Ampk phosphorylation of Ulk1 is required for targeting of mitochondria to lysosomes in exercise-induced mitophagy methods
Aim. Evidence-backed execution summary for Ampk phosphorylation of Ulk1 is required for targeting of mitochondria to lysosomes in exercise-induced mitophagy methods from Ampk phosphorylation of Ulk1 is required for targeting of mitochondria to lysosomes in exercise-induced mitophagy.
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mouse
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Western blotting
reagent used in the protocol.
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- Total protein or mitochondrial-enriched lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membrane. Mitochondrial-enriched lysates were isolated via differential centrifugation from frozen gastrocnemius muscle. Briefly, muscles were pulverized by...
Tissue preparation and confocal microscopy
At the time of muscle harvest, the FDB was immediately fixed in 4% paraformaldehyde for 20 min and whole mounted on gelatin coated slides with 50% glycerol in phosphate-buffered saline with a coverslip. Images were acquired at × 100 magnification under a confocal microscope (Olympus Fluoview FV1000) using...
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- At the time of muscle harvest, the FDB was immediately fixed in 4% paraformaldehyde for 20 min and whole mounted on gelatin coated slides with 50% glycerol in phosphate-buffered saline with a coverslip. Images were acquired at × 100 magnification under a confocal microscope (Olympus Fluoview FV1000) using...
Western blotting
Membranes were probed with the following primary antibodies at 1:1000 dilution, targeting Lc3A/B (CST #4108), T172 Ampk (CST #2535), Ampk α1/2 (CST #2532), Ampk γ1 (Abcam #32508), S79 Acc (CST #3661), Acc (CST #3662), S555 Ulk1 (CST #5869), S757 Ulk1 (CST #6888), S467 Ulk1 (CST #4634), Ulk1 (Sigma-Aldrich...
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- Membranes were probed with the following primary antibodies at 1:1000 dilution, targeting Lc3A/B (CST #4108), T172 Ampk (CST #2535), Ampk α1/2 (CST #2532), Ampk γ1 (Abcam #32508), S79 Acc (CST #3661), Acc (CST #3662), S555 Ulk1 (CST #5869), S757 Ulk1 (CST #6888), S467 Ulk1 (CST #4634), Ulk1 (Sigma-Aldrich...
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Activation of mitophagy signaling in response to exercise
It is well known that Ampk can be activated by an acute bout of exercise,. We found that treadmill running increased phosphorylation of Ampk at tyrosine (T) 172 and its downstream substrate Acetyl-CoA Carboxylase (Acc) at serine (S) 79 in gastrocnemius muscle immediately following the exercise bout (Fig. ). This effect was transient and not observed at later time points during the recovery period. Concurrently, acute exercise increased phosphorylation of Ulk1 only at the activating S555 site (Fig. ). The inhibitory phosphorylation sites of S467 or S757 were unaltered (Fig. ). These findings suggest that exercise leads to a transient phosphorylation of Ampk and Ulk1 on activating sites, prior to mitophagic degradation of targeted mitochondria during recovery from exercise (Fig., Supplementary Fig. ). Fig. 2 Ampk, Ulk1 and Drp1, are transiently activated...
Activation of mitophagy signaling in response to exercise
Mitochondrial fission is essential to separate damaged/dysfunctional areas of mitochondria from the mitochondrial reticulum and thus facilitates mitophagy. The dynamin-related protein 1 (Drp1) is an important GTPase that promotes mitochondrial fission when phosphorylated at S616. We found that phosphorylation of Drp1 at this site (S616) was transiently increased immediately following treadmill running (Fig. ). We also found that phosphorylation at site S637 was elevated following acute exercise, reaching significance 3 and 6 hours after the cessation of exercise (Fig. ). S637 phosphorylation has been shown to increase fission in response to Ca 2+ signaling, yet can also inhibit Drp1 activity under conditions of protein kinase A (Pka)-dependent phosphorylation -, and has been suggested as an Ampk target,. This discrepancy in the literature highlights the importan...
Ampk is required for exercise-induced phosphorylation of Ulk1
To determine whether Ampk is required for exercise-induced activation of Ulk1, we subjected muscle-specific transgenic mice of a dominant-negative form of the catalytic α2 subunit of Ampk (dnTG) to acute exercise. These mice were able to complete the exercise bout to the same capacity as the wild type littermates. Ampk phosphorylation at T172 was blunted in dnTG mice in response to exercise (two-way analysis of variance (ANOVA) interaction effect p = 0.044, F = 4.45, DF = 1) (Fig. ); however, exercise-induced phosphorylation of Acc was not disturbed (Fig. ), as has previously been reported for this Ampkα mouse model and others -. Importantly, Ulk1 S555 phosphorylation was completely ablated in dnTG skeletal muscle following exercise (two-way ANOVA interaction effect p = 0.015, F = 6.71, DFR...
Ampk is required for exercise-induced phosphorylation of Ulk1
To investigate whether mitochondrial dynamics were regulated through Ampk, we also measured Drp1 phosphorylation and Mfn2 protein in dnTG and caTG Ampk mice. As observed in WT exercised mice, phosphorylation of Drp1 was significantly enhanced following acute exercise in Ampk dnTG mice at both S616 and S637 (Fig. ). In fact, phosphorylation of S637 was induced to a level almost twofold higher than that of WT mice by acute exercise (two-way ANOVA interaction effect p = 0.007, F = 8.69, DF = 1) (Fig. ). Furthermore, under sedentary conditions, caTG mice displayed similar levels of Drp1 phosphorylation to their WT littermates (Fig. ) and there were no changes in Mfn2 protein, regardless of genotype or exercise (Fig. ). These findings suggest that exercise-induced phosphorylation of Drp1 occurs independently of Ampk in skeletal...
Ampk is required for exercise-induced mitophagy in muscle
Since Ampk is required for activation of Ulk1 by exercise (Fig. ), we investigated whether exercise-induced mitophagy is dependent on Ampk. We performed co-transfection in FDB muscles with pMitoTimer and pLamp1-YFP in dnTG mice. As already described, dnTG mice were subjected to treadmill running and the FDB was harvested 6 h post-exercise, corresponding with the peak of mitochondrial oxidative stress and mitophagy (Fig., Supplementary Fig. ). Consistent with our previous experiment, we observed an overall increase of red:green ratio 6 h following exercise in WT mice (Fig. ). In contrast, dnTG mice showed moderately elevated oxidative stress, but there was no further increase following exercise (two-way ANOVA interaction effect p = 0.037, F = 4.92, DF = 1) (Fig. ). The increase in MitoTimer pure red puncta...
Ulk1 is required for exercise-induced lysosomal targeting
Since Ulk1 is a critical regulator of autophagy and is activated by acute exercise (Fig. ), Ulk1 may be important for mitophagy to remove damaged/dysfunctional mitochondria during recovery in response to acute exercise (Fig., Supplementary Fig. ). To address this question, we again co-transfected FDB muscles with pMitoTimer and pLamp1-YFP in muscle-specific Ulk1 knockout (MKO) mice. As described above, mice were then subjected to acute exercise, and FDB muscles were harvested 6 h later (Fig., Fig., and Supplementary Fig. ). There was no difference in basal red:green ratio of MitoTimer between the WT and MKO mice (Fig. ). Furthermore, the overall increase of the red:green ratio 6 h after exercise was similar between genotypes (Fig. ), indicating that deletion of Ulk1 did not alter the responsiveness of mitochondria to exerc...
Ulk1 is required for exercise-induced lysosomal targeting
Ulk1 in skeletal muscle is important for exercise training-induced metabolic adaptation. MKO and iMKO mice displayed normal adaptation to exercise training as assessed by daily running distance a, d, exhaustive exercise test b, e and blood lactate levels c, f. WT mice exhibited improved glucose tolerance following 4 weeks of exercise training as assessed by blood glucose profile during GTT g and AUC i. In contrast, iMKO mice failed to show improved glucose tolerance following exercise training during GTT h and AUC i. Data presented as mean ± standard error of the mean. For MKO study n = 7-8 WT and n = 6-7 MKO (12-13 weeks). Two-tailed t -tests were used for (A: t = 0.84 and DF = 9) and (C: WT Sed t = 3.73 and DF = 12, WT Ex t = 2.97 and DF = 14...
Ulk1 is important for metabolic adaptation to exercise
Since we reported that Ulk1 is critical for acute exercise-induced mitophagy, we reasoned that it would also play a critical role in exercise training adaptation and endurance capacity. Therefore, we subjected MKO and WT mice to 6 weeks of voluntary wheel running and performed an exhaustive exercise test at the cessation of exercise training. Unexpectedly, daily voluntary running distance was similar between genotypes, and both genotypes improved their endurance running capacity after 6 weeks of voluntary running compared to sedentary controls (Fig. ), suggesting that Ulk1 is not required for enhanced physical performance in response to exercise training. In order to further validate these findings and rule out developmental compensation to loss of Ulk1 from early life, we generated tamoxifen-inducible, skeletal muscle-specific Ulk1 knockout (iMKO) mice and subjected them to the...
Measurement outputs
What raw and processed outputs should exist?
To determine whether Ampk is required for exercise-induced activation of Ulk1, we subjected muscle-specific transgenic mice of a dominant-negative form of the catalytic α2...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Since Ampk is required for activation of Ulk1 by exercise (Fig. ), we investigated whether exercise-induced mitophagy is dependent on Ampk. We performed co-transfection in...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
We designed and validated pMitoTimer as a useful tool for measuring mitochondrial health and damage as previously published. pLamp1-YFP was purchased from Addgene (Plasmid #181...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Total protein or mitochondrial-enriched lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membrane. Mitocho...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Analysis plan
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Acquisition
Collect the raw assay or blot output and retain identifiers for each sample and experimental group.
inferred from protocolPreprocessing / cleaning
It is well known that Ampk can be activated by an acute bout of exercise,.
from paperScoring or quantification
Quantify the primary readouts for this experiment: To determine whether Ampk is required for exercise-induced activation of Ulk1, we subjected muscle-specific transgenic mice of a dominant-negative form of the catalytic α2...; Since Ampk is required for activation of Ulk1 by exercise (Fig. ), we investigated whether exercise-induced mitophagy is dependent on Ampk. We performed co-transfection in...; We designed and validated pMitoTimer as a useful tool for measuring mitochondrial health and damage as previously published. pLamp1-YFP was purchased from Addgene (Plasmid #181...; Total protein or mitochondrial-enriched lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membrane. Mitocho....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
It is well known that Ampk can be activated by an acute bout of exercise,. We found that treadmill running increased phosphorylation of Ampk at tyrosine (T) 172 and its downst...; To determine whether Ampk is required for exercise-induced activation of Ulk1, we subjected muscle-specific transgenic mice of a dominant-negative form of the catalytic α2...; To investigate whether mitochondrial dynamics were regulated through Ampk, we also measured Drp1 phosphorylation and Mfn2 protein in dnTG and caTG Ampk mice. As observed in WT e...; Since Ampk is required for activation of Ulk1 by exercise (Fig. ), we investigated whether exercise-induced mitophagy is dependent on Ampk. We performed co-transfection in...
from paperReporting output
Report representative outputs alongside summary comparisons for To determine whether Ampk is required for exercise-induced activation of Ulk1, we subjected muscle-specific transgenic mice of a dominant-negative form of the catalytic α2..., Since Ampk is required for activation of Ulk1 by exercise (Fig. ), we investigated whether exercise-induced mitophagy is dependent on Ampk. We performed co-transfection in..., We designed and validated pMitoTimer as a useful tool for measuring mitochondrial health and damage as previously published. pLamp1-YFP was purchased from Addgene (Plasmid #181..., Total protein or mitochondrial-enriched lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membrane. Mitocho....
inferred from protocolStructured statistical methods
It is well known that Ampk can be activated by an acute bout of exercise,. We found that treadmill running increased phosphorylation of Ampk at tyrosine (T) 172 and its downst...; To determine whether Ampk is required for exercise-induced activation of Ulk1, we subjected muscle-specific transgenic mice of a dominant-negative form of the catalytic α2...; To investigate whether mitochondrial dynamics were regulated through Ampk, we also measured Drp1 phosphorylation and Mfn2 protein in dnTG and caTG Ampk mice. As observed in WT e...; Since Ampk is required for activation of Ulk1 by exercise (Fig. ), we investigated whether exercise-induced mitophagy is dependent on Ampk. We performed co-transfection in...
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Evidence quotes (8)
It is well known that Ampk can be activated by an acute bout of exercise,. We found that treadmill running increased phosphorylation of Ampk at tyrosine (T) 172 and its downstream substrate Acetyl-CoA Carboxylase (Acc) at serine (S) 79 in gastrocnemius muscle immediately following the exercise bout (Fig. ). This effect was transient and not observed at later time points during the recovery period. Concurrently, acute exercise increased phosphorylation of Ulk1 only at the activating S555 site (Fig. ). The inhibitory phosphorylation sites of S467 or S757 were unaltered (Fig. ). These findings suggest that exercise leads to a transient phosphorylation of Ampk and Ulk1 on activating sites, prior to mitophagic degradation of targeted mitochondria during recovery from exercise (Fig., Supplementary Fig. ). Fig. 2 Ampk, Ulk1 and Drp1, are transiently activated immediately following acute exercise. Ampk and its substrate Acc are phosphorylated at Thr172 a, b and S79 a, c, respectively, immediately after the cessation of exercise and return to baseline values by 3 h post-exercise in C57BL/6 J mice aged 10-12 weeks. Ulk1 is phosphorylat...
Mitochondrial fission is essential to separate damaged/dysfunctional areas of mitochondria from the mitochondrial reticulum and thus facilitates mitophagy. The dynamin-related protein 1 (Drp1) is an important GTPase that promotes mitochondrial fission when phosphorylated at S616. We found that phosphorylation of Drp1 at this site (S616) was transiently increased immediately following treadmill running (Fig. ). We also found that phosphorylation at site S637 was elevated following acute exercise, reaching significance 3 and 6 hours after the cessation of exercise (Fig. ). S637 phosphorylation has been shown to increase fission in response to Ca 2+ signaling, yet can also inhibit Drp1 activity under conditions of protein kinase A (Pka)-dependent phosphorylation -, and has been suggested as an Ampk target,. This discrepancy in the literature highlights the importance of context specificity of molecular signaling. Finally, we found no alterations in the mitochondrial fusion protein Mfn2 across the 24 h time course following acute exercise (Fig. ).
To determine whether Ampk is required for exercise-induced activation of Ulk1, we subjected muscle-specific transgenic mice of a dominant-negative form of the catalytic α2 subunit of Ampk (dnTG) to acute exercise. These mice were able to complete the exercise bout to the same capacity as the wild type littermates. Ampk phosphorylation at T172 was blunted in dnTG mice in response to exercise (two-way analysis of variance (ANOVA) interaction effect p = 0.044, F = 4.45, DF = 1) (Fig. ); however, exercise-induced phosphorylation of Acc was not disturbed (Fig. ), as has previously been reported for this Ampkα mouse model and others -. Importantly, Ulk1 S555 phosphorylation was completely ablated in dnTG skeletal muscle following exercise (two-way ANOVA interaction effect p = 0.015, F = 6.71, DF = 1) (Fig. ). Phosphorylation of Ulk1 at the inhibiting site S757 was unchanged by exercise in both WT and dnTG mice, however, there was a genotype effect of lower phosphorylation in the dnTG (Fig. ). Consistently, muscle-specific transgenic mice expressing a constitutively acti...
To investigate whether mitochondrial dynamics were regulated through Ampk, we also measured Drp1 phosphorylation and Mfn2 protein in dnTG and caTG Ampk mice. As observed in WT exercised mice, phosphorylation of Drp1 was significantly enhanced following acute exercise in Ampk dnTG mice at both S616 and S637 (Fig. ). In fact, phosphorylation of S637 was induced to a level almost twofold higher than that of WT mice by acute exercise (two-way ANOVA interaction effect p = 0.007, F = 8.69, DF = 1) (Fig. ). Furthermore, under sedentary conditions, caTG mice displayed similar levels of Drp1 phosphorylation to their WT littermates (Fig. ) and there were no changes in Mfn2 protein, regardless of genotype or exercise (Fig. ). These findings suggest that exercise-induced phosphorylation of Drp1 occurs independently of Ampk in skeletal muscle. Fig. 4 Ampk is not required for exercise-induced phosphorylation of Drp1. Phosphorylation of Drp1 at S616 and S637 is significantly increased in dnTG mice post-exercise ( a - c ), while there was no effect of caTG Ampk on phosphorylation of Drp1 at either S616 e, f or S637 e, g under...
Since Ampk is required for activation of Ulk1 by exercise (Fig. ), we investigated whether exercise-induced mitophagy is dependent on Ampk. We performed co-transfection in FDB muscles with pMitoTimer and pLamp1-YFP in dnTG mice. As already described, dnTG mice were subjected to treadmill running and the FDB was harvested 6 h post-exercise, corresponding with the peak of mitochondrial oxidative stress and mitophagy (Fig., Supplementary Fig. ). Consistent with our previous experiment, we observed an overall increase of red:green ratio 6 h following exercise in WT mice (Fig. ). In contrast, dnTG mice showed moderately elevated oxidative stress, but there was no further increase following exercise (two-way ANOVA interaction effect p = 0.037, F = 4.92, DF = 1) (Fig. ). The increase in MitoTimer pure red puncta 6 h post-exercise observed in WT mice was completely abolished in dnTG mice (two-way ANOVA interaction effect p < 0.0001, F = 36.26, DF = 1) (Fig. ), demonstrating that exercise-induced mitophagy is blocked in dnTG mice. Importantly, acute exercise...
Since Ulk1 is a critical regulator of autophagy and is activated by acute exercise (Fig. ), Ulk1 may be important for mitophagy to remove damaged/dysfunctional mitochondria during recovery in response to acute exercise (Fig., Supplementary Fig. ). To address this question, we again co-transfected FDB muscles with pMitoTimer and pLamp1-YFP in muscle-specific Ulk1 knockout (MKO) mice. As described above, mice were then subjected to acute exercise, and FDB muscles were harvested 6 h later (Fig., Fig., and Supplementary Fig. ). There was no difference in basal red:green ratio of MitoTimer between the WT and MKO mice (Fig. ). Furthermore, the overall increase of the red:green ratio 6 h after exercise was similar between genotypes (Fig. ), indicating that deletion of Ulk1 did not alter the responsiveness of mitochondria to exercise with regard to oxidative stress. In WT mice, there was an increase in MitoTimer pure red puncta after exercise (Fig. ), which was concurrent with both increases in Lamp1 puncta (Fig. ) and co-localization between the two signals (Fig. ). These findings consolidate those from ou...
Ulk1 in skeletal muscle is important for exercise training-induced metabolic adaptation. MKO and iMKO mice displayed normal adaptation to exercise training as assessed by daily running distance a, d, exhaustive exercise test b, e and blood lactate levels c, f. WT mice exhibited improved glucose tolerance following 4 weeks of exercise training as assessed by blood glucose profile during GTT g and AUC i. In contrast, iMKO mice failed to show improved glucose tolerance following exercise training during GTT h and AUC i. Data presented as mean ± standard error of the mean. For MKO study n = 7-8 WT and n = 6-7 MKO (12-13 weeks). Two-tailed t -tests were used for (A: t = 0.84 and DF = 9) and (C: WT Sed t = 3.73 and DF = 12, WT Ex t = 2.97 and DF = 14, MKO Sed t = 6.35 and DF = 10, MKO Ex t = 5.25, DF = 12). Lactate data c was 1/Y transformed to account for unequal variance. A two-way ANOVA was used for b with ** p < 0.01 for sedentary vs. exercise comparison, F = 11.50,...
Since we reported that Ulk1 is critical for acute exercise-induced mitophagy, we reasoned that it would also play a critical role in exercise training adaptation and endurance capacity. Therefore, we subjected MKO and WT mice to 6 weeks of voluntary wheel running and performed an exhaustive exercise test at the cessation of exercise training. Unexpectedly, daily voluntary running distance was similar between genotypes, and both genotypes improved their endurance running capacity after 6 weeks of voluntary running compared to sedentary controls (Fig. ), suggesting that Ulk1 is not required for enhanced physical performance in response to exercise training. In order to further validate these findings and rule out developmental compensation to loss of Ulk1 from early life, we generated tamoxifen-inducible, skeletal muscle-specific Ulk1 knockout (iMKO) mice and subjected them to the same exercise training protocol after 5 days of tamoxifen injection (to delete the Ulk1 gene in skeletal muscle). Consistently, iMKO mice demonstrated similar daily voluntary running distance compared with WT mice, as well as similar improvement in endurance capacity following 6 weeks of voluntary...
Machine-readable layer
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"name": "Ampk phosphorylation of Ulk1 is required for targeting of mitochondria to lysosomes in exercise-induced mitophagy methods",
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"name": "Activation of mitophagy signaling in response to exercise",
"text": "It is well known that Ampk can be activated by an acute bout of exercise,. We found that treadmill running increased phosphorylation of Ampk at tyrosine (T) 172 and its downstream substrate Acetyl-CoA Carboxylase (Acc) at serine (S) 79 in gastrocnemius muscle immediately following the exercise bout (Fig. ). This effect was transient and not observed at later time points during the recovery period. Concurrently, acute exercise increased phosphorylation of Ulk1 only at the activating S555 site (Fig. ). The inhibitory phosphorylation sites of S467 or S757 were unaltered (Fig. ). These findings suggest that exercise leads to a transient phosphorylation of Ampk and Ulk1 on activating sites, prior to mitophagic degradation of targeted mitochondria during recovery from exercise (Fig., Supplementary Fig. ). Fig. 2 Ampk, Ulk1 and Drp1, are transiently activated..."
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"name": "Activation of mitophagy signaling in response to exercise",
"text": "Mitochondrial fission is essential to separate damaged/dysfunctional areas of mitochondria from the mitochondrial reticulum and thus facilitates mitophagy. The dynamin-related protein 1 (Drp1) is an important GTPase that promotes mitochondrial fission when phosphorylated at S616. We found that phosphorylation of Drp1 at this site (S616) was transiently increased immediately following treadmill running (Fig. ). We also found that phosphorylation at site S637 was elevated following acute exercise, reaching significance 3 and 6 hours after the cessation of exercise (Fig. ). S637 phosphorylation has been shown to increase fission in response to Ca 2+ signaling, yet can also inhibit Drp1 activity under conditions of protein kinase A (Pka)-dependent phosphorylation -, and has been suggested as an Ampk target,. This discrepancy in the literature highlights the importan..."
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"name": "Ampk is required for exercise-induced phosphorylation of Ulk1",
"text": "To determine whether Ampk is required for exercise-induced activation of Ulk1, we subjected muscle-specific transgenic mice of a dominant-negative form of the catalytic α2 subunit of Ampk (dnTG) to acute exercise. These mice were able to complete the exercise bout to the same capacity as the wild type littermates. Ampk phosphorylation at T172 was blunted in dnTG mice in response to exercise (two-way analysis of variance (ANOVA) interaction effect p = 0.044, F = 4.45, DF = 1) (Fig. ); however, exercise-induced phosphorylation of Acc was not disturbed (Fig. ), as has previously been reported for this Ampkα mouse model and others -. Importantly, Ulk1 S555 phosphorylation was completely ablated in dnTG skeletal muscle following exercise (two-way ANOVA interaction effect p = 0.015, F = 6.71, DFR..."
},
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"name": "Ampk is required for exercise-induced phosphorylation of Ulk1",
"text": "To investigate whether mitochondrial dynamics were regulated through Ampk, we also measured Drp1 phosphorylation and Mfn2 protein in dnTG and caTG Ampk mice. As observed in WT exercised mice, phosphorylation of Drp1 was significantly enhanced following acute exercise in Ampk dnTG mice at both S616 and S637 (Fig. ). In fact, phosphorylation of S637 was induced to a level almost twofold higher than that of WT mice by acute exercise (two-way ANOVA interaction effect p = 0.007, F = 8.69, DF = 1) (Fig. ). Furthermore, under sedentary conditions, caTG mice displayed similar levels of Drp1 phosphorylation to their WT littermates (Fig. ) and there were no changes in Mfn2 protein, regardless of genotype or exercise (Fig. ). These findings suggest that exercise-induced phosphorylation of Drp1 occurs independently of Ampk in skeletal..."
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"name": "Ampk is required for exercise-induced mitophagy in muscle",
"text": "Since Ampk is required for activation of Ulk1 by exercise (Fig. ), we investigated whether exercise-induced mitophagy is dependent on Ampk. We performed co-transfection in FDB muscles with pMitoTimer and pLamp1-YFP in dnTG mice. As already described, dnTG mice were subjected to treadmill running and the FDB was harvested 6 h post-exercise, corresponding with the peak of mitochondrial oxidative stress and mitophagy (Fig., Supplementary Fig. ). Consistent with our previous experiment, we observed an overall increase of red:green ratio 6 h following exercise in WT mice (Fig. ). In contrast, dnTG mice showed moderately elevated oxidative stress, but there was no further increase following exercise (two-way ANOVA interaction effect p = 0.037, F = 4.92, DF = 1) (Fig. ). The increase in MitoTimer pure red puncta..."
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"name": "Ulk1 is required for exercise-induced lysosomal targeting",
"text": "Since Ulk1 is a critical regulator of autophagy and is activated by acute exercise (Fig. ), Ulk1 may be important for mitophagy to remove damaged/dysfunctional mitochondria during recovery in response to acute exercise (Fig., Supplementary Fig. ). To address this question, we again co-transfected FDB muscles with pMitoTimer and pLamp1-YFP in muscle-specific Ulk1 knockout (MKO) mice. As described above, mice were then subjected to acute exercise, and FDB muscles were harvested 6 h later (Fig., Fig., and Supplementary Fig. ). There was no difference in basal red:green ratio of MitoTimer between the WT and MKO mice (Fig. ). Furthermore, the overall increase of the red:green ratio 6 h after exercise was similar between genotypes (Fig. ), indicating that deletion of Ulk1 did not alter the responsiveness of mitochondria to exerc..."
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"name": "Ulk1 is required for exercise-induced lysosomal targeting",
"text": "Ulk1 in skeletal muscle is important for exercise training-induced metabolic adaptation. MKO and iMKO mice displayed normal adaptation to exercise training as assessed by daily running distance a, d, exhaustive exercise test b, e and blood lactate levels c, f. WT mice exhibited improved glucose tolerance following 4 weeks of exercise training as assessed by blood glucose profile during GTT g and AUC i. In contrast, iMKO mice failed to show improved glucose tolerance following exercise training during GTT h and AUC i. Data presented as mean ± standard error of the mean. For MKO study n = 7-8 WT and n = 6-7 MKO (12-13 weeks). Two-tailed t -tests were used for (A: t = 0.84 and DF = 9) and (C: WT Sed t = 3.73 and DF = 12, WT Ex t = 2.97 and DF = 14..."
},
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"name": "Ulk1 is important for metabolic adaptation to exercise",
"text": "Since we reported that Ulk1 is critical for acute exercise-induced mitophagy, we reasoned that it would also play a critical role in exercise training adaptation and endurance capacity. Therefore, we subjected MKO and WT mice to 6 weeks of voluntary wheel running and performed an exhaustive exercise test at the cessation of exercise training. Unexpectedly, daily voluntary running distance was similar between genotypes, and both genotypes improved their endurance running capacity after 6 weeks of voluntary running compared to sedentary controls (Fig. ), suggesting that Ulk1 is not required for enhanced physical performance in response to exercise training. In order to further validate these findings and rule out developmental compensation to loss of Ulk1 from early life, we generated tamoxifen-inducible, skeletal muscle-specific Ulk1 knockout (iMKO) mice and subjected them to the..."
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"headline": "Ampk phosphorylation of Ulk1 is required for targeting of mitochondria to lysosomes in exercise-induced mitophagy",
"datePublished": "2017",
"author": [
{
"@type": "Person",
"name": "Rhianna C. Laker"
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"name": "Joshua C. Drake"
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"name": "Rebecca J. Wilson"
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"name": "Vitor A. Lira"
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"name": "Bevan M. Lewellen"
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"name": "Karen A. Ryall"
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"name": "Carleigh C. Fisher"
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"@type": "Person",
"name": "Mei Zhang"
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"name": "Jeffrey J. Saucerman"
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"name": "Laurie J. Goodyear"
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"name": "Mondira Kundu"
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"name": "Zhen Yan"
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"identifier": "10.1038/s41467-017-00520-9"
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