Biomedical subjects

Robert H Lane

Publications and source records attributed to Robert H Lane.

13 recordsLinked to original sources

PubMed2006

Growth retardation alters the epigenetic characteristics of hepatic dual specificity phosphatase 5.

Uteroplacental insufficiency leads to intrauterine growth retardation (IUGR) and adult onset insulin resistance in both humans and rats. IUGR rat liver is characterized by persistent changes in histone 3 lysine 9 and lysine 14 acetylation, which may induce postnatal changes in gene expression. We hypothesized that it would be possible to identify hepatic genes whose epigenetic characteristics and mRNA levels are altered due to IUGR using chromatin immunoprecipitation (ChIP) coupled with random primed differential display polymerase chain reaction (PCR). One of the isolated sequences identified contained exon 2 of the dual specificity phosphatase-5 gene (DUSP5). IUGR affected hepatic DUSP5 mRNA levels and exon 2 DNA methylation into adulthood in the rat. DUSP5 dephosphorylates Erk1 and Erk2 within the MAPK signaling cascade, which in turn affects serine 612 phosphorylation of insulin receptor substrate-1 (p612 IRS-1). In adult rat liver, IUGR increased Erk1/Erk2 phosphorylation and p612 IRS-1 phosphorylation. Increased serine phosphorylation of hepatic IRS-1 may contribute to the insulin resistance that characterizes these animals. We conclude that intrauterine growth retardation induced by uteroplacental insufficiency 1) affects the hepatic epigenetic characteristics and mRNA of the DUSP-5 and 2) increases hepatic insulin receptor substrate-1 phosphorylation at serine 612 in adult rats.

Animals↗

PubMed2006

Uteroplacental insufficiency increases p53 phosphorylation without triggering the p53-MDM2 functional circuit response in the IUGR rat kidney.

Uteroplacental insufficiency (UPI) leads to intrauterine growth restriction (IUGR), which predisposes infants toward renal insufficiency early in life and increases the risk of kidney-related adult morbidities, such as hypertension. This compromised in utero environment has been demonstrated to impair nephrogenesis, as evidenced by a reduced nephron endowment in humans and in rats rendered IUGR by UPI. Concordantly, we have observed that IUGR rats have increased kidney p53 protein levels associated with increased apoptosis. Several factors can regulate p53 gene expression and activity, including posttranslational modifications and protein-protein interactions in the cell. Among these, two important mechanisms are 1) phosphorylation of the amino terminal serine 15 [phospho-p53 (Ser15)], which increases p53 stability and apoptotic activity, and 2) the murine double-minute (MDM2) functional circuit that limits further p53-induced apoptosis by promoting proteosomal degradation of p53. We hypothesize that UPI induces an increase in phospho-p53 (Ser15) in association with an absent MDM2 response, predisposing the kidney to increased apoptosis. To test our hypothesis, we induced IUGR through bilateral uterine artery ligation of the pregnant rat. UPI significantly increased phospho-p53 (Ser15), as well as ataxia teleangiectasia-mutated kinase/A-T-related kinase and dsDNA-activated protein kinase kinase levels, which induce phosphorylation of p53. In contrast, UPI induced no increase in kidney MDM2 mRNA and protein levels in IUGR pups. We conclude that among multiple mechanisms that affect nephrogenesis, UPI induces an increase in p53 phosphorylation without a corresponding increase in MDM2 expression, and we speculate that this response may contribute to the increased apoptosis previously described in the IUGR kidney.

Animals↗

PubMed2005

Uteroplacental insufficiency alters hepatic expression, phosphorylation, and activity of the glucocorticoid receptor in fetal IUGR rats.

Uteroplacental insufficiency (UPI) induces persistent changes in hepatic gene expression secondary to altered chromatin dynamics in the intrauterine growth- restricted (IUGR) rat liver. The glucocorticoid receptor (GR) is a transcription factor that when activated can induce changes in chromatin structure. To begin the process of identifying pathways by which IUGR affects chromatin structure, we hypothesized that UPI in the rat induces a significant increase in endogenous glucocorticoids (corticosterone) and increases GR expression and activation. To prove our hypothesis, we induced IUGR through bilateral uterine artery ligation of the pregnant rat. At day 1, UPI significantly increased corticosterone levels and was associated with increased total GR mRNA and protein levels in the liver, as well as increased hepatic phosphorylation of GR serine 211. Moreover, cyclin-dependent kinase 2 (CDK2) cyclinA/CDK2 protein levels, which selectively phosphorylate GR serine 211, were also significantly increased. To assess activity of the GR, we measured protein levels of the transcription factor p53 whose levels are downregulated, at least in part, by active GR. In this study, UPI decreased p53 protein and its downstream target Bax mRNA levels. We conclude that UPI in rats affects GR expression and activity in the liver. We speculate that these alterations early in life may contribute to the changes in chromatin structure and gene expression previously described in the IUGR liver.

Animals↗

PubMed2004

Nonresponsiveness of cerebral p53-MDM2 functional circuit in newborn rat pups rendered IUGR via uteroplacental insufficiency.

Severe uteroplacental insufficiency causes cerebral apoptosis in the fetus. Moderate uteroplacental insufficiency causes intrauterine growth retardation (IUGR) and increases the risk of postnatal neurological morbidity. In the rat, uteroplacental insufficiency and IUGR affect cerebral gene expression of Bcl-2 and predispose the newborn IUGR rat toward cerebral apoptosis when challenged with perinatal hypoxia. Expression of Bcl-2, as well as the proapoptotic protein Bax, is regulated by p53. p53 also induces MDM2 transcription, which functions to limit further p53-induced apoptosis. The predisposition of the IUGR fetus toward cerebral apoptosis suggests that the p53-MDM2 "functional" circuit may be perturbed in the newborn IUGR rat brain. We hypothesized that MDM2 cerebral expression does not increase in response to increased p53 expression or increased levels of phospho-p53 (Ser15), an activated form of p53. To prove this hypothesis, we induced IUGR through bilateral uterine ligation of the pregnant rat. Uteroplacental insufficiency significantly increased p53 mRNA, total p53 protein, and phospho-p53 (Ser15) protein levels in the brain at term. Increased expression of phospho-p53 (Ser15) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells were localized to the CA1 region of the hippocampus, the subcortical and periventricular white matter, and the amygdala of the IUGR rat brain. In contrast, uteroplacental insufficiency decreased cerebral MDM2 mRNA and phospho-MDM2 (Ser166) protein levels in the IUGR rat pups. We conclude that the cerebral MDM2 response to increased p53 expression is not present in the newborn IUGR rat pup, and we speculate that this contributes to the predisposition of the IUGR fetus toward perinatal and long-term neurodevelopmental morbidities.

Animals↗

PubMed2004

Epidermal growth factor reduces intestinal apoptosis in an experimental model of necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating intestinal disease of premature infants. Although end-stage NEC is characterized histopathologically as extensive necrosis, apoptosis may account for the initial loss of epithelium before full development of disease. We have previously shown that epidermal growth factor (EGF) reduces the incidence of NEC in a rat model. Although EGF has been shown to protect intestinal enterocytes from apoptosis, the mechanism of EGF-mediated protection against NEC is not known. The aim of this study was to investigate if EGF treatment elicits changes in expression of apoptotic markers in the ileum during the development of NEC. With the use of a well-established neonatal rat model of NEC, rats were divided into the following three experimental groups: dam fed (DF), milk formula fed (NEC), or fed with formula supplemented with 500 ng/ml EGF (NEC+EGF). Changes in ileal morphology, gene and protein expression, and histological localization of apoptotic regulators were evaluated. Anti-apoptotic Bcl-2 mRNA levels were markedly reduced and pro-apoptotic Bax mRNA levels were markedly elevated in the NEC group compared with DF controls. Supplementation of EGF into formula significantly increased anti-apoptotic Bcl-2 mRNA, whereas pro-apoptotic Bax was significantly decreased. The Bax-to-Bcl-2 ratio for mRNA and protein was markedly decreased in NEC+EGF animals compared with the NEC group. The presence of caspase-3-positive epithelial cells was markedly reduced in EGF-treated rats. These data suggest that alteration of the balance between pro-and anti-apoptotic proteins in the site of injury is a possible mechanism by which EGF maintains intestinal integrity and protects intestinal epithelium against NEC injury.

Animals↗

PubMed2004

Uteroplacental insufficiency induces site-specific changes in histone H3 covalent modifications and affects DNA-histone H3 positioning in day 0 IUGR rat liver.

Uteroplacental insufficiency and subsequent intrauterine growth retardation (IUGR) increase the risk of adult onset insulin resistance and dyslipidemia in humans and rats. IUGR rats are further characterized by postnatal alterations in hepatic PPAR-gamma coactivator (PGC-1) and carnitine-palmitoyl-transferase I (CPTI) expression, as well as overall hyperacetylation of histone H3. However, it is unknown whether the histone H3 hyperacetylation is site specific or relates to the changes in gene expression previously described in IUGR rats. We therefore hypothesized that uteroplacental insufficiency causes site-specific modifications in hepatic H3 acetylation and affects the association of acetylated histone H3 with PGC-1 and CPTI promoter sequences. Uteroplacental insufficiency was used to produce asymmetrical IUGR rats. IUGR significantly increased acetylation of H3 lysine-9 (H3/K9), lysine-14 (H3/K14), and lysine-18 (H3/K18) at day 0 of life, and these changes occurred in association with decreased nuclear protein levels of histone deacetylase 1 (HDAC1) and HDAC activity. Chromatin immunoprecipitation using acetyl-H3/K9 antibody and day 0 chromatin revealed that uteroplacental insufficiency affected the association between acetylated H3/K9 and the promoters of PGC-1 and CPTI, respectively, in IUGR liver. At day 21 of life, the neonatal pattern of H3 hyperacetylation persisted only in the IUGR males. We conclude that uteroplacental insufficiency increases H3 acetylation in a site-specific manner in IUGR liver and that these changes persist in male IUGR animals. The altered association of the PGC-1 and CPTI promoters with acetylated H3/K9 correlates with previous reports of IUGR altering the expression of these genes. We speculate that in utero alterations of chromatin structure contribute to fetal programming.

Animals↗

PubMed2004

Uteroplacental insufficiency alters DNA methylation, one-carbon metabolism, and histone acetylation in IUGR rats.

Uteroplacental insufficiency leads to intrauterine growth retardation (IUGR) and increases the risk of insulin resistance and hypertriglyceridemia in both humans and rats. Postnatal changes in hepatic gene expression characterize the postnatal IUGR rat, despite the transient nature of the initial in utero insult. Phenomena such as DNA methylation and histone acetylation can induce a relatively static reprogramming of gene transcription by altering chromatin infrastructure. We therefore hypothesized that uteroplacental insufficiency persistently affects DNA methylation and histone acetylation in the IUGR rat liver. IUGR rat pups were created by inducing uteroplacental insufficiency through bilateral uterine artery ligation of the pregnant dam on day 19 of gestation. The SssI methyltransferase assay and two-dimensional thin-layer chromatography demonstrated genome-wide DNA hypomethylation in postnatal IUGR liver. To investigate a possible mechanism for this hypomethylation, levels of hepatic metabolites and enzyme mRNAs involved in one-carbon metabolism were measured using HPLC with coulometric electrochemical detection and real-time RT-PCR, respectively. Uteroplacental insufficiency increased IUGR levels of S-adenosylhomocysteine, homocysteine, and methionine in association with decreased mRNA levels of methionine adenosyltransferase and cystathionine-beta-synthase. Western blotting further demonstrated that increased quantities of acetylated histone H3 also characterized the IUGR liver. Increased hepatic levels of S-adenosylhomocysteine can promote DNA hypomethylation, which is often associated with histone hyperacetylation. We speculate that the altered intrauterine milieu associated with uteroplacental insufficiency affects hepatic one-carbon metabolism and subsequent DNA methylation, which thereby alters chromatin dynamics and leads to persistent changes in hepatic gene expression.

Acetylation↗

PubMed2004

Uteroplacental insufficiency decreases small intestine growth and alters apoptotic homeostasis in term intrauterine growth retarded rats.

Human and animal studies demonstrate that uteroplacental insufficiency and subsequent intrauterine growth retardation (IUGR) decrease intestinal growth and lead to both an increased incidence of feeding intolerance and necrotizing enterocolitis. Our objective was to determine the effects of uteroplacental insufficiency upon small intestine growth, histology, gene expression of the apoptosis related proteins Bcl-2, Bax and p53, and caspase-3 activity. For this purpose, we induced uteroplacental insufficiency through bilateral uterine artery ligation on day 19 of gestation in fully anesthetized pregnant Sprague-Dawley rats and harvested pups at term 2 days latter. Pups from sham surgeries served as controls. Uteroplacental insufficiency reduced cell count per crypt and decreased small intestinal weight. In association with these changes, IUGR intestinal Bcl-2 mRNA levels were decreased significantly, and Bax and p53 mRNA were significantly increased in distal ileum. Immunohistochemistry for Bcl-2, Bax, and p53 revealed similar findings. In association with the decreased Bcl-2 and the increased Bax gene expression, increased caspase-3 activity characterized the IUGR distal ileum. We conclude that uteroplacental insufficiency affects intestinal growth and morphology in association with altered gene expression of apoptosis related proteins. We speculate that the morphological change and associated altered apoptotic homeostasis contribute to the increased morbidity of infants affected by uteroplacental insufficiency.

Animals↗

PubMed2003

Uteroplacental insufficiency increases apoptosis and alters p53 gene methylation in the full-term IUGR rat kidney.

Uteroplacental insufficiency causes intrauterine growth retardation (IUGR), which is associated with adult onset diseases such as hypertension. Previous studies demonstrate that growth retardation in humans and rats decreases glomeruli number; however, the molecular mechanisms responsible for this reduction are unknown. Apoptosis plays a key role in renal organogenesis. We therefore hypothesized that the in utero deprivation associated with uteroplacental insufficiency decreases glomeruli, increases apoptosis, and alters the mRNA levels of key apoptosis-related proteins in full-term IUGR kidneys. To prove this hypothesis, we induced asymmetric IUGR through bilateral uterine artery ligation of the pregnant rat. We found that uteroplacental insufficiency significantly reduced glomeruli number while increasing TUNEL staining and caspase-3 activity in the IUGR kidney. A significant decrease in Bcl-2 mRNA and a significant increase in Bax and p53 mRNA further characterized the IUGR kidney. Because altered p53 CpG methylation affects p53 expression, we analyzed p53 promoter CpG methylation using methylation-sensitive restriction enzymes and real-time PCR. Uteroplacental insufficiency specifically decreased CpG methylation of the renal p53 BstU I site promoter without affecting the Hha I or the Aci I sites. Uteroplacental insufficiency also induced a relative hypomethylation from exon 5 to exon 8, which was associated with deceased mRNA levels of DNMT1. We conclude that uteroplacental insufficiency alters p53 DNA CpG methylation, affects mRNA levels of key apoptosis-related proteins, increases renal apoptosis, and reduces glomeruli number in the IUGR kidney. We speculate that these changes represent mechanisms that contribute to the fetal origins of adult disease.

Animals↗

PubMed2003

IUGR alters postnatal rat skeletal muscle peroxisome proliferator-activated receptor-gamma coactivator-1 gene expression in a fiber specific manner.

Uteroplacental insufficiency and subsequent intrauterine growth retardation (IUGR) increase the risk of insulin resistance in humans and rats. Aberrant skeletal muscle lipid metabolism contributes to the pathogenesis of insulin resistance. Peroxisome proliferator-activated receptor-gamma co-activator-1 (PGC-1) is a transcriptional co-activator that affects gene expression of key lipid metabolizing enzymes such as carnitine palmitoyl-transferase I (mCPTI). Because gene expression of lipid metabolizing enzymes is altered in IUGR postnatal skeletal muscle, and we hypothesized that PGC-1 expression would be similarly affected. To prove this hypothesis, bilateral uterine artery ligation and sham surgery were used to produce IUGR and control rats respectively. Western Blotting demonstrated that PGC-1 hind limb skeletal muscle protein levels were increased in perinatal and postnatal IUGR rats. Conventional RT-PCR demonstrated that PGC-1 mRNA levels were similarly increased in perinatal hind limb skeletal muscle and juvenile extensor digitorum longus (EDL), but were decreased in juvenile soleus. Because a gender specific trend was noted in PGC-1 mRNA levels, real time RT-PCR was used for further differentiation. Real time RT-PCR revealed that changes in postnatal skeletal muscle PGC-1 expression were more marked in male IUGR rats versus female IUGR rats. Down stream targets of PGC-1 followed a similar pattern of expression. We conclude that PGC-1 expression is altered in rat IUGR skeletal muscle and speculate that it contributes to the pathogenesis of insulin resistance in the IUGR rat.

Female↗

PubMed2002

IGF alters jejunal glucose transporter expression and serum glucose levels in immature rats.

Milk-borne insulin-like growth factors (IGFs) enhance nutrient absorption in the immature intestine, which is characterized by low levels of glucose oxidation. We therefore hypothesized that feeding a rat milk substitute (RMS) devoid of growth factors to rat pups would lower serum glucose levels relative to dam-fed control rats and that supplementation of RMS with physiological doses of either IGF-I or IGF-II would normalize serum glucose levels via increased jejunal glucose transporter 2 (GLUT2) and high-affinity Na(+)-glucose cotransporter (SGLT1) expression. We found lower serum glucose concentrations in RMS-fed pups; in contrast, serum glucose levels in the IGF-supplemented pups were similar to those of dam-fed controls. RT-PCR and laser scanning confocal microscopy similarly demonstrated that IGF supplementation increased expression of jejunal glucose transporters. Further experiments demonstrated that IGF supplementation altered mRNA levels of key mitochondrial enzymes without altering jejunal lactase activity. We conclude that IGF-I and IGF-II supplementation increases serum glucose levels in the immature rat pup fed artificial formula and alters gene expression of the jejunal glucose transporters.

Animal Feed↗

PubMed2002

P-glycoprotein expression in mouse brain increases with maturation.

The mdr1a isoform of P-glycoprotein (Pgp) is an integral plasma membrane efflux pump expressed in adult brain capillary endothelial cells and astrocytes of the blood-brain barrier. We determined the developmental pattern of Pgp expression in brain tissue at embryonic day 16 (E16), day of life 0 (D0), day of life 7 (D7), day of life 21 (D21), and adults (Ad). The relative expression of Pgp mRNA and protein was indexed as a percent (mean +/- SEM) of D0 levels. Pgp mRNA levels increased significantly (p < 0.01) with maturation (E16: 75 +/- 8%; D21: 303 +/- 37%, and Ad: 1,160 +/- 120%). Similarly, Pgp protein expression was observed at E16 and increased significantly (p < 0.01) during development (E16: 52 +/- 8%; D7: 187 +/- 23%; D21: 440 +/- 48%, and Ad: 441 +/- 56%). This developmental pattern of enhanced blood-brain barrier Pgp expression with maturation was confirmed by immunohistochemistry. We conclude that (i) Pgp expression in mouse brain is limited during late embryogenesis and the newborn period; (ii) Pgp expression increases markedly with postnatal maturation, and (iii) by D21 brain Pgp protein expression approximates adult levels.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2002

Increased hepatic peroxisome proliferator-activated receptor-gamma coactivator-1 gene expression in a rat model of intrauterine growth retardation and subsequent insulin resistance.

Uteroplacental insufficiency and subsequent intrauterine growth retardation (IUGR) increase the risk of type 2 diabetes in humans and rats. Unsuppressed endogenous hepatic glucose production is a common component of the insulin resistance associated with type 2 diabetes. Peroxisome proliferator-activated receptor-gamma coactivator-1 (PGC-1) mediates hepatic glucose production by controlling mRNA levels of glucose-6-phosphatase (G-6-Pase), phosphoenolpyruvate carboxykinase (PEPCK), and fructose-1,6-bisphosphatase (FBPase). We therefore hypothesized that gene expression of PGC-1 would be increased in juvenile IUGR rat livers, and this increase would directly correlate with hepatic mRNA levels of PEPCK, G-6-Pase, and FBPase, but not glucokinase. We found that IUGR hepatic PGC-1 protein levels were increased to 230 +/- 32% and 310 +/- 47% of control values at d 0 and d 21 of life, respectively. Similarly, IUGR hepatic PGC-1 mRNA levels were significantly elevated at both ages. Concurrent with the increased PGC-1 gene expression, IUGR hepatic mRNA levels of G-6-Pase, PEPCK, and FBPase were also significantly increased, whereas glucokinase mRNA levels were significantly decreased. These data suggest that increased PGC-1 expression and subsequent hepatic glucose production contribute to the insulin resistance observed in the IUGR juvenile rat.

Animals↗