RESUMO
OBJECTIVE: AUP1 (ancient ubiquitous protein 1) is an endoplasmic reticulum-associated protein that also localizes to the surface of lipid droplets (LDs), with dual role in protein quality control and LD regulation. Here, we investigated the role of AUP1 in hepatic lipid mobilization and demonstrate critical roles in intracellular biogenesis of apoB100 (apolipoprotein B-100), LD mobilization, and very-low-density lipoprotein (VLDL) assembly and secretion. APPROACH AND RESULTS: siRNA (short/small interfering RNA) knockdown of AUP1 significantly increased secretion of VLDL-sized apoB100-containing particles from HepG2 cells, correcting a key metabolic defect in these cells that normally do not secrete much VLDL. Secreted particles contained higher levels of metabolically labeled triglyceride, and AUP1-deficient cells displayed a larger average size of LDs, suggesting a role for AUP1 in lipid mobilization. Importantly, AUP1 was also found to directly interact with apoB100, and this interaction was enhanced with proteasomal inhibition. Knockdown of AUP1 reduced apoB100 ubiquitination, decreased intracellular degradation of newly synthesized apoB100, and enhanced extracellular apoB100 secretion. Interestingly, the stimulatory effect of AUP1 knockdown on VLDL assembly was reminiscent of the effect previously observed after MEK-ERK (mitogen-activated protein kinase kinase-extracellular signal-regulated kinase) inhibition; however, further studies indicated that the AUP1 effect was independent of MEK-ERK signaling. CONCLUSIONS: In summary, our findings reveal an important role for AUP1 as a regulator of apoB100 stability, hepatic LD metabolism, and intracellular lipidation of VLDL particles. AUP1 may be a crucial factor in apoB100 quality control, determining the rate at which apoB100 is degraded or lipidated to enable VLDL particle assembly and secretion.
Assuntos
Apolipoproteína B-100/metabolismo , Proteínas de Transporte/metabolismo , Hepatócitos/metabolismo , Lipoproteínas VLDL/metabolismo , Fígado/metabolismo , Triglicerídeos/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Transporte/genética , Células Hep G2 , Humanos , Gotículas Lipídicas/metabolismo , Proteínas de Membrana , Tamanho da Partícula , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Estabilidade Proteica , Proteólise , Interferência de RNA , Transfecção , UbiquitinaçãoRESUMO
Recent cell culture and animal studies have suggested that expression of human apo C-III in the liver has a profound impact on the triacylglycerol (TAG)-rich VLDL1 production under lipid-rich conditions. The apoC-III Gln38Lys variant was identified in subjects of Mexican origin with moderate hypertriglyceridemia. We postulated that Gln38Lys (C3QK), being a gain-of-function mutation, promotes hepatic VLDL1 assembly/secretion. To test this hypothesis, we expressed C3QK in McA-RH7777 cells and apoc3-null mice to contrast its effect with WT apoC-III (C3WT). In both model systems, C3QK expression increased the secretion of VLDL1-TAG (by 230%) under lipid-rich conditions. Metabolic labeling experiments with C3QK cells showed an increase in de novo lipogenesis (DNL). Fasting plasma concentration of TAG, cholesterol, cholesteryl ester, and FA were increased in C3QK mice as compared with C3WT mice. Liver of C3QK mice also displayed an increase in DNL and expression of lipogenic genes as compared with that in C3WT mice. These results suggest that C3QK variant is a gain-of-function mutation that can stimulate VLDL1 production, through enhanced DNL.