Lost-in-Translation of Metabolic Effects of Inorganic Nitrate in Type 2 Diabetes: Is Ascorbic Acid the Answer?

Beneficial metabolic effects of inorganic nitrate (NO3-) and nitrite (NO2-) in type 2 diabetes mellitus (T2DM) have been documented in animal experiments; however, this is not the case for humans. Although it has remained an open question, the redox environment affecting the conversion of NO3- to NO2- and then to NO is suggested as a potential reason for this lost-in-translation. Ascorbic acid (AA) has a critical role in the gastric conversion of NO2- to NO following ingestion of NO3-. In contrast to AA-synthesizing species like rats, the lack of ability to synthesize AA and a lower AA body pool and plasma concentrations may partly explain why humans with T2DM do not benefit from NO3-/NO2- supplementation. Rats also have higher AA concentrations in their stomach tissue and gastric juice that can significantly potentiate gastric NO2--to-NO conversion. Here, we hypothesized that the lack of beneficial metabolic effects of inorganic NO3- in patients with T2DM may be at least in part attributed to species differences in AA metabolism and also abnormal metabolism of AA in patients with T2DM. If this hypothesis is proved to be correct, then patients with T2DM may need supplementation of AA to attain the beneficial metabolic effects of inorganic NO3- therapy.

Impact of CAD-deficiency in flax on biogas production.

Global warming and the reduction in our fossil fuel reservoir have forced humanity to look for new means of energy production. Agricultural waste remains a large source for biofuel and bioenergy production. Flax shives are a waste product obtained during the processing of flax fibers. We investigated the possibility of using low-lignin flax shives for biogas production, specifically by assessing the impact of CAD deficiency on the biochemical and structural properties of shives. The study used genetically modified flax plants with a silenced CAD gene, which encodes the key enzyme for lignin synthesis. Reducing the lignin content modified cellulose crystallinity, improved flax shive fermentation and optimized biogas production. Chemical pretreatment of the shive biomass further increased biogas production efficiency.

Systems pharmacology identifies drug targets for Stargardt disease-associated retinal degeneration.

A systems pharmacological approach that capitalizes on the characterization of intracellular signaling networks can transform our understanding of human diseases and lead to therapy development. Here, we applied this strategy to identify pharmacological targets for the treatment of Stargardt disease, a severe juvenile form of macular degeneration. Diverse GPCRs have previously been implicated in neuronal cell survival, and crosstalk between GPCR signaling pathways represents an unexplored avenue for pharmacological intervention. We focused on this receptor family for potential therapeutic interventions in macular disease. Complete transcriptomes of mouse and human samples were analyzed to assess the expression of GPCRs in the retina. Focusing on adrenergic (AR) and serotonin (5-HT) receptors, we found that adrenoceptor α 2C (Adra2c) and serotonin receptor 2a (Htr2a) were the most highly expressed. Using a mouse model of Stargardt disease, we found that pharmacological interventions that targeted both GPCR signaling pathways and adenylate cyclases (ACs) improved photoreceptor cell survival, preserved photoreceptor function, and attenuated the accumulation of pathological fluorescent deposits in the retina. These findings demonstrate a strategy for the identification of new drug candidates and FDA-approved drugs for the treatment of monogenic and complex diseases.

Amelioration of behavioral abnormalities in BH(4)-deficient mice by dietary supplementation of tyrosine.

This study reports an amelioration of abnormal motor behaviors in tetrahydrobiopterin (BH4)-deficient Spr (-/-) mice by the dietary supplementation of tyrosine. Since BH4 is an essential cofactor for the conversion of phenylalanine into tyrosine as well as the synthesis of dopamine neurotransmitter within the central nervous system, the levels of tyrosine and dopamine were severely reduced in brains of BH4-deficient Spr (-/-) mice. We found that Spr (-/-) mice display variable 'open-field' behaviors, impaired motor functions on the 'rotating rod', and dystonic 'hind-limb clasping'. In this study, we report that these aberrant motor deficits displayed by Spr (-/-) mice were ameliorated by the therapeutic tyrosine diet for 10 days. This study also suggests that dopamine deficiency in brains of Spr (-/-) mice may not be the biological feature of aberrant motor behaviors associated with BH4 deficiency. Brain levels of dopamine (DA) and its metabolites in Spr (-/-) mice were not substantially increased by the dietary tyrosine therapy. However, we found that mTORC1 activity severely suppressed in brains of Spr (-/-) mice fed a normal diet was restored 10 days after feeding the mice the tyrosine diet. The present study proposes that brain mTORC1 signaling pathway is one of the potential targets in understanding abnormal motor behaviors associated with BH4-deficiency.

Sleep and rhythm consequences of a genetically induced loss of serotonin.

BACKGROUND: A genetic deficiency in sepiapterin reductase leads to a combined deficit of serotonin and dopamine. The motor phenotype is characterized by a dopa-responsive fluctuating generalized dystonia-parkinsonism. The non-motor symptoms are poorly recognized. In particular, the effects of brain serotonin deficiency on sleep have not been thoroughly studied. OBJECTIVE: We examine the sleep, sleep-wake rhythms, CSF neurotransmitters, and melatonin profile in a patient with sepiapterin reductase deficiency. PATIENT: The patient was a 28-year-old man with fluctuating generalized dystonia-parkinsonism caused by sepiapterin reductase deficiency. METHODS: A sleep interview, wrist actigraphy, sleep log over 14 days, 48-h continuous sleep and core temperature monitoring, and measurement of CSF neurotransmitters and circadian serum melatonin and cortisol levels before and after treatment with 5-hydroxytryptophan (the precursor of serotonin) and levodopa were performed. RESULTS: Before treatment, the patient had mild hypersomnia with long sleep time (704 min), ultradian sleep-wake rhythm (sleep occurred every 11.8 +/- 5.3 h), organic hyperphagia, attentionlexecutive dysfunction, and no depression. The serotonin metabolism in the CSF was reduced, and the serum melatonin profile was flat, while cortisol and core temperature profiles were normal. Supplementation with 5-hydroxytryptophan, but not with levodopa, normalized serotonin metabolism in the CSF, reduced sleep time to 540 min, normalized the eating disorder and the melatonin profile, restored a circadian sleep-wake rhythm (sleep occurred every 24 +/- 1.7 h, P < 0.0001), and improved cognition. CONCLUSION: In this unique genetic paradigm, the melatonin deficiency (caused by a lack of its substrate, serotonin) may cause the ultradian sleep-wake rhythm.

A murine model for human sepiapterin-reductase deficiency.

Tetrahydrobiopterin (BH(4)) is an essential cofactor for several enzymes, including all three forms of nitric oxide synthases, the three aromatic hydroxylases, and glyceryl-ether mono-oxygenase. A proper level of BH(4) is, therefore, necessary for the metabolism of phenylalanine and the production of nitric oxide, catecholamines, and serotonin. BH(4) deficiency has been shown to be closely associated with diverse neurological psychiatric disorders. Sepiapterin reductase (SPR) is an enzyme that catalyzes the final step of BH(4) biosynthesis. Whereas the number of cases of neuropsychological disorders resulting from deficiencies of other catalytic enzymes involved in BH(4) biosynthesis and metabolism has been increasing, only a handful of cases of SPR deficiency have been reported, and the role of SPR in BH(4) biosynthesis in vivo has been poorly understood. Here, we report that mice deficient in the Spr gene (Spr(-/-)) display disturbed pterin profiles and greatly diminished levels of dopamine, norepinephrine, and serotonin, indicating that SPR is essential for homeostasis of BH(4) and for the normal functions of BH(4)-dependent enzymes. The Spr(-/-) mice exhibit phenylketonuria, dwarfism, and impaired body movement. Oral supplementation of BH(4) and neurotransmitter precursors completely rescued dwarfism and phenylalanine metabolism. The biochemical and behavioral characteristics of Spr(-/-) mice share striking similarities with the symptoms observed in SPR-deficient patients. This Spr mutant strain of mice will be an invaluable resource to elucidate many important issues regarding SPR and BH(4) deficiencies.

Temporal response of hepatic threonine dehydrogenase in chickens to the initial consumption of a threonine-imbalanced diet.

Amino acid imbalances contribute to higher requirements of amino acids than would occur if the dietary profile of amino acids perfectly matched the requirements. The mechanisms of imbalances have not been fully elucidated. Because threonine dehydrogenase (TDH) activity in liver mitochondria increases in chicks and rats subjected to threonine imbalance, the current study was carried out to determine whether the change in TDH activity occurs rapidly enough after the consumption of an imbalanced diet to be considered a possible primary metabolic response. In a series of experiments, Leghorn chicks were allowed free access to a semipurified basal diet marginally limited in threonine or the same diet containing a mixture of indispensable amino acids (IAA) lacking threonine to cause a threonine imbalance. In the first experiment, dietary supplements of 5.5 and 11.1% IAA were used to determine a level of supplement that would cause a robust response in the specific activity of TDH. Feed intake, body weight gains and efficiency of feed utilization were lower and specific activities of TDH were higher in chicks fed 11.1% IAA than in those fed 5.5% IAA. In subsequent experiments, hepatic TDH activities and plasma amino acid profiles of the control and experimental groups were determined at 1. 5, 3, 6, 12 and 24 h after the first offering of the diet containing 11.1% IAA. The specific activities of TDH in chicks fed the IAA supplement were 40-150% higher (P < 0.05) and plasma threonine concentrations were 42-53% lower (P < 0.05) than in chicks fed the basal diet at all times except 1.5 h. These results indicate that changes in the capacity for threonine degradation via TDH may occur in the liver within a few hours after the consumption of a threonine-imbalanced diet and suggest the possibility that altered TDH activity may contribute to the increased threonine requirement associated with threonine imbalance.

Identification of mutations causing 6-pyruvoyl-tetrahydropterin synthase deficiency in four Italian families.

6-Pyruvoyl-tetrahydrobiopterin synthase (PTPS) is involved in tetrahydrobiopterin (BH4) biosynthesis, the cofactor for various enzymes including the hepatic phenylalanine hydroxylase. Inherited PTPS deficiency leads to BH4 depletion, causes hyperphenylalaninemia, and requires cofactor replacement therapy for treatment. We previously isolated the human PTPS cDNA and recently characterized its corresponding gene, PTS. Here we developed PCR-based mutation analysis with newly designed primers to detect genomic alterations and describe five mutations, four of which are novel, in the PTS gene of four Italian families with affected individuals. The mutant alleles found included three missense mutations (T67M, K129E, D136V), a previously described triplet deletion (delta V57), and a single c-3-->g transversion in the 3'-acceptor splice site of intron 1, leading to cryptic splice site usage that resulted in a 12 bp deletion (mutant allele delta (K29-S32)). Except for K129E, all mutant alleles were inactive and/or unstable proteins, as shown by recombinant expression and Western blot analysis of patients' fibroblasts. The PTPS-deficient patient with the homozygous K129E allele had transient hyperphenylalaninemia, did not depend on BH4 replacement therapy, and showed normal PTPS immunoreactivity, but no enzyme activity in primary fibroblasts and red blood cells. In contrast to its inactivity in these cells, the K129E mutant was 2-3 fold more active than wild-type PTPS when transfected into COS-1 or the human hepatoma cell line Hep G2. K129E appears thus as a mutant PTPS whose activity depends on the cell type.

Hyperphenylalaninemia due to defects in tetrahydrobiopterin metabolism: molecular characterization of mutations in 6-pyruvoyl-tetrahydropterin synthase.

A variant type of hyperphenylalaninemia is caused by a deficiency of tetrahydrobiopterin (BH4), the obligatory cofactor for phenylalanine hydroxylase. The most frequent form of this cofactor deficiency is due to lack of 6-pyruvoyl-tetrahydropterin synthase (PTPS) activity, the second enzyme in the biosynthetic pathway for BH4. The human liver cDNA for PTPS was previously isolated, and the recombinant protein was found to be active when expressed in Escherichia coli. We now have investigated two patients for their molecular nature of this autosomal recessive disorder. Both patients were diagnosed as PTPS deficient, one with the central and one with the peripheral form, on the basis of an elevated serum phenylalanine concentration concomitant with lowered levels of urinary biopterin and PTPS activity in erythrocytes. Molecular analysis was performed on the patients' cultured primary skin fibroblasts. PTPS activities were found in vitro to be reduced to background activity. Direct cDNA sequence analysis using reverse transcriptase-PCR technology showed for the patient with the central from a homozygous G-to-A transition at codon 25, causing the replacement of an arginine by glutamine (R25Q). Expression of this mutant allele in E. coli revealed 14% activity when compared with the wild-type enzyme. The patient with the peripheral form exhibited compound heterozygosity, having on one allele a C-to-T transition resulting in the substitution of arginine 16 for cysteine (R16C) in the enzyme and having on the second allele a 14-bp deletion (delta 14bp), leading to a frameshift at lysine 120 and a premature stop codon (K120-->Stop). Heterologous expression of the enzyme with the single-amino-acid exchange R16C revealed only 7% enzyme activity, whereas expression of the deletion allele delta 14bp exhibited no detectable activity. All three mutations, R25Q, R16C, and K120-->Stop, affect evolutionarily conserved residues in PTPS, result in reduced enzymatic activity when reconstituted in E. coli, and are thus believed to be the molecular cause for the BH4 deficiency. This is the first report describing mutations in PTPS that lead to BH4 deficiency.

Tetrahydrobiopterin deficiencies: preliminary analysis from an international survey.

Tetrahydrobiopterin deficiency is a rare cause of hyperphenylalaninemic syndromes. The natural history of the disease is characterized by progressive neurologic illness unresponsive to a phenylalanine-restricted diet. Fifty patients have been reported. From the documented cases, the following statements can be made: (1) An incidence of 2% among hyperphenylalaninemic babies can be reasonably estimated. (2) Most patients have high neonatal blood phenylalanine concentrations, but some have only mild elevations. (3) Among the available diagnostic tests, measurement of urine pteridines should be proposed in all hyperphenylalaninemic babies, (4) The tolerance to dietary phenylalanine is generally high. (5) The results of neurotransmitter replacement therapy are encouraging, but treatment should be started within the first month and requires a strict follow-up protocol. Consequently, in every newborn infant with positive Guthrie test results, a rapid investigation of BH4 metabolism should be accomplished in order to differentiate between phenylalanine-hydroxylase deficiencies (phenylketonuria, mild hyperphenylalaninemia, transient hyperphenylalaninemia) and BH4 deficiencies.