RESUMO
A high-glucose diet (HGD) is associated with the development of metabolic diseases that decrease life expectancy, including obesity and type-2 diabetes (T2D); however, the mechanism through which a HGD does so is still unclear. Autophagy, an evolutionarily conserved mechanism, has been shown to promote both cell and organismal survival. The goal of this study was to determine whether exposure of Caenorhabditis elegans to a HGD affects autophagy and thus contributes to the observed lifespan reduction under a HGD. Unexpectedly, nematodes exposed to a HGD showed increased autophagic flux via an HLH-30/TFEB-dependent mechanism because animals with loss of HLH-30/TFEB, even those with high glucose exposure, had an extended lifespan, suggesting that HLH-30/TFEB might have detrimental effects on longevity through autophagy under this stress condition. Interestingly, pharmacological treatment with okadaic acid, an inhibitor of the PP2A and PP1 protein phosphatases, blocked HLH-30 nuclear translocation, but not TAX-6/calcineurin suppression by RNAi, during glucose exposure. Together, our data support the suggested dual role of HLH-30/TFEB and autophagy, which, depending on the cellular context, may promote either organismal survival or death.
Assuntos
Autofagia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Dieta , Glucose/metabolismo , Longevidade , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 2/metabolismo , Transdução de SinaisRESUMO
Chronic exposure to elevated glucose levels leads to fatty acid accumulation, which promotes the development of metabolic diseases such as obesity and type 2 diabetes. MXL-3 is a conserved transcriptional factor that modulates the inhibition of lipolysis in Caenorhabditis elegans. However, the role of MXL-3 in lipid metabolism during nutrient excess remains unknown. We hypothesized that inhibition of MXL-3 prevents glucose-dependent fat accumulation. Nematodes from wild-type N2, MXL-3::GFP and sbp-1 or mxl-3 null strains were grown on standard, high glucose or high glucose plus metformin plates for 24 h. Using laser-scanning confocal microscopy, we monitored the glucose-induced activation of MXL-3 labeled with GFP (MXL-3::GFP). Lipid levels were determined by Oil Red O (ORO) staining and gas chromatography/mass spectrometry, and gene expression was assessed by qRT-PCR. We found that high glucose activated MXL-3 by increasing its rate of nuclear entry, which in turn increased lipid levels via sterol regulatory element-binding protein (SBP-1). This activated critical genes that synthesize long chain unsaturated fatty acids (MUFAs and PUFAs) and repress lipolytic genes. Interestingly, the anti-diabetic drug metformin inhibited MXL-3 activation and subsequently prevented glucose-dependent fat accumulation. These findings highlight the importance of the MXL-3/SBP-1 axis in the regulation of lipid metabolism during nutritional excess and provide new insight into the mechanism by which metformin prevents lipid accumulation. This study also suggests that inhibition of MXL-3 may serve as a potential target for the treatment of chronic metabolic diseases, including obesity, type 2 diabetes, and cardiovascular disease.
RESUMO
Two different types of vaccines were developed against poliomyelitis: The Salk vaccine using inactivated virus and the Sabin one, that was used later, after investigations assured its safety. The first one was made in Mexico with its own resources since 1957 thanks to the efforts of young researchers and technicians coordinated by Luis Gutiérrez-Villegas, M.D., who was a Clinical Pathologist, University Professor and President of the Mexican National Academy of Mexico.
Assuntos
Poliomielite/prevenção & controle , Vacina Antipólio de Vírus Inativado/história , Vacina Antipólio Oral/história , História do Século XX , Humanos , México , Poliomielite/história , Vacina Antipólio de Vírus Inativado/administração & dosagem , Vacina Antipólio Oral/administração & dosagemRESUMO
Thiamine is one of several essential cofactors for ATP generation. Its deficiency, like in beriberi and in the Wernicke-Korsakoff syndrome, has been studied for many decades. However, its mechanism of action is still not completely understood at the cellular and molecular levels. Since it acts as a coenzyme for dehydrogenases of pyruvate, branched-chain keto acids, and ketoglutarate, its nutritional privation is partly a phenocopy of inborn errors of metabolism, among them maple syrup urine disease. In the present paper, we report metabolic and genomic findings in mice deprived of thiamine. They are similar to the ones we have previously found in biotin deficiency, another ATP generation cofactor. Here we show that thiamine deficiency substantially reduced the energy state in the liver and activated the energy sensor AMP-activated kinase. With this vitamin deficiency, several metabolic parameters changed: blood glucose was diminished and serum lactate was increased, but insulin, triglycerides, and cholesterol, as well as liver glycogen, were reduced. These results indicate a severe change in the energy status of the whole organism. Our findings were associated with modified hepatic levels of the mRNAs of several carbon metabolism genes: a reduction of transcripts for liver glucokinase and fatty acid synthase and augmentation of those for carnitine palmitoyl transferase 1 and phosphoenolpyruvate carboxykinase as markers for glycolysis, fatty acid synthesis, beta-oxidation, and gluconeogenesis, respectively. Glucose tolerance was initially increased, suggesting augmented insulin sensitivity, as we had found in biotin deficiency; however, in the case of thiamine, it was diminished from the 3rd week on, when the deficient animals became undernourished, and paralleled the changes in AKT and mTOR, 2 main proteins in the insulin signaling pathway. Since many of the metabolic and gene expression effects on mice deprived of thiamine are similar to those in biotin deficiency, it may be that they result from a more general impairment of oxidative phosphorylation due to a shortage of ATP generation cofactors. These findings may be relevant to energy-related disorders, among them several inborn errors of metabolism, as well as common energy disorders like obesity, diabetes, and neurodegenerative illnesses.
Assuntos
Trifosfato de Adenosina/metabolismo , Deficiência de Biotinidase , Metabolismo Energético , Fígado/metabolismo , Doenças Metabólicas/etiologia , Deficiência de Tiamina/genética , Deficiência de Tiamina/metabolismo , Trifosfato de Adenosina/deficiência , Animais , Deficiência de Biotinidase/genética , Deficiência de Biotinidase/metabolismo , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/genética , Interação Gene-Ambiente , Genoma/efeitos dos fármacos , Gluconeogênese/efeitos dos fármacos , Gluconeogênese/genética , Fígado/efeitos dos fármacos , Masculino , Doenças Metabólicas/genética , Doenças Metabólicas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Tiamina/farmacologiaRESUMO
INTRODUCTION: Methylmalonic acidemia (MMA) is a genetically determined human metabolic disease, characterized by deficient activity of the mitochondrial enzyme, methylmalonyl CoA mutase (MCM). This enzyme catalyzes the isomerization of L-methylmalonyl CoA to succinyl CoA and requires adenosylcobalamin as cofactor. Several mutations have been identified in the unique genetic locus encoding the MCM apoenzyme (mut) which causes MMA. AIM: To identify the mutations present in Mexican patients diagnosed with MMA. RESULTS: Complete nucleotide sequencing of mut gene exons of 10 Mexican patients with methylmalonic acidemia (MMA) identified one novel mutation and eight mutations previously reported in the methylmalonyl-CoA mutase (mut) gene. The new mutation c.406G > T (p.V136F) was found in one patient combined with the deletion c.1891delG (p.A631QfsX17). The missense mutation c.322C > T (p.R108C) was found in six non-related patients; in addition, the mutations c.ins671-678dupAATTTATG (p.V227NfsX16), c.682C > T (p.R228X), c1022-1023dupA (p. N341KfsX20), c.1846C > T (p.R616C), c.2080C > T (p.R694W), and c.385+3insTAAGGGT (splice) were found. This work reveals that Mexican patients with MMA have new (p.V136F) as well as worldwide and hispanic reported mutations. The mutation R108C is the most frequent change (40% of total alleles) mainly in patients from León, Guanajuato.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Análise Mutacional de DNA , Metilmalonil-CoA Mutase/genética , Feminino , Humanos , Masculino , MéxicoRESUMO
We recently showed that in biotin starvation in yeast Saccharomyces cerevisiae, nematode Caenorhabditis elegans and rat Rattus norvegicus, despite abundant glucose provision, the expression of genes for glucose utilization and lipogenesis were lowered, and for fatty acid ß-oxidation and gluconeogenesis were raised, and glycolytic/fermentative flow was reduced. This work explored the mechanisms of these results. We show that they are associated with ATP deficit and activation of the energy stress sensor AMP kinase (AMPK; Snf1 in yeast). Analysis of microarray results revealed extensive changes of transcripts for signal transduction pathways and transcription factors AMPK, SREBP-1c, ChREBP, NAMPT, PGC-1α, mTORC1 in rat, and their homologs in worm. In yeast the altered factor transcripts were Adr1, Cat8, Sip4, Mig1, HXK2, and Rgt1. The insulin pathway was negatively enriched (in rat and worm), whereas the adiponectins and JAK/STAT pathways were increased (present only in the rat; they activate AMPK). Together, all these changes explain the effects of biotin starvation on glucose utilization, energy status and carbon metabolism gene expression in a coherent manner across three phylogenetically distant eukaryotes and may have clinical significance in humans, since the effects are reminiscent of insulin resistance. We propose a general model for integrating these results in regulatory circuitries, according to the biology of each species, based on impaired anaplerosis due to pyruvate carboxylase deficiency, that have a basic underlying logic. In a preliminary test in yeast, aspartate corrects all the alterations produced by biotin starvation.
Assuntos
Biotina/deficiência , Caenorhabditis elegans/metabolismo , Glucose/metabolismo , Redes e Vias Metabólicas/genética , Saccharomyces cerevisiae/metabolismo , Adenilato Quinase/química , Adenilato Quinase/metabolismo , Animais , Ácido Aspártico/metabolismo , Biotina/metabolismo , Perfilação da Expressão Gênica , Masculino , Oxigênio/metabolismo , Fosforilação , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Transcrição GênicaRESUMO
Hexokinase-catalyzed glucose phosphorylation is the first and crucial step for glucose utilization. Although there are reported studies on glucose metabolism in commercial species, knowledge on it is almost nil in zebrafish (Danio rerio), an important model organism for biological research. We have searched these fish hexokinase genes by BLAST analysis; determined their expression in liver, muscle, brain and heart; measured their response to fasting and glucose administration; and performed homology sequences studies to glimpse their evolutionary history. We have confirmed by RT-qPCR studies that the six DNA sequences annotated as possible hexokinases in the NCBI GenBank are transcribed. The organ distribution of the HXK genes is similar in zebrafish as in mammals, to which they are distantly related. Of these, DrGLK and DrSHXK1 are expressed in the fish liver, DrHXK1 in brain and heart, and DrHXK2 in muscle. The only gene responsive to glucose was liver DrGLK. Its expression is induced approximately 1 h after glucose intraperitoneal injection, but not after saline solution injection. The comparison of the fish sequences and the corresponding mammalian ones imply that in both taxa the main muscle and brain isoforms are fusion products of the ancestral gene, their amino halves having separated before than their carboxy ones, followed by the fusion event, whereas fish and mammalian glucokinase genes remained unduplicated.
Assuntos
Hexoquinase/química , Hexoquinase/genética , Família Multigênica , Filogenia , Peixe-Zebra/genética , Animais , Jejum , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Genoma/genética , Glucose/administração & dosagem , Glucose/farmacologia , Hexoquinase/metabolismo , Humanos , Especificidade de Órgãos/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Fatores de TempoRESUMO
The tricarboxylic acid (TCA) cycle is the main ATP provider for the heart. TCA carbons must be replenished by anaplerosis for normal cardiac function. Biotin is cofactor of the anaplerotic enzymes pyruvate and propionyl-CoA carboxylases. Here, we found that in biotin deficient rats, both carboxylases decreased 90% in adipose tissue, jejunum and spleen, but in heart they conserved about 60% residual activity. We then investigated if under biotin deficiency (BtDEF), the heart is able to maintain its function in vivo and in isolated conditions, and during ischemia and reperfusion, where metabolism drastically shifts from oxidative to mainly glycolytic. Neither glucose nor octanoate oxidation were severely affected in BtDEF hearts, as assessed by mechanical performance, oxygen uptake or high-energy metabolite content; however, myocardial hexokinase activity and lactate concentration were reduced in deficient hearts. When challenged by ischemia and reperfusion injury, BtDEF hearts did not suffer more damage than the controls, although they lowered significantly their performance, when changed to ischemic conditions, which may have clinical implications. Post-ischemic increase in ADP/ATP ratio was similar in both groups, but during reperfusion there was higher rhythm perturbation in BtDEF hearts. By being relatively insensitive to biotin deficiency, cardiac tissue seems to be able to replenish TCA cycle intermediates and to maintain ATP synthesis.
Assuntos
Biotina/deficiência , Coração/fisiopatologia , Miocárdio/metabolismo , Animais , Humanos , Técnicas In Vitro , Masculino , Metilmalonil-CoA Descarboxilase/metabolismo , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/fisiopatologia , Miocárdio/enzimologia , Piruvato Carboxilase/metabolismo , Ratos , Ratos WistarRESUMO
With the advent of nutrigenomics, a more mechanistic view of the variable host responses to nutrients is beginning to emerge. Proteomics is central to nutrigenomics since studies on the effect of nutrients on the proteome have the potential to explain, at the molecular level, many of the physiological changes associated with nutritional stimuli. Proteomics aims at the resolution, identification and quantitation of complex protein mixtures, discovery of interactions of proteins with other molecules, as well as their cellular localization and their role in metabolism. In this article, recent studies on proteomic effects of two vitamins, biotin and folic acid, will be considered as examples of this novel approach to nutriology.
Assuntos
Valor Nutritivo , Proteômica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
Pyruvate carboxylase (PC) is a biotin-dependent enzyme that plays a crucial role in gluconeogenesis, lipogenesis, Krebs cycle anaplerosis and amino acid catabolism. Biotin deficiency reduces its mass besides its activity. Enzyme mass is the result of its cellular turnover, i.e., its rates of synthesis and degradation. We have now investigated, by a pulse and chase approach in cultured primary hepatocytes, the effects of biotin deficiency on these rates. Wistar rats were fed a biotin-deficient diet and the controls were fed the same diet supplemented with biotin; their biotin status was monitored measuring lymphocytes propionyl-CoA carboxylase activity and urinary 3-hydroxyisovaleric acid. After 6-7 weeks primary hepatocytes were cultured in biotin-deficient or complete DMEM. PC activity was determined by measuring the incorporation of (14)C-bicarbonate into acid-non-volatile products, and its mass by streptavidin Western blots. Its synthesis rate was estimated from [(35)S] methionine incorporation into anti-PC antibody immunoprecipitate. Its degradation rate was calculated from the loss of radioactivity from previously labeled hepatocytes, in a medium containing an excess of non-radioactive methionine. PC synthesis rate in biotin-deficient hepatocytes was approximately 4.5-fold lower than in the controls, and its degradation rate was 5.1-fold higher. Therefore, the decrement of PC mass during biotin deficiency results both from a decrease in its synthesis and an increase in its degradation rates. To our knowledge, this is the first instance where a mammalian enzyme cofactor is necessary to sustain both processes.