Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Feb;356(2):341-53.
doi: 10.1124/jpet.115.229740. Epub 2015 Nov 16.

Creation of Apolipoprotein C-II (ApoC-II) Mutant Mice and Correction of Their Hypertriglyceridemia with an ApoC-II Mimetic Peptide

Toshihiro Sakurai  1 , Akiko Sakurai  1 , Boris L Vaisman  1 , Marcelo J Amar  1 , Chengyu Liu  1 , Scott M Gordon  1 , Steven K Drake  1 , Milton Pryor  1 , Maureen L Sampson  1 , Ling Yang  1 , Lita A Freeman  1 , Alan T Remaley  2
Affiliations

Creation of Apolipoprotein C-II (ApoC-II) Mutant Mice and Correction of Their Hypertriglyceridemia with an ApoC-II Mimetic Peptide

Toshihiro Sakurai et al. J Pharmacol Exp Ther. 2016 Feb.

Abstract

Apolipoprotein C-II (apoC-II) is a cofactor for lipoprotein lipase, a plasma enzyme that hydrolyzes triglycerides (TGs). ApoC-II deficiency in humans results in hypertriglyceridemia. We used zinc finger nucleases to create Apoc2 mutant mice to investigate the use of C-II-a, a short apoC-II mimetic peptide, as a therapy for apoC-II deficiency. Mutant mice produced a form of apoC-II with an uncleaved signal peptide that preferentially binds high-density lipoproteins (HDLs) due to a 3-amino acid deletion at the signal peptide cleavage site. Homozygous Apoc2 mutant mice had increased plasma TG (757.5 ± 281.2 mg/dl) and low HDL cholesterol (31.4 ± 14.7 mg/dl) compared with wild-type mice (TG, 55.9 ± 13.3 mg/dl; HDL cholesterol, 55.9 ± 14.3 mg/dl). TGs were found in light (density < 1.063 g/ml) lipoproteins in the size range of very-low-density lipoprotein and chylomicron remnants (40-200 nm). Intravenous injection of C-II-a (0.2, 1, and 5 μmol/kg) reduced plasma TG in a dose-dependent manner, with a maximum decrease of 90% occurring 30 minutes after the high dose. Plasma TG did not return to baseline until 48 hours later. Similar results were found with subcutaneous or intramuscular injections. Plasma half-life of C-II-a is 1.33 ± 0.72 hours, indicating that C-II-a only acutely activates lipolysis, and the sustained TG reduction is due to the relatively slow rate of new TG-rich lipoprotein synthesis. In summary, we describe a novel mouse model of apoC-II deficiency and show that an apoC-II mimetic peptide can reverse the hypertriglyceridemia in these mice, and thus could be a potential new therapy for apoC-II deficiency.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
Targeting strategy of ZFN-mediated targeted genome editing for creating Apoc2 mutant mice. (A) Uppercase and lowercase letters delineate exons and introns, respectively. Bold black letters (GGGAAC), red letters (tccctcagAGGTCCAG), and blue letters (CAGGAAGATGACTCGGGC) indicate the site targeted to cut by FokI and sites for ZFN-1 and ZFN-2 DNA binding, respectively. Numbers above the base indicate nucleotide position for the Apoc2 gene. The amino acid sequences of apoC-II in both WT mice (top) and in mutant mice with a 9-bp deletion in Apoc2 gene and the deletion of 3 amino acids in the mutant protein (bottom) are also shown. Numbers in subscript below each amino acid indicate amino acid position of the prosequence form of protein. Bold blue arrow indicates position of signal peptide cleavage site for WT protein. Dotted lines in the sequence indicate either missing nucleotides or amino acids for the mutant apoC-II protein. (B) A PCR–restriction fragment length polymorphism assay for genotyping of the 9-bp deletion. DNA fragments cleaved by the BspLI restriction enzyme were resolved on a 4% agarose gel and stained with ethidium bromide. The following bands were observed for the three possible genotypes: 1) WT (50, 84, and 197 bp), 2) heterozygous mice (50, 84, 197, and 238 bp), and 3) homozygous mice (84 and 238 bp). (C) A cleavable signal peptide site was predicted by online software (SignalP). The C-score (red) was calculated to be high at the position immediately after the (+1) residue, the known cleavage site for the WT protein but not for the mutant protein. The S-score (green) indicates the likelihood of the presence of a signal peptide and was similar for WT and mutant protein. The Y-score (blue) is a composite of the C- and S-scores and indicates the correct cleavage site of the WT sequence but a low likelihood of signal peptide cleavage for the mutant protein (below purple threshold line).
Fig. 2.
Fig. 2.
WT and mutant apoC-II protein abundance and size. (A) Immunoblotting of apoC-II from plasma of three representative individual mice (WT, heterozygous, and homozygous mice). (B) Immunoblotting of apoC-II from FPLC fractions [VLDL/chylos (20 μl), IDL (20 μl), LDL (7 μl), HDL (7 μl), and albumin (20 μl)] of an apoC-II heterozygous mouse. (C) Quantitation of WT versus mutant apoC-II in FPLC fractions [from (B)] by densitometry, expressed as a percentage of the total signal (WT plus mutant) for apoC-II in all fractions combined. The percentages were corrected for the different volumes applied to the gel.
Fig. 3.
Fig. 3.
Effect of Apoc2 9-bp deletion on plasma lipids. A 5-hour fasting plasma was obtained from 2-month-old mice (n = 10–19 per groups). Results are represented as the mean ± S.D. **P < 0.01; ***P < 0.005; ****P < 0.0001. EC, esterified cholesterol; FC, free cholesterol.
Fig. 4.
Fig. 4.
Analysis of lipoprotein profiles by FPLC (A–C), agarose gel electrophoresis (D), and electron microscopy (E) in Apoc2 mutant mice. For (A), (B), and (C), pooled, 5-hour-fasting plasma (0.25 ml; n = 3–5 per group) was separated by FPLC. (A) WT sibling control mice. (B) Apoc2 heterozygous mutant mice. (C) Apoc2 homozygous mutant mice. (D) Pooled fasting plasma samples (10 μl; n = 3–5 per group) were analyzed by Sebia Hydragel lipoprotein(e) gel electrophoresis, followed by Sudan Black staining. (E) A lipoprotein fraction enriched in chylomicrons, VLDL, IDL, and LDL was isolated by ultracentrifugation (d < 1.063 kg/l) of plasma from mice (n = 3–5) to analyze lipoprotein particle size by electron microscopy. (F) Distribution of lipoprotein particle size greater than and less than 30 nm was presented as a percentage of total lipoprotein particle counts. EC, esterified cholesterol; FC, free cholesterol.
Fig. 5.
Fig. 5.
Proteome contents of lipoprotein fractions. Pooled plasma (0.25 ml; n = 3–5 per group) was separated by FPLC, as shown in Fig. 4, A–C. Fractions corresponding to VLDL (elution volumes: 14.0–16.0 ml), LDL (21.5–24.0 ml), and HDL (27.0–31.0 ml) were pooled for analysis. Lipid-bound proteins were isolated by LRA, trypsinized, and then analyzed by LC-MS/MS. For each sample, individual spectral counts for each protein were normalized to the total spectral count. The results are presented as the percent change in normalized spectrum counts for each identified protein in the mutant mice compared with the wild type for each lipoprotein class. HRG, histidine-rich glycoprotein.
Fig. 6.
Fig. 6.
Effects of C-II-a on in vitro lipolysis. Pooled plasma from homozygous Apoc2 mutant mice (n = 3; mean ± S.D. of plasma TG: 627.5 ± 76.7 mg/dl) or human plasma from an apoC-II–deficient patient (TG: 658 mg/dl) was incubated in vitro with LPL alone or in combination with C-II-a, and the production of FFA was measured. Results are represented as the mean of triplicates ± S.D. ****P < 0.0001.
Fig. 7.
Fig. 7.
Effects of i.v. injection of C-II-a into Apoc2 mutant mice. (A) Analysis of plasma lipids. 200 microliters of C-II-a peptide (0.2, 1.0, and 5.0 μmol/kg of body weight) was injected into the retro-orbital sinus of Apoc2 mutant mice (n = 3 per groups). Lipid levels were measured in the plasma at 0, 0.5, 1, 2, 4, 7, 24, and 48 hours after C-II-a injection. Data are the mean ± S.E.M. (B) Distribution of lipids in plasma analyzed by FPLC. 50 microliters of plasma pooled from three mice was separated by FPLC. The level of TC and TG in the fractions was measured at 0, 2, 24, and 48 hours after C-II-a injection (5.0 μmol/kg of body weight).
Fig. 8.
Fig. 8.
Decreased TG and C-II-a in Apoc2 mutant mice after i.v. injection of C-II-a. (A) Changes in plasma levels of TG and C-II-a after C-II-a injection. C-II-a was injected into four Apoc2 mutant mice (5.0 μmol/kg of body weight). C-II-a concentrations in plasma were analyzed by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) using 5A-R peptide solution (molecular weight 4330) as an internal standard. Data shown are the mean ± S.E.M. (B) The change of plasma levels of TG and C-II-a after heparin injection (0.5 U/g of body weight) at 6.5 hours after the C-II-a injection. Data shown are the mean ± S.E.M. (Insets) Half-time of C-II-a without (A) or with (B) heparin injection at t = 6.5 hours after log transformation of the y-axis.
Fig. 9.
Fig. 9.
Effects of different routes of C-II-a administration on plasma lipids in Apoc2 mutant mice. 50 microliters of C-II-a peptide (1.0 μmol/kg of body weight) was injected subcutaneously (SQ) into the abdomen, intramuscularly (IM) into the thigh muscle, or intravenously (IV) to the retro-orbital sinus of Apoc2 mutant mice (n = 3 per groups). Plasma TC and TG levels were measured at 0, 0.5, 1, 4, 7, 24, and 48 hours after C-II-a injection. Data are shown as the mean ± S.E.M.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Amar MJA, D’Souza W, Turner S, Demosky S, Sviridov D, Stonik J, Luchoomun J, Voogt J, Hellerstein M, Sviridov D, et al. (2010) 5A apolipoprotein mimetic peptide promotes cholesterol efflux and reduces atherosclerosis in mice. J Pharmacol Exp Ther 334:634–641. - PMC - PubMed
    1. Amar MJA, Sakurai T, Sakurai-Ikuta A, Sviridov D, Freeman L, Ahsan L, Remaley AT. (2015) A novel apolipoprotein C-II mimetic peptide that activates lipoprotein lipase and decreases serum triglycerides in apolipoprotein E-knockout mice. J Pharmacol Exp Ther 352:227–235. - PMC - PubMed
    1. Anantharamaiah GM, Jones JL, Brouillette CG, Schmidt CF, Chung BH, Hughes TA, Bhown AS, Segrest JP. (1985) Studies of synthetic peptide analogs of the amphipathic helix. Structure of complexes with dimyristoyl phosphatidylcholine. J Biol Chem 260:10248–10255. - PubMed
    1. Athyros VG, Giouleme OI, Nikolaidis NL, Vasiliadis TV, Bouloukos VI, Kontopoulos AG, Eugenidis NP. (2002) Long-term follow-up of patients with acute hypertriglyceridemia-induced pancreatitis. J Clin Gastroenterol 34:472–475. - PubMed
    1. Auclair SM, Bhanu MK, Kendall DA. (2012) Signal peptidase I: cleaving the way to mature proteins. Protein Sci 21:13–25. - PMC - PubMed

Publication types