DNA Methylation Near CPT1A and Changes in Triglyceride-rich Lipoproteins in Response to Weight-loss Diet Interventions

Abstract

Context

Carnitine palmitoyltransferase-1A, encoded by the CPT1A gene, plays a key role in the oxidation of long-chain fatty acids in the mitochondria and may be important in triglyceride metabolism. Previous work has shown that high fat intake was negatively associated with CPT1A methylation and positively associated with CPT1A expression.

Objective

We aim to investigate the association of DNA methylation (DNAm) at the CPT1A gene with reductions in triglycerides and triglyceride-rich lipoproteins (TRLs) in response to weight-loss diet interventions.

Methods

The current study included 538 White participants, who were randomly assigned to 1 of 4 diets varying in macronutrient components. We defined the regional DNAm at CPT1A as the average methylation level over CpGs within 500 bp of the 3 triglyceride-related DNAm sites.

Results

Dietary fat intake significantly modified the association between baseline DNAm at CPT1A and 2-year changes in total plasma triglycerides, independent of concurrent weight loss. Among participants assigned to a low-fat diet, a higher regional DNAm level at CPT1A was associated with a greater reduction in total plasma triglycerides at 2 years (P = .01), compared with those assigned to a high-fat diet (P = .64) (P interaction = .018). Further investigation on lipids and apolipoproteins in very low-density lipoprotein (VLDL) revealed similar interaction patterns for 2-year changes in VLDL-triglycerides, VLDL-cholesterol, and VLDL-apolipoprotein B (P interaction = .009, .002, and .016, respectively), but not for VLDL-apoC-III (P interaction = .36).

Conclusion

Participants with a higher regional DNAm level at CPT1A benefit more in long-term improvement in triglycerides, particularly in the TRLs and related apolipoproteins when consuming a low-fat weight-loss diet.

Obesity is featured by high circulating triglycerides and triglyceride-rich lipoproteins (TRLs), such as very low-density lipoprotein (VLDL), which contribute to elevated risks of cardiometabolic abnormalities (1). With the persistent rise in the prevalence of obesity, there is growing interest in the role of TRLs in the prevention and treatment of obesity-related cardiometabolic disorders (2, 3). Plasma triglycerides is a marker of the abundance of TRLs (3). Multiple recent epigenome-wide association studies (EWAS) consistently reported that high levels of methylation of several 5′-cytosine-phosphate-guanine-3′ (CpG) sites near the CPT1A gene were associated with lower blood levels of triglycerides (4-11). The CPT1A gene encodes carnitine palmitoyltransferase 1 A, which is a key enzyme in the oxidation of the long-chain fatty acids in the mitochondria and may play an important role in triglycerides metabolism (12, 13).

Various weight-loss diets lower plasma triglycerides and TRLs (14-16). DNA methylation (DNAm) serves as an important bridge linking environmental factors, including diets, with gene expression (17, 18). Previous evidence has particularly linked dietary fat with DNAm at CPT1A (19, 20), showing that high fat intake was negatively associated with CPT1A methylation and positively associated with CPT1A expression. Therefore, we hypothesized that weight-loss diets varying in fat might modify the relations between DNAm and changes in triglycerides and TRLs. Moreover, VLDL, the main carrier of plasma triglycerides, is a complex particle, containing triglycerides, cholesterol, as well as attachments of apolipoproteins, such as apolipoprotein C-III (apoC-III) and apolipoprotein B (apoB), which determine functions of the lipoprotein particles such as lipolysis, binding to liver receptors, and clearance from the circulation (21-23).

In the present study, we investigated the relationship of DNAm levels at the CPT1A gene region with long-term changes in total plasma triglycerides, lipids, and apolipoproteins in the VLDL fraction, including VLDL-cholesterol (VLDL-c), VLDL-triglycerides, VLDL-apoB, VLDL-apoC-III, VLDL-apoE, as well as subspecies with or without apoC-III in the Preventing Overweight Using Novel Dietary Strategies (POUNDS Lost) trial (15). We particularly examined the interaction between the regional DNAm level at CPT1A and dietary fat on long-term changes in the above outcomes.

Methods

Study Population

The POUNDS Lost trial (clinical trial reg. no. NCT00072995, clinicaltrials.gov) is a 2-year randomized clinical trial, designed to compare the effects of 4 energy-reduced diets with varying macronutrient compositions (carbohydrate, fat, and protein) on weight change (15). The study was conducted at 2 sites: (1) the Harvard T.H. Chan School of Public Health and Brigham and Women's Hospital (BWH), Boston, MA; and (2) the Pennington Biomedical Research Center (PBRC) of the Louisiana State University System, Baton Rouge, LA, from 2004 to 2007. Detailed information on study design and methods has been described previously (15). In brief, a total of 811 participants who were overweight or obese were randomized to one of 4 weight-loss diets. The targeted percentages of energy derived from fat, protein, and carbohydrate in the 4 diets were: (1) 20% fat, 15% protein, and 65% carbohydrate; (2) 20% fat, 25% protein, and 55% carbohydrate; (3) 40% fat, 15% protein, and 45% carbohydrate; and 4) 40% fat, 25% protein, and 35% carbohydrate. Two diets were low-fat diets (20%) and 2 diets were high-fat (40%), and 2 diets were average-protein (15%) and 2 were high-protein (25%), which constituted a 2-by-2 factorial design. To assess the adherence to the assigned diet groups, dietary intake and 2 biomarkers of adherence (urinary nitrogen and respiratory quotient) were assessed in a random sample of 50% of the entire participants. The 4 diets consisted of similar foods and were in accordance with the cardiovascular health guidelines. All the participants provided written informed consent. Of these participants, 50% were randomly selected for lipoprotein subspecies measurement at baseline and 2 years. The study was approved by the human subjects committee at the Harvard School of Public Health and Brigham and Women's Hospital, Boston, MA; the Pennington Biomedical Research Center of the Louisiana State University, Baton Rouge, LA; and a data and safety monitoring board appointed by the National Heart, Lung, and Blood Institute.

In the current analysis, we included 538 White participants based on the availability of blood samples and measurement of DNAm at CPT1A. We only included White participants because the DNAm at CPT1A was primarily found among European ancestries. Of the included 538 participants, 472 were followed up at 6 months, and 408 were followed up at 2 years. The follow-up rate is comparable to that of the entire POUNDS Lost trial.

Measurement of DNA Methylation

Fasting blood samples were collected and stored at −80°C. Baseline DNAm in peripheral blood was sequenced on the IlluminaNovaSeq6000 platform by a high-resolution methyl-capture sequencing (MCC-Seq) approach at Children's Mercy Research Institute (CMRI) (24). The sequenced reads were trimmed for Illumina adapters and quality (phred33 ≥ 20) using trimgalore v.0.4.2, a wrapper tool combining Cutadapt (25) and FastQC. The trimmed reads were then aligned to the bisulfite-converted human reference genome build 37 (GRCh37) using Illumina's DRAGEN Bio-IT Platform v3.6 in paired end mode and the highest quality unique alignment was retained. Duplicates were flagged using Picard Tool's MarkDuplicates v2.17.8. Methylation calls were extracted by Bismark's bismark_methylation_extractor v0.20.0. The resulting CpG sites were further removed using BEDTool's subtract v2.29.2 if: (1) CpGs with total coverage <10 reads, (2) CpGs overlapping an SNP from dbSNP build 151, or (3) CpGs overlapping regions in the ENCODE Blacklist (https://hgwdev.gi.ucsc.edu/cgi-bin/hgFileUi?db=hg19&g=wgEncodeMapability). Previous evidence has shown that region-based DNAm is more strongly associated with phenotype than individual CpG sites (26). In addition, because DNAm is correlated, a region-based approach is more suitable for the next-generation sequencing–based method, such as the MCC-Seq in our study (27). Therefore, we fetched the regional DNAm (chr11: 68,607,372-68,607,985) within the ±250-bp window of the 3 CpG sites (cg00574958, cg09737197, and cg17058475) that are closely located within CPT1A and confirmed in previous studies. The regional DNAm level was calculated as the percentage of the total number of pooled methylation reads of the total number of pooled sequencing reads covering CpGs over the region.

Measurements of Lipids and Lipoprotein Subspecies

Fasting concentrations of common lipid profiles, including total triglycerides, total cholesterol, low-density lipoprotein cholesterol (LDL-c), and high-density lipoprotein cholesterol (HDL-c), at baseline, 6 months, and 2 years were measured on the Synchron CX7 (Beckman Coulter, Brea CA). Specialized lipid measurements were measured in a random sample (N = 283) of the 538 (53%) participants at baseline and 2 years at the Lipid Core Laboratory, Harvard T.H. Chan School of Public Health. The methods for immunoaffinity chromatography, ultracentrifugation, and determination of lipids and apolipoprotein have been described previously (28, 29). Briefly, plasma was first incubated in anti-apoC-III immuno-affinity columns to separate lipoproteins that contain and lack apoC-III. The bound and unbound fractions were then ultracentrifuged to further separate particles by density. In the current study, we focused on VLDL with or without apoC-III. Sandwich enzyme-linked immunoabsorbent assays (ELISAs) were performed to measure the concentrations of apoB, apoC-III, and apoE (Antibodies-Online Cat# ABIN236810, RRID:AB_10781309, Academy Biomedical, Houston, TX) in VLDL. Concentrations of cholesterol and triglycerides in VLDL were determined by enzymatic assays (Thermo Electron Corp, Waltham, MA).

Statistical Analysis

The primary outcome was 2-year changes in total plasma triglycerides. Secondary outcomes were 2-year changes in lipids and apolipoproteins in the VLDL fraction, including VLDL-triglycerides, VLDL-c, VLDL-apoB, VLDL-apoC-III, and VLDL-apoE. The level of total triglycerides was log-transformed to improve normality. Characteristics across the tertiles of the regional DNAm were compared by F test with adjustment for age and sex for continuous variables and chi-square test for categorical variables. We chose tertiles for data presentation, considering the relatively small sample size in subgroups, consistent with previous studies (27). Generalized linear regression models were used to examine the prospective association between baseline regional DNAm at CPT1A and 2-year changes in lipid traits, with adjustment for age, sex, baseline body mass index (BMI), use of lipid-lowering medication, and baseline values of respective outcomes in Model 1. We further controlled for concurrent weight loss in Model 2. To test the potential interaction between the regional DNAm at CPT1A and the weight-loss diet, a DNAm-diet product term was included in the above model. Regional DNAm was analyzed as a continuous variable. All statistical analyses were performed by SAS version 9.4 (SAS Institute). All P values were 2-sided, and a P value <.05 was considered statistically significant.

Results

The baseline characteristics of the study participants according to the tertile categories of the blood regional DNAm at CPT1A are shown in Table 1. No significant differences were observed for age, sex, BMI, glucose, lipid traits, and use of lipid-lowering medications. Participants with a higher level of regional DNAm at CPT1A tended to have a lower level of total plasma triglycerides, although not statistically significant. Supplementary Table S1 describes the baseline characteristics of those who have completed the 2-year follow-up according to the tertiles of the regional DNAm at CPT1A (30). Baseline characteristics according to the dietary interventions are described in Supplementary Table S2 (30). All the measured variables of the included study participants were balanced across the 4 dietary interventions, except for the use of lipid-lowering medications. Nutrient intake and biomarkers of adherence at 6 months and 2 years are presented in Supplementary Table S3 (30). In line with the entire participants in POUNDS Lost, the study participants in the current analysis showed changes in the macronutrient intake in the direction of the intervention, although the targets were not fully achieved (15). There was no significant difference in nutrient intake and adherence across the tertiles at 6 months and 2 years (P > .05 for all). Among the included participants, 300 of them achieved successful weight loss (weight loss ≥5%, 168 were defined as weight stability (weight change less than ±5%), and 4 had weight gain (weight increased ≥5%) at 6 months. At 2 years, 189 participants had successful weight loss, 200 were weight stability, and 19 had weight gain.

Table 1.

Baseline characteristics of study population according to the tertiles of the blood regional DNAm level at CPT1A (n = 538)

	Tertiles of regional DNAm
	T1 (N = 179)	T2 (N = 180)	T3 (N = 179)	P
Age, years	52.2 (8.8)	52.9 (8.9)	50.8 (9.2)	.07
Female	94 (52.5)	104 (57.8)	107 (59.8)	.36
BMI, kg/m2	32.7 (4.0)	32.6 (3.9)	32.3 (3.8)	.74
Weight, kg	94.4 (15.2)	93.6 (15.5)	92.7 (17.1)	.77
Waist circumference, cm	105.5 (12.6)	104.3 (13.4)	103.1 (14.2)	.64
LDL-c, mg/dL	125.0 (31.6)	126.1 (33.2)	126.5 (30.9)	.96
HDL-c, mg/dL	48.1 (13.9)	48.4 (15.9)	49.8 (14.1)	.46
Total cholesterol, mg/dL	203.9 (36.3)	203.9 (39.7)	204.3 (35.3)	.97
Total triglycerides, mg/dL	138.0 [100.0, 204.0]	126.0 [85.0, 185.5]	120.0 [92.0, 190.0]	.08
Lipid-lowering medication	41 (22.9)	37 (20.6)	30 (16.8)	.34
Glucose, mg/dL	93.0 (11.6)	92.0 (10.4)	92.5 (13.5)	.57
Insulin, μU/mL	11.2 [7.2, 15.4]	10.5 [7.0, 15.0]	9.4 [6.9, 13.6]	.14
HbA1c, %	5.3% (0.3)	5.4% (0.4)	5.3% (0.4)	.62
HOMA-IR	2.5 [1.6, 3.5]	2.3 [1.5, 3.7]	2.1 [1.5, 3.2]	.17
Lipids and apolipoproteins in VLDL
VLDL-triglycerides	81.2 (49.1)	76.2 (50.8)	70.4 (49.3)	.36
VLDL-c	15.6 (9.2)	16.0 (12.0)	14.9 (9.9)	.78
VLDL-apoB	5.9 (4.1)	5.5 (4.2)	6.0 (4.8)	.73
VLDL-apoC-III	0.8 (0.7)	0.7 (0.6)	0.8 (0.7)	.56
VLDL-apoE	0.5 (0.4)	0.5 (0.4)	0.5 (0.4)	.99
Diet group				.14
High-fat group	104 (58.1)	89 (49.4)	87 (48.6)
Low-fat group	75 (41.9)	91 (50.6)	92 (51.4)
Dietary intake per day
Energy, kcal	1948.0 (535.6)	1969.9 (548.3)	1998.1 (518.7)	.80
Fat, %	37.3% (6.7)	37.1% (6.0)	36.7% (5.4)	.76
Protein, %	18.1% (3.3)	18.5% (3.8)	17.9% (2.9)	.47
Carbohydrate, %	44.0% (7.6)	44.1% (8.2)	44.7% (7.1)	.75
Biomarkers of adherence
Respiratory quotient	0.8 (0.04)	0.8 (0.04)	0.8 (0.04)	.54
Urinary nitrogen, g	12.6 (4.1)	12.4 (5.0)	12.7 (4.7)	.68

Data are mean (SD), median [IQR], or N (%).

P values were calculated by χ² test for categorical variables and an F test after adjusting for age and sex for continuous variables. Lipids and lipoprotein in VLDL fraction: T1 (N = 94); T2 (N = 102), T3 (N = 87).

Abbreviations: BMI, body mass index; LDL-c, low-density lipoprotein cholesterol; HDL-c, high-density lipoprotein cholesterol; HOMA-IR:,homeostatic model assessment of insulin resistance.

The baseline association of regional DNAm at CPT1A and total plasma triglycerides was consistent with previous studies (4-10), confirming the validity of using the DNAm at CPT1A as an epigenetic marker for triglycerides in the POUNDS Lost population (Supplementary Table S4) (30). Higher regional DNAm at CPT1A was associated with a lower level of log-transformed total triglycerides (β [SE] per SD: −.05 [.02], P = .03).

We found significant interactions between the regional DNAm at CPT1A and dietary fat intake on 2-year changes in total plasma triglycerides (Fig. 1 & Supplementary Table S5) (30). In the low-fat diet group, a higher level of regional DNAm at CPT1A was associated with significantly greater reductions in total triglycerides (P = .002, Supplementary Table S5, model 1), after adjustment for age, sex, use of lipid-lowering medication, BMI, and baseline value of triglycerides; while no association was found in the high-fat diet group (P = .92, Supplementary Table S5, model 1) (30). Further adjustment for concurrent weight loss did not appreciably change the association (P interaction = .018, Fig. 1 and Supplementary Table S5, model 2) (30). Such interaction patterns did not depend on the choice of tertiles, quartiles, or quantiles. Similar results were also observed if reclassifying the DNAm at CPT1A into quartiles or quantiles.

Figure 1.

Changes in triglycerides from baseline to 2 years according to tertiles of blood regional DNA methylation level at CPT1A in low- and high-fat diet group. Models were adjusted for age, sex, use of lipid-lowering medication, baseline body mass index, baseline values of respective outcomes, and concurrent weight loss. Triglycerides were log-transformed before analysis. Sample size in low-fat group: T1, n = 55; T2, n = 74; T3, n = 69; high-fat group: T1, n = 76; T2, n = 70; T3, n = 64.

Considering that VLDL is a complex TRL particle containing triglycerides, cholesterol, and apolipoproteins, we then investigated the association between DNAm at CPT1A and 2-year changes in lipids and apolipoproteins within the VLDL fraction, including VLDL-triglycerides, VLDL-c, VLDL-apoB, VLDL-apoC-III, and VLDL-apoE (Table 2). We observed significant interactions between the regional DNAm at CPT1A and dietary fat intake on 2-year changes in VLDL-triglycerides, VLDL-c, and VLDL-apoB, but not for VLDL-apoC-III, after adjusting for age, sex, baseline BMI, use of lipid-lowering medication, and baseline values of respective outcomes (P interaction = .018, .003, .017, and .37 respectively, Table 2, model 1). In the low-fat diet group, participants with a higher regional DNAm at CPT1A exhibited greater reductions in VLDL-triglycerides (P = .016), VLDL-c (P = .011), and VLDL-apoB (P = .032), while no significant associations were found in the high-fat diet group (P > .05 for all). Such associations did not change materially after further adjustment for concurrent weight loss, indicating the independence of concurrent weight loss (Table 2, model 2). The interaction between regional DNAm at CPT1A and apoE became borderline significant (P interaction = .045), after additional adjustment for concurrent weight loss, although subgroup analyses were not significant.

Table 2.

Association of regional DNAm at CPT1A and changes in lipids and apolipoprotein in VLDL fraction in response to low- or high-fat diet at 2 years of diet intervention

	Low-fat group		High-fat group		P interaction
	β (SE)	P	β (SE)	P
Model 1
VLDL-triglycerides	−7.68 (3.16)	.016	2.03 (2.21)	.36	.018
VLDL-c	−1.82 (0.70)	.011	0.62 (0.46)	.18	.003
VLDL-apoB	−0.66 (0.30)	.028	0.23 (0.20)	.26	.017
VLDL-apoC-III	−0.06 (0.07)	.40	0.02 (0.05)	.76	.37
VLDL-apoE	−0.06 (0.03)	.032	0.01 (0.02)	.75	.06
Model 2
VLDL-triglycerides	−6.37 (2.95)	.033	3.20 (1.98)	.11	.009
VLDL-c	−1.55 (0.67)	.022	0.80 (0.44)	.07	.002
VLDL-apoB	−0.55 (0.29)	.06	0.26 (0.20)	.19	.016
VLDL-apoC-III	−0.04 (0.07)	.55	0.03 (0.05)	.51	.36
VLDL-apoE	−0.05 (0.03)	.063	0.01 (0.02)	.47	.045

β (SE) represents the changes in each outcome per 1 SD higher methylation percentage at CPT1A.

Model 1 was adjusted for age, sex, baseline BMI, use of lipid-lowering medications, and baseline values for the respective outcome; Model 2: Model 1 + concurrent weight loss.

Abbreviations: apo, apoprotein; VLDL, very low-density lipoprotein; VLDL-c, VLDL-cholesterol.

VLDL is also heterogeneous in apoC-III containment, which is an independent risk factor for cardiovascular disease (31). Therefore, we further examined the associations of the regional DNAm at CPT1A with 2-year changes in lipids and apolipoprotein subspecies defined by the presence or absence of apoC-III in the VLDL fraction in response to a high-/low-fat diet (Fig. 2). We found that dietary fat intake significantly modified the association between regional DNAm at CPT1A and 2-year changes in triglycerides in VLDL without apoC-III, cholesterol in VLDL without apoC-III, apoB in VLDL without apoC-III, and apoE in VLDL with apoC-III (P interaction = .01, .002, .002, and .035, respectively). In response to the low-fat diet, participants with a higher regional DNAm at CPT1A was associated with significantly greater reductions in triglycerides in VLDL without apoC-III (P = .028), cholesterol in VLDL without apoC-III (P = .023), apoE in VLDL with apoC-III (P = .025), and borderline decrease in apoB in VLDL without apoC-III (P = .07), while no association was found in the high-fat diet group (P > .05).

Figure 2.

Changes in lipids and apolipoproteins subspecies in the VLDL fraction from baseline to 2 years according to tertiles of blood regional DNA methylation level at CPT1A in low- and high-fat diet group. Models were adjusted for age, sex, use of lipid-lowering medication, baseline body mass index, baseline values of the respective outcomes, and concurrent weight loss. Panel A: Triglycerides in VLDL with apoC-III; B: Cholesterol in VLDL with apoC-III; C: apoB in VLDL with apoC-III; D: apoE in VLDL with apoC-III; E: Triglycerides in VLDL without apoC-III; F: Cholesterol in VLDL without apoC-III; G: apoB in VLDL without apoC-III; H: apoE in VLDL without apoC-III. Sample size for lipids and apolipoproteins subspecies in low-fat group: T1, n = 40; T2, n = 48; T3, n = 44 (except for TG in VLDL with apoC-III, n = 43); high-fat group: T1, n = 53; T2, n = 54 (except for apoB in VLDL with apoC-III, n = 53); T3, n = 44.

Discussion

In this 2-year weight-loss diet intervention trial, we found that higher regional DNAm at CPT1A was associated with lower levels of total plasma triglycerides at baseline. Moreover, we found that dietary fat intake significantly modified the association between regional DNAm at CPT1A and 2-year changes in total plasma triglycerides. In response to the low-fat diet, a higher regional level of DNAm at CPT1A was associated with greater reductions in total plasma triglycerides. Further investigation of the lipids and apolipoproteins in TRL revealed similar interaction patterns for the 2-year changes in VLDL-triglycerides, VLDL-c, VLDL-apoB, and VLDL-apoE.

Our observation of the associations between regional DNAm at CPT1A and total triglycerides at baseline was consistent with previous findings in genome-wide association studies (GWAS) (4, 5, 7, 8), confirming the validity of analyzing the regional DNAm at CPT1A in our study population. For example, previous studies have reported that greater DNAm at CPT1A was associated with lower triglycerides in the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN) study and the Framingham Heart Study (FHS) (4, 5). Gagnon et al also successfully validated 2 CpG sites annotated to CPT1A with triglycerides in 2 independent studies (6).

For the first time, we found that the pretreatment DNAm at CPT1A was differentially associated with long-term changes in total plasma triglycerides and TRLs in response to weight-loss diets varying in fat intakes. Such association was particularly driven by the triglycerides in VLDL. Of note, our findings of the relationship between DNAm at CPT1A and plasma triglycerids and TRLs are independent of weight loss. Weight loss has long been associated with improvement in lipid profiles.(32) Our study suggests pretreatment DNAm of CPT1A may predict the lipid outcome independent of weight loss. Our previous studies in POUNDS Lost and other weight loss trial also reported that lipids changes could be independent of weight loss (33, 34). These observations suggest that other biological changes during weight loss may be also involved in determining the changes of lipids. In addition to VLDL-triglycerides, we also found that higher DNAm at CPT1A was associated with decreases in VLDL-c and VLDL-apoB. Our findings provide novel insight into the role of DNAm at CPT1A in TRL metabolism. ApoB is the primary apolipoprotein in VLDL particles, responsible for transporting triglycerides and cholesterol esters in the circulation (35). Growing evidence has shown that apoB is more strongly associated with cardiovascular risk than LDL-c or non-HDL-c (36-39). In addition, since each VLDL particle contains one molecule of apoB, VLDL-apoB indicates the concentration of the VLDL particle in the plasma (38). Therefore, our findings on DNAm at CPT1A and reduction of VLDL-apoB among individuals with a low-fat weight-loss diet provide novel evidence for the tailored precision interventions according to DNAm. Because DNAm is modifiable and reversible, potential novel dietary interventions targeting on the DNAm at CPT1A could be employed in the prevention and treatment of dyslipidemia, particularly abnormal TRLs. Beyond that, we also found that the higher DNAm at CPT1A was particularly associated with greater reductions in the lipids and apolipoprotein subspecies in terms of with and without apoC-III in response to dietary fat intake. ApoC-III is mainly present on TRLs (3), particularly on most VLDL that contain apoE (40). The observation of the apoE in VLDL with apoC-III might have potentially important implications, as apoC-III impairs the apoE-mediated clearance of VLDL (40).

The observed interaction between dietary fat intake and DNAm at CPT1A is biologically possible. Previous gene expression analysis has shown that lower DNAm at CPT1A was associated with increased gene expression of CPT1A, which is subsequently linked to a higher level of triglycerides (4, 41). Of note, evidence from a longitudinal study in rats suggested that a high-fat diet could increase CPT1A expression (42). In humans, a lower level of DNAm at CPT1A was also associated with higher triglycerides after a high-fat meal challenge (19). Taken together, it is plausible that the favorable effects of higher DNAm at CPT1A on triglycerides may be offset when high fat presents, accounting for the null association of DNAm with lipids in the high-fat diet group in our study.

Our study has several strengths. First, to our knowledge, this is the first study to examine the association between regional DNAm at CPT1A and long-term changes in lipids and lipoproteins in one of the largest and longest weight-loss diet intervention trials. Second, we measured the regional DNAm using a high-resolution MCC-Seq approach, which comprehensively captured the DNAm profiles in the target region, rather than probe-based methods (24). Third, we repeatedly measured the specialized lipids and apolipoproteins before and after the diet invention, which allowed us to take a further investigation into the subspecies of lipoproteins. However, several limitations should also be acknowledged. First, we only measured the pretreatment DNAm level at baseline, thus we could not examine whether the DNAm profiles changed with diet interventions and how the changes in DNAm at CPT1A would be associated with changes in lipids. Second, our analyses were confined to the White population only, because the DNAm at CPT1A was primarily found in populations with European ancestry, and the POUNDS Lost participants are 80% White. The relatively small sample size for populations other than the White population in POUNDS Lost limits our ability to test the generalizability to other populations. Further investigations are warranted. Lastly, we only measured the DNAm in peripheral blood, not in liver tissue. However, DNAm at CPT1A has been consistently reported in both blood samples and across tissues, including liver samples, suggesting that blood-based DNAm reflects functional variations in DNAm at the tissue level (9, 43, 44). Indeed, assessment of DNAm in blood samples has been widely accepted as a surrogate for methylation in closely-related tissues in clinical and population studies (45).

In conclusion, in this 2-year diet intervention trial, we found that individuals who were overweight or obese and with a higher level of regional DNAm at CPT1A are associated with long-term favorable changes in triglycerides and TRLs profile by consuming a low-fat weight-loss diet. Given that DNAm modifications are potentially reversible, our findings provide important information for the development of novel, precision dietary intervention approaches targeting on DNAm at CPT1A in the management of dyslipidemia, especially abnormal TRLs, in people with obesity.

Abbreviations

apoB

apoprotein B

apoC-III

apolipoprotein C-III

BMI

body mass index

BWH

Brigham and Women’s Hospital/Harvard School of Public Health

CpG

5′-cytosine-phosphate-guanine-3′

CPT1A

carnitine palmitoyltransferase-1A

DNAm

DNA methylation

HDL-c

high-density lipoprotein cholesterol

LDL-c

low-density lipoprotein cholesterol

PBRC

Pennington Biomedical Research Center Louisiana State University

POUNDS Lost

Preventing Overweight Using Novel Dietary Strategies study

TRL

triglyceride-rich lipoprotein

VLDL

very low-density lipoprotein

VLDL-c

VLDL-cholesterol

Funding

The study was supported by grants from the National Heart, Lung, and Blood Institute (HL071981, HL034594, HL126024), the National Institute of Diabetes and Digestive and Kidney Diseases (DK115679, DK091718, DK100383), the Fogarty International Center (TW010790), and Tulane Research Centers of Excellence Awards. Xiang Li was the recipient of the American Heart Association Predoctoral Fellowship Award (19PRE34380036).

Author Contributions

X.L. contributed to the study concept and design, analysis, and interpretation of the data, and drafting and revising the manuscript. X.S. contributed to the statistical analysis with critical input. X.S., Q.X., M.K., C.M.C., L.A.B., B.S.K., Y.H., and E.G. contributed to the interpretation of data and critical revision of the manuscript for important intellectual content. G.A.B. and F.M.S. contributed to the interpretation of data, critical revision of the manuscript for important intellectual content, and supervision. L.Q. contributed to the study concept and design, acquisition of the data, analysis, and interpretation of the data, and funding and study supervision. L.Q. is the guarantor and takes responsibility for the integrity of the data and the accuracy of the data analyses.

Disclosures

All authors declared no conflict of interest. The sponsoring institutes have no role in the study design or in the collection, analysis, and interpretation of the data.

Data Availability Statement

Restrictions apply to the availability of data generated or analyzed during this study to preserve patient confidentiality or because they were used under license. The corresponding author will on request detail the restrictions and any conditions under which access to some data may be provided.