Preliminary evidence that blocking the uptake of placenta-derived preeclamptic extracellular vesicles protects the vascular endothelium and prevents vasoconstriction.

Preeclampsia (PE) is a pregnancy syndrome characterized by hypertension and organ damage manifesting after 20 gestational weeks. The etiology is of multifactorial origin, where placental stress causes increased levels of placenta-derived extracellular vesicles (STBEVs) in the maternal circulation, shown to cause inflammation, endothelial activation, vasoconstriction, and anti-angiogenic activity. General endothelial dysfunction is believed to be initiated by endothelial insult during pregnancy that alters vascular function resulting in increased arterial stiffness, cardiac dysfunction, and increased risk of cardiovascular disease later in life. We compared the effect of normal and PE derived STBEVs in vitro on vascular contractility of human subcutaneous arteries using wire myography. Cellular structures of exposed vessels were investigated by transmission electron microscopy. We explored strategies to pharmacologically block the effects of the STBEVs on human vessels. The PE STBEVs caused significantly stronger angiotensin II-mediated contractions and extended structural damage to human subcutaneous arteries compared to normal STBEVs. These negative effects could be reduced by blocking vesicle uptake by endothelial cells, using chlorpromazine or specific antibodies towards the LOX-1 receptor. The therapeutic potential of blocking vesicle uptake should be further explored, to reduce the permanent damage caused on the vasculature during PE pregnancy to prevent future cardiovascular risk.

Short-term exposure to urban PM2.5 particles induces histopathological and inflammatory changes in the rat small intestine.

Air pollution and exposure to fine airborne particles with aerodynamic diameter <2.5 µm (PM2.5 ) negatively impacts human health. Airways constitute a primary route of exposure but PM2.5 -contaminated food, drinks as well as mucociliary and hepatobiliary clearance all constitute potential entry points into the intestine. This study evaluated intestinal histopathological and inflammatory changes as well as enteric neuronal numbers after short- or long-term exposure to urban PM2.5 . Using a nebulizer, male rats were exposed to a mist with a concentration of 5.3mg PM2.5 /m3 for 8 h (short term) or 1.8 mg PM2.5 /m3 for 3 h/day, 5 days/week for 8 weeks (long-term) with controls run in parallel. Samples were taken from three regions of the small intestine as well as the colon. Results showed that short-term exposure to PM2.5 induces mucosal lesions and reduces IL1ß levels in the small intestine but not colon. No significant changes were observed after long-term exposure, suggesting the presence of intestinal adaptation to environmental stressors in the PM2.5 . To our knowledge, this is the first study to systematically characterize regional effects along the intestine.

Digestion and Absorption of Milk Phospholipids in Newborns and Adults.

Milk polar lipids provide choline, ethanolamine, and polyunsaturated fatty acids, which are needed for the growth and plasticity of the tissues in a suckling child. They may also inhibit cholesterol absorption by interacting with cholesterol during micelle formation. They may also have beneficial luminal, mucosal, and metabolic effects in both the neonate and the adult. The milk fat globule membrane contains large proportions of sphingomyelin (SM), phosphatidylcholine (PC), and phosphatidylethanolamine (PE), and some phosphatidylserine (PS), phosphatidylinositol (PI), and glycosphingolipids. Large-scale technical procedures are available for the enrichment of milk fat globule membrane (MFGM) in milk replacement formulations and food additives. Pancreatic phospholipase A2 (PLA2) and mucosal phospholipase B digest glycero-phospholipids in the adult. In the neonate, where these enzymes may be poorly expressed, pancreatic lipase-related protein 2 probably has a more important role. Mucosal alkaline SM-ase and ceramidase catalyze the digestion of SM in both the neonate and the adult. In the mucosa, the sphingosine is converted into sphingosine-1-phosphate, which is both an intermediate in the conversion to palmitic acid and a signaling molecule. This reaction sequence also generates ethanolamine. Here, we summarize the pathways by which digestion and absorption may be linked to the biological effects of milk polar lipids. In addition to the inhibition of cholesterol absorption and the generation of lipid signals in the gut, the utilization of absorbed choline and ethanolamine for mucosal and hepatic phospholipid synthesis and the acylation of absorbed lyso-PC with polyunsaturated fatty acids to chylomicron and mucosal phospholipids are important.

Erenumab (AMG 334), a monoclonal antagonist antibody against the canonical CGRP receptor, does not impair vasodilatory or contractile responses to other vasoactive agents in human isolated cranial arteries.

BACKGROUND: Calcitonin gene-related peptide (CGRP) is a neuronal transmitter present in intracranial sensory nerves, where it is involved in migraine pathophysiology as well as other biological functions. Recently, the fully human monoclonal antibody erenumab (AMG 334), which targets the canonical calcitonin gene-related peptide receptor, showed significant prophylactic efficacy and favourable safety in phase II and III clinical trials for episodic and chronic migraine and is now approved for migraine prevention in several countries. OBJECTIVE: Given that calcitonin gene-related peptide can mediate vasodilation, we investigated the effect of erenumab on vasoactive responses in the presence or absence of various vasodilatory and vasocontractile mediators in a model using isolated human cerebral and meningeal arteries. METHODS: Ring segments of human isolated cerebral and meningeal arteries were mounted in a sensitive myograph. On arterial segments pre-contracted with 30 mM potassium chloride, vasoactive responses to calcitonin gene-related peptide were studied in the presence of different concentrations of erenumab. At the maximal tested inhibitory concentration of erenumab (100 nM), functional arterial relaxation in response to nicardipine or substance P, and the contractile responses to sumatriptan and dihydroergotamine were examined. RESULTS: 30 mM potassium chloride produced a stable contraction of the vessel segments and calcitonin gene-related peptide induced a concentration-dependent relaxation. We observed that (i) erenumab had no direct contractile or relaxant effects per se (by itself), (ii) pre-treatment with erenumab antagonized the calcitonin gene-related peptide-induced relaxation in a competitive manner, (iii) the relaxant responses to nicardipine or substance P were unaffected in the presence of erenumab and (iv) the contraction induced by sumatriptan or dihydroergotamine was not modified by erenumab. CONCLUSION: Our findings demonstrate that erenumab, while not associated with vasoactive properties per se, specifically inhibits calcitonin gene-related peptide-induced relaxation of cranial arteries without impacting vasodilatory responses or contractile responses of endogenous or pharmacological vasoactive compounds.

Linoleic acid esters of hydroxy linoleic acids are anti-inflammatory lipids found in plants and mammals.

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a recently discovered class of biologically active lipids. Here we identify the linoleic acid ester of 13-hydroxy linoleic acid (13-LAHLA) as an anti-inflammatory lipid. An oat oil fraction and FAHFA-enriched extract from this fraction showed anti-inflammatory activity in a lipopolysaccharide-induced cytokine secretion assay. Structural studies identified three LAHLA isomers (15-, 13-, and 9-LAHLA) as being the most abundant FAHFAs in the oat oil fraction. Of these LAHLAs, 13-LAHLA is the most abundant LAHLA isomer in human serum after ingestion of liposomes made of fractionated oat oil, and it is also the most abundant endogenous LAHLA in mouse and human adipose tissue. As a result, we chemically synthesized 13-LAHLA for biological assays. 13-LAHLA suppresses lipopolysaccharide-stimulated secretion of cytokines and expression of pro-inflammatory genes. These studies identify LAHLAs as an evolutionarily conserved lipid with anti-inflammatory activity in mammalian cells.

MEK/ERK/1/2 sensitive vascular changes coincide with retinal functional deficit, following transient ophthalmic artery occlusion.

Retinal ischemia remains a major cause of blindness in the world with few acute treatments available. Recent emphasis on retinal vasculature and the ophthalmic artery's vascular properties after ischemia has shown an increase in vasoconstrictive functionality, as previously observed in cerebral arteries following stroke. Specifically, endothelin-1 (ET-1) receptor-mediated vasoconstriction regulated by the MEK/ERK1/2 pathway. In this study, the ophthalmic artery of rats was occluded for 2 h with the middle cerebral artery occlusion model. MEK/ERK1/2 inhibitor U0126 was administered at 0, 6, and 24 h following reperfusion and the functional properties of the ophthalmic artery were evaluated at 48 h post reperfusion. Additionally, retinal function was evaluated at day 1, 4, and 7 after reperfusion. Occlusion of the ophthalmic artery led to a significant increase of endothelin-1 mediated vasoconstriction which can be attenuated by U0126 treatment, most evident at higher ET-1 concentrations of 10-7 M (Emax151.0 ±â€¯22.0% of 60 mM K+), vs non-treated ischemic arteries Emax 212.1 ±â€¯14.7% of 60 mM K+). Retinal function also deteriorated following ischemia and was improved with treatment with a-wave amplitudes of 725 ± 36 µV in control, 560 ± 21 µV in non-treated, and 668 ± 73 µV in U0126 treated at 2 log cd*s/m2 luminance in the acute stages (1 days post-ischemia). Full spontaneous retinal recovery was observed at day 7 regardless of treatment. In conclusion, this is the first study to show a beneficial in vivo effect of U0126 on vascular contractility following ischemia in the ophthalmic artery. Coupled with the knowledge obtained from cerebral vasculature, these results point towards a novel therapeutic approach following ischemia-related injuries to the eye.

Expression of Pituitary Adenylate Cyclase-activating Peptide, Calcitonin Gene-related Peptide and Headache Targets in the Trigeminal Ganglia of Rats and Humans.

Neurotransmitter and headache target localization in the trigeminal ganglia (TG) might increase the understanding of sites of action, and mechanisms related to headache therapy. The overall aim of the study was to investigate the presence of migraine targets in the TG with particular emphasis on pituitary adenylate cyclase-activating peptide (PACAP) and calcitonin gene-related peptide (CGRP), known to be involved in cranial pain processing, and selected headache targets. Rat- and human TG were processed for immunohistochemistry. PACAP-38, CGRP and the headache targets were expressed in rat and human TG. PACAP receptors were confined to neurons and satellite glial cells (SGCs), however with variability between the receptor subtypes PACAP type I receptor (PAC1) and vasoactive intestinal peptide/PACAP receptors 1/2 (VPAC1/2). 5-Hydroxytryptamine (5-HT) receptors were expressed in neuronal somas in rat and human TG (human TG frequency: 5-HT1D > 5-HT1B/1F). Synaptosomal-associated protein 25 kDa (SNAP25) was primarily expressed in SGCs in humans, and neurons in rats, while synaptic vesicle glycoprotein 2A (SV2-A) was confined to SGCs and some neurons in rats and humans. Occasionally, PACAP-38-positive cells also expressed VPAC1, SNAP25 and SV2-A. VPAC1 was generally detected in SGCs enveloping PACAP-38-positive and -negative neuronal somas. Our study revealed potential sites of actions for anti-headache drugs such as PACAP receptor antagonists, Lasmiditan (5-HT1F agonist) and Botox (blocks exocytosis through SV2-A/SNAP25) in rat and human TG and considerable overlap between species in expression to specific cell types, except for VPAC1 and SNAP25.

Fremanezumab blocks CGRP induced dilatation in human cerebral, middle meningeal and abdominal arteries.

BACKGROUND: Fremanezumab (TEV-48125) is a fully humanized anti-calcitonin gene-related peptide (CGRP) monoclonal antibody (mAb) that has shown positive results in the prevention of episodic migraine and chronic migraine. Previous preclinical studies have revealed CGRP antagonistic effects on intracranial arteries (ICA). The aim of the study was to evaluate the in vitro antagonistic effects of fremanezumab on human arteries. METHODS: Arteries were removed in conjunction with neurosurgery (cerebral, CA, and middle meningeal artery, MMA, n = 7) or reconstructive abdominal surgery (abdominal artery, AA, n = 6). Ring segments of the vessels were mounted in a sensitive myograph, the functional responses of vasoactive intestinal peptide (VIP), substance P and CGRP in increasing concentrations (10- 10-10- 7 M) were studied using pre-contraction with 30 mM potassium chloride (KCl). The concentrations of fremanezumab or isotype control antibody (66.7 nM, 0.33 µM, 0.67 µM) were given 30 min prior to CGRP administration. RESULTS: All included arteries responded with a strong stable contraction to the application of 30 mM KCl. During this pre-contraction, CGRP caused a concentration-dependent relaxation which differed slightly in maximum effect (Imax) between the types of arteries (ICA = 100%; AA 80%). Fremanezumab (66.7 nM) showed a shift in the IC50 value of CGRP, but no significant change in Imax. At higher doses there was also a reduction of Imax. For AA, the Imax decreased from 71% at 66.7 nM, to 4.5% with 0.33 µM of fremanezumab. Isotype control antibody did not modify the responses. There was no effect on concentration-dependent relaxation with VIP with 66.7 nM of fremanezumab or isotype control. CONCLUSION: CGRP relaxes pre-contracted human arteries by 80-100%, but with different IC50; the potency range was ICA < AA. The antagonistic effect and potency of fremanezumab was similar, suggesting that there are vasodilatory CGRP receptors present in all studied arteries and that the antibody may have effect in all studied vessels.

Faster Protocol for Endogenous Fatty Acid Esters of Hydroxy Fatty Acid (FAHFA) Measurements.

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a recently discovered class of endogenous lipids with antidiabetic and anti-inflammatory activities. Interest in these lipids is due to their unique biological activites and the observation that insulin-resistant people have lower palmitic acid esters of hydroxystearic acid (PAHSA) levels, suggesting that a FAHFA deficiency may contribute to metabolic disease. Rigorous testing of this hypothesis will require the measurement of many clinical samples; however, current analytical workflows are too slow to enable samples to be analyzed quickly. Here we describe the development of a significantly faster workflow to measure FAHFAs that optimizes the fractionation and chromatography of these lipids. We can measure FAHFAs in 30 min with this new protocol versus 90 min using the older protocol with comparable performance in regioisomer detection and quantitation. We also discovered through this optimization that oleic acid esters of hydroxystearic acids (OAHSAs), another family of FAHFAs, have a much lower background signal than PAHSAs, which makes them easier to measure. Our faster workflow was able to quantify changes in PAHSAs and OAHSAs in mouse tissues and human plasma, highlighting the potential of this protocol for basic and clinical applications.

Increased endothelin-1-mediated vasoconstriction after organ culture in rat and pig ocular arteries can be suppressed with MEK/ERK1/2 inhibitors.

PURPOSE: Even though retinal vascular changes following ischaemia have been poorly understood, the upregulation of vasoconstrictive endothelin-1 (ET-1) receptors (ETA /ETB ) following global cerebral ischaemia has been described. The aim of this study was to investigate whether or not the MEK/ERK1/2 pathway is involved in the observed upregulation and whether specific MEK/ERK1/2 inhibitors U0126 and trametinib can prevent it. METHODS: The aim was also to localize ETA and ETB receptors using immunohistochemistry in both fresh rat ophthalmic arteries and after 24-hr organ culture and study the receptors functionally using myography. Pig retinal arteries also underwent 24-hr organ culture to validate similar responses across species and the retinal vasculature. RESULTS: Results showed that following organ culture there is a significant increase in ET-1-mediated vasoconstriction, in particular via the ETB receptor. Furthermore, immunohistochemistry revealed a clear increase in pERK in the smooth muscle cells of rat ophthalmic artery. U0126 and trametinib were successful in attenuating the functional vasoconstriction in both rat and pig, as well as restoring immunofluorescence of pERK to fresh levels and counteracting ETB expression in the smooth muscle cells of the rat ophthalmic artery. CONCLUSION: This is the first study to show that the MEK/ERK1/2 pathway in responsible for the increase in functional vasoconstriction via ET-1 receptor in rat ophthalmic and pig retinal arteries. Furthermore, this study is the first to suggest a way of inhibiting and preventing such an increase. With these results, we suggest a novel approach in retinal ischaemia therapy.

Postprandial effects on plasma lipids and satiety hormones from intake of liposomes made from fractionated oat oil: two randomized crossover studies.

BACKGROUND: The composition and surface structure of dietary lipids influence their intestinal degradation. Intake of liposomes made of fractionated oat oil (LOO) is suggested to affect the digestion process and postprandial lipemia and also induce satiety. OBJECTIVE: In the present study, the metabolic effects on plasma lipids and gut hormones related to satiety were investigated in healthy individuals after intake of LOO, with dairy lipids as placebo. DESIGN: Two blinded randomized studies with crossover design were performed. In the first study, 19 subjects consumed 35 g lipids from LOO or yoghurt in a breakfast meal. In a follow-up study, 15 women consumed 14 or 1.8 g lipids from LOO mixed in yoghurt. Blood samples were analyzed for plasma lipids, insulin, glucose, and intestinal hormones CCK, PYY, GLP-1, and GLP-2 before and four times after the meal. Subjective analysis of satiety was measured using a visual analog scale questionnaire. Participants recorded their food intake during the rest of the day. RESULTS: Intake of 35 and 14 g lipids from LOO significantly increased plasma concentrations of CCK, GLP-1, GLP-2, and PYY postprandially. This coincided with a prolonged elevation of triglycerides and large cholesterol-containing particles. Non-esterified fatty acids decreased after intake of 14 and 1.8 g lipids from LOO. The subjective sensation of satiety in women was increased 7 h after intake of 35 g lipids from LOO without any difference in food intake. Our results indicate that intake of 14 g lipids from LOO at breakfast substantially reduced energy intake during the rest of the day. CONCLUSIONS: This study suggests that intake of LOO prolong lipid digestion, affect postprandial plasma lipids and have an effect on satiety. The effect of LOO on GLP-2 indicates that intake of LOO also improve gut health.

GLP-1 released to the mesenteric lymph duct in mice: effects of glucose and fat.

Using a newly developed in vivo model measuring glucagon-like peptide-1 (GLP-1) in gut lymphatics in mice, we quantified GLP-1 secretion in vivo after glucose versus fat ingestion with and without concomitant DPP-4 inhibition. The mesenteric lymphatic duct was cannulated in anesthetized C57BL6/J mice and lymph was collected in 30 min intervals. Glucose or fat emulsion (Intralipid®) (0.03, 0.1 or 0.3 kcal) with or without DPP-4-inhibition (NVP DPP728; 10 µmol/kg) was administered by gastric gavage. Basal intact GLP-1 levels were 0.37±0.04 pmol/l (n=61) in lymph compared to 0.07±0.03 in plasma (n=6; P=0.04) and basal DPP-4 activity was 4.7±0.3 pmol/min/µl in lymph (n=23) compared to 22.3±0.9 pmol/min/µl in plasma (n=8; P<0.001). Lymph flow increased from 1.2±0.1 µl/min to 2.3±02µl/min at 30 min after glucose and fat administration, with no difference between type of challenge or dose (n=81). Lymph GLP-1 levels increased calorie-dependently after both glucose and fat but with different time courses in that glucose induced a transient increase which had returned to baseline after 90 min whereas the lipid induced a sustained increase which was still elevated above baseline after 210 min. Lymph GLP-1 appearance during 210 min was two to three-fold higher after glucose (7.4±2.3 fmol at 0.3 kcal) than after isocaloric fat (2.9±0.8 fmol at 0.3 kcal; P<0.001). The slope between caloric load and lymph GLP-1 appearance was, however, identical after glucose and fat. We conclude that lymph GLP-1 is higher than plasma GLP-1 whereas lymph DPP-4 activity is lower than plasma DPP-4 activity and that both glucose and fat clearly stimulate GLP-1 secretion calorie-dependently in vivo but with different time courses.

Alkaline sphingomyelinase (NPP7) promotes cholesterol absorption by affecting sphingomyelin levels in the gut: A study with NPP7 knockout mice.

We previously showed that dietary sphingomyelin (SM) inhibited cholesterol absorption in animals. The key enzyme hydrolyzing SM in the gut is alkaline sphingomyelinase (alk-SMase, nucleotide pyrophosphatase/phosphodiesterase 7). Here using the fecal dual-isotope ratio method we compared cholesterol absorption in the wild-type (WT) and alk-SMase knockout (KO) mice. The animals were fed an emulsion containing [(14)C]cholesterol and [(3)H]sitosterol. The radioactivities in the lipids of the fecal samples collected 4, 8, and 24 h thereafter were determined, and the ratio of (14)C/(3)H was calculated. We found that the fecal [(14)C]cholesterol recovery in the KO mice was significantly higher than in the WT mice. A maximal 92% increase occurred 8 h after feeding. Recovery of [(3)H]sitosterol did not differ between the two groups. Accordingly, the (14)C-to-(3)H ratio of fecal lipids was 133% higher at 8 h and 75% higher at 24 h in the KO than in the WT mice. Decreased [(14)C]cholesterol was also found in the serum of the KO mice 4 h after feeding. Supplement of SM in the emulsion reduced the differences in fecal [(14)C]cholesterol recovery between the WT and KO mice because of a greater increase of [(14)C]cholesterol recovery in the WT mice. Without treatment, the KO mice had significantly higher SM levels in the intestinal content and feces, but not in the intestinal mucosa or serum. The expression of Niemann-Pick C1 like 1 protein in the small intestine was not changed. In conclusion, alk-SMase is a physiological factor promoting cholesterol absorption by reducing SM levels in the intestinal lumen.

Postprandial lipid responses to an alpha-linolenic acid-rich oil, olive oil and butter in women: a randomized crossover trial.

BACKGROUND: Postprandial lipaemia varies with gender and the composition of dietary fat due to the partitioning of fatty acids between beta-oxidation and incorporation into triacylglycerols (TAGs). Increasing evidence highlights the importance of postprandial measurements to evaluate atherogenic risk. Postprandial effects of alpha-linolenic acid (ALA) in women are poorly characterized. We therefore studied the postprandial lipid response of women to an ALA-rich oil in comparison with olive oil and butter, and characterized the fatty acid composition of total lipids, TAGs, and non-esterified fatty acids (NEFAs) in plasma. METHODS: A randomized crossover design (n = 19) was used to compare the postprandial effects of 3 meals containing 35 g fat. Blood samples were collected at regular intervals for 7 h. Statistical analysis was carried out with ANOVA (significant difference = P < 0.05). RESULTS: No significant difference was seen in incremental area under the curve (iAUC) plasma-TAG between the meals. ALA and oleic acid levels were significantly increased in plasma after ALA-rich oil and olive oil meals, respectively. Palmitic acid was significantly increased in plasma-TAG after the butter meal. The ratios of 18:2 n-6 to18:3 n-3 in plasma-TAGs, three and seven hours after the ALA-rich oil meal, were 1.5 and 2.4, respectively. The corresponding values after the olive oil meal were: 13.8 and 16.9; and after the butter meal: 9.0 and 11.6. CONCLUSIONS: The postprandial p-TAG and NEFA response in healthy pre-menopausal women was not significantly different after the intake of an ALA-rich oil, olive oil and butter. The ALA-rich oil significantly affected different plasma lipid fractions and improved the ratio of n-6 to n-3 fatty acids several hours postprandially.

Crucial role of alkaline sphingomyelinase in sphingomyelin digestion: a study on enzyme knockout mice.

Alkaline sphingomyelinase (alk-SMase) hydrolyses sphingomyelin (SM) to ceramide in the gut. To evaluate the physiological importance of the enzyme, we generated alk-SMase knockout (KO) mice by the Cre-recombinase-Locus of X-over P1(Cre-LoxP) system and studied SM digestion. Both wild-type (WT) and KO mice were fed ³H-palmitic acid labeled SM together with milk SM by gavage. The lipids in intestinal content, intestinal tissues, serum, and liver were analyzed by TLC. In KO mice, nondigested ³H-SM in the intestinal content increased by 6-fold and the formation of ³H-ceramide decreased markedly, resulting in 98% reduction of ³H-ceramide/³H-SM ratio 1 h after gavage. The absorbed ³H-palmitic acid portion was decreased by 95%. After 3 h, a small increase in ³H-ceramide was identified in distal intestine in KO mice. In feces, ³H-SM was increased by 243% and ceramide decreased by 74% in the KO mice. The KO mice also showed significantly decreased radioactivity in liver and serum. Furthermore, alkaline phosphatase activity in the mucosa was reduced by 50% and histological comparison of two female littermates preliminarily suggested mucosal hypertrophy in KO mice. This study provides definite proof for crucial roles of alk-SMase in SM digestion and points to possible roles in regulating mucosal growth and alkaline phosphatase function.

Dairy products and plasma cholesterol levels.

Cholesterol synthesized in the body or ingested is an essential lipid component for human survival from our earliest life. Newborns ingest about 3-4 times the amount per body weight through mother's milk compared to the dietary intake of adults. A birth level of 1.7 mmol/L plasma total cholesterol will increase to 4-4.5 mmol/L during the nursing period and continue to increase from adulthood around 40% throughout life. Coronary artery disease and other metabolic disorders are strongly associated with low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol as well as triacylglycerol concentration. Milk fat contains a broad range of fatty acids and some have a negative impact on the cholesterol rich lipoproteins. The saturated fatty acids (SFAs), such as palmitic acid (C16:0), myristic acid (C14:0), and lauric acid (C12:0), increase total plasma cholesterol, especially LDL, and constitute 11.3 g/L of bovine milk, which is 44.8% of total fatty acid in milk fat. Replacement of dairy SFA and trans-fatty acids with polyunsaturated fatty acids decreases plasma cholesterol, especially LDL cholesterol, and is associated with a reduced risk of cardiovascular disease. Available data shows different effects on lipoproteins for different dairy products and there is uncertainty as to the impact a reasonable intake amount of dairy items has on cardiovascular risk. The aim of this review is to elucidate the effect of milk components and dairy products on total cholesterol, LDL, HDL, and the LDL/HDL quotients. Based on eight recent randomized control trials of parallel or cross-over design and recent reviews it can be concluded that replacement of saturated fat mainly (but not exclusively) derived from high-fat dairy products with low-fat dairy products lowers LDL/HDL cholesterol and total/HDL cholesterol ratios. Whey, dairy fractions enriched in polar lipids, and techniques such as fermentation, or fortification of cows feeding can be used to produce dairy products with more beneficial effects on plasma lipid profile.

Generating ceramide from sphingomyelin by alkaline sphingomyelinase in the gut enhances sphingomyelin-induced inhibition of cholesterol uptake in Caco-2 cells.

Background Sphingomyelin (SM) is present in dietary products and cell plasma membranes. We previously showed that dietary SM inhibited cholesterol absorption in rats. In the intestinal tract, SM is mainly hydrolyzed by alkaline sphingomyelinase (alk-SMase) to ceramide.Aims We investigated the influence of SM and its hydrolytic products ceramide and sphingosine on cholesterol uptake in intestinal Caco-2 cells.Methods Micelles containing bile salt, monoolein, and (14)C-cholesterol were prepared with or without SM, ceramide,or sphingosine. The micelles were incubated with Caco-2 cells, and uptake of radioactive cholesterol was quantified.Results We found that confluent monolayer Caco-2 cells expressed NPC1L1, and the uptake of cholesterol in the cells was inhibited by ezetimibe, a specific inhibitor of NPC1L1. Incorporation of SM in the cholesterol micelles inhibited cholesterol uptake dose-dependently; 38% inhibition occurred at an equal mole ratio of SM and cholesterol.The inhibition was further enhanced to 45% by pretreating the cholesterol/SM micelles with recombinant alk-SMase, which hydrolyzed SM in the micelles by 85%, indicating ceramide has stronger inhibitory effects on cholesterol uptake. To confirm this, we further replaced SM in the micelles with ceramide and sphingosine, and found that at equal mole ratio to cholesterol, ceramide exhibited stronger inhibitory effect (50% vs 38%) on cholesterol uptake than SM, whereas sphingosine only had a weak effect at high concentrations.Conclusion Both SM and ceramide inhibit cholesterol uptake, the effect of ceramide being stronger than that of SM. Alk-SMase enhances SM-induced inhibition of cholesterol uptake by generating ceramide in the intestinal lumen.

Curcumin inhibits cholesterol uptake in Caco-2 cells by down-regulation of NPC1L1 expression.

BACKGROUND: Curcumin is a polyphenol and the one of the principle curcuminoids of the spice turmeric. Its antioxidant, anti-cancer and anti-inflammatory effects have been intensively studied. Previous in vivo studies showed that administration of curcumin also decreased cholesterol levels in the blood, and the effects were considered to be related to upregulation of LDL receptor. However, since plasma cholesterol levels are also influenced by the uptake of cholesterol in the gut, which is mediated by a specific transporter Niemann-Pick Cl-like 1 (NPC1L1) protein, the present study is to investigate whether curcumin affects cholesterol uptake in the intestinal Caco-2 cells. METHODS: Caco-2 cells were cultured to confluence. The micelles composed of bile salt, monoolein, and 14C-cholesterol were prepared. We first incubated the cells with the micelles in the presence and absence of ezetimibe, the specific inhibitor of NPC1L1, to see whether the uptake of the cholesterol in the cells was mediated by NPC1L1. We then pretreated the cells with curcumin at different concentrations for 24 h followed by examination of the changes of cholesterol uptake in these curcumin-treated cells. Finally we determined whether curcumin affects the expression of NPC1L1 by both Western blot analysis and qPCR quantification. RESULTS: We found that the uptake of radioactive cholesterol in Caco-2 cells was inhibited by ezetimibe in a dose-dependent manner. The results indicate that the uptake of cholesterol in this study was mediated by NPC1L1. We then pretreated the cells with 25-100 muM curcumin for 24 h and found that such a treatment dose-dependently inhibited cholesterol uptake with 40% inhibition obtained by 100 muM curcumin. In addition, we found that the curcumin-induced inhibition of cholesterol uptake was associated with significant decrease in the levels of NPC1L1 protein and NPC1L1 mRNA, as analyzed by Western blot and qPCR, respectively. CONCLUSION: Curcumin inhibits cholesterol uptake through suppression of NPC1L1 expression in the intestinal cells.

Sphingolipids in human ileostomy content after meals containing milk sphingomyelin.

BACKGROUND: Sphingomyelin occurs in modest amounts in the diet, in sloughed mucosal cells, and in bile. It is digested by the mucosal enzymes alkaline sphingomyelinase and ceramidase. In humans, alkaline sphingomyelinase is also secreted in bile. The digestion of sphingomyelin is slow and incomplete, which has been linked to the inhibition of cholesterol absorption and colonic carcinogenesis. OBJECTIVE: We evaluated whether the supply of moderate amounts of milk sphingomyelin increases the exposure of the colon to sphingomyelin and its metabolites. DESIGN: Two experimental series were performed. In experiment A, we measured the content of sphingomyelin and ceramide in human ileostomy content by HPLC during 8 h after consumption of a test meal containing 250 mg milk sphingomyelin. In experiment B, we measured the molecular species of sphingomyelin and ceramide by HPLC-tandem mass spectrometry after doses of 50, 100, or 200 mg sphingomyelin. RESULTS: In experiment A, the average increase in ileostomy content of ceramide plus sphingomyelin amounted to 19% of the fed dose of sphingomyelin. In experiment B, the output of C-22:0-sphingomyelin, C-23:0-sphingomyelin, C-24:0-sphingomyelin, and sphingosine increased significantly, and palmitoyl-sphingomyelin increased proportionally less. Outputs and concentrations of palmitoyl-ceramide and sphingosine showed great individual variation, and stearoyl-sphingomyelin and stearoyl-ceramide did not increase after the meals. Although the output of long-chain sphingomyelin species increased significantly, the data indicated that >81% of all measured sphingomyelin species had been digested. CONCLUSIONS: Humans digest and absorb most of the sphingomyelin in normal diets. The amount of sphingolipid metabolites to which the colon is exposed can, however, be influenced by realistic amounts of dietary sphingomyelin.

Long term effects on human plasma lipoproteins of a formulation enriched in butter milk polar lipid.

BACKGROUND: Sphingolipids (SL), in particular sphingomyelin (SM) are important components of milk fat polar lipids. Dietary SM inhibits cholesterol absorption in rats (Nyberg et al. J Nutr Biochem. 2000) and SLs decrease both cholesterol and TG concentrations in lipid- and cholesterol fed APOE*3Leiden mice (Duivenvoorden et al. Am J Clin Nutr. 2006). This human study examines effects of a butter milk formulation enriched in milk fat globule membrane material, and thereby in SLs, on blood lipids in healthy volunteers. In a four week parallel group study with 33 men and 15 women we examined the effects of an SL-enriched butter milk formulation (A) and an equivalent control formulation (B) on plasma lipid levels. Plasma concentrations of HDL and LDL cholesterol, triacylglycerols (TG), apolipoproteins AI and B, and lipoprotein (a) were measured. The daily dose of SL in A was 975 mg of which 700 mg was SM. The participants registered food and drink intake four days before introducing the test formula and the last four days of the test period. RESULTS: A daily increase of SL intake did not significantly influence fasting plasma lipids or lipoproteins. In group B TG, cholesterol, LDL, HDL and apolipoprotein B concentrations increased, however, but not in group A after four weeks. The difference in LDL cholesterol was seen primarily in women and difference in TG primarily in men. No significant side effects were observed. CONCLUSION: The study did not show any significant decrease on plasma lipids or lipoprotein levels of an SL-enriched formulation containing 2-3 times more SL than the normal dietary intake on cholesterol, other plasma lipids or on energy intake. The formulation A may, however, have counteracted the trend towards increased blood lipid concentrations caused by increased energy intake that was seen with the B formulation.