Dietary n-3 long-chain polyunsaturated fatty acids upregulate energy dissipating metabolic pathways conveying anti-obesogenic effects in mice.

BACKGROUND: We previously reported on the anti-obesogenic and anti-inflammatory effects associated with n-3 long-chain polyunsaturated fatty acids (LCPUFA) in our diet-induced obesity (DIO) mouse model. Two isocaloric high-fat diets (HFDs; 48 kJ% fat), HFD (HF) and n-3 LCPUFA-enriched HFD (HF/n-3), and a control diet (C; 13 kJ% fat) were used. The underlying mechanisms however have largely remained unclear. Here, we assessed whether the reduced fat mass reflected n-3 LCPUFA-induced expression changes in lipid metabolism of the intestine, liver, and interscapular brown adipose tissue (iBAT), as well as increased iBAT thermogenic capacity. METHODS: For HF/n-3, saturated and monounsaturated fatty acids were partially substituted by n-3 LCPUFA eicosapentaenoic acid and docosahexaenoic acid to achieve a balanced n-6/n-3 PUFA ratio (0.84) compared to the unbalanced ratios of HF (13.5) and C (9.85). Intestine, liver and iBAT from male C57BL/6 J mice, fed defined soybean/palm oil-based diets for 12 weeks, were further analysed. Gene and protein expression analyses, immunohistochemistry and correlation analyses for metabolic interactions were performed. RESULTS: Compared to HF and C, our analyses suggest significantly diminished de novo lipogenesis (DNL) and/or increased hepatic and intestinal fatty acid oxidation (ω-oxidation and peroxisomal ß-oxidation) in HF/n-3 mice. For iBAT, the thermogenic potential was enhanced upon HF/n-3 consistent with upregulated expression for uncoupling protein-1 and genes involved in mitochondrial biogenesis. In addition, a higher capacity for the supply and oxidation of fatty acids was observed and expression and correlation analyses indicated a coordinated regulation of energy metabolism and futile cycling of triacylglycerol (TAG). Moreover, HF/n-3 significantly increased the number of anti-inflammatory macrophages and eosinophils and significantly enhanced the levels of activated AMP-activated protein kinase α (AMPKα), peroxisome proliferator-activated receptor α (PPARα) and fibroblast growth factor 21 (FGF21). CONCLUSIONS: Our data suggest that by targeting transcriptional regulatory pathways, AMPKα, and FGF21 as potential mediators, HF/n-3 activated less efficient pathways for energy production, such as peroxisomal ß-oxidation, increased ATP consumption upon the induction of futile cycling of TAG, and additionally increased the thermogenic and oxidative potential of iBAT. Therefore, we consider n-3 LCPUFA as the potent inducer for upregulating energy dissipating metabolic pathways conveying anti-obesogenic effects in mice.

Medium-chain dicarboxylic acylcarnitines as markers of n-3 PUFA-induced peroxisomal oxidation of fatty acids.

SCOPE: Omega-3 polyunsaturated fatty acids (n-3 PUFA) found in fish oil activate PPAR-α, stimulate peroxisomal fatty acid (FA) ß-oxidation and prevent impairments on glucose homeostasis. METHODS AND RESULTS: Glucose metabolism and FA oxidation were studied in C57/Bl6 mice fed with diets containing either 3.6 and 31.5% fish oil or lard. To assess the effects of peroxisomal proliferation on FA oxidation independent of n-3 PUFA intake, mice were treated with the PPAR-α agonist WY-14643. n-3 PUFA-fed mice were protected from glucose intolerance and dyslipidemia compared to animals fed a lard-based high-fat diet. Most importantly, mice fed on the hyperlipidic diet based on fish oil as well as the WY-14643 treated mice showed twofold increase of odd, medium-chain, dicarboxylic acylcarnitines in the liver suggesting that not only ß-oxidation, but also α- and ω-oxidation of FA were increased. Finally, an oxidation assay using liver homogenates and palmitic acid as substrate revealed an over tenfold increased production of similar acylcarnitines, indicating that FA are their precursors. CONCLUSION: This study shows at the metabolite level that peroxisome proliferation induced either by fish oil or WY-14643 is associated with increased α- and ω-oxidation of FA producing specific acylcarnitines that can be utilized as biomarkers of peroxisomal FA oxidation.

Methyl-donor supplementation in obese mice prevents the progression of NAFLD, activates AMPK and decreases acyl-carnitine levels.

Non-alcoholic fatty liver disease (NAFLD) results from increased hepatic lipid accumulation and steatosis, and is closely linked to liver one-carbon (C1) metabolism. We assessed in C57BL6/N mice whether NAFLD induced by a high-fat (HF) diet over 8 weeks can be reversed by additional 4 weeks of a dietary methyl-donor supplementation (MDS). MDS in the obese mice failed to reverse NAFLD, but prevented the progression of hepatic steatosis associated with major changes in key hepatic C1-metabolites, e.g. S-adenosyl-methionine and S-adenosyl-homocysteine. Increased phosphorylation of AMPK-α together with enhanced ß-HAD activity suggested an increased flux through fatty acid oxidation pathways. This was supported by concomitantly decreased hepatic free fatty acid and acyl-carnitines levels. Although HF diet changed the hepatic phospholipid pattern, MDS did not. Our findings suggest that dietary methyl-donors activate AMPK, a key enzyme in fatty acid ß-oxidation control, that mediates increased fatty acid utilization and thereby prevents further hepatic lipid accumulation.

Metabolic and immunomodulatory effects of n-3 fatty acids are different in mesenteric and epididymal adipose tissue of diet-induced obese mice.

In studies emphasizing antiobesogenic and anti-inflammatory effects of long-chain n-3 polyunsaturated fatty acids (LC-n-3 PUFA), diets with very high fat content, not well-defined fat quality, and extreme n-6/n-3 PUFA ratios have been applied frequently. Additionally, comparative analyses of visceral adipose tissues (VAT) were neglected. Considering the link of visceral obesity to insulin resistance or inflammatory bowel diseases, we hypothesized that VAT, especially mesenteric adipose tissue (MAT), may exhibit differential responsiveness to diets through modulation of metabolic and inflammatory processes. Here, we aimed to assess dietary LC-n-3 PUFA effects on MAT and epididymal adipose tissue (EAT) and on MAT-adjacent liver and intestine in diet-induced obese mice fed defined soybean/palm oil-based diets. High-fat (HF) and LC-n-3 PUFA-enriched high-fat diet (HF/n-3) contained moderately high fat with unbalanced and balanced n-6/n-3 PUFA ratios, respectively. Body composition/organ analyses, glucose tolerance test, measurements of insulin, lipids, mRNA and protein expression, and immunohistochemistry were applied. Compared with HF, HF/n-3 mice showed reduced fat mass, smaller adipocytes in MAT than EAT, improved insulin level, and lower hepatic triacylglycerol and plasma NEFA levels, consistent with liver and brown fat gene expression. Gene expression arrays pointed to immune cell activation in MAT and alleviation of intestinal endothelial cell activation. Validations demonstrated simultaneously upregulated pro- (TNFα, MCP-1) and anti-inflammatory (IL-10) cytokines and M1/M2-macrophage markers in VAT and reduced CD4/CD8α expression in MAT and spleen. Our data revealed differential responsiveness to diets for VAT through preferentially metabolic alterations in MAT and inflammatory processes in EAT. LC-n-3 PUFA effects were pro- and anti-inflammatory and disclose T cell-immunosuppressive potential.

Adaptation of AmtR-controlled gene expression by modulation of AmtR binding activity in Corynebacterium glutamicum.

In corynebacteria, nitrogen regulation is controlled by the TetR family protein AmtR, which was extensively studied in the last years. In frame of these studies a number of AmtR binding sites were identified and characterized and it became obvious that for distinct genes the number and sequences of these sites varied significantly. In this study, the influence of numbers and alterations of AmtR binding sites were addressed by in vivo and in vitro studies. It can be concluded that in general a single highly conserved AmtR site is sufficient for stringent regulation and that non-conserved binding sites have a very limited influence, despite the fact that binding of AmtR was shown for several of these sites, e.g. upstream of amtA, amtB and gdh. Furthermore, the reason for and consequences of the lack of AmtR autoregulation were addressed in vivo. The introduction of a spacing nucleotide between the two conserved half sites of the AmtR binding box alone is sufficient to restore AmtR autoregulation. The main differences observed between wild type and an AmtR autoregulation strain were a slightly enhanced background of transcription of AmtR-controlled genes and a slightly slower response to nitrogen limitation.