Background: We (and others) have previously identified five clinically distinct diabetes subtypes. Currently, few models to identify diabetes subtypes are readily accessible. Further, while COVID-19 has been associated with increased risk of new-onset diabetes, it remains unknown whether the pandemic is also associated with changes in diabetes subtype distribution. Methods: We used the electronic health records of patients diagnosed with diabetes from 2010 to 2019 at the Kirklin Clinic of the University of Alabama at Birmingham (UAB) to train models to assign diabetes subtypes previously identified by hierarchical clustering. We then applied the trained model to conduct a retrospective cluster analysis of electronic health records of patients diagnosed with diabetes from 2020 to 2024 at UAB. We further validated our findings using data from the 2015-2023 National Health and Nutrition Examination Surveys (NHANES). Results: The trained classification model had an average specificity of 98% and an average sensitivity of 93%. Using the model, we identified a significant difference in the distribution of type 2 diabetes subtypes in patients at UAB and in participants in NHANES. In particular, the proportion of patients with severe insulin-dependent diabetes or severe insulin-resistant diabetes subtypes increased from 42% to 61% and 31% to 40% at the UAB and in NHANES, respectively. Conclusions: The model presented here can facilitate the identification of diabetes subtypes. The proportions of patients with severe subtypes of diabetes have seemed to increase in the more recent years following the pandemic. Further studies are required to determine the potential causes of this phenomenon.

Introduction and Objective: We have discovered that verapamil inhibits pancreatic islet expression of beta cell toxic thioredoxin-interacting protein (TXNIP) and preserves beta cell function in mice and in subjects with T1D and these clinical findings have been independently validated. However, as a calcium channel blocker, verapamil has many additional effects. We therefore aimed to explore the genes/proteins regulated by verapamil in the context of diabetes. Methods: We performed proteomics on serum samples of T1D subjects treated with placebo or verapamil. We also conducted transcriptomics on human islets treated with or without verapamil. Common genes/proteins significantly downregulated by verapamil in both omics were selected and analyzed in islets in the context of diabetes and inflammation. In addition, we analyzed the effect of verapamil on these genes in islets and islet endothelial cells. Results: We found five common proteins/genes (VCAM1, SPINK1, SYCN, PI16 and CD14) significantly decreased by verapamil in both omics. Besides its important role in immune cell transmigration and in diabetes, we chose VCAM1 for further study because SPINK1 is exclusively expressed in exocrine cells while the other genes are very lowly expressed in islets. We found that the serum levels of VCAM1 were significantly decreased in T1D subjects after 1 year of verapamil but not placebo treatment. In addition, VCAM1 expression was increased in islets of diabetic NOD mice, T2D subjects and in human islets treated with T1D-associated cytokines. Furthermore, we demonstrated that the expression of VCAM1 and two other adhesion molecules (PECAM1 and ESAM) was significantly decreased by verapamil in human islets and mouse islet endothelial cells. Conclusion: VCAM1 is significantly increased in islets in the context of diabetes and inflammation, which may play an important role in T cell infiltration into islets in T1D. The inhibition of VCAM1 and other vascular adhesion molecules by verapamil may contribute to the potential protective effects of this drug against islet inflammation. Disclosure G. Xu: None. J. Chen: None. B. Lu: None. A. Shalev: Other Relationship; TIXiMED, Inc.

Context Diabetes is a heterogenic disease and distinct clusters have emerged, but the implications for diverse populations have remained understudied. Objective Apply cluster analysis to a diverse diabetes cohort in the US Deep South. Design Retrospective hierarchical cluster analysis of electronic health records from 89 875 patients diagnosed with diabetes between January 1, 2010, and December 31, 2019, at the Kirklin Clinic of the University of Alabama at Birmingham, an ambulatory referral center. Patients Adult patients with International Classification of Diseases diabetes codes were selected based on available data for 6 established clustering parameters (glutamic acid decarboxylase autoantibody; hemoglobin A1c; body mass index; diagnosis age; HOMA2-B; HOMA2-IR); ∼42% were Black/African American. Main Outcome Measure(s) Diabetes subtypes and their associated characteristics in a diverse adult population based on clustering analysis. We hypothesized that racial background would affect the distribution of subtypes. Outcome and hypothesis were formulated prior to data collection. Results Diabetes cluster distribution was significantly different in Black/African Americans compared to Whites (P < .001). Black/African Americans were more likely to have severe insulin-deficient diabetes (OR, 1.83; 95% CI, 1.36-2.45; P < .001), associated with more serious metabolic perturbations and a higher risk for complications (OR, 1.42; 95% CI, 1.06-1.90; P = .020). Surprisingly, Black/African Americans specifically had more severe impairment of β-cell function (homoeostatic model assessment 2 estimates of β-cell function, C-peptide) (P < .001) but not being more obese or insulin resistant. Conclusion Racial background greatly influences diabetes cluster distribution and Black/African Americans are more frequently and more severely affected by severe insulin-deficient diabetes. This may further help explain the disparity in outcomes and have implications for treatment choice.

Introduction and Objective: We and others have previously identified 5 clinically distinct diabetes subtypes. While COVID-19 has been associated with increased risk of new-onset diabetes, it remains unknown whether the pandemic is also associated with changes in diabetes subtype distribution. Methods: We conducted a retrospective cluster analysis of electronic health records of 89,875 patients diagnosed with diabetes between January 1, 2010 and December 31, 2019 and 51,858 patients diagnosed with diabetes between January 1, 2020 and May 23, 2024 at the Kirklin Clinic of the University of Alabama at Birmingham. Adult patients with ICD diabetes codes were selected based on availability of data for 6 established clustering parameters (GAD autoantibody, HbA1c, BMI, diagnosis age, HOMA2-B, and HOMA2-IR), resulting in 1,194 and 165 patients for analysis, respectively. The differences in the distribution and proportions of diabetes subtypes between the 2 cohorts were assessed using chi-squared tests and Z-tests, respectively. Results: The 2010-2019 cohort and the 2020-2024 cohort had similar proportions of males (41% and 45%) and Black/African Americans (42% and 39%). However, the distribution of diabetes subtypes was significantly different between the 2 cohorts (p<0.001). In particular, the proportion of patients with severe insulin-dependent diabetes or severe insulin-resistant diabetes subtypes increased from 38% to 56% (p<0.001). In contrast, the proportion of patients with mild obesity-related diabetes or mild age-related diabetes subtypes decreased from 53% to just 38% (p<0.001). No significant changes were observed in the severe autoimmune diabetes. Conclusion: The proportions of patients with severe subtypes of diabetes seem to increase in the more recent years following the pandemic. Further studies are required to determine the potential causes of this phenomenon. B. Lu: None. P. Li: None. A. Crouse: None. T. Grimes: None. A.N. Smith: None. M. Might: Advisory Panel; Praxis Precision Medicines. Stock/Shareholder; Actio Biosciences. Advisory Panel; Alloy Therapeutics. F. Ovalle: None. A. Shalev: Other Relationship; TIXiMED, Inc. National Institutes of Health (R01DK137506), National Institutes of Health (UM1TR004771)

Introduction and Objective: We have identified inhibition of thioredoxin-interacting protein as an attractive therapeutic target for diabetes. Using the calcium channel blocker, verapamil to non-specifically lower TXNIP expression, we were able to establish a proof of concept demonstrating that TXNIP inhibition protects endogenous beta cell function in patients with recent onset T1D. However, based on the limitations of verapamil as a calcium channel blocker including the risk for cardiovascular side effects such as arrhythmias, heart block and hypotension and multiple ‘off-target’ effects, a novel chemical entity, TIX100, was specifically developed. This orally available TXNIP inhibitor was recently approved by the FDA as an investigational new drug. The goal of the current studies was to compare the TXNIP inhibitory and antidiabetic effects of TIX100 and verapamil. Methods: We used TIX100 and verapamil dose response experiments and assessment of TXNIP expression in INS-1 beta-like cells as well as human islets and performed RNA sequencing of treated human islets. In addition, we conducted in vivo experiments treating diabetic mice with oral TIX100 or verapamil. Results: Interestingly, we found that TIX100 was ~100-times more potent than verapamil in inhibiting TXNIP expression in INS-1 cells and human islets. TIX100 was also twice as effective in normalizing beta cell TXNIP levels and RNA sequencing confirmed its specificity. Moreover, in the context of beta cell destruction, the antidiabetic effects of TIX100 were more pronounced and sustained than those with verapamil. Finally, unlike verapamil, TIX100 was also found to protect against hyperglucagonemia, another important aspect of diabetic islet dysfunction. Conclusion: Thus, in these preclinical studies, TIX100 provided more potent, effective and specific TXNIP inhibition as well as antidiabetic effects than verapamil, suggesting that this may ultimately translate into a safer and more effective diabetes therapy. G. Jing: None. J. Chen: None. B. Lu: None. G. Xu: None. A. Shalev: Other Relationship; TIXiMED, Inc.

IntroductionWe reported that Ca2+-independent phospholipase A2β (iPLA2β)–derived lipids (iDLs) contribute to type 1 diabetes (T1D) onset. As CD4+ and CD8+ T cells are critical in promoting β-cell death, we tested the hypothesis that iDL signaling from these cells participates in T1D development. MethodsCD4+ and CD8+ T cells from wild-type non-obese diabetic (NOD) and NOD.iPLA2β+/- (NOD.HET) mice were administered in different combinations to immunodeficient NOD.scid. ResultsIn mice receiving only NOD T cells, T1D onset was rapid (5 weeks), incidence 100% by 20 weeks, and islets absent. In contrast, onset was delayed 1 week and incidence reduced 40%–50% in mice receiving combinations that included NOD.HET T cells. Consistently, islets from these non-diabetic mice were devoid of infiltrate and contained insulin-positive β-cells. Reduced iPLA2β led to decreased production of proinflammatory lipids from CD4+ T cells including prostaglandins and dihydroxyeicosatrienoic acids (DHETs), products of soluble epoxide hydrolase (sEH), and inhibition of their signaling decreased (by 82%) IFNγ+CD4+ cells abundance. However, only DHETs production was reduced from CD8+ T cells and was accompanied by decreases in sEH and granzyme B. DiscussionThese findings suggest that differential select iDL signaling in CD4+ and CD8+ T cells contributes to T1D development, and that therapeutics targeting such signaling might be considered to counter T1D.

We have previously found that the calcium channel blocker and anti-hypertensive, verapamil, promotes functional beta cell mass and improves glucose homeostasis in diabetic mice and in humans with recent-onset type 1 diabetes (T1D). Surprisingly, we also discovered that verapamil reversed the T1D-associated elevation in T-follicular-helper cell markers in peripheral blood monocytes, suggesting that it also has immunomodulatory effects. Now, a global proteomics analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) of serum samples from T1D subjects at baseline and after 1 year of receiving verapamil or placebo identified insulin-like growth factor 1 (IGF-1) as one of the top proteins with significantly changed abundance over time. Specifically, IGF-1 abundance decreased during year 1 after diagnosis in subjects with T1D receiving placebo, which is consistent with the lower IGF-1 levels reported previously in individuals with T1D as compared to age- and sex-matched healthy subjects. Interestingly, this decline was blunted in subjects receiving verapamil. To further confirm the LC-MS/MS results, we also measured patient serum levels of IGF-1 by ELISA. The results were very much in alignment and again revealed significantly decreased IGF-1 levels in the placebo group a year after diagnosis, but no such effect in the verapamil treatment group, suggesting that verapamil prevents the decline of IGF-1 in subjects with T1D. Of note, IGF-1, which is primarily produced in liver, acts on many tissues, including pancreatic islets promoting pathways involved in cell growth, differentiation and glucose metabolism. In beta cells, IGF-1 has also been shown to control apoptosis. Thus, our results reveal yet another previously unappreciated pathway affected by verapamil that may contribute to the beneficial effects observed with this treatment in the context of T1D. Disclosure G.Xu: None. J.Chen: None. B.Lu: None. W.Qian: None. A.Shalev: Other Relationship; TIXiMED. Funding National Institutes of Health (R01DK078752, U01DK120379)

Verapamil promotes functional beta-cell mass and improves glucose homeostasis in diabetic mice and humans with type 1 diabetes (T1D). Now, our global proteomics analysis of serum from T1D subjects at baseline and after 1 year of receiving verapamil or placebo revealed insulin-like growth factor 1 (IGF-1) as a protein with significantly changed abundance over time. IGF-1, which promotes beta-cell survival and insulin secretion, decreased during disease progression and this decline was blunted by verapamil. In addition, we found that verapamil reduces beta-cell expression of IGF-binding protein 3 (IGFBP3), whereas IGFBP3 was increased in human islets exposed to T1D-associated cytokines and diabetic NOD mouse islets. IGFBP3 binds IGF-1 and blocks its downstream signaling, which has been associated with increased beta-cell apoptosis and impaired glucose homeostasis. Consistent with the downregulation of IGFBP3, we have now discovered that verapamil increases beta-cell IGF-1 signaling and phosphorylation/activation of the IGF-1 receptor (IGF1R). Moreover, we found that thioredoxin interacting protein (TXNIP), a pro-apoptotic factor downregulated by verapamil, promotes IGFBP3 expression and inhibits the phosphorylation/activation of IGF1R. Thus, our results reveal IGF-1 signaling as yet another previously unappreciated pathway affected by verapamil and TXNIP that may contribute to the beneficial verapamil effects in the context of T1D.

Thioredoxin-interacting protein (Txnip) has emerged as a key factor in pancreatic beta cell biology and its upregulation by glucose and diabetes contributes to the impairment in functional beta cell mass and glucose homeostasis. In addition, beta cell deletion of Txnip protects against diabetes in different mouse models. However, while Txnip is ubiquitously expressed, its role in pancreatic alpha cells has remained elusive. We therefore now generated an alpha cell Txnip knockout (aTKO) mouse and assessed the effects on glucose homeostasis. While no significant changes were observed on regular chow, after a 30-week high-fat diet, aTKO animals showed improvement in glucose tolerance and lower blood glucose levels compared to their control littermates. Moreover, in the context of streptozotocin (STZ)-induced diabetes, aTKO mice showed significantly lower blood glucose levels compared to controls. While serum insulin levels were reduced in both control and aTKO mice, STZ-diabetes significantly increased glucagon levels in control mice, but this effect was blunted in aTKO mice. Moreover, glucagon secretion from aTKO islets was >2-fold lower than from control islets, while insulin secretion was unchanged in aTKO islets. At the same time, no change in alpha cell or beta cell numbers or mass was observed and glucagon and insulin expression and content were comparable in isolated islets from aTKO and control mice. Thus, together the current studies suggest that downregulation of alpha cell Txnip is associated with reduced glucagon secretion and that this may contribute to the glucose-lowering effects observed in diabetic aTKO mice.

Thioredoxin-interacting protein (TXNIP) has emerged as a key factor in pancreatic beta cell biology, and its upregulation by glucose and diabetes contributes to the impairment in functional beta cell mass and glucose homeostasis. In addition, beta cell deletion of TXNIP protects against diabetes in different mouse models. However, while TXNIP is ubiquitously expressed, its role in pancreatic alpha cells has remained elusive. We generated an alpha cell TXNIP knockout (aTKO) mouse and assessed the effects on glucose homeostasis. While no significant changes were observed on regular chow, after a 30-week high-fat diet, aTKO animals showed improvement in glucose tolerance and lower blood glucose levels compared to their control littermates. Moreover, in the context of streptozotocin (STZ)-induced diabetes, aTKO mice showed significantly lower blood glucose levels compared to controls. While serum insulin levels were reduced in both control and aTKO mice, STZ-induced diabetes significantly increased glucagon levels in control mice, but this effect was blunted in aTKO mice. Moreover, glucagon secretion from aTKO islets was >2-fold lower than from control islets, while insulin secretion was unchanged in aTKO islets. At the same time, no change in alpha cell or beta cell numbers or mass was observed, and glucagon and insulin expression and content were comparable in isolated islets from aTKO and control mice. Thus together the current studies suggest that downregulation of alpha cell TXNIP is associated with reduced glucagon secretion and that this may contribute to the glucose-lowering effects observed in diabetic aTKO mice.

Diabetes is characterized by hyperglycemia, loss of functional islet beta cell mass, deficiency of glucose-lowering insulin, and persistent alpha cell secretion of gluconeogenic glucagon. Still, no therapies that target these underlying processes are available. We therefore performed high-throughput screening of 300,000 compounds and extensive medicinal chemistry optimization and here report the discovery of SRI-37330, an orally bioavailable, non-toxic small molecule, which effectively rescued mice from streptozotocin- and obesity-induced (db/db) diabetes. Interestingly, in rat cells and in mouse and human islets, SRI-37330 inhibited expression and signaling of thioredoxin-interacting protein, which we have previously found to be elevated in diabetes and to have detrimental effects on islet function. In addition, SRI-37330 treatment inhibited glucagon secretion and function, reduced hepatic glucose production, and reversed hepatic steatosis. Thus, these studies describe a newly designed chemical compound that, compared to currently available therapies, may provide a distinct and effective approach to treating diabetes.