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During pregnancy, women often become insulin resistant, thus requiring an expansion of pancreatic beta cell mass to provide more insulin. Michael German and his colleagues now report that lactogenic hormones drive the expression of serotonin in the beta cells to induce this increase in beta cell mass. During pregnancy, the energy requirements of the fetus impose changes in maternal metabolism. Increasing insulin resistance in the mother maintains nutrient flow to the growing fetus, whereas prolactin and placental lactogen counterbalance this resistance and prevent maternal hyperglycemia by driving expansion of the maternal population of insulin-producing beta cells 1 , 2 , 3 . However, the exact mechanisms by which the lactogenic hormones drive beta cell expansion remain uncertain. Here we show that serotonin acts downstream of lactogen signaling to stimulate beta cell proliferation. Expression of serotonin synthetic enzyme tryptophan hydroxylase-1 (Tph1) and serotonin production rose sharply in beta cells during pregnancy or after treatment with lactogens in vitro . Inhibition of serotonin synthesis by dietary tryptophan restriction or Tph inhibition blocked beta cell expansion and induced glucose intolerance in pregnant mice without affecting insulin sensitivity. Expression of the Gα q -linked serotonin receptor 5-hydroxytryptamine receptor-2b (Htr2b) in maternal islets increased during pregnancy and normalized just before parturition, whereas expression of the Gα i -linked receptor Htr1d increased at the end of pregnancy and postpartum. Blocking Htr2b signaling in pregnant mice also blocked beta cell expansion and caused glucose intolerance. These studies reveal an integrated signaling pathway linking beta cell mass to anticipated insulin need during pregnancy. Modulators of this pathway, including medications and diet, may affect the risk of gestational diabetes 4 .

In preparation for the metabolic demands of pregnancy, β cells in the maternal pancreatic islets increase both in number and in glucose-stimulated insulin secretion (GSIS) per cell. Mechanisms have been proposed for the increased β cell mass, but not for the increased GSIS. Because serotonin production increases dramatically during pregnancy, we tested whether flux through the ionotropic 5-HT3 receptor (Htr3) affects GSIS during pregnancy. Pregnant Htr3a ⁻/⁻ mice exhibited impaired glucose tolerance despite normally increased β cell mass, and their islets lacked the increase in GSIS seen in islets from pregnant wild-type mice. Electrophysiological studies showed that activation of Htr3 decreased the resting membrane potential in β cells, which increased Ca ²⁺ uptake and insulin exocytosis in response to glucose. Thus, our data indicate that serotonin, acting in a paracrine/autocrine manner through Htr3, lowers the β cell threshold for glucose and plays an essential role in the increased GSIS of pregnancy.

Short-term treatment with cytokines reprograms acinar cells into beta cells and restores durable normoglycemia in diabetic mice. Reprogramming of pancreatic exocrine cells into cells resembling beta cells may provide a strategy for treating diabetes. Here we show that transient administration of epidermal growth factor and ciliary neurotrophic factor to adult mice with chronic hyperglycemia efficiently stimulates the conversion of terminally differentiated acinar cells to beta-like cells. Newly generated beta-like cells are epigenetically reprogrammed, functional and glucose responsive, and they reinstate normal glycemic control for up to 248 d. The regenerative process depends on Stat3 signaling and requires a threshold number of Neurogenin 3 (Ngn3)-expressing acinar cells. In contrast to previous work demonstrating in vivo conversion of acinar cells to beta-like cells by viral delivery of exogenous transcription factors, our approach achieves acinar-to-beta-cell reprogramming through transient cytokine exposure rather than genetic modification.

MicroRNA Expression Is Required for Pancreatic Islet Cell Genesis in the Mouse Francis C. Lynn 1 , Peter Skewes-Cox 1 , Yasuhiro Kosaka 1 , Michael T. McManus 1 2 , Brian D. Harfe 3 and Michael S. German 1 4 1 Diabetes Center, Hormone Research Institute, University of California San Francisco, San Francisco, California 2 Department of Microbiology and Immunology, University of California San Francisco, San Francisco, California 3 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida 4 Department of Medicine, University of California San Francisco, San Francisco, California Address correspondence and reprint requests to Michael S. German, MD, University of California San Francisco Diabetes Center, University of California San Francisco, 513 Parnassus Ave., San Francisco, CA 94143-0534. E-mail: mgerman{at}diabetes.ucsf.edu Abstract OBJECTIVE— The generation of distinct cell types during the development of the pancreas depends on sequential changes in gene expression. We tested the hypothesis that microRNAs (miRNAs), which limit gene expression through posttranscriptional silencing, modulate the gene expression cascades involved in pancreas development. RESEARCH DESIGN AND METHODS— miRNAs were cloned and sequenced from developing pancreata, and expression of a subset of these genes was tested using locked nucleic acid in situ analyses. To assess the overall contribution of miRNAs to pancreatic development, Dicer1, an enzyme required for miRNA processing, was conditionally deleted from the developing pancreas. RESULTS— Sequencing of small RNAs identified over 125 miRNAs, including 18 novel sequences, with distinct expression domains within the developing pancreas. To test the developmental contribution of these miRNAs, we conditionally deleted the miRNA processing enzyme Dicer1 early in pancreas development. Dicer-null animals displayed gross defects in all pancreatic lineages, although the endocrine cells, and especially the insulin-producing β-cells, were most dramatically reduced. The endocrine defect was associated with an increase in the notch-signaling target Hes1 and a reduction in the formation of endocrine cell progenitors expressing the Hes1 target gene neurogenin3. CONCLUSIONS— The expression of a unique profile of miRNAs is required during pancreas development and is necessary for β-cell formation. LNA, locked nucleic acid miRNA, micoRNA Pdx-1, pancreatic duodenal homeobox-1 RIP2, rat insulin promoter 2 Footnotes Published ahead of print at http://diabetes.diabetesjournals.org on 5 September 2007. DOI: 10.2337/db07-0175. Additional information for this article can be found in an online appendix at http://dx.doi.org/10.2337/db07-0175 . The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Accepted August 26, 2007. Received February 7, 2007. DIABETES

During organogenesis, the final size of mature cell populations depends on their rates of differentiation and expansion. Because transient expression of Neurogenin3 (Neurog3) in progenitor cells in the developing pancreas initiates their differentiation to mature islet cells, we examined the role of Neurog3 in cell cycle control during this process. We found that mitotically active pancreatic progenitor cells in mouse embryos exited the cell cycle after the initiation of Neurog3 expression. Transcriptome analysis demonstrated that the Neurog3-expressing cells dramatically up-regulated the mRNA encoding cyclin-dependent kinase inhibitor 1a (Cdkn1a). In Neurog3 null mice, the islet progenitor cells failed to activate Cdkn1a expression and continued to proliferate, showing that their exit from the cell cycle requires Neurog3. Furthermore, induced transgenic expression of Neurog3 in mouse β-cells in vivo markedly decreased their proliferation, increased Cdkn1a levels, and eventually caused profound hyperglycemia. In contrast, in Cdkn1a null mice, proliferation was incompletely suppressed in the Neurog3-expressing cells. These studies reveal a crucial role for Neurog3 in regulating the cell cycle during the differentiation of islet cells and demonstrate that the subsequent down-regulation of Neurog3 allows the mature islet cell population to expand.

Insulin from the β-cells of the pancreatic islets of Langerhans controls energy homeostasis in vertebrates, and its deficiency causes diabetes mellitus. During embryonic development, the transcription factor neurogenin 3 (Neurog3) initiates the differentiation of the β-cells and other islet cell types from pancreatic endoderm, but the genetic program that subsequently completes this differentiation remains incompletely understood. Here we show that the transcription factor Rfx6 directs islet cell differentiation downstream of Neurog3. Mice lacking Rfx6 failed to generate any of the normal islet cell types except for pancreatic-polypeptide-producing cells. In human infants with a similar autosomal recessive syndrome of neonatal diabetes, genetic mapping and subsequent sequencing identified mutations in the human RFX6 gene. These studies demonstrate a unique position for Rfx6 in the hierarchy of factors that coordinate pancreatic islet development in both mice and humans. Rfx6 could prove useful in efforts to generate β-cells for patients with diabetes. Insulin production boosted by Rfx6 The transcription factor neurogenin 3 (Neurog3) initiates the differentiation of insulin-producing β-cells and other islet cell types from pancreatic endoderm in the developing embryo, but the genetic program that subsequently completes this differentiation is incompletely understood. German et al . now show that the transcription factor Rfx6 directs islet cell differentiation downstream of Neurog3. Loss of Rfx6 function in mice leads to specific loss of pancreatic-polypeptide-producing cells, while in human infants mutations in RFX6 underlie a recessive syndrome of neonatal diabetes. These studies demonstrate a unique position for Rfx6 in the hierarchy of factors coordinating pancreatic islet development. Rfx6 could prove useful in efforts to generate β-cells for patients with diabetes. Pancreatic β-cells release insulin, which controls energy homeostasis in vertebrates, and its lack causes diabetes mellitus. The transcription factor neurogenin 3 (Neurog3) initiates differentiation of β-cells and other islet cell types from pancreatic endoderm; here, the transcription factor Rfx6 is shown to direct islet cell differentiation downstream of Neurog3 in mice and humans. This may be useful in efforts to generate β-cells for patients with diabetes.

The prevalence of type 2 diabetes in the United States is projected to double or triple by 2050. We reasoned that the genes that modulate insulin production might be new targets for diabetes therapeutics. Therefore, we developed an siRNA screening system to identify genes important for the activity of the insulin promoter in beta cells. We created a subclone of the MIN6 mouse pancreatic beta cell line that expresses destabilized GFP under the control of a 362 base pair fragment of the human insulin promoter and the mCherry red fluorescent protein under the control of the constitutively active rous sarcoma virus promoter. The ratio of the GFP to mCherry fluorescence of a cell indicates its insulin promoter activity. As G protein coupled receptors (GPCRs) have emerged as novel targets for diabetes therapies, we used this cell line to screen an siRNA library targeting all known mouse GPCRs. We identified several known GPCR regulators of insulin secretion as regulators of the insulin promoter. One of the top positive regulators was Gpr27, an orphan GPCR with no known role in beta cell function. We show that knockdown of Gpr27 reduces endogenous mouse insulin promoter activity and glucose stimulated insulin secretion. Furthermore, we show that Pdx1 is important for Gpr27's effect on the insulin promoter and insulin secretion. Finally, the over-expression of Gpr27 in 293T cells increases inositol phosphate levels, while knockdown of Gpr27 in MIN6 cells reduces inositol phosphate levels, suggesting this orphan GPCR might couple to Gq/11. In summary, we demonstrate a MIN6-based siRNA screening system that allows rapid identification of novel positive and negative regulators of the insulin promoter. Using this system, we identify Gpr27 as a positive regulator of insulin production.

Significance This paper shows that a class of receptors known to modulate insulin release by pancreatic β cells also regulates the proliferation of these cells and restrains the perinatal β-cell expansion that establishes adult β-cell mass, suggesting that alterations in signaling by these receptors could contribute to the decreased β-cell numbers seen in patients with type 2 diabetes. Further, inhibition of signaling through these receptors potentially could be used to generate more β cells for people with diabetes. Gi-GPCRs, G protein-coupled receptors that signal via Gα proteins of the i/o class (Gα ᵢ/ₒ), acutely regulate cellular behaviors widely in mammalian tissues, but their impact on the development and growth of these tissues is less clear. For example, Gi-GPCRs acutely regulate insulin release from pancreatic β cells, and variants in genes encoding several Gi-GPCRs—including the α-2a adrenergic receptor, ADRA2A—increase the risk of type 2 diabetes mellitus. However, type 2 diabetes also is associated with reduced total β-cell mass, and the role of Gi-GPCRs in establishing β-cell mass is unknown. Therefore, we asked whether Gi-GPCR signaling regulates β-cell mass. Here we show that Gi-GPCRs limit the proliferation of the insulin-producing pancreatic β cells and especially their expansion during the critical perinatal period. Increased Gi-GPCR activity in perinatal β cells decreased β-cell proliferation, reduced adult β-cell mass, and impaired glucose homeostasis. In contrast, Gi-GPCR inhibition enhanced perinatal β-cell proliferation, increased adult β-cell mass, and improved glucose homeostasis. Transcriptome analysis detected the expression of multiple Gi-GPCRs in developing and adult β cells, and gene-deletion experiments identified ADRA2A as a key Gi-GPCR regulator of β-cell replication. These studies link Gi-GPCR signaling to β-cell mass and diabetes risk and identify it as a potential target for therapies to protect and increase β-cell mass in patients with diabetes.

Atonal Homolog 8 (Atoh8) is a basic helix-loop-helix (bHLH) transcription factor that is highly conserved across species and expressed in multiple tissues during embryogenesis. In the developing pancreas, Atoh8 is expressed in endocrine progenitors but declines in hormone-positive cells, suggesting a role during early stages of the endocrine differentiation program. We previously generated a whole-body Atoh8 knockout but early lethality of null embryos precluded assessment of Atoh8 functions during organ development. Here we report the generation of a conditional Atoh8 knockout mouse strain by insertion of two loxP sites flanking exon 1 of the Atoh8 gene. Pancreas-specific Atoh8 knockout (Atoh8 Δpanc) mice were obtained by mating this strain with a Pdx1-Cre transgenic line. Atoh8 Δpanc mice were born at the expected mendelian ratio and showed normal appearance and fertility. Pancreas weight and gross pancreatic morphology were normal. All pancreatic cell lineages were present, although endocrine δ (somatostatin) cells were modestly augmented in Atoh8 Δpanc as compared to control neonates. This increase did not affect whole-body glucose tolerance in adult knockout animals. Gene expression analysis in embryonic pancreases at the time of the major endocrine differentiation wave revealed modest alterations in several early endocrine differentiation markers. Together, these data argue that Atoh8 modulates activation of the endocrine program but it is not essential for pancreas formation or endocrine differentiation in the mouse. Given the ubiquitous expression pattern of Atoh8, the availability of a mouse strain carrying a conditional allele for this gene warrants further studies using temporally regulated Cre transgenic lines to elucidate time or cell-autonomous functions of Atoh8 during development and in the adult.

Chronology of Islet Differentiation Revealed By Temporal Cell Labeling Takeshi Miyatsuka , Zhongmei Li and Michael S. German From the Diabetes Center and Department of Medicine, University of California, San Francisco, San Francisco, California. Corresponding author: Michael S. German, mgerman{at}diabetes.ucsf.edu . Abstract OBJECTIVE Neurogenin 3 plays a pivotal role in pancreatic endocrine differentiation. Whereas mouse models expressing reporters such as eGFP or LacZ under the control of the Neurog3 gene enable us to label cells in the pancreatic endocrine lineage, the long half-life of most reporter proteins makes it difficult to distinguish cells actively expressing neurogenin 3 from differentiated cells that have stopped transcribing the gene. RESEARCH DESIGN AND METHODS In order to separate the transient neurogenin 3 –expressing endocrine progenitor cells from the differentiating endocrine cells, we developed a mouse model (Ngn3-Timer) in which DsRed-E5, a fluorescent protein that shifts its emission spectrum from green to red over time, was expressed transgenically from the NEUROG3 locus. RESULTS In the Ngn3-Timer embryos, green-dominant cells could be readily detected by microscopy or flow cytometry and distinguished from green/red double-positive cells. When fluorescent cells were sorted into three different populations by a fluorescence-activated cell sorter, placed in culture, and then reanalyzed by flow cytometry, green-dominant cells converted to green/red double-positive cells within 6 h. The sorted cell populations were then used to determine the temporal patterns of expression for 145 transcriptional regulators in the developing pancreas. CONCLUSIONS The precise temporal resolution of this model defines the narrow window of neurogenin 3 expression in islet progenitor cells and permits sequential analyses of sorted cells as well as the testing of gene regulatory models for the differentiation of pancreatic islet cells. Footnotes The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received March 13, 2009. Accepted May 5, 2009. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details. © 2009 by the American Diabetes Association.