Episodic ß-cell death and dedifferentiation during diet-induced obesity and dysglycemia in male mice.

Loss of functional islet ß-cell mass through cellular death or dedifferentiation is thought to lead to dysglycemia during the progression from obesity to type 2 diabetes. To assess these processes in a mouse model of obesity, we performed measures of circulating cell-free differentially methylated insulin II ( Ins2) DNA as a biomarker of ß-cell death and aldehyde dehydrogenase 1 family member A3 (ALDH1A3) and forkhead box 01 (Foxo1) immunostaining as markers of ß-cell dedifferentiation. Eight-week-old, C57BL/6J mice were fed a low-fat diet (LFD; 10% kcal from fat) or a high-fat diet (HFD; 60% kcal from fat) and were followed longitudinally for up to 13 wk to measure glycemic control and ß-cell mass, death, and dedifferentiation. Compared with LFD controls, ß-cell mass increased during the feeding period in HFD animals, and statistically greater ß-cell death (unmethylated Ins2) was detectable at 2 and 6 wk after diet initiation. Those times correspond to periods when significant step increases in fasting glucose and glucose intolerance, respectively, were detected. ALDH1A3 and Foxo1 immunostaining of the pancreas revealed evidence of ß-cell dedifferentiation by 13 wk when fed an HFD, but not in LFD controls. In conclusion, early episodic ß-cell death may be a feature of cellular turnover correlated with changes in glycemia during ß-cell mass accrual in obesity, whereas ß-cell dedifferentiation may be a feature seen later in established disease.-Tersey, S. A., Levasseur, E. M., Syed, F., Farb, T. B., Orr, K. S., Nelson, J. B., Shaw, J. L., Bokvist, K., Mather, K. J., Mirmira, R. G. Episodic ß-cell death and dedifferentiation during diet-induced obesity and dysglycemia in male mice.

Peroxisome Proliferator-activated Receptor-γ Activation Augments the ß-Cell Unfolded Protein Response and Rescues Early Glycemic Deterioration and ß Cell Death in Non-obese Diabetic Mice.

Type 1 diabetes is an autoimmune disorder that is characterized by a failure of the unfolded protein response in islet ß cells with subsequent endoplasmic reticulum stress and cellular death. Thiazolidinediones are insulin sensitizers that activate the nuclear receptor PPAR-γ and have been shown to partially ameliorate autoimmune type 1 diabetes in humans and non-obese diabetic (NOD) mice. We hypothesized that thiazolidinediones reduce ß cell stress and death independently of insulin sensitivity. To test this hypothesis, female NOD mice were administered pioglitazone during the pre-diabetic phase and assessed for insulin sensitivity and ß cell function relative to controls. Pioglitazone-treated mice showed identical weight gain, body fat distribution, and insulin sensitivity compared with controls. However, treated mice showed significantly improved glucose tolerance with enhanced serum insulin levels, reduced ß cell death, and increased ß cell mass. The effect of pioglitazone was independent of actions on T cells, as pancreatic lymph node T cell populations were unaltered and T cell proliferation was unaffected by pioglitazone. Isolated islets of treated mice showed a more robust unfolded protein response, with increases in Bip and ATF4 and reductions in spliced Xbp1 mRNA. The effect of pioglitazone appears to be a direct action on ß cells, as islets from mice treated with pioglitazone showed reductions in PPAR-γ (Ser-273) phosphorylation. Our results demonstrate that PPAR-γ activation directly improves ß cell function and survival in NOD mice by enhancing the unfolded protein response and suggest that blockade of PPAR-γ (Ser-273) phosphorylation may prevent type 1 diabetes.

Discordant Effects of Polyamine Depletion by DENSpm and DFMO on ß-cell Cytokine Stress and Diabetes Outcomes in Mice.

Type 1 diabetes (T1D) is an autoimmune disease leading to dysfunction and loss of insulin-secreting ß cells. In ß cells, polyamines have been implicated in causing cellular stress and dysfunction. An inhibitor of polyamine biosynthesis, difluoromethylornithine (DFMO), has been shown to delay T1D in mouse models and preserve ß-cell function in humans with recent-onset T1D. Another small molecule, N1,N11-diethylnorspermine (DENSpm), both inhibits polyamine biosynthesis and accelerates polyamine metabolism and is being tested for efficacy in cancer clinical trials. In this study, we show that DENSpm depletes intracellular polyamines as effectively as DFMO in mouse ß cells. RNA-sequencing analysis, however, suggests that the cellular responses to DENSpm and DFMO differ, with both showing effects on cellular proliferation but the latter showing additional effects on mRNA translation and protein-folding pathways. In the low-dose streptozotocin-induced mouse model of T1D, DENSpm, unlike DFMO, did not prevent or delay diabetes outcomes but did result in improvements in glucose tolerance and reductions in islet oxidative stress. In nonobese diabetic (NOD) mice, short-term DENSpm administration resulted in a slight reduction in insulitis and proinflammatory Th1 cells in the pancreatic lymph nodes. Longer term treatment resulted in a dose-dependent increase in mortality. Notwithstanding the efficacy of both DFMO and DENSpm in reducing potentially toxic polyamine levels in ß cells, our results highlight the discordant T1D outcomes that result from differing mechanisms of polyamine depletion and, more importantly, that toxic effects of DENSpm may limit its utility in T1D treatment.

12-Lipoxygenase inhibition suppresses islet immune and inflammatory responses and delays autoimmune diabetes in human gene replacement mice.

Type 1 diabetes (T1D) is characterized by the autoimmune destruction of insulin-producing ß cells and involves an interplay between ß cells and cells of the innate and adaptive immune systems. We investigated the therapeutic potential of targeting 12-lipoxygenase (12-LOX), an enzyme implicated in inflammatory pathways in ß cells and macrophages, using a mouse model in which the endogenous mouse Alox15 gene is replaced by the human ALOX12 gene. Our findings demonstrate that VLX-1005, a potent 12-LOX inhibitor, effectively delays the onset of autoimmune diabetes in human gene replacement non-obese diabetic mice. By spatial proteomics analysis, VLX-1005 treatment resulted in marked reductions in infiltrating T and B cells and macrophages with accompanying increases in immune checkpoint molecule PD-L1, suggesting a shift towards an immune-suppressive microenvironment. RNA sequencing analysis of isolated islets and polarized proinflammatory macrophages revealed significant alteration of cytokine-responsive pathways and a reduction in interferon response after VLX-1005 treatment. Our studies demonstrate that the ALOX12 human replacement gene mouse provides a platform for the preclinical evaluation of LOX inhibitors and supports VLX-1005 as an inhibitor of human 12-LOX that engages the enzymatic target and alters the inflammatory phenotypes of islets and macrophages to promote the delay of autoimmune diabetes.

Inhibition of the Eukaryotic Initiation Factor-2-α Kinase PERK Decreases Risk of Autoimmune Diabetes in Mice.

Preventing the onset of autoimmune type 1 diabetes (T1D) is feasible through pharmacological interventions that target molecular stress-responsive mechanisms. Cellular stresses, such as nutrient deficiency, viral infection, or unfolded proteins, trigger the integrated stress response (ISR), which curtails protein synthesis by phosphorylating eIF2α. In T1D, maladaptive unfolded protein response (UPR) in insulin-producing ß cells renders these cells susceptible to autoimmunity. We show that inhibition of the eIF2α kinase PERK, a common component of the UPR and ISR, reverses the mRNA translation block in stressed human islets and delays the onset of diabetes, reduces islet inflammation, and preserves ß cell mass in T1D-susceptible mice. Single-cell RNA sequencing of islets from PERK-inhibited mice shows reductions in the UPR and PERK signaling pathways and alterations in antigen processing and presentation pathways in ß cells. Spatial proteomics of islets from these mice shows an increase in the immune checkpoint protein PD-L1 in ß cells. Golgi membrane protein 1, whose levels increase following PERK inhibition in human islets and EndoC-ßH1 human ß cells, interacts with and stabilizes PD-L1. Collectively, our studies show that PERK activity enhances ß cell immunogenicity, and inhibition of PERK may offer a strategy to prevent or delay the development of T1D.

Inhibition of the eukaryotic initiation factor-2α kinase PERK decreases risk of autoimmune diabetes in mice.

Preventing the onset of autoimmune type 1 diabetes (T1D) is feasible through pharmacological interventions that target molecular stress-responsive mechanisms. Cellular stresses, such as nutrient deficiency, viral infection, or unfolded proteins, trigger the integrated stress response (ISR), which curtails protein synthesis by phosphorylating eukaryotic translation initiation factor-2α (eIF2α). In T1D, maladaptive unfolded protein response (UPR) in insulin-producing ß cells renders these cells susceptible to autoimmunity. We found that inhibition of the eIF2α kinase PKR-like ER kinase (PERK), a common component of the UPR and ISR, reversed the mRNA translation block in stressed human islets and delayed the onset of diabetes, reduced islet inflammation, and preserved ß cell mass in T1D-susceptible mice. Single-cell RNA-Seq of islets from PERK-inhibited mice showed reductions in the UPR and PERK signaling pathways and alterations in antigen-processing and presentation pathways in ß cells. Spatial proteomics of islets from these mice showed an increase in the immune checkpoint protein programmed death-ligand 1 (PD-L1) in ß cells. Golgi membrane protein 1, whose levels increased following PERK inhibition in human islets and EndoC-ßH1 human ß cells, interacted with and stabilized PD-L1. Collectively, our studies show that PERK activity enhances ß cell immunogenicity and that inhibition of PERK may offer a strategy for preventing or delaying the development of T1D.

Identification of a naturally-occurring canine model for early detection and intervention research in high grade urothelial carcinoma.

Background: Early detection and intervention research is expected to improve the outcomes for patients with high grade muscle invasive urothelial carcinoma (InvUC). With limited patients in suitable high-risk study cohorts, relevant animal model research is critical. Experimental animal models often fail to adequately represent human cancer. The purpose of this study was to determine the suitability of dogs with high breed-associated risk for naturally-occurring InvUC to serve as relevant models for early detection and intervention research. The feasibility of screening and early intervention, and similarities and differences between canine and human tumors, and early and later canine tumors were determined. Methods: STs (n=120) ≥ 6 years old with no outward evidence of urinary disease were screened at 6-month intervals for 3 years with physical exam, ultrasonography, and urinalysis with sediment exam. Cystoscopic biopsy was performed in dogs with positive screening tests. The pathological, clinical, and molecular characteristics of the "early" cancer detected by screening were determined. Transcriptomic signatures were compared between the early tumors and published findings in human InvUC, and to more advanced "later" canine tumors from STs who had the typical presentation of hematuria and urinary dysfunction. An early intervention trial of an oral cyclooxygenase inhibitor, deracoxib, was conducted in dogs with cancer detected through screening. Results: Biopsy-confirmed bladder cancer was detected in 32 (27%) of 120 STs including InvUC (n=29, three starting as dysplasia), grade 1 noninvasive cancer (n=2), and carcinoma in situ (n=1). Transcriptomic signatures including druggable targets such as EGFR and the PI3K-AKT-mTOR pathway, were very similar between canine and human InvUC, especially within luminal and basal molecular subtypes. Marked transcriptomic differences were noted between early and later canine tumors, particularly within luminal subtype tumors. The deracoxib remission rate (42% CR+PR) compared very favorably to that with single-agent cyclooxygenase inhibitors in more advanced canine InvUC (17-25%), supporting the value of early intervention. Conclusions: The study defined a novel naturally-occurring animal model to complement experimental models for early detection and intervention research in InvUC. Research incorporating the canine model is expected to lead to improved outcomes for humans, as well as pet dogs, facing bladder cancer.

Circulating unmethylated CHTOP and INS DNA fragments provide evidence of possible islet cell death in youth with obesity and diabetes.

BACKGROUND: Identification of islet ß cell death prior to the onset of type 1 diabetes (T1D) or type 2 diabetes (T2D) might allow for interventions to protect ß cells and reduce diabetes risk. Circulating unmethylated DNA fragments arising from the human INS gene have been proposed as biomarkers of ß cell death, but this gene alone may not be sufficiently specific to report ß cell death. RESULTS: To identify new candidate genes whose CpG sites may show greater specificity for ß cells, we performed unbiased DNA methylation analysis using the Infinium HumanMethylation 450 array on 64 human islet preparations and 27 non-islet human tissues. For verification of array results, bisulfite DNA sequencing of human ß cells and 11 non-ß cell tissues was performed on 5 of the top 10 CpG sites that were found to be differentially methylated. We identified the CHTOP gene as a candidate whose CpGs show a greater frequency of unmethylation in human islets. A digital PCR strategy was used to determine the methylation pattern of CHTOP and INS CpG sites in primary human tissues. Although both INS and CHTOP contained unmethylated CpG sites in non-islet tissues, they occurred in a non-overlapping pattern. Based on Naïve Bayes classifier analysis, the two genes together report 100% specificity for islet damage. Digital PCR was then performed on cell-free DNA from serum from human subjects. Compared to healthy controls (N = 10), differentially methylated CHTOP and INS levels were higher in youth with new onset T1D (N = 43) and, unexpectedly, in healthy autoantibody-negative youth who have first-degree relatives with T1D (N = 23). When tested in lean (N = 32) and obese (N = 118) youth, increased levels of unmethylated INS and CHTOP were observed in obese individuals. CONCLUSION: Our data suggest that concurrent measurement of circulating unmethylated INS and CHTOP has the potential to detect islet death in youth at risk for both T1D and T2D. Our data also support the use of multiple parameters to increase the confidence of detecting islet damage in individuals at risk for developing diabetes.

Hypoglycemia in a Patient With a Polyhormonal Pancreatic Neuroendocrine Tumor With Evidence of Endocrine Progenitors.

A 55-year-old woman with a large polyhormonal neuroendocrine tumor with unusual pathology is described. The patient presented with intermittent neuroglycopenic symptoms between more protracted asymptomatic periods occurring over the preceding 4 years. During a diagnostic 72-hour inpatient fast, she exhibited hypoglycemia at 70 hours after initiation. On computed tomography scan, a 6-cm mass was identified at the pancreatic head. The patient underwent a pylorus-preserving pancreaticoduodenectomy, and pathology was positive for cells staining for pancreatic polypeptide, insulin, and occasional double hormone (insulin plus pancreatic polypeptide)-positive cells. In addition, the tumor exhibited broad staining for ALDH1A3, a new marker of endocrine progenitors. This case serves to highlight the clinical and pathologic variability of insulin-producing tumors and raises the potential for cells in these tumors to exhibit hormone interconversion and progenitor-like states.

Inhibition of 12/15-Lipoxygenase Protects Against ß-Cell Oxidative Stress and Glycemic Deterioration in Mouse Models of Type 1 Diabetes.

Islet ß-cell dysfunction and aggressive macrophage activity are early features in the pathogenesis of type 1 diabetes (T1D). 12/15-Lipoxygenase (12/15-LOX) is induced in ß-cells and macrophages during T1D and produces proinflammatory lipids and lipid peroxides that exacerbate ß-cell dysfunction and macrophage activity. Inhibition of 12/15-LOX provides a potential therapeutic approach to prevent glycemic deterioration in T1D. Two inhibitors recently identified by our groups through screening efforts, ML127 and ML351, have been shown to selectively target 12/15-LOX with high potency. Only ML351 exhibited no apparent toxicity across a range of concentrations in mouse islets, and molecular modeling has suggested reduced promiscuity of ML351 compared with ML127. In mouse islets, incubation with ML351 improved glucose-stimulated insulin secretion in the presence of proinflammatory cytokines and triggered gene expression pathways responsive to oxidative stress and cell death. Consistent with a role for 12/15-LOX in promoting oxidative stress, its chemical inhibition reduced production of reactive oxygen species in both mouse and human islets in vitro. In a streptozotocin-induced model of T1D in mice, ML351 prevented the development of diabetes, with coincident enhancement of nuclear Nrf2 in islet cells, reduced ß-cell oxidative stress, and preservation of ß-cell mass. In the nonobese diabetic mouse model of T1D, administration of ML351 during the prediabetic phase prevented dysglycemia, reduced ß-cell oxidative stress, and increased the proportion of anti-inflammatory macrophages in insulitis. The data provide the first evidence to date that small molecules that target 12/15-LOX can prevent progression of ß-cell dysfunction and glycemic deterioration in models of T1D.

Measurement of Differentially Methylated INS DNA Species in Human Serum Samples as a Biomarker of Islet ß Cell Death.

The death of islet ß cells is thought to underlie the pathogenesis of virtually all forms of diabetes and to precede the development of frank hyperglycemia, especially in type 1 diabetes. The development of sensitive and reliable biomarkers of ß cell death may allow for early therapeutic intervention to prevent or delay the development of diabetes. Recently, several groups including our own have reported that cell-free, differentially methylated DNA encoding preproinsulin (INS) in the circulation is correlated to ß cell death in pre-type 1 diabetes and new-onset type 1 diabetes. Here, we present a step-by-step protocol using digital PCR for the measurement of cell-free INS DNA that is differentially methylated at cytosine at position -69 bp (relative to the transcriptional start site). We demonstrate that the assay can distinguish between methylated and unmethylated cytosine at position -69 bp, is linear across several orders of magnitude, provides absolute quantitation of DNA copy numbers, and can be applied to samples of human serum from individuals with new-onset type 1 diabetes and disease-free controls. The protocol described here can be adapted to any DNA species for which detection of differentially methylated cytosines is desired, whether from circulation or from isolated cells and tissues, and can provide absolute quantitation of DNA fragments.