Introduction and Objective: Glucagon-like peptide 1 (GLP1) receptor agonists such as semaglutide have made medical weight loss possible, but for many the continued treatment necessary to maintain the weight loss is not sustainable. Reasons include gastrointestinal side effects, loss of lean/muscle mass, need for injections, and high cost. This results in half of the people stopping the medication within one year, which in most cases results in the regaining of the weight. Thus, there is an unmet need for non-GLP1 based, oral approaches to prevent this weight rebound that are affordable, well tolerated and do not impair muscle mass. The oral thioredoxin-interacting protein (TXNIP) inhibitor, TIX100, has recently been approved as an investigational new drug for diabetes by the FDA and found to be safe and well tolerated in a human Phase 1 trial. Interestingly, we recently discovered that TIX100 also has beneficial effects on weight control in mice. The goal of the current studies was therefore to determine whether TIX100 can prevent post-GLP1 weight regain. Methods: C57BL/6J male mice were rendered obese by high-fat diet (HFD), followed by 2 weeks of subcutaneous semaglutide treatment and weight and body composition, by quantitative magnetic resonance (QMR), were assessed 4 weeks after semaglutide discontinuation with or without oral TIX100 treatment. Results: HFD resulted in marked obesity and semaglutide led to a significant >6g weight loss; however, 4 weeks after semaglutide discontinuation untreated mice had regained almost 8g back, whereas mice receiving oral TIX100 maintained their weight loss despite ongoing HFD. QMR further showed that this difference was due to reduced fat mass as lean mass was preserved during TIX100 treatment. Conclusion: TIX100 may represent an attractive novel, oral, muscle-sparing, non-GLP1 approach for weight maintenance after GLP1-induced weight loss, in addition to its promising antidiabetic properties. Disclosure S. Jo: None. J. Chen: None. G. Jing: None. A. Shalev: Other - Co-founder, CSO/CMO; Current; TIXiMED, Inc.

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.

Introduction and Objective: While glucagon-like peptide 1 receptor agonists have revolutionized the treatment landscape of obesity and type 2 diabetes (T2D), significant limitations have emerged due to their gastrointestinal side effects, loss of lean mass, and necessity for ongoing subcutaneous injections. This underlines the need for different, better tolerated oral medications to improve obesity-associated impairment in glucose homeostasis. TIX100, a novel orally available thioredoxin-interacting protein (TXNIP) inhibitor showed beneficial effects in diabetic mice with streptozotocin-induced beta-cell destruction, but its effects in diet-induced obesity as a more relevant model of human T2D and glucose intolerance remained elusive. We therefore now aimed at defining the TIX100 effects on obesity and high-fat diet (HFD)-induced glucose intolerance. Methods: We employed diet-induced and genetically obese mouse models to define the effects of oral treatment with the investigational new drug, TIX100, on glucose intolerance and weight control using measurements of glucose homeostasis, plasma hormone levels, metabolic parameters, quantitative magnetic resonance, and indirect calorimetry. Results: Interestingly, we found that TIX100 treatment protected against hyperinsulinemia, hyperglucagonemia and obesity associated glucose intolerance. TIX100 also reduced diet-induced adiposity in wild-type mice, while preserving lean mass and leaving gastric emptying unaffected. In severe, genetically obese leptin-deficient ob/ob mice these effects on weight control were lost, yet the TIX100-mediated dramatic improvement in HbA1C was maintained independent of any weight loss. Conclusion: Taken together, TIX100 may provide a novel, oral therapy for T2D that, without aggressive weight loss, provides weight control, targets underlying disease pathology and promotes metabolic health. S. Jo: None. G. Jing: None. J. Chen: None. G. Xu: None. A. Shalev: Other Relationship; TIXiMED, Inc. NIH (R01DK078752 and U01DK120379)

Aims: Glucagon-like peptide 1 receptor agonists and dual agonists have changed the treatment landscape of obesity and type 2 diabetes (T2D), but significant limitations have emerged due to their gastrointestinal side effects, loss of lean mass, and necessity for ongoing subcutaneous injections. Our objective was, therefore, to test a novel small molecule as a different and potentially better tolerated oral medications to improve obesity-associated impairment in glucose homeostasis. Materials and Methods: High-fat diet (HFD)-fed mice or severely obese, leptin-deficient ob/ob mice were randomly assigned to serve as controls or receive oral TIX100, a novel thioredoxin-interacting protein (TXNIP) inhibitor just approved by the FDA as an investigational new drug for type 1 diabetes (T1D). The TIX100 effects on glucose intolerance and weight control were then assessed. Results: TIX100 protected against HFD-induced glucose intolerance, hyperinsulinemia, and hyperglucagonemia. TIX100 also reduced diet-induced adiposity resulting in 15% lower weight in treated mice as compared with controls on HFD (p <0.05), while preserving lean mass. Even though the TIX100 weight effects were lost in ob/ob mice, TIX100 improved glucose control leading to a dramatic 2.3% reduction in HbA1C (p <0.05), independent of any weight loss. This is consistent with the beneficial effects of TIX100 in non-obese diabetes models and its protection against elevated TXNIP and islet cell stress common to all diabetes types. Conclusions: Thus, TIX100 may provide a novel, oral therapy for T2D that targets underlying disease pathology including islet cell dysfunction and hyperglucagonemia and promotes metabolic health and weight control without aggressive weight loss.

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.

Abstract Disclosure: S. Jo: None. J. Chen: None. G. Jing: None. K.M. Habegger: None. A. Shalev: Other; Self; TIXiMED, Inc. Founder & CSO. Metabolic dysfunction-associated steatotic liver disease (MASLD) and steatohepatitis (MASH) (aka NAFLD and NASH) affect over 1 billion people worldwide, yet no approved therapy is currently available. MASLD/MASH is often associated with diabetes and obesity and some diabetes medications have shown some beneficial effects in this context. However, beyond steatosis these drugs are in general unable to prevent all the problems associated with MASH, such as inflammation, hepatocyte injury and especially fibrosis. Interestingly, we have now discovered that the new antidiabetic small molecule thioredoxin-interacting protein (TXNIP) inhibitor, TIX100, effectively protects against high-fat diet-induced hepatic steatosis. Oral TIX100 also protected obese and diabetic db/db mice against steatosis and significantly reduced the elevated expression levels of lipogenic genes found in db/db livers, many of which have been shown to be elevated in human MASLD. In addition though, TIX100 also normalized the blood glucose of db/db mice to the levels observed in lean control animals. Therefore, to address the question of whether TIX100 would still be effective in the absence of diabetes, obesity and any genetic leptin-receptor deficiency, we used a validated choline-deficient, amino acid-defined, high-fat diet to induce MASH. Indeed, compared to control chow, this diet significantly increased plasma liver enzymes indicating liver injury without causing diabetes or obesity. It also led to elevated hepatic expression of inflammatory and fibrogenic markers and histology revealed lipid accumulation and steatosis as well as pronounced fibrosis and upregulation of liver macrophages. Excitingly, all these pathological changes were blunted or improved by oral TIX100 treatment. Taken together, oral TIX100 may provide an attractive and effective novel approach for the treatment of MASLD/MASH that goes beyond the correction of diabetes, obesity, and steatosis. Presentation: 6/3/2024

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.

Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are often associated with diabetes, but are becoming an even bigger world-wide epidemic with over 1 billion people affected. Nevertheless, while some diabetes medications such as glucagon-like peptide 1 and peroxisome proliferator-activated receptor agonists have shown some beneficial effects in the context of NAFLD with diabetes, no approved therapy is currently available for NAFLD/NASH. Interestingly, the new antidiabetic small molecule thioredoxin-interacting protein (Txnip) inhibitor, TIX100, effectively protected obese and diabetic db/db mice against steatosis. TIX100 also significantly reduced the elevated expression levels of lipogenic genes found in db/db livers, many of which have also been shown to be elevated in human NAFLD and are being pursued as potential therapeutic targets. Importantly, the beneficial effects of TIX100 were not due to any weight loss as the severe obesity of the db/db mice was not affected by the treatment. In contrast, TIX100 did demonstrated strong antidiabetic effects and normalized the blood glucose of db/db mice to the levels observed in lean control animals. Therefore, to address the question of whether TIX100 would still be effective in the absence of diabetes, obesity and any genetic leptin-receptor deficiency, we next used a validated choline-deficient, amino acid-defined, high-fat diet to induce NASH. Indeed, compared to control chow, this diet significantly increased plasma liver enzymes indicating liver injury, led to elevated hepatic expression of inflammatory and fibrogenic markers and resulted in clear histological changes, demonstrating lipid accumulation and steatosis as well as pronounced fibrosis and upregulation of liver macrophages. Excitingly, TIX100 treatment blunted or improved all these pathological changes. Thus, TIX100 may not only be effective in diabetes, but may also provide an attractive novel approach for NAFLD/NASH. Disclosure S. Jo: None. J. Chen: None. G. Jing: None. K.M. Habegger: Research Support; Eli Lilly and Company, Novo Nordisk. Consultant; Glyscend Inc. Stock/Shareholder; Glyscend Inc. Research Support; Glyscend Inc. A. Shalev: Other Relationship; TIXiMED. Funding National Institutes of Health (R01DK078752); Human Islet Research Network (U01DK120379)

Currently, no oral medications are available for type 1 diabetes (T1D). While our recent randomized placebo-controlled T1D trial revealed that oral verapamil had short-term beneficial effects, their duration and underlying mechanisms remained elusive. Now, our global T1D serum proteomics analysis identified chromogranin A (CHGA), a T1D-autoantigen, as the top protein altered by verapamil and as a potential therapeutic marker and revealed that verapamil normalizes serum CHGA levels and reverses T1D-induced elevations in circulating proinflammatory T-follicular-helper cell markers. RNA-sequencing further confirmed that verapamil regulates the thioredoxin system and promotes an anti-oxidative, anti-apoptotic and immunomodulatory gene expression profile in human islets. Moreover, continuous use of oral verapamil delayed T1D progression, promoted endogenous beta-cell function and lowered insulin requirements and serum CHGA levels for at least 2 years and these benefits were lost upon discontinuation. Thus, the current studies provide crucial mechanistic and clinical insight into the beneficial effects of verapamil in 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.

Endoplasmic reticulum (ER) stress contributes to pancreatic beta-cell apoptosis in diabetes, but the factors involved are still not fully elucidated. Growth differentiation factor 15 (GDF15) is a stress response gene and has been reported to be increased and play an important role in various diseases. However, the role of GDF15 in beta cells in the context of ER stress and diabetes is still unclear. In this study, we have discovered that GDF15 promotes ER stress-induced beta-cell apoptosis and that downregulation of GDF15 has beneficial effects on beta-cell survival in diabetes. Specifically, we found that GDF15 is induced by ER stress in beta cells and human islets, and that the transcription factor C/EBPβ is involved in this process. Interestingly, ER stress-induced apoptosis was significantly reduced in INS-1 cells with Gdf15 knockdown and in isolated Gdf15 knockout mouse islets. In vivo, we found that Gdf15 deletion attenuates streptozotocin-induced diabetes by preserving beta cells and insulin levels. Moreover, deletion of Gdf15 significantly delayed diabetes development in spontaneous ER stress-prone Akita mice. Thus, our findings suggest that GDF15 contributes to ER stress-induced beta-cell apoptosis and that inhibition of GDF15 may represent a novel strategy to promote beta-cell survival and treat diabetes.

Increased glucagon is a hallmark of diabetes and leads to worsening of the hyperglycemia, but the molecular mechanisms causing it are still unknown. We therefore investigated the possibility that microRNAs might be involved in the regulation of glucagon. Indeed, analysis of the glucagon 3' untranslated region (UTR) revealed potential binding sites for miR-320a, and using luciferase reporter assays we found that miR-320a directly targets the 3' UTRs of human and rodent glucagon. In addition, endogenous glucagon mRNA and protein expression as well as glucagon secretion were reduced in response to miR-320a overexpression, whereas inhibition of miR-320a upregulated glucagon expression. Interestingly, miR-320a expression was decreased by high glucose, and this was associated with an increase in glucagon expression in human islets and mouse αTC1-6 cells. Moreover, miR-320a overexpression completely blunted these effects. Importantly, miR-320a was also significantly downregulated in human islets of subjects with type 2 diabetes and this was accompanied by increased glucagon expression. Thus, our data suggest that glucose-induced downregulation of miR-320a may contribute to the paradoxical increase in glucagon observed in type 2 diabetes and reveal for the first time that glucagon expression is under the control by a microRNA providing novel insight into the abnormal regulation of glucagon in diabetes.

Endoplasmic reticulum (ER) stress has been shown to play an important role in beta cell loss in diabetes. However, the factors involved in this process are still not fully understood. Growth differentiation factor 15 (GDF15), a member of the transforming growth factor beta (TGFβ) family, is a stress response gene and is involved in various diseases. GDF15 is expressed in adult human beta cells; however, its role in beta cells and diabetes had not been elucidated. Interestingly, we now have identified GDF15 as a key factor involved in ER stress-induced beta cell loss. We found that GDF15 mRNA, protein levels and promoter activity were significantly induced by ER stress in INS-1 cells, Akita beta cells, and primary human islets and we identified the transcription factor involved. Moreover, we discovered that overexpression of GDF15 exacerbated, while knockdown of GDF15 inhibited ER stress-induced beta cell apoptosis, at least in part by regulating the pro-apoptotic factor CHOP. Most importantly, we found that GDF15 knockout mice were protected from STZ-induced diabetes, maintained insulin producing beta cells and exhibited improved serum insulin levels. Taken together, our findings suggest that GDF15 plays an important role in ER stress-induced beta cell death and that inhibition of GDF15 may represent a novel strategy to reduce beta cell loss and treat diabetes. Disclosure G. Xu: None. S. Jo: None. J. Chen: None. T. Grayson: None. A. Shalev: None.