BCAA-nitrogen flux in brown fat controls metabolic health independent of thermogenesis.

Brown adipose tissue (BAT) is best known for thermogenesis. Rodent studies demonstrated that enhanced BAT thermogenesis is tightly associated with increased energy expenditure, reduced body weight, and improved glucose homeostasis. However, human BAT is protective against type 2 diabetes, independent of body weight. The mechanism underlying this dissociation remains unclear. Here, we report that impaired mitochondrial catabolism of branched-chain amino acids (BCAAs) in BAT, by deleting mitochondrial BCAA carriers (MBCs), caused systemic insulin resistance without affecting energy expenditure and body weight. Brown adipocytes catabolized BCAA in the mitochondria as nitrogen donors for the biosynthesis of non-essential amino acids and glutathione. Impaired mitochondrial BCAA-nitrogen flux in BAT resulted in increased oxidative stress, decreased hepatic insulin signaling, and decreased circulating BCAA-derived metabolites. A high-fat diet attenuated BCAA-nitrogen flux and metabolite synthesis in BAT, whereas cold-activated BAT enhanced the synthesis. This work uncovers a metabolite-mediated pathway through which BAT controls metabolic health beyond thermogenesis.

Skeletal muscle-enriched miRNAs are highly unstable in vivo and may be regulated in a Dicer-independent manner.

MicroRNAs (miRNAs) are small noncoding RNAs that control essential cellular processes. For several decades, the molecular mechanisms underlying the functions and biogenesis of miRNAs have been clarified, whereas the molecular dynamics of miRNAs are poorly understood. We recently found that muscle-enriched miRNAs were reduced by only 20 ~ 50% in the skeletal muscles even 4 weeks after the suppression of miRNA processing through an inducible depletion of Dicer1 gene. These data suggest that miRNAs are stably expressed in skeletal muscle. In this study, we investigated the half-lives of those miRNAs in adult skeletal muscle with an in vivo metabolic labeling strategy and a genetic mouse model. In contrast to the hypothesis, in vivo metabolic labeling revealed that the half-lives of skeletal-muscle-enriched miRNAs were approximately 11-20 h. Furthermore, the levels of mature miR-23a decreased rapidly in the skeletal muscle of mice lacking miR-23 clusters in a tamoxifen-inducible manner. These data suggest that skeletal-muscle-enriched miRNAs are not highly stable in vivo. We also observed that the transfer of miR-150 into Dicer1-deficient muscle increased the miR-150 level to the same as that in control muscle. Taken together, our data demonstrate that miRNAs are degraded within a few days in adult skeletal muscle and that a Dicer-independent biogenetic pathway may produce mature miRNAs.

Functional Analysis of MicroRNAs in Skeletal Muscle.

MicroRNAs (miRNAs) are small non-coding RNAs that are highly conserved in vertebrates and play important roles in diverse biological processes. miRNAs function to fine-tune gene expression by accelerating the degradation of mRNA and/or by inhibiting protein translation. Identification of muscle-specific miRNAs has extended our knowledge of the molecular network in skeletal muscle. Here we describe methods that are commonly used to analyze the function of miRNAs in skeletal muscle.

The SLC25A47 locus controls gluconeogenesis and energy expenditure.

Mitochondria provide essential metabolites and adenosine triphosphate (ATP) for the regulation of energy homeostasis. For instance, liver mitochondria are a vital source of gluconeogenic precursors under a fasted state. However, the regulatory mechanisms at the level of mitochondrial membrane transport are not fully understood. Here, we report that a liver-specific mitochondrial inner-membrane carrier SLC25A47 is required for hepatic gluconeogenesis and energy homeostasis. Genome-wide association studies found significant associations between SLC25A47 and fasting glucose, HbA1c, and cholesterol levels in humans. In mice, we demonstrated that liver-specific depletion of SLC25A47 impaired hepatic gluconeogenesis selectively from lactate, while significantly enhancing whole-body energy expenditure and the hepatic expression of FGF21. These metabolic changes were not a consequence of general liver dysfunction because acute SLC25A47 depletion in adult mice was sufficient to enhance hepatic FGF21 production, pyruvate tolerance, and insulin tolerance independent of liver damage and mitochondrial dysfunction. Mechanistically, SLC25A47 depletion leads to impaired hepatic pyruvate flux and malate accumulation in the mitochondria, thereby restricting hepatic gluconeogenesis. Together, the present study identified a crucial node in the liver mitochondria that regulates fasting-induced gluconeogenesis and energy homeostasis.

Effects of lung volume and trigeminal nerve stimulation on diving response in breath-hold divers and non-divers.

PURPOSE: This study investigated the effects of lung volume and trigeminal nerve stimulation (TS) on diving responses in breath-hold divers (BHDs) and non-divers (NDs). METHODS: Eight BHDs and nine NDs performed four breath-hold trials at different lung volumes, with or without TS, and one trial of TS. Haemodynamic parameters and electrocardiograms were measured for each trial. RESULTS: During the TS trial, the total peripheral resistance increased more in BHDs. Breath-hold performed at total lung capacity showed a more pronounced decrease in stroke volume and cardiac output in BHDs. The decrease in heart rate and increase in total peripheral resistance were more pronounced in BHDs when breath-holding was performed with TS. CONCLUSION: The more pronounced diving response in BHDs was attributed to the greater increase in total peripheral resistance caused by TS. Furthermore, the lower stroke volume and cardiac output in BH performed at total lung capacity could also cause a more pronounced diving response in BHDs.

Live-cell imaging of microRNA expression with post-transcriptional feedback control.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate complex gene expression networks in eukaryotic cells. Because of their unique expression patterns, miRNAs are potential molecular markers for specific cell states. Although a system capable of imaging miRNA in living cells is needed to visually detect miRNA expression, very few fluorescence signal-on sensors that respond to expression of target miRNA (miR-ON sensors) are available. Here we report an miR-ON sensor containing a bidirectional promoter-driven Csy4 endoribonuclease and green fluorescent protein, ZsGreen1, for live-cell imaging of miRNAs with post-transcriptional feedback control. Csy4-assisted miR-ON (Csy4-miR-ON) sensors generate negligible background but respond sensitively to target miRNAs, allowing high-contrast fluorescence detection of miRNAs in various human cells. We show that Csy4-miR-ON sensors enabled imaging of various miRNAs, including miR-21, miR-302a, and miR-133, in vitro as well as in vivo. This robust tool can be used to evaluate miRNA expression in diverse biological and medical applications.

Effects of aerobic exercise training on mental health and arterial stiffness in middle-aged and older adults.

BACKGROUND: Aging is associated with deterioration of arterial function and mental health, which are known as cardiovascular risk factors. The present study investigated the effect of aerobic exercise training on mental health and arterial stiffness in middle-aged and older adults. METHODS: Twenty-nine healthy middle-aged and older adults were assigned to either the aerobic exercise training (N.=14) or the control groups (N.=15). The aerobic exercise training group completed 12 weeks of moderate aerobic exercise training for 3-4 session per week (30-60 minutes). The control group did not change their levels of physical activity. Before and after the 12-week period, the General Health Questionnaire (GHQ) and carotid ß-stiffness index, peak oxygen uptake were measured. RESULTS: At the onset of the 12-week period, the GHQ score, Carotid Β-Stiffness Index, and other key variables did not differ significantly between the aerobic exercise and control groups. The 12-week of aerobic exercise training increased peak oxygen uptake. The GHQ score and Carotid Β-Stiffness Index were decreased after the 12-week period in the aerobic exercise training group; however, no significant improvements were observed in the control group. CONCLUSIONS: We conclude that 12 weeks of aerobic exercise enhance mental health and decrease arterial stiffness in healthy middle-aged and older adults.

Dicer-mediated miRNA processing is not involved in controlling muscle mass during muscle atrophy.

Muscle atrophy occurs in a variety of physiological and pathological conditions. Specific molecular networks that govern protein synthesis and degradation play important roles in controlling muscle mass under diverse catabolic states. MicroRNAs (miRNAs) were previously found to be regulators of protein synthesis and degradation, and their expressions in skeletal muscle were altered in muscle wasting conditions. However, functional roles of miRNAs in muscle atrophy are poorly understood. In this study, we generated tamoxifen-inducible Dicer knockout (iDicer KO) mice and subjected them to 2 weeks of single hindlimb denervation. The expression of Dicer mRNA was significantly reduced in muscle of the iDicer KO mice compared to that of WT mice. The loss of Dicer moderately reduced levels of muscle-enriched miRNAs, miR-1, miR-133a and miR-206 in both innervated and denervated muscles of the iDicer KO mice. We also found that the extent of denervation-induced muscle atrophy as well as changes of signaling molecules related to protein synthesis/degradation pathways in the iDicer KO mice were comparable to these in WT mice. Taken together, Dicer knockout in adult skeletal muscle did not affect denervation-induced muscle atrophy.

Millimeter-wave radio-over-fiber system using optical phase modulation and photonic downconversion for uplink fronthaul transmission.

This Letter proposes a high-performance radio-over-fiber (RoF) system for high-speed and high-fidelity analog waveform transmission of radio signals in the millimeter-wave band in the uplink direction. At the antenna site, the system utilizes a newly fabricated low half-wave voltage broadband phase modulator to convert a millimeter-wave radio signal into an optical signal. At the receiver, by using photonic downconversion and optical filtering technology, a simple direct detection and downconversion of the signal to the microwave band can be achieved simultaneously. As a demonstration of proof of concept, we successfully transmitted a 1024-quadrature amplitude modulation (QAM) narrowband orthogonal frequency-division multiplexing signal at 38 GHz and a 60 Gb/s 64-QAM single-carrier signal at 26.5 GHz over a 20 km RoF system. The system is promising for facilitating the deployment of ultra-dense small cells in high-frequency bands in 5G and beyond networks.

An acute eccentric exercise increases circulating myomesin 3 fragments.

Discovery of blood biomarkers to evaluate exercise-induced muscle damage have attracted many researchers and coaches. This study aimed to determine changes in circulating myomesin 3 fragments as a novel biomarker for exercise-induced muscle damage. Nine healthy males performed 10 sets of 40 repetitions of one-leg calf-raise exercise by the load corresponding to the half of their body weight. Muscle symptoms were evaluated by a visual analog scale (VAS). Blood samples were collected before and 2, 4, 24, 48, 72, and 96 h post-exercise. Plasma myomesin 3 fragments levels were significantly increased at 96 h after the eccentric exercise. The myomesin 3 fragments levels were correlated with other biomarkers of muscle damage and the muscle symptoms. These results suggest that the circulating myomesin 3 fragments levels are potential biomarkers reflecting eccentric exercise-induced muscle damage.

The impact of aerobic fitness on arterial stiffness and adrenal cortex hormones in middle-aged and older adults.

An increase in arterial stiffness with advance aging is a risk for cardiovascular disease. Cardiovascular dysfunction is associated with the imbalance of adrenal cortex hormones, especially with the cortisol/dehydroepiandrosterone sulfate (DHEAs) ratio. However, the impact of aerobic fitness on arterial stiffness and cortisol/DHEAs ratio is unclear. The aim of this study was to investigate the relationship between aerobic fitness, arterial stiffness, and cortisol/DHEAs ratio. A total of 198 middle-aged and older adults (aged 50-79 years old) participated in this study. The aerobic fitness evaluated by peak oxygen consumption (VO2peak), carotid-femoral pulse wave velocity (cfPWV) as an indicator of arterial stiffness, and serum cortisol and DHEAs and their ratio were measured. The subjects were divided into the lower (n = 100) and the higher (n = 98) aerobic fitness groups based on the median value of VO2peak. There were no significant differences in serum cortisol and DHEAs concentration alone between the lower and higher fitness groups. However, the cortisol/DEHAs ratio and cfPWV in the higher fitness group was smaller than in the lower fitness group (p < 0.05). The cortisol/DHEAs ratio was significantly correlated with cfPWV (r = 0.159, p < 0.05). These findings suggest that the cortisol/DHEAs ratio is associated with aerobic fitness and arterial stiffness in middle-aged and older adults.

Conditional Deletion of Dicer in Adult Mice Impairs Skeletal Muscle Regeneration.

Skeletal muscle has a remarkable regenerative capacity, which is orchestrated by multiple processes, including the proliferation, fusion, and differentiation of the resident stem cells in muscle. MicroRNAs (miRNAs) are small noncoding RNAs that mediate the translational repression or degradation of mRNA to regulate diverse biological functions. Previous studies have suggested that several miRNAs play important roles in myoblast proliferation and differentiation in vitro. However, their potential roles in skeletal muscle regeneration in vivo have not been fully established. In this study, we generated a mouse in which the Dicer gene, which encodes an enzyme essential in miRNA processing, was knocked out in a tamoxifen-inducible way (iDicer KO mouse) and determined its regenerative potential after cardiotoxin-induced acute muscle injury. Dicer mRNA expression was significantly reduced in the tibialis anterior muscle of the iDicer KO mice, whereas the expression of muscle-enriched miRNAs was only slightly reduced in the Dicer-deficient muscles. After cardiotoxin injection, the iDicer KO mice showed impaired muscle regeneration. We also demonstrated that the number of PAX7+ cells, cell proliferation, and the myogenic differentiation capacity of the primary myoblasts did not differ between the wild-type and the iDicer KO mice. Taken together, these data demonstrate that Dicer is a critical factor for muscle regeneration in vivo.

Effects of Exercise Training on Growth and Differentiation Factor 11 Expression in Aged Mice.

Exercise training is considered an effective way to prevent age-related skeletal muscle loss. However, the molecular mechanism has not been clarified. Growth and differentiation factor 11 (GDF11) has been controversially considered a regulator of skeletal muscle aging. In this study, we examined whether GDF11 is associated with skeletal muscle aging and the effects of exercise training on age-related skeletal muscle loss. First, we observed that Gdf11 mRNA and protein expression levels in young (5-month-old, n = 6) and aged (22-to 26-month-old, n = 5) mice were not significantly different. Aged mice were then divided into sedentary (n = 5) and exercise (n = 6) groups. The exercise group performed moderate-intensity treadmill running for 6 weeks. Treadmill exercise training increased Gdf11 mRNA expression in the soleus muscle, but its protein expression was not altered. In contrast, the GDF11 level in the plantaris muscle was not changed at either the mRNA or protein level. Collectively, our data demonstrate that GDF11 levels do not change during aging, and that treadmill exercise training increased Gdf11 mRNA expression in a predominantly slow-twitch muscle.

Loss of microRNA-23-27-24 clusters in skeletal muscle is not influential in skeletal muscle development and exercise-induced muscle adaptation.

MicroRNAs are small regulatory noncoding RNAs that repress gene expression at the posttranscriptional level. Previous studies have reported that the expression of miR-23, miR-27, and miR-24, driven from two miR-23-27-24 clusters, is altered by various pathophysiological conditions. However, their functions in skeletal muscle have not been clarified. To define the roles of the miR-23-27-24 clusters in skeletal muscle, we generated double-knockout (dKO) mice muscle-specifically lacking the miR-23-27-24 clusters. The dKO mice were viable and showed normal growth. The contractile and metabolic features of the muscles, represented by the expression of the myosin heavy chain and the oxidative markers PGC1-α and COX IV, were not altered in the dKO mice compared with wild-type mice. The dKO mice showed increased cross-sectional areas of the oxidative fibers. However, this dKO did not induce functional changes in the muscles. The dKO mice also showed normal adaptation to voluntary wheel running for 4 weeks, including the glycolytic-to-oxidative fiber type switch, and increases in mitochondrial markers, succinate dehydrogenase activity, and angiogenesis. In conclusion, our data demonstrate that the miR-23-27-24 clusters have subtle effects on skeletal muscle development and endurance-exercise-induced muscle adaptation.

An inducible knockout of Dicer in adult mice does not affect endurance exercise-induced muscle adaptation.

The contractile and metabolic properties of adult skeletal muscle change in response to endurance exercise. The mechanisms of transcriptional regulation in exercise-induced skeletal muscle adaptation, including fiber-type switching and mitochondrial biogenesis, have been investigated intensively, whereas the role of microRNA (miRNA)-mediated posttranscriptional gene regulation is less well understood. We used tamoxifen-inducible Dicer1 knockout (iDicer KO) mice to reduce the global expression of miRNAs in adult skeletal muscle and subjected these mice to 2 wk of voluntary wheel running. Dicer mRNA expression was completely depleted in fast-twitch plantaris muscle after tamoxifen injection. However, several muscle-enriched miRNAs, including miR-1 and miR-133a, were reduced by only 30-50% in both the slow and fast muscles. The endurance exercise-induced changes that occurred for many parameters (i.e., fast-to-slow fiber-type switch and increases in succinate dehydrogenase, respiratory chain complex II, and citrate synthase activity) in wild type (WT) also occurred in the iDicer KO mice. Protein expression of myosin heavy chain IIa, peroxisome proliferator-activated receptor-γ coactivator-1α, and cytochrome c complex IV was also increased in the iDicer KO mice by the voluntary running. Furthermore, there was no significant difference in oxygen consumption rate in the isolated mitochondria between the WT and iDicer KO mice. These data indicate that muscle-enriched miRNAs were detectable even after 4 wk of tamoxifen treatment and there was no apparent specific endurance-exercise-induced muscle phenotype in the iDicer KO mice.

Rer1-mediated quality control system is required for neural stem cell maintenance during cerebral cortex development.

Rer1 is a retrieval receptor for endoplasmic reticulum (ER) retention of various ER membrane proteins and unassembled or immature components of membrane protein complexes. However, its physiological functions during mammalian development remain unclear. This study aimed to investigate the role of Rer1-mediated quality control system in mammalian development. We show that Rer1 is required for the sufficient cell surface expression and activity of γ-secretase complex, which modulates Notch signaling during mouse cerebral cortex development. When Rer1 was depleted in the mouse cerebral cortex, the number of neural stem cells decreased significantly, and malformation of the cerebral cortex was observed. Rer1 loss reduced γ-secretase activity and downregulated Notch signaling in the developing cerebral cortex. In Rer1-deficient cells, a subpopulation of γ-secretase complexes and components was transported to and degraded in lysosomes, thereby significantly reducing the amount of γ-secretase complex on the cell surface. These results suggest that Rer1 maintains Notch signaling by maintaining sufficient expression of the γ-secretase complex on the cell surface and regulating neural stem cell maintenance during cerebral cortex development.

Role of endothelial microRNA-23 clusters in angiogenesis in vivo.

The capillary network is distributed throughout the body, and its reconstruction is induced under various pathophysiological conditions. MicroRNAs are small noncoding RNAs that regulate gene expression via posttranscriptional mechanisms and are involved in many biological functions, including angiogenesis. Previous studies have shown that each microRNA of miR-23 clusters, composed of the miR-23a cluster (miR-23a~27a~24-2) and miR-23b cluster (miR-23b~27b~24-1), regulates angiogenesis in vitro. However, the role of miR-23 clusters, located within a single transcription unit, in angiogenesis in vivo has not been elucidated. In the present study, we generated vascular endothelial cell (EC)-specific miR-23 cluster double-knockout (DKO) mice and demonstrated sprouting angiogenesis under various conditions, including voluntary running exercise, hindlimb ischemia, skin wound healing, and EC sprouting from aorta explants. Here, we demonstrated that EC-specific miR-23 DKO mice are viable and fertile, with no gross abnormalities observed in pups or adults. The capillary number was normally increased in the muscles of these DKO mice in response to 2 wk of voluntary running and hindlimb ischemia. Furthermore, we did not observe any abnormalities in skin wound closure or EC sprouting from aortic ring explants in EC-specific miR-23 cluster DKO mice. Our results suggest that endothelial miR-23 clusters are dispensable for embryonic development and postnatal angiogenesis in vivo. NEW & NOTEWORTHY We generated vascular endothelial cell (EC)-specific miR-23a/b cluster double-knockout mice and determined sprouting angiogenesis under various conditions, including voluntary running exercise, hindlimb ischemia, skin wound healing, and EC sprouting from aorta explants. We demonstrated that the double-knockout mice were viable and fertile, with no gross abnormalities in exercise- and ischemia-induced angiogenesis and skin wound closure or EC sprouting from aortic ring explants.

Plasma ADMA concentrations associate with aerobic fitness in postmenopausal women.

AIMS: Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of nitric oxide (NO) synthase, an enzyme responsible for the generation of NO. Plasma concentrations of ADMA increase in the elderly and in postmenopausal women. In fact, an elevated ADMA level is a risk factor of cardiovascular disease. Aerobic exercise has a beneficial effect on cardiovascular disease. However, the relationship between ADMA and aerobic fitness is unknown. The aim of this study was to determine whether plasma ADMA concentrations correlate with aerobic fitness levels in postmenopausal women. MAIN METHODS: Thirty healthy postmenopausal women aged 50-76 years participated in this study. We measured plasma concentrations of ADMA and oxygen consumption at the ventilatory threshold (VO2VT) as an index of aerobic fitness. Subjects were divided into the low aerobic fitness (Low fitness) and high aerobic fitness (High fitness) groups, and the dividing line was set at the median VO2VT value. KEY FINDINGS: VO2VT was significantly higher in the High fitness group than in the Low fitness group (P<0.01). The plasma ADMA concentrations in the High fitness group were significantly lower than those in the Low fitness group (P<0.05). There was a negative correlation between plasma ADMA concentrations and VO2VT (r=-0.532, P<0.01). SIGNIFICANCE: We found that plasma ADMA concentrations were associated with aerobic fitness in postmenopausal women. The results of this study suggest that habitual aerobic exercise may decrease plasma ADMA concentrations.