High-glucose diets induce mitochondrial dysfunction in Caenorhabditis elegans.

Glucose is an important nutrient that dictates the development, fertility and lifespan of all organisms. In humans, a deficit in its homeostatic control might lead to hyperglucemia and the development of obesity and type 2 diabetes, which show a decreased ability to respond to and metabolize glucose. Previously, we have reported that high-glucose diets (HGD) induce alterations in triglyceride content, body size, progeny, and the mRNA accumulation of key regulators of carbohydrate and lipid metabolism, and longevity in Caenorhabditis elegans (PLoS ONE 13(7): e0199888). Herein, we show that increasing amounts of glucose in the diet induce the swelling of both mitochondria in germ and muscle cells. Additionally, HGD alter the enzymatic activities of the different respiratory complexes in an intricate pattern. Finally, we observed a downregulation of ceramide synthases (hyl-1 and hyl-2) and antioxidant genes (gcs-1 and gst-4), while mitophagy genes (pink-1 and dct-1) were upregulated, probably as part of a mitohormetic mechanism in response to glucose toxicity.

A high glucose diet induces autophagy in a HLH-30/TFEB-dependent manner and impairs the normal lifespan of C. elegans.

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.

A forgotten chapter of Mexican technology and science: Luis Gutiérrez Villegas and poliomyelitis in Mexico. / Un capítulo olvidado de la tecnología y la ciencia mexicanas: Luis Gutiérrez Villegas y la poliomielitis en México.

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.

The MXL-3/SBP-1 Axis Is Responsible for Glucose-Dependent Fat Accumulation in C. elegans.

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.

Guide and Position of the International Society of Nutrigenetics/Nutrigenomics on Personalised Nutrition: Part 1 - Fields of Precision Nutrition.

Diversity in the genetic profile between individuals and specific ethnic groups affects nutrient requirements, metabolism and response to nutritional and dietary interventions. Indeed, individuals respond differently to lifestyle interventions (diet, physical activity, smoking, etc.). The sequencing of the human genome and subsequent increased knowledge regarding human genetic variation is contributing to the emergence of personalized nutrition. These advances in genetic science are raising numerous questions regarding the mode that precision nutrition can contribute solutions to emerging problems in public health, by reducing the risk and prevalence of nutrition-related diseases. Current views on personalized nutrition encompass omics technologies (nutrigenomics, transcriptomics, epigenomics, foodomics, metabolomics, metagenomics, etc.), functional food development and challenges related to legal and ethical aspects, application in clinical practice, and population scope, in terms of guidelines and epidemiological factors. In this context, precision nutrition can be considered as occurring at three levels: (1) conventional nutrition based on general guidelines for population groups by age, gender and social determinants; (2) individualized nutrition that adds phenotypic information about the person's current nutritional status (e.g. anthropometry, biochemical and metabolic analysis, physical activity, among others), and (3) genotype-directed nutrition based on rare or common gene variation. Research and appropriate translation into medical practice and dietary recommendations must be based on a solid foundation of knowledge derived from studies on nutrigenetics and nutrigenomics. A scientific society, such as the International Society of Nutrigenetics/Nutrigenomics (ISNN), internationally devoted to the study of nutrigenetics/nutrigenomics, can indeed serve the commendable roles of (1) promoting science and favoring scientific communication and (2) permanently working as a 'clearing house' to prevent disqualifying logical jumps, correct or stop unwarranted claims, and prevent the creation of unwarranted expectations in patients and in the general public. In this statement, we are focusing on the scientific aspects of disciplines covering nutrigenetics and nutrigenomics issues. Genetic screening and the ethical, legal, social and economic aspects will be dealt with in subsequent statements of the Society.

Thiamine Deprivation Produces a Liver ATP Deficit and Metabolic and Genomic Effects in Mice: Findings Are Parallel to Those of Biotin Deficiency and Have Implications for Energy Disorders.

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.

Biotin deprivation impairs mitochondrial structure and function and has implications for inherited metabolic disorders.

Certain inborn errors of metabolism result from deficiencies in biotin containing enzymes. These disorders are mimicked by dietary absence or insufficiency of biotin, ATP deficit being a major effect,whose responsible mechanisms have not been thoroughly studied. Here we show that in rats and cultured cells it is the result of reduced TCA cycle flow, partly due to deficient anaplerotic biotin-dependent pyruvate carboxylase. This is accompanied by diminished flow through the electron transport chain, augmented by deficient cytochrome c oxidase (complex IV) activity with decreased cytochromes and reduced oxidative phosphorylation. There was also severe mitochondrial damage accompanied by decrease of mitochondria, associated with toxic levels of propionyl CoA as shown by carnitine supplementation studies, which explains the apparently paradoxical mitochondrial diminution in the face of the energy sensor AMPK activation, known to induce mitochondria biogenesis. This idea was supported by experiments on AMPK knockout mouse embryonic fibroblasts (MEFs). The multifactorial ATP deficit also provides a plausible basis for the cardiomyopathy in patients with propionic acidemia, and other diseases.Additionally, systemic inflammation concomitant to the toxic state might explain our findings of enhanced IL-6, STAT3 and HIF-1α, associated with an increase of mitophagic BNIP3 and PINK proteins, which may further increase mitophagy. Together our results imply core mechanisms of energy deficit in several inherited metabolic disorders.

Insulin sensitivity is inversely related to cellular energy status, as revealed by biotin deprivation.

We have reported an early decrease in glycemia in rats fed a biotin-deficient diet with reduced cellular ATP levels, suggesting increased insulin sensitivity. Here, we show that biotin-deprived rats are more tolerant of glucose, as shown by both oral and intraperitoneal glucose tolerance tests, during which insulin plasma levels were significantly diminished in deficient rats compared with controls. Biotin-deficient rats had lower blood glucose concentrations during intraperitoneal insulin sensitivity tests than controls. Furthermore, more glucose was infused to maintain euglycemia in the biotin-deficient rats during hyperinsulinemic euglycemic clamp studies. These results demonstrate augmented sensitivity to insulin in biotin-deprived rats. They are most likely the consequence of an insulin-independent effect of AMPK activation on GLUT4 membrane translocation with increased glucose uptake. In biotin-deficient cultured L6 muscle cells, there was increased phosphorylation of the energy sensor AMPK. We have now confirmed the augmented AMPK activation in both biotin-deprived in vivo muscle and cultured muscle cells. In these cells, glucose uptake is increased by AMPK activation by AICAR and diminished by its knockdown by the specific siRNAs directed against its α1- and α2-catalytic subunits, with all of these effects being largely independent of the activity of the insulin-signaling pathway that was inhibited with wortmannin. The enhanced insulin sensitivity in biotin deficiency likely has adaptive value for organisms due to the hormone promotion of uptake and utilization of not only glucose but other nutrients such as branched-chain amino acids, whose deficiency has been reported to increase insulin tolerance.

[Mutation analysis of the methylmalonyl-CoA mutase gene in ten Mexican patients with methylmalonic acidemia]. / Análisis de mutaciones en el gen de la metilmalonilCoA mutasa en diez pacientes mexicanos con acidemia metilmalónica aislada.

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.

Temporal development of genetic and metabolic effects of biotin deprivation. A search for the optimum time to study a vitamin deficiency.

Biotin deficiency (Bt-D) is usually studied at the point at which the animal model exhibits the signs of full-blown deficiency symptoms; in rats, this typically occurs at 6-8 weeks of feeding a deficient diet. To differentiate specific deficiency effects from those of undernutrition, biotin sufficient and deficient rats were studied at 2, 3, 4, and 5 weeks on the deficiency diet, before the onset of weight loss and deficiency signs. The deficiency state was confirmed by biochemical and molecular analyses. Blood and liver metabolites were determined and western blots of signaling proteins, and qRT-PCR gene expression studies. The main effects of Bt-D were already well established by the fourth week on the diet; thus, we consider the fourth week as the optimum time to study the consequences of biotin depletion. Early effects, which were already apparent at week 2, included cellular energy deficit (as assessed by increased AMP/ATP ratio), activation of the AMPK energy sensor, and changes of carbon metabolism gene transcripts (e.g., phosphoenolpyruvate carboxykinase, carnitine palmitoyl transferase 1, liver glucokinase and fatty acid synthetase). Reduced post-prandial blood concentrations of glucose were also observed early; we speculate that these are attributable to augmented sensitivity to insulin and increased glucose utilization, a likely effect of AMPK induction of translocation of glucose transporter GLUT4 to the cell membranes and increased hexokinase expression. Other late-onset changes (week 4) included increased serum concentrations of lactate and free fatty acids and decreased liver glycogen and serum concentrations of triglycerides and total cholesterol. The identification of the early specific molecular and metabolic disturbances of biotin deficiency might be useful in identifying individuals with marginal deficiency of this vitamin, which appears to be common in normal human pregnancy. The study of time-course of other vitamin deficiencies, such as this one, might help to better understand and cope with their effects.

A heuristic model for paradoxical effects of biotin starvation on carbon metabolism genes in the presence of abundant glucose.

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.

The hexokinase gene family in the zebrafish: structure, expression, functional and phylogenetic analysis.

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.

Functional and metabolic implications of biotin deficiency for the rat heart.

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.

Role of proteomics in the study of nutrition.

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.

Biotin deficiency affects both synthesis and degradation of pyruvate carboxylase in rat primary hepatocyte cultures.

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.

From an inborn error patient to a search for regulatory meaning: a biotin conducted voyage.

This article summarizes some findings of a research that I have pursued for the past 25 years, whose roots are immersed in the field of inherited metabolic disorders, and deal with different aspects of the vitamin biotin, starting with a patient with multiple carboxylase deficiency (MCD). Several of MCD clinical manifestations resemble those of infant malnutrition; we demonstrated that about one-third of infants with this common nutritional disorder were indeed biotin-deficient, and that this deficiency is metabolically significant, by studying urine instead of blood, studying urinary organic acids by gas chromatography-mass spectrometry. Remarkably, the metabolic abnormalities became apparent only after protein feeding was started, suggesting that this phenomenon may contribute to the worsening of malnourished individuals when they are abruptly fed. Afterwards, we studied biotin deficiency at the tissue level. Carboxylase activities and masses were significantly reduced in liver, kidney, muscle, adipose tissue, intestine, and spleen, but brain and heart were spared; their mRNAs remained unchanged. On the other hand, holocarboxylase synthetase (HCS) mRNA levels were markedly low in the deficient animals, and increased upon biotin injection. Over 2000 human genes have been identified that depend on biotin for expression. To probe into the "logic" of this enigma, we have started comparative studies among evolutionarily distant organisms, such as mouse and Saccharomyces cerevisiae, and we are now looking for biotin effects on specific genes and proteins, such as HCS and hexokinases, and on their proteomes.

Effects of biotin on growth and protein biotinylation in Saccharomyces cerevisiae.

In mammals, biotin, well known for its role as the cofactor of carboxylases, also controls the expression not only of proteins involved in this function, but also of a large number and variety of other different proteins. As a first step towards looking for a rationale for these phenomena, we intend to compare these regulatory functions of biotin between the rat and the much less evolutionized eukaryote, Saccharomyces cerevisiae. Thus far, we have measured growth in yeast cultured on different concentrations of biotin to choose the experimental conditions to be used (2, 200 and 2000 microM) and have found that a band corresponding to the biotinylated S. cerevisiae Arc1p protein appears at streptavidin Western blots at a biotin concentration above 2000 muM, its density increasing with higher biotin amounts. We will now study changes in yeast transcriptome with these varying concentrations and compare them with changes observed in the rat.