Selective regulation of UGT1A1 and SREBP‐1c mRNA expression by docosahexaenoic,eicosapentaenoic, and arachidonic acids

We evaluated, in human cell line HepG2, the action of individual dietary polyunsaturated fatty acids (PUFAs) on the expression of several lipid metabolism genes. The effects of docosahexaenoic acid, 22:6, n‐3 (DHA), eicosapentaenoic acid, 20:5, n‐3 (EPA), and arachidonic acid, 20:4, n‐6 (AA) were studied alone and with vitamin E (Vit.E). DHA, EPA, and AA down‐regulated mRNAs and encoded proteins of stearoyl‐CoA desaturase (SCD) and sterol regulatory element binding protein (SREBP‐1c), two major factors involved in unsaturated fatty acids synthesis. DHA affected SREBP‐1c mRNA less markedly than EPA and AA. Vit.E did not affect these products, both when individually added or together with fatty acids. The expression of UDP‐glucuronosyl transferase 1A1 (UGT1A1) mRNA, an enzyme of phase II drug metabolism with relevant actions within lipid metabolism, resulted also differentially regulated. DHA did not essentially reduce UGT1A1 mRNA expression while EPA and AA produced a considerable decrease. Nevertheless, when these PUFAs were combined with Vit.E, which by itself did not produce any effect, the result was a reduction of UGT1A1 mRNA with DHA, an increase reverting to basal level with EPA and no variation with AA. Observed regulations did not result to be mediated by peroxisome proliferator‐activated receptor (PPAR). Our data indicate that major dietary PUFAs and Vit.E are differentially and selectively able to affect the expression of genes involved in lipid metabolism. The different actions of these slightly different molecules could be associated with their physiological role as relevant nutrient molecules. J. Cell. Physiol. 226: 187–193, 2010. © 2010 Wiley‐Liss, Inc.     

Selective action of human sera differing in fatty acids and cholesterol content on in vitro gene expression

Serum constituents might directly affect metabolic diseases pathogenesis and are commonly used as diagnostic tool. The aim of this study was to investigate the human serum effect on in vitro gene expression, related to nutrients action and involved in lipid metabolism. In detail, 40 human sera were firstly analyzed in fatty acids profile by gas-chromatography. Then samples were tested through direct addition within culture medium on Hep G2 human hepatoma cells, comparing samples from hypercholesterolemic (average 273 mg/dl) versus normocholesterolemic male subjects (average 155 mg/dl), since this condition is a relevant disease risk factor and is typically consequent to nutritional style. Hypercholesterolemic sera produced a 0.4-fold reduction of sterol regulatory element binding protein 1c (SREBP-1c) mRNA (P < 0.05) and a 1.5-fold increase of UDP-glucuronosyltransferase 1A1 (UGT1A1) mRNA (P < 0.01). Samples with higher concentrations of n-6 fatty acids produced a higher expression of UGT1A1 mRNA. Total fatty acids [docosahexaenoic, eicosopentanoic, arachidonic, linolenic, and linoleic acid (DHA, EPA, AA, LNA, and LA, respectively)] in each serum resulted roughly inverse with trend of SREBP-1c mRNA expression. Serum AA, LA, and trans fatty acids were more abundant in hypercholesterolemic subjects (P < 0.01) while DHA as quota of detected fatty acids was significantly higher in normocholesterolemic subjects (P < 0.05). While it is not possible to indicate which component was responsible for the observed gene modulations, our data indicate that sera differing in lipid profiles, mainly associated with dietary behavior, differentially affect gene expression known to be involved in metabolic and nutritional related conditions.     

DHA Supplemented in Peptamen Diet Offers No Advantage in Pathways to Amyloidosis: Is It Time to Evaluate Composite Lipid Diet?

Numerous reports have documented the beneficial effects of dietary docosahexaenoic acid (DHA) on beta-amyloid production and Alzheimer's disease (AD). However, none of these studies have examined and compared DHA, in combination with other dietary nutrients, for its effects on plaque pathogenesis. Potential interactions of DHA with other dietary nutrients and fatty acids are conventionally ignored. Here we investigated DHA with two dietary regimes; peptamen (pep+DHA) and low fat diet (low fat+DHA). Peptamen base liquid diet is a standard sole-source nutrition for patients with gastrointestinal dysfunction. Here we demonstrate that a robust AD transgenic mouse model shows an increased tendency to produce beta-amyloid peptides and amyloid plaques when fed a pep+DHA diet. The increase in beta-amyloid peptides was due to an elevated trend in the levels of beta-secretase amyloid precursor protein (APP) cleaving enzyme (BACE), the proteolytic C-terminal fragment beta of APP and reduced levels of insulin degrading enzyme that endoproteolyse beta-amyloid. On the contrary, TgCRND8 mice on low fat+DHA diet (based on an approximately 18% reduction of fat intake) ameliorate the production of abeta peptides and consequently amyloid plaques. Our work not only demonstrates that DHA when taken with peptamen may have a tendency to confer a detrimental affect on the amyloid plaque build up but also reinforces the importance of studying composite lipids or nutrients rather than single lipids or nutrients for their effects on pathways important to plaque development.     

Stearoyl-CoA desaturase-1 is associated with insulin resistance in morbidly obese subjects

Animal studies have revealed the association between stearoyl-CoA desaturase 1 (SCD1) and obesity and insulin resistance. However, only a few studies have been undertaken in humans. We studied SCD1 in visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) from morbidly obese patients and their association with insulin resistance, sterol regulatory element binding protein-1 (SREBP-1) and ATPase p97, proteins involved in SCD1 synthesis and degradation. The insulin resistance was calculated in 40 morbidly obese patients and 11 overweight controls. Measurements were made of VAT and SAT SCD1, SREBP-1 and ATPase p97 mRNA expression and protein levels. VAT and SAT SCD1 mRNA expression levels in the morbidly obese patients were significantly lower than in the controls (P = 0.006), whereas SCD1 protein levels were significantly higher (P < 0.001). In the morbidly obese patients, the VAT SCD1 protein levels were decreased in patients with higher insulin resistance (P = 0.007). However, SAT SCD1 protein levels were increased in morbidly obese patients with higher insulin resistance (P < 0.05). Multiple linear regressions in the morbidly obese patients showed that the variable associated with the SCD1 protein levels in VAT was insulin resistance, and the variables associated with SCD1 protein levels in SAT were body mass index (BMI) and ATPase p97. In conclusion, these data suggest that the regulation of SCD1 is altered in individuals with morbid obesity and that the SCD1 protein has a different regulation in the two adipose tissues, as well as being closely linked to the degree of insulin resistance.     

Docosapentaenoic acid (22:5n-3) down-regulates the expression of genes involved in fat synthesis in liver cells

Previous studies have shown that Eicosapentaenoic Acid (EPA) and Docosahexaenoic Acid (DHA) exhibit triacylglycerol (TAG) lowering effect in vitro and in vivo by down-regulating the Sterol Regulating Element Binding Protein (SREBP-1c) and reducing the expression levels of lipogenic genes. However, there is no evidence on the effect of Docosapentaenoic Acid (DPA) on SREBP-1c expression levels. DPA is a long chain n-3 fatty acid present in our diet through fish, red meat and milk of ruminant animals. Therefore, this study aimed to elucidate the effect of DPA on liver fatty acid synthesis in an in vitro model using rat liver cells. Our results suggested that DPA incubation (50μM) for 48h (like EPA and DHA) caused a significant decrease in the mRNA expression levels of SREBP-1c, 3-Hydroxy-3-Methyl-Glutaryl-Coenzyme A reductase (HMG-CoA reductase), Acetyl Coenzyme A Carboxylase (ACC-1) and Fatty Acid Synthase (FASn) compared with Oleic Acid (OA) and also a decrease in the protein levels of SREBP-1 and ACC-1. A time-course fatty acid analysis showed that DPA and EPA are interconvertable in the cells; however, after 8h of incubation with DPA, the cell phospholipids contained mainly DPA. The gene expression profiling of the lipogenic genes repeated at 8h confirmed that the inhibitory effect of DPA on mRNA expression levels of the lipogenic genes was most likely due to DPA itself and not due to its conversion into EPA.     

Highly purified eicosapentaenoic acid prevents the progression of hepatic steatosis by repressing monounsaturated fatty acid synthesis in high-fat/high-sucrose diet-fed mice

Eicosapentaenoic acid (EPA) is a member of the family of n-3 polyunsaturated fatty acids (PUFAs) that are clinically used to treat hypertriglyceridemia. The triglyceride (TG) lowering effect is likely due to an alteration in lipid metabolism in the liver, but details have not been fully elucidated. To assess the effects of EPA on hepatic TG metabolism, mice were fed a high-fat and high-sucrose diet (HFHSD) for 2 weeks and were given highly purified EPA ethyl ester (EPA-E) daily by gavage. The HFHSD diet increased the hepatic TG content and the composition of monounsaturated fatty acids (MUFAs). EPA significantly suppressed the hepatic TG content that was increased by the HFHSD diet. EPA also altered the composition of fatty acids by lowering the MUFAs C16:1 and C18:1 and increasing n-3 PUFAs, including EPA and docosahexaenoic acid (DHA). Linear regression analysis revealed that hepatic TG content was significantly correlated with the ratios of C16:1/C16:0, C18:1/C18:0, and MUFA/n-3 PUFA, but was not correlated with the n-6/n-3 PUFA ratio. EPA also decreased the hepatic mRNA expression and nuclear protein level of sterol regulatory element binding protein-1c (SREBP-1c). This was reflected in the levels of lipogenic genes, such as acetyl-CoA carboxylase α (ACCα), fatty acid synthase, stearoyl-CoA desaturase 1 (SCD1), and glycerol-3-phosphate acyltransferase (GPAT), which are regulated by SREBP-1c. In conclusion, oral administration of EPA-E ameliorates hepatic fat accumulation by suppressing TG synthesis enzymes regulated by SREBP-1 and decreases hepatic MUFAs accumulation by SCD1.     

A specific multi-nutrient formulation enhances M1 muscarinic acetylcholine receptor responses in vitro

Recent evidence indicates that supplementation with a specific combination of nutrients may affect cell membrane synthesis and composition. To investigate whether such nutrients may also modify the physical properties of membranes, and affect membrane-bound processes involved in signal transduction pathways, we studied the effects of nutrient supplementation on G protein-coupled receptor activation in vitro. In particular, we investigated muscarinic receptors, which are important for the progression of memory deterioration and pathology of Alzheimer's disease. Nerve growth factor differentiated pheochromocytoma cells that were supplemented with specific combinations of nutrients showed enhanced responses to muscarinic receptor agonists in a membrane potential assay. The largest effects were obtained with a combination of nutrients known as Fortasyn? Connect, comprising docosahexaenoic acid, eicosapentaenoic acid, uridine monophosphate as a uridine source, choline, vitamin B6, vitamin B12, folic acid, phospholipids, vitamin C, vitamin E, and selenium. In subsequent experiments, it was shown that the effects of supplementation could not be attributed to single nutrients. In addition, it was shown that the agonist-induced response and the supplement-induced enhancement of the response were blocked with the muscarinic receptor antagonists atropine, telenzepine, and AF-DX 384. In order to determine whether the effects of Fortasyn? Connect supplementation were receptor subtype specific, we investigated binding properties and activation of human muscarinic M1, M2 and M4 receptors in stably transfected Chinese hamster ovary cells after supplementation. Multi-nutrient supplementation did not change M1 receptor density in plasma membranes. However, M1 receptor-mediated G protein activation was significantly enhanced. In contrast, supplementation of M2- or M4-expressing cells did not affect receptor signaling. Taken together, these results indicate that a specific combination of nutrients acts synergistically in enhancing muscarinic M1 receptor responses, probably by facilitating receptor-mediated G protein activation.     

Inhibition of cholesterol absorption associated with a PPAR alpha-dependent increase in ABC binding cassette transporter A1 in mice

Dietary supplementation with the peroxisome proliferator-activated receptor alpha (PPAR alpha) ligand WY 14,643 gave rise to a 4- to 5-fold increase in the expression of mRNA for the ATP binding cassette transporter A1 (ABCA1) in the intestine of normal mice. There was no effect in the intestine of PPAR alpha-null mice. Consumption of a high-cholesterol diet also increased intestinal ABCA1 expression. The effects of WY 14,643 and the high-cholesterol diet were not additive. WY 14,643 feeding reduced intestinal absorption of cholesterol in the normal mice, irrespective of the dietary cholesterol concentration, and this resulted in lower diet-derived cholesterol and cholesteryl ester concentrations in plasma and liver. At each concentration of dietary cholesterol, there was a similar significant inverse correlation between intestinal ABCA1 mRNA content and the amount of cholesterol absorbed. The fibrate-induced changes in the intestines of the normal mice were accompanied by an increased concentration of the mRNA encoding the sterol-regulatory element binding protein-1c gene (SREBP-1c), a known target gene for the oxysterol receptor liver X receptor alpha (LXR alpha). There was a correlation between intestinal ABCA1 mRNA and SREBP-1c mRNA contents, but not between SREBP-1c mRNA content and cholesterol absorption. These results suggest that PPAR alpha influences cholesterol absorption through modulating ABCA1 activity in the intestine by a mechanism involving LXR alpha.     

Versatile roles of docosahexaenoic acid in the prenatal brain: from pro- and anti-oxidant features to regulation of gene expression

Docosahexaenoic acid (DHA) is the most ubiquitous polyunsaturated fatty acid (FA) in brain tissue. It is selectively esterified to amino phospholipids (PL) and therefore it is highly prevalent at the cytofacial site of the plasma membrane where it may specifically participate in intracellular events. A highly selective DHA accumulation prior to birth is the result of maternal supply via the placenta through a bio-magnification process. Supplements of DHA via the intra-amniotic route to the fetal rat increase brain DHA levels and also confer neuroprotection to fetuses subjected to global ischemic stress. The protective effect has been attributed to an enhanced free radical scavenging capacity of DHA. Dietary deprivation of linolenic acid (LNA) during the perinatal life on the other hand, resulted in losses of DHA from cerebral PLs [M. Schiefermeier, E. Yavin, n-3 deficient and DHA-enriched diets during critical periods of the developing prenatal rat brain, J. Lipid Res. 43 (2002) 124-131]. LNA deprivation also caused changes in a number of gene markers the identification of which was attained by a labor-intensive suppression subtractive hybridization protocol using mRNA from 2-week-old postnatal brains [E. Yakubov, P. Dinerman, F. Kuperstein, S. Saban, E. Yavin, Improved representation of gene markers on microarray by PCR-select subtracted cDNA targets, Mol. Brain Res. 137 (2005) 110-118]. Most notable was a remarkable elevation of dopamine (DA) receptor (D1 and D2) genes as evaluated by quantitative RT-PCR, SDS-PAGE gel electrophoresis and immunochemical staining [F. Kuperstein, E. Yakubov, P. Dinerman, S. Gil, R. Eylam, N. Salem Jr., E. Yavin, Overexpression of dopamine receptor genes and their products in the postnatal rat brain following maternal n-3 FA dietary deficiency, J. Neurochem. 95 (2005) 1550-1562]. Over-expression of DA receptors has been attributed to a compensatory mechanism resulting from impairment in DA neurotransmitter production, storage and processing. In conclusion, DHA is a versatile molecule with a wide range of actions spanning from participation in cellular oxidative processes and intracellular signaling to modulatory roles in gene expression and growth regulation.     

Lipid metabolism related gene-expression profiling in liver,skeletal muscle and adipose tissue in crossbred Duroc and Pietrain Pigs

Body-weight differences in animals may be ascribed to genetic and environmental factors. Here we utilized two divergent porcine genotypes, the highly muscled, leaner Pietrian × Yorkshire pigs and less muscled, fatter Duroc × Yorkshire growing pigs (75–110 kg), to examine the role of genetic background on expression of genes associated with anabolic (Fatty acid synthase, FAS; glucose transporter 4, GLUT-4; stearoyl CoA desaturase, SCD; Sterol regulatory binding protein-1, SREBP-1; leptin) and catabolic lipid metabolism (Carnitine palmitoyltransferase-1B, CPT-1B; acyl-CoA dehydrogenase, ACDH) in adipose tissue (AT), liver (L) and skeletal muscle (SKM). Pietrain pigs had lower mRNA abundance for FAS, SREBP-1, SCD and leptin in AT and L, but higher mRNA abundance for L ACDH and SKM ACDH and CPT-1B than Durocs. Duroc pigs exhibited higher expression of FAS, SREBP-1, SCD, leptin in AT and FAS in L and lower expression of ACDH and CPT-1B in L SKM. GLUT-4 expression did not differ in SKM between the two genotypes. Feeding of a beta adrenergic agonist (Paylean) for 52 days lowered expression of lipid anabolic and enhanced lipid catabolic genes expressions similarly in both genotypes. Overall, the lipid metabolism genes differential expression patterns documented here showed that in Pietrain pigs mRNA abundances of synthesis genes were lower and of catabolic genes were higher than in Duroc pigs.     

Vitamin A regulates obesity in WNIN/Ob obese rat; independent of stearoyl-CoA desaturase-1

Stearoyl-CoA desaturase 1 (SCD1), an important enzyme involved in monounsaturated fatty acid biosynthesis is a key player in energy homeostasis. Here, we tested the impact of vitamin A on hepatic and adipose tissue SCD1 expression and adiposity per se, using an obese mutant rat strain namely, WNIN/Ob developed at National Center for Laboratory Animal Sciences of National Institute of Nutrition, India. Seven months-old 24 male lean and obese rats of WNIN/Ob strain were divided into two groups; each group was subdivided into two subgroups having 6 lean and 6 obese rats and received diets containing either 2.6 mg or 129 mg vitamin A/kg diet for two months. Feeding of high (but non-toxic) doses of vitamin A resulted in significant reduction in body weight gain, and retroperitoneal white adipose tissue weight (RPWAT) in obese rats. Further, vitamin A feeding resulted in augmented expression of SCD1 in liver and RPWAT of lean rats, while no such effect was seen in obese rats. Taken together, the present data suggest that vitamin A decreases body weight gain in obese rat model independent of SCD1 gene regulation.     

Omega-3 PUFA profoundly affect neural,physiological, and behavioural competences – implications for systemic changes in trophic interactions

In recent decades, much conceptual thinking in trophic ecology has been guided by theories of nutrient limitation and the flow of elements, such as carbon and nitrogen, within and among ecosystems. More recently, ecologists have also turned their attention to examining the value of specific dietary nutrients, in particular polyunsaturated fatty acids (PUFA), among which the omega-3 PUFA, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) play a central role as essential components of neuronal cell membranes in many organisms. This review focuses on a new neuro-ecological approach stemming from the biochemical (mechanistic) and physiological (functional) role of DHA in neuronal cell membranes, in particular in conjunction with G-protein coupled receptors (GPCRs). We link the co-evolution of these neurological functions to metabolic dependency on dietary omega-3 PUFA. We outline ways in which deficiencies in dietary DHA supply may affect, cognition, vision, and behaviour, and ultimately, the biological fitness of consumers. We then review emerging evidence that changes in access to dietary omega-3 PUFA may ultimately have profound impacts on trophic interactions leading to potential changes in community structure and ecosystem functioning that, in turn, may affect the supply of DHA within and across ecosystems, including the supply for human consumption.