β2-Adrenergic activation increases glycogen synthesis in L6 skeletal muscle cells through a signalling pathway independent of cyclic AMP

Abstract

Aims/hypothesis In skeletal muscle, the storage of glycogen by insulin is regulated by glycogen synthase, which is regulated by glycogen synthase kinase 3 (GSK3). Here we examined whether adrenergic receptor activation, which can increase glucose uptake, regulates glycogen synthesis in L6 skeletal muscle cells. Methods We used L6 cells and measured glycogen synthesis (as incorporation of D-[U-14 C]glucose into glycogen) and GSK3 phosphorylation following adrenergic activation. Results Insulin (negative logarithm of median effective concentration [pEC 50 ] 8.2± 0.3) and the β-adrenergic agonist isoprenaline (pEC 50 7.5±0.3) induced a twofold increase in glycogen synthesis in a concentration-dependent manner. The α 1 -adrenergic agonist cirazoline and α 2 -adrenergic agonist clonidine had no effect. Both insulin and isoprenaline phosphorylated GSK3. The β-adrenergic effect on glycogen synthesis is mediated by β 2 -adrenoceptors and not β 1 -/β 3 -adrenoceptors, and was not mimicked by 8-bromo-cyclic AMP or cholera toxin, and also was insensitive to pertussis toxin, indicating no involvement of cyclic AMP or inhibitory G-protein (G i ) signalling in the β 2 -adrenergic effect on glycogen synthesis. 12-O-tetradecanoylphorbol-13-acetate (TPA) increased glycogen synthesis 2.5-fold and phosphorylated GSK3 fourfold.Inhibition of protein kinase C (PKC) isoforms with 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrollo(3,4-c)-carbazole (Gö6976; inhibits conventional and novel PKCs) or 2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl)maleimide (Gö6983; inhibits conventional, novel and atypical PKCs) inhibited the stimulatory TPA effect, but did not significantly inhibit glycogen synthesis mediated by insulin or isoprenaline. Inhibition of phosphatidylinositol 3-kinase (PI3K) with wortmannin inhibited the effects of insulin and isoprenaline on glycogen synthesis. Conclusions/interpretation These results demonstrate that in L6 skeletal muscle cells adrenergic stimulation through β 2 -adrenoceptors, but not involving cyclic AMP or G i , activates a PI3K pathway that stimulates glycogen synthesis through GSK3.

Role of β-Adrenoceptor Signaling in Skeletal Muscle: Implications for Muscle Wasting and Disease

“…To further complicate skeletal muscle ␤ 2 -adrenoceptor signaling, the G␤␥ dimer has been found to initiate intracellular signaling pathways independent of the G␣ subunit (471). Specifically, G␤␥ activates the phosphoinositol 3-kinase (PI3K)-AKT signaling pathway (Fig.…”

“…Specifically, G␤␥ activates the phosphoinositol 3-kinase (PI3K)-AKT signaling pathway (Fig. 2) (263,471). PI3K is thought to phosphorylate the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PIP 2 ), generating phosphatidylinositol-3,4,5-trisphosphate (PIP 3 ), and creating two lipid-binding sites on the cell membrane for the serine/threonine kinase AKT (also referred to as protein kinase B) and 3Ј-phosphoinositide-dependent protein kinase 1 (PDK).…”

Improving Type 2 Diabetes Through a Distinct Adrenergic Signaling Pathway Involving mTORC2 That Mediates Glucose Uptake in Skeletal Muscle

“…Mechanisms that have been proposed to explain adrenergically mediated glucose uptake include stimulation of the transcription and translation of proteins, non-carrier mediated and unspecific effects, and translocation or activation of GLUTs (10,19,20), emphasizing the lack of a unifying concept for the mechanisms involved. Previously, we showed that b-adrenoceptors profoundly affect glucose uptake by an AMPK-independent pathway (3) that is not secondary to the lowering of ATP levels or energy deficiency, inasmuch as activation also increases glycogen formation in L6 skeletal muscle cells (4,21). Based on these studies and others (22), we have examined the mechanism used by b-adrenoceptors to increase glucose uptake in skeletal muscle and whether activation of this pathway could improve the condition of individuals with type 2 diabetes.…”

Anabolic and Catabolic Signaling Pathways that Regulate Skeletal Muscle Mass

“…In skeletal muscle, signaling through the β 2 -AR occurs primarily via coupling with the G protein Gα s , though there are reports describing signaling events involving Gα i and, more recently, Gβγ [58,59]. β 2 -AR coupling to Gα s activates adenylyl cyclase production of cyclic-AMP (cAMP) which in turn activates downstream signaling through protein kinase A (PKA) [60].…”