Phosphatidylcholine Transfer Protein Interacts with Thioesterase Superfamily Member 2 to Attenuate Insulin Signaling

Abstract

Phosphatidylcholine transfer protein (PC-TP) is a phospholipid-binding protein that is enriched in liver and that interacts with thioesterase superfamily member 2 (THEM2). Mice lacking either protein exhibit improved hepatic glucose homeostasis and are resistant to diet-induced diabetes. Insulin receptor substrate 2 (IRS2) and mammalian target of rapamycin complex 1 (mTORC1) are key effectors of insulin signaling, which is attenuated in diabetes. We found that PC-TP inhibited IRS2, as evidenced by insulin-independent IRS2 activation following knockdown, genetic ablation, or chemical inhibition of PC-TP. In addition, IRS2 was activated after knockdown of THEM2, providing support for a role for the interaction of PC-TP with THEM2 in suppressing insulin signaling. Additionally, we showed that PC-TP bound to tuberous sclerosis complex 2 (TSC2) and stabilized the components of the TSC1-TSC2 complex, which functions to inhibit mTORC1. Preventing phosphatidylcholine from binding to PC-TP disrupted interactions of PC-TP with THEM2 and TSC2, and disruption of the PC-TP–THEM2 complex was associated with increased activation of both IRS2 and mTORC1. In livers of mice with genetic ablation of PC-TP or that had been treated with a PC-TP inhibitor, steady-state amounts of IRS2 were increased, whereas those of TSC2 were decreased. These findings reveal a phospholipid-dependent mechanism that suppresses insulin signaling downstream of its receptor.

Thioesterase-mediated control of cellular calcium homeostasis enables hepatic ER stress

“…However, knockdown of Them2 or PC-TP expression did not impair IP 3 -mediated ER calcium release ( Figure 3L). We previously demonstrated that knockdown of Them2 or PC-TP expression in HEK 293E cells activates Akt and the mammalian target of rapamycin (mTOR) (16). We found that the regulation of calcium fluxes by Them2 and PC-TP occurs independently of Akt and mTOR activity, since inhibiting Akt or mTOR activity with GDC-0941 or rapamycin, respectively, did not abolish the effects of Them2 and PC-TP on ER calcium fluxes (Supplemental Figure 7).…”

“…To validate the importance of Them2-PC-TP interactions in regulating the ER stress response, we treated mice with the PC-TP inhibitor compound A1 (16,17,19,23). In Them2 +/+ mice, inhibition of PC-TP by compound A1 before tunicamycin administration led to increased PERK activity and reduced activities of IRE1α and eIF2α relative to vehicle-treated controls (Supplemental Figure 4).…”

“…Experiments were conducted 48 hours following transfection after overnight serum starvation. Mouse primary hepatocytes were isolated and cultured from 6-to 9-week-old mice (16,18). Briefly, mice were anesthetized with an i.p.…”

“…Human embryonic kidney (HEK) 293E (52) and Hepa1-6 (ATCC) cells were cultured in DMEM supplemented with 10% (vol/vol) FBS (Invitrogen). siRNAs (Ambion) targeting human PC-TP (ID: 121081) and THEM2 (ID: 119801; Ambion) as well as a scramble control (ID: AM4611; Ambion) were used to knock down protein expression (16). A custom Silencer Select siRNA that targets human IP 3 R3 was designed using the sense 5′-UGAGAAGCAGAAGAAGGAGtt-3′ and antisense 5′-CUCCUUCUUCUGCUUCUCAcU-3′ sequences (Invitrogen, catalog .…”

“…PC-TP comprises a single lipidbinding domain with high specificity for phosphatidylcholines (PCs), particularly those containing more unsaturated fatty acyl chains (13). Binding of PCs to PC-TP promotes its interaction with Them2 (16), which in turn increases thioesterase activity (13). Mice lacking either Them2 (Them2 -/-) or PC-TP (Pctp -/-) exhibit altered fatty acid metabolism (15,17,18).…”