Fiber Specific Changes in Sphingolipid Metabolism in Skeletal Muscles of Hyperthyroid Rats

Thyroid hormones (T₃, T₄) are well known modulators of different cellular signals including the sphingomyelin pathway. However, studies regarding downstream effects of T₃ on sphingolipid metabolism in skeletal muscle are scarce. In the present work we sought to investigate the effects of hyperthyroidism on the activity of the key enzymes of ceramide metabolism as well as the content of fundamental sphingolipids. Based on fiber/metabolic differences, we chose three different skeletal muscles, with diverse fiber compositions: soleus (slow-twitch oxidative), red (fast-twitch oxidative-glycolytic) and white (fast-twitch glycolytic) section of gastrocnemius. We demonstrated that T₃ induced accumulation of sphinganine, ceramide, sphingosine, as well as sphingomyelin, mostly in soleus and in red, but not white section of gastrocnemius. Concomitantly, the activity of serine palmitoyltransferase and acid/neutral ceramidase was increased in more oxidative muscles. In conclusion, hyperthyroidism induced fiber specific changes in the content of sphingolipids that were relatively more related to de novo synthesis of ceramide rather than to its generation via hydrolysis of sphingomyelin.     

N-取代苄基-N’-苯甲酰氧乙基哌嗪类化合物的合成及其生物活性

合成了15个新的N-取代苄基-N’-苯甲酰氧乙基哌嗪类化合物,其结构经红外光谱、核磁共振氢谱、元素分析及质谱确证,平初步测定了目标化合物的生物活性。结果表明,目标化合物均表现出一定的对血清诱导的血管平滑肌细胞增殖的抑制活性,其中化合物ⅣC、Ⅳd、Ⅳh活性较高。     

Analysis of the Binding Sites of Porcine Sialoadhesin Receptor with PRRSV

Porcine reproductive and respiratory syndrome virus (PRRSV) can infect pigs and cause enormous economic losses to the pig industry worldwide. Porcine sialoadhesin (pSN) and CD163 have been identified as key viral receptors on porcine alveolar macrophages (PAM), a main target cell infected by PRRSV. In this study, the protein structures of amino acids 1–119 from the pSN and cSN (cattle sialoadhesin) N-termini (excluding the 19-amino acid signal peptide) were modeled via homology modeling based on mSN (mouse sialoadhesin) template structures using bioinformatics tools. Subsequently, pSN and cSN homology structures were superposed onto the mSN protein structure to predict the binding sites of pSN. As a validation experiment, the SN N-terminus (including the wild-type and site-directed-mutant-types of pSN and cSN) was cloned and expressed as a SN-GFP chimera protein. The binding activity between SN and PRRSV was confirmed by WB (Western blotting), FAR-WB (far Western blotting), ELISA (enzyme-linked immunosorbent assay) and immunofluorescence assay. We found that the S107 amino acid residue in the pSN N-terminal played a crucial role in forming a special cavity, as well as a hydrogen bond for enhancing PRRSV binding during PRRSV infection. S107 may be glycosylated during PRRSV infection and may also be involved in forming the cavity for binding PRRSV along with other sites, including W2, Y44, S45, R97, R105, W106 and V109. Additionally, S107 might also be important for pSN binding with PRRSV. However, the function of these binding sites must be confirmed by further studies.     

Effect of grass vs. concentrate feeding on the fatty acid profile of different fat depots in lambs

The aim of this study was to produce high‐quality meat from lambs under different feeding conditions, as measured by the accumulation of n‐3 fatty acids and conjugated linoleic acids (CLA) in muscle and subcutaneous fat. In total, 13 male crossbred lambs (Black Head×Gotland), each at 24 kg live weight, were divided into two feeding groups. Lambs were kept either on pasture (pasture grazing, n = 6) or in the stable (concentrate feeding, n = 7). The linolenic acid (C18:3n‐3) contained in the grass was absorbed and deposited into the different lipid classes of muscle and subcutaneous fat. The proportion of total n‐3 fatty acids in the different lipids of grazing lambs was significantly (p = 0.05) higher compared to that in concentrate‐fed lambs. The n‐6/n‐3 ratio (mean ± SEM) in muscle of grazing lambs was 1.2 ± 0.09 in contrast to 2.3 ± 0.09 (p = 0.05) of the animals kept in the stable. In subcutaneous fat, this ratio was 0.9 ± 0.2 in lambs kept on pasture versus 3.5 ± 0.2 (p = 0.05) after indoor keeping. The relative concentration of C18:1trans‐11 in total muscle lipids, phospholipids, triacylglycerols and subcutaneous fat was significantly increased by grass feeding compared to concentrate feeding. Significant influences of feeding were shown for saturated fatty acids. In concentrate‐fed lambs, a lower content of saturated fatty acids was detected. The proportion of CLAcis‐9,trans‐11 (1.9 ± 0.2% vs. 1.1 ± 0.1% in muscle, 2.5 ± 0.2% vs. 1.4 ± 0.2% in subcutaneous fat, 0.7 ± 0.04% vs. 0.4 ± 0.04% in phospholipids) in lambs was significantly (p = 0.05) higher after grazing than after concentrate feeding, respectively.     

Therapeutic Effect of Losartan,an Angiotensin II Type 1 Receptor Antagonist,on CCl4-Induced Skeletal Muscle Injury

TGF-β1 is known to inhibit muscle regeneration after muscle injury. However, it is unknown if high systemic levels of TGF-β can affect the muscle regeneration process. In the present study, we demonstrated the effect of a CCl₄ intra-peritoneal injection and losartan (an angiotensin II type 1 receptor antagonist) on skeletal muscle (gastrocnemius muscle) injury and regeneration. Male C57BL/6 mice were grouped randomly as follows: control (n = 7), CCl₄-treatment group (n = 7), and CCl₄ + losartan treatment group (n = 7). After CCl₄ treatment for a 16-week period, the animals were sacrificed and analyzed. The expression of dystrophin significantly decreased in the muscle tissues of the control group, as compared with that of the CCl₄ + losartan group (p < 0.01). p(phospho)-Smad2/3 expression significantly increased in the muscles of the control group compared to that in the CCl₄ + losartan group (p < 0.01). The expressions of Pax7, MyoD, and myogenin increased in skeletal muscles of the CCl₄ + losartan group compared to the corresponding levels in the control group (p < 0.01). We hypothesize that systemically elevated TGF-β1 as a result of CCl₄-induced liver injury causes skeletal muscle injury, while losartan promotes muscle repair from injury via blockade of TGF-β1 signaling.     

Inducing Energetic Switching Using Klotho Improves Vascular Smooth Muscle Cell Phenotype

The cardiovascular disease of atherosclerosis is characterised by aged vascular smooth muscle cells and compromised cell survival. Analysis of human and murine plaques highlights markers of DNA damage such as p53, Ataxia telangiectasia mutated (ATM), and defects in mitochondrial oxidative metabolism as significant observations. The antiageing protein Klotho could prolong VSMC survival in the atherosclerotic plaque and delay the consequences of plaque rupture by improving VSMC phenotype to delay heart attacks and stroke. Comparing wild-type VSMCs from an ApoE model of atherosclerosis with a flox’d Pink1 knockout of inducible mitochondrial dysfunction we show WT Pink1 is essential for normal cell viability, while Klotho mediates energetic switching which may preserve cell survival. Methods: Wild-type ApoE VSMCs were screened to identify potential drug candidates that could improve longevity without inducing cytotoxicity. The central regulator of cell metabolism AMP Kinase was used as a readout of energy homeostasis. Functional energetic switching between oxidative and glycolytic metabolism was assessed using XF24 technology. Live cell imaging was then used as a functional readout for the WT drug response, compared with Pink1 (phosphatase-and-tensin-homolog (PTEN)-induced kinase-1) knockout cells. Results: Candidate drugs were assessed to induce pACC, pAMPK, and pLKB1 before selecting Klotho for its improved ability to perform energetic switching. Klotho mediated an inverse dose-dependent effect and was able to switch between oxidative and glycolytic metabolism. Klotho mediated improved glycolytic energetics in wild-type cells which were not present in Pink1 knockout cells that model mitochondrial dysfunction. Klotho improved WT cell survival and migration, increasing proliferation and decreasing necrosis independent of effects on apoptosis. Conclusions: Klotho plays an important role in VSMC energetics which requires Pink1 to mediate energetic switching between oxidative and glycolytic metabolism. Klotho improved VSMC phenotype and, if targeted to the plaque early in the disease, could be a useful strategy to delay the effects of plaque ageing and improve VSMC survival.     

The Effect of Long-Lasting Swimming on Rats Skeletal Muscles Energy Metabolism after Nine Days of Dexamethasone Treatment

This study investigates the effect of Dexamethasone (Dex) treatment on blood and skeletal muscle metabolites level and skeletal muscle activity of enzymes related to energy metabolism after long-duration swimming. To evaluate whether Dex treatment, swimming, and combining these factors act on analyzed data, rats were randomly divided into four groups: saline treatment non-exercise and exercise and Dex treatment non-exercised and exercised. Animals in both exercised groups underwent long-lasting swimming. The concentration of lipids metabolites, glucose, and lactate were measured in skeletal muscles and blood according to standard colorimetric and fluorimetric methods. Also, activities of enzymes related to aerobic and anaerobic metabolism were measured in skeletal muscles. The results indicated that Dex treatment induced body mass loss and increased lipid metabolites in the rats’ blood but did not alter these changes in skeletal muscles. Interestingly, prolonged swimming applied after 9 days of Dex treatment significantly intensified changes induced by Dex; however, there was no difference in skeletal muscle enzymatic activities. This study shows for the first time the cumulative effect of exercise and Dex on selected elements of lipid metabolism, which seems to be essential for the patient’s health due to the common use of glucocorticoids like Dex.     

Transforming Growth Factor-Beta in Skeletal Muscle Wasting

Transforming growth factor-beta (TGF-β) is part of a family of molecules that is present in many body tissues and performs many different functions. Evidence has been obtained from mice and human cancer patients with bony metastases and non-metastatic disease, as well as pediatric burn patients, that inflammation leads to bone resorption and release of TGF-β from the bone matrix with paracrine effects on muscle protein balance, possibly mediated by the generation of reactive oxygen species. Whether immobilization, which confounds the etiology of bone resorption in burn injury, also leads to the release of TGF-β from bone contributing to muscle wasting in other conditions is unclear. The use of anti-resorptive therapy in both metastatic cancer patients and pediatric burn patients has been successful in the prevention of muscle wasting, thereby creating an additional therapeutic niche for this class of drugs. The liberation of TGF-β may be one way in which bone helps to control muscle mass, but further investigation will be necessary to assess whether the rate of bone resorption is the determining factor for the release of TGF-β. Moreover, whether different resorptive conditions, such as immobilization and hyperparathyroidism, also involve TGF-β release in the pathogenesis of muscle wasting needs to be investigated.     

Effect of Porcine Whole Blood Protein Hydrolysate on Slow-Twitch Muscle Fiber Expression and Mitochondrial Biogenesis via the AMPK/SIRT1 Pathway

Skeletal muscle is a heterogeneous tissue composed of a variety of functionally different fiber types. Slow-twitch type I muscle fibers are rich with mitochondria, and mitochondrial biogenesis promotes a shift towards more slow fibers. Leucine, a branched-chain amino acid (BCAA), regulates slow-twitch muscle fiber expression and mitochondrial function. The BCAA content is increased in porcine whole-blood protein hydrolysates (PWBPH) but the effect of PWBPH on muscle fiber type conversion is unknown. Supplementation with PWBPH (250 and 500 mg/kg for 5 weeks) increased time to exhaustion in the forced swimming test and the mass of the quadriceps femoris muscle but decreased the levels of blood markers of exercise-induced fatigue. PWBPH also promoted fast-twitch to slow-twitch muscle fiber conversion, elevated the levels of mitochondrial biogenesis markers (SIRT1, p-AMPK, PGC-1α, NRF1 and TFAM) and increased succinate dehydrogenase and malate dehydrogenase activities in ICR mice. Similarly, PWBPH induced markers of slow-twitch muscle fibers and mitochondrial biogenesis in C2C12 myotubes. Moreover, AMPK and SIRT1 inhibition blocked the PWBPH-induced muscle fiber type conversion in C2C12 myotubes. These results indicate that PWBPH enhances exercise performance by promoting slow-twitch muscle fiber expression and mitochondrial function via the AMPK/SIRT1 signaling pathway.     

吐温修饰脂肪酶的研究

分别用甲醛和戊二醛作为偶联剂,共价连接吐温80和猪胰脂肪酶,回收共价修饰脂肪酶,并且比较修饰酶与自然酶在性质上的差异。当甲醛和戊二醛浓度相同时,用甲醛连接的修饰酶的活力最高,而用戊二醛连接的修饰酶的氨基连接效率高。当用不同浓度的甲醛修饰时,发现浓度为1．25mg·mL的甲醛连接的修饰酶的活力最高。在碱性条件下,修饰酶比自然酶的活力高,在中性、酸性条件下,自然酶的活力高