Exon 9-deleted CETP inhibits full length-CETP synthesis and promotes cellular triglyceride storage

Abstract

Cholesteryl ester transfer protein (CETP) exists as full-length (FL) and exon 9 (E9)-deleted isoforms. The function of E9-deleted CETP is poorly understood. Here, we investigated the role of E9-deleted CETP in regulating the secretion of FL-CETP by cells and explored its possible role in intracellular lipid metabolism. CETP overexpression in cells that naturally express CETP confirmed that E9-deleted CETP is not secreted, and showed that cellular FL- and E9-deleted CETP form an isolatable complex. Coexpression of CETP isoforms lowered cellular levels of both proteins and impaired FL-CETP secretion. These effects were due to reduced synthesis of both isoforms; however, the predominate consequence of FL- and E9-deleted CETP coexpression is impaired FL-CETP synthesis. We reported previously that reducing both CETP isoforms or overexpressing FL-CETP impairs cellular triglyceride (TG) storage. To investigate this further, E9-deleted CETP was expressed in SW872 cells that naturally synthesize CETP and in mouse 3T3-L1 cells that do not. E9-deleted CETP overexpression stimulated SW872 triglyceride synthesis and increased stored TG 2-fold. Expression of E9-deleted CETP in mouse 3T3-L1 cells produced a similar lipid phenotype. In vitro, FL-CETP promotes the transfer of TG from ER-enriched membranes to lipid droplets. E9-deleted CETP also promoted this transfer, although less effectively, and it inhibited the transfer driven by FL-CETP. We conclude that FL- and E9-deleted CETP isoforms interact to mutually decrease their intracellular levels and impair FL-CETP secretion by reducing CETP biosynthesis. E9-deleted CETP, like FL-CETP, alters cellular TG metabolism and storage but in a contrary manner.

Study of effect modifiers of genetically predicted CETP reduction

“…CETP may also have an important intracellular role in storing triglycerides and cholesteryl esters in lipid droplets. 45,46 . Whether this activity was altered in our genetic models or played a role in the effect modification by BMI remains to be determined.…”

Both full length‐cholesteryl ester transfer protein and exon 9‐deleted cholesteryl ester transfer protein promote triacylglycerol storage in cultured hepatocytes

“…In HepG2/C3A cells, transient CETP expression was achieved by transduction with recombinant adenoviruses (type 5, E1 and E3 regions deleted). Adenovirus constructs containing FL‐CETP or ∆E9‐CETP were prepared as previously described (Izem et al, 2020). For transduction, cells at 70% confluence were incubated in media plus adenovirus (30 pfu/cell).…”

“…The EcoRI/NotI fragment containing CETP was subcloned into pcDNA3. Cells at 70% confluence were transfected with 10 μg native pCDNA3 or pCDNA3‐FL‐CETP plasmid using Lipofectamine 2000 transfection reagent (Life Technologies) following the manufacturer's instructions (Izem et al, 2020). After 5 h, media containing 10% FBS was added and cells were allowed to recover overnight before use in experiments.…”

“…In SW872 adipocytes, overexpression of FL‐CETP impairs TAG storage capacity (Izem et al, 2015). In contrast, ∆E9‐deleted CETP overexpression stimulates TAG synthesis and storage (Izem et al, 2020). Consistent with a role for CETP in adipocyte lipid storage, several studies in animals have shown an association between increased FL‐CETP expression and decreased adiposity.…”

“…Taken together, the above data suggest that the impact of CETP on cellular lipid homeostasis may be different in cells whose primary function is to store lipid (adipocytes) versus those involved in lipoprotein assembly and secretion (hepatocytes, intestinal epithelium). While the impact of CETP on lipid homeostasis has been directly explored in adipocytes (Izem et al, 2015, 2020; Izem & Morton, 2007), such information is lacking in other cell types. In this study, we directly examined the impact of CETP overexpression in cultured hepatocytes and intestinal epithelial cells on lipid content and on the activities of metabolic pathways such as fatty acid uptake, lipogenesis, lipid secretion, and lipolysis that control cellular lipid homeostasis.…”