RESUMO
OBJECTIVE: Consumption of a high-fat (HF) diet is a contributing factor for the development of obesity. HF diet per se acts as a stressor, stimulating hypothalamo-pituitary-adrenal (HPA) axis activity resulting in elevated glucocorticoid levels; however, the mechanism behind this activation is unclear. We hypothesized that consumption of an HF diet activates HPA axis by increasing norepinephrine (NE) in the paraventricular nucleus (PVN) of the hypothalamus, leading to elevation in corticotrophin-releasing hormone (CRH) concentration in the median eminence (ME) resulting in elevated serum corticosterone (CORT). SUBJECTS: To test this hypothesis, diet-induced obese (DIO) and diet-resistant (DR) rats were exposed to either chow or HF diet for 6 weeks. MEASUREMENTS: At the end of 6 weeks, NE in the PVN was measured using HPLC, CRH in the ME, and CORT and leptin levels in the serum were measured using RIA and ELISA, respectively. The gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in NE synthesis, and leptin receptor in brainstem noradrenergic nuclei were also measured. RESULTS: HF diet increased PVN NE in both DIO and DR rats (P<0.05). However, this was accompanied by increases in CRH and CORT secretion only in DR animals, but not in DIO rats. Leptin receptor mRNA levels in the brainstem noradrenergic areas were not affected in both DIO and DR rats. However, HF diet increased TH mRNA levels only in DIO rats. CONCLUSION: Significant differences occur in all the arms of HPA axis function between DIO and DR rats. Further studies are needed to determine whether this could be a causative factor or a consequence to obesity.
Assuntos
Peso Corporal/fisiologia , Hormônio Liberador da Corticotropina/metabolismo , Hipotálamo/fisiopatologia , Norepinefrina/metabolismo , Obesidade/fisiopatologia , Sistema Hipófise-Suprarrenal/fisiopatologia , Hormônio Adrenocorticotrópico/sangue , Animais , Hormônio Liberador da Corticotropina/genética , Dieta , Hipotálamo/metabolismo , Leptina/sangue , Masculino , Norepinefrina/genética , Obesidade/genética , Obesidade/metabolismo , Sistema Hipófise-Suprarrenal/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Free radicals and reactive oxygen species (ROS) which are generated continuously cause mutagenic alterations resulting in cancer, aging and abnormalities in the nervous system. Accumulating evidence indicates that Vitamin E, the most potent lipid peroxyl radical scavenger, may reduce free radical induced chromosomal damages through inhibition of free radical formation, and activation of endonuclease that can be triggered by intracellular oxidative stress, and by increasing the rate of removal of damaged DNA. Although some studies suggest a potential usefulness of Vitamin E in the prevention of mutagenic effects caused by genotoxic free radicals, other studies report no effects. Thus the data are not conclusive enough to be used as a basis to change the current recommended dietary allowances (RDA). Future research should address molecular mechanisms underlying the protective effects of Vitamin E and develop appropriate biologically relevant biomarkers of DNA damage to further help in determining the dietary levels of Vitamin E needed to protect the genetic pool from internally and externally induced DNA damages.
Assuntos
Dano ao DNA , Vitamina E/farmacologia , Animais , DNA/efeitos dos fármacos , DNA/genética , DNA/metabolismo , Suplementos Nutricionais , Humanos , Política NutricionalRESUMO
Dominant mutations at the mouse Agouti locus lead to ectopic expression of the Agouti gene and exhibit diabetes, obesity, and yellow coat color. Obese yellow mice are hyperinsulinemic and hyperleptinemic, and we hypothesized that Agouti directly induces leptin secretion. Accordingly, we used transgenic mice expressing agouti in adipocytes (under the control of aP2 promoter, aP212) to examine changes in leptin levels. Agouti expression in adipose tissue did not significantly alter food intake, weight gain, fat pad weight, or insulinemia; however, the transgenic mice were hyperglycemic. We demonstrated that plasma leptin levels are approximately twofold higher in aP212 transgenic mice compared with their respective controls, whereas ubiquitous expression of agouti (under the control of beta-actin promoter, BAP20) led to a sixfold increase in leptin. Insulin treatment of aP212 mice increased adipocyte leptin content without affecting plasma leptin levels. These findings were further confirmed in vitro in 3T3-L1 adipocytes treated with recombinant Agouti protein and/or insulin. Agouti but not insulin significantly increased leptin secretion, indicating that insulin enhances leptin synthesis but not secretion while Agouti increases both leptin synthesis and secretion. This increased leptin synthesis and secretion was due to increased leptin mRNA levels by Agouti. Interestingly, agouti regulation of leptin was not mediated by melanocortin receptor 4, previously implicated in agouti regulation of food intake. These results suggest that increased leptin secretion by agouti may serve to limit agouti-induced obesity, independent of melanocortin receptor antagonism, and indicate that interaction between obesity genes may play a key role in obesity.
Assuntos
Adipócitos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Leptina/sangue , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Proteínas/metabolismo , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Tecido Adiposo/química , Tecido Adiposo/citologia , Tecido Adiposo/efeitos dos fármacos , Proteína Agouti Sinalizadora , Animais , Proteínas de Transporte , Células Cultivadas , Proteínas de Ligação a DNA , Diabetes Mellitus/genética , Esquema de Medicação , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Homozigoto , Injeções Subcutâneas , Insulina/administração & dosagem , Leptina/análise , Leptina/genética , Masculino , Hormônios Estimuladores de Melanócitos/farmacologia , Camundongos , Camundongos Transgênicos , Obesidade , Regiões Promotoras Genéticas/genética , Proteínas/genética , Proteínas/farmacologia , RNA Mensageiro/metabolismo , Receptor Tipo 4 de Melanocortina , Receptores da Corticotropina/agonistas , Receptores para Leptina , Receptores de Melanocortina , Receptores de Peptídeos/antagonistas & inibidoresRESUMO
Mice carrying dominant mutations at the agouti locus exhibit ectopic expression of agouti gene transcripts, obesity, and type II diabetes through unknown mechanisms. To gain insight into the role of agouti protein in modulating adiposity, we investigated regulation of a key lipogenic gene, fatty acid synthase (FAS) by agouti alone and in combination with insulin. Both agouti and insulin increase FAS activity in 3T3-L1 and in human adipocytes. Agouti and insulin independently and additively increase FAS activity in 3T3-L1 adipocytes. We further investigated the mechanism responsible for the agouti-induced FAS expression in these cells and demonstrated that both insulin (3-fold increase) and agouti (2-fold) increased FAS gene expression at the transcriptional level. Furthermore, insulin and agouti together exerted additive effects (5-fold increase) on FAS gene transcription. Transfection assays of FAS promoter-luciferase fusion gene constructs into 3T3-L1 adipocytes indicated that the agouti response element(s) is (are) located in the -435 to -415 region (-435/-415) of the FAS promoter. Nuclear proteins binding to this novel sequence are adipocyte specific. Thus the agouti response sequences mapped to a region upstream of the insulin-responsive element (which we previously reported to be located at -67/-52), consistent with additive effects of these two factors on FAS gene transcription.
Assuntos
Adipócitos/enzimologia , Ácido Graxo Sintases/genética , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas/farmacologia , Células 3T3 , Adipócitos/citologia , Proteína Agouti Sinalizadora , Animais , DNA/genética , Interações Medicamentosas , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Insulina/farmacologia , Luciferases/genética , Luciferases/metabolismo , Camundongos , Proteínas Nucleares/metabolismo , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Elementos de Resposta/genética , Transcrição Gênica/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
The yellow mouse obesity syndrome is due to dominant mutations at the Agouti locus, which is characterized by obesity, hyperinsulinemia, insulin resistance, hyperglycemia, hyperleptinemia, increased linear growth, and yellow coat color. This syndrome is caused by ectopic expression of Agouti in multiple tissues. Mechanisms of Agouti action in obesity seem to involve, at least in part, competitive melanocortin antagonism. Both central and peripheral effects have been implicated in Agouti-induced obesity. An Agouti-Related Protein (AGRP) has been described recently. It has been shown to be expressed in mice hypothalamus and to act similarly to agouti as a potent antagonist to central melanocortin receptor MC4-R, suggesting that AGRP is an endogenous MC4-R ligand. Mice lacking MC4-R become hyperphagic and develop obesity, implying that agouti may lead to obesity by interfering with MC4-R signaling in the brain and consequently regulating food intake. Furthermore, food intake is inhibited by intracerebroventricular injection of a potent melanocortin agonist and was reversed by administration of an MC4-R antagonist. The direct cellular actions of Agouti include stimulation of fatty acid and triglyceride synthesis via a Ca(2+)-dependent mechanism. Agouti and insulin act in an additive manner to increase lipogenesis. This additive effect of agouti and insulin is demonstrated by the necessity of insulin in eliciting weight gain in transgenic mice expressing agouti specifically in adipose tissue. This suggests that agouti expression in adipose tissue combined with hyperinsulinemia may lead to increased adiposity. The roles of melanocortin receptors or agouti-specific receptor(s) in agouti regulation of adipocyte metabolism and other peripheral effects remain to be determined. In conclusion, both central and peripheral actions of agouti contribute to the yellow mouse obesity syndrome and this action is mediated at least in part by antagonism with melanocortin receptors and/or regulation of intracellular calcium.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular , Obesidade/genética , Proteínas/genética , Proteína Agouti Sinalizadora , Proteína Relacionada com Agouti , Animais , Modelos Animais de Doenças , Insulina/fisiologia , Leptina/fisiologia , Hormônios Estimuladores de Melanócitos/antagonistas & inibidores , Camundongos , Camundongos Obesos , Mutação , Proteínas/fisiologia , Receptores da Corticotropina/fisiologia , Receptores de Melanocortina , Transdução de Sinais , SíndromeRESUMO
Stearoyl-CoA desaturase (SCD; EC 1.14.99.5) is a key enzyme in the synthesis polyunsaturated fatty acids. Liver and ose tissue are the predominant sites of SCD expression. Regulation of tic SCD by various nutritional and hormonal ors, such as insulin, dietary carbohydrates and polyunsaturated fatty s, has been well documented. Little is known, ver, about adipocyte SCD regulation despite high levels of SCD activity adipose tissue. The present study was gned to investigate SCD regulation in adipocytes by examining the cts of glucose and insulin on SCD expression. We rt here that glucose availability directly increased SCD gene scription in 3T3-L1 adipocytes. This response was pendent of insulin, and insulin alone in the absence of glucose had no ct on SCD mRNA levels. SCD thus represents a l model in which to investigate the mechanisms of direct regulation of expression by glucose in adipose cells.
Assuntos
Adipócitos/enzimologia , Glucose/farmacologia , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismo , Células 3T3 , Adipócitos/efeitos dos fármacos , Animais , Metabolismo dos Carboidratos , Carboidratos/farmacologia , Desoxiglucose/farmacologia , Ácido Graxo Sintases/efeitos dos fármacos , Ácido Graxo Sintases/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Insulina/metabolismo , Insulina/farmacologia , Camundongos , Estearoil-CoA Dessaturase/efeitos dos fármacos , Transcrição Gênica , Regulação para CimaRESUMO
The purpose of this study was to investigate the molecular mechanism whereby insulin increases expression of a key de novo lipogenic gene, fatty acid synthase (FAS), in cultured human adipocytes and hepatoma cells. RNA isolated from cultured adipocytes or from Hep G2 cells treated with or without insulin (20 nM) was analyzed. In addition, run-on transcription assays and measurements of RNA half-life were performed to determine the controlled step in FAS gene regulation by insulin. We demonstrated that FAS mRNA was expressed in both Hep G2 cells and human adipocytes. Insulin induced an approximately five- and three-fold increase in FAS mRNA content in adipocytes and hepatoma cells, respectively. Similar regulation of FAS was observed in adipocytes from lean and obese human subjects. Furthermore, we demonstrated that the induction of human FAS expression by insulin was due to increased transcription rate of the FAS gene in human adipocytes, whereas mRNA stabilization accounted for increased FAS mRNA content in hepatoma cells. In conclusion, we report here for the first time expression of human FAS mRNA and its specific transcriptional induction by insulin in cultured human adipocytes.
Assuntos
Adipócitos/enzimologia , Ácido Graxo Sintases/biossíntese , Ácido Graxo Sintases/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Transcrição Gênica/efeitos dos fármacos , Células Cultivadas , Humanos , RNA Mensageiro/análiseRESUMO
OBJECTIVES: Proinflammatory eicosanoids (cyclooxgenase and lipoxygenase metabolites of arachidonic acid) released by alveolar macrophages play an important role in endotoxin-induced acute lung injury. We investigated the effect of prefeeding rats for 21 days with enteral diets that provided the anti-inflammatory fatty acids, eicosapentaenoic acid and gamma-linolenic acid (derived from fish oil and borage oil, respectively), as compared with an n-6 fatty acid-enriched diet (corn oil) on the following: a) lung microvascular protein permeability, arterial blood pressure, and platelet and white blood cells in a model of endotoxin-induced acute lung injury; b) alveolar macrophage prostaglandin and leukotriene synthesis; and c) liver and alveolar macrophage phospholipid fatty acid composition. DESIGN: Prospective, randomized, controlled, double-blind study. SETTING: Research laboratory at a university medical center. SUBJECTS: Male Long-Evans rats, weighing 250 g. INTERVENTIONS: Rats were randomized into four dietary treatment groups and fed nutritionally complete diets (300 kcal/kg/day), containing 55.2% of the total calories from fat with either 97% corn oil, 20% fish oil, 20% fish and 5% borage oil, or 20% fish and 20% borage oil for 21 days. On day 22, lung microvascular protein permeability, mean arterial pressure, and platelet and white blood cell counts were determined for 2 hrs after an intravenous injection of Salmonella enteritidis endotoxin (10 mg/kg). In a second group of prefed rats, the phospholipid fatty acid composition was determined in liver and alveolar macrophages. Alveolar macrophages were harvested by bronchoalveolar lavage and stimulated in vitro with a calcium ionophore (A23187), and the concentrations of leukotrienes B4 and B5, thromboxane A2, prostaglandin E2, and 6-keto-prostaglandin F1 alpha were measured in a third group of prefed rats. MEASUREMENT AND MAIN RESULTS: Lung permeability was greatest with corn oil and was significantly attenuated with 20% fish oil and 20% fish and 5% borage oil, and this effect approached significance with 20% fish and 20% borage oil (p = .06). The early and late hypotensive effects of endotoxin were attenuated with 20% fish oil, 20% fish and 5% borage oil, and 20% fish and 20% borage oil, as compared with corn oil. Concentrations of leukotriene B4, prostaglandin E2, and thromboxane B2 released from A23187-stimulated alveolar macrophages were significantly lower with 20% fish oil and 20% fish and 20% borage oil, as compared with corn oil. The increase in lung microvascular protein permeability with 20% fish and 20% borage oil was not significantly different than the lung microvascular protein permeability that was found in animals receiving 20% fish oil (p = .20) and 20% fish and 5% borage oil (p = .31). Alveolar macrophage and liver phospholipid concentrations of arachidonic acid were lower, and the concentrations of eicosapentaenoic acid and docosahexaenic acid were higher, with 20% fish oil, and 5% borage oil, and 20% fish and 20% borage oil, as compared with corn oil. Dihomo-gamma-linolenic acid, the desaturated and elongated intermediate of gamma-linolenic acid, was increased with 20% fish and 20% borage oil, as compared with 20% fish oil and 20% fish and 5% borage oil. CONCLUSIONS: The severity of pulmonary microvascular protein permeability and the degree of hypotension were reduced with fish or fish and borage oil diets, as compared with corn oil, in endotoxic rats. The reduced synthesis of the proinflammatory arachidonic acid-derived mediators, leukotriene B4, thromboxane B2, and prostaglandin E2 from stimulated alveolar macrophages was indicative of a decrease in arachidonic acid and an increase in eicosapentaenoic acid and docosahexaenoic acid in cell membrane phospholipids.
Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Ácido Eicosapentaenoico/farmacologia , Pulmão/irrigação sanguínea , Macrófagos Alveolares/efeitos dos fármacos , Síndrome do Desconforto Respiratório/tratamento farmacológico , Ácido gama-Linolênico/farmacologia , Animais , Modelos Animais de Doenças , Método Duplo-Cego , Avaliação Pré-Clínica de Medicamentos , Endotoxemia/complicações , Masculino , Distribuição Aleatória , Ratos , Ratos Endogâmicos , Síndrome do Desconforto Respiratório/imunologia , Síndrome do Desconforto Respiratório/microbiologiaRESUMO
Atrial natriuretic peptide (ANP) is released from the heart and participates in regulating blood pressure and volume. We recently developed a reverse hemolytic plaque assay to measure the release of ANP from individual rat atrial cardiocytes. This assay determines the total proportion of atrial cardiocytes committed to releasing ANP. We combined the plaque assay with immunocytochemistry (IC) and in situ hybridization (IS). Combining the plaque assay with IC for myosin revealed that 13.5 +/- 0.9% (%myosin+ and plaque forming; mean +/- SE, n = 4) of atrial cardiocytes are cardiomyocytes that release ANP. Combination of the plaque assay with IS for ANP mRNA showed that 16.6 +/- 0.6% (%in situ+ and plaque forming; mean +/- SE, n = 4) of the cardiocytes in the rat atria synthesize and release the hormone. Incubation of atrial cardiocytes with dexamethasone to stimulate ANP gene expression did not alter the total proportion of in situ-positive ANP-secreting cells. These data suggest that, within the total ANP-secreting population of the rat atria, only 33% of the secreting cells are cardiomyocytes. In addition, 68% of the ANP-secreting cells do not appear to synthesize the hormone. These results imply that muscle and nonmuscle cells are involved in secreting ANP and that cardiomyocytes synthesize the hormone.