AMP Deaminase Research Articles

Effects of glutamine and alanine supplementation on muscle fatigue parameters of rats submitted to resistance training.

Although glutamine and alanine have properties that could delay fatigue, recent evidence showed that these amino acids impaired central fatigue markers. Nevertheless, the effect of this intervention on muscle fatigue is unknown. The aim of this study was to investigate the effects of glutamine and alanine supplementation on muscle fatigue parameters in rats submitted to resistance training (RT). Wistar rats were distributed into the following groups: sedentary (SED), exercised (CON), exercised and supplemented with alanine (ALA), glutamine and alanine in their free form (G+A) or l-alanyl-l-glutamine (DIP). Trained groups underwent a ladder-climbing exercise for 8 wk. In the last 3 wk of RT, supplementations were offered in water with a 4% concentration. G+A and DIP supplementation increased the muscle content of glutamine and glutamate. DIP administration increased glycogen and lactate dehydrogenase (LDH) concentrations in muscle, whereas ALA and G+A supplementation reduced plasma LDH and creatine kinase levels. All trained groups presented higher levels of muscle glutathione (GSH) than SED. There was no difference between groups in lactate, xanthine, hypoxanthine, thiobarbituric acid reactive substances, 8-isoprostane and GSH in plasma; adenosine monophosphate deaminase, citrate synthase and monocarboxylate transporters 1 and 4 in muscle; and glycogen and GSH in the liver. Moreover, physical performance did not differ between groups. Glutamine and alanine supplementation improved muscle fatigue markers without affecting exercise performance.

Identification of Two Novel Single Nucleotide Polymorphisms in the Promoter Region of the Pig AMP Deaminase 1 Gene Associated with Carcass Traits.

The AMP deaminase 1 (AMPD1) gene plays an important role in purine nucleotide interconversion and energy metabolism. In this study, two novel single nucleotide polymorphisms (SNPs) (g.-626 G > A and g.-566 A>G) were detected in the proximal promoter region of the AMPD1 gene. The Chinese indigenous pig breeds (Meishan and Tongcheng) had higher frequencies of the G and A alleles than Western meat-type breeds (Landrace and Large White) at the g.-626 G > A and g.-566 A>G loci. The transcriptional activity of the AMPD1 promoter carrying the haplotype H1 (A-626G-566) was significantly (p < 0.05) higher than that of the haplotype H2 (G-626A-566). In addition, pigs with the haplotype combination H1H1 had significantly (p < 0.05) higher mRNA expression levels of the AMPD1 gene than those with haplotype combinations H1H2 and H2H2 in two different skeletal muscles. Association analyses revealed that the pigs with the haplotype combination H1H1 had significantly higher lean meat percentage values but lower average backfat thickness (ABT, cm), buttock fat thickness (cm), and thorax-waist fat thickness (cm) values than the pigs with the haplotype combinations H1H2 and H2H2. These findings demonstrate that the two novel SNPs in the promoter region of the AMPD1 gene have significant associations with pig fat deposition traits.

Role of the HPRG Component of Striated Muscle AMP Deaminase in the Stability and Cellular Behaviour of the Enzyme.

Multiple muscle-specific isoforms of the Zn2+ metalloenzyme AMP deaminase (AMPD) have been identified based on their biochemical and genetic differences. Our previous observations suggested that the metal binding protein histidine-proline-rich glycoprotein (HPRG) participates in the assembly and maintenance of skeletal muscle AMP deaminase (AMPD1) by acting as a zinc chaperone. The evidence of a role of millimolar-strength phosphate in stabilizing the AMPD-HPRG complex of both AMPD1 and cardiac AMP deaminase (AMPD3) is suggestive of a physiological mutual dependence between the two subunit components with regard to the stability of the two isoforms of striated muscle AMPD. The observed influence of the HPRG content on the catalytic behavior of the two enzymes further strengthens this hypothesis. Based on the preferential localization of HPRG at the sarcomeric I-band and on the presence of a Zn2+ binding motif in the N-terminal regions of fast TnT and of the AMPD1 catalytic subunit, we advance the hypothesis that the Zn binding properties of HPRG could promote the association of AMPD1 to the thin filament.

Screening of exosomal miRNAs derived from subcutaneous and visceral adipose tissues: Determination of targets for the treatment of obesity and associated metabolic disorders.

Exosomal micro (mi)RNAs have been suggested to have important roles in abdominal obesity, and to be associated with metabolic alterations via posttranscriptional regulation of target genes. However, exosomal miRNA profiles in subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) have rarely been investigated. In the present study, microarray data were obtained from the Gene Expression Omnibus database with the following accession numbers: GSE68885 (exosomal miRNAs in SAT obtained from seven patients with obesity and five lean patients), GSE50574 (exosomal miRNAs in VAT obtained from seven patients with obesity and five lean patients) and GSE29718 [mRNAs in SAT (obtained from seven patients with obesity and eight lean patients) and VAT (obtained from three patients with obesity and two lean patients)]. Differentially expressed (DE)-miRNAs and differentially expressed genes (DEGs) were identified using the Linear Models for Microarray Data method, and mRNA targets of DE-miRNAs were predicted using the miRWalk2.0 database. Potential functions of DE-miRNA target genes were determined using the Database for Annotation, Visualization and Integrated Discovery. As a result, 10 exosomal DE-miRNAs were identified in SAT between patients with obesity and lean patients, while 58 DE-miRNAs were identified in VAT between patients with obesity and lean patients. miRNA (miR)-4517 was revealed to be a downregulated exosomal miRNA between SAT and VAT, while the other DE-miRNAs were SAT-(e.g. hsa-miR-3156-5p and hsa-miR-4460) or VAT-(e.g. hsa-miR-582-5p, hsa-miR-566 and miR-548) specific. Following overlapping with the target genes of DE-miRNAs, only one DEG [cluster of differentiation 86 (CD86)] was identified in SAT samples, whereas 25 DEGs (e.g. fibroblast growth factor 2 (FGF2), FOS like 2, AP-1 transcription factor subunit (FOSL2); and adenosine monophosphate deaminase 3 (AMPD3)] were identified in VAT samples. CD86 was revealed to be regulated by hsa-miR-3156-5p; whereas FGF2, FOSL2 and AMPD3 were revealed to be regulated by hsa-miR-582-5p, hsa-miR-566 and miR-548, respectively. Functional enrichment analysis demonstrated that these target genes may be associated with inflammation. In conclusion, exosomal miRNAs may represent underlying therapeutic targets for the treatment of abdominal obesity and metabolic disorders via regulation of inflammatory genes.

Metabolic homeostasis: oxidative phosphorylation and the metabolic requirements of higher plants and animals.

A model of oxidative phosphorylation and its regulation is presented, which is consistent with the experimental data on metabolism in higher plants and animals. The variables that provide real-time control of metabolic status are: intramitochondrial [NAD+]/[NADH], energy state ([ATP]/[ADP][Pi]), and local oxygen concentration ([O2]). ATP consumption and respiratory chain enzyme content are tissue specific (liver vs. heart muscle), and the latter is modulated by chronic alterations in ATP consumption (i.e., endurance training etc.). ATP consumption affects the energy state, which increases or decreases as necessary to match synthesis with consumption. [NAD+]/[NADH], local [O2], and respiratory chain content determine the energy state at which match of synthesis and utilization is achieved. Tissues vary widely in their ranges of ATP consumption. Expressed as the turnover of cytochrome c, the rates may change little (7 to 12/s) (liver) or a lot (1 to >300/s) (flight muscle of birds, bats, and insects). Ancillary metabolic pathways, including creatine or arginine kinase, glycerol phosphate shuttle, fatty acid, and citric acid cycle dehydrogenases, are responsible for meeting tissue-specific differences in maximal rate and range in ATP utilization without displacing metabolic homeostasis. Intramitochondrial [NAD+]/[NADH], [ATP], and [Pi] are adjusted to keep [ADP] and [AMP] similar for all tissues despite large differences in ranges in ATP utilization. This is essential because [ADP] and [AMP], particularly the latter, have major roles in regulating the activity of many enzymes and signaling pathways (AMP deaminase, AMP dependent protein kinases, etc.) common to all higher plants and animals.NEW & NOTEWORTHY Oxidative phosphorylation has an intrinsic program that sets and stabilizes cellular energy state ([ATP]/[ADP][Pi]), and thereby metabolic homeostasis. A computational model consistent with regulation of oxidative phosphorylation in higher plants and animals is presented. Focus is on metabolism ancillary to oxidative phosphorylation by which it was integrated into preexisting metabolic regulation and adapted by evolution to develop cells and tissues with differing rates of ATP utilization: i.e., liver versus brain versus muscle.

Translational regulation by miR-301b upregulates AMP deaminase in diabetic hearts

AMP deaminase (AMPD) plays a crucial role in adenine nucleotide metabolism. Recently we found that upregulated AMPD activity is associated with ATP depletion and contractile dysfunction under the condition of pressure overloading in the heart of a rat model of type 2 diabetes mellitus (T2DM), OLETF. Here we examined the mechanism of AMPD upregulation by T2DM. The protein level of 90-kDa full-length AMPD3 was increased in whole myocardial lysates by 55% in OLETF compared to those in LETO, a non-diabetic control. In contrast, the mRNA levels of AMPD3 in the myocardium were similar in OLETF and LETO. AMPD3 was comparably ubiquitinated in OLETF and LETO, and its degradation ex vivo was more sensitive to MG-132, a proteasome inhibitor, in OLETF than in LETO. MicroRNA array analysis revealed downregulation (>50%) of 57 microRNAs in OLETF compared to those in LETO, among which miR-301b was predicted to interact with the 3’UTR of AMPD3 mRNA. AMPD3 protein level was significantly increased by a miR-301b inhibitor and was decreased by a miR-301b mimetic in H9c2 cells. A luciferase reporter assay confirmed binding of miR-301b to the 3′UTR of AMPD3 mRNA. Transfection of neonatal rat cardiomyocytes with a miR-301b inhibitor increased 90-kDa AMPD3 and reduced ATP level. The results indicate that translational regulation by miR-301b mediates upregulated expression of cardiac AMPD3 protein in OLETF, which potentially reduces the adenine nucleotide pool at the time of increased work load. The miR-301b-AMPD3 axis may be a novel therapeutic target for intervening enegy metabolism in diabetic hearts.

AMP Deaminase 3 Knockout Does Not Reduce Mitochondrial Content Loss in Denervation Induced Inactivity

Muscle atrophy leads to decrements in muscle function, partly attributable to reductions in mitochondrial content. One controller of mitochondrial content is AMP‐activated protein kinase (AMPK), which when bound by AMP stimulates PGC‐1α leading to mitochondrial biogenesis, keeping mitochondrial content elevated. AMP Deaminase 1 is the dominant isoform of AMPD in skeletal muscle, yet AMP Deaminase 3 (AMPD3) is robustly upregulated during atrophy conditions in skeletal muscle, and degrades AMP (AMP à IMP + NH3). The purpose of this study is to determine if loss of AMPD3 gene will reduce mitochondrial content loss typically seen with atrophy.MethodsWhole body AMPD3 knock out mice on a C57BL6 strain background were surgically denervated on the left hind limb by removal of ~2 mm of the sciatic nerve. A sham surgery was performed on the right hind limb. Two weeks later, mice were weighed, and extensor digitorum longus (EDL) muscles were collected, weighed, and analyzed for citrate synthase enzyme activity as a measure of mitochondrial content. ANOVA analyses were made between three genotypes (Wild Type ‐ WT, AMPD3 heterozygous ‐ HET, and AMPD3 knockout ‐ KO) of 12 week old littermate female mice (n = 5–8).ResultsBody weights were not significantly different among genotypes (22.9 ± 0.7 g WT, 22.3 ± 0.4 HET, 22.0 ± 1.0 KO)(p = .74). Likewise, non‐denervated EDL muscle weights were not significantly different among genotypes (8.75 ± 0.2 mg WT, 8.47 ± 0.2 HET, 8.77 ± 0.3 KO)(p = 0.67). As expected, surgical denervation for two weeks led to substantial muscle atrophy versus innervated muscle (22.07 ± 1.42% WT, 23.69 ± 4.84 HET, 19.2 ± 3.67 KO), which was not different among genotypes (p = 0.68). Finally, citrate synthase activity in innervated EDL was not significantly different among genotypes (10.27 ± 0.36 WT, 9.3 ± 0.77 HET, 8.93 ± 0.38 KO)(p = 0.16). The decrease in citrate synthase activity due to denervation was not significantly different between wild type and heterozygous, but there was a trend for heterozygous group to have less activity (20.32 ± 2.02% WT, 4.38 ± 5.83 HET)(p = 0.059).ConclusionThese data suggest AMPD3 does not affect mitochondrial content loss in this atrophy model. However, since AMPD3 was not present during development in our AMPD3 knockout mice, we cannot exclude compensation from other AMPD isoforms. Therefore, future research should investigate either inducible muscle specific AMPD3 knockouts or loss of both AMPD1 and AMPD3.Support or Funding InformationNIH R01 AR 070200This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

AMP Deaminase 3 Overexpression Reduces Mitochondrial Content in C2C12 Myotubes by Decreasing PGC‐1α Promotor Activation

Skeletal muscles undergoing atrophy have decreased intracellular [ATP], PGC‐1α expression, and mitochondrial content. This combination of findings is unexpected considering that decreased [ATP] is typically associated with increases in [ADP] and [AMP], activation of AMPK and PGC‐1α, and subsequent mitochondrial biogenesis in order to increase energy production. Therefore, it is unclear why atrophying muscles display deficits in PGC‐1α and mitochondrial content despite having decreased [ATP]. A possible explanation is increased activity of the enzyme AMP Deaminase 3 (AMP→IMP+NH3), which is highly upregulated during muscle atrophy. We tested the hypothesis that increased expression of AMPD3 can reduce mitochondrial content in skeletal muscle cells by decreasing the AMPK→ PGC‐1α→ mitochondrial biogenesis intracellular signaling cascade by decreasing [AMP].MethodsC2C12 myotubes were transduced with adenovirus encoding AMPD3 or GFP (control) and allowed to differentiate further for 5 days. Myotubes were then collected and analyzed for nucleotide concentrations (UPLC), protein expression of AMPK, phosphorylated AMPK(Thr172), AMPKα substrates, mitochondrial OXPHOS proteins (Western Blot), 2 Kb PGC‐1α promotor activity (PGC‐1α‐Luciferase assay), and citrate synthase enzyme activity (marker of mitochondrial content).ResultsIncreased AMPD activity was confirmed by the presence of IMP (0.20 μmol/g) in AMPD3 groups compared to undetectable in GFP controls. Overexpression of AMPD3 decreased [ATP] by 32%, [ADP] by 19%, [AMP] by 28%, and the total adenine nucleotide pool + IMP by 31% (p&lt;0.001 for all). Notably, despite the pronounced decrease in [ATP], the [AMP]/[ATP] did not differ between groups. This was reflected by no differences in protein expression of AMPK, pAMPK(Thr172), and AMPKα substrate. However, PGC‐1α‐luciferase activity was significantly lower after 1 and 5 days of AMPD3 overexpression compared to GFP (p&lt;0.0001, p&lt;0.05), suggesting decreased PGC‐1α promotor region activation despite no measurable difference in pAMPK(Thr172). Furthermore, citrate synthase activity was 17% lower in myotubes overexpressing AMPD3 compared to GFP (p=0.01), yet no differences in mitochondrial OXPHOS proteins were detected.Summary/ConclusionsOur results show that increased expression of AMPD3 can decrease adenine nucleotide concentrations without disrupting the relative ATP:ADP:AMP ratios. Consequently, we do not detect measurable differences in AMPK activation. However, we do find significantly lower PGC‐1α promotor activity and citrate synthase activity. These findings indicate that increased expression of AMPD3, as occurs during muscle atrophy, can reduce PGC‐1α activation and mitochondrial biogenesis, ultimately resulting in reduced mitochondrial content. Interestingly, our data suggests that the reduction in PGC‐1α activity occurs independent of AMPK activation. Future studies should be conducted to determine the specific mechanism of PGC‐1α activation along with direct measures of mitochondrial biogenesis, such as, mitochondrial protein synthesis rate.Support or Funding InformationFunded by NIH R01 AR070200This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impact of Metal Toxicity on Oxidative Balance and Mitochondrial Enzyme Function in Muscle of Tilapia.

Present study investigates the effect of metal accumulation on antioxidant level and mitochondrial enzymes function in muscle of Oreochromis mossambicus. Metal accumulation in muscle upregulated stress marker malondialdehyde and the activity of different antioxidant enzymes with no significant alteration in glutathione system. Metal exposure to fish muscle decreased the activity of mitochondrial enzymes. AMP deaminase, aldolase, cytochrome C oxidase and lipoamide reductase showed positive correlation with acetylcholinesterase, glutathione reductase, reduced glutathione and glutathione peroxidase, but negative correlation with superoxide dismutase, catalase, glutathione S-transferase and thiobarbituric acid reactive substance. Analysis of these biomarkers clearly indicates the change in oxidative load in muscle tissues and provides insight to muscle response to the metal exposure. Therefore, the study outlines the potential use of biomarkers in context of muscle mitochondrial enzymes relating to oxidative processes that take place in the fish muscle following metal exposure and toxicity.

Clinical and genetic spectrum of AMPD2-related pontocerebellar hypoplasia type 9.

Pontocerebellar hypoplasia (PCH) represents a group of autosomal-recessive progressive neurodegenerative disorders of prenatal onset. Eleven PCH subtypes are classified according to clinical, neuroimaging and genetic findings. Individuals with PCH type 9 (PCH9) have a unique combination of postnatal microcephaly, hypoplastic cerebellum and pons, and hypoplastic or absent corpus callosum. PCH9 is caused by biallelic variants in AMPD2 encoding adenosine monophosphate deaminase 2; however, a homozygous AMPD2 frameshift variant has recently been reported in two family members with spastic paraplegia type 63 (SPG63). We identified homozygous or compound heterozygous AMPD2 variants in eight PCH-affected individuals from six families. The eight variants likely affect function and comprise one frameshift, one nonsense and six missense variants; seven of which were novel. The main clinical manifestations in the eight new patients and 17 previously reported individuals with biallelic AMPD2 variants were postnatal microcephaly, severe global developmental delay, spasticity, and central visual impairment. Brain imaging data identified hypomyelination, hypoplasia of the cerebellum and pons, atrophy of the cerebral cortex, complete or partial agenesis of the corpus callosum and the "figure 8" shape of the hypoplastic midbrain as consistent features. We broaden the AMPD2-related clinical spectrum by describing one individual without microcephaly and absence of the characteristic "figure 8" shape of the midbrain. The existence of various AMPD2 isoforms with different functions possibly explains the variability in phenotypes associated with AMPD2 variants: variants leaving some of the isoforms intact may cause SPG63, while those affecting all isoforms may result in the severe and early-onset PCH9.

Expression of serum AMPD1 in thyroid carcinoma and its clinical significance.

This study investigated the expression of adenosine monophosphate deaminase 1 (AMPD1) in serum of patients with papillary thyroid carcinoma (PTC) and its clinical significance. The expression levels of AMPD1 mRNA in serum of 157 patients with PTC and 100 normal controls were detected by real-time fluorescent quantitative polymerase chain reaction (PCR), and the relationships between expression level of AMPD1 in serum of PTC patients and clinicopathological factors as well as prognosis were analyzed. The results of real-time fluorescent quantitative PCR showed that the expression of AMPD1 mRNA in serum of PTC patients was lower than that in normal human serum (P<0.01). The expression of AMPD1 in serum of PTC patients was not significantly different from the clinicopathological features such as sex, age, lymph node metastasis and the number of lesions (P>0.05); there were distinct differences between its expression and tumor-node-metastasis (TNM) staging and tumor diameter (P<0.05). The single factor Cox analysis revealed that sex, age, number of lesions, TNM staging and the occurrence of lymph node metastasis were significantly correlated with the prognosis of patients (P<0.05). Multivariate Cox analysis showed that TNM staging hazard ratio (HR)=2.93, 95% confidence interval (CI): 1.52–7.04, P=0.015 was an independent prognostic factor in PTC patients. Survival analysis indicated that there was a statistically significant difference in the 5-year overall survival rate between patients with high expression of AMPD1 and those with low expression (P=0.007). In conclusion, the expression of AMPD1 in serum of patients with PTC is closely related to the malignant evolution of PTC and clinical prognosis of patients. AMPD1 is expected to become an important molecule in judging the clinical prognosis of PTC patients, and may become a new target for molecular targeted therapy of PTC.

Clinical Pharmacogenetic Models of Treatment Response to Methotrexate Monotherapy in Slovenian and Serbian Rheumatoid Arthritis Patients: Differences in Patient's Management May Preclude Generalization of the Models.

Objectives: Methotrexate (MTX) is the first line treatment for rheumatoid arthritis (RA), but nevertheless 30% of patients experience MTX inefficacy. Our aim was to develop a clinical pharmacogenetic model to predict which RA patients will not respond to MTX monotherapy. We also assessed whether this model can be generalized to other populations by validating it on a group of Serbian RA patients.Methods: In 110 RA Slovenian patients, data on clinical factors and 34 polymorphisms in MTX pathway were analyzed by Least Absolute Shrinkage and Selection Operator (LASSO) penalized regression to select variables associated with the disease activity as measured by Disease Activity Score (DAS28) score after 6 months of MTX monotherapy. A clinical pharmacogenetic index was constructed from penalized regression coefficients with absolute value above 0.05. This index was cross-validated and also independently validated on 133 Serbian RA patients.Results: A clinical pharmacogenetic index for prediction of DAS28 after 6 months of MTX monotherapy in Slovenian RA patients consisted of DAS28 score at diagnosis, presence of erosions, MTX dose, Solute Carrier Family 19 Member 1 (SLC19A1) rs1051266, Solute Carrier Organic Anion Transporter Family Member 1B1 (SLCO1B1) rs2306283, Thymidylate Synthase (TYMS), and Adenosine Monophosphate Deaminase 1 (AMPD1) rs17602729. It correctly classified 69% of Slovenian patients as responders or nonresponders and explained 30% of variability in DAS28 after 6 months of MTX monotherapy. Testing for validity in another population showed that it classified correctly only 22.5% of Serbian RA patients.Conclusions: We developed a clinical pharmacogenetic model for DAS28 after 6 months of MTX monotherapy in Slovenian RA patients by combining clinical and genetic variables. The clinical pharmacogenetic index developed for Slovenian patients did not perform well on Serbian patients, presumably due to the differences in patients' characteristics and clinical management between the two groups.

Competitive cation binding computations of proton balance for reactions of the phosphagen and glycolytic energy systems within skeletal muscle.

Limited research and data has been published for the H+ coefficients for the metabolites and reactions involved in non-mitochondrial energy metabolism. The purpose of this investigation was to compute the fractional binding of H+, K+, Na+ and Mg2+ to 21 metabolites of skeletal muscle non-mitochondrial energy metabolism, resulting in 104 different metabolite-cation complexes. Fractional binding of H+ to these metabolite-cation complexes were applied to 17 reactions of skeletal muscle non-mitochondrial energy metabolism, and 8 conditions of the glycolytic pathway based on the source of substrate (glycogen vs. glucose), completeness of glycolytic flux, and the end-point of pyruvate vs. lactate. For pH conditions of 6.0 and 7.0, respectively, H+ coefficients (-‘ve values = H+ release) for the creatine kinase, adenylate kinase, AMP deaminase and ATPase reactions were 0.8 and 0.97, -0.13 and -0.02, 1.2 and 1.09, and -0.01 and -0.66, respectively. The glycolytic pathway is net H+ releasing, regardless of lactate production, which consumes 1 H+. For glycolysis fueled by glycogen and ending in either pyruvate or lactate, H+ coefficients for pH 6.0 and 7.0 were -3.97 and -2.01 (pyruvate), and -1.96 and -0.01 (lactate), respectively. When starting with glucose, the same conditions result in H+ coefficients of -3.98 and -2.67, and -1.97 and –0.67, respectively. The most H+ releasing reaction of glycolysis is the glyceraldehyde-3-phosphate dehydrogenase reaction, with H+ coefficients for pH 6.0 and 7.0 of -1.58 and -0.76, respectively. Incomplete flux of substrate through glycolysis would increase net H+ release due to the absence of the pyruvate kinase and lactate dehydrogenase reactions, which collectively result in H+ coefficients for pH 6.0 and 7.0 of 1.35 and 1.88, respectively. The data presented provide an extensive reference source for academics and researchers to accurately profile the balance of protons for all metabolites and reactions of non-mitochondrial energy metabolism, and reveal the greater role of glycolysis in net H+ release than previously assumed. The data can also be used to improve the understanding of the cause of metabolic acidosis, and reveal mechanistic connections between H+ release within and from muscle and the electrochemical neutrality concepts that further refine acid-base balance in biological solutions.

High-pressure effects on the mechanism of accumulated inosine 5′-monophosphate

Abstract This study was conducted to determine whether the application of ultrahigh pressure could modify the content of inosine 5′-monophosphate (IMP) in fresh pork and give direct detection of each relevant enzymatic activity to affirm the enzymatic mechanism. Pressure (0–400 MPa) and time (10 min) were applied to two types of samples: adenosine monophosphate deaminase, acid phosphatase, and alkaline phosphatase in normal saline and IMP in pork tenderloin. The results showed that varied degrees of susceptibility depended upon the pressure level. The pressure of 300 MPa could promote the formation of IMP to 1250%, at which the activity of adenosine monophosphate deaminase, an enzyme catalyzing adenosine monophosphate (AMP) to IMP, was maximized. In contrast, the activities of acid phosphatase and alkaline phosphatase were suppressed at this extreme pressure, resulting in decomposition of IMP. Finally, IMP accumulated to its maximum level at 300 MPa.

Effects of AMPD1 common mutation on the metabolic-chronotropic relationship: Insights from patients with myoadenylate deaminase deficiency.

PurposeCurrent evidence indicates that the common AMPD1 gene variant is associated with improved survival in patients with advanced heart failure. Whilst adenosine has been recognized to mediate the cardioprotective effect of C34T AMPD1, the precise pathophysiologic mechanism involved remains undefined to date. To address this issue, we used cardio-pulmonary exercise testing data (CPX) from subjects with myoadenylate deaminase (MAD) defects.MethodsFrom 2009 to 2013, all the patients referred in our laboratory to perform a metabolic exercise testing, i.e. a CPX with measurements of muscle metabolites in plasma during and after exercise testing, were prospectively enrolled. Subjects that also underwent an open muscle biopsy for diagnosis purpose were finally included. The metabolic-chronotropic response was assessed by calculating the slope of the linear relationship between the percent heart rate reserve and the percent metabolic reserve throughout exercise. MAD activity was measured using the Fishbein’s technique in muscle biopsy sample. The common AMPD1 mutation was genotyped and the AMPD1 gene was sequenced to screen rare variants from blood DNA.ResultsSixty-seven patients were included in the study; 5 had complete MAD deficiency, 11 had partial MAD deficiency, and 51 had normal MAD activity. Compared with normal MAD activity subjects, MAD deficient subjects appeared to have a lower-than-expected metabolic-chronotopic response during exercise. The metabolic-chronotropic relationship is more closely correlated with MAD activity in skeletal muscle (Rs = 0.57, p = 5.93E-7, Spearman correlation) than the presence of the common AMPD1 gene variant (Rs = 0.34, p = 0.005). Age-predicted O2 pulse ratio is significantly increased in MAD deficient subjects, indicating a greater efficiency of the cardiovascular system to deliver O2 (p < 0.01, Scheffé’s post hoc test).ConclusionThe metabolic-chronotropic response is decreased in skeletal muscle MAD deficiency, suggesting a biological mechanism by which AMPD1 gene exerts cardiac effect.

The State of the Adenyl Nucleotide System in the Liver of Rats with Toxic Hepatitis under Conditions of Protein Deficiency

Adenyl nucleotide levels and activity of AMP catabolism enzymes in the cytosolic liver fraction of rats with acetaminophen-induced hepatitis have been studied under different dietary protein regimens. It was found that in animals with toxic hepatitis maintained on a diet rich in protein the ATP and ADP levels decreased, while AMP levels were similar to those in control animals. At the same time, in the cytosolic liver fraction of rats with acetaminophen-induced hepatitis kept under conditions of protein deficiency, ATP and AMP pools were depleted. Changes in the adenyl nucleotides content were accompanied by altered activity of AMP catabolism enzymes, such as 5′-nucleotidase and AMP deaminase. It was found that in rats with toxic hepatitis that were fed a complete diet, AMP deaminase activity increased in comparison to the control level along with 5′-nucleotidase stimulation. At the same time, in protein-restricted rats with toxic liver damage, AMP deaminase activity decreased, while 5′-nucleotidase activity was elevated in comparison to control values. These results indicate depletion of energy sources in the liver cells of rats with acetaminophen-induced hepatitis that were fed a low-protein diet. The observed changes in the activity of AMP catabolism enzymes may be considered as one of the mechanisms that regulate the cellular energy function.

Hypoxia modulates the purine salvage pathway and decreases red blood cell and supernatant levels of hypoxanthine during refrigerated storage.

Hypoxanthine catabolism in vivo is potentially dangerous as it fuels production of urate and, most importantly, hydrogen peroxide. However, it is unclear whether accumulation of intracellular and supernatant hypoxanthine in stored red blood cell units is clinically relevant for transfused recipients. Leukoreduced red blood cells from glucose-6-phosphate dehydrogenase-normal or -deficient human volunteers were stored in AS-3 under normoxic, hyperoxic, or hypoxic conditions (with oxygen saturation ranging from <3% to >95%). Red blood cells from healthy human volunteers were also collected at sea level or after 1–7 days at high altitude (>5000 m). Finally, C57BL/6J mouse red blood cells were incubated in vitro with 13C1-aspartate or 13C5-adenosine under normoxic or hypoxic conditions, with or without deoxycoformycin, a purine deaminase inhibitor. Metabolomics analyses were performed on human and mouse red blood cells stored for up to 42 or 14 days, respectively, and correlated with 24 h post-transfusion red blood cell recovery. Hypoxanthine increased in stored red blood cell units as a function of oxygen levels. Stored red blood cells from human glucose-6-phosphate dehydrogenase-deficient donors had higher levels of deaminated purines. Hypoxia in vitro and in vivo decreased purine oxidation and enhanced purine salvage reactions in human and mouse red blood cells, which was partly explained by decreased adenosine monophosphate deaminase activity. In addition, hypoxanthine levels negatively correlated with post-transfusion red blood cell recovery in mice and – preliminarily albeit significantly - in humans. In conclusion, hypoxanthine is an in vitro metabolic marker of the red blood cell storage lesion that negatively correlates with post-transfusion recovery in vivo. Storage-dependent hypoxanthine accumulation is ameliorated by hypoxia-induced decreases in purine deamination reaction rates.

Enhanced pyruvate production in Candida glabrata by overexpressing the CgAMD1 gene to improve acid tolerance.

To enhance acid tolerance of Candida glabrata for pyruvate production by engineering AMP metabolism. The physiological function of AMP deaminase in AMP metabolism from C. glabrata was investigated by deleting or overexpresseing the corresponding gene, CgAMD1. At pH 4, CgAMD1 overexpression resulted in 59 and 51% increases in biomass and cell viability compared to those of wild type strain, respectively. In addition, the intracellular ATP level of strain Cgamd1Δ/CgAMD1 was down-regulated by 22%, which led to a 94% increase in pyruvate production. Further, various strengths of CgAMD1 expression cassettes were designed, thus resulting in a 59% increase in pyruvate production at pH 4. Strain Cgamd1Δ/CgAMD1 (H) was grown in a 30l batch bioreactor at pH 4, and pyruvate reached 46.1g/l. CgAMD1 overexpression plays an active role in improving acid tolerance and pyruvate fermentation performance of C. glabrata at pH 4.

Teaching Neuro Images : Figure of 8

A 2-year-old girl, born after uneventful pregnancy from healthy nonconsanguineous parents, presented with failure to thrive, microcephaly, facial dysmorphism, strabismus, nystagmus, axial hypotonia, lower limb spasticity/hyperreflexia, dystonic movements, and no active postures. Since the first months of life, developmental delay without any motor skills acquisition, drug-resistant epilepsy, and progressive spasticity had emerged. Neuroimaging revealed pontocerebellar hypoplasia with the figure of 8 midbrain appearance (figure), a distinctive sign for PCH8 or AMPD2 deficiency.1,2 Sanger sequencing of the adenosine monophosphate deaminase 2 enzyme gene ( AMPD2 ) revealed the first compound heterozygous mutation (c.1664C>T; c.1112+1 G>A). The patient died at age 3 years due to respiratory insufficiency.

O22-3 - Upregulation of AMP Deaminase in the Vicinity of the SR via Translational Regulation by miR-301b Contributes to Diabetic Cardiomyopathy

Background: We recently reported that ATP loss by upregulated activity of AMP deaminase (AMPD) contributes to afterload-induced diastolic dysfunction in OLETF, type 2 diabetic rats. Here we examined the mechanism underlying the upregulation of AMPD. Methods and Results: Western blot analysis revealed that protein level of 90-kDa full-length AMPD3 was significantly increased in the sarcoplasmic reticulum (SR) fraction (117%) as well as in whole cell lysates (171%) in OLETF compared to its level in LETO, non-diabetic control rats. Levels of AMPD3 mRNA determined by RT-PCR was comparable in OLETF and LETO. Inhibitory effect of MG-132, a proteasome inhibitor, on degradation of the 90-kDa AMPD3 ex vivo was larger in OLETF than in LETO, arguing against suppressed degradation of AMPD3 in OLETF. MicroRNA array analysis revealed downregulation (>50%) of 57 microRNAs in OLETF compared to those in LETO. Transfection of H9c2 cells with an inhibitor of miR-301b, one of the 57 microRNAs, significantly increased level of the 90-kDa AMPD3. Binding of miR-301b to the 3'UTR of AMPD3 mRNA was confirmed by a luciferase reporter assay. Conclusion: Up-regulated AMPD3 translation by reduced miR-301b mediates increased AMPD3 in the diabetic heart. Since ATP generated by SR-associated glycolytic enzymes contributes to Ca2+ uptake by SERCA2a and promote diastolic relaxation, AMPD3 in the vicinity of the SR is a therapeutic target for diabetic cardiomyopathy.