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, while prolactin and placental lactogen counterbalance this resistance and prevent maternal hyperglycemia by driving expansion of the maternal population of insulin-producing β-cells1–3. However, the exact mechanisms by which the lactogenic hormones drive β-cell expansion remain uncertain. Here we show that serotonin acts downstream of lactogen signaling to drive β-cell proliferation. Serotonin synthetic enzyme Tph1 and serotonin production increased sharply in β-cells during pregnancy or after treatment with lactogens in vitro. Inhibition of serotonin synthesis by dietary tryptophan restriction or Tph inhibition blocked β-cell expansion and induced glucose intolerance in pregnant mice without affecting insulin sensitivity. Expression of the Gαq-linked serotonin receptor Htr2b in maternal islets increased during pregnancy and normalized just prior to parturition, while expression of the Gαi-linked receptor Htr1d increased at the end of pregnancy and postpartum. Blocking Htr2b signaling in pregnant mice also blocked β-cell expansion and caused glucose intolerance. These studies reveal an integrated signaling pathway linking β-cell mass to anticipated insulin need during pregnancy. Modulators of this pathway, including medications and diet, may affect the risk of gestational diabetes4.

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 ␤-cells, were most dramatically reduced. The endocrine defect was associated with an increase in the notchsignaling 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 ␤-cell formation. Diabetes 56:2938-2945, 2007 P ancreatic organogenesis begins at embryonic day (e)9.5 in the mouse embryo with the budding of the dorsal anlagen from the prospective gut endoderm (1). Pancreatic duodenal homeobox-1 (Pdx-1) appears in the same area 1 day earlier and is expressed in all pancreas progenitors (2,3). As the pancreatic program continues, the expression of the basic helixloop-helix factor neurogenin3 initiates the differentiation of the endocrine cells. Neurogenin3 expression peaks coincidently with the "secondary transition," a time of rapid endocrine cell generation that occurs between e13.5 and e14.5 (4,5). The cell-specific factors Onecut1, Tcf1, Tcf2, and Sox9, as well as the Notch signaling pathway, regulate Neurog3 expression during development (5-9).Many tissues of the developing organism express microRNAs (miRNAs), which are small (ϳ20 nt) RNAs that mediate posttranscriptional gene silencing by interacting with the RNA-induced silencing complex and binding to the 3Ј untranslated region of their cognate messenger RNA targets (10,11). Here, we hypothesize that miRNAs are expressed in the developing pancreas, that they are necessary for the normal development of pancreatic ␤-cells, and that they may be involved in regulating genes important for normal pancreas morphogenesis. Ultimately, an intimate understanding of how miRNAs govern pancreas development may be necessary for stem cell-based therapies for all forms of diabetes. RESEARCH DESIGN AND METHODSMice were housed on a 12-h light/dark cycle in a controlled climate accordin...

Evolving evidence indicates that platelets and megakaryocytes (MKs) have unexpected activities in inflammation and infection; whether viral infections upregulate biologically active, antiviral immune genes in platelets and MKs is unknown, however. We examined antiviral immune genes in these cells in dengue and influenza infections, viruses that are global public health threats. Using complementary biochemical, pharmacological, and genetic approaches, we examined the regulation and function of interferon-induced transmembrane protein 3 (IFITM3), an antiviral immune effector gene not previously studied in human platelets and MKs. IFITM3 was markedly upregulated in platelets isolated from patients during clinical influenza and dengue virus (DENV) infections. Lower IFITM3 expression in platelets correlated with increased illness severity and mortality in patients. Administering a live, attenuated DENV vaccine to healthy subjects significantly increased platelet IFITM3 expression. Infecting human MKs with DENV selectively increased type I interferons and IFITM3. Overexpression of IFITM3 in MKs was sufficient to prevent DENV infection. In naturally occurring, genetic loss-of-function studies, MKs from healthy subjects harboring a homozygous mutation in IFITM3 (rs12252-C, a common single-nucleotide polymorphism in areas of the world where DENV is endemic) were significantly more susceptible to DENV infection. DENV-induced MK secretion of interferons prevented infection of bystander MKs and hematopoietic stem cells. Thus, viral infections upregulate IFITM3 in human platelets and MKs, and IFITM3 expression is associated with adverse clinical outcomes. These observations establish, for the first time, that human MKs possess antiviral functions, preventing DENV infection of MKs and hematopoietic stem cells after local immune signaling.

In a Plenary Paper, Middleton and colleagues describe important transcriptional and translational changes in murine and human platelets during sepsis, elucidating the emerging role of platelets in the complications of systemic inflammatory illness.

OBJECTIVEDespite their origins in different germ layers, pancreatic islet cells share many common developmental features with neurons, especially serotonin-producing neurons in the hindbrain. Therefore, we tested whether these developmental parallels have functional consequences.RESEARCH DESIGN AND METHODSWe used transcriptional profiling, immunohistochemistry, DNA-binding analyses, and mouse genetic models to assess the expression and function of key serotonergic genes in the pancreas.RESULTSWe found that islet cells expressed the genes encoding all of the products necessary for synthesizing, packaging, and secreting serotonin, including both isoforms of the serotonin synthetic enzyme tryptophan hydroxylase and the archetypal serotonergic transcription factor Pet1. As in serotonergic neurons, Pet1 expression in islets required homeodomain transcription factor Nkx2.2 but not Nkx6.1. In β-cells, Pet1 bound to the serotonergic genes but also to a conserved insulin gene regulatory element. Mice lacking Pet1 displayed reduced insulin production and secretion and impaired glucose tolerance.CONCLUSIONSThese studies demonstrate that a common transcriptional cascade drives the differentiation of β-cells and serotonergic neurons and imparts the shared ability to produce serotonin. The interrelated biology of these two cell types has important implications for the pathology and treatment of 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.T he mature structure of an organ depends on its constituent cell populations and how those populations expand and organize as the organ grows. Within the pancreas, the size of the islets of Langerhans and especially how many insulin-producing β-cells they contain is a critical determinant of pancreatic function and the risk of developing diabetes. The number of islet cells depends on the rate at which new endocrine cells [α-, β-, δ-, ε-, and pancreatic polypeptide (PP) cells] differentiate from progenitors, the size of the progenitor population, and the rates of proliferation of the progenitors and mature endocrine cells. Understanding the mechanisms that control these rates will help explain how distinct cell populations assemble into functional organs.The coordinated activity of numerous transcription factors regulates the differentiation of the islet cells (1, 2). Among these factors, Neurogenin3 (Neurog3/Neurog3), a member of the basic helix-loop-helix (bHLH) transcription factor family, transiently marks the progenitor cells that will become islet cells and initiates endocrine differentiation during embryonic development, regeneration, and transdifferentiation (3-10).Although we know that most descendants of Neurog3-expressing cells exit the cell cycle (6, 11), we do not know whether or how Neurog3 might drive cell cycle exit. To address these questions, we used several mouse models with loss-and gain-offunction mutations of Neurog3 and demonstrated that Neurog3 is both necessary and sufficient to promote cellular quiescence in pancreatic progenitors. Furthermore, transcriptome analysis with high time resolution using the Neurog3-Timer mouse model identified the cell...

To investigate the role of the Sry/hydroxymethylglutaryl box (Sox) transcription factors in the development of the pancreas, we determined the expression pattern of Sox factors in the developing mouse pancreas. By RT-PCR, we detected the presence of multiple Sox family members in both the developing pancreas and mature islets and then focused on two factors, Sox2 and Sox4. The expression field of Sox2, which plays a role in the maintenance of some stem cell populations, included the developing duodenum, but Sox2 was specifically excluded from the pancreatic buds. In contrast, Sox4 was detected broadly in the early pancreatic buds and eventually became restricted to the nuclei of all islet cells in the adult mouse. Mice homozygous for a null mutation of the sox4 gene showed normal pancreatic bud formation and endocrine cell differentiation up to embryonic day 12.5. Beyond that date, cultured pancreatic explants lacking sox4 failed to form normal islets. Instead, a markedly reduced number of endocrine cells were found scattered through the explant. We show here that several Sox transcription factors are expressed in the developing pancreas and in the islet, and that one of these factors, Sox4, is required for the normal development of pancreatic islets. Diabetes 54:3402-3409, 2005 S cattered through the exocrine pancreas, the islets of Langerhans are highly organized clusters of endocrine cells comprised of four distinct cell types: the glucagon-secreting ␣-cells, the insulinsecreting ␤-cells, the somatostatin-secreting ␦-cells, and the pancreatic polypeptide-secreting cells. Because of the essential role of the islet hormones in energy metabolism, defects in the development, maintenance, or function of the endocrine pancreas have serious consequences, including diabetes.The pancreas forms from the endoderm in the region of the foregut/midgut junction and is first visible in mice at embryonic day 9.5 (e9.5) (1). The first endocrine cells of the pancreas appear at around e9.5 in the dorsal pancreatic bud. These cells express glucagon; a few insulinexpressing cells appear ϳ1 day later. At ϳe13-14, the pancreas undergoes a distinct change termed the secondary transition, characterized by the appearance of ductal cells, exocrine cells, and delta cells and a rapid expansion of the insulin-expressing cells. The endocrine cells found in the pancreas before the secondary transition have clear differences from mature islet cells and may result from developmental pathways that are distinct from those that produce the more mature endocrine cells that arise following the secondary transition (2-6). By e18, the first pancreatic polypeptide-expressing cells appear, and the endocrine cells organize into distinct islets with the ␤-cells forming the central core. Around the same time, ␤-cell neogenesis declines, but the simultaneous onset of ␤-cell replication continues to expand the ␤-cell population and enlarge the forming islets (7).This process of endocrine cell determination and differentiation depends on the proper sequentia...

Concurrent chemoradiation therapy (CCRT) is the treatment of choice for locally advanced non-small cell lung cancer (LA-NSCLC). Several clinical trials that combine programmed cell death 1 (PD-1) axis inhibitors with radiotherapy are in development for patients with LA-NSCLC. However, the effect of CCRT on programmed cell death ligand-1 (PD-L1) expression on tumor cells is unknown. In this study, we analysed paired NSCLC specimens that had been obtained pre- and post-CCRT. PD-L1 expression on tumor cells was studied by immunohistochemistry. A total of 45 patients with LA-NSCLC were included, among which there were sufficient pre- and post-CCRT specimens in 35 patients. Overall, the percentage of tumor cells with PD-L1 expression significantly decreased between pre- and post-CCRT specimens (P = 0.024). Sixteen, 15, and 4 patients had decreased, unchanged, or increased PD-L1 expression after CCRT, respectively. Median OS of patients with decreased, unchanged, or increased PD-L1 expression was 85.1, 92.8, and 14.6 months, respectively (P < 0.001). In conclusion, the percentage of PD-L1-positive tumor cells significantly decreased after CCRT. Alteration of PD-L1 expression after neoadjuvant CCRT was associated with prognosis in patients with LA-NSCLC. These data should be considered when developing the optimal approach of integrating PD-1 axis inhibitors with CCRT.