The apolipoprotein C-III (Gln38Lys) variant associated with human hypertriglyceridemia is a gain-of-function mutation

Abstract

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 (C3^QK), being a gain-of-function mutation, promotes hepatic VLDL₁ assembly/secretion. To test this hypothesis, we expressed C3^QK in McA-RH7777 cells and apoc3-null mice to contrast its effect with WT apoC-III (C3^WT). In both model systems, C3^QK expression increased the secretion of VLDL₁-TAG (by 230%) under lipid-rich conditions. Metabolic labeling experiments with C3^QK cells showed an increase in de novo lipogenesis (DNL). Fasting plasma concentration of TAG, cholesterol, cholesteryl ester, and FA were increased in C3^QK mice as compared with C3^WT mice. Liver of C3^QK mice also displayed an increase in DNL and expression of lipogenic genes as compared with that in C3^WT mice. These results suggest that C3^QK variant is a gain-of-function mutation that can stimulate VLDL₁ production, through enhanced DNL.

Keywords: VLDL; de novo lipogenesis; lipogenic gene expression.

Fig. 1.

ApoC-III Q38K variant is a gain-of-function mutation that enhances secretion of VLDL₁-associated TAG and apoB-100. A: Six-helix (H1–H6) structure of human apoC-III (left) and position of Gln³⁸ at the putative water-lipid interface of helix 3 (right). Apolar residues are colored in green, positively charged residues in blue, polar residues in gray, and Gly residue in yellow. The Q38K mutation introduces a positively charged Arg residue at the putative water-lipid interface, thus presumably rendering helix 3 a type A amphipathic helix (26). B: Immunoblots of human apoC-III and endogenous rat apoE in the medium of neo, C3^WT, or C3^QK cells. C: [³H]TAG in the medium of neo, C3^WT, or C3^QK cells labeled with [³H]glycerol for 2 h under lipid-rich conditions. * P < 0.05; ** P < 0.01; *** P < 0.001 (n = 3 dishes per cell line). D: [³H]TAG (left) and [³H]PC (right) in fractionated lipoproteins secreted from cells 2 h after labeling with [³H]glycerol. E: [³⁵S]ApoB-100 in fractionated lipoproteins secreted from cells 3 h after labeling with [³⁵S]methionine/cysteine. F: Fluorography of [³⁵S]apoB-100, [³⁵S]apoE, and [³⁵S]apoA-I in fractionated lipoproteins.

Fig. 2.

Expression of C3^QK mutant increases DNL. A, B: Cells were labeled with [³H]acetic acid (25 µCi/ml) for up to 2 h under lipid-poor (A) or lipid-rich (B) conditions. C: Cells were labeled with [³H]glycerol for up to 2 h under lipid-rich conditions. At the end of labeling, cell-associated lipids were quantified. Data are expressed as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001 (n = 3 dishes per cell line).

Fig. 3.

Expression of C3^QK mutant in apoc3-null mice promotes TAG-rich VLDL₁ production. Apoc3-null mice were fed with a high-fat diet for a week and then injected with adenovirus constructs encoding C3^WT, C3^QK, or empty vector. At 48 h after injection, mice were fasted for 12 h, and blood samples were collected (time zero) and then injected with P407 intraperitoneally. At 1 and 2 h after P407 injection, plasma samples from three mice per time point per group were collected, pooled, and analyzed for lipids. A: Immunoblot of C3^WT or C3^QK (top) along with endogenous mouse apoE (bottom). B: Total TAG in pooled plasma samples at 0, 1, and 2 h after P407 injection are shown. C: Total CE in pooled plasma samples at 0, 1, and 2 h after P407 injection are shown. D–F: Pooled plasma at time zero (D), at 1 h (E), and at 2 h (F) were fractionated by cumulative rate flotation ultracentrifugation, mass of TAG (left), CE (middle), and PC (right) in each fraction was quantified and plotted. The lipid-rich VLDL₁ fractions collected at 2 h after P407 injection are shown in F, left (inset).

Fig. 4.

Expression of C3^QK mutant in apoc3-null mice promotes the association of apoB100, apoE, and apoC3 to plasma VLDL. Plasma collected from mice at 2 h after P407 injection were pooled and fractionated by size exclusion chromatography (FPLC). A: Western blot images of ApoB-100, apoB-48, apoE, and apoA-I (left, C3^WT; right, C3^QK). B: Western blots of apoC-III in FPLC fractionated plasma of C3^WT and C3^QK mice. C: Comparison of lipid-binding properties of apoC-III protein from C3^WT and C3^QK.

Fig. 5.

Expression of C3^QK mutant under high-fat diet condition in apoc3-null mice promotes hepatic steatosis. A: Oil Red O staining of liver sections of apoc3-null mice fed with a high-fat diet for 1 week and then injected with adenovirus constructs encoding C3^WT or C3^QK. Liver samples collected from the mice at 48 h after injection were frozen fresh and sectioned by using cryostat and then stained with Oil Red O and visualized under light microscope. Images of intrahepatic lipid droplets from three different mice liver sections per group are shown. Scale bar, 20 µm. B, C: Lipid mass measurements of liver TAG (B) and CE (C) from C3^QK and C3^WT mice are shown. Data are expressed as mean ± SD. * P < 0.05; *** P < 0.001 (n = 3–6 mice per group).

Fig. 6.

Expression of C3^QK in mice increases the expression of lipogenesis genes and hepatic DNL. A: Relative mRNA expression profile of genes involved in lipogenesis in the livers of mice injected with adenovirus constructs of vector control or C3^WT or C3^QK. Data are expressed as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001 (n = 3–5 mice per treatment). B: In vivo hepatic DNL in mice. Bar graph shows the radioactivity associated with the indicated lipids extracted from the livers of mice 3 h after tail vein injection of [³H] acetic acid. Data are expressed as mean ± SD. *** P < 0.001 (n = 3–5 mice per treatment).

Fig. 7.

Expression of C3^QK in mice causes hyperlipidemia. Twelve week old apoC3-null mice maintained on normal chow diet were injected with adenovirus constructs encoding C3^WT or C3^QK or empty vector. At 48 h after injection, mice were fasted for 8 h, blood was collected, and then levels of FA (A), TAG (B), Chol (C), and CE (D) in the plasma were quantified. Data are expressed as mean ± SD. ** P < 0.01; *** P < 0.001 (n = 5 mice per treatment).