Dietary intake of n-3 and n-6 polyunsaturated fatty acids (PUFAs) may have beneficial effects on cardiovascular health, although some controversy exists in this regard. For example, observational and intervention studies have shown that long chain n-3 PUFAs tend to reduce serum triglycerides (TG), but may increase low density lipoprotein (LDL) cholesterol (C) (1, 2). Further, cross-sectional analysis and controlled trials show diets rich in n-6 PUFAs lower LDL-C, but may also lower high density lipoprotein (HDL)-C (3, 4). Increasing PUFA dietary intake has been associated with positive health outcomes in healthy individuals without diabetes (1, 2, 4). Current recommendations for individuals at high risk for cardiovascular disease (CVD), including those with diabetes, support increasing food sources of n-3 PUFAs including α-linolenic acid (ALA, 18:3n-3), eicosapentanoic acid (EPA, 20:5n-3), and docosahexanoic acid (DHA, 22:6 n-3) to favorably alter serum lipids and for the prevention of heart disease (5). However few studies exist showing health benefits in individuals with diabetes, particularly those with type 1 diabetes (T1D).

PUFAs modify plasma lipids through several intracellular mechanisms including modulation of cellular signaling pathways and transcription factors that control fatty acid synthesis, beta oxidation, and lipid transport and clearance (3, 6). Total plasma PUFAs can alter lipid metabolism, but individual circulating PUFAs exert differential effects such that the relative proportion of one PUFA compared to another, even within the same class (n-3 or n-6), may impact health and well-being (3, 4). As evidence, several clinical studies have shown that the n-6 PUFA di-homo- γ - linolenic acid (DGLA, 20:3n-6) but not the less saturated n-6 PUFA arachidonic acid (AA, 20:4n-6) was related to dyslipidemia and a high degree of insulin resistance in patients with type 2 diabetes (T2D) (3, 4). While dietary intake of n-3 and n-6 PUFA is an important determinate of the relative amount of these fatty acids in vivo, long chain PUFAs can be further metabolized endogenously (see Figure 1). Therefore, activities of enzymes that regulate fatty acid desaturation processes also play a critical role in the availability of long chain n-3 and n-6 PUFAs. Product-to-precursor ratios of certain PUFAs can be used to estimate enzyme activity of desaturases involved in PUFA metabolism (7), and applying these ratios may be of particular relevance to diabetes risk management, as insulin induces the genes encoding desaturase enzymes (7, 8). For example, acute hypoinsulinemia that often accompanies T1D has been shown to contribute to altered desaturase levels and lower endogenous formation of PUFAs (9). Whether these metabolic perturbations resulting in dysregulation of PUFA metabolism also have adverse effects on plasma lipids of individuals with T1D has not been investigated.

Presently, longitudinal studies are lacking on relationships between long chain PUFAs, desaturase activities and fasting plasma lipids in youth with T1D. In the current study, we examined these associations in a large, multi-ethnic sample of youth with T1D. In these analyses, consideration was given to relevant confounders such as hemoglobin A1c (HbA1c), insulin treatment regimen, and body mass index (BMI) to provide a more precise estimate of the relationships between n-3 and n-6 PUFAs and each lipid parameter.

Demographics and clinical characteristics of the population studied are shown in Table 1. Our sample consisted of 914 youth with T1D who were predominately Caucasians with a mean age of 11 years and who were, on average, 10.9 months post-diagnosis. Forty-five percent were managed with basal bolus regimens including insulin pump therapy or a combination of long + short/rapid acting insulin with injections 3 or more times per day. Average HbA1c values were 7.8% with 11.2% (N=100) having HbA1c in excess of 9.5%. Table 2 shows the relative proportion of individual PUFAs in plasma PL, D5D and D6D activities estimated by fatty acid product-to-precursor ratios, and lipid profiles of the sample.

Results of mixed effects models to examine associations between PUFAs, desaturases and lipid outcomes are shown in Table 3 and Table 4. In unadjusted models (Table 3), favorable associations were found for the following n-6 PUFAs, desaturases and lipids: LA was positively related with HDL-C (p<0.01); AA was negatively associated with TG (p<0.05), total C (p<0.01) and TG/HDL-C ratio (p<0.05); DGLA and D6D were negatively associated with total C (p<0.01 for both); and D5D was negatively related with TG (p<0.05) and TG/HDL-C ratio (p<0.05) and positively associated with HDL-C (p<0.01). Of the n-6 PUFAs, only DGLA showed an adverse lipid association with HDL-C (p<0.05) and TG/HDL-C (p<0.05). For the n-3 PUFAs, adverse lipid associations were found for ALA and EPA. Specifically, ALA was positively associated with TG (p<0.05), total C (p<0.01) and TG/HDL-C ratio (p<0.05) and EPA was positively associated with total C (p<0.01) and LDL–C (p<0.01).

In adjusted models (Table 4), the overall finding of favorable lipid associations with LA, AA and D5D remained; mixed effects for the n-3 PUFAs, DGLA and D6D were identified. Specifically, LA was negatively associated with LDL-C (p<0.05); AA was negatively associated with TG (p<0.05) and TG/HDL-C ratio (p<0.05); and D5D was inversely associated with TG (p<0.05) and TG/HDL-C ratio (p<0.05). For the n-3 PUFAs, ALA was positively associated with TG (p<0.05) and HDL-C (p<0.05); EPA was positively associated with total C (p<0.05), LDL–C (p<0.01), and HDL-C (p<0.05); and DHA was positively associated with LDL-C (p<0.05). For DGLA and D6D, positive associations with TG/HDL-C ratio (p<0.05) were identified.

Given the unexpected adverse relationships between the n-3 PUFAs and plasma lipids and to determine whether saturated fat intake could be a potential mediator for these observed associations, adjusted models were re-fit to include dietary saturated fat intake (grams/day) in the subset of the sample with food frequency data available. The adjustment for saturated fat did not change any of the findings of significance as reported in Table 4 (data not shown). For all models except AA and total C, the beta coefficients changed less than 0.5% and the p-values for these relationships remained unchanged to 3 decimal places. For AA and total C, the beta coefficient changed 13%, however the effect was not significant.

There is ongoing debate regarding the cardiovascular effects of n-3 and n-6 PUFAs and key enzymes involved in their metabolism. The debate stems from controversial evidence regarding whether too much of these fatty acids and the relative dietary proportion of each may have beneficial or adverse effects on the cardiovascular system (1–4). This debate is clinically relevant to youth with T1D who are prone to CVD and its complications (20). A major finding from the present analyses was that the n-6 PUFAs LA and AA were favorably associated with plasma lipids after adjustment for important demographic and clinical characteristics, whereas findings were mixed relative to DGLA and the n-3 PUFAs. Desaturase activities were related to plasma lipids, but not in a similar manner, suggesting different functional roles for D5D and D6D relative to lipid metabolism. These results suggest that desaturase activities and plasma PL PUFA profile of youth with T1D may be important targets for CVD lipid risk factor reduction in this at-risk population.

In this study, a higher proportion of the n-6 PUFAs LA and AA were related to lower LDL-C and TG concentrations, respectively, suggesting a CVD protective effect. AA was also inversely related to TG/HDL-C ratio, a predictor of atherogenic small, dense LDL-C (12). In contrast, the n-6 PUFA DGLA, which is the precursor to AA (see Figure 1), was directly related to the TG/HDL-C ratio. This finding suggests that regulation of D5D activity, which catalyzes the desaturation of DGLA to AA, may be an important determinant of the TG/HDL-C ratio and LDL particle size in T1D. In the literature, some (21, 22) but not all studies (4, 23) have shown n-6 PUFAs are related to beneficial effects on plasma lipids. Recent data from the HEPFAT trial (24) showed that high intake of n-6 PUFAs were related to a decrease in cholesterol biosynthesis and low hepatic fat content and exportation, which may explain our findings of favorable n-6 PUFA relationships with plasma lipids. From our results, the calculated LDL-cholesterol lowering effect from LA was small (1.6 mg/dl change in LDL-cholesterol per unit change in LA). Whether this effect is clinically meaningful remains to be determined. Dietary LA is the most abundant of the n-6 PUFA (25), derived from vegetable oils. In randomized controlled trials, safflower and corn oil, which are richest in LA, were shown to have the most potent cholesterol lowering effects of the vegetable oils (22). Preformed AA is limited and derived from animal products such as meats, poultry and eggs. LA can be endogenously converted to AA after successive desaturation and elongation reactions, but this conversion is limited in humans (Figure 1) (26). Therefore, determining alternative methods to increase plasma PL AA may be important for CV risk reduction, although some caution is needed as AA can also be endogenously converted to bioactive eicosanoids, which produce chemical mediators that have diverse inflammatory and immune modulating effects (4, 27).

The unfavorable positive association between the n-3 PUFA ALA and blood TG in our study was unexpected, but very little research has been done with ALA specifically (28). The literature supports a TG lowering effect of the longer chain n-3 PUFAs EPA and DHA (1, 2); however the TG lowering effects usually occur with supplemental doses of EPA and DHA in the range of 2–4 g/day (29, 30) and ALA is not typically evaluated. No prior information exists between ALA and plasma lipids in youth with T1D, although several researchers have shown higher levels of plasma PL ALA and lower EPA and DHA in T1D adults compared to healthy controls suggesting altered synthesis of LC n-3 PUFAs from the precursor ALA (31, 32). Therefore, imbalances in EPA/DHA to ALA ratios in this sample due to disease related factors may provide some explanation for the unexpected findings. Alternatively, ALA may have its own specific physiological effects relative to lipid metabolism that are independent of being a precursor for EPA (33, 34). Others have shown that in contrast to EPA and DHA, fasting VLDL concentrations were not reduced with ALA feeding (34). Decreased hepatic synthesis of VLDL is the primary mechanism whereby EPA and DHA exert TG lowering effects (6). A related increase in the activity of lecithin-cholesterol acyl-transfer protein by ALA, as seen by others (35), could explain the favorable positive association between ALA and HDL-cholesterol in the present study. This enzyme promotes the esterification and internalization of cholesterol into HDL, thereby increasing HDL-C levels.

Our findings that PL EPA and DHA were unfavorably related to LDL-C and that PL EPA was favorably related to HDL-C and unrelated to TG is in line with several recent reviews of the controversial effects of EPA and DHA on plasma lipids in healthy individuals (36, 37). LC n-3 PUFAs are thought to modify plasma lipids in a number of ways, including decreasing hepatic VLDL TG synthesis through modulation of transcription factors (e.g., SREBP-1c and PPARα) involved in lipogenic and beta-oxidation pathways (38). N-3 PUFAs also reportedly increase lipoprotein lipase activity, promoting increased clearance of TG and conversion of VLDL to LDL (39). This is thought to be the likely mechanism for the LDL-raising effect of LC n-3 PUFAs (37). Additionally, cholesterol ester transfer protein has been shown to be increased by EPA and DHA leading to enhanced lipid exchange between LDL and HDL particles (39). In most studies, alterations in the function of these enzymes and transcription factors are achieved when supplemental doses of EPA and DHA are provided (3). Although supplementation does not explain the findings of the present investigation, it is possible that those with relatively high EPA in the sample also had a higher proportion of saturated fat in the diet. A positive relationship between saturated fat, LDL-C and HDL-C is well established (40). To determine whether the relationship between EPA and LDL- and HDL-C observed in the present study could be mediated by dietary saturated fat intake, we re-fit our adjusted repeated measures models for EPA and plasma lipids to include saturated fat intake from a subset of participants with available food frequency data (n=497). Findings from this sub-analysis do not support saturated fat as a likely explanation of our results.

Similar to previous studies in healthy adolescents (20), our results show a favorable association between estimated D5D and plasma lipids, whereas adverse associations were found for D6D. D5D and D6D activities may have divergent effects on the FA composition of cell membranes, which affect many aspects of glucose control including insulin receptor binding, glucose transport activity, and intracellular signaling (9). The activities of D5D and D6D appear to be of high relevance for the development of T2D, as higher D5D activity has been associated with decreased disease risk, while D6D activity has been directly linked to increased risk (41, 42). To our knowledge, this is the first study to examine estimated desaturase activities and CVD lipid risk factors in T1D. Given the favorable association between D5D and plasma lipids in our sample, further studies are required to better understand how the desaturase enzymes relate to cardiovascular health in T1D.

The strengths of the study include the large, diverse sample of adolescents with T1D studied. Additionally, confounding was assessed in our statistical models by including variables known to influence fatty acid metabolism-CVD lipid risk factor associations in youth with diabetes; however the possibility for residual confounding remains. The repeated measures analysis used in this investigation provides increased power over existing cross-sectional analyses in examining associations between fatty acid exposures and plasma lipid risk factors. Intra-individual variation as well as inter-individual variation was taken into account in mixed models, thus providing increased validity for the associations identified. It should be noted, however, that the repeated measures analysis is limited in deducing causality. Another limitation was the fact that desaturase activities were not directly measured but rather estimated using product-to-precursor ratios. Although desaturase activity can be measured directly in animals by measurement of the rate of conversion of radiolabeled precursor fatty acids to their respective products (7), for ethical and practical reasons it cannot be measured in humans. Finally, imputation was used to replace undetectable plasma PL PUFAs to avoid bias in the results; best practices were used in this regard.

The present study provides novel findings regarding relationships between n-3 and n-6 PUFAs, desaturases, and plasma lipids/lipoproteins in T1D. Our findings of potential beneficial effects of LA, AA and D5D and mixed effects of the n-3 PUFAs, DGLA and D6D on plasma lipids suggest interplay between the types of dietary fat consumed, endogenous fatty acid synthesis, and diabetes that is important in the development and management of the CVD risk profile. The practical interpretation of these data is that vegetable oils rich in the most abundant n-6 PUFAs, such as LA rich-corn oil and safflower oil, may provide some benefit relative to lipid profile in T1D. Future studies are needed to clarify whether the observed associations are related to long-term health outcomes in youth with T1D.