Maternal folic acid supplementation with vitamin B12 deficiency during pregnancy and lactation affects the metabolic health of adult female offspring but is dependent on offspring diet.

Epidemiologic studies have reported relationships between maternal high folate and/or low B12 status during pregnancy and greater adiposity and insulin resistance in children. The goal of this study was to determine the effects of maternal folic acid supplementation (10 mg/kg diet), with (50 µg/kg diet) and without B12, on adult female offspring adiposity and glucose homeostasis. Female C57BL/6J mice were fed 1 of 3 diets from weaning and throughout breeding, pregnancy, and lactation: control (2 mg/kg diet folic acid, 50 µg/kg diet B12), supplemental folic acid with no B12 (SFA-B12), or supplemental folic acid with adequate B12 (SFA+B12). Female offspring were weaned onto the control diet or a Western diet (45% energy fat, 2 mg/kg diet folic acid, 50 µg/kg diet B12) for 35 wk. After weaning, control diet-fed offspring with SFA-B12 dams had fasting hyperglycemia, glucose intolerance, lower ß cell mass, and greater islet hepatocyte nuclear factor 1 homeobox α and nuclear receptor subfamily 1 group H member 3 mRNA than did offspring from control dams. In Western diet-fed offspring, those with SFA-B12 dams had lower fasting blood glucose and plasma insulin concentrations, and were smaller than control offspring. Our findings suggest that maternal folic acid supplementation with B12 deficiency during pregnancy/lactation programs the metabolic health of adult female offspring but is dependent on offspring diet.-Henderson, A. M., Tai, D. C., Aleliunas, R. E., Aljaadi, A. M., Glier, M. B., Xu, E. E., Miller, J. W., Verchere, C. B., Green, T. J., Devlin, A. M. Maternal folic acid supplementation with vitamin B12 deficiency during pregnancy and lactation affects the metabolic health of adult female offspring but is dependent on offspring diet.

SOX4 cooperates with neurogenin 3 to regulate endocrine pancreas formation in mouse models.

AIMS/HYPOTHESIS: The sex-determining region Y (SRY)-related high mobility group (HMG) box (SOX) family of transcription factors is essential for normal organismal development. Despite the longstanding knowledge that many SOX family members are expressed during pancreas development, a role for many of these factors in the establishment of insulin-producing beta cell fate remains to be determined. The aim of this study is to elucidate the role of SOX4 during beta cell development. METHODS: We used pancreas and endocrine progenitor mouse knockouts of Sox4 to uncover the roles of SOX4 during pancreas development. Lineage tracing and in vitro models were used to determine how SOX4 regulates beta cell formation and understand the fate of Sox4-null endocrine lineage cells. RESULTS: This study demonstrates a progenitor cell-autonomous role for SOX4 in regulating the genesis of beta cells and shows that it is required at multiple stages of the process. SOX4 deletion in the multipotent pancreatic progenitors resulted in impaired endocrine progenitor cell differentiation. Deletion of SOX4 later in the Neurog3-expressing cells also caused reductions in beta cells. Lineage studies showed loss of Sox4 in endocrine progenitors resulted in a block in terminal islet cell differentiation that was attributed to reduction in the production of key beta cell specification factors. CONCLUSIONS/INTERPRETATION: These results demonstrate that SOX4 is essential for normal endocrine pancreas development both concomitant with, and downstream of, the endocrine fate decision. In conclusion, these studies position Sox4 temporally in the endocrine differentiation programme and provide a new target for improving in vitro differentiation of glucose-responsive pancreatic beta cells.

Type 2 diabetes susceptibility gene GRK5 regulates physiological pancreatic ß-cell proliferation via phosphorylation of HDAC5.

Restoring functional ß cell mass is a potential therapy for those with diabetes. However, the pathways regulating ß cell mass are not fully understood. Previously, we demonstrated that Sox4 is required for ß cell proliferation during prediabetes. Here, we report that Sox4 regulates ß cell mass through modulating expression of the type 2 diabetes (T2D) susceptibility gene GRK5. ß cell-specific Grk5 knockout mice showed impaired glucose tolerance with reduced ß cell mass, which was accompanied by upregulation of cell cycle inhibitor gene Cdkn1a. Furthermore, we found that Grk5 may drive ß cell proliferation through a pathway that includes phosphorylation of HDAC5 and subsequent transcription of immediate-early genes (IEGs) such as Nr4a1, Fosb, Junb, Arc, Egr1, and Srf. Together, these studies suggest GRK5 is linked to T2D through regulation of ß cell growth and that it may be a target to preserve ß cells during the development of T2D.

Single-Cell Transcriptome Profiling of Mouse and hESC-Derived Pancreatic Progenitors.

Human embryonic stem cells (hESCs) are a potential unlimited source of insulin-producing ß cells for diabetes treatment. A greater understanding of how ß cells form during embryonic development will improve current hESC differentiation protocols. All pancreatic endocrine cells, including ß cells, are derived from Neurog3-expressing endocrine progenitors. This study characterizes the single-cell transcriptomes of 6,905 mouse embryonic day (E) 15.5 and 6,626 E18.5 pancreatic cells isolated from Neurog3-Cre; Rosa26mT/mG embryos, allowing for enrichment of endocrine progenitors (yellow; tdTomato + EGFP) and endocrine cells (green; EGFP). Using a NEUROG3-2A-eGFP CyT49 hESC reporter line (N5-5), 4,462 hESC-derived GFP+ cells were sequenced. Differential expression analysis revealed enrichment of markers that are consistent with progenitor, endocrine, or previously undescribed cell-state populations. This study characterizes the single-cell transcriptomes of mouse and hESC-derived endocrine progenitors and serves as a resource (https://lynnlab.shinyapps.io/embryonic_pancreas) for improving the formation of functional ß-like cells from hESCs.

SOX4 Allows Facultative ß-Cell Proliferation Through Repression of Cdkn1a.

The high-mobility group box transcription factor SOX4 is the most highly expressed SOX family protein in pancreatic islets, and mutations in Sox4 are associated with an increased risk of developing type 2 diabetes. We used an inducible ß-cell knockout mouse model to test the hypothesis that Sox4 is essential for the maintenance of ß-cell number during the development of type 2 diabetes. Knockout of Sox4 at 6 weeks of age resulted in time-dependent worsening of glucose tolerance, impairment of insulin secretion, and diabetes by 30 weeks of age. Immunostaining revealed a decrease in ß-cell mass in knockout mice that was caused by a 39% reduction in ß-cell proliferation. Gene expression studies revealed that induction of the cell cycle inhibitor Cdkn1a was responsible for the decreased proliferation in the knockout animals. Altogether, this study demonstrates that SOX4 is necessary for adult ß-cell replication through direct regulation of the ß-cell cycle.

Reduced Insulin Production Relieves Endoplasmic Reticulum Stress and Induces ß Cell Proliferation.

Pancreatic ß cells are mostly post-mitotic, but it is unclear what locks them in this state. Perturbations including uncontrolled hyperglycemia can drive ß cells into more pliable states with reduced cellular insulin levels, increased ß cell proliferation, and hormone mis-expression, but it is unknown whether reduced insulin production itself plays a role. Here, we define the effects of ∼50% reduced insulin production in Ins1(-/-):Ins2(f/f):Pdx1Cre(ERT):mTmG mice prior to robust hyperglycemia. Transcriptome, proteome, and network analysis revealed alleviation of chronic endoplasmic reticulum (ER) stress, indicated by reduced Ddit3, Trib3, and Atf4 expression; reduced Xbp1 splicing; and reduced phospho-eIF2α. This state was associated with hyper-phosphorylation of Akt, which is negatively regulated by Trib3, and with cyclinD1 upregulation. Remarkably, ß cell proliferation was increased 2-fold after reduced insulin production independently of hyperglycemia. Eventually, recombined cells mis-expressed glucagon in the hyperglycemic state. We conclude that the normally high rate of insulin production suppresses ß cell proliferation in a cell-autonomous manner.