Mouse hepatic lipase alleles with variable effects on lipoprotein composition and size

Abstract

The structural features responsible for the activities of hepatic lipase (HL) can be clarified by in vivo comparisons of naturally occurring variants. The coding sequence of HL from C57BL/6J (B6) and SPRET/EiJ (SPRET) mice differs by four amino acids (S106N, A156V, L416V, S480T); however, these changes are not predicted to influence HL function. To test for allelic effects, we generated SPRET-HL transgenics with physiological levels of HL mRNA and HL activity that was parallel in female transgenics and about 70% higher in male transgenics, toward tri-[3H]oleate, compared with B6 controls. We found no correlation between activity levels and plasma lipids. However, significant allelic effects on plasma lipids were observed. Compared with B6-HL, SPRET-HL mediated reductions in total cholesterol (TC) and VLDL-, LDL- and HDL-cholesterol and HDL-triglyceride (TG) in fed males, and SPRET-HL decreased total TG and VLDL- and HDL-TG levels in fasted males. Fasted female transgenics had reduced TC compared with controls. We also found allele and sex effects on lipoprotein particle size. Male transgenic mice had increased VLDL and decreased LDL size, and female transgenic mice had decreased HDL size compared with control animals. These findings demonstrate highly divergent effects of naturally occurring HL coding sequence variants on lipid and lipoprotein metabolism.

Fig. 1.

Diagram of BAC with BAC-end and genotyping primers mapped using Ensembl genome browser v53. See Table 1 for sequences and bp positions of genotyping primers. Black rectangles = exons 1–9; gray rectangle = vector (pTARBAC2.1). Sequence from T7 mapped to Chr 9: 70,612,052-70,612,192 bp and BAC End-1 to Chr 9:70,801,301-70,801,753 bp. Ensembl gene ENSMUSG00000078305 maps to Chr:9 70,700,357-70,702,269 bp. BAC, bacterial artificial chromosome.

Fig. 2.

Relative expression fold change of liver HL mRNA as determined by qPCR in fasted mice sacrificed at 140 ± 14 days. For HL expression, 6 biologic replicates were used per genotype except for HL^+/+ mice, which had 4 biologic replicates. For ENSMUSG00000078305 expression, a minimum of 8 and a maximum of 12 biological replicates were used. HL^−/− = mice lacking endogenous HL; HL^+/− = F1 (C57BL6/J X HL^−/− mice); HL^+/+ = C57BL6/J mice; HL^−/−,+S = mice lacking endogenous HL and positive for the SPRET-HL transgene; HL^+/+,+S = C57BL6/J mice positive for the SPRET-HL transgene; Relative expression = Ct(specified HL genotype)-Ct(control gene)/ Ct(HL^+/+) – Ct(Gusb). Data are means ± SEM. Bars not sharing a symbol are different by P < 0.05 by Sidak's t-test for multiple comparisons.

Fig. 3.

Hepatic lipase activity in fasted mice measured in pre and postheparin plasma. Activity units are mU/ul. HL^−/− = mice lacking endogenous HL; HL^+/− = F1 (C57BL6/J X HL^−/− mice); HL^+/+ = C57BL6/J mice; HL^−/−,+S = mice lacking endogenous HL and positive for the SPRET-HL transgene; HL^+/+,+S = C57BL6/J mice positive for the SPRET-HL transgene. Ns are as follows for preheparin plasma: females HL^−/− 14; HL^+/− 9; HL^+/+ 15; HL^−/−,+S 12; HL^+/+,+S 10; and males HL^−/− 13; HL^+/− 7; HL^+/+ 10; HL^−/−,+S 9; HL^+/+,+S 9. For postheparin plasma, N = 3 for HL^+/+ and N = 4 for HL^−/−,+S mice. Data shown are means ± SEM. Sexes were analyzed separately. **P < 0.0001 compared with all other HL genotypes and *P ≤ 0.008 compared with HL^+/+; HL^−/−,+S; and HL^+/+,+S for females; and HL^−/−,+S and HL^+/+,+S for males by Sidak's t-test for multiple comparisons.

Fig. 4.

Lipoprotein composition determined in 20 continuously eluted fractions of fasted male and female plasma determined by dual detection HPLC. Filled circles = HL^−/−; Filled triangles = HL^+/+; white circles = HL^−.-,+S; white triangles = HL^+/+,+S; HL^−/− = mice lacking endogenous HL; HL^−/−,+S = mice lacking endogenous HL and positive for the SPRET-HL transgene; HL^+/+ = C57BL6/J mice; HL^+/+,+S = C57BL6/J mice positive for the SPRET-HL transgene. A: Male cholesterol. B: Female cholesterol. C: Male triglyceride. D: Female triglyceride. Elution fractions correspond to the following lipoprotein subfractions: VLDL, large, 3–5; medium, 6; and small, 7. LDL, large, 8; medium, 9; small, 10; and very small, 11–13. HDL, very large, 14–15; large, 16; medium, 17; small 18; very small, 19–20. Sample numbers are as follows: HL^−/− 6, HL^+/+ 8, HL^−/−,+S 7, HL^+/+,+S 5. Data shown are means ± SEM and significance determined by Scheffe's posthoc test. A: * P < 0.005 HL^+/+ compared to all other genotypes; ^# P < 0.005 HL^+/+ compared to HL^−/− and HL^−/−,+S; ^§ P < 0.05 HL^+/+ compared to HL^−/−,+S and HL^+/+,+S; † P < 0.0001 HL^−/−,+S compared to HL^+/+ and HL^+/+,+S; ‡ P < 0.01 HL^+/+,+S compared to all other genotypes; ** P < 0.05 HL^−/− compared to HL^+/+,+S; ^## P < 0.05 HL^−/− compared to HL^−/−,+S; ^§§ P < 0.05 HL^−/− compared to HL^−/−,+S and HL^+/+,+S; ^†† P < 0.05 HL^−/− compared to all other genotypes; ^‡‡ P < 0.01 HL^+/+,+S compared to HL^−/− and HL^+/+. B: * P < 0.0001 HL^+/+ compared to HL^−/− and HL^−/−,+S; ^# P < 0.05 HL^−/− compared to HL^+/+ and HL^+/+,+S; ^§ P < 0.05 HL^−/− compared to all other genotypes; ^† P < 0.005 HL^−/− compared to all other genotypes; ^‡ P < 0.0001 HL^−/− compared to all other genotypes; ** P < 0.001 HL^−/− compared to HL^+/+ and HL^+/+,+S; ^#,# P < 0.01 HL^−/− compared to HL^+/+,+S. C: * P < 0.05 HL^+/+ compared to all other genotypes; ^# P < 0.005 HL^+/+ compared to all other genotypes; ^§ P < 0.05 HL^+/+ compared to HL^−/−,+S and HL^+/+,+S; ^† P < 0.05 HL^+/+ compared to HL^−/−,+S; ^‡ P < 0.005 HL^+/+,+S compared to HL^−/− and HL^−/−,+S; ** P < 0.0001 HL^+/+,+S compared to all genotypes; ^## P < 0.05 HL^−/−,+S compared to HL^+/+ and HL^+/+.+S; ^§§ P < 0.001 HL^−/− compared to all other genotypes; ^†† P < 0.02 HL^−/− compared to HL^−/−,+S and HL^+/+,+S; ^‡‡ P < 0.05 HL^+/+,+S compared to HL^+/+ and HL^−/−,+S. D: * P < 0.05 HL^+/+,+S compared to HL^−/− and HL^−/−,+S; ^# P < 0.05 HL^+/+,+S compared to HL^−/−; ^§ P < 0.05 HL^+/+ compared to HL^−/− and HL^−/−,+S; ^† P < 0.05 HL^+/+,+S compared to HL^−/− and HL^−/−,+S; ^‡ P < 0.05 HL^+/+ compared to HL^−/−; ** P < 0.05 HL^−/− compared to all other genotypes; ^## P < 0.01 HL^−/− compared to all other genotypes; ^§§ P < 0.0001 HL^−/− compared to all other genotypes; ^†† P < 0.0001 HL^−/− compared to HL^+/+ and HL^+/+,+S.