The Arg59Trp variant in ANGPTL8 (betatrophin) is associated with total and HDL-cholesterol in American Indians and Mexican Americans and differentially affects cleavage of ANGPTL3

Abstract

We previously identified a locus linked to total cholesterol (TC) concentration in Pima Indians on chromosome 19p. To characterize this locus, we genotyped >2000 SNPs in 1838 Pimas and assessed association with log(TC). We observed evidence for association with log(TC) with rs2278426 (3.5% decrease/copy of the T allele; P=5.045×10(-6)) in the ANGPTL8 (angiopoietin-like 8) gene. We replicated this association in 2413 participants of the San Antonio Mexican American Family Study (SAMAFS: 2.0% decrease per copy of the T allele; P=0.005842). In a meta-analysis of the combined data, we found the strongest estimated effect with rs2278426 (P=2.563×10(-7)). The variant T allele at rs2278426 predicts an Arg59Trp substitution and has previously been associated with LDL-C and HDL-C. In Pimas and SAMAFS participants, the T allele of rs2278426 was associated with reduced HDL-C levels (P=0.000741 and 0.00002, respectively), and the combined estimated effect for the two cohorts was -3.8% (P=8.526×10(-8)). ANGPTL8 transcript and protein levels increased in response to both glucose and insulin. The variant allele was associated with increased levels of cleaved ANGPTL3. We conclude that individuals with the variant allele may have lower TC and HDL-C levels due to increased activation of ANGPTL3 by ANGPTL8.

Keywords: Betatrophin; C19ORF80; Cholesterol; Diabetes; Dyslipidemias; Genetics; HDL; LOC55908; Lipasin; RIFL.

Figure 1. General test of association of all markers genotyped on Chr 19 with total cholesterol levels in Pima Indians

Data were analyzed under an additive model and adjusted for age, sex, diabetes status, and exam date. Only SNPs with a MAF ≥0.01 were analyzed (N=1838).

Figure 2. Effect of high glucose treatment on ANGPTL8 transcript (A) and protein (B) expression

Hep G2 cells were grown to 80% confluence and starved overnight with serum-free media before treatment with conditioned media over 48 hrs. Total RNA and protein were extracted and expression levels assessed as described in the Methods section. mRNA levels were normalized against ACTB. Protein concentrations are shown as percentage of total soluble protein (TSP). Results represent averages from three independent experiments. Data are shown as means ± standard deviation; a two-tailed t-test was used to determine p-values; *p<0.05.

Figure 3. Effect of insulin concentration on ANGPTL8 transcript (A) and protein (B) expression

Hep G2 Cells were grown to 80% confluence and starved overnight with serum-free media before treatment with varying concentrations of insulin over 12 hours. Total RNA and protein were extracted and expression levels assessed as described in the Methods section. mRNA levels were normalized against ACTB. Protein concentrations are shown as percentage of total soluble protein (TSP). Results represent averages from three independent experiments. Data are shown as means ± standard deviation; a two-tailed t-test was used to determine p-values; *p<0.05.

Figure 4. Effect of insulin over time on ANGPTL8 transcript (A) and protein (B) expression

Hep G2 cells were grown to 80% confluence and starved overnight with serum-free media before treatment with 100 mM insulin over 48 hours. mRNA levels were normalized against ACTB. Protein concentrations are shown as percentage of total soluble protein (TSP). Results represent averages from three independent experiments. Data are shown as means ± standard deviation; the two-tailed t-test was used to determine p-values; *p<0.05.

Figure 5. Combined effect of insulin and glucose on ANGPTL8 transcript (A) and protein (B) expression

Hep G2 cells were grown to 80% confluence and starved overnight with serum-free media before treatment with 30 mM glucose, 100 nM insulin, or both over 24 hrs. Total RNA and protein were extracted and expression levels assessed as described in the Methods section. mRNA levels were normalized against ACTB. Protein concentrations are shown as percentage of total soluble protein (TSP). Results represent averages from three independent experiments. Data are shown as means ± standard deviation; two-tailed t-test was used to determine p-values; *p<0.05.

Figure 6. Differential expression of ANGPTL3 and ANGPTL8 in conditioned media and cell lysates

HEK293 cells were grown in 60 mm dishes to 90% confluence and then serum-starved for 24 hours prior to transfection with the appropriate vector for 6 hours with Lipofectamine 2000. Cells were incubated with either glucose (30 mM) or insulin (100 nM) for an additional 8 hours. Lane 1: ANGPTL3 only; Lane 2: ANGPTL3 plus the C allele (wild-type) of rs2278426; Lane 3: ANGPTL3 plus the T allele (variant) of rs2278426; and Lane 4: empty vector. The top panels for each set of conditions represent ANGPTL3 levels in conditioned media, while the bottom panels represent ANGPTL8 levels in cell lysates.