Rhianna C. Laker

Autophagy is required for exercise training-induced skeletal muscle adaptation and improvement of physical performance

Pathological and physiological stimuli, including acute exercise, activate autophagy; however, it is unknown whether exercise training alters basal levels of autophagy and whether autophagy is required for skeletal muscle adaptation to training. We observed greater autophagy flux (i.e., a combination of increased LC3‐II/LC3‐I ratio and LC3‐II levels and reduced p62 protein content indicating a higher rate of initiation and resolution of autophagic events), autophagy protein expression (i.e., Atg6/Beclin1, Atg7, and Atg8/LC3) and mitophagy protein Bnip3 expression in tonic, oxidative muscle compared to muscles of either mixed fiber types or of predominant glycolytic fibers in mice. Long‐term voluntary running (4 wk) resulted in increased basal autophagy flux and expression of autophagy proteins and Bnip3 in parallel to mitochondrial biogenesis in plantaris muscle with mixed fiber types. Conversely, exercise training promoted autophagy protein expression with no significant increases of autophagy flux and mitochondrial biogenesis in the oxidative soleus muscle. We also observed increased basal autophagy flux and Bnip3 content without increases in autophagy protein expression in the plantaris muscle of sedentary muscle‐specific Pgc‐1α. transgenic mice, a genetic model of augmented mitochondrial biogenesis. These findings reveal that endurance exercise training‐induced increases in basal autophagy, including mitophagy, only take place if an enhanced oxidative phenotype is achieved. However, autophagy protein expression is mainly dictated by contractile activity independently of enhancements in oxidative phenotype. Exercise‐trained mice heterozygous for the critical autophagy protein Atg6 showed attenuated increases of basal autophagy flux, mitochondrial content, and angiogenesis in skeletal muscle, along with impaired improvement of endurance capacity. These results demonstrate that increased basal autophagy is required for endurance exercise training‐induced skeletal muscle adaptation and improvement of physical performance.—Lira, V. A., Okutsu, M., Zhang, M., Greene, N. P., Laker, R. C., Breen, D. S., Hoehn, K. L., Yan, Z., Autophagy is required for exercise training‐induced skeletal muscle adaptation and improvement of physical performance. FASEB J. 27, 4184–4193 (2013). www.fasebj.org

Exercise Prevents Maternal High-Fat Diet–Induced Hypermethylation of the Pgc-1α Gene and Age-Dependent Metabolic Dysfunction in the Offspring

Abnormal conditions during early development adversely affect later health. We investigated whether maternal exercise could protect offspring from adverse effects of a maternal high-fat diet (HFD) with a focus on the metabolic outcomes and epigenetic regulation of the metabolic master regulator, peroxisome proliferator-activated receptor γ coactivator-1α (Pgc-1α). Female C57BL/6 mice were exposed to normal chow, an HFD, or an HFD with voluntary wheel exercise for 6 weeks before and throughout pregnancy. Methylation of the Pgc-1α promoter at CpG site −260 and expression of Pgc-1α mRNA were assessed in skeletal muscle from neonatal and 12-month-old offspring, and glucose and insulin tolerance tests were performed in the female offspring at 6, 9, and 12 months. Hypermethylation of the Pgc-1α promoter caused by a maternal HFD was detected at birth and was maintained until 12 months of age with a trend of reduced expression of Pgc-1α mRNA (P = 0.065) and its target genes. Maternal exercise prevented maternal HFD-induced Pgc-1α hypermethylation and enhanced Pgc-1α and its target gene expression, concurrent with amelioration of age-associated metabolic dysfunction at 9 months of age in the offspring. Therefore, maternal exercise is a powerful lifestyle intervention for preventing maternal HFD-induced epigenetic and metabolic dysregulation in the offspring.

A Novel MitoTimer Reporter Gene for Mitochondrial Content, Structure, Stress, and Damage in Vivo

Background: Mitochondrial health is difficult to assess in vivo. Results: We have generated a reporter gene, MitoTimer, which targets mitochondria, and fluoresces green and shifts to red when oxidized, for assessment of mitochondrial content, structure, stress, and damage under physiological and pathological conditions. Conclusion: MitoTimer is useful for assessment of mitochondrial health in vivo. Significance: MitoTimer could advance mitochondrial research in multiple disciplines. Mitochondrial dysfunction plays important roles in many diseases, but there is no satisfactory method to assess mitochondrial health in vivo. Here, we engineered a MitoTimer reporter gene from the existing Timer reporter gene. MitoTimer encodes a mitochondria-targeted green fluorescent protein when newly synthesized, which shifts irreversibly to red fluorescence when oxidized. Confocal microscopy confirmed targeting of the MitoTimer protein to mitochondria in cultured cells, Caenorhabditis elegans touch receptor neurons, Drosophila melanogaster heart and indirect flight muscle, and mouse skeletal muscle. A ratiometric algorithm revealed that conditions that cause mitochondrial stress led to a significant shift toward red fluorescence as well as accumulation of pure red fluorescent puncta of damaged mitochondria targeted for mitophagy. Long term voluntary exercise resulted in a significant fluorescence shift toward green, in mice and D. melanogaster, as well as significantly improved structure and increased content in mouse FDB muscle. In contrast, high-fat feeding in mice resulted in a significant shift toward red fluorescence and accumulation of pure red puncta in skeletal muscle, which were completely ameliorated by voluntary wheel running. Hence, MitoTimer allows for robust analysis of multiple parameters of mitochondrial health under both physiological and pathological conditions and will be highly useful for future research of mitochondrial health in multiple disciplines in vivo.

Opening of the mitochondrial permeability transition pore links mitochondrial dysfunction to insulin resistance in skeletal muscle.

Insulin resistance is associated with mitochondrial dysfunction, but the mechanism by which mitochondria inhibit insulin-stimulated glucose uptake into the cytoplasm is unclear. The mitochondrial permeability transition pore (mPTP) is a protein complex that facilitates the exchange of molecules between the mitochondrial matrix and cytoplasm, and opening of the mPTP occurs in response to physiological stressors that are associated with insulin resistance. In this study, we investigated whether mPTP opening provides a link between mitochondrial dysfunction and insulin resistance by inhibiting the mPTP gatekeeper protein cyclophilin D (CypD) in vivo and in vitro. Mice lacking CypD were protected from high fat diet-induced glucose intolerance due to increased glucose uptake in skeletal muscle. The mitochondria in CypD knockout muscle were resistant to diet-induced swelling and had improved calcium retention capacity compared to controls; however, no changes were observed in muscle oxidative damage, insulin signaling, lipotoxic lipid accumulation or mitochondrial bioenergetics. In vitro, we tested 4 models of insulin resistance that are linked to mitochondrial dysfunction in cultured skeletal muscle cells including antimycin A, C2-ceramide, ferutinin, and palmitate. In all models, we observed that pharmacological inhibition of mPTP opening with the CypD inhibitor cyclosporin A was sufficient to prevent insulin resistance at the level of insulin-stimulated GLUT4 translocation to the plasma membrane. The protective effects of mPTP inhibition on insulin sensitivity were associated with improved mitochondrial calcium retention capacity but did not involve changes in insulin signaling both in vitro and in vivo. In sum, these data place the mPTP at a critical intersection between alterations in mitochondrial function and insulin resistance in skeletal muscle.

Short-term exercise training early in life restores deficits in pancreatic β-cell mass associated with growth restriction in adult male rats

Fetal growth restriction is associated with reduced pancreatic β-cell mass, contributing to impaired glucose tolerance and diabetes. Exercise training increases β-cell mass in animals with diabetes and has long-lasting metabolic benefits in rodents and humans. We studied the effect of exercise training on islet and β-cell morphology and plasma insulin and glucose, following an intraperitoneal glucose tolerance test (IPGTT) in juvenile and adult male Wistar-Kyoto rats born small. Bilateral uterine vessel ligation performed on day 18 of pregnancy resulted in Restricted offspring born small compared with sham-operated Controls and also sham-operated Reduced litter offspring that had their litter size reduced to five pups at birth. Restricted, Control, and Reduced litter offspring remained sedentary or underwent treadmill running from 5 to 9 or 20 to 24 wk of age. Early life exercise increased relative islet surface area and β-cell mass across all groups at 9 wk, partially restoring the 60-68% deficit (P < 0.05) in Restricted offspring. Remarkably, despite no further exercise training after 9 wk, β-cell mass was restored in Restricted at 24 wk, while sedentary littermates retained a 45% deficit (P = 0.05) in relative β-cell mass. Later exercise training also restored Restricted β-cell mass to Control levels. In conclusion, early life exercise training in rats born small restored β-cell mass in adulthood and may have beneficial consequences for later metabolic health and disease.

Ampk phosphorylation of Ulk1 is required for targeting of mitochondria to lysosomes in exercise-induced mitophagy

Mitochondrial health is critical for skeletal muscle function and is improved by exercise training through both mitochondrial biogenesis and removal of damaged/dysfunctional mitochondria via mitophagy. The mechanisms underlying exercise-induced mitophagy have not been fully elucidated. Here, we show that acute treadmill running in mice causes mitochondrial oxidative stress at 3–12 h and mitophagy at 6 h post-exercise in skeletal muscle. These changes were monitored using a novel fluorescent reporter gene, pMitoTimer, that allows assessment of mitochondrial oxidative stress and mitophagy in vivo, and were preceded by increased phosphorylation of AMP activated protein kinase (Ampk) at tyrosine 172 and of unc-51 like autophagy activating kinase 1 (Ulk1) at serine 555. Using mice expressing dominant negative and constitutively active Ampk in skeletal muscle, we demonstrate that Ulk1 activation is dependent on Ampk. Furthermore, exercise-induced metabolic adaptation requires Ulk1. These findings provide direct evidence of exercise-induced mitophagy and demonstrate the importance of Ampk-Ulk1 signaling in skeletal muscle.Exercise is associated with biogenesis and removal of dysfunctional mitochondria. Here the authors use a mitochondrial reporter gene to demonstrate the occurrence of mitophagy following exercise in mice, and show this is dependent on AMPK and ULK1 signaling.

Progesterone Withdrawal, and Not Increased Circulating Relaxin, Mediates the Decrease in Myometrial Relaxin Receptor (RXFP1) Expression in Late Gestation in Rats

In pregnant rats, a significant decrease in myometrial relaxin family peptide receptor 1 (RXFP1) expression, indicative of a functional relaxin withdrawal for activation of myometrial contractions, occurs in late gestation and during spontaneous labor. This coincides with the highest level of circulating relaxin and a decrease in progesterone. We investigated the potential regulatory role of these two systemic factors on myometrial RXFP1 expression by examining the effects of the antiprogestin RU486 and a monoclonal antibody against rat relaxin (MCA1) in pregnant rats. Rats were injected with RU486 on Gestational Day (GD) 7, 16, or 19 and were killed on GD 8, 17, or 20. RU486 caused a significant reduction in myometrial RXFP1. Plasma progesterone and 17beta-estradiol levels were increased in RU486-treated animals compared with controls. RU486 treatment also caused significant increases in myometrial Esr1 and Vegf and a decrease in Esr2. MCA1 was administered i.v. to rats from GD 17 to GD 19. On GD 20, no significant effect of MCA1 treatment on myometrial RXFP1 expression was observed compared with controls. Furthermore, there was no change in Esr1 or Esr2. A significant reduction in myometrial Vegf, however, was observed. We suggest that blocking progesterone action with RU486 increases plasma 17beta-estradiol and myometrial Esr1 and results in decreased RXFP1 expression. In summary, myometrial RXFP1 expression is mediated mainly by progesterone and not circulating relaxin in pregnant rats.

Central infusion of leptin does not increase AMPK signaling in skeletal muscle of sheep

In sheep, central leptin infusion reduces food intake and increases energy expenditure in adipose tissue and skeletal muscle. The mechanisms for these peripheral effects of central leptin in sheep are not known but, on the basis of rodent studies, may involve AMPK. In mice, central leptin acutely increases both skeletal muscle AMPK activation and glucose uptake. Thus, to investigate whether these effects exist in higher-order mammals, ovariectomized Corriedale ewes (n = 4 per group) received a continuous lateral ventricular infusion (60 μl/h) of either leptin (50 μg/h) or artificial cerebrospinal fluid (aCSF; CON) for 8 days. Tritiated glucose (3-(3)H-glucose) was infused intravenously for calculation of whole body glucose turnover during both acute (6 h) and chronic (7-8 days) leptin/aCSF infusion. Muscle biopsies were also obtained. Leptin infusion reduced (P < 0.05) food intake and body weight, and it also increased plasma epinephrine concentration at 6 h and 7 days, suggesting increased sympathetic nerve activity. Despite this, and in contrast to rodent studies, central leptin infusion did not increase skeletal muscle AMPKα Thr(172) phosphorylation or ACCβ Ser(221) phosphorylation. Surprisingly, the glucose rate of appearance (glucose Ra) and rate of disappearance (glucose Rd) were reduced by both acute and chronic leptin infusion. Direct infusion of the AMPK activator 5-aminoimidazole-4-carboxyamide-ribonucleoside (AICAR) into the femoral artery increased skeletal muscle AMPK phosphorylation. In conclusion, although central leptin infusion in sheep caused the predicted reduction in food intake and increases plasma epinephrine concentration, it had no effect on AMPK activation in skeletal muscle and actually reduced glucose disposal. This suggests that there are species differences in the peripheral responses to central leptin infusion.

Ulk1-mediated autophagy plays an essential role in mitochondrial remodeling and functional regeneration of skeletal muscle

Autophagy is a conserved cellular process for degrading aggregate proteins and dysfunctional organelle. It is still debatable if autophagy and mitophagy (a specific process of autophagy of mitochondria) play important roles in myogenic differentiation and functional regeneration of skeletal muscle. We tested the hypothesis that autophagy is critical for functional regeneration of skeletal muscle. We first observed time-dependent increases (3- to 6-fold) of autophagy-related proteins (Atgs), including Ulk1, Beclin1, and LC3, along with reduced p62 expression during C2C12 differentiation, suggesting increased autophagy capacity and flux during myogenic differentiation. We then used cardiotoxin (Ctx) or ischemia-reperfusion (I/R) to induce muscle injury and regeneration and observed increases in Atgs between days 2 and 7 in adult skeletal muscle followed by increased autophagy flux after day 7 Since Ulk1 has been shown to be essential for mitophagy, we asked if Ulk1 is critical for functional regeneration in skeletal muscle. We subjected skeletal muscle-specific Ulk1 knockout mice (MKO) to Ctx or I/R. MKO mice had significantly impaired recovery of muscle strength and mitochondrial protein content post-Ctx or I/R. Imaging analysis showed that MKO mice have significantly attenuated recovery of mitochondrial network at 7 and 14 days post-Ctx. These findings suggest that increased autophagy protein and flux occur during muscle regeneration and Ulk1-mediated mitophagy is critical for recovery for the mitochondrial network and hence functional regeneration.

Endurance training in early life results in long‐term programming of heart mass in rats

Being born small for gestational age increases the risk of developing adult cardiovascular and metabolic diseases. This study aimed to examine if early‐life exercise could increase heart mass in the adult hearts from growth restricted rats. Bilateral uterine vessel ligation to induce uteroplacental insufficiency and fetal growth restriction in the offspring (Restricted) or sham surgery (Control) was performed on day 18 of gestation in WKY rats. A separate group of sham litters had litter size reduced to five pups at birth (Reduced litter), which restricted postnatal growth. Male offspring remained sedentary or underwent treadmill running from 5 to 9 weeks (early exercise) or 20 to 24 weeks of age (later exercise). Remarkably, in Control, Restricted, and Reduced litter groups, early exercise increased (P < 0.05) absolute and relative (to body mass) heart mass in adulthood. This was despite the animals being sedentary for ~4 months after exercise. Later exercise also increased adult absolute and relative heart mass (P < 0.05). Blood pressure was not significantly altered between groups or by early or later exercise. Phosphorylation of Akt Ser473 in adulthood was increased in the early exercise groups but not the later exercise groups. Microarray gene analysis and validation by real‐time PCR did not reveal any long‐term effects of early exercise on the expression of any individual genes. In summary, early exercise programs the heart for increased mass into adulthood, perhaps by an upregulation of protein synthesis based on greater phosphorylation of Akt Ser473.