Surviving winter on the Qinghai-Xizang Plateau: Extensive reversible protein phosphorylation plays a dominant role in regulating hypometabolism in hibernating Nanorana parkeri.

Changes in protein abundance and reversible protein phosphorylation (RPP) play important roles in regulating hypometabolism but have never been documented in overwintering frogs at high altitudes. To test the hypothesis that protein abundance and phosphorylation change in response to winter hibernation, we conducted a comprehensive and quantitative proteomic and phosphoproteomic analysis of the liver of the Xizang plateau frog, Nanorana parkeri, living on the Qinghai-Xizang (Tibet) Plateau (QTP). In total, 5 170 proteins and 5 695 phosphorylation sites in 1 938 proteins were quantified. Based on proteomic analysis, 674 differentially expressed proteins (438 up-regulated, 236 down-regulated) were screened in hibernating N. parkeri versus summer individuals. Functional enrichment analysis revealed that higher expressed proteins in winter were significantly enriched in immune-related signaling pathways, whereas lower expressed proteins were mainly involved in metabolic processes. A total of 4 251 modified sites (4 147 up-regulated, 104 down-regulated) belonging to 1 638 phosphoproteins (1 555 up-regulated, 83 down-regulated) were significantly changed in the liver. During hibernation, RPP regulated a diverse array of proteins involved in multiple functions, including metabolic enzymatic activity, ion transport, protein turnover, signal transduction, and alternative splicing. These changes contribute to enhancing protection, suppressing energy-consuming processes, and inducing metabolic depression. Moreover, the activities of phosphofructokinase, glutamate dehydrogenase, and ATPase were all significantly lower in winter compared to summer. In conclusion, our results support the hypothesis and demonstrate the importance of RPP as a regulatory mechanism when animals transition into a hypometabolic state.

Hepatic transcriptome and gut microbiome provide insights into freeze tolerance in the high-altitude frog, Nanorana parkeri.

Among amphibians, freeze tolerance is a low-temperature survival strategy that has been well studied in several species. One influence on animal health and survival under adverse conditions is the gut microbiome. Gut microbes can be greatly affected by temperature fluctuations but, to date, this has not been addressed in high-altitude species. Nanorana parkeri (Anura: Dicroglossidae) lives at high altitudes on the Tibetan plateau and shows a good freeze tolerance. In the present study, we addressed two goals: (1) analysis of the effects of whole body freezing on the liver transcriptome, and (2) assess modifications of the gut microbiome as a consequence of freezing. We found that up-regulated genes in liver were significantly enriched in lipid and fatty acid metabolism that could contribute to accumulating the cryoprotectant glycerol and raising levels of unsaturated fatty acids. The results suggest the crucial importance of membrane adaptations and fuel reserves for freezing survival of these frogs. Down-regulated genes were significantly enriched in the immune response and inflammatory response, suggesting that energy-consuming processes are inhibited to maintain metabolic depression during freezing. Moreover, freezing had a significant effect on intestinal microbiota. The abundance of bacteria in the family Lachnospiraceae was significantly increased after freezing exposure, which likely supports freezing survival of N. parkeri. The lower abundance of bacteria in the family Peptostreptococcaceae in frozen frogs may be associated with the hypometabolic state and decreased immune response. In summary, these findings provide insights into the regulatory mechanisms of freeze tolerance in a high-altitude amphibian at the level of gene expression and gut microbiome, and contribute to enhancing our understanding of the adaptations that support frog survival in high-altitude extreme environments.

[Responses of blood parameters and hemoglobin subtypes in plateau zokors and plateau pikas to different altitude habitats].

The plateau zokor (Myospalax baileyi) and plateau pika (Ochotona curzoniae) are native species unique to the Qinghai-Tibetan Plateau with successful adaptation to the hypoxic environment. In this study, the number of red blood cells, hemoglobin concentration, mean hematocrit and mean volume of red blood cells were measured in plateau zokors and plateau pikas at different altitudes. Hemoglobin subtypes of two plateau animals were identified by mass spectrometry sequencing. The forward selection sites in two animals' hemoglobin subunits were analyzed by PAML4.8 program. Homologous modeling was used to analyze the effect of forward selection sites on the affinity of hemoglobin to oxygen. The adapting strategies of plateau zokors and plateau pikas to hypoxia at different altitudes were analyzed through comparing blood parameters between the two species. The results indicated that, with increasing altitudes, plateau zokors responded to hypoxia by increasing red blood cell count and decreasing red blood cell volume, while plateau pikas took the opposite strategies to plateau zokors. In erythrocytes of plateau pikas, both adult α2ß2 and fetal α2ε2 hemoglobins were identified, while erythrocytes of plateau zokors only had adult α2ß2 hemoglobin, however the affinities and the allosteric effects of the hemoglobin of plateau zokors were significantly higher than those of plateau pikas. Mechanistically, in the α and ß subunits of hemoglobin of plateau zokors and pikas, the numbers and the sites of the positively selected amino acids as well as the side chain groups polarities and orientations of the amino acids differed significantly, which may result in the difference of the affinities to oxygen of hemoglobin between plateau zokors and pikas. In conclusion, the adaptive mechanisms to respond to hypoxia in blood properties of plateau zokors and plateau pikas are species-specific.

A homotetrameric hemoglobin expressed in alveolar epithelial cells increases blood oxygenation in high-altitude plateau pika (Ochotona curzoniae).

The plateau pika (Ochotona curzoniae) is native to the Qinghai-Tibet Plateau. In this study, the gene that encodes a heme-binding protein in the pulmonary surfactant (PS) of the pika is identified. The protein is a homotetrameric hemoglobin (δ4) encoded by HBD (δ). HBD is expressed in alveolar epithelial type II (ATII) and type I (ATI) cells, upregulated by hypoxia. δ4 is secreted into alveolar cavities through osmiophilic multilamellar bodies. HBD expression is downregulated by RNAi, which significantly increases hypoxia-inducible factor 1α expression in lung tissue and red blood cells and hemoglobin and blood lactate concentrations but significantly decreases arterial partial pressure of oxygen (PaO2). Our results indicate that plateau pikas physiologically show hypoxemia when HBD expression is downregulated. Therefore, specific HBD expression in the lungs helps plateau pikas to obtain oxygen under hypoxia by maintaining higher PaO2. These findings provide insights into the adaptive mechanisms of plateau pikas to withstand high-altitude environments.

Vitamin D3 Metabolic Enzymes in Plateau Zokor (Myospalax baileyi) and Plateau Pika (Ochotona curzoniae): Expression and Response to Hypoxia.

Vitamin D3 (D3) is produced endogenously from 7-dehydrocholesterol by irradiation and is an important secosteroid for the absorption of calcium and phosphate. Lithocholic acid (LCA) increases intestinal paracellular calcium absorption in a vitamin D receptor-dependent manner in vitamin D-deficient rats. The plateau zokor (Myospalax baileyi), a strictly subterranean species, and plateau pika are endemic to the Qinghai-Tibet Plateau. To verify whether the zokors were deficient in D3 and reveal the effects of hypoxia on D3 metabolism in the zokors and pikas, we measured the levels of 25(OH)D3, calcium, and LCA, and quantified the expression levels of D3 metabolism-related genes. The results showed an undetectable serum level of 25(OH)D3 and a significantly higher concentration of LCA in the serum of plateau zokor, but its calcium concentration was within the normal range compared with that of plateau pika and Sprague-Dawley rats. With increasing altitude, the serum 25(OH)D3 levels in plateau pika decreased significantly, and the mRNA and protein levels of CYP2R1 (in the liver) and CYP27B1 (in the kidney) in plateau pika decreased significantly. Our results indicate that plateau zokors were deficient in D3 and abundant in LCA, which might be a substitution of D3 in the zokor. Furthermore, hypoxia suppresses the metabolism of D3 by down-regulating the expression of CYP2R1 and CYP27B1 in plateau pika.

The Bile Acid Membrane Receptor TGR5 in Cancer: Friend or Foe?

The G-protein-coupled bile acid receptor, Gpbar1 or TGR5, is characterized as a membrane receptor specifically activated by bile acids. A series of evidence shows that TGR5 induces protein kinase B (AKT), nuclear factor kappa-B (NF-κB), extracellular regulated protein kinases (ERK1/2), signal transducer and activator of transcription 3 (STAT3), cyclic adenosine monophosphate (cAMP), Ras homolog family member A (RhoA), exchange protein activated by cAMP (Epac), and transient receptor potential ankyrin subtype 1 protein (TRPA1) signaling pathways, thereby regulating proliferation, inflammation, adhesion, migration, insulin release, muscle relaxation, and cancer development. TGR5 is widely distributed in the brain, lung, heart, liver, spleen, pancreas, kidney, stomach, jejunum, ileum, colon, brown adipose tissue (BAT), white adipose tissue (WAT), and skeletal muscle. Several recent studies have demonstrated that TGR5 exerts inconsistent effects in different cancer cells upon activating via TGR5 agonists, such as INT-777, ursodeoxycholic acid (UDCA), and taurolithocholic acid (TLCA). In this review, we discuss both the 'friend' and 'foe' features of TGR5 by summarizing its tumor-suppressing and oncogenic functions and mechanisms.

Effects of Fdft 1 gene silencing and VD3 intervention on lung injury in hypoxia-stressed rats.

BACKGROUND: Hypoxia can induce lung injury such as pulmonary arterial hypertension and pulmonary edema. And in the rat model of hypoxia-induced lung injury, the expression of Farnesyl diphosphate farnesyl transferase 1 (Fdft 1) was highly expressed and the steroid biosynthesis pathway was activated. However, the role of Fdft 1 and steroid biosynthesis pathway in hypoxia-induced lung injury remains unclear. OBJECTIVE: The study aimed to further investigate the relationship between Fdft1 and steroid biosynthesis pathway with hypoxia-induced lung injury. METHODS: A rat model of lung injury was constructed by hypobaric chamber with hypoxic stress, the adenovirus interference vector was used to silence the expression of Fdft 1, and the exogenous steroid biosynthesis metabolite Vitamin D3 (VD3) was used to treat acute hypoxia-induced lung injury in rats. RESULTS: Sh-Fdft 1 and exogenous VD3 significantly inhibited the expression of Fdft 1 and the activation of the steroid pathway in hypoxia-induced lung injury rats, which showed a synergistic effect on the steroid activation pathway. In addition, sh-Fdft 1 promoted the increase of pulmonary artery pressure and lung water content, the decrease of oxygen partial pressure and oxygen saturation, and leaded to the increase of lung cell apoptosis and the aggravation of mitochondrial damage in hypoxia-stressed rats. And VD3 could significantly improve the lung injury induced by hypoxia and sh-Fdft 1 in rats. CONCLUSIONS: Fdft 1 gene silencing can promote hypoxic-induced lung injury, and exogenous supplement of VD3 has an antagonistic effect on lung injury induced by Fdft 1 gene silencing and hypoxic in rats, suggesting that VD3 has a preventive and protective effect on the occurrence and development of hypoxia-induced lung injury.

A New Homotetramer Hemoglobin in the Pulmonary Surfactant of Plateau Zokors (Myospalax Baileyi).

The plateau zokor (Myospalax baileyi) is a native species to the Qinghai-Tibetan Plateau, inhabiting hypoxia and hypercapnia sealed subterranean burrows that pose several unique physiological challenges. In this study, we observed a novel heme-containing protein in the pulmonary surfactant (PS) of plateau zokor, identified the encoding gene of the protein, predicted its origination and structure, verified its expression in alveolar epithelial cells, and determined the protein's affinity to oxygen and its effect on the oxygen-dissolving capability in the PS of plateau zokors. The protein is an unusual homotetramer hemoglobin consisting of four γ-like subunits, and the subunit is encoded by a paralog gene of γ, that is γ-like. The divergence time of γ-like from γ is estimated by the molecular clock to be about 2.45 Mya. The generation of γ-like in plateau zokors might well relate to long-time stress of the high land hypoxia. Unlike γ, the γ-like has a hypoxia response element (HRE) and a lung tissue-specific enhancer in its upstream region, and it is expressed specifically in lung tissues and up-regulated by hypoxia. The protein is named as γ4-like which is expressed specifically in Alveolar epithelial type II (ATII) cells and secreted into the alveolar cavities through the osmiophilic multilamellar body (LBs). The γ4-like has a higher affinity to oxygen, and that increases significantly oxygen-dissolving capability in the PS of plateau zokors by its oxygenation function, which might be beneficial for the plateau zokors to obtain oxygen from the severe hypoxia environments by facilitating oxygen diffusion from alveoli to blood.

[Comparison of the composition and content of pulmonary surfactant among plateau zokors, plateau pikas and rats].

In the present study, the composition and content of pulmonary surfactant (PS) were analyzed to explore the hypoxia adaptation mechanism in plateau zokors (Myospalax baileyi) and plateau pikas (Ochotona curzoniae). 36 plateau zokors and plateau pikas were trapped alive at the Laji Mountain in Guide County, Qinghai Province (at the altitude of about 3 600 m), and 36 Sprague-Dawley (SD) rats were purchased from the experimental animal center of Lanzhou University (at the altitude of about 1 500 m). All animals were lavaged after laboratory anesthesia, the blood in lung tissues was fully washed out and the lung tissues were then taken out to obtain the bronchoalveolar lavage fluid by bronchoalveolar lavage. The composition and content of phospholipids in the PS of three different kinds of animals were analyzed by using high performance liquid chromatography; the protein composition, content and types in the PS were analyzed by G-250 Coomassie brilliant blue method, polyacrylamide gel electrophoresis (PAGE) and mass spectrometry; the dissolved oxygen in the PS solutions were determined by using dissolved oxygen electrode. The results showed that the total contents of phospholipids in the PS were successively increased among plateau zokors, plateau pikas and SD rats (P < 0.05), while the total content of proteins successively decreased (P < 0.05). There were five phospholipids identified in the PS, including linoleic palmitoylphosphatidylcholine (LPPC), dipalmitoylphosphatidylcholine (DPPC), phosphatidylglyerol (PG), phosphatidylinositol (PI) and phosphatidylserine (PSe), but the relative contents of these phospholipids were different. The relative content of LPPC was successively increased among plateau zokors, plateau pikas and SD rats (P < 0.01). The relative contents of DPPC, PG and PI in the PS of plateau zokors were significantly higher than those of plateau pikas (P < 0.01), while insignificant differences between plateau pikas and SD rats (P > 0.05). The relative content of PSe had no significant differences between plateau zokors and plateau pikas (P > 0.05), but both were significantly higher than that of SD rats (P < 0.01). The serum albumin (SA) was identified in the PS of three kinds of animals, including homologous tetramer protein containing heme, which is composed of hemoglobin ß subunit, in plateau zokors and plateau pikas. Immunoglobulin (Ig) heavy chain was found in PS of plateau zokors and SD rats. The content of Ig heavy chain in plateau zokor was significantly higher than that in SD rats (P < 0.01), and the content of protein containing heme was significantly higher than that in plateau pikas (P < 0.05). The amount of dissolved oxygen was successively decreased in the PS among plateau zokors, plateau pikas and SD rats (P < 0.01), but it was significantly higher than that in saline (P < 0.01). These results suggest that the total content of proteins in the PS of plateau zokors and plateau pikas was significantly higher, while the total content of phospholipids was significantly decreased. There were high content of homologous tetramer protein containing heme in the PS of plateau zokors and plateau pikas. The relative content of DPPC, the main component of phospholipids, was significantly increased in plateau zokors. The changes of PS component and content improve the adaptability of the two plateau animals in hypoxia environment.

Evolutionary analysis of TSP-1 gene in Plateau zokor (MyospalaxBaileyi) and its expression pattern under hypoxia.

The plateau zokor (Myospalaxbaileyi) is a specialized subterranean rodent that lives on the Qinghai-Tibet Plateau, and has successfully adapted to hypoxic environment. Raised expression of vascular endothelial growth factor (VEGF) and enhanced microvessel density (MVD) in tissues enable subterranean rodents to adapt to hypoxic sealed burrows. However, the expression of VEGF is inhibited by decreases in oxygen content, which is different from what obtains in Sprague Dawley (SD)rats. Thromspondin-1(TSP-1) is the first endogenous angiogenesis inhibitor identified inp53 pathway. It has several domains that bind to different proteins which regulate cell-to-cell interactions, inhibit endothelial cell proliferation and induce endothelial cell apoptosis (anti-angiogenesis). In this study, we analyzed the coding region and the expression pattern of TSP-1 gene in plateau zokor under different oxygen partial pressures using bioinformatics and qRT-PCR, respectively. Our results showed that the base and amino acid homologies between plateau zokor and Northern Israeli blind subterranean mole rat (Nannospalaxgalili) were 95.08 and 97.61%, respectively. There were eight parallel evolution sites with Nannospalaxgalili. Evaluation by 'Sorting Tolerant From Intolerant' (SIFT) algorithm showed four sites with significant effects on the function of TSP-1. Three-dimensional (3D) structures revealed that Asp185 and Thr270 were located in the NH2 terminal domain, with Glu536 in the Type I repeat domain, and Thr1092 in the COOH terminal domain. Compared to SD rats, the polarities of these four mutation sites changed. The expression levels of TSP-1 in plateau zokor tissues increased significantly from 2 260 m(16.12kPa) to 3 300 m(14.13kPa), but there was no significant difference in TSP-1 expression in SD rats. In conclusion, due to long-term adaption to the hypoxic environment of sealed burrows, plateau zokor upregulates the expression of TSP-1 to effect anti-angiogenesis. Moreover, mutations in gene structure of TSP-1 may play an important role in inhibiting angiogenesis.

Effect of Hypoxia on Ldh-c Expression in Somatic Cells of Plateau Pika.

Sperm specific lactate dehydrogenases (LDH-C4) is a lactate dehydrogenase that catalyzes the conversion of pyruvate to lactate. In mammals, Ldh-c was originally thought to be expressed only in testes and spermatozoa. Plateau pika (Ochotona curzoniae), which belongs to the genus Ochotona of the Ochotonidea family, is a hypoxia-tolerant mammal living 3000-5000 m above sea level on the Qinghai-Tibet Plateau, an environment which is strongly hypoxic. Ldh-c is expressed not only in testes and sperm, but also in the somatic tissues of plateau pika. To reveal the effect of hypoxia on pika Ldh-c expression, we investigated the mRNA and protein level of Ldh-c as well as the biochemical index of anaerobic glycolysis in pika somatic tissues at the altitudes of 2200 m, 3200 m and 3900 m. Our results showed that mRNA and protein expression levels of Ldh-c in the tissues of pika's heart, liver, brain and skeletal muscle were increased significantly from 2200 m to 3200 m, but had no difference from 3200 m to 3900 m; the activities of LDH and the contents of lactate showed no difference from 2200 m to 3200 m, but were increased significantly from 3200 m to 3900 m. Hypoxia up-regulated and maintained the expression levels of Ldh-c in the pika somatic cells. Under the hypoxia condition, plateau pikas increased anaerobic glycolysis in somatic cells by LDH-C4, and that may have reduced their dependence on oxygen and enhanced their adaptation to the hypoxic environment.

Enzymatic Kinetic Properties of the Lactate Dehydrogenase Isoenzyme C4 of the Plateau Pika (Ochotona curzoniae).

Testis-specific lactate dehydrogenase (LDH-C4) is one of the lactate dehydrogenase (LDH) isozymes that catalyze the terminal reaction of pyruvate to lactate in the glycolytic pathway. LDH-C4 in mammals was previously thought to be expressed only in spermatozoa and testis and not in other tissues. Plateau pika (Ochotona curzoniae) belongs to the genus Ochotona of the Ochotonidea family. It is a hypoxia-tolerant species living in remote mountain areas at altitudes of 3000-5000 m above sea level on the Qinghai-Tibet Plateau. Surprisingly, Ldh-c is expressed not only in its testis and sperm, but also in somatic tissues of plateau pika. To shed light on the function of LDH-C4 in somatic cells, Ldh-a, Ldh-b, and Ldh-c of plateau pika were subcloned into bacterial expression vectors. The pure enzymes of Lactate Dehydrogenase A4 (LDH-A4), Lactate Dehydrogenase B4 (LDH-B4), and LDH-C4 were prepared by a series of expression and purification processes, and the three enzymes were identified by the method of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis (PAGE). The enzymatic kinetics properties of these enzymes were studied by Lineweaver-Burk double-reciprocal plots. The results showed the Michaelis constant (Km) of LDH-C4 for pyruvate and lactate was 0.052 and 4.934 mmol/L, respectively, with an approximate 90 times higher affinity of LDH-C4 for pyruvate than for lactate. At relatively high concentrations of lactate, the inhibition constant (Ki) of the LDH isoenzymes varied: LDH-A4 (Ki = 26.900 mmol/L), LDH-B4 (Ki = 23.800 mmol/L), and LDH-C4 (Ki = 65.500 mmol/L). These data suggest that inhibition of lactate by LDH-A4 and LDH-B4 were stronger than LDH-C4. In light of the enzymatic kinetics properties, we suggest that the plateau pika can reduce reliance on oxygen supply and enhance its adaptation to the hypoxic environments due to increased anaerobic glycolysis by LDH-C4.

Testis-Specific Lactate Dehydrogenase (LDH-C4) in Skeletal Muscle Enhances a Pika's Sprint-Running Capacity in Hypoxic Environment.

LDH-C4 is a lactate dehydrogenase that catalyzes the conversion of pyruvate to lactate. In mammals, ldh-c was originally thought to be expressed only in testis and spermatozoa. Plateau pika (Ochotona curzoniae), which belongs to the genus Ochotona of the Ochotonidea family, is a hypoxia tolerant mammal living 3000-5000 m above sea level on the Qinghai-Tibet Plateau, an environment which is strongly hypoxic. Ldh-c is expressed not only in testis and sperm but also in somatic tissues of plateau pika. In this study, the effects of N-propyl oxamate and N-isopropyl oxamate on LDH isozyme kinetics were compared to screens for a selective inhibitor of LDH-C4. To reveal the role and physiological mechanism of LDH-C4 in skeletal muscle of plateau pika, we investigated the effect of N-isopropyl oxamate on the pika exercise tolerance as well as the physiological mechanism. Our results show that Ki of N-propyl oxamate and N-isopropyl oxamate for LDH-A4, LDH-B4, and LDH-C4 were 0.094 mmol/L and 0.462 mmol/L, 0.119 mmol/L and 0.248 mmol/L, and 0.015 mmol/L and 0.013 mmol/L, respectively. N-isopropyl oxamate is a powerful selective inhibitor of plateau pika LDH-C4. In our exercise tolerance experiment, groups treated with inhibitors had significantly lower swimming times than the uninhibited control group. The inhibition rates of LDH, LD, and ATP were 37.12%, 66.27%, and 32.42%, respectively. Our results suggested that ldh-c is expressed in the skeletal muscle of plateau pika, and at least 32.42% of ATP in the skeletal muscle is catalyzed by LDH-C4 by anaerobic glycolysis. This suggests that pika has reduced dependence on oxygen and enhanced adaptation to hypoxic environment due to increased anaerobic glycolysis by LDH-C4 in skeletal muscle. LDH-C4 in plateau pika plays the crucial role in anaerobic glycolysis and generates ATP rapidly since this is the role of LDH-A4 in most species on plain land, which provide evidence that the native humans and animals in Qinghai-Tibet plateau can adapt to the hypoxia environment.

[The expression of the sperm-specific lactate dehydrogenase gene Ldh-c in plateau pika (Ochotona curzoniae) cardiac muscle and its effect on the anaerobic glycolysis].

The plateau pika (Ochotona curzoniae) has a strong adaptability to hypoxic plateau environment. We found that the sperm-specific lactate dehydrogenase (LDH-C4) gene Ldh-c expressed in plateau pika cardiac muscle. In order to shed light on the effect of LDH-C4 on the anaerobic glycolysis in plateau pika cardiac muscle, 20 pikas were randomly divided into the inhibitor group and the control group, and the sample size of each group was 10. The pikas of inhibitor group were injected with 1 mL 1 mol/L N-isopropyl oxamate, a specific LDH-C4 inhibitor, in biceps femoris muscle of hind legs, each leg with 500 µL. The pikas of control group were injected with the same volume of normal saline (0.9% NaCl). The mRNA and protein expression levels of Ldh-c gene in plateau pika cardiac muscle were determined by real-time PCR and Western blot. The activities of LDH, and the contents of lactate (LD) and ATP in cardiac muscle were compared between the inhibitor group and the control group. The results showed that 1) the expression levels of Ldh-c mRNA and protein were 0.47 ± 0.06 and 0.68 ± 0.08, respectively; 2) 30 min after injection of 1 mL 1 mol/L N-isopropyl oxamate in biceps femoris muscle, the concentration of N-isopropyl oxamate in blood was 0.08 mmol/L; 3) in cardiac muscle of the inhibitor group and the control group, the LDH activities were (6.18 ± 0.48) U/mg and (9.08 ± 0.58) U/mg, the contents of LD were (0.21 ± 0.03) mmol/g and (0.26 ± 0.04) mmol/g, and the contents of ATP were (4.40 ± 0.69) nmol/mg and (6.18 ± 0.73) nmol/mg (P < 0.01); 5) the inhibition rates of N-isopropyl oxamate to LDH, LD and ATP were 31.98%, 20.90% and 28.70%, respectively. The results suggest that Ldh-c expresses in cardiac muscle of plateau pika, and the pika cardiac muscle may get at least 28% ATP for its activities by LDH-C4 catalyzed anaerobic glycolysis, which reduces the dependence on oxygen and enhances the adaptation to the hypoxic environments.

[Establishment of RP-HPLC detection method of N-isopropyl oxamate in the serum of plateau pikas].

OBJECTIVE: To explore the intergrating of N-isopropyl oxamate and serum protein and establish a high performance liquid chromatography (HPLC) detection method of N-isopropyl oxamate (specific inhibitor of testis-specific lactate dehydrogenase (LDH-C4)) in the blood of plateau pikas. METHODS: Twenty highland pika 150-200 g, were randomly divided into two groups (n = 10): control group and inhibitor group. Different concentrations of N-isopropyl oxamate were added to examine the intergrating of N-isopropyl oxamate and serum protein. In order to determine its concentration in the pika blood accurately, we used the method of adding trypsin to incubate the serum first, followed by trichloroacetic acid treatment and detecting by HPLC. Results: When the concentrations of N-isopropyl oxamate in the pika serum were added to 0.05 mmol/L, 0.1 mmol/L, 1 mmol/L, 10 mmol/L, 16.7 mmol/L, 33.3 mmol/L and 100 mmol/L, the intergrating rates between N-isopropyl oxamate and plateau pika serum were 100%, 100%, 100%, 86.84%, 54.11%, 40.10% and 20.18%, respectively. The method established in this paper was good on recovery rates, precision and stability. A good linearity was obtained in the range of 0.0125-0.25 mmol/L. When the concentrations of N-isopropyl oxamate in the serum were added to 0. 15 mmol/L,0.3 mmol/L and 1 mmol/L, the recovery rates were 98.05%, 98.98% and 98.12%, respectively; the precision relative standard deviation( RSD) of concentrations were 1.17%, 0.92% and 0.83%, respectively; the stability relative standard deviation (RSD) of concentrations were 1.38%, 1.40% and 0.88%, respectively. The repeatability RSD of the method was 1.76%. Quantitative limit was 0.0125 mmol/L. CONCLUSION: N-isopropyl oxamate has a strong affinity with plateau pika serum protein that can't be accurately determined with common HIPLC method. It can be accurately determined in the blood by adding trypsinto digest the serum protein first, followed by adding trichloroacetic acid to precipitate the protein.

The plateau zokors' learning and memory ability is related to the high expression levels of foxP2 in the brain.

Plateau zokor (Myospalax baileyi) is a subterranean mammal. Plateau zokor has high learning and memory ability, and can determine the location of blocking obstacles in their tunnels. Forkhead box p2 (FOXP2) is a transcription factor implicated in the neural control of orofacial coordination and sensory-motor integration, particularly with respect to learning, memory and vocalization. To explore the association of foxP2 with the high learning and memory ability of plateau zokor, the cDNA of foxP2 of plateau zokor was sequenced; by using plateau pika as control, the expression levels of foxP2 mRNA and FOXP2 protein in brain of plateau zokor were determined by real-time PCR and Western blot, respectively; and the location of FOXP2 protein in the brain of plateau zokor was determined by immunohistochemistry. The result showed that the cDNA sequence of plateau zokor foxP2 was similar to that of other mammals and the amino acid sequences showed a relatively high degree of conservation, with the exception of two particular amino acid substitutions [a Gln (Q)-to-His (H) change at position 231 and a Ser (S)-to-Ile (I) change at position 235]. Higher expression levels of foxP2 mRNA (3-fold higher) and FOXP2 protein (>2-fold higher) were detected in plateau zokor brain relative to plateau pika brain. In plateau zokor brain, FOXP2 protein was highly expressed in the cerebral cortex, thalamus and the striatum (a basal ganglia brain region). The results suggest that the high learning and memory ability of plateau zokor is related to the high expression levels of foxP2 in the brain.

Testis-specific lactate dehydrogenase is expressed in somatic tissues of plateau pikas.

LDH-C4 is a lactate dehydrogenase that catalyzes the interconversion of pyruvate with lactate. In mammals the, Ldh-c gene was originally thought to be expressed only in testis and spermatozoa. Plateau pika (Ochotona curzoniae), belonging to the genus Ochotona of the Ochotonidea family, is a hypoxia tolerant mammal living at 3000-5000 m above sea levelon the Qinghai-Tibet Plateau. We found that the expression pattern of six LDH isoenzymes in the somatic tissues of female and male plateau pikas to be the same as those in testis and sperm, suggesting that LDH-C4 was expressed in somatic tissues of plateau pika. Here we report the detection of LDHC in the somatic tissues of plateau pika using RT-PCR, Western blotting and immunohistochemistry. Our results indicate that Ldh-c mRNA is transcribed in the heart, liver, lung, kidney, brain, skeletal muscle and testis. In somatic tissues LDHC was translated in the cytoplasm, while in testis it was expressed in both cytoplasm and mitochondria. The third band from cathode to anode in LDH isoenzymes was identified as LDH-C4. The finding that Ldh-c is expressed in both somatic tissues and testis of plateau pika provides important implications for more in-depth research into the Ldh-c function in mammals.

Differences of glycolysis in skeletal muscle and lactate metabolism in liver between plateau zokor (Myospalax baileyi) and plateau pika (Ochotona curzoniae).

The plateau pika (Ochotona curzoniae) and plateau zokor (Myospalax baileyi) are specialized native species of the Qinghai-Tibetan plateau. The goal of this study was to examine physiological differences in skeletal muscle glycolysis and hepatic lactate metabolism between these two species. The partial sequence of pyruvate carboxylase (PC) gene was cloned and sequenced. The mRNA expression levels of PC and lactate dehydrogenases (LDH-A, LDH-B) were determined by real-time PCR. The enzymatic activity of PC was measured using malic acid coupling method. The concentration of lactic acid (LD) and the specific activities of LDH in liver and skeletal muscle of two species were measured. The different isoenzymes of LDH were determined by native polyacrylamide gel electrophoresis (PAGE). The results showed that, (1) LDH-B mRNA level in skeletal muscle of plateau zokor was significantly higher than that of plateau pika (P < 0.01), but no differences was found at LDH-A mRNA levels between them (P > 0.05); (2) PC, LDH-A and LDH-B mRNA levels in liver of plateau pika were significantly higher than those of plateau zokor (P < 0.01); (3) The LDH activity and concentration of LD in skeletal muscle and liver, as well as the PC activity in liver of plateau pika were significantly higher than those of plateau zokor (P < 0.01); (4) The isoenzymatic spectrum of lactate dehydrogenase showed that the main LDH isoenzymes were LDH-A4, LDH-A3B and LDH-A2B2 in skeletal muscle of plateau pika, while the main LDH isoenzymes were LDH-AB3 and LDH-B4 in skeletal muscle of plateau zokor; the main isoenzymes were LDH-A3B, LDH-A2B2, LDH-AB3 and LDH-B4 in liver of plateau pika, while LDH-A4 was the only isoenzyme in liver of plateau zokor. These results indicate that the plateau pika gets most of its energy for sprint running through enhancing anaerobic glycolysis, producing more lactate in their skeletal muscle, and converting lactate into glucose and glycogen in the liver by enhancing gluconeogenesis. As a result, the plateau pika has a reduced dependence on oxygen in its hypoxic environment. In contrast, plateau zokor derives most of its energy used for digging activity by enhancing aerobic oxidation in their skeletal muscle, although they inhabit hypoxic underground burrows.

[Functional difference of malate-aspartate shuttle system in liver between plateau zokor (Myospalax baileyi) and plateau pika (Ochotona curzoniae)].

To explore the adaptive mechanisms of plateau zokor (Myospalax baileyi) to the enduring digging activity in the hypoxic environment and of plateau pika (Ochotona curzoniae) to the sprint running activity, the functional differences of malate-aspartate shuttle system (MA) in liver of plateau zokor and plateau pika were studied. The ratio of liver weight to body weight, the parameters of mitochondria in hepatocyte and the contents of lactic acid in serum were measured; the open reading frame of cytoplasmic malate dehydrogenase (MDH1), mitochondrial malate dehydrogenase (MDH2), and the partial sequence of aspartate glutamate carrier (AGC) and oxoglutarate malate carrier (OMC) genes were cloned and sequenced; MDH1, MDH2, AGC and OMC mRNA levels were determined by real-time PCR; the specific activities of MDH1 and MDH2 in liver of plateau zokor and plateau pika were measured using enzymatic methods. The results showed that, (1) the ratio of liver weight to body weight, the number and the specific surface of mitochondria in hepatocyte of plateau zokor were markedly higher than those of plateau pika (P < 0.01 or P < 0.05), but the content of lactic acid in serum of plateau pika was significantly higher than that of plateau zokor (P < 0.01); (2) MDH1 and MDH2 mRNA levels as well as their enzymatic activities in liver of plateau zokor were significantly higher than those of plateau pika (P < 0.01 or 0.05), AGC mRNA level of the zokor was significantly higher than that of the pika (P < 0.01), while no difference was found at OMC mRNA level between them (P > 0.05); (3) mRNA level and enzymatic activity of MDH1 was significantly lower than those of MDH2 in the pika liver (P < 0.01), MDH1 mRNA level of plateau zokor was markedly higher than that of MDH2 (P < 0.01), but the activities had no difference between MDH1 and MDH2 in liver of the zokor (P > 0.05). These results indicate that the plateau zokor obtains ATP in the enduring digging activity by enhancing the function of MA, while plateau pika gets glycogen for their sprint running activity by increasing the process of gluconeogenesis. As a result, plateau pika converts the lactic acid quickly produced in their skeletal muscle by anaerobic glycolysis and reduces dependence on the oxygen.

[Gene coding and mRNA expression of vascular endothelial growth factor as well as microvessel density in brain of plateau zokor: comparison with other rodents].

Vascular endothelial growth factor (VEGF) plays an important role in tissues angiogenesis. The adaptation of animals to hypoxic environment is relative to the microvessel density (MVD) in tissues. To further explore the adaptation mechanisms of plateau zokor (Myospalax baileyi) to the hypoxic-hypercapnic burrows, the VEGF mRNA and the MVD in cerebral tissues of the plateau zokor were studied. Total RNA was isolated from liver, and VEGF cDNA was obtained by RT-PCR, then the VEGF cDNA was cloned and sequenced. The coding sequence of plateau pika (Ochotona curzniae), rat (Rattus norvegicus) and mouse (Mus musculus) VEGF cDNA are obtained from GenBank, and the nucleotide and amino acid sequence homology of plateau zokor VEGF cDNA coding sequence with that of plateau pika, rat and mouse were analyzed and compared by using of bioinformatics software. The VEGF mRNA was detected by real-time PCR, and the MVDs in cerebral tissues of the plateau zokor, plateau pika and Sprague-Dawley (SD) rat were measured by immunohistochemical staining. The results showed that the open reading frame of the plateau zokor VEGF was 645 bp, and the coding sequence of the plateau zokor VEGF cDNA shared 92.1%, 93.6% and 93.8% nucleotide sequence homology to that of the plateau pika, rat and mouse, respectively. The deduced amino acid sequence of the plateau zokor VEGF cDNA was composed of 188 amino acids and the amino acids from 1 to 26 were signal peptide sequence. The plateau zokor VEGF188 was 90.2%, 94.9% and 94.4% homologous to that of plateau pika, rat and mouse. The level of VEGF mRNA in brain of the plateau zokor was significantly lower than that of SD rat, but there was no obvious difference in VEGF mRNA level between plateau zokor and plateau pika. The MVD in brain of the plateau zokor was markedly higher than that of plateau pika and SD rat. In conclusion, plateau zokor enhances its adaptation to the hypoxic environment by increasing the MVD. The level of VEGF mRNA in the brain of plateau zokor is lower than that of SD rat, which may be as a result of inhibition by the higher concentration of carbon dioxide in the burrow.