RESUMO
Air pollution (AP) is detrimental to pregnancies including increasing risk factors of gestational diabetes mellitus. We hypothesized that exposure to AP causes cardiovascular and metabolic disruption thereby altering placental gene expression, which in turn affects the placental phenotype and thereby embryonic/fetal development. To test this hypothesis, we investigated the impact of intra-nasal instilled AP upon gestational day 16-19 maternal mouse cardiovascular and metabolic status, placental nutrient transporters, and placental-fetal size and morphology. To further unravel mechanisms, we also examined placental total DNA 5'-hydroxymethylation and bulk RNA sequenced gene expression profiles. AP exposed pregnant mice and fetuses were tachycardic with a reduction in maternal left ventricular fractional shortening and increased uterine artery with decreased umbilical artery systolic peak velocities. In addition, they were hyperglycemic, glucose intolerant and insulin resistant, with changes in placental glucose (Glut3) and fatty acid (Fatp1 & Cd36) transporters, and a spatial disruption of cells expressing Glut10 that imports L-dehydroascorbic acid in protecting against oxidative stress. Placentas revealed inflammatory cellular infiltration with associated cellular edema and necrosis, with dilated vascular spaces and hemorrhage. Placental and fetal body weights decreased in mid-gestation with a reduction in brain cortical thickness emerging in late gestation. Placental total DNA 5'-hydroxymethylation was 2.5-fold higher, with perturbed gene expression profiles involving key metabolic, inflammatory, transcriptional, cellular polarizing and processing genes and pathways. We conclude that gestational exposure to AP incites a maternal inflammatory response resulting in features mimicking maternal gestational diabetes mellitus with altered placental DNA 5'-hydroxymethylation, gene expression, and associated injury.
RESUMO
Glucose is the primary energy source for most mammalian cells and its transport is affected by a family of facilitative glucose transporters (GLUTs) encoded by the SLC2 gene. GLUT1 and GLUT3, highly expressed isoforms in the blood-brain barrier and neuronal membranes, respectively, are associated with multiple neurodevelopmental disorders including epilepsy, dyslexia, ADHD, and autism spectrum disorder (ASD). Dietary therapies, such as the ketogenic diet, are widely accepted treatments for patients with the GLUT1 deficiency syndrome, while ameliorating certain symptoms associated with GLUT3 deficiency in animal models. A ketogenic diet, high-fat diet, and calorie/energy restriction during prenatal and postnatal stages can also alter the placental and brain GLUTs expression with long-term consequences on neurobehavior. This review focuses primarily on the role of diet/energy perturbations upon GLUT isoform-mediated emergence of neurodevelopmental and neurodegenerative disorders.
Assuntos
Encéfalo , Transportador de Glucose Tipo 1 , Transportador de Glucose Tipo 3 , Transtornos do Neurodesenvolvimento , Placenta , Transportador de Glucose Tipo 3/metabolismo , Transportador de Glucose Tipo 3/genética , Humanos , Gravidez , Encéfalo/metabolismo , Placenta/metabolismo , Feminino , Transportador de Glucose Tipo 1/metabolismo , Transtornos do Neurodesenvolvimento/etiologia , Animais , Fenômenos Fisiológicos da Nutrição Materna , Dieta Cetogênica , Dieta Hiperlipídica/efeitos adversos , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/metabolismo , Glucose/metabolismoRESUMO
Introduction: Serotonin (5-HT) is critical for neurodevelopment and the serotonin transporter (SERT) modulates serotonin levels. Perturbed prenatal and postnatal dietary exposures affect the developing offspring predisposing to neurobehavioral disorders in the adult. We hypothesized that the postnatal brain 5-HT-SERT imbalance associated with gut dysbiosis forms the contributing gut-brain axis dependent mechanism responsible for such ultimate phenotypes. Methods: Employing maternal diet restricted (IUGR, n=8) and high fat+high fructose (HFhf, n=6) dietary modifications, rodent brain serotonin was assessed temporally by ELISA and SERT by quantitative Western blot analysis. Simultaneously, colonic microbiome studies were performed. Results: At early postnatal (P) day 2 no changes in the IUGR, but a ~24% reduction in serotonin (p = 0.00005) in the HFhf group occurred, particularly in the males (p = 0.000007) revealing a male versus female difference (p = 0.006). No such changes in SERT concentrations emerged. At late P21 the IUGR group reared on HFhf (IUGR/HFhf, (n = 4) diet revealed increased serotonin by ~53% in males (p = 0.0001) and 36% in females (p = 0.023). While only females demonstrated a ~40% decrease in serotonin (p = 0.010), the males only trended lower without a significant change within the HFhf group (p = 0.146). SERT on the other hand was no different in HFhf or IUGR/RC, with only the female IUGR/HFhf revealing a 28% decrease (p = 0.036). In colonic microbiome studies, serotonin-producing Bacteriodes increased with decreased Lactobacillus at P2, while the serotonin-producing Streptococcus species increased in IUGR/HFhf at P21. Sex-specific changes emerged in association with brain serotonin or SERT in the case of Alistipase, Anaeroplasma, Blautia, Doria, Lactococcus, Proteus, and Roseburia genera. Discussion: We conclude that an imbalanced 5-HT-SERT axis during postnatal brain development is sex-specific and induced by maternal dietary modifications related to postnatal gut dysbiosis. We speculate that these early changes albeit transient may permanently alter critical neural maturational processes affecting circuitry formation, thereby perturbing the neuropsychiatric equipoise.
RESUMO
Intra-Uterine Growth Restriction (IUGR) is a risk factor for many adult-onset chronic diseases, such as diabetes and obesity. These diseases are associated with intestinal microbiome perturbations (dysbiosis). The establishment of an intestinal microbiome begins in utero and continues postnatally (PN). Hypercaloric diet-induced dysbiosis is a major driver of childhood obesity. We hypothesized that different postnatal diets superimposed on IUGR will alter the postnatal intestinal microbiome. We compared four experimental rat groups: (1) Ad lib fed regular chow diet pre- and postnatally (CON), (2-3) IUGR induced by maternal caloric restriction prenatally followed postnatally (PN) by either (2) the control diet (IUGR-RC) or (3) High-Fat-high-fructose (IUGR-HFhf) diet, and lastly (4) HFhf ad lib pre- and postnatally (HFhf). Fecal samples were collected from dams and male and female rat offspring at postnatal day 2, 21, and adult day 180 for 16S rRNA gene sequencing. Maternal diet induced IUGR led to dysbiosis of the intestinal microbiome at PN21. Postnatal HFhf diet significantly reduced microbial diversity and worsened dysbiosis reflected by an increased Gammaproteobacteria/Clostridia ratio. Dysbiosis arising from a mismatch between IUGR and a postnatal HFhf diet may contribute to increased risk of the IUGR offspring for subsequent detrimental health problems.
Assuntos
Microbioma Gastrointestinal , Obesidade Infantil , Criança , Humanos , Animais , Ratos , Masculino , Feminino , Disbiose/complicações , RNA Ribossômico 16S/genética , Obesidade Infantil/complicações , Retardo do Crescimento Fetal/etiologia , Dieta , Dieta Hiperlipídica/efeitos adversosRESUMO
Perturbed maternal diet and prenatal exposure to air pollution (AP) affect the fetal brain, predisposing to postnatal neurobehavioral disorders. Glucose transporters (GLUTs) are key in fueling neurotransmission; deficiency of the neuronal isoform GLUT3 culminates in autism spectrum disorders. Along with the different neurotransmitters, serotonin (5-HT) and oxytocin (OXT) are critical for the development of neural connectivity. Serotonin transporter (SERT) modulates synaptic 5-HT levels, while the OXT receptor (OXTR) mediates OXT action. We hypothesized that perturbed brain GLUT1/GLUT3 regulated 5-HT-SERT imbalance, which serves as a contributing factor to postnatal neuropsychiatric phenotypes, with OXT/OXTR providing a counterbalance. Employing maternal diet restriction (intrauterine growth restriction [IUGR]), high-fat (HF) dietary modifications, and prenatal exposure to simulated AP, fetal (E19) murine brain 5-HT was assessed by ELISA with SERT and OXTR being localized by immunohistochemistry and measured by quantitative Western blot analysis. IUGR with lower head weights led to a 48% reduction in male and female fetal brain GLUT3 with no change in GLUT1, when compared to age- and sex-matched controls, with no significant change in OXTR. In addition, a â¼50% (p = 0.005) decrease in 5-HT and SERT concentrations was displayed in fetal IUGR brains. In contrast, despite emergence of microcephaly, exposure to a maternal HF diet or AP caused no significant changes. We conclude that in the IUGR during fetal brain development, reduced GLUT3 is associated with an imbalanced 5-HT-SERT axis. We speculate that these early changes may set the stage for altering the 5HT-SERT neural axis with postnatal emergence of associated neurodevelopmental disorders.
Assuntos
Poluentes Atmosféricos , Transportador de Glucose Tipo 3 , Receptores de Ocitocina , Proteínas da Membrana Plasmática de Transporte de Serotonina , Serotonina , Animais , Encéfalo , Dieta , Feminino , Proteínas Facilitadoras de Transporte de Glucose , Transportador de Glucose Tipo 1 , Masculino , Camundongos , Ocitocina , GravidezRESUMO
Since GLUT3 is vital for fueling neurotransmission, we examined in-vivo the adult phenotype carrying the conditional homozygous glut3 gene mutation (KO) in glutamate-excitatory neurons. These KO mice demonstrated sex-specific differences in brain and body weights (p = 0.0001 and p = 0.01 each) with reduced GLUT3 protein in cerebral cortices and brain stem (p = 0.005). In patch clamp studies the glut3 KO mice displayed a shorter latency to and enhanced paroxysmal activity (p = 0.01 and p = 0.015 each) in pyramidal neurons upon application of a GABAA antagonist, supporting hyperexcitability. Further, associated changes in neurobehavior consisted of reduced latency to fall in the rotorod motor test related to incoordination, increased distance traveled in total and periphery versus center in open field testing suggesting hyperactivity with anxiety (p = 0.0013 in male, p = 0.045 in female), reduced time freezing reminiscent of disrupted contextual fear conditioning (p = 0.0033), decreased time in target quadrant seen with spatial cognitive memory water maze testing (p = 0.034), and enhanced sociability particularly for novelty reflecting a lack of inhibition/impulsivity (p = 0.038). Some of these features were equally pronounced in males and females (cognitive) while others were seen in females (anxiety and impulsivity). We conclude that GLUT3 in adult glutamate-excitatory neurons is essential for maintaining neurotransmitory equipoise regulating excitation with maintenance of motor coordination and activity, cognition, spatial memory and normal fear for both contextual events and novelty with tempered sociability. While sex-specificity was forthcoming for some of these behaviors, our findings collectively suggest that loss-of-function glut3 gene mutations or polymorphisms may underlie an endophenotype of attention deficit-hyperactivity disorder.
Assuntos
Comportamento Animal/fisiologia , Encéfalo/fisiopatologia , Transportador de Glucose Tipo 3/genética , Transtornos do Neurodesenvolvimento/genética , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Feminino , Transportador de Glucose Tipo 3/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transtornos do Neurodesenvolvimento/metabolismo , Transtornos do Neurodesenvolvimento/fisiopatologia , Células Piramidais/metabolismoRESUMO
To understand the cellular basis for the neurodevelopmental effects of intrauterine growth restriction (IUGR), we examined the global and regional expression of various cell types within murine (Mus musculus) fetal brain. Our model employed maternal calorie restriction to 50% daily food intake from gestation day 10-19, producing IUGR offspring. Offspring had smaller head sizes with larger head:body ratios indicating a head sparing IUGR effect. IUGR fetuses at embryonic day 19 (E19) had reduced nestin (progenitors), ß-III tubulin (immature neurons), Glial fibrillary acidic protein (astrocytes), and O4 (oligodendrocytes) cell lineages via immunofluorescence quantification and a 30% reduction in cortical thickness. No difference was found in Bcl-2 or Bax (apoptosis) between controls and IUGR, though qualitatively, immunoreactivity of doublecortin (migration) and Ki67 (proliferation) was decreased. In the interest of examining a potential therapeutic peptide, we next investigated a novel pro-survival peptide, mouse Humanin (mHN). Ontogeny examination revealed highest mHN expression at E19, diminishing by postnatal day 15 (P15), and nearly absent in adult (3 months). Subanalysis by sex at E19 yielded higher mHN expression among males during fetal life, without significant difference between sexes postnatally. Furthermore, female IUGR mice at E19 had a greater increase in cortical mHN versus the male fetus over their respective controls. We conclude that maternal dietary restriction-associated IUGR interferes with neural progenitors differentiating into the various cellular components populating the cerebral cortex, and reduces cerebral cortical size. mHN expression is developmental stage and sex specific, with IUGR, particularly in the females, adaptively increasing its expression toward mediating a pro-survival approach against nutritional adversity.
Assuntos
Córtex Cerebral/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fenômenos Fisiológicos da Nutrição Materna/fisiologia , Neurônios/metabolismo , Animais , Restrição Calórica , Feminino , Retardo do Crescimento Fetal/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Masculino , CamundongosRESUMO
We examined the role of hepatocyte micro-RNA-122 and hypothalamic neuropeptides, in weanling (21d) female rats exposed to calorie restriction induced growth restriction either prenatally (IUGR), postnatally (PNGR) or both (IPGR) vs. ad lib fed controls (CON). IUGR were hyperinsulinemic, hyperleptinemic and dyslipidemic with high circulating miR-122. In contrast, PNGR and IPGR displayed insufficient glucose, insulin and leptin amidst high ketones with a dichotomy in circulating miR-122 of PNGRAssuntos
Restrição Calórica/efeitos adversos
, Metabolismo dos Lipídeos/genética
, Metabolismo dos Lipídeos/fisiologia
, MicroRNAs/fisiologia
, Animais
, Peso Corporal
, Ritmo Circadiano
, Metabolismo Energético
, Ácidos Graxos/genética
, Ácidos Graxos/metabolismo
, Feminino
, Retardo do Crescimento Fetal/etiologia
, Expressão Gênica
, Transtornos do Crescimento/etiologia
, Hipotálamo/química
, Fígado/química
, Fígado/metabolismo
, MicroRNAs/genética
, Tamanho do Órgão
, Gravidez
, RNA Mensageiro/análise
, Ratos
, Ratos Sprague-Dawley
, Desmame
RESUMO
Diet during pregnancy has long lasting consequences on the offspring, warranting a study on the impact of early exposure to a high fat diet on the adult offspring. We hypothesized that a prenatal n-6 enriched diet will have adverse metabolic outcomes on the adult offspring that may be reversed with a postnatal n-3 enriched diet. To test this hypothesis, we examined the adult offspring from three groups: (1) n-6 group: during gestation and lactation, dams consumed an n-6 polyunsaturated fatty acid enriched diet, (2) n-3 group: gestational n-6 diet was followed by an n-3 enriched diet during lactation, and (3) a control (CD) group that received standard diet throughout gestation and lactation. Offspring from all groups weaned to a control diet ad libitum. Beginning at postnatal day 2 (Pâ¯<â¯.03) and persisting at 360 days in males (Pâ¯<â¯.04), an increase in hypothalamic AgRP expression occurred in the n-6 and n-3 groups, with an increase in food intake (Pâ¯=â¯.01), and the n-3 group displaying lower body (Pâ¯<â¯.03) and brain (Pâ¯<â¯.05) weights. At 360 days, the n-6 and n-3 groups remained glucose tolerant and insulin sensitive, with increased phosphorylated-AMP-activated protein kinase (Pâ¯<â¯.05). n-6 group developed hepatic steatosis with reduced hepatic reflected as higher plasma microRNA-122 (Pâ¯<â¯.04) that targets pAMPK. We conclude that early life exposure to n-6 and n-3 led to hypothalamic AgRP-related higher food intake, with n-6 culminating in a fatty liver partially mitigated by postnatal n-3. While both diets preserved glucose tolerance and insulin sensitivity, postnatal n-3 displayed detrimental effects on the brain.
Assuntos
Encéfalo/patologia , Dieta Hiperlipídica/efeitos adversos , Fígado Gorduroso/metabolismo , Glucose/metabolismo , Resistência à Insulina , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Animais , Animais Lactentes , Dieta Hiperlipídica/métodos , Modelos Animais de Doenças , Fígado Gorduroso/patologia , Feminino , Masculino , Tamanho do Órgão , Gravidez , Efeitos Tardios da Exposição Pré-Natal/patologia , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: We examined Red Blood Cell (RBC) Glucose Transporter isoform 1 (GLUT1) and White Blood Cell (WBC) Glucose Transporter isoform 3 (GLUT3) protein concentrations to assess their potential as surrogate biomarkers for the presence of hypoxic-ischemic encephalopathy (HIE) and response to therapeutic hypothermia (TH), with respect to the neurodevelopmental prognosis. STUDY DESIGN: A prospective feasibility study of 10 infants with HIE and 8 age-matched control subjects was undertaken. Following parental consent, blood samples were obtained at baseline before institution of TH (<6â¯h of life), during TH, at rewarming and post-TH in the HIE group with a baseline sample from the control group. GLUT1 and GLUT3 were measured by Enzyme-linked immunosorbent assay (ELISA) with brain biomarkers, Neuron-Specific Enolase (NSE) and Glial Fibrillary Acidic Protein (GFAP). Novel "HIE-high risk" and "Neurological" scores were developed to help identify HIE and to assess severity and prognosis, respectively. RESULTS: RBC GLUT1 concentrations were increased at the baseline pre-TH time point in HIE versus control subjects (pâ¯=â¯.006), normalizing after TH (pâ¯=â¯.05). An association between GLUT1 and NSE concentrations (which was reflective of the HIE-high risk and the Neuro-scores) in controls and HIE pre-TH was seen (R2â¯=â¯0.36, pâ¯=â¯.008), with GLUT1 demonstrating 90% sensitivity and 88% specificity for presence of HIE identified by Sarnat Staging. WBC GLUT3 concentrations were low and no different in HIE versus control, and GFAP concentrations trended higher during re-warming (pâ¯=â¯.11) and post-TH (pâ¯=â¯.16). We demonstrated a significant difference between HIE and controls for both the "HIE-high risk" and the "Neurological" Scores. The latter score revealing the severity of clinical neurological illness correlated with the corresponding RBC GLUT1 (R2 valueâ¯=â¯0.39; pâ¯=â¯.006). CONCLUSION: Circulating RBC GLUT1 concentrations with NSE demonstrate a significant potential in reflecting the severity of HIE pre-TH and gauging effectiveness of TH. In contrast, the low neonatal WBC GLUT3 concentrations make discerning differences between degrees of HIE as well as assessing effectiveness of TH difficult. The HIE-high risk and Neurological scores may extend the "Sarnat staging" towards assessing severity and neuro-developmental prognosis of HIE.
Assuntos
Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 3/metabolismo , Hipóxia-Isquemia Encefálica/diagnóstico , Biomarcadores , Eritrócitos/metabolismo , Estudos de Viabilidade , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Recém-Nascido , Unidades de Terapia Intensiva Neonatal , Masculino , Fosfopiruvato Hidratase/metabolismo , Projetos Piloto , Prognóstico , Estudos Prospectivos , Sensibilidade e Especificidade , Índice de Gravidade de DoençaRESUMO
We created a neural-specific conditional murine glut3 (Slc2A3) deletion (glut3flox/flox/nestin-Cre+) to examine the effect of a lack of Glut3 on neurodevelopment. Compared with age-matched glut3flox/flox = WT and heterozygotes (glut3flox/+/nestin-Cre+), we found that a >90% reduction in male and female brain Glut3 occurred by postnatal day 15 (PN15) in glut3flox/flox/nestin-Cre+ This genetic manipulation caused a diminution in brain weight and cortical thickness at PN15, a reduced number of dendritic spines, and fewer ultrasonic vocalizations. Patch-clamp recordings of cortical pyramidal neurons revealed increased frequency of bicuculline-induced paroxysmal discharges as well as reduced latency, attesting to a functional synaptic and cortical hyperexcitability. Concomitant stunting with lower glucose concentrations despite increased milk intake shortened the lifespan, failing rescue by a ketogenic diet. This led to creating glut3flox/flox/CaMK2α-Cre+ mice lacking Glut3 in the adult male limbic system. These mice had normal lifespan, displayed reduced IPSCs in cortical pyramidal neurons, less anxiety/fear, and lowered spatial memory and motor abilities but heightened exploratory and social responses. These distinct postnatal and adult phenotypes, based upon whether glut3 gene is globally or restrictively absent, have implications for humans who carry copy number variations and present with neurodevelopmental disorders.SIGNIFICANCE STATEMENT Lack of the key brain-specific glucose transporter 3 gene found in neurons during early postnatal life results in significant stunting, a reduction in dendritic spines found on neuronal processes and brain size, heightened neuronal excitability, along with a shortened lifespan. When occurring in the adult and limited to the limbic system alone, lack of this gene in neurons reduces the fear of spatial exploration and socialization but does not affect the lifespan. These features are distinct heralding differences between postnatal and adult phenotypes based upon whether the same gene is globally or restrictively lacking. These findings have implications for humans who carry copy number variations pertinent to this gene and have been described to present with neurodevelopmental disorders.
Assuntos
Encéfalo/metabolismo , Comportamento Exploratório/fisiologia , Deleção de Genes , Transportador de Glucose Tipo 3/deficiência , Transportador de Glucose Tipo 3/genética , Fenótipo , Fatores Etários , Animais , Animais Recém-Nascidos , Encéfalo/patologia , Espinhas Dendríticas/genética , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/patologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gravidez , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/genéticaRESUMO
Intrauterine growth restriction is linked to decreased lean body mass and insulin resistance. The mammalian target of rapamycin (mTOR) regulates muscle mass and glucose metabolism; however, little is known about maternal dietary restriction and skeletal muscle mTOR in offspring. We hypothesized that early dietary restriction would decrease skeletal muscle mass and mTOR in the suckling rat. To test this hypothesis, ab libitum access to food or dietary restriction during gestation followed by postnatal cross-fostering to a dietary-restricted or ad libitum-fed rat dam during lactation generated 4 groups: control (CON), intrauterine dietary restricted (IUDR), postnatal dietary restricted (PNDR), and IUDR+PNDR (IPDR). At day 21, when compared to CON, the IUDR group demonstrated "catchup" growth, but no changes were observed in the mTOR pathway. Despite having less muscle mass than CON and IUDR (Pâ¯<â¯.001), in IPDR and PNDR rats mTOR remained unchanged. IPDR and PNDR (p)-tuberous sclerosis complex 2 was less than the IUDR group (Pâ¯<â¯.05). Downstream, IPDR's and PNDR's phosphorylated (p)-ribosomal s6 (rs6)/rs6 was less than that of CON (Pâ¯<â¯.05). However, male IPDR's and PNDR's p-mitogen activated protein kinase MAPK/MAPK was greater than CON (Pâ¯<â¯.05) without a change in p90 ribosomal s6 kinase (p90RSK). In contrast, in females, MAPK was unchanged, but IPDR p-p90RSK/p90RSK was less than CON (Pâ¯=â¯.01). In conclusion, IPDR and PNDR reduced skeletal muscle mass but did not decrease mTOR. In IPDR and PNDR, a reduction in tuberous sclerosis complex 2 may explain why mTOR was unchanged, whereas, in males, an increase in MAPK with a decrease in rs6 may suggest a block in MAPK signaling.
Assuntos
Restrição Calórica , Ingestão de Energia , Fenômenos Fisiológicos da Nutrição Materna , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Esquelético/crescimento & desenvolvimento , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Esclerose Tuberosa/metabolismo , Animais , Composição Corporal , Feminino , Retardo do Crescimento Fetal , Resistência à Insulina , Lactação , Masculino , Fosforilação , Ratos Sprague-Dawley , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismoRESUMO
BACKGROUND: Intrauterine growth restriction (IUGR) results from a lack of nutrients transferred to the developing fetus, particularly oxygen and glucose. Increased expression of the cytoprotective mitochondrial peptide, humanin (HN), and the glucose transporter 8, GLUT8, has been reported under conditions of hypoxic stress. However, the presence and cellular localization of HN and GLUT8 in IUGR-related placental pathology remain unexplored. Thus, we undertook this study to investigate placental expression of HN and GLUT8 in IUGR-affected versus normal pregnancies. RESULTS: We found 1) increased HN expression in human IUGR-affected pregnancies on the maternal aspect of the placenta (extravillous trophoblastic (EVT) cytoplasm) compared to control (i.e. appropriate for gestational age) pregnancies, and a concomitant increase in GLUT8 expression in the same compartment, 2) HN and GLUT8 showed a protein-protein interaction by co-immunoprecipitation, 3) elevated HN and GLUT8 levels in vitro under simulated hypoxia in human EVT cells, HTR8/SVneo, and 4) increased HN expression but attenuated GLUT8 expression in vitro under serum deprivation in HTR8/SVneo cells. CONCLUSIONS: There was elevated HN expression with cytoplasmic localization to EVTs on the maternal aspect of the human placenta affected by IUGR, also associated with increased GLUT8 expression. We found that while hypoxia increased both HN and GLUT8, serum deprivation increased HN expression alone. Also, a protein-protein interaction between HN and GLUT8 suggests that their interaction may fulfill a biologic role that requires interdependency. Future investigations delineating molecular interactions between these proteins are required to fully uncover their role in IUGR-affected pregnancies.
Assuntos
Retardo do Crescimento Fetal/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Placenta/metabolismo , Adulto , Citoplasma/metabolismo , Feminino , Retardo do Crescimento Fetal/genética , Retardo do Crescimento Fetal/patologia , Proteínas Facilitadoras de Transporte de Glucose/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Placenta/patologia , Gravidez , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Trofoblastos/patologia , Regulação para CimaRESUMO
We tested the hypothesis that exposure of glut3+/- mice to a ketogenic diet ameliorates autism-like features, which include aberrant behavior and electrographic seizures. We first investigated the life course sex-specific changes in basal plasma-cerebrospinal fluid (CSF)-brain metabolic profile, brain glucose transport/uptake, glucose and monocarboxylate transporter proteins, and adenosine triphosphate (ATP) in the presence or absence of systemic insulin administration. Glut3+/- male but not female mice (5 months of age) displayed reduced CSF glucose/lactate concentrations with no change in brain Glut1, Mct2, glucose uptake or ATP. Exogenous insulin-induced hypoglycemia increased brain glucose uptake in glut3+/- males alone. Higher plasma-CSF ketones (ß-hydroxybutyrate) and lower brain Glut3 in females vs males proved protective in the former while enhancing vulnerability in the latter. As a consequence, increased synaptic proteins (neuroligin4 and SAPAP1) with spontaneous excitatory postsynaptic activity subsequently reduced hippocampal glucose content and increased brain amyloid ß1-40 deposition in an age-dependent manner in glut3+/- males but not females (4 to 24 months of age). We then explored the protective effect of a ketogenic diet on ultrasonic vocalization, sociability, spatial learning and memory, and electroencephalogram seizures in male mice (7 days to 6 to 8 months of age) alone. A ketogenic diet partially restored sociability without affecting perturbed vocalization, spatial learning and memory, and reduced seizure events. We conclude that (1) sex-specific and age-dependent perturbations underlie the phenotype of glut3+/- mice, and (2) a ketogenic diet ameliorates seizures caused by increased cortical excitation and improves sociability, but fails to rescue vocalization and cognitive deficits in glut3+/- male mice.
Assuntos
Comportamento Animal/fisiologia , Encéfalo/metabolismo , Dieta Cetogênica , Transportador de Glucose Tipo 3/metabolismo , Convulsões/dietoterapia , Comportamento Social , Animais , Encéfalo/fisiopatologia , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Eletroencefalografia , Feminino , Glucose/metabolismo , Transportador de Glucose Tipo 3/genética , Masculino , Memória/fisiologia , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Convulsões/metabolismo , Convulsões/fisiopatologia , Fatores Sexuais , Aprendizagem Espacial/fisiologia , Vocalização Animal/fisiologiaRESUMO
The purpose of this study is to clarify the subcellular localizations of multidrug resistance protein 1 (MDR1)/ABCB1 and breast cancer resistance protein (BCRP)/ABCG2 in the rodent placental SynT bilayer, i.e., a maternal-facing (SynT-I) layer and a fetal-facing (SynT-II) layer. In double immunofluorescence staining, the signals of MDR1 and BCRP appeared midway between the signals of glucose transporter 1 on the apical membrane of SynT-I and the basal plasma membrane of SynT-II, and mostly overlapped with signals of connexin 26, which forms gap junctions between SynT-I and SynT-II. In detail, median intensities (pixels) of the MDR1 and BCRP signals were significantly closer to the fetal circulation as compared to the location of connexin 26 signals. In double in situ hybridization studies, the signals of Mdr1b mRNA mostly overlapped with those of Syncytin-B, a SynT-II marker. In conclusion, MDR1 and BCRP are expressed on apical membranes of the rodent placental SynT-II layer.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Placenta/metabolismo , Trofoblastos/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Feminino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Placenta/citologia , Gravidez , RNA Mensageiro/metabolismo , Ratos Sprague-DawleyRESUMO
Pre- and postnatal calorie restriction is associated with postnatal growth restriction, reduced circulating leptin concentrations, and perturbed energy balance. Hypothalamic regulation of energy balance demonstrates enhanced orexigenic (NPY, AgRP) and diminished anorexigenic (POMC, CART) neuropeptide expression (PN21), setting the stage for subsequent development of obesity in female Sprague-Dawley rats. Leptin replenishment during the early postnatal period (PN2-PN8) led to reversal of the hypothalamic orexigenic:anorexigenic neuropeptide ratio at PN21 by reducing only the orexigenic (NPY, AgRP), without affecting the anorexigenic (POMC, CART) neuropeptide expression. This hypothalamic effect was mediated via enhanced leptin receptor (ObRb) signaling that involved increased pSTAT3/STAT3 but reduced PTP1B. This was further confirmed by an increase in body weight at PN21 in response to intracerebroventricular administration of antisense ObRb oligonucleotides (PN2-PN8). The change in the hypothalamic neuropeptide balance in response to leptin administration was associated with increased oxygen consumption, carbon dioxide production, and physical activity, which resulted in increased milk intake (PN14) with no change in body weight. This is in contrast to the reduction in milk intake with no effect on energy expenditure and physical activity observed in controls. We conclude that pre- and postnatal calorie restriction perturbs hypothalamic neuropeptide regulation of energy balance, setting the stage for hyperphagia and reduced energy expenditure, hallmarks of obesity. Leptin in turn reverses this phenotype by increasing hypothalamic ObRb signaling (sensitivity) and affecting only the orexigenic arm of the neuropeptide balance.
Assuntos
Restrição Calórica , Ingestão de Energia/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Hipotálamo/efeitos dos fármacos , Leptina/farmacologia , Neuropeptídeos/metabolismo , Efeitos Tardios da Exposição Pré-Natal/patologia , Fatores Etários , Proteína Relacionada com Agouti/metabolismo , Animais , Animais Recém-Nascidos , Feminino , Hipotálamo/metabolismo , Masculino , Proteínas do Tecido Nervoso , Neuropeptídeo Y/metabolismo , Gravidez , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Pró-Opiomelanocortina/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Intrauterine growth restriction leads to the development of adult onset obesity/metabolic syndrome, diabetes mellitus, cardiovascular disease, hypertension, stroke, dyslipidemia, and non-alcoholic fatty liver disease/steatohepatitis. Continued postnatal growth restriction has been shown to ameliorate many of these sequelae. To further our understanding of the mechanism of how intrauterine and early postnatal growth affects adult health we have employed Affymetrix microarray-based expression profiling to characterize hepatic gene expression of male offspring in a rat model of maternal nutrient restriction in early and late life. At day 21 of life (p21) combined intrauterine and postnatal calorie restriction treatment led to expression changes in circadian, metabolic, and insulin-like growth factor genes as part of a larger transcriptional response that encompasses 144 genes. Independent and controlled experiments at p21 confirm the early life circadian, metabolic, and growth factor perturbations. In contrast to the p21 transcriptional response, at day 450 of life (d450) only seven genes, largely uncharacterized, were differentially expressed. This lack of a transcriptional response identifies non-transcriptional mechanisms mediating the adult sequelae of intrauterine growth restriction. Independent experiments at d450 identify a circadian defect as well as validate expression changes to four of the genes identified by the microarray screen which have a novel association with growth restriction. Emerging from this rich dataset is a portrait of how the liver responds to growth restriction through circadian dysregulation, energy/substrate management, and growth factor modulation.
Assuntos
Restrição Calórica/efeitos adversos , Retardo do Crescimento Fetal/genética , Perfilação da Expressão Gênica/métodos , Fígado/crescimento & desenvolvimento , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Peso Corporal , Ritmo Circadiano , Feminino , Retardo do Crescimento Fetal/etiologia , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Gravidez , Efeitos Tardios da Exposição Pré-Natal/genética , Ratos , Ratos Sprague-DawleyRESUMO
Diminished transplacental glucose transport plays an important role in prenatal calorie restriction (CR) induced reduction in fetal growth. Fetal growth restriction (FGR) has an impact in shaping the adult phenotype with transgenerational implications. To understand the mechanisms underlying prenatal CR-induced transplacental glucose transport, we examined the epigenetic regulation of placental glucose transporter (Glut1 and Glut3) expression. We restricted calories by 50% in C57BL6 pregnant mice from gestational days 10 to 19 (CR; n=8) vs. controls (CON; n=8) and observed a 50% diminution in placental Glut3 expression (P<.05) with no effect on Glut1 expression by reverse transcription and quantitative real-time polymerase chain reaction (PCR). CR enhanced DNA methylation of a CpG island situated ~1000 bp upstream from the transcriptional start site of the glut3 gene, with no such effect on the glut1 gene as assessed by methylation-sensitive PCR and bisulfite sequencing. Chromatin immunoprecipitation (ChIP) assays demonstrated enhanced MeCP2 binding to the CpG island of the glut3 gene in response to CR vs. CON (P<.05). Sequential ChIP demonstrated that enhanced MeCP2 binding of the glut3-(m)CpG island enhanced histone deacetylase 2 recruitment (P<.05) but interfered with Sp1 binding (P<.001), although it did not affect Sp3 or Creb/pCreb interaction. We conclude that late-gestation CR enhanced DNA methylation of the placental glut3 gene. This epigenetic change augmented specific nuclear protein-DNA complex formation that was associated with prenatal CR-induced reduction of placental glut3 expression and thereby transplacental glucose transport. This molecular complex provides novel targets for developing therapeutic interventions aimed at reversing FGR.
Assuntos
Restrição Calórica , Metilação de DNA , Transportador de Glucose Tipo 3/metabolismo , Metiltransferases/metabolismo , Placenta/metabolismo , Animais , Sequência de Bases , Ilhas de CpG , Primers do DNA , Feminino , Camundongos , Gravidez , Reação em Cadeia da Polimerase em Tempo RealRESUMO
DNA methylation and microRNAs (miRNAs) play crucial roles in maturation of postnatal mouse neurons. Aberrant DNA methylation and/or altered miRNA expression cause postnatal neurodevelopmental disorders. In general, DNA methylation in the 5'-flanking region suppresses gene expression through recruitment of methyl-CpG binding domain proteins (MBPs) to the cytosine residues of CpG dinucleotides. X-linked MeCP2 (methyl-CpG binding protein 2), a member of MBPs, is a methylation-associated transcriptional repressor with other functions in the central nervous system (CNS). miRNAs negatively regulate gene expression by targeting the 3'-untranslated region (3'UTR). Some miRNA genes harboring or being embedded in CpG islands undergo methylation-mediated silencing. One such miRNA is miR-7b which is differentially expressed through stages of neurodevelopment. In our present study, we focused on a canonical CpG island located in the 5'-flanking region of the murine miR-7b gene. Hypermethylation of this CpG island down-regulates miR-7b while recruiting MeCP2 to the methylated CpG dinucleotides. Meanwhile, Mecp2, a target of miR-7b, was up-regulated due to lack of restrain exerted by miR-7b during maturation of postnatal (PN) mouse neurons between PN3 and PN14. Our results indicate that miR-7b is a direct downstream gene transcriptional target while also being a negative post-transcriptional regulator of Mecp2 expression. We speculate that this bidirectional feed-back autoregulatory function of miR-7b and Mecp2 while linking DNA methylation and miRNA action maintains the homeostatic control of gene expression necessary during postnatal maturation of mammalian neurons.
Assuntos
Encéfalo/crescimento & desenvolvimento , Encéfalo/fisiologia , Expressão Gênica , Proteína 2 de Ligação a Metil-CpG/metabolismo , MicroRNAs/metabolismo , Neurônios/fisiologia , Regiões 3' não Traduzidas , Região 5'-Flanqueadora , Animais , Sequência de Bases , Linhagem Celular , Células Cultivadas , Ilhas de CpG , Hipocampo/crescimento & desenvolvimento , Hipocampo/fisiologia , Metilação , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Dados de Sequência MolecularRESUMO
The murine neuronal facilitative glucose transporter isoform 3 (Glut3) is developmentally regulated, peaking in expression at postnatal day (PN)14. In the present study, we characterized a canonical CpG island spanning the 5'-flanking region of the glut3 gene. Methylation-specific PCR and bisulfite sequencing identified methylation of this CpG ((m)CpG) island of the glut3 gene, frequency of methylation increasing 2.5-fold with a 1.6-fold increase in DNA methyl transferase 3a concentrations noted with advancing postnatal age (PN14 vs PN3). 5'-flanking region of glut3-luciferase reporter transient transfection in HT22 hippocampal neurons demonstrated that (m)CpGs inhibit glut3 transcription. Contrary to this biological function, glut3 expression rises synchronously with (m)CpGs in PN14 vs PN3 neurons. Chromatin immunoprecipitation (IP) revealed that methyl-CpG binding protein 2 (Mecp2) bound the glut3-(m)CpGs. Depending on association with specific coregulators, Mecp2, a dual regulator of gene transcription, may repress or activate a downstream gene. Sequential chromatin IP uncovered the glut3-(m)CpGs to bind Mecp2 exponentially upon recruitment of Creb1 rather than histone deacetylase 1. Co-IP and coimmunolocalization confirmed that Creb1 associated with Mecp2 and cotransfection with glut3-(m)CpG in HT22 cells enhanced glut3 transcription. Separate 5-aza-2'-deoxycytidine pretreatment or in combination with trichostatin A reduced (m)CpG and specific small interference RNAs targeting Mecp2 and Creb1 separately or together depleting Mecp2 and/or Creb1 binding of glut3-(m)CpGs reduced glut3 expression in HT22 cells. We conclude that Glut3 is a methylation-sensitive neuronal gene that recruits Mecp2. Recruitment of Creb1-Mecp2 by glut3-(m)CpG contributes towards transactivation, formulating an escape from (m)CpG-induced gene suppression, and thereby promoting developmental neuronal glut3 gene transcription and expression.