Trib1 is a lipid- and myocardial infarction-associated gene that regulates hepatic lipogenesis and VLDL production in mice

Abstract

Recent genome-wide association studies have identified a genetic locus at human chromosome 8q24 as having minor alleles associated with lower levels of plasma triglyceride (TG) and LDL cholesterol (LDL-C), higher levels of HDL-C, as well as decreased risk for myocardial infarction. This locus contains only one annotated gene, tribbles homolog 1 (TRIB1), which has not previously been implicated in lipoprotein metabolism. Here we demonstrate a role for Trib1 as a regulator of lipoprotein metabolism in mice. Hepatic-specific overexpression of Trib1 reduced levels of plasma TG and cholesterol by reducing VLDL production; conversely, Trib1-knockout mice showed elevated levels of plasma TG and cholesterol due to increased VLDL production. Hepatic Trib1 expression was inversely associated with the expression of key lipogenic genes and measures of lipogenesis. Thus, we provide functional evidence for what we believe to be a novel gene regulating hepatic lipogenesis and VLDL production in mice that influences plasma lipids and risk for myocardial infarction in humans.

Figure 1. Hepatic Trib1 overexpression reduces plasma lipid levels in mice.

C57BL/6 mice (n = 5 per group) were injected with 10¹² genome copies (gc) control (AAV_null) or 10¹⁰, 10¹¹, or 10¹² gc AAV_trib1. (A) Plasma cholesterol and TG levels were measured 2 weeks after injection. Plasma lipoproteins were isolated by sequential ultracentrifugation, and cholesterol (B) and TG (C) levels were determined in the VLDL, LDL, and HDL fractions. (D) Pooled plasma samples were fractionated by FPLC, and TG levels were determined in collected fractions. IDL, intermediate-density lipoprotein. (E) Effects of AAV_trib1 injection (10¹² gc) on plasma total cholesterol (TC) and TG in Ldlr^–/– mice and (F) LAhB-H mice (n = 6 per group). Human plasma apoB was determined in LAhB-H mice and was reduced after AAV_trib1 injection. (G) Distribution of cholesterol and TG in FPLC-fractionated plasma of AAV-injected LAhB-H mice. *P < 0.05, **P < 0.005, AAV_null versus AAV_trib1 groups.

Figure 2. Trib1 deficiency increases plasma lipid levels in mice.

Chow-fed Trib1^–/– mice (n = 8) and littermate controls (Trib1^+/+, n = 13) were phenotyped at 8 weeks of age (*P < 0.05, **P < 0.01, ***P < 0.001). (A) Plasma cholesterol and TG were elevated in Trib1^–/– mice. VLDL, LDL, and HDL were isolated by sequential ultracentrifugation, and significant increases in cholesterol (B) and TG (C) were observed in VLDL and LDL of Trib1^–/– mice. (D) Pooled plasma samples were separated by FPLC, and TG was determined in collected fractions. (E) Trib1^–/– mice (n = 4) were injected with 10¹¹ gc AAV_trib1 to reconstitute hepatic expression of Trib1. Plasma TG was measured 2 weeks after injection and the percentage change compared with that in control mice receiving AAV_null.

Figure 3. Effects of Trib1 on hepatic VLDL production, gene expression, and lipogenesis.

VLDL-TG production was determined over 4 hours after Pluronic-407 injection in (A) AAV_null– or AAV_trib1–treated C57BL/6 (WT), Ldlr^–/–, and LAhB-H mice (n = 6 per group) and in (B) Trib1^+/+, Trib1^–/– (n = 6 per group), and AAV_null– or AAV_trib1–treated Trib1^+/+ and Trib1^–/– mice (n = 4 per group). Values are expressed as relative changes compared with respective controls. (C) Quantitative RT-PCR: Control values (Trib1^+/+ and AAV_null) were defined as 1, and changes in Trib1^–/– and AAV_trib1 groups expressed as relative amounts compared with controls (n = 6–8 per group). (D and E) Ex vivo TG synthesis and secretion in primary hepatocytes and HepG2 cells overexpressing TRIB1 (HepG2_trib1) or control vector (HepG2_control). Hepatocytes of AAV_trib1– and AAV_null–treated mice and HepG2_trib1 and HepG2_control cells (experiments were performed in triplicate) were radiolabeled with [³H]1,2,3-glycerol. (D) [³H]TG was determined in media and cellular lysates after 30, 60, and 120 minutes in primary hepatocytes. (E) HepG2 cells were labeled in the presence of either BSA or BSA plus oleic acid (OA, 0.4 mM) for 4 hours before measurement of [³H]TG. (*P < 0.05, **P < 0.01).