Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology最新文献

For small mammals, such as mice, cannulation procedures can be quite challenging, limiting research associated with tracing isotopically labelled substrates at the whole-animal level. When cannulation in mice is possible, assessment of substrate use is further limited to when mice are either under anesthesia or are at rest, as there are no studies directly quantifying substrate use during exercise in mice. The use of isotopic tracer techniques has greatly advanced our knowledge in understanding how metabolic substrates (carbohydrates, amino acids, and fatty acids) contribute to whole-body metabolism. However, research regarding tissue-specific fuel use contributions to whole-body energy expenditure in mice at varying metabolic intensities (i.e., exercise) is lacking, despite the popularity of using mice in a variety of metabolic models. In this commentary, we briefly discuss the methodologies, advantages, and disadvantages of using radiolabelled, positron emission, and stable isotopes with a specific focus on fatty acids. We highlight recent mouse studies that have used creative experimental designs employing the use of isotopic tracer techniques and we briefly discuss how these methodologies can be further pursued to deepen our understanding of substrate use during exercise. Lastly, we show findings of a recent study we performed using a radiolabelled fatty acid tracer (¹⁴C-bromopalmitic acid) to determine fatty acid uptake in 16 muscles, two brown and two white adipose tissue depots during submaximal exercise in deer mice.

n-3 Long-chain polyunsaturated fatty acids (n-3 LC-PUFAs), including eicosapentaenoic acid (EPA), are essential multifunctional nutrients in animals. Microorganisms such as microalgae are known to be n-3 LC-PUFA producers in aquatic environments. Various aquatic invertebrates, including Harpacticoida copepods, and a few terrestrial invertebrates, such as the nematode Caenorhabditis elegans, possess n-3 LC-PUFA biosynthetic enzymes. However, the capacity for n-3 LC-PUFA biosynthesis and the underlying molecular mechanisms in terrestrial insects are largely unclear. In this study, we investigated the fatty acid biosynthetic pathway in the silkworm Bombyx mori and found that EPA was present in silkworms throughout their development. Stable isotope tracing revealed that dietary α-linolenic acid (ALA) was metabolized to EPA in silkworm larvae. These results indicated that silkworms synthesize EPA from ALA. Given that EPA is enriched in the central nervous system, we propose that EPA confers optimal neuronal functions, similar to docosahexaenoic acid, in the mammalian nervous system.

Psalidodon bifasciatus is a fish species sensitive to physical and chemical changes in water. It serves as a good bioindicator of temperature variations and is utilized in environmental monitoring studies in Brazilian rivers. The objective of this study was to evaluate antioxidant defense biomarkers in the heart, brain, and muscle of P. bifasciatus exposed to a 10 °C thermal increase. P. bifasciatus were collected and divided into a control group (21 °C) and groups subjected to thermal shock (31 °C) for periods of 2, 6, 12, 24, and 48h. Two-way ANOVA indicated that a 10 °C temperature increase caused oxidative stress in P. bifasciatus. This was evidenced by altered levels of lipid peroxidation (LPO), carbonylated proteins (PCO), and glutathione peroxidase (GPx) in the heart, catalase (CAT) and LPO in the brain, and LPO in the muscle. Principal component analysis (PCA) and integrated biomarker response (IBR) analysis indicated that, compared to the heart and muscle, the brain exhibited a greater activation of the antioxidant response. Sensitivity analysis indicated that the muscle was the most sensitive organ, followed by the brain and heart. Our results indicate that the stress response is tissue-specific through the activation of distinct mechanisms. These responses may be associated with the tissue's function as well as its energy demand. As expected, P. bifasciatus showed changes in response to thermal stress, with the brain showing the greatest alteration in antioxidant defenses and the muscle being the most sensitive tissue.

Wood frogs are freeze-tolerant vertebrates that can endure weeks to months frozen during the winter without breathing and with as much as 65% of total body water frozen as extracellular ice. Underlying tolerances of anoxia and of cellular dehydration support whole body freezing. One pro-survival mechanism employed by these frogs is epigenetic modifications via DNA hypomethylation processes facilitating transcriptional repression or activation. These processes involve proteins such as DNA Methyltransferases (DNMTs), Methyl Binding Domain proteins (MBDs), Ten-Eleven Translocases (TETs), and Thymine Deglycosylase (TDG). The present study evaluates the responses of these proteins to dehydration and anoxia stresses in wood frog liver. DNMT relative protein expression was reduced in liver, but nuclear DNMT activity did not change significantly under anoxia stress. By contrast, liver DNMTs and nuclear DNMT activity were upregulated under dehydration stress. These stress-specific differences were speculated to arise from Post-Translational Modifications (PTMs). DNMT3A and DNMT3B showed increased relative protein expression during recovery from dehydration and anoxia. Further, MBD1 was elevated during both conditions suggesting transcriptional repression. TET proteins showed varying responses to anoxia likely due to the absence of oxygen, a main substrate required by TETs. Similarly, TDG, an enzyme that corrects DNA damage, was downregulated under anoxia potentially due to lower levels of reactive oxygen species that damage DNA, but levels returned to normal during reperfusion of oxygen. Our results indicate differential stress-specific responses that indicate the need for more research in the DNA hypomethylation mechanisms employed by the wood frog during stress.

Animals living at high-altitude are faced with unremitting low oxygen availability. This can make it difficult to perform daily tasks that require increases in aerobic metabolism. An activity important for survival is aerobic locomotion, and the rapid recovery of muscle metabolism post exercise. Past work shows that hypoxia acclimated high-altitude mice (Peromyscus maniculatus) have a greater reliance on carbohydrates to power exercise than low altitude mice. However, it is unclear how quickly after aerobic exercise these mice can recovery and replenish muscle glycogen stores. The gastrocnemius muscle of high-altitude deer mice has a more aerobic phenotype and a greater capacity to oxidize lipids than low altitude deer mice. This suggests that high altitude mice may recover more rapidly from exercise than their lowland counterparts due to a greater capacity to support glycogen replenishment using intramuscular triglycerides (IMTG). To explore this possibility, we used low- and high-altitude native deer mice born and raised in common lab conditions and acclimated to chronic hypoxia. We determined changes in oxygen consumption following 15 min of aerobic exercise in 12% O₂ and sampled skeletal muscles and liver at various time points during recovery to examine changes in key metabolites, including glycogen and IMTG. We found depletion in glycogen stores during exercise only in lowlanders, which returned to resting levels following 90 min of recovery. In contrast, IMTG did not change significantly with exercise or during recovery in either population. These data suggest that exercise recovery is influenced by altitude ancestry in deer mice.

Myotis davidii cystatin A (MdCSTA), a stefin A-like from the Chinese native bat species M. davidii, was expressed as a recombinant protein and functionally characterized as a strong inhibitor of the cysteine proteases papain, human cathepsins L and B and the tick cathepsin L-like BmCL1. Despite the highly conserved amino acid sequences among stefins A from different vertebrates, MdCSTA presents a Methionine-2 residue at the N-terminal region and the second binding loop (pos 73–79) that differs from human stefin A (HsCSTA) and might be related to the lower inhibition constant (K_i) value presented by this inhibitor in comparison to human stefin A inhibition to cathepsin B. Therefore, to investigate the importance of these variable regions in cathepsin B inhibition, recombinant stefins A MdCSTA and HsCSTA containing mutations at the second amino acid residue and second binding loop were expressed and evaluated in kinetic assays. Enzymatic inhibition assays with cathepsin B revealed that switching the amino acid residues at position 2 and second binding loop region between bat and human CSTAs improved the HsCSTA's and reduced MdCSTA's inhibitory activity. Additionally, molecular docking analysis estimated lower energy values for the complex between MdCSTA-cathepsin B, in comparison to human CSTA-cathepsin B, while the mutants presented intermediate values, suggesting that other regions might contribute to the higher inhibitory activity against cathepsin B by MdCSTA. In conclusion, MdCSTA, the first bat's stefin A-like inhibitor to be functionally characterized, presented a higher inhibitory activity against cathepsin B in comparison to the human inhibitor, which is partially related to the glutamine-rich second binding loop and Met-2. Further structural analysis should be performed to elucidate potential inhibitor effects on cysteine proteinases.

Projected increases in temperature and decreases in salinity associated with global climate change will likely have detrimental impacts on eastern oyster, Crassostrea virginica, as these variables can influence physiological processes in these keystone species. We set out to determine how the interactive effects of temperature (20 °C or 27 °C) and/or salinity (27‰ or 17‰) impacted the energetic reserves, aerobic and anaerobic metabolism, and changes to oxidative stress or total antioxidant potential as a consequence of an altered environment over a 21-day exposure. Gill and adductor muscle were used to quantify changes in total glycogen and lipid content, Electron Transport System and Citrate Synthase activities, Malate Dehydrogenase activity, Protein Carbonyl formation, lipid peroxidation, and total antioxidant potential. A second exposure was performed to determine if these environmental factors influenced the ingestion of microfibers, which are now one of the leading forms of marine debris. Elevated temperature and the combination of elevated temperature and decreased salinity led to an overall decline in oyster mass, which was exacerbated by the presence of microfibers. Changes in metabolism and oxidative stress were largely influenced by time, but exposure to elevated temperature, decreased salinity, the combination of these stressors or exposure to microfibers had small impacts on oyster physiology and survival. Overall these studies demonstrate that oyster are fairly resilient to changes in salinity in short-term exposures, and elevations in temperature or temperature combined with salinity result in changes to the oyster energetic response, which can be further impacted by the presence of microfibers.

Mannose-binding lectin (MBL) is a vital member of the lectin family, crucial for mediating functions within the complement lectin pathway. In this study, following the cloning of the mannose-binding lectin (MBL) gene in the ridgetail white prawn, Exopalaemon carinicauda, we examined its expression patterns across various tissues and its role in combating challenges posed by Vibrio parahaemolyticus. The results revealed that the MBL gene spans 1342 bp, featuring an open reading frame of 972 bp. It encodes a protein comprising 323 amino acids, with a predicted relative molecular weight of 36 kDa and a theoretical isoelectric point of 6.18. The gene exhibited expression across various tissues including the eyestalk, heart, gill, hepatopancreas, stomach, intestine, ventral nerve cord, muscle, and hemolymph, with the highest expression detected in the hepatopancreas. Upon challenge with V. parahaemolyticus, RT-PCR analysis revealed a trend of MBL expression in hepatopancreatic tissues, characterized by an initial increase followed by a subsequent decrease, peaking at 24 h post-infection. Employing RNA interference to disrupt MBL gene expression resulted in a significant increase in mortality rates among individuals challenged with V. parahaemolyticus. Furthermore, we successfully generated the Pet32a-MBL recombinant protein through the construction of a prokaryotic expression vector for conducting in vitro bacterial inhibition assays, which demonstrated the inhibitory effect of the recombinant protein on V. parahaemolyticus, laying a foundation for further exploration into its immune mechanism in response to V. parahaemolyticus challenges.

The thermogenic capacity of brown adipose tissue (BAT) in rodents decreases with prolonged heat exposure. However, the underlying mechanisms are not well understood. In this study, Kunming mice were acclimated at 23 ± 1 °C and 33 ± 1 °C for four weeks each to examine the body heat balance and BAT alterations. Results showed that heat-acclimated Kunming mice exhibited reduced body mass and elevated body temperature. Additionally, they displayed lower resting metabolic rates, diminished non-shivering thermogenesis, and reduced BAT thermogenic function. Metabolically, there was a significant reduction in several key metabolites involved in energy metabolism in BAT, including thiamine pyrophosphate, citric acid, cis-Aconitate, isocitric acid, oxoglutaric acid, succinate, fumarate, L-Malic acid, oxaloacetate, flavin mononucleotide, nicotinamide adenine dinucleotide, and adenosine 5′-triphosphate. These findings suggest that BAT adapts to heat acclimation by regulating pathways related to pyruvate oxidation, tricarboxylic acid cycle, and oxidative phosphorylation, which may help maintain thermal homeostasis in Kunming mice.

Non-blood-feeding leeches, Whitmania pigra, have evolved unique digestive structures and physiological mechanisms to cope with fasting. However, the metabolic changes and molecular mechanisms induced by fasting remain unclear. Therefore, this study recorded the weights of leeches during the fasting process. The weight changes were divided into two stages: a rapid decline period (1–9 weeks) and a fluctuating decline period (9–24 weeks). Leeches fasted for 4 (H4), 11 (H11), and 24 (H24) weeks were selected for transcriptome sequencing. Compared to the control group (H0), 436, 1157, and 337 differentially expressed genes (DEGs) were identified, which were mainly related to glycolysis/gluconeogenesis, amino acid metabolism, and the lipid metabolism pathway. The 6-phosphofructokinase (Pfk), pyruvate kinase (PK), and phosphoenolpyruvate carboxykinase (Pck) transcription levels revealed glycolysis/gluconeogenesis activation during the early stage of fasting and peaked at 11 weeks. Decreased expression of the rate-limiting enzyme acetyl-CoA carboxylase (ACC) in fatty acid synthesis during fasting may impede fatty acid synthesis. These results indicated that the nutrient storage and energy-supplying pathways in W. pigra were modified to improve fasting resistance. The findings of this study provided guidance for exploring the mechanism underlying fasting metabolism and laid a foundation for artificial breeding to improve the resistance of leeches.