Abstract
To examine the role of AMP-activated protein kinase (AMPK) in muscle glucose transport, we generated muscle-specific transgenic mice (TG) carrying cDNAs of inactive alpha2 (alpha2i TG) and alpha1 (alpha1i TG) catalytic subunits. Extensor digitorum longus (EDL) muscles from wild type and TG mice were isolated and subjected to a series of in vitro incubation experiments. In alpha2i TG mice basal alpha2 activity was barely detectable, whereas basal alpha1 activity was only partially reduced. Known AMPK stimuli including 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), rotenone (a Complex I inhibitor), dinitrophenol (a mitochondrial uncoupler), muscle contraction, and sorbitol (producing hyperosmolar shock) did not increase AMPK alpha2 activity in alpha2i TG mice, whereas alpha1 activation was attenuated by only 30-50%. Glucose transport was measured in vitro using isolated EDL muscles from alpha2i TG mice. AICAR- and rotenone-stimulated glucose transport was fully inhibited in alpha2i TG mice; however, the lack of AMPK alpha2 activity had no effect on contraction- or sorbitol-induced glucose transport. Similar to these observations in vitro, contraction-stimulated glucose transport, assessed in vivo by 2-deoxy-d-[(3)H]glucose incorporation into EDL, tibialis anterior, and gastrocnemius muscles, was normal in alpha2i TG mice. Thus, AMPK alpha2 activation is essential for some, but not all, insulin-independent glucose transport. Muscle contraction- and hyperosmolarity-induced glucose transport may be regulated by a redundant mechanism in which AMPK alpha2 is one of multiple signaling pathways.
Highlights
AMPK by pharmacological stimulation and transient expression of an AMPK-active mutant increases glucose transport [3,4,5,6]
Because skeletal muscle accounts for ϳ80% of disposal of an oral glucose load [9, 10] and because type 2 diabetes is associated with reduced muscle glucose disposal [11], AMPK may be critical in the control of metabolic homeostasis and perhaps the development of type 2 diabetes [12, 13]
aminoimidazole-4-carboxamide riboside (AICAR)-stimulated ␣1 activity was reduced by 50% in ␣2i transgenic mice (TG) mice, but this still represented a significant 2-fold increase above basal
Summary
Generation of Transgenic Mice—To render the catalytic subunit inactive, the aspartic acid at amino acid residue 157 of rat AMPK ␣2 subunit was substituted to alanine by a PCR-based site direct mutagenesis as described by Stein et al [30]. Transgenic mouse founders were identified by polymerase chain reaction-based methods, and transgene product expression was confirmed by immunoblots with an antibody for each tagged epitope Mice expressing both transgenes were generated by breeding an ␣2i TG line to an ␣1i TG line (␣1/2i TG mice). In the second series of experiments muscles from wild type mice were stimulated 2–3 times before the 10-min contraction period to find an optimum voltage to match muscle force production with the ␣2i TG mice. Radioactivity in aliquots of the digested muscles was determined by liquid scintillation counting for dual labels, and the extracellular and intracellular spaces were calculated as previously described [4]. After injection of the tracer, mice were subjected to the 15 min in situ muscle contraction protocol described above. When analysis of variance revealed significant differences, the Bonferroni t test was used as a post hoc test for multiple comparisons
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