Role of AMPK2 in basal, training-, and AICAR-induced GLUT4, hexokinase II, and mitochondrial protein expression in mouse muscle

Am J Physiol Endocrinol Metab 292: E331–E339, 2007. First published September 5, 2006;

We investigated the role of AMPK2 in basal, exercise training-, and AICAR-induced protein expression of GLUT4, hexokinase II (HKII), mitochondrial markers, and AMPK subunits. This was conducted in red (RG) and white gastrocnemius (WG) muscle from wild-type (WT) and 2-knockout (KO) mice after 28 days of activity wheel running or daily AICAR injection. Additional experiments were conducted to measure acute activation of AMPK by exercise and AICAR. At basal, mitochondrial markers were reduced by 20% in 2-KO muscles compared with WT. In both muscle types, AMPK2 activity was increased in response to both stimuli, whereas AMPK1
activity was increased only in response to exercise. Furthermore, AMPK signaling was estimated to be 60–70% lower in 2-KO compared with WT muscles. In WG, AICAR treatment increased HKII, GLUT4, cytochrome
c, COX-1, and CS, and the 2-KO abolished the AICARinduced increases, whereas no AICAR responses were observed in RG. Exercise training increased GLUT4, HKII, COX-1, CS, and HAD protein in WG, but the 2-KO did not affect training-induced increases. Furthermore, AMPK1, -2, -1, -2, and -3 subunits were reduced in RG, but not in WG, by 30–60% in response to exercise training. In conclusion, the 2-KO was associated with an 20% reduction in mitochondrial markers in both muscle types and abolished AICAR-induced increases in protein expression in WG. However, the 2-KO did not reduce traininginduced increases in HKII, GLUT4, COX-1, HAD, or CS protein in WG, suggesting that AMPK2 may not be essential for metabolic adaptations of skeletal muscles to exercise training.

5-adenosine monophosphate-activated protein kinase-2; glucose transporter-4; 5-aminoimidazole-1--D-ribofuranoside; exercise training; mitochondrial proteins; skeletal muscle

BIOCHEMICAL ADAPTATIONS of skeletal muscle to regular physical activity include an increase in mitochondrial oxidative enzyme capacity and an increase in key proteins involved in glucose uptake, such as glucose transporter-4 (GLUT4) and hexokinase II (HKII) (41, 51). The intracellular pathways involved in eliciting these exercise training-induced increases in protein expression and mitochondrial content remain largely unknown. The 5-AMPactivated protein kinase (AMPK) has been proposed as a signaling molecule involved in transmitting an “exercise signal” to the nuclei of the muscle cell (15, 49). This is in part based on the finding that AMPK is activated by in vitro electrical induced contractions of rodent skeletal muscle (8, 44) and by in vivo exercise in human (11, 50) and rodent skeletal muscle (48).
Chronic activation of AMPK by the adenosine analog 5-aminoimidazole- 4-carboxamide-1--D-ribofuranoside (AICAR) or the creatine analog -guanadinopropionic acid (-GPA) in resting rat and mouse muscles increases transcription of metabolic genes and expression of metabolic enzymes as well as mitochondrial density mimicking effects of exercise training (4, 15, 20, 49, 52). The observation that pharmacologically induced upregulation of peroxisome proliferator-activated receptor-  coactivator-1 (PGC-1) and -aminolevulinate synthase mRNA, cytochrome c (cyt c) protein, and mitochondrial density is abolished in mouse muscle overexpressing a kinasedead AMPK construct suggests a causal role of AMPK in these responses (52). The AMPK-dependent increase in PGC-1 is of particular interest because this nuclear transcription modulator is shown to be important in coordinating muscular adaptations in lipid oxidation and mitochondrial function to exercise training (23, 24) and to be involved in regulating GLUT4 protein expression (28, 30).
Several studies indicate that AMPK increases content of target proteins by increasing transcriptional activity of their respective genes. For instance, arterial infusion of AICAR in rodent hindlimb muscle stimulates HKII and uncoupling protein- 3 (UCP3) gene transcription (43), and an acute injection of AICAR increases muscle GLUT4 mRNA in rodents (5). Furthermore, a chronic treatment of rat with -GPA treatment is followed by an increase in nuclear respiratory factor-1 (NRF-1) DNA binding activity correlated by an increase in expression of NRF-1 targets and mitochondrial density in muscle (4).
NRF-1 is activated by binding with PGC-1, which in turn leads to increased transcription of nuclear genes encoding subunits of the mitochondrial respiratory chain and components of the mitochondrial transcription and replication machinery (for review, see Refs. 22 and 42).
Altogether, it seems evident that activation of AMPK in resting muscle specifically increases the protein content of several metabolic enzymes as well as mitochondrial density.
Therefore, the purpose of the present study was to investigate the importance of the catalytic AMPK2 subunit in expression of HKII and GLUT4 protein, as well as mitochondrial enzymes in skeletal muscle in the basal state, and after exercise training and chronic AICAR treatment. This was investigated in AMPK2 whole body knockout (2- KO) and corresponding wild-type (WT) mice that had undertaken a 28-day program of activity wheel exercise training or daily AICAR injections.