AMP-activated protein kinase is required for exercise-induced peroxisome proliferator-activated receptor co-activator 1 translocation to subsarcolemmal mitochondria in skeletal muscle - PubMed (original) (raw)

AMP-activated protein kinase is required for exercise-induced peroxisome proliferator-activated receptor co-activator 1 translocation to subsarcolemmal mitochondria in skeletal muscle

Brennan K Smith et al. J Physiol. 2013.

Abstract

In skeletal muscle, mitochondria exist as two subcellular populations known as subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. SS mitochondria preferentially respond to exercise training, suggesting divergent transcriptional control of the mitochondrial genomes. The transcriptional co-activator peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) and mitochondrial transcription factor A (Tfam) have been implicated in the direct regulation of the mitochondrial genome in mice, although SS and IMF differences may exist, and the potential signalling events regulating the mitochondrial content of these proteins have not been elucidated. Therefore, we examined the potential for PGC-1α and Tfam to translocate to SS and IMF mitochondria in human subjects, and performed experiments in rodents to identify signalling mechanisms regulating these translocation events. Acute exercise in humans and rats increased PGC-1α content in SS but not IMF mitochondria. Acute exposure to 5-aminoimidazole-4-carboxamide-1-β-ribofuranoside in rats recapitulated the exercise effect of increased PGC-1α protein within SS mitochondria only, suggesting that AMP-activated protein kinase (AMPK) signalling is involved. In addition, rendering AMPK inactive (AMPK kinase dead mice) prevented exercise-induced PGC-1α translocation to SS mitochondria, further suggesting that AMPK plays an integral role in these translocation events. In contrast to the conserved PGC-1α translocation to SS mitochondria across species (humans, rats and mice), acute exercise only increased mitochondrial Tfam in rats. Nevertheless, in rat resting muscle PGC-1α and Tfam co-immunoprecipate with α-tubulin, suggesting a common cytosolic localization. These data suggest that exercise causes translocation of PGC-1α preferentially to SS mitochondria in an AMPK-dependent manner.

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Figures

Figure 1

Figure 1. Mitochondrial isolation cleanliness checks

A, to ensure that our mitochondrial isolation procedures yielded sample devoid of nuclear contamination we loaded 5, 10, 20 30 μg of isolated mitochondrial protein (subsarcolemmal (SS) and intermyofibrillar (IMF)) and probed for H2B (nuclear protein) and COXIV (mitochondrial protein). This check was performed on the same membrane and indicates that our isolation procedure yields highly purified mitochondria. B, to confirm that PGC-1α exists within both the SS and the IMF fraction, 10 μg of mitochondrial protein was loaded and detected in concert with other mitochondrial proteins and the absence of non-mitochondrial proteins. Thirty micrograms of muscle homogenate protein was loaded.

Figure 2

Figure 2. In human skeletal muscle, exercise promotes PGC-1α translocation to subsarcolemmal mitochondria

A, representative control blots to ensure purified mitochondrial samples. B, following an acute bout of exercise at 60%formula image for 120 min, PGC-1α is increased in subsarcolemmal (SS) mitochondria 3 h post-exercise. C, in contrast, Tfam is not increased in either mitochondrial population; n = 8 for all independent experiments. Data are expressed as mean ± SEM. *P < 0.05, significantly different from rest. Ten micrograms of mitochondrial protein was loaded for all conditions. IMF, intermyofibrillar mitochondria.

Figure 3

Figure 3. Changes in rat skeletal muscle PGC-1α and Tfam subcellular localization following exhaustive exercise

A, following 3 h of recovery PGC-1α is increased in subsarcolemmal (SS) mitochondria. B, immediately post-exercise, Tfam is transiently increased in both the SS and the intermyofibrillar (IMF) mitochondria; n = 6 for all independent experiments. Data are expressed as mean ± SEM. *P < 0.05, significantly different from rest. Ten micrograms of mitochondrial protein was loaded for all conditions.

Figure 4

Figure 4. Acute 5-aminoimidazole-4-carboxamide-1-β-ribofuranoside (AICAR) treatment increases PGC-1α and Tfam in a mitochondrial population-specific manner

Rat skeletal muscle was harvested 60 min after an intraperitoneal injection (1 mg g body weight−1) of AICAR. A, acute AICAR treatment increases PGC-1α in SS mitochondria. B, acute AICAR treatment increases Tfam in both SS and intermyofibrillar (IMF) mitochondria; n = 4 for all independent experiments. Data are expressed as mean ± SEM. *P < 0.05, significantly different from rest. Ten micrograms of mitochondrial protein was loaded for all conditions.

Figure 5

Figure 5. Differences in exercise-induced PGC-1α translocation between AMPK KD and WT mice

A, following exhaustive exercise, PGC-1α is increased in the subsarcolemmal (SS) mitochondria of WT mice immediately post and 3 h post exercise. B, in contrast, in AMPK KD mice, exercise did not induce PGC-1α translocation to either mitochondrial population. C and D, Tfam is not increased in either mitochondrial population following exhaustive exercise; n = 4 for all independent experiments. Data are expressed as mean ± SEM. *P < 0.05, significantly different from rest. Ten micrograms of mitochondrial protein was loaded for all conditions. IMF, intermyofibrillar mitochondria.

Figure 6

Figure 6. PGC-1α and Tfam co-localize with α-tubulin in the cytosol

PGC-1α and Tfam co-immunoprecipitate (IP) with each other and with α-tubulin. In contrast, lactate dehydrogenase (LDH), cytochrome c oxidase complex IV (COXIV) and pyruvate dehydrogenase (PDHE1α) do not co-immunoprecipitate with Tfam. Gels were cut simultaneously; n = 4 for all independent experiments. Two independent representative blots shown. C, control elutant wash-through.

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