PLD1 regulates adipogenic differentiation through mTOR - IRS-1 phosphorylation at serine 636/639.

Hae-In Song,Mee-Sup Yoon

doi:10.1038/srep36968

Abstract

Phospholipase D1 (PLD1) plays a known role in several differentiation processes, but its role in adipogenic differentiation remains unknown. In the present study, we identified PLD1 as a negative regulator of adipogenic differentiation. We showed that PLD activity was downregulated by both 3-Isobutyl-1-methylxanthine (IBMX) and insulin upon induction of differentiation in 3T3-L1 adipogenic cells. In line with this observation, PLD activity decreased in both high fat diet (HFD)-fed mice and ob/ob mice. We also found that differentiation of 3T3-L1 preadipocytes was enhanced by the depletion of PLD1 levels or inhibition of PLD1 activity by VU0155069, a PLD1-specific inhibitor. Conversely, treatment with phosphatidic acid (PA), a PLD product, and overexpression of PLD1 both caused a decrease in adipogenic differentiation. Moreover, the elevated differentiation in PLD1-knockdown 3T3-L1 cells was reduced by either PA treatment or PLD1 expression, confirming negative roles of PLD1 and PA in adipogenic differentiation. Further investigation revealed that PA displaces DEP domain-containing mTOR-interacting protein (DEPTOR) from mTORC1, which subsequently phosphorylates insulin receptor substrate-1 (IRS-1) at serine 636/639 in 3T3-L1 cells. Taken together, our findings provide convincing evidence for a direct role of PLD1 in adipogenic differentiation by regulating IRS-1 phosphorylation at serine 636/639 through DEPTOR displacement and mTOR activation.

Highlights

Obesity is defined by excessive accumulation of white adipose tissue above the normal level of adipocyte differentiation owing to an energy imbalance
Phospholipase D1 (PLD1) during adipogenic differentiation to examine its role in this process. 3T3-L1 preadipocytes were stimulated to differentiate by addition of differentiation medium 1 (DM1) with dexamethasone, IBMX, and insulin for two days, and with differentiation medium 2 (DM2) for the following 4–6 days
Measurements showed that total Phospholipase D (PLD) activity during differentiation began to decrease on day 2 (Fig. 1a), and by day 4 PLD activity had dropped to almost 50% of the level seen in undifferentiated adipocytes

Summary

Introduction

Obesity is defined by excessive accumulation of white adipose tissue above the normal level of adipocyte differentiation owing to an energy imbalance. Expression of C/EBPβand C/EBPδis induced in preadipocytes during very early differentiation These regulators activate PPARγand C/EBPα,which upregulate each other and maintain their expression to govern the entire adipogenic process by activating additional transcription factors[4]. Even though recent study has shown that PLD deficiency promotes adiposity by up-regulating appetite[35], the role of PLD/PA in adipogenic differentiation remains unclear. It is not known whether PLD/PA is involved in the autonomous role of mTOR in adipogenesis, despite the fact that PLD and PA are well established as critical regulators of mTOR

Methods

Results

Conclusion