中国汉族人群PNPLA3 I148M 多态性影响脂联素水平及非酒精性脂肪性 肝病遗传易感性的相关性研究*

目的:探讨在中国人群中PNPLA3 I148M基因型、脂联素与非酒精性脂肪性肝病的遗传易感性的相关性,及PNPLA3基因型与空腹血清脂联素水平的关系。方法:对96例NAFLD患者和76名正常对照,采用多聚酶链反应(PCR)及直接测序法检测PNPLA3基因型。计量资料结果均用均数±标准差(X±S)表示,经方差齐性检验后,行t检验;性别、基因型及等位基因频率的比较行X2检验。结果:中国汉族人群中,存在PNPLA3基因I148M多态性,I148M G等位基因频率分布在NAFLD(64.89%)与正常对照组(34.87%)、NASH组(71.70%)与SS组(56.09%)中比较差异均有统计学意义(P<0.05)。病例对照分析显示:148GG基因携带者与148CC基因携带者相比较,前者发生NAFLD的比值比(OR)为3.45(95%CI:2.21~5.41,P<0.05),发生NASH的OR为1.98(95%CI=1.08~3.64,P<0.05)。PNPLA3基因rs738409多态性与血清ALT水平有关(P<0.05),对NASH组分层分析,148GG基因型BMI、ALT、FINS均高于148CC基因型(P<0.05),血清HDL水平低于148CC基因型和148GC基因型(P<0.05),这些结果提示等位基因G与肝脏炎症和肝脏脂肪增加有相关性.Ordinal Logistic回归分析显示PNPLA3 I148M多态性与低浓度血清脂联素水平相关(<6μg/ml)(OR=2.78,95%CI=1.765~4.384,P<0.05)。结论:中国汉族人群中,PNPLA3基因I148M多态性与NAFLD的遗传易感性及脂联素的分泌调节相关,是决定NAFLD个体遗传易感性的重要因素。     

Associations of maternal prenatal dietary intake of n-3 and n-6 fatty acids with maternal and umbilical cord blood levels

Maternal n-3 and n-6 polyunsaturated fatty acid (PUFA) status may influence birth outcomes and child health. We assessed second trimester maternal diet with food frequency questionnaires (FFQs) (n=1666), mid-pregnancy maternal erythrocyte PUFA concentrations (n=1550), and umbilical cord plasma PUFA concentrations (n=449). Mean (SD) maternal intake of total n-3 PUFA was 1.17 g/d (0.43), docosahexaenoic and eicosapentaenoic acids (DHA+EPA) 0.16 g/d (0.17), and total n-6 PUFA 12.25 g/d (3.25). Mean maternal erythrocyte and cord plasma PUFA concentrations were 7.0% and 5.2% (total n-3), 5.0% and 4.6% (DHA+EPA), and 27.9% and 31.4% (total n-6). Mid-pregnancy diet–blood and blood–blood correlations were strongest for DHA+EPA (r=0.38 for diet with maternal blood, r=0.34 for diet with cord blood, r=0.36 for maternal blood with cord blood), and less strong for n-6 PUFA. The FFQ is a reliable measure of elongated PUFA intake, although inter-individual variation is present     

PNPLA3在实验性非酒精性脂肪性肝病中的表达及其CpG岛甲基化状态的研究

目的:探讨PNPLA3在非酒精性脂肪性肝病中的表达水平及其Cp G岛甲基化状态,探讨PNPLA3在NAFLD发生、发展中的作用。方法:1.采用10μg/m L的油酸作用于人正常肝细胞株L02建立脂肪肝细胞模型,72 h后经油红O染色,观察正常组、脂肪肝模型组及给药组细胞内脂滴形成情况,并用荧光定量PCR检测PNPLA3在三组肝细胞中的表达情况。2.采用专用DNA提取试剂盒分别提取正常组、脂肪肝模型组、给药组细胞中DNA,经亚硫酸转化后行PCR扩增目的基因,并用焦磷酸测序方法检测不同组PNPLA3 Cp G岛中甲基化水平,探讨其在非酒精性脂肪肝中的作用。结果:1、脂肪肝模型组肝细胞PNPLA3 m RNA的表达高于正常组,经姜黄素给药后,其表达降低,差异均有统计学意义(P0.05)。2、在PNPLA3启动子区6个检测位点中,脂肪肝组位点2甲基化水平高于正常组,姜黄素给药后甲基化水平降低,差异均有统计学意义(P0.05),在其余各检测位点中甲基化水平无差异。结论:1、油红O染色后,脂肪肝模型组细胞内发现有大量红色脂滴积聚,而正常组基本上未见脂滴,姜黄素给药组脂滴较模型组减少,表明用10μg/m L的油酸诱导能成功建立体外肝细胞模型。2、PNPLA3 m RNA在肝细胞中高表达及其启动子区甲基化状态异常参与非酒精性脂肪肝发病。3、抑制肝脏PNPLA3活性或降低肝细胞PNPLA3 m RNA的表达水平可能是今后治疗非酒精性脂肪性肝病(NAFLD)的一个新的治疗方法。     

Association of the I148M/PNPLA3 variant with elevated alanine transaminase levels in normal-weight and overweight/obese Mexican children

Background and aims

Non-alcoholic fatty liver disease (NAFLD) and elevated alanine transaminase (ALT) levels are common in obese Hispanic adults and children. Recently, a PNPLA3 gene variant (I148M) was strongly associated with NAFLD and higher ALT levels in obese adults, including Hispanics. The aims of this study were to estimate the frequency of elevated ALT levels, and to address the influence of obesity and PNPLA3/I148M on ALT levels in a general population sample of Mexican school-aged children.

Methods

A total of 1037 non-related Mexican children aged 6 to 12 years were genotyped for the I148M variant. Anthropometric, clinical and metabolic parameters were collected from all participants.

Results

Elevated ALT levels (> 35 U/L) were more frequent in obese (26.9%) and overweight (9.3%) than in normal weight children (2.2%). The M148M genotype was significantly associated with elevated ALT levels in this population (OR = 3.7, 95% CI 2.3–5.9; P = 3.7 × 10^− 8), and children carrying the M148M genotype showed significantly lower HDL cholesterol levels and BMI z-core (P = 0.036 and 0.015, respectively). On stratifying by BMI percentile, this genotype conferred a much greater risk of elevated ALT levels in normal weight (OR = 19.9, 95% CI 2.5–157.7; P = 0.005) than overweight and obese children (OR = 3.4, 95% CI 1.3–8.9; P = 0.014 and OR = 3.1, 95% CI 1.7–5.5; P = 1.4 x10^− 4, respectively).

Conclusions

The I148M PNPLA3 variant is strongly associated with elevated ALT levels in normal weight and overweight/obese Mexican children. Thus, the M148M genotype may be considered as an important risk factor for liver damage in this population.     

Expression and characterization of a PNPLA3 protein isoform (I148M) associated with nonalcoholic fatty liver disease

A genetic variant of PNPLA3 (patatin-like phospholipase domain-containing 3; PNPLA3-I148M), a serine protease of unknown function, is associated with accumulation of triacylglycerol (TAG) in the liver. To determine the biological substrates of PNPLA3 and the effect of the I148M substitution on enzymatic activity and substrate specificity, we purified and characterized recombinant human PNPLA3 and PNPLA3-I148M. Maximal hydrolytic activity of PNPLA3 was observed against the three major glycerolipids, TAG, diacylglycerol, and monoacylglycerol, with a strong preference for oleic acid as the acyl moiety. Substitution of methionine for isoleucine at position 148 markedly decreased the V(max) of the enzyme for glycerolipids but had only a modest effect on the K(m). Purified PNPLA3 also catalyzed the hydrolysis of oleoyl-CoA, but the V(max) was 100-fold lower for oleoyl-CoA than for triolein. The thioesterase activity required the catalytic serine but was only modestly decreased by the I148M substitution. The enzyme had little or no hydrolytic activity against the other lipid substrates tested, including phospholipids, cholesteryl ester, and retinyl esters. Neither the wild-type nor mutant enzyme catalyzed transfer of oleic acid from oleoyl-CoA to glycerophosphate, lysophosphatidic acid, or diacylglycerol, suggesting that the enzyme does not promote de novo TAG synthesis. Taken together, our results are consistent with the notion that PNPLA3 plays a role in the hydrolysis of glycerolipids and that the I148M substitution causes a loss of function, although we cannot exclude the possibility that the enzyme has additional substrates or activities.     

Recovery of Altered Fatty Acid Composition Induced by a Diet Devoid of n-3 Fatty Acids in Myelin, Synaptosomes, Mitochondria, and Microsomes of Developing Rat Brain

Rats were fed a semisynthetic diet containing either sunflower oil or soya oil. Half the litter fed with sunflower oil diet was changed to a soya oil diet when the pups were 15 days old (during active myelination). Fatty acid analysis was then performed on subcellular fractions of the animals fed (a) soya oil, (b) sunflower oil, and (c) soya oil replacing sunflower oil from the 15th day, to determine the speed of the recovery. All material from animals fed sunflower oil showed an important reduction in docosahexaenoic acid (22:6 n-3), compensated by an increase in docosapentaenoic acid (22:5 n-6), whereas arachidonic acid (20:4 n-6) was not affected. In all fractions examined, when sunflower oil was replaced by soya oil in 15-day-old pups the recovery started from the very first day but lasted more than 2 months (this recovery was determined by the increase of 22:6 n-3 up to the normal value and decrease of the 22:5 n-6). In addition a delay was found for myelin recovery, starting only from the 25th day.     

Recombinant PNPLA3 protein shows triglyceride hydrolase activity and its I148M mutation results in loss of function

The patatin-like phospholipase domain containing 3 (PNPLA3, also called adiponutrin, ADPN) is a membrane-bound protein highly expressed in the liver. The genetic variant I148M (rs738409) was found to be associated with progression of chronic liver disease. We aimed to establish a protein purification protocol in a yeast system (Pichia pastoris) and to examine the human PNPLA3 enzymatic activity, substrate specificity and the I148M mutation effect. hPNPLA3 148I wild type and 148M mutant cDNA were cloned into P. pastoris expression vectors. Yeast cells were grown in 3 L fermentors. PNPLA3 protein was purified from membrane fractions by Ni-affinity chromatography. Enzymatic activity was assessed using radiolabeled substrates. Both 148I wild type and 148M mutant proteins are localized to the membrane. The wild type protein shows a predominant lipase activity with mild lysophosphatidic acid acyl transferase activity (LPAAT) and the I148M mutation results in a loss of function of both these activities. Our data show that PNPLA3 has a predominant lipase activity and I148M mutation results in a loss of function.     

Influence of dietary pattern,physical activity,and I148M PNPLA3 on steatosis severity in at-risk adolescents

Evidence relating dietary patterns to obesity and related disorders such as non-alcoholic fatty liver disease (NAFLD) is limited in pediatric age. Aim of this study was to analyze the association between dietary patterns, obesity and development of severe steatosis and the metabolic syndrome in a series of children and adolescents referred for suspected NAFLD, and the interaction with the rs738409 I148M PNPLA3 polymorphism. Two hundred patients (112 females) had completed a food frequency and demographic questionnaire. Nearly 58 % were obese, and 32 % were overweight. Mild, moderate, and severe fatty liver was present in 60 (30 %), 87 (44 %), and 51 (26 %) participants, respectively. A great proportion of overweight/obese children and adolescents reported a correct dietary pattern. At multivariate ordinal regression analysis considering demographic, anthropometric, genetic, and behavioral determinants, the major determinant of steatosis severity was PNPLA3 I148M genotype (p < 0.0001), followed by older age (p = 0.017), higher waist circumference (p = 0.016), and less time spent practising physical exercise (p = 0.034). Furthermore, there was a significant interaction between PNPLA3 I148M and intake of sweetened beverages (p = 0.033) and of vegetables (p = 0.038). In conclusion, although dietary pattern was reportedly correct in at-risk overweight adolescents with NAFLD, we report a novel interaction between PNPLA3 I148M and dietary components with the severity of steatosis.     

Bioavailability and spatial distribution of fatty acids in the rat retina after dietary omega-3 supplementation

Spatial changes of FAs in the retina in response to different dietary n-3 formulations have never been explored, although a diet rich in EPA and DHA is recommended to protect the retina against the effects of aging. In this study, Wistar rats were fed for 8 weeks with balanced diet including either EPA-containing phospholipids (PLs), EPA-containing TGs, DHA-containing PLs, or DHA-containing TGs. Qualitative changes in FA composition of plasma, erythrocytes, and retina were evaluated by gas chromatography-flame ionization detector. Following the different dietary intakes, changes to the quantity and spatial organization of PC and PE species in retina were determined by LC coupled to MS/MS and MALDI coupled to MS imaging. The omega-3 content in the lipids of plasma and erythrocytes suggests that PLs as well as TGs are good omega-3 carriers for retina. However, a significant increase in DHA content in retina was observed, especially molecular species as di-DHA-containing PC and PE, as well as an increase in very long chain PUFAs (more than 28 carbons) following PL-EPA and TG-DHA diets only. All supplemented diets triggered spatial organization changes of DHA in the photoreceptor layer around the optic nerve. Taken together, these findings suggest that dietary omega-3 supplementation can modify the content of FAs in the rat retina.     

The effects on boar sperm quality of dietary supplementation with omega-3 polyunsaturated fatty acids differ among porcine breeds

The present study was undertaken to shed light on the relationship between boar sperm quality and dietary supplementation with omega-3 polyunsaturated fatty acids, which has been reported inconsistently in the literature. With this aim, such effects were evaluated and compared among three different porcine breeds: Duroc, Large-White, and Pietrain. Animals were randomly separated into two groups and fed either with a control diet or with a diet supplemented with omega-3. Sperm quality of these boar (ejaculate volume, sperm concentration, sperm viability, acrosome and mitochondrial sheath integrity, sperm motility, sperm morphology, and osmotic resistance of spermatozoa) was assessed every week for a 26-week period. Supplementing boar's diet with omega-3 did not affect ejaculate volume, sperm concentration, sperm motility, sperm viability, and acrosome and mitochondrial sheath integrity. In contrast, supplemented diet positively affected both sperm morphology in Large-White and Pietrain breeds and the osmotic resistance of Pietrain spermatozoa. No effects were seen for the same sperm parameters in Duroc breed. These breed-differences in boar fed with the supplemented diet could explain the contradictions in literature and might be related with differences in the composition of plasma membrane among breeds reported by other authors. Because no harmful effects were observed in the three evaluated breeds, but positive effects in Large-White and Pietrain boar, we can conclude that omega-3 fatty acids may be added to boar's diet at the levels used in this study to improve their sperm quality. More research is, however, needed to determine how these fatty acids differently affect the morphology and the osmotic resistance of the spermatozoa in these breeds.     

Fatty acid profiles in hepatic membranes of rats with different levels of circulating estrogen and prolactin

Plasma and liver microsomal fatty acid patterns of female rats (Rattus norvegicus) with either low or high serum levels of prolactin (PRL) were studied. Hyperprolactinemia was achieved by grafting anterior pituitary glands or by estradiol administration. One group treated with estradiol also received bromocriptine to inhibit PRL secretion. Ovariectomized (OVX) rats showed a decrease in PRL levels as compared with intact animals (controls). Rats possessing high levels of circulating PRL showed a significant decrease of linoleic acid in the fatty acid pattern of total and polar liver microsomal lipids. High PRL levels in the presence of normal estrogen levels significantly increased arachidonic acid in the same group of lipids. The group of rats treated with estrogen evidenced a decrease in arachidonic acid and in the unsaturation index. From these results it is possible to infer a decrease in the activity of the desaturases. The changes observed in the estradiol-treated group were not modified by bromocriptine administration. OVX rats showed no changes when compared with controls. It is concluded that, while PRL decreases the microsomal unsaturation index, estrogen administration causes a decrease in poly-unsaturated fatty acid biosynthesis and that this effect is independent of PRL levels.     

A review of nutritional effects on fat composition of animal products with special emphasis on n-3 polyunsaturated fatty acids

The fatty acid composition of animal products (eggs, milk and meat) is the reflect of both the tissue fatty acid biosynthesis and the fatty acid composition of ingested lipids. This relationship is stronger in monogastrics (pigs, poultry and rabbits) than in ruminants, where dietary fatty acids are hydrogenated in the rumen. There is an increasing recognition of the health benefits of polyunsaturated fatty acids (PUFA), because these fatty acids are essential for humans. In addition, the ratio n-6/n-3 fatty acids in the human diet is important. This ratio by far exceeds the recommended value of 5. Therefore, inclusion of fish meals, or n-3 PUFA rich oils, or linseed in animal diets is a valid means of meeting consumer demand for animal products that are nutritionally beneficial.The studies that are undertaken on animals mainly use diets supplemented with linseed, as a source of n-3 fatty acids. The use of linseed diets generally leads to an increased n-3 fatty acid content in animal products (egg, meat, milk) in ruminants and monogastrics. Recent studies have also demonstrated that neither the processing nor the cooking affects the PUFA content of pork meat or meat products.The ability of unsaturated fatty acids, especially those with more than two double bonds, to rapidly oxidise, is important in regulating the shelf life of animal products (rancidity and colour deterioration); however, a good way to avoid such problems is to use antioxidant products (such as vitamin E) in the diet.Some studies also show that it is not necessary to feed animals with linseed-supplemented diets for a long time to have the highest increase in PUFA content of the products. So, short-term diet manipulation can be a practical reality for industry.As the market for n-3 PUFA enriched products is today limited in most countries, other studies must be undertaken to develop this kind of production.     

Beneficial effects of enrichment of chicken meat with n-3 polyunsaturated fatty acids,vitamin E and selenium on health parameters: a study on male rats

Consumption of chicken meat enriched with bioactive compounds such as n-3 polyunsaturated fatty acids (PUFAn-3), vitamin E (vE) and selenium (Se) can help prevent many diseases and can be used to deliver those substances to humans. This might be of importance as chicken meat consumption is increasing worldwide. The effects of enriching chicken meat with PUFAn-3, vE and Se through dietary interventions were studied in rats. Four groups of Ross 308 female broilers from day 22 to day 35 of age were fed control diet (L) that contained lard and 80 mg vE and 0.3 mg Se/kg, or diets that contained rape seeds and fish oil with the same level of Se and vE as in the control diet, the same level of Se as in the control and 150 mg vE/kg, or 150 mg of vE and 0.7 mg Se/kg. Broiler carcasses were boiled, deboned, lyophilized and pooled by group. Boiled edible components of chicken carcass (BECC) were included (240 g/kg) in the diets fed to four groups of ten 10-week-old Wistar male rats for 8 weeks. Inclusion of BECCs modulated dietary fatty acid profile in the rat diets. Feeding these diets did not influence parameters related to growth or relative weights of internal organs in the rats. Feeding BECCs with lower PUFAn-6/n-3 decreased the n-6/n-3 ratio in the rat brain and liver, and increased the proportion of docosahexaenoic acid in the brain lipids. Liver cholesterol level was similar among the experimental groups, whereas the concentration of vE in the liver of rats fed BECC with increased vE levels was higher than that in the rats fed BECC with the basal vE level. Haematological and biochemical parameters in blood were within the normal range for rats, but a few rats showed a tendency towards increased levels because of the higher vE and Se level. The health-promoting effect of feeding rats PUFAn-3 enriched BECC was more pronounced when an increased dietary level of vE was used, but the increased level of Se did not provide the rats with additional benefits. Thus, the findings indicate that BECC enriched with PUFAn-3 and vE by a dietary intervention is a functional food with great potential of implementation.     

The effects of aspirin and fish oil consumption on lysophosphatidylcholines and lysophosphatidic acids and their correlates with platelet aggregation in adults with diabetes mellitus

Many diabetics are insensitive to aspirin's platelet anti-aggregation effects. The influence of co-administration of aspirin and fish oil (FO) on plasma lysophospholipids in subjects with diabetes is poorly characterized. Thirty adults with type 2 diabetes mellitus were treated with aspirin (81 mg/day) for seven days, then with FO (4 g/day) for 28 days, then in combination for another seven days. Lysophospholipids and platelet measures were determined after acute (4 h) and chronic (7 days) ingestion of aspirin, FO, or both in combination. FO ingestion reduced all lysophosphatidic acid (LPA) concentrations, while EPA (20:5n−3) and DHA (22:6n−3) lysophosphatidylcholine (LPC) concentrations significantly increased after FO alone and in combination with aspirin. In vitro arachidonic acid-induced platelet aggregation was most strongly correlated with palmitoleic (16:1) and oleic (18:1) LPA and LPC concentrations at all time points. The ingestion of these agents may reduce cardiovascular disease risk in diabetic adults, with a disrupted lipid milieu, via lysolipid mediated mechanisms.     

Marked variability in hepatic expression of cytochromes CYP7A1 and CYP27A1 as compared to cerebral CYP46A1. Lessons from a dietary study with omega 3 fatty acids in hamsters

Two diets simulating the recommendations of the American Heart Association to increase the intake of n-3 polyunsaturated fatty acids (n-3 PUFAs) were tested on Golden Syrian hamsters and compared to the diet simulating the current estimated consumption of fat in the United States. N-3 PUFAs were evaluated for their effects on serum and brain lipids and on the three cytochrome P450 enzymes (CYPs 7A1, 27A1, and 46A1) that play key roles in cholesterol elimination from different organs. Hamsters on the highest concentration of n-3 PUFAs had a statistically significant decrease in LDL and HDL cholesterol and no change in serum total cholesterol and triglycerides levels. CYP27A1 and CYP46A1 mRNA levels were increased in the liver and brain, respectively, whereas possible effects on CYP7A1 were obscured by a marked intergroup variability at mRNA, protein, and sterol product levels. Increased levels of CYP46A1 mRNA in the brain did not lead to significant changes in the levels of lathosterol, 24S-hydroxycholesterol or cholesterol in this organ. The data obtained are discussed in relation to inconsistent effects of n-3 PUFAs on serum lipids in human trials and reported positive effects of fish oil on cognitive function.     

Partial replacement of dietary fish oil with blends of vegetable oils (rapeseed, linseed and palm oils) in diets for European sea bass (Dicentrarchus labrax L.) over a long term growth study: effects on muscle and liver fatty acid composition and effectiveness of a fish oil finishing diet

Triplicate groups of European sea bass (Dicentrarchus labrax L.), of initial mass 5 g, were fed one of three practical type diets for 64 weeks. The three diets differed only in the added oil and were 100% fish oil (FO; diet A), 40% FO/60% vegetable oil blend (VO; diet B) where the VO blend was rapeseed oil, linseed oil and palm oil in the ratio 10/35/15 by weight and 40% FO/60% VO blend (diet C) where the ratio was 24/24/12 by weight. After final sample collection the remaining fish were switched to a 100% FO finishing diet for a further 20 weeks. After 64 weeks fish fed 60% VO diet B had significantly lower live mass and liver mass than fish fed diets A and C although SGR, FCR and length were not different between groups. There were no differences in any of the above parameters after either 14 or 20 weeks on the FO finishing diet. Fatty acid compositions of flesh were correlated to dietary fatty acids although there was selective retention of docosahexaenoic acid (22:6n-3; DHA) regardless of dietary input. Inclusion of dietary VO resulted in significantly reduced flesh levels of DHA and eicosapentaenoic acid (20:5n-3; EPA) while 18:1n-9, 18:2n-6 and 18:3n-3 were all significantly increased in fish fed the 60% VO diets. Fatty acid compositions of liver showed broadly similar changes, as a result of dietary fatty acid composition, as was seen in flesh. However, the response of flesh and liver to feeding a FO finishing diet was different. In flesh, DHA and EPA values were not restored after 14 or 20 weeks of feeding a FO finishing diet with the values in fish fed the two 60% VO diets being around 70% of the values seen in fish fed FO throughout. Conversely, and despite liver DHA and EPA levels being reduced to only 40% of the value seen in fish fed 100% FO after 64 weeks, the levels of liver DHA and EPA were not significantly different between treatments after feeding the FO finishing diet for 14 weeks. However, a 200 g portion of sea bass flesh, after feeding the experimental diets for 64 weeks followed by a FO diet for 14 weeks, contained 1.22 and 0.95 g of EPA + DHA for fish fed FO or 60% VO, respectively. Therefore, sea bass grown for most of the production cycle using diets containing 60% VO can still contribute a significant quantity of healthy n-3 HUFA to the human consumer.