Fads2 knockout mice reveal that ALA prevention of hepatic steatosis is dependent on delta-6 desaturase activity.

The production of the omega-3 long-chain polyunsaturated fatty acids (n-3 LCPUFA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from alpha-linolenic acid (ALA) relies on the delta-6 desaturase (D6D) enzyme encoded by the Fads2 gene. While EPA and DHA reduce hepatic triacylglycerol (TAG) storage and regulate lipogenesis, the independent impact of ALA is less understood. To address this gap in knowledge, hepatic fatty acid metabolism was investigated in male wildtype (WT) and Fads2 knockout (KO) mice fed diets (16% kcal from fat) containing either lard (no n-3 LCPUFA), flaxseed oil (ALA rich), or menhaden oil (EPA/DHA rich) for 21 weeks. Fat content and composition, as well as markers of lipogenesis, glyceroneogenesis, and TAG synthesis, were analyzed using histology, gas chromatography, and reverse transcription quantitative PCR (RT-qPCR). Mice fed the menhaden diet had significantly lower hepatic TAG compared to both lard- and flax-fed mice, concomitant with changes in n-3 and n-6 LCPUFA in both TAG and phospholipid (PL) fractions (all p < 0.05). Flax-fed WT mice had lower liver TAG content compared to their KO counterparts. Menhaden-fed mice had significantly lower expression of key lipogenic (Scd1, Srebp-1c, Fasn, Fads1, Fads2), glyceroneogenic (Pck1), and TAG synthesis (Agpat3) genes compared to lard, with flax-fed mice showing some intermediate effects. Gene expression effects were independent of D6D activity, since no differences were detected between WT and KO mice fed the same diet. This study demonstrates that EPA/DHA and not ALA itself is critical for the prevention of hepatic steatosis.

Delta-6 desaturase (Fads2) deficiency alters triacylglycerol/fatty acid cycling in murine white adipose tissue.

The Δ-6 desaturase (D6D) enzyme is not only critical for the synthesis of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from α-linolenic acid (ALA), but recent evidence suggests that it also plays a role in adipocyte lipid metabolism and body weight; however, the mechanisms remain largely unexplored. The goal of this study was to investigate if a D6D deficiency would inhibit triacylglycerol storage and alter lipolytic and lipogenic pathways in mouse white adipose tissue (WAT) depots due to a disruption in EPA and DHA production. Male C57BL/6J D6D knockout (KO) and wild-type (WT) mice were fed either a 7% w/w lard or flax (ALA rich) diet for 21 weeks. Energy expenditure, physical activity, and substrate utilization were measured with metabolic caging. Inguinal and epididymal WAT depots were analyzed for changes in tissue weight, fatty acid composition, adipocyte size, and markers of lipogenesis, lipolysis, and insulin signaling. KO mice had lower body weight, higher serum nonesterified fatty acids, smaller WAT depots, and reduced adipocyte size compared to WT mice without altered food intake, energy expenditure, or physical activity, regardless of the diet. Markers of lipogenesis and lipolysis were more highly expressed in KO mice compared to WT mice in both depots, regardless of the diet. These changes were concomitant with lower basal insulin signaling in WAT. Collectively, a D6D deficiency alters triacylglycerol/fatty acid cycling in WAT by promoting lipolysis and reducing fatty acid re-esterification, which may be partially attributed to a reduction in WAT insulin signaling.

A reduction of skeletal muscle DHA content does not result in impaired whole body glucose tolerance or skeletal muscle basal insulin signaling in otherwise healthy mice.

Delta-6 desaturase (D6D), encoded by the Fads2 gene, catalyzes the first step in the conversion of α-linolenic acid to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The ablation of D6D in whole body Fads2-/- knockout (KO) mice results in an inability to endogenously produce EPA and DHA. Evidence supports a beneficial role for EPA and DHA on insulin-stimulated glucose disposal in skeletal muscle in the context of a metabolic challenge; however, it is unknown how low EPA and DHA levels impact skeletal muscle fatty acid composition and insulin signaling in a healthy context. The objective of this study was to examine the impact of ablating the endogenous production of EPA and DHA on skeletal muscle fatty acid composition, whole body glucose and insulin tolerance, and a key marker of skeletal muscle insulin signaling (pAkt). Male C57BL/6J wild-type (WT), Fads2+/- heterozygous, and Fads2-/- KO mice were fed a low-fat diet (16% kcal from fat) modified to contain either 7% w/w lard or 7% w/w flaxseed for 21 wk. No differences in total phospholipid (PL), triacylglycerol, or reactive lipid content were observed between genotypes. As expected, KO mice on both diets had significantly less DHA content in skeletal muscle PL. Despite this, KO mice did not have significantly different glucose or insulin tolerance compared with WT mice on either diet. Basal pAktSer473 was not significantly different between the genotypes within each diet. Ultimately, this study shows for the first time, to our knowledge, that the reduction of DHA in skeletal muscle is not necessarily detrimental to glucose homeostasis in otherwise healthy animals.NEW & NOTEWORTHY Skeletal muscle is the primary location of insulin-stimulated glucose uptake. EPA and DHA supplementation has been observed to improve skeletal muscle insulin-stimulated glucose uptake in models of metabolic dysfunction. Fads2-/- knockout mice cannot endogenously produce long-chain n-3 polyunsaturated fatty acids. Our results show that the absence of DHA in skeletal muscle is not detrimental to whole body glucose homeostasis in healthy mice.

Compensatory induction of Fads1 gene expression in heterozygous Fads2-null mice and by diet with a high n-6/n-3 PUFA ratio.

In mammals, because they share a single synthetic pathway, n-6/n-3 ratios of dietary PUFAs impact tissue arachidonic acid (ARA) and DHA content. Likewise, SNPs in the human fatty acid desaturase (FADS) gene cluster impact tissue ARA and DHA. Here we tested the feasibility of using heterozygous Fads2-null-mice (HET) as an animal model of human FADS polymorphisms. WT and HET mice were fed diets with linoleate/α-linolenate ratios of 1:1, 7:1, and 44:1 at 7% of diet. In WT liver, ARA and DHA in phospholipids varied >2× among dietary groups, reflecting precursor ratios. Unexpectedly, ARA content was only <10% lower in HET than in WT livers, when fed the 44:1 diet, likely due to increased Fads1 mRNA in response to reduced Fads2 mRNA in HET. Consistent with the RNA data, C20:3n-6, which is elevated in minor FADS haplotypes in humans, was lower in HET than WT. Diet and genotype had little effect on brain PUFAs even though brain Fads2 mRNA was low in HET. No differences in cytokine mRNA were found among groups under unstimulated conditions. In conclusion, differential PUFA profiles between HET mice and human FADS SNPs suggest low expression of both FADS1 and 2 genes in human minor haplotypes.

Successful dietary changes correlate with weight-loss outcomes in a new dietary weight-loss program.

Background: Currently available behavioral and dietary weight-loss programs lack magnitude and sustainability compared with bariatric surgery. A novel dietary weight-loss program was developed to assist participants in achieving sustainable diet changes by building knowledge and skills in food self-selection. Although the approach worked, a large variation was observed in outcome among participants. Objective: Determine factors affecting weight-loss outcomes among participants to further improve the efficacy of the program. Methods: Participants attended 19 dietary educational sessions during a 1-year intervention which included prescribed homework. Changes in weight, diet, and body composition were assessed. Results: Participants (n = 22) achieved mean body weight loss of -6.49(8.37%, p < 0.001) from baseline at 12 months. Nine participants (41%) achieved weight loss >5% of initial bodyweight; two reached a Body Mass Index 25 kg/m2. A large divergence in weight loss among participants was observed; successful (n = 9) achieved -12.9(9.6)% while unsuccessful achieved -2.03(2.78)%. Dietary protein and fiber density by 24-h records showed a significant and inverse correlation with weight loss (%) throughout the program. Weight loss at 3 months and 12 months showed a strong correlation (r = 0.84). Participants with self-reported depression lost significantly less weight than those without depression at 12 months (p < 0.03). Conclusions: Divergence in weight-loss outcomes among the participants is likely due to a difference in successful dietary implementation. Intra-cohort analysis indicates early weight-loss success and early dietary implementation was predictive of long-term success.

Oils rich in α-linolenic acid independently protect against characteristics of fatty liver disease in the Δ6-desaturase null mouse.

Alpha-linolenic acid's (ALA) biological activity is poorly understood and primarily associated with its conversion to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Delta-6 desaturase (D6D) initiates the metabolism of linoleic acid (LA) and ALA to arachidonic acid, EPA, and DHA, respectively. In this study, D6D knock-out (D6KO) mice were used to evaluate the effects of ALA-rich oils in preventing hepatic steatosis and inflammation. D6KO and wild-type mice were fed 1 of 4 high-fat (14% w/w) diets: (i) lard (LD, 0% n-3 PUFA), (ii) canola oil + ARASCO (CD, 8% ALA), (iii) flax seed oil + ARASCO (FD, 55% ALA), (iv) menhaden oil (MD, 30% EPA/DHA) for 8 or 20 weeks. Livers of D6KO mice consuming CD and FD were depleted of EPA/DHA, and enriched in ALA. Markers of fat accumulation and inflammation were lowest in the MD-fed mice, at 8 and 20 weeks, regardless of genotype. CD- and FD-fed D6KO groups were found to have lower liver lipid accumulation and lower hepatic inflammation relative to the LD-fed mice at 8 weeks. In conclusion, while MD was the most protective, this study shows that ALA can act independently on risk factors associated with the development of fatty liver disease.

Menhaden oil, but not safflower or soybean oil, aids in restoring the polyunsaturated fatty acid profile in the novel delta-6-desaturase null mouse.

BACKGROUND: Polyunsaturated fatty acids (PUFA) have diverse biological effects, from promoting inflammation to preventing cancer and heart disease. Growing evidence suggests that individual PUFA may have independent effects in health and disease. The individual roles of the two essential PUFA, linoleic acid (LA) and α-linolenic acid (ALA), have been difficult to discern from the actions of their highly unsaturated fatty acid (HUFA) downstream metabolites. This issue has recently been addressed through the development of the Δ-6 desaturase knock out (D6KO) mouse, which lacks the rate limiting Δ-6 desaturase enzyme and therefore cannot metabolize LA or ALA. However, a potential confounder in this model is the production of novel Δ-5 desaturase (D5D) derived fatty acids when D6KO mice are fed diets containing LA and ALA, but void of arachidonic acid. OBJECTIVE: The aim of the present study was to characterize how the D6KO model differentially responds to diets containing the essential n-6 and n-3 PUFA, and whether the direct provision of downstream HUFA can rescue the phenotype and prevent the production of D5D fatty acids. METHODOLOGY: Liver and serum phospholipid (PL) fatty acid composition was examined in D6KO and wild type mice fed i) 10% safflower oil diet (SF, LA rich) ii) 10% soy diet (SO, LA+ALA) or iii) 3% menhaden oil +7% SF diet (MD, HUFA rich) for 28 days (n = 3-7/group). RESULTS: Novel D5D fatty acids were found in liver PL of D6KO fed SF or SO-fed mice, but differed in the type of D5D fatty acid depending on diet. Conversely, MD-fed D6KO mice had a liver PL fatty acid profile similar to wild-type mice. CONCLUSIONS: Through careful consideration of the dietary fatty acid composition, and especially the HUFA content in order to prevent the synthesis of D5D fatty acids, the D6KO model has the potential to elucidate the independent biological and health effects of the parent n-6 and n-3 fatty acids, LA and ALA.

A feasibility study to test a novel approach to dietary weight loss with a focus on assisting informed decision making in food selection.

Obesity is a significant contributor to the development of chronic diseases, some of which can be prevented or reversed by weight loss. However, dietary weight loss programs have shortcomings in the success rate, magnitude, or sustainability of weight loss. The Individualized Diet Improvement Program's (iDip) objective was to test the feasibility of a novel approach that helps individuals self-select a sustainable diet for weight loss and maintenance instead of providing weight loss products or rigid diet instructions to follow. The iDip study consisted of 22 dietary improvement sessions over 12 months with six months of follow-up. Daily weights were collected, and a chart summarizing progress was provided weekly. Six 24-hour dietary records were collected, and dietary feedback was provided in the form of a protein-fiber plot, in which protein/energy and fiber/energy of foods were plotted two-dimensionally together with a target box specific to weight loss or maintenance. An exit survey was conducted at 12 months. Twelve (nine female, 46.3±3.1 years (mean±SE)) of the initial 14 participants (BMI>28 kg/m2) completed all sessions. Mean percent weight loss (n = 12) at six and 12 months was -4.9%±1.1 (p = 0.001) and -5.4%±1.7 (p = 0.007), respectively. Weight loss varied among individuals at 12 months; top and bottom halves (n = 6 each) achieved -9.7%±1.7 (p = 0.0008) and -1.0%±1.4 weight loss, respectively. The 24-hour records showed a significant increase in protein density from baseline to final (4.1g/100kcal±0.3 vs. 5.7g/100kcal±0.5; p = 0.008). Although mean fiber density showed no significant change from the first month (1.3g/100kcal±0.1), the top half had significantly higher fiber/energy intake than the bottom half group. The survey suggested that all participants valued the program and its self-guided diet approach. In conclusion, half of the participants successfully lost >5% and maintained the lost weight for 12 months without strict diet instructions, showing the feasibility of the informed decision-making approach.

Deficiency in the omega-3 fatty acid pathway results in failure of acrosome biogenesis in mice.

An omega-3 fatty acid, docosahexaenoic acid (DHA), is enriched in testicular membrane phospholipids, but its function is not well understood. The Fads2 gene encodes an enzyme required for the endogenous synthesis of DHA. Using Fads2-null mice (Fads2-/-), we found in our preceding studies that DHA deficiency caused the arrest of spermiogenesis and male infertility, both of which were reversed by dietary DHA. In this study, we investigated a cellular mechanism underlying the DHA essentiality in spermiogenesis. Periodic acid-Schiff staining and acrosin immunohistochemistry revealed the absence of acrosomes in Fads2-/- round spermatids. Acrosin, an acrosomal marker, was scattered throughout the cytoplasm of the Fads2-/- spermatids, and electron microscopy showed that proacrosomal granules were formed on the trans-face of the Golgi. However, excessive endoplasmic reticulum and vesicles were present on the cis-face of the Golgi in Fads2-/- spermatids. The presence of proacrosomal vesicles but lack of a developed acrosome in Fads2-/- spermatids suggested failed vesicle fusion. Syntaxin 2, a protein involved in vesicle fusion, colocalized with acrosin in the acrosome of wild-type mice. In contrast, syntaxin 2 remained scattered in reticular structures and showed no extensive colocalization with acrosin in the Fads2-/- spermatids, suggesting failed fusion with acrosin-containing vesicles or failed transport and release of syntaxin 2 vesicles from Golgi. Dietary supplementation of DHA in Fads2-/- mice restored an intact acrosome. In conclusion, acrosome biogenesis under DHA deficiency is halted after release of proacrosomal granules. Misplaced syntaxin 2 suggests an essential role of DHA in proper delivery of membrane proteins required for proacrosomal vesicle fusion.

Docosahexaenoic acid supplementation fully restores fertility and spermatogenesis in male delta-6 desaturase-null mice.

Delta-6 desaturase-null mice ((-/-)) are unable to synthesize highly unsaturated fatty acids (HUFAs): arachidonic acid (AA), docosahexaenoic acid (DHA), and n6-docosapentaenoic acid (DPAn6). The (-/-) males exhibit infertility and arrest of spermatogenesis at late spermiogenesis. To determine which HUFA is essential for spermiogenesis, a diet supplemented with either 0.2% (w/w) AA or DHA was fed to wild-type ((+/+)) and (-/-) males at weaning until 16 weeks of age (n = 3-5). A breeding success rate of DHA-supplemented (-/-) was comparable to (+/+). DHA-fed (-/-) showed normal sperm counts and spermiogenesis. Dietary AA was less effective in restoring fertility, sperm count, and spermiogenesis than DHA. Testis fatty acid analysis showed restored DHA in DHA-fed (-/-), but DPAn6 remained depleted. In AA-fed (-/-), AA was restored at the (+/+) level, and 22:4n6, an AA elongated product, accumulated in testis. Cholesta-3,5-diene was present in testis of (+/+) and DHA-fed (-/-), whereas it diminished in (-/-) and AA-fed (-/-), suggesting impaired sterol metabolism in these groups. Expression of spermiogenesis marker genes was largely normal in all groups. In conclusion, DHA was capable of restoring all observed impairment in male reproduction, whereas 22:4n6 formed from dietary AA may act as an inferior substitute for DHA.

Changes in behavior and fatty acid composition induced by long-term reduction in murine Δ6-desaturation activity.

Polyunsaturated fatty acids (PUFAs), especially arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), play an important role in biological regulation. In our previous study using mice deficient in Δ6 desaturase (D6D), we reported that ARA is required for body growth, while DHA is necessary for functional development. In mammals, ARA and DHA are supplied directly or by synthesis from linoleic acid (LA) and α-linolenic acid (ALA). However, as desaturase enzyme activity is immature or low in newborns, and humans with minor alleles of the gene encoding desaturase, respectively, they require dietary supplementation with ARA and DHA. To investigate how the body reacts to a long-term reduction in fatty acid synthesis, we measured behavioral changes and fatty acid composition in mice heterozygous for the D6D null mutation with reduced D6D activity fed a diet containing only LA and ALA as PUFAs. During the growth-maturity period, heterozygous mice showed a slightly change in interest and curiosity compared with the wild-type group. ARA levels were decreased in the brain and liver in the heterozygous group, especially during the growth-maturity period, whereas DHA levels were decreased in the liver only in the old age period, suggesting that there are differences in the synthesis of and demand for ARA and DHA during life. For newborns, and humans with minor alleles with low desaturase activity, direct ARA intake is particularly important during the growth-maturity period, but they may need to be supplemented with DHA in the old age period. Further research is needed to determine the optimal intake and duration of these fatty acids.

Hydrolyzed Fat Formula Increases Brain White Matter in Small for Gestational Age and Appropriate for Gestational Age Neonatal Piglets.

Background: Intrauterine growth restriction is a common cause of small for gestational age (SGA) infants worldwide. SGA infants are deficient in digestive enzymes required for fat digestion and absorption compared to appropriate for gestational age (AGA) infants, putting them at risk for impaired neurocognitive development. Objective: The objective was to determine if a hydrolyzed fat (HF) infant formula containing soy free fatty acids, 2-monoacylglycerolpalmitate, cholesterol, and soy lecithin could increase brain tissue incorporation of essential fatty acids or white matter to enhance brain development in SGA and AGA neonatal piglet models. Methods: Sex-matched, littermate pairs of SGA (0.5-0.9 kg) and AGA (1.2-1.8 kg) 2 days old piglets (N = 60) were randomly assigned to control (CON) or HF formula diets in a 2 × 2 factorial design. On day 14, 24 piglets were used for hippocampal RNA-sequencing; the rest began a spatial learning task. On days 26-29, brain structure was assessed by magnetic resonance imaging (MRI). Cerebellum and hippocampus were analyzed for fatty acid content. Results: SGA piglets grew more slowly than AGA piglets, with no effect of diet on daily weight gain or weight at MRI. HF diet did not affect brain weight. HF diet increased relative volumes of 7 brain regions and white matter (WM) volume in both SGA and AGA piglets. However, HF did not ameliorate SGA total WM integrity deficits. RNA sequencing revealed SGA piglets had increased gene expression of synapse and cell signaling pathways and decreased expression of ribosome pathways in the hippocampus compared to AGA. HF decreased expression of immune response related genes in the hippocampus of AGA and SGA piglets, but did not correct gene expression patterns in SGA piglets. Piglets learned the T-maze task at the same rate, but SGA HF, SGA CON, and AGA HF piglets had more accurate performance than AGA CON piglets on reversal day 2. HF increased arachidonic acid (ARA) percentage in the cerebellum and total ARA in the hippocampus. Conclusions: HF enhanced brain development in the neonatal piglet measured by brain volume and WM volume in specific brain regions; however, more studies are needed to assess long-term outcomes.

Disruption of FADS2 gene in mice impairs male reproduction and causes dermal and intestinal ulceration.

Delta-6 desaturase (D6D) catalyzes the first step in the synthesis of highly unsaturated fatty acids (HUFA) such as arachidonic (AA), docosapentaenoic (DPAn-6), and docosahexaenoic (DHA) acids, as well as the last desaturation of DPAn-6 and DHA. We created D6D-null mice (-/-), which enabled us to study HUFA deficiency without depleting their precursors. In -/-, no in vivo AA synthesis was detected after administration of [U-(13)C]linoleic acid (LA), indicating absence of D6D isozyme. Unexpectedly, all of the -/- developed ulcerative dermatitis when fed a purified diet lacking D6D products but containing ample LA. The -/- also exhibited splenomegaly and ulceration in duodenum and ileocecal junction. Male -/- lacked normal spermatozoa with a severe impairment of spermiogenesis. Tissue HUFAs in -/- declined differentially: liver AA and DHA by 95%, and a smaller decrease in brain and testes. Dietary AA completely prevented dermatitis and intestinal ulcers in -/-. DPAn-6 was absent in -/- brain under AA supplementation, indicating absence of D6D isozyme for DPAn-6 synthesis from AA. This study demonstrated a distinct advantage of the D6D-null mice (-/-) to elucidate (1) AA function without complication of LA deprivation and (2) DHA function in the nervous system without AA depletion or DPAn-6 replacement seen in traditional models.

Dietary fructose induces a wide range of genes with distinct shift in carbohydrate and lipid metabolism in fed and fasted rat liver.

Dietary fructose has been suspected to contribute to development of metabolic syndrome. However, underlying mechanisms of fructose effects are not well characterized. We investigated metabolic outcomes and hepatic expression of key regulatory genes upon fructose feeding under well defined conditions. Rats were fed a 63% (w/w) glucose or fructose diet for 4 h/day for 2 weeks, and were killed after feeding or 24-hour fasting. Liver glycogen was higher in the fructose-fed rats, indicating robust conversion of fructose to glycogen through gluconeogenesis despite simultaneous induction of genes for de novo lipogenesis and increased liver triglycerides. Fructose feeding increased mRNA of previously unidentified genes involved in macronutrient metabolism including fructokinase, aldolase B, phosphofructokinase-1, fructose-1,6-bisphosphatase and carbohydrate response element binding protein (ChREBP). Activity of glucose-6-phosphate dehydrogenase, a key enzyme for ChREBP activation, remained elevated in both fed and fasted fructose groups. In the fasted liver, the fructose group showed lower non-esterified fatty acids, triglycerides and microsomal triglyceride transfer protein mRNA, suggesting low VLDL synthesis even though plasma VLDL triglycerides were higher. In conclusion, fructose feeding induced a broader range of genes than previously identified with simultaneous increase in glycogen and triglycerides in liver. The induction may be in part mediated by ChREBP.

Replacing dietary glucose with fructose increases ChREBP activity and SREBP-1 protein in rat liver nucleus.

Diets high in fructose cause hypertriglyceridemia and insulin resistance in part due to simultaneous induction of gluconeogenic and lipogenic genes in liver. We investigated the mechanism underlying the unique pattern of gene induction by dietary fructose. Male Sprague-Dawley rats (n=6 per group) were meal-fed (4h/d) either 63% (w/w) glucose or 63% fructose diet. After two weeks, animals were killed at the end of the last meal. Nuclear SREBP-1 was 2.2 times higher in fructose-fed rats than glucose-fed rats. Nuclear FoxO1 was elevated 1.7 times in fructose group, but did not reach significance (P=0.08). Unexpectedly, no difference was observed in nuclear ChREBP between two groups. However, ChREBP DNA binding was 3.9x higher in fructose-fed animals without an increase in xylulose-5-phospate, a proposed ChREBP activator. In conclusion, the gene induction by dietary fructose is likely to be mediated in part by simultaneously increased ChREBP activity, SREBP-1 and possibly FoxO1 protein in nucleus.

Reduced delta-6 desaturase activity partially protects against high-fat diet-induced impairment in whole-body glucose tolerance.

Delta-6 desaturase (D6D), which is encoded by the fatty acid desaturase (Fads2) gene, is the rate-limiting enzyme for the endogenous production of n-3 long-chain polyunsaturated fatty acids. The absence of D6D activity in Fads2-/- knockout mice results in the inability to produce eicosapentaenoic acid and docosahexaenoic acid, and has previously been associated with altered glucose and lipid metabolism. Skeletal muscle is a major site for insulin-stimulated glucose disposal; however, the consequences of reduced D6D activity on skeletal muscle metabolism are unknown. The objective of this study was to examine the role of a partial reduction in D6D activity on whole-body glucose tolerance, skeletal muscle fatty acid profiles and protein content of key markers of carbohydrate and fat signaling pathways in the context of both low- and high-fat diets. Male C57BL/6J heterozygous (Fads2+/-) and wild-type (WT) mice were fed either a low-fat (16% kcal from fat) or high-fat (HFD; 45% kcal from fat) diet for 21 weeks. Fads2+/- mice were protected from the HFD-induced impairment in glucose tolerance. Unexpectedly, HFD-fed Fads2+/- mice had reduced GLUT4 skeletal muscle protein content compared to their WT counterparts. No changes were detected in total protein content of key markers of fatty acid uptake, glycogen formation or substrate oxidation. This study shows that reduced D6D activity is partially protective against HFD-induced impairments in whole-body glucose tolerance but does not appear to be due to increased muscle GLUT4 content or total content of proteins regulating substrate utilization.

Dietary EPA and DHA prevent changes in white adipose tissue omega-3 PUFA and oxylipin content associated with a Fads2 deficiency.

Fatty acid desaturase 2 (Fads2) encodes the delta-6 desaturase (D6D) enzyme, which is rate-limiting for the endogenous production of omega-3 long-chain polyunsaturated fatty acids (LC-PUFA). Numerous studies have reported the cardiometabolic health benefits of omega-3 LC-PUFA. Humans carrying genetic variants in the FADS2 gene have reduced levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), as well as oxylipins, in blood, erythrocytes and white adipose tissue (WAT). Similar findings have been reported in whole-body Fads2-/- mice fed a diet deficient in omega-3 LC-PUFA. The objective of this study was to determine if a diet containing EPA and DHA would prevent the deficiencies in WAT lipid profiles seen in Fads2-/- mice fed a diet containing only ALA. Male C57BL/6 J Fads2-/- and wild type (WT) mice were fed a low fat (7% w/w) diet for 9 weeks containing either flaxseed oil + ARASCO (FD, containing~53% ALA) or menhaden oil (MD, containing~14% EPA and 10% DHA). Fads2-/- mice fed an ALA-enriched diet had reduced body weight, little-to-no omega-3 LC-PUFA and a near complete loss of all omega-3 derived oxylipins in both epididymal and inguinal WAT (P<.05) compared to their WT counterparts, as well as altered expression of key regulators of the fatty acid desaturase pathway. However, Fads2-/- mice fed a diet containing EPA and DHA prevented most of these changes. This study provides evidence that a diet containing EPA and DHA provides a nutritional strategy to prevent alterations in WAT lipid content caused by reduced D6D activity.

Differentiating the biological effects of linoleic acid from arachidonic acid in health and disease.

Dietary fatty acids are associated with the development of many chronic diseases, such as obesity, diabetes, cardiovascular disease, metabolic syndrome, and several cancers. This review explores the literature surrounding the combined and individual roles of n-6 PUFAs linoleic acid (LA) and arachidonic acid (AA) as they relate to immune and inflammatory response, cardiovascular health, liver health, and cancer. The evidence suggests that a pro-inflammatory view of LA and AA may be over simplified. Overall, this review highlights gaps in our understanding of the biological roles of LA, AA and their complex relationship with n-3 PUFA and the need for future studies that examine the roles of individual fatty acids, rather than groups.

Serum Lipid Concentrations and FADS Genetic Variants in Young Mexican College Students: The UP-AMIGOS Cohort Study.

BACKGROUND: Recent genome-wide association studies in the Mexican population have identified several genetic loci associated with blood lipid levels in adults. However, studies focusing on the fatty acid desaturase (FADS) gene cluster have been understudied in this population, even though it seems associated with lipid profiles in other ethnicities. The aim of this study was to test associations between single nucleotide polymorphisms (SNPs) in the FADS cluster (rs174546, rs1535, rs174548, rs174550, rs174450, and rs174618) and serum lipid profiles in young Mexicans. METHODS: Anthropometrics, serum lipid profiles, and FADS SNPs were measured in 998 subjects in the UP-AMIGOS cohort study. Genotype-phenotype (total cholesterol [TC], triglyceride [TG], high-density lipoprotein cholesterol [HDL-C], low-density lipoprotein cholesterol [LDL-C], and very-low-density lipoprotein [VLDL]) associations were assessed using PLINK adjusted for sex, age, and body mass index (BMI). RESULTS: Among 6 FADS SNPs, we found that carriers of the C-allele of the FADS1-rs174546 showed a significant association with lower TG concentrations (ß = -12.6 mg/dL, p = 0.009) and lower VLDL concentrations (ß = -2.52 mg/dL, p = 0.005). We found that rs174546, rs1535, and rs174550 were in high linkage disequilibrium (r2 > 0.80). There were no significant associations between rs174550, rs174548, and rs174618 and lipid profiles. CONCLUSION: A genetic variant in the FADS1 (rs174546) gene is a major contributor of plasma TG and VLDL concentrations in healthy young Mexicans.

Hepatocyte nuclear factor-4alpha contributes to carbohydrate-induced transcriptional activation of hepatic fatty acid synthase.

Refeeding a carbohydrate-rich meal after a fast produces a co-ordinated induction of key glycolytic and lipogenic genes in the liver. The transcriptional response is mediated by insulin and increased glucose oxidation, and both signals are necessary for optimal induction of FAS (fatty acid synthase). The glucose-regulated component of FAS promoter activation is mediated in part by ChREBP [ChoRE (carbohydrate response element)-binding protein], which binds to a ChoRE between -7300 and -7000 base-pairs in a carbohydrate-dependent manner. Using in vivo footprinting with nuclei from fasted and refed rats, we identify an imperfect DR-1 (direct repeat-1) element between -7110 and -7090 bp that is protected upon carbohydrate refeeding. Electrophoretic mobility-shift assays establish that this DR-1 element binds HNF-4alpha (hepatocyte nuclear factor 4alpha), and chromatin immunoprecipitation establishes that HNF-4alpha binding to this site is increased approx. 3-fold by glucose refeeding. HNF-4alpha transactivates reporter constructs containing the distal FAS promoter in a DR-1-dependent manner, and this DR-1 is required for full glucose induction of the FAS promoter in primary hepatocytes. In addition, a 3-fold knockdown of hepatocyte HNF-4alpha by small interfering RNA produces a corresponding decrease in FAS gene induction by glucose. Co-immunoprecipitation experiments demonstrate a physical interaction between HNF-4alpha and ChREBP in primary hepatocytes, further supporting an important complementary role for HNF-4alpha in glucose-induced activation of FAS transcription. Taken together, these observations establish for the first time that HNF-4alpha functions in vivo through a DR-1 element in the distal FAS promoter to enhance gene transcription following refeeding of glucose to fasted rats. The findings support the broader view that HNF-4alpha is an integral component of the hepatic nutrient sensing system that co-ordinates transcriptional responses to transitions between nutritional states.