Skeletal muscle insulin resistance is induced by 4-hydroxy-2-hexenal, a by-product of n-3 fatty acid peroxidation.

AIMS/HYPOTHESIS: Oxidative stress is involved in the pathophysiology of insulin resistance and its progression towards type 2 diabetes. The peroxidation of n-3 polyunsaturated fatty acids produces 4-hydroxy-2-hexenal (4-HHE), a lipid aldehyde with potent electrophilic properties able to interfere with many pathophysiological processes. The aim of the present study was to investigate the role of 4-HHE in the development of insulin resistance. METHODS: 4-HHE concentration was measured in plasma from humans and rats by GC-MS. Insulin resistance was estimated in healthy rats after administration of 4-HHE using hyperinsulinaemic-euglycaemic clamps. In muscle cells, glucose uptake was measured using 2-deoxy-D-glucose and signalling pathways were investigated by western blotting. Intracellular glutathione was measured using a fluorimetric assay kit and boosted using 1,2-dithiole-3-thione (D3T). RESULTS: Circulating levels of 4-HHE in type 2 diabetic humans and a rat model of diabetes (obese Zucker diabetic fatty rats), were twice those in their non-diabetic counterparts (33 vs 14 nmol/l, p < 0.001), and positively correlated with blood glucose levels. During hyperinsulinaemic-euglycaemic clamps in rats, acute intravenous injection of 4-HHE significantly altered whole-body insulin sensitivity and decreased glucose infusion rate (24.2 vs 9.9 mg kg-1 min-1, p < 0.001). In vitro, 4-HHE impaired insulin-stimulated glucose uptake and signalling (protein kinase B/Akt and IRS1) in L6 muscle cells. Insulin-induced glucose uptake was reduced from 186 to 141.9 pmol mg-1 min-1 (p < 0.05). 4-HHE induced carbonylation of cell proteins and reduced glutathione concentration from 6.3 to 4.5 nmol/mg protein. Increasing intracellular glutathione pools using D3T prevented 4-HHE-induced carbonyl stress and insulin resistance. CONCLUSIONS/INTERPRETATION: 4-HHE is produced in type 2 diabetic humans and Zucker diabetic fatty rats and blunts insulin action in skeletal muscle. 4-HHE therefore plays a causal role in the pathophysiology of type 2 diabetes and might constitute a potential therapeutic target to taper oxidative stress-induced insulin resistance.

Unveiling anti-oxidative and anti-inflammatory effects of docosahexaenoic acid and its lipid peroxidation product on lipopolysaccharide-stimulated BV-2 microglial cells.

BACKGROUND: Phospholipids in the central nervous system are enriched in n-3 and n-6 polyunsaturated fatty acids (PUFA), especially docosahexaenoic acid (DHA) and arachidonic acid (ARA). These PUFA can undergo enzymatic reactions to produce lipid mediators, as well as reaction with oxygen free radicals to produce 4-hydroxyhexenal (4-HHE) from DHA and 4-hydroxynonenal (4-HNE) from ARA. Recent studies demonstrated pleiotropic properties of these peroxidation products through interaction with oxidative and anti-oxidant response pathways. In this study, BV-2 microglial cells were used to investigate ability for DHA, 4-HHE, and 4-HNE to stimulate the anti-oxidant stress responses involving the nuclear factor erythroid-2-related factor 2 (Nrf2) pathway and synthesis of heme oxygenase (HO-1), as well as to mitigate lipopolysaccharide (LPS)-induced nitric oxide (NO), reactive oxygen species (ROS), and cytosolic phospholipase A2 (cPLA2). In addition, LC-MS/MS analysis was carried out to examine effects of exogenous DHA and LPS stimulation on endogenous 4-HHE and 4-HNE levels in BV-2 microglial cells. METHODS: Effects of DHA, 4-HHE, and 4-HNE on LPS-induced NO production was determined using the Griess reagent. LPS-induced ROS production was measured using CM-H2DCFDA. Western blots were used to analyze expression of p-cPLA2, Nrf2, and HO-1. Cell viability and cytotoxicity were measured using the WST-1 assay, and cell protein concentrations were measured using the BCA protein assay kit. An ultra-high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was used to determine levels of free 4-HHE and 4-HNE in cells. RESULTS: DHA (12.5-100 µM), 4-HHE (1.25-10 µM), and 4-HNE (1.25-10 µM) dose dependently suppressed LPS-induced production of NO, ROS, and as p-cPLA2 in BV-2 microglial cells. With the same concentrations, these compounds could enhance Nrf2 and HO-1 expression in these cells. Based on the estimated IC50 values, 4-HHE and 4-HNE were five- to tenfold more potent than DHA in inhibiting LPS-induced NO, ROS, and p-cPLA2. LC-MS/MS analysis indicated ability for DHA (10-50 µM) to increase levels of 4-HHE and attenuate levels of 4-HNE in BV-2 microglial cells. Stimulation of cells with LPS caused an increase in 4-HNE which could be abrogated by cPLA2 inhibitor. In contrast, bromoenol lactone (BEL), a specific inhibitor for the Ca2+-independent phospholipase A2 (iPLA2), could only partially suppress levels of 4-HHE induced by DHA or DHA + LPS. CONCLUSIONS: This study demonstrated the ability of DHA and its lipid peroxidation products, namely, 4-HHE and 4-HNE at 1.25-10 µM, to enhance Nrf2/HO-1 and mitigate LPS-induced NO, ROS, and p-cPLA2 in BV-2 microglial cells. In addition, LC-MS/MS analysis of the levels of 4-HHE and 4-HNE in microglial cells demonstrates that increases in production of 4-HHE from DHA and 4-HNE from LPS are mediated by different mechanisms.

Lipid Aldehydes 4-Hydroxynonenal and 4-Hydroxyhexenal Exposure Differentially Impact Lipogenic Pathways in Human Placenta.

Long chain polyunsaturated fatty acids (LCPUFAs), such as the omega-6 (n-6) arachidonic acid (AA) and n-3 docosahexanoic acid (DHA), have a vital role in normal fetal development and placental function. Optimal supply of these LCPUFAs to the fetus is critical for improving birth outcomes and preventing programming of metabolic diseases in later life. Although not explicitly required/recommended, many pregnant women take n-3 LCPUFA supplements. Oxidative stress can cause these LCPUFAs to undergo lipid peroxidation, creating toxic compounds called lipid aldehydes. These by-products can lead to an inflammatory state and negatively impact tissue function, though little is known about their effects on the placenta. Placental exposure to two major lipid aldehydes, 4-hydroxynonenal (4-HNE) and 4-hydroxyhexenal (4-HHE), caused by peroxidation of the AA and DHA, respectively, was examined in the context of lipid metabolism. We assessed the impact of exposure to 25 µM, 50 µM and 100 µM of 4-HNE or 4-HHE on 40 lipid metabolism genes in full-term human placenta. 4-HNE increased gene expression associated with lipogenesis and lipid uptake (ACC, FASN, ACAT1, FATP4), and 4-HHE decreased gene expression associated with lipogenesis and lipid uptake (SREBP1, SREBP2, LDLR, SCD1, MFSD2a). These results demonstrate that these lipid aldehydes differentially affect expression of placental FA metabolism genes in the human placenta and may have implications for the impact of LCPUFA supplementation in environments of oxidative stress.

Analysis of reactive aldehydes in urine and plasma of type-2 diabetes mellitus patients through liquid chromatography-mass spectrometry: Reactive aldehydes as potential markers of diabetic nephropathy.

Introduction: Diabetes is a major public health issue that is approaching epidemic proportions globally. Diabetes mortality is increasing in all ethnic groups, irrespective of socio-economic class. Obesity is often seen as the main contributor to an increasing prevalence of diabetes. Oxidative stress has been shown to trigger obesity by stimulating the deposition of white adipose tissue. In this study, we measured reactive aldehydes by liquid chromatography-mass spectrometry (LC-MS), in the urine and plasma of type-2 diabetic mellitus (T2DM) patients, as potential surrogates of oxidative stress. Our hypothesis was that reactive aldehydes play a significant role in the pathophysiology of diabetes, and these reactive species, may present potential drug targets for patient treatment. Materials and methods: Study participants [N = 86; control n = 26; T2DM n = 32, and diabetic nephropathy (DN) n = 28] were recruited between 2019 and 2020. Urine and blood samples were collected from all participants, including a detailed clinical history, to include patient behaviours, medications, and co-morbidities. Reactive aldehyde concentrations in urine and plasma were measured using pre-column derivatisation and LC-MS, for control, T2DM and DN patients. Results: Reactive aldehydes were measured in the urine and plasma of control subjects and patients with T2DM and DN. In all cases, the reactive aldehydes under investigation; 4-HNE, 4-ONE, 4-HHE, pentanal, methylglyoxal, and glyoxal, were significantly elevated in the urine and serum of the patients with T2DM and DN, compared to controls (p < 0.001) (Kruskal-Wallis). Urine and serum reactive aldehydes were significantly correlated (≥0.7) (p < 0.001) (Spearman rho). The concentrations of the reactive aldehydes were significantly higher in plasma samples, when compared to urine, suggesting that plasma is the optimal matrix for screening T2DM and DN patients for oxidative stress. Conclusion: Reactive aldehydes are elevated in the urine and plasma of T2DM and DN patients. Reactive aldehydes have been implicated in the pathobiology of T2DM. Therefore, if reactive aldehydes are surrogates of oxidative stress, these reactive aldehyde species could be therapeutic targets for potential drug development.

Nonenzymatic oxygenated metabolite of docosahexaenoic acid, 4(RS)-4-F4t -neuroprostane, acts as a bioactive lipid molecule in neuronal cells.

Docosahexaenoic acid (DHA), an abundant fatty acid in the brain, is susceptible to auto-oxidation in situ and releases metabolites such as F4 -neuroprostane (4-F4t -NeuroP). The presence of 4-F4t -NeuroP in the brain is not well explored. In this study, 4-F4t -NeuroP was introduced into neuroblastoma cells (SH-SY5Y) and, by in vivo infusion, into rodents. Targeted lipidomic analysis of liver and brain tissues shows significant elevation of anti-inflammatory hydroxylated DHA metabolites and an isomer of neuroprotectin D1, suggesting potential beneficial bioactivities of 4-F4t -NeuroP. Additionally, 4-F4t -NeuroP treatment in SH-SY5Y cells and primary neuronal culture consistently upregulates the transcriptional level of the antioxidant enzyme heme oxygenase-1, but the effect is reduced when 4-F4t -NeuroP is further oxidized. Our data suggest that 4-F4t -NeuroP could be neuroprotective in the native state but may have disadvantageous bioactivity when oxidized extensively.

Histone Carbonylation Is a Redox-Regulated Epigenomic Mark That Accumulates with Obesity and Aging.

Oxidative stress is a hallmark of metabolic disease, though the mechanisms that define this link are not fully understood. Irreversible modification of proteins by reactive lipid aldehydes (protein carbonylation) is a major consequence of oxidative stress in adipose tissue and the substrates and specificity of this modification are largely unexplored. Here we show that histones are avidly modified by 4-hydroxynonenal (4-HNE) in vitro and in vivo. Carbonylation of histones by 4-HNE increased with age in male flies and visceral fat depots of mice and was potentiated in genetic (ob/ob) and high-fat feeding models of obesity. Proteomic evaluation of in vitro 4-HNE- modified histones led to the identification of both Michael and Schiff base adducts. In contrast, mapping of sites in vivo from obese mice exclusively revealed Michael adducts. In total, we identified 11 sites of 4-hydroxy hexenal (4-HHE) and 10 sites of 4-HNE histone modification in visceral adipose tissue. In summary, these results characterize adipose histone carbonylation as a redox-linked epigenomic mark associated with metabolic disease and aging.

Antioxidant activity of milk and polyphenol-rich beverages during simulated gastrointestinal digestion of linseed oil emulsions.

Polyunsaturated fatty acids (PUFA) are associated with health benefits. However, high PUFA intake increases the risk of lipid oxidation and formation of potentially toxic lipid oxidation species. The objective of this study was to determine the antioxidant activity of milk fractions (whole milk, skim milk, acid whey, ultrafiltration (UF) permeate) and polyphenol-rich beverages (green tea, grape juice) during simulated gastrointestinal digestion. We also determined the effect of milk and polyphenol-rich beverages on the formation of advanced oxidation species during in vitro digestion of PUFA-rich emulsion. Antioxidant activity during digestion of milk fractions emphasized the important role of proteins (more specifically caseins) and the contribution of fat to the antioxidant capacity of milk. In comparison to milk, the antioxidant activity of polyphenol-rich beverages was at least four times higher. During digestion of a PUFA-rich emulsion, the formation of 4-hydroxyhexanal (4-HHE) and 4-hydroxynonenal (4-HNE) in the intestinal phase were respectively reduced by 60% and 75%, in the presence of milk or polyphenol-rich beverages. Further reduction was observed when the emulsion was co-digested with both, milk and polyphenol-rich beverages (89% for 4-HHE and 93% for 4-HNE). These results suggest that the combination of milk and polyphenol-rich beverages increases the antioxidant activity and synergistically reduces the formation of toxic lipid oxidation species during simulated digestion of PUFA-rich foods.

Effect of milk proteins and food-grade surfactants on oxidation of linseed oil-in-water emulsions during in vitro digestion.

Health benefits are associated with polyunsaturated fatty acids, but their sensitivity to oxidation may generate toxic oxidation species. The objective of this study was to compare the effect of milk proteins (casein, whey protein) and surfactants (Citrem, Tween 20) on the in vitro digestion and oxidation of linseed oil emulsions. The emulsion produced with Tween 20 resisted coalescence in the gastric phase and showed the highest concentrations of free fatty acids and reactive carbonyl compounds in the intestinal digestion phase. The Citrem-stabilized emulsion showed extensive coalescence in the gastric environment, which reduced lipolysis and the formation of advanced oxidation species. The protein-stabilized emulsions showed aggregation with some coalescence in the gastric phase, and casein provided better protection than whey protein against oxidation. This study suggests that the mechanism of emulsion destabilization in the gastric environment and the type of protein can modulate lipolysis and oxidation during in vitro digestion.

Docosahexaenoic acid (DHA): An essential nutrient and a nutraceutical for brain health and diseases.

Docosahexaenoic acid (DHA), a polyunsaturated fatty acid (PUFA) enriched in phospholipids in the brain and retina, is known to play multi-functional roles in brain health and diseases. While arachidonic acid (AA) is released from membrane phospholipids by cytosolic phospholipase A2 (cPLA2), DHA is linked to action of the Ca2+-independent iPLA2. DHA undergoes enzymatic conversion by 15-lipoxygenase (Alox 15) to form oxylipins including resolvins and neuroprotectins, which are powerful lipid mediators. DHA can also undergo non-enzymatic conversion by reacting with oxygen free radicals (ROS), which cause the production of 4-hydoxyhexenal (4-HHE), an aldehyde derivative which can form adducts with DNA, proteins and lipids. In studies with both animal models and humans, there is evidence that inadequate intake of maternal n-3 PUFA may lead to aberrant development and function of the central nervous system (CNS). What is less certain is whether consumption of n-3 PUFA is important in maintaining brain health throughout one's life span. Evidence mostly from non-human studies suggests that DHA intake above normal nutritional requirements might modify the risk/course of a number of diseases of the brain. This concept has fueled much of the present interest in DHA research, in particular, in attempts to delineate mechanisms whereby DHA may serve as a nutraceutical and confer neuroprotective effects. Current studies have revealed ability for the oxylipins to regulation of cell redox homeostasis through the Nuclear factor (erythroid-derived 2)-like 2/Antioxidant response element (Nrf2/ARE) anti-oxidant pathway, and impact signaling pathways associated with neurotransmitters, and modulation of neuronal functions involving brain-derived neurotropic factor (BDNF). This review is aimed at describing recent studies elaborating these mechanisms with special regard to aging and Alzheimer's disease, autism spectrum disorder, schizophrenia, traumatic brain injury, and stroke.

The initial characteristics of marine oil emulsions and the composition of the media inflect lipid oxidation during in vitro gastrointestinal digestion.

This work aimed at evaluating the effect of simulated digestive fluids, interface and lipid droplet sizes on the oxidation of oil-in-water emulsions containing long chain n-3 fatty acyls. Emulsions stabilised by a protein or by phosphatidyl-choline/Tween 80 were submitted to gastro-intestinal in vitro conditions in presence of metmyoglobin. The gastric phase was characterised by a decrease of tocopherol amounts and moderate O2 uptake and aldehyde formation. Oxidation developed further during the intestinal phase, with tocopherols tending to zero, oxygen uptake and production of aldehydes at potentially toxic concentrations. The simulated digestive fluids reduced oxygen uptake and MDA formation only during the intestinal step of the phospholipid-stabilised emulsion. Quantitative losses of PUFA (e.g. EPA, DHA) were less than 10% even significant at the end of the digestion.

Signaling properties of 4-hydroxyalkenals formed by lipid peroxidation in diabetes.

Peroxidation of polyunsaturated fatty acids is intensified in cells subjected to oxidative stress and results in the generation of various bioactive compounds, of which 4-hydroxyalkenals are prominent. During the progression of type 2 diabetes mellitus, the ensuing hyperglycemia promotes the generation of reactive oxygen species (ROS) that contribute to the development of diabetic complications. It has been suggested that ROS-induced lipid peroxidation and the resulting 4-hydroxyalkenals markedly contribute to the development and progression of these pathologies. Recent findings, however, also suggest that noncytotoxic levels of 4-hydroxyalkenals play important signaling functions in the early phase of diabetes and act as hormetic factors to induce adaptive and protective responses in cells, enabling them to function in the hyperglycemic milieu. Our studies and others' have proposed such regulatory functions for 4-hydroxynonenal and 4-hydroxydodecadienal in insulin secreting ß-cells and vascular endothelial cells, respectively. This review presents and discusses the mechanisms regulating the generation of 4-hydroxyalkenals under high glucose conditions and the molecular interactions underlying the reciprocal transition from hormetic to cytotoxic agents.

High-fat diet induces changes in adipose tissue trans-4-oxo-2-nonenal and trans-4-hydroxy-2-nonenal levels in a depot-specific manner.

Protein carbonylation is the covalent modification of proteins by α,ß-unsaturated aldehydes produced by nonenzymatic lipid peroxidation of polyunsaturated fatty acids. The most widely studied aldehyde product of lipid peroxidation, trans-4-hydroxy-2-nonenal (4-HNE), is associated with obesity-induced metabolic dysfunction and has demonstrated reactivity toward key proteins involved in cellular function. However, 4-HNE is only one of many lipid peroxidation products and the lipid aldehyde profile in adipose tissue has not been characterized. To further understand the role of oxidative stress in obesity-induced metabolic dysfunction, a novel LC-MS/MS method was developed to evaluate aldehyde products of lipid peroxidation and applied to the analysis of adipose tissue. 4-HNE and trans-4-oxo-2-nonenal (4-ONE) were the most abundant aldehydes present in adipose tissue. In high fat-fed C57Bl/6J and ob/ob mice the levels of lipid peroxidation products were increased 5- to 11-fold in epididymal adipose, unchanged in brown adipose, but decreased in subcutaneous adipose tissue. Epididymal adipose tissue of high fat-fed mice also exhibited increased levels of proteins modified by 4-HNE and 4-ONE, whereas subcutaneous adipose tissue levels of these modifications were decreased. High fat feeding of C57Bl/6J mice resulted in decreased expression of a number of genes linked to antioxidant biology selectively in epididymal adipose tissue. Moreover, TNFα treatment of 3T3-L1 adipocytes resulted in decreased expression of GSTA4, GPx4, and Prdx3 while upregulating the expression of SOD2. These results suggest that inflammatory cytokines selectively downregulate antioxidant gene expression in visceral adipose tissue, resulting in elevated lipid aldehydes and increased protein carbonylation.