Francis C. Lynn

HIP1, a human homologue of S. cerevisiae Sla2p, interacts with membrane-associated huntingtin in the brain

Huntington disease (HD) is associated with the expansion of a polyglutamine tract, greater than 35 repeats, in the HD gene product, huntingtin. Here we describe a novel huntingtin interacting protein, HIP1, which co-localizes with huntingtin and shares sequence homology and biochemical characteristics with Sla2p, a protein essential for function of the cytoskeleton in Saccharomyces cerevisiae. The huntingtin–HIP1 interaction is restricted to the brain and is inversely correlated to the polyglutamine length in huntingtin. This provides the first molecular link between huntingtin and the neuronal cytoskeleton and suggests that, in HD, loss of normal huntingtin–HIP1 interaction may contribute to a defect in membrane-cytoskeletal integrity in the brain.

Serotonin regulates pancreatic beta cell mass during pregnancy

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. 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 Gαq-linked serotonin receptor 5-hydroxytryptamine receptor-2b (Htr2b) in maternal islets increased during pregnancy and normalized just before parturition, whereas expression of the Gαi-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.

MicroRNA Expression is Required for Pancreatic Islet Cell Genesis in the Mouse

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.

Rfx6 directs islet formation and insulin production in mice and humans

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.

Sox9 coordinates a transcriptional network in pancreatic progenitor cells

During pancreas development, both the exocrine and endocrine lineages differentiate from a common pool of progenitor cells with similarities to mature pancreatic duct cells. A small set of transcription factors, including Tcf2, Onecut1, and Foxa2, has been identified in these pancreatic progenitor cells. The Sry/HMG box transcription factor Sox9 is also expressed in the early pancreatic epithelium and is required for normal pancreatic exocrine and endocrine development in humans. In this study, we found Sox9 in mice specifically expressed with the other progenitor transcription factors in both pancreatic progenitor cells and duct cells in the adult pancreas. Sox9 directly bound to all three genes in vitro and in intact cells, and regulated their expression. In turn, both Foxa2 and Tcf2 regulated Sox9 expression, demonstrating feedback circuits between these genes. Furthermore, Sox9 activated the expression of the proendocrine factor Neurogenin3, which also depends on the other members of the progenitor transcription network. These studies indicate that Sox9 plays a dual role in pancreatic progenitor cells: both maintaining a stable transcriptional network and supporting the programs by which these cells differentiate into distinct lineages.

Meta-regulation: microRNA regulation of glucose and lipid metabolism.

Maintenance of homeostasis during environmental flux requires constant metabolic adjustment, achieved partly through the fine regulation of gene expression. MicroRNAs are key players in this regulatory milieu; they have been implicated in regulating gene expression within several metabolically active tissues including the endocrine pancreas, liver and adipose tissue. Recent studies, for example, implicate miR-375 in pancreatic islet cell viability and function, and removal or overexpression of miR-375 profoundly affects glucose metabolism. In the liver, miR-122 is important for normal lipid metabolism. In fact, misexpression of miRNAs can occur in some diseases, suggesting that restoring miRNA expression is a potential therapeutic approach for both metabolic syndrome and diabetes.

Improved glucose tolerance in rats treated with the dipeptidyl peptidase IV (CD26) inhibitor Ile-thiazolidide.

The incretins glucose-dependent insulinotropic polypeptide (GIP1-42) and truncated forms of glucagon-like peptide-1 (GLP-1) are hormones released from the gut in response to ingested nutrients, which act on the pancreas to potentiate glucose-induced insulin secretion. These hormones are rapidly inactivated by the circulating enzyme dipeptidyl peptidase IV ([DPIV] CD26). This study describes the effect on glucose tolerance and insulin secretion of inhibiting endogenous DPIV in the rat using Ile-thiazolidide, a specific DPIV inhibitor. High-performance liquid chromatography (HPLC) analysis of plasma following in vivo administration of 125I-labeled peptides showed that inhibition of DPIV by about 70% prevented the degradation of 90.0% of injected 125I-GLP-17-36 after 5 minutes, while only 13.4% remained unhydrolyzed in rats not treated with the DPIV-inhibiting agent after only 2 minutes. Ile-thiazolidide treatment also increased the circulating half-life of intact GLP-17-36 released in response to intraduodenal (ID) glucose (as measured by N-terminal specific radioimmunoassay [RIA]). In addition, inhibition of DPIV in vivo resulted in an earlier increase and peak of plasma insulin and a more rapid clearance of blood glucose in response to ID glucose challenge. When considered with the HPLC data, these results suggest that the altered insulin profile is an incretin-mediated response. DPIV inhibition resulting in improved glucose tolerance may have therapeutic potential for the management of type 2 diabetes mellitus.

A novel pathway for regulation of glucose-dependent insulinotropic polypeptide (GIP) receptor expression in beta cells.

Glucose‐dependent insulinotropic polypeptide (GIP) is secreted postprandially and acts in concert with glucose to stimulate insulin secretion from the pancreas. Here, we describe a novel pathway for the regulation of GIP receptor (GIPR) expression within clonal β‐cell lines, pancreatic islets, and in vivo. High (25 mM) glucose was able to significantly reduce GIPR mRNA levels in INS(832/13) cells after only 6 h. In contrast, palmitic acid (2 mM) and WY 14643 (100 μM) stimulated approximate doublings of GIPR expression in INS(832/13) cells under low (5.5 mM), but not high (25 mM), glucose conditons, suggesting that fat can regulate GIPR expression via PPARα in a glucose‐dependent manner. Both MK‐886, an antagonist of PPARα, and a dominant negative form of PPARα transfected into INS(832/13) cells caused a significant reduction in GIPR expression in low, but not high, glucose conditions. Finally, in hyperglycemic clamped rats, there was a 70% reduction in GIPR expression in the islets and a 71% reduction in GIP‐stimulated insulin secretion from the perfused pancreas. Thus, evidence is presented that the GIPR is controlled at normoglycemia by the fatty acid load on the islet; however, when exposed to hyperglycemic conditions, the GIPR is down‐regulated, which may contribute to the decreased responsiveness to GIP that is observed in type 2 diabetes.

Characterization of polyhormonal insulin-producing cells derived in vitro from human embryonic stem cells.

Human embryonic stem cells (hESCs) were used as a model system of human pancreas development to study characteristics of the polyhormonal cells that arise during fetal pancreas development. HESCs were differentiated into fetal-like pancreatic cells in vitro using a 33-day, 7-stage protocol. Cultures were ~90-95% PDX1-positive by day (d) 11 and 70-75% NKX6.1-positive by d17. Polyhormonal cells were scattered at d17, but developed into islet-like clusters that expressed key transcription factors by d33. Human C-peptide and glucagon secretion were first detected at d17 and increased thereafter in parallel with INS and GCG transcript levels. HESC-derived cells were responsive to KCl and arginine, but not glucose in perifusion studies. Compared to adult human islets, hESC-derived cells expressed ~10-fold higher levels of glucose transporter 1 (GLUT1) mRNA, but similar levels of glucokinase (GCK). In situ hybridization confirmed the presence of GLUT1 transcript within endocrine cells. However, GLUT1 protein was excluded from this population and was instead observed predominantly in non-endocrine cells, whereas GCK was co-expressed in insulin-positive cells. In rubidium efflux assays, hESC-derived cells displayed mild potassium channel activity, but no responsiveness to glucose, metabolic inhibitors or glibenclamide. Western blotting experiments revealed that the higher molecular weight SUR1 band was absent in hESC-derived cells, suggesting a lack of functional KATP channels at the cell surface. In addition, KATP channel subunit transcript levels were not at a 1:1 ratio, as would be expected (SUR1 levels were ~5-fold lower than KIR6.2). Various ratios of SUR1:KIR6.2 plasmids were transfected into COSM6 cells and rubidium efflux was found to be particularly sensitive to a reduction in SUR1. These data suggest that an impaired ratio of SUR1:KIR6.2 may contribute to the observed KATP channel defects in hESC-derived islet endocrine cells, and along with lack of GLUT1, may explain the absence of glucose-stimulated insulin secretion.

Maintenance of β-Cell Maturity and Plasticity in the Adult Pancreas: Developmental Biology Concepts in Adult Physiology

Diabetes occurs when the pancreas fails to produce and secrete sufficient insulin for the maintenance of glucose homeostasis. Although many factors required for β-cell development have been elucidated, we know surprisingly little regarding the mechanisms that maintain the differentiated state of adult β-cells. Despite years of fruitful research, many hurdles remain before we can replace failing β-cells in patients. Efforts to produce new β-cells will benefit from detailed knowledge of their differentiation, maturation, maintenance, heterogeneity, and plasticity. Studies of single cells and conditional knockout mice reveal surprising relationships between the different islet cell types and previously unappreciated roles for transcription factors and soluble factors in β-cell maintenance. Herein we highlight numerous innovative efforts made to identify the core mechanisms responsible for β-cell phenotypic maintenance and compare these with other long-lived cell types. In the adult pancreas, physical β-cell mass is determined by the balance of β-cell birth, differentiation, size, and death. The prenatal origins and development of β-cells (1,2), the molecular mechanisms of adult β-cell proliferation (3,4), and programmed β-cell death (5) have all been reviewed recently and will not be revisited here. The maintenance and adaptation of β-cell mass after birth involves the addition of new β-cells (6). Cells with robust staining for insulin have been shown to slowly proliferate in vivo and in vitro (7–9). It has been reported that β-cells have an extremely long life span under typical conditions and proliferation plays a diminishing role with advancing age (7,10,11). The apparent low rate of β-cell replication has led some groups to suggest that adult β-cells arise from a pool of non–β-cell progenitors (12). Because this topic has been hotly debated and reviewed recently (13), we will only briefly discuss studies relevant to other aspects of this review. …