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.

ß-cell Jagged1 is sufficient but not necessary for islet Notch activity and insulin secretory defects in obese mice.

OBJECTIVE: Notch signaling, re-activated in ß cells from obese mice and causal to ß cell dysfunction, is determined in part by transmembrane ligand availability in a neighboring cell. We hypothesized that ß cell expression of Jagged1 determines the maladaptive Notch response and resultant insulin secretory defects in obese mice. METHODS: We assessed expression of Notch pathway components in high-fat diet-fed (HFD) or leptin receptor-deficient (db/db) mice, and performed single-cell RNA sequencing (scRNA-Seq) in islets from patients with and without type 2 diabetes (T2D). We generated and performed glucose tolerance testing in inducible, ß cell-specific Jagged1 gain-of- and loss-of-function mice. We also tested effects of monoclonal neutralizing antibodies to Jagged1 in glucose-stimulated insulin secretion (GSIS) assays in isolated islets. RESULTS: Jag1 was the only Notch ligand that tracked with increased Notch activity in HFD-fed and db/db mice, as well as in metabolically-inflexible ß cells enriched in patients with T2D. Neutralizing antibodies to block Jagged1 in islets isolated from HFD-fed and db/db mice potentiated GSIS ex vivo. To demonstrate if ß cell Jagged1 is sufficient to cause glucose tolerance in vivo, we generated inducible ß cell-specific Jag1 transgenic (ß-Jag1TG) and loss-of-function (iß-Jag1KO) mice. While forced Jagged1 impaired glucose intolerance due to reduced GSIS, loss of ß cell Jagged1 did not protect against HFD-induced insulin secretory defects. CONCLUSIONS: Jagged1 is increased in islets from obese mice and in patients with T2D, and neutralizing Jagged1 antibodies lead to improved GSIS, suggesting that inhibition of Jagged1-Notch signaling may have therapeutic benefit. However, genetic loss-of-function experiments suggest that ß cells are not a likely source of the Jagged1 signal.

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.

MAD2-Dependent Insulin Receptor Endocytosis Regulates Metabolic Homeostasis.

Insulin activates insulin receptor (IR) signaling and subsequently triggers IR endocytosis to attenuate signaling. Cell division regulators MAD2, BUBR1, and p31comet promote IR endocytosis on insulin stimulation. Here, we show that genetic ablation of the IR-MAD2 interaction in mice delays IR endocytosis, increases IR levels, and prolongs insulin action at the cell surface. This in turn causes a defect in insulin clearance and increases circulating insulin levels, unexpectedly increasing glucagon levels, which alters glucose metabolism modestly. Disruption of the IR-MAD2 interaction increases serum fatty acid concentrations and hepatic fat accumulation in fasted male mice. Furthermore, disruption of the IR-MAD2 interaction distinctly changes metabolic and transcriptomic profiles in the liver and adipose tissues. Our findings establish the function of cell division regulators in insulin signaling and provide insights into the metabolic functions of IR endocytosis. ARTICLE HIGHLIGHTS: The physiological role of IR endocytosis in insulin sensitivity remains unclear. Disruption of the IR-MAD2 interaction delays IR endocytosis and prolongs insulin signaling. IR-MAD2 controls insulin clearance and glucose metabolism. IR-MAD2 maintains energy homeostasis.

Reversing pancreatic ß-cell dedifferentiation in the treatment of type 2 diabetes.

The maintenance of glucose homeostasis is fundamental for survival and health. Diabetes develops when glucose homeostasis fails. Type 2 diabetes (T2D) is characterized by insulin resistance and pancreatic ß-cell failure. The failure of ß-cells to compensate for insulin resistance results in hyperglycemia, which in turn drives altered lipid metabolism and ß-cell failure. Thus, insulin secretion by pancreatic ß-cells is a primary component of glucose homeostasis. Impaired ß-cell function and reduced ß-cell mass are found in diabetes. Both features stem from a failure to maintain ß-cell identity, which causes ß-cells to dedifferentiate into nonfunctional endocrine progenitor-like cells or to trans-differentiate into other endocrine cell types. In this regard, one of the key issues in achieving disease modification is how to reestablish ß-cell identity. In this review, we focus on the causes and implications of ß-cell failure, as well as its potential reversibility as a T2D treatment.

Unraveling the mysteries of hepatic insulin signaling: deconvoluting the nuclear targets of insulin.

Over 100 years have passed since insulin was first administered to a diabetic patient. Since then great strides have been made in diabetes research. It has determined where insulin is secreted from, which organs it acts on, how it is transferred into the cell and is delivered to the nucleus, how it orchestrates the expression pattern of the genes, and how it works with each organ to maintain systemic metabolism. Any breakdown in this system leads to diabetes. Thanks to the numerous researchers who have dedicated their lives to cure diabetes, we now know that there are three major organs where insulin acts to maintain glucose/lipid metabolism: the liver, muscles, and fat. The failure of insulin action on these organs, such as insulin resistance, result in hyperglycemia and/or dyslipidemia. The primary trigger of this condition and its association among these tissues still remain to be uncovered. Among the major organs, the liver finely tunes the glucose/lipid metabolism to maintain metabolic flexibility, and plays a crucial role in glucose/lipid abnormality due to insulin resistance. Insulin resistance disrupts this tuning, and selective insulin resistance arises. The glucose metabolism loses its sensitivity to insulin, while the lipid metabolism maintains it. The clarification of its mechanism is warranted to reverse the metabolic abnormalities due to insulin resistance. This review will provide a brief historical review for the progress of the pathophysiology of diabetes since the discovery of insulin, followed by a review of the current research clarifying our understanding of selective insulin resistance.

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.

Cyb5r3-based mechanism and reversal of secondary failure to sulfonylurea in diabetes.

Sulfonylureas (SUs) are effective and affordable antidiabetic drugs. However, chronic use leads to secondary failure, limiting their utilization. Here, we identify cytochrome b5 reductase 3 (Cyb5r3) down-regulation as a mechanism of secondary SU failure and successfully reverse it. Chronic exposure to SU lowered Cyb5r3 abundance and reduced islet glucose utilization in mice in vivo and in ex vivo murine islets. Cyb5r3 ß cell-specific knockout mice phenocopied SU failure. Cyb5r3 engaged in a glucose-dependent interaction that stabilizes glucokinase (Gck) to maintain glucose utilization. Hence, Gck activators can circumvent Cyb5r3-dependent SU failure. A Cyb5r3 activator rescued secondary SU failure in mice in vivo and restored insulin secretion in ex vivo human islets. We conclude that Cyb5r3 is a key factor in the secondary failure to SU and a potential target for its prevention, which might rehabilitate SU use in diabetes.

Genetic and pharmacologic inhibition of ALDH1A3 as a treatment of ß-cell failure.

Type 2 diabetes (T2D) is associated with ß-cell dedifferentiation. Aldehyde dehydrogenase 1 isoform A3 (ALHD1A3) is a marker of ß-cell dedifferentiation and correlates with T2D progression. However, it is unknown whether ALDH1A3 activity contributes to ß-cell failure, and whether the decrease of ALDH1A3-positive ß-cells (A+) following pair-feeding of diabetic animals is due to ß-cell restoration. To tackle these questions, we (i) investigated the fate of A+ cells during pair-feeding by lineage-tracing, (ii) somatically ablated ALDH1A3 in diabetic ß-cells, and (iii) used a novel selective ALDH1A3 inhibitor to treat diabetes. Lineage tracing and functional characterization show that A+ cells can be reconverted to functional, mature ß-cells. Genetic or pharmacological inhibition of ALDH1A3 in diabetic mice lowers glycemia and increases insulin secretion. Characterization of ß-cells following ALDH1A3 inhibition shows reactivation of differentiation as well as regeneration pathways. We conclude that ALDH1A3 inhibition offers a therapeutic strategy against ß-cell dysfunction in diabetes.

Reflections on the state of diabetes research and prospects for treatment.

Research on the etiology and treatment of diabetes has made substantial progress. As a result, several new classes of anti-diabetic drugs have been introduced in clinical practice. Nonetheless, the number of patients achieving glycemic control targets has not increased for the past 20 years. Two areas of unmet medical need are the restoration of insulin sensitivity and the reversal of pancreatic beta cell failure. In this review, we integrate research advances in transcriptional regulation of insulin action and pathophysiology of beta cell dedifferentiation with their potential impact on prospects of a durable "cure" for patients suffering from type 2 diabetes.

PPARγ (Peroxisome Proliferator-Activated Receptor γ) Deacetylation Suppresses Aging-Associated Atherosclerosis and Hypercholesterolemia.

BACKGROUND: Atherosclerosis is a medical urgency manifesting at the onset of hypercholesterolemia and is associated with aging. Activation of PPARγ (peroxisome proliferator-activated receptor γ) counteracts metabolic dysfunction influenced by aging, and its deacetylation displays an atheroprotective property. Despite the marked increase of PPARγ acetylation during aging, it is unknown whether PPARγ acetylation is a pathogenic contributor to aging-associated atherosclerosis. METHODS: Mice with constitutive deacetylation-mimetic PPARγ mutations on lysine residues K268 and K293 (2KR) in an LDL (low-density lipoprotein)-receptor knockout (Ldlr-/-) background (2KR:Ldlr-/-) were aged for 18 months on a standard laboratory diet to examine the cardiometabolic phenotype, which was confirmed in Western-type diet-fed 2KR:Ldlr+/- mice. Whole-liver RNA-sequencing and in vitro studies in bone marrow-derived macrophages were conducted to decipher the mechanism. RESULTS: In contrast to severe atherosclerosis in WT:Ldlr-/- mice, aged 2KR:Ldlr-/- mice developed little to no plaque, which was underlain by a significantly improved plasma lipid profile, with particular reductions in circulating LDL. The protection from hypercholesterolemia was recapitulated in Western-type diet-fed 2KR:Ldlr+/- mice. Liver RNA-sequencing analysis revealed suppression of liver inflammation rather than changes in cholesterol metabolism. This anti-inflammatory effect of 2KR was attributed to polarized M2 activation of macrophages. Additionally, the upregulation of core circadian component Bmal1 (brain and muscle ARNT-like 1), perceived to be involved in anti-inflammatory immunity, was observed in the liver and bone marrow-derived macrophages. CONCLUSIONS: PPARγ deacetylation in mice prevents the development of aging-associated atherosclerosis and hypercholesterolemia, in association with the anti-inflammatory phenotype of 2KR macrophages.

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.

Deletion of skeletal muscle Akt1/2 causes osteosarcopenia and reduces lifespan in mice.

Aging is considered to be accelerated by insulin signaling in lower organisms, but it remained unclear whether this could hold true for mammals. Here we show that mice with skeletal muscle-specific double knockout of Akt1/2, key downstream molecules of insulin signaling, serve as a model of premature sarcopenia with insulin resistance. The knockout mice exhibit a progressive reduction in skeletal muscle mass, impairment of motor function and systemic insulin sensitivity. They also show osteopenia, and reduced lifespan largely due to death from debilitation on normal chow and death from tumor on high-fat diet. These phenotypes are almost reversed by additional knocking out of Foxo1/4, but only partially by additional knocking out of Tsc2 to activate the mTOR pathway. Overall, our data suggest that, unlike in lower organisms, suppression of Akt activity in skeletal muscle of mammals associated with insulin resistance and aging could accelerate osteosarcopenia and consequently reduce lifespan.

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.

Single-agent FOXO1 inhibition normalizes glycemia and induces gut ß-like cells in streptozotocin-diabetic mice.

OBJECTIVES: Insulin treatment remains the sole effective intervention for Type 1 Diabetes. Here, we investigated the therapeutic potential of converting intestinal epithelial cells to insulin-producing, glucose-responsive ß-like cells by targeted inhibition of FOXO1. We have previously shown that this can be achieved by genetic ablation in gut Neurogenin3 progenitors, adenoviral or shRNA-mediated inhibition in human gut organoids, and chemical inhibition in Akita mice, a model of insulin-deficient diabetes. METHODS: We profiled two novel FOXO1 inhibitors in reporter gene assays, and hepatocyte gene expression studies, and in vivo pyruvate tolerance test (PTT) for their activity and specificity. We evaluated their glucose-lowering effect in mice rendered insulin-deficient by administration of streptozotocin. RESULTS: We provide evidence that two novel FOXO1 inhibitors, FBT432 and FBT374 have glucose-lowering and gut ß-like cell-inducing properties in mice. FBT432 is also highly effective in combination with a Notch inhibitor in this model. CONCLUSION: The data add to a growing body of evidence suggesting that FOXO1 inhibition be pursued as an alternative treatment to insulin administration in diabetes.

Activation of the insulin receptor by an insulin mimetic peptide.

Insulin receptor (IR) signaling defects cause a variety of metabolic diseases including diabetes. Moreover, inherited mutations of the IR cause severe insulin resistance, leading to early morbidity and mortality with limited therapeutic options. A previously reported selective IR agonist without sequence homology to insulin, S597, activates IR and mimics insulin's action on glycemic control. To elucidate the mechanism of IR activation by S597, we determine cryo-EM structures of the mouse IR/S597 complex. Unlike the compact T-shaped active IR resulting from the binding of four insulins to two distinct sites, two S597 molecules induce and stabilize an extended T-shaped IR through the simultaneous binding to both the L1 domain of one protomer and the FnIII-1 domain of another. Importantly, S597 fully activates IR mutants that disrupt insulin binding or destabilize the insulin-induced compact T-shape, thus eliciting insulin-like signaling. S597 also selectively activates IR signaling among different tissues and triggers IR endocytosis in the liver. Overall, our structural and functional studies guide future efforts to develop insulin mimetics targeting insulin resistance caused by defects in insulin binding and stabilization of insulin-activated state of IR, demonstrating the potential of structure-based drug design for insulin-resistant diseases.

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.

Notch-mediated Ephrin signaling disrupts islet architecture and ß cell function.

Altered islet architecture is associated with ß cell dysfunction and type 2 diabetes (T2D) progression, but molecular effectors of islet spatial organization remain mostly unknown. Although Notch signaling is known to regulate pancreatic development, we observed "reactivated" ß cell Notch activity in obese mouse models. To test the repercussions and reversibility of Notch effects, we generated doxycycline-dependent, ß cell-specific Notch gain-of-function mice. As predicted, we found that Notch activation in postnatal ß cells impaired glucose-stimulated insulin secretion and glucose intolerance, but we observed a surprising remnant glucose intolerance after doxycycline withdrawal and cessation of Notch activity, associated with a marked disruption of normal islet architecture. Transcriptomic screening of Notch-active islets revealed increased Ephrin signaling. Commensurately, exposure to Ephrin ligands increased ß cell repulsion and impaired murine and human pseudoislet formation. Consistent with our mouse data, Notch and Ephrin signaling were increased in metabolically inflexible ß cells in patients with T2D. These studies suggest that ß cell Notch/Ephrin signaling can permanently alter islet architecture during a morphogenetic window in early life.

TOX4, an insulin receptor-independent regulator of hepatic glucose production, is activated in diabetic liver.

Increased hepatic glucose production (HGP) contributes to hyperglycemia in type 2 diabetes. Hormonal regulation of this process is primarily, but not exclusively, mediated by the AKT-FoxO1 pathway. Here, we show that cAMP and dexamethasone regulate the high-mobility group superfamily member TOX4 to mediate HGP, independent of the insulin receptor/FoxO1 pathway. TOX4 inhibition decreases glucose production in primary hepatocytes and liver and increases glucose tolerance. Combined genetic ablation of TOX4 and FoxO1 in liver has additive effects on glucose tolerance and gluconeogenesis. Moreover, TOX4 ablation fails to reverse the metabolic derangement brought by insulin receptor knockout. TOX4 expression is increased in livers of patients with steatosis and diabetes and in diet-induced obese and db/db mice. In the latter two murine models, knockdown Tox4 decreases glycemia and improves glucose tolerance. We conclude that TOX4 is an insulin receptor-independent regulator of HGP and a candidate contributor to the pathophysiology of diabetes.