Metabolic control of adaptive ß-cell proliferation by the protein deacetylase SIRT2.

Selective and controlled expansion of endogenous ß-cells has been pursued as a potential therapy for diabetes. Ideally, such therapies would preserve feedback control of ß-cell proliferation to avoid excessive ß-cell expansion and an increased risk of hypoglycemia. Here, we identified a regulator of ß-cell proliferation whose inactivation results in controlled ß-cell expansion: the protein deacetylase Sirtuin 2 (SIRT2). Sirt2 deletion in ß-cells of mice increased ß-cell proliferation during hyperglycemia with little effect in homeostatic conditions, indicating preservation of feedback control of ß-cell mass. SIRT2 restrains proliferation of human islet ß-cells cultured in glucose concentrations above the glycemic set point, demonstrating conserved SIRT2 function. Analysis of acetylated proteins in islets treated with a SIRT2 inhibitor revealed that SIRT2 deacetylates enzymes involved in oxidative phosphorylation, dampening the adaptive increase in oxygen consumption during hyperglycemia. At the transcriptomic level, Sirt2 inactivation has context-dependent effects on ß-cells, with Sirt2 controlling how ß-cells interpret hyperglycemia as a stress. Finally, we provide proof-of-principle that systemic administration of a GLP1-coupled Sirt2-targeting antisense oligonucleotide achieves ß-cell selective Sirt2 inactivation and stimulates ß-cell proliferation under hyperglycemic conditions. Overall, these studies identify a therapeutic strategy for increasing ß-cell mass in diabetes without circumventing feedback control of ß-cell proliferation.

A vascularized 3D model of the human pancreatic islet forex vivostudy of immune cell-islet interaction.

Insulin is an essential regulator of blood glucose homeostasis that is produced exclusively byßcells within the pancreatic islets of healthy individuals. In those affected by diabetes, immune inflammation, damage, and destruction of isletßcells leads to insulin deficiency and hyperglycemia. Current efforts to understand the mechanisms underlyingßcell damage in diabetes rely onin vitro-cultured cadaveric islets. However, isolation of these islets involves removal of crucial matrix and vasculature that supports islets in the intact pancreas. Unsurprisingly, these islets demonstrate reduced functionality over time in standard culture conditions, thereby limiting their value for understanding native islet biology. Leveraging a novel, vascularized micro-organ (VMO) approach, we have recapitulated elements of the native pancreas by incorporating isolated human islets within a three-dimensional matrix nourished by living, perfusable blood vessels. Importantly, these islets show long-term viability and maintain robust glucose-stimulated insulin responses. Furthermore, vessel-mediated delivery of immune cells to these tissues provides a model to assess islet-immune cell interactions and subsequent islet killing-key steps in type 1 diabetes pathogenesis. Together, these results establish the islet-VMO as a novel,ex vivoplatform for studying human islet biology in both health and disease.

Beta cell dysfunction induced by bone morphogenetic protein (BMP)-2 is associated with histone modifications and decreased NeuroD1 chromatin binding.

Insufficient insulin secretion is a hallmark of type 2 diabetes and has been attributed to beta cell identity loss characterized by decreased expression of several key beta cell genes. The pro-inflammatory factor BMP-2 is upregulated in islets of Langerhans from individuals with diabetes and acts as an inhibitor of beta cell function and proliferation. Exposure to BMP-2 induces expression of Id1-4, Hes-1, and Hey-1 which are transcriptional regulators associated with loss of differentiation. The aim of this study was to investigate the mechanism by which BMP-2 induces beta cell dysfunction and loss of cell maturity. Mouse islets exposed to BMP-2 for 10 days showed impaired glucose-stimulated insulin secretion and beta cell proliferation. BMP-2-induced beta cell dysfunction was associated with decreased expression of cell maturity and proliferation markers specific to the beta cell such as Ins1, Ucn3, and Ki67 and increased expression of Id1-4, Hes-1, and Hey-1. The top 30 most regulated proteins significantly correlated with corresponding mRNA expression. BMP-2-induced gene expression changes were associated with a predominant reduction in acetylation of H3K27 and a decrease in NeuroD1 chromatin binding activity. These results show that BMP-2 induces loss of beta cell maturity and suggest that remodeling of H3K27ac and decreased NeuroD1 DNA binding activity participate in the effect of BMP-2 on beta cell dysfunction.

Nutrient regulation of the islet epigenome controls adaptive insulin secretion.

Adaptation of the islet ß cell insulin-secretory response to changing insulin demand is critical for blood glucose homeostasis, yet the mechanisms underlying this adaptation are unknown. Here, we have shown that nutrient-stimulated histone acetylation plays a key role in adapting insulin secretion through regulation of genes involved in ß cell nutrient sensing and metabolism. Nutrient regulation of the epigenome occurred at sites occupied by the chromatin-modifying enzyme lysine-specific demethylase 1 (Lsd1) in islets. ß Cell-specific deletion of Lsd1 led to insulin hypersecretion, aberrant expression of nutrient-response genes, and histone hyperacetylation. Islets from mice adapted to chronically increased insulin demand exhibited shared epigenetic and transcriptional changes. Moreover, we found that genetic variants associated with type 2 diabetes were enriched at LSD1-bound sites in human islets, suggesting that interpretation of nutrient signals is genetically determined and clinically relevant. Overall, these studies revealed that adaptive insulin secretion involves Lsd1-mediated coupling of nutrient state to regulation of the islet epigenome.

Systemic LSD1 Inhibition Prevents Aberrant Remodeling of Metabolism in Obesity.

The transition from lean to obese states involves systemic metabolic remodeling that impacts insulin sensitivity, lipid partitioning, inflammation, and glycemic control. Here, we have taken a pharmacological approach to test the role of a nutrient-regulated chromatin modifier, lysine-specific demethylase (LSD1), in obesity-associated metabolic reprogramming. We show that systemic administration of an LSD1 inhibitor (GSK-LSD1) reduces food intake and body weight, ameliorates nonalcoholic fatty liver disease (NAFLD), and improves insulin sensitivity and glycemic control in mouse models of obesity. GSK-LSD1 has little effect on systemic metabolism of lean mice, suggesting that LSD1 has a context-dependent role in promoting maladaptive changes in obesity. In analysis of insulin target tissues we identified white adipose tissue as the major site of insulin sensitization by GSK-LSD1, where it reduces adipocyte inflammation and lipolysis. We demonstrate that GSK-LSD1 reverses NAFLD in a non-hepatocyte-autonomous manner, suggesting an indirect mechanism potentially via inhibition of adipocyte lipolysis and subsequent effects on lipid partitioning. Pair-feeding experiments further revealed that effects of GSK-LSD1 on hyperglycemia and NAFLD are not a consequence of reduced food intake and weight loss. These findings suggest that targeting LSD1 could be a strategy for treatment of obesity and its associated complications including type 2 diabetes and NAFLD.

Deletion of ABCB10 in beta-cells protects from high-fat diet induced insulin resistance.

OBJECTIVE: The contribution of beta-cell dysfunction to type 2 diabetes (T2D) is not restricted to insulinopenia in the late stages of the disease. Elevated fasting insulinemia in normoglycemic humans is a major factor predicting the onset of insulin resistance and T2D, demonstrating an early alteration of beta-cell function in T2D. Moreover, an early and chronic increase in fasting insulinemia contributes to insulin resistance in high-fat diet (HFD)-fed mice. However, whether there are genetic factors that promote beta-cell-initiated insulin resistance remains undefined. Human variants of the mitochondrial transporter ABCB10, which regulates redox by increasing bilirubin synthesis, have been associated with an elevated risk of T2D. The effects of T2D ABCB10 variants on ABCB10 expression and the actions of ABCB10 in beta-cells are unknown. METHODS: The expression of beta-cell ABCB10 was analyzed in published transcriptome datasets from human beta-cells carrying the T2D-risk ABCB10 variant. Insulin sensitivity, beta-cell proliferation, and secretory function were measured in beta-cell-specific ABCB10 KO mice (Ins1Cre-Abcb10flox/flox). The short-term role of beta-cell ABCB10 activity on glucose-stimulated insulin secretion (GSIS) was determined in isolated islets. RESULTS: Carrying the T2Drisk allele G of ABCB10 rs348330 variant was associated with increased ABCB10 expression in human beta-cells. Constitutive deletion of Abcb10 in beta-cells protected mice from hyperinsulinemia and insulin resistance by limiting HFD-induced beta-cell expansion. An early limitation in GSIS and H2O2-mediated signaling caused by elevated ABCB10 activity can initiate an over-compensatory expansion of beta-cell mass in response to HFD. Accordingly, increasing ABCB10 expression was sufficient to limit GSIS capacity. In health, ABCB10 protein was decreased during islet maturation, with maturation restricting beta-cell proliferation and elevating GSIS. Finally, ex-vivo and short-term deletion of ABCB10 in islets isolated from HFD-fed mice increased H2O2 and GSIS, which was reversed by bilirubin treatments. CONCLUSIONS: Beta-cell ABCB10 is required for HFD to induce insulin resistance in mice by amplifying beta-cell mass expansion to maladaptive levels that cause fasting hyperinsulinemia.

Endoplasmic reticulum tubules limit the size of misfolded protein condensates.

The endoplasmic reticulum (ER) is composed of sheets and tubules. Here we report that the COPII coat subunit, SEC24C, works with the long form of the tubular ER-phagy receptor, RTN3, to target dominant-interfering mutant proinsulin Akita puncta to lysosomes. When the delivery of Akita puncta to lysosomes was disrupted, large puncta accumulated in the ER. Unexpectedly, photobleach analysis indicated that Akita puncta behaved as condensates and not aggregates, as previously suggested. Akita puncta enlarged when either RTN3 or SEC24C were depleted, or when ER sheets were proliferated by either knocking out Lunapark or overexpressing CLIMP63. Other ER-phagy substrates that are segregated into tubules behaved like Akita, while a substrate (type I procollagen) that is degraded by the ER-phagy sheets receptor, FAM134B, did not. Conversely, when ER tubules were augmented in Lunapark knock-out cells by overexpressing reticulons, ER-phagy increased and the number of large Akita puncta was reduced. Our findings imply that segregating cargoes into tubules has two beneficial roles. First, it localizes mutant misfolded proteins, the receptor, and SEC24C to the same ER domain. Second, physically restraining condensates within tubules, before they undergo ER-phagy, prevents them from enlarging and impacting cell health.

Transcriptional mechanisms of pancreatic ß-cell maturation and functional adaptation.

Pancreatic ß-cells secrete insulin commensurate to circulating nutrient levels to maintain normoglycemia. The ability of ß-cells to couple insulin secretion to nutrient stimuli is acquired during a postnatal maturation process. In mature ß-cells the insulin secretory response adapts to changes in nutrient state. Both ß-cell maturation and functional adaptation rely on the interplay between extracellular cues and cell type-specific transcriptional programs. Here we review emerging evidence that developmental and homeostatic regulation of ß-cell function involves collaboration between lineage-determining and signal-dependent transcription factors (LDTFs and SDTFs, respectively). A deeper understanding of ß-cell SDTFs and their cognate signals would delineate mechanisms of ß-cell maturation and functional adaptation, which has direct implications for diabetes therapies and for generating mature ß-cells from stem cells.

LSD1-mediated enhancer silencing attenuates retinoic acid signalling during pancreatic endocrine cell development.

Developmental progression depends on temporally defined changes in gene expression mediated by transient exposure of lineage intermediates to signals in the progenitor niche. To determine whether cell-intrinsic epigenetic mechanisms contribute to signal-induced transcriptional responses, here we manipulate the signalling environment and activity of the histone demethylase LSD1 during differentiation of hESC-gut tube intermediates into pancreatic endocrine cells. We identify a transient requirement for LSD1 in endocrine cell differentiation spanning a short time-window early in pancreas development, a phenotype we reproduced in mice. Examination of enhancer and transcriptome landscapes revealed that LSD1 silences transiently active retinoic acid (RA)-induced enhancers and their target genes. Furthermore, prolonged RA exposure phenocopies LSD1 inhibition, suggesting that LSD1 regulates endocrine cell differentiation by limiting the duration of RA signalling. Our findings identify LSD1-mediated enhancer silencing as a cell-intrinsic epigenetic feedback mechanism by which the duration of the transcriptional response to a developmental signal is limited.

Beta-cell dysfunction induced by non-cytotoxic concentrations of Interleukin-1ß is associated with changes in expression of beta-cell maturity genes and associated histone modifications.

Decreased insulin secretory capacity in Type 2 diabetes mellitus is associated with beta-cell dedifferentiation and inflammation. We hypothesize that prolonged exposure of beta-cells to low concentrations of IL-1ß induce beta-cell dedifferentiation characterized by impaired glucose-stimulated insulin secretion, reduced expression of key beta-cell genes and changes in histone modifications at gene loci known to affect beta-cell function. Ten days exposure to IL-1ß at non-cytotoxic concentrations reduced insulin secretion and beta-cell proliferation and decreased expression of key beta-cell identity genes, including MafA and Ucn3 and decreased H3K27ac at the gene loci, suggesting that inflammatory cytokines directly affects the epigenome. Following removal of IL-1ß, beta-cell function was normalized and mRNA expression of beta-cell identity genes, such as insulin and Ucn3 returned to pre-stimulation levels. Our findings indicate that prolonged exposure to low concentrations of IL-1ß induces epigenetic changes associated with loss of beta-cell identity as observed in Type 2 diabetes.

Integrated In Vivo Quantitative Proteomics and Nutrient Tracing Reveals Age-Related Metabolic Rewiring of Pancreatic ß Cell Function.

Pancreatic ß cell physiology changes substantially throughout life, yet the mechanisms that drive these changes are poorly understood. Here, we performed comprehensive in vivo quantitative proteomic profiling of pancreatic islets from juvenile and 1-year-old mice. The analysis revealed striking differences in abundance of enzymes controlling glucose metabolism. We show that these changes in protein abundance are associated with higher activities of glucose metabolic enzymes involved in coupling factor generation as well as increased activity of the coupling factor-dependent amplifying pathway of insulin secretion. Nutrient tracing and targeted metabolomics demonstrated accelerated accumulation of glucose-derived metabolites and coupling factors in islets from 1-year-old mice, indicating that age-related changes in glucose metabolism contribute to improved glucose-stimulated insulin secretion with age. Together, our study provides an in-depth characterization of age-related changes in the islet proteome and establishes metabolic rewiring as an important mechanism for age-associated changes in ß cell function.

Evaluation of Different Decellularization Protocols on the Generation of Pancreas-Derived Hydrogels.

Different approaches have investigated the effects of different extracellular matrices (ECMs) and three-dimensional (3D) culture on islet function, showing encouraging results. Ideally, the proper scaffold should mimic the biochemical composition of the native tissue as it drives numerous signaling pathways involved in tissue homeostasis and functionality. Tissue-derived decellularized biomaterials can preserve the ECM composition of the native tissue making it an ideal scaffold for 3D tissue engineering applications. However, the decellularization process may affect the retention of specific components, and the choice of a proper detergent is fundamental in preserving the native ECM composition. In this study, we evaluated the effect of different decellularization protocols on the mechanical properties and biochemical composition of pancreatic ECM (pECM) hydrogels. Fresh porcine pancreas tissue was harvested, cut into small pieces, rinsed in water, and treated with two different detergents (sodium dodecyl sulfate [SDS] or Triton X-100) for 1 day followed by 3 days in water. Effective decellularization was confirmed by PicoGreen assay, Hoescht, and H&E staining, showing no differences among groups. Use of a protease inhibitor (PI) was also evaluated. Effective decellularization was confirmed by PicoGreen assay and hematoxylin and eosin (H&E) staining, showing no differences among groups. Triton-treated samples were able to form a firm hydrogel under appropriate conditions, while the use of SDS had detrimental effects on the gelation properties of the hydrogels. ECM biochemical composition was characterized both in the fresh porcine pancreas and all decellularized pECM hydrogels by quantitative mass spectrometry analysis. Fibrillar collagen was the major ECM component in all groups, with all generated hydrogels having a higher amount compared with fresh pancreas. This effect was more pronounced in the SDS-treated hydrogels when compared with the Triton groups, showing very little retention of other ECM molecules. Conversely, basement membrane and matricellular proteins were better retained when the tissue was pretreated with a PI and decellularized in Triton X-100, making the hydrogel more similar to the native tissue. In conclusion, we showed that all the protocols evaluated in the study showed effective tissue decellularization, but only when the tissue was pretreated with a PI and decellularized in Triton detergent, the biochemical composition of the hydrogel was closer to the native tissue ECM. Impact Statement The article compares different methodologies for the generation of a pancreas-derived hydrogel for tissue engineering applications. The biochemical characterization of the newly generated hydrogel shows that the material retains all the extracellular molecules of the native tissue and is capable of sustaining functionality of the encapsulated beta-cells.

High T Gives ß Cells a Boost.

Androgen deficiency in men is associated with metabolic syndrome and diabetes, which has been attributed to actions of androgens upon insulin target tissues. In this issue of Cell Metabolism, Navarro et al. (2016) report a role for androgens and their receptor in the regulation of insulin secretion in ß cells.

Chromatin accessibility mapping identifies mediators of basal transcription and retinoid-induced repression of OTX2 in medulloblastoma.

Despite an emerging understanding of the genetic alterations giving rise to various tumors, the mechanisms whereby most oncogenes are overexpressed remain unclear. Here we have utilized an integrated approach of genomewide regulatory element mapping via DNase-seq followed by conventional reporter assays and transcription factor binding site discovery to characterize the transcriptional regulation of the medulloblastoma oncogene Orthodenticle Homeobox 2 (OTX2). Through these studies we have revealed that OTX2 is differentially regulated in medulloblastoma at the level of chromatin accessibility, which is in part mediated by DNA methylation. In cell lines exhibiting chromatin accessibility of OTX2 regulatory regions, we found that autoregulation maintains OTX2 expression. Comparison of medulloblastoma regulatory elements with those of the developing brain reveals that these tumors engage a developmental regulatory program to drive OTX2 transcription. Finally, we have identified a transcriptional regulatory element mediating retinoid-induced OTX2 repression in these tumors. This work characterizes for the first time the mechanisms of OTX2 overexpression in medulloblastoma. Furthermore, this study establishes proof of principle for applying ENCODE datasets towards the characterization of upstream trans-acting factors mediating expression of individual genes.

Global identification of MLL2-targeted loci reveals MLL2's role in diverse signaling pathways.

Myeloid/lymphoid or mixed-lineage leukemia (MLL)-family genes encode histone lysine methyltransferases that play important roles in epigenetic regulation of gene transcription. MLL genes are frequently mutated in human cancers. Unlike MLL1, MLL2 (also known as ALR/MLL4) and its homolog MLL3 are not well-understood. Specifically, little is known regarding the extent of global MLL2 involvement in the regulation of gene expression and the mechanism underlying its alterations in driving tumorigenesis. Here we profile the global loci targeted by MLL2. A combinatorial analysis of the MLL2 binding profile and gene expression in MLL2 wild-type versus MLL2-null isogenic cell lines identified direct transcriptional target genes and revealed the connection of MLL2 to multiple cellular signaling pathways, including the p53 pathway, cAMP-mediated signaling, and cholestasis signaling. In particular, we demonstrate that MLL2 participates in retinoic acid receptor signaling by promoting retinoic acid-responsive gene transcription. Our results present a genome-wide integrative analysis of the MLL2 target loci and suggest potential mechanisms underlying tumorigenesis driven by MLL2 alterations.

Extensive evolutionary changes in regulatory element activity during human origins are associated with altered gene expression and positive selection.

Understanding the molecular basis for phenotypic differences between humans and other primates remains an outstanding challenge. Mutations in non-coding regulatory DNA that alter gene expression have been hypothesized as a key driver of these phenotypic differences. This has been supported by differential gene expression analyses in general, but not by the identification of specific regulatory elements responsible for changes in transcription and phenotype. To identify the genetic source of regulatory differences, we mapped DNaseI hypersensitive (DHS) sites, which mark all types of active gene regulatory elements, genome-wide in the same cell type isolated from human, chimpanzee, and macaque. Most DHS sites were conserved among all three species, as expected based on their central role in regulating transcription. However, we found evidence that several hundred DHS sites were gained or lost on the lineages leading to modern human and chimpanzee. Species-specific DHS site gains are enriched near differentially expressed genes, are positively correlated with increased transcription, show evidence of branch-specific positive selection, and overlap with active chromatin marks. Species-specific sequence differences in transcription factor motifs found within these DHS sites are linked with species-specific changes in chromatin accessibility. Together, these indicate that the regulatory elements identified here are genetic contributors to transcriptional and phenotypic differences among primate species.

Apples to origins: identifying brain tumor stem cell genes by comparing transcriptomes of normal and cancer stem cells.

The mechanisms whereby medulloblastoma stem cells coordinate tumor propagation are poorly understood. Using microarray analysis, Corno and colleagues draw parallels and distinctions between medulloblastoma stem cells from the Ptch(+/-) mouse and normal neural stem cells, identifying Ebf3 as a cancer stem cell-specific transcript critical for tumor growth.

Aberrant Otx2 expression enhances migration and induces ectopic proliferation of hindbrain neuronal progenitor cells.

Dysregulation of Otx2 is a hallmark of the pediatric brain tumor medulloblastoma, yet its functional significance in the establishment of these tumors is unknown. Here we have sought to determine the functional consequences of Otx2 overexpression in the mouse hindbrain to characterize its potential role in medulloblastoma tumorigenesis and identify the cell types responsive to this lineage-specific oncogene. Expression of Otx2 broadly in the mouse hindbrain resulted in the accumulation of proliferative clusters of cells in the cerebellar white matter and dorsal brainstem of postnatal mice. We found that brainstem ectopia were derived from neuronal progenitors of the rhombic lip and that cerebellar ectopia were derived from granule neuron precursors (GNPs) that had migrated inwards from the external granule layer (EGL). These hyperplasias exhibited various characteristics of medulloblastoma precursor cells identified in animal models of Shh or Wnt group tumors, including aberrant localization and altered spatiotemporal control of proliferation. However, ectopia induced by Otx2 differentiated and dispersed as the animals reached adulthood, indicating that factors restricting proliferative lifespan were a limiting factor to full transformation of these cells. These studies implicate a role for Otx2 in altering the dynamics of neuronal progenitor cell proliferation.

OTX2 is critical for the maintenance and progression of Shh-independent medulloblastomas.

OTX2 is a developmentally regulated transcription factor involved in early morphogenesis of the central nervous system. This gene is amplified and overexpressed in medulloblastoma cell lines, but the nature and extent of its genetic alterations in primary tumors have not been evaluated. Analysis of a large cohort of primary medulloblastomas revealed frequent focal copy number gain of a region minimally containing OTX2 as a single gene. OTX2 copy number gain was restricted to tumor subtypes that did not express a molecular signature of Wnt or Shh pathway activation. FISH analysis revealed copy number gain in a subset of cells within medulloblastoma samples, suggesting a late event in tumor progression. Gain of OTX2 copy number was associated with the presence of anaplastic histologic features and shorter survival in medulloblastoma patients. In support of a functional role, ectopic OTX2 expression enhanced proliferation and tumorigenicity of immortalized primary cells, whereas OTX2 knockdown in medulloblastoma cells prolonged the survival of animals bearing xenograft tumors. Mechanistic investigations revealed upregulation of MYC as a potential mechanism whereby OTX2 promotes tumor progression. Our findings define OTX2 as an important oncogenic driver in medulloblastoma.