to treat diabetes. However, promising compounds induce profound proliferation of islet non-β-cells in available screening platforms, calling for both β-cell targeted drug discovery and rigorous identification of β-cell identity to reduce false positivity. First, we used an established drug discovery dispersed islet cell-based platform(2D) to test the replication-promoting activity of GNF2133, a selective DYRK1A inhibitor that promotes β-cell proliferation, in isolated islets from human, rat and mouse. After 72 h in vitro culture, replication of non-β cells (PDX/Insulin negative) contributed to over 60% of the total replicating cells (Ki67+) in all species (human: 64%, rat: 71%, mouse: 67%). Mouse islets, which exhibit the greatest non-β-cell replication response upon GNF2133 treatment (4-fold above baseline, [50 nM]), were selected for subsequent studies. Next, we evaluated identification efficiency of β cell markers in this image-based platform. β cells, selected by the quantification value of insulin intensity, comprised 60-70% of total cell population at 48 h, 50-60% at 72 h and 20-30% at 96 h (p< 0.0001 for both comparisons: 48 h vs 72 h, 72 h vs 96 h). Basal replication of β-cells (Insulin+Ki67+) was 2.80%, 2.32%, 0.85% at 48 h, 72 h and 96 h, respectively. By comparison, the proportion of non-β cells showed an increasing trend as 2.48%, 6.49%, 8.68%, correspondingly. Identification with PDX1, another β cell marker, exhibited the same findings. Prolonged culture of dispersed islet cells, as these results indicate, favors growth of non-β-cells over β-cells. In addition, proliferating β-cells express lower levels of differentiation markers makes their discernment from replicating non-β cells more challenging. 窗体顶端 To minimize the duration of monolayer culture, replication induction was measured using intact islets with addition of BrdU during the last 24 h of culture. Then, islets were either directly fixed (3D) or dispersed and cultured for an additional 36 hour before being fixed (3D+2D). Immunostaining showed 70-80% PDX1 positive cells from both methods, which resembles in vivo β cell distribution within primary mouse islets. Basal β-cell replication in 3D+2D method was 0.30%, similar to in vivo BrdU labelling level. Upon mitogen treatment, PDX1+BrdU+ cells increased to 6.57% in 3D+2D method, slightly lower than 10.02% in 3D assay (p<0.05) yet significantly higher than 1.15% in 2D assay (<0.01). In summary, we developed a 3D+2D assay that enhances β-cell survival and the β-cell replication-induction response. This platform, while offering reduced throughput, provides a robust platform for discovery of β-cell replication-promoting compounds. Further validation with human islets is needed. Presentation: 6/2/2024

In vivo and in silico insights into the antidiabetic efficacy of EVOO and hydroxytyrosol in a rat model

Extra virgin olive oil (EVOO) has a putative antidiabetic activity mostly attributed to its polyphenol Hydroxytyrosol. In this study, we explored the antidiabetic effects of EVOO and Hydroxytyrosol on an in vivo T2D-simulated rat model as well as in in silico study. Wistar rats were divided into four groups. The first group served as a normal control (NC), while type 2 diabetes (T2D) was induced in the remaining groups using a high-fat diet (HFD) for 12 weeks followed by a single dose of streptozotocin (STZ, 30 mg/kg). One diabetic group remained untreated (DC), while the other two groups received an 8-week treatment with either EVOO (90 g/kg of the diet) (DO) or Hydroxytyrosol (17.3 mg/kg of the diet) (DH). The DC group exhibited hallmark features of established T2D, including elevated fasting blood glucose levels, impaired glucose tolerance, increased HOMA-IR, widespread downregulation of insulin receptor expression, heightened oxidative stress, and impaired β-cell function. In contrast, treatments with EVOO and Hydroxytyrosol elicited an antidiabetic response, characterized by improved glucose tolerance, as indicated by accelerated blood glucose clearance. Systematic analysis revealed the underlying antidiabetic mechanisms: both treatments enhanced insulin receptor expression in the liver and skeletal muscles, increased adiponectin levels, and mitigated oxidative stress. Moreover, while EVOO reduced intramyocellular lipids, Hydroxytyrosol restored adipose tissue insulin sensitivity and enhanced β-cell survival. Molecular docking and dynamics confirm Hydroxytyrosol's high affinity binding to PGC-1α, IRE-1α, and PPAR-γ, particularly IRE-1α, highlighting its potential to modulate diabetic signaling pathways. Collectively, these mechanisms highlight the putative antidiabetic role of EVOO and Hydroxytyrosol. Moreover, the favorable docking scores of Hydroxytyrosol with PGC-1α, IRE-1α, and PPAR-γ support the antidiabetic potential and offer promising avenues for further research and the development of novel antidiabetic therapies.

A-064 Improved Insulin production in Advanced glycation end-products (AGEs)-induced RIN-m5F pancreatic β-cells by using a Flavonoid-Rich Medicinal Plant, Kaempferia parviflora treatment

Abstract Background Advanced glycation end-products (AGEs) are glycated proteins that react with reducing sugar which causes the damages of function and cellular signaling of insulin production in insulinoma cells. AGEs also contribute to hyperglycemia-induced insulin resistance in type 2 diabetes. AGEs gradually form during the chronic high blood glucose condition, which is a major risk factor and harmful to β-cells function and survival, and could alter the insulin signaling and downregulate the insulin-responsive transcription factors including PDX-1, Glut-2, GCK, and Pre-INS. Kaempferia parviflora, also known as black ginger and flavonoid-rich medicinal plant, has beneficial properties for type 2 diabetes patients as a counteracts the detrimental effects of AGEs and improves the insulin production in pancreatic β-cells. Thus, we hypothesize that treated β-cells with K. parviflora, which is the most effective flavonoid-containing plant might protect against the damage of AGEs-induced insulin impairment, and these effects are also responsible for improved insulin synthesis and the essential transcription factors in pancreatic β-cells. Methods Rat pancreatic β-cell line RIN-m5F (ATCC CRL-2058) were maintained and cultured in RPMI 1640 medium containing 10% (v/v) FBS, 1% penicillin-streptomycin at 37◦C in a humidified atmosphere containing 5% CO2. RIN-m5F cells were pre-treated with the K. parviflora at concentrations 0, 25, and 50 µg/ml for 48 hours, and afterward, exposure to 250 µg/ml AGEs treatment. To quantify the bioactive compound of K. parviflora, total phenolic and flavonoid content assays were performed and the transcriptional changes of insulin-related genes (PDX-1, Glut-2, GCK, and Pre-INS) were determined by using real-time qRT-PCR. Results K. parviflora has shown a high phenolic content and flavonoid content was 50.43 ± 1.74, 26.66 ± 1.18 mg GAE/g Dry weight of sample, respectively. For the transcriptional changes of insulin-related genes, the levels of gene expression in the regulators of insulin secreting pathway (PDX-1, Glut-2, GCK, and Pre-INS) were significantly downregulated after exposure to AGEs-treated, while the pre-treatment of K. parviflora was showed statistically difference. PDX-1, Glut-2, GCK, and Pre-INS expressions were significantly upregulated after pre-treated with 50 µg/ml K. parviflora. However, under the AGEs-treated alone condition, the transcriptional levels were significantly decreased as compared to the control (untreated). Taken together, these results suggest K. parviflora could be a potential natural medicine to ameliorate the damage of insulin production in AGEs-induced β-cell damage and highlight promising alternatives for therapeutic avenue against diabetes. Conclusions Treated RIN-m5F pancreatic β-cells with K. parviflora, a flavonoid-rich medicinal plant, could upregulate the insulin-responsive transcription factors and improve the insulin production in damaging AGEs-induced pancreatic β-cells. Our findings provide a novel strategy related metabolic disease and insights into the regulation of insulin synthesis by using K. parviflora treatment as a therapeutic agent against type 2 diabetes.

Metabolic Stress Levels Influence the Ability of Myelin Transcription Factors to Regulate β-Cell Identity and Survival.

A hallmark of type 2 diabetes (T2D) is endocrine islet β-cell failure, which can occur via cell dysfunction, loss-of-identity, and/or death. How each is induced remains largely unknown. Here, we use mouse β-cells that are deficient for Myelin transcription factors (Myt TFs, including Myt1, 2, and 3) to address this question. We have reported that inactivating all three Myt genes in pancreatic progenitor cells (MytPancΔ) causes β-cell failure and late onset diabetes in mice. Their lower expression in human β-cells is correlated with β-cell dysfunction and SNPs in MYT2 and MYT3 are associated in higher risk of T2D. We now show that these Myt TF-deficient postnatal β-cells also de-differentiate by reactivating several progenitor markers. Intriguingly, mosaic Myt TF inactivation in only a portion of islet β-cells does not results in overt diabetes, but this creates a condition where Myt TF-deficient β-cells stay alive while activating several markers of Ppy-expressing islet cells. By transplanting MytPancΔ islets into the anterior eye chambers of immune-compromised mice, we directly show that glycemic and obesity-related conditions influence cell fate, with euglycemia inducing several Ppy+ cell markers while hyperglycemia and insulin resistance inducing additional cell death. These findings suggest that the observed β-cell defects in T2D depend on not only their inherent genetic/epigenetic defects, but also the metabolic load.

Cafeteria diet compromises natural adaptations of islet cell transdifferentiation and turnover in pregnancy.

Pancreatic islet β-cell mass expands during pregnancy, but underlying mechanisms are not fully understood. This study examines the impact of pregnancy and cafeteria diet on islet morphology, associated cellular proliferation/apoptosis rates as well as β-cell lineage. Non-pregnant and pregnant Ins1Cre/+;Rosa26-eYFP transgenic mice were maintained on either normal or high-fat cafeteria diet, with pancreatic tissue obtained at 18 days gestation. Immunohistochemical changes in islet morphology, β-/α-cell proliferation and apoptosis, as well as islet cell identity, neogenesis and ductal cell transdifferentiation were assessed. Pregnant normal diet mice displayed an increase in body weight and glycaemia. Cafeteria feeding attenuated this weight gain while causing overt hyperglycaemia. Pregnant mice maintained on a normal diet exhibited typical expansion in islet and β-cell area, owing to increased β-cell proliferation and survival as well as ductal to β-cell transdifferentiation and β-cell neogenesis, alongside decreased β-cell dedifferentiation. Such pregnancy-induced islet adaptations were severely restricted by cafeteria diet. Accordingly, islets from these mice displayed high levels of β-cell apoptosis and dedifferentiation, together with diminished β-cell proliferation and lack of pregnancy-induced β-cell neogenesis and transdifferentiation, entirely opposing islet cell modifications observed in pregnant mice maintained on a normal diet. Augmentation of β-cell mass during gestation arises through various mechanisms that include proliferation and survival of existing β-cells, transdifferentiation of ductal cells as well as β-cell neogenesis. Remarkably, cafeteria feeding almost entirely annuls pregnancy-induced islet adaptations, which may contribute to the development of gestational diabetes in the setting of dietary provoked metabolic stress.

Monocarbonyl analogs of curcumin C66 and B2BrBC modulate oxidative stress, JNK activity, and pancreatic gene expression in rats with streptozotocin-induced diabetes

The pathogenesis of type 1 diabetes mellitus (T1DM) involves oxidative stress and inflammation. Curcumin, a natural polyphenolic compound found in turmeric, known to exhibit antioxidative and anti-inflammatory properties, is characterized by poor chemical stability, low bioavailability, and rapid metabolism. Monocarbonyl analogs of curcumin (MACs) with a structural absence of β-diketone and enhanced stability and bioavailability present a potential solution to the challenges associated with the use of curcumin. This study aimed to evaluate the effect of two MACs, C66 and B2BrBC, on oxidative stress markers, antioxidant enzyme activity, expression of diabetes-associated genes, and signaling pathway proteins in the context of T1DM. Streptozotocin (STZ)-induced male Wistar rats or rat pancreatic RIN-m cells were used for in vivo and in vitro experiments, respectively. C66 or B2BrBC were given either before or after STZ treatment. Oxidative stress markers and antioxidant enzyme activities were determined in various tissues. Expression of diabetes-associated genes was assessed using RT-qPCR, and the activity of signaling pathway proteins in the pancreas was determined through Western blot analysis. Treatment with C66 and B2BrBC significantly reduced oxidative stress markers and positively influenced antioxidant enzyme activities. Moreover, both compounds inhibited JNK activity in the pancreas while enhancing the expression of genes crucial for β-cell survival and glucose and redox homeostasis. The findings highlight the multifaceted potential of C66 and B2BrBC in ameliorating oxidative stress, influencing gene expression patterns linked to diabetes, and modulating key signaling pathways in the pancreas. The findings suggest that these compounds can potentially address diabetes-related pathological processes.

Phenotypic quantification of Nphs1-deficient mice.

Steroid-resistant nephrotic syndrome (SRNS) is the second most frequent cause of chronic kidney disease in children and young adults. The most severe form of steroid-resistant nephrotic syndrome is congenital nephrotic syndrome Finnish type (CNSF), caused by biallelic loss-of-function variants inNPHS1,encoding nephrin. Since each of the 68 monogenic causes of steroid-resistant nephrotic syndrome represents a rare cause of the disease, tailoring therapeutic interventions to multiple molecular targets remains challenging, suggesting gene replacement therapy (GRT) as a viable alternative. To set the ground for a gene replacement study in vivo, we established rigorous, quantifiable, and reproducible phenotypic assessment of a conditional Nphs1 knockout mouse model. By breeding a floxed Nphs1fl/- mouse (Nphs1tm1Afrn/J) previously studied for pancreatic β-cell survival with a podocin promoter-driven Cre recombinase mouse model (Tg(NPHS2-Cre)295Lbh/J), we generated mice with podocyte-specific nephrin deficiency (Nphs1fl/fl NPHS2-Cre +). We observed a median survival to postnatal day P5 in nephrin-deficient mice, whereas heterozygous control mice and wild type (WT) control group showed 90% and 100% survival, respectively (at P50 days). Light microscopy analysis showed a significantly higher number of renal-tubular microcysts per kidney section in nephrin-deficient mice compared to the control groups (P < 0.0022). Transmission electron microscopy demonstrated reduced foot process (FP) density in nephrin-deficient mice compared to controls (P < 0.0001). Additionally, proteinuria quantitation using urine albumin-to-creatinine ratio (UACR) was significantly higher in nephrin-deficient mice compared to controls. This study represents the first comprehensive description of the kidney phenotype in anephrin-deficient mouse model, laying the foundation for future gene replacement therapy endeavors.

Hyperglycemia impairs EAAT2 glutamate transporter trafficking and glutamate clearance in islets of Langerhans: implications for type 2 diabetes pathogenesis and treatment.

Pancreatic endocrine cells employ a sophisticated system of paracrine and autocrine signals to synchronize their activities, including glutamate, which controls hormone release and β-cell viability by acting on glutamate receptors expressed by endocrine cells. We here investigate whether alteration of the excitatory amino acid transporter 2 (EAAT2), the major glutamate clearance system in the islet, may occur in type 2 diabetes mellitus and contribute to β-cell dysfunction. Increased EAAT2 intracellular localization was evident in islets of Langerhans from T2DM subjects as compared with healthy control subjects, despite similar expression levels. Chronic treatment of islets from healthy donors with high-glucose concentrations led to the transporter internalization in vesicular compartments and reduced [H3]-d-glutamate uptake (65 ± 5% inhibition), phenocopying the findings in T2DM pancreatic sections. The transporter relocalization was associated with decreased Akt phosphorylation protein levels, suggesting an involvement of the phosphoinositide 3-kinase (PI3K)/Akt pathway in the process. In line with this, PI3K inhibition by a 100-µM LY294002 treatment in human and clonal β-cells caused the transporter relocalization in intracellular compartments and significantly reduced the glutamate uptake compared to control conditions, suggesting that hyperglycemia changes the trafficking of the transporter to the plasma membrane. Upregulation of the glutamate transporter upon treatment with the antibiotic ceftriaxone rescued hyperglycemia-induced β-cells dysfunction and death. Our data underscore the significance of EAAT2 in regulating islet physiology and provide a rationale for potential therapeutic targeting of this transporter to preserve β-cell survival and function in diabetes.NEW & NOTEWORTHY The glutamate transporter SLC1A2/excitatory amino acid transporter 2 (EAAT2) is expressed on the plasma membrane of pancreatic β-cells and controls islet glutamate clearance and β-cells survival. We found that the EAAT2 membrane expression is lost in the islets of Langerhans from type 2 diabetes mellitus (T2DM) patients due to hyperglycemia-induced downregulation of the phosphoinositide 3-kinase/Akt pathway and modification of its intracellular trafficking. Pharmacological rescue of EAAT2 expression prevents β-cell dysfunction and death, suggesting EAAT2 as a new potential target of intervention in T2DM.

Chronic Epinephrine-Induced Endoplasmic Reticulum and Oxidative Stress Impairs Pancreatic β-Cells Function and Fate.

Epinephrine influences the function of pancreatic β-cells, primarily through the α2A-adrenergic receptor (α2A-AR) on their plasma membrane. Previous studies indicate that epinephrine transiently suppresses insulin secretion, whereas prolonged exposure induces its compensatory secretion. Nonetheless, the impact of epinephrine-induced α2A-AR signaling on the survival and function of pancreatic β-cells, particularly the impact of reprogramming after their removal from sustained epinephrine stimulation, remains elusive. In the present study, we applied MIN6, a murine insulinoma cell line, with 3 days of high concentration epinephrine incubation and 2 days of standard incubation, explored cell function and activity, and analyzed relevant regulatory pathways. The results showed that chronic epinephrine incubation led to the desensitization of α2A-AR and enhanced insulin secretion. An increased number of docked insulin granules and impaired Syntaxin-2 was found after chronic epinephrine exposure. Growth curve and cell cycle analyses showed the inhibition of cell proliferation. Transcriptome analysis showed the occurrence of endoplasmic reticulum stress (ER stress) and oxidative stress, such as the presence of BiP, CHOP, IRE1, ATF4, and XBP, affecting cellular endoplasmic reticulum function and survival, along with UCP2, OPA1, PINK, and PRKN, associated with mitochondrial dysfunction. Consequently, we conclude that chronic exposure to epinephrine induces α2A-AR desensitization and leads to ER and oxidative stress, impairing protein processing and mitochondrial function, leading to modified pancreatic β-cell secretory function and cell fate.

Pancreatic stellate cells support human pancreatic β-cell viability in vitro and enhance survival of immunoisolated human islets exposed to cytokines

Pancreatic islet transplantation is proposed as a cure for type 1 diabetes mellitus (T1D). Despite its success in optimal regulation of glucose levels, limitations in longevity of islet grafts still require innovative solutions. Inflammatory stress post-transplantation and loss of extracellular matrix attribute to the limited β-cell survival. Pancreatic stellate cells (PSCs), identified as pancreatic-specific stromal cells, have the potential to play a crucial role in preserving islet survival. Our study aimed to determine the effects of PSCs co-cultured with human CM β-cells and human islets under inflammatory stress induced by a cytokine cocktail of IFN-γ, TNF-α and IL-1β. Transwell culture inserts were utilized to assess the paracrine impact of PSCs on β-cells, alongside co-cultures enabling direct interaction between PSCs and human islets. We found that co-culturing PSCs with human CM β-cells and human cadaveric islets had rescuing effects on cytokine-induced stress. Effects were different under normoglycemic and hyperglycemic conditions. PSCs were associated with upregulation of β-cell mitochondrial activity and suppression of inflammatory gene expression. The rescuing effects exist both in indirect and direct co-culture methods. Furthermore, we tested whether PSCs have rescuing effects on human islets in conventional alginate-based microcapsules and in composite microcapsules composed of alginate-pectin collagen type IV, laminin sequence RGD, Nec-1, and amino acid. PSCs partially prevented cytokine-induced stress in both systems, but beneficial effects were stronger in composite capsules. Our findings show novel effects of PSCs on islet health. Islets and PSCs coculturing or co-transplantation might mitigate the inflammation stress and improve islet transplantation outcomes.

Unraveling Verapamil's Multidimensional Role in Diabetes Therapy: From β-Cell Regeneration to Cholecystokinin Induction in Zebrafish and MIN6 Cell-Line Models.

This study unveils verapamil's compelling cytoprotective and proliferative effects on pancreatic β-cells amidst diabetic stressors, spotlighting its unforeseen role in augmenting cholecystokinin (CCK) expression. Through rigorous investigations employing MIN6 β-cells and zebrafish models under type 1 and type 2 diabetic conditions, we demonstrate verapamil's capacity to significantly boost β-cell proliferation, enhance glucose-stimulated insulin secretion, and fortify cellular resilience. A pivotal revelation of our research is verapamil's induction of CCK, a peptide hormone known for its role in nutrient digestion and insulin secretion, which signifies a novel pathway through which verapamil exerts its therapeutic effects. Furthermore, our mechanistic insights reveal that verapamil orchestrates a broad spectrum of gene and protein expressions pivotal for β-cell survival and adaptation to immune-metabolic challenges. In vivo validation in a zebrafish larvae model confirms verapamil's efficacy in fostering β-cell recovery post-metronidazole infliction. Collectively, our findings advocate for verapamil's reevaluation as a multifaceted agent in diabetes therapy, highlighting its novel function in CCK upregulation alongside enhancing β-cell proliferation, glucose sensing, and oxidative respiration. This research enriches the therapeutic landscape, proposing verapamil not only as a cytoprotector but also as a promoter of β-cell regeneration, thereby offering fresh avenues for diabetes management strategies aimed at preserving and augmenting β-cell functionality.

Diabetes and cancer: two epidemic diseases requiring an opposite therapeutic approach to target cells

Diabetes and cancer are two chronic metabolic diseases with ever-increasing incidence rates worldwide. These disorders can often occur together, as diabetes presents an important risk factor for cancer and some cancers could in turn lead to diabetes. In this perspective article, many more commonalities between diabetes and cancer are highlighted, including the role of lifestyle and environmental factors in the pathogenesis, the presence of a rather long latency period before clinical diagnosis of invasive disease, as well as the ultimate progression to diabetic complications or malignant metastases. Moreover, both of these devastating disorders still lack curative treatment options, whereas several currently approved antidiabetic and anticancer drugs have been originally derived from different natural sources. However, while in the case of diabetes, the main therapeutic goal is to maintain the pancreatic islet mass by preserving β-cells survival, the major purpose of cancer therapy is to kill malignant cells and reduce the neoplastic mass of solid tumors. It is expected that both diabetes and cancer, two systemic diseases with epidemic proportions, would be managed more effectively through an integral approach, considering many different aspects related to their pathogenesis, including also lifestyle changes and dietary modifications.

Gαz-independent and -dependent Improvements With EPA Supplementation on the Early Type 1 Diabetes Phenotype of NOD Mice.

Prostaglandin E2 (PGE2) is a key mediator of inflammation and is derived from the omega-6 polyunsaturated fatty acid, arachidonic acid (AA). In the β-cell, the PGE2 receptor, Prostaglandin EP3 receptor (EP3), is coupled to the unique heterotrimeric G protein alpha subunit, Gɑz to reduce the production of cyclic adenosine monophosphate (cAMP), a key signaling molecule that activates β-cell function, proliferation, and survival pathways. Nonobese diabetic (NOD) mice are a strong model of type 1 diabetes (T1D), and NOD mice lacking Gɑz are protected from hyperglycemia. Therefore, limiting systemic PGE2 production could potentially improve both the inflammatory and β-cell dysfunction phenotype of T1D. Here, we sought to evaluate the effect of eicosapentaenoic acid (EPA) feeding, which limits PGE2 production, on the early T1D phenotype of NOD mice in the presence and absence of Gαz. Wild-type and Gαz knockout NOD mice were fed a control or EPA-enriched diet for 12 weeks, beginning at age 4 to 5 weeks. Oral glucose tolerance, splenic T-cell populations, islet cytokine/chemokine gene expression, islet insulitis, measurements of β-cell mass, and measurements of β-cell function were quantified. EPA diet feeding and Gɑz loss independently improved different aspects of the early NOD T1D phenotype and coordinated to alter the expression of certain cytokine/chemokine genes and enhance incretin-potentiated insulin secretion. Our results shed critical light on the Gαz-dependent and -independent effects of dietary EPA enrichment and provide a rationale for future research into novel pharmacological and dietary adjuvant therapies for T1D.

Bioengineered Artificial Extracellular Vesicles Presenting PD-L1 and Gal-9 Ameliorate New-Onset Type 1 Diabetes.

An important factor in the development of Type 1 diabetes (T1D) is the deficiency of inhibitory immune checkpoint ligands, specifically programmed cell death ligand 1 (PD-L1) and Galectin-9 (Gal-9), in β-cells. Hence, modulation of the pancreas infiltrated T lymphocytes by exogenous PD-L1 or Gal-9 is an ideal approach for treating the new-onset T1D. Herein, we genetic engineered the macrophage cells to generate artificial extracellular vesicles (aEVs) overexpressing PD-L1 and Gal-9, which could restrict the islets autoreactive T lymphocytes and protect β-cells from destruction. Intriguingly, overexpressing Gal-9 spurred macrophage polarization to M2 phenotype with immune suppressive attribute. Alternatively, both of PD-L1 and Gal-9 presenting aEVs (PD-L1-Gal-9 aEVs) favorably adhere to T cells via the interaction of programmed cell death protein 1 (PD-1)/PD-L1 or T cell immunoglobulin mucin 3 (TIM-3)/Gal-9. Moreover, PD-L1-Gal-9 aEVs prominently promoted effector T cell apoptosis and splenic regulatory T cells (Treg) cells differentiation in vitro. Virtually, PD-L1-Gal-9 aEVs efficaciously reversed the new-onset hyperglycemia in the NOD mice, prevented T1D progress, and declined the proportion and activation of CD4+ and CD8+ T cells infiltrating the pancreas notably, which together contributed to preserving the residual β-cells survival and mitigating the hyperglycemia.

PAX4 gene delivery improves β-cell function in human islets of Type II diabetes.

Aim: Type II diabetes (T2D) stems from insulin resistance, with β-cell dysfunction as a hallmark in its progression. Studies reveal that β cells undergo apoptosis or dedifferentiation during T2D development. The transcription factor PAX4 is vital for β differentiation and survival, thus may be a potential enhancer of β-cell function in T2D islets. Materials & methods: Human PAX4 cDNA was delivered into T2D human islets with an adenoviral vector, and its effects on β cells were examined. Results: PAX4 gene delivery significantly improved β-cell survival, and increased β-cell composition in the T2D human islets. Basal insulin and glucose-stimulated insulin secretion in PAX4-expressing islets were substantially higher than untreated or control-treated T2D human islets. Conclusion: Introduced PAX4 expression in T2D human islets improves β-cell function, thus could provide therapeutic benefits for T2D treatment.

The prostaglandin E2 EP3 receptor has disparate effects on islet insulin secretion and content in β-cells in a high-fat diet-induced mouse model of obesity.

Signaling through prostaglandin E2 EP3 receptor (EP3) actively contributes to the β-cell dysfunction of type 2 diabetes (T2D). In T2D models, full-body EP3 knockout mice have a significantly worse metabolic phenotype than wild-type controls due to hyperphagia and severe insulin resistance resulting from loss of EP3 in extra-pancreatic tissues, masking any potential beneficial effects of EP3 loss in the β cell. We hypothesized β-cell-specific EP3 knockout (EP3 βKO) mice would be protected from high-fat diet (HFD)-induced glucose intolerance, phenocopying mice lacking the EP3 effector, Gαz, which is much more limited in its tissue distribution. When fed a HFD for 16 wk, though, EP3 βKO mice were partially, but not fully, protected from glucose intolerance. In addition, exendin-4, an analog of the incretin hormone, glucagon-like peptide 1, more strongly potentiated glucose-stimulated insulin secretion in islets from both control diet- and HFD-fed EP3 βKO mice as compared with wild-type controls, with no effect of β-cell-specific EP3 loss on islet insulin content or markers of replication and survival. However, after 26 wk of diet feeding, islets from both control diet- and HFD-fed EP3 βKO mice secreted significantly less insulin as a percent of content in response to stimulatory glucose, with or without exendin-4, with elevated total insulin content unrelated to markers of β-cell replication and survival, revealing severe β-cell dysfunction. Our results suggest that EP3 serves a critical role in temporally regulating β-cell function along the progression to T2D and that there exist Gαz-independent mechanisms behind its effects.NEW & NOTEWORTHY The EP3 receptor is a strong inhibitor of β-cell function and replication, suggesting it as a potential therapeutic target for the disease. Yet, EP3 has protective roles in extrapancreatic tissues. To address this, we designed β-cell-specific EP3 knockout mice and subjected them to high-fat diet feeding to induce glucose intolerance. The negative metabolic phenotype of full-body knockout mice was ablated, and EP3 loss improved glucose tolerance, with converse effects on islet insulin secretion and content.