Acquisition of Unprecedented Phosphatidylinositol 3,5-Bisphosphate Rise in Hyperosmotically Stressed 3T3-L1 Adipocytes, Mediated by ArPIKfyve-PIKfyve Pathway

Diego Sbrissa,Assia Shisheva

doi:10.1074/jbc.m412729200

Abstract

Unlike yeast, where hyperosmotic stress induces a dramatic increase in phosphatidylinositol 3,5-bisphosphate (PtdIns 3,5-P(2)) synthesis, in mammalian cells, although activating a complex array of signaling events, hyperosmotic stress fails to up-regulate PtdIns 3,5-P(2), indicating the PtdIns 3,5-P(2) pathway is not involved in mammalian osmo-protective responses. Here we report an unexpected and marked PtdIns 3,5-P(2) increase in response to hyperosmotic stress in differentiated 3T3-L1 adipocytes. Because this effect was not observed in the precursor preadipocytes, a specific role during acquisition of the adipocyte phenotype and transition into insulin-responsive cells could be suggested. However, acute insulin action did not result in a measurable PtdIns 3,5-P(2) rise, indicating the PtdIns 3,5-P(2) pathway is a specific hyperosmotically activated signaling cascade selectively operating in differentiated 3T3-L1 adipocytes. Hyperosmolarity activates different components of several kinase cascades, including p38 mitogen-activated protein and tyrosine kinases, but these appear to be separate from the activated PtdIns 3,5-P(2) pathway. Because PtdIns 3,5-P(2) is primarily produced by PIKfyve-catalyzed synthesis and requires the upstream activator hVac14 (called herein ArPIKfyve) that physically associates with and activates PIKfyve, we examined the contribution of ArPIKfyve-PIKfyve for the hyperosmotic stress-induced rise in PtdIns 3,5-P(2). Small interfering RNA-directed gene silencing to selectively deplete ArPIKfyve or PIKfyve in 3T3-L1 adipocytes determined the ArPIKfyve-PIKfyve axis fully accountable for the hyperosmotically activated PtdIns 3,5-P(2). Together these results reveal a previously uncharacterized PtdIns 3,5-P(2) pathway activated selectively in hyperosmotically stressed 3T3-L1 adipocytes and suggest a plausible role for PtdIns 3,5-P(2) in the osmo-protective response mechanism in this cell type.

Highlights

Unlike yeast, where hyperosmotic stress induces a dramatic increase in phosphatidylinositol 3,5-bisphosphate (PtdIns 3,5-P2) synthesis, in mammalian cells, activating a complex array of signaling events, hyperosmotic stress fails to up-regulate PtdIns 3,5-P2, indicating the PtdIns 3,5-P2 pathway is not involved in mammalian osmo-protective responses
To assess the possibility that the elevated PtdIns 3,5-P2 is part of a novel adipocyte-specific hyperosmotic regulatory mechanism, we examined the effect of hyperosmolarity on radiolabeled PtdIns 3,5-P2 accumulation in 3T3-L1 preadipocytes metabolically labeled with [32P]orthophosphate just before initiation of the differentiation program
Whereas PtdIns 3,5-P2 levels were changed in opposite directions, hyperosmolarity increased the levels of PtdIns 3-P in both 3T3-L1 adipocytes and 3T3-L1 preadipocytes (Table I; Fig. 1)

Summary

EXPERIMENTAL PROCEDURES

[32P]Orthophosphate Cell Labeling, Lipid Extraction, and HPLC— Cells (60-mm plates) were phosphate/serum-deprived for 1 h and labeled with [32P]orthophosphate in phosphate/serum-free Dulbecco’s modified Eagle’s medium for 2.5 h at 37 °C as described previously [35]. In siRNA experiments, after electroporation, 3T3-L1 adipocytes were seeded on 6-well plates and labeled (2.5 h) with [32P]orthophosphate 72 h post-electroporation subsequent to overnight starvation in serum/ phosphate-free media supplemented with 0.5% bovine serum albumin. Myo-[2-3H]Inositol Labeling of 3T3-L1 Adipocytes—Fully differentiated 3T3-L1 adipocytes (60-mm plates) were maintained for h in glucose- and inositol-free Dulbecco’s modified Eagle’s medium containing 0.5% bovine serum albumin, 5% dialyzed fetal bovine serum, 2 mM pyruvate, mM HEPES, pH 7.4, 100 units/ml penicillin, and 100 ␮g/ml streptomycin. 3H-Labeled lipids were co-injected on the HPLC column (Whatman 5-␮m Partisphere SAX) with the above-specified [32P]GroPIns as internal standards. Several films of different exposure times were quantified to assure the signals were within the linear range

RESULTS

PI spc

DISCUSSION