Inhibition of Inositol hexakisphosphate Kinase 1 (IP6K1) Does Not Increase Akt or mTOR Activity in vitro.

Abstract

BackgroundInositol hexakisphosphate Kinase 1 (IP6K1) has been identified as a key signalling enzyme that inhibits Akt activity in hepatic cell lines and diabetic rodent models (Chakraborty et al., 2010; 2012; Goshal et al., 2016). Given both glucose transport and protein synthesis require Akt activation, we investigate if IP6K1 altered the IGF ‐ Akt – mTOR signalling cascade.MethodsC2C12 mouse skeletal myoblast cells were cultured in 12mL growth media (500mL DMEM, 10% FBS, 1% anti ‐ anti, 1% penicillin ‐ streptomycin) under standard conditions, changing media every 24 – 48 hours. Once 80% confluence was reached myoblasts were incubated in 12mL differentiation media (500mL DMEM, 2% donor equine serum, 1% anti ‐anti, 1% penicillin ‐ streptomycin) for 96 hours, changing media every 24 – 48 hours, to form mature myotubes. Myotubes were treated with IGF −1 (10ng/ml) +/− N2‐(m‐(trifluoromethy)lbenzyl) N6‐(p‐nitrobenzyl)purine (TNP; 10μm) for 24 hours before being lysed (1X cell lysis buffer supplemented with protease and phosphatase inhibitor cocktail) and total protein content determined using Lowry method. Western blot analysis was used to quantify t‐Akt, p‐Akt308, p‐Akt473, t‐mTOR, p‐mTOR2448 and IP6K1.ResultsA significant condition x IP6K1 effect was noted (P < 0.05) between IGF – 1 treatment and control in C2C12 myotubes, whilst IGF – 1 + TNP decreased IP6K1 content compared to IGF – 1 alone (P < 0.05). Significant differences were observed between IGF – 1 + TNP and control in p‐Akt308 and p‐Akt473 (P < 0.05), however no significant differences were observed between IGF – 1 + TNP and control in p/Akt308, p/Akt473 and p/mTOR2448 (P > 0.05).ConclusionsThis research is the first to characterize IP6K1 signalling in the IGF – 1, Akt – mTOR signalling cascade in C2C12 muscle cells. A reduction in IP6K1 did not increase p‐Akt or p‐mTOR activity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.