Ji Yao Zhang

Positive selection on a regulatory insertion-deletion polymorphism in FADS2 influences apparent endogenous synthesis of arachidonic acid

Long chain polyunsaturated fatty acids (LCPUFA) are bioactive components of membrane phospholipids and serve as substrates for signaling molecules. LCPUFA can be obtained directly from animal foods or synthesized endogenously from 18 carbon precursors via the FADS2 coded enzyme. Vegans rely almost exclusively on endogenous synthesis to generate LCPUFA and we hypothesized that an adaptive genetic polymorphism would confer advantage. The rs66698963 polymorphism, a 22-bp insertion–deletion within FADS2, is associated with basal FADS1 expression, and coordinated induction of FADS1 and FADS2 in vitro. Here, we determined rs66698963 genotype frequencies from 234 individuals of a primarily vegetarian Indian population and 311 individuals from the US. A much higher I/I genotype frequency was found in Indians (68%) than in the US (18%). Analysis using 1000 Genomes Project data confirmed our observation, revealing a global I/I genotype of 70% in South Asians, 53% in Africans, 29% in East Asians, and 17% in Europeans. Tests based on population divergence, site frequency spectrum, and long-range haplotype consistently point to positive selection encompassing rs66698963 in South Asian, African, and some East Asian populations. Basal plasma phospholipid arachidonic acid (ARA) status was 8% greater in I/I compared with D/D individuals. The biochemical pathway product–precursor difference, ARA minus linoleic acid, was 31% and 13% greater for I/I and I/D compared with D/D, respectively. This study is consistent with previous in vitro data suggesting that the insertion allele enhances n-6 LCPUFA synthesis and may confer an adaptive advantage in South Asians because of the traditional plant-based diet practice.

Desaturase and elongase-limiting endogenous long-chain polyunsaturated fatty acid biosynthesis.

Purpose of reviewEndogenous synthesis of the long-chain polyunsaturated fatty acids (LCPUFAs) is mediated by the fatty acid desaturase (FADS) gene cluster (11q12-13.1) and elongation of very long-chain fatty acids 2 (ELOVL2) (6p24.2) and ELOVL5 (6p12.1). Although older biochemical work identified the product of one gene, FADS2, rate limiting for LCPUFA synthesis, recent studies suggest that polymorphisms in any of these genes can limit accumulation of product LCPUFA. Recent findingsGenome-wide association study (GWAS) of Greenland Inuit shows strong adaptation signals within FADS gene cluster, attributed to high omega-3 fatty acid intake, while GWAS found ELOVL2 associated with sleep duration, age and DNA methylation. ELOVL5 coding mutations cause spinocerebellar ataxia 38, and epigenetic marks were associated with depression and suicide risk. Two sterol response element binding sites were found on ELOVL5, a SREBP-1c target gene. Minor allele carriers of a 3 single nucleotide polymorphism (SNP) haplotype in ELOVL2 have decreased 22 : 6n-3 levels. Unequivocal molecular evidence shows mammalian FADS2 catalyzes direct &Dgr;4–desaturation to yield 22 : 6n-3 and 22 : 5n-6. An SNP near FADS1 influences the levels of 5-lipoxygenase products and epigenetic alteration. SummaryGenetic polymorphisms within FADS and ELOVL can limit LCPUFA product accumulation at any step of the biosynthetic pathway.

Brown but not white adipose cells synthesize omega-3 docosahexaenoic acid in culture

Adipose tissue is a complex endocrine organ which coordinates several crucial biological functions including fatty acid metabolism, glucose metabolism, energy homeostasis, and immune function. Brown adipose tissue (BAT) is most abundant in young infants during the brain growth spurt when demands for omega-3 docosahexaenoic acid (DHA, 22:6n-3) is greatest for brain structure. Our aim was to characterize relative biosynthesis of omega-3 long chain polyunsaturated fatty acids (LCPUFA) from precursors in cultured white (WAT) and brown (BAT) cells and study relevant gene expression. Mouse WAT and BAT cells were grown in regular DMEM media to confluence, and differentiation was induced. At days 0 and 8 cells were treated with albumin bound d5-18:3n-3 (d5-ALA) and analyzed 24h later. d5-ALA increased cellular eicosapentaenoic acid (EPA, 20:5n-3) and docosapentaenoic acid (DPA, 22:5n-3) in undifferentiated BAT cells, whereas differentiated BAT cells accumulated 20:4n-3, EPA and DPA. DHA as a fraction of total omega-3 LCPUFA was greatest in differentiated BAT cells compared to undifferentiated cells. Undifferentiated WAT cells accumulated EPA, whereas differentiated cells accumulated DPA. WAT accumulated trace newly synthesized DHA. Zic1 a classical brown marker and Prdm16 a key driver of brown fat cell fate are expressed only in BAT cells. Ppargc1a is 15 fold higher in differentiated BAT cells. We conclude that in differentiated adipose cells accumulating fat, BAT cells but not WAT cells synthesize DHA, supporting the hypothesis that BAT is a net producer of DHA.

A novel FADS2 isoform identified in human milk fat globule suppresses FADS2 mediated Δ6-desaturation of omega-3 fatty acids

INTRODUCTION The only known non-pharmacological means to alter long chain polyunsaturated fatty acid (LCPUFA) abundance in mammalian tissue is by altering substrate fatty acid ratios. Alternative mRNA splicing is increasingly recognized as a modulator of protein structure and function. Here we report identification of a novel alternative transcript (AT) of fatty acid desaturase 2 (FADS2) that inhibits production of omega-3 but not omega-6 LCPUFA, discovered during study of ATs in human milk fat globules (MFG). METHODS Human breastmilk collected from a single donor was used to isolate MFG. An mRNA-sequencing library was constructed from the total RNA isolated from the MFG. The constructed library was sequenced using an Illumina HiSeq instrument operating in high output mode. Expression levels of evolutionary conserved FADSAT were measured using cDNA from MFG by semi-quantitative RT-PCR assay. RESULTS RNA sequencing revealed >15,000 transcripts, including moderate expression of the FADS2 classical transcript (CS). A novel FADS2 alternative transcript (FADS2AT2) with 386 amino acids was discovered. When FADS2AT2 was transiently transfected into MCF7 cells stably expressing FADS2, delta-6 desaturation (D6D) of alpha-linolenic acid 18:3n-3 → 18:4n-3 was suppressed as were downstream products 20:4n-3 and 20:5n-3. In contrast, no significant effect on D6D of linoleic acid 18:2n-6 → 18:3n-6 or downstream products was observed. FADS2, FADS2AT1 and 5 out of 8 known FADS3AT were expressed in MFG. FADS1, FADS3AT3, and FADS3AT5 are undetectable. CONCLUSION The novel, noncatalytic FADS2AT2 regulates FADS2CS-mediated Δ6-desaturation of omega-3 but not omega-6 PUFA biosynthesis. This spliced isoform mediated interaction is the first molecular mechanism by which desaturation of one PUFA family but not the other is modulated.

Fads3 modulates docosahexaenoic acid in liver and brain

Fatty acid desaturase 3 (FADS3) is the third member of the FADS gene cluster. FADS1 and FADS2 code for enzymes required for highly unsaturated fatty acid (HUFA) biosynthesis, but FADS3 function remains elusive. We generated the first Fads3 knockout (KO) mouse with an aim to characterize its metabolic phenotype and clues to in vivo function. All mice (wild type (WT) and KO) were fed facility rodent chow devoid of HUFA. No differences in overt phenotypes (survival, fertility, growth rate) were observed. Docosahexaenoic acid (DHA, 22:6n-3) levels in the brain of postnatal day 1 (P1) KO mice were lower than the WT (P < 0.05). The ratio of docosapentaenoic acid (DPA, 22:5n-3) to DHA in P1 KO liver was higher than in WT suggesting lower desaturase activity. Concomitantly, 20:4n-6 was lower but its elongation product 22:4n-6 was greater in the liver of P1 KO mice. P1 KO liver Fads1 and Fads2 mRNA levels were significantly downregulated whereas expression levels of elongation of very long chain 2 (Elovl2) and Elovl5 genes were upregulated compared to age-matched WT. No Δ13-desaturation of vaccenic acid was observed in liver or heart in WT mice expressing FADS3 as was reported in vitro. Taken together, the fatty acid compositional results suggest that Fads3 enhances liver-mediated 22:6n-3 synthesis to support brain 22:6n-3 accretion before and during the brain growth spurt.