Disialylated apolipoprotein C-III proteoform is associated with improved lipids in prediabetes and type 2 diabetes

Abstract

The apoC-III proteoform containing two sialic acid residues (apoC-III2) has different in vitro effects on lipid metabolism compared with asialylated (apoC-III0) or the most abundant monosialylated (apoC-III1) proteoforms. Cross-sectional and longitudinal associations between plasma apoC-III proteoforms (by mass spectrometric immunoassay) and plasma lipids were tested in two randomized clinical trials: ACT NOW, a study of pioglitazone in subjects with impaired glucose tolerance (n = 531), and RACED (n = 296), a study of intensive glycemic control and atherosclerosis in type 2 diabetes patients. At baseline, higher relative apoC-III2 and apoC-III2/apoC-III1 ratios were associated with lower triglycerides and total cholesterol in both cohorts, and with lower small dense LDL in the RACED. Longitudinally, changes in apoC-III2/apoC-III1 were inversely associated with changes in triglycerides in both cohorts, and with total and small dense LDL in the RACED. apoC-III2/apoC-III1 was also higher in patients treated with PPAR-γ agonists and was associated with reduced cardiovascular events in the RACED control group. Ex vivo studies of apoC-III complexes with higher apoC-III2/apoC-III1 showed attenuated inhibition of VLDL uptake by HepG2 cells and LPL-mediated lipolysis, providing possible functional explanations for the inverse association between a higher apoC-III2/apoC-III1 and hypertriglyceridemia, proatherogenic plasma lipid profiles, and cardiovascular risk.

Keywords: lipoproteins; mass spectrometry; proteomics; triglycerides; very low density lipoprotein.

Fig. 1.

RPAs of all apoC-III proteoforms in the ACT NOW (n = 531) and RACED (n = 296) cohorts at baseline (A, B). 0a, native apoC-III; 0b, glycosylated nonsialylated apoC-III; 1, monosialylated apoC-III; 2, disialylated apoC-III; d, des-Alanine; Gal, galactose; GalNAc, N-acetylgalactosamine; Fuc, fucose. Major apoC-III proteoforms by race and ethnicity (C, D) and lipid-lowering therapy (Lipid-lowering th.) (E, F), and longitudinal changes in the major apoC-III proteoforms (G, H) (median follow-up 33 months ACT NOW and 9 months RACED). Data are medians ± quartiles; *P < 0.05, ^†P < 0.01, ^‡P < 0.001 by Wilcoxon-rank-sum test or Kruskal-Wallis test [(E) vs. Non-Hispanic Whites].

Fig. 2.

Associations between the apoC-III₂/apoC-III₁ ratio and plasma triglycerides. Scatter plots (with regression lines) and Spearman correlation coefficients of baseline values of (A, B) and longitudinal changes in (C, D) plasma triglycerides with apoC-III₂/apoC-III₁ in the ACT NOW and RACED cohorts. ^a, partially adjusted for treatment. Pio, pioglitazone; Plc, placebo; Int, intensive glucose-lowering therapy; Std, standard glucose-lowering therapy.

Fig. 3.

Spearman correlations between baseline values (n = 258) and longitudinal changes (Δ, follow-up minus baseline, n = 253) of apoC-III₂/apoC-III₁ ratio and fasting LDL-cholesterol subclass concentrations [LDL1 (A, B); LDL2 (C, D); LDL3 (E, F); LDL4 (G, H)] in the RACED cohort. Data are Spearman correlation coefficients. ^apartially adjusted for treatment. Int, intensive glucose-lowering therapy; Std, standard glucose-lowering therapy.

Fig. 4.

Associations between apoC-III proteoforms and MACEs in the RACED cohort. A: Cox proportional HRs and 95% CIs for treatment-adjusted effects of total apoC-III, RPAs of apoC-III₁ and apoC-III₂, and apoC-III₂/apoC-III₁ ratio. ^§P < 0.15 interaction with treatment. B, C: Kaplan-Meier curves of the time to MACE in the standard and intensive glucose lowering groups of the RACED cohort by median apoC-III₂/apoC-III₁ ratio.

Fig. 5.

Effect of higher apoC-III₂/apoC-III₁ ratio on VLDL uptake and LPL activity. VLDL was isolated from pooled plasma of subjects within the lower (Lo) and upper (Hi) quartiles of plasma apoC-III₂/apoC-III₁. A: Triglyceride content in HepG2 cells incubated with isolated VLDL in the presence of LPL. B, C: Mass spectra of apoC-III proteoforms in immunoprecipitated apoC-III complexes from Lo and Hi apoC-III₂/apoC-III₁ VLDL pools. D: Images obtained by fluorescent microscopy of HepG2 cells after addition of DiI-control VLDL without and with apoC-III complexes from Lo and Hi VLDL pools. DiI appears in orange, the lysosomes are stained in green and the nucleus in blue. E: Quantification of HepG2 DiI-VLDL uptake. F: LPL-mediated lipolysis in control VLDL without addition of apoC-III, and with addition of 30 μg/ml pure apoC-III and apoC-III complexes prepared from Lo and Hi VLDL pools. Data are mean ± SD, n = 4 [n = 2 (F)] repeats. *P < 0.05, ^‡P < 0.001.