Increased apolipoprotein C3 drives cardiovascular risk in type 1 diabetes

Abstract

Type 1 diabetes mellitus (T1DM) increases the risk of atherosclerotic cardiovascular disease (CVD) in humans by poorly understood mechanisms. Using mouse models of T1DM-accelerated atherosclerosis, we found that relative insulin deficiency rather than hyperglycemia elevated levels of apolipoprotein C3 (APOC3), an apolipoprotein that prevents clearance of triglyceride-rich lipoproteins (TRLs) and their remnants. We then showed that serum APOC3 levels predict incident CVD events in subjects with T1DM in the Coronary Artery Calcification in Type 1 Diabetes (CACTI) study. To explore underlying mechanisms, we investigated the impact of Apoc3 antisense oligonucleotides (ASOs) on lipoprotein metabolism and atherosclerosis in a mouse model of T1DM. Apoc3 ASO treatment abolished the increased hepatic Apoc3 expression in diabetic mice - resulting in lower levels of TRLs - without improving glycemic control. APOC3 suppression also prevented arterial accumulation of APOC3-containing lipoprotein particles, macrophage foam cell formation, and the accelerated atherosclerosis in diabetic mice. Our observations demonstrate that relative insulin deficiency increases APOC3 and that this results in elevated levels of TRLs and accelerated atherosclerosis in a mouse model of T1DM. Because serum levels of APOC3 predicted incident CVD events in the CACTI study, inhibiting APOC3 might reduce CVD risk in T1DM patients.

Keywords: Atherosclerosis; Metabolism.

Conflict of interest statement

Conflict of interest: MJG and RMC are employed by Ionis Pharmaceuticals and provided antisense oligonucleotides for the study. KEB and JEK have received research support from Novo Nordisk A/S. KB is a cofounder of and equity holder in Matchstick Technologies Inc., which has licensed the PIXUL technology from the University of Washington.

Figures

Figure 1. Plasma APOC3 is a strong predictor of CAD events in subjects with T1DM and is independent of traditional CAD risk factors.

HRs for CAD events per 1 SD increase in log fasting plasma TGs or log serum APOC3, as calculated by Cox proportional hazards models (47 subjects with events; 181 total subjects). Also shown are 95% CIs and P values. A total of 47 participants had a primary endpoint CAD event, defined as a first nonfatal myocardial infarction, coronary revascularization, or death from CAD. Model 1 is a model adjusted for age, sex, and diabetes duration. Model 2 is model 1 further adjusted for nonlipid risk factors: HbA1c, systolic and diastolic blood pressure, and current smoking status. Model 3 is model 1 further adjusted for lipid risk factors: LDL-C and HDL-C. Model 4 is model 1 further adjusted for log fasting TGs.

Figure 2. Diabetes increases APOC3 levels relative to plasma TG levels through a lack of sufficient insulin.

(A) Female Ldlr–/–GpTg mice were rendered diabetic using LCMV. Saline was used as a control in nondiabetic littermates. At the onset of diabetes, the mice were switched to a low-fat, semipurified diet and maintained on the diet for 4 weeks. Plasma TGs were compared with plasma APOC3 levels measured by ELISA using data from 3 separate cohorts of mice (n = 42–43). Ranges and averages of TG levels and blood glucose (mg/dL) in diabetic and nondiabetic mice are listed below the graph. (B–E) Diabetes was induced by STZ treatment in male Ldlr–/–GpTg mice. Following induction of diabetes, half of the diabetic cohort received the SGLT2 inhibitor dapagliflozin in their drinking water for 4 weeks. n = 6–8. (B) Blood glucose levels at the end of the study. (C) Plasma cholesterol levels. (D) Plasma TGs. (E) Plasma APOC3 levels. Diabetes was induced using LCMV in Ldlr–/–GpTg mice. (F–I) Following development of diabetes, half of the diabetic cohort was subjected to intense insulin therapy with the goal of normalizing blood glucose, whereas the other half was maintained on traditional insulin therapy. n = 5–6. (F) Blood glucose at the end of the study. (G) Plasma cholesterol. (H) Plasma TGs. (I) Plasma APOC3. ND, nondiabetic mice; D, diabetic mice. D + int. ins., diabetes plus intense insulin therapy. *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-tailed, unpaired Student’s t test (A) or 1-way ANOVA followed by Tukey’s multiple comparisons tests (B–I).

Figure 3. Reducing APOC3 expression with an ASO normalizes TRL levels in diabetic mice.

Female Ldlr–/–GpTg mice were rendered diabetic using LCMV. Saline was used as a control in nondiabetic mice. The mice were maintained for 12 weeks. At the onset of diabetes, the animals were switched to a low-fat, semipurified diet. The mice were treated twice weekly with 25 mg/kg (i.p. injections) Apoc3 ASO or cASO starting 2 days after the onset of diabetes. Doses were adjusted every 2 weeks on the basis of body weight. Animals were bled every 4 weeks for glucose and lipid measurements. (A) Blood glucose levels. (B) Plasma cholesterol levels. Note that time point 0 is before animals were initiated on the low-fat, semipurified diet but after they had developed diabetes. (C) Plasma TGs (A–C; n = 16–20). (D) Hepatic mRNA was isolated, and Apoc3 mRNA was measured by real-time PCR (n = 6–18). (E and F) At the end of the study, cholesterol and TG lipoprotein profiles were analyzed in a subset (n = 4) of mice. (G) Plasma levels of APOC3, measured by ELISA (n = 12–16) at the end of the study. (H) The APOC3 ELISA was validated by targeted MS (n = 5–6). *P < 0.05, and ***P < 0.001, unless otherwise indicated, by 1-way ANOVA followed by Tukey’s multiple comparisons test (D, G, and H) or 2-way ANOVA followed by Bonferroni’s multiple comparisons test (E and F). #P < 0.01 compared with D cASO and §P < 0.01 compared with ND cASO, by 2-way ANOVA followed by Bonferroni’s multiple comparisons test (B and C).

Figure 4. Diabetes results in increased plasma levels of APOE, which are normalized by Apoc3 ASO treatment.

Diabetes was induced in female Ldlr–/–GpTg mice using LCMV. Saline was used as a control in nondiabetic mice. The mice were maintained for 12 weeks and treated with cASO or Apoc3 ASO. At the end of the study, plasma levels of APOE (A), APOB100 (B), APOB48 plus APOB100 (C), APOA1 (D), APOA4 (E), and APOC2 (F) were measured by targeted MS. The results are expressed as AU. n = 4–5. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA followed by Tukey’s multiple comparisons tests.

Figure 5. Diabetes-accelerated atherosclerosis is prevented by Apoc3 ASO treatment.

Female Ldlr–/–GpTg mice were rendered diabetic using LCMV. Saline was used as a control in nondiabetic mice. The mice were maintained for 12 weeks. At the onset of diabetes, the mice were switched to a low-fat, semipurified diet. Animals were treated twice weekly with 25 mg/kg (i.p. injections) of Apoc3 ASO or cASO starting 2 days after the onset of diabetes. (A and D) En face aortic atherosclerosis (n = 15–19). (B and C) Examples of early lesions in the BCA. Inset in B shows Mac-2 staining at magnified 2-fold from the image above. The internal elastic lamina is indicated by arrows. (E) Quantification of the maximal lesion area in the BCA (n = 9–11). (F) Mac-2+ lesion area in BCA cross sections (n = 7–11). (G) Quantification of APOC3 immunoreactivity in the BCA (n = 3–8). (H) Quantification of APOE immunoreactivity in the BCA (n = 6–11). (I) Quantification of APOB immunoreactivity in the BCA (n = 7–11) *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA followed by Tukey’s multiple comparisons test. Scale bar: 0.5 cm (A) and 100 μm (B and C).

Figure 6. Diabetes increases APOC3 levels in interstitial fluid concomitant with macrophage cholesteryl ester accumulation, both of which are prevented by Apoc3 ASO.

Female Ldlr–/–GpTg mice were rendered diabetic using LCMV. Saline was used as a control in nondiabetic mice. The mice were maintained for 4 weeks. At the onset of diabetes, the animals were switched to a low-fat, semipurified diet. The mice were treated twice weekly with 25 mg/kg (i.p. injections) of Apoc3 ASO or cASO starting 2 days after the onset of diabetes. At the end of the study, resident macrophages were isolated by peritoneal lavage, and interstitial peritoneal fluid was collected. (A) Total cholesterol in macrophages. (B) Cholesteryl ester (CE) in macrophages. (C) Cholesterol levels in peritoneal fluid. (D) APOC3 levels in peritoneal fluid. n = 10–14 (A–D) (2 statistical outliers were removed from B: 1 in the ND Apoc3 ASO group and 1 in the D cASO group). *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA followed by Tukey’s multiple comparisons test.

Figure 7. Apoc3 ASO treatment reduces necrotic cores in preexisting lesions in diabetic mice.

Female Ldlr–/–GpTg mice were fed a high-fat diet containing 1.25% cholesterol for 12 weeks, switched to chow for 2 weeks, and then injected with LCMV to induce diabetes (D) or with saline (ND). Once diabetic, the mice were maintained for 4 weeks on a low-fat, semipurified diet. The mice were treated twice weekly with 25 mg/kg (i.p. injections) Apoc3 ASO or cASO starting 2 days after the onset of diabetes. (A) Blood glucose. (B) Plasma cholesterol. (C) Plasma TGs. (D) Plasma APOC3. (E) IHC images of aortic sinus lesions stained with Movat’s pentachrome, Mac-2, and APOC3. Arrow indicates the necrotic core. Scale bar: 100 μm. (F) Quantification of aortic sinus lesion size at +180 μm (largest lesion) after the appearance of all 3 aortic valve leaflets. There were no differences in lesion size at 0 or +90 μm. (G) Quantification of aortic lesion Mac-2 staining at +180 μm. (H and I) Quantification of APOC3 IHC staining at +180 μm. (J and K) Quantification of aortic lesion necrotic cores at +180 μm. Similar results were observed at +90 μm. n = 9–10. *P < 0.05, and ***P < 0.001, by 2-tailed, unpaired Student’s t test.

Figure 8. Schematic model.

Suboptimally controlled diabetes associated with relative insulin deficiency increases hepatic APOC3 production and, to a larger extent, plasma APOC3 levels. The increased levels of APOC3 prevent clearance of plasma TLRs and their remnant lipoproteins (RLPs). As a result, lipoproteins containing APOC3, APOE, and APOB accumulate in the artery wall and accelerate macrophage accumulation, foam cell formation, atherogenesis, and CAD risk.