Beta-cell Apoptosis Research Articles

The Interplay of Adipokines and Pancreatic Beta Cells in Metabolic Regulation and Diabetes.

The interplay between adipokines and pancreatic beta cells, often referred to as the adipo-insular axis, plays a crucial role in regulating metabolic homeostasis. Adipokines are signaling molecules secreted by adipocytes that have profound effects on several physiological processes. Adipokines such as adiponectin, leptin, resistin, and visfatin influence the function of pancreatic beta cells. The reciprocal communication between adipocytes and beta cells is remarkable. Insulin secreted by beta cells affects adipose tissue metabolism, influencing lipid storage and lipolysis. Conversely, adipokines released from adipocytes can influence beta cell function and survival. Chronic obesity and insulin resistance can lead to the release of excess fatty acids and inflammatory molecules from the adipose tissue, contributing to beta cell dysfunction and apoptosis, which are key factors in developing type 2 diabetes. Understanding the complex interplay of the adipo-insular axis provides insights into the mechanisms underlying metabolic regulation and pathogenesis of metabolic disorders. By elucidating the molecular mediators involved in this interaction, new therapeutic targets and strategies may emerge to reduce the risk and progression of diseases, such as type 2 diabetes and its associated complications. This review summarizes the interactions between adipokines and pancreatic beta cells, and their roles in the pathogenesis of diabetes and metabolic diseases.

Sex-based impact of pancreatic islet stressors in GluCreERT2/Rosa26-eYFP mice.

The present study examines differences in metabolic and pancreatic islet adaptative responses following streptozotocin (STZ) and hydrocortisone (HC) administration in male and female transgenic GluCreERT2/Rosa26-eYFP mice. Mice received five daily doses of STZ (50 mg/kg, i.p.) or 10 daily doses of HC (70 mg/kg, i.p.), with parameters assessed on day 11. STZ-induced hyperglycaemia was evident in both sexes, alongside impaired glucose tolerance and reduced insulin concentrations. HC also had similar metabolic effects in male and female mice resulting in classical increases of circulating insulin indicative of insulin resistance. Control male mice had larger pancreatic islets than females and displayed a greater reduction of islet and beta-cell area in response to STZ insult. In addition, female STZ mice had lower levels of beta-cell apoptosis than male counterparts. Following HC administration, female mouse islets contained a greater proportion of alpha cells when compared to males. All HC mice presented with relatively comparable increases in beta- and alpha-cell turnover rates, with female mice being slightly more susceptible to HC-induced beta-cell apoptosis. Interestingly, healthy control female mice had inherently increased alpha-to-beta-cell transdifferentiation rates, which was decreased by HC treatment. The number of glucagon-positive alpha cells altering their lineage to insulin-positive beta cells was increased in male, but not female, STZ mice. Taken together, although there was no obvious sex-specific alteration of metabolic profile in STZ or HC mice, subtle differences in pancreatic islet morphology emphasises the impact of sex hormones on islets and importance of taking care when interpreting observations between males and females.

Thyroid dysfunction and glycaemic control among Type 2 diabetes mellitus patients in Ghana: A comparative cross-sectional study.

Thyroid disorders and diabetes mellitus coexist and are prevalent endocrinopathies among adult population. Thyroid dysfunction contributes to metabolic imbalances, increase beta-cell apoptosis and glucose intolerance. There is paucity of data and contradicting findings on how thyroid dysfunction influence glycaemic control. Therefore, we evaluated thyroid dysfunction and glycaemic control among Type 2 diabetes mellitus (T2DM) patients in Ghana. A comparative cross-sectional study was conducted among 192 T2DM patients from Effia Nkwanta Regional Hospital. Three consecutive monthly fasting plasma glucose (FBG) and glycated haemoglobin (HbA1c) were analysed and the results were classified as, moderate hyperglycaemia (MH) (FBG = 6.1-12.0 mmol/L, HbA1c < 7%), severe hyperglycaemia (SH) (FBG ≥ 12.1 mmol/L, HbA1c > 7%) and good glycaemic controls (GC) (FBG = 4.1-6.0 mmol/L, HbA1c < 7%). Thyroid-stimulating hormone (TSH), free triiodothyronine (FT3) and free thyroxine (FT4), body mass index (BMI) and other clinical parameters were measured. Data analysis was done using R language version 4.0.2 and p < .05 was considered statistically significant. There were no significant differences in age (years) between patients in the various glycaemic groups (p = .9053). The overall prevalence of thyroid disorders was 7.8% among T2DM patients. The prevalence of thyroid disorders was higher in patients with SH (11.7%) followed by those with MH (7.5%) and then those with GC (5.4%). Serum levels of TSH and FT3/FT4 ratio were significantly lower in T2DM patients with SH compared to those with MH and the GC (p < .0001). However, FT4 was significantly higher in SH patients compared to the good glycaemic controls (p < .01). The first tertiles of TSH [aOR = 10.51, 95% CI (4.04-17.36), p < .0001] and FT3 [aOR = 2.77, 95% CI (1.11-6.92), p = .0290] were significantly and independently associated with increased odds of hyperglycaemia. The prevalence of thyroid dysfunction is high in T2DM and increases with hyperglycaemia. Reduced TSH and T3 may worsen glycaemic control. Periodic monitoring of thyroid function should be incorporated into management guidelines among T2DM patients in Ghana.

Mitochondrial protein MPV17 promotes beta-cell apoptosis in diabetogenesis.

MPV17 is a mitochondrial inner membrane protein, and its deficiency can cause mitochondrial DNA (mtDNA) depletion, increase reactive oxygen species (ROS), and promote apoptosis in several cell types, suggesting that MPV17 plays a protective role in cells although the underlying mechanism remains unknown. To test whether MPV17 is also protective in diabetic kidney disease, we treated MPV17-deficient mice with streptozotocin (STZ) and surprisingly found that they were resistant to diabetes. MPV17 deficiency was also found to confer resistance to the diabetes induced by an insulin mutation (Ins2Akita), which represents a mouse model of monogenic diabetes characterized by proinsulin misfolding and beta-cell failure. In both STZ and Ins2Akita models, Mpv17 mutants had significantly less severe beta-cell loss and apoptosis compared with the wild-type mice. We next showed that Mpv17 is expressed in beta-cells of mice normally, suggesting that MPV17 acts beta-cells autonomously to facilitate apoptosis. Consistently, Mpv17 knockdown improved the viability and ameliorated the apoptosis of cultured MIN6 cells treated with STZ and palmitic acid (PA), respectively, accompanied by prevention of caspase 3 activation. The proapoptotic effect of MPV17 in beta-cells is in contrast with its known anti-apoptotic effect in other cell types. Thus, we have identified a novel regulator of beta-cell death in diabetes development.

Synergistic effect of schizandrin A and DNase I knockdown on high glucose induced beta cell apoptosis by decreasing intracellular calcium concentration.

To explore the synergistic effect of deoxyribonuclease I (DNase I) knockdown combined with Schizandrin A (Sch A) in protecting islet beta-cells (β-cells) from apoptosis under high-glucose (HG) conditions. The concentration of Sch A was detected by Cell Counting Kit-8 (CCK-8). High glucose-cultured rat insulinoma beta cell line (RIN-M5F) cells were treated with Sch A and transfected with DNase I small interfering RNA (siRNA). Cell apoptosis rate and apoptosis-related protein level were examined by flow cytometry and Western blot method respectively. In addition, Na-K-adenosine triphosphatease (Na-K-ATPase) and Ca-Mg-ATPase activity, cell membrane potential, and intracellular Ca concentration was also examined respectively. Our study revealed that HG stimulation can cause a significant increase in DNase I level and cell apoptosis rate. However, Sch A combined with DNase I knockdown can significantly decrease the cell apoptosis rate and apoptosis-related protein levels such as BAX ( 0.05) and Caspase-3 ( 0.01). In addition, we also found that the combination of Sch A and DNase I knockdown can dramatically increase cell membrane potential level, Na-K-ATPase, and Ca-Mg-ATPase activity. Meanwhile, intracellular Ca concentration was also found to be significantly decreased by the synergistic effect of Sch A and DNase I knockdown. Overall, our study reveals a synergistic effect of Sch A and DNase I knockdown in protecting β-cells from HG-induced apoptosis.

ER stress increases expression of intracellular calcium channel RyR1 to modify Ca2+ homeostasis in pancreatic beta cells

Pancreatic beta cells maintain glucose homeostasis by secreting pulses of insulin in response to a rise in plasma glucose. Pulsatile insulin secretion occurs as a result of glucose-induced oscillations in beta-cell cytosolic Ca2+. The endoplasmic reticulum (ER) helps regulate beta-cell cytosolic Ca2+, and ER stress can lead to ER Ca2+ reduction, beta-cell dysfunction, and an increased risk of type 2 diabetes. However, the mechanistic effects of ER stress on individual calcium channels are not well understood. To determine the effects oftunicamycin-induced ER stress on ER inositol 1,4,5-triphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) and their involvement in subsequent Ca2+ dysregulation, we treated INS-1 832/13cells and primary mouse islets with ER stress inducer tunicamycin (TM). We showed TM treatment increased RyR1 mRNA without affecting RyR2 mRNA and decreased both IP3R1 and IP3R3 mRNA. Furthermore, we found stress reduced ER Ca2+ levels, triggered oscillations in cytosolic Ca2+ under subthreshold glucose conditions, and increased apoptosis and that these changes were prevented by cotreatment with the RyR1 inhibitor dantrolene. In addition, we demonstrated silencing RyR1-suppressed TM-induced subthreshold cytosolic Ca2+ oscillations, but silencing RyR2 did not affect these oscillations. In contrast, inhibiting IP3Rs with xestospongin-C failed to suppress the TM-induced cytosolic Ca2+ oscillations and did not protect beta cells from TM-induced apoptosis although xestospongin-C inclusion did prevent ER Ca2+ reduction. Taken together, these resultsshowchanges in RyR1 play a critical role in ER stress-induced Ca2+ dysfunction and beta-cell apoptosis.

Luteolin Protects Pancreatic β Cells against Apoptosis through Regulation of Autophagy and ROS Clearance.

Diabetes, which is mainly characterized by increased apoptosis and dysfunction of beta (β) cells, is a metabolic disease caused by impairment of pancreatic islet function. Previous studies have demonstrated that death-associated protein kinase-related apoptosis-inducing kinase-2 (Drak2) is involved in regulating β cell survival. Since natural products have multiple targets and often are multifunctional, making them promising compounds for the treatment of diabetes, we identified Drak2 inhibitors from a natural product library. Among the identified products, luteolin, a flavonoid, was found to be the most effective compound. In vitro, luteolin effectively alleviated palmitate (PA)-induced apoptosis of β cells and PA-induced impairment of primary islet function. In vivo, luteolin showed a tendency to lower blood glucose levels. It also alleviated STZ-induced apoptosis of β cells and metabolic disruption in mice. This function of luteolin partially relied on Drak2 inhibition. Furthermore, luteolin was also found to effectively relieve oxidative stress and promote autophagy in β cells, possibly improving β cell function and slowing the progression of diabetes. In conclusion, our findings show the promising effect of Drak2 inhibitors in relieving diabetes and offer a potential therapeutic target for the protection of β cells. We also reveal some of the underlying mechanisms of luteolin's cytoprotective function.

Determination of molecular mechanisms of development and course of experimental diabetes mellitus in Wistar rats

The development and progression of diabetes involves several molecular mechanisms, in particular: insulin resistance, dysfunction of beta cells, inflammatory processes. These mechanisms can disrupt insulin signaling pathways, contribute to beta-cell apoptosis, and are not necessarily dependent on the intervention of cytokines and chemokines. Additionally, genetics play a role, as some forms of diabetes are caused by genetic mutations affecting insulin production or sensitivity. The molecular mechanisms underlying the development and progression of diabetes are complex and encompass various aspects of the body’s physiology and biochemistry. Understanding these mechanisms is crucial for the development of effective methods for treating and preventing diabetes. The aim of the work is analysis of the expression of genes, related to diabetes in pancreatic tissue samples of Wistar rats. Materials and methods. The polymerase chain reaction method with real-time reverse transcription was used to analyze gene expression using the RTI Profiler™ PCR Array Rat Diabetes kit (QIAGEN, Germany), where the pancreas was the object of research in experimental animals. Results. RTI Profiler™ PCR Array Rat Diabetes profiles the expression of 84 genes, associated with the onset, development, and progression of diabetes. The panel contains genes that contribute to obesity, insulin resistance, early-onset diabetes, and its late complications. These genes are represented by functional categories: receptors, transporters, and channels; nuclear receptors; metabolic enzymes; secretion factors; signal transduction proteins; transcription factors. According to the PCR results of the study of the control group of animals and animals with experimental diabetes, we established the activity of the Nkx2-1 genes; Pik3r1; Slc14a2 with high expression compared to control animals. Conclusions. The Nkx2-1 gene has been implicated in the pathogenesis of diabetes, as evidenced by its high expression activity compared to the control group of animals. Additionally, the Pik3r1 protein shows elevated expression levels in the group of animals with experimental diabetes. These changes are believed to be part of a compensatory mechanism aimed at preserving the cellular function of the pancreatic endocrine system. Furthermore, the observed increase in Slc14a2 protein expression likely indicates the onset of late complications associated with kidney pathology in the course of diabetes.

283-OR: ADA Presidents' Select Abstract: An Isogenic hPSC-Based Platform for Functional Interrogation of the Role of Multiple T2D-Associated Genes in Pancreatic Beta-Cell Failure

Type 2 diabetes (T2D) mellitus is a complex polygenic disease. Multiple genome-wide association studies (GWAS) have identified hundreds of T2D-related genetic variants, the vast majority of which are non-coding. Pinpointing the actual causal variants and their target gene transcripts has been challenging. Building on recent advances in CRISPR technology and protocols that allow differentiation into functional pancreatic beta cells, we created isogenic hPSCs encompassing knockout (KO) mutations in 20 GWAS-identified T2D risk genes, and systematically examined their roles in beta cell differentiation, insulin production, glucose response, and apoptosis. As a result, loss of TCF7L2, HNF4A, TLE4, TGFB1, GIPR and COBLL1 severely impaired the differentiation capacities of hPSCs into functional beta cells and over half of the 20 T2D genes shared implications in beta cell dysfunctions including decreased insulin content, impaired insulin secretion and high susceptibility to lipotoxicity. Next, we sorted beta cells from each KO and performed RNA-seq and ATAC-seq assays. An integrative analysis revealed KO of HNF4A, HNF1A, ABCC8, KCNJ11, TLE4 and WDR13 resulted in extensive transcriptomic and epigenetic changes in the beta cells. We identify that KO of HNF4A results in dramatic expressional changes of dozens of known T2D effector genes and additional ones critical to beta cell functions and biology. An analysis of T2D GWAS credible set of SNPs using ATAC-seq and RNA-seq coupled with functional assays, helped us to nominate a single likely T2D causal SNP (rs7132908) at FAIM2 locus. Finally, a correlation analysis of gene expression levels and insulin content across KO lines identified 1,867 differential genes including PCSK1N that likely serves as a hub gene by mediating insulin production in beta cells. This study thus offers a powerful strategy to interrogate shared molecular pathways among multiple risk genes for diabetes. Disclosure D.Xue: None. J.Vandana: None. A.Chong: None. F.S.Collins: None. S.Chen: Consultant; Vesalius Therapeutics, Stock/Shareholder; Oncobeat Theraputics. N.Narisu: None. T.Yan: None. M.Zhang: None. C.Grenko: None. X.Tang: None. J.Zhu: None. L.L.Bonnycastle: None. M.Erdos: None. Funding American Diabetes Association (9-22-PDFPM-06 to D.X.)

Evaluation of adiponectin and TNF-α expression in diabetic patients and its relationship with cardiovascular diseases.

Diabetes is caused by peripheral insulin resistance and lack of insulin secretion due to the apoptosis of pancreatic beta cells. Tumor necrosis factor alpha (TNF-α), a pro-inflammatory cytokine secreted from the tissue on the insulin signaling pathway, can play a role in causing fat resistance to insulin in type 2 diabetes patients. Adiponectin is a specific protein of adipose tissue. It belongs to the collectin family, which is present in human plasma at a high level and can protect against vascular lesions. Considering the importance of epigenetic changes in the development of multifactorial diseases, this study was conducted to investigate the methylation of TNF-α gene promoter in patients with type diabetes with cardiovascular disease and compare it with diabetic people without cardiovascular disease. Also, the serum concentration of adiponectin was investigated in diabetic patients with and without cardiovascular disease. For this purpose, 95 patients with type 2 diabetes referred to Isfahan Endocrine and Metabolism Research Center were divided into two groups: cardiovascular disease and without cardiovascular disease, based on the angiography results. Serum adiponectin level was measured by the RIA method. In addition to adiponectin, indicators such as FBS, cholesterol, triglycerides, and HDL were also measured in these patients. Then, the promoter region of the TNF-α gene was investigated by bisulfite treatment method, nested PCR, and finally, sequence determination. The results showed that the serum level of adiponectin was higher in diabetic patients without cardiovascular disease than in diabetic patients with cardiovascular disease, but this difference was not statistically significant. Also, no change was observed between men and women in TNF-α gene promoter methylation in diabetic and non-diabetic groups. In general, the decrease in adiponectin concentration in diabetic people can be a factor in causing macroangiopathy, so it can be predicted that the production of recombinant adiponectin can be helpful in the treatment and protection of cardiovascular disease in these patients. Also, it seems that the epigenetic changes of cytokines that play a role in causing insulin resistance in type 2 diabetic patients are not noticeable in the peripheral blood sample. In this regard, other tissues should probably be investigated.

Glucolipotoxic Stress-Induced Mig6 Desensitizes EGFR Signaling and Promotes Pancreatic Beta Cell Death.

A loss of functional beta cell mass is a final etiological event in the development of frank type 2 diabetes (T2D). To preserve or expand beta cells and therefore treat/prevent T2D, growth factors have been considered therapeutically but have largely failed to achieve robust clinical success. The molecular mechanisms preventing the activation of mitogenic signaling pathways from maintaining functional beta cell mass during the development of T2D remain unknown. We speculated that endogenous negative effectors of mitogenic signaling cascades impede beta cell survival/expansion. Thus, we tested the hypothesis that a stress-inducible epidermal growth factor receptor (EGFR) inhibitor, mitogen-inducible gene 6 (Mig6), regulates beta cell fate in a T2D milieu. To this end, we determined that: (1) glucolipotoxicity (GLT) induces Mig6, thereby blunting EGFR signaling cascades, and (2) Mig6 mediates molecular events regulating beta cell survival/death. We discovered that GLT impairs EGFR activation, and Mig6 is elevated in human islets from T2D donors as well as GLT-treated rodent islets and 832/13 INS-1 beta cells. Mig6 is essential for GLT-induced EGFR desensitization, as Mig6 suppression rescued the GLT-impaired EGFR and ERK1/2 activation. Further, Mig6 mediated EGFR but not insulin-like growth factor-1 receptor nor hepatocyte growth factor receptor activity in beta cells. Finally, we identified that elevated Mig6 augmented beta cell apoptosis, as Mig6 suppression reduced apoptosis during GLT. In conclusion, we established that T2D and GLT induce Mig6 in beta cells; the elevated Mig6 desensitizes EGFR signaling and induces beta cell death, suggesting Mig6 could be a novel therapeutic target for T2D.

The glucagon receptor antagonist desHis1Pro4Glu9-glucagon(Lys12PAL) alters alpha-cell turnover and lineage in mice, but does not cause alpha-cell hyperplasia

ObjectiveGlucagon receptor (GCGR) antagonism elicits antihyperglycemic effects in rodents and humans. The present study investigates whether the well characterised peptide-based GCGR antagonist, desHis1Pro4Glu9-glucagon (Lys12PAL), alters alpha-cell turnover or identity in mice. MethodsMultiple low-dose streptozotocin (STZ) treated (50 mg/kg bw, 5 days) transgenic GluCreERT2;ROSA26-eYFP mice were employed. STZ mice received twice daily administration of saline vehicle or desHis1Pro4Glu9-glucagon (Lys12PAL), at low- or high-dose (25 and 100 nmol/kg, respectively) for 11 days. ResultsNo GCGR antagonist induced changes in food or fluid intake, body weight or glucose homeostasis were observed. As expected, STZ dramatically reduced (P < 0.001) islet numbers and increased (P < 0.01) alpha-to beta-cell ratio, which was linked to elevated (P < 0.05) levels of beta-cell apoptosis. Whilst treatment with desHis1Pro4Glu9-glucagon (Lys12PAL) decreased (P < 0.05-P < 0.001) alpha- and beta-cell areas, it also helped restore the classic rodent islet alpha-cell mantle in STZ mice. Interestingly, low-dose desHis1Pro4Glu9-glucagon (Lys12PAL) increased (P < 0.05) alpha-cell apoptosis rates whilst high dose decreased (p < 0.05) this parameter. This difference reflects substantially increased (P < 0.001) alpha-to beta-cell transdifferentiation following high dose desHis1Pro4Glu9-glucagon (Lys12PAL) treatment, which was not fully manifest with low-dose therapy. ConclusionsTaken together, the present study indicates that peptidic GCGR antagonists can positively influence alpha-cell turnover and lineage in identity in multiple low-dose STZ mice, but that such effects are dose-related.

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

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

Deficiency of WTAP in islet beta cells results in beta cell failure and diabetes in mice.

N6-methyladenosine (m6A) mRNA methylation and m6A-related proteins (methyltransferase-like 3 [METTL3], methyltransferase-like 14 [METTL14] and YTH domain containing 1 [YTHDC1]) have been shown to regulate islet beta cell function and the pathogenesis of diabetes. However, whether Wilms' tumour 1-associating protein (WTAP), a key regulator of the m6A RNA methyltransferase complex, regulates islet beta cell failure during pathogenesis of diabetes is largely unknown. The present study aimed to investigate the role of WTAP in the regulation of islet beta cell failure and diabetes. Islet beta cell-specific Wtap-knockout and beta cell-specific Mettl3-overexpressing mice were generated for this study. Blood glucose, glucose tolerance, serum insulin, glucose-stimulated insulin secretion (both in vivo and in vitro), insulin levels, glucagon levels and beta cell apoptosis were examined. RNA-seq and MeRIP-seq were performed, and the data were well analysed. WTAP was downregulated in islet beta cells in type 2 diabetes, due to lipotoxicity and chronic inflammation, and islet beta cell-specific deletion of Wtap (Wtap-betaKO) induced beta cell failure and diabetes. Wtap-betaKO mice showed severe hyperglycaemia (above 20 mmol/l [360 mg/dl]) from 8 weeks of age onwards. Mechanistically, WTAP deficiency decreased m6A mRNA modification and reduced the expression of islet beta cell-specific transcription factors and insulin secretion-related genes by reducing METTL3 protein levels. Islet beta cell-specific overexpression of Mettl3 partially reversed the abnormalities observed in Wtap-betaKO mice. WTAP plays a key role in maintaining beta cell function by regulating m6A mRNA modification depending on METTL3, and the downregulation of WTAP leads to beta cell failure and diabetes. The RNA-seq and MeRIP-seq datasets generated during the current study are available in the Gene Expression Omnibus database repository ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE215156 ; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE215360 ).

Phytocompound inhibitors of caspase 3 as beta-cell apoptosis treatment development option: An In-silico approach

The prevalence of Diabetes mellitus (DM) is continuously rising worldwide. Among its types, type I is characterized by the destruction of beta cells triggered by various mechanisms, including the activation of Caspase 3. Studies have demonstrated the crucial role of Caspase 3 in initiating the apoptosis of beta cells in DM. Our research aims to identify possible phytocompounds inhibitors of Caspase 3 using computational approach. We obtained 3D structures of Caspase 3 and 6511 phytocompounds from the Protein Data Bank and the African Natural Products Database, respectively. The phytocompounds were assessed for druglikeness properties, topological polar surface area, and preliminary toxicity using DataWarrior. The phytocompounds were subjected to molecular docking simulation (MDS) at Caspase 3 active site using AutoDock-Vina. The frontrunner phytocompounds obtained from the MDS were subjected to protease inhibition prediction on Molinspiration. The pharmacokinetics of the phytocompounds were assessed on SwissADME. The in-depth computational toxicity profile of the phytocompounds was evaluated on the pkCSM web. The binding interactions of the phytocompounds with Caspase 3 were assessed with Discovery Studio Visualizer and Maestro. Seventeen phytocompounds were found to have no violation of Lipinski's rule and had no toxicity based on the preliminary assessment, have better binding affinity and protease inhibitory prediction scores than the references, have optimistic bioactivity radar prediction and similar amino acids interaction, in comparison with the references. Further studies, which include in-vitro and in-vivo studies, will be carried out to validate the results of this study.

Effect of Verapamil on Pancreatic Beta Cell Function in Newly Diagnosed Pediatric Type 1 Diabetes

In preclinical studies, thioredoxin-interacting protein overexpression induces pancreatic beta cell apoptosis and is involved in glucotoxicity-induced beta cell death. Calcium channel blockers reduce these effects and may be beneficial to beta cell preservation in type 1 diabetes. To determine the effect of verapamil on pancreatic beta cell function in children and adolescents with newly diagnosed type 1 diabetes. This double-blind, randomized clinical trial including children and adolescents aged 7 to 17 years with newly diagnosed type 1 diabetes who weighed 30 kg or greater was conducted at 6 centers in the US (randomized participants between July 20, 2020, and October 13, 2021) and follow-up was completed on September 15, 2022. Participants were randomly assigned 1:1 to once-daily oral verapamil (n = 47) or placebo (n = 41) as part of a factorial design in which participants also were assigned to receive either intensive diabetes management or standard diabetes care. The primary outcome was area under the curve values for C-peptide level (a measure of pancreatic beta cell function) stimulated by a mixed-meal tolerance test at 52 weeks from diagnosis of type 1 diabetes. Among 88 participants (mean age, 12.7 [SD, 2.4] years; 36 were female [41%]; and the mean time from diagnosis to randomization was 24 [SD, 4] days), 83 (94%) completed the trial. In the verapamil group, the mean C-peptide area under the curve was 0.66 pmol/mL at baseline and 0.65 pmol/mL at 52 weeks compared with 0.60 pmol/mL at baseline and 0.44 pmol/mL at 52 weeks in the placebo group (adjusted between-group difference, 0.14 pmol/mL [95% CI, 0.01 to 0.27 pmol/mL]; P = .04). This equates to a 30% higher C-peptide level at 52 weeks with verapamil. The percentage of participants with a 52-week peak C-peptide level of 0.2 pmol/mL or greater was 95% (41 of 43 participants) in the verapamil group vs 71% (27 of 38 participants) in the placebo group. At 52 weeks, hemoglobin A1c was 6.6% in the verapamil group vs 6.9% in the placebo group (adjusted between-group difference, -0.3% [95% CI, -1.0% to 0.4%]). Eight participants (17%) in the verapamil group and 8 participants (20%) in the placebo group had a nonserious adverse event considered to be related to treatment. In children and adolescents with newly diagnosed type 1 diabetes, verapamil partially preserved stimulated C-peptide secretion at 52 weeks from diagnosis compared with placebo. Further studies are needed to determine the longitudinal durability of C-peptide improvement and the optimal length of therapy. ClinicalTrials.gov Identifier: NCT04233034.

TXNIP overexpression in mice enhances streptozotocin-induced diabetes severity

Thioredoxin-interacting protein (TXNIP) is a key player in the endocrine pancreas; it induces beta cell apoptosis, such that TXNIP deficiency promotes beta cell survival. To study its function in more detail, we generated transgenic mice with ubiquitous overexpression of TXNIP. CBATXNIP/+ mice were investigated under basal conditions and after being challenged in diet-induced obesity (DIO) and streptozotocin-induced type 1 diabetes mellitus (T1DM) models. TXNIP overexpression caused no effect in the DIO model, contrasting to the already reported TXNIP-deficient mice. However, in the T1DM background, CBATXNIP/+ animals showed significantly enhanced blood glucose and increased glucose levels in a glucose tolerance test. Finally, the beta cell mass of CBATXNIP/+ transgenic animals in the T1DM model was significantly reduced compared to control littermates. Our study demonstrates that overexpression of TXNIP doesn't affect blood glucose parameters under basal conditions. However, overexpression of TXNIP in a T1DM model enhances the severity of the disease.

The Adaptor Protein NumbL Is Involved in the Control of Glucolipotoxicity-Induced Pancreatic Beta Cell Apoptosis.

Avoiding the loss of functional beta cell mass is critical for preventing or treating diabetes. Currently, the molecular mechanisms underlying beta cell death are partially understood, and there is a need to identify new targets for developing novel therapeutics to treat diabetes. Previously, our group established that Mig6, an inhibitor of EGF signaling, mediates beta cell death under diabetogenic conditions. The objective here was to clarify the mechanisms linking diabetogenic stimuli to beta cell death by investigating Mig6-interacting proteins. Using co-immunoprecipitation and mass spectrometry, we evaluated the binding partners of Mig6 under both normal glucose (NG) and glucolipotoxic (GLT) conditions in beta cells. We identified that Mig6 interacted dynamically with NumbL, whereas Mig6 associated with NumbL under NG, and this interaction was disrupted under GLT conditions. Further, we demonstrated that the siRNA-mediated suppression of NumbL expression in beta cells prevented apoptosis under GLT conditions by blocking the activation of NF-κB signaling. Using co-immunoprecipitation experiments, we observed that NumbL's interactions with TRAF6, a key component of NFκB signaling, were increased under GLT conditions. The interactions among Mig6, NumbL, and TRAF6 were dynamic and context-dependent. We proposed a model wherein these interactions activated pro-apoptotic NF-κB signaling while blocking pro-survival EGF signaling under diabetogenic conditions, leading to beta cell apoptosis. These findings indicated that NumbL should be further investigated as a candidate anti-diabetic therapeutic target.

Metabolic stress-induced human beta-cell death is mediated by increased intracellular levels of adenosine.

High intracellular concentrations of adenosine and 2'-deoxyadenosine have been suggested to be an important mediator of cell death. The aim of the present study was to characterize adenosine-induced death in insulin-producing beta-cells, at control and high glucose + palmitate-induced stress conditions. Human insulin-producing EndoC-betaH1 cells were treated with adenosine, 2'-deoxyadenosine, inosine and high glucose + sodium palmitate, and death rates using flow cytometry were studied. We observed that adenosine and the non-receptor-activating analogue 2-deoxyadenosine, but not the adenosine deamination product inosine, promoted beta-cell apoptosis at concentrations exceeding maximal adenosine-receptor stimulating concentrations. Both adenosine and inosine were efficiently taken up by EndoC-betaH1 cells, and inosine counteracted the cell death promoting effect of adenosine by competing with adenosine for uptake. Both adenosine and 2'-deoxyadenosine promptly reduced insulin-stimulated production of plasma membrane PI(3,4,5)P3, an effect that was reversed upon wash out of adenosine. In line with this, adenosine, but not inosine, rapidly diminished Akt phosphorylation. Both pharmacological Bax inhibition and Akt activation blocked adenosine-induced beta-cell apoptosis, indicating that adenosine/2'-deoxyadenosine inhibits the PI3K/Akt/BAD anti-apoptotic pathway. High glucose + palmitate-induced cell death was paralleled by increased intracellular adenosine and inosine levels. Overexpression of adenosine deaminase-1 (ADA1) in EndoC-betaH1 cells, which increased Akt phosphorylation, prevented both adenosine-induced apoptosis and high glucose + palmitate-induced necrosis. ADA2 overexpression not only failed to protect against adenosine and high glucose + palmitate-activated cell death, but instead potentiated the apoptosis-stimulating effect of adenosine. In line with this, ADA1 overexpression increased inosine production from adenosine-exposed cells, whereas ADA2 did not. Knockdown of ADA1 resulted in increased cell death rates in response to both adenosine and high glucose + palmitate. Inhibition of miR-30e-3p binding to the ADA1 mRNA 3'-UTR promoted the opposite effects on cell death rates and reduced intracellular adenosine contents. It is concluded that intracellular adenosine/2'-deoxyadenosine regulates negatively the PI3K pathway and is therefore an important mediator of beta-cell apoptosis. Adenosine levels are controlled, at least in part, by ADA1, and strategies to upregulate ADA1 activity, during conditions of metabolic stress, could be useful in attempts to preserve beta-cell mass in diabetes.

Prolonged culture of human pancreatic islets under glucotoxic conditions changes their acute beta cell calcium and insulin secretion glucose response curves from sigmoid to bell-shaped.

The rapid remission of type 2 diabetes by a diet very low in energy correlates with a marked improvement in glucose-stimulated insulin secretion (GSIS), emphasising the role of beta cell dysfunction in the early stages of the disease. In search of novel mechanisms of beta cell dysfunction after long-term exposure to mild to severe glucotoxic conditions, we extensively characterised the alterations in insulin secretion and upstream coupling events in human islets cultured for 1-3 weeks at ~5, 8, 10 or 20 mmol/l glucose and subsequently stimulated by an acute stepwise increase in glucose concentration. Human islets from 49 non-diabetic donors (ND-islets) and six type 2 diabetic donors (T2D-islets) were obtained from five isolation centres. After shipment, the islets were precultured for 3-7 days in RPMI medium containing ~5 mmol/l glucose and 10% (vol/vol) heat-inactivated FBS with selective islet picking at each medium renewal. Islets were then cultured for 1-3 weeks in RPMI containing ~5, 8, 10 or 20 mmol/l glucose before measurement of insulin secretion during culture, islet insulin and DNA content, beta cell apoptosis and cytosolic and mitochondrial glutathione redox state, and assessment of dynamic insulin secretion and upstream coupling events during acute stepwise stimulation with glucose [NAD(P)H autofluorescence, ATP/(ATP+ADP) ratio, electrical activity, cytosolic Ca2+ concentration ([Ca2+]c)]. Culture of ND-islets for 1-3 weeks at 8, 10 or 20 vs 5 mmol/l glucose did not significantly increase beta cell apoptosis or oxidative stress but decreased insulin content in a concentration-dependent manner and increased beta cell sensitivity to subsequent acute stimulation with glucose. Islet glucose responsiveness was higher after culture at 8 or 10 vs 5 mmol/l glucose and markedly reduced after culture at 20 vs 5 mmol/l glucose. In addition, the [Ca2+]c and insulin secretion responses to acute stepwise stimulation with glucose were no longer sigmoid but bell-shaped, with maximal stimulation at 5 or 10 mmol/l glucose and rapid sustained inhibition above that concentration. Such paradoxical inhibition was, however, no longer observed when islets were acutely depolarised by 30 mmol/l extracellular K+. The glucotoxic alterations of beta cell function were fully reversible after culture at 5 mmol/l glucose and were mimicked by pharmacological activation of glucokinase during culture at 5 mmol/l glucose. Similar results to those seen in ND-islets were obtained in T2D-islets, except that their rate of insulin secretion during culture at 8 and 20 mmol/l glucose was lower, their cytosolic glutathione oxidation increased after culture at 8 and 20 mmol/l glucose, and the alterations in GSIS and upstream coupling events were greater after culture at 8 mmol/l glucose. Prolonged culture of human islets under moderate to severe glucotoxic conditions markedly increased their glucose sensitivity and revealed a bell-shaped acute glucose response curve for changes in [Ca2+]c and insulin secretion, with maximal stimulation at 5 or 10 mmol/l glucose and rapid inhibition above that concentration. This novel glucotoxic alteration may contribute to beta cell dysfunction in type 2 diabetes independently from a detectable increase in beta cell apoptosis.