Complex effects of inhibiting hepatic apolipoprotein B100 synthesis in humans

Abstract

Mipomersen is a 20mer antisense oligonucleotide (ASO) that inhibits apolipoprotein B (apoB) synthesis; its low-density lipoprotein (LDL)-lowering effects should therefore result from reduced secretion of very-low-density lipoprotein (VLDL). We enrolled 17 healthy volunteers who received placebo injections weekly for 3 weeks followed by mipomersen weekly for 7 to 9 weeks. Stable isotopes were used after each treatment to determine fractional catabolic rates and production rates of apoB in VLDL, IDL (intermediate-density lipoprotein), and LDL, and of triglycerides in VLDL. Mipomersen significantly reduced apoB in VLDL, IDL, and LDL, which was associated with increases in fractional catabolic rates of VLDL and LDL apoB and reductions in production rates of IDL and LDL apoB. Unexpectedly, the production rates of VLDL apoB and VLDL triglycerides were unaffected. Small interfering RNA-mediated knockdown of apoB expression in human liver cells demonstrated preservation of apoB secretion across a range of apoB synthesis. Titrated ASO knockdown of apoB mRNA in chow-fed mice preserved both apoB and triglyceride secretion. In contrast, titrated ASO knockdown of apoB mRNA in high-fat-fed mice resulted in stepwise reductions in both apoB and triglyceride secretion. Mipomersen lowered all apoB lipoproteins without reducing the production rate of either VLDL apoB or triglyceride. Our human data are consistent with long-standing models of posttranscriptional and posttranslational regulation of apoB secretion and are supported by in vitro and in vivo experiments. Targeting apoB synthesis may lower levels of apoB lipoproteins without necessarily reducing VLDL secretion, thereby lowering the risk of steatosis associated with this therapeutic strategy.

Fig. 1. Effects of mipomersen treatment on human lipoprotein lipids and apoB levels

Blood samples were obtained from subjects during their kinetic studies performed after 3 weeks of treatment with placebo and after 7 weeks of treatment with mipomersen. Plasma was separated from those samples, and VLDL, IDL, and LDL were isolated by sequential ultracentrifugation. (A and B) Cholesterol and TG were measured by enzymatic methods. (C) ApoB was determined by commercial enzyme-linked immunosorbent assay. Data are means ± SD (n = 5 samples obtained from each of the 17 subjects during their two kinetic studies). P values determined by paired t tests after determining that the data were normally distributed using the Kolmogorov-Smirnov normality test.

Fig. 2. Effects of siRNA-mediated knockdown of APOB on apoB secretion in human HepG2 cells

(A) APOB siRNA (10 nM) inhibits the synthesis and secretion of apoB in HepG2 cells. HepG2 cells were transfected with 10 nM APOB or 10 nM irrelevant control (Con) siRNA for 2 days, radiolabeled continuously with [³⁵S]methionine/cysteine for 2 hours, and radioactivity [counts per minute (CPM)] in cellular or media apoB was determined. (B) APOB siRNA knocks down APOB mRNA in a dose-dependent manner. HepG2 cells were transfected at varying doses of APOB siRNA for 2 days. Total RNA was extracted and APOB mRNA levels were analyzed by quantitative polymerase chain reaction (qPCR). (C) APOB siRNA reduces initial synthesis rates of apoB in a dose-dependent manner. Cells were labeled with [³⁵S]methionine/cysteine for 10 min and chased for 10 min with or without proteasome inhibitor ALLN pretreatment for 1 hour. (D and E) The effect of APOB siRNA doses on cellular and secreted apoB in HepG2 cells transfected as in (B), and apoB synthesis and secretion analyzed as in (A). (F) Efficiency of apoB secretion as a function of APOB siRNA knockdown. Efficiency was defined as the percentage of apoB secreted into the medium compared with the amount of total newly synthesized apoB (cellular and secreted apoB over 2 hours). (G and H) Cellular and secreted apoB in HepG2 cell culture after treatment with an MTP inhibitor (MTPI) for 1 hour (before steady-state labeling for 2 hours). (I) Efficiency of apoB secretion as a function of MTP inhibition. Data were plotted using the cell and secreted apoB data, as described in (F). All data are mean percentage of control [siRNA (1 nM) in (B) to (F); dimethyl sulfoxide (DMSO) in (G) to (I)] ± SD (n = 3). *P < 0.05; **P < 0.01 versus respective control, unless otherwise noted, one-way analysis of variance (ANOVA) followed by post hoc comparisons using the Fisher LSD (least significant difference) method.

Fig. 3. Effects of ApoB ASO treatment on apoB and TG secretion in Apobec-1 knockout mice

Chow-and HFD-fed mice were treated for 3 weeks with ApoB ASO. (A) ApoB mRNA expression was determined using qPCR of livers from both chow and HFD mice and normalized to liver β-actin mRNA. In each diet group, irrelevant, control (Ctrl) ASO-treated mRNA levels were set as 100%. (B) Mice were fasted 4 hours and injected with Tyloxapol and [³⁵S]methionine intravenously. Plasma ³⁵S-labeled apoB levels in CPM were determined in samples obtained at 120 min after the start of the study. ApoB radioactivity in mice receiving the irrelevant control ASO was set as 100%. (C) Blood samples were collected at 0, 30, 60, 90, and 120 min for measurement of TG levels. TG secretion rate was determined by the change in plasma TG concentration between 30 and 120 min after Tyloxapol injection and divided by 1.5. The secretion rate of the mice receiving irrelevant control ASO was set at 100%. Data in (A) to (C) are means ± SD (n noted on figure). *P < 0.05 versus control, unless otherwise noted, one-way ANOVA and Tukey’s post hoc test.