Introduction
The fatty acid desaturase (FADS) gene cluster plays a pivotal role in the metabolism of polyunsaturated fatty acids (PUFAs), which are essential for various physiological functions, including immunity, inflammation, and brain development. This article explores the intricate relationship between genetic variations within the FADS cluster, dietary PUFA intake, and their combined impact on health outcomes, particularly in diverse populations. The focus is on the FADS1 gene and its interaction with linoleic acid (LA), an omega-6 (n-6) PUFA, in the context of adipose tissue inflammation and overall metabolic health.
The Role of FADS Genes in PUFA Metabolism
The FADS Cluster and LC-PUFA Biosynthesis
Key determinants of the efficiency with which long chain polyunsaturated fatty acids (LC-PUFA) are synthesized, as well as LC-PUFA levels themselves, have been established within the fatty acid desaturase cluster (FADS) on chromosome 11q12.2. The FADS cluster, comprising three genes (FADS1, FADS2, and FADS3), is essentially a single large expanse of high linkage disequilibrium (LD) in populations of European ancestry, which explains the numerous single nucleotide polymorphisms (SNPs) in the literature that have been shown to have exceedingly strong effects on PUFA metabolism. These genes encode enzymes that catalyze rate-limiting desaturation steps in the biosynthesis of LC-PUFAs from essential 18-carbon (18C) PUFAs, linoleic acid (LA, 18:2n-6) and α-linolenic acid (ALA, 18:3n-3). The desaturation steps have long been recognized as the rate-limiting steps in the pathway. Specifically, FADS2 converts LA and ALA to gamma (γ)-linolenic acid (GLA, 18:3, n-6) and stearidonic acid (SDA, 18:4, n-3), respectively. Subsequently, FADS1 encodes delta-5 desaturase (D5D), which is crucial for the production of arachidonic acid (AA) from dihomo-γ-linolenic acid (DGLA).
Competition Between n-6 and n-3 PUFAs
Both n-6 and n-3 substrates compete for early enzymatic steps in the pathway. Since LA and ALA compete in early steps of the pathway and there is a limited synthetic capacity of the pathway, both human and animal models indicate that this shift has markedly reduced the synthesis and bioavailability of n-3 LC-PUFAs. The balance between n-6 and n-3 PUFA intake is critical because they have different, and often opposing, effects on inflammation. While some oxidation products of AA can be anti-inflammatory, many studies have shown that eicosanoids (prostaglandins and leukotrienes) derived from AA are pro-inflammatory. Conversely, n-3 LC-PUFAs and their metabolites, such as resolvins, protectins, and maresins, are known for their anti-inflammatory and "pro-resolution" properties.
Genetic Variation in FADS Genes and Its Impact
Genetic variants within the FADS cluster are determinants of long chain polyunsaturated fatty acid (LC-PUFA) levels in circulation, cells, and tissues. The FADS cluster comprising three genes (FADS1, FADS2 and FADS3) is essentially a single large expanse of high linkage disequilibrium (LD) in populations of European ancestry which explains the numerous single nucleotide polymorphisms (SNPs) in the literature that have been shown to have exceedingly strong effects on PUFA metabolism. Subsequent to the first candidate gene study by Shaeffer et al., there have been close to 20 candidate gene studies lending support to the role these variants play, with a particular note that the dramatic effects are not only for the inter-individual variation in a single PUFA level (e.g., ARA or DGLA) but rather in those product-precursor ratios such as ARA/DGLA that specifically serve a surrogate markers for the efficiency by which PUFAs are moving through a given step (FADS1 in this case) within the biosynthetic pathway. In fact, these effects described by candidate genes studies have found strong support in the genome-wide association (GWA) approach as well; the peak associated SNP, rs174537, yielded a p-value of p = 5.95 × 10-46 and accounted for 18.6% of the additive variance in ARA, perhaps one the strongest GWAS-identified allelic effects to date.
These genetic variations influence the efficiency of desaturase enzymes, leading to differences in LC-PUFA levels among individuals. For example, the single nucleotide polymorphism (SNP) rs174537 is strongly associated with arachidonic acid (ARA) levels. Moreover, the G allele at rs174537 is the derived allele and swept to fixation within African, is maintained at intermediate levels in Europe and European ancestry populations and at very low frequencies in Native American in the US. A common haplotype associated with the enhanced enzymatic efficiency of FADS1 is specific to humans appearing after the split of the common ancestor of humans and Neanderthals. This haplotype shows evidence of positive selection in African populations.
Read also: The Hoxsey Diet
Ethnic and Ancestral Differences in FADS Allele Frequencies
Evidence clearly supports that the associations are robust to ethnicity. However ∼80% of African Americans carry two copies of the alleles associated with increased levels of arachidonic acid, compared to only ∼45% of European Americans raising important questions of whether gene-PUFA interactions induced by a modern western diet are differentially driving the risk of diseases of inflammation in diverse populations, and are these interactions leading to health disparities. There appears to be compelling evidence that the G allele at rs174537 is the derived allele and swept to fixation within African, maintained at intermediate levels in Europe and European ancestry populations and at very low frequencies in Native American in the US.
Dietary Recommendations and Considerations
Modern Western Diet (MWD) and PUFA Intake
There has been a dramatic (∼3-fold) increase in the dietary access to the 18C n-6 PUFA, LA (currently 6-8% of calories) in the MWD in the past 50 years with the increase in LA-containing vegetable oil products (soybean, corn, palm, and canola oils as well as margarine and shortenings). In contrast, ALA levels (typically 0.5-1.5% of calories) found in green plants, nuts and botanical oils, such as flax seed oil have remained relatively constant over that same period of time. Given that humans ingest much lower quantities of LC-PUFAs (typically less than 200mg/day) than 18C PUFAs, LA and ALA, the capacity to synthesize LC-PUFAs from these 18C PUFAs is critical in determining circulating and cellular levels of LC-PUFAs. Additionally, the dramatic increase of dietary LA has not only increased total PUFAs in the diet, but has dramatically shifted the ratio of LA to ALA that enters the pathway. Because LA and ALA compete in early steps of the pathway and there is a limited synthetic capacity of the pathway, both human and animal models indicate that this shift has markedly reduced the synthesis and bioavailablity of n-3 LC-PUFAs. It is important to note that the current levels and ratios of 18C PUFAs in the MWD are in line in in many ways resulted from with recommendations by the American Heart Association to consume 5-10% of daily calories as PUFAs.
Biological Functions of n-6 and n-3 LC-PUFAs
For example while a few oxidation products of ARA have been shown to be anti-inflammatory, hundreds of laboratories around the world have been able to find eicosanoids (prostaglandins and leukotrienes) at concentrations that are pharmacologically active as pro-inflammatory, and this has resulted in thousands of publications establishing the role of these ARA metabolites in inflammation. Importantly there is the emerging story of n-3 LC-PUFAs and their products as anti-inflammatory, “pro-resolution” metabolites termed resolvins, protectins, and maresins. Consequently, there is a consistent scientific literature that supports the concept that n-6 and n-3 LC-PUFAs and their products have not only different, but often opposing effects, with regard to inflammation. Many in the field of eicosanoid biology believe that this component has been underappreciated in the current recommendations by groups such as the AHA.
Re-evaluation of Dietary Recommendations
For example, the current AHA dietary recommendations have been made largely based on several randomized controlled trials and population cohort studies that measured cardiovascular disease biomarkers such as serum lipids and lipoproteins. These data show that PUFAs can have cardiovascular benefits when viewed from the perspective of measuring cardiovascular disease biomarkers such as serum lipids and lipoproteins. However, recent sets of data have raised important questions about this approach. Ramsden and colleagues recently re-examined studies utilized to support this recommendation and found that many of the oils used in the aforementioned clinical trials were mixtures of n-6 and n-3 PUFAs. Their data suggests that only substituting n-6 PUFAs for saturated and trans-fatty acids actually trended toward increased risk of death from all causes. This same group also recently reexamined the Sydney Diet Heart Study (458 men 30-59 with a recent coronary event), which replaced dietary saturated fatty acids with a high LA-containing life the aforementioned studies found that the LA intervention group had lower levels of total cholesterol; however, the n-6 PUFA group had unexpectedly higher rates of death than controls.
Impact of 18C PUFAs on LC-PUFA Biosynthesis
Most dietary PUFA recommendations made to date have been based on a simple assumption that there is a limited capacity (somewhere between 2-3% of calories) of humans to synthesize LC-PUFAs from dietary 18C PUFAs such as LA and ALA. This line of reasoning assumes that higher (than 3% calories) dietary quantities of 18C PUFAs are irrelevant since it is beyond the capacity of the PUFA biosynthetic pathway to utilize them. The second critical assumption in this line of reasoning is that the biosynthetic capacity is equivalent for all human populations. As mentioned above, the desaturase enzymes encoded by genes in the FADS cluster are consistently recognized as a bottleneck that determines the levels of LC-PUFA through the biosynthetic pathway.
Read also: Walnut Keto Guide
FADS1 and Adipose Tissue Inflammation
FADS1 Variants and Inflammatory Responses
Fatty acid desaturase (FADS) variants associate with fatty acid (FA) and adipose tissue (AT) metabolism and inflammation. Dietary fatty acids (FA) have important roles in the regulation of adipose tissue (AT) metabolism, gene expression and inflammation. The dietary intake of the essential omega-6 (n-6) polyunsaturated fatty acid (PUFA) linoleic acid (LA) associates with reduced inflammation whereas arachidonic acid (AA) intake has been shown to associate with increased inflammation. Previous studies by others and us have shown that the health outcomes of dietary LA intake are regulated by the FADS1 variants. Dietary LA intake has shown to modify the association between FADS1 variants and clinical phenotypes, including HDL and anthropometric measures. This suggests that the variants of FADS genes could regulate the effect of dietary n-6 PUFA on AT inflammation.
Study Design and Methodology
Subjects homozygotes for the TT and CC genotypes of the FADS1-rs174550 (TT, n = 25 and CC, n = 28) or -rs174547 (TT, n = 42 and CC, n = 28), were either recruited from the METabolic Syndrome In Men cohort to participate in an intervention with LA-enriched diet (FADSDIET) or from the Kuopio Obesity Surgery (KOBS) study. The study design of the FADSDIET intervention (NCT02543216) has been previously described. Briefly, 255 healthy normal weight or borderline overweight men homozygous for the FADS1-rs174550 (minor allele frequency 0.2977) were invited from the METabolic Syndrome In Men (METSIM) study to participate in the FADSDIET intervention study. The subjects using an anticoagulant treatment and having severe chronic diseases were excluded from the study. Altogether, 62 men participated in the intervention and 59 of them completed the intervention. During the 4-week run-in period they consumed habitual diet but stopped consumption of oil supplements, e.g. fish oil supplements, or plant stanol or sterol enriched products, which were not allowed during the study. Patients accepted to obesity surgery at the Kuopio University Hospital have been recruited to the ongoing Kuopio Obesity Surgery (KOBS) study since 2005. The present analysis contains cross‐sectional baseline data from a sub-cohort of 70 individuals (males: n = 23 and females: n = 47), having FADS1-rs174547 variant (minor allele frequency 0.2979) genotyped (only CC: n = 28 and TT: n = 42) genotypes included in the analysis), gene expression data available from AT and plasma FA composition (clinical characteristics presented in Supplementary Table S1). The study participants were genotyped for the FADS1 variants rs174550 (FADSDIET) and rs174547 (KOBS), that are in complete linkage disequilibrium, using the TaqMan SNP Genotyping Assay according to the protocol provided by the manufacturer (Applied Biosystems, Foster City, CA, USA). For the gene expression, AT samples were immediately frozen in liquid nitrogen. TruSeq Targeted RNA Expression (TREx) platform with the MiSeq system (Illumina, San Diego, CA) was used for measuring gene expression levels in AT, as previously described. Additionally, the interleukin 1 beta (IL1β) gene expression in obese subjects (n = 70) of the KOBS study was measured, as described previously. Commercial kits (Thermo Fisher Scientific) were used for measuring concentrations of serum total, LDL, and HDL cholesterol and total triglycerides. The Konelab 20XTi Clinical Chemistry Analyzer (Thermo Fisher Scientific) and Enzymatic photometric (glucose hexokinase) method (Thermo Fisher Scientific) was used for the measurement of plasma glucose concentration. FA composition in plasma lipid fractions was analyzed according to previously described method. Briefly, plasma samples were extracted with chloroform-methanol (2:1), and the different lipid fractions, cholesteryl esters (CE), triglycerides (TG), and phospholipids (PL) were separated by solid phase extraction with an aminopropyl column. Enzyme activities in different lipid fractions were estimated as product-to-precursor ratios of individual FAs as follows: D5D = 20:4 n-6/20:3 n-6 and D6D = 18:3 n-6/18:2 n-6. Total n-6 PUFA and total n-3 PUFA composition was calculated as a sum of corresponding individual FA proportion. Plasma PL fraction was chosen due to its rapid response to dietary changes in fat, and the fact that CEs are produced by the transfer of FAs from PLs. Thus, PL fraction was chosen for further analyses. Statistical analyses were conducted with the SPSS software (version 25, IBM Corp., Armonk, NY, USA) and GraphPad Prism 5.03 for Windows (GraphPad Software, San Diego, CA, USA).
Key Findings
In the KOBS study, interleukin (IL)1 beta mRNA expression in AT was increased in subjects with the TT genotype and highest LA proportion. In the FADSDIET, n-6/LA proportions correlated positively with AT-InSc in those with the TT genotype but not with the CC genotype after LA-enriched diet. LA-enriched diet increases inflammatory AT gene expression in subjects with the TT genotype, while CC genotype could play a protective role against LA-induced AT inflammation. We observed a diet-genotype interaction between LA-enriched diet and AT-InSc in the FADSDIET. There was a non-significant trend towards a reduced AT-InSc in subjects with the CC genotype (p = 0.067), while the AT-InSc did not change in subjects with the TT genotype (p = 0.130) of the FADS1-rs174550 in response to a LA-enriched diet. In line with this, we could additionally demonstrate that IL-1β mRNA expression in subcutaneous AT was statistically different between the groups of LA proportion in plasma PL fraction in subjects with the TT genotype (ANOVA, p = 0.019). AT-InSc correlated negatively with total n-6 PUFA proportion in plasma PL fraction at baseline (r = − 0.449, p = 0.028) but positively after LA-enriched diet (r = 0.494, p = 0.014) in subjects with the TT genotype of the FADS1-rs174550. Additionally, AT-InSc correlated positively with the proportion of LA in plasma PL fraction after LA-enriched diet (r = 0.544, p = 0.006) in subjects with the TT genotype of the FADS1-rs174550. In contrast, there were no significant correlations in subjects with the CC genotype of the FADS1-rs174550 between AT-InSc and n-6 or LA. There was on average a positive correlation (r = 0.153, p = 0.008) between the AT-InSc and all LA-derived eicosanoids at the end of the intervention in subjects with the TT genotype of the FADS1-rs174550. There was also a significant difference in the correlation between the AT-InSc and LA-derived lipid mediators between the two timepoints (baseline vs. the end of the intervention) within TT genotype (p = 0.024), but not within CC genotype, of the FADS1-rs174550. There was on average a positive correlation (r = 0.081, p = 0.008) between AT-InSc and all AA-derived eicosanoids at the end of the intervention in subjects with the TT genotype of the FADS1-rs174550. In contrast, there was a positive correlation at baseline (r = 0.116, p = 0.003, corrected for multiple comparison p = 0.042) and a negative correlation (r = − 0.138, p = 0.011) at the end of the intervention between AT-InSc and all AA-derived eicosanoids in subjects with the CC genotype. Moreover, there was a significant difference in the correlation between the AT-InSc and AA-derived eicosanoids between the two timepoints (baseline vs.
Implications of FADS1 Genotype on Metabolic Health
Fasting plasma glucose concentration decreased in participants with the CC genotype (paired-samples t test, p = 0.01; diet-genotype interaction, p = 0.012) but not in participants with the TT genotype during the intervention. Additionally, serum total cholesterol concentration decreased in participants with the CC genotype (p = 0.017) but not in those with TT genotype. Serum LDL cholesterol concentration decreased in both genotype groups (p = 0.011, p = 0.010) during the intervention.
Associations with Complex Diseases
While it is clear that FADS variants have an important impact on LC-PUFA levels and ratios of PUFA products to precursors, it is likely that resulting additional molecular and clinical phenotypes associated with FADS variation place individuals at varying risk. For example, there are studies that document the role FADS variants in complex lipid and inflammatory phenotypes and coronary artery disease (CAD). Over the past decade, genome-wide association studies (GWAS) have identified a number of genetic polymorphisms that convey increased risk for coronary artery disease, diabetes, cancer, and other common diseases, including some that implicate FADS variants. Associations have also been documented between FADS variants and traditional markers of cardiovascular disease, including LDL-cholesterol, triglycerides, HDL-cholesterol, and total cholesterol levels. Associations have also been documented for phospholipid metabolites with four double bonds (i.e. ARA) for all major phospholipid species. Perhaps it is not surprising that strong associations are noted with FADS SNPs and LC-PUFA-containing glycerolipids as well as total cholesterol, LDL-cholesterol, HDL-cholesterol and triglycerides given that PUFA-containing gylcerolipids are key molecular components (intermediate phenotypes) of lipoprotein particles. However, there are also now several studies that indicate specific FADS haplotypes favoring high desaturase activity lead to enhanced levels of inflammatory and CAD biomarkers including oxidative products of ARA (104; 116; 117). In one of the earliest studies in this area, Martinelli and colleagues demonstrated that a higher ARA/LA ratio (presumably representing more conversion of L…
Read also: Weight Loss with Low-FODMAP