CREBH normalizes dyslipidemia and halts atherosclerosis in diabetes by decreasing circulating remnant lipoproteins

Abstract

Loss-of-function mutations in the transcription factor CREB3L3 (CREBH) associate with severe hypertriglyceridemia in humans. CREBH is believed to lower plasma triglycerides by augmenting the activity of lipoprotein lipase (LPL). However, by using a mouse model of type 1 diabetes mellitus (T1DM), we found that greater liver expression of active CREBH normalized both elevated plasma triglycerides and cholesterol. Residual triglyceride-rich lipoprotein (TRL) remnants were enriched in apolipoprotein E (APOE) and impoverished in APOC3, an apolipoprotein composition indicative of increased hepatic clearance. The underlying mechanism was independent of LPL, as CREBH reduced both triglycerides and cholesterol in LPL-deficient mice. Instead, APOE was critical for CREBH's ability to lower circulating remnant lipoproteins because it failed to reduce TRL cholesterol in Apoe-/- mice. Importantly, individuals with CREB3L3 loss-of-function mutations exhibited increased levels of remnant lipoproteins that were deprived of APOE. Recent evidence suggests that impaired clearance of TRL remnants promotes cardiovascular disease in patients with T1DM. Consistently, we found that hepatic expression of CREBH prevented the progression of diabetes-accelerated atherosclerosis. Our results support the proposal that CREBH acts through an APOE-dependent pathway to increase hepatic clearance of remnant lipoproteins. They also implicate elevated levels of remnants in the pathogenesis of atherosclerosis in T1DM.

Keywords: Atherosclerosis; Diabetes; Endocrinology; Lipoproteins; Metabolism.

Figure 1. Liver-specific expression of active CREBH induces hepatic expression of genes involved in hepatic clearance of remnant lipoproteins.

(A) Male Ldlr^–/– Gp^Tg mice were fed a high-fat diet (HFD) for 16 weeks, followed by a regular chow to normalize lipids for 1 week. Empty control TGB-AAV-DJ/8 (cAAV, 5 × 10¹⁰ GC) or TBG-AAV-DJ/8 containing the active form of mouse CREBH (CREBH AAV, 5 × 10¹⁰ GC) was then injected i.v. After 1 week, the mice were rendered diabetic using lymphocytic choriomeningitis virus (LCMV). Saline was used as control in nondiabetic littermates. At the time of LCMV injection, the mice were switched to a low-fat, semipurified diet (LFD) and maintained for an additional 4 weeks after the onset of diabetes. At the end of the study, the liver was collected for measurements of gene expression by real-time PCR (B and D) and liver TG content (C). Plasma was used for measurements of FGF21 (E) and alanine transaminase (ALT) to confirm lack of liver toxicity (F) by ELISA. ND, nondiabetic mice; D, diabetic mice. Mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by 2-way ANOVA (overall effects shown above panels) followed by Tukey’s multiple comparisons test (B, D, and E: n = 17 ND cAAV, n = 16 ND CREBH AAV, n = 18 D cAAV, n = 17 D CREBH AAV; C and F: n = 13 ND cAAV, n = 14 ND CREBH AAV, n = 10 D cAAV, n = 12 D CREBH AAV).

Figure 2. Hepatic expression of active CREBH reduces plasma lipids and APOE.

Diabetic (D) and nondiabetic (ND) mice with hepatic expression of active CREBH were generated as described in the Figure 1 legend. (A) Blood glucose was measured 1 day before the mice were euthanized. Plasma collected at the end point was used for measurement of TG (B) and cholesterol (C) (n = 17 ND cAAV, n = 16 ND CREBH AAV, n = 18 D cAAV, n = 17 D CREBH AAV). (D) Plasma apolipoproteins were analyzed by targeted mass spectrometry and normalized to ¹⁵N-APOA1 (n = 8/group). Mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by 2-way ANOVA (overall effects shown above panels) followed by Tukey’s multiple comparisons test.

Figure 3. Hepatic CREBH activity results in APOE enrichment and APOC3 impoverishment of TRLs and remnants in diabetic mice.

Plasma samples were separated by FPLC (pooled plasma of 2–3 mice/n, n = 3/group). Cholesterol (A) and TG (B) were measured in each fraction, generating lipoprotein profiles. (C–K) TGs, phospholipids, APOB, APOC3, APOC2, APOA5, and APOE were measured in the VLDL peak fraction (#17) and the IDL/LDL peak fraction (#22). Apolipoproteins were measured by ELISA. The relative levels of APOE and APOC3 in VLDL and IDL/LDL peak fractions were normalized to APOB (G and K), providing an estimate of APOE and APOC3 molecules/lipoprotein particle. (L and M) VLDL was isolated by ultracentrifugation (UC) and APOC3 and APOE were analyzed by ELISA and normalized to APOB (n = 3). Mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by 2-way ANOVA followed by Tukey’s multiple comparisons test. D, diabetic mice; ND, nondiabetic mice.

Figure 4. CREBH promotes lipid clearance through an APOE-dependent mechanism.

After 3 weeks of diabetes, plasma was collected 5 minutes after heparin i.v. injection. (A) Heparin-releasable LPL activity was determined by subtracting pre-heparin plasma lipase activity in each mouse (n = 9 nondiabetic [ND] cAAV, n = 8 ND CREBH AAV, n = 10 diabetic [D] cAAV and D CREBH AAV). (B) LPL activity in brown adipose tissue (BAT) 4 weeks after diabetes induction (n = 14 ND cAAV; n = 13 ND CREBH AAV, D cAAV, and D CREBH AAV). (C) Plasma lipid levels at indicated times in mice with inducible LPL deficiency (iLpl^–/–) injected with CREBH AAV or cAAV (n = 7 cAAV, n = 9 CREBH AAV). (D) Diabetic and nondiabetic mice expressing CREBH were maintained for 4 weeks. Whole-liver lysate (n = 8 ND cAAV, n = 9 ND CREBH AAV, n = 7 D cAAV, n = 8 D CREBH AAV) and plasma membranes (n = 10 ND cAAV, ND CREBH AAV, and D CREBH AAV; n = 9 D cAAV) were used for LRP1 immunoblot. (E) Ldlr^–/– mice were injected with CREBH AAV or cAAV (5 × 10¹⁰ GC) and LCMV injected 1 week later. Mice were treated with a liver-targeted GalNAc LRP1 antisense oligonucleotide (ASO) or control ASO (cASO) once a week for 4 weeks from the onset of diabetes. (F) Plasma glucose was measured 3 weeks after diabetes induction. CRH, CREBH. (G and H) Plasma TG and cholesterol were measured at week 2 after induction of diabetes (n values shown in E). (I) Plasma lipids were measured at the indicated times after cAAV or CREBH AAV injection in male Apoe^–/– mice (n = 6). Plasma lipid profiles at week 3 (pooled plasma of 2 mice/n, n = 3/group). Mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by 2-way ANOVA (overall effects shown above panels) followed by Tukey’s multiple comparisons test or 2-tailed unpaired t test. ^#P < 0.05; ^##P < 0.01; ^###P < 0.001; ^####P < 0.0001 denote significance versus the corresponding nondiabetic group.

Figure 5. Hepatic expression of CREBH prevents the effects of diabetes on lesion necrotic core expansion and APOE accumulation.

Diabetic (D) and nondiabetic (ND) mice with hepatic expression of active CREBH were generated as described in the Figure 1 legend. Atherosclerotic lesions in the aortic sinus were analyzed for cross-sectional lesion area (A) and necrotic core area as a percentage of lesion area (B) in sections stained using a Movat’s pentachrome stain (C). In C, necrotic cores are marked by dashed lines. Immunohistochemistry was used to evaluate APOE-positive (D and F) and APOC3-positive (E and G) lesion areas. Rabbit IgG and lesions from Apoe^–/– mice were used as negative controls for APOE immunohistochemistry. Rabbit IgG and lesions from mice treated with an APOC3 ASO21 were used as negative controls for APOC3 immunohistochemistry. (H) Cell death and efferocytosis were measured by TUNEL assay. Mean ± SEM. *P < 0.05; **P < 0.01 by 1-way ANOVA followed by Tukey’s multiple comparisons test (n = 14 baseline, n = 17 ND cAAV, n = 16 ND CREBH AAV, n = 18 D cAAV, n = 17 D CREBH AAV). Scale bars: 100 μm.

Figure 6. Human subjects with CREB3L3 loss-of-function or missense mutations exhibit increased concentrations of small VLDL and IDL and reduced APOE in LDL/IDL.

Age-matched human subjects with rare loss-of-function or missense mutations in CREB3L3 and controls with normotriglyceridemia (NTG) were identified. (A) Plasma total cholesterol. (B) Plasma TG levels (logTG). (C) HDL-cholesterol levels. (D–E) VLDL and IDL were separated by density ultracentrifugation (d > 1.019 g/mL) after removal of chylomicrons. Total VLDL and IDL particle concentrations and sizes were measured by calibrated ion mobility analysis. (F–L) Apolipoproteins were quantified in the VLDL+IDL fraction (d < 1.019 g/mL) by targeted mass spectrometry. Data are shown as box-and-whisker plots, with boxes showing 25th to 75th percentile, horizontal lines showing medians, and whiskers showing minimum to maximum. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by unpaired, 2-tailed Mann-Whitney test (n = 8 for NTG and n = 10 for subjects with CREB3L3 loss-of-function or missense mutations). (M) Schematic representation of the effects of diabetes and active CREBH on lipoproteins, apolipoproteins, and atherosclerosis. Poorly controlled T1DM results in increased hepatic production of APOC3, which leads to increased APOC3 loading of VLDL and remnants. The increased APOC3 load on VLDL mediates a reduced ability of LPL to hydrolyze VLDL and IDL. Diabetes also suppresses plasma membrane translocation of LRP1, further slowing clearance of remnants and leading to an increased accumulation of remnants in the artery wall, promoting lesion progression. Hepatic CREBH increases hepatic clearance of VLDL and IDL by enhancing the APOE loading of TRL remnants and depleting these particles of APOC3, thereby preventing the effects of diabetes on remnant accumulation and lesion progression (generated with BioRender.com).