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.

Differential Gene Expression Profile Induced by Valproic Acid (VPA) in Pediatric Epileptic Patients.

Epilepsy is a neuronal disease that affects up to 70 million people worldwide. The development of effective therapies to combat childhood epilepsy requires early biomarkers. Here, we performed a whole-genome microarray analysis in blood cells to identify genes differentially expressed between epileptic and epileptic valproic acid (VPA)-treated children versus normal children to obtain information about the gene expression to help us to understand genetic aspects of this disease. We found that the most significant differentially expressed genes were related to the transcriptional factor cAMP-response element binding protein (CREB) that is overexpressed in children with epilepsy compared with normal children, and 6 and 12 months of VPA treatment reversed several of these changes. Interestingly, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), a type I transmembrane glycoprotein that binds collagen proteins and contains CREB binding sites, was one of the more up-regulated genes in epileptic patients, and treatment with VPA strongly reversed its up-regulation. CREB up-regulates genes related to epilepsy; here, we suggest that LAIR1 could activate CREB, and together, they trigger epilepsy. After VPA treatment, LAIR1 repressed genes by disrupting the functional LAIR1⁻CREB complex, resulting in successful treatment. A functional microarray analysis offers new information that could open novel avenues of research in biomarker discovery, which may be useful for the early identification of children with a predisposition to epilepsy.

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.

AMP-Activated Protein Kinase Regulates Oxidative Metabolism in Caenorhabditis elegans through the NHR-49 and MDT-15 Transcriptional Regulators.

Cellular energy regulation relies on complex signaling pathways that respond to fuel availability and metabolic demands. Dysregulation of these networks is implicated in the development of human metabolic diseases such as obesity and metabolic syndrome. In Caenorhabditis elegans the AMP-activated protein kinase, AAK, has been associated with longevity and stress resistance; nevertheless its precise role in energy metabolism remains elusive. In the present study, we find an evolutionary conserved role of AAK in oxidative metabolism. Similar to mammals, AAK is activated by AICAR and metformin and leads to increased glycolytic and oxidative metabolic fluxes evidenced by an increase in lactate levels and mitochondrial oxygen consumption and a decrease in total fatty acids and lipid storage, whereas augmented glucose availability has the opposite effects. We found that these changes were largely dependent on the catalytic subunit AAK-2, since the aak-2 null strain lost the observed metabolic actions. Further results demonstrate that the effects due to AAK activation are associated to SBP-1 and NHR-49 transcriptional factors and MDT-15 transcriptional co-activator, suggesting a regulatory pathway that controls oxidative metabolism. Our findings establish C. elegans as a tractable model system to dissect the relationship between distinct molecules that play a critical role in the regulation of energy metabolism in human metabolic diseases.

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.

Caenorhabditis elegans: A useful model for studying metabolic disorders in which oxidative stress is a contributing factor.

Caenorhabditis elegans is a powerful model organism that is invaluable for experimental research because it can be used to recapitulate most human diseases at either the metabolic or genomic level in vivo. This organism contains many key components related to metabolic and oxidative stress networks that could conceivably allow us to increase and integrate information to understand the causes and mechanisms of complex diseases. Oxidative stress is an etiological factor that influences numerous human diseases, including diabetes. C. elegans displays remarkably similar molecular bases and cellular pathways to those of mammals. Defects in the insulin/insulin-like growth factor-1 signaling pathway or increased ROS levels induce the conserved phase II detoxification response via the SKN-1 pathway to fight against oxidative stress. However, it is noteworthy that, aside from the detrimental effects of ROS, they have been proposed as second messengers that trigger the mitohormetic response to attenuate the adverse effects of oxidative stress. Herein, we briefly describe the importance of C. elegans as an experimental model system for studying metabolic disorders related to oxidative stress and the molecular mechanisms that underlie their pathophysiology.

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.