The Ldb1 transcriptional co-regulator is required for establishment and maintenance of the pancreatic endocrine lineage.

Pancreatic islet cell development is regulated by transcription factors (TFs) that mediate embryonic progenitor differentiation toward mature endocrine cells. Prior studies from our lab and others showed that the islet-enriched TF, Islet-1 (Isl1), interacts with the broadly-expressed transcriptional co-regulator, Ldb1, to regulate islet cell maturation and postnhyperatal function (by embryonic day (E)18.5). However, Ldb1 is expressed in the developing pancreas prior to Isl1 expression, notably in multipotent progenitor cells (MPCs) marked by Pdx1 and endocrine progenitors (EPs) expressing Neurogenin-3 (Ngn3). MPCs give rise to the endocrine and exocrine pancreas, while Ngn3+ EPs specify pancreatic islet endocrine cells. We hypothesized that Ldb1 is required for progenitor identity in MPC and EP populations during development to impact islet appearance and function. To test this, we generated a whole-pancreas Ldb1 knockout, termed Ldb1ΔPanc , and observed severe developmental and postnatal pancreas defects including disorganized progenitor pools, a significant reduction of Ngn3-expressing EPs, Pdx1HI ß-cells, and early hormone+ cells. Ldb1ΔPanc neonates presented with severe hyperglycemia, hypoinsulinemia, and drastically reduced hormone expression in islets, yet no change in total pancreas mass. This supports the endocrine-specific actions of Ldb1. Considering this, we also developed an endocrine-enriched model of Ldb1 loss, termed Ldb1ΔEndo . We observed similar dysglycemia in this model, as well as a loss of islet identity markers. Through in vitro and in vivo chromatin immunoprecipitation experiments, we found that Ldb1 occupies key Pdx1 and Ngn3 promoter domains. Our findings provide insight into novel regulation of endocrine cell differentiation that may be vital toward improving cell-based diabetes therapies.

Xenotransplantation of tannic acid-encapsulated neonatal porcine islets decreases proinflammatory innate immune responses.

BACKGROUND: Islet transplantation with neonatal porcine islets (NPIs) is a promising treatment for type 1 diabetes (T1D), but immune rejection poses a major hurdle for clinical use. Innate immune-derived reactive oxygen species (ROS) synthesis can facilitate islet xenograft destruction and enhance adaptive immune responses. METHODS: To suppress ROS-mediated xenograft destruction, we utilized nanothin encapsulation materials composed of multilayers of tannic acid (TA), an antioxidant, and a neutral polymer, poly(N-vinylpyrrolidone) (PVPON). We hypothesized that (PVPON/TA)-encapsulated NPIs will maintain euglycemia and dampen proinflammatory innate immune responses following xenotransplantation. RESULTS: (PVPON/TA)-encapsulated NPIs were viable and glucose-responsive similar to non-encapsulated NPIs. Transplantation of (PVPON/TA)-encapsulated NPIs into hyperglycemic C57BL/6.Rag or NOD.Rag mice restored euglycemia, exhibited glucose tolerance, and maintained islet-specific transcription factor levels similar to non-encapsulated NPIs. Gene expression analysis of (PVPON/TA)-encapsulated grafts post-transplantation displayed reduced proinflammatory Ccl5, Cxcl10, Tnf, and Stat1 while enhancing alternatively activated macrophage Retnla, Arg1, and Stat6 mRNA accumulation compared with controls. Flow cytometry analysis demonstrated significantly reduced innate immune infiltration, MHC-II, co-stimulatory molecule, and TNF expression with concomitant increases in arginase-1+ macrophages and dendritic cells. Similar alterations in immune responses were observed following xenotransplantation into immunocompetent NOD mice. CONCLUSION: Our data suggest that (PVPON/TA) encapsulation of NPIs is an effective strategy to decrease inflammatory innate immune signals involved in NPI xenograft responses through STAT1/6 modulation without compromising islet function.

LIM-domain transcription complexes interact with ring-finger ubiquitin ligases and thereby impact islet ß-cell function.

Diabetes is characterized by a loss of ß-cell mass, and a greater understanding of the transcriptional mechanisms governing ß-cell function is required for future therapies. Previously, we reported that a complex of the Islet-1 (Isl1) transcription factor and the co-regulator single-stranded DNA-binding protein 3 (SSBP3) regulates the genes necessary for ß-cell function, but few proteins are known to interact with this complex in ß-cells. To identify additional components, here we performed SSBP3 reverse-cross-linked immunoprecipitation (ReCLIP)- and MS-based experiments with mouse ß-cell extracts and compared the results with those from our previous Isl1 ReCLIP study. Our analysis identified the E3 ubiquitin ligases ring finger protein 20 (RNF20) and RNF40, factors that in nonpancreatic cells regulate transcription through imparting monoubiquitin marks on histone H2B (H2Bub1), a precursor to histone H3 lysine 4 trimethylation (H3K4me3). We hypothesized that RNF20 and RNF40 regulate similar genes as those regulated by Isl1 and SSBP3 and are important for ß-cell function. We observed that Rnf20 and Rnf40 depletion reduces ß-cell H2Bub1 marks and uncovered several target genes, including glucose transporter 2 (Glut2), MAF BZIP transcription factor A (MafA), and uncoupling protein 2 (Ucp2). Strikingly, we also observed that Isl1 and SSBP3 depletion reduces H2Bub1 and H3K4me3 marks, suggesting that they have epigenetic roles. We noted that the RNF complex is required for glucose-stimulated insulin secretion and normal mitochondrial reactive oxygen species levels. These findings indicate that RNF20 and RNF40 regulate ß-cell gene expression and insulin secretion and establish a link between Isl1 complexes and global cellular epigenetics.

The islet-expressed Lhx1 transcription factor interacts with Islet-1 and contributes to glucose homeostasis.

The LIM-homeodomain (LIM-HD) transcription factor Islet-1 (Isl1) interacts with the LIM domain-binding protein 1 (Ldb1) coregulator to control expression of key pancreatic ß-cell genes. However, Ldb1 also has Isl1-independent effects, supporting that another LIM-HD factor interacts with Ldb1 to impact ß-cell development and/or function. LIM homeobox 1 (Lhx1) is an Isl1-related LIM-HD transcription factor that appears to be expressed in the developing mouse pancreas and in adult islets. However, roles for this factor in the pancreas are unknown. This study aimed to determine Lhx1 interactions and elucidate gene regulatory and physiological roles in the pancreas. Co-immunoprecipitation using ß-cell extracts demonstrated an interaction between Lhx1 and Isl1, and thus we hypothesized that Lhx1 and Isl1 regulate similar target genes. To test this, we employed siRNA-mediated Lhx1 knockdown in ß-cell lines and discovered reduced Glp1R mRNA. Chromatin immunoprecipitation revealed Lhx1 occupancy at a domain also known to be occupied by Isl1 and Ldb1. Through development of a pancreas-wide knockout mouse model ( Lhx1∆Panc), we demonstrate that aged Lhx1∆Panc mice have elevated fasting blood glucose levels, altered intraperitoneal and oral glucose tolerance, and significantly upregulated glucagon, somatostatin, pancreatic polypeptide, MafB, and Arx islet mRNAs. Additionally, Lhx1∆Panc mice exhibit significantly reduced Glp1R, an mRNA encoding the insulinotropic receptor for glucagon-like peptide 1 along with a concomitant dampened Glp1 response and mild glucose intolerance in mice challenged with oral glucose. These data are the first to reveal that the Lhx1 transcription factor contributes to normal glucose homeostasis and Glp1 responses.

Transcriptional activity of the islet ß cell factor Pdx1 is augmented by lysine methylation catalyzed by the methyltransferase Set7/9.

The transcription factor Pdx1 is crucial to islet ß cell function and regulates target genes in part through interaction with coregulatory factors. Set7/9 is a Lys methyltransferase that interacts with Pdx1. Here we tested the hypothesis that Lys methylation of Pdx1 by Set7/9 augments Pdx1 transcriptional activity. Using mass spectrometry and mutational analysis of purified proteins, we found that Set7/9 methylates the N-terminal residues Lys-123 and Lys-131 of Pdx1. Methylation of these residues occurred only in the context of intact, full-length Pdx1, suggesting a specific requirement of secondary and/or tertiary structural elements for catalysis by Set7/9. Immunoprecipitation assays and mass spectrometric analysis using ß cells verified Lys methylation of endogenous Pdx1. Cell-based luciferase reporter assays using wild-type and mutant transgenes revealed a requirement of Pdx1 residue Lys-131, but not Lys-123, for transcriptional augmentation by Set7/9. Lys-131 was not required for high-affinity interactions with DNA in vitro, suggesting that its methylation likely enhances post-DNA binding events. To define the role of Set7/9 in ß cell function, we generated mutant mice in which the gene encoding Set7/9 was conditionally deleted in ß cells (Set(Δ)ß). Set(Δ)ß mice exhibited glucose intolerance similar to Pdx1-deficient mice, and their isolated islets showed impaired glucose-stimulated insulin secretion with reductions in expression of Pdx1 target genes. Our results suggest a previously unappreciated role for Set7/9-mediated methylation in the maintenance of Pdx1 activity and ß cell function.

The FOXP1, FOXP2 and FOXP4 transcription factors are required for islet alpha cell proliferation and function in mice.

AIMS/HYPOTHESIS: Several forkhead box (FOX) transcription factor family members have important roles in controlling pancreatic cell fates and maintaining beta cell mass and function, including FOXA1, FOXA2 and FOXM1. In this study we have examined the importance of FOXP1, FOXP2 and FOXP4 of the FOXP subfamily in islet cell development and function. METHODS: Mice harbouring floxed alleles for Foxp1, Foxp2 and Foxp4 were crossed with pan-endocrine Pax6-Cre transgenic mice to generate single and compound Foxp mutant mice. Mice were monitored for changes in glucose tolerance by IPGTT, serum insulin and glucagon levels by radioimmunoassay, and endocrine cell development and proliferation by immunohistochemistry. Gene expression and glucose-stimulated hormone secretion experiments were performed with isolated islets. RESULTS: Only the triple-compound Foxp1/2/4 conditional knockout (cKO) mutant had an overt islet phenotype, manifested physiologically by hypoglycaemia and hypoglucagonaemia. This resulted from the reduction in glucagon-secreting alpha cell mass and function. The proliferation of alpha cells was profoundly reduced in Foxp1/2/4 cKO islets through the effects on mediators of replication (i.e. decreased Ccna2, Ccnb1 and Ccnd2 activators, and increased Cdkn1a inhibitor). Adult islet Foxp1/2/4 cKO beta cells secrete insulin normally while the remaining alpha cells have impaired glucagon secretion. CONCLUSIONS/INTERPRETATION: Collectively, these findings reveal an important role for the FOXP1, 2, and 4 proteins in governing postnatal alpha cell expansion and function.

Characterization of an apparently novel ß-cell line-enriched 80-88 kDa transcriptional activator of the MafA and Pdx1 genes.

MafA and Pdx1 represent critical transcriptional regulators required for the maintenance of pancreatic islet ß-cell function. The in vivo ß-cell-enriched expression pattern of these genes is principally directed by islet transcription factors binding within conserved Region 3 (base pairs (bp) -8118/-7750) of MafA and Area II (bp -2153/-1923) of the Pdx1 gene. Comprehensive mutational analysis of conserved MafA Region 3 revealed two new ß-cell line-specific cis-activation elements, termed Site 4 (bp -7997 to -7988) and Site 12 (bp -7835 to -7826). Gel mobility and antibody super-shift analysis identified Pdx1 as the Site 4 binding factor, while an 80-88 kilodalton (kDa) ß-cell line-enriched protein complex bound Site 12 and similar aligned nucleotides within Pdx1 Area II. The 80-88 kDa activator was also found in adult mouse islet extract. Strikingly, the molecular weight, DNA binding, and antibody recognition properties of this activator were unique when compared with all other key islet transcription factors tested, including Prox1 (83 kDa), Hnf1α (67 kDa), FoxA2 (48 kDa), MafA (46 kDa), Isl1 (44 kDa), Pdx1 (42 kDa), and Nkx2.2 (30 kDa). Collectively, these data define an apparently novel MafA Region 3 and Pdx1 Area II activator contributing to expression in ß-cells.

The SSBP3 co-regulator is required for glucose homeostasis, pancreatic islet architecture, and beta-cell identity.

OBJECTIVE: Transcriptional complex activity drives the development and function of pancreatic islet cells to allow for proper glucose regulation. Prior studies from our lab and others highlighted that the LIM-homeodomain transcription factor (TF), Islet-1 (Isl1), and its interacting co-regulator, Ldb1, are vital effectors of developing and adult ß-cells. We further found that a member of the Single Stranded DNA-Binding Protein (SSBP) co-regulator family, SSBP3, interacts with Isl1 and Ldb1 in ß-cells and primary islets (mouse and human) to impact ß-cell target genes MafA and Glp1R in vitro. Members of the SSBP family stabilize TF complexes by binding directly to Ldb1 and protecting the complex from ubiquitin-mediated turnover. In this study, we hypothesized that SSBP3 has critical roles in pancreatic islet cell function in vivo, similar to the Isl1::Ldb1 complex. METHODS: We first developed a novel SSBP3 LoxP allele mouse line, where Cre-mediated recombination imparts a predicted early protein termination. We bred this mouse with constitutive Cre lines (Pdx1- and Pax6-driven) to recombine SSBP3 in the developing pancreas and islet (SSBP3ΔPanc and SSBP3ΔIslet), respectively. We assessed glucose tolerance and used immunofluorescence to detect changes in islet cell abundance and markers of ß-cell identity and function. Using an inducible Cre system, we also deleted SSBP3 in the adult ß-cell, a model termed SSBP3Δß-cell. We measured glucose tolerance as well as glucose-stimulated insulin secretion (GSIS), both in vivo and in isolated islets in vitro. Using islets from control and SSBP3Δß-cell we conducted RNA-Seq and compared our results to published datasets for similar ß-cell specific Ldb1 and Isl1 knockouts to identify commonly regulated target genes. RESULTS: SSBP3ΔPanc and SSBP3ΔIslet neonates present with hyperglycemia. SSBP3ΔIslet mice are glucose intolerant by P21 and exhibit a reduction of ß-cell maturity markers MafA, Pdx1, and UCN3. We observe disruptions in islet cell architecture with an increase in glucagon+ α-cells and ghrelin+ ε-cells at P10. Inducible loss of ß-cell SSBP3 in SSBP3Δß-cell causes hyperglycemia, glucose intolerance, and reduced GSIS. Transcriptomic analysis of 14-week-old SSBP3Δß-cell islets revealed a decrease in ß-cell function gene expression (Ins, MafA, Ucn3), increased stress and dedifferentiation markers (Neurogenin-3, Aldh1a3, Gastrin), and shared differentially expressed genes between SSBP3, Ldb1, and Isl1 in adult ß-cells. CONCLUSIONS: SSBP3 drives proper islet identity and function, where its loss causes altered islet-cell abundance and glucose homeostasis. ß-Cell SSBP3 is required for GSIS and glucose homeostasis, at least partially through shared regulation of Ldb1 and Isl1 target genes.

ST6GAL1 sialyltransferase promotes acinar to ductal metaplasia and pancreatic cancer progression.

The role of aberrant glycosylation in pancreatic ductal adenocarcinoma (PDAC) remains an under-investigated area of research. In this study, we determined that ST6 ß-galactoside α2,6 sialyltransferase 1 (ST6GAL1), which adds α2,6-linked sialic acids to N-glycosylated proteins, was upregulated in patients with early-stage PDAC and was further increased in advanced disease. A tumor-promoting function for ST6GAL1 was elucidated using tumor xenograft experiments with human PDAC cells. Additionally, we developed a genetically engineered mouse (GEM) model with transgenic expression of ST6GAL1 in the pancreas and found that mice with dual expression of ST6GAL1 and oncogenic KRASG12D had greatly accelerated PDAC progression compared with mice expressing KRASG12D alone. As ST6GAL1 imparts progenitor-like characteristics, we interrogated ST6GAL1's role in acinar to ductal metaplasia (ADM), a process that fosters neoplasia by reprogramming acinar cells into ductal, progenitor-like cells. We verified ST6GAL1 promotes ADM using multiple models including the 266-6 cell line, GEM-derived organoids and tissues, and an in vivo model of inflammation-induced ADM. EGFR is a key driver of ADM and is known to be activated by ST6GAL1-mediated sialylation. Importantly, EGFR activation was dramatically increased in acinar cells and organoids from mice with transgenic ST6GAL1 expression. These collective results highlight a glycosylation-dependent mechanism involved in early stages of pancreatic neoplasia.

Islet-1 regulates Arx transcription during pancreatic islet alpha-cell development.

Aristaless related homeodomain protein (Arx) specifies the formation of the pancreatic islet α-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 α-cells, a process mediated by Isl-1 in overexpression, knockdown, and site-directed mutation experiments. Moreover, Arx mRNA levels were up-regulated in islet α-cells upon Isl-1 overexpression in vivo. Isl-1 represents the first known activator of Arx transcription in α-cells, here established to be acting through the conserved Re1 and Re2 control domains.

Islet transplantation into brown adipose tissue can delay immune rejection.

Type 1 diabetes is an autoimmune disease characterized by insulin-producing ß cell destruction. Although islet transplantation restores euglycemia and improves patient outcomes, an ideal transplant site remains elusive. Brown adipose tissue (BAT) has a highly vascularized and antiinflammatory microenvironment. Because these tissue features can promote islet graft survival, we hypothesized that islets transplanted into BAT will maintain islet graft and BAT function while delaying immune-mediated rejection. We transplanted syngeneic and allogeneic islets into BAT or under the kidney capsule of streptozotocin-induced diabetic NOD.Rag and NOD mice to investigate islet graft function, BAT function, metabolism, and immune-mediated rejection. Islet grafts within BAT restored euglycemia similarly to kidney capsule controls. Islets transplanted in BAT maintained expression of islet hormones and transcription factors and were vascularized. Compared with those in kidney capsule and euglycemic mock-surgery controls, no differences in glucose or insulin tolerance, thermogenic regulation, or energy expenditure were observed with islet grafts in BAT. Immune profiling of BAT revealed enriched antiinflammatory macrophages and T cells. Compared with the kidney capsule control, there were significant delays in autoimmune and allograft rejection of islets transplanted in BAT, possibly due to increased antiinflammatory immune populations. Our data support BAT as an alternative islet transplant site that may improve graft survival.

Partners in Crime: Beta-Cells and Autoimmune Responses Complicit in Type 1 Diabetes Pathogenesis.

Type 1 diabetes (T1D) is an autoimmune disease characterized by autoreactive T cell-mediated destruction of insulin-producing pancreatic beta-cells. Loss of beta-cells leads to insulin insufficiency and hyperglycemia, with patients eventually requiring lifelong insulin therapy to maintain normal glycemic control. Since T1D has been historically defined as a disease of immune system dysregulation, there has been little focus on the state and response of beta-cells and how they may also contribute to their own demise. Major hurdles to identifying a cure for T1D include a limited understanding of disease etiology and how functional and transcriptional beta-cell heterogeneity may be involved in disease progression. Recent studies indicate that the beta-cell response is not simply a passive aspect of T1D pathogenesis, but rather an interplay between the beta-cell and the immune system actively contributing to disease. Here, we comprehensively review the current literature describing beta-cell vulnerability, heterogeneity, and contributions to pathophysiology of T1D, how these responses are influenced by autoimmunity, and describe pathways that can potentially be exploited to delay T1D.

The transcriptional co-regulator LDB1 is required for brown adipose function.

OBJECTIVE: Brown adipose tissue (BAT) is critical for thermogenesis and glucose/lipid homeostasis. Exploiting the energy uncoupling capacity of BAT may reveal targets for obesity therapies. This exploitation requires a greater understanding of the transcriptional mechanisms underlying BAT function. One potential regulator of BAT is the transcriptional co-regulator LIM domain-binding protein 1 (LDB1), which acts as a dimerized scaffold, allowing for the assembly of transcriptional complexes. Utilizing a global LDB1 heterozygous mouse model, we recently reported that LDB1 might have novel roles in regulating BAT function. However, direct evidence for the LDB1 regulation of BAT thermogenesis and substrate utilization has not been elucidated. We hypothesize that brown adipocyte-expressed LDB1 is required for BAT function. METHODS: LDB1-deficient primary cells and brown adipocyte cell lines were assessed via qRT-PCR and western blotting for altered mRNA and protein levels to define the brown adipose-specific roles. We conducted chromatin immunoprecipitation with primary BAT tissue and immortalized cell lines. Potential transcriptional partners of LDB1 were revealed by conducting LIM factor surveys via qRT-PCR in mouse and human brown adipocytes. We developed a Ucp1-Cre-driven LDB1-deficiency mouse model, termed Ldb1ΔBAT, to test LDB1 function in vivo. Glucose tolerance and uptake were assessed at thermoneutrality via intraperitoneal glucose challenge and glucose tracer studies. Insulin tolerance was measured at thermoneutrality and after stimulation with cold or the administration of the ß3-adrenergic receptor (ß3-AR) agonist CL316,243. Additionally, we analyzed plasma insulin via ELISA and insulin signaling via western blotting. Lipid metabolism was evaluated via BAT weight, histology, lipid droplet morphometry, and the examination of lipid-associated mRNA. Finally, energy expenditure and cold tolerance were evaluated via indirect calorimetry and cold challenges. RESULTS: Reducing Ldb1 in vitro and in vivo resulted in altered BAT-selective mRNA, including Ucp1, Elovl3, and Dio2. In addition, there was reduced Ucp1 induction in vitro. Impacts on gene expression may be due, in part, to LDB1 occupying Ucp1 upstream regulatory domains. We also identified BAT-expressed LIM-domain factors Lmo2, Lmo4, and Lhx8, which may partner with LDB1 to mediate activity in brown adipocytes. Additionally, we observed LDB1 enrichment in human brown adipose. In vivo analysis revealed LDB1 is required for whole-body glucose and insulin tolerance, in part through reduced glucose uptake into BAT. In Ldb1ΔBAT tissue, we found significant alterations in insulin-signaling effectors. An assessment of brown adipocyte morphology and lipid droplet size revealed larger and more unilocular brown adipocytes in Ldb1ΔBAT mice, particularly after a cold challenge. Alterations in lipid handling were further supported by reductions in mRNA associated with fatty acid oxidation and mitochondrial respiration. Finally, LDB1 is required for energy expenditure and cold tolerance in both male and female mice. CONCLUSIONS: Our findings support LDB1 as a regulator of BAT function. Furthermore, given LDB1 enrichment in human brown adipose, this co-regulator may have conserved roles in human BAT.

Glucagon receptor signaling regulates weight loss via central KLB receptor complexes.

Glucagon regulates glucose and lipid metabolism and promotes weight loss. Thus, therapeutics stimulating glucagon receptor (GCGR) signaling are promising for obesity treatment; however, the underlying mechanism(s) have yet to be fully elucidated. We previously identified that hepatic GCGR signaling increases circulating fibroblast growth factor 21 (FGF21), a potent regulator of energy balance. We reported that mice deficient for liver Fgf21 are partially resistant to GCGR-mediated weight loss, implicating FGF21 as a regulator of glucagon's weight loss effects. FGF21 signaling requires an obligate coreceptor (ß-Klotho, KLB), with expression limited to adipose tissue, liver, pancreas, and brain. We hypothesized that the GCGR-FGF21 system mediates weight loss through a central mechanism. Mice deficient for neuronal Klb exhibited a partial reduction in body weight with chronic GCGR agonism (via IUB288) compared with controls, supporting a role for central FGF21 signaling in GCGR-mediated weight loss. Substantiating these results, mice with central KLB inhibition via a pharmacological KLB antagonist, 1153, also displayed partial weight loss. Central KLB, however, is dispensable for GCGR-mediated improvements in plasma cholesterol and liver triglycerides. Together, these data suggest GCGR agonism mediates part of its weight loss properties through central KLB and has implications for future treatments of obesity and metabolic syndrome.

Glucagon-Receptor Signaling Reverses Hepatic Steatosis Independent of Leptin Receptor Expression.

Glucagon (GCG) is an essential regulator of glucose and lipid metabolism that also promotes weight loss. We have shown that glucagon-receptor (GCGR) signaling increases fatty acid oxidation (FAOx) in primary hepatocytes and reduces liver triglycerides in diet-induced obese (DIO) mice; however, the mechanisms underlying this aspect of GCG biology remains unclear. Investigation of hepatic GCGR targets elucidated a potent and previously unknown induction of leptin receptor (Lepr) expression. Liver leptin signaling is known to increase FAOx and decrease liver triglycerides, similar to glucagon action. Therefore, we hypothesized that glucagon increases hepatic LEPR, which is necessary for glucagon-mediated reversal of hepatic steatosis. Eight-week-old control and liver-specific LEPR-deficient mice (LeprΔliver) were placed on a high-fat diet for 12 weeks and then treated with a selective GCGR agonist (IUB288) for 14 days. Liver triglycerides and gene expression were assessed in liver tissue homogenates. Administration of IUB288 in both lean and DIO mice increased hepatic Lepr isoforms a-e in acute (4 hours) and chronic (72 hours,16 days) (P < 0.05) settings. LeprΔliver mice displayed increased hepatic triglycerides on a chow diet alone (P < 0.05), which persisted in a DIO state (P < 0.001), with no differences in body weight or composition. Surprisingly, chronic administration of IUB288 in DIO control and LeprΔliver mice reduced liver triglycerides regardless of genotype (P < 0.05). Together, these data suggest that GCGR activation induces hepatic Lepr expression and, although hepatic glucagon and leptin signaling have similar liver lipid targets, these appear to be 2 distinct pathways.

Prolactin Receptor Signaling Regulates a Pregnancy-Specific Transcriptional Program in Mouse Islets.

Pancreatic ß-cells undergo profound hyperplasia during pregnancy to maintain maternal euglycemia. Failure to reprogram ß-cells into a more replicative state has been found to underlie susceptibility to gestational diabetes mellitus (GDM). We recently identified a requirement for prolactin receptor (PRLR) signaling in the metabolic adaptations to pregnancy, where ß-cell-specific PRLR knockout (ßPRLRKO) mice exhibit a metabolic phenotype consistent with GDM. However, the underlying transcriptional program that is responsible for the PRLR-dependent metabolic adaptations during gestation remains incompletely understood. To identify PRLR signaling gene regulatory networks and target genes within ß-cells during pregnancy, we performed a transcriptomic analysis of pancreatic islets isolated from either ßPRLRKO mice or littermate controls in late gestation. Gene set enrichment analysis identified forkhead box protein M1 and polycomb repressor complex 2 subunits, Suz12 and enhancer of zeste homolog 2 (Ezh2), as novel candidate regulators of PRLR-dependent ß-cell adaptation. Gene ontology term pathway enrichment revealed both established and novel PRLR signaling target genes that together promote a state of increased cellular metabolism and/or proliferation. In contrast to the requirement for ß-cell PRLR signaling in maintaining euglycemia during pregnancy, PRLR target genes were not induced following high-fat diet feeding. Collectively, the current study expands our understanding of which transcriptional regulators and networks mediate gene expression required for islet adaptation during pregnancy. The current work also supports the presence of pregnancy-specific adaptive mechanisms distinct from those activated by nutritional stress.

Three novel missense mutations within the LHX4 gene are associated with variable pituitary hormone deficiencies.

CONTEXT: The LHX4 LIM-homeodomain transcription factor has essential roles in pituitary gland and nervous system development. Heterozygous mutations in LHX4 are associated with combined pituitary hormone deficiency. OBJECTIVES: Our objectives were to determine the nature and frequency of LHX4 mutations in patients with pituitary hormone deficiency and to examine the functional outcomes of observed mutations. DESIGN: The LHX4 gene sequence was determined from patient DNA. The biochemical and gene regulatory properties of aberrant LHX4 proteins were characterized using structural predictions, pituitary gene transcription assays, and DNA binding experiments. PATIENTS: A total of 253 patients from 245 pedigrees with GH deficiency and deficiency of at least one additional pituitary hormone was included in the study. RESULTS: In five patients, three types of heterozygous missense mutations in LHX4 that result in substitution of conserved amino acids were identified. One substitution is between the LIM domains (R84C); the others are in the homeodomain (L190R; A210P). The patients have GH deficiency; some also display reductions in TSH, LH, FSH, or ACTH, and aberrant pituitary morphology. Structural models predict that the aberrant L190R and A210P LHX4 proteins would have impaired DNA binding and gene activation properties. Consistent with these models, EMSAs and transfection experiments using pituitary gene promoters demonstrate that whereas the R84C form has reduced activity, the L190R and A210P proteins are inactive. CONCLUSIONS: LHX4 mutations are a relatively rare cause of combined pituitary hormone deficiency. This report extends the range of phenotypes associated with LHX4 gene mutations and describes three novel exonic mutations in the gene.

Four novel mutations of the LHX3 gene cause combined pituitary hormone deficiencies with or without limited neck rotation.

CONTEXT: The Lhx3 LIM-homeodomain transcription factor gene is required for development of the pituitary and motoneurons in mice. Human LHX3 gene mutations have been reported in five subjects with a phenotype consisting of GH, prolactin, TSH, LH, and FSH deficiency; abnormal pituitary morphology; and limited neck rotation. OBJECTIVE: The objective of the study was to determine the frequency and nature of LHX3 mutations in patients with isolated GH deficiency or combined pituitary hormone deficiency (CPHD) and characterize the molecular consequences of mutations. DESIGN: The LHX3 sequence was determined. The biochemical properties of aberrant LHX3 proteins resulting from observed mutations were characterized using reporter gene and DNA binding experiments. PATIENTS: The study included 366 patients with isolated GH deficiency or CPHD. RESULTS: In seven patients with CPHD from four consanguineous pedigrees, four novel, recessive mutations were identified: a deletion of the entire gene (del/del), mutations causing truncated proteins (E173ter, W224ter), and a mutation causing a substitution in the homeodomain (A210V). The mutations were associated with diminished DNA binding and pituitary gene activation, consistent with observed hormone deficiencies. Whereas subjects with del/del, E173ter, and A210V mutations had limited neck rotation, patients with the W224ter mutation did not. CONCLUSIONS: LHX3 mutations are a rare cause of CPHD involving deficiencies for GH, prolactin, TSH, and LH/FSH in all patients. Whereas most patients have a severe hormone deficiency manifesting after birth, milder forms can be observed, and limited neck rotation is not a universal feature of patients with LHX3 mutations. This study extends the known molecular defects and range of phenotypes found in LHX3-associated diseases.

Mutations in the LHX3 gene cause dysregulation of pituitary and neural target genes that reflect patient phenotypes.

The LHX3 LIM-homeodomain transcription factor is required for correct development of the mammalian pituitary gland and spinal motoneurons. Mutations in the LHX3 gene underlie complex diseases featuring combined anterior pituitary hormone deficiency and, in specific cases, loss of neck rotation considered to result from nervous system abnormalities. The molecular basis for LHX3 protein actions in both normal and aberrant pituitary and nervous system development is poorly understood. In this study, the gene regulatory abilities of mutant LHX3 proteins associated with distinct types of diseases (LHX3a A210V, LHX3a E173Ter, and LHX3a W224Ter) were investigated. The capacity of these proteins to activate pituitary hormone and transcription factor gene promoters, nervous system target genes, and to localize to the nucleus of pituitary cells was measured. Consistent with the symptoms of patients with these mutations, the abnormal proteins displayed diminished capacities to activate the promoters of genes expressed in the pituitary gland. On nervous system promoters, several mutant proteins retained some activity. The ability of the mutant proteins to concentrate in the nucleus of pituitary cells was correlated with the retention of defined nuclear localization signals in the protein sequence, except for the E173Ter protein which unexpectedly localizes to the nucleus, likely due to the insertion of cryptic nuclear localization signals by a frame shift caused by the mutation. This study extends the molecular characterization of the severe neuroendocrine diseases associated with LHX3 gene mutations.

Roles of the LHX3 and LHX4 LIM-homeodomain factors in pituitary development.

The LHX3 and LHX4 LIM-homeodomain transcription factors play essential roles in pituitary gland and nervous system development. Mutations in the genes encoding these regulatory proteins are associated with combined hormone deficiency diseases in humans and animal models. Patients with these diseases have complex syndromes involving short stature, and reproductive and metabolic disorders. Analyses of the features of these diseases and the biochemical properties of the LHX3 and LHX4 proteins will facilitate a better understanding of the molecular pathways that regulate the development of the specialized hormone-secreting cells of the mammalian anterior pituitary gland.