Sandrine Gremlich

Fatty Acids Decrease IDX-1 Expression in Rat Pancreatic Islets and Reduce GLUT2, Glucokinase, Insulin, and Somatostatin Levels

IDX-1 (islet/duodenum homeobox-1) is a transcription factor expressed in the duodenum and pancreatic beta and delta cells. It is required for embryonic development of the pancreas and transactivates theGlut2, glucokinase, insulin, and somatostatin genes. Here we show that exposure of isolated rat pancreatic islets to palmitic acid induced a ∼70% decrease in IDX-1 mRNA and protein expression as well as 40 and 65% decreases in the binding activity of IDX-1 for its cognate cis-regulatory elements of theGlut2 and insulin promoters, respectively. The inhibitory effect of palmitic acid required its mitochondrial oxidation since it was prevented by the carnitine palmitoyltransferase I inhibitor bromopalmitic acid. The palmitic acid effect on IDX-1 was correlated with decreases in GLUT2 and glucokinase expression of 40 and 25%, respectively, at both the mRNA and protein levels. Insulin and somatostatin mRNA expression was also decreased by 40 and 60%, whereas glucagon mRNA expression was not modified. After 48 h of exposure to fatty acids, total islet insulin, somatostatin, and glucagon contents were decreased by 85, 55, and 65%, respectively. At the same time, total hormone release was strongly stimulated (13-fold) for glucagon, whereas its was only marginally increased for insulin and somatostatin (1.5- and 1.7-fold, respectively). These results indicate that elevated fatty acid levels 1) negatively regulateIdx-1 expression; 2) decrease the expression of genes transactivated by IDX-1 such as those for GLUT2, glucokinase, insulin, and somatostatin; and 3) lead to an important increase in glucagon synthesis and secretion. Fatty acids thus have pleiotropic effects on pancreatic islet gene expression, and the negative control ofIdx-1 expression may be an initial event in the development of these multiple defects.

Expression and Functional Activity of Glucagon, Glucagon-Like Peptide I, and Glucose-Dependent Insulinotropic Peptide Receptors in Rat Pancreatic Islet Cells

Rat pancreatic α- and β-cells are critically dependent on hormonal signals generating cyclic AMP (cAMP) as a synergistic messenger for nutrient-induced hormone release. Several peptides of the glucagon-secretin family have been proposed as physiological ligands for cAMP production in β-cells, but their relative importance for islet function is still unknown. The present study shows expression at the RNA level in β-cells of receptors for glucagon, glucose-dependent insulinotropic polypeptide (GIP), and glucagon-like peptide I(7-36) amide (GLP-I), while RNA from islet α-cells hybridized only with GIP receptor cDNA. Western blots confirmed that GLP-I receptors were expressed in β-cells and not in α-cells. Receptor activity, measured as cellular cAMP production after exposing islet β-cells for 15 min to a range of peptide concentrations, was already detected using 10 pmol/l GLP-I and 50 pmol/l GIP but required 1 nmol/l glucagon. EC50 values of GLP-I- and GIP-induced cAMP formation were comparable (0.2 nmol/l) and 45-fold lower than the EC50 of glucagon (9 nmol/l). Maximal stimulation of cAMP production was comparable for the three peptides. In purified α-cells, 1 nmol/l GLP-I failed to increase cAMP levels, while 10 pmol/l to 10 nmol/l GIP exerted similar stimulatory effects as in β-cells. In conclusion, these data show that stimulation of glucagon, GLP-I, and GIP receptors in rat β-cells causes cAMP production required for insulin release, while adenylate cyclase in α-cells is positively regulated by GIP.

Cloning, Functional Expression, and Chromosomal Localization of the Human Pancreatic Islet Glucose-Dependent Insulinotropic Polypeptide Receptor

Glucose-dependent insulinotropic polypeptide (GIP) is a hormone secreted by the endocrine K-cells from the duodenum that stimulates glucose-induced insulin secretion. Here, we present the molecular characterization of the human pancreatic islet GIP receptor. cDNA clones for the GIP receptor were isolated from a human pancreatic islet cDNA library. They encoded two different forms of the receptor, which differed by a 27–amino acid insertion in the COOH-terminal cytoplasmic tail. The receptor protein sequence was 81% identical to that of the rat GIP receptor. When expressed in Chinese hamster lung fibroblasts, both forms of the receptor displayed high-affinity binding for GIP (180 and 600 µmol/l). GIP binding was displaced by <20% by 1 µmol/l glucagon, glucagon-like peptide (GLP-I)(7–36) amide, vasoactive intestinal peptide, and secretin. However exendin-4 and exendin-(9–39) at 1 μumol/l displaced binding by ∼70 and ∼100% at 10 µmol/l. GIP binding to both forms of the receptor induced a dose-dependent increase in intracellular cAMP levels (EC50 values of 0.6–0.8 µmol/l) but no elevation of cytoplasmic calcium concentrations. Interestingly, both exendin-4 and exendin-(9–39) were antagonists of the receptor, inhibiting GIP-induced cAMP formation by up to 60% when present at a concentration of 10 μmol/l. Finally, the physical and genetic chromosomal localization of the receptor gene was determined to be on 19q13.3, close to the ApoC2 gene. These data will help study the physiology and pathophysiology of the human GIP receptor.

Dexamethasone induces posttranslational degradation of GLUT2 and inhibition of insulin secretion in isolated pancreatic beta cells. Comparison with the effects of fatty acids

GLUT2 expression is strongly decreased in glucose-unresponsive pancreatic β cells of diabetic rodents. This decreased expression is due to circulating factors distinct from insulin or glucose. Here we evaluated the effect of palmitic acid and the synthetic glucocorticoid dexamethasone on GLUT2 expression by in vitro cultured rat pancreatic islets. Palmitic acid induced a 40% decrease in GLUT2 mRNA levels with, however, no consistent effect on protein expression. Dexamethasone, in contrast, had no effect on GLUT2 mRNA, but decreased GLUT2 protein by about 65%. The effect of dexamethasone was more pronounced at high glucose concentrations and was inhibited by the glucocorticoid antagonist RU-486. Biosynthetic labeling experiments revealed that GLUT2 translation rate was only minimally affected by dexamethasone, but that its half-life was decreased by 50%, indicating that glucocorticoids activated a posttranslational degradation mechanism. This degradation mechanism was not affecting all membrane proteins, since the α subunit of the Na+/K+-ATPase was unaffected. Glucose-induced insulin secretion was strongly decreased by treatment with palmitic acid and/or dexamethasone. The insulin content was decreased (∼55 percent) in the presence of palmitic acid, but increased (∼180%) in the presence of dexamethasone. We conclude that a combination of elevated fatty acids and glucocorticoids can induce two common features observed in diabetic β cells, decreased GLUT2 expression, and loss of glucose-induced insulin secretion.

The loss of GLUT2 expression in the pancreatic β-cells of diabetic db/db mice is associated with an impaired DNA-binding activity of islet-specific trans-acting factors

GLUT2 expression is reduced in the pancreatic beta-cells of several diabetic animals. The transcriptional control of the gene in beta-cells involves at least two islet-specific DNA-binding proteins, GTIIa and PDX-1, which also transactivates the insulin, somatostatin and glucokinase genes. In this report, we assessed the DNA-binding activities of GTIIa and PDX-1 to their respective cis-elements of the GLUT2 promoter using nuclear extracts prepared from pancreatic islets of 12 week old db/db diabetic mice. We show that the decreased GLUT2 mRNA expression correlates with a decrease of the GTIIa DNA-binding activity, whereas the PDX-1 binding activity is increased. In these diabetic animals, insulin mRNA expression remains normal. The adjunction of dexamethasone to isolated pancreatic islets, a treatment previously shown to decrease PDX-1 expression in the insulin-secreting HIT-T15 cells, has no effect on the GTIIa and PDX-1 DNA-binding activities. These data suggest that the decreased activity of GTIIa, in contrast to PDX-1, may be a major initial step in the development of the beta-cell dysfunction in this model of diabetes.

Evolution of gene expression changes in newborn rats after mechanical ventilation with reversible intubation

Mechanical ventilation (MV) is life‐saving but potentially harmful for lungs of premature infants. So far, animal models dealt with the acute impact of MV on immature lungs, but less with its delayed effects. We used a newborn rodent model including non‐surgical and therefore reversible intubation with moderate ventilation and hypothesized that there might be distinct gene expression patterns after a ventilation‐free recovery period compared to acute effects directly after MV. Newborn rat pups were subjected to 8 hr of MV with 60% oxygen (O2), 24 hr after injection of lipopolysaccharide (LPS), intended to create a low inflammatory background as often recognized in preterm infants. Animals were separated in controls (CTRL), LPS injection (LPS), or full intervention with LPS and MV with 60% O2 (LPS + MV + O2). Lungs were recovered either directly following (T:0 hr) or 48 hr after MV (T:48 hr). Histologically, signs of ventilator‐induced lung injury (VILI) were observed in LPS + MV + O2 lungs at T:0 hr, while changes appeared similar to those known from patients with chronic lung disease (CLD) with fewer albeit larger gas exchange units, at T:48 hr. At T:0 hr, LPS + MV + O2 increased gene expression of pro‐inflammatory MIP‐2. In parallel anti‐inflammatory IL‐1Ra gene expression was increased in LPS and LPS + MV + O2 groups. At T:48 hr, pro‐ and anti‐inflammatory genes had returned to their basal expression. MMP‐2 gene expression was decreased in LPS and LPS + MV + O2 groups at T:0 hr, but no longer at T:48 hr. MMP‐9 gene expression levels were unchanged directly after MV. However, at T:48 hr, gene and protein expression increased in LPS + MV + O2 group. In conclusion, this study demonstrates the feasibility of delayed outcome measurements after a ventilation‐free period in newborn rats and may help to further understand the time‐course of molecular changes following MV. The differences obtained from the two time points could be interpreted as an initial transitory increase of inflammation and a delayed impact of the intervention on structure‐related genes. Pediatr Pulmonol. 2012; 47:1204–1214.

Amniotic fluid insulin-like growth factor binding protein 3 concentration as early indicator of fetal growth restriction

OBJECTIVE Insulin-like growth factor-I (IGF-I) is an important regulator of fetal growth and its bioavailability depends on insulin-like growth factor binding proteins (IGFBPs). Genes coding for IGF-I and IGFBP3 are polymorphic. We hypothesized that either amniotic fluid protein concentration at the beginning of the second trimester or genotype of one of these two genes could be predictive of abnormal fetal growth. STUDY DESIGN Amniotic fluid samples (14-18 weeks of pregnancy) from 123 patients with appropriate for gestational age (AGA) fetuses, 39 patients with small for gestational age (SGA) fetuses and 34 patients with large for gestational age (LGA) were analyzed. Protein concentrations were evaluated by ELISA and gene polymorphisms by PCR. RESULTS Amniotic fluid IGFBP3 concentrations were significantly higher in SGA compared to AGA group (P=0.030), and this was even more significant when adjusted to gestational age at the time of amniocentesis and other covariates (ANCOVA analysis: P=0.009). Genotypic distribution of IGF-I variable number of tandem repeats (VNTR) polymorphism was significantly different in SGA compared to AGA group (P=0.029). 19CA/20CA genotype frequency was threefold decreased in SGA compared to AGA group and the risk of SGA occurrence of this genotype was decreased accordingly: OR=0.289, 95%CI=0.1-0.9, P=0.032. Genotype distribution of IGFBP3(A-202C) polymorphism was similar in all three groups. CONCLUSIONS High IGFBP3 concentrations in amniotic fluid at the beginning of the second trimester are associated with increased risks of SGA while 19CA/20CA genotype at IGF-I VNTR polymorphism is associated with reduced risks of SGA. Neither IGFBP3 concentrations, nor IGF-I/IGFBP3 polymorphisms are associated with modified risks of LGA.

LIF and sIL-2R plasma concentrations in IVF patients on the day of embryo transfer: predictive markers of IVF outcome

Successful implantation is still the limiting step in IVF. We hypothesized that maternal plasma concentrations of certain cytokines at the time of embryo transfer could predict the likelihood of successful implantation and pregnancy. sIL-2R, IL-6, LIF, and MMP2 concentrations were measured in plasma from 160 IVF patients (natural and stimulated IVF cycles) on the morning of the embryo transfer (ET0) and 14 days later (ET+14). Patients were ultimately subdivided into four groups depending on the IVF treatment outcome (pregnancy failure, biochemical pregnancy, first-trimester miscarriage and normal term delivery). In natural and stimulated IVF cycles at ET0, sIL-2R concentrations were threefold higher in biochemical pregnancies than in pregnancy failures (P=0.020), and in natural cycles only, 2.5-fold higher in normal term deliveries than in pregnancy failures (P=0.023). Conversely, in natural and stimulated IVF cycles at ET0, LIF concentrations were one third lower in biochemical pregnancies/first-trimester miscarriages compared with pregnancy failures (P=0.042). We suggest that high sIL-2R and low LIF concentrations in maternal plasma on the morning of the embryo transfer might be associated with increased risks of early pregnancy loss, while a basal level of sIL-2R is necessary for normal term delivery outcome. Both cytokine measurements might therefore be useful in the management of IVF patients, and modulation of their concentrations could be investigated as a therapeutic alternative for women with abnormal concentrations at the time of embryo transfer.

Gene expression profile in newborn rat lungs after two days of recovery of mechanical ventilation

Background:Preterm infants having immature lungs often require respiratory support, potentially leading to bronchopulmonary dysplasia (BPD). Conventional BPD rodent models based on mechanical ventilation (MV) present outcome measured at the end of the ventilation period. A reversible intubation and ventilation model in newborn rats recently allowed discovering that different sets of genes modified their expression related to time after MV. In a newborn rat model, the expression profile 48 h after MV was analyzed with gene arrays to detect potentially interesting candidates with an impact on BPD development.Methods:Rat pups were injected P4-5 with 2 mg/kg lipopolysaccharide (LPS). One day later, MV with 21 or 60% oxygen was applied during 6 h. Animals were sacrified 48 h after end of ventilation. Affymetrix gene arrays assessed the total gene expression profile in lung tissue.Results:In fully treated animals (LPS + MV + 60% O2) vs. controls, 271 genes changed expression significantly. All modified genes could be classified in six pathways: tissue remodeling/wound repair, immune system and inflammatory response, hematopoiesis, vasodilatation, and oxidative stress. Major alterations were found in the MMP and complement system.Conclusion:MMPs and complement factors play a central role in several of the pathways identified and may represent interesting targets for BPD treatment/prevention.Bronchopulmonary dysplasia (BPD) is a chronic lung disease occurring in ~30% of preterm infants born less than 30 wk of gestation (1). Its main risk factors include lung immaturity due to preterm delivery, mechanical ventilation (MV), oxygen toxicity, chorioamnionitis, and sepsis. The main feature is an arrest of alveolar and capillary formation (2). Models trying to decipher genes involved in the pathophysiology of BPD are mainly based on MV and oxygen application to young mammals with immature lungs of different species (3). In newborn rodent models, analyses of lung structure and gene and protein expression are performed for practical reasons directly at the end of MV (4,5,6). However, later appearing changes of gene expression might also have an impact on lung development and the evolution towards BPD and cannot be discovered by such models. Recently, we developed a newborn rat model of MV using an atraumatic (orotracheal) intubation technique that allows the weaning of the newborn animal off anesthesia and MV, the extubation to spontaneous breathing, and therefore allows the evaluation of effects of MV after a ventilation-free period of recovery (7). Indeed, applying this concept of atraumatic intubation by direct laryngoscopy, we recently were able to show significant differences between gene expression changes appearing directly after MV compared to those measured after a ventilation-free interval of 48 h. Immediately after MV, inflammation-related genes showed a transitory modified expression, while another set of more structurally related genes changed their expression only after a delay of 2 d (7). Lung structure, analyzed by conventional 2D histology and also by 3D reconstruction using synchrotron x-ray tomographic microscopy revealed, 48 h after end of MV, a reduced complexity of lung architecture compared to the nonventilated rat lungs, similar to the typical findings in BPD. To extend these observations about late gene expression modifications, we performed with a similar model a full gene expression profile of lung tissue 48 h after the end of MV with either room air or 60% oxygen. Essentially, we measured changes in the expression of genes related to the MMPs and complement system which played a role in many of the six identified mostly affected pathways.

Analysis of Potential Biomarkers of Bronchopulmonary Dysplasia (BPD) in a Newborn Rat Ventilation Model

Background: BPD is a chronic lung disease affecting preterm infants leading to increased morbidity and mortality. Main risk factors include premature birth, mechanical ventilation (MV), hyperoxia and inflammation. A newborn rat ventilation model with reversible intubation was used to study delayed gene expression modifications to increase our understanding of its pathophysiology.Methods: LPS was injected intraperitoneally (2mg/kg) to male rat pups on postnatal day 4 or 5 to mimic systemic inflammation. Twenty-four hours after injection they were intubated and ventilated for 6h with tidal volume of 15ml/kg and 21% or 60%O2. After weaning from anesthesia, they were returned to their mothers for 48h. Gene expression was measured by Affymetrix®Gene-Arrays in four groups (n=9/Group) and verified by qPCR.Results: Expression changes were mainly found in genes involved in inflammation and extracellular matrix remodeling. Among them MMP-9 and several of its regulator genes were significantly modified.Table 1Conclusion: MMP-9 is known to be important in lung development, angiogenesis and tissue repair. In our BPD-model, MMP-9 pathway seemed to be a central target. Several genes increasing MMP-9 activity were synergistically upregulated, highlighting its potential role in lung injury and repair. Therefore, the role of MMP-9 in the pathophysiology of BPD deserves further investigation.