Biochemical Response to Freezing in the Siberian Salamander Salamandrella keyserlingii

The Siberian salamander Salamandrella keyserlingii Dybowski, 1870 is a unique amphibian that is capable to survive long-term freezing at −55 °C. Nothing is known on the biochemical basis of this remarkable freezing tolerance, except for the fact that it uses glycerol as a low molecular weight cryoprotectant. We used 1H-NMR analysis to study quantitative changes of multiple metabolites in liver and hindlimb muscle of S. keyserlingii in response to freezing. For the majority of molecules we observed significant changes in concentrations. Glycerol content in frozen organs was as high as 2% w/w, which confirms its role as a cryoprotectant. No other putative cryoprotectants were detected. Freezing resulted in ischemia manifested as increased concentrations of glycolysis products: lactate and alanine. Unexpectedly, we detected no increase in concentrations of succinate, which accumulates under ischemia in various tetrapods. Freezing proved to be a dramatic stress with reduced adenosine phosphate pool and high levels of nucleotide degradation products (hypoxanthine, β-alanine, and β-aminoisobutyrate). There was also significant increase in the concentrations of choline and glycerophosphocholine, which may be interpreted as the degradation of biomembranes. Thus, we found that freezing results not only in macroscopical damage due to ice formation, but also to degradation of DNA and biomembranes.

Sensing soluble uric acid by Naip1-Nlrp3 platform

Uric acid (UA), a product of purine nucleotide degradation able to initiate an immune response, represents a breakpoint in the evolutionary history of humans, when uricase, the enzyme required for UA cleavage, was lost. Despite being inert in human cells, UA in its soluble form (sUA) can increase the level of interleukin-1β (IL-1β) in murine macrophages. We, therefore, hypothesized that the recognition of sUA is achieved by the Naip1-Nlrp3 inflammasome platform. Through structural modelling predictions and transcriptome and functional analyses, we found that murine Naip1 expression in human macrophages induces IL-1β expression, fatty acid production and an inflammation-related response upon sUA stimulation, a process reversed by the pharmacological and genetic inhibition of Nlrp3. Moreover, molecular interaction experiments showed that Naip1 directly recognizes sUA. Accordingly, Naip may be the sUA receptor lost through the human evolutionary process, and a better understanding of its recognition may lead to novel anti-hyperuricaemia therapies.

Abstract MP121: Deleting the 2-oxoglutarate- and Iron-dependent Prolyl Hydroxylase Ogfod1 Alters Purine Nucleotide Regulation and is Cardioprotective

Prolyl hydroxylation is a post-translational modification that regulates protein stability, turnover, and activity. The proteins that catalyze prolyl hydroxylation belong to the 2-oxoglutarate- and iron-dependent oxygenase family of enzymes. A newly-described member of this family is 2-oxoglutarate- and iron-dependent oxygenase domain containing protein 1 (Ogfod1), which catalyzes prolyl hydroxylation of the ribosomal protein s23 (Rps23). To investigate the cardiovascular function of Ogfod1, we isolated hearts from 5 Ogfod1 -WT and 5 Ogfod1 -KO mice and used Liquid Chromatography and Tandem Mass Spectrometry (LC-MS/MS) to identify proteomic changes. Ingenuity Pathway Analysis (IPA) identified “Purine Nucleotides Degradation II (Aerobic)” ( P = 0.00017) to be one of the most significantly-enriched pathways. We then did metabolomics and found that Inosine 5’-monophosphate (IMP) was 3.5x higher in Ogfod1 -KO hearts ( P = 0.011), further supporting a role for Ogfod1 in regulating purine nucleotide metabolism. Recent evidence has shown that altering purine nucleotide degradation protects against diet-induced obesity and insulin resistance, so we tested this hypothesis in Ogfod1 -KO mice by feeding them high-fat diets. Ogfod1 ablation protects against high-fat diet-induced obesity and insulin resistance. Altering purine nucleotide degradation has also been shown to be protective against cardiac injury, so we tested the hypothesis that Ogfod1 loss protects the heart from ischemia-reperfusion (I/R) injury by subjecting perfused hearts from 6 Ogfod1 -WT and 6 Ogfod1 -KO mice to ischemia and reperfusion and assessed tissue death. We found a 37% decrease in infarct size in Ogfod1 -KO hearts (56% in Ogfod1 -WT and 35% in Ogfod1 -KO, P = 0.0003). In a separate set of experiments, we treated Ogofd1 -KO mice with isoproterenol to induce hypertrophy, and Ogfod1 -KO hearts showed protection against hypertrophic remodeling. Interestingly, OGFOD1 transcripts were up-regulated in human heart failure, indicating a potential role for OGFOD1 in the human failing heart. Altogether, these data show that Ogfod1 deletion alters the myocardial proteome and myocardial metabolism and protects against obesity, insulin sensitivity, I/R injury, and hypertrophy.

Increased Adenine Nucleotide Degradation in Skeletal Muscle Atrophy

Adenine nucleotides (AdNs: ATP, ADP, AMP) are essential biological compounds that facilitate many necessary cellular processes by providing chemical energy, mediating intracellular signaling, and regulating protein metabolism and solubilization. A dramatic reduction in total AdNs is observed in atrophic skeletal muscle across numerous disease states and conditions, such as cancer, diabetes, chronic kidney disease, heart failure, COPD, sepsis, muscular dystrophy, denervation, disuse, and sarcopenia. The reduced AdNs in atrophic skeletal muscle are accompanied by increased expression/activities of AdN degrading enzymes and the accumulation of degradation products (IMP, hypoxanthine, xanthine, uric acid), suggesting that the lower AdN content is largely the result of increased nucleotide degradation. Furthermore, this characteristic decrease of AdNs suggests that increased nucleotide degradation contributes to the general pathophysiology of skeletal muscle atrophy. In view of the numerous energetic, and non-energetic, roles of AdNs in skeletal muscle, investigations into the physiological consequences of AdN degradation may provide valuable insight into the mechanisms of muscle atrophy.

Effect of Chilled Ageing Conditioning at 4°C in Lamb Longissimus Dorsi Muscles on Water-Soluble Flavour Precursors as Revealed by a Metabolomic Approach

The objective of this study was to investigate, by a metabolomic approach, the effects of chilled ageing conditioning at 4°C in lamb longissimus dorsi (LD) muscles on water-soluble flavour precursors. The results showed that the content of nucleotide degradation products significantly increased (P<0.05) due to the adjusted biosynthesis of alkaloids derived from histidine and purine from day 0 to day 4. Additionally, the content of glycolytic compounds significantly increased (P<0.05) due to enhanced glycolysis, and the content of organic acid increased (P<0.05) because of the altered tricarboxylic acid cycle (TCA) from day 0 to day 4. In addition, the content of total free amino acids significantly increased (P<0.05), owing to the altered biosynthesis of amino acids from day 4 to day 8. These results are significant proof that there were quantitative changes observed in lamb flavour precursors during chilled ageing.

Seric and hepatic NTPDase and 5′ nucleotidase activities of rats experimentally infected byFasciola hepatica

SUMMARYThe enzymatic activities of NTPDase and 5′nucleotidase are important to regulate the concentration of adenine nucleotides, known molecules involved in many physiological functions. Therefore, the objective of this study was to evaluate the activity of NTPDase and 5′nucleotidase in serum and liver tissue of rats infected byFasciola hepatica. Rats were divided into two groups: uninfected control and infected. NTPDase activity for adenosine triphosphate (ATP) and ADP substrates in the liver was higher compared with the control group at 15 days post-infection (PI), while seric activity was lower. In addition, seric and hepatic samples did not show changes for 5′nucleotidase activity at this time. On the other hand, either NTPDase or 5′nucleotidase activities in liver homogenate and serum were higher at 87 days PI. Early in the infection, low NTPDase activity maintains an increase of ATP in the bloodstream in order to activate host immune response, while in hepatic tissue it decreases extracellular ATP to maintain a low inflammatory response in the tissue. As stated, higher NTPDase and 5′nucleotidase activities 87 days after infection in serum and tissue, probably results on an increased concentration of adenosine molecule which stimulates a Th2 immune response. Thus, it is possible to conclude thatF. hepaticainfections lead to different levels of nucleotide degradation when considering the two stages of infection studied, which influences the inflammatory and pathological processes developed by the purinergic system.