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Glutamatergic neurotransmission entails a tonic loss of glutamate from nerve endings into the synapse. Replacement of neuronal glutamate is essential in order to avoid depletion of the internal pool. In brain this occurs primarily via the glutamate-glutamine cycle, which invokes astrocytic synthesis of glutamine and hydrolysis of this amino acid via neuronal phosphate-dependent glutaminase. This cycle maintains constancy of internal pools, but it does not provide a mechanism for inevitable losses of glutamate N from brain. Import of glutamine or glutamate from blood does not occur to any appreciable extent. However, the branched-chain amino acids (BCAA) cross the blood–brain barrier swiftly. The brain possesses abundant branched-chain amino acid transaminase activity which replenishes brain glutamate and also generates branched-chain ketoacids. It seems probable that the branched-chain amino acids and ketoacids participate in a “glutamate-BCAA cycle” which involves shuttling of branched-chain amino acids and ketoacids between astrocytes and neurons. This mechanism not only supports the synthesis of glutamate, it also may constitute a mechanism by which high (and potentially toxic) concentrations of glutamate can be avoided by the re-amination of branched-chain ketoacids.

Tumor cell proliferation requires rapid synthesis of macromolecules including lipids, proteins, and nucleotides. Many tumor cells exhibit rapid glucose consumption, with most of the glucose-derived carbon being secreted as lactate despite abundant oxygen availability (the Warburg effect). Here, we used ¹³C NMR spectroscopy to examine the metabolism of glioblastoma cells exhibiting aerobic glycolysis. In these cells, the tricarboxylic acid (TCA) cycle was active but was characterized by an efflux of substrates for use in biosynthetic pathways, particularly fatty acid synthesis. The success of this synthetic activity depends on activation of pathways to generate reductive power (NADPH) and to restore oxaloacetate for continued TCA cycle function (anaplerosis). Surprisingly, both these needs were met by a high rate of glutamine metabolism. First, conversion of glutamine to lactate (glutaminolysis) was rapid enough to produce sufficient NADPH to support fatty acid synthesis. Second, despite substantial mitochondrial pyruvate metabolism, pyruvate carboxylation was suppressed, and anaplerotic oxaloacetate was derived from glutamine. Glutamine catabolism was accompanied by secretion of alanine and ammonia, such that most of the amino groups from glutamine were lost from the cell rather than incorporated into other molecules. These data demonstrate that transformed cells exhibit a high rate of glutamine consumption that cannot be explained by the nitrogen demand imposed by nucleotide synthesis or maintenance of nonessential amino acid pools. Rather, glutamine metabolism provides a carbon source that facilitates the cell's ability to use glucose-derived carbon and TCA cycle intermediates as biosynthetic precursors.

Mammalian cells fuel their growth and proliferation through the catabolism of two main substrates: glucose and glutamine. Most of the remaining metabolites taken up by proliferating cells are not catabolized, but instead are used as building blocks during anabolic macromolecular synthesis. Investigations of phosphoinositol 3-kinase (PI3K) and its downstream effector AKT have confirmed that these oncogenes play a direct role in stimulating glucose uptake and metabolism, rendering the transformed cell addicted to glucose for the maintenance of survival. In contrast, less is known about the regulation of glutamine uptake and metabolism. Here, we report that the transcriptional regulatory properties of the oncogene Myc coordinate the expression of genes necessary for cells to engage in glutamine catabolism that exceeds the cellular requirement for protein and nucleotide biosynthesis. A consequence of this Myc-dependent glutaminolysis is the reprogramming of mitochondrial metabolism to depend on glutamine catabolism to sustain cellular viability and TCA cycle anapleurosis. The ability of Myc-expressing cells to engage in glutaminolysis does not depend on concomitant activation of PI3K or AKT. The stimulation of mitochondrial glutamine metabolism resulted in reduced glucose carbon entering the TCA cycle and a decreased contribution of glucose to the mitochondrial-dependent synthesis of phospholipids. These data suggest that oncogenic levels of Myc induce a transcriptional program that promotes glutaminolysis and triggers cellular addiction to glutamine as a bioenergetic substrate.

Argininosuccinate lyase (ASL) is essential for the NO-dependent regulation of tyrosine hydroxylase (TH) and thus for catecholamine production. Using a conditional mouse model with loss of ASL in catecholamine neurons, we demonstrate that ASL is expressed in dopaminergic neurons in the substantia nigra pars compacta, including the ALDH1A1 + subpopulation that is pivotal for the pathogenesis of Parkinson disease (PD). Neuronal loss of ASL results in catecholamine deficiency, in accumulation and formation of tyrosine aggregates, in elevation of α-synuclein, and phenotypically in motor and cognitive deficits. NO supplementation rescues the formation of aggregates as well as the motor deficiencies. Our data point to a potential metabolic link between accumulations of tyrosine and seeding of pathological aggregates in neurons as initiators for the pathological processes involved in neurodegeneration. Hence, interventions in tyrosine metabolism via regulation of NO levels may be therapeutic beneficial for the treatment of catecholamine-related neurodegenerative disorders.

Background Lysosomal acid lipase deficiency (LAL-D) is an autosomal recessive disorder that can present as a severe, infantile form also known as Wolman disease. We sought to determine the outcomes and clinical needs of infants diagnosed with LAL-D, treated with enzyme replacement therapy (ERT). Methods A chart review was conducted on two infantile-onset LAL-D patients to determine clinical outcomes based on laboratory results, abdominal imaging, growth and dietary records, cardiology, endocrinology, ophthalmology, hematology, and neurocognitive evaluations. Results Two patients, both diagnosed and treated before 6 months old, demonstrated clinical improvement following weekly ERT. They required dosage increases to optimize growth and symptomatology. Both received a formula low in long chain triglycerides and high in medium chain triglycerides, an intervention that allowed significant catch-up growth. Patient 1 required treatment for partial adrenal insufficiency and hypothyroidism. Both patients demonstrated reduction in liver and spleen size and varying degrees of improved liver function. Neither experienced serious adverse reactions to ERT. Conclusion ERT has led to longer and healthier survival of affected infants. It is imperative that dietary interventions and systemic clinical care become integral to the management. Continued evidence of survival and clinical improvement in this population, coupled with available mass spectrometry enzyme assay from dried blood spots, raises the question of this rare and possibly underdiagnosed disorder’s candidacy for newborn screening.

Coenzyme Q (CoQ) is an essential electron carrier in the respiratory chain whose deficiency has been implicated in a wide variety of human mitochondrial disease manifestations. Its multi-step biosynthesis involves production of polyisoprenoid diphosphate in a reaction that requires the enzymes be encoded by PDSS1 and PDSS2. Homozygous mutations in either of these genes, in humans, lead to severe neuromuscular disease, with nephrotic syndrome seen in PDSS2 deficiency. We now show that a presumed autoimmune kidney disease in mice with the missense Pdss2(kd/kd) genotype can be attributed to a mitochondrial CoQ biosynthetic defect. Levels of CoQ9 and CoQ10 in kidney homogenates from B6.Pdss2(kd/kd) mutants were significantly lower than those in B6 control mice. Disease manifestations originate specifically in glomerular podocytes, as renal disease is seen in Podocin/cre,Pdss2(loxP/loxP) knockout mice but not in conditional knockouts targeted to renal tubular epithelium, monocytes, or hepatocytes. Liver-conditional B6.Alb/cre,Pdss2(loxP/loxP) knockout mice have no overt disease despite demonstration that their livers have undetectable CoQ9 levels, impaired respiratory capacity, and significantly altered intermediary metabolism as evidenced by transcriptional profiling and amino acid quantitation. These data suggest that disease manifestations of CoQ deficiency relate to tissue-specific respiratory capacity thresholds, with glomerular podocytes displaying the greatest sensitivity to Pdss2 impairment.

We describe here a new strategy for the treatment of stroke, through the inhibition of NAALADase (N-acetylated-α-linked-acidic dipeptidase), an enzyme responsible for the hydrolysis of the neuropeptide NAAG (N-acetyl-aspartyl-glutamate) to N-acetyl-aspartate and glutamate. We demonstrate that the newly described NAALADase inhibitor 2-PMPA (2-(phosphonomethyl)pentanedioic acid) robustly protects against ischemic injury in a neuronal culture model of stroke and in rats after transient middle cerebral artery occlusion. Consistent with inhibition of NAALADase, we show that 2-PMPA increases NAAG and attenuates the ischemia-induced rise in glutamate. Both effects could contribute to neuroprotection. These data indicate that NAALADase inhibition may have use in neurological disorders in which excessive excitatory amino acid transmission is pathogenic.

Increasing evidence indicates that the gut microbiota can be altered to ameliorate or prevent disease states, and engineering the gut microbiota to therapeutically modulate host metabolism is an emerging goal of microbiome research. In the intestine, bacterial urease converts host-derived urea to ammonia and carbon dioxide, contributing to hyperammonemia-associated neurotoxicity and encephalopathy in patients with liver disease. Here, we engineered murine gut microbiota to reduce urease activity. Animals were depleted of their preexisting gut microbiota and then inoculated with altered Schaedler flora (ASF), a defined consortium of 8 bacteria with minimal urease gene content. This protocol resulted in establishment of a persistent new community that promoted a long-term reduction in fecal urease activity and ammonia production. Moreover, in a murine model of hepatic injury, ASF transplantation was associated with decreased morbidity and mortality. These results provide proof of concept that inoculation of a prepared host with a defined gut microbiota can lead to durable metabolic changes with therapeutic utility.

One of the many biological functions of nitric oxide is the ability to protect cells from oxidative stress. To investigate the potential contribution of low steady state levels of nitric oxide generated by endothelial nitric oxide synthase (eNOS) and the mechanisms of protection against H2O 2, spontaneously transformed human ECV304 cells, which normally do not express eNOS, were stably transfected with a green fluorescent-tagged eNOS cDNA. The eNOS-transfected cells were found to be resistant to injury and delayed death following a 2-h exposure to H2O 2(50-150 µM). Inhibition of nitric oxide synthesis abolished the protective effect against H2O 2exposure. The ability of nitric oxide to protect cells depended on the presence of respiring mitochondria as ECV304+eNOS cells with diminished mitochondria respiration (ρ-)are injured to the same extent as nontransfected ECV304 cells and recovery of mitochondrial respiration restores the ability of nitric oxide to protect against H2O 2-induced death. Nitric oxide also found to have a profound effect in cell metabolism, because ECV304+eNOS cells had lower steady state levels of ATP and higher utilization of glucose via the glycolytic pathway than ECV304 cells. However, the protective effect of nitric oxide against H2O 2exposure is not reproduced in ECV304 cells after treatment with azide and oligomycin suggesting that the dynamic regulation of respiration by nitric oxide represent a critical and unrecognized primary line of defense against oxidative stress.

We sought to prospectively characterize the nutritional status of adults ≥ 19 years (n = 22, 27% males) and children (n = 38, 61% male) with genetically-confirmed primary mitochondrial disease (PMD) to guide development of precision nutritional support strategies to be tested in future clinical trials. We excluded subjects who were exclusively tube-fed. Daily caloric requirements were estimated using World Health Organization (WHO) equations to predict resting energy expenditure (REE) multiplied by an activity factor (AF) based on individual activity levels. We developed a Mitochondrial Disease Activity Factors (MOTIVATOR) score to encompass the impact of muscle fatigue typical of PMD on physical activity levels. PMD cohort daily diet intake was estimated to be 1,143 ± 104.1 kcal in adults (mean ± SEM, 76.2% of WHO-MOTIVATOR predicted requirement), and 1,114 ± 62.3 kcal in children (86.4% predicted). A total of 11/22 (50%) adults and 18/38 (47.4%) children with PMD consumed ≤ 75% predicted daily Kcal needs. Malnutrition was identified in 16/60 (26.7%) PMD subjects. Increased protein and fat intake correlated with improved muscle strength in those with insufficient daily Kcal intake (≤ 75% predicted); higher protein and fat intake correlated with decreased muscle fatigue; and higher protein, fat, and carbohydrate intake correlated with improved quality of life (QoL). These data demonstrate the frequent occurrence of malnutrition in PMD and emphasize the critical need to devise nutritional interventions to optimize clinical outcomes.