Distinct roles of adipose triglyceride lipase and hormone-sensitive lipase in the catabolism of triacylglycerol estolides.

Branched esters of palmitic acid and hydroxy stearic acid are antiinflammatory and antidiabetic lipokines that belong to a family of fatty acid (FA) esters of hydroxy fatty acids (HFAs) called FAHFAs. FAHFAs themselves belong to oligomeric FA esters, known as estolides. Glycerol-bound FAHFAs in triacylglycerols (TAGs), named TAG estolides, serve as metabolite reservoir of FAHFAs mobilized by lipases upon demand. Here, we characterized the involvement of two major metabolic lipases, adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL), in TAG estolide and FAHFA degradation. We synthesized a library of 20 TAG estolide isomers with FAHFAs varying in branching position, chain length, saturation grade, and position on the glycerol backbone and developed an in silico mass spectra library of all predicted catabolic intermediates. We found that ATGL alone or coactivated by comparative gene identification-58 efficiently liberated FAHFAs from TAG estolides with a preference for more compact substrates where the estolide branching point is located near the glycerol ester bond. ATGL was further involved in transesterification and remodeling reactions leading to the formation of TAG estolides with alternative acyl compositions. HSL represented a much more potent estolide bond hydrolase for both TAG estolides and free FAHFAs. FAHFA and TAG estolide accumulation in white adipose tissue of mice lacking HSL argued for a functional role of HSL in estolide catabolism in vivo. Our data show that ATGL and HSL participate in the metabolism of estolides and TAG estolides in distinct manners and are likely to affect the lipokine function of FAHFAs.

Intramuscular short-chain acylcarnitines in elderly people are decreased in (pre-)frail females, but not in males.

This study tested the hypothesis that in human aging, a decreased intramuscular acylcarnitine status is associated with (pre-)frailty, reduced physical performance, and altered mitochondrial function. We used a cross-sectional study design with well-matched fit and (pre-)frail old males and females, using young males and females as healthy controls. Frailty was assessed according to the Fried criteria and physical performance was determined by 400 m walk test, short physical performance battery and handgrip strength. Muscle and plasma acylcarnitine status, and muscle mitochondrial gene expression was analyzed. Results showed that intramuscular total carnitine levels and short-chain acylcarnitine levels were lower in (pre-)frail old females compared to fit old females and young females, whereas no differences were observed in males. The low intramuscular short-chain acylcarnitine levels in females correlated with low physical performance, even after correction for muscle mass (%), and were accompanied with lowered expression of genes involved in mitochondrial energy production and functionality. It is, therefore, concluded that in (pre-)frail old females, intramuscular total carnitine levels and short-chain acylcarnitine levels are decreased, and this decrease is associated with reduced physical performance and low expression of a wide range of genes critical for mitochondrial function. The results stress the importance of taking sex differences into account in aging research.

Branched and linear fatty acid esters of hydroxy fatty acids (FAHFA) relevant to human health.

Fatty acid esters of hydroxy fatty acids (FAHFAs) represent a complex lipid class that contains both signaling mediators and structural components of lipid biofilms in humans. The majority of endogenous FAHFAs share a common chemical architecture, characterized by an estolide bond that links the hydroxy fatty acid (HFA) backbone and the fatty acid (FA). Two structurally and functionally distinct FAHFA superfamilies are recognized based on the position of the estolide bond: omega-FAHFAs and in-chain branched FAHFAs. The existing variety of possible HFAs and FAs combined with the position of the estolide bond generates a vast quantity of unique structures identified in FAHFA families. In this review, we discuss the anti-diabetic and anti-inflammatory effects of branched FAHFAs and the role of omega-FAHFA-derived lipids as surfactants in the tear film lipid layer and dry eye disease. To emphasize potential pharmacological targets, we recapitulate the biosynthesis of the HFA backbone within the superfamilies together with the degradation pathways and the FAHFA regioisomer distribution in human and mouse adipose tissue. We propose a theoretical involvement of cytochrome P450 enzymes in the generation and degradation of saturated HFA backbones and present an overview of small-molecule inhibitors used in FAHFA research. The FAHFA lipid class is huge and largely unexplored. Besides the unknown biological effects of individual FAHFAs, also the enigmatic enzymatic machinery behind their synthesis could provide new therapeutic approaches for inflammatory metabolic or eye diseases. Therefore, understanding the mechanisms of (FA)HFA synthesis at the molecular level should be the next step in FAHFA research.

The role of peroxiredoxin 6 in biosynthesis of FAHFAs.

Peroxiredoxin 6 (Prdx6) is a multifunctional enzyme, a unique member of the peroxiredoxin family, with an important role in antioxidant defense. Moreover, it has also been linked with the biosynthesis of anti-inflammatory and anti-diabetic lipids called fatty acid esters of hydroxy fatty acids (FAHFAs) and many diseases, including cancer, inflammation, and metabolic disorders. Here, we performed metabolomic and lipidomic profiling of subcutaneous adipose tissue from mouse models with genetically modified Prdx6. Deletion of Prdx6 resulted in reduced levels of FAHFAs containing 13-hydroxylinoleic acid (13-HLA). Mutation of Prdx6 C47S impaired the glutathione peroxidase activity and reduced FAHFA levels, while D140A mutation, responsible for phospholipase A2 activity, showed only minor effects. Targeted analysis of oxidized phospholipids and triacylglycerols in adipocytes highlighted a correlation between FAHFA and hydroxy fatty acid production by Prdx6 or glutathione peroxidase 4. FAHFA regioisomer abundance was negatively affected by the Prdx6 deletion, and this effect was more pronounced in longer and more unsaturated FAHFAs. The predicted protein model of Prdx6 suggested that the monomer-dimer transition mechanism might be involved in the repair of longer-chain peroxidized phospholipids bound over two monomers and that the role of Prdx6 in FAHFA synthesis might be restricted to branching positions further from carbon 9. In conclusion, our work linked the peroxidase activity of Prdx6 with the levels of FAHFAs in adipose tissue.

Triacylglycerols containing branched palmitic acid ester of hydroxystearic acid (PAHSA) are present in the breast milk and hydrolyzed by carboxyl ester lipase.

Breast milk is a complex mixture containing underexplored bioactive lipids. We performed an observational case-control study to compare the impact of delivery mode: caesarean section (CS) and vaginal birth (VB); and term (preterm and term delivery) on the levels of lipokines in human milk at different stages of lactation. Metabolomic analysis of the milk identified triacylglycerol estolides as a metabolic reservoir of the anti-inflammatory lipid mediator 5-palmitic acid ester of hydroxystearic acid (5-PAHSA). We found that triacylglycerol estolides were substrates of carboxyl ester lipase and 5-PAHSA-containing lipids were the least preferred substrates among tested triacylglycerol estolide isomers. This explained exceptionally high colostrum levels of 5-PAHSA in the VB group. CS and preterm birth negatively affected colostrum lipidome, including 5-PAHSA levels, but the lipidomic profiles normalized in mature milk. Mothers delivering term babies vaginally produce colostrum rich in 5-PAHSA, which could contribute to the prevention of intestinal inflammation in newborns.

Understanding FAHFAs: From structure to metabolic regulation.

The discovery of branched fatty acid esters of hydroxy fatty acids (FAHFAs) in humans draw attention of many researches to their biological effects. Although FAHFAs were originally discovered in insects and plants, their introduction into the mammalian realm opened new horizons in bioactive lipid research. Hundreds of isomers from different families have been identified so far and their role in (patho) physiological processes is currently being explored. The family of palmitic acid esters of hydroxy stearic acids (PAHSAs), especially 5-PAHSA and 9-PAHSA regioisomers, stands out in the crowd of other FAHFAs for their anti-inflammatory and anti-diabetic effects. Beneficial effects of PAHSAs have been linked to metabolic disorders such as type 1 and type 2 diabetes, colitis, and chronic inflammation. Besides PAHSAs, a growing family of polyunsaturated FAHFAs exerts mainly immunomodulatory effects and biological roles of many other FAHFAs remain currently unknown. Therefore, FAHFAs represent unique lipid messengers capable of affecting many immunometabolic processes. The objective of this review is to summarize the knowledge concerning the diversity of FAHFAs, nomenclature, and their analysis and detection. Special attention is paid to the total syntheses of FAHFAs, optimal strategies, and to the formation of the stereocenter required for optically active molecules. Biosynthetic pathways of saturated and polyunsaturated FAHFAs in mammals and plants are reviewed together with their metabolism and degradation. Moreover, an overview of biological effects of branched FAHFAs is provided and many unanswered questions regarding FAHFAs are discussed.

Triacylglycerol-Rich Oils of Marine Origin are Optimal Nutrients for Induction of Polyunsaturated Docosahexaenoic Acid Ester of Hydroxy Linoleic Acid (13-DHAHLA) with Anti-Inflammatory Properties in Mice.

SCOPE: The docosahexaenoic acid ester of hydroxy linoleic acid (13-DHAHLA) is a bioactive lipid with anti-inflammatory properties from the family of fatty acid esters of hydroxy fatty acids (FAHFA). METHODS AND RESULTS: To explore the biosynthesis of 13-DHAHLA from dietary oils, C57BL/6N mice are gavaged for 8 days with various corn oil/marine oil mixtures containing the same amount of DHA. Plasma levels of omega-3 FAHFAs are influenced by the lipid composition of the mixtures but do not reflect the changes in bioavailability of polyunsaturated fatty acids in plasma. Triacylglycerol-bound DHA and linoleic acid serve as more effective precursors for 13-DHAHLA synthesis than DHA bound in phospholipids or wax esters. Both 13(S)- and 13(R)-DHAHLA inhibit antigen and PGE2 -induced chemotaxis and degranulation of mast cells to a comparable extent and 13(S)-DHAHLA is identified as the predominant isomer in mouse adipose tissue. CONCLUSION: Here, the optimal nutritional source of DHA is identified, which supports production of anti-inflammatory FAHFAs, as triacylglycerol-based marine oil and also reveals a possible role of triacylglycerols in the synthesis of FAHFA lipokines.

Lipokine 5-PAHSA Is Regulated by Adipose Triglyceride Lipase and Primes Adipocytes for De Novo Lipogenesis in Mice.

Branched esters of palmitic acid and hydroxystearic acid (PAHSA) are anti-inflammatory and antidiabetic lipokines that connect glucose and lipid metabolism. We aimed to characterize involvement of the 5-PAHSA regioisomer in the adaptive metabolic response of white adipose tissue (WAT) to cold exposure (CE) in mice, exploring the cross talk between glucose utilization and lipid metabolism. CE promoted local production of 5- and 9-PAHSAs in WAT. Metabolic labeling of de novo lipogenesis (DNL) using 2H2O revealed that 5-PAHSA potentiated the effects of CE and stimulated triacylglycerol (TAG)/fatty acid (FA) cycling in WAT through impacting lipogenesis and lipolysis. Adipocyte lipolytic products were altered by 5-PAHSA through selective FA re-esterification. The impaired lipolysis in global adipose triglyceride lipase (ATGL) knockout mice reduced free PAHSA levels and uncovered a metabolite reservoir of TAG-bound PAHSAs (TAG estolides) in WAT. Utilization of 13C isotope tracers and dynamic metabolomics documented that 5-PAHSA primes adipocytes for glucose metabolism in a different way from insulin, promoting DNL and impeding TAG synthesis. In summary, our data reveal new cellular and physiological mechanisms underlying the beneficial effects of 5-PAHSA and its relation to insulin action in adipocytes and independently confirm a PAHSA metabolite reservoir linked to ATGL-mediated lipolysis.

Long-term high intake of 9-PAHPA or 9-OAHPA increases basal metabolism and insulin sensitivity but disrupts liver homeostasis in healthy mice.

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are a new family of endogenous lipids recently discovered. Several studies reported that some FAHFAs have antidiabetic and anti-inflammatory effects. The objective of this study was to explore the impact of two FAHFAs, 9-PAHPA or 9-OAHPA, on the metabolism of mice. C57Bl/6J male mice, 6 weeks old, were divided into 3 groups of 10 mice each. One group received a control diet and the two others groups received the control diet supplemented with 9-PAHPA or 9-OAHPA for 12 weeks. Mouse weight and body composition were monitored throughout the study. Some days before euthanasia, energy expenditure, glucose tolerance and insulin sensitivity were also determined. After sacrifice, blood and organs were collected for relevant molecular, biochemical and histological analyses. Although high intake of 9-PAHPA or 9-OAHPA increased basal metabolism, it had no direct effect on body weight. Interestingly, the 9-PAHPA or 9-OAHPA intake increased insulin sensitivity but without modifying glucose tolerance. Nevertheless, 9-PAHPA intake induced a loss of glucose-stimulated insulin secretion. Surprisingly, both studied FAHFAs induced hepatic steatosis and fibrosis in some mice, which were more marked with 9-PAHPA. Finally, a slight remodeling of white adipose tissue was also observed with 9-PAHPA intake. In conclusion, the long-term high intake of 9-PAHPA or 9-OAHPA increased basal metabolism and insulin sensitivity in healthy mice. However, this effect, highly likely beneficial in a diabetic state, was accompanied by manifest liver damage in certain mice that should deserve special attention in both healthy and pathological studies.

Omega-3 index in the Czech Republic: No difference between urban and rural populations.

Naturally occurring long-chain omega-3 PUFA such as eicosapentaenoic acid (EPA; 20:5 ω-3) and docosahexaenoic acid (DHA; 22:6 ω-3) exert multiple effects on health, which are related to the intake of these lipids in the diet and correlate with the levels of omega-3 PUFA in the body. These levels are reflected by the omega-3 PUFA index, i.e. the EPA and DHA content as % of all fatty acids in red blood cells. The aim of this study was to evaluate omega-3 index in the Czech Republic, using blood samples collected from the capital city (n = 476) and the rural region (n = 388). The mean omega-3 index was 3.56 mol % with a maximal value of 8.10% and a minimal value of 1.12%. There was no difference in the index value between rural and urban / industrial regions, but this value was higher in subjects who reported eating fish or omega-3 PUFA supplements. In conclusion, the results indicated suboptimal values of the omega-3 index in the Czech population independent of the sampling region.