Dietary DHA reduces downstream endocannabinoid and inflammatory gene expression and epididymal fat mass while improving aspects of glucose use in muscle in C57BL/6J mice.

OBJECTIVES: Endocannabinoid system (ECS) overactivation is associated with increased adiposity and likely contributes to type 2 diabetes risk. Elevated tissue cannabinoid receptor 1 (CB1) and circulating endocannabinoids (ECs) derived from the n-6 polyunsaturated acid (PUFA) arachidonic acid (AA) occur in obese and diabetic patients. Here we investigate whether the n-3 PUFA docosahexaenoic acid (DHA) in the diet can reduce ECS overactivation (that is, action of ligands, receptors and enzymes of EC synthesis and degradation) to influence glycemic control. This study targets the ECS tonal regulation of circulating glucose uptake by skeletal muscle as its primary end point. DESIGN: Male C57BL/6J mice were fed a semipurified diet containing DHA or the control lipid. Serum, skeletal muscle, epididymal fat pads and liver were collected after 62 and 118 days of feeding. Metabolites, genes and gene products associated with the ECS, glucose uptake and metabolism and inflammatory status were measured. RESULTS: Dietary DHA enrichment reduced epididymal fat pad mass and increased ECS-related genes, whereas it reduced downstream ECS activation markers, indicating that ECS activation was diminished. The mRNA of glucose-related genes and proteins elevated in mice fed the DHA diet with increases in DHA-derived and reductions in AA-derived EC and EC-like compounds. In addition, DHA feeding reduced plasma levels of various inflammatory cytokines, 5-lipoxygenase-dependent inflammatory mediators and the vasoconstrictive 20-HETE. CONCLUSIONS: This study provides evidence that DHA feeding altered ECS gene expression to reduce CB1 activation and reduce fat accretion. Furthermore, the DHA diet led to higher expression of genes associated with glucose use by muscle in mice, and reduced those associated with systemic inflammatory status.

Low to moderate alcohol consumption on serum vitamin D and other indicators of bone health in postmenopausal women in a controlled feeding study.

The objective of this study was to evaluate the effect of 8 weeks of no alcohol, low (1 drink or 15 g/day) and moderate (2 drinks or 30 g/day) alcohol consumption on markers of bone health: fasting serum 25-hydroxy vitamin D (25(OH)D), osteocalcin (OC), bone-specific alkaline phosphatase (BSAP), urine deoxypyridinoline (DPD) and helical peptide (HP) in postmenopausal women (n=51). Compared with no alcohol, 1 or 2 drinks/day for 8 weeks had no significant impact on any of the bone markers. Within each alcohol group, obese women had significantly lower serum 25(OH)D and higher DPD concentrations than normal weight women. Season significantly affected the concentrations of serum 25(OH)D, but there was no significant interaction between alcohol and season on serum 25(OH)D concentrations. Low or moderate alcohol consumption for 8 weeks had no significant impact on markers of bone health in postmenopausal women.

Improvement of bone quality in gonad-intact middle-aged male rats by long-chain n-3 polyunsaturated fatty acid.

The effect of long-chain n-3 polyunsaturated fatty acid (PUFA) on bone measurements was evaluated in gonad-intact middle-aged male rats. Seven rats were killed on day 0 of dietary intervention to determine bone parameters at baseline. Experimental rats (7/group) were fed one of the following lipid treatments (g/kg diet): 167 g safflower oil + 33 g menhaden oil (N6+N3 diet, control), 200 g safflower oil (N6 diet), or 190 menhaden oil + 10 g corn oil (N3 diet). After 20 weeks of dietary treatment, all groups had lower values for peak load and ultimate stiffness in femurs compared to baseline values. Rats fed the N3 diet had the highest values for peak load, ultimate stiffness, and Young's modulus compared with those fed the N6 and control diets. Compared to baseline, all dietary treatment groups had significantly lower values for trabecular thickness and number in proximal tibia but higher values for trabecular separation and formation rate in proximal tibia and endocortical bone formation rate in tibial shaft. Compared with the control group, rats fed the N3 diet had lower values for formation rate, osteoclast number, and eroded surface in proximal tibia but higher values for periosteal mineral apposition and formation rates in tibia shaft. These findings indicate that a diet rich in long-chain n-3 PUFA mitigate aging-induced loss of bone integrity in intact middle-aged male rats through reducing bone turnover rate by suppressing both bone formation and resorption as a result of a larger net bone volume and modulating endocortical and cancellous bone compartments.

Anthocyanin quantification and radical scavenging capacity of Concord, Norton, and Marechal Foch grapes and wines.

The anthocyanin content and the radical scavenging capacity of three non-Vitis vinifera grapes (Marechal Foch, Norton, and Concord varieties) were determined. Analyses of anthocyanins in the skin (S) and wine (W) of these grape varieties were performed by spectrophotometry, HPLC with electrochemical detection, and matrix-assisted laser desorption ionization (MALDI). The total anthocyanin contents of S samples were 258 +/- 37 mg/100 g of wet weight for Foch, 888 +/- 78 mg/100 g for Norton, and 326 +/- 5.9 mg/100 g for Concord grapes. The malvidin 3,5-diglucoside content quantified by HPLC indicated that Norton S had the highest amount of the compound (327 +/- 110 mg/100 g). The MALDI mass spectrometric analysis indicated an abundance of malvidin glucosides in W of Foch grapes and in S and W of Norton grapes and of cyanidin aglycon in S and W of Concord grapes. S samples were subjected to a radical scavenging capacity test using the 2,2-diphenyl-1-picrylhydrazyl radical and compared to Trolox. The radical scavenging capacity for Foch S was 0.78 mM Trolox equiv, that of Concord S, 0.80 Trolox equiv, and that of Norton S was highest at 0.95 Trolox equiv. The higher concentrations of malvidin 3,5-diglucoside in S of grape varieties were associated with greater radical scavenging capacity.

Fatty acid analysis of wild ruminant tissues: evolutionary implications for reducing diet-related chronic disease.

HYPOTHESES: Consumption of wild ruminant fat represented the primary lipid source for pre-agricultural humans. Hence, the lipid composition of these animals' tissues may provide insight into dietary requirements that offer protection from chronic disease in modern humans. METHOD: We examined the lipid composition of muscle, brain, marrow and subcutaneous adipose tissue (AT) from 17 elk (Cervus elaphus), 15 mule deer (Odocoileus hemionus), and 17 antelope (Antilicapra americana) and contrasted them to wild African ruminants and pasture and grain-fed cattle. RESULTS: Muscle fatty acid (FA) was similar among North American species with polyunsaturated fatty acids/saturated fatty acids (P/S) values from 0.80 to 1.09 and n-6/n-3 FA from 2.32 to 2.60. Marrow FA was similar among North American species with high levels (59.3-67.0%) of monounsaturated FA; a low P/S (0.24-0.33), and an n-6/n-3 of 2.24-2.88. Brain had the lowest n-6/n-3 (1.20-1.29), the highest concentration of 22:6 n-3 (elk, 8.90%; deer, 9.62%; antelope, 9.25%) and a P/S of 0.69. AT had the lowest P/S (0.05-0.09) and n-6/n-3 (2.25-2.96). Conjugated linoleic acid (CLA) isomers were found in marrow of antelope (1.5%), elk (1.0%) and deer (1.0%), in AT (deer, 0.3%; antelope, 0.3%) in muscle (antelope, 0.4%; elk, trace), but not in brain. CONCLUSIONS: Literature comparisons showed tissue lipids of North American and African ruminants were similar to pasture-fed cattle, but dissimilar to grain-fed cattle. The lipid composition of wild ruminant tissues may serve as a model for dietary lipid recommendations in treating and preventing chronic disease.

Supercritical fluid extraction of lycopene from tomato processing byproducts.

Tomato seeds and skins acquired from the byproduct of a local tomato processing facility were studied for supercritical fluid extraction (SFE) of phytochemicals. The extracts were analyzed for lycopene, beta-carotene, alpha-carotene, alpha-tocopherol, gamma-tocopherol, and delta-tocopherol content using high-performance liquid chromatography-electrochemical detection and compared to a chemically extracted control. SFEs were carried out using CO(2) at seven temperatures (32-86 degrees C) and six pressures (13.78-48.26 MPa). The effect of CO(2) flow rate and volume also was investigated. The results indicated that the percentage of lycopene extracted increased with elevated temperature and pressure until a maximum recovery of 38.8% was reached at 86 degrees C and 34.47 MPa, after which the amount of lycopene extracted decreased. Conditions for the optimum extraction of lycopene from 3 g of raw material were determined to be 86 degrees C, 34.47 MPa, and 500 mL of CO(2) at a flow rate of 2.5 mL/min. These conditions resulted in the extraction of 61.0% of the lycopene (7.19 microg lycopene/g).

Nutraceutical fatty acids as biochemical and molecular modulators of skeletal biology.

Several systemic hormones and localized growth factors coordinate events of bone formation and resorption to support bone growth in the young and maintain bone mineral content in the adult. Some of the more important factors produced in the bone microenvironment that impact skeletal biology include prostaglandins, cytokines, and insulin-like growth factors. Dietary fat sources that exert potent biological effects on the skeletal tissues belong to the omega-6 and omega-3 families of essential fatty acids. Specific long-chain polyunsaturated fatty acids (PUFA) belonging to these families are substrates for prostanoids that influence the differentiation and activity of cells in bone and cartilage tissues. These PUFA appear to alter prostanoid formation, cell-to-cell signaling processes, and impact transcription factors in vivo. Hence, these biologically active PUFA can be called nutraceutical fatty acids. This review highlights the role of nutraceutical fatty acids on bone metabolism and joint disease. The recent discovery of transcription factors controlling osteoblast function, and soluble proteins directing osteoclastogenesis and osteoblastogenesis offer new research opportunities for studying nutraceutical fatty acids in skeletal biology.

Dietary conjugated linoleic acids and lipid source alter fatty acid composition of juvenile yellow perch, Perca flavescens.

A study was conducted to examine the effects of dietary conjugated linoleic acids (CLA; 0, 0.5 or 1.0 g/100 g total CLA) and lipid source (menhaden oil, soybean oil or a 1:1 mixture of menhaden:soybean oil) on growth rates and fatty acid composition of yellow perch. Dietary treatments were fed to apparent satiation to triplicate groups of fish initially weighing 37.9 g/fish. At the end of the 9-wk feeding trial, no significant differences were detected in weight gain or feed intake among fish fed any of the dietary treatments. Dietary CLA, lipid source and/or their interaction significantly affected feed efficiency, total liver lipid concentration, and muscle and liver fatty acid concentrations. Feed efficiency (g gain/g feed) was significantly lower in fish fed diets containing soybean oil (0.51) compared with fish fed menhaden oil (0.58) or menhaden:soybean oil (0.60). Liver total lipid concentrations were significantly reduced in fish fed 0.5 and 1.0 g/100 g CLA compared with fish fed the diets containing no CLA and in fish fed menhaden oil compared with those fed soybean oil or a 1:1 mixture of menhaden:soybean oil. Total CLA levels increased in both liver and muscle as dietary CLA concentration increased, irrespective of lipid source. However, total CLA concentrations were significantly lower in liver and muscle of fish fed soybean oil. Total muscle CLA concentrations were 0, 1.26 and 2.92 g/100 g fatty acids in fish fed diets containing menhaden oil and 0, 0.5 and 1.0 g/100 g CLA, respectively. Mono- and polyunsaturated fatty acid (PUFA) concentrations were significantly lower in muscle and liver of fish fed CLA compared with fish fed the diets containing no CLA. In contrast, liver concentrations of saturated fatty acids, 14:0, 16:0 and 18:0, were significantly higher in fish fed 1.0 g/100 g CLA.

Effects of conjugated linoleic acids and docosahexaenoic acid on rat liver and reproductive tissue fatty acids, prostaglandins and matrix metalloproteinase production.

Long chain n-6 and n-3 fatty acids play important roles in labor and delivery. These effects may be mediated by prostaglandin (PG) synthesis and by regulation of matrix metalloproteinases (MMPs), both of which play roles in uterine contraction, cervical ripening and rupture of fetal membranes. The effects of altering dietary n-6:n-3 long chain fatty acid ratios, and the addition of dietary conjugated linoleic acids (CLA) and docosahexaenoic acid (DHA) on fatty acid composition of reproductive tissues, PG synthesis in liver and reproductive tissue and serum MMP levels were examined in pregnant rats. Modified AIN-96G diets with n-6:n-3 ratios of 7:1 and 34:1 with and without added 1.1% (by weight) conjugated linoleic acid (CLA) and/or 0.3% (by weight) DHA were fed through day 20 of gestation. Reproductive tissues readily incorporated both DHA and CLA. CLA significantly (P<0.05) depressed PGF(2 alpha)synthesis in placenta, uterus and liver by 50% when the n-6:n-3 ratio was 7:1 and by 66% at 34:1 ratio. Significant differences (P<0.05) in PGE(2)synthesis in uterus and liver were seen only between groups fed the high ratio of n-6:n-3 without CLA, and the low ratio with CLA. Addition of CLA to DHA containing diets depressed PGF(2alpha) by one-third in uterus and liver (P<0.05). Serum MMP-9 and active MMP-2 were suppressed (P<0.05) by addition of either CLA or DHA.

Omega-3 polyunsaturated fatty acids and skeletal health.

This minireview on skeletal biology describes the actions of prostaglandins and cytokines involved in the local regulation of bone metabolism, it documents the role of lipids in bone biology, and it presents relationships between fatty acids and other factors that impact skeletal metabolism. The data presented herein show consistent and reproducible beneficial effects of omega-3 (n-3) fatty acids on bone metabolism and bone/joint diseases. Polyunsaturated fatty acids modulate eicosanoid biosynthesis in numerous tissues and cell types, alter signal transduction, and influence gene expression. These effects have not been explored in the skeletal system. Future research on n-3 fatty acids in bone biology should focus on the following two aspects. First, the further elucidation of how n-3 fatty acids alter biochemical and molecular processes involved in bone modeling and bone cell differentiation, and second, the evaluation of the potential pharmaceutical applications of these nutraceutical fatty acids in maintaining bone mineral status and controlling inflammatory bone/joint diseases.

Metabolic and behavioral responses in pre-weanling rats following alteration of maternal diet.

In rat pups, blockade of fatty acid oxidation by administration of 2-mercaptoacetate (MA) produces an increase in independent ingestion by 12 days of age. In contrast, administration of methyl palmoxirate (MP) fails to increase intake or to alter oxidation of fatty acids in young rats due to the high proportion of medium chain triglycerides (MCT) in rat milk. In the present experiments, the composition of rat milk was altered by placing dams on a high fat (HF) diet to examine the development of ingestive responding in rat pups following administration of MP. Following delivery of MP (0.5--10 mg/kg), pups were placed in a cage inside an incubator for 1, 3, or 6 h, and then received an intake test consuming a commercial half-and-half milk diet from the floor of test containers. Blood was collected from additional groups of pups for measurement of beta-hydroxybutyrate (beta HBA) and glucose levels. The results demonstrated that administration of MP produced significant reductions in beta HBA levels after 3 h in 12- and 15-day-old pups, but behavioral responses were noted only in pups aged 15 days. Similar results were obtained following administration of MA to pups reared by dams on HF diets; physiological responses were observed at 12 and 15 days of age, but behavioral responses were not observed after administration of MA until 15 days of age. Taken together, these results suggest that (1) changes in fatty acid oxidation may represent an early metabolic signal that can influence intake in rat pups and (2) alteration of the dam's diet produces physiological and behavioral changes in the pups.

The role of methyl-linoleic acid epoxide and diol metabolites in the amplified toxicity of linoleic acid and polychlorinated biphenyls to vascular endothelial cells.

Selected dietary lipids may increase the atherogenic effects of environmental chemicals, such as polychlorinated biphenyls (PCBs), by cross-amplifying mechanisms leading to dysfunction of the vascular endothelium. We have shown previously that the omega-6 parent fatty acid, linoleic acid, or 3,3',4,4'-tetrachlorobiphenyl (PCB 77), an aryl hydrocarbon (Ah) receptor agonist, independently can cause disruption of endothelial barrier function. Furthermore, cellular enrichment with linoleic acid can amplify PCB-induced endothelial cell dysfunction. We hypothesize that the amplified toxicity of linoleic acid and PCBs to endothelial cells could be mediated in part by cytotoxic epoxide metabolites of linoleic acid called leukotoxins (LTX) or their diol derivatives (LTXD). Exposure to LTXD resulted in a dose-dependent increase in albumin transfer across endothelial cell monolayers, whereas this disruption of endothelial barrier function was observed only at a high concentration of LTX. Pretreatment with the cytosolic epoxide hydrolase inhibitor 1-cyclohexyl-3-dodecyl urea partially protected against the observed LTX-induced endothelial dysfunction. Endothelial cell activation mediated by LTX and/or LTXD also enhanced nuclear translocation of the transcription factor NF-kappa B and gene expression of the inflammatory cytokine IL-6. Inhibiting cytosolic epoxide hydrolase decreased the LTX-mediated induction of both NF-kappa B and the IL-6 gene, whereas the antioxidant vitamin E did not block LTX-induced endothelial cell activation. Most importantly, inhibition of cytosolic epoxide hydrolase blocked both linoleic acid-induced cytotoxicity, as well as the additive toxicity of linoleic acid plus PCB 77 to endothelial cells. Interestingly, cellular uptake and accumulation of linoleic acid was markedly enhanced in the presence of PCB 77. These data suggest that cytotoxic epoxide metabolites of linoleic acid play a critical role in linoleic acid-induced endothelial cell dysfunction. Furthermore, the severe toxicity of PCBs in the presence of linoleic acid may be due in part to the generation of epoxide and diol metabolites. These findings have implications in understanding interactive mechanisms of how dietary fats can modulate dysfunction of the vascular endothelium mediated by certain environmental contaminants.

Lipids as modulators of bone remodelling.

Bone remodelling processes are regulated by systemic hormones and a multitude of local and systemic factors, including prostaglandins, cytokines, and growth factors. Dietary fatty acids and their derivatives (eicosanoids) have been a recent focus of investigation on bone and cartilage metabolism. Specific fatty acids are recognized modulators of eicosanoid biosynthesis, signal transduction, and gene expression. The actions of polyunsaturated fatty acids have not been extensively examined in the skeletal system. Promising research on fatty acids and bone remodelling should evaluate the potential effects on pathways for osteoclastogenesis and osteoblastogenesis.

Bioactive fatty acids: role in bone biology and bone cell function.

Bone is a unique tissue providing support, movement, and mineral balance for the body. Bone growth is achieved in the young by a process called modeling, and maintained during adulthood by a process termed remodeling. Three types of cells are responsible for the formation of cartilage and bone; the chondrocyte, osteoblast, and osteoclast. These cells are under the influence of a plethora of regulatory molecules, which govern their action to provide an individual optimal bone mass. Interruption of this homeostatic machinery, especially in the elderly, often results in a loss of bone mass (osteoporosis) or cartilage damage (rheumatoid arthritis). Many pharmacological agents have been made available in an effort to prevent or alleviate these pathologies, however, one vector often overlooked is the diet. This review focuses on the relationship between dietary polyunsaturated fatty acids and bone biology, both in vivo and in vitro.

Perturbations in factors that modulate osteoblast functions in vitamin B6 deficiency.

It was hypothesized that the widespread structural defect of collagen in connective tissue of vitamin B6 deficient-animals and the consequent alteration in bone biomechanical properties cause an additional stress to their inflamed swollen tibiotarsometatarsal joints. The present study showed a 32% elevation (P < 0.02) in mean plasma free cortisol concentration. Vitamin D metabolism was impaired but without changing plasma calcium homeostasis and bone mineral content. Mean plasma calcitriol [1,25(OH)2D] concentration was significantly reduced (P < 0.001). Because plasma calcidiol concentration did not change, we speculated that either renal 25-hydroxycalciferol-1alpha-hydroxylase activity was reduced or 1,25(OH)2D turnover was increased. Plasma osteocalcin, an index of osteoblast function related to bone formation, was significantly decreased (P < 0.05). This adverse effect on osteoblasts was consistent with the reduction of bone specific alkaline phosphatase activity (another index of bone formation) found in a previous study. The excess of cortisol may have impaired these bone cells functions directly and (or) indirectly via the decline in calcitriol synthesis. Plasma hydroxyproline concentrations in B6-deficient animals were found to be significantly reduced (P < 0.001), suggesting that cortisol in excess had also a suppressive effect on another hydroxylase, namely tissue (mainly bone and liver) prolyl hydroxylase. The bone uncoupling (in formation and resorption) associated with vitamin B6 deficiency seems to be due to secondary hypercortisolism and (or) another unknown factors but not related to a change in bone modulators such as IGF-1 and eicosanoids.

Dietary ratio of (n-6)/(n-3) polyunsaturated fatty acids alters the fatty acid composition of bone compartments and biomarkers of bone formation in rats.

The effects of dietary polyunsaturated fatty acids (PUFA) on ex vivo bone prostaglandin E(2) (PGE(2)) production and bone formation rate were evaluated in rats. Weanling male Sprague-Dawley rats were fed AIN-93G diet containing 70 g/kg of added fat for 42 d. The dietary lipid treatments were formulated with safflower oil and menhaden oil to provide the following ratios of (n-6)/(n-3) fatty acids: 23.8 (SMI), 9.8 (SMII), 2.6 (SMIII), and 1.2 (SMIV). Ex vivo PGE(2) production in liver homogenates and bone organ cultures (right femur and tibia) were significantly lower in rats fed diets with a lower dietary ratio of (n-6)/(n-3) fatty acids than in those fed diets with a higher dietary ratio. Regression analysis revealed a significant positive correlation between bone PGE(2) and the ratio of arachidonic acid (AA)/eicosapentaenoic acid (EPA), but significant negative correlations between bone formation rate and either the ratio of AA/EPA or PGE(2) in bone. Activities of serum alkaline phosphatase isoenzymes, including the bone-specific isoenzyme (BALP), were greater in rats fed a diet high in (n-3) or a low ratio of (n-6)/(n-3), further supporting the positive action of (n-3) fatty acids on bone formation. These results demonstrated that the dietary ratio of (n-6)/(n-3) modulates bone PGE(2) production and the activity of serum BALP in growing rats.

Conjugated linoleic acid and bone biology.

Osteoporosis, osteoarthritis and inflammatory joint disease afflict millions of people worldwide. Inflammatory cytokines inhibit chondrocyte proliferation and induce cartilage degradation for which part of the response is mediated by PGE2. Excess production of PGE2 is linked to osteoporosis and arthritis and is associated with bone and proteoglycan loss. PGE2 also influences the IGF-I/IGFBP axis to facilitate bone and cartilage formation. Recent investigations with growing rats given butter fat and supplements of CLA demonstrated an increased rate of bone formation and reduced ex vivo bone PGE2 production, respectively. Furthermore, the supplements of CLA isomers resulted in their enrichment in lipids of various bone compartments of animals. The effects of CLA on bone biology in rats (IGF action and cytokines) appear to be dependent on the level of n-6 and n-3 fatty acids in the diet; however, these studies generally showed that CLA decreased ex vivo bone PGE2 production and in osteoblast-like cultures. Anti-inflammatory diets, including nutraceutical applications of CLA, may be beneficial in moderating cyclooygenase 2 (COX-2) activity or expression (influencing PGE2 biosynthesis) and might help to reduce rheumatoid arthritis (secondary osteoporosis). This review summarizes findings of CLA on bone modeling in rats and effects on cellular functions of osteoblasts and chondrocytes. These experiments indicate that CLA isomers possess anti-inflammatory activity in bone by moderating prostanoid formation.