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.

The effects of diet and chronic exercise on skeletal muscle ghrelin response.

Background: Recent findings indicate that ghrelin, particularly the unacylated form (UnAG), acutely stimulates skeletal muscle fatty acid oxidation (FAO) and can preserve insulin signaling and insulin-stimulated glucose uptake in the presence of high concentrations of saturated fatty acids. However, we recently reported that the stimulatory effect of ghrelin on FAO and subsequent ability to protect insulin stimulated glucose uptake was lost following 6-weeks (6w) of chronic high fat feeding. In the current study we examined the effects of both short-term 5 day (5d) and chronic 6w high-fat diet (HFD) on muscle ghrelin response, and whether exercise training could prevent the development of muscle ghrelin resistance with 6w of HFD. Methods and Results: Soleus muscle strips were isolated from male rats to determine the direct effects of acylated (AG) and UnAG isoforms on FAO and glucose uptake. A 5d HFD did not alter the response of soleus muscle to AG or UnAG. Conversely, 6w of HFD was associated with a loss of ghrelin's ability to stimulate FAO and protect insulin stimulated glucose uptake. Muscle response to UnAG remained intact following the 6w HFD with chronic exercise training. Unexpectedly, muscle response to both AG and UnAG was also lost after 6w of low-fat diet (LFD) consumption. Protein content of the classic ghrelin receptor, GHS-R1a, was not affected by diet or training. Corticotropin-releasing hormone receptor-2 (CRF-2R) content, a putative receptor for ghrelin in muscle, was significantly decreased in soleus from 6w HFD-fed animals and increased following exercise training. This may explain the protection of UnAG response with training in HFD-fed rats but does not explain why ghrelin response was also lost in LFD-fed animals. Conclusions: UnAG protects muscle glucose uptake during acute lipid oversupply, likely due to its ability to stimulate FAO. This effect is lost in 6w HFD-fed animals but protected with exercise training. Unexpectedly, ghrelin response was lost in 6w LFD-fed animals. The loss of ghrelin response in muscle with a LFD cannot be explained by a change in putative ghrelin receptor content. We believe that the sedentary nature of the animals is a major factor in the development of muscle ghrelin resistance and warrants further research.

Plant and marine N3-PUFA regulation of fatty acid trafficking along the adipose tissue-liver axis varies according to nutritional state.

Marine sourced N3-PUFA regulate lipid metabolism in adipose tissue and liver; however, less is known about plant sourced N3-PUFA. The goal of this study was to investigate plant and marine N3-PUFA regulation of fatty acid trafficking along the adipose tissue-liver axis according to nutritional state. Mice were fed low-fat diets (7% w/w) containing either lard, flaxseed, or menhaden oils for 8 weeks, and were euthanized in either fed or fasted states. Substrate utilization and physical activity were assessed during the transition from a fed to fasted state. Plasma biomarkers (triacylglycerol [TAG], non-esterified fatty acids [NEFA]), as well as liver and epididymal adipose tissue (eWAT) lipogenic and lipolytic markers, were measured. Neither plant nor marine N3-PUFA influenced substrate utilization or activity during the transition from a fed to fasted state. In the fed state, marine N3-PUFA reduced plasma TAG levels compared to the other diets, with no further reduction seen in fasted mice. Hepatic lipogenic markers (Fasn, Acc, Scd1, and Elovl6) were reduced in the fed state with marine N3-PUFA, but not plant N3-PUFA. In the fasted state, mice fed either N3-PUFA accumulated less liver TAG, had lower plasma NEFA, and suppressed eWAT HSL activity compared to lard. Marine N3-PUFA are more potent regulators of lipogenesis than plant N3-PUFA in the fed state, whereas both N3-PUFA influence eWAT lipolysis and plasma NEFA in the fasted state. This work provides novel insights regarding N3-PUFA regulation of fatty acid trafficking along the adipose tissue-liver axis according to nutritional state.

Regulation of adipose tissue lipolysis by ghrelin is impaired with high-fat diet feeding and is not restored with exercise.

Ghrelin is released from the stomach as an anticipatory signal prior to a meal and decreases immediately after. Previous research has shown that both acylated (AG) and unacylated (UnAG) ghrelin blunt adrenoreceptor-stimulated lipolysis in rat white adipose tissue (WAT) ex vivo. We investigated whether acute or chronic consumption of a high fat diet (HFD) impaired the ability of ghrelin to regulate adipose tissue lipolysis, and if this impairment could be restored with exercise. After 5 days (5d) of a HFD, or 6 weeks (6 w) of a HFD (60% kcal from fat) with or without exercise training, inguinal and retroperitoneal WAT was collected from anesthetized rats for adipose tissue organ culture. Samples were treated with 1 µM CL 316,243 (CL; lipolytic control), 1 µM CL+150 ng/ml AG or 1 µM CL+150 ng/ml UnAG. Incubation media and tissue were collected after 2 hours. Colorometric assays were used to determine glycerol and free fatty acid (FFA) concentrations in media. Western blots were used to quantify the protein content of lipolytic enzymes and ghrelin receptors in both depots. CL stimulated lipolysis was evidenced by increases in glycerol (p < 0.0001) and FFA (p < 0.0001) concentrations in media compared to control. AG decreased CL-stimulated glycerol release in inguinal WAT from 5d LFD rats (p = 0.0097). Neither AG nor UnAG blunted lipolysis in adipose tissue from 5d or 6 w HFD-fed rats, and exercise did not restore ghrelin's anti-lipolytic ability in 6 w HFD-fed rats. Overall, this study demonstrates that HFD consumption impairs ghrelin's ability to regulate adipose tissue lipolysis.

Unacylated ghrelin stimulates fatty acid oxidation to protect skeletal muscle against palmitate-induced impairment of insulin action in lean but not high-fat fed rats.

BACKGROUND: Ghrelin is a gut hormone that spikes in circulation before mealtime. Recent findings suggest that both ghrelin isoforms stimulate skeletal muscle fatty acid oxidation, lending to the possibility that it may regulate skeletal muscle's handling of meal-derived substrates. It was hypothesized in the current study that ghrelin may preserve muscle insulin response during conditions of elevated saturated fatty acid (palmitate) availability by promoting its oxidation. METHODS AND RESULTS: Soleus muscle strips were isolated from male rats to determine the direct effects of ghrelin isoforms on fatty acid oxidation, glucose uptake and insulin signaling. We demonstrate that unacylated ghrelin (UnAG) is the more potent stimulator of skeletal muscle fatty acid oxidation. Both isoforms of ghrelin generally protected muscle from impaired insulin-mediated phosphorylation of AKT Ser473 and Thr308, as well as downstream phosphorylation of AS160 Ser588 during high palmitate exposure. However, only UnAG was able to preserve insulin-stimulated glucose uptake during exposure to high palmitate concentrations. The use of etomoxir, an irreversible inhibitor of carnitine palmitoyltransferase (CPT-1) abolished this protection, strongly suggesting that UnAG's stimulation of fatty acid oxidation may be essential to this protection. To our knowledge, we are also the first to investigate the impact of a chronic high-fat diet on ghrelin's actions in muscle. Following 6 wks of a high-fat diet, UnAG was unable to preserve insulin-stimulated signaling or glucose transport during an acute high palmitate exposure. UnAG was also unable to further stimulate 5' AMP-activated protein kinase (AMPK) or fatty acid oxidation during high palmitate exposure. Corticotropin-releasing hormone receptor-2 (CRF-2R) content was significantly decreased in muscle from high-fat fed animals, which may partially account for the loss of UnAG's effects. CONCLUSIONS: UnAG is able to protect muscle from acute lipid exposure, likely due to its ability to stimulation fatty acid oxidation. This effect is lost in high-fat fed animals, implying a resistance to ghrelin at the level of the muscle. The underlying mechanisms accounting for ghrelin resistance in high fat-fed animals remain to be discovered.

Dietary n-6/n-3 Ratio Influences Brain Fatty Acid Composition in Adult Rats.

There is mounting evidence that diets supplemented with polyunsaturated fatty acids (PUFA) can impact brain biology and functions. This study investigated whether moderately high-fat diets differing in n-6/n-3 fatty acid ratio could impact fatty acid composition in regions of the brain linked to various psychopathologies. Adult male Sprague Dawley rats consumed isocaloric diets (35% kcal from fat) containing different ratios of linoleic acid (n-6) and alpha-linolenic acid (n-3) for 2 months. It was found that the profiles of PUFA in the prefrontal cortex, hippocampus, and hypothalamus reflected the fatty acid composition of the diet. In addition, region-specific changes in saturated fatty acids and monounsaturated fatty acids were detected in the hypothalamus, but not in the hippocampus or prefrontal cortex. This study in adult rats demonstrates that fatty acid remodeling in the brain by diet can occur within months and provides additional evidence for the suggestion that diet could impact mental health.

Her-2 Breast Cancer Outcomes Are Mitigated by Consuming n-3 Polyunsaturated, Saturated, and Monounsaturated Fatty Acids Compared to n-6 Polyunsaturated Fatty Acids.

Lifestyle habits, such as the consumption of a healthy diet, may prevent up to 30-50% of breast cancer (BC) cases. Dietary fats are of specific interest, as research provides strong evidence regarding the association of dietary fats and BC. However, there is limited research on the role of different types of fats including polyunsaturated (PUFA), monounsaturated (MUFA), and saturated fatty acids (SFA). The objective of this study was to determine the effects of lifelong exposure to various dietary fats on mammary tumour development over a 20-week period. Female heterozygous MMTV-neu (ndl) YD5 mouse models were fed five maternal diets containing (1) 10% safflower oil (n-6 PUFA, control), (2) 3% menhaden oil + 7% safflower oil (marine n-3 PUFA, control), (3) 3% flaxseed + 7% safflower oil (plant-based n-3 PUFA), (4) 10% olive oil (MUFA), or (5) 10% lard (SFA). The primary measures, tumour latency, volume, and multiplicity differed by diet treatment in the following general order, n-6 PUFA > plant n-3 PUFA, SFA, MUFA > marine n-3 PUFA. Overall, these findings show that the quality of the diet plays a significant role influencing mammary tumour outcomes.

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.

Marine fish oil is more potent than plant-based n-3 polyunsaturated fatty acids in the prevention of mammary tumors.

Marine-derived n-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have been shown to inhibit mammary carcinogenesis. However, evidence regarding plant-based α-linolenic acid (ALA), the major n-3 PUFA in the Western diet, remains equivocal. The objective of this study was to examine the effect of lifelong exposure to plant- or marine-derived n-3 PUFAs on pubertal mammary gland and tumor development in MMTV-neu(ndl)-YD5 mice. It is hypothesized that lifelong exposure to n-3 PUFA reduces terminal end buds during puberty leading to delayed tumor onset, volume and multiplicity. It is further hypothesized that plant-derived n-3 PUFAs will exert dose-dependent effects. Harems of MMTV-FVB males were bred with wild-type females and fed either a (1) 10% safflower (10% SF, n-6 PUFA, control), (2) 10% flaxseed (10% FS), (3) 7% safflower plus 3% flaxseed (3% FS) or (4) 7% safflower plus 3% menhaden (3% FO) diet. Female offspring were maintained on parental diets. Compared to SF, 10% FS and 3% FO reduced (P<.05) terminal end buds at 6 weeks and tumor volume and multiplicity at 20 weeks. A dose-dependent reduction of tumor volume and multiplicity was observed in mice fed 3% and 10% FS. Antitumorigenic effects were associated with altered HER2, pHER-2, pAkt and Ki-67 protein expression. Compared to 10% SF, 3% FO significantly down-regulated expression of genes involved in eicosanoid synthesis and inflammation. From this, it can be estimated that ALA was 1/8 as potent as EPA+DHA. Thus, marine-derived n-3 PUFAs have greater potency versus plant-based n-3 PUFAs.