Gain-of-Function R225W Mutation in Human AMPKγ3 Causing Increased Glycogen and Decreased Triglyceride in Skeletal Muscle

Abstract

BackgroundAMP-activated protein kinase (AMPK) is a heterotrimeric enzyme that is evolutionarily conserved from yeast to mammals and functions to maintain cellular and whole body energy homeostasis. Studies in experimental animals demonstrate that activation of AMPK in skeletal muscle protects against insulin resistance, type 2 diabetes and obesity. The regulatory γ3 subunit of AMPK is expressed exclusively in skeletal muscle; however, its importance in controlling overall AMPK activity is unknown. While evidence is emerging that gamma subunit mutations interfere specifically with AMP activation, there remains some controversy regarding the impact of gamma subunit mutations [1]–[3]. Here we report the first gain-of-function mutation in the muscle-specific regulatory γ3 subunit in humans.Methods and FindingsWe sequenced the exons and splice junctions of the AMPK γ3 gene (PRKAG3) in 761 obese and 759 lean individuals, identifying 87 sequence variants including a novel R225W mutation in subjects from two unrelated families. The γ3 R225W mutation is homologous in location to the γ2R302Q mutation in patients with Wolf-Parkinson-White syndrome and to the γ3R225Q mutation originally linked to an increase in muscle glycogen content in purebred Hampshire Rendement Napole (RN-) pigs. We demonstrate in differentiated muscle satellite cells obtained from the vastus lateralis of R225W carriers that the mutation is associated with an approximate doubling of both basal and AMP-activated AMPK activities. Moreover, subjects bearing the R225W mutation exhibit a ∼90% increase of skeletal muscle glycogen content and a ∼30% decrease in intramuscular triglyceride (IMTG).ConclusionsWe have identified for the first time a mutation in the skeletal muscle-specific regulatory γ3 subunit of AMPK in humans. The γ3R225W mutation has significant functional effects as demonstrated by increases in basal and AMP-activated AMPK activities, increased muscle glycogen and decreased IMTG. Overall, these findings are consistent with an important regulatory role for AMPK γ3 in human muscle energy metabolism.

Increased susceptibility to oxidative damage in post-diabetic human myotubes

“…Muscle biopsies Biopsies of vastus lateralis were obtained from seven post-diabetes mellitus and seven non-diabetic participants using a 5 mm Bergstrom needle (Opitek, Glostrup, Denmark) as described previously [34]. Participants had refrained from physical activity for 3 days and had fasted for 12 h prior to biopsy.…”

“…), 0.5 mg/ml BSA, 1 μmol/l dexamethazone, 10 ng/ml EGF, 0.5 mg/ml fetuin and 0.25 pmol/l insulin. Muscle satellite cells were isolated using an immuno-based magnetic sorting technique as previously described [34]. Myoblasts were differentiated for 7 days prior to experimentation in LGI DMEM (5.5 mmol/l glucose, supplemented with 0.25 pmol/l insulin, 2% horse serum (vol./vol.…”

“…Ex vivo determinations were conducted as described previously [34]. Frozen tissues were sectioned, mounted on Superfrost slides and fixed in 10% neutral buffered formalin (vol./vol.).…”

Naturally occurring R225W mutation of the gene encoding AMP-activated protein kinase (AMPK)γ3 results in increased oxidative capacity and glucose uptake in human primary myotubes

“…The rare but naturally occurring R225W mutation in the gene (PRKAG3) encoding the AMPKγ 3 subunit in humans was previously identified by our laboratory as a gain-offunction mutation that results in a doubling of basal and AMP-stimulated AMPK activity [18]. In addition to increased AMPK activity, participants carrying the R225W mutation were shown to have ∼90% higher glycogen content and ∼30% lower intramuscular triacylglycerol (IMTG) content in the vastus lateralis muscle [18].…”

“…In addition to increased AMPK activity, participants carrying the R225W mutation were shown to have ∼90% higher glycogen content and ∼30% lower intramuscular triacylglycerol (IMTG) content in the vastus lateralis muscle [18]. This mutation is homologous to the PRKAG3 R225Q mutation in RN − Hampshire pigs, which causes high muscle glycogen content [19], as well as to the PRKAG2 R302Q mutation in humans, which is associated with WolffParkinson-White syndrome [20][21][22].…”