Insulin induces phosphorylation of pyruvate dehydrogenase through RhoA activation pathway in HepG2 cells

Abstract

Insulin is a critical signaling molecule in reducing blood glucose levels, and pyruvate dehydrogenase (PDH) is an essential enzyme in regulating glucose metabolism. However, the insulin effect on PDH function has not been well established. We observed that insulin attenuated the phosphorylation (p) of Ser264 (p-Ser264) in the PDH E1α subunit (PDHA1) in normal rat hepatocyte. In contrast, insulin induced an increase of p-Ser264 PDHA1 levels in hepatocellular carcinoma HepG2 and Huh7 cells. Insulin activated RhoA and Rho-dependent coiled coil kinase, an effector protein of active RhoA, which regulated p-Ser264 PDHA1 levels, along with both p-Ser9 and p-Tyr216 forms of glycogen synthase kinase-3β (GSK-3β) in HepG2 cells. Only p-Tyr216 GSK-3β, the active form was involved in an increase of p-Ser264 PDHA1. Akt was also engaged in p-Ser9 of GSK-3β, but neither in p-Tyr216 of GSK-3β nor p-Ser264 of PDHA1 upon insulin. Reconstituted dephospho-mimic forms PDHA1 S264A and GSK-3β Y216F impaired, but wild-types PDHA1 and GSK-3β and phospho-mimic forms PDHA1 S264D and GSK-3β Y216E increased cell proliferation upon insulin through expression of c-Myc and cyclin D1. Therefore, we propose that insulin-mediated p-PDHA1 is involved in the regulation of HepG2 cell proliferation through RhoA signaling pathway.-Islam, R., Kim, J.-G., Park, Y., Cho, J.-Y., Cap, K.-C., Kho, A.-R., Chung, W.-S., Suh, S.-W., Park, J.-B. Insulin induces phosphorylation of pyruvate dehydrogenase through RhoA activation pathway in HepG2 cells.

GSK3 as a Regulator of Cytoskeleton Architecture: Consequences for Health and Disease

“…In the human hepatocellular cancer cell line HEPG2, treatment with insulin, an upstream effector downregulating GSK3β, led to the activation of RhoA. In the same study, RhoA/ROCK1 was identified as a non-canonical pathway leading to GSK3β inactivation by insulin [131]. In contrast, it has been shown that in fibroblasts, GSK3β indirectly activated RhoA by phosphorylation and inhibition of RhoGAP, an upstream effector inactivating RhoA [111,132].…”

Pyruvate Dehydrogenase A1 Phosphorylated by Insulin Associates with Pyruvate Kinase M2 and Induces LINC00273 through Histone Acetylation

“…Insulin was reported to increase p-Ser293 PDHA1 (p-PDHA1) levels in HepG2 and Huh7 hepatocellular carcinoma cells [ 14 ]. To elucidate a novel function of p-PDHA1 in HepG2 hepatocellular carcinoma cells, which are sensitive to insulin signaling, we identified the binding protein that may interact with p-PDHA1 using a recombinant GST phospho-mimic mutant PDHA1 Ser293Glu (S293D).…”

“…Endogenous expression levels of PKM2 and p-PDHA1 in each cell were different depending on the cell types, and they did not correlate with each other ( Figure 1 D). Insulin enhanced p-AKT level in HepG2 cells, suggesting insulin stimulates HepG2 cells in the classical pathway ( Figure 1 E) [ 14 ]. Of note, insulin augmented p-PDHA1 and PKM2 levels in HepG2 and Hepa1c1c7 ( Figure 1 F,G).…”

“…Human hepatocellular carcinoma cell lines HepG2, Huh7, mouse hepatocellular carcinoma cell line Hepa1c1c7, and 4T1 mouse breast cancer cell line (Korean Cell Line Bank, Seoul, Korea) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing high glucose, 4.5 g/L D-glucose, L-glutamine, 110 mg/L sodium pyruvate and sodium bicarbonate and PC12 cells were maintained in RPMI 1640 medium 1× with low glucose (2.0 g/L), sodium bicarbonate (2.0 mg/L), sodium chloride (6.0 g/L) supplemented with 10% heat-inactivated fetal bovine serum (FBS) with 1% penicillin/streptomycin antibiotics. Cell culture flasks were maintained under 5% CO 2 , 95% ambient air, and humidified conditions at 37 °C [ 14 ]. Experimental plate cells were first serum starvation for 12 h without serum and antibiotics and finally treated with insulin (100 nM) and other stimulants such as LPS (10 µg/mL) and NGF (100 ng/mL).…”

“…Recently, we reported that insulin induces an increase in p-Ser293 PDHA1 along with p-Ser473 Akt levels in HepG2 hepatocellular carcinoma cells. However, insulin reduced p-Ser293 PDHA1 in insulin-treated rat liver tissue and rat primary hepatocyte [ 14 ]. However, the role of p-PDHA1 in hepatocellular carcinoma HepG2 cells due to insulin is poorly understood.…”