A 7-day high-PUFA diet reduces angiopoietin-like protein 3 and 8 responses and postprandial triglyceride levels in healthy females but not males: a randomized control trial

Hypertriglyceridemia is a form of dyslipidemia that is categorized by elevated blood triglyceride (TG) concentrations and affects nearly 30% of the US population [1–3]. High plasma TG levels are associated with type 2 diabetes mellitus and obesity, possibly due to lipid-induced insulin resistance, glucose intolerance, and increased very low-density lipoprotein (VLDL) production [1, 2, 4, 5]. Elevated postprandial blood TGs are a public health concern since they have shown to be an independent risk factor for cardiovascular disease and atherosclerosis [4, 5].

In addition to chronic high fat (HF) diets, previous studies have shown the impact of individual HF meals on cardiovascular disease (CVD) risk by measuring markers such as inflammatory factors, thrombosis, and blood pressure responses [6–8]. The majority of the detrimental effects of dietary fat consumption appears to be from saturated fat with mono- and polyunsaturated fat appearing to be protective, or at least not harmful. A diet high in long chain omega-3 polyunsaturated fatty acids (PUFA), specifically α-linolenic acid (18:3n3), eicosapentaenoic acid (EPA) (20:5n3) and docosahexaenoic acid (DHA) (22:6n3), is linked with reduced fasting blood TG levels, increased high density lipoprotein (HDL) cholesterol, and decreased low density lipoprotein (LDL) cholesterol [9–12]. Contrary to the beneficial role of PUFAs on plasma TG, consuming saturated fatty acids (SFAs) has been shown to increase LDL cholesterol, decrease insulin sensitivity and may promote inflammation if consumed as part of a hypercaloric diet, therefore increasing the risk of CVD [13–15].

Some of the effects of dietary PUFAs may be from increased oxidation compared to SFAs, however the mechanism by which this occurs is not known [16, 17]. A newly identified model for the systemic regulation of lipid metabolism is through three members of the eight-member family of angiopoietin-like proteins (ANGPTLs) including ANGPTL3, − 4, and − 8 and their tissue specific regulation of lipoprotein lipase (LPL) activity [18]. ANGPTL3, for instance, decreases the clearance of VLDL triglyceride through constraining LPL activity [19], directly affecting adipocytes to activate lipolysis [20]. A consequent increase in the release of free fatty acid (FFA) and glycerol from adipocytes as a result of ANGPTL3 functioning indicates that it might be an important regulatory agent in lipid metabolism [21].

Several genetic studies have shown distinct effects of common variants in the ANGPTL3, ANGPTL4 and ANGPTL8 genes on plasma lipid levels [22, 23]. Functional studies have revealed that these three ANGPTLs influence plasma lipid levels by inhibiting the activity of extracellular lipases, including the structurally related LPL, as well as hepatic, endothelial and pancreatic lipases during fasting and fed states [18, 19, 22–28]. Hence, these three ANGPTLs are postulated to generate a framework for how TG trafficking is regulated given their LPL-inhibitory roles [25]. Consequently, it is reasonable to conclude that deficiency of any of these ANGPTLs could result in hypotriglyceridemia [25]. Altogether, it would be sensible to hypothesize that replacing dietary SFAs with PUFAs may aid in reducing plasma TG levels following a possible diminished ANGPTL3, 4, and 8 response, especially after acute consumption of SFA-rich meals. However, whether these ANGPTLs are affected differently by various dietary fatty acids in humans remains unknown.

The purpose of this study was to 1) determine whether a high PUFA diet could mitigate the effects of a high SFA meal through reduced postprandial TG levels, and 2) to explore the mechanisms behind any plasma changes in plasma TG levels by examining the changes in ANGPTL3, − 4, and − 8. Based on previous work on the impact of dietary FA composition on plasma TG levels, we hypothesized that consuming a high PUFA diet may aid in reducing plasma TG levels. We found that postprandial TG levels were decreased following a PUFA-rich diet. In order to examine the possible mechanisms for this reduction, we measured changes in plasma ANGPTL3, − 4, and − 8 levels. Despite the fact that there are limited data on the mechanisms of how FA composition changes in the diet can lead to alterations in plasma TG levels, we were able to provide clear evidence that a PUFA-rich diet will decrease postprandial TG levels in women.

The aim of this study was to determine whether a high-PUFA diet could mitigate the effects of a high-fatSFA-rich meal by reducing postprandial TG levels. Research suggests that both the amount and type of dietary fat influences postprandial lipemia, showing that test meals containing up to 50 g of fat can substantially increase TG concentrations [32]. Furthermore, previous studies have reported reduced postprandial TG concentrations in response to n-3 PUFA consumption, while SFA consumption leads to increased postprandial TG concentrations [33, 34]. However, to our knowledge no study has utilized a whole-food based, longer-term PUFA diet to measure postprandial TG response to high fat meals rich in SFA. Therefore, for the first time, we are showing that a PUFA-rich diet reduces postprandial TG concentrations in females, following a SFA-rich meal. While this study design is unique, our results are consistent with previous literature showing that postprandial TG concentrations are lower after consuming either a PUFA or SFA meal while following a PUFA background diet compared to a SFA background diet [35]. These findings suggest that a diet rich in PUFAs may provide metabolic protection from high-fat SFA-rich meals. Also, since the pre and post-diet high-SFA meals were of identical composition, the differences observed from pre to post-diet intervention are due to the chronic effect of the 7 days of high PUFA consumption.

Furthermore, the current study demonstrates that ANGPTL3 and − 8 are both decreased in females following a high PUFA diet. ANGPTL8 has been reported to downregulate adipose triglyceride lipase (ATGL) that catalyzes the initial step in hydrolyzing triglyceride in adipocytes, hence plays an important role in degrading lipid droplet/adipose in mammalian cells [36–39]. Several studies have shown postprandial effects on all three ANGPTLs, but thus far, no studies have demonstrated a diet-induced effect on ANGPTL3, − 4, or − 8. One previous study compared tissue-specific uptake of dietary fat in male versus female wild-type and Angptl4−/− mice and reported no sex differences [40], which is in agreement with our human data in the baseline measures. However, our study looked at the impact of a high PUFA diet in males and females independently and found that only females displayed a diet-induced change in ANGPTL3, and − 8. From these data, it is worth assessing whether the presence of estrogen or the absence of androgens may influence ANGPTL3 or − 8 activity in females, especially pre- versus post-menopausal. While there was no observed change in ANGPTL4, it is possible that cleaved versus N-terminal forms of the protein were differentially affected by diet. Therefore, further research is needed to separately investigate different forms of the protein since the ELISA-based assay that we used to measure ANGPTL4 could not differentiate between the cleaved and the N-terminal form of this protein. Moreover, ANGPTL4 is thought to be associated with HDL in plasma [41] and our measurements did not distinguish between HDL-associated and HDL-free ANGPTL4. There is limited evidence in the literature on the complex functionality of ANGPTL4 in response to diet. Thus, it is possible that ANGPTL4 may differ by diet, form of the protein and differential HDL association, although it would require a more detailed analysis of its status in various plasma fractions to be sure.

The lack of reduction in TG concentrations from pre- to post-diet in the male PUFA diet group participants was unexpected. Although this was not a mechanistic study, and we do not know the exact mechanism by which PUFAs reduce postprandial TG concentrations differently for males and females. There are noticeable physiological differences between males and females and sex as a biological variable (SABV) should be accounted for in basic and clinical research [42]. In looking at the differences between the way males and females metabolize and store fat, previous research is consistent with our results in finding that males and females do not respond to meal ingestions in the same way [43]. It is believed that the gonadal hormones, estrogen and androgen, are responsible for the differences in energy balance and meal response between sexes [44]. Specifically, studies suggest that estradiol suppresses free fatty acid and TG synthesis and accumulation in the blood and tissues, and increases fat oxidation in females, compared to males. Some studies have also shown that females are more resistant to metabolic disturbances caused by high-fat diets compared to males due to the presence and abundance of estrogen receptor-α (ERα) and estrogen receptor-β (ERβ) [44–46]. ERα is the main receptor in hepatocytes and in conjunction with the presence of estradiol, they control genes involved in glucose, lipid, protein and cholesterol homeostasis [46].Taken together, these findings might help explain the differences seen in TG concentrations in our study between the male and female participants. A longer period study would be beneficial to highlight more prominent gender specific differences if any, in order to deduce more physiologically meaningful conclusions and comparisons. Finally, we acknowledged that the reduction in body weight occurred in the control group from pre- to post-diet visit, however it is likely to be clinically non-significant. In spite of our internal speculations about the cause for this, we cannot say with certainty how or why this happened. Future studies on longer term interventions might provide more accurate information on the effects of different dietary fatty acids on body weight changes.

Altogether, the results of this study indicate that consuming a high PUFA diet is capable of reducing the detrimental effects of high SFA meals. Both males and females improved plasma TG levels, albeit to a far greater extent in females. Further studies are needed to assess whether the presence or absence of estrogen or testosterone are responsible for these effects and to what extent PUFA intake can be reduced while still providing protection. Finally, these results provide further support for the role of SABV in physiology and should be included in study design when feasible.