Diabetes treatment by conversion of gut epithelial cells to insulin-producing cells.

Insulin-deficient (type 1) diabetes is treated by providing insulin to maintain euglycemia. The current standard of care is a quasi-closed loop integrating automated insulin delivery with a continuous glucose monitoring sensor. Cell replacement technologies are advancing as an alternative treatment and have been tested as surrogates to cadaveric islets in transplants. In addition, immunomodulatory treatments to delay the onset of type 1 diabetes in high-risk (stage 2) individuals have gained regulatory approval. We have pioneered a cell conversion approach to restore insulin production through pharmacological conversion of intestinal epithelial cells into insulin-producing cells. We have advanced this approach along a translational trajectory through the discovery of small molecule forkhead box protein O1 inhibitors. When administered to different rodent models of insulin-deficient diabetes, these inhibitors have resulted in robust glucose-lowering responses and generation of insulin-producing cells in the gut epithelium. We review past work and delineate a path to human clinical trials.

Cyb5r3 activation rescues secondary failure to sulfonylurea but not ß-cell dedifferentiation.

Diabetes mellitus is characterized by insulin resistance and ß-cell failure. The latter involves impaired insulin secretion and ß-cell dedifferentiation. Sulfonylurea (SU) is used to improve insulin secretion in diabetes, but it suffers from secondary failure. The relationship between SU secondary failure and ß-cell dedifferentiation has not been examined. Using a model of SU secondary failure, we have previously shown that functional loss of oxidoreductase Cyb5r3 mediates effects of SU failure through interactions with glucokinase. Here we demonstrate that SU failure is associated with partial ß-cell dedifferentiation. Cyb5r3 knockout mice show more pronounced ß-cell dedifferentiation and glucose intolerance after chronic SU administration, high-fat diet feeding, and during aging. A Cyb5r3 activator improves impaired insulin secretion caused by chronic SU treatment, but not ß-cell dedifferentiation. We conclude that chronic SU administration affects progression of ß-cell dedifferentiation and that Cyb5r3 activation reverses secondary failure to SU without restoring ß-cell dedifferentiation.

Calorie Restriction activates a gastric Notch-FOXO1 pathway to expand Ghrelin cells.

Calorie restriction increases lifespan. While some tissue-specific protective effects of calorie restriction have been described, the impact of calorie restriction on the gastrointestinal tract remains unclear. We found increased abundance of chromogranin A+, including orexigenic ghrelin+, endocrine cells in the stomach of calorie-restricted mice. This effect coincided with increased Notch target Hes1 and Notch ligand Jag1 and was reversed when Notch signaling was blocked using the γ-secretase inhibitor DAPT. Using primary cultures and genetically-modified reporter mice, we determined that increased endocrine cell abundance was due to altered stem and progenitor proliferation. Different from the intestine, calorie restriction decreased gastric Lgr5+ stem cells, while increasing a FOXO1/Neurog3+ subpopulation of endocrine progenitors in a Notch-dependent manner. Further, calorie restriction triggered nuclear localization of FOXO1, which was sufficient to promote endocrine cell differentiation. Taken together, the data indicate that calorie restriction promotes gastric endocrine cell differentiation triggered by active Notch signaling and regulated by FOXO1.

Chemical induction of gut ß-like-cells by combined FoxO1/Notch inhibition as a glucose-lowering treatment for diabetes.

OBJECTIVE: Lifelong insulin replacement remains the mainstay of type 1 diabetes treatment. Genetic FoxO1 ablation promotes enteroendocrine cell (EECs) conversion into glucose-responsive ß-like cells. Here, we tested whether chemical FoxO1 inhibitors can generate ß-like gut cells. METHODS: We used Ngn3-or Villin-driven FoxO1 ablation to capture the distinctive developmental effects of FoxO1 on EEC pool. We combined FoxO1 ablation with Notch inhibition to enhance the expansion of EEC pool. We tested the ability of an orally available small molecule of FoxO1 inhibitor, Cpd10, to phenocopy genetic ablation of FoxO1. We evaluated the therapeutic impact of genetic ablation or chemical inhibition of FoxO1 on insulin-deficient diabetes in Ins2Akita/+ mice. RESULTS: Pan-intestinal epithelial FoxO1 ablation expanded the EEC pool, induced ß-like cells, and improved glucose tolerance in Ins2Akita/+ mice. This genetic effect was phenocopied by Cpd10. Cpd10 induced ß-like cells that released insulin in response to glucose in gut organoids, and this effect was enhanced by the Notch inhibitor, DBZ. In Ins2Akita/+ mice, a five-day course of either Cpd10 or DBZ induced intestinal insulin-immunoreactive ß-like cells, lowered glycemia, and increased plasma insulin levels without apparent adverse effects. CONCLUSION: These results provide proof of principle of gut cell conversion into ß-like cells by a small molecule FoxO1 inhibitor, paving the way for clinical applications.

Pharmacological conversion of gut epithelial cells into insulin-producing cells lowers glycemia in diabetic animals.

As a highly regenerative organ, the intestine is a promising source for cellular reprogramming for replacing lost pancreatic ß cells in diabetes. Gut enterochromaffin cells can be converted to insulin-producing cells by forkhead box O1 (FoxO1) ablation, but their numbers are limited. In this study, we report that insulin-immunoreactive cells with Paneth/goblet cell features are present in human fetal intestine. Accordingly, lineage-tracing experiments show that, upon genetic or pharmacologic FoxO1 ablation, the Paneth/goblet lineage can also undergo conversion to the insulin lineage. We designed a screening platform in gut organoids to accurately quantitate ß-like cell reprogramming and fine-tune a combination treatment to increase the efficiency of the conversion process in mice and human adult intestinal organoids. We identified a triple blockade of FOXO1, Notch, and TGF-ß that, when tested in insulin-deficient streptozotocin (STZ) or NOD diabetic animals, resulted in near normalization of glucose levels, associated with the generation of intestinal insulin-producing cells. The findings illustrate a therapeutic approach for replacing insulin treatment in diabetes.

FOXO1 Is Present in Stomach Epithelium and Determines Gastric Cell Distribution.

BACKGROUND AND AIMS: Stomach cells can be converted to insulin-producing cells by Neurog3, MafA, and Pdxl over-expression. Enteroendocrine cells can be similarly made to produce insulin by the deletion of FOXO1. Characteristics and functional properties of FOXO1-expressing stomach cells are not known. METHODS: Using mice bearing a FOXO1-GFP knock-in allele and primary cell cultures, we examined the identity of FOXO1-expressing stomach cells and analyzed their features through loss-of-function studies with red-to-green fluorescent reporters. RESULTS: FOXO1 localizes to a subset of Neurog3 and parietal cells. FOXO1 deletion ex vivo or in vivo using Neurog3-cre or Atp4b-cre increased numbers of parietal cells, generated insulin- and C-peptide-immunoreactive cells, and raised Neurog3 messenger RNA. Gene expression and ChIP- seq experiments identified the cell cycle regulator cyclin E1 (CCNE1) as a FOXO1 target. CONCLUSION: FOXO1 is expressed in a subset of stomach cells. Its ablation increases parietal cells and yields insulin-immunoreactive cells, consistent with a role in lineage determination.

Inducing and measuring apoptotic cell death in mouse pancreatic ß-cells and in isolated islets.

Type 2 diabetes is mediated by insulin resistance and pancreatic ß-cell failure, the latter reflecting a combination of ß-cell dysfunction, dedifferentiation, and apoptosis. Quantification of ß-cell apoptosis in diabetes can be challenging both with respect to methodology and selection of clinically relevant inducers and readouts. This protocol describes approaches to measure cell death in immortalized ß-cells, primary mouse islet preparations, and pancreatic tissue. The resulting information may be useful for mechanistic studies and assessment of the contribution of ß-cell death to pathogenesis. For complete details on the use and execution of this protocol, please refer to McKimpson et al. (2021).

Conversion of the death inhibitor ARC to a killer activates pancreatic ß cell death in diabetes.

Loss of insulin-secreting pancreatic ß cells through apoptosis contributes to the progression of type 2 diabetes, but underlying mechanisms remain elusive. Here, we identify a pathway in which the cell death inhibitor ARC paradoxically becomes a killer during diabetes. While cytoplasmic ARC maintains ß cell viability and pancreatic architecture, a pool of ARC relocates to the nucleus to induce ß cell apoptosis in humans with diabetes and several pathophysiologically distinct mouse models. ß cell death results through the coordinate downregulation of serpins (serine protease inhibitors) not previously known to be synthesized and secreted by ß cells. Loss of the serpin α1-antitrypsin from the extracellular space unleashes elastase, triggering the disruption of ß cell anchorage and subsequent cell death. Administration of α1-antitrypsin to mice with diabetes prevents ß cell death and metabolic abnormalities. These data uncover a pathway for ß cell loss in type 2 diabetes and identify an FDA-approved drug that may impede progression of this syndrome.

Reprogramming Cells to Make Insulin.

Type 1 diabetes is a disease characterized by the destruction of insulin-secreting ß-cells in the pancreas. Individuals are treated for this disease with lifelong insulin replacement. However, one attractive treatment possibility is to reprogram an individual's endogenous cells to acquire the ability to secrete insulin, essentially replacing destroyed ß-cells. Herein, we review the literature on the topic of reprogramming endodermal cells to produce insulin.

A fluorescent reporter assay of differential gene expression response to insulin in hepatocytes.

Insulin regulates multiple hepatic metabolic pathways in a seemingly heterogeneous manner. To understand this heterogeneity, we hypothesized that different subpopulations of hepatocytes have different sensitivity to insulin. To test this hypothesis, we developed a fluorescent reporter in which the insulin-responsive fatty acid synthase (FAS) promoter drove expression of a time-dependent fluorescent protein ("timer") and characterized timer expression in flow-sorted cell populations. In Hepa1c1c7 and AML12 hepatocytes, we found that different cell populations express distinct timer fluorescence following insulin treatment, consistent with cellular heterogeneity in the response to insulin. RNA measurements indicated an enrichment of forkhead box O transcription factors in cells with a greater response to insulin. Moreover, we found evidence of increased Akt activation. These data are consistent with a heterogeneous cellular response to insulin and raise the possibility that these different subpopulations underlie the peculiar pathophysiology of hepatic insulin resistance.

An Akt3 Splice Variant Lacking the Serine 472 Phosphorylation Site Promotes Apoptosis and Suppresses Mammary Tumorigenesis.

The Akt pathway is a well-known promoter of tumor malignancy. Akt3 is expressed as two alternatively spliced variants, one of which lacks the key regulatory serine 472 phosphorylation site. Whereas the function of full-length Akt3 isoform (Akt3/+S472) is well-characterized, that of Akt3/-S472 isoform remains unknown. Despite being expressed at a substantially lower level than Akt3/+S472 in triple-negative breast cancer cells, specific ablation of Akt3/-S472 enhanced, whereas overexpression, suppressed mammary tumor growth, consistent with a significant association with patient survival duration relative to Akt3/+S472. These effects were due to striking induction of apoptosis, which was mediated by Bim upregulation, leading to conformational activation of Bax and caspase-3 processing. Bim accumulation was caused by marked endocytosis of EGF receptors with concomitant ERK attenuation, which stabilizes BIM. These findings demonstrate an unexpected function of an endogenously expressed Akt isoform in promoting, as opposed to suppressing, apoptosis, underscoring that Akt isoforms may exert dissonant functions in malignancy.Significance: These results illuminate an unexpected function for an endogenously expressed Akt isoform in promoting apoptosis, underscoring the likelihood that different Akt isoforms exert distinct functions in human cancer. Cancer Res; 78(1); 103-14. ©2017 AACR.

ARC is essential for maintaining pancreatic islet structure and ß-cell viability during type 2 diabetes.

Pancreatic ß-cell loss through apoptosis is an important disease mechanism in type 2 diabetes. Apoptosis Repressor with CARD (ARC) is a cell death inhibitor that antagonizes multiple death programs. We previously reported that ARC is abundant in pancreatic ß-cells and modulates survival of these cells in vitro. Herein we assessed the importance of endogenous ARC in maintaining islet structure and function in vivo. While generalized loss of ARC did not result in detectable abnormalities, its absence in ob/ob mice, a model of type 2 diabetes, induced a striking pancreatic phenotype: marked ß-cell death, loss of ß-cell mass, derangements of islet architecture, and impaired glucose-stimulated insulin secretion in vivo. These abnormalities contributed to worsening of hyperglycemia and glucose-intolerance in these mice. Mechanistically, the absence of ARC increased levels of C/EBP homologous protein (CHOP) in wild type isolated islets stimulated with ER stress and in ob/ob isolated islets at baseline. Deletion of CHOP in ob/ob; ARC -/- mice led to reversal of ß-cell death and abnormalities in islet architecture. These data indicate that suppression of CHOP by endogenous levels of ARC is critical for ß-cell viability and maintenance of normal islet structure in this model of type 2 diabetes.

A myeloid tumor suppressor role for NOL3.

Despite the identification of several oncogenic driver mutations leading to constitutive JAK-STAT activation, the cellular and molecular biology of myeloproliferative neoplasms (MPN) remains incompletely understood. Recent discoveries have identified underlying disease-modifying molecular aberrations contributing to disease initiation and progression. Here, we report that deletion of Nol3 (Nucleolar protein 3) in mice leads to an MPN resembling primary myelofibrosis (PMF). Nol3-/- MPN mice harbor an expanded Thy1+LSK stem cell population exhibiting increased cell cycling and a myelomonocytic differentiation bias. Molecularly, this phenotype is mediated by Nol3-/--induced JAK-STAT activation and downstream activation of cyclin-dependent kinase 6 (Cdk6) and MycNol3-/- MPN Thy1+LSK cells share significant molecular similarities with primary CD34+ cells from PMF patients. NOL3 levels are decreased in CD34+ cells from PMF patients, and the NOL3 locus is deleted in a subset of patients with myeloid malignancies. Our results reveal a novel genetic PMF-like mouse model and identify a tumor suppressor role for NOL3 in the pathogenesis of myeloid malignancies.

The Cell Death Inhibitor ARC Is Induced in a Tissue-Specific Manner by Deletion of the Tumor Suppressor Gene Men1, but Not Required for Tumor Development and Growth.

Multiple endocrine neoplasia type 1 (MEN1) is a genetic disorder characterized by tissue-specific tumors in the endocrine pancreas, parathyroid, and pituitary glands. Although tumor development in these tissues is dependent upon genetic inactivation of the tumor suppressor Men1, loss of both alleles of this gene is not sufficient to induce these cancers. Men1 encodes menin, a nuclear protein that influences transcription. A previous ChIP on chip analysis suggested that menin binds promoter sequences of nol3, encoding ARC, which is a cell death inhibitor that has been implicated in cancer pathogenesis. We hypothesized that ARC functions as a co-factor with Men1 loss to induce the tissue-restricted distribution of tumors seen in MEN1. Using mouse models that recapitulate this syndrome, we found that biallelic deletion of Men1 results in selective induction of ARC expression in tissues that develop tumors. Specifically, loss of Men1 in all cells of the pancreas resulted in marked increases in ARC mRNA and protein in the endocrine, but not exocrine, pancreas. Similarly, ARC expression increased in the parathyroid with inactivation of Men1 in that tissue. To test if ARC contributes to MEN1 tumor development in the endocrine pancreas, we generated mice that lacked none, one, or both copies of ARC in the context of Men1 deletion. Studies in a cohort of 126 mice demonstrated that, although mice lacking Men1 developed insulinomas as expected, elimination of ARC in this context did not significantly alter tumor load. Cellular rates of proliferation and death in these tumors were also not perturbed in the absence of ARC. These results indicate that ARC is upregulated by loss Men1 in the tissue-restricted distribution of MEN1 tumors, but that ARC is not required for tumor development in this syndrome.

MacroH2A1 and ATM Play Opposing Roles in Paracrine Senescence and the Senescence-Associated Secretory Phenotype.

Oncogene-induced senescence (OIS) is a tumor-suppressive mechanism typified by stable proliferative arrest, a persistent DNA damage response, and the senescence-associated secretory phenotype (SASP), which helps to maintain the senescent state and triggers bystander senescence in a paracrine fashion. Here, we demonstrate that the tumor suppressive histone variant macroH2A1 is a critical component of the positive feedback loop that maintains SASP gene expression and triggers the induction of paracrine senescence. MacroH2A1 undergoes dramatic genome-wide relocalization during OIS, including its removal from SASP gene chromatin. The removal of macroH2A1 from SASP genes results from a negative feedback loop activated by SASP-mediated endoplasmic reticulum (ER) stress. ER stress leads to increased reactive oxygen species and persistent DNA damage response including activation of ATM, which mediates removal macroH2A1 from SASP genes. Together, our findings indicate that macroH2A1 is a critical control point for the regulation of SASP gene expression during senescence.

The apoptosis inhibitor ARC alleviates the ER stress response to promote ß-cell survival.

Type 2 diabetes involves insulin resistance and ß-cell failure leading to inadequate insulin secretion. An important component of ß-cell failure is cell loss by apoptosis. Apoptosis repressor with caspase recruitment domain (ARC) is an inhibitor of apoptosis that is expressed in cardiac and skeletal myocytes and neurons. ARC possesses the unusual property of antagonizing both the extrinsic (death receptor) and intrinsic (mitochondria/endoplasmic reticulum [ER]) cell death pathways. Here we report that ARC protein is abundant in cells of the endocrine pancreas, including >99.5% of mouse and 73% of human ß-cells. Using genetic gain- and loss-of-function approaches, our data demonstrate that ARC inhibits ß-cell apoptosis elicited by multiple inducers of cell death, including ER stressors tunicamycin, thapsigargin, and physiological concentrations of palmitate. Unexpectedly, ARC diminishes the ER stress response, acting distal to protein kinase RNA-like ER kinase (PERK) and inositol-requiring protein 1α, to suppress C/EBP homologous protein (CHOP) induction. Depletion of ARC in isolated islets augments palmitate-induced apoptosis, which is dramatically rescued by deletion of CHOP. These data demonstrate that ARC is a previously unrecognized inhibitor of apoptosis in ß-cells and that its protective effects are mediated through suppression of the ER stress response pathway.