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Background: Omega-3 fatty acids, including DHA and α-linolenic acid (ALA), are proposed to improve metabolic health by reducing obesity-associated inflammation. Their effects are mediated in part by conversion to oxylipins. ALA is relatively understudied, and direct comparisons to other omega-3 fatty acids are limited.Objectives: We compared the effects of equal doses of ALA and DHA on plasma oxylipins and markers of metabolic health in women with obesity.Methods: We carried out a randomized, double-blind, crossover clinical trial where women aged 20-51 with a BMI of 30-51 kg/m2 were supplemented with 4 g/day of ALA or DHA for 4 weeks in the form of ALA-rich flaxseed oil or DHA-rich fish oil. The primary outcome, the plasma oxylipin profile, was assessed at Days 0 and 28 of each phase by HPLC-MS/MS. Plasma fatty acids, inflammatory markers, and the monocyte glucose metabolism were key secondary outcomes. Data were analyzed using a mixed model.Results: Compared to the baseline visit, there were higher plasma levels of nearly all oxylipins derived from DHA (3.8-fold overall; P < 0.001) and EPA (2.7-fold overall; P < 0.05) after 28 days of fish-oil supplementation, while there were no changes to oxylipins after flaxseed-oil supplementation. Neither supplement altered plasma cytokines; however, adiponectin was increased (1.1-fold; P < 0.05) at the end of the fish-oil phase. Compared to the baseline visit, 28 days of flaxseed-oil supplementation reduced ATP-linked oxygen consumption (0.75-fold; P < 0.05) and increased spare respiratory capacity (1.4-fold; P < 0.05) in monocytes, and countered the shift in oxygen consumption induced by LPS.Conclusions: Flaxseed oil and fish oil each had unique effects on metabolic parameters in women with obesity. The supplementation regimens were insufficient to reduce inflammatory markers but adequate to elicit increases in omega-3 oxylipins and adiponectin in response to fish oil and to alter monocyte bioenergetics in response to flaxseed oil. This trial was registered at clinicaltrials.gov as NCT03583281. [ABSTRACT FROM AUTHOR]

Background: Differences in health effects of dietary α-linolenic acid (ALA) and DHA are mediated at least in part by differences in their effects on oxylipins.Objectives: Time course and sex differences of plasma oxylipins in response to ALA- compared with DHA-rich supplements were examined.Methods: Healthy men and women, aged 19-34 y and BMI 18-28 kg/m2, were provided with capsules containing ∼4 g/d of ALA or DHA in a randomized double-blind crossover study with >6-wk wash-in and wash-out phases. Plasma PUFA and oxylipin (primary outcome) concentrations at days 0, 1, 3, 7, 14, and 28 of supplementation were analyzed by GC and HPLC-MS/MS, respectively. Sex differences, supplementation and time effects, and days to plateau were analyzed.Results: ALA supplementation doubled ALA concentrations, but had no effects on ALA oxylipins after 28 d, whereas DHA supplementation tripled both DHA and its oxylipins. Increases in DHA oxylipins were detected as early as day 1, and a plateau was reached by days 5-7 for 11 of 12 individual DHA oxylipins and for total DHA oxylipins. Nine individual DHA oxylipins reached a plateau in females with DHA supplementation, compared with only 4 in males. A similar time course and sex difference pattern occurred with EPA and its oxylipins with DHA supplementation. DHA compared with ALA supplementation also resulted in higher concentrations of 4 individual arachidonic acids, 1 linoleic acid, and 1 dihomo-γ-linolenic acid oxylipin, despite not increasing the concentrations of these fatty acids, further demonstrating that oxylipins do not always reflect their precursor PUFA.Conclusions: DHA compared with a similar dose of ALA has greater effects on both n-3 and n-6 oxylipins in young, healthy adults, with differences in response to DHA supplementation occurring earlier and being greater in females. These findings can help explain differences in dietary effects of ALA and DHA.This study was registered at clinicaltrials.gov as NCT02317588. [ABSTRACT FROM AUTHOR]

Background and Aims: Consuming pulses (dry beans, dry peas, chickpeas, lentils) over several weeks can improve vascular function and decrease cardiovascular disease risk; however, it is unknown whether pulses can modulate postprandial vascular responses. The objective of this study was to compare different bean varieties (black, navy, pinto, red kidney) and white rice for their acute postprandial effects on vascular and metabolic responses in healthy individuals.Methods and Results: The study was designed as a single-blinded, randomized crossover trial with a minimum 6 days between consumption of the food articles. Vascular tone (primary endpoint), haemodynamics and serum biochemistry (secondary endpoints) were measured in 8 healthy adults before and at 1, 2, and 6 h after eating ¾ cup of beans or rice. Blood pressure and pulse wave velocity (PWV) were lower at 2 h following red kidney bean and pinto bean consumption compared to rice and navy bean, respectively (p < 0.05). There was greater vasorelaxation 6 h following consumption of darker-coloured beans, as shown by decreased vascular tone: PWV was lower after consuming black bean compared to pinto bean, augmentation pressure was lower after consuming black bean compared to rice and pinto bean, and wave reflection magnitude was lower after consuming red kidney bean and black bean compared to rice, navy bean, and pinto bean (p < 0.05). LDL-cholesterol concentrations were lower 6 h after black bean consumption compared to rice (p < 0.05).Conclusion: Overall, red kidney and black beans, the darker-coloured beans, elicited a positive effect on the tensile properties of blood vessels, and this acute response may provide insight for how pulses modify vascular function. [ABSTRACT FROM AUTHOR]

Background: Different fatty acids (FAs) can vary in their obesogenic effect, and genetic makeup can contribute to fat deposition in response to dietary FA composition. However, the antiobesogenic effects of the interactions between dietary MUFAs and genetics have scarcely been tested in intervention studies.Objective: We evaluated the overall (primary outcome) and genetically modulated (secondary outcome) response in body weight and fat mass to different levels of MUFA consumption.Methods: In the Canola Oil Multicenter Intervention Trial II, a randomized, crossover, isocaloric, controlled-feeding multicenter trial, 44 men and 71 women with a mean age of 44 y and an increased waist circumference (men ∼108 cm and women ∼102 cm) consumed each of 3 oils for 6 wk, separated by four 12-wk washout periods. Oils included 2 high-MUFA oils-conventional canola and high-oleic canola (<7% SFAs, >65% MUFAs)-and 1 low-MUFA/high-SFA oil blend (40.2% SFAs, 22.0% MUFAs). Body fat was measured using DXA. Five candidate single-nucleotide polymorphisms (SNPs) were genotyped using qualitative PCR. Data were analyzed using a repeated measures mixed model.Results: No significant differences were observed in adiposity measures following the consumption of either high-MUFA diet compared with the low-MUFA/high-SFA treatment. However, when stratified by genotype, 3 SNPs within lipoprotein lipase (LPL), adiponectin, and apoE genes influenced, separately, fat mass changes in response to treatment (n = 101). Mainly, the LPL rs13702-CC genotype was associated with lower visceral fat (high-MUFA: -216.2 ± 58.6 g; low-MUFA: 17.2 ± 81.1 g; P = 0.017) and android fat mass (high-MUFA: -267.3 ± 76.4 g; low-MUFA: -21.7 ± 102.2 g; P = 0.037) following average consumption of the 2 high-MUFA diets.Conclusions: Common variants in LPL, adiponectin, and apoE genes modulated body fat mass response to dietary MUFAs in an isocaloric diet in adults with abdominal obesity. These findings might eventually help in developing personalized dietary recommendations for weight control. The trial was registered at clinicaltrials.gov as NCT02029833 (https://www.clinicaltrials.gov/ct2/show/NCT02029833?cond=NCT02029833&rank=1). [ABSTRACT FROM AUTHOR]

Background: Novel oils high in monounsaturated fatty acids (MUFAs) and low in saturated fatty acids (SFAs) are an alternative to partially hydrogenated oils high in trans-unsaturated fatty acids. There is widespread use of high-MUFA oils across the food industry; however, limited knowledge of their cardiovascular impact exists.Objectives: We investigated the effects of diets containing canola oil, high-oleic acid canola oil (HOCO), and a control oil blend (diet formulated to emulate a Western fat profile) on lipids, lipoproteins, and apolipoproteins (apos), as secondary outcomes of the trial.Methods: In a multi-center, double-blind, randomized, 3-period crossover, controlled feeding trial, men (n = 44) and women (n = 75) with a mean age of 44 y, mean body mass index (BMI; in kg/m2) of 31.7, and an increased waist circumference plus ≥1 metabolic syndrome criteria consumed prepared, weight-maintenance diets containing canola oil [17.5% MUFAs, 9.2% polyunsaturated fatty acids (PUFAs), 6.6% SFAs], HOCO (19.1% MUFAs, 7.0% PUFAs, 6.4% SFAs), or control oil (10.5% MUFAs, 10.0% PUFAs, 12.3% SFAs) for 6 wk with ≥4-wk washouts. Fasting serum lipids were assessed at baseline and 6 wk. Diet effects were examined using a repeated measures mixed model.Results: Compared with the control, canola and HOCO diets resulted in lower endpoint total cholesterol (TC; -4.2% and -3.4%; P < 0.0001), LDL cholesterol (-6.6% and -5.6%; P < 0.0001), apoB (-3.7% and -3.4%; P = 0.002), and non-HDL cholesterol (-4.5% and -4.0%; P = 0.001), with no differences between canola diets. The TC:HDL cholesterol and apoB:apoA1 ratios were lower after the HOCO diet than after the control diet (-3.7% and -3.4%, respectively). There were no diet effects on triglyceride, HDL cholesterol, or apoA1 concentrations.Conclusions: HOCO, with increased MUFAs at the expense of decreased PUFAs, elicited beneficial effects on lipids and lipoproteins comparable to conventional canola oil and consistent with reduced cardiovascular disease risk in adults with central adiposity. This trial was registered at www.clinicaltrials.gov as NCT02029833. [ABSTRACT FROM AUTHOR]

Highlights • Oxylipin profiles of four rat adipose depots were determined. • Depots differed in 20–22 carbon oxylipins from cytochrome P450 and cyclooxygenase. • Linoleic acid derived lipoxygenase products made up the majority of oxylipin mass. • Dietary linoleic acid increased n-6 PUFA derived oxylipins. • Dietary linoleic acid with α-linolenic acid reduced n-6 PUFA oxylipins to control. Abstract Dietary PUFA and their effects on adipose tissue have been well studied, but oxylipins, the oxygenated metabolites of PUFA, have been sparsely studied in adipose tissue. To determine the oxylipin profile and to examine their potential importance in various adipose sites, female and male rats were provided control, high linoleic acid (LA), or high LA and high α-linolenic acid (LA + ALA) diets for six weeks. Analysis of gonadal (GAT), mesenteric (MAT), perirenal (PAT), and subcutaneous adipose tissues (SAT) revealed higher numbers of oxylipins in MAT and SAT, primarily due to 20–22 carbon cytochrome P450 oxylipins, as well as metabolites of cyclooxygenase derived oxylipins. LA oxylipins made up 75–96% of the total oxylipin mass and largely determined the total relative amounts between depots (GAT > MAT > PAT > SAT). However, when the two most abundant LA oxylipins (TriHOMEs) were excluded, MAT had the highest mass of oxylipins and exhibited the most sex differences. These differences existed despite comparable PUFA composition between depots. Dietary LA increased oxylipins derived from n-6 PUFA, and the addition of ALA generally returned n-6 PUFA oxylipins to levels similar to control and elevated some n-3 oxylipins. These data on oxylipin profiles in adipose depots from different anatomical sites and the effects of diet and sex provide fundamental knowledge that will aid future studies investigating the physiological effects of adipose tissue. [ABSTRACT FROM AUTHOR]

Dietary EPA and DHA given together alter oxylipins in adipose tissue. To compare the separate effects of individual dietary n-3 PUFA on oxylipins in different adipose depots (gonadal, mesenteric, perirenal, subcutaneous) in males and females, rats were provided diets containing higher levels of α-linolenic acid (ALA), EPA or DHA. Each n-3 PUFA enhanced its respective oxylipins the most, while effects on other n-3 oxylipins varied. For example: in perirenal and subcutaneous depots, more DHA oxylipins were higher with dietary ALA than with EPA; dietary EPA uniquely decreased 14-hydroxy-docosahexaenoic acid, in contrast to increasing many other DHA oxylipins. The n-3 PUFAs also reduced oxylipins from n-6 PUFAs in order of effectiveness: DHA > EPA > ALA. Diet by sex interactions in all depots except the perirenal depot resulted in higher oxylipins in males given DHA, and higher oxylipins in females given the other diets. Diet and sex effects on oxylipins did not necessarily reflect effects on either their tissue phospholipid or neutral lipid PUFA precursors. These varying diet and sex effects on oxylipins in the different adipose sites indicate that they may have distinct effects on adipose function. [ABSTRACT FROM AUTHOR]

Caloric restriction (CR) is one of the most promising strategies for weight loss but is associated with loss of lean mass, whereas compounds such as trans-10,cis-12 conjugated linoleic acid (t10-c12 CLA) have been promoted as antiobesity agents. To compare the mechanisms of weight reduction by CR and t10-c12 CLA, body composition, glucose control, and characteristics of adipose tissue with respect to cell turnover (stem cells and preadipocytes, apoptosis and autophagy) and Tbx-1 localization were examined in obese db/db mice and lean C57BL/6J mice undergoing CR or fed CLA isomers (0.4% w/w c9-t11 or t10-c12) for 4 weeks. Our findings show that the t10-c12 CLA reduced whole-body fat mass by decreasing all fat depots (visceral, inguinal, brown/interscapular), while CR lowered both whole-body fat and lean mass in obese mice. t10-c12 CLA elevated blood glucose in both obese and lean mice, while glycemia was not altered by CR. The adipocyte stem cell population remained unchanged; however, t10-c12 CLA reduced and CR elevated the proportion of immature adipocytes in obese mice, suggesting differential effects on adipocyte maturation. t10-c12 CLA reduced apoptosis (activated caspase-3) in both obese and lean mice but did not alter autophagy (LC3II/LC3I). Nuclear Tbx-1, a marker of metabolically active beige adipocytes, was greater in the adipose of t10-c12 CLA-fed animals. Thus, weight loss achieved via t10-c12 CLA primarily involves fat loss and more cells with Tbx-1 localized to the nucleus, while CR operates through a mechanism that reduces both lean and fat mass and blocks adipocyte differentiation. [ABSTRACT FROM AUTHOR]

Generation of oxylipins (oxygenated metabolites of fatty acids) by lipoxygenases may be responsible for the beneficial effects of 20- and 22-carbon n-3 fatty acids on adipose tissue dysfunction in obesity, but the potential actions of oxylipins derived from 18-carbon fatty acids, which are generally at higher levels in the diet, are unknown. We therefore compared the effects of select lipoxygenase-derived oxylipins produced from α-linolenic acid (ALA, C18:3 n-3), linoleic acid (LA, C18:2 n-6), and arachidonic acid (AA, C20:4 n-6) on key adipocyte functions that are altered in obesity. Individual oxylipins were added to the culture medium of differentiating 3T3-L1 preadipocytes for 6 days. Lipid accumulation was subsequently determined by Oil Red O staining, while Western blotting was used to measure levels of proteins associated with lipid metabolism and characteristics of adipocyte functionality. Addition of all oxylipins at 30 nM was sufficient to significantly decrease triglyceride accumulation in lipid droplets, and higher levels completely blocked lipid production. Our results establish that lipoxygenase-derived oxylipins produced from 18-carbon PUFA differentially affect multiple adipocyte processes associated with lipid storage and adipokine production. However, these effects are not due to the oxylipins blocking adipocyte maturation and thus globally suppressing all adipocyte characteristics. Furthermore, these oxylipin species decrease the lipid content of adipocytes regardless from which precursor fatty acid or lipoxygenase they were derived. Consequently, adipocyte characteristics can be altered through the ability of oxylipins to selectively modulate levels of proteins involved in both lipid metabolism and adipokine production. [ABSTRACT FROM AUTHOR]

Abstract: This study investigates the effects of monounsaturated and polyunsaturated fatty acids from different fat sources (High Oleic Canola, Canola, Canola–Flaxseed (3:1 blend), Safflower, or Soybean Oil, or a Lard-based diet) on adipose tissue function and markers of inflammation in Obese Prone rats fed high-fat (55% energy) diets for 12 weeks. Adipose tissue fatty acid composition reflected the dietary fatty acid profiles. Protein levels of fatty acid synthase, but not mRNA levels, were lower in adipose tissue of all groups compared to the Lard group. Adiponectin and fatty acid receptors GPR41 and GPR43 protein levels were also altered, but other metabolic and inflammatory mediators in adipose tissue and serum were unchanged among groups. Overall, rats fed vegetable oil- or lard-based high-fat diets appear to be largely resistant to major phenotypic changes when the dietary fat composition is altered, providing little support for the importance of specific fatty acid profiles in the context of a high-fat diet. [Copyright &y& Elsevier]

Abstract: This study investigated the efficacy of the plant-based n−3 fatty acid, α-linolenic acid (ALA), a dietary precursor of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), for modulating hepatic steatosis. Rats were fed high fat (55% energy) diets containing high oleic canola oil, canola oil, a canola/flax oil blend (C/F, 3:1), safflower oil, soybean oil, or lard. After 12 weeks, C/F and weight-matched (WM) groups had 20% less liver lipid. Body mass, liver weight, glucose and lipid metabolism, inflammation and molecular markers of fatty acid oxidation, synthesis, desaturation and elongation did not account for this effect. The C/F group had the highest total n−3 and EPA in hepatic phospholipids (PL), as well as one of the highest DHA and lowest arachidonic acid (n−6) concentrations. In conclusion, the C/F diet with the highest content of the plant-based n−3 ALA attenuated hepatic steatosis and altered the hepatic PL fatty acid profile. [Copyright &y& Elsevier]

Abstract: Objective: Peripheral artery disease (PAD) results from a decrease in blood flow to the limbs due to the presence of atherosclerotic plaque. It has been reported that isoflavones isolated from soybeans reduce arterial stiffness, a component of atherosclerotic disease. This study examined the effect of consuming whole legumes (non-soy) on arterial function in humans with PAD. Methods: Twenty-six individuals with PAD consumed ½ cup/day cooked legumes (beans, peas, lentils, chickpeas) daily for 8 weeks. Measurements of circulating factors and vascular function at baseline and study conclusion were compared. Results: No changes in were detected relative to baseline values for most parameters. Total and LDL-cholesterol were reduced by 5.0% and 8.7%, respectively. The ankle-brachial index (ABI) showed a 5.5% increase. Changes in ABI and LDL-cholesterol did not correlate. Serum markers of endothelial dysfunction and inflammation were unchanged, but short-chain acylcarnitine concentrations were significantly decreased. Conclusions: A legume-rich diet can elicit major improvements in arterial function and serum cholesterol in the absence of changes in either body mass or blood pressure, although the improvements in vascular function and serum lipids were unrelated. Although the positive results obtained with this dietary intervention were not explained by biomarkers of endothelial function and inflammation, altered acylcarnitine levels indicate an improvement in skeletal muscle metabolism due to enhanced tissue perfusion. [Copyright &y& Elsevier]

Analysis of oxylipins derived from fatty acids may provide insight into the biological effects of dietary lipids beyond their effects on tissue fatty acid profiles. We have previously observed that diets with higher amounts of e-linolenic acid (ALA; 18:3n3) are associated with reduced obesity-related glomerulopathy (ORG). Therefore, to examine the renal oxylipin profile, the effects of dietary linoleic acid (LA; 18:2n6) and ALA on oxytipins and renal phospholipid fatty acid composition, and the relationship between oxylipins and ORG, diet-induced obese rats displaying ORG were fed 8 different diets for 8 wk as follows (oil/oil = combination of two oils) [shown as ALA/LA (in g) per 100 g oil]: canola/flax (20/18), canola (8/18), soy (9/53), high-oleic canola/canola (5/16), high-oleic canola (2/15), lard/soy (1/8), and safflower (0.2/73). Targeted lipidomic analysis by HPLC-tandem mass spectrometry revealed that LA and ALA oxylipins comprised 60% of the total renal oxylipin profile examined. Of the >60 oxylipins screened, only those derived either directly or indirectly from ALA were associated with less glomerulomegaly, indicative of reduced ORG progression. Both the amount and ratio of dietary LA and ALA influenced renal polyunsaturated fatty acids (PUFAs); in contrast, only fatty acid amount altered oxylipins derived from these fatty acids, but there was no apparent competition by LA or ALA on their formation. Dietary LA incorporation into renal phospholipids was higher than for ALA, but ALA oxylipin:ALA ratios were higher than the analogous LA ratios for select lipoxygenase reactions. This indicates that the effect of dietary ALA on renal oxylipins exceeded what was reflected in renal PUFA composition, In conclusion, dietary LA and ALA have differential effects on renal oxylipins and PUFAs, and ALA-derived oxylipins are associated with renoprotection in this model of ORG. [ABSTRACT FROM AUTHOR]

Abstract: Purpose: In healthy participants and those with diet-controlled type 2 diabetes (T2DM), to (1) compare the acute 3-hour post-prandial response of glucose, insulin and other gastrointestinal hormones known to influence food intake and glucose metabolism after consumption of a food product made from whole grain buckwheat flour versus rice flour; (2) determine the effect of daily consumption of a food product made from whole grain buckwheat flour on fasting glucose, lipids and apolipoproteins. Methods: Healthy participants or those with T2DM consumed either buckwheat or rice crackers. Blood samples were collected at baseline and 15, 30, 45, 60, 120 and 180minutes after consumption. In a second phase of the study, participants consumed one serving of buckwheat crackers daily for 1week; fasting blood samples from day 1 and day 7 were analyzed. Results: Post-prandial plasma glucagon-like peptide-1, glucose-dependent insulinotropic peptide and pancreatic polypeptide were altered after consuming buckwheat versus rice crackers. Interestingly, changes in these hormones did not lead to changes in post-prandial glucose, insulin or C-peptide concentrations. Significant correlations were observed between both fasting concentrations and post-prandial responses of several of the hormones examined. Interestingly, certain correlations were present only in the healthy participant group or the T2DM group. There was no effect of consuming buckwheat for one week on fasting glucose, lipids or apolipoproteins in either the healthy participants or those with T2DM. Conclusions: Although the buckwheat cracker did not modify acute glycemia or insulinemia, it was sufficient to modulate gastrointestinal satiety hormones. [Copyright &y& Elsevier]

Abstract: Background: Flaxseed is an important source of alpha-linolenic acid an essential omega-3 fatty acid. The possibility that a supplementation of the diet with foods rich in alpha-linolenic acid, antioxidants and fiber (like flaxseed) has not been investigated. Methods/design: The primary objective is to determine whether consumption of a diet rich in FLAXseed over a one year period has any beneficial cardiovascular effects in patients with Peripheral Arterial Disease (FLAX-PAD study). This is a single center, prospective, double blinded, randomized controlled clinical trial aimed at in 110 patients over 40years old and with peripheral arterial disease. Patients will receive 30g of milled flaxseed (or placebo) per day. Primary endpoints are incidence of myocardial infarction and stroke. Secondary measures include: requirement for surgical interventions, exercise and cardiopulmonary performance, cardiac arrhythmias, serum lipid profile, arterial sufficiency, blood pressure, inflammatory profile, platelet function, changes in drug dosage levels, as well as nutrigenomic and biomarker profiles in the blood. Recruitment and baseline examinations started in October 2008. Baseline data of the 110 patients is shown. Conclusions: FLAX-PAD will generate data on the safety, tolerability, cardiovascular efficacy and genomic response to a diet rich in flaxseed. It will determine the effects on primary and secondary events (stroke, myocardial infarctions, angina pectoris, cardiac arrhythmias) as well as in secondary endpoints (exercise performance, blood pressure and circulating lipid levels) in patients with PAD. [Copyright &y& Elsevier]

Abstract: The isomer-specific effects of conjugated linoleic acid (CLA) on hepatic steatosis were assessed in fa/fa Zucker rats, a model for insulin resistance and the metabolic syndrome. Eight weeks of feeding trans-10,cis-12 CLA significantly improved glucose tolerance without changing body weight or visceral adipose mass. The trans-10,cis-12 isomer was also associated with reduced liver lipid content, improved liver function and reduced inflammation; these effects were not observed in rats fed the cis-9,trans-11 CLA isomer. Reduced liver lipid content did not correlate with activation of AMP-activated protein kinase or suppressed activation of sterol-regulatory element binding protein-1, two key regulators of hepatic lipid metabolism. Interestingly, rats fed cis-9,trans-11 CLA had fewer cytoplasmic lipid droplets in hepatocytes compared to rats fed control diet, but these droplets were larger in size. Conversely, fa/fa rats fed the trans-10,cis-12 CLA isomer had greater numbers of hepatic lipid droplets that were smaller in size, resulting in overall lower total lipid within these droplets. Changes in lipid droplets were associated with lower hepatic levels of PERILIPIN-2 (formerly known as adipophilin) in rats fed trans-10,cis-12 CLA, whereas amounts of other members of the PERILIPIN family of lipid droplet proteins were unaffected by dietary CLA. However, CLA isomers differentially affected the subcellular localization of these proteins. Treatment of H4IIE rat hepatoma cells with CLA isomers neither prevented nor reversed, but rather induced cytoplasmic lipid droplet formation, suggesting that the anti-steatotic effects of trans-10,cis-12 CLA are likely indirect and potentially mediated via increased lipid utilization by peripheral tissues. [Copyright &y& Elsevier]

Conjugated linoleic acid (CLA) slows the progression of disease in models of chronic kidney disease. Because obesity is associated with nephropathy and increased renal cyclooxygenase (COX) levels, the effects of dietary CLA on kidney function, morphology, and COX protein levels in the kidneys of young obese (fa/fa) Zucker rats, a model of metabolic syndrome, were examined. In study 1,6-wk-old fa/fa and lean Zucker rats were given a mixture of CLA isomers (1.5% CLA, wt:wt) or the control diet (CTL) with no CLA for 8 wk. To examine specific isomer effects, study 2 used the same model with the following diets: 0.4% (g/g) cis-9, trans-11 (c9,t11) CLA; 0.4% trans-10, cis-12 (t10,c12) CLA; a combination of these 2 isomers (0.4% each); or CTL diets with no CLA. In study 1, fa/fa rats given the CLA mixture had 11% smaller kidney weights and 28% smaller glomeruli, and feed intake and body weight did not differ from the CTL rats. In study 2, diet also did not affect body weights, but fa/fa rats given a diet containing t10, c12 CLA had 7% lower kidney weights, 20% smaller glomeruli, and 39% lower COX-2 protein levels than CTL rats. In conclusion, dietary t10, C12 CLA reduces the enlargement of glomeruli in young obesity-associated nephropathy and is associated with lower protein levels of renal COX-2. Long-term studies with CLA supplementation are required to determine whether these changes would lead to reduction in development of renal disease associated with obesity. [ABSTRACT FROM AUTHOR]

Abstract: Conjugated linoleic acids (CLA) have been shown to alter adiposity in some species with varying effects on insulin resistance. The objective of this 8-week study was to investigate the effects of feeding a CLA mixture (1.5%, wt/wt) on adipocyte size, insulin sensitivity, adipokine status, and adipose lipid composition in fa/fa vs lean Zucker rats. The fa/fa CLA-fed rats had smaller adipocytes and improved insulin sensitivity compared with fa/fa rats fed the control diet. Conjugated linoleic acids did not affect select markers of adipose differentiation, lipid filling, lipid uptake, or oxidation. Dietary CLA, compared with the control diet, reduced circulating leptin and elevated fasting serum adiponectin concentrations in fa/fa rats. Adipose resistin messenger RNA levels were greater in fa/fa CLA-fed rats compared with fa/fa control rats. CLA did not markedly alter adipose phospholipid fatty acid composition, and the changes in the triacylglycerol fatty acid composition reflected a lower delta-9 desaturase index of CLA-fed vs control-fed rats. In conclusion, CLA reduced adipocyte size and favorably modified adipokine status and insulin sensitivity in fa/fa Zucker rats. [Copyright &y& Elsevier]

Abstract: Pancreatic preservation is an important part of diabetes management that may occur with improved peripheral insulin sensitivity and attenuated low-grade adipose tissue inflammation. The objective of the current study was to determine the response of obese, insulin-resistant fa/fa Zucker rats vs lean controls to dietary conjugated linoleic acid (CLA) supplementation with respect to pancreatic islet size, insulin resistance, and markers of inflammation and adipose glucose uptake. Six-week-old fa/fa and lean Zucker rats (n = 20 per genotype) were fed either a 1.5% CLA mixture or control diet for 8 weeks. Oral glucose tolerance testing was conducted at 7.5 weeks. Fasting serum haptoglobin, insulin, and C-peptide were assayed, and select messenger RNA (mRNA) and protein markers of inflammation and glucose metabolism were measured in adipose and liver tissues. CLA-fed fa/fa Zucker rats had smaller islet cell size, improved oral glucose tolerance and insulinemia, and attenuated serum haptoglobin levels compared with control-fed fa/fa Zucker rats, despite no differences in body weight and a slightly higher visceral adipose mass. CLA did not alter insulin sensitivity or islet size in lean Zucker rats. The CLA-fed fa/fa rats also had greater adipose glucose transporter-4 mRNA and less adipose tumor necrosis factor α mRNA and protein compared with control-fed fa/fa rats. In contrast, other markers of inflammation and glucose metabolism including adipose macrophage inflammatory factor, macrophage inflammatory protein-2, and liver pyruvate carboxylase and pyruvate dehydrogenase kinase 4 were not significantly changed. These results suggest that CLA supplementation preserved pancreatic function in conjunction with improved peripheral glucose use and reduced inflammation in fa/fa Zucker rats. [Copyright &y& Elsevier]

Dietary conjugated linoleic acid (CLA) is being investigated for beneficial effects for disease prevention and treatment in a variety of experimental models, including obesity and type 2 diabetes. To date, rodent studies suggest that trans-10,cis-12 (t10,c12) CLA is associated with greater insulin resistance, despite lower body fat, and that a CLA mixture (and perhaps c9,t11) could be beneficial for the management of insulin resistance. Studies investigating the mechanisms by which CLA operates at the cellular level show that the primary targets for CLA are members of the nuclear receptor family, particularly the lipostat transcription factors peroxisome proliferator-activated receptor α (PPARα), PPARγ, sterol regulatory element-binding protein 1c, and liver X receptor α. Consequently, the effects of CLA on glucose metabolism are likely secondary effects mediated through factors such as PPARγ coactivator 1 that are controlled by these nuclear receptors. The different responses of normal compared with insulin-resistant obese rodents suggest that interactions of CLA isomers with the cellular components that contribute to development of metabolic syndrome require further investigation. [ABSTRACT FROM AUTHOR]

Prostaglandins released by injured vascular tissue can modulate smooth muscle cell (SMC) proliferation. The mechanism of action of PGE2 was investigated with porcine coronary artery SMCs obtained by explant culture. DNA and RNA syntheses exhibited a concentration-dependent increase following treatment of quiescent SMCs with PGE2, while PGI2 had no effect. PGE2 also elevated PCNA expression, bromodeoxyuridine incorporation, and cell number, indicative of a hyperplastic growth response. Furthermore, induction of c-fos expression required activation of both phosphatidylinositol 3-kinase and mitogen-activated protein kinase. Contrary to these data, treatment of proliferating cells with PGE2 caused a reduction in DNA synthesis. A role for PKA in either growth stimulation or inhibition was excluded. Interestingly, only quiescent SMCs expressed EP2 receptors, and the selective EP2 receptor agonist butaprost confirmed that this receptor was essential for growth stimulation and possibly inhibition. These data suggest that the growth state-dependent actions of PGE2 on SMC proliferation may be mediated via the EP2 receptor. [Copyright &y& Elsevier]

Background. The internal mammary artery (IMA) has been shown to have a significantly superior long-term patency rate when compared with the saphenous vein (SV) graft. Cultured smooth muscle cells (SMCs) from the IMA are more resistant to the mitogenic effects of platelet-derived growth factor (PDGF), when compared with SMCs that are derived from the SV. The radial artery (RA) is currently being used as an alternative to the SV. However, no long-term patency data are available for the RA, and there is no information on the biological behavior of RA-derived SMCs in culture.Methods. Smooth muscle cell cultures were taken from patients who underwent coronary artery bypass grafting with the IMA, RA, and SV. A quiescent state was induced by serum deprivation for 5 days. Thereafter cells were induced to proliferate by exposure to PDGF-BB. Levels of c-fos expression and 3H-thymidine incorporation were used as markers of cell proliferation.Results. We found that even after serum deprivation, c-fos was still detectable; however, basal levels were higher in cells from the SV than cells from either the RA (p = 0.003) or IMA (p = 0.008). After stimulation with PDGF-BB, c-fos expression was greater in SMCs from the SV relative to the RA (p < 0.001 or the IMA (p = 0.02). Finally, relative to the SV, 3H-thymidine in the RA was 0.76 ± 0.22 (p < 0.05) and 0.39 ± 0.24 (p < 0.002) in the IMA, respectively.Conclusions. The data indicate that SMCs from arterial conduits are more resistant to the mitogenic effects of PDGF-BB than those from venous conduits. Our results offer a mechanistic explanation of why arterial conduits might demonstrate patency superior to that of the SV. [Copyright &y& Elsevier]

• Rationale, design, and procedures of the OXBIO study. • A double-blind, cross-over study examining 4-week ALA and DHA supplementation. • Compares effects of n-3 supplements on oxylipin profiles in females with obesity. • Examines relationships between oxylipins & inflammation during n-3 supplementation. Consumption of omega-3 polyunsaturated fatty acids (n-3 PUFAs) has been reported to provide health benefits, but it remains unknown whether the fatty acids themselves or their oxygenated metabolites, oxylipins, are responsible for the beneficial effects. This paper describes the design and rationale of a randomized, double-blinded, cross-over study comparing the effects of α-linolenic acid (ALA)-rich flax oil and docosahexaenoic acid (DHA)-rich fish oil supplementation on circulating oxylipin profiles in females with obesity, in relation to obesity-induced inflammation. Pre-menopausal females (n = 24) aged 20-55 with a BMI ≥30, will consume capsules containing flaxseed oil (4 g ALA/day) or fish oil (4 g DHA + 0.8 g EPA/day) during 4-week supplementation phases, with a minimum 4-week washout. The primary outcome is alterations in plasma oxylipin profiles. Secondary outcomes include effects of supplementation on circulating markers of inflammation, adipokines, plasma fatty acid composition, blood lipid profile, anthropometrics, oxylipin and cytokine profiles of stimulated immune cells, monocyte glucose metabolism, blood pressure and pulse wave velocity. This trial has been approved by the University of Manitoba Biomedical Research Ethics Board and the St. Boniface Hospital Research Review Committee. The study will provide information regarding the effects of ALA and DHA supplementation on oxylipin profiles in obese but otherwise healthy females. Additionally, it will improve our understanding of the response of circulating inflammatory mediators originating from immune cells, adipose tissue and the liver to n-3 PUFA supplementation in relation to the metabolic features of obesity. [ABSTRACT FROM AUTHOR]

• Summary of effects of oxylipins on endothelial cells in vitro, ex vivo & in vivo. • Describes key mechanisms by which oxylipins modulate endothelial cell function. • DHA-derived oxylipins show beneficial effects on endothelial cells. • Studies on ALA-derived oxylipins and endothelial cells are lacking. Oxylipins, which are circulating bioactive lipids generated from polyunsaturated fatty acids (PUFAs) by cyclooxygenase, lipooxygenase and cytochrome P450 enzymes, have diverse effects on endothelial cells. Although studies of the effects of oxylipins on endothelial cell function are accumulating, a review that provides a comprehensive compilation of current knowledge and recent advances in the context of vascular homeostasis is lacking. This is the first compilation of the various in vitro, ex vivo and in vivo reports to examine the effects and potential mechanisms of action of oxylipins on endothelial cells. The aggregate data indicate docosahexaenoic acid-derived oxylipins consistently show beneficial effects related to key endothelial cell functions, whereas oxylipins derived from other PUFAs exhibit both positive and negative effects. Furthermore, information is lacking for certain oxylipin classes, such as those derived from α-linolenic acid, which suggests additional studies are required to achieve a full understanding of how oxylipins affect endothelial cells. [ABSTRACT FROM AUTHOR]

Abstract: In skeletal muscle, the molecular mechanisms by which insulin stimulates glucose transport remains incompletely understood. Our study investigated the cellular dynamics of intracellular Ca2+ mobilisation and Ca2+/calmodulin-dependent protein kinase II (CaMKII) activation on insulin-induced skeletal muscle glucose transport. L6 myotubes were treated without or with insulin [100nM] for 15min and subsequently monitored for glucose uptake using isotope-labelled 2-deoxyglucose (I-2DOG), intracellular Ca2+ (Cai 2+) release using Fluo-4AM and protein phosphorylation using Western blotting. Acute exposure of myotubes to insulin increased both Akt substrate-160kDa (AS160) phosphorylation and I-2DOG uptake. Insulin concurrently increased Cai 2+ and activated CaMKII. Exposing myotubes to either BAPTA/AM to sequester Cai 2+ or KN-93 to inhibit CaMKII activity, decreased insulin-induced glucose uptake without affecting AS160 phosphorylation. On the other hand, blocking either calmodulin or the autoregulatory domain of CaMKII blocked the effect of insulin on both AS160 phosphorylation and glucose transport. Likewise, genetic knockdown of CaMKII in myotubes using siRNA completely abolished insulin-mediated glucose uptake. These results illustrate impairments in Cai 2+ mobilisation and CaMKII activation are sufficient to negatively influence insulin-dependent glucose transport by L6 myotubes. Additionally, our results show for the first time that Cai 2+ and domain-dependent CaMKII signalling differentially affect insulin-induced AS160 phosphorylation, and establish that Ca2+ and CaMKII are components of the insulin signalling pathway in L6 myotubes. [Copyright &y& Elsevier]

Abstract: With the rising incidence of obesity and the metabolic syndrome, obesity-associated nephropathy also has increased. One of the earliest pathologies in the development of this nephropathy is glomerular hyperfiltration and hypertrophy. Dietary soy protein (SP) ameliorates disease progression in several models of renal disease, and vegetable sources of protein, as compared to animal sources of protein, alter renal hemodynamics. Therefore, the effect of dietary SP on early renal disease and prostanoid production was examined in the obese fa/fa Zucker rat. Rats, 6 weeks of age, were given diets containing 17% protein from either SP or egg white (EW) for 8 weeks. Feed consumption and body and kidney weights were significantly greater in fa/fa rats as compared to lean rats. The fa/fa rats also had 139% more proteinuria and kidneys with 43% larger glomeruli. SP feeding did not alter body weights or proteinuria but did result in 6% lower kidney weights (g/100 g body weight) and 16% smaller glomeruli in fa/fa rats. Cyclooxygenase activity as determined by 6-keto prostaglandin F1α (6-keto PGF1α) synthesis was lower in fa/fa rats given SP-based diets as compared to those given EW-based diets. Ratios of renal thromboxane (TX) B2/6-keto PGF1α and PGE2/6-keto PGF1α were higher, while TXB2/PGE2 levels were not different in rats given SP diets as compared to those given EW diets, also indicating that dietary SP reduced renal 6-keto PGF1α levels. These findings suggest that attenuation of early glomerular hypertrophy in young obese fa/fa rats by dietary SP may be mediated by the lower levels of 6-keto PGF1α since this would be expected to reduce glomerular hyperfiltration. [Copyright &y& Elsevier]

The arachidonate 12-lipoxygenase (ALOX12) enzyme catalyzes polyunsaturated fatty acids and facilitates generation of bioactive lipid mediators associated with various biological processes and disease pathologies. The human genome assembly revealed that the ALOX12 gene overlaps an antisense non-coding gene designated as ALOX12-antisense 1 (ALOX12-AS1). This arrangement indicates that the uncharacterized ALOX12-AS1 long non-coding RNA (lncRNA) may bind to the sense coding ALOX12 mRNA to form an antisense-sense duplex providing the basis of a novel ALOX12 regulatory mechanism. Therefore, this study was designed to determine whether the interaction of ALOX12-AS1 with ALOX12 mRNA functions as an anti-sense/sense duplex-mediated regulatory mechanism controlling the cellular content of ALOX12. Our findings indicate that two major isoforms of ALOX12-AS1 lncRNA are ubiquitously expressed in a variety of primary adult human tissues and different transformed cell types. RNA-FISH revealed cell-type-specific cytosolic as well as nuclear and nucleolar localization of the lncRNA. Interestingly, phorbol ester-induced nucleo-cytoplasmic translocation of the lncRNA in monocytic THP-1 cells resulted in a reduction of ALOX12 protein without a concomitant change in its mRNA level. This indicated ALOX12-AS1 operates via an antisense-sense duplex-mediated translational downregulation mechanism. This deduction was validated by demonstrating sense/antisense duplex formation and an association of the duplex with ribosomal proteins in HEK293 cells. Overall, this study revealed a hitherto unknown mechanism of antisense lncRNA-mediated translational downregulation of ALOX12 that adds to the existing regulatory mechanisms for the modulation of potent bioactive lipid mediators that contribute to both health and disease. [Display omitted] • ALOX12-AS1 lncRNA is ubiquitously expressed in primary adult human tissues. • The cytosolic and nuclear localization of the lncRNA is cell-type-specific and controlled by cell signaling. • The sense-antisense duplex is associated with ribosomal proteins. • ALOX12-AS1 modulates ALOX12 protein levels by translational downregulation. [ABSTRACT FROM AUTHOR]

We recently reported that loss of calcium/calmodulin-dependent protein kinase kinase-2 (CAMKK2), a serine/threonine kinase activated by intracellular calcium, in mice leads to tissue-specific aberrant turnover of transferrin (TF), a receptor-mediated iron-transporter that supplies iron to tissues. Iron dyshomeostasis is associated with the pathogenesis of several diseases, making TF transport relevant to health. In this study, we used hemizygous CAMKK2 hypomorphic human endothelial cell (EA.hy926) clones to demonstrate that cells with reduced CAMKK2 exhibit increased TF uptake and transcytosis, and decreased intracellular trafficking to subcellular organelles compared to wild-type. The abnormal TF trafficking in CAMKK2 hypomorphic cells correlated with a reduction in intracellular iron content and defective glucose metabolism including glycolysis and mitochondrial respiration. CAMKK2 deficiency also caused reduction in GAPDH and VDAC1 protein level which correlated to defective bioenergetics function. These findings have identified a novel mechanistic link between abnormal calcium signaling, iron dyshomeostasis and metabolic dysfunction involving CAMKK2. Unlabelled Image • Hemizygous deletion created CAMKK2 hypomorphic (CAMKK2+/−) Ea.hy926 endothelial cells. • Reduced (50%) CAMKK2 disrupted uptake and transcytosis of TF in Ea.hy926 cells. • CAMKK2 reduction also disrupted subcellular trafficking of TF and iron content. • Iron dyshomeostasis in CAMKK2+/− cells lead to defective glucose metabolism • A fraction of cytosolic CAMKK2 is associated with TF and TFRC. [ABSTRACT FROM AUTHOR]

Chronic inflammation, mediated in large part by proinflammatory macrophage populations, contributes directly to the induction and perpetuation of metabolic diseases, including obesity, insulin resistance and type 2 diabetes. Polyunsaturated fatty acids (PUFAs) can have profound effects on inflammation through the formation of bioactive oxygenated metabolites called oxylipins. The objective of this study was to determine if exposure to the dietary omega-3 PUFA α-linolenic acid (ALA) can dampen the inflammatory properties of classically activated (M1-like) macrophages derived from the human THP-1 cell line and to examine the accompanying alterations in oxylipin secretion. We find that ALA treatment leads to a reduction in lipopolysaccharide (LPS)-induced interleukin (IL)-1β, IL-6 and tumor necrosis factor-α production. Although ALA is known to be converted to longer-chain PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), DHA oxylipins were reduced overall by ALA treatment, as was LPS-induced secretion of EPA oxylipins. In contrast, we observed profound increases in oxylipins directly derived from ALA. Lipoxygenase products of linoleic acid were also dramatically increased, and LPS-induced production of AA oxylipins, particularly prostaglandin D2, was reduced. These results suggest that ALA may act to dampen the inflammatory phenotype of M1-like macrophages by a unique set of mechanisms distinct from those used by the long-chain omega-3 fatty acids EPA and DHA. Thus, there is strong rationale for investigating the functions of ALA oxylipins and lesser-known LA oxylipins since they hold promise as anti-inflammatory agents. [ABSTRACT FROM AUTHOR]

Monocytes play a large role in chronic inflammatory conditions such as obesity, atherosclerosis and infection. Marine-derived omega-3 fatty acids such as docosahexaenoic acid (DHA) beneficially alter immune function and attenuate chronic inflammation in part by modifying gene expression. Comparisons with plant-derived omega-3 α-linolenic acid (ALA) on immune cell gene expression and function are limited. Transcriptome analysis was performed on THP-1 human monocytes treated with ALA, DHA or vehicle for 48 hr using fold change analysis, principal component analysis (PCA), partial least squares-discriminant analysis (PLS-DA), variable importance analysis (VIP), and ingenuity pathway analysis (IPA). Candidate genes were validated by qPCR. Functional assays evaluated the transcriptomic predictions. Expression of candidate transcripts identified in THP-1 cells were examined in PBMC from clinical trial (OXBIO; NCT 03583281) participants consuming ALA- or DHA-rich oil supplements. ALA and DHA-treated monocytes presented distinct transcriptomic profiles as per VIP and PLS-DA. Both fatty acids were predicted to reduce cellular cholesterol content, while ALA would uniquely increase response to infection and chemotactic signals. Functional assays revealed ALA and DHA decreased cholesterol content. DHA significantly decreased the response to infection and chemotaxis, but ALA had no effect. Candidate transcripts responded similarly in PBMC from n-3 PUFA supplemented women with obesity. ALA and DHA differentially alter the transcription profiles and functions associated with the response to infection, chemotaxis, and cholesterol metabolism in mononuclear immune cells. Thus, they may uniquely affect related disease processes contributing to obesity, atherosclerosis, and the response to infection. [Display omitted] • ALA and DHA treatment decrease cholesterol metabolism genes in THP-1 monocytes. • ALA increases expression of genes related to chemotaxis and response to infection. • OXBIO participants Immune cells have similar transcription patterns as THP-1 cells. • ALA and DHA uniquely affect processes associated with obesity and atherosclerosis. [ABSTRACT FROM AUTHOR]

We previously established that lentils were able to significantly attenuate the development of hypertension in spontaneously hypertensive rats (SHRs), but the mechanism was not investigated. The current study was therefore designed to examine the effect of lentils on arterial function in relation to arterial stiffness, lipid biochemistry and activation of select aortic proteins. Seventeen-week-old male SHRs were randomly assigned to groups (n=10/group) fed (a) 30% w/w green lentils, (b) 30% red lentils, (c) 30% mixed lentils (red and green) or (d) no lentils for 8 weeks. Normotensive Wistar Kyoto (WKY) groups (n=10/group) received either the mixed lentil or no lentil diet. Blood pressure, pulse wave velocity and serum lipids were measured at baseline and 8 weeks, while pressure myography, arterial morphology and aortic proteins were measured after termination. There were no dietary-related changes in pulse wave velocity or blood pressure for any SHR or WKY group. Low-density lipoprotein cholesterol and high-density lipoprotein cholesterol were significantly lower in only SHR red lentil and WKY mixed lentil groups compared to their controls. The lentil diets reduced the media:lumen ratio of SHRs relative to control-fed SHRs but had no effect on WKYs. Both red and green lentils reduced arterial stiffness of SHRs but not WKYs. SHR lentil groups showed lower aortic p38 mitogen-activated protein kinase (p38MAPK) phosphorylation, thus implying that p38MAPK activation is suppressed with lentil feeding. Lentil-based diets suppress pathological vascular remodeling in SHRs, while green lentils maintain the vascular function of SHRs similar to normotensive WKYs despite the presence of high blood pressure. [ABSTRACT FROM AUTHOR]

In this study, we hypothesize that the biologically active isomers of conjugated linoleic acid (CLA), cis -9, trans -11 (c9,t11) and trans -10, cis -12 (t10,c12) CLA, have different effects on early and late stages 3T3-L1 preadipocyte differentiation. Both c9-t11 and t10-c12CLA stimulated early stage pre-adipocyte differentiation (day 2), while t10-c12CLA inhibited late differentiation (day 8) as determined by lipid droplet numbers and both perilipin-1 levels and phosphorylation state. At day 8, the adipokines adiponectin, chemerin and adipsin were all reduced in t10-c12CLA treated cells versus control cells. Immunofluorescence microscopy showed perilipin-1 was present solely on lipid droplets on day 8 in t10-c12 treated 3T3-L1 cells, whereas preilipin-1 was also located in the perinuclear region in control and c9-t11 treated cells. The t10-c12CLA isomer also decreased levels of hormone-sensitive lipase and inhibited lipolysis. These findings indicate that the decrease in lipid droplets caused by t10-c12CLA is the result of an inhibition of lipid droplet production during adipogenesis rather than a stimulation of lipolysis. Additionally, treatment with Gö6976 blocked the effect of t10-c12CLA on perilipin-1 phosphorylation, implicating PKCα in perilipin-1 phosphorylation, and thus a regulator of triglyceride catabolism. These data are supported by evidence that t10-c12CLA activated PKCα. These are the first data to show that CLA isomers can affect lipid droplet dynamics in adipocytes through PKCα. [ABSTRACT FROM AUTHOR]

Background Trans- 10, cis -12 ( t 10 -c 12) CLA treatment reduces lipid accumulation in differentiating mouse and human adipocytes, and decreases fat mass in mice, yet the mechanism of action remains unknown. Objective This study investigated the effect of the cis- 9, trans- 11 ( c 9- t 11) and t 10 -c 12 CLA isomers on the Wnt/β-catenin pathway, which has been reported to inhibit adipogenesis by down-regulating PPARγ. Results We observed that t 10 -c 12 CLA treatment of 3T3-L1 adipocytes increases the levels of β-catenin and Ser-675 phosphorylated β-catenin due to inhibition of its degradation. These changes in β-catenin were not linked to either the Wnt/β-catenin agonist Wnt10b or other upstream effectors such as SFRP-5. Paradoxically, the presence of higher amounts of β-catenin did not elevate cyclin D1 levels, which is recognized as a critical target gene. Neither of the CLA isomers affected the localization of β-catenin in the cytosol and nucleus as determined by immunofluorescence microscopy. However, subcellular fractionation suggested the level of cytosolic β-catenin was reduced in t 10 -c 12 CLA treated cells. Immunoprecipitation revealed that t 10 -c 12 CLA increased the interaction of β-catenin and PPARγ. Conclusions t 10 -c 12-CLA inhibits adipocyte differentiation by increasing β-catenin stability in 3T3-L1 adipocytes, thus enhancing sequestration of PPARγ in an inactive complex, which prevents progression of adipogenesis. [ABSTRACT FROM AUTHOR]

Dietary conjugated linoleic acid (CLA) reduces indicators of early renal disease progression and the associated elevated cyclooxygenase (COX) levels in young obese rats with obesity-associated nephropathy (OAN). Therefore, renal function and injury and COX and its metabolites were assessed in obese fa/fa Zucker rats with more advanced renal disease. Obese rats at 16 weeks of age were provided with either cis (c)9, trans (t)11 ( fa/fa -9,11) or t10,c12 ( fa/fa -10,12) CLA for 8 weeks, and compared to lean (lean-CTL) and obese ( fa/fa -CTL) rats provided the control diet without CLA. Obese rats displayed significantly reduced renal function and increased renal injury compared to lean rats. In the obese rat groups, glomerular hypertrophy was reduced in both CLA-supplemented groups. While all other measures of renal function or injury were not different in fa/fa -9,11 compared to fa/fa -CTL rats, the fa/fa -10,12 rats had greater renal hypertrophy, glomerular fibrosis, fibrosis, tubular casts and macrophage infiltration compared to the fa/fa -CTL and fa/fa -9,11 groups. The fa/fa -10,12 group also had elevated levels of renal COX1, which was associated with increased levels of two oxylipins produced by this enzyme, 6-keto-prostaglandin F 1α , and thromboxane B 2 . Renal linoleic acid and its lipoxygenase products also were lower in obese compared to lean rats, but CLA supplementation had no effect on these or any other lipoxygenase oxylipins. In summary, supplementation with c9,t11 CLA did not improve more advanced OAN and t10,c12 CLA worsened the renal pathology. Altered production of select COX1 derived oxylipins was associated with the detrimental effect of the t10,c12 isomer. [ABSTRACT FROM AUTHOR]

Oxylipins are oxygenated derivatives of polyunsaturated fatty acids, generated by COX, LOX and CYP enzymes, that regulate various aspects of endothelial cell physiology. Although 15-LOX and its products are positively associated with atherosclerosis, the relevant mechanisms have not been explored. The current study examined the effects of PD146176 (PD), a putative 15-LOX inhibitor, on EA.hy926 endothelial cell functions in the growing and confluent states. The effects of PD on endothelial cell oxylipin production (profiled by LC/MS/MS), cell viability, proliferation, eNOS activity, ICAM-1 and VE-cadherin levels were assessed. The contribution of signaling pathways relevant to endothelial function (p38 MAPK, Akt, PPARα) were also investigated. PD treatment for 30 min did not block formation of individual 15-LOX oxylipins, but 20 μM PD stimulated the accumulation of total LOX and COX products, while reducing several individual CYP products generated by epoxygenase. At 20 μM, the accumulated total oxylipins were primarily LOX-derived (86%) followed by COX (12%) and CYP (2%). PD altered cell functions by upregulating p38 MAPK and PPARα and downregulating Akt in a dose-dependent fashion. These observations suggest a link between PD-induced changes in oxylipins and altered endothelial cell functions via specific signaling pathways. In conclusion, the results of this study imply that PD does not function as a 15-LOX inhibitor in EA.hy926 endothelial cells, and instead inhibits CYP epoxygenase. These findings suggest that the cellular function changes induced by PD may be contingent upon its ability to modulate total oxylipin production, particularly by the LOX and CYP families. • PD146176 (PD) does not block formation of individual oxylipins by 15-LOX. • 30 min PD treatment stimulates total oxylipin production by LOX and COX families. • LOX-derived oxylipins are more abundant than from COX and CYP after PD treatment. • 20 μM PD blocks total oxylipin production by CYP epoxygenases after 30 min. • PD146176 modulates endothelial cell function through p38 MAPK, PPARα and Akt. [ABSTRACT FROM AUTHOR]

Abstract: Conjugated linoleic acid (CLA), a dietary lipid, has been proposed as an antidiabetic agent. However, studies specifically addressing the molecular dynamics of CLA on skeletal muscle glucose transport and differences between the key isomers are limited. We demonstrate that acute exposure of L6 myotubes to cis-9, trans-11 (c9,t11) and trans-10, cis-12 (t10,c12) CLA isomers mimics insulin action by stimulating glucose uptake and glucose transporter-4 (GLUT4) trafficking. Both c9,t10-CLA and t10,c12-CLA stimulate the phosphorylation of phosphatidylinositol 3-kinase (PI3-kinase) p85 subunit and Akt substrate-160 kDa (AS160), while showing isomer-specific effects on AMP-activated protein kinase (AMPK). CLA isomers showed synergistic effects with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR). Blocking PI3-kinase and AMPK prevented the stimulatory effects of t10,c12-CLA on AS160 phosphorylation and glucose uptake, indicating that this isomer acts via a PI3-kinase and AMPK-dependent mechanism, whereas the mechanism of c9,t11-CLA remains unclear. Intriguingly, CLA isomers sensitized insulin-Akt-responsive glucose uptake and prevented high insulin-induced Akt desensitisation. Together, these results establish that CLA exhibits isomer-specific effects on GLUT4 trafficking and the increase in glucose uptake induced by CLA treatment of L6 myotubes occurs via pathways that are distinctive from those utilised by insulin. [Copyright &y& Elsevier]

Abstract: Connexin-43 (Cx43) is a membrane phosphoprotein that mediates direct inter-cellular communication by forming gap junctions. In this way Cx43 can influence gene expression, differentiation and growth. Its role in adipogenesis, however, is poorly understood. In this study, we established that Cx43 becomes highly phosphorylated in early adipocyte differentiation and translocates to the plasma membrane from the endoplasmic reticulum. As preadipocytes differentiate, Cx43 phosphorylation declines, the protein is displaced from the plasma membrane, and total cellular levels are reduced via proteosomal degradation. Notably, we show that inhibiting Cx43 degradation or constitutively over-expressing Cx43 blocks adipocyte differentiation. These data reveal that transient activation of Cx43 via phosphorylation followed by its degradation is vital for preadipocyte differentiation and maturation of functional adipocytes. [Copyright &y& Elsevier]

Abstract: We hypothesized that a low-dose resveratrol will reverse cardiovascular abnormalities in rats fed a high-fat (HF) diet. Obese prone (OP) and obese resistant (OR) rats were fed an HF diet for 17 weeks; Sprague–Dawley rats fed laboratory chow served as control animals. During the last 5 weeks of study, treatment group received resveratrol daily by oral gavage at a dosage of 2.5 mg/kg body weight. Assessments included echocardiography, blood pressure, adiposity, glycemia, insulinemia, lipidemia, and inflammatory and oxidative stress markers. Body weight and adiposity were significantly higher in OP rats when compared to OR rats. Echocardiographic measurements showed prolonged isovolumic relaxation time in HF-fed OP and OR rats. Treatment with resveratrol significantly improved diastolic function in OP but not in OR rats without affecting adiposity. OP and OR rats had increased blood pressure which remained unchanged with treatment. OP rats had elevated fasting serum glucose and insulin, whereas OR rats had increased serum glucose and normal insulin concentrations. Resveratrol treatment significantly reduced serum glucose while increasing serum insulin in both OP and OR rats. Inflammatory and oxidative stress markers, serum triglycerides and low-density lipoprotein were higher in OP rats, which were significantly reduced with treatment. In conclusion, HF induced cardiac dysfunction in both OP and OR rats. Treatment reversed abnormalities in diastolic heart function associated with HF feeding in OP rats, but not in OR rats. The beneficial effects of resveratrol may be mediated through regression of hyperglycemia, oxidative stress and inflammation. [Copyright &y& Elsevier]

Abstract: Conjugated linoleic acid (CLA), a dietary fat, has been considered beneficial in metabolic syndrome. Despite several findings indicating that CLA improves glucose clearance, little information is available regarding the cellular dynamics of CLA on skeletal muscle. We sought to investigate the role of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in cis-9, trans-11(c9,t11) and trans-10, cis-12 (t10,c12) CLA isomer-mediated glucose transport by L6 myotubes. t10,c12-CLA stimulated both intracellular Ca2+ release (Ca i 2+) and CaMKII phosphorylation, whereas c9,t11-CLA showed only modest effects on both. Sequestering Ca i 2+ with BAPTA/AM abrogated the effect of both CLA isomers on Akt substrate-160kDa (AS160) phosphorylation and glucose uptake by myotubes. Exposing myotubes to KN-93 or autocamtide 2-related inhibitory peptide to block CaMKII activity prevented both CLA isomers from inducing AS160 phosphorylation and glucose transport. Likewise, genetic knockdown of CaMKII in myotubes using siRNA completely abolished CLA isomer-mediated glucose uptake. These results indicate that CLA isomers require Ca i 2+–CaMKII to mediate glucose uptake. Evidence that CaMKII blockers inhibit t10,c12-CLA-mediated AMP-activated protein kinase (AMPK) activation indicated that CaMKII acts upstream of AMPK in response to t10,c12-CLA. Lastly, CLA isomers stimulated the formation of reactive oxygen species but had no effect on stress-activated protein kinase/c-jun NH2-terminal kinase. These data establish that t10,c12-CLA acts via Ca i 2+–CaMKII–AMPK–AS160 to stimulate skeletal muscle glucose transport, whereas the mechanism of c9,t11-CLA remains unclear. Given that impairments in muscle glucose utilisation are apparent in metabolic syndrome, delineating the molecular mechanisms by which CLA isomers mediate muscle glucose uptake may identify new approaches to manage this condition. [Copyright &y& Elsevier]

Abstract: Conjugated linoleic acid (CLA) has been reported to reduce blood pressure in obese insulin-resistant rats, but its mechanism of action has not been identified. The objective of this study was to determine whether CLA isomers can reduce obesity-related hypertension in the fa/fa Zucker rat in relation to adiponectin production and endothelial nitric oxide synthase (eNOS) activation. Obese fa/fa Zucker rats were randomly assigned to one of four groups: (1) cis-9,trans-11-CLA, (2) trans-10,cis-12 (t10,c12)-CLA, (3) control or (4) captopril. After 8 weeks, systolic blood pressure increased 30% in control obese rats. This increase was attenuated 11%–13% in the t10,c12-CLA isomer and captopril groups, respectively. The t10,c12-CLA isomer concurrently elevated adiponectin levels in both plasma and adipose tissue and increased phosphorylated eNOS in adipose tissue as well as the aorta. Although a direct effect of CLA was not observed in cultured endothelial cells, direct adiponectin treatment increased phosphorylation of eNOS. Endothelial nitric oxide synthase phosphorylation was also increased in adipose of fa/fa Zucker rats infused with adiponectin in parallel with improvements in blood pressure. Our results suggest that the t10,c12-CLA isomer attenuates development of obesity-related hypertension, at least in part, by stimulating adiponectin production, which subsequently activates vascular eNOS. [Copyright &y& Elsevier]

Abstract: Increased adrenergic drive is a major factor influencing the development of pathological cardiac hypertrophy, a stage which precedes overt heart failure. We examined the effect of resveratrol, a polyphenol (found predominantly in grapes), in preventing norepinephrine induced hypertrophy of adult cardiomyocyte, and the role of nitric oxide (NO) and adenosine monophosphate kinase (AMPK) in the effects of resveratrol. Cardiomyocytes isolated from adult rats were pretreated, or not, with resveratrol and then exposed to norepinephrine for 24h. In other experiments cardiomyocytes were also treated with different pharmacological inhibitors of NO synthase, AMPK and sirtuin for elucidating the signaling pathways underlying the effect of resveratrol. In order to validate the role of these signaling molecules in the in vivo settings, we also examined hearts from resveratrol treated spontaneously hypertensive rats (SHR), a genetic model of essential hypertension. Cardiomyocyte hypertrophy was determined by morphometry and 3H-phenylalanine incorporation assay. NO levels and AMPK activity were measured using a specific assay kit and western blot analysis respectively. In vitro, resveratrol prevented the norepinephrine-induced increase in cardiomyocytes size and protein synthesis. Pharmacological inhibition of NO-AMPK signaling abolished the anti-hypertrophic action of resveratrol. Consistent with the in vitro findings, the anti-hypertrophic effect of resveratrol in the SHR model was associated with increases in NO and AMPK activity. This study demonstrates that NO-AMPK signaling is linked to the anti-hypertrophic effect of resveratrol in adult cardiomyocytes in vitro, and in the SHR model in vivo. [Copyright &y& Elsevier]

Abstract: The extracellular-regulated kinase (ERK1/2) is a key conduit for transduction of signals from growth factor receptors to the nucleus. Previous work has shown that ERK1/2 activation in response to IGF-1 may require the participation of G proteins, but the role of the receptor tyrosine kinase in this process has not been clearly resolved. This investigation of IGF-1 receptor function was therefore designed to examine the contribution of the receptor tyrosine kinase to ERK1/2 activation. Phosphorylation of ERK1/2 in smooth muscle cells following treatment with IGF-1 was not blocked by pretreatment with AG1024 or picropodophylin, inhibitors of the IGF-1 receptor tyrosine kinase. Likewise, IGF-1 activated ERK1/2 in cells expressing a kinase-dead mutant of the IGF-1 receptor. ERK1/2 activation was unaffected by the phosphatidylinositol 3-kinase inhibitor LY-294002, but was sensitive to inhibitors of Src kinase, phospholipase C and Gβγ subunit signalling. Treatment with αIR-3, a neutralizing monoclonal antibody, also stimulated ERK1/2 phosphorylation without concomitant activation of the receptor tyrosine kinase. Phosphoprotein mapping of IGF-1 and αIR-3 treated cells confirmed that antibody-induced ERK1/2 phosphorylation occurred in the absence of tyrosine kinase phosphorylation, and enabled extension of these findings to p38 MAPK. These results suggest that stimulation of ERK1/2 phosphorylation by IGF-1 does not require activation of the receptor tyrosine kinase. [Copyright &y& Elsevier]

[Display omitted] • Loss of β-Arrestin or six Gα proteins failed to prevent P4-induced Warburg effect. • Progesterone-induced PGRMC1 degradation is β-Arrestin or Gα protein-dependent. • P4induced subcellular translocation of PGRMC1, HK1, and GAPDH in HEK293 cells. • β-Arrestin/ six Gα loss altered subcellular distribution of PGRMC1, HK1, and GAPDH. • Loss of β-Arrestin/six Gα differentially affected P4-induced protein translocation. Hormonal dysregulation plays a significant role in the metabolic switching during malignant transformation. Progesterone Receptor Membrane Component 1 (PGRMC1) is a single-pass transmembrane receptor activated by the binding of progesterone (P4), a sex hormone. In a previous study, P4 treatment caused rapid (within 30 min) induction of aerobic glycolysis in transformed HEK293 cells, a hallmark malignant phenotype known as the Warburg effect. This metabolic reprogramming was associated with the proteasomal degradation of a 70 kilodalton (kDa) PGRMC1. PGRMC1 interacts with a variety of proteins, including G protein-coupled receptors (GPCRs) and P4-PGRMC1 signaling modulates cyclic adenosine monophosphate (cAMP) production. Therefore, we hypothesized that the P4-induced Warburg effect and proteasomal degradation of PGRMC1 involve G proteins and β-Arrestins (ARRBs). In the present study, we investigated P4-induced aerobic glycolysis, proteasomal degradation of p70 PGRMC1, as well as abundance and subcellular translocation of PGRMC1 along with two key glycolytic enzymes Hexokinase 1 (HK1) and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) in six Gα subunit (Gsix) proteins or ARRB1/2-deficient HEK293 cells. Loss of ARRB1/2 or Gsix proteins inhibited P4-induced p70 PGRMC1 degradation but failed to prevent the P4-induced Warburg effect. Also, deficiency of ARRB1/2 or Gsix proteins differentially affected the basal as well as P4-induced abundance and subcellular translocation of PGRMC1, HK1, and GAPDH proteins. Overall, the findings indicate that P4-PGRMC1-mediated metabolic reprogramming in HEK293 cells depends on β-Arrestins and Gα proteins suggesting the involvement of an underlying GPCR signal transduction pathway. [ABSTRACT FROM AUTHOR]

Endothelial cells, which help to maintain vascular homeostasis, can be functionally modulated by polyunsaturated fatty acids. Previously, we reported that docosahexaenoic acid (DHA) reduced the viability of confluent EA.hy926 endothelial cells with caspase-3 activation. This study therefore examined the molecular mechanism by which DHA affects the viability of confluent cells, with a focus on the interaction between caspase-9, caspase-8, caspase-3, p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) by Western blotting. Our results revealed that DHA induces apoptosis of confluent cells through both intrinsic and extrinsic pathways, which requires activation of p38 MAPK, and involves activation of JNK, caspase-9, caspase-8 and caspase-3 with the exception that cleavage of caspase-8 was incomplete and truncated BID was not detected at the maximum time (8 h) examined. Apoptosis induced by high levels of DHA in healthy endothelial cells is achieved through positive feedback loops linking these MAPKs to multiple caspases, as well as negative feedback from p38 MAPK to JNK. However, only p38 MAPK is crucial in apoptosis induction in comparison with JNK or any other caspase examined. This study has expanded the knowledge on the molecular mechanism of DHA-induced apoptosis in human endothelial cells and has also implied the differential roles of MAP kinases and caspases in apoptosis. Unlabelled Image • DHA induces apoptosis in confluent EA.hy926 cells. • DHA treatment leads to partial cleavage of caspase-8 (p43 subunit). • DHA-induced apoptosis does not involve participation of cleaved BID. • DHA upregulates multiple caspases via p38 MAPK and JNK. • p38 MAPK, instead of caspase-8, -9, -3 or JNK, is critical in apoptosis regulation. [ABSTRACT FROM AUTHOR]

Alternatively activated macrophages are innate immune cells that contribute to resolution of inflammation and maintenance of homeostasis. Modulation of available fatty acid sources is thought to affect cellular physiology through a variety of mechanisms, including through alterations to the profile of oxygenated free fatty acid metabolites, called oxylipins, produced in a cell type specific manner. Here, we investigated how treatment with the plant-sourced omega-3 fatty acid α-linolenic acid (ALA) affects the oxylipin profile and functional capacity of a cell culture model of human alternatively activated (M2a-like) macrophages. In a targeted but unbiased screen, ALA enhanced the production of oxylipins from all polyunsaturated fatty acid (PUFA) precursors, with oxylipins derived from ALA being enhanced the most. Consistently, ALA treatment enhanced the expression of both cytoplasmic and calcium-independent phospholipase A2. At a functional level, ALA treatment increased phagocytic activity and altered production of the chemokine MCP-1 by M2a-like cells in a manner dependent on the time of treatment. ALA treatment during polarization increased MCP-1 secretion, which was sensitive to pharmacological inhibition of 15-LOX-1 by ML351. Thus, ALA modulates the phenotype of alternatively activated macrophages, likely through its own LOX-derived oxylipins and/or through general modulation of oxylipin biosynthesis. These effects likely contribute to the overall anti-inflammatory benefit observed with ALA supplementation. [ABSTRACT FROM AUTHOR]