ß-Cell Insulin Resistance Plays a Causal Role in Fat-Induced ß-Cell Dysfunction In Vitro and In Vivo.

In the classical insulin target tissues of liver, muscle, and adipose tissue, chronically elevated levels of free fatty acids (FFA) impair insulin signaling. Insulin signaling molecules are also present in ß-cells where they play a role in ß-cell function. Therefore, inhibition of the insulin/insulin-like growth factor 1 pathway may be involved in fat-induced ß-cell dysfunction. To address the role of ß-cell insulin resistance in FFA-induced ß-cell dysfunction we co-infused bisperoxovanadate (BPV) with oleate or olive oil for 48 hours in rats. BPV, a tyrosine phosphatase inhibitor, acts as an insulin mimetic and is devoid of any antioxidant effect that could prevent ß-cell dysfunction, unlike most insulin sensitizers. Following fat infusion, rats either underwent hyperglycemic clamps for assessment of ß-cell function in vivo or islets were isolated for ex vivo assessment of glucose-stimulated insulin secretion (GSIS). We also incubated islets with oleate or palmitate and BPV for in vitro assessment of GSIS and Akt (protein kinase B) phosphorylation. Next, mice with ß-cell specific deletion of PTEN (phosphatase and tensin homolog; negative regulator of insulin signaling) and littermate controls were infused with oleate for 48 hours, followed by hyperglycemic clamps or ex vivo evaluation of GSIS. In rat experiments, BPV protected against fat-induced impairment of ß-cell function in vivo, ex vivo, and in vitro. In mice, ß-cell specific deletion of PTEN protected against oleate-induced ß-cell dysfunction in vivo and ex vivo. These data support the hypothesis that ß-cell insulin resistance plays a causal role in FFA-induced ß-cell dysfunction.

High-Throughput Methods for the Discovery of Small Molecule Modulators of Pancreatic Beta-Cell Function and Regeneration.

The progression of type II diabetes (T2D) is characterized by a complex and highly variable loss of beta-cell mass, resulting in impaired insulin secretion. Many T2D drug discovery efforts aimed at discovering molecules that can protect or restore beta-cell mass and function have been developed using limited beta-cell lines and primary rodent/human pancreatic islets. Various high-throughput screening methods have been used in the context of drug discovery, including luciferase-based reporter assays, glucose-stimulated insulin secretion, and high-content screening. In this context, a cornerstone of small molecule discovery has been the use of immortalized rodent beta-cell lines. Although insightful, this usage has led to a more comprehensive understanding of rodent beta-cell proliferation pathways rather than their human counterparts. Advantages gained in enhanced physiological relevance are offered by three-dimensional (3D) primary islets and pseudoislets in contrast to monolayer cultures, but these approaches have been limited to use in low-throughput experiments. Emerging methods, such as high-throughput 3D islet imaging coupled with machine learning, aim to increase the feasibility of integrating 3D microtissue structures into high-throughput screening. This review explores the current methods used in high-throughput screening for small molecule modulators of beta-cell mass and function, a potentially pivotal strategy for diabetes drug discovery.

You may delay, but time will not. Beta cells lost are never found again: a case for timely initiation of basal insulin in type 2 diabetes.

Since its first use just over a century ago, insulin treatment has evolved dramatically, such that the molecules are physiologic in nature, and treatment can now closely resemble the natural hormone response over 24 hours. Newer, longer-acting basal insulin analogs have provided insulin therapies with improved characteristics and, therefore, ease of use, and can readily be incorporated as part of routine treatment for type 2 diabetes (T2D), but evidence suggests that insulin remains underused in people with T2D. We review the barriers to initiation of basal insulin and the education needed to address these barriers, and we provide practical pointers, supported by evidence, for primary care physicians and advanced practice providers to facilitate timely initiation of basal insulin in the people with T2D who will benefit from such treatment.

Type 2 diabetes is a complex disease. It causes increased amounts of sugar in the blood, which can cause damage to the body. Medications are given to people with type 2 diabetes to keep their blood sugar at normal levels. Unfortunately, type 2 diabetes worsens over time, so regular adjustments to medications are needed to keep blood sugar levels controlled.Basal insulin, which is a type of insulin that works over the entire day, is a key treatment for type 2 diabetes. It works best if it is started as soon as other medications (tablets or non-insulin injections) are not working to control blood sugar levels. Unfortunately, delays in starting basal insulin are common. Some healthcare professionals and people with type 2 diabetes believe insulin is difficult to use. False information on insulin is common; for example, some people with diabetes believe that their symptoms are caused by insulin treatment rather than high blood sugar.This review summarizes key information to encourage effective conversations between healthcare professionals and people with type 2 diabetes about starting basal insulin. Proactive, positive, early discussion of the benefits of basal insulin can help to: 1) address concerns, 2) set appropriate, individual treatment targets, and 3) provide practical information and training to help with injecting insulin. This will give people living with type 2 diabetes the knowledge and confidence to take an active part in managing their diabetes and overcome any barriers to using basal insulin.

Baricitinib and ß-Cell Function in Patients with New-Onset Type 1 Diabetes.

BACKGROUND: Janus kinase (JAK) inhibitors, including baricitinib, block cytokine signaling and are effective disease-modifying treatments for several autoimmune diseases. Whether baricitinib preserves ß-cell function in type 1 diabetes is unclear. METHODS: In this phase 2, double-blind, randomized, placebo-controlled trial, we assigned patients with type 1 diabetes diagnosed during the previous 100 days to receive baricitinib (4 mg once per day) or matched placebo orally for 48 weeks. The primary outcome was the mean C-peptide level, determined from the area under the concentration-time curve, during a 2-hour mixed-meal tolerance test at week 48. Secondary outcomes included the change from baseline in the glycated hemoglobin level, the daily insulin dose, and measures of glycemic control assessed with the use of continuous glucose monitoring. RESULTS: A total of 91 patients received baricitinib (60 patients) or placebo (31 patients). The median of the mixed-meal-stimulated mean C-peptide level at week 48 was 0.65 nmol per liter per minute (interquartile range, 0.31 to 0.82) in the baricitinib group and 0.43 nmol per liter per minute (interquartile range, 0.13 to 0.63) in the placebo group (P = 0.001). The mean daily insulin dose at 48 weeks was 0.41 U per kilogram of body weight per day (95% confidence interval [CI], 0.35 to 0.48) in the baricitinib group and 0.52 U per kilogram per day (95% CI, 0.44 to 0.60) in the placebo group. The levels of glycated hemoglobin were similar in the two trial groups. However, the mean coefficient of variation of the glucose level at 48 weeks, as measured by continuous glucose monitoring, was 29.6% (95% CI, 27.8 to 31.3) in the baricitinib group and 33.8% (95% CI, 31.5 to 36.2) in the placebo group. The frequency and severity of adverse events were similar in the two trial groups, and no serious adverse events were attributed to baricitinib or placebo. CONCLUSIONS: In patients with type 1 diabetes of recent onset, daily treatment with baricitinib over 48 weeks appeared to preserve ß-cell function as estimated by the mixed-meal-stimulated mean C-peptide level. (Funded by JDRF International and others; BANDIT Australian New Zealand Clinical Trials Registry number, ACTRN12620000239965.).

Zarzio Ameliorates Non-alcoholic Fatty Liver Induced By a High Fat Diet in a Rat Experimental Model: A Histological and Immunohistochemical Study

SUMMARY: The therapeutic effect of a granulocyte-colony stimulating factor (G-CSF) biosimilar drug, zarzio, on non-alcoholic fatty liver disease (NAFLD) in a rat model was investigated in this study. Thirty-two rats were randomly divided into four groups. Groups I and II were fed a standard laboratory diet, whereas groups III and IV were fed a high fat diet (HFD) for 14 weeks. After 12 weeks of feeding, groups I and III were administered normal saline, and groups II and IV were intraperitoneally administered zarzio (200 mg/kg/day) for two consecutive weeks. Hematoxylin-eosin (H&E) staining was used to assess hepatic and pancreatic morphology in all groups, oil red O (ORO) staining for lipid accumulation, Masson's staining for fibrosis, and immunohistochemistry assay for hepatic protein expression of insulin receptor substrate 1 (IRS1), nuclear factor erythroid 2-related factor 2 (Nrf2), tumour necrosis factor alpha (TNF-α) and pancreatic caspase-3. The NAFLD rats (group III) developed hepatic steatosis with increased lipid accumulation, perisinusoidal fibrosis, upregulated IRS1, TNF-α (all P<0.05) without a significant increase in Nrf2 protein expression compared with normal control. In comparison, model rats treated with zarzio (group IV) showed significant rejuvenation of the hepatic architecture, reduction of fat accumulation, and fibrosis. This was accompanied by the upregulation of Nrf2, downregulation of IRS1 and TNF-α protein expression (all P<0.05). No correlation was detected between NAFLD and non-alcoholic fatty pancreas disease (NAFPD). However, the pancreatic β-cells in group III showed increased caspase-3 expression, which was decreased (P<0.05) in group IV. In conclusion, zarzio ameliorates NAFLD by improving the antioxidant capacity of liver cells, reducing hepatic IRS1, TNF-α protein expression and pancreatic β-cells apoptosis, suggesting that zarzio could be used as a potential therapy for NAFLD.

En este estudio se investigó el efecto terapéutico de un fármaco biosimilar del factor estimulante de colonias de granulocitos (G-CSF), zarzio, sobre la enfermedaddel hígado graso no alcohólico (NAFLD) en un modelo de rata. Treinta y dos ratas se dividieron aleatoriamente en cuatro grupos. Los grupos I y II fueron alimentados con una dieta estándar de laboratorio, mientras que los grupos III y IV fueron alimentados con una dieta alta en grasas (HFD) durante 14 semanas. Después de 12 semanas de alimentación, a los grupos I y III se les administró solución salina normal, y a los grupos II y IV se les administró zarzio por vía intraperitoneal (200 mg/kg/ día) durante dos semanas consecutivas. Se utilizó tinción de hematoxilina-eosina (H&E) para evaluar la morfología hepática y pancreática en todos los grupos, tinción con rojo aceite O (ORO) para la acumulación de lípidos, tinción de Masson para la fibrosis y ensayo de inmunohistoquímica para la expresión de la proteína hepática del sustrato 1 del receptor de insulina (IRS1), factor nuclear eritroide 2 relacionado con el factor 2 (Nrf2), factor de necrosis tumoral alfa (TNF-α) y caspasa-3 pancreática. Las ratas NAFLD (grupo III) desarrollaron esteatosis hepática con aumento de la acumulación de lípidos, fibrosis perisinusoidal, IRS1 y TNF-α regulados positivamente (todos P <0,05) sin un aumento significativo en la expresión de la proteína Nrf2 en comparación con el control normal. En comparación, las ratas modelo tratadas con zarzio (grupo IV) mostraron un rejuvenecimiento significativo de la arquitectura hepática, una reducción de la acumulación de grasa y fibrosis. Esto estuvo acompañado por la regulación positiva de Nrf2, la regulación negativa de la expresión de la proteína IRS1 y TNF-α (todas P <0,05). No se detectó correlación entre NAFLD y la enfermedad del páncreas graso no alcohólico (NAFPD). Sin embargo, las células β pancreáticas en el grupo III mostraron una mayor expresión de caspasa-3, que disminuyó (P <0,05) en el grupo IV. En conclusión, zarzio mejora la NAFLD al mejorar la capacidad antioxidante de las células hepáticas, reduciendo el IRS1 hepático, la expresión de la proteína TNF-α y la apoptosis de las células β pancreáticas, lo que sugiere que zarzio podría usarse como una terapia potencial para la NAFLD.

Teplizumab and ß-Cell Function in Newly Diagnosed Type 1 Diabetes.

BACKGROUND: Teplizumab, a humanized monoclonal antibody to CD3 on T cells, is approved by the Food and Drug Administration to delay the onset of clinical type 1 diabetes (stage 3) in patients 8 years of age or older with preclinical (stage 2) disease. Whether treatment with intravenous teplizumab in patients with newly diagnosed type 1 diabetes can prevent disease progression is unknown. METHODS: In this phase 3, randomized, placebo-controlled trial, we assessed ß-cell preservation, clinical end points, and safety in children and adolescents who were assigned to receive teplizumab or placebo for two 12-day courses. The primary end point was the change from baseline in ß-cell function, as measured by stimulated C-peptide levels at week 78. The key secondary end points were the insulin doses that were required to meet glycemic goals, glycated hemoglobin levels, time in the target glucose range, and clinically important hypoglycemic events. RESULTS: Patients treated with teplizumab (217 patients) had significantly higher stimulated C-peptide levels than patients receiving placebo (111 patients) at week 78 (least-squares mean difference, 0.13 pmol per milliliter; 95% confidence interval [CI], 0.09 to 0.17; P<0.001), and 94.9% (95% CI, 89.5 to 97.6) of patients treated with teplizumab maintained a clinically meaningful peak C-peptide level of 0.2 pmol per milliliter or greater, as compared with 79.2% (95% CI, 67.7 to 87.4) of those receiving placebo. The groups did not differ significantly with regard to the key secondary end points. Adverse events occurred primarily in association with administration of teplizumab or placebo and included headache, gastrointestinal symptoms, rash, lymphopenia, and mild cytokine release syndrome. CONCLUSIONS: Two 12-day courses of teplizumab in children and adolescents with newly diagnosed type 1 diabetes showed benefit with respect to the primary end point of preservation of ß-cell function, but no significant differences between the groups were observed with respect to the secondary end points. (Funded by Provention Bio and Sanofi; PROTECT ClinicalTrials.gov number, NCT03875729.).

Energy status regulates levels of the RAR/RXR ligand 9-cis-retinoic acid in mammalian tissues: Glucose reduces its synthesis in ß-cells.

9-cis-retinoic acid (9cRA) binds retinoic acid receptors (RAR) and retinoid X receptors (RXR) with nanomolar affinities, in contrast to all-trans-retinoic acid (atRA), which binds only RAR with nanomolar affinities. RXR heterodimerize with type II nuclear receptors, including RAR, to regulate a vast gene array. Despite much effort, 9cRA has not been identified as an endogenous retinoid, other than in pancreas. By revising tissue analysis methods, 9cRA quantification by liquid chromatography-tandem mass spectrometry becomes possible in all mouse tissues analyzed. 9cRA occurs in concentrations similar to or greater than atRA. Fasting increases 9cRA in white and brown adipose, brain and pancreas, while increasing atRA in white adipose, liver and pancreas. 9cRA supports FoxO1 actions in pancreas ß-cells and counteracts glucose actions that lead to glucotoxicity; in part by inducing Atg7 mRNA, which encodes the key enzyme essential for autophagy. Glucose suppresses 9cRA biosynthesis in the ß-cell lines 832/13 and MIN6. Glucose reduces 9cRA biosynthesis in 832/13 cells by inhibiting Rdh5 transcription, unconnected to insulin, through cAMP and Akt, and inhibiting FoxO1. Through adapting tissue specifically to fasting, 9cRA would act independent of atRA. Widespread occurrence of 9cRA in vivo, and its self-sufficient adaptation to energy status, provides new perspectives into regulation of energy balance, attenuation of insulin and glucose actions, regulation of type II nuclear receptors, and retinoid biology.

Denosumab Attenuates Glucolipotoxicity-Induced ß-Cell Dysfunction and Apoptosis by Attenuating RANK/RANKL Signals.

Obesity is strongly associated with insulin sensitivity in type 2 diabetes (T2D), mainly because free fatty acids (FFAs) are released from excess fat tissue. Long-term exposure to high levels of FFAs and glucose leads to glucolipotoxicity, causing damage to pancreatic ß-cells, thus accelerating the progression of T2D. Therefore, the prevention of ß-cell dysfunction and apoptosis is essential to prevent the development of T2D. Unfortunately, there are currently no specific clinical strategies for protecting ß-cells, highlighting the need for effective therapies or preventive approaches to improve the survival of ß-cells in T2D. Interestingly, recent studies have shown that the monoclonal antibody denosumab (DMB), used in osteoporosis, displays a positive effect on blood glucose regulation in patients with T2D. DMB acts as an osteoprotegerin (OPG) by inhibiting the receptor activator of the NF-κB ligand (RANKL), preventing the maturation and function of osteoclasts. However, the exact mechanism by which the RANK/RANKL signal affects glucose homeostasis has not been fully explained. The present study used human 1.4 × 107 ß-cells to simulate the T2D metabolic condition of high glucose and free fatty acids (FFAs), and it investigated the ability of DMB to protect ß-cells from glucolipotoxicity. Our results show that DMB effectively attenuated the cell dysfunction and apoptosis caused by high glucose and FFAs in ß-cells. This may be caused by blocking the RANK/RANKL pathway that reduced mammalian sterile 20-like kinase 1 (MST1) activation and indirectly increased pancreatic and duodenal homeobox 1 (PDX-1) expression. Furthermore, the increase in inflammatory cytokines and ROS caused by the RANK/RANKL signal also played an important role in glucolipotoxicity-induced cytotoxicity, and DMB can also protect ß-cells by reducing the mechanisms mentioned above. These findings provide detailed molecular mechanisms for the future development of DMB as a potential protective agent of ß-cells.

Stress-induced pseudokinase TRB3 augments IL1ß signaling by interacting with Flightless homolog 1.

Interleukin-1ß is one of the most potent inducers of beta cell inflammation in the lead-up to type 1 diabetes. We have previously reported that IL1ß-stimulated pancreatic islets from mice with genetic ablation of stress-induced pseudokinase TRB3(TRB3KO) show attenuated activation kinetics for the MAP3K MLK3 and JNK stress kinases. However, JNK signaling constitutes only a portion of the cytokine-induced inflammatory response. Here we report that TRB3KO islets also show a decrease in amplitude and duration of IL1ß-induced phosphorylation of TAK1 and IKK, kinases that drive the potent NF-κB proinflammatory signaling pathway. We observed that TRB3KO islets display decreased cytokine-induced beta cell death, preceded by a decrease in select downstream NF-κB targets, including iNOS/NOS2 (inducible nitric oxide synthase), a mediator of beta cell dysfunction and death. Thus, loss of TRB3 attenuates both pathways required for a cytokine-inducible, proapoptotic response in beta cells. In order to better understand the molecular basis of TRB3-enhanced, post-receptor IL1ß signaling, we interrogated the TRB3 interactome using coimmunoprecipitation followed by mass spectrometry to identify immunomodulatory protein Flightless homolog 1 (Fli1) as a novel, TRB3-interacting protein. We show that TRB3 binds and disrupts Fli1-dependent sequestration of MyD88, thereby increasing availability of this most proximal adaptor required for IL1ß receptor-dependent signaling. Fli1 sequesters MyD88 in a multiprotein complex resulting in a brake on the assembly of downstream signaling complexes. By interacting with Fli1, we propose that TRB3 lifts the brake on IL1ß signaling to augment the proinflammatory response in beta cells.

Nutrient-dependent regulation of ß-cell proinsulin content.

Insulin is made from proinsulin, but the extent to which fasting/feeding controls the homeostatically regulated proinsulin pool in pancreatic ß-cells remains largely unknown. Here, we first examined ß-cell lines (INS1E and Min6, which proliferate slowly and are routinely fed fresh medium every 2-3 days) and found that the proinsulin pool size responds to each feeding within 1 to 2 h, affected both by the quantity of fresh nutrients and the frequency with which they are provided. We observed no effect of nutrient feeding on the overall rate of proinsulin turnover as quantified from cycloheximide-chase experiments. We show that nutrient feeding is primarily linked to rapid dephosphorylation of translation initiation factor eIF2α, presaging increased proinsulin levels (and thereafter, insulin levels), followed by its rephosphorylation during the ensuing hours that correspond to a fall in proinsulin levels. The decline of proinsulin levels is blunted by the integrated stress response inhibitor, ISRIB, or by inhibition of eIF2α rephosphorylation with a general control nonderepressible 2 (not PERK) kinase inhibitor. In addition, we demonstrate that amino acids contribute importantly to the proinsulin pool; mass spectrometry shows that ß-cells avidly consume extracellular glutamine, serine, and cysteine. Finally, we show that in both rodent and human pancreatic islets, fresh nutrient availability dynamically increases preproinsulin, which can be quantified without pulse-labeling. Thus, the proinsulin available for insulin biosynthesis is rhythmically controlled by fasting/feeding cycles.

A peptide encoded by lncRNA MIR7-3 host gene (MIR7-3HG) alleviates dexamethasone-induced dysfunction in pancreatic ß-cells through the PI3K/AKT signaling pathway.

BACKGROUND: Dysfunction of pancreatic ß-cells induced by glucocorticoids contributes to diabetes mellitus development. Long noncoding RNAs (lncRNAs) have been recognized to contain short open reading frames (ORFs) that can be translated into functional small peptides. Here, we investigated whether the short peptide encoded by the lncRNA MIR7-3 host gene (MIR7-3HG) can affect dexamethasone (DEX)-induced ß-cell dysfunction. METHODS: Bioinformatics analysis was used for selection of MIR7-3HG and prediction of its protein encoding potential. The small peptide was identified by a western blot method. The cell-permeable TAT was fused into MIR7-3HG ORF to produce the cell-permeable fusion peptide (TAT-MIR7-3HG-ORF). The effects of TAT-MIR7-3HG-ORF on DEX-induced ß-cell dysfunction were evaluated by examining cell viability, apoptosis, insulin secretion, and reactive oxygen species (ROS) generation. RESULTS: DEX induced ß-TC6 cell dysfunction by impairing cell viability, insulin secretion and promoting cell apoptosis and ROS generation. The MIR7-3HG ORF could encode a 125-amino-acid-long short peptide. TAT-MIR7-3HG-ORF effectively transduced into ß-TC6 cells and attenuated DEX-induced dysfunction in ß-TC6 cells. Moreover, transduced TAT-MIR7-3HG-ORF reversed DEX-mediated inhibition of the activation of the PI3K/AKT signaling pathway. The inhibitor of the PI3K/AKT pathway partially abolished the alleviative effect of transduced TAT-MIR7-3HG-ORF on DEX-induced ß-TC6 cell dysfunction. CONCLUSION: The lncRNA MIR7-3HG encodes a short peptide, which can protect pancreatic ß-cells from DEX-induced dysfunction by activating the PI3K/AKT pathway. Our study broadens the diversity and breadth of lncRNAs in human disorders.

Effect of Tight Glycemic Control on Pancreatic Beta Cell Function in Newly Diagnosed Pediatric Type 1 Diabetes: A Randomized Clinical Trial.

Importance: Near normalization of glucose levels instituted immediately after diagnosis of type 1 diabetes has been postulated to preserve pancreatic beta cell function by reducing glucotoxicity. Previous studies have been hampered by an inability to achieve tight glycemic goals. Objective: To determine the effectiveness of intensive diabetes management to achieve near normalization of glucose levels on preservation of pancreatic beta cell function in youth with newly diagnosed type 1 diabetes. Design, Setting, and Participants: This randomized, double-blind, clinical trial was conducted at 6 centers in the US (randomizations from July 20, 2020, to October 13, 2021; follow-up completed September 15, 2022) and included youths with newly diagnosed type 1 diabetes aged 7 to 17 years. Interventions: Random assignment to intensive diabetes management, which included use of an automated insulin delivery system (n = 61), or standard care, which included use of a continuous glucose monitor (n = 52), as part of a factorial design in which participants weighing 30 kg or more also were assigned to receive either oral verapamil or placebo. Main Outcomes and Measures: The primary outcome was mixed-meal tolerance test-stimulated C-peptide area under the curve (a measure of pancreatic beta cell function) 52 weeks from diagnosis. Results: Among 113 participants (mean [SD] age, 11.8 [2.8] years; 49 females [43%]; mean [SD] time from diagnosis to randomization, 24 [5] days), 108 (96%) completed the trial. The mean C-peptide area under the curve decreased from 0.57 pmol/mL at baseline to 0.45 pmol/mL at 52 weeks in the intensive management group, and from 0.60 to 0.50 pmol/mL in the standard care group (treatment group difference, -0.01 [95% CI, -0.11 to 0.10]; P = .89). The mean time in the target range of 70 to 180 mg/dL, measured with continuous glucose monitoring, at 52 weeks was 78% in the intensive management group vs 64% in the standard care group (adjusted difference, 16% [95% CI, 10% to 22%]). One severe hypoglycemia event and 1 diabetic ketoacidosis event occurred in each group. Conclusions and Relevance: In youths with newly diagnosed type 1 diabetes, intensive diabetes management, which included automated insulin delivery, achieved excellent glucose control but did not affect the decline in pancreatic C-peptide secretion at 52 weeks. Trial Registration: ClinicalTrials.gov Identifier: NCT04233034.

Effect of Verapamil on Pancreatic Beta Cell Function in Newly Diagnosed Pediatric Type 1 Diabetes: A Randomized Clinical Trial.

Importance: 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. Objective: To determine the effect of verapamil on pancreatic beta cell function in children and adolescents with newly diagnosed type 1 diabetes. Design, Setting, and Participants: 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. Interventions: 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. Main Outcomes and Measures: 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. Results: 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. Conclusions and Relevance: 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. Trial Registration: ClinicalTrials.gov Identifier: NCT04233034.

Butyrate inhibits IL-1ß-induced inflammatory gene expression by suppression of NF-κB activity in pancreatic beta cells.

Cytokine-induced beta cell dysfunction is a hallmark of type 2 diabetes (T2D). Chronic exposure of beta cells to inflammatory cytokines affects gene expression and impairs insulin secretion. Thus, identification of anti-inflammatory factors that preserve beta cell function represents an opportunity to prevent or treat T2D. Butyrate is a gut microbial metabolite with anti-inflammatory properties for which we recently showed a role in preventing interleukin-1ß (IL-1ß)-induced beta cell dysfunction, but how prevention is accomplished is unclear. Here, we investigated the mechanisms by which butyrate exerts anti-inflammatory activity in beta cells. We exposed mouse islets and INS-1E cells to a low dose of IL-1ß and/or butyrate and measured expression of inflammatory genes and nitric oxide (NO) production. Additionally, we explored the molecular mechanisms underlying butyrate activity by dissecting the activation of the nuclear factor-κB (NF-κB) pathway. We found that butyrate suppressed IL-1ß-induced expression of inflammatory genes, such as Nos2, Cxcl1, and Ptgs2, and reduced NO production. Butyrate did not inhibit IκBα degradation nor NF-κB p65 nuclear translocation. Furthermore, butyrate did not affect binding of NF-κB p65 to target sequences in synthetic DNA but inhibited NF-κB p65 binding and RNA polymerase II recruitment to inflammatory gene promoters in the context of native DNA. We found this was concurrent with increased acetylation of NF-κB p65 and histone H4, suggesting butyrate affects NF-κB activity via inhibition of histone deacetylases. Together, our results show butyrate inhibits IL-1ß-induced inflammatory gene expression and NO production through suppression of NF-κB activation and thereby possibly preserves beta cell function.

The m6A Methyltransferase METTL3 Ameliorates Methylglyoxal-Induced Impairment of Insulin Secretion in Pancreatic ß Cells by Regulating MafA Expression.

Methylglyoxal, a major precursor of advanced glycation end products, is elevated in the plasma of patients with type 2 diabetes mellitus. Islet ß-cell function was recently shown to be regulated by N6-methyladenosine (m6A), an RNA modification consisting of methylation at the N6 position of adenosine. However, the role of m6A methylation modification in methylglyoxal-induced impairment of insulin secretion in pancreatic ß cells has not been clarified. In this study, we showed that treatment of two ß-cell lines, NIT-1 and ß-TC-6, with methylglyoxal reduced m6A RNA content and methyltransferase-like 3 (METTL3) expression levels. We also showed that silencing of METTL3 inhibited glucose-stimulated insulin secretion (GSIS) from NIT-1 cells, whereas upregulation of METTL3 significantly reversed the methylglyoxal-induced decrease in GSIS. The methylglyoxal-induced decreases in m6A RNA levels and METTL3 expression were not altered by knockdown of the receptor for the advanced glycation end product but were further decreased by silencing of glyoxalase 1. Mechanistic investigations revealed that silencing of METTL3 reduced m6A levels, mRNA stability, and the mRNA and protein expression levels of musculoaponeurotic fibrosarcoma oncogene family A (MafA). Overexpression of MafA greatly improved the decrease in GSIS induced by METTL3 silencing; silencing of MafA blocked the reversal of the MG-induced decrease in GSIS caused by METTL3 overexpression. The current study demonstrated that METTL3 ameliorates MG-induced impairment of insulin secretion in pancreatic ß cells by regulating MafA.

Artesunate protects pancreatic ß-cells from streptozotocin-induced diabetes via inhibition of the NLRP3/caspase-1/GSDMD pathway.

BACKGROUND: Reports in recent years have shown that pancreatic ß-cell pyroptosis represents a critical mechanism involved with the progressive failure of pancreatic function. Previous research from our laboratory has indicated that artemether can increase the number of cells in pancreatic islets of db/db mice. In this study, we further examined whether artesunate (ART) protects pancreatic ß-cells from the damage of streptozotocin (STZ) by inhibiting pyroptosis. MATERIALS AND METHODS: In vitro, MIN6 cells exposed to 1 mM STZ were treated with ART (0.8 or 1.6 µM). The effects of ART on STZ-treated cells were evaluated through CCK-8 assay, flow cytometry and western blot, and further compared the effects of ART with the NLRP3 inhibitor, Mcc950 upon pyroptosis pathway proteins using western blot. In vivo, Male C57 mice were administered with a single intraperitoneal injection of STZ, and those with confirmed diabetes mellitus were given ART (0.5 or 1.0 mg/ml in drinking water) for 18 days. The effects of ART on STZ-induced diabetes were assessed by the observation of the general situation, glucose tolerance test, hematoxylin-eosin (HE) staining and immunohistochemistry. RESULTS: In MIN6 cells treated with STZ, we found that ART increased cell viability, decreased the number of late apoptotic cells (including pyroptosis cells) and inhibited the expression of proteins associated with the pyroptosis pathway. In STZ-induced animal model, the administration of ART reduced blood glucose levels, improved the consumption status within this diabetic mouse model and inhibited the expression of proteins include in the pyroptosis pathway in mice pancreats. CONCLUSIONS: Inhibition of pyroptosis may be a critical mechanism through which artesunate exerts protective effects upon pancreatic ß cells.

TAZ promotes PDX1-mediated insulinogenesis.

Transcriptional co-activator with PDZ-binding motif (TAZ) is a key mediator of the Hippo signaling pathway and regulates structural and functional homeostasis in various tissues. TAZ activation is associated with the development of pancreatic cancer in humans, but it is unclear whether TAZ directly affects the structure and function of the pancreas. So we sought to identify the TAZ function in the normal pancreas. TAZ defect caused structural changes in the pancreas, particularly islet cell shrinkage and decreased insulin production and ß-cell markers expression, leading to hyperglycemia. Interestingly, TAZ physically interacted with the pancreatic and duodenal homeobox 1 (PDX1), a key insulin transcription factor, through the N-terminal domain of TAZ and the homeodomain of PDX1. TAZ deficiency decreased the DNA-binding and transcriptional activity of PDX1, whereas TAZ overexpression promoted PDX1 activity and increased insulin production even in a low glucose environment. Indeed, high glucose increased insulin production by turning off the Hippo pathway and inducing TAZ activation in pancreatic ß-cells. Ectopic TAZ overexpression along with PDX1 activation was sufficient to produce insulin in non-ß-cells. TAZ deficiency impaired the mesenchymal stem cell differentiation into insulin-producing cells (IPCs), whereas TAZ recovery restored normal IPCs differentiation. Compared to WT control, body weight increased in TAZ-deficient mice with age and even more with a high-fat diet (HFD). TAZ deficiency significantly exacerbated HFD-induced glucose intolerance and insulin resistance. Therefore, TAZ deficiency impaired pancreatic insulin production, causing hyperglycemia and exacerbating HFD-induced insulin resistance, indicating that TAZ may have a beneficial effect in treating insulin deficiency in diabetes.

SGLT2 inhibitors therapy protects glucotoxicity-induced ß-cell failure in a mouse model of human KATP-induced diabetes through mitigation of oxidative and ER stress.

Progressive loss of pancreatic ß-cell functional mass and anti-diabetic drug responsivity are classic findings in diabetes, frequently attributed to compensatory insulin hypersecretion and ß-cell exhaustion. However, loss of ß-cell mass and identity still occurs in mouse models of human KATP-gain-of-function induced Neonatal Diabetes Mellitus (NDM), in the absence of insulin secretion. Here we studied the temporal progression and mechanisms underlying glucotoxicity-induced loss of functional ß-cell mass in NDM mice, and the effects of sodium-glucose transporter 2 inhibitors (SGLT2i) therapy. Upon tamoxifen induction of transgene expression, NDM mice rapidly developed severe diabetes followed by an unexpected loss of insulin content, decreased proinsulin processing and increased proinsulin at 2-weeks of diabetes. These early events were accompanied by a marked increase in ß-cell oxidative and ER stress, without changes in islet cell identity. Strikingly, treatment with the SGLT2 inhibitor dapagliflozin restored insulin content, decreased proinsulin:insulin ratio and reduced oxidative and ER stress. However, despite reduction of blood glucose, dapagliflozin therapy was ineffective in restoring ß-cell function in NDM mice when it was initiated at >40 days of diabetes, when loss of ß-cell mass and identity had already occurred. Our data from mouse models demonstrate that: i) hyperglycemia per se, and not insulin hypersecretion, drives ß-cell failure in diabetes, ii) recovery of ß-cell function by SGLT2 inhibitors is potentially through reduction of oxidative and ER stress, iii) SGLT2 inhibitors revert/prevent ß-cell failure when used in early stages of diabetes, but not when loss of ß-cell mass/identity already occurred, iv) common execution pathways may underlie loss and recovery of ß-cell function in different forms of diabetes. These results may have important clinical implications for optimal therapeutic interventions in individuals with diabetes, particularly for those with long-standing diabetes.