The Effects of AS160 Modulation on Fatty Acid Transporters Expression and Lipid Profile in L6 Myotubes

Abstract

Background/Aims: AS160 is a key intracellular regulator of energy utilization in cells. It was shown to regulate GLUT4 translocation from intracellular depots to the plasma membrane, with subsequent changes in facilitated glucose uptake into the skeletal muscles. Similarly, also free fatty acids (FFAs) transmembrane transport seems to be largely protein-mediated. Therefore, the objective of this study was to examine the effects of moderate AS160 depletion (-82% mRNA, -25% of protein content) on the expression of fatty acid transporters and subsequent changes in lipid profile in L6 myotubes. Results: Surprisingly, moderate down regulation of AS160 expression was followed by increased AS160 phosphorylation (∼40%). These resulted in a greater expression of fatty acid transporters, namely FABPpm and FAT/CD36, with subsequently increased FAs cellular influx. No changes in the expression of FATP1 and 4 were noticed. Accordingly, we have observed a reduction in total TAG content. This was mainly caused by a significant changes in TAG fatty acids composition favouring a decrease in the amount of palmitic and stearic fatty acid moieties. In contrast, our experimental intervention led to distinctively increased total content of DAG and PL, but concomitantly decreased the content of all measured sphingolipids, e.g. SFA, SA1P, CER, SFO and S1P, in the AS160 knockdown group. Conclusions: Modulation of AS160 level and activity led to significant increase in the concentration of DAG and PL, which was associated with changes in FAs composition and expression of fatty acid transporters. Interestingly, the intervention also simultaneously decreased the content of sphingolipids.

Challenging of AS160/TBC1D4 Alters Intracellular Lipid milieu in L6 Myotubes Incubated With Palmitate

“…In the aforementioned research, we showed that modest, temporary knockdown of AS160 (−25% of protein content) caused increased contents of DAG and PL, which was mainly due to the elevation of saturated fatty acid lipid species. On the contrary, we observed a decrease in the amount of TAG caused by a reduction in the palmitate and stearate acids levels (Miklosz et al, ). The data presented herein showed that pre‐ and post‐silencing of AS160 combined with the palmitate treatment had different impact on lipid pool(s) when compared with the palmitate group alone.…”

“…Apart from that study, also our previous work documented the importance of AS160 modulation with respect to fatty acids transporters expression and lipid profile in L6 myotubes (Miklosz et al, 2016). We demonstrated that moderate inactivation of AS160 resulted in greater expression of fatty acids transporters, namely FABPpm and FAT/CD36, together with a slightly increased FAs cellular influx (Miklosz et al, 2016). However, molecular regulation of fatty acids uptake by AS160 and its potential contribution to the etiology of insulin resistance in skeletal muscle still remains elusive.…”

“…It is well known that immunofluorescence is a valuable technique that can provide (semi)quantitative data as well as yield informative images showing the localization of the proteins of interest (Bradley et al, ). In our previous paper, we demonstrated that in normal conditions in L6 myotubes moderate silencing of AS160 increased the total expression of FAT/CD36 and FABPpm, and did not change FATP‐1 and 4 contents (Miklosz et al, ). The data presented herein demonstrate that pre‐ and post‐silencing of AS160 combined with palmitate treatment increased the expression of fatty acid transporters, namely: FAT/CD36, FATP‐1, 4, but not FABPpm.…”

“…However, the role of AS160 in the fatty acid induced insulin resistance remains poorly explored. To date, it seems that only one research (our own previous study) has demonstrated that AS160 modulation may induce changes with respect to lipid content and composition in L6 myotubes (Miklosz et al, ). In the aforementioned research, we showed that modest, temporary knockdown of AS160 (−25% of protein content) caused increased contents of DAG and PL, which was mainly due to the elevation of saturated fatty acid lipid species.…”

“…So far, only one study conducted in cardiomyocytes indicated that trafficking of CD36, a most important facilitator of FA uptake, is regulated by AS160 in a similar way to that of GLUT4 (Samovski et al, 2012). Apart from that study, also our previous work documented the importance of AS160 modulation with respect to fatty acids transporters expression and lipid profile in L6 myotubes (Miklosz et al, 2016). We demonstrated that moderate inactivation of AS160 resulted in greater expression of fatty acids transporters, namely FABPpm and FAT/CD36, together with a slightly increased FAs cellular influx (Miklosz et al, 2016).…”