Sortilin facilitates VLDL-B100 secretion by insulin sensitive McArdle RH7777 cells

Abstract

Studies examining the relationship between cellular sortilin and VLDL-B100 secretion demonstrate inconsistent results. Current studies explore the possibility that discrepancies may be related to insulin sensitivity. McArdle RH7777 cells (McA cells) cultured under serum enriched conditions lose sensitivity to insulin. Following incubation in serum-free DMEM containing 1% BSA, McA cells become insulin responsive and demonstrate reduced apo B secretion. Current studies indicate that insulin sensitive McA cells express lower cellular sortilin that corresponds with reduction in VLDL-B100 secretion without changes in mRNA of either sortilin or apo B. When sortilin expression is further reduced by siRNA knockdown (KD), there are additional decreases in VLDL-B100 secretion. A crystal structure of human sortilin (hsortilin) identifies two binding sites on the luminal domain for the N- and C-termini of neurotensin (NT). A small organic compound (cpd984) was identified that has strong theoretical binding to the N-terminal site. Both cpd984 and NT bind hsortilin by surface plasmon resonance. In incubations with insulin sensitive McA cells, cpd984 was shown to enhance VLDL-B100 secretion at each level of sortilin KD suggesting cpd984 acted through sortilin in mediating its effect. Current results support a role for sortilin to facilitate VLDL-B100 secretion which is limited to insulin sensitive McA cells. Inconsistent reports of the relationship between VLDL-B100 secretion and sortilin in previous studies may relate to differing functions of sortilin in VLDL-B100 secretion depending upon insulin sensitivity.

Phosphatidylinositol (3,4,5)-trisphosphate binds to sortilin and competes with neurotensin: Implications for very low density lipoprotein binding

“…Compound 98477898 (2S)-1-methyl-N-{3-[(3-phenylpropanoyl)-amino]phenyl} pyrrolidine-2-carboxamide (cpd984) was obtained from ChemBridge Corp. (San Diego, CA). All other materials and reagents were essentially as described previously [5]. …”

“…Docking of diC4-PIP3 on a grid centered at the C5 term NT (site1) of hsortilin crystal structure at excess concentration of NT PDB ID: 4PO7 was performed using Schrödinger’s Glide software [9] as described [5]. …”

“…HBS running buffer with and without 1% (v/v) DMSO (10 mM HEPES pH 7.4, 150 mM NaCl) was used at a flow rate of 30 µl/min and injections had an association time of 90 sec, dissociation time of 300 sec, and binding was measured in relative RU or difference in response between the immobilized protein flow cell and the corresponding control flow cell subtracting relevant blank injections of buffer alone [5]. Regeneration buffer of pH 3.0 glycine was injected after each injection for PIP3 nanodiscs and C-term NT flowed in the presence of cpd984.…”

“…50 µl PBS and 20 µL 5X SDS sample buffer was added to the final liposome pellet. Bound proteins were resolved by SDS-PAGE and detected by western immunoblotting following transfer to PVDF membranes [5]. …”

“…Sortilin also binds B100-containing very low density lipoprotein (VLDL) and LDL [3, 4]. We previously demonstrated that sortilin facilitates the secretion of VLDL-B100 by insulin-sensitive McArdle RH7777 (McA) cells [5]. We have further shown that insulin enhances B100 binding to sortilin in McA cells [6] preceding autophagic destruction [7].…”