EPA prevents fat mass expansion and metabolic disturbances in mice fed with a Western diet.

The impact of alpha linolenic acid (ALA), EPA, and DHA on obesity and metabolic complications was studied in mice fed a high-fat, high-sucrose (HF) diet. HF diets were supplemented with ALA, EPA, or DHA (1% w/w) and given to C57BL/6J mice for 16 weeks and to Ob/Ob mice for 6 weeks. In C57BL/6J mice, EPA reduced plasma cholesterol (-20%), limited fat mass accumulation (-23%) and adipose cell hypertrophy (-50%), and reduced plasma leptin concentration (-60%) compared with HF-fed mice. Furthermore, mice supplemented with EPA exhibited a higher insulin sensitivity (+24%) and glucose tolerance (+20%) compared with HF-fed mice. Similar effects were observed in EPA-supplemented Ob/Ob mice, although fat mass accumulation was not prevented. By contrast, in comparison with HF-fed mice, DHA did not prevent fat mass accumulation, increased plasma leptin concentration (+128%) in C57BL/6J mice, and did not improve glucose homeostasis in C57BL/6J and Ob/Ob mice. In 3T3-L1 adipocytes, DHA stimulated leptin expression whereas EPA induced adiponectin expression, suggesting that improved leptin/adiponectin balance may contribute to the protective effect of EPA. In conclusion, supplementation with EPA, but not ALA and DHA, could preserve glucose homeostasis in an obesogenic environment and limit fat mass accumulation in the early stage of weight gain.

A substrate-based approach to convert SerpinB1 into a specific inhibitor of proteinase 3, the Wegener's granulomatosis autoantigen.

The physiological and pathological functions of proteinase 3 (PR3) are not well understood due to its close similarity to human neutrophil elastase (HNE) and the lack of a specific inhibitor. Based on structural analysis of the active sites of PR3 and HNE, we generated mutants derived from the polyvalent inhibitor SerpinB1 (monocyte/neutrophil elastase inhibitor) that specifically inhibit PR3 and that differ from wt-SerpinB1 by only 3 or 4 residues in the reactive center loop. The rate constant of association between the best SerpinB1 mutant and PR3 is 1.4 × 107 M⁻¹ · s⁻¹, which is ∼100-fold higher than that observed with wt-SerpinB1 and compares with that of α1-protease inhibitor (α1-PI) toward HNE. SerpinB1(S/DAR) is cleaved by HNE, but due to differences in rate, inhibition of PR3 by SerpinB1(S/DAR) is only minimally affected by the presence of HNE even when the latter is in excess. SerpinB1(S/DAR) inhibits soluble PR3 and also membrane-bound PR3 at the surface of activated neutrophils. Moreover, SerpinB1(S/DAR) clears induced PR3 from the surface of activated neutrophils. Overall, these specific inhibitors of PR3 will be valuable for defining biological functions of the protease and may prove useful as therapeutics for PR3-related inflammatory diseases, such as Wegener's granulomatosis.

Dietary canolol protects the heart against the deleterious effects induced by the association of rapeseed oil, vitamin E and coenzyme Q10 in the context of a high-fat diet.

BACKGROUND: Obesity progressively leads to cardiac failure. Omega-3 polyunsaturated fatty acids (PUFA) have been shown to have cardio-protective effects in numerous pathological situations. It is not known whether rapeseed oil, which contains α-linolenic acid (ALA), has a similar protective effect. Omega-3 PUFAs are sensitive to attack by reactive oxygen species (ROS), and lipid peroxidation products could damage cardiac cells. We thus tested whether dietary refined rapeseed oil (RSO) associated with or without different antioxidants (vitamin E, coenzyme Q10 and canolol) is cardio-protective in a situation of abdominal obesity. METHODS: Sixty male Wistar rats were subdivided into 5 groups. Each group was fed a specific diet for 11 weeks: a low-fat diet (3% of lipids, C diet) with compositionally-balanced PUFAs; a high-fat diet rich in palm oil (30% of lipids, PS diet); the PS diet in which 40% of lipids were replaced by RSO (R diet); the R diet supplemented with coenzyme Q10 (CoQ10) and vitamin E (RTC diet); and the RTC diet supplemented with canolol (RTCC diet). At the end of the diet period, the rats were sacrificed and the heart was collected and immediately frozen. Fatty acid composition of cardiac phospholipids was then determined. Several features of cardiac function (fibrosis, inflammation, oxidative stress, apoptosis, metabolism, mitochondrial biogenesis) were also estimated. RESULTS: Abdominal obesity reduced cardiac oxidative stress and apoptosis rate by increasing the proportion of arachidonic acid (AA) in membrane phospholipids. Dietary RSO had the same effect, though it normalized the proportion of AA. Adding vitamin E and CoQ10 in the RSO-rich high fat diet had a deleterious effect, increasing fibrosis by increasing angiotensin-2 receptor-1b (Ag2R-1b) mRNA expression. Overexpression of these receptors triggers coronary vasoconstriction, which probably induced ischemia. Canolol supplementation counteracted this deleterious effect by reducing coronary vasoconstriction. CONCLUSION: Canolol was found to counteract the fibrotic effects of vitamin E + CoQ10 on cardiac fibrosis in the context of a high-fat diet enriched with RSO. This effect occurred through a restoration of cardiac Ag2R-1b mRNA expression and decreased ischemia.

Early sepsis does not stimulate reactive oxygen species production and does not reduce cardiac function despite an increased inflammation status.

If it is sustained for several days, sepsis can trigger severe abnormalities of cardiac function which leads to death in 50% of cases. This probably occurs through activation of toll-like receptor-9 by bacterial lipopolysaccharides and overproduction of proinflammatory cytokines such as TNF-α and IL-1ß In contrast, early sepsis is characterized by the development of tachycardia. This study aimed at determining the early changes in the cardiac function during sepsis and at finding the mechanism responsible for the observed changes. Sixty male Wistar rats were randomly assigned to two groups, the first one being made septic by cecal ligation and puncture (sepsis group) and the second one being subjected to the same surgery without cecal ligation and puncture (sham-operated group). The cardiac function was assessed in vivo and ex vivo in standard conditions. Several parameters involved in the oxidative stress and inflammation were determined in the plasma and heart. As evidenced by the plasma level of TNF-α and gene expression of IL-1ß and TNF-α in the heart, inflammation was developed in the sepsis group. The cardiac function was also slightly stimulated by sepsis in the in vivo and ex vivo situations. This was associated with unchanged levels of oxidative stress, but several parameters indicated a lower cardiac production of reactive oxygen species in the septic group. In conclusion, despite the development of inflammation, early sepsis did not increase reactive oxygen species production and did not reduce myocardial function. The depressant effect of TNF-α and IL-1ß on the cardiac function is known to occur at very high concentrations. The influence of low- to moderate-grade inflammation on the myocardial mechanical behavior must thus be revisited.

Combining citrulline with atorvastatin preserves glucose homeostasis in a murine model of diet-induced obesity.

BACKGROUND AND PURPOSE: NO is a crucial regulator of energy and lipid metabolism, whose homeostasis is compromised during obesity. Combination of citrulline and atorvastatin potentiated NO production in vitro. Here we have assessed the effects of this combination in mice with diet-induced obesity (DIO). EXPERIMENTAL APPROACH: C57BL/6J male mice were given a standard diet (control) or a high fat-high sucrose diet (DIO) for 8 weeks. DIO mice were then treated with DIO alone, DIO with citrulline, DIO with atorvastatin or DIO with citrulline and atorvastatin (DIOcit-stat) for 3 weeks. Thereafter, body composition, glucose tolerance, insulin sensitivity and liver fat metabolism were measured. KEY RESULTS: DIOcit-stat mice showed lower body weight, fat mass and epididymal fat depots compared with other DIO groups. Unlike other DIO groups, glucose tolerance and insulin sensitivity of DIOcit-stat, along with blood glucose and insulin concentrations in response to feeding, were restored to control values. Refeeding-induced changes in liver lipogenic activity were also reduced in DIOcit-stat mice compared with those of DIO animals. This was associated with decreased gene expression of the transcription factor SREBP-1, liver X receptor α, ChREBP and of target lipogenic enzymes in the liver of DIOcit-stat mice compared with those of other DIO groups. CONCLUSIONS AND IMPLICATIONS: The citrulline-atorvastatin combination prevented fat mass accumulation and maintained glucose homeostasis in DIO mice. Furthermore, it potentiated inhibition of hepatic de novo lipogenesis activity. This combination has potential for preservation of glucose homeostasis in patients receiving statin therapy.

DHA at nutritional doses restores insulin sensitivity in skeletal muscle by preventing lipotoxicity and inflammation.

Skeletal muscle plays a major role in the control of whole body glucose disposal in response to insulin stimulus. Excessive supply of fatty acids to this tissue triggers cellular and molecular disturbances leading to lipotoxicity, inflammation, mitochondrial dysfunctions, impaired insulin response and decreased glucose uptake. This study was conducted to analyze the preventive effect of docosahexaenoic acid (DHA), a long-chain polyunsaturated n-3 fatty acid, against insulin resistance, lipotoxicity and inflammation in skeletal muscle at doses compatible with nutritional supplementation. DHA (30 µM) prevented insulin resistance in C2C12 myotubes exposed to palmitate (500 µM) by decreasing protein kinase C (PKC)-θ activation and restoring cellular acylcarnitine profile, insulin-dependent AKT phosphorylation and glucose uptake. Furthermore, DHA protected C2C12 myotubes from palmitate- or lipopolysaccharide-induced increase in Ptgs2, interleukin 6 and tumor necrosis factor-α mRNA level, probably through the inhibition of p38 MAP kinase and c-Jun amino-terminal kinase. In LDLR -/- mice fed a high-cholesterol-high-sucrose diet, supplementation with DHA reaching up to 2% of daily energy intake enhanced the insulin-dependent AKT phosphorylation and reduced the PKC-θ activation in skeletal muscle. Therefore, DHA used at physiological doses participates in the regulation of muscle lipid and glucose metabolisms by preventing lipotoxicity and inflammation.

Measurement of neutrophil elastase, proteinase 3, and cathepsin G activities using intramolecularly quenched fluorogenic substrates.

Neutrophil elastase, proteinase 3, and cathepsin G are three hematopoietic serine proteases, large quantities of which are stored in neutrophil cytoplasmic azurophilic granules. They act in combination with reactive oxygen species to degrade engulfed microorganisms inside phagolysosomes. Active forms of these proteases are also externalized during neutrophil activation at inflammatory sites, thus helping to regulate inflammatory and immune responses. A fraction of secreted neutrophil serine proteases (NSPs) remains bound to the external plasma membrane, where they remain enzymatically active. This protocol describes the spectrofluorometric measurement of NSP activities using sensitive ortho-aminobenzoyl-peptidyl-N-(2,4-dinitrophenyl) ethylenediamine fluorescence resonance energy transfer (FRET) substrates that fully discriminate between the three human NSPs. These are used to measure subnanomolar concentrations of free or membrane-bound NSPs in low-binding microplates and to quantify the activities of individual proteases in biological fluids. We describe the synthesis of FRET substrate, neutrophil purification, and kinetic experiments on activated neutrophils. The protocol for measuring NSP activity on the surface of activated neutrophils can be adapted to measure NSP activities in whole biological fluids. Such data clarify the contributions of individual NSPs to the development of inflammatory diseases. Ultimately, these proteases may be shown to be targets for therapeutic inhibitors.

Discriminating between the activities of human cathepsin G and chymase using fluorogenic substrates.

Cathepsin G (CG) (EC 3.4.21.20) and chymase (EC 3.4.21.39) are two closely-related chymotrypsin-like proteases that are released from cytoplasmic granules of activated mast cells and/or neutrophils. We investigated the potential for their substrate-binding subsites to discriminate between their substrate specificities, aiming to better understand their respective role during the progression of inflammatory diseases. In addition to their preference for large aromatic residues at P1, both preferentially accommodate small hydrophilic residues at the S1' subsite. Despite significant structural differences in the S2' subsite, both prefer an acidic residue at that position. The Ala226/Glu substitution at the bottom of the CG S1 pocket, which allows CG but not chymase to accommodate a Lys residue at P1, is the main structural difference, allowing discrimination between the activities of these two proteases. However, a Lys at P1 is accommodated much less efficiently than a Phe, and the corresponding substrate is cleaved by ß2-tryptase (EC 3.4.21.59). We optimized a P1 Lys-containing substrate to enhance sensitivity towards CG and prevent cleavage by chymase and ß2-tryptase. The resulting substrate (ABZ-GIEPKSDPMPEQ-EDDnp) [where ABZ is O-aminobenzoic acid and EDDnp is N-(2,4-dinitrophenyl)-ethylenediamine] was cleaved by CG but not by chymase and tryptase, with a specificity constant of 190 mM(-1)·s(-1). This allows the quantification of active CG in cells or tissue extracts where it may be present together with chymase and tryptase, as we have shown using a HMC-1 cell homogenate and a sputum sample from a patient with severe asthma.