Cardiotonic Steroids Stimulate Glycogen Synthesis in Human Skeletal Muscle Cells via a Src- and ERK1/2-dependent Mechanism

Abstract

The cardiotonic steroid, ouabain, a specific inhibitor of Na ؉ ,K ؉ -ATPase, initiates protein-protein interactions that lead to an increase in growth and proliferation in different cell types. We explored the effects of ouabain on glucose metabolism in human skeletal muscle cells (HSMC) and clarified the mechanisms of ouabain signal transduction. In HSMC, ouabain increased glycogen synthesis in a concentration-dependent manner reaching the maximum at 100 nM. The effect of ouabain was additive to the effect of insulin and was independent of phosphatidylinositol 3-kinase inhibitor LY294002 but was abolished in the presence of a MEK1/2 inhibitor (PD98059) or a Src inhibitor (PP2). Ouabain increased Src-dependent tyrosine phosphorylation of ␣ 1 -and ␣ 2 -subunits of Na ؉ ,K ؉ -ATPase and promoted interaction of ␣ 1 -and ␣ 2 -subunits with Src, as assessed by co-immunoprecipitation with Src. Phosphorylation of ERK1/2 and GSK3␣/␤, as well as p90rsk activity, was increased in response to ouabain in HSMC, and these responses were prevented in the presence of PD98059 and PP2. Incubation of HSMC with 100 nM ouabain increased phosphorylation of the ␣-subunits of the Na-pump at a MAPK-specific Thr-Pro motif. Ouabain treatment decreased the surface abundance of ␣ 2 -subunit, whereas abundance of the ␣ 1 -subunit was unchanged. Marinobufagenin, an endogenous vertebrate bufadienolide cardiotonic steroid, increased glycogen synthesis in HSMC at 10 nM concentration, similarly to 100 nM ouabain. In conclusion, ouabain and marinobufagenin stimulate glycogen synthesis in skeletal muscle. This effect is mediated by activation of a Src-, ERK1/2-, p90rsk-, and GSK3-dependent signaling pathway.

A-769662 activates AMPK β1-containing complexes but induces glucose uptake through a PI3-kinase-dependent pathway in mouse skeletal muscle

“…Unpublished observations from Dr. Alexander Chibalin and coworkers show that A-769662 can inhibit the Na ϩ -K ϩ -ATPase (personal communication). In addition, it has been shown in some (11,21,29,30,50) but not all models (23,24) that inhibition of the Na ϩ -K ϩ -ATPase by ouabain increases PI3-kinase activity, possibly through an association between p85 and the ␣ 1 -subunit of the Na ϩ -K ϩ -ATPase, leading to increased Akt phosphorylation. This effect can be inhibited by wortmannin (30).…”

The cardiotonic steroid hormone marinobufagenin induces renal fibrosis: implication of epithelial-to-mesenchymal transition

“…Moreover, elevated levels of endogenous CTSs including marinobufagenin (MBG) have been associated with various pathological conditions: essential hypertension (4,24,73), preeclampsia (3,45,83), experimental diabetes (7), uremic cardiomyopathy (33), and cardiac failure (20,48,49,66,74). MBG, like other CTSs, binds to the extracellular domain of the ␣-subunit of Na ϩ -K ϩ -ATPase (16,21), which, in addition to its well-known function of maintaining cellular electrochemical balance through pumping sodium and potassium ions, can act as a typical membrane receptor (14,28,35,55,56,73,74,84,85,88).…”

“…First, because the elevated levels of MBG, or its analogs, accompany the conditions at which EMT occurs, including end stage renal disease (34), normal pregnancy, and preeclampsia (3,45,68,83). Second, the interaction of MBG with the Na ϩ -K ϩ -ATPase activates Src and ERK1/2 protein kinase pathways and increases the production of reac-tive oxygen species (20,33,35,80,81). Finally, as it was shown for ouabain, MBG possibly can trigger phosphoinositide 3-kinase/protein kinase B (Akt) axis, stimulate NF-B, and elevate the concentration of intracellular Ca 2ϩ concentration (44,55,84,88).…”

Marinobufagenin interferes with the function of the mineralocorticoid receptor

“…Thus, intracellular signaling pathways originating from distinct classes of molecules can have profound influences on MR and SRC-3 function and consequently steroid receptor-dependent gene expression. MBG increases the activities of Src kinase, ERK1/2 and GSK3 in muscle cells [32], and several of these signaling molecules (e.g. ERK1/2 and GSK3) can affect the activity of SRC-3 [18].…”