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.

An integrative transcriptional logic model of hepatic insulin resistance.

Abnormalities of lipid/lipoprotein and glucose metabolism are hallmarks of hepatic insulin resistance in type 2 diabetes. The former antedate the latter, but the latter become progressively refractory to treatment and contribute to therapeutic failures. It's unclear whether the two processes share a common pathogenesis and what underlies their progressive nature. In this study, we investigated the hypothesis that genes in the lipid/lipoprotein pathway and those in the glucose metabolic pathway are governed by different transcriptional regulatory logics that affect their response to physiologic (fasting/refeeding) as well as pathophysiologic cues (insulin resistance and hyperglycemia). To this end, we obtained genomic and transcriptomic maps of the key insulin-regulated transcription factor, FoxO1, and integrated them with those of CREB, PPAR-α, and glucocorticoid receptor. We found that glucose metabolic genes are primarily regulated by promoter and intergenic enhancers in a fasting-dependent manner, while lipid genes are regulated through fasting-dependent intron enhancers and fasting-independent enhancerless introns. Glucose genes also showed a remarkable transcriptional resiliency (i.e., the ability to compensate following constitutive FoxO1 ablation through an enrichment of active marks at shared PPAR-α/FoxO1 regulatory elements). Unexpectedly, insulin resistance and hyperglycemia were associated with a "spreading" of FoxO1 binding to enhancers and the emergence of unique target sites. We surmise that this unusual pattern correlates with the progressively intractable nature of hepatic insulin resistance. This transcriptional logic provides an integrated model to interpret the combined lipid and glucose abnormalities of type 2 diabetes.

Antagonistic epistasis of Hnf4α and FoxO1 metabolic networks through enhancer interactions in ß-cell function.

OBJECTIVE: Genetic and acquired abnormalities contribute to pancreatic ß-cell failure in diabetes. Transcription factors Hnf4α (MODY1) and FoxO1 are respective examples of these two components and act through ß-cell-specific enhancers. However, their relationship is unclear. METHODS: In this report, we show by genome-wide interrogation of chromatin modifications that ablation of FoxO1 in mature ß-cells enriches active Hnf4α enhancers according to a HOMER analysis. RESULTS: To model the functional significance of this predicted unusual enhancer utilization, we generated single and compound knockouts of FoxO1 and Hnf4α in ß-cells. Single knockout of either gene impaired insulin secretion in mechanistically distinct fashions as indicated by their responses to sulfonylurea and calcium fluxes. Surprisingly, the defective ß-cell secretory function of either single mutant in hyperglycemic clamps and isolated islets treated with various secretagogues was completely reversed in double mutants lacking FoxO1 and Hnf4α. Gene expression analyses revealed distinct epistatic modalities by which the two transcription factors regulate networks associated with reversal of ß-cell dysfunction. An antagonistic network regulating glycolysis, including ß-cell "disallowed" genes, and a synergistic network regulating protocadherins emerged as likely mediators of the functional restoration of insulin secretion. CONCLUSIONS: The findings provide evidence of antagonistic epistasis as a model of gene/environment interactions in the pathogenesis of ß-cell dysfunction.

The role of striated muscle Pik3r1 in glucose and protein metabolism following chronic glucocorticoid exposure.

Chronic glucocorticoid exposure causes insulin resistance and muscle atrophy in skeletal muscle. We previously identified phosphoinositide-3-kinase regulatory subunit 1 (Pik3r1) as a primary target gene of skeletal muscle glucocorticoid receptors involved in the glucocorticoid-mediated suppression of insulin action. However, the in vivo functions of Pik3r1 remain unclear. Here, we generated striated muscle-specific Pik3r1 knockout (MKO) mice and treated them with a dexamethasone (DEX), a synthetic glucocorticoid. Treating wildtype (WT) mice with DEX attenuated insulin activated Akt activity in liver, epididymal white adipose tissue, and gastrocnemius (GA) muscle. This DEX effect was diminished in GA muscle of MKO mice, therefore, resulting in improved glucose and insulin tolerance in DEX-treated MKO mice. Stable isotope labeling techniques revealed that in WT mice, DEX treatment decreased protein fractional synthesis rates in GA muscle. Furthermore, histology showed that in WT mice, DEX treatment reduced GA myotube diameters. In MKO mice, myotube diameters were smaller than in WT mice, and there were more fast oxidative fibers. Importantly, DEX failed to further reduce myotube diameters. Pik3r1 knockout also decreased basal protein synthesis rate (likely caused by lower 4E-BP1 phosphorylation at Thr37/Thr46) and curbed the ability of DEX to attenuate protein synthesis rate. Finally, the ability of DEX to inhibit eIF2α phosphorylation and insulin-induced 4E-BP1 phosphorylation was reduced in MKO mice. Taken together, these results demonstrate the role of Pik3r1 in glucocorticoid-mediated effects on glucose and protein metabolism in skeletal muscle.

Cyb5r3 links FoxO1-dependent mitochondrial dysfunction with ß-cell failure.

OBJECTIVE: Diabetes is characterized by pancreatic ß-cell dedifferentiation. Dedifferentiating ß cells inappropriately metabolize lipids over carbohydrates and exhibit impaired mitochondrial oxidative phosphorylation. However, the mechanism linking the ß-cell's response to an adverse metabolic environment with impaired mitochondrial function remains unclear. METHODS: Here we report that the oxidoreductase cytochrome b5 reductase 3 (Cyb5r3) links FoxO1 signaling to ß-cell stimulus/secretion coupling by regulating mitochondrial function, reactive oxygen species generation, and nicotinamide actin dysfunction (NAD)/reduced nicotinamide actin dysfunction (NADH) ratios. RESULTS: The expression of Cyb5r3 is decreased in FoxO1-deficient ß cells. Mice with ß-cell-specific deletion of Cyb5r3 have impaired insulin secretion, resulting in glucose intolerance and diet-induced hyperglycemia. Cyb5r3-deficient ß cells have a blunted respiratory response to glucose and display extensive mitochondrial and secretory granule abnormalities, consistent with altered differentiation. Moreover, FoxO1 is unable to maintain expression of key differentiation markers in Cyb5r3-deficient ß cells, suggesting that Cyb5r3 is required for FoxO1-dependent lineage stability. CONCLUSIONS: The findings highlight a pathway linking FoxO1 to mitochondrial dysfunction that can mediate ß-cell failure.

Identification of C2CD4A as a human diabetes susceptibility gene with a role in ß cell insulin secretion.

Fine mapping and validation of genes causing ß cell failure from susceptibility loci identified in type 2 diabetes genome-wide association studies (GWAS) poses a significant challenge. The VPS13C-C2CD4A-C2CD4B locus on chromosome 15 confers diabetes susceptibility in every ethnic group studied to date. However, the causative gene is unknown. FoxO1 is involved in the pathogenesis of ß cell dysfunction, but its link to human diabetes GWAS has not been explored. Here we generated a genome-wide map of FoxO1 superenhancers in chemically identified ß cells using 2-photon live-cell imaging to monitor FoxO1 localization. When parsed against human superenhancers and GWAS-derived diabetes susceptibility alleles, this map revealed a conserved superenhancer in C2CD4A, a gene encoding a ß cell/stomach-enriched nuclear protein of unknown function. Genetic ablation of C2cd4a in ß cells of mice phenocopied the metabolic abnormalities of human carriers of C2CD4A-linked polymorphisms, resulting in impaired insulin secretion during glucose tolerance tests as well as hyperglycemic clamps. C2CD4A regulates glycolytic genes, and notably represses key ß cell "disallowed" genes, such as lactate dehydrogenase A We propose that C2CD4A is a transcriptional coregulator of the glycolytic pathway whose dysfunction accounts for the diabetes susceptibility associated with the chromosome 15 GWAS locus.

Induction of α cell-restricted Gc in dedifferentiating ß cells contributes to stress-induced ß-cell dysfunction.

Diabetic ß cell failure is associated with ß cell dedifferentiation. To identify effector genes of dedifferentiation, we integrated analyses of histone methylation as a surrogate of gene activation status and RNA expression in ß cells sorted from mice with multiparity-induced diabetes. Interestingly, only a narrow subset of genes demonstrated concordant changes to histone methylation and RNA levels in dedifferentiating ß cells. Notable among them was the α cell signature gene Gc, encoding a vitamin D-binding protein. While diabetes was associated with Gc induction, Gc-deficient islets did not induce ß cell dedifferentiation markers and maintained normal ex vivo insulin secretion in the face of metabolic challenge. Moreover, Gc-deficient mice exhibited a more robust insulin secretory response than normal controls during hyperglycemic clamps. The data are consistent with a functional role of Gc activation in ß cell dysfunction, and indicate that multiparity-induced diabetes is associated with altered ß cell fate.

Pik3r1 Is Required for Glucocorticoid-Induced Perilipin 1 Phosphorylation in Lipid Droplet for Adipocyte Lipolysis.

Glucocorticoids promote lipolysis in white adipose tissue (WAT) to adapt to energy demands under stress, whereas superfluous lipolysis causes metabolic disorders, including dyslipidemia and hepatic steatosis. Glucocorticoid-induced lipolysis requires the phosphorylation of cytosolic hormone-sensitive lipase (HSL) and perilipin 1 (Plin1) in the lipid droplet by protein kinase A (PKA). We previously identified Pik3r1 (also called p85α) as a glucocorticoid receptor target gene. Here, we found that glucocorticoids increased HSL phosphorylation, but not Plin1 phosphorylation, in adipose tissue-specific Pik3r1-null (AKO) mice. Furthermore, in lipid droplets, the phosphorylation of HSL and Plin1 and the levels of catalytic and regulatory subunits of PKA were increased by glucocorticoids in wild-type mice. However, these effects were attenuated in AKO mice. In agreement with reduced WAT lipolysis, glucocorticoid- initiated hepatic steatosis and hypertriglyceridemia were improved in AKO mice. Our data demonstrated a novel role of Pik3r1 that was independent of the regulatory function of phosphoinositide 3-kinase in mediating the metabolic action of glucocorticoids. Thus, the inhibition of Pik3r1 in adipocytes could alleviate lipid disorders caused by excess glucocorticoid exposure.

Cloche is a bHLH-PAS transcription factor that drives haemato-vascular specification.

Vascular and haematopoietic cells organize into specialized tissues during early embryogenesis to supply essential nutrients to all organs and thus play critical roles in development and disease. At the top of the haemato-vascular specification cascade lies cloche, a gene that when mutated in zebrafish leads to the striking phenotype of loss of most endothelial and haematopoietic cells and a significant increase in cardiomyocyte numbers. Although this mutant has been analysed extensively to investigate mesoderm diversification and differentiation and continues to be broadly used as a unique avascular model, the isolation of the cloche gene has been challenging due to its telomeric location. Here we used a deletion allele of cloche to identify several new cloche candidate genes within this genomic region, and systematically genome-edited each candidate. Through this comprehensive interrogation, we succeeded in isolating the cloche gene and discovered that it encodes a PAS-domain-containing bHLH transcription factor, and that it is expressed in a highly specific spatiotemporal pattern starting during late gastrulation. Gain-of-function experiments show that it can potently induce endothelial gene expression. Epistasis experiments reveal that it functions upstream of etv2 and tal1, the earliest expressed endothelial and haematopoietic transcription factor genes identified to date. A mammalian cloche orthologue can also rescue blood vessel formation in zebrafish cloche mutants, indicating a highly conserved role in vertebrate vasculogenesis and haematopoiesis. The identification of this master regulator of endothelial and haematopoietic fate enhances our understanding of early mesoderm diversification and may lead to improved protocols for the generation of endothelial and haematopoietic cells in vivo and in vitro.

Transcriptional regulation of FoxO3 gene by glucocorticoids in murine myotubes.

Glucocorticoids and FoxO3 exert similar metabolic effects in skeletal muscle. FoxO3 gene expression was increased by dexamethasone (Dex), a synthetic glucocorticoid, both in vitro and in vivo. In C2C12 myotubes the increased expression is due to, at least in part, the elevated rate of FoxO3 gene transcription. In the mouse FoxO3 gene, we identified three glucocorticoid receptor (GR) binding regions (GBRs): one being upstream of the transcription start site, -17kbGBR; and two in introns, +45kbGBR and +71kbGBR. Together, these three GBRs contain four 15-bp glucocorticoid response elements (GREs). Micrococcal nuclease (MNase) assay revealed that Dex treatment increased the sensitivity to MNase in the GRE of +45kbGBR and +71kbGBR upon 30- and 60-min Dex treatment, respectively. Conversely, Dex treatment did not affect the chromatin structure near the -17kbGBR, in which the GRE is located in the linker region. Dex treatment also increased histone H3 and/or H4 acetylation in genomic regions near all three GBRs. Moreover, using chromatin conformation capture (3C) assay, we showed that Dex treatment increased the interaction between the -17kbGBR and two genomic regions: one located around +500 bp and the other around +73 kb. Finally, the transcriptional coregulator p300 was recruited to all three GBRs upon Dex treatment. The reduction of p300 expression decreased FoxO3 gene expression and Dex-stimulated interaction between distinct genomic regions of FoxO3 gene identified by 3C. Overall, our results demonstrate that glucocorticoids activated FoxO3 gene transcription through multiple GREs by chromatin structural change and DNA looping.

Repression of glucocorticoid-stimulated angiopoietin-like 4 gene transcription by insulin.

Angiopoietin-like 4 (Angptl4) is a glucocorticoid receptor (GR) primary target gene in hepatocytes and adipocytes. It encodes a secreted protein that inhibits extracellular LPL and promotes adipocyte lipolysis. In Angptl4 null mice, glucocorticoid-induced adipocyte lipolysis and hepatic steatosis are compromised. Markedly, insulin suppressed glucocorticoid-induced Angptl4 transcription. To unravel the mechanism, we utilized small molecules to inhibit insulin signaling components and found that phosphatidylinositol 3-kinase and Akt were vital for the suppression in H4IIE cells. A forkhead box transcription factor response element (FRE) was found near the 15 bp Angptl4 glucocorticoid response element (GRE). Mutating the Angptl4 FRE significantly reduced glucocorticoid-induced reporter gene expression in cells. Moreover, chromatin immunoprecipitation revealed that GR and FoxO1 were recruited to Angptl4 GRE and FRE in a glucocorticoid-dependent manner, and cotreatment with insulin abolished both recruitments. Furthermore, in 24 h fasted mice, significant occupancy of GR and FoxO1 at the Angptl4 GRE and FRE was found in the liver. In contrast, both occupancies were diminished after 24 h refeeding. Finally, overexpression of dominant negative FoxO1 mutant abolished glucocorticoid-induced Angptl4 expression, mimicking the insulin suppression. Overall, we demonstrate that both GR and FoxO1 are required for Angptl4 transcription activation, and that FoxO1 negatively mediates the suppressive effect of insulin.

Angiopoietin-like 4 (ANGPTL4, fasting-induced adipose factor) is a direct glucocorticoid receptor target and participates in glucocorticoid-regulated triglyceride metabolism.

Glucocorticoids are important regulators of lipid homeostasis, and chronically elevated glucocorticoid levels induce hypertriglyceridemia, hepatic steatosis, and visceral obesity. The occupied glucocorticoid receptor (GR) is a transcription factor. However, those genes regulating lipid metabolism under GR control are not fully known. Angiopoietin-like 4 (ANGPTL4, fasting-induced adipose factor), a protein inhibitor of lipoprotein lipase, is synthesized and secreted during fasting, when circulating glucocorticoid levels are physiologically increased. We therefore tested whether the ANGPTL4 gene (Angptl4) is transcriptionally controlled by GR. We show that treatment with the synthetic glucocorticoid dexamethasone increased Angptl4 mRNA levels in primary hepatocytes and adipocytes (2-3-fold) and in the livers and white adipose tissue of mice (approximately 4-fold). We tested the mechanism of this increase in H4IIE hepatoma cells and found that dexamethasone treatment increased the transcriptional rate of Angptl4. Using bioinformatics and chromatin immunoprecipitation, we identified a GR binding site within the rat Angptl4 sequence. A reporter plasmid containing this site was markedly activated by dexamethasone, indicative of a functional glucocorticoid response element. Dexamethasone treatment also increased histone H4 acetylation and DNase I accessibility in genomic regions near this site, further supporting that it is a glucocorticoid response element. Glucocorticoids promote the flux of triglycerides from white adipose tissue to liver. We found that mice lacking ANGPTL4 (Angptl4(-/-)) had reductions in dexamethasone-induced hypertriglyceridemia and hepatic steatosis, suggesting that ANGPTL4 is required for this flux. Overall, we establish that ANGPTL4 is a direct GR target that participates in glucocorticoid-regulated triglyceride metabolism.