RESUMO
BACKGROUND: Clinical recommendations to limit gestational weight gain (GWG) imply high GWG is causally related to adverse outcomes in mother or offspring, but GWG is the sum of several inter-related complex phenotypes (maternal fat deposition and vascular expansion, placenta, amniotic fluid and fetal growth). Understanding the genetic contribution to GWG could help clarify the potential effect of its different components on maternal and offspring health. Here we explore the genetic contribution to total, early and late GWG. PARTICIPANTS AND METHODS: A genome-wide association study was used to identify maternal and fetal variants contributing to GWG in up to 10 543 mothers and 16 317 offspring of European origin, with replication in 10 660 mothers and 7561 offspring. Additional analyses determined the proportion of variability in GWG from maternal and fetal common genetic variants and the overlap of established genome-wide significant variants for phenotypes relevant to GWG (for example, maternal body mass index (BMI) and glucose, birth weight). RESULTS: Approximately 20% of the variability in GWG was tagged by common maternal genetic variants, and the fetal genome made a surprisingly minor contribution to explain variation in GWG. Variants near the pregnancy-specific beta-1 glycoprotein 5 (PSG5) gene reached genome-wide significance (P=1.71 × 10-8) for total GWG in the offspring genome, but did not replicate. Some established variants associated with increased BMI, fasting glucose and type 2 diabetes were associated with lower early, and higher later GWG. Maternal variants related to higher systolic blood pressure were related to lower late GWG. Established maternal and fetal birth weight variants were largely unrelated to GWG. CONCLUSIONS: We found a modest contribution of maternal common variants to GWG and some overlap of maternal BMI, glucose and type 2 diabetes variants with GWG. These findings suggest that associations between GWG and later offspring/maternal outcomes may be due to the relationship of maternal BMI and diabetes with GWG.
Assuntos
Feto/fisiologia , Ganho de Peso na Gestação/genética , Gravidez/genética , Feminino , Estudo de Associação Genômica Ampla , Ganho de Peso na Gestação/fisiologia , Humanos , Gravidez/fisiologia , Gravidez/estatística & dados numéricosRESUMO
Islet transplantation represents a potential cure for type 1 diabetes, yet the clinical approach of intrahepatic delivery is limited by the microenvironment. Microporous scaffolds enable extrahepatic transplantation, and the microenvironment can be designed to enhance islet engraftment and function. We investigated localized trophic factor delivery in a xenogeneic human islet to mouse model of islet transplantation. Double emulsion microspheres containing exendin-4 (Ex4) or insulin-like growth factor-1 (IGF-1) were incorporated into a layered scaffold design consisting of porous outer layers for islet transplantation and a center layer for sustained factor release. Protein encapsulation and release were dependent on both the polymer concentration and the identity of the protein. Proteins retained bioactivity upon release from scaffolds in vitro. A minimal human islet mass transplanted on Ex4-releasing scaffolds demonstrated significant improvement and prolongation of graft function relative to blank scaffolds carrying no protein, and the release profile significantly impacted the duration over which the graft functioned. Ex4-releasing scaffolds enabled better glycemic control in animals subjected to an intraperitoneal glucose tolerance test. Scaffolds releasing IGF-1 lowered blood glucose levels, yet the reduction was insufficient to achieve euglycemia. Ex4-delivering scaffolds provide an extrahepatic transplantation site for modulating the islet microenvironment to enhance islet function posttransplant.
Assuntos
Diabetes Mellitus Experimental/prevenção & controle , Sobrevivência de Enxerto/fisiologia , Fator de Crescimento Insulin-Like I/metabolismo , Transplante das Ilhotas Pancreáticas , Peptídeos/metabolismo , Poliglactina 910/química , Peçonhas/metabolismo , Animais , Glicemia/metabolismo , Diabetes Mellitus Experimental/metabolismo , Exenatida , Feminino , Teste de Tolerância a Glucose , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Microesferas , Pessoa de Meia-IdadeRESUMO
Maternal glucose metabolism during pregnancy differs from the non-gravid state to allow the mother to meet her own and the growing fetus's energy needs. New insights into the mechanisms underlying maternal metabolism during pregnancy are being gained through the use of new 'omics' technologies. This review focuses on the application of genetics/genomics and metabolomics to the study of maternal metabolism during pregnancy. Following the identification of susceptibility genes for Type 2 diabetes through genome-wide association studies, association has been demonstrated of some Type 2 diabetes susceptibility genes with gestational diabetes mellitus, suggesting that the genetic architecture of Type 2 diabetes and gestational diabetes are, in part, similar. More recent genome-wide association studies examining maternal metabolism during pregnancy have demonstrated overlap of genes associated with metabolic traits in the gravid and non-gravid population, as well as genes that appear to be relatively unique to pregnancy. Metabolomics has also been used to profile the metabolic state of women during pregnancy through the multiplexed measurement of many low molecular weight metabolites. Measurement of amino acids and conventional metabolites have demonstrated changes in mothers with higher insulin resistance and glucose similar to changes in non-gravid, insulin-resistant populations, suggesting similarities in the metabolic profile characteristic of insulin resistance and hyperglycaemia in pregnant and non-pregnant populations. Metabolomics and genomics are but a few of the now available high-throughput 'omics' technologies. Future studies that integrate data from multiple technologies will allow an integrated systems biology approach to maternal metabolism during pregnancy.
Assuntos
Glicemia/metabolismo , Diabetes Gestacional/genética , Hemoglobinas Glicadas/metabolismo , Metabolômica/tendências , Gestantes , Biomarcadores , Diabetes Gestacional/epidemiologia , Feminino , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Recém-Nascido , Resistência à Insulina , GravidezRESUMO
Altered sex hormone levels are thought to play an important role in adult-onset diseases including obesity, cardiovascular disease, and diabetes. They contribute to these complex diseases through changes in their availability, which is influenced, in part, by binding proteins. Insulin resistance, which is characteristic of these diseases, along with increased insulin secretion, is a physiologic change that occurs normally during pregnancy. To determine the relationship between insulin resistance and sex hormone levels, we examined the associations of sex hormone-binding globulin (SHBG) and testosterone with measures of glycemia and insulinemia in a healthy pregnant population. We measured fasting serum SHBG and testosterone levels in 215 Hispanic mothers of Mexican ancestry from the HAPO Study cohort and tested for associations between SHBG and testosterone levels and maternal plasma glucose and C-peptide. After adjusting for confounding variables, serum total testosterone (TT) was positively associated with fasting C-peptide (0.18 µg/l higher for TT higher by 1 SD, p=0.001) and 1-h C-peptide (0.79 µg/l higher for TT higher by 1 SD, p<0.001). Free testosterone (FT) was also positively associated with fasting C-peptide (0.19 µg/l higher for FT higher by 1 SD, p<0.001), and 1-h C-peptide (0.83 µg/l higher for FT higher by 1 SD, p<0.001). Although these findings are from a single cohort, this study provides evidence for an association between testosterone and C-peptide during pregnancy in a nondiabetic Hispanic obstetric population.
Assuntos
Peptídeo C/sangue , Hiperglicemia/sangue , Complicações na Gravidez/sangue , Testosterona/sangue , Glicemia/metabolismo , Estudos de Coortes , Feminino , Humanos , Hiperglicemia/etnologia , Insulina/sangue , Americanos Mexicanos , México/etnologia , Gravidez , Complicações na Gravidez/etnologia , Resultado da Gravidez , Globulina de Ligação a Hormônio Sexual/metabolismo , Estados UnidosRESUMO
The goal of this study was to examine associations of measures of maternal glucose metabolism and blood pressure during pregnancy with blood pressure at follow-up in the Hyperglycemia and Adverse Pregnancy Outcome (HAPO) cohort. The HAPO Follow-Up Study included 4747 women who had a 75-g oral glucose tolerance test (OGTT) at ~28 weeks' gestation. Of these, 4572 women who did not have chronic hypertension during their pregnancy or other excluding factors, had blood pressure evaluation 10-14 years after the birth of their HAPO child. Primary outcomes were systolic blood pressure (SBP), diastolic blood pressure (DBP), and hypertension (SBP ≥ 140 and/or DBP ≥ 90 or treatment for hypertension) at follow-up. Blood pressure during pregnancy was associated with all blood pressure outcomes at follow-up independent of glucose and insulin sensitivity during pregnancy. The sum of glucose z-scores was associated with blood pressure outcomes at follow-up but associations were attenuated in models that included pregnancy blood pressure measures. Associations with SBP were significant in adjusted models, while associations with DBP and hypertension were not. Insulin sensitivity during pregnancy was associated with all blood pressure outcomes at follow-up, and although attenuated after adjustments, remained statistically significant (hypertension OR 0.79, 95%CI 0.68-0.92; SBP beta -0.91, 95% CI -1.34 to -0.49; DBP beta -0.50, 95% CI -0.81 to -0.19). In conclusion, maternal glucose values at the pregnancy OGTT were not independently associated with maternal blood pressure outcomes 10-14 years postpartum; however, insulin sensitivity during pregnancy was associated independently of blood pressure, BMI, and other covariates measured during pregnancy.
Assuntos
Glicemia , Pressão Sanguínea , Hiperglicemia , Glicemia/metabolismo , Feminino , Seguimentos , Glucose , Humanos , Período Pós-Parto , Gravidez , Resultado da GravidezRESUMO
The promise and progress of islet transplantation for treating type 1 diabetes has been challenged by obstacles to patient accessibility and long-term graft function that may be overcome by integrating emerging technologies in biomaterials, drug delivery and immunomodulation. The hepatic microenvironment and traditional systemic immunosuppression stress the vulnerable islets and contribute to the limited success of transplantation. Locally delivering extracellular matrix proteins and trophic factors can enhance transplantation at extrahepatic sites by promoting islet engraftment, revascularisation and long-term function while avoiding unintended systemic effects. Cell- and cytokine-based therapies for immune cell recruitment and reprogramming can inhibit local and systemic immune system activation that normally attacks transplanted islets. Combined with antigen-specific immunotherapies, states of operational tolerance may be achievable, reducing or eliminating the long-term pharmaceutical burden. Integration of these technologies to enhance engraftment and combat rejection may help to advance the therapeutic efficacy and availability of islet transplantation.
Assuntos
Imunomodulação/imunologia , Transplante das Ilhotas Pancreáticas/imunologia , Biotecnologia , Humanos , Engenharia TecidualRESUMO
The insulin receptor from human brain tumors of glial origin was examined for the first time using intact cells (from an established cultured human glioblastoma cell line) and partially purified solubilized membranes (from cultured cells and freshly isolated human brain tumors). The structure of the glial insulin receptor subunits was assessed by affinity cross-linking of 125I-insulin with the alpha-subunit of the receptor, neuraminidase treatment of the cross-linked receptor, behavior of the receptor on lectin columns, and electrophoretic mobility of the phosphorylated beta-subunit. The functions of the insulin receptor were examined by measuring specific 125I-insulin binding (receptor concentration, affinity, specificity, pH-, time-, and temperature dependence), insulin-induced down-regulation of the receptor, insulin-stimulated autophosphorylation of the beta-subunit, and phosphorylation of exogenous substrates as well as insulin-stimulated glucose uptake in glioblastoma cells. All of these properties were typical for the insulin receptor from target tissues for insulin action. The insulin receptor of the normal human brain showed the altered electrophoretic mobility and lack of neuraminidase sensitivity of its alpha-subunit previously reported for the rat brain receptor. There was no difference, however, in the functions of the receptor subunits (binding, phosphorylation) from the normal brain tissue and the eight human gliomal tumors. Since the glial elements compose a majority of the brain cells, the "normal" structure and function of their insulin receptor might provide a key to understanding the role of insulin in the carbohydrate metabolism of the human central nervous system.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Receptor de Insulina/metabolismo , Transporte Biológico , Células Cultivadas , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB , Glucose/metabolismo , Hormônio do Crescimento/metabolismo , Humanos , Insulina/metabolismo , Substâncias Macromoleculares , Peso Molecular , Neuraminidase/metabolismo , Receptores de Superfície Celular/metabolismoRESUMO
We have examined, in liver and extrahepatic tissues, the effects of fasting on total insulin-like growth factor I (IGF-I) mRNA levels, on levels of different IGF-I mRNAs generated by alternative splicing of the primary IGF-I transcript, and on IGF-I receptor binding and mRNA levels. A 48-h fast decreased total IGF-I mRNA levels by approximately 80% in lung and liver, approximately 60% in kidney and muscle, and only approximately 30-40% in stomach, brain, and testes. In heart, IGF-I mRNA levels did not change. The levels of the different splicing variants, however, were essentially coordinately regulated within a given tissue. Specific 125I-IGF-I binding in lung, testes, stomach, kidney, and heart was increased by fasting by approximately 30-100%, whereas in brain 125I-IGF-I binding did not change in response to fasting. In tissues in which fasting increased IGF-I receptor number, receptor mRNA levels increased approximately 1.6- to 2.5-fold, whereas when IGF-I receptor number was unchanged in response to fasting, receptor mRNA levels did not change. These data demonstrate that the change in IGF-I and IGF-I receptor mRNA levels during fasting is quantitatively different in different tissues and suggest that regulation of IGF-I and IGF-I receptor gene expression by fasting is discoordinate.
Assuntos
Jejum , Regulação da Expressão Gênica , Fator de Crescimento Insulin-Like I/biossíntese , Receptores de Superfície Celular/biossíntese , Somatomedinas/biossíntese , Animais , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos , Receptores de Superfície Celular/genética , Receptores de SomatomedinaRESUMO
AIM: We hypothesised that some of the genetic risk for gestational diabetes (GDM) is due to the fetal genome affecting maternal glucose concentrations. Previously, we found associations between fetal IGF2 gene variants and maternal glucose concentrations in late pregnancy. METHODS: In the present study, we tested associations between SNP alleles from 15 fetal imprinted genes and maternal glucose concentrations in late pregnancy in the Cambridge Baby Growth and Wellbeing cohorts (1160 DNA trios). RESULTS: Four fetal SNP alleles with the strongest univariate associations: paternally-transmitted IGF2 rs10770125 (P-value=2×10-4) and INS rs2585 (P-value=7×10-4), and maternally-transmitted KCNQ1(OT1) rs231841 (P-value=1×10-3) and KCNQ1(OT1) rs7929804 (P-value=4×10-3), were used to construct a composite fetal imprinted gene allele score which was associated with maternal glucose concentrations (P-value=4.3×10-6, n=981, r2=2.0%) and GDM prevalence (odds ratio per allele 1.44 (1.15, 1.80), P-value=1×10-3, n=89 cases and 899 controls). Meta-analysis of the associations including data from 1367 Hyperglycaemia and Adverse Pregnancy Outcome Study participants confirmed the paternally-transmitted fetal IGF2/INS SNP associations (rs10770125, P-value=3.2×10-8, rs2585, P-value=3.6×10-5) and the composite fetal imprinted gene allele score association (P-value=1.3×10-8), but not the maternally-transmitted fetal KCNQ1(OT1) associations (rs231841, P-value=0.4; rs7929804, P-value=0.2). CONCLUSION: This study suggests that polymorphic variation in fetal imprinted genes, particularly in the IGF2/INS region, contribute a small but significant part to the risk of raised late pregnancy maternal glucose concentrations.
Assuntos
Alelos , Glicemia/genética , Diabetes Gestacional/genética , Impressão Genômica , Polimorfismo de Nucleotídeo Único , Adulto , Diabetes Gestacional/sangue , Feminino , Humanos , Insulina/genética , Fator de Crescimento Insulin-Like II/genética , Canal de Potássio KCNQ1/genética , Gravidez , Resultado da GravidezAssuntos
Proteínas de Transporte de Cátions/metabolismo , Células Secretoras de Glucagon/metabolismo , Células Secretoras de Insulina/metabolismo , Animais , Proteínas de Transporte de Cátions/genética , Grânulos Citoplasmáticos/genética , Grânulos Citoplasmáticos/metabolismo , Glucose/metabolismo , Insulina/genética , Insulina/metabolismo , Secreção de Insulina , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Zinco/metabolismoRESUMO
We have previously reported that myo-inositol uptake and metabolism is reduced in human fibroblasts derived from patients with ataxia telangiectasia (AT). Treating normal fibroblasts with 10-100 microM wortmannin duplicates some of the phenotypic properties of AT fibroblasts including the decrease in myo-inositol accumulation. In the present study we examined whether treatment of other types of mammalian cells with wortmannin or LY294002 altered myo-inositol uptake. Cultured bovine aorta endothelial cells or 3T3-L1 adipocytes were incubated with either wortmannin or LY294002, and afterwards, myo-inositol uptake and SMIT mRNA levels were determined. Incubating cultured bovine aorta endothelial cells and 3T3-L1 adipocytes with either wortmannin or LY294002 caused a time- and concentration-dependent decrease in myo-inositol accumulation that was independent of changes in SMIT mRNA levels. The effect of wortmannin and LY294002 on myo-inositol accumulation was not due to an increase in myo-inositol secretion. The effect of LY294002 on myo-inositol accumulation was reversible. Furthermore, the LY294002-induced decrease in myo-inositol accumulation was specific since the uptake of serine or choline by cultured bovine aorta endothelial cells and 3T3-L1 adipocytes treated with LY294002 was not significantly decreased. Co-incubation of cultured bovine aorta endothelial cells and 3T3-L1 adipocytes with either wortmannin or LY294002 and hyperosmotic medium caused a significant decrease in the induction of myo-inositol accumulation by hyperosmolarity without significantly affecting the hyperosmotic-induced increase in SMIT mRNA levels. These data suggest that myo-inositol accumulation is regulated post-translationally by wortmannin and LY294002.
Assuntos
Adipócitos/efeitos dos fármacos , Androstadienos/farmacologia , Cromonas/farmacologia , Endotélio Vascular/efeitos dos fármacos , Inositol/metabolismo , Proteínas de Membrana , Morfolinas/farmacologia , Simportadores , Células 3T3 , Adipócitos/metabolismo , Animais , Proteínas de Transporte/genética , Bovinos , Células Cultivadas , Colina/metabolismo , Endotélio Vascular/metabolismo , Proteínas de Choque Térmico/genética , Camundongos , Concentração Osmolar , Fosfatidilinositol 3-Quinases/análise , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Serina/metabolismo , Fatores de Tempo , WortmaninaRESUMO
Because basic fibroblast growth factor (bFGF) is recognized as an angiogenic factor and diabetes is characterized by multiple vascular complications, including diabetic microangiopathy, we examined the regulation of tissue bFGF mRNA levels by diabetes. Diabetes was induced in male Sprague-Dawley rats by injection of 125 mg/kg body wt i.v. streptozocin (STZ), with intensive insulin therapy initiated in half of the diabetic rats. Rats were killed 96 h postinjection of STZ. Tissue bFGF and insulinlike growth factor I (IGF-I) mRNA levels were measured simultaneously with a solution hybridization-RNase protection assay. bFGF mRNA levels increased from 1.7- to 2.7-fold in eye, heart, lung, and brain from diabetic compared with buffer-injected control rats. In skeletal muscle, bFGF mRNA levels decreased to 23% of control levels, whereas bFGF mRNA levels were unchanged in kidneys from diabetic versus control rats. Changes in tissue bFGF mRNA levels were partially reversed by insulin treatment in all tissues. In contrast, IGF-I mRNA levels were significantly decreased from 15 to 50% of control levels in all tissues studied except those in brain, which decreased to only 85% of control levels. These data demonstrate that bFGF mRNA levels are altered by diabetes in a tissue-specific fashion and are consistent with the hypothesis that increased production of bFGF may contribute to the development of diabetic microangiopathy in some tissues.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Angiopatias Diabéticas/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , RNA Mensageiro/metabolismo , Animais , Diabetes Mellitus Experimental/tratamento farmacológico , Regulação da Expressão Gênica/efeitos dos fármacos , Insulina/farmacologia , Fator de Crescimento Insulin-Like I/genética , Masculino , Especificidade de Órgãos , Ratos , Ratos EndogâmicosRESUMO
Alternative splicing of insulin-like growth factor I (IGF-I)/somatomedin C mRNAs generates two IGF-I mRNAs coding for IGF-I peptides with different sequences in the E domain of the IGF-I prohormone. These two mRNAs encode alternative E peptides due to the presence (IGF-Ib) or absence (IGF-Ia) of a 52-base insert in the region coding for the E domain. We have used a solution hybridization/RNase protection assay to determine the tissue distribution and regulation by GH of the expression of these alternative IGF-I mRNAs. IGF-Ib mRNAs are present in low abundance (representing approximately 2.5% of the total IGF-I mRNA) in heart, lung, muscle, testes, stomach, kidney, and brain, but represent approximately 13% of the IGF-I mRNA in liver. GH treatment of hypophysectomized rats increased steady-state IGF-I mRNA levels in liver, kidney, lung, and heart. In kidney, lung, and heart, IGF-Ia and IGF-Ib mRNA levels were coordinately regulated by GH, but, in liver, the fold increase in IGF-Ib mRNA levels was approximately three times greater than the fold increase in IGF-Ia mRNA levels. These data suggest that the processing of IGF-I mRNA in liver is different than in nonhepatic tissues. These results also further elucidate the organization of the rat IGF-I gene as well as the generation of multiple IGF-I mRNAs by alternative splicing.
Assuntos
Regulação da Expressão Gênica , Fator de Crescimento Insulin-Like I/genética , Fígado/metabolismo , Fragmentos de Peptídeos/genética , RNA Mensageiro/metabolismo , Somatomedinas/genética , Animais , Hormônio do Crescimento/farmacologia , Hipofisectomia , Masculino , Hibridização de Ácido Nucleico , Splicing de RNA , Ratos , Ratos Endogâmicos , Ribonucleases , Soluções , Distribuição TecidualRESUMO
Developmental regulation of somatostatin (SRIF) gene expression was studied in five regions of rat brain and in rat stomach. Total RNA was isolated from hypothalamus, cortex, brainstem, cerebellum, and olfactory bulb, as well as stomach at eight stages of development from prenatal day 16 to postnatal day 82. Hybridization of a 32P-labeled rat SRIF cDNA probe to Northern blots of total RNA from the above tissues during development demonstrated a single hybridizing band approximately 670 base pairs in length. When SRIF mRNA levels from each stage of development were quantified and normalized by the amount of poly (A)+ RNA present at that stage of development, a unique pattern of SRIF gene expression was seen in each region. In brainstem and cerebellum, SRIF mRNA levels peaked early in development between prenatal day 21 and postnatal day 8 and then declined until postnatal day 82. Hypothalamus and cortex, on the other hand, showed a progressive increase during development with peak levels occurring between postnatal days 13 and 82. In contrast, stomach and olfactory bulb showed SRIF mRNA levels which were low during early development and which rose late in development (postnatal days 13 to 82). Marked differences in the amount of SRIF mRNA within each region were present as well. These data suggest that there is differential expression of the SRIF gene in different regions of the brain and in the stomach during development. Further study of this phenomenon may provide insight into the in vivo control of SRIF gene expression and the role of SRIF in the developing brain.
Assuntos
Encéfalo/embriologia , Somatostatina/genética , Animais , Química Encefálica , Feminino , Regulação da Expressão Gênica , Masculino , Especificidade de Órgãos , Gravidez , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos , Somatostatina/análise , Somatostatina/fisiologia , Estômago/análise , Estômago/embriologiaRESUMO
The developmental regulation of rat brain-derived/Hep G2 glucose transporter gene expression was studied by means of Northern blot hybridization, using a rat brain glucose transporter cDNA probe, in order to directly quantify steady state glucose transporter mRNA levels. The results obtained showed different tissue-specific patterns of glucose transporter mRNA levels during ontogenesis; while in brain there was a sustained increase in the levels of the message from 20 days embryogenesis until 50 days postnatal, other organs such as heart, lung, liver, and muscle expressed maximal levels of the glucose transporter mRNA in 20-day fetuses and 1-day neonates, decreasing subsequently to very low levels. The relative expression of the glucose transporter mRNA in the different tissues, at both fetal and adult stages, was analyzed using a solution hybridization-RNase protection assay. This approach revealed that, while the heart expresses the highest levels of glucose transporter mRNA at 20 days of fetal life, the brain shows the highest levels at the adult stage. These results indicate a tissue-specific ontogenic pattern of glucose transporter gene expression, suggesting a developmental role for this glucose transporter gene product.
Assuntos
Encéfalo/metabolismo , Regulação da Expressão Gênica , Proteínas de Transporte de Monossacarídeos/genética , Animais , Encéfalo/embriologia , Sondas de DNA , Proteínas de Transporte de Monossacarídeos/análise , RNA Mensageiro/genética , Ratos , Ratos EndogâmicosRESUMO
Two classes of insulin-like growth factor I (IGF-I) cDNAs were isolated from an adult rat liver library using a human IGF-I cDNA probe. The two types of rat IGF-I cDNA differed by the presence or absence of a 52-base pair insert which altered the derived C-terminal amino acid sequence of the E peptide, but not the 3'-untranslated region or the sequence coding for the mature IGF-I protein. When probes derived from these cDNA clones were hybridized to Northern blots of rat mRNA, specific bands of 8.6, 2.1, and 1.0-1.4 kilobases were seen. Hybridization to poly(A)+ RNA from various tissues from GH-treated and control rats demonstrated an increase in IGF-I mRNA due to GH treatment in all tissues examined.
Assuntos
DNA/genética , Hormônio do Crescimento/farmacologia , Fator de Crescimento Insulin-Like I/genética , Fígado/metabolismo , RNA Mensageiro/efeitos dos fármacos , Somatomedinas/genética , Animais , Sequência de Bases , Clonagem Molecular/métodos , DNA/isolamento & purificação , Fator de Crescimento Insulin-Like I/isolamento & purificação , Fígado/análise , Masculino , Dados de Sequência Molecular , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos , Transcrição GênicaRESUMO
Cultured endothelial cells have been shown to produce insulin-like growth factor-binding proteins (IGFBPs); however, the identity of these BPs has not been defined. We now demonstrate that cultured bovine endothelial cells produce IGFBP2, IGFBP3, and IGFBP4 and have mRNA specific for IGFBP2, -3, -4, -5 and -6. DNA probes for bovine IGFBP2-6 were obtained by polymerase chain reaction (PCR) amplification of cDNA from bovine large vessel pulmonary artery and aortic endothelial cells as well as omental and periaortic fat microvessel cells, using oligonucleotide primers whose sequences were based on the reported cDNA sequences of IGFBP2-6. The PCR-derived probes were labeled with 32P and used for Northern blot analysis of RNAs obtained from the four bovine endothelial cell types. Transcripts corresponding to IGFBP2-6 were found in RNA from large vessel endothelial cells (bovine pulmonary artery and bovine aorta) and microvessel cells (periaortic and omental fat). The PCR-derived probe for IGFBP4 was used to screen a bovine pulmonary artery cDNA library for a full-length bovine IGFBP4 cDNA clone. One positive clone, containing a single EcoRI insert of approximately 2.0 kilobases, was selected for further characterization by DNA sequence analysis. This clone contained an open reading frame encoding a 258-amino acid protein that was 97% identical to human IGFBP4, 268 basepairs of 5'-untranslated region, and a longer 1044 basepairs of 3'-untranslated region. IGFBP4 protein was purified from bovine pulmonary artery-conditioned medium, shown to have N-terminal amino acid sequence DEAIHCPPCSEEKLARCR (identical to human IGFBP4) and to be secreted in glycosylated and nonglycosylated forms. Immunoblots further demonstrated that microvessel cells, at early passage, secrete predominantly IGFBP2 and IGFBP3, while large vessel cells, at early and late passages, secrete IGFBP3 and IGFBP4. Thus, cultured bovine endothelial cells synthesize and secrete IGFBP2, IGFBP3, and IGFBP4 and have mRNA encoding IGFBP2-6. The production of specific IGFBPs by endothelial cells raises the interesting possibility that the vascular endothelium contributes to circulating and tissue levels of specific IGFBPs in vivo.
Assuntos
Proteínas de Transporte/biossíntese , Endotélio/metabolismo , Tecido Adiposo , Sequência de Aminoácidos , Animais , Aorta , Sequência de Bases , Bovinos , Células Cultivadas , DNA/genética , Endotélio/citologia , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina , Dados de Sequência Molecular , Especificidade de Órgãos , Artéria Pulmonar , RNA Mensageiro/biossíntese , Ratos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
Fibroblasts represent one of the in vivo sites of insulin-like growth factor-I (IGF-I) production. In this study rat dermal fibroblasts in culture were used as a model system to assess the effect of activation of protein kinase-C on the levels of the mRNAs encoding IGF-I and another growth factor, basic fibroblast growth factor (bFGF). IGF-I and bFGF mRNA levels were determined using a solution hybridization/RNase protection assay. Treatment of cells in serum-free medium containing 0.25% BSA (MEM + BSA) with the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) decreased IGF-I and increased bFGF mRNA levels in a time- and dose-dependent fashion. The peak effect of 100 nM PMA on IGF-I mRNA levels occurred at 9 h, whereas the peak effect on bFGF mRNA levels occurred after 3 h of incubation. In dose-response studies, half-maximal inhibition of IGF-I mRNA levels was achieved with approximately 0.08 nM PMA, while half-maximal stimulation of bFGF mRNA levels was achieved with approximately 3 nM PMA. Inhibition of protein synthesis with cycloheximide abrogated the effect of PMA on bFGF mRNA levels, but only partially inhibited the effect of PMA on IGF-I mRNA levels. Studies employing sphingosine or staurosporine to inhibit protein kinase-C or preincubation in high doses of PMA to down-regulate protein kinase-C suggested that the effect of PMA on IGF-I and bFGF mRNA levels was mediated by activation of protein kinase-C, although both staurosporine and sphingosine had independent effects on the levels of these mRNAs and down-regulation of protein kinase-C had a sustained effect on IGF-I mRNA levels. Ligands known to activate protein kinase-C were then tested. Treatment of cells with 100 micrograms/ml of the synthetic diacylglycerol 1-oleoyl-2-acetyl-sn-glycerol decreased IGF-I mRNA levels to 25% and increased bFGF mRNA levels to 520% of the level present in cells maintained in MEM + BSA. Treatment of cells with thrombin or bradykinin also decreased IGF-I mRNA levels and increased bFGF mRNA levels, but whereas the effect of thrombin on IGF-I mRNA levels was marked, the effect of bradykinin was minimal, and whereas the effect of thrombin on bFGF mRNA levels was sustained, the effect of bradykinin was transient.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Fator 2 de Crescimento de Fibroblastos/genética , Fator de Crescimento Insulin-Like I/genética , Proteína Quinase C/fisiologia , Animais , Bradicinina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Técnicas In Vitro , RNA Mensageiro/biossíntese , Ratos , Acetato de Tetradecanoilforbol/farmacologia , Trombina/farmacologiaRESUMO
Cellular proliferation is a dominant aspect of ovarian follicular development in the rat, and insulin-like growth factor I (IGF-I) has been proposed as a mediator of cellular growth and differentiation in the ovary. An SV40-transformed rat granulosa cell line (RGA-41S) has been established as a model for studies on dividing cells of granulosa origin. Granulosa cells from the ovaries of immature diethylstilbestrol-treated rats were infected with the tsA255 mutant of SV40, followed by cloning in serum-free medium to select transformed cell lines which were serum independent. At the permissive temperature (33 C), RGA-41S cells exhibited a transformed phenotype and rapidly formed high density multilayers of compact cells that readily overgrew nontransformed cells. At the nonpermissive temperature (40 C) cell replication declined and division ceased after 4 days. Furthermore, at 40 C the cells grew as a monolayer and assumed a tetrahedral shape with a high cytoplasm-to-nucleus ratio, and displayed reduced ability to overgrow nontransformed cells. The transformed ovarian cells did not express detectable gonadotropin receptors and steroidogenic activity but retained their epithelial phenotype as demonstrated by cytokeratin staining of the cytoskeleton, the presence of microvilli, and the formation of tight junctions between cells. In support of the proposed autocrine-paracrine actions of IGF-I in the ovary, assay of conditioned serum-free culture medium revealed secretion of IGF-I-immunoreactive material by RGA-41S cells. HPLC-purified IGF-I immunoreactivity from these cells eluted with the same retention time as recombinant human IGF-I. When hybridized with a 32P-labeled rat IGF-I cDNA probe, poly(A)+ mRNA prepared from RGA-41S cells grown at both temperatures showed the typical three size classes of IGF-I mRNA on Northern blots (7.5, 1.7, and 0.8-1.2 kilobase kb), although the levels were somewhat higher at 33 C. The presence of IGF-I receptors in transformed cells was demonstrated by specific 125I-IGF-I binding to intact cells. Scatchard analysis indicated a single class of high affinity receptors at a density of 10(5) binding sites per cell and a dissociation constant (Kd) = 0.52 x 10(-9) M. Furthermore, hybridization of a 32P-labeled IGF-I receptor probe to Northern blots of poly(A+) RNA prepared from cells grown at 33 C and 40 C revealed an 11-kilobase rat IGF-I receptor mRNA. Physiological concentrations of IGF-I increased [3H]aminoisobutyric acid uptake by RGA-41S cells grown at either temperature, attesting to the retention of responsiveness to IGF-I in these transformed granulosa cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Células da Granulosa/metabolismo , Fator de Crescimento Insulin-Like I/biossíntese , Receptores de Superfície Celular/análise , Vírus 40 dos Símios/genética , Somatomedinas/biossíntese , Aminobutiratos/metabolismo , Animais , Ligação Competitiva , Northern Blotting , Feminino , Técnicas In Vitro , Queratinas , Ratos , Receptores de Somatomedina , Transformação GenéticaRESUMO
Despite an improved understanding of the molecular mechanisms of insulin-like growth factor-I (IGF-I) signaling and the recognition that IGF-I mediates many effects in endothelial cells, some of which may be important for atherosclerosis, little is known about the signal transduction pathways that mediate the effects of IGF-I in endothelial cells. To that end, we examined the signaling pathways activated by IGF-I in endothelial cells and their contribution to IGF-I-stimulated endothelial cell migration and nuclear factor (NF)-kappaB-dependent transcription. Treatment of bovine pulmonary artery endothelial cells (PAEC) with IGF-I activated the mitogen-activated protein kinases extracellular signal-regulated kinase (ERK)1/2 and ERK5. In contrast, IGF-I had no effect on either c-Jun amino-terminal kinase or p38 kinase activity. IGF-I also activated phosphatidylinositol (PI) 3-kinase, as reflected by increased phosphorylation of AKT: There was no evidence of cross-talk between the ERK and PI 3-kinase pathways in PAEC. In PAEC transiently transfected with pTK81-NFkappaB-Luc, which contained four copies of the NF-kappaB DNA binding site 5' to a minimal promoter and the luciferase gene, treatment with 50 ng/ml IGF-I increased luciferase activity 1.8-fold. Inhibition of ERK activity using PD98059 and PI 3-kinase activity with LY 294002 abrogated the induction of NF-kappaB-dependent transcription by IGF-I, suggesting that both pathways contribute to the effect of IGF-I on NF-kappaBdependent transcription. In contrast to the effect of tumor necrosis factor-alpha on NF-kappaB activation, Western blot analyses demonstrated that IGF-I had no effect on IkappaB phosphorylation and degradation or nuclear translocation and DNA binding of NF-kappaB. These data suggest a direct of effect of IGF-I on nuclear NF-kappaB. IGF-I also increased endothelial cell migration approximately 2-fold, as demonstrated using a Boyden chamber apparatus. IGF-I-induced endothelial cell migration was inhibited, in part, by LY 294002 but not PD98059. Together, these studies demonstrate that IGF-I activates multiple signaling pathways in endothelial cells with little evidence for cross-talk between the pathways. Moreover, these pathways appear to mediate both overlapping and distinct effects in that activation of both PI 3-kinase and the ERKs contributed to the stimulation of NF-kappaB-dependent transcription by IGF-I, whereas only PI 3-kinase mediated IGF-I-stimulated endothelial cell migration.