Patterns of alpha-linolenic acid incorporation into phospholipids in H4IIE cells.

Alpha linolenic acid (ALA) is an 18-carbon essential fatty acid found in plant-based foods and oils. While much attention has been placed on conversion of ALA to long chain polyunsaturated fatty acids, alternative routes of ALA metabolism exist and may lead to formation of other bioactive metabolites of ALA. The current study employed a non-targeted metabolomics approach to profile ALA metabolites that are significantly upregulated by ALA treatment. H4IIE hepatoma cells (n=3 samples per time point) were treated with 60 µM ALA or vehicle for 0, 0.25, 0.5, 1, 2, 3, 4, 6, 8, and 12 h. Samples were then extracted with methanol and analyzed using high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry. We observed selective changes in ALA incorporation into phospholipid classes and subclasses over the 12 h following ALA treatment. While levels of specific molecular species of ALA-containing phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and lysophospholipids were elevated with ALA treatment, others were not affected. Of the phospholipids that were increased, some (e.g., PC[18:3/18:1], PC[18:3/18:4], PE[18:3/18:2], PE[18:3/18:3]) were elevated almost immediately after exposure to ALA, while others [e.g., PE[18:1/18:3] PA[18:3/22:6], and PA[18:3/18:2]) were not elevated until several hours after ALA treatment. Overall, these results suggest that ALA incorporation into phospholipids is selective and support a metabolic hierarchy for ALA incorporation into specific phospholipids. Given the functionality of phospholipids based on their fatty acid composition, future studies will need to investigate the implications of ALA incorporation into specific phospholipids on cell function.

Alteration of the dietary methionine: Cysteine ratio modulates the inflammatory response to an inter-peritoneal injection of lipopolysaccharide in wistar rats.

Sulphur amino acids (SAA) are essential for multiple physiological/metabolic processes, with the ratio of dietary methionine: cysteine (Met:Cys) being an important contributor to pro-inflammatory responses, including TNF-α activity. The current study was designed to determine the effect an altered dietary SAA ratio, and the resulting reliance on the transsulfuration pathway to supply Cys, will have on the inflammatory response. In the present study, 100 µg/kg of an intraperitoneal (IP) injection of lipopolysaccharide (LPS) was used as a model for systemic inflammation. Male Wistar rats were randomized to one of two amino acid-defined diets, (100Met:0Cys or 50Met:50Cys) and subdivided to receive either IP LPS or saline injections. LPS significantly increased total plasma Cys, homocysteine (Hcy) and glutathione (GSH) 240 min post-IP injection in rats fed a 50Met:50Cys ratio compared to other treatments. The TNF-α area under the curve for LPS-treated rats consuming a dietary 50Met:50Cys ratio was significantly higher (P < .004) compared to those consuming a dietary 100Met:0Cys ratio. A significant increase in the percentage of leukocytes that were neutrophils was observed in rats injected with LPS when compared to saline with no effect of diet. These results demonstrate that an alteration of the dietary Met:Cys ratio did not attenuate the inflammatory response to an IP injection of LPS in Wistar rats; however, a diet with a balanced Met:Cys ratio increased concentrations of Cys and GSH which may result in a more rapid response to an LPS challenge.

Effects of cyanidin 3-O-glucoside and hydrochlorothiazide on T-cell phenotypes and function in spontaneously hypertensive rats.

Immune system dysfunction may contribute to the pathogenesis of hypertension in spontaneously hypertensive rats (SHR). We examined the effects of the anthocyanin, cyanidin 3-O-glucoside (C3G), and the diuretic, hydrochlorothiazide (HCT), on T-cell function in SHR. Five-week-old male SHR and Wistar-Kyoto (WKY) rats received water (n = 8/SHR; n = 8/WKY), 10 mg kg-1 day-1 C3G (n = 8/SHR; n = 8/WKY), 10 mg kg-1 day-1 HCT (n = 8/SHR; n = 8/WKY), or 10 mg kg-1 day-1 C3G + 10 mg kg-1 day-1 HCT (n = 8/SHR; n = 8/WKY) by oral gavage for 15 weeks. Spleens were used to assess T-cell phenotypes via flow cytometry and concanavalin A stimulated ex vivo cytokine production (IL-2, IL-10, TNFα, IFNγ) using a cytometric bead array. SHR had lower proportions of helper T-cells (Th) that were T-regulatory, CD62Llo, CD62L- and CD25+ compared to WKY. C3G treated SHR had higher proportions of Th that were CD62Llo and CD62L-, while HCT treated rats had higher CD62Lhi and CD62Llo and lower CD62L- compared to SHR control. The proportion of T-regulatory and Th that were CD25+ were not affected by treatment in SHR. Stimulated splenocytes from SHR produced lower concentrations of cytokines compared to WKY. C3G treated SHR produced higher while HCT treated SHR produced lower TNFα and IFNγ concentrations compared to controls. Our findings suggest that C3G has positive effects, whereas HCT further suppresses T-cell function in SHR.

Ginseng Berry Extract Rich in Phenolic Compounds Attenuates Oxidative Stress but not Cardiac Remodeling post Myocardial Infarction.

The cardioprotective effects of ginseng root extracts have been reported. However, nothing is known about the myocardial actions of the phenolic compounds enriched in ginseng berry. Therefore, this study was undertaken to investigate the effects of American ginseng berry extract (GBE) in an experimental model of myocardial infarction (MI). Coronary artery ligation was performed on Sprague⁻Dawley male rats to induce MI after which animals were randomized into groups receiving either distilled water or GBE intragastrically for 8 weeks. Echocardiography and assays for malondialdehyde (MDA) and TNF-α were conducted. Flow cytometry was used to test the effects of GBE on T cell phenotypes and cytokine production. Although GBE did not improve the cardiac functional parameters, it significantly attenuated oxidative stress in post-MI rat hearts. GBE treatment also resulted in lower than control levels of TNF-α in post-MI rat hearts indicating a strong neutralizing effect of GBE on this cytokine. However, there was no effect of GBE on the proportion of different T cell subsets or ex-vivo cytokine production. Taken together, the present study demonstrates GBE reduces oxidative stress, however no effect on cardiac structure and function in post-MI rats. Moreover, reduction of TNF-α levels below baseline raises concern regarding its use as prophylactic or preventive adjunct therapy in cardiovascular disease.

A randomized, controlled, crossover study of appetite-related sensations after consuming snacks made from buckwheat.

With the rising incidence of overweight and obesity in developed countries, there is an interest in developing food products that may aid in satiety and reduce energy intake. Buckwheat (Fagopyrum esculentum) is a gluten-free edible seed that has been previously shown to induce changes in postprandial concentrations of satiety hormones; however, subjective measures of appetite-related sensations and objective measures of energy intake at subsequent meals following buckwheat consumption have not been measured. Thirty-eight healthy adults were recruited to participate in a randomized, controlled, crossover trial with the main objective to determine if consuming snacks made from buckwheat would increase satiety and reduce energy intake compared with snacks comparable in serving size, physical characteristics, and nutrient composition. Water was included as a no-kilocalorie control. Participants received each of the treatments once separated by at least 7 days. Appetite related sensations were assessed using visual analog scales at fasting and after consuming the snack at 30-min intervals for 180 min. Lunch was provided at the clinic and the amount of food consumed was weighed. Participants recorded food intake for the rest of the day. Consuming buckwheat groats (32 g serving; 141 kcal) or pita bread made from buckwheat flour (50 g serving; 135 kcal) was not associated with changes in appetite related sensations or energy consumption compared with reference snack products made from corn or rice flour. Sensory questionnaires revealed that snacks made from buckwheat were liked to a similar degree or more as reference snack products, which shows commercial promise for developing buckwheat-containing snacks.

Lingonberry anthocyanins protect cardiac cells from oxidative-stress-induced apoptosis.

Lingonberry grown in northern Manitoba, Canada, contains exceptionally high levels of anthocyanins and other polyphenols. Previous studies from our lab have shown that lingonberry anthocyanins can protect H9c2 cells from ischemia-reperfusion injury and anthocyanin-rich diets have been shown to be associated with decreased cardiovascular disease and mortality. Oxidative stress can impair function and trigger apoptosis in cardiomyocytes. This study investigated the protective effects of physiologically relevant doses of lingonberry extracts and pure anthocyanins against hydrogen-peroxide-induced cell death. Apoptosis and necrosis were detected in H9c2 cells after hydrogen peroxide treatment via flow cytometry using FLICA 660 caspase 3/7 combined with YO-PRO-1 and then confirmed with Hoechst staining and fluorescence microscopy. Each of the 3 major anthocyanins found in lingonberry (cyanidin-3-galactoside, cyanidin-3-glucoside, and cyanidin-3-arabinoside) was protective against hydrogen-peroxide-induced apoptosis in H9c2 cells at 10 ng·mL-1 (20 nmol·L-1) and restored the number of viable cells to match the control group. A combination of the 3 anthocyanins was also protective and a lingonberry extract tested at 3 concentrations produced a dose-dependent protective effect. Lingonberry anthocyanins protected cardiac cells from oxidative-stress-induced apoptosis and may have cardioprotective effects as a dietary modification.

Manitoba Lingonberry (Vaccinium vitis-idaea) Bioactivities in Ischemia-Reperfusion Injury.

Evidence for the efficacy of dietary interventions in protecting against cardiovascular disease has grown significantly, with flavonoids and anthocyanins receiving special attention. Lingonberry (Vaccinium vitis-idaea L.) is a good source of these compounds, and this study examined the protective effects of wild lingonberry found in Manitoba, Canada, against ischemia-reperfusion (IR) injury. Manitoba lingonberry contained 3793 ± 27 mg gallic acid equiv, 120,501 ± 7651 µmol trolox equiv, and 575 ± 20 mg cyanidin-3-glucoside equiv per 100 g dry weight, which correspond with high total phenolic content, antioxidant activity, and anthocyanin content, respectively. A complete methanolic extract and both anthocyanin-rich and phenolic-rich fractions inhibited apoptosis in H9c2 cells during simulated IR. Lingonberry extract and fractions significantly inhibited several markers of apoptosis induced by IR, including nuclei condensation, caspase-3 activation, and MAP kinase signaling. These results provide the first analysis of Manitoba lingonberry and highlight the mechanistic importance of dietary berry compounds for cardiovascular health.

Salidroside and tyrosol from Rhodiola protect H9c2 cells from ischemia/reperfusion-induced apoptosis.

AIMS: Heart disease is the leading cause of death worldwide. Ischemia-reperfusion injury can lead to apoptotic death of heart cells and subsequently heart failure. Rhodiola is an herbal medicine with two main bioactive compounds--salidroside (SAL) and tyrosol (TYR). This study aimed to investigate whether these two compounds can prevent ischemia/reperfusion-induced apoptosis in H9c2 cells. MAIN METHODS: Assays for total phenolics assay and Oxygen Radical Absorbance Capacity showed high antioxidant capacity of SAL and TYR. H9c2 cells were subjected to simulated ischemia/reperfusion (IR) in the presence and absence of SAL and/or TYR, and nuclei condensation, caspase-3 activity, cytochrome c release and JNK phosphorylation were determined. KEY FINDINGS: In H9c2 cells, IR can lead to a 5-fold increase in p-JNK level. Apoptotic nuclei condensation, caspase-3 activity and cytochrome c release were markedly elevated, indicating the occurrence of apoptosis. SAL and TYR caused a dose-dependent inhibition of nuclear condensation. Furthermore, SAL and TYR, separately and in combination, significantly reduced caspase-3 activity, cytochrome c release and JNK activation. The anti-apoptotic effect of the combination was markedly higher than that of SAL or TYR alone. SIGNIFICANCE: The inhibition of the JNK signaling pathway is the key mechanism for the cytoprotective effect of SAL and TYR in IR-induced apoptosis.