Exercise combined with a probiotics treatment alters the microbiome, but moderately affects signalling pathways in the liver of male APP/PS1 transgenic mice.

It has been demonstrated that physical exercise and probiotic supplementation delay the progress of Alzheimer's Disease (AD) in male APP/PS1TG mice. However, it has also been suggested that both exercise and AD have systemic effects. We have studied the effects of exercise training and probiotic treatment on microbiome and biochemical signalling proteins in the liver. The results suggest that liver is under oxidative stress, since SOD2 levels of APP/PS1 mice were decreased when compared to a wild type of mice. Exercise training prevented this decrease. We did not find significant changes in COX4, SIRT3, PGC-1a or GLUT4 levels, while the changes in pAMPK/AMPK, pmTOR/mTOR, pS6/S6 and NRF2 levels were randomly modulated. The data suggest that exercise and probiotics-induced changes in microbiome do not strongly affect mitochondrial density or protein synthesis-related AMPK/mTOR/S6 pathways in the liver of these animals.

Overexpression of SRS5 improves grain size of brassinosteroid-related dwarf mutants in rice (Oryza sativa L.).

Grain size is a trait that is important for rice (Oryza sativa L.) yield potential. Many genes regulating grain size have been identified, deepening our understanding of molecular mechanisms of grain size determination in rice. Previously, we cloned SMALL AND ROUND SEED 5 (SRS5) gene (encoding alpha-tubulin) from a small and round seed mutant and revealed that this gene regulates grain length independently of the brassinosteroid (BR) signaling pathway, although BR-related mutants set small grain. In this study, we showed that overexpression of SRS5 can promote grain length and demonstrated that the overexpression of SRS5 in BR-related mutants rescued the shortened grain length, which is an unfavorable phenotype in the yield potential of BR-related mutants, while preserving the useful semi-dwarf and erect leaf phenotypes.

Decreased pancreatic amylase activity after acute high-intensity exercise and its effects on post-exercise muscle glycogen recovery.

Our prior results showed that an acute bout of endurance exercise for 6 h, but not 1 h, decreased pancreatic amylase activity, indicating that acute endurance exercise may affect carbohydrate digestive capacity in an exercise duration-dependent manner. Here, we investigated the effects of acute endurance exercise of different intensities on mouse pancreatic amylase activity. Male C57BL/6J mice performed low- or high-intensity running exercise for 60 min at either 10 (Ex-Low group) or 20 m/min (Ex-High group). The control group comprised sedentary mice. Immediately after acute exercise, pancreatic amylase activity was significantly decreased in the Ex-High group and not the Ex-Low group in comparison with the control group. To determine whether the decreased amylase activity induced by high-intensity exercise influenced muscle glycogen recovery after exercise, we investigated the rates of muscle glycogen resynthesis in Ex-High group mice administered either oral glucose or starch solution (2.0 mg/g body weight) immediately after exercise. The starch-fed mice exhibited significantly lower post-exercise glycogen accumulation rates in the 2-h recovery period compared with the glucose-fed mice. This difference in the glycogen accumulation rate was absent for starch- and glucose-fed mice in the sedentary (no exercise) control group. Furthermore, the plasma glucose AUC during early post-exercise recovery (0-60 min) was significantly lower in the starch-fed mice than in the glucose-fed mice. Thus, our findings suggest that acute endurance exercise diminishes the carbohydrate digestive capacity of the pancreas in a manner dependent on exercise intensity, with polysaccharides leading to delayed muscle glycogen recovery after exercise.

The omega-3 postbiotic trans-10-cis-15-octadecadienoic acid attenuates contact hypersensitivity in mice through downregulation of vascular endothelial growth factor A.

Intestinal bacteria metabolize dietary substances to produce bioactive postbiotics, among which some are recognized for their role in promoting host health. We here explored the postbiotic potential of two omega-3 α-linolenic acid-derived metabolites: trans-10-cis-15-octadecadienoic acid (t10,c15-18:2) and cis-9-cis-15-octadecadienoic acid (c9,c15-18:2). Dietary intake of lipids rich in omega-3 α-linolenic acid elevated levels of t10,c15-18:2 and c9,c15-18:2 in the serum and feces of mice, an effect dependent on the presence of intestinal bacteria. Notably, t10,c15-18:2 mitigated skin inflammation in mice that became hypersensitive after exposure to 2,4-dinitrofluorobenzene, an experimental model for allergic contact dermatitis. In particular, t10,c15-18:2-but not c9,c15-18:2-attenuated ear swelling and edema, characteristic symptoms of contact hypersensitivity. The anti-inflammatory effects of t10,c15-18:2 were due to its ability to suppress the release of vascular endothelial growth factor A from keratinocytes, thereby mitigating the enhanced vascular permeability induced by hapten stimulation. Our study identified retinoid X receptor as a functional receptor that mediates the downregulation of skin inflammation upon treatment with t10,c15-18:2. Our results suggest that t10,c15-18:2 holds promise as an omega-3 fatty acid-derived postbiotic with potential therapeutic implications for alleviating the skin edema seen in allergic contact dermatitis-induced inflammation.

High-level expression by tissue/cancer-specific promoter with strict specificity using a single-adenoviral vector.

Tissue-/cancer-specific promoters for use in adenovirus vectors (AdVs) are valuable for elucidating specific gene functions and for use in gene therapy. However, low activity, non-specific expression and size limitations in the vector are always problems. Here, we developed a 'double-unit' AdV containing the Cre gene under the control of an α-fetoprotein promoter near the right end of its genome and bearing a compact 'excisional-expression' unit consisting of a target cDNA 'upstream' of a potent promoter between two loxPs near the left end of its genome. When Cre was expressed, the expression unit was excised as a circular molecule and strongly expressed. Undesired leak expression of Cre during virus preparation was completely suppressed by a dominant-negative Cre and a short-hairpin RNA against Cre. Using this novel construct, a very strict specificity was maintained while achieving a 40- to 90-fold higher expression level, compared with that attainable using a direct specific promoter. Therefore, the 'double-unit' AdV enabled us to produce a tissue-/cancer-specific promoter in an AdV with a high expression level and strict specificity.

[Recent progress in adenovirus vectors: focusing on VA-deleted AdV].

First-generation adenovirus vectors (FG-AdVs) are widely used because transduction efficiency of the vectors is very high. However, severe immune responses especially to the liver have been a serious problem of this vector. We succeeded to identify a viral protein that cause the immune responses and reported ''low-inflammatory AdVs'' that mostly solve this problem. However, to develop the ultimate form of this vector, it is necessary to remove virus-associated RNA (VA RNA) genes from the AdV vector genome. VA RNAs are transcribed by polymerase III; they are not essential for viral growth but have important roles to make appropriate circumstances for this virus. Large amount of VA RNAs are required in the late phase to support viral growth. Hence it is difficult to establish 293 cell lines that can support replication of AdVs lacking VA RNA genes (VA-deleted AdVs) supplying sufficient amount of VA RNA in trans. Recently we have developed a method for efficient production of VA-deleted AdVs and succeeded to obtain a high titer of VA-deleted AdVs. Then we construct VA-deleted AdVs expressing shRNA that knockdown the replication of hepatitis C virus (HCV). In fact, VA-deleted AdVs expressing these shRNAs suppressed HCV replication more effectively than conventional FG-AdV. Therefore, we showed that VA RNAs expressed from FG-AdVs probably compete with shRNA in the maturation pathway and reduce the effect of shRNAs. We think that VA-deleted AdV may substitute for current FG-AdVs and become a standard AdV.

The TAS1R2 sweet taste receptor regulates skeletal muscle mass and fitness.

Muscle fitness and mass deteriorate under the conditions of obesity and aging for reasons yet to be fully elucidated. Herein, we describe a novel pathway linking peripheral nutrient sensing and skeletal muscle function through the sweet taste receptor TAS1R2 and the involvement of ERK2-PARP1-NAD signaling axis. Muscle-specific deletion of TAS1R2 (mKO) in mice produced elevated NAD levels due to suppressed PARP1 activity, improved mitochondrial function, increased muscle mass and strength, and prolonged running endurance. Deletion of TAS1R2 in obese or aged mice also ameliorated the decline in muscle mass and fitness arising from these conditions. Remarkably, partial loss-of-function of TAS1R2 (rs35874116) in older, obese humans recapitulated the healthier muscle phenotype displayed by mKO mice in response to exercise training. Our findings show that inhibition of the TAS1R2 signaling in skeletal muscle is a promising therapeutic approach to preserve muscle mass and function.

Copy number of adenoviral vector genome transduced into target cells can be measured using quantitative PCR: application to vector titration.

Both transfection and adenovirus vectors are commonly used in studies measuring gene expression. However, the real DNA copy number that is actually transduced into target cells cannot be measured using quantitative PCR because attached DNA present on the cell surface is difficult to distinguish from successfully transduced DNA. Here, we used Cre/loxP system to show that most of the transfected DNA was in fact attached to the cell surface; in contrast, most of the viral vector DNA used to infect the target cells was present inside the cells after the cells were washed according to the conventional infection protocol. We applied this characteristic to adenoviral vector titration. Current methods of vector titration using the growth of 293 cells are influenced by the effect of the expressed gene product as well as the cell conditions and culture techniques. The titration method proposed here indicates the copy numbers introduced to the target cells using a control vector that is infected in parallel (relative vector titer: rVT). Moreover, the new titration method is simple and reliable and may replace the current titration methods of viral vectors.

Comparison of efficiency between FLPe and Cre for recombinase-mediated cassette exchange in vitro and in adenovirus vector production.

Cre and FLP recombinases mediate not only specific deletions and insertions, but also the recombinase-mediated cassette exchange (RMCE) reaction, which is used in cell biotechnology including ES cells and mouse genetics. However, comparison of efficiencies for Cre and FLP in RMCE has not been made. We here examined the detailed process of RMCE with Cre and FLP in vitro using mutant loxP 2272 and three mutant FRTs (FRT G, FRT H, and FRT F3) and then quantitatively compared the RMCE reactions in vitro. Interestingly, in the in vitro reactions, the RMCE efficiency of Cre reached a plateau level of approximately 5% and did not proceed further, whereas that of FLPe reached approximately 12-13%, showing that FLPe reached a higher level of efficiency than Cre possibly when they were supplied at a very high concentration. Moreover, we quantitatively compared the production efficiency of E1-deleted adenovirus vector using the RMCE method with Cre or FLP. The results showed that FLPe was again found more efficient than Cre in RMCE reaction. Thus, although Cre is considered more active than, or similar to, FLPe, it may not be necessarily true for RMCE reaction. Possible reasons explaining these results are discussed.

Influence of loxP insertion upstream of the cis-acting packaging domain on adenovirus packaging efficiency.

First-generation AdV enables efficient gene transduction, although its immunogenicity is an important problem in vivo. Helper-dependent AdV (HD-AdV) is one possible solution to this problem. The construction of HD-AdV requires a helper virus, in which the viral packaging domain is flanked by two inserted loxP to hamper its packaging in Cre-expressing 293 cells. Here, we constructed 19L viruses containing loxP at 191 nt from the left end of the genome upstream of the packaging domain, 15L viruses bearing loxP at 143 nt, and a control ΔL virus lacking loxP at these positions. The 19L position is used worldwide, and the 15L position has been reported to result in a lower titer than that of 19L. When the titers were compared for six pairs of 19L and 15L AdV, the 19L AdV produced titers similar to, or sometimes lower than, the 15L and ΔL AdV, unlike the results of previous reports. We next chose one pair of 15L and 19L AdV that produced titers similar to that of ΔL and a competitor AdV lacking loxP for use in a competition assay. When a small amount of the competitor AdV was co-infected, both the 15L and the 19L AdV, but not ΔL, gradually became minority components during subsequent viral passages. Therefore, the loxP insertions at 143 nt and 191 nt decreased the viral packaging efficiency.

Effects of a Very High-Carbohydrate Diet and Endurance Exercise Training on Pancreatic Amylase Activity and Intestinal Glucose Transporter Content in Rats.

We previously reported that the combination of a very high-carbohydrate diet and endurance training increased glucose transporter 4 and glycogen concentration in skeletal muscle. However, it remains unclear whether they also affect the digestive and absorptive capacity in the pancreas and small intestine, which are suggested to be rate-limiting steps in the delivery of exogenous carbohydrates to skeletal muscle and muscle glycogen synthesis. Thus, we aimed to evaluate the effects of a very high-carbohydrate diet and endurance training on pancreatic amylase activity and intestinal glucose transporters in rats and to examine the relationship between these adaptations and their influence on muscle glycogen concentration. Male Sprague-Dawley rats (n=29) were fed a high-carbohydrate diet (59% carbohydrate) or a very high-carbohydrate diet (76% carbohydrate) for 4 wk. Half of the rats in each dietary group were subjected to 6-h swimming exercise training (two 3-h sessions separated by 45 min of rest) for 4 wk. Although there was no significant effect of diet or endurance training on sodium-dependent glucose transporter 1 and glucose transporter 2 contents in the intestine, the rats fed a very high-carbohydrate diet in combination with endurance training had substantially higher pancreatic amylase activity and muscle glycogen concentration. Furthermore, there was a positive correlation between pancreatic amylase activity and muscle glycogen concentration (r=0.599, p=0.001). In conclusion, intake of a very high-carbohydrate diet and endurance training synergistically elevated carbohydrate digestive capacity, which partially accounted for the higher muscle glycogen accumulation.

Oral administration of Blautia wexlerae ameliorates obesity and type 2 diabetes via metabolic remodeling of the gut microbiota.

The gut microbiome is an important determinant in various diseases. Here we perform a cross-sectional study of Japanese adults and identify the Blautia genus, especially B. wexlerae, as a commensal bacterium that is inversely correlated with obesity and type 2 diabetes mellitus. Oral administration of B. wexlerae to mice induce metabolic changes and anti-inflammatory effects that decrease both high-fat diet-induced obesity and diabetes. The beneficial effects of B. wexlerae are correlated with unique amino-acid metabolism to produce S-adenosylmethionine, acetylcholine, and L-ornithine and carbohydrate metabolism resulting in the accumulation of amylopectin and production of succinate, lactate, and acetate, with simultaneous modification of the gut bacterial composition. These findings reveal unique regulatory pathways of host and microbial metabolism that may provide novel strategies in preventive and therapeutic approaches for metabolic disorders.

Effects of Dietary Fat Restriction on Endurance Training-induced Metabolic Adaptations in Rat Skeletal Muscle.

Endurance exercise training enhances muscle fat oxidation while concomitantly reducing carbohydrate (glycogen) utilization during exercise, thereby delaying the onset of fatigue. This study examined the effects of dietary fat restriction on endurance training-induced metabolic adaptations in rat skeletal muscle. Male Sprague-Dawley rats were placed on either a control diet (CON: 19.2% protein, 21.6% fat, and 59.2% carbohydrate as a percentage of total energy) or a fat-restricted diet (FR: 21.5% protein, 2.4% fat, and 76.1% carbohydrate as a percentage of total energy) for 4 wks. Half the rats in each dietary group performed daily 6-h swimming exercise (two 3-h sessions separated by 45 min of rest) on 5 days each wk. Endurance training significantly increased the expression of ß-hydroxyacyl CoA dehydrogenase (ßHAD), a key enzyme of fat oxidation, and pyruvate dehydrogenase kinase 4 (PDK4), an inhibitory regulator of glycolytic flux, in the skeletal muscle of rats fed the CON diet. However, such endurance training-induced increases in muscle ßHAD and PDK4 were partially suppressed by the FR diet, suggesting that a FR diet may diminish the endurance training-induced enhancement of fat oxidation and reduction in glycogen utilization during exercise. We then assessed the muscle glycogen utilization rate during an acute bout of swimming exercise in the trained rats fed either the CON or the FR diet and consequently found that rats fed the FR diet had a significantly higher muscle glycogen utilization rate during exercise compared with rats fed the CON diet. In conclusion, dietary fat restriction may attenuate the endurance training-induced metabolic adaptations in skeletal muscle.

Acetophenone 4-nitrophenylhydrazone inhibits Hepatitis B virus replication by modulating capsid assembly.

Hepatitis B virus (HBV) is the causative agent of chronic liver disease and is correlated with the development of subsequent hepatic cirrhosis and hepatocellular carcinoma. Current antiviral therapy using nucleos(t)ide analogs is effective in suppressing viral replication and interrupting disease progression, but HBV is rarely cured completely. Thus, there remains an unmet need for the development of novel anti-HBV drugs. Here, we report the identification of N-(4-Nitrophenyl)-1-phenylethanone hydrazone (ANPH) as a novel structural class of selective inhibitors targeting the replication of the HBV genome using adenovirus vector-mediated HBV genome transduction. ANPH inhibited viral genome replication in HepG2.2.15 cells by inducing the formation of empty capsids devoid of pregenomic RNA without affecting its transcription and translation. Biochemical assays using a truncated core protein consisting of the assembly domain showed that ANPH accelerates the formation of morphologically intact capsids. Taken together, we propose that ANPH might provide a new structural scaffold to design a new anti-HBV drug in medicinal chemistry as well as chemical probes for HBV core protein functions in the future.

The Safety of Very-long-term Intake of a Ketogenic Diet Containing Medium-chain Triacylglycerols.

We previously reported that consuming a ketogenic diet containing medium-chain triacylglycerols (MCTs) might be a valuable dietary strategy for endurance athletes. However, the long-term safety of the diet has not been established, and there is a concern that a higher intake of MCTs increases the liver triacylglycerol content. In this study, we found that consuming an MCT-containing ketogenic diet for 24 weeks decreased, rather than increased, the liver triacylglycerol concentration and did not aggravate safety-related blood biomarkers in male Wistar rats. Our results may therefore suggest that the long-term intake of a ketogenic diet containing MCTs may have no deleterious effects on physiological functions.

Effects of a Ketogenic Diet Containing Medium-Chain Triglycerides and Endurance Training on Metabolic Enzyme Adaptations in Rat Skeletal Muscle.

Long-term intake of a ketogenic diet enhances utilization of ketone bodies, a particularly energy-efficient substrate, during exercise. However, physiological adaptation to an extremely low-carbohydrate diet has been shown to upregulate pyruvate dehydrogenase kinase 4 (PDK4, a negative regulator of glycolytic flux) content in skeletal muscle, resulting in impaired high-intensity exercise capacity. This study aimed to examine the effects of a long-term ketogenic diet containing medium-chain triglycerides (MCTs) on endurance training-induced adaptations in ketolytic and glycolytic enzymes of rat skeletal muscle. Male Sprague-Dawley rats were placed on either a standard diet (CON), a long-chain triglyceride-containing ketogenic diet (LKD), or an MCT-containing ketogenic diet (MKD). Half the rats in each group performed a 2-h swimming exercise, 5 days a week, for 8 weeks. Endurance training significantly increased 3-oxoacid CoA transferase (OXCT, a ketolytic enzyme) protein content in epitrochlearis muscle tissue, and MKD intake additively enhanced endurance training-induced increases in OXCT protein content. LKD consumption substantially increased muscle PDK4 protein level. However, such PDK4 increases were not observed in the MKD-fed rats. In conclusion, long-term intake of ketogenic diets containing MCTs may additively enhance endurance training-induced increases in ketolytic capacity in skeletal muscle without exerting inhibitory effects on carbohydrate metabolism.

Effects of long-term exercise training for different durations on pancreatic amylase activity and intestinal glucose transporter content in rats.

Long-term endurance training for a relatively short duration (~1 h) is reported to increase pancreatic amylase activity in rats, suggesting that chronic exercise training enhances carbohydrate digestive capacity. However, it remains unknown whether longer exercise training duration results in greater adaptation in the pancreas and small intestine. Thus, this study aimed to examine the effects of long-term endurance training for a longer duration on pancreatic amylase activity and intestinal glucose transporter content in rats. Male Sprague-Dawley rats were subjected to swimming exercise training for 1 h (Ex-1h group) or 6 h (Ex-6h group, two 3-h sessions separated by 1 h of rest) each day, 5 days a week, for 6 weeks. Sedentary rats were used as a control (Con group). Total pancreatic amylase activity in the Ex-6h group was significantly lower than that in the Con and Ex-1h groups immediately after the last training session. After 24 h of recovery, total pancreatic amylase activity was significantly higher in the Ex-1h group (~46%) than in the Con group, and a further increase was observed in the Ex-6h group (~98%). In addition, the Ex-6h group, but not the Ex-1h group, showed significantly greater intestinal sodium-dependent glucose transporter 1 (SGLT1) content compared with the Con group after 24 h of recovery. However, no significant difference was observed in glucose transporter 2 (GLUT2) content among the three groups. In conclusion, chronic endurance exercise training for a longer duration results in larger increases in pancreatic amylase activity and intestinal SGLT1 content in rats.

Preexercise Carbohydrate Ingestion and Transient Hypoglycemia: Fasting versus Feeding.

PURPOSE: Carbohydrate (CHO) ingestion 30 to 45 min before exercise results in transient hypoglycemia after starting the exercise in some, but not all, subjects. However, whether transient hypoglycemia is more likely to occur under fed or fasted condition remains unknown. This study aimed to directly compare the effects of fasting versus feeding on plasma glucose responses after preexercise CHO intake and to examine the relationship between insulin responses and onset of transient hypoglycemia. METHODS: Sixteen subjects performed 60-min cycle ergometer exercises at 75% maximal oxygen uptake (V˙O2max) under overnight fasted and fed (4 h after breakfast) conditions. In both conditions, they consumed 500 mL of beverage (150 g of glucose) 30 min before beginning exercise. RESULTS: The mean plasma glucose concentrations 15 min after starting the exercise did not fall below 4.0 mmol·L (criteria for hypoglycemia) in both states; however, individual differences in the occurrence of transient hypoglycemia were noted. In the fasted state, plasma glucose levels transiently dropped below 4.0 mmol·L in five subjects, who had substantially higher serum insulin levels at the start of exercise, compared with those who did not develop hypoglycemia. Although seven subjects developed transient hypoglycemia in the fed state, no relationship was observed between insulin responses and hypoglycemia. Three subjects developed hypoglycemia in both fasted and fed states. CONCLUSIONS: These results suggest that transient hypoglycemia after preexercise CHO ingestion occurs in some, but not all, subjects, under both conditions. Furthermore, subjects with enhanced insulin responses seem to be more prone to transient hypoglycemia in the fasted condition.

Effects of Ingestion of Different Amounts of Carbohydrate after Endurance Exercise on Circulating Cytokines and Markers of Neutrophil Activation.

We aimed to examine the effects of ingestion of different amounts of carbohydrate (CHO) after endurance exercise on neutrophil count, circulating cytokine levels, and the markers of neutrophil activation and muscle damage. Nine participants completed three separate experimental trials consisting of 1 h of cycling exercise at 70% V · O2 max, followed by ingestion of 1.2 g CHO·kg body mass-1·h-1 (HCHO trial), 0.2 g CHO·kg body mass-1·h-1 (LCHO trial), or placebo (PLA trial) during the 2 h recovery phase in random order. Circulating glucose, insulin, and cytokine levels, blood cell counts, and the markers of neutrophil activation and muscle damage were measured. The concentrations of plasma glucose and serum insulin at 1 h after exercise were higher in the HCHO trial than in the LCHO and PLA trials. Although there were significant main effects of time on several variables, including neutrophil count, cytokine levels, and the markers of neutrophil activation and muscle damage, significant time × trial interactions were not observed for any variables. These results suggest that CHO ingestion after endurance exercise does not enhance exercise-induced increase in circulating neutrophil and cytokine levels and markers of neutrophil activation and muscle damage, regardless of the amount of CHO ingested.

UTX-mediated enhancer and chromatin remodeling suppresses myeloid leukemogenesis through noncatalytic inverse regulation of ETS and GATA programs.

The histone H3 Lys27-specific demethylase UTX (or KDM6A) is targeted by loss-of-function mutations in multiple cancers. Here, we demonstrate that UTX suppresses myeloid leukemogenesis through noncatalytic functions, a property shared with its catalytically inactive Y-chromosome paralog, UTY (or KDM6C). In keeping with this, we demonstrate concomitant loss/mutation of KDM6A (UTX) and UTY in multiple human cancers. Mechanistically, global genomic profiling showed only minor changes in H3K27me3 but significant and bidirectional alterations in H3K27ac and chromatin accessibility; a predominant loss of H3K4me1 modifications; alterations in ETS and GATA-factor binding; and altered gene expression after Utx loss. By integrating proteomic and genomic analyses, we link these changes to UTX regulation of ATP-dependent chromatin remodeling, coordination of the COMPASS complex and enhanced pioneering activity of ETS factors during evolution to AML. Collectively, our findings identify a dual role for UTX in suppressing acute myeloid leukemia via repression of oncogenic ETS and upregulation of tumor-suppressive GATA programs.