Female athlete triad affects rat intestinal morphology and sucrase-isomaltase expression.

Female athletes follow a strict diet and perform rigorous exercise to boost their performance, which induces health issues called the female athlete triad (FAT), defined as the combination of disordered eating, amenorrhoea and low bone mineral density. It is known to have a significant effect on bones. However, its effects on the small intestine, which is responsible for nutrient uptake into the body, remain unclear. In this study, we created an animal model of FAT to examine its effects on digestive and absorptive molecules in the small intestine. Thirty 5-week-old female Sprague-Dawley (sd) rats with an initial body weight of about 147 g were divided into control (Con, n = 7), exercise (Ex, n = 7), food restriction (FR, n = 8) and exercise plus food restriction (FAT, n = 8) groups. The rats were subjected to 4 weeks of wheel running (Ex, FAT) and 50-40 % food restriction (FR, FAT) to examine the effects on bone and typical digestive enzymes and transporters in the jejunum. Two-way ANOVA and the Kruskal-Wallis test were used for statistical analysis of normal and non-normal data, respectively. Four weeks of exercise and food restriction decreased bone weight (vs. other group P < 0·01) and bone breaking power (vs. other group P < 0·01). Villus height decreased in the jejunum (vs. other group P < 0·01), but the expression of typical macronutrients digestive enzyme and absorptive molecules remained unchanged. In contrast, sucrase-isomaltase gene (v. Ex P = 0·02) and protein expression were increased (vs. other group P < 0·05). The study findings show that FAT affects sucrase-isomaltase without histone methylation changes.

Acute cold stress induces transient MuRF1 upregulation in the skeletal muscle of zebrafish.

Cryotherapy is one of the most common treatments for trauma or fatigue in the field of sports medicine. However, the molecular biological effects of acute cold exposure on skeletal muscle remain unclear. Therefore, we used zebrafish, which have recently been utilized as an animal model for skeletal muscle, to comprehensively investigate and selectively clarify the time-course changes induced by cryotherapy. Zebrafish were exposed intermittently to cold stimulation three times for 15 min each. Thereafter, skeletal muscle samples were collected after 15 min and 1, 2, 4, and 6 h. mRNA sequencing revealed the involvement of trim63a, fbxo32, fbxo30a, and klhl38b in "protein ubiquitination" from the top 10 most upregulated genes. Subsequently, we examined the time-course changes of the four genes by quantitative PCR, and their expression peaked 2 h after cryotherapy and returned to baseline after 6 h. Moreover, the proteins encoded by trim63a and fbxo32 (muscle-specific RING finger protein 1 [MuRF1] and muscle atrophy F-box, respectively), which are known to be major genes encoding E3 ubiquitin ligases, were examined by western blotting, and MuRF1 expression displayed similar temporal changes as trim63a expression. These findings suggest that acute cold exposure transiently upregulates E3 ubiquitin ligases, especially MuRF1; thus, cryotherapy may contribute to the treatment of trauma or fatigue by promoting protein processing.

One Week of CDAHFD Induces Steatohepatitis and Mitochondrial Dysfunction with Oxidative Stress in Liver.

The prevalence of nonalcoholic fatty liver disease (NAFLD) has been rapidly increasing worldwide. A choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) has been used to create a mouse model of nonalcoholic steatohepatitis (NASH). There are some reports on the effects on mice of being fed a CDAHFD for long periods of 1 to 3 months. However, the effect of this diet over a short period is unknown. Therefore, we examined the effect of 1-week CDAHFD feeding on the mouse liver. Feeding a CDAHFD diet for only 1-week induced lipid droplet deposition in the liver with increasing activity of liver-derived enzymes in the plasma. On the other hand, it did not induce fibrosis or cirrhosis. Additionally, it was demonstrated that CDAHFD significantly impaired mitochondrial respiration with severe oxidative stress to the liver, which is associated with a decreasing mitochondrial DNA copy number and complex proteins. In the gene expression analysis of the liver, inflammatory and oxidative stress markers were significantly increased by CDAHFD. These results demonstrated that 1 week of feeding CDAHFD to mice induces steatohepatitis with mitochondrial dysfunction and severe oxidative stress, without fibrosis, which can partially mimic the early stage of NASH in humans.

Improvement of low-intensity long-time running performance in rats by intake of glucosyl hesperidin.

Recently, the use of ergogenic aids in sports by both athletes and fans has increased. Moreover, the overall demand for new ergogenic aids has increased. Hesperidin is a polyphenol that is useful for improving exercise performance by activating energy generation through ß-oxidation and oxidative phosphorylation in skeletal muscles. However, it is difficult to use this compound as an ergogenic aid because of its poor water solubility and low bioavailability. Glucosyl hesperidin is formed when one molecule of glucose is transferred to hesperidin via glycosyl-transferase. It is 10,000× more soluble and has 3.7× higher bioavailability than hesperidin. In this study, we assessed whether continuous (14 days) intake of glucosyl hesperidin improves the aerobic exercise capacity of rats during long-term acute exercise. Although glucosyl hesperidin intake did not improve the performance of high-intensity running (30 m/min), we did observe improvement in low-intensity running (15 m/min) (p < 0.05). We demonstrate that in sedentary rats, glucosyl hesperidin intake increased ß-oxidation and oxidative phosphorylation in the skeletal muscle (p < 0.05 and p < 0.01, respectively). Glucosyl hesperidin intake may have created a metabolic state useful for long-term exercise. In conclusion, the continuous intake of glucosyl hesperidin improved the aerobic exercise capacity of rats during long-term acute exercise.

Cyclic-AMP response element binding protein (CREB) and microRNA miR-29b regulate renalase gene expression under catecholamine excess conditions.

AIMS: Renalase, a key mediator of cross-talk between kidneys and sympathetic nervous system, exerts protective roles in various cardiovascular/renal disease states. However, molecular mechanisms underpinning renalase gene expression remain incompletely understood. Here, we sought to identify the key molecular regulators of renalase under basal/catecholamine-excess conditions. MATERIALS AND METHODS: Identification of the core promoter domain of renalase was carried out by promoter-reporter assays in N2a/HEK-293/H9c2 cells. Computational analysis of the renalase core promoter domain, over-expression of cyclic-AMP-response-element-binding-protein (CREB)/dominant negative mutant of CREB, ChIP assays were performed to determine the role of CREB in transcription regulation. Role of the miR-29b-mediated-suppression of renalase was validated in-vivo by using locked-nucleic-acid-inhibitors of miR-29. qRT-PCR and Western-blot analyses measured the expression of renalase, CREB, miR-29b and normalization controls in cell lysates/ tissue samples under basal/epinephrine-treated conditions. KEY FINDINGS: CREB, a downstream effector in epinephrine signaling, activated renalase expression via its binding to the renalase-promoter. Physiological doses of epinephrine and isoproterenol enhanced renalase-promoter activity and endogenous renalase protein level while propranolol diminished the promoter activity and endogenous renalase protein level indicating a potential role of beta-adrenergic receptor in renalase gene regulation. Multiple animal models (acute exercise, genetically hypertensive/stroke-prone mice/rat) displayed directionally-concordant expression of CREB and renalase. Administration of miR-29b inhibitor in mice upregulated endogenous renalase expression. Moreover, epinephrine treatment down-regulated miR-29b promoter-activity/transcript levels. SIGNIFICANCE: This study provides evidence for renalase gene regulation by concomitant transcriptional activation via CREB and post-transcriptional attenuation via miR-29b under excess epinephrine conditions. These findings have implications for disease states with dysregulated catecholamines.

Age-related morphological and functional changes in the small intestine of senescence-accelerated mouse.

The ability of the small intestine to perform various functions, such as digestion/absorption of nutrients, gradually declines with age. However, the mechanism that causes intestinal senescence remains unclear. Therefore, age-related changes in the jejunum and ileum were evaluated using senescence-accelerated mouse (SAM) strains that possess characteristic phenotypes of aging. In particular, to understand how senescence affects the small intestine, we investigated whether age-related changes in the morphology of the intestinal villi and its capability to digest/absorb nutrients are associated with the senescence phenotypes identified in specific SAM strains. Four SAM strains were selected (SAMP1, SAMP6, SAMP10, and SAMR1; of which SAMR1 served as a control of SAMP strain) and age-related changes in the small intestine were evaluated for each strain. A villus morphological analysis, mRNA expression level analysis of the small intestine-specific molecules, and disaccharidase activity measurement were performed. We observed that the mRNA expression levels of the genes involved in the differentiation of intestinal epithelial cells and in the digestion/absorption of nutrients were markedly decreased in all the SAM strains, especially in the SAMP10 strain. Our results revealed that all the SAM strains spontaneously induced senescence of the small intestine, which occurred due to the disorders affecting the differentiation/maturation system of intestinal epithelial cells. In addition, it was evident that senile phenotypes, such as brain dysfunction, enhanced intestinal senescence in the SAMP10 strain. The results of this study suggest that the brain-intestinal nervous system may play role in maintenance of villous morphology and nutrients uptake via the GLP-2 and IGF-2 signaling pathway.

The Oral Administration of Honey Recovers the Function of the Small Intestine for Digestion and Absorption of Nutrients in a Rat Model of Total Parenteral Nutrition.

The oral administration of pure monosaccharides is effective for improving intestinal function such as nutrient digestion and absorption. However, day-to-day diets tend not to include high purity monosaccharides for intestinal health. Honey possesses large amounts of monosaccharides including glucose and fructose in the same ratio. In this study, we have evaluated the nutritional properties of honey and examined the effects of its oral ingestion on the recovery of intestinal function in the total parenteral nutrition (TPN) rat model. It was observed that honey remarkably recovered the function of the small intestine including the villous morphology, nutrient digestion, and absorption capabilities. In particular, the expression of disaccharidase was significantly enhanced by the ingestion of honey after TPN treatment. Therefore, oral intake of honey is effective in recovering and maintaining small intestinal functions and can potentially be used as a supplement for promoting small intestinal function recovery.

Comparative Effects of Allulose, Fructose, and Glucose on the Small Intestine.

Despite numerous studies on the health benefits of the rare sugar allulose, its effects on intestinal mucosal morphology and function are unclear. We therefore first determined its acute effects on the small intestinal transcriptome using DNA microarray analysis following intestinal allulose, fructose and glucose perfusion in rats. Expression levels of about 8-fold more genes were altered by allulose compared to fructose and glucose perfusion, suggesting a much greater impact on the intestinal transcriptome. Subsequent pathway analysis indicated that nutrient transport, metabolism, and digestive system development were markedly upregulated, suggesting allulose may acutely stimulate these functions. We then evaluated whether allulose can restore rat small intestinal structure and function when ingested orally following total parenteral nutrition (TPN). We also monitored allulose effects on blood levels of glucagon-like peptides (GLP) 1 and 2 in TPN rats and normal mice. Expression levels of fatty acid binding and gut barrier proteins were reduced by TPN but rescued by allulose ingestion, and paralleled GLP-2 secretion potentially acting as the mechanism mediating the rescue effect. Thus, allulose can potentially enhance disrupted gut mucosal barriers as it can more extensively modulate the intestinal transcriptome relative to glucose and fructose considered risk factors of metabolic disease.

Long-Term Habitual Exercise and Combination of ß-Hydroxy-ß-Methylbutyrate plus Black Ginger Alter the Autophagy and Mitochondria Related Genes in SAMP8 Mice.

Muscle mass and strength decrease with aging; however, habitual exercise can maintain muscle health. ß-Hydroxy-ß-methyl butyrate calcium (HMB) and black ginger (BG) improve muscle protein metabolism and energy production. Combining these two molecules, which have similar effects, may have a synergistic effect. Senescence-accelerated mouse-prone 8 (SAMP8) is a useful model of muscle aging. Therefore, we explored how the combination of habitual exercise, HMB, and BG affected muscle aging. We used 28-wk-old (28w) SAMP8 mice divided into six groups: 28 wk (28w), 44 wk (44w, Con), exercise (Ex), Ex+BG, Ex+HMB, and Ex+BG+HMB (Ex+Comb). Mice were required to run on a treadmill for 16 wk for 5 d per week. In 28w and 44w mice, grip strength tests and dissection were conducted. Muscle weight was measured, and qPCR and immunoblotting were conducted. Muscle mass and strength were declined in the 44w group. Exercise with HMB or BG alone had no effect, whereas muscle mass and strength were augmented in the Ex+Comb group. Similarly, levels of mitochondrial function- and biogenesis-related genes were increased. Autophagy-related protein (Atg3, 7, 16L1 and Beclin1) were altered in the Ex+Comb group. These results suggest that Ex+Comb affects autophagy. Overall, the combination of habitual exercise and HMB+BG may enhance muscle mass and strength by affecting the mitochondrial and autophagy systems in SAMP8.

Ultraviolet and Deep-Ultraviolet Excitation Photothermal Heterodyne Interferometer Combined with Semi-Micro HPLC.

A detection system consisting of a photothermal heterodyne interferometer (PHI) combined with semi-micro HPLC (high-performance liquid chromatography) has been designed and investigated. An ultraviolet (UV) or deep-UV laser emitting at 375 or 213 nm, respectively, was used for the excitation of nitro-polycyclic aromatic hydrocarbons (NPAHs) and amino acids. A photothermally induced change in the refractive index of the solvent causes an optical phase difference between two arms of the interferometer, one beam passing through the photoexcited region and another used as a reference, which was sensitively detected with the PHI. The separation and detection of NPAHs and amino acids were successfully demonstrated via semi-micro HPLC with the PHI and a UV detector. The detection limits of the UV-excitation PHI for NPAHs were 1.2 - 5.2 times better than that of the commercial UV detector, although the first demonstration of deep-UV excitation suffered from significant baseline fluctuation.

Gene expression level of renalase in the skeletal muscles is increased with high-intensity exercise training in mice on a high-fat diet.

INTRODUCTION: Exercise training is beneficial for reducing obesity. In particular, exercise training can lower the catecholamine concentration in circulation. Renalase, whose expression was first confirmed in the kidneys, is a physiologically active substance that decomposes circulating catecholamines; additionally, it has been reported to be present in the skeletal muscles. The aim of this study was to clarify the expression of renalase in the skeletal muscles and kidneys after high-intensity exercise training in obese mice. MATERIAL AND METHODS: The mice were divided into four groups: normal diet and sedentary, normal diet and exercise training, high-fat diet and sedentary, and high-fat diet and exercise training, and the test was performed for 8 weeks. RESULTS: Body weight and skeletal muscle wet weight were reduced by high-fat diet intake but were rescued by training. Skeletal muscle renalase gene expression was significantly increased by exercise training. However, in the kidneys the gene expression of renalase was significantly increased by high-fat diet intake and exercise training. No significant changes were observed in the gene expression of catecholamine-degrading enzymes, catechol-O-methyltransferase and monoamine oxidase A and B. CONCLUSION: We demonstrated that exercise training increased the gene expression of renalase in the skeletal muscles and kidneys, thus lowering circulating catecholamine levels. This may lead to amelioration of obesity as catecholamines are lipolytic.

Development of a gene doping detection method to detect overexpressed human follistatin using an adenovirus vector in mice.

BACKGROUND: Gene doping is the misuse of genome editing and gene therapy technologies for the purpose of manipulating specific genes or gene functions in order to improve athletic performance. However, a non-invasive detection method for gene doping using recombinant adenoviral (rAdV) vectors containing human follistatin (hFST) genes (rAdV) has not yet been developed. Therefore, the aim of this study was to develop a method to detect gene doping using rAdV. METHODS: First, we generated rAdV and evaluated the overexpression of the hFST gene, FST protein, and muscle protein synthesis signaling using cell lines. Next, rAdV was injected intravenously or intramuscularly into mice, and whole blood was collected, and hFST and cytomegalovirus promoter (CMVp) gene fragments were detected using TaqMan-quantitative polymerase chain reaction (qPCR). Finally, to confirm the specificity of the primers and the TaqMan probes, samples from each experiment were pooled, amplified using TaqMan-qPCR, and sequenced using the Sanger sequencing. RESULTS: The expression of hFST and FST proteins and muscle protein synthesis signaling significantly increased in C2C12 cells. In long-term, transgene fragments could be detected until 4 days after intravenous injection and 3 days after intramuscular injection. Finally, the Sanger sequencing confirmed that the primers and TaqMan probe specifically amplified the gene sequence of interest. CONCLUSIONS: These results indicate the possibility of detecting gene doping using rAdV using TaqMan-qPCR in blood samples. This study may contribute to the development of detection methods for gene doping using rAdV.

Effects of renalase deficiency on liver fibrosis markers in a nonalcoholic steatohepatitis mouse model.

Progression of nonalcoholic steatohepatitis (NASH) is attributed to several factors, including inflammation and oxidative stress. In recent years, renalase has been reported to suppress oxidative stress, apoptosis and inflammation. A number of studies have suggested that renalase may be associated with protecting the liver from injury. The present study aimed to clarify the effects of renalase knockout (KO) in mice with NASH that were induced with a choline­deficient high­fat diet (CDAHFD) supplemented with 0.1% methionine. Wild type (WT) and KO mice (6­week­old) were fed a normal diet (ND) or CDAHFD for 6 weeks, followed by analysis of the blood liver function markers and liver tissues. CDAHFD intake was revealed to increase blood hepatic function markers, lipid accumulation and oxidative stress compared with ND, but no significant differences were observed between the WT and KO mice. However, in the KO­CDAHFD group, the Adgre1 and Tgfb1 mRNA levels were significantly higher, and α­SMA expression was significantly lower compared with the WT­CDAHFD group. Furthermore, the Gclc mRNA and phosphorylated protein kinase B (Akt) levels were significantly lower in the KO­ND group compared with the WT­ND group. The results of the current study indicated that as NASH progressed in the absence of renalase, oxidative stress, macrophage infiltration and TGF­ß expression were enhanced, while α­SMA expression in NASH may be partly suppressed due to the decreased phosphorylation of Akt level.

Renalase is localized to the small intestine crypt and expressed upon the activation of NF-κB p65 in mice model of fasting-induced oxidative stress.

AIMS: Renalase expression is regulated by Nuclear Factor (NF)-κB and hypoxia inducible factor (HIF)-1α, and antioxidative stress function in renal cells were reported. However, dynamics of renalase and localizes in intestine were remain unknown. We evaluated the effects of oxidative stress on renalase expression and localization using model of fasting induced oxidative stress and Caco-2 cell, and examined the its physiological effects. MAIN METHODS: 24 male mice were divided into three groups: Control (Con), 72 h fasting (Fast), and 24 h refeeding after fasting (Refeed). Jejunum and ileum were collected respectively. The structure of jejunum and ileum were observed by hematoxylin and eosin (HE) stain. The expression levels of carbonylated protein, renalase, NF-κB p65 and HIF-1α were measured by immunoblotting. Localization of renalase was observed by immunofluorescent. in vitro assay was performed using Caco-2 cell. Renalase was overexpressed using adenovirus. After that, Caco-2 cell was treated with 2 mM H2O2 for 30 min or 24 h. KEY FINDINGS: Renalase was increased in Fast and it was localized in crypt. HIF-1α did not increase, but NF-κB p65 increased in Fast. Renalase overexpression protects the Caco-2 cells against H2O2 induced oxidative stress. SIGNIFICANCE: Renalase was localized in crypt and increased in Fast. This increase suggested protect response to oxidative stress because undifferenced cells were localized in crypt and need to be protected. Actually, renalase protected Caco-2 cells against H2O2 induced oxidative stress. Small intestinal renalase expression was regulated by NF-κB p65 and was considered to be a defense mechanism against oxidative stress.

Acute Low-Intensity Treadmill Running Upregulates the Expression of Intestinal Glucose Transporters via GLP-2 in Mice.

The effects of exercise on nutrient digestion and absorption in the intestinal tract are not well understood. A few studies have reported that exercise training increases the expression of molecules involved in carbohydrate digestion and absorption. Exercise was also shown to increase the blood concentration of glucagon-like peptide-2 (GLP-2), which regulates carbohydrate digestion and absorption in the small intestine. Therefore, we investigated the effects of exercise on the expression of molecules involved in intestinal digestion and absorption, including GLP-2. Six-week-old male mice were divided into a sedentary (SED) and low-intensity exercise (LEx) group. LEx mice were required to run on a treadmill (12.5 m/min, 1 h), whereas SED mice rested. All mice were euthanized 1 h after exercise or rest, and plasma, jejunum, ileum, and colon samples were collected, followed by analysis via IHC, EIA, and immunoblotting. The levels of plasma GLP-2 and the jejunum expression of the GLP-2 receptor, sucrase-isomaltase (SI), and glucose transporter 2 (GLUT2) were higher in LEx mice. Thus, we showed that acute low-intensity exercise affects the expression of molecules involved in intestinal carbohydrate digestion and absorption via GLP-2. Our results suggest that exercise might be beneficial for small intestine function in individuals with intestinal frailty.

Proof of Gene Doping in a Mouse Model with a Human Erythropoietin Gene Transferred Using an Adenoviral Vector.

Despite the World Anti-Doping Agency (WADA) ban on gene doping in the context of advancements in gene therapy, the risk of EPO gene-based doping among athletes is still present. To address this and similar risks, gene-doping tests are being developed in doping control laboratories worldwide. In this regard, the present study was performed with two objectives: to develop a robust gene-doping mouse model with the human EPO gene (hEPO) transferred using recombinant adenovirus (rAdV) as a vector and to develop a detection method to identify gene doping by using this model. The rAdV including the hEPO gene was injected intravenously to transfer the gene to the liver. After injection, the mice showed significantly increased whole-blood red blood cell counts and increased expression of hematopoietic marker genes in the spleen, indicating successful development of the gene-doping model. Next, direct and potentially indirect proof of gene doping were evaluated in whole-blood DNA and RNA by using a quantitative PCR assay and RNA sequencing. Proof of doping could be detected in DNA and RNA samples from one drop of whole blood for approximately a month; furthermore, the overall RNA expression profiles showed significant changes, allowing advanced detection of hEPO gene doping.

Detection of Multiple Transgene Fragments in a Mouse Model of Gene Doping Based on Plasmid Vector Using TaqMan-qPCR Assay.

The World Anti-Doping Agency has prohibited gene doping in the context of progress in gene therapy. There is a risk that the augmentation of genes using plasmids could be applied for gene doping. However, no gold standard method to detect this has been established. Here, we aimed to develop a method to detect multiple transgene fragments as proof of gene doping. Firstly, gene delivery model mice as a mimic of gene doping were created by injecting firefly luciferase plasmid with polyethylenimine (PEI) into the abdominal cavity. The results confirmed successful establishment of the model, with sufficient luminescence upon in vivo imaging. Next, multiple transgene fragments in the model were detected in plasma cell-free (cf)DNA, blood-cell-fraction DNA, and stool DNA using the TaqMan- quantitative real-time PCR(qPCR) assay, with the highest levels in plasma cfDNA. Using just a single drop of whole blood from the model, we also attempted long-term detection. The results showed that multiple transgene fragments were detected until 11 days. These findings indicate that the combination of plasma cfDNA or just one drop of whole blood with TaqMan-qPCR assay is feasible to detect plasmid-PEI-based gene doping. Our findings could accelerate the development of methods for detecting gene doping in humans.

Habitual Aerobic Exercise Diminishes the Effects of Sarcopenia in Senescence-Accelerated Mice Prone8 Model.

Loss of muscle mass and strength are progressing with aging. Exercise is a beneficial method to prevent physical dysfunction, and habitual exercise can improve the muscle quality. Therefore, we evaluated the effects of long-term habitual exercise's impact on sarcopenia utilizing the senescence-accelerated mice prone8 (SAMP8) model. Notably, 27 w SAMP8 were used in this study. Mice were classified into 28 (28 w) and 44 weeks old. The 44-week group was divided into the sedentary group (44 w) and a group exercising for 16 weeks (44 w + Ex). The 44 w + Ex performed habitual exercise from 28 to 44 weeks. Additionally, grip strength tests were performed with mice aged 28 and 44 weeks. Muscles were harvested and measured muscle weight at 44 w. Gastrocnemius decreased in 44 w, but was unchanged in 44 w + Ex. There was a trend for lower muscle grip strength in the 44 w group, but there was no change in 44 w + Ex. The phosphorylation levels of Akt and p70S6K as a protein synthesis marker were decreased in 44 w. Cytochrome c oxidase subunit IV (CoxIV) mRNA and protein levels decreased in 44 w. These results suggested that long-term habitual exercise attenuates muscle mass and strength decline, possibly through maintenance of muscle protein synthesis and mitochondrial maintenance.

High Salt Diet Impacts the Risk of Sarcopenia Associated with Reduction of Skeletal Muscle Performance in the Japanese Population.

The World Health Organization has recommended 5 g/day as dietary reference intakes for salt. In Japan, the averages for men and women were 11.0 g/day and 9.3 g/day, respectively. Recently, it was reported that amounts of sodium accumulation in skeletal muscles of older people were significantly higher than those in younger people. The purpose of this study was to investigate whether the risk of sarcopenia with decreased muscle mass and strength was related to the amount of salt intake. In addition, we investigated its involvement with renalase. Four groups based on age and salt intake ("younger low-salt," "younger high-salt," "older low-salt," and "older high-salt") were compared. Stratifying by age category, body fat percentage significantly increased in high-salt groups in both younger and older people. Handgrip strength/body weight and chair rise tests of the older high-salt group showed significant reduction compared to the older low-salt group. However, there was no significant difference in renalase concentrations in plasma. The results suggest that high-salt intake may lead to fat accumulation and muscle weakness associated with sarcopenia. Therefore, efforts to reduce salt intake may prevent sarcopenia.

The detection of trans gene fragments of hEPO in gene doping model mice by Taqman qPCR assay.

BACKGROUND: With the rapid progress of genetic engineering and gene therapy methods, the World Anti-Doping Agency has raised concerns regarding gene doping, which is prohibited in sports. However, there is no standard method available for detecting transgenes delivered by injection of naked plasmids. Here, we developed a detection method for detecting transgenes delivered by injection of naked plasmids in a mouse model that mimics gene doping. METHODS: Whole blood from the tail tip and one piece of stool were used as pre-samples of injection. Next, a plasmid vector containing the human erythropoietin (hEPO) gene was injected into mice through intravenous (IV), intraperitoneal (IP), or local muscular (IM) injection. At 1, 2, 3, 6, 12, 24, and 48 h after injection, approximately 50 µL whole blood was collected from the tail tip. One piece of stool was collected at 6, 12, 24, and 48 h. From each sample, total DNA was extracted and transgene fragments were analyzed by Taqman quantitative PCR (qPCR) and SYBR green qPCR. RESULTS: In whole blood DNA samples evaluated by Taqman qPCR, the transgene fragments were detected at all time points in the IP sample and at 1, 2, 3, 6, and 12 h in the IV and IM samples. In the stool-DNA samples, the transgene fragments were detected at 6, 12, 24, and 48 h in the IV and IM samples by Taqman qPCR. In the analysis by SYBR green qPCR, the transgene fragments were detected at some time point in both specimens; however, many non-specific amplicons were detected. CONCLUSIONS: These results indicate that transgene fragments evaluated after each injection method of naked plasmids were detected in whole-blood and stool DNA samples. These findings may facilitate the development of methods for detecting gene doping.