Islet-1 (Isl-1) is essential for the survival and ensuing differentiation of pancreatic endocrine progenitors. Isl-1 remains expressed in all adult pancreatic endocrine lineages; however, its specific function in the postnatal pancreas is unclear. Here we determine whether Isl-1 plays a distinct role in the postnatal β-cell by performing physiological and morphometric analyses of a tamoxifen-inducible, β-cell–specific Isl-1 loss-of-function mouse: Isl-1L/L; Pdx1-CreERTm. Ablating Isl-1 in postnatal β-cells reduced glucose tolerance without significantly reducing β-cell mass or increasing β-cell apoptosis. Rather, islets from Isl-1L/L; Pdx1-CreERTm mice showed impaired insulin secretion. To identify direct targets of Isl-1, we integrated high-throughput gene expression and Isl-1 chromatin occupancy using islets from Isl-1L/L; Pdx1-CreERTm mice and βTC3 insulinoma cells, respectively. Ablating Isl-1 significantly affected the β-cell transcriptome, including known targets Insulin and MafA as well as novel targets Pdx1 and Slc2a2. Using chromatin immunoprecipitation sequencing and luciferase reporter assays, we found that Isl-1 directly occupies functional regulatory elements of Pdx1 and Slc2a2. Thus Isl-1 is essential for postnatal β-cell function, directly regulates Pdx1 and Slc2a2, and has a mature β-cell cistrome distinct from that of pancreatic endocrine progenitors.

The recognition of β cell dedifferentiation in type 2 diabetes raises the translational relevance of mechanisms that direct and maintain β cell identity. LIM domain-binding protein 1 (LDB1) nucleates multimeric transcriptional complexes and establishes promoter-enhancer looping, thereby directing fate assignment and maturation of progenitor populations. Many terminally differentiated endocrine cell types, however, remain enriched for LDB1, but its role is unknown. Here, we have demonstrated a requirement for LDB1 in maintaining the terminally differentiated status of pancreatic β cells. Inducible ablation of LDB1 in mature β cells impaired insulin secretion and glucose homeostasis. Transcriptomic analysis of LDB1-depleted β cells revealed the collapse of the terminally differentiated gene program, indicated by a loss of β cell identity genes and induction of the endocrine progenitor factor neurogenin 3 (NEUROG3). Lineage tracing confirmed that LDB1-depleted, insulin-negative β cells express NEUROG3 but do not adopt alternate endocrine cell fates. In primary mouse islets, LDB1 and its LIM homeodomain-binding partner islet 1 (ISL1) were coenriched at chromatin sites occupied by pancreatic and duodenal homeobox 1 (PDX1), NK6 homeobox 1 (NKX6.1), forkhead box A2 (FOXA2), and NK2 homeobox 2 (NKX2.2) - factors that co-occupy active enhancers in 3D chromatin domains in human islets. Indeed, LDB1 was enriched at active enhancers in human islets. Thus, LDB1 maintains the terminally differentiated state of β cells and is a component of active enhancers in both murine and human islets.

Ldb1 and Ldb2 are coregulators that mediate Lin11-Isl1-Mec3 (LIM)–homeodomain (HD) and LIM-only transcription factor–driven gene regulation. Although both Ldb1 and Ldb2 mRNA were produced in the developing and adult pancreas, immunohistochemical analysis illustrated a broad Ldb1 protein expression pattern during early pancreatogenesis, which subsequently became enriched in islet and ductal cells perinatally. The islet-enriched pattern of Ldb1 was similar to pan-endocrine cell–expressed Islet-1 (Isl1), which was demonstrated in this study to be the primary LIM-HD transcription factor in developing and adult islet cells. Endocrine cell–specific removal of Ldb1 during mouse development resulted in a severe reduction of hormone+ cell numbers (i.e., α, β, and δ) and overt postnatal hyperglycemia, reminiscent of the phenotype described for the Isl1 conditional mutant. In contrast, neither endocrine cell development nor function was affected in the pancreas of Ldb2−/− mice. Gene expression and chromatin immunoprecipitation (ChIP) analyses demonstrated that many important Isl1-activated genes were coregulated by Ldb1, including MafA, Arx, insulin, and Glp1r. However, some genes (i.e., Hb9 and Glut2) only appeared to be impacted by Ldb1 during development. These findings establish Ldb1 as a critical transcriptional coregulator during islet α-, β-, and δ-cell development through Isl1-dependent and potentially Isl1-independent control.

Aristaless related homeodomain protein (Arx) specifies the formation of the pancreatic islet ␣-cell during development. This cell type produces glucagon, a major counteracting hormone to insulin in regulating glucose homeostasis in adults. However, little is known about the factors that regulate Arx transcription in the pancreas. In this study, we showed that the number of Arx ؉ cells was significantly reduced in the pancreata of embryos deficient for the Islet-1 (Isl-1) transcription factor, which was also supported by the reduction in Arx mRNA levels. Chromatin immunoprecipitation analysis localized Isl-1 activator binding sites within two highly conserved noncoding regulatory regions (Re) in the Arx locus, termed Re1 (؉5.6 to ؉6.1 kb) and Re2 (؉23.6 to ؉24 kb). Using cell line-based transfection assays, we demonstrated that a Re1-and Re2-driven reporter was selectively activated in islet ␣-cells, a process mediated by Isl-1 in overexpression, knockdown, and site-directed mutation experiments. Moreover, Arx mRNA levels were upregulated in islet ␣-cells upon Isl-1 overexpression in vivo. Isl-1 represents the first known activator of Arx transcription in ␣-cells, here established to be acting through the conserved Re1 and Re2 control domains.Pancreatic islets play an important role in regulating carbohydrate metabolism through the production and secretion of hormones. The five cell types found in the islet are ␣-, ␤-, ␦-, ⑀-, and pancreatic polypeptide cells that, respectively, produce the hormones glucagon, insulin, somatostatin, ghrelin, and pancreatic polypeptide (1). The hormonal products of the predominant ␣-and ␤-cells act in peripheral tissues in a counter-regulatory manner to control blood glucose homeostasis, with insulin promoting cellular glucose uptake and storage and glucagon promoting its release. Notably, although insulin resistance and ␤-cell dysfunction are the major causes of diabetes, the sustained, unregulated secretion of glucagon from ␣-cells also contributes to hyperglycemia and the associated complications (2-6). In fact, suppression of glucagon activity or levels has been shown to be a promising treatment for diabetics (3, 6 -9). Unfortunately, and in contrast to islet ␤-cells, our understanding of the factors involved in controlling ␣-cell differentiation and function is quite limited (1, 10).Glucagon ϩ cells first appear at embryonic day (E) 9.5 3 in mice, followed by insulin ϩ cells a day later (1). At around E13.5, a major expansion of a distinct population of hormone-producing cells begins to occur, with only these cells becoming mature islet ␣-and ␤-cells (11). A variety of distinct transcription factors are required during development for their production (1), including those that are necessary early in specifying the ␣-(e.g. Arx) and ␤-(e.g. Pax4 and Nkx6.1) lineages and others acting later in cell maintenance and maturation (e.g. MafB, Foxa2, Isl-1, and Pdx1) (12)(13)(14)(15)(16)(17)(18)(19)(20).Arx plays an essential role in islet ␣-cell formation. Expression of this transcrip...

ObjectiveHistone deacetylases are epigenetic regulators known to control gene transcription in various tissues. A member of this family, histone deacetylase 3 (HDAC3), has been shown to regulate metabolic genes. Cell culture studies with HDAC-specific inhibitors and siRNA suggest that HDAC3 plays a role in pancreatic β-cell function, but a recent genetic study in mice has been contradictory. Here we address the functional role of HDAC3 in β-cells of adult mice.MethodsAn HDAC3 β-cell specific knockout was generated in adult MIP-CreERT transgenic mice using the Cre-loxP system. Induction of HDAC3 deletion was initiated at 8 weeks of age with administration of tamoxifen in corn oil (2 mg/day for 5 days). Mice were assayed for glucose tolerance, glucose-stimulated insulin secretion, and islet function 2 weeks after induction of the knockout. Transcriptional functions of HDAC3 were assessed by ChIP-seq as well as RNA-seq comparing control and β-cell knockout islets.ResultsHDAC3 β-cell specific knockout (HDAC3βKO) did not increase total pancreatic insulin content or β-cell mass. However, HDAC3βKO mice demonstrated markedly improved glucose tolerance. This improved glucose metabolism coincided with increased basal and glucose-stimulated insulin secretion in vivo as well as in isolated islets. Cistromic and transcriptomic analyses of pancreatic islets revealed that HDAC3 regulates multiple genes that contribute to glucose-stimulated insulin secretion.ConclusionsHDAC3 plays an important role in regulating insulin secretion in vivo, and therapeutic intervention may improve glucose homeostasis.

V-ATPases are molecular motors that reversibly disassemble in vivo. Anchored in the membrane is subunit a. Subunit a has a movable N terminus that switches positions during disassembly and reassembly. Deletions were made at residues securing the N terminus of subunit a (yeast isoform Vph1) to its membranebound C-terminal domain in order to understand the role of this conserved region for V-ATPase function. Shrinking of the tether made cells pH-sensitive (vma phenotype) because assembly of V 0 subunit d was harmed. Subunit d did not co-immunoprecipitate with subunit a and the c-ring. Cells contained pools of V 1 and V 0 (؊d) that failed to form V 1 V 0 , and very low levels of V-ATPase subunits were found at the membrane. Although subunit d expression was stable and at wild-type levels, growth defects were rescued by exogenous VMA6 (subunit d). Stable V 1 V 0 assembled after yeast cells were co-transformed with VMA6 and mutant VPH1. Tether-less V 1 V 0 was delivered to the vacuole and active. It retained 63-71% of the wild-type activity and was responsive to glucose. Tether-less V 1 V 0 disassembled and reassembled after brief glucose depletion and readdition. The N terminus retained binding to V 1 subunits and the C terminus to phosphofructokinase. Thus, no major structural change was generated at the N and C termini of subunit a. We concluded that early steps of V 0 assembly and trafficking were likely impaired by shorter tethers and rescued by VMA6. V-ATPase4 proton pumps are highly conserved proteins fundamental for pH homeostasis (for review, See Refs. 1-6). Located in the endomembrane system, V-ATPases establish and maintain the low pH essential for endocytic and exocytic vesicular transport, zymogen activation, and protein sorting (for review, see Refs. 1-3). Cells specialized for active proton secretion, like kidney epithelial cells and osteoclasts, also express V-ATPases at the plasma membrane, where they transfer protons from the cytosol to the extracellular milieu (4, 5). In the kidney, plasma membrane V-ATPases of the intercalated cells are critical for regulation of the systemic acid-base balance (5, 6). Mutations in human kidney V-ATPase cause distal-renal tubular acidosis (6). V-ATPases at the plasma membrane of osteoclasts are essential for bone resorption, and mutations result in osteopetrosis, a disease characterized by thickening of the bones (1, 4, 7). Complete loss of V-ATPase activity is lethal in eukaryotes other than fungi (3).V-ATPases are multisubunit complexes that consist of two domains, V 1 (peripheral) and V 0 (membrane-bound) (1, 2). Each of the subunits in the V-ATPase complex is critical for function and V 1 V 0 assembly (8). Deletion of a peripheral V 1 subunit leads to disruption of the entire V 1 domain in yeast. Loss of a V 0 subunit does not affect V 1 assembly but disrupts the entire V 0 domain, which also prevents V 1 from associating with the membrane. An exception is subunit a for which two functional isoforms (Vph1, Stv1) exist in yeast (9). Disruption of subunit a requi...

The broadly expressed transcriptional coregulator LDB1 is essential for β-cell development and glucose homeostasis. However, it is unclear whether LDB1 has metabolic roles beyond the β-cell, especially under metabolic stress. Global Ldb1 deletion results in early embryonic lethality; thus, we used global heterozygous Ldb1+/- and inducible β-cell-specific Ldb1-deficient (Ldb1Δβ-cell) mice. We assessed glucose and insulin tolerance, body composition, feeding, and energy expenditure during high-fat diet exposure. Brown adipose tissue (BAT) biology was evaluated by thermogenic gene expression and LDB1 chromatin immunoprecipitation analysis. We found that partial loss of Ldb1 does not impair the maintenance of glucose homeostasis; rather, we observed improved insulin sensitivity in these mice. Partial loss of Ldb1 also uncovered defects in energy expenditure in lean and diet-induced obese (DIO) mice. This decreased energy expenditure during DIO was associated with significantly altered BAT gene expression, specifically Cidea, Elovl3, Cox7a1, and Dio2. Remarkably, the observed changes in energy balance during DIO were absent in Ldb1Δβ-cell mice, despite a similar reduction in plasma insulin, suggesting a role for LDB1 in BAT. Indeed, LDB1 is expressed in brown adipocytes and occupies a regulatory domain of Elovl3, a gene crucial to normal BAT function. We conclude that LDB1 regulates energy homeostasis, in part through transcriptional modulation of critical regulators in BAT function.

V1V0‐ATPase proton pumps are multisubunit proteins consisting of integral and peripheral subunits regulated by reversible disassembly in response to the extracellular concentration of glucose; it is hypothesized that disassembly is an energy conserving strategy to preserve ATP due to limited glucose. In the absence of glucose, the peropheral sub‐complex (V1) is released into the cytoplasm and the membrane sub‐complex (Vo) remains integral. The disassembled enzyme is catalytically inactive but addition of glucose to glucose‐deprived cells yields reassembly of a functional holoenzyme. This project studies subunit d, a key subunit coupling ATP hydrolysis to proton transport. Subunit d remains bound to the membrane upon disassembly and interacts with different V1 and Vo subunits depending on the V‐ATPase's state of assembly. We used computational models of subunit d to introduce cysteine residues that will be photo‐chemically cross‐linked to neighboring V‐ATPase subunits using the sulfhydryl reagent MBP. This study will map subunit interactions involving subunit d in the assembled and disassembled states. It will generate new information regarding the roles of subunit d for V‐ATPase function and the mechanism of glucose‐dependent dissociation.Supported by NSF