Research ArticleMetabolism

Complex effects of inhibiting hepatic apolipoprotein B100 synthesis in humans

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Science Translational Medicine  27 Jan 2016:
Vol. 8, Issue 323, pp. 323ra12
DOI: 10.1126/scitranslmed.aad2195
  • 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.

  • Table 1. Subject baseline characteristics.

    Twenty individuals were enrolled and 17 completed the study (table S1).

    Mean age, years (±SD)43.5 (14.2)
    Gender, n (%)
      Male8 (47.1)
      Female9 (52.9)
    Race (%)
      White29.4
      Black47.1
      Asian5.9
      Unknown/mixed race17.6
      Mean BMI (±SD)27.6 (3.2)
  • Table 2. Plasma lipid levels after 3 weeks of placebo and 7 weeks of mipomersen treatment.

    Data are means (±SD) of one sample obtained from each subject (n = 17) just before the kinetic study in each period. Percent change is based on the difference between levels during mipomersen treatment and during placebo treatment for each subject. The significance of percent change for each endpoint was examined by paired t test after determining that the data were normally distributed using the Kolmogorov-Smirnov normality test.

    ParameterPlacebo
    (mg/dl)
    Mipomersen
    (mg/dl)
    Change
    (%)
    P value
    Total cholesterol187.2 (25.7)132.4 (29.7)−28.6 (16.1)<0.001
    TG86.9 (49.3)57.1 (30.4)−28.0 (28.0)<0.001
    LDL-C114.7 (22.3)61.9 (24)−45.5 (18.9)<0.001
    ApoB69.6 (18.9)40.8 (14.1)−40.3 (17.5)<0.001
    HDL-C55.2 (14.3)59.1 (17.9)6.8 (19.3)0.17
  • Table 3. ApoB kinetics.

    The effects of mipomersen on apoB metabolism were determined from 17 kinetic studies at the end of the placebo and mipomersen treatment periods. Absolute values for the placebo and mipomersen periods are shown. If the data were found to be normally distributed by the Kolmogorov-Smirnov test, they are presented as means (±SD), and statistical analyses were performed on percent change for each endpoint on mipomersen from placebo using paired t tests. If the data were not normally distributed, the median and interquartile values (in parentheses) are shown for percent change, and the Wilcoxon rank-sum test was performed to test for significance.

    ParameterPlaceboMipomersenChange
    (%)
    P value
    VLDL apoB FCR (pools/day)7.6 (3.6)9.9 (4.6)23.0
    (−7.4, 55.6)
    0.023
    VLDL apoB PR (mg kg−1 day−1)21.7 (11.3)19.0 (10.9)−17.6
    (−32.7, −0.9)
    0.15
    VLDL to IDL (% conversion)*70.2 (24.2)58.2 (24.5)−12.10.09
    IDL apoB FCR (pools/day)5.5 (3.5)5.8 (3.6)11.10.30
    IDL apoB PR (mg kg−1 day−1)15.0 (8.1)10.2 (6.9)−32.7
    (−53.5, −5.7)
    0.05
    IDL to LDL (% conversion)*76.2 (23)74.4 (26)−1.70.84
    LDL apoB FCR (pools/day)0.52 (0.14)0.65 (0.19)29.7<0.001
    LDL apoB PR (mg kg−1 day−1)12.7 (4.6)8.8 (3.1)−26.50.001
    Direct LDL PR (mg kg−1 day−1)2.6 (3.5)2.2 (1.5)0.0
    (−0.8, 1.5)
    0.89
    LDL apoB PR from direct LDL secretion (%)*21.3 (22)26.8 (18)5.50.38
    Total apoB PR (sum VLDL PR and direct LDL PR) (mg kg−1 day−1)24.3 (11.5)21.3 (10.7)−15.1
    (−34.5, −7.4)
    0.26
    VLDL TG FCR (pools/hour)10.5 (7.9)13.2 (8.4)46.30.03
    VLDL TG PR (mg kg−1 hour−1)13.1 (6.4)13.3 (7.0)10.00.40
    VLDL TG PR/VLDL apoB PR ratio22.9 (27.0)26.2 (37.0)16.5
    (−7.8, 93.2)
    0.05
    Plasma VLDL TG/VLDL apoB ratio14.5 (13.7)15.3 (9.0)24.60.03

    *For the percent conversions (VLDL to IDL; IDL to LDL), absolute differences between placebo and mipomersen results were used to test for significance.

    †Some direct LDL secretion rates were zero, so no percent change was calculated for this parameter; instead, values are absolute differences.

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/8/323/323ra12/DC1

      Methods

      Fig. S1. Study flow diagram.

      Fig. S2. No changes in LDLR, IDOL, or PCSK9 mRNA levels in HepG2 cells after knockdown of APOB.

      Fig. S3. ApoB ASO inhibits hepatic secretion of TG and apoB from mice on either chow or HFD diet.

      Fig. S4. ApoB secretion is increased in Apobec-1 knockout mice on HFD.

      Fig. S5. ApoB siRNA treatment does not affect expression of Ldlr, Idol, or Pcsk9 in mouse livers.

      Fig. S6. Hepatic LDL receptor was not affected by ApoB ASO.

      Fig. S7. Relationship between mipomersen-induced reductions in VLDL apoB PR and baseline VLDL PR.

      Table S1. Participant information.

      Table S2. Effect of mipomersen on apoB lipoprotein particle number by ion mobility analysis.

      Table S3. Effect of mipomersen on apoB lipoprotein size distribution by ion mobility analysis.

      Table S4. Effect of mipomersen on LDL size subfraction particle numbers by ion mobility analysis.

      Table S5. Markers of cholesterol synthesis and absorption.

      Table S6. Hepatic lipase, lipoprotein lipase, FFA, and β-hydroxybutyrate levels.

      Table S7. Individual participant apoB kinetics of VLDL, IDL, LDL, and VLDL TG FCR.

      References (4349)

    • Supplementary Material for:

      Complex effects of inhibiting hepatic apolipoprotein B100 synthesis in humans

      Gissette Reyes-Soffer,* Byoung Moon, Antonio Hernandez-Ono, Marija Dionizovick-Dimanovski, Jhonsua Jimenez, Joseph Obunike, Tiffany Thomas, Colleen Ngai, Nelson Fontanez, Daniel S. Donovan, Wahida Karmally, Stephen Holleran, Rajasekhar Ramakrishnan, Robert S. Mittleman, Henry N. Ginsberg*

      *Corresponding author. E-mail: gr2104{at}cumc.columbia.edu (G.R.-S.); hng1{at}cumc.columbia.edu (H.N.G.)

      Published 27 January 2016, Sci. Transl. Med. 8, 323ra12 (2016)
      DOI: 10.1126/scitranslmed.aad2195

      This PDF file includes:

      • Methods
      • Fig. S1. Study flow diagram.
      • Fig. S2. No changes in LDLR, IDOL, or PCSK9 mRNA levels in HepG2 cells after knockdown of APOB.
      • Fig. S3. ApoB ASO inhibits hepatic secretion of TG and apoB from mice on either chow or HFD diet.
      • Fig. S4. ApoB secretion is increased in Apobec-1 knockout mice on HFD.
      • Fig. S5. ApoB siRNA treatment does not affect expression of Ldlr, Idol, or csk9P in mouse livers.
      • Fig. S6. Hepatic LDL receptor was not affected by ApoB ASO.
      • Fig. S7. Relationship between mipomersen-induced reductions in VLDL apoB PR and baseline VLDL PR.
      • Table S1. Participant information.
      • Table S2. Effect of mipomersen on apoB lipoprotein particle number by ion mobility analysis.
      • Table S3. Effect of mipomersen on apoB lipoprotein size distribution by ion mobility analysis.
      • Table S4. Effect of mipomersen on LDL size subfraction particle numbers by ion mobility analysis.
      • Table S5. Markers of cholesterol synthesis and absorption.
      • Table S6. Hepatic lipase, lipoprotein lipase, FFA, and β-hydroxybutyrate levels.
      • Table S7. Individual participant apoB kinetics of VLDL, IDL, LDL, and VLDL TG FCR.
      • References (4349)

      [Download PDF]

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