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Background. Excessive consumption of sucrose or protein deficiency in the diet can induce metabolic disorders in the kidney, whose functioning requires significant ATP energy expenditure. The study investigated the levels of the purine nucleotides ATP, ADP, AMP, and the activity of the enzymes FoF1-ATPase, 5′-nucleotidase, and AMP deaminase in the kidneys of rats exposed to different levels of protein and sucrose in their diet. Materials and Methods. The research was conducted on white non-linear rats, which were kept under different dietary regimens for a period of 4 weeks. Quantitative evaluation of the ATP, ADP, and AMP content was performed by thin-layer chromatography on Silufol sheets. FoF1-ATPase activity was determined by the accumulation of Pi. 5′-nucleotidase activity was measured based on the amount of inorganic phosphorus released in AMP hydrolysis. AMP deaminase activity was determined by the accumulation of ammonia. Results and Discussion. Research results revealed that in the mitochondria of the animals’ kidneys under conditions of low-protein diet, a significant reduction in AMP content was observed compared to the control, while ATP and ADP content remained unchanged. Simultaneously, the activities of 5′-nucleotidase, AMP deaminase, and FoF1-ATPase in the kidneys of animals on a low-protein diet were maintained at control levels. However, in rats maintained on a low-protein/high-sucrose diet, depletion of all adenine nucleotides is observed against an increase in the hydrolytic activity of FoF1-ATPase, AMP deaminase, and 5′-nucleotidase activities. The 5′-nucleotidase activity in animals of this group reaches maximum values in comparison with the control, indica­ting an enhanced AMP degradation mediated by 5′-nucleotidase in the conditions of low-protein/high-sucrose diet consumption. Conclusion. Excessive sucrose consumption in the context of dietary protein deficiency is accompanied by a depletion of the adenine nucleotides pool in the mitochondrial fraction and a significantly increased activity of purine catabolism enzymes in the cytosolic fraction of rat kidneys. This may result in an imbalance in the energy supply of renal cells. The obtained results open up prospects for developing a strategy for correc­ting energy metabolism disorders in the conditions of nutritional imbalance.

The food enzyme AMP deaminase (AMP aminohydrolase; EC 3.5.4.6) is produced with the non‐genetically modified microorganism Aspergillus sp. strain DEA 56‐111 by Shin Nihon Chemical Co., Ltd. The food enzyme was considered free from viable cells of the production organism. It is intended to be used in the processing of yeast and yeast products. Dietary exposure to the food enzyme‐total organic solids (TOS) was estimated to be up to 0.005 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The Panel identified a no observed adverse effect level of 1984 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 396,800. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use. [ABSTRACT FROM AUTHOR]

The N-terminal region of troponin T (TnT) does not bind any protein of the contractile machinery and the role of its hypervariability remains uncertain. In this review we report the evidence of the interaction between TnT and AMP deaminase (AMPD), a regulated zinc enzyme localized on the myofibril. In periods of intense muscular activity, a decrease in the ATP/ADP ratio, together with a decrease in the tissue pH, is the stimulus for the activation of the enzyme that deaminating AMP to IMP and NH3 displaces the myokinase reaction towards the formation of ATP. In skeletal muscle subjected to strong tetanic contractions, a calpain-like proteolytic activity produces the removal in vivo of a 97-residue N-terminal fragment from the enzyme that becomes desensitized towards the inhibition by ATP, leading to an unrestrained production of NH3. When a 95-residue N-terminal fragment is removed from AMPD by trypsin, simulating in vitro the calpain action, rabbit fast TnT or its phosphorylated 50-residue N-terminal peptide binds AMPD restoring the inhibition by ATP. Taking in consideration that the N-terminus of TnT expressed in human as well as rabbit white muscle contains a zinc-binding motif, we suggest that TnT might mimic the regulatory action of the inhibitory N-terminal domain of AMPD due to the presence of a zinc ion connecting the N-terminal and C-terminal regions of the enzyme, indicating that the two proteins might physiologically associate to modulate muscle contraction and ammonia production in fast-twitching muscle under strenuous conditions. [ABSTRACT FROM AUTHOR]

Abstract The food enzyme AMP deaminase (AMP aminohydrolase; EC 3.5.4.6) is produced with the non‐genetically modified microorganism Aspergillus sp. strain DEA 56‐111 by Shin Nihon Chemical Co., Ltd. The food enzyme was considered free from viable cells of the production organism. It is intended to be used in the processing of yeast and yeast products. Dietary exposure to the food enzyme‐total organic solids (TOS) was estimated to be up to 0.005 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The Panel identified a no observed adverse effect level of 1984 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 396,800. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

The loss of cognitive function in Alzheimer's disease is pathologically linked with neurofibrillary tangles, amyloid deposition, and loss of neuronal communication. Cerebral insulin resistance and mitochondrial dysfunction have emerged as important contributors to pathogenesis supporting our hypothesis that cerebral fructose metabolism is a key initiating pathway for Alzheimer's disease. Fructose is unique among nutrients because it activates a survival pathway to protect animals from starvation by lowering energy in cells in association with adenosine monophosphate degradation to uric acid. The fall in energy from fructose metabolism stimulates foraging and food intake while reducing energy and oxygen needs by decreasing mitochondrial function, stimulating glycolysis, and inducing insulin resistance. When fructose metabolism is overactivated systemically, such as from excessive fructose intake, this can lead to obesity and diabetes. Herein, we present evidence that Alzheimer's disease may be driven by overactivation of cerebral fructose metabolism, in which the source of fructose is largely from endogenous production in the brain. Thus, the reduction in mitochondrial energy production is hampered by neuronal glycolysis that is inadequate, resulting in progressive loss of cerebral energy levels required for neurons to remain functional and viable. In essence, we propose that Alzheimer's disease is a modern disease driven by changes in dietary lifestyle in which fructose can disrupt cerebral metabolism and neuronal function. Inhibition of intracerebral fructose metabolism could provide a novel way to prevent and treat this disease.

The food enzyme AMP deaminase (AMP aminohydrolase; EC 3.5.4.6) is produced with the non‐genetically modified Streptomyces murinus strain AE‐DNTS by Amano Enzyme Inc. The food enzyme is free from viable cells. It is intended to be used in yeast processing and the production of mushroom extracts. Dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 0.0004 mg TOS/kg body weight (bw) per day in European populations. The food enzyme batches, including the batch used in the toxicological studies, were not fully characterised. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. In the absence of adequate toxicological data, the Panel could not conclude on the safety of the food enzyme AMP deaminase from the non‐genetically modified Streptomyces murinus strain AE‐DNTS. [ABSTRACT FROM AUTHOR]

Systemic branched‐chain amino acid (BCAA) metabolism is dysregulated in cardiometabolic diseases. We previously demonstrated that upregulated AMP deaminase 3 (AMPD3) impairs cardiac energetics in a rat model of obese type 2 diabetes, Otsuka Long‐Evans‐Tokushima fatty (OLETF). Here, we hypothesized that the cardiac BCAA levels and the activity of branched‐chain α‐keto acid dehydrogenase (BCKDH), a rate‐limiting enzyme in BCAA metabolism, are altered by type 2 diabetes (T2DM), and that upregulated AMPD3 expression is involved in the alteration. Performing proteomic analysis combined with immunoblotting, we discovered that BCKDH localizes not only to mitochondria but also to the endoplasmic reticulum (ER), where it interacts with AMPD3. Knocking down AMPD3 in neonatal rat cardiomyocytes (NRCMs) increased BCKDH activity, suggesting that AMPD3 negatively regulates BCKDH. Compared with control rats (Long‐Evans Tokushima Otsuka [LETO] rats), OLETF rats exhibited 49% higher cardiac BCAA levels and 49% lower BCKDH activity. In the cardiac ER of the OLETF rats, BCKDH‐E1α subunit expression was downregulated, while AMPD3 expression was upregulated, resulting in an 80% lower AMPD3‐E1α interaction compared to LETO rats. Knocking down E1α expression in NRCMs upregulated AMPD3 expression and recapitulated the imbalanced AMPD3‐BCKDH expressions observed in OLETF rat hearts. E1α knockdown in NRCMs inhibited glucose oxidation in response to insulin, palmitate oxidation, and lipid droplet biogenesis under oleate loading. Collectively, these data revealed previously unrecognized extramitochondrial localization of BCKDH in the heart and its reciprocal regulation with AMPD3 and imbalanced AMPD3‐BCKDH interactions in OLETF. Downregulation of BCKDH in cardiomyocytes induced profound metabolic changes that are observed in OLETF hearts, providing insight into mechanisms contributing to the development of diabetic cardiomyopathy. [ABSTRACT FROM AUTHOR]

Abstract The food enzyme AMP deaminase (AMP aminohydrolase; EC 3.5.4.6) is produced with the non‐genetically modified Streptomyces murinus strain AE‐DNTS by Amano Enzyme Inc. The food enzyme is free from viable cells. It is intended to be used in yeast processing and the production of mushroom extracts. Dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 0.0004 mg TOS/kg body weight (bw) per day in European populations. The food enzyme batches, including the batch used in the toxicological studies, were not fully characterised. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. In the absence of adequate toxicological data, the Panel could not conclude on the safety of the food enzyme AMP deaminase from the non‐genetically modified Streptomyces murinus strain AE‐DNTS.

Abstract Systemic branched‐chain amino acid (BCAA) metabolism is dysregulated in cardiometabolic diseases. We previously demonstrated that upregulated AMP deaminase 3 (AMPD3) impairs cardiac energetics in a rat model of obese type 2 diabetes, Otsuka Long‐Evans‐Tokushima fatty (OLETF). Here, we hypothesized that the cardiac BCAA levels and the activity of branched‐chain α‐keto acid dehydrogenase (BCKDH), a rate‐limiting enzyme in BCAA metabolism, are altered by type 2 diabetes (T2DM), and that upregulated AMPD3 expression is involved in the alteration. Performing proteomic analysis combined with immunoblotting, we discovered that BCKDH localizes not only to mitochondria but also to the endoplasmic reticulum (ER), where it interacts with AMPD3. Knocking down AMPD3 in neonatal rat cardiomyocytes (NRCMs) increased BCKDH activity, suggesting that AMPD3 negatively regulates BCKDH. Compared with control rats (Long‐Evans Tokushima Otsuka [LETO] rats), OLETF rats exhibited 49% higher cardiac BCAA levels and 49% lower BCKDH activity. In the cardiac ER of the OLETF rats, BCKDH‐E1α subunit expression was downregulated, while AMPD3 expression was upregulated, resulting in an 80% lower AMPD3‐E1α interaction compared to LETO rats. Knocking down E1α expression in NRCMs upregulated AMPD3 expression and recapitulated the imbalanced AMPD3‐BCKDH expressions observed in OLETF rat hearts. E1α knockdown in NRCMs inhibited glucose oxidation in response to insulin, palmitate oxidation, and lipid droplet biogenesis under oleate loading. Collectively, these data revealed previously unrecognized extramitochondrial localization of BCKDH in the heart and its reciprocal regulation with AMPD3 and imbalanced AMPD3‐BCKDH interactions in OLETF. Downregulation of BCKDH in cardiomyocytes induced profound metabolic changes that are observed in OLETF hearts, providing insight into mechanisms contributing to the development of diabetic cardiomyopathy.

AMP deaminase 1 (AMPD1; AMP → IMP + NH3) deficiency in skeletal muscle results in an inordinate accumulation of AMP during strenuous exercise, with some but not all studies reporting premature fatigue and reduced work capacity. To further explore these inconsistencies, we investigated the extent to which AMPD1 deficiency impacts skeletal muscle contractile function of different muscles and the [AMP]/AMPK responses to different intensities of fatiguing contractions. To reduce AMPD1 protein, we electroporated either an inhibitory AMPD1-specific miRNA encoding plasmid or a control plasmid, into contralateral EDL and SOL muscles of C57BL/6J mice (n = 48 males, 24 females). After 10 days, isolated muscles were assessed for isometric twitch, tetanic, and repeated fatiguing contraction characteristics using one of four (None, LOW, MOD, and HIGH) duty cycles. AMPD1 knockdown (~35%) had no effect on twitch force or twitch contraction/relaxation kinetics. However, during maximal tetanic contractions, AMPD1 knockdown impaired both time-to-peak tension (TPT) and half-relaxation time (1/2 RT) in EDL, but not SOL muscle. In addition, AMPD1 knockdown in EDL exaggerated the AMP response to contractions at LOW (+100%) and MOD (+54%) duty cycles, but not at HIGH duty cycle. This accumulation of AMP was accompanied by increased AMPK phosphorylation (Thr-172; LOW +25%, MOD +34%) and downstream substrate phosphorylation (LOW +15%, MOD +17%). These responses to AMPD1 knockdown were not different between males and females. Our findings demonstrate that AMPD1 plays a role in maintaining skeletal muscle contractile function and regulating the energetic responses associated with repeated contractions in a muscle- but not sex-specific manner.NEW & NOTEWORTHY AMP deaminase 1 (AMPD1) deficiency has been associated with premature muscle fatigue and reduced work capacity, but this finding has been inconsistent. Herein, we report that although AMPD1 knockdown in mouse skeletal muscle does not change maximal isometric force, it negatively impacts muscle function by slowing contraction and relaxation kinetics in EDL muscle but not SOL muscle. Furthermore, AMPD1 knockdown differentially affects the [AMP]/AMPK responses to fatiguing contractions in an intensity-dependent manner in EDL muscle. [ABSTRACT FROM AUTHOR]

We previously reported that upregulated AMP deaminase (AMPD) contributes to diastolic ventricular dysfunction via depletion of the adenine nucleotide pool in a rat model of type 2 diabetes (T2DM), Otsuka Long-Evans-Tokushima Fatty rats (OLETF). Meanwhile, AMPD promotes the formation of substrates of xanthine oxidoreductase (XOR), which produces ROS as a byproduct. Here, we tested the hypothesis that a functional link between upregulated AMPD and XOR is involved in ventricular dysfunction in T2DM rats. Pressure-volume loop analysis revealed that pressure overloading by phenylephrine infusion induced severer left ventricular diastolic dysfunction (tau: 14.7 ± 0.8 vs 12.5 ± 0.7 msec, left ventricular end-diastolic pressure: 18.3 ± 1.5 vs 12.2 ± 1.3 mmHg, p < 0.05) and ventricular-arterial uncoupling in OLETF than in LETO, non-diabetic rats, though the baseline parameters were comparable in the two groups. While the pressure overload did not affect AMPD activity, it increased XOR activity both in OLETF and LETO, with OLETF showing significantly higher XOR activity than that in LETO (347.2 ± 17.9 vs 243.2 ± 6.1 μg/min/mg). Under the condition of pressure overload, myocardial ATP level was lower, and levels of xanthine and uric acid were higher in OLETF than in LETO. Addition of exogenous inosine, a product of AMP deamination, to the heart homogenates augmented XOR activity. OLETF showed 68% higher tissue ROS levels and 47% reduction in mitochondrial state 3 respiration compared with those in LETO. Overexpression of AMPD3 in H9c2 cells elevated levels of hypoxanthine and ROS and reduced the level of ATP. Inhibition of XOR suppressed the production of tissue ROS and mitochondrial dysfunction and improved ventricular function under the condition of pressure overload in OLETF. The results suggest that increases in the activity of XOR and the formation of XOR substrates by upregulated AMPD contribute to ROS-mediated diastolic ventricular dysfunction at the time of increased cardiac workload in diabetic hearts. [Display omitted] • AMPD is upregulated in type 2 diabetic (T2DM) hearts. • Pressure overload unmasked latent diastolic dysfunction in T2DM hearts. • Activity and substrates of XOR were increased in pressure-overloaded T2DM hearts. • Upregulated AMPD leads to ATP depletion and ROS production. • Inhibition of XOR restored mitochondrial and ventricular functions in T2DM hearts. [ABSTRACT FROM AUTHOR]

Summary: Ascospores generated during sexual reproduction are the primary inoculum for the wheat scab fungus Fusarium graminearum. Purine metabolism is known to play important roles in fungal pathogens but its lifecycle stage‐specific regulation is unclear.By characterizing the genes involved in purine de novo and salvage biosynthesis pathways, we showed that de novo syntheses of inosine, adenosine and guanosine monophosphates (IMP, AMP and GMP) are important for vegetative growth, sexual/asexual reproduction, and infectious growth, whereas purine salvage synthesis is dispensable for these stages in F. graminearum.Addition of GMP rescued the defects of the Fgimd1 mutant in vegetative growth and conidiation but not sexual reproduction, whereas addition of AMP rescued all of these defects of the Fgade12 mutant, suggesting that the function of de novo synthesis of GMP rather than AMP is distinct in sexual stages. Moreover, Acd1, an ortholog of AMP deaminase, is dispensable for growth but essential for ascosporogenesis and pathogenesis, suggesting that AMP catabolism has stage‐specific functions during sexual reproduction and infectious growth. The expression of almost all the genes involved in de novo purine synthesis is downregulated during sexual reproduction and infectious growth relative to vegetative growth.This study revealed that F. graminearum has stage‐specific regulation of purine metabolism during infectious growth and sexual reproduction. [ABSTRACT FROM AUTHOR]

Lipid biosynthesis is a significant metabolic response to nitrogen starvation in oleaginous fungi. The oleaginous fungus Mucor circinelloides copes with nitrogen stress by degrading AMP through AMP deaminase (AMPD). However, the mechanism of AMPD in regulating lipogenesis remains largely unclear. To elucidate the mechanism of AMPD in lipid synthesis in this M. circinelloides , we identified two genes (ampd1 and ampd2) encoding AMPD and constructed an ampd double knockout mutant. The engineered M. circinelloides strain elevated cell growth and lipid accumulation, as well as the content of oleic acid (OA) and gamma-linolenic acid (GLA). In addition to the expected increase in transcription levels of genes associated with lipid and TAG synthesis, we observed suppression of lipid degradation and reduced amino acid biosynthesis. This suggested that the deletion of AMPD genes induces the redirection of carbon towards lipid synthesis pathways. Moreover, the pathways related to nitrogen metabolism, including nitrogen assimilation and purine metabolism (especially energy level), were also affected in order to maintain homeostasis. Further analysis discovered that the transcription factors (TFs) related to lipid accumulation were also regulated. This study provides new insights into lipid biosynthesis in M. circinelloides , indicating that the trigger for lipid accumulation is not entirely AMPD-dependent and suggest that there may be additional mechanisms involved in the initiation of lipogenesis. [Display omitted] • Deletion of ampd genes increases lipogenesis in Mucor circinelloides. • AMPD plays an essential role in the regulation of lipid and nitrogen metabolism. • Deletion of ampd genes regulates energy metabolism to maintain homeostasis. • TFs involved in lipogenesis are regulated after knockout of ampd genes. [ABSTRACT FROM AUTHOR]

Because metabolism is a complex balanced process involving multiple enzymes, understanding how organisms compensate for transient or permanent metabolic imbalance is a challenging task that can be more easily achieved in simpler unicellular organisms. The metabolic balance results not only from the combination of individual enzymatic properties, regulation of enzyme abundance, but also from the architecture of the metabolic network offering multiple interconversion alternatives. Although metabolic networks are generally highly resilient to perturbations, metabolic imbalance resulting from enzymatic defect and specific environmental conditions can be designed experimentally and studied. Starting with a double amd1 aah1 mutant that severely and conditionally affects yeast growth, we carefully characterized the metabolic shuffle associated with this defect. We established that the GTP decrease resulting in an adenylic/guanylic nucleotide imbalance was responsible for the growth defect. Identification of several gene dosage suppressors revealed that TAT1, encoding an amino acid transporter, is a robust suppressor of the amd1 aah1 growth defect. We show that TAT1 suppression occurs through replenishment of the GTP pool in a process requiring the histidine biosynthesis pathway. Importantly, we establish that a tat1 mutant exhibits synthetic sickness when combined with an amd1 mutant and that both components of this synthetic phenotype can be suppressed by specific gene dosage suppressors. Together our data point to a strong phenotypic connection between amino acid uptake and GTP synthesis, a connection that could open perspectives for future treatment of related human defects, previously reported as etiologically highly conserved. [ABSTRACT FROM AUTHOR]

The loss of cognitive function in Alzheimer’s disease is pathologically linked with neurofibrillary tangles, amyloid deposition, and loss of neuronal communication. Cerebral insulin resistance and mitochondrial dysfunction have emerged as important contributors to pathogenesis supporting our hypothesis that cerebral fructose metabolism is a key initiating pathway for Alzheimer’s disease. Fructose is unique among nutrients because it activates a survival pathway to protect animals from starvation by lowering energy in cells in association with adenosine monophosphate degradation to uric acid. The fall in energy from fructose metabolism stimulates foraging and food intake while reducing energy and oxygen needs by decreasing mitochondrial function, stimulating glycolysis, and inducing insulin resistance. When fructose metabolism is overactivated systemically, such as from excessive fructose intake, this can lead to obesity and diabetes. Herein, we present evidence that Alzheimer’s disease may be driven by overactivation of cerebral fructose metabolism, in which the source of fructose is largely from endogenous production in the brain. Thus, the reduction in mitochondrial energy production is hampered by neuronal glycolysis that is inadequate, resulting in progressive loss of cerebral energy levels required for neurons to remain functional and viable. In essence, we propose that Alzheimer’s disease is a modern disease driven by changes in dietary lifestyle in which fructose can disrupt cerebral metabolism and neuronal function. Inhibition of intracerebral fructose metabolism could provide a novel way to prevent and treat this disease.

The loss of cognitive function in Alzheimer's disease is pathologically linked with neurofibrillary tangles, amyloid deposition, and loss of neuronal communication. Cerebral insulin resistance and mitochondrial dysfunction have emerged as important contributors to pathogenesis supporting our hypothesis that cerebral fructose metabolism is a key initiating pathway for Alzheimer's disease. Fructose is unique among nutrients because it activates a survival pathway to protect animals from starvation by lowering energy in cells in association with adenosine monophosphate degradation to uric acid. The fall in energy from fructose metabolism stimulates foraging and food intake while reducing energy and oxygen needs by decreasing mitochondrial function, stimulating glycolysis, and inducing insulin resistance. When fructose metabolism is overactivated systemically, such as from excessive fructose intake, this can lead to obesity and diabetes. Herein, we present evidence that Alzheimer's disease may be driven by overactivation of cerebral fructose metabolism, in which the source of fructose is largely from endogenous production in the brain. Thus, the reduction in mitochondrial energy production is hampered by neuronal glycolysis that is inadequate, resulting in progressive loss of cerebral energy levels required for neurons to remain functional and viable. In essence, we propose that Alzheimer's disease is a modern disease driven by changes in dietary lifestyle in which fructose can disrupt cerebral metabolism and neuronal function. Inhibition of intracerebral fructose metabolism could provide a novel way to prevent and treat this disease. [ABSTRACT FROM AUTHOR]

Non-shivering thermogenesis in mammalian brown adipose tissue is a powerful mechanism to defend normothermia in cold climates. To minimize the loss of chemical energy, the central functional component, mitochondrial uncoupling protein 1, UCP1, must be tightly regulated. The canonical pathway of UCP1 activation includes lipolytic release of free fatty acids in response to an adrenergic signal. Activating fatty acids overcome constitutive inhibition of UCP1 by the di- and triphosphate forms of purine nucleotides, i.e., ATP, ADP, GTP, and GDP. Cellular concentrations of inhibitory, free nucleotides are subject to significant, adrenergically induced alterations. The regulatory components involved may constitute novel drug targets to manipulate brown fat thermogenesis and thereby organismic energy balance. We here review evidence for and against a dominant role of nucleotides in thermogenic control, describe conceptual routes to endogenously and pharmacologically alter free nucleotide pool size, speculate on a signaling role of degradation products released from active brown fat, and highlight gaps in our understanding of signaling and metabolic pathways involved. [ABSTRACT FROM AUTHOR]

Histidine-rich glycoprotein (HRG) is a plasma protein synthesized by the liver. We have given the first evidence of a tissue localization of HRG demonstrating its presence in skeletal muscle, associated with the zinc enzyme AMP deaminase (AMPD1). Moreover, we have shown that muscle cells do not synthesize HRG, but they can internalize it from plasma. We have recently demonstrated by confocal laser scanning microscopy that in human skeletal muscle, HRG is mainly localized in the myofibrils, preferentially at the I-band of the sarcomere, in the sarcoplasm, and in the nuclei. Using transmission electron microscopy and immunogold analysis, we carried out this study on human and rat normal skeletal muscles with the purpose to deepen the ultrastructural localization of HRG in skeletal muscle fibers. The immunogold analysis evidenced the presence of HRG in the sarcomeres, mainly in the I-band and to a less extent in the A-band, in the heterochromatin of nuclei, and in the sarcoplasmic reticulum. The colocalization of HRG and skeletal muscle AMPD1 was also analyzed. A colabeling of HRG and AMPD1 was evident at sarcomeric, sarcoplasmic reticulum, and nuclear levels. The significance of these interesting and new results is discussed in this article. [ABSTRACT FROM AUTHOR]

Lipid accumulation in oleaginous organisms is initiated by AMP deaminase (AMPD) after nitrogen depletion because it mediates the concentration of intracellular adenosine monophosphate (AMP). However, the role of AMPD in lipogenesis in the oleaginous fungus Mucor circinelloide s is largely unknown. Therefore, we identified the genes ( ampd1 and ampd2 ) encoding AMPD and investigated the role of AMPD in lipid synthesis in this fungus by overexpressing and deleting ampd genes. Deletion of ampd1 and ampd2 caused 21 and 28% increments in lipid contents under N-limited conditions, respectively. These increases were correlated with the activation of enzymes involved in lipogenesis and the alteration of energy balance. Unexpectedly, overexpression of ampd genes affected nitrogen consumption in both N-limited and N-excess media, which resulted in an increase in cell growth and lipid accumulation compared with the control strain when nitrogen was available. Furthermore, the increased lipid accumulation in the ampd -overexpressing mutants in N-excess media was accompanied by enhanced activities of lipid biosynthetic enzymes. These data suggested that nitrogen metabolism and energy metabolism are affected by AMPD, and overexpression of ampd genes induced lipid accumulation under nitrogen-rich conditions by mimicking the nitrogen limitation response. This highlights an intriguing function of AMPD in M. circinelloides .

Highlights • Bioengineering of Y. lipolytica guaranteed lycopene production. • Adjusting crt genes copy numbers led to a high lycopene yield. • AMPD overexpression improved lycopene production. • Fed-batch fermentation showed feasibility for large scale lycopene production. Abstract Lycopene has been broadly studied in recent decades due to its health benefits including cancer prevention, anti-atherogenic and anti-obesity effects, and modulation of the immune system. To obtain efficient synthesis of lycopene, extensive researches have been conducted in various microbial cells, including Yarrowia lipolytica , to heterologously produce lycopene using various genetic and metabolic engineering methods. In this study, the effects of copy numbers of lycopene synthesis genes, a variety of key central metabolic genes (especially AMP deaminase-encoding gene AMPD), and 5-L fermenter cultivation on lycopene production in Y. lipolytica were investigated and the engineered strains with significantly enhanced lycopene content (46–60 mg/g DCW) were achieved. It is therefore possible to make use of the obtained strains to meet the industrial demand of lycopene production on the basis of further genetic and process optimization. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We report here the first occurrence of an adenosine deaminase-related growth factor (ADGF) that deaminates adenosine 5' monophosphate (AMP) in preference to adenosine. The ADGFs are a group of secreted deaminases found throughout the animal kingdom that affect the extracellular concentration of adenosine by converting it to inosine. The AMP deaminase studied here was first isolated and biochemically characterized from the roman snail Helix pomatia in 1983. Determination of the amino acid sequence of the AMP deaminase enabled sequence comparisons to protein databases and revealed it as a member of the ADGF family. Cloning and expression of its cDNA in Pichia pastoris allowed the comparison of the biochemical characteristics of the native and recombinant forms of the enzyme and confirmed they correspond to the previously reported activity. Uncharacteristically, the H. pomatia AMP deaminase was determined to be dissimilar to the AMP deaminase family by sequence comparison while demonstrating similarity to the ADGFs despite having AMP as its preferred substrate rather than adenosine., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests. George Tzertzinis, Mehul B. Ganatra, Cristian Ruse, Christopher H. Taron, Bryce Causey, Liang Wang, Ira Schildkraut are/were employees of New England Biolabs, a manufacturer and vendor of molecular biology reagents. New England Biolabs funded the work and paid the salaries of all authors. There are no patents, products in development or marketed products to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2023 Tzertzinis et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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. [ABSTRACT FROM AUTHOR]

Objectives: To enhance acid tolerance of Candida glabrata for pyruvate production by engineering AMP metabolism.Results: 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 30 l batch bioreactor at pH 4, and pyruvate reached 46.1 g/l.Conclusion: CgAMD1 overexpression plays an active role in improving acid tolerance and pyruvate fermentation performance of C. glabrata at pH 4. [ABSTRACT FROM AUTHOR]

Histidine-rich glycoprotein (HRG) is a relatively abundant plasma protein that is synthesized by parenchymal liver cells. Using Western blot analysis and immunoperoxidase techniques, we have previously shown the presence of HRG in human skeletal muscle. This paper reports the results of immunofluorescence experiments carried out on sections of human normal skeletal muscle biopsies to investigate the subcellular localization of HRG. The HRG localization was also compared with that of skeletal muscle AMP deaminase (AMPD1), since we have previously described an association of the enzyme with the protein. The obtained results give evidence for a preferential localization of HRG at the I-band level, where it shows the same distribution of actin and where AMPD1 is present in major concentration. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Metallochaperones function as intracellular shuttles for metal ions. At present, no evidence for the existence of any eukaryotic zinc-chaperone has been provided although metallochaperones could be critical for the physiological functions of Zn2+ metalloenzymes. We propose that the complex formed in skeletal muscle by the Zn2+ metalloenzyme AMP deaminase (AMPD) and the metal binding protein histidine-proline-rich glycoprotein (HPRG) acts in this manner. HPRG is a major plasma protein. Recent investigations have reported that skeletal muscle cells do not synthesize HPRG but instead actively internalize plasma HPRG. X-ray absorption spectroscopy (XAS) performed on fresh preparations of rabbit skeletal muscle AMPD provided evidence for a dinuclear zinc site in the enzyme compatible with a (μ-aqua)(μ-carboxylato)dizinc(II) core with two histidine residues at each metal site. XAS on HPRG isolated from the AMPD complex showed that zinc is bound to the protein in a dinuclear cluster where each Zn2+ ion is coordinated by three histidine and one heavier ligand, likely sulfur from cysteine. We describe the existence in mammalian HPRG of a specific zinc binding site distinct from the His-Pro-rich region. The participation of HPRG in the assembly and maintenance of skeletal muscle AMPD by acting as a zinc chaperone is also demonstrated.

Mutation of the AMP deaminase 1 (AMPD1) gene, the predominate AMPD gene expressed in skeletal muscle, is one of the most common inherited defects in the Caucasian population; 2–3% of individuals in this ethnic group are homozygous for defects in the AMPD1 gene. Several studies of human subjects have reported variable results with some studies suggesting this gene defect may cause symptoms of a metabolic myopathy and/or easy fatigability while others indicate individuals with this inherited defect are completely asymptomatic. Because of confounding problems in assessing muscle symptoms and performance in human subjects with different genetic backgrounds and different environmental experiences such as prior exercise conditioning and diet, a strain of inbred mice with selective disruption of the AMPD1 was developed to study the consequences of muscle AMPD deficiency in isolation. Studies reported here demonstrate that these animals are a good metabolic phenocopy of human AMPD1 deficiency but they exhibit no abnormalities in muscle performance in three different exercise protocols.

In fast muscle, isoforms of troponin T (TnT) contain an N-terminal hypervariable region that does not bind any protein of the thin filament. The N-terminal domain of TnT is removed by calpain during stress conditions and so could modulate the role of TnT in the regulation of contraction by affecting the TnT-binding affinity for tropomyosin (Tm) depending on the sequence and charge within the domain. During skeletal muscle contraction, the myokinase reaction is displaced by AMP deaminase (AMPD), an allosteric metalloenzyme, toward the formation of ATP. An unrestrained AMPD activity follows the proteolytic cleavage of the enzyme in vivo that releases a 97 aa N-terminal fragment, removing the inhibition exerted by the binding of ATP to a zinc site in the N-terminal region. Rabbit fast TnT or its phosphorylated 50-aa residue N-terminal peptide restores in AMPD the inhibition by ATP, removed in vitro by the release of a 95 aa N-terminal fragment by trypsin. Since the N-terminal region of fast rabbit TnT contains a putative zinc-binding motif, it can be inferred that TnT mimics the regulatory action exerted in native AMPD by the N-terminal domain that holds the enzyme in a less active conformation due to the presence of a zinc ion connecting the N-terminal and C-terminal regions. Together with evidence that AMPD is localized on the myofibril, the data reported in this review on the interactions between AMPD and TnT strongly suggest that these proteins mutually combine to fine-tune the regulation of muscle contraction in fast muscle. [ABSTRACT FROM AUTHOR]

5′-adenylic acid deaminase (AMP deaminase), an important enzyme for the food industry, can catalyze the irreversible hydrolysis of adenosine monophosphate (AMP) to inosine monophosphate (IMP) and ammonia. In this study, a new strain was screened that efficiently produces 3191.6 U/g of AMP deaminase at 32 °C. After purification, the optimal temperature and pH of the AMP deaminase were found to be 40 °C and 6.0, respectively, but it was partially inhibited by Fe 3+ , Cu 2+ , Al 3+ , and Zn 2+ . With amplification of the AMP deaminase production system, 6 mL of crude enzyme could produce 2.00 mg/g of IMP from 2.04 mg/g of dried yeast with an 84.8% molar yield after 40 min. These results provide a new insight into AMP deaminase production and offer a potential platform for producing 5′-IMP. [ABSTRACT FROM AUTHOR]

Similar symptoms observed in Myasthenia gravis (MG) can be also detected in the case of skeletal muscle AMP-deaminase deficiency. We compared the activity and expression of AMP- deaminase (AMPD) products in skeletal muscles of MG patients and MG-free individuals. The activity of AMP-deaminase in the muscles of MG patients was significantly higher than in the controls and was 2.05 µmol/min/mg protein (±0.31). The two groups differ in level of AMPD product expression. Furthermore in MG-group molecular size of isoform AMPD1 is 90 kDa in contrast to MG-free group where is present 70 kDa isoform of enzyme. The data suggests that the disturbances in transmission of neuronal signaling, taking place in the skeletal muscles of MG patients, may also change energetic metabolism of the affected muscles by changing molecular mass of isoform. [ABSTRACT FROM AUTHOR]

During physical activity in McArdle patients, little or no lactate is released in the skeletal muscle. However, excessive ammonia production has frequently been reported in these patients. Production of ammonia is catalysed by AMP deaminase (AMPD) and adenylate kinase (AK). The activities of AMPD and AK along with housekeeping enzyme phosphoglucoisomerase (PGI) were measured in 11 genetically confirmed McArdle patients and compared with 27 healthy controls. The AMPD and AK activities were not significantly different in patients and controls. The activity of PGI was significantly higher in patients than in controls suggesting compensation of the impaired glycogenolysis in McArdle. The ratios of activities of AMPD and AK over PGI were significantly lower in patients than in controls. High ammonia production in McArdle patients is not based on enzyme induction of AMPD and AK but possibly due to kinetic activation of the enzyme AMPD by increased concentration of the substrate AMP. [ABSTRACT FROM AUTHOR]

Huntington's disease (HD) is a monogenic neurodegenerative disorder with a significant peripheral component to the disease pathology. This includes an HD-related cardiomyopathy, with an unknown pathological mechanism. In this study, we aimed to define changes in the metabolism of cardiac nucleotides using the well-established R6/2 mouse model. In particular, we focused on measuring the activity of enzymes that control ATP and other adenine nucleotides in the cardiac pool, including eNTPD, AMPD, e5′NT, ADA, and PNP. We employed HPLC to assay the activities of these enzymes by measuring the concentrations of adenine nucleotide catabolites in the hearts of symptomatic R6/2 mice. We found a reduced activity of AMPD (12.9 ± 1.9 nmol/min/mg protein in control; 7.5 ± 0.5 nmol/min/mg protein in R6/2) and e5′NT (11.9 ± 1.7 nmol/min/mg protein in control; 6.7 ± 0.7 nmol/min/mg protein in R6/2). Moreover, we detected an increased activity of ADA (1.3 ± 0.2 nmol/min/mg protein in control; 5.2 ± 0.5 nmol/min/mg protein in R6/2), while no changes in eNTPD and PNP activities were observed. Analysis of cardiac adenine nucleotide catabolite levels revealed an increased inosine level (0.7 ± 0.01 nmol/mg dry tissue in control; 2.7 ±0.8 nmol/mg dry tissue in R6/2) and a reduced concentration of cardiac adenosine (0.9 ± 0.2 nmol/mg dry tissue in control; 0.2 ± 0.08 nmol/mg dry tissue in R6/2). This study highlights a decreased rate of degradation of cardiac nucleotides in HD mouse model hearts, and an increased capacity for adenosine deamination, that may alter adenosine signaling. [ABSTRACT FROM AUTHOR]

Background: Inflammation plays a major role in the development and progression of atherosclerosis and coronary artery disease (CAD). Inflammation markers, including white blood cell (WBC) count, C-reactive protein (CRP) and interleukin-6 (IL-6), are widely used for cardiovascular risk prediction. The aim of the study was to establish factors associated with WBC, CRP and IL-6 in patients with CAD. Two functional polymorphisms in genes encoding enzymes participating in adenosine metabolism were analyzed (C34T AMPD1, G22A ADA). Methods: Plasma concentrations of IL-6 were measured using high-sensitivity ELISA kits, and the nephelometric method was used for high-sensitivity CRP (hs-CRP) measurement in 167 CAD patients. Results: Presence of metabolic syndrome (MS) and its components, presence of heart failure, severity of CAD symptoms, severe past ventricular arrhythmia (sustained ventricular tachycardia [sVT] or ventricular fibrillation [VF]), lower left ventricle ejection fraction, higher left ventricle mass index, higher end-diastolic volume and higher number of smoking pack-years were significantly associated with higher WBC, CRP and IL-6. Strong associations with arrhythmia were observed for IL-6 (median 3.90 vs 1.89 pg/mL, p<0.00001) and CRP concentration (6.32 vs 1.47 mg/L, p=0.00009), while MS was associated most strongly with IL-6. CRP and IL-6 were independent markers discriminating patients with sVT or VF. There were no associations between AMPD1 or ADA genotypes and inflammation markers. Conclusions: WBC, CRP and IL-6 are strongly associated with components of the metabolic syndrome. Their strong association with life-threatening ventricular arrhythmia emphasizes the proarrhythmic role of inflammation in the increased cardiovascular risk of CAD patients. [ABSTRACT FROM AUTHOR]