CaMKK is an upstream signal of AMP-activated protein kinase in regulation of substrate metabolism in contracting skeletal muscle

Abstract

Multiple signals have been shown to be involved in regulation of fatty acid (FA) and glucose metabolism in contracting skeletal muscle. This study aimed to determine whether a Ca(2+)-stimulated kinase, CaMKK, is involved in regulation of contraction-induced substrate metabolism and whether it does so in an AMP-activated protein kinase (AMPK)-dependent manner. Rat hindlimbs were perfused at rest (n = 16), with 3 mM caffeine (n = 15), with 2 mM 5-aminoimidazole-4-carboxamide 1-beta-d-ribofuranoside (AICAR; n = 16), or during moderate-intensity muscle contraction (MC; n = 14) and with or without 5 microM STO-609, a CaMKK inhibitor. FA uptake and oxidation increased (P < 0.05) 64% and 71% by caffeine, 42% and 93% by AICAR, and 65% and 143% by MC. STO-609 abolished (P < 0.05) caffeine- and MC-induced FA uptake and oxidation but had no effect with AICAR treatment. Glucose uptake increased (P < 0.05) 104% by caffeine, 85% by AICAR, and 130% by MC, and STO-609 prevented the increase in glucose uptake in caffeine and muscle contraction groups. CaMKKbeta activity increased (P < 0.05) 113% by caffeine treatment and 145% by MC but was not affected by AICAR treatment. STO-609 prevented the caffeine- and MC-induced increase in CaMKKbeta activity. Caffeine, AICAR, and MC increased (P < 0.05) AMPKalpha2 activity by 295%, 11-fold, and 7-fold but did not affect AMPKalpha1 activity. STO-609 decreased (P < 0.05) AMPKalpha2 activity induced by caffeine treatment and MC by 60% and 61% but did not affect AICAR-induced activity. Plasma membrane transport protein content of CD36 and glucose transporter 4 (GLUT4) increased (P < 0.05) with caffeine, AICAR, and MC, and STO-609 prevented caffeine- and MC-induced increases in protein content. These results show the importance of Ca(2+)-dependent signaling via CaMKK activation in the regulation of substrate uptake and FA oxidation in contracting rat skeletal muscle and agree with the notion that CaMKK is an upstream kinase of AMPK in the regulation of substrate metabolism in skeletal muscle.

AMPKα2deficiency uncovers time dependency in the regulation of contraction-induced palmitate and glucose uptake in mouse muscle

“…Indeed, we have shown that when CaMKII or CaMKK was inhibited during caffeine stimulation, there were subsequent decreases in AMPK␣ 2 activity along with decreases in the rates of FA uptake, glucose uptake, and FA oxidation (1,35). In line with these previous results, we measured an increase in AMPK␣ 2 activity in caffeine-treated WT mice in this study.…”

“…It has been shown by us and others that caffeine-induced increases in Ca 2ϩ signaling regulate substrate metabolism, possibly via AMPK activation (1,6,22,35). Indeed, we have shown that when CaMKII or CaMKK was inhibited during caffeine stimulation, there were subsequent decreases in AMPK␣ 2 activity along with decreases in the rates of FA uptake, glucose uptake, and FA oxidation (1,35).…”

“…The total cell homogenate was then transferred to a microcentrifuge tube and vortexed frequently for 1 h, whereupon the samples were centrifuged at 4,500 g at 4°C for 1 h. For immunoprecipitation procedures, ϳ90 mg of powdered muscle samples were homogenized in HEPES buffer and centrifuged at 15,000 g for 5 min. Supernatants (200 g) were incubated with antibodies for AMPK␣ 1 or AMPK␣2 (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h at 4°C with gentle agitation (35). After the incubation, protein A/G-agarose (Santa Cruz Biotechnology) was added to the tubes, which were gently agitated overnight (4°C).…”

“…There is evidence that Ca 2ϩ /calmodulin-dependent protein kinase (CaMK) kinase (CaMKK) activates AMPK in vitro in cells, whole skeletal muscle, and yeast (17,20,21,55), and we recently showed that activation of CaMKK and CaMKII leads to activation of AMPK in perfused rat hindlimbs (1,35). Additionally, we showed that inhibition of CaMKK or CaMKII reduces glucose uptake, FA uptake, and FA oxidation (1,35). However, it is unclear whether Ca 2ϩ signaling relies extensively on the activation of AMPK to mediate its metabolic effects or whether there are Ca 2ϩ -dependent AMPK-independent regulators of glucose uptake, FA uptake, and FA oxidation.…”

Contraction-induced skeletal muscle FAT/CD36 trafficking and FA uptake is AMPK independent

“…Based on the fi ndings from studies using muscle contractions and/or pharmacological approaches, it has been suggested that AMPK could be a major regulator of contraction-induced FAT/CD36 translocation in skeletal muscle ( 11,17 ). In the present skeletal muscle and heart muscle ( 17,18,36,37 ). During muscle contractions, FAT/CD36 translocation to the plasma membrane has been shown in rat gastrocnemius muscle ( 17,18 ), and the increase of FAT/CD36 in the plasma membrane has been associated with an increase in Fig.…”