Establishment of Real-Time TaqMan-Fluorescence Quantitative RT-PCR Assay for Detection and Quantification of Porcine Lipoprotein Lipase mRNA

Porcine lipoprotein lipase （LPL） cDNA was cloned as the standard for real-time quantifying LPL mRNA and the TaqMan-fluorescence quantitative PCR assay for detection was established. The total RNA extracted from Longissimus dorsi of porcine was reverse-transcribed to cDNA. LPL cDNA was ligated with pGM-T vector and transformed into Escherichia coli TOP10. Plasmid DNA extracted from positive clones was verified by PCR amplification and sequenced. LPL was amplified by real-time fluorescence quantitative PCR from the plasmid DNA. The concentration of DNA template purified was detected by analyzing absorbance in 260 nm and then the combined plasmid was diluted to series as standard for fluorescence quantitative PCR （FQ-PCR）. The method of LPL mRNA real-time PCR was well established, which detected as low as 103 with the linear range 10^3 to 10^10 copies. The standard curves showed high correlations （R2 = 0.9871）. A series of standards for real-time PCR analysis have been constructed successfially, and real-time TaqMan-fluorescence quantitative RT-PCR is reliable to quantitatively evaluate FQ-PCR mRNA in L. dorsi of porcine.

Correlation between betatrophin/angiogenin-likeprotein3/lipoprotein lipase pathway and severity of coronary artery disease in Kazakh patients with coronary heart disease

BACKGROUND The results of previous animal experiments and clinical studies have shown that there is a correlation between expression of betatrophin and blood lipid levels.However,there are still differences studies on the correlation and interaction mechanism between betatrophin,angiogenin-likeprotein3(ANGPTL3)and lipoprotein lipase(LPL).In our previous studies,we found an increase in serum ANGPTL3 Levels in Chinese patients with coronary heart disease(CHD).Therefore,we retrospectively studied Kazakh CHD patients.AIM To explore the correlation between the betatrophin/ANGPTL3/LPL pathway and severity of coronary artery disease(CAD)in patients with CHD.METHODS Nondiabetic patients diagnosed with CHD were selected as the case group;79 were of Kazakh descent and 72 were of Han descent.The control groups comprised of 61 Kazakh and 65 Han individuals.The serum levels of betatrophin and LPL were detected by enzyme-linked immunosorbent assay(ELISA),and the double antibody sandwich ELISA was used to detect serum level of ANGPTL3.The levels of triglycerides,total cholesterol,and fasting blood glucose in each group were determined by an automatic biochemical analyzer.At the same time,the clinical baseline data of patients in each group were included.RESULTS Betatrophin,ANGPTL3 and LPL levels of Kazakh patients were significantly higher than those of Han patients(P=0.031,0.038,0.021 respectively).There was a positive correlation between the Gensini score and total cholesterol(TC),triglycerides(TG),low-density lipoprotein cholesterol(LDL-C),betatrophin,and LPL in Kazakh patients(r=0.204,0.453,0.352,0.471,and 0.382 respectively),(P=0.043,0.009,0.048,0.001,and P<0.001 respectively).A positive correlation was found between the Gensini score and body mass index(BMI),TC,TG,LDL-C,LPL,betatrophin in Han patients(r=0.438,0.195,0.296,0.357,0.328,and 0.446 respectively),(P=0.044,0.026,0.003,0.20,0.004,and P<0.001).TG and betatrophin were the risk factors of coronary artery disease in Kazakh patients,while BMI and betatrophin were the risk factors in Han patients.CONCLUSION There was a correlation between the betatrophin/ANGPTL3/LPL pathway and severity of CAD in patients with CHD.

Effect of Lipoprotein Lipase Gene Polymorphism on Plasma Lipid Levels, BMI and Subcutaneous Fat Distribution in Simple Obesity Children

Objective To study the effects of Hind Ⅲ DNA polymorphism in the lipoprotein lipase (LPL)gene on plasma lipid levels, body mass index (BMI) and subcutaneous fat distribution in simple obesity children.Methods The LPL Hind Ⅲ genotypes were detected with the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) techniques in 92 children with simple obesity. The levels of the plasma lipid, plasma lipoproteins, BMI and skinfold thickness in three regions (biceps, subscapular and abdominal wall) were also measured. Results It was shown that the levels of TG, TC, LDL C, ApoB, BMI, biceps and subscapular skinfold thickness, and the average value of the skinfold thickness in three regions were significantly higher in the obese children with H+H+ genotype than those with H+H-genotype. Conclusions It can be concluded that LPL Hind Ⅲ polymorphism may modify the levels of plasma lipid, plasma lipoprotein and BMI in children with simple obesity, and meanwhile alters the distribution of subcutaneous fat.

Hypotriglyceridemic Effects of Apple Polyphenols Extract via Up-Regulation of Lipoprotein Lipase in Triton WR-1339-Induced Mice

Objective：To investigate the anti-hyperlipidemic effects of apple polyphenols extract（APE）in Triton WR-1339-induced endogenous hyperlipidemic model.Methods：Firstly,APE was isolated and purified from the pomace of Red Fuji Apple and contents of individual polyphenols in APE were determined using highperformance liquid chromatography-mass spectrometry（HPLC-MS）.Secondly,forty male National Institude of Health（NIH）mice were randomly divided into 5 groups with 8 animals in each group.The Fenofibrate Capsules（FC）group and APE groups received oral administration of respective drugs for 7 consecutive days.All mice except those in the normal group were intravenously injected through tail vein with Triton WR-1339 on the6th day.Serum and livers from all the mice were obtained 18 h after the injection.The changes in serum total cholesterol（TC）,triglyceride（TG）,lipoprotein lipase（LPL）and hepatic triglyceride lipase（HTGL）were measured by respective kits.Finally,expression of hepatic peroxisome proliferator-activated receptor alpha（PPARα）mRNA was measured by real-time reverse transcription-polymerase chain reaction（RT-PCR）method.Results：Serum TC and TG levels significantly increased in Triton WR-1339-induced model group compared with the normal group（P〈0.01）.Oral administration of APE[200 and 400 mg/（kg·day）]dose-dependently reduced the serum level of TG in hyperlipidemic mice（P〈0.01）.Serum LPL and HTGL activities significantly decreased in Triton WR-1339-induced model group compared with the normal group（P〈0.05）.Oral administration of APE[200 and 400 mg/（kg·day）]dose-dependently elevated the serum activity of LPL in hyperlipidemic mice（P〈0.05or P〈0.01）.Furthermore,compared with the normal group,hepatic mRNA level of PPARαin the model group significantly decreased（P〈0.01）.Oral administration of APE[200 and 400 mg/（kg·day）]dose-dependently elevated the expression of PPARαin hyperlipidemic mice（P〈0.05 or P〈0.01）.Conclusion：APE could reduce TG level via up-regulation of LPL activity,which provides new evidence to elucidate the anti-hyperlipidemic effects of APE.

Effect of Astragalus-Angelica Mixture on Lipoprotein Lipase and Lecithin-Cholesterol Acyltransferase of Nephrotic Rats

Objective: To investigate the mechanism of Astragalus-Angelica Mixture (AAM) ’s effects on lipid metabolism disturbance in nephrotic rats.Methods: To examine the effects of AAM on serum albumin, lipid levels, and activities of lipoprotein lipase (LPL) and lecithin-cholesterol acyltransferase (LCAT), which are key enzymes for metabolism of lipid in immune-induced nephrotic hyperlipidemia rats and exogenous hyperlipidemia rats.Results: In nephrotic rats, serum albumin was reduced, lipid increased significantly, LPL activity decreased markedly and the LCAT activity was relatively insufficient. Activities of LPL and LCAT increased obviously in AAM treated nephrotic rats. There were no change of activities of LPL and LCAT in exogenous hyperlipidemia rats and AAM showed no effect on the activities of these two enzymes.Conclusion: The effect of AAM on regulating lipid metabolism might be due to enhance the clearance of both triglyceride-rich and cholesterol-rich apoB containing lipoprotein by improving the activities of LPL and LCAT.

Knockout of lipoprotein lipase with CRISPR/Cas9 causes severe developmental defects and affects lipid deposition in Japanese medaka(Oryzias latipes)

In mammals,lipoprotein lipase(LPL)has been found to play an important role in lipid mentalismand deposition.LPL deficiency in humans(Homo sapiens)and mice(Mus musculus)tends to cause hypertriglyceridemia.The lpl gene is not expressed in adult mammalian liver,but is in adult fish liver.The functions provided by the lpl gene are diverse in vertebrates.Here,we knocked out the lpl gene in Japanese medaka(Oryzias latipes)with the CRISPR/Cas9 system.The lpl-knockout(KO)homozygous individuals showed severe developmental defects with an extremely emaciated and deformedbody and onlyaccounted for about5%of the F2fish.This is consistentwiththefindings inmicebut disaccords with the results for zebrafish(Danio rerio).Compared with wild-type(WT)madaka,the mRNA level of lpl in lpl-KO heterozygous mutant was significantly higher in the muscle,showed no significant difference in the liver,and was significantly lower in the heart.Under lpl heterozygous deficiency,the relative area of Oil RedO and triglycerides(TG)level in the liver,heart and muscle tissue covaried with levels of lpl mRNA in medaka.The lpl heterozygous deficiency did not affect the levelsofTG,low-density lipoprotein cholesterol(LDL-C),high-density lipoprotein cholesterol(HDL-C)and total cholesterol(TC)in the plasma of medaka,which is inconsistent with the findings in mammals.In general,the lpl gene plays an important role in the growth and development and is closely related to lipid deposition of medaka.

Apolipoprotein A-V gene therapy for disease prevention/treatment:a critical analysis

Apolipoprotein（apo） A-V is a novel member of the class of exchangeable apo＇s involved in triacylglycerol（TG）homeostasis.Whereas a portion of hepatic-derived apoA-V is secreted into plasma and functions to facilitate lipoprotein Iipase-mediated TG hydrolysis,another portion is recovered intracellularly,in association with cytosolic lipid droplets.Loss of apo A-V function is positively correlated with elevated plasma TG and increased risk of cardiovascular disease.Single nucleotide polymorphisms（SNP） in the APOA5 locus can affect transcription efficiency or introduce deleterious amino acid substitutions.Likewise,rare mutations in APOA5 that compromise functionality are associated with increased plasma TG and premature myocardial infarction.Genetically engineered mouse models and human population studies suggest that,in certain instances,supplementation with wild type（WT） apoA-V may have therapeutic benefit.It is hypothesized that individuals that manifest elevated plasma TG owing to deleterious APOA5 SNPs or rare mutations would respond to WT apoA-V supplementation with improved plasma TG clearance.On the other hand,subjects with hypertriglyceridemia of independent origin（unrelated to apoA-V function） may not respond to apoA-V augmentation in this manner.Improvement in the ability to identify individuals predicted to benefit,advances in gene transfer technology and the strong connection between HTG and heart disease,point to apoA-V supplementation as a viable disease prevention / therapeutic strategy.Candidates would include individuals that manifest chronic TG elevation,have low plasma apoA-V due to an APOA5 mutation/polymorphism and not have deleterious mutations/polymorphisms in other genes known to influence plasma TG levels.

Developmental Changes of the LPL mRNA Expression and Its Effect on IMF Content in Sheep Muscle

This study investigates the developmental changes of the lipoprotein lipase （LPL） mRNA level in sheep muscle and its effect on intramuscular fat （IMF） accumulation. Male Kazak and Xinjiang Merino sheep at 2-120 days old were selected. Six animals of each breed were slaughtered at 2, 30, 60, 90, and 120 days （only the Xinjiang Merino sheep at 120-day old were available） to collect samples from longissimus dorsi muscle for the purpose of determining the IMF content and extracting total RNA that was used to investigate the developmental changes of the LPL mRNA expression by real-time PCR. The results showed that in male Kazak sheep, the IMF content increased with the progress of development and there were significant differences （P〈0.05） between the age groups. However, there was no difference （P〉0.05） between age groups in Xinjiang Merino sheep. Furthermore, the IMF content of the male Kazak sheep was significantly higher （P 〈 0.01） than that of the Xinjiang Merino sheep aged from 30 to 90 days. The highest LPL mRNA expression appeared at day 2 and it was significantly higher than that of all other age groups （P 〈 0.01）, while animals at 60-day old had the lowest LPL mRNA expression in the male Kazak sheep. In Xinjiang Merino sheep, the highest one occurred at 30-day old （P〈0.01）, followed by a continuous decrease to the lowest level at 90-day old, and then it started to increase slightly. At 2 to 60-day old, the LPL mRNA expression was negatively correlated to the IMF content （r=-0.625, P 〈 0.05） in male Kazak sheep, but no such relationship was detected in the male Xinjiang Merino sheep.

Effects of high ambient temperature on lipid metabolism in finishing pigs

In this study,we investigated the effects of high ambient temperature on lipid metabolism in finishing pigs.Sixteen pigs（（79.6±1.2） kg） were randomly assigned to two groups：（1） ambient temperature of 30℃ with ad libitum access to feed（HT;n=8） and（2） ambient temperature of 22℃ and fed the average amount consumed by eight pigs in HT group on the previous day（PF;n=8）.After 21 days of constant exposure to different environmental conditions,all the pigs were euthanized,and blood and tissue samples were obtained.High ambient temperature increased the proportion of backfat（P=0.04,＋21.6%）and flare fat（P〈0.01,＋43.3%）.Compared with pair-fed pigs,the activities of fatty acid synthase（FAS） and malic enzyme in backfat and flare fat were lower（P〈0.05） in heat-stressed pigs,as were the amounts of acetyl-CoA-carboxylase and FAS in the longissimus muscle（LM）,the amount of FAS in backfat（P〈0.01）,and FAS activity in the liver（P〈0.01）.Ambient temperature did not affect the amount of hormone-sensitive lipase in different tissues.The amount of lipoprotein lipase in flare fat tended to be higher（P=0.09,＋28.3%）,and the activities of β-hydroxyacyl coenzyme A dehydrogenase in front and back of LM were lower（P〈0.01,-48.3 and-49.8%,respectively） at 30℃ than at 22℃.The plasma concentration of high-density lipoprotein tended to be lower（P=0.08）,but the plasma concentrations of very low-density lipoprotein（VLDL）（P=0.09,＋50.0%） and nonestesterified fatty acid（NEFA）（P=0.04,＋44.2%） were higher in heat-stressed pigs.We concluded that high ambient temperature depressed de novo fatty acid synthesis in both adipose tissues and the liver.However,β-oxidation of fatty acid in skeletal muscles was also inhibited in the high-temperature environment.As a result,more plasma NEFAs were used to synthesize VLDLs in the liver and were absorbed by adipose tissues.This may be one reason that high ambient temperature enhances the accumulation of backfat and flare fat in finishing pigs.

The interplay between non-esterified fatty acids and bovine peroxisome proliferator-activated receptors: results of an in vitro hybrid approach

Background: In dairy cows circulating non-esterified fatty acids(NEFA) increase early post-partum while liver and other tissues undergo adaptation to greater lipid metabolism, mainly regulated by peroxisome proliferator-activated receptors(PPAR). PPAR are activated by fatty acids(FA), but it remains to be demonstrated that circulating NEFA or dietary FA activate bovine PPAR. We hypothesized that circulating NEFA and dietary FA activate PPAR in dairy cows.Methods: The dose-response activation of PPAR by NEFA or dietary FA was assessed using HP300 e digital dispenser and luciferase reporter in several bovine cell types. Cells were treated with blood plasma isolated from Jersey cows before and after parturition, NEFA isolated from the blood plasma, FA released from lipoproteins using milk lipoprotein lipase(LPL), and palmitic acid(C16:0). Effect on each PPAR isotype was assessed using specific synthetic inhibitors.Results: NEFA isolated from blood serum activate PPAR linearly up to ~ 4-fold at 400 μmol/L in MAC-T cells but had cytotoxic effect. Addition of albumin to the culture media decreases cytotoxic effects of NEFA but also PPAR activation by ~ 2-fold. Treating cells with serum from peripartum cows reveals that much of the PPAR activation can be explained by the amount of NEFA in the serum(R~2 = 0.91) and that the response to serum NEFA follows a quadratic tendency, with peak activation around 1.4 mmol/L. Analysis of PPAR activation by serum in MAC-T, BFH-12 and BPAEC cells revealed that most of the activation is explained by the activity of PPARδ and PPARγ, but not PPARα. Palmitic acid activated PPAR when added in culture media or blood serum but the activation was limited to PPARδ and PPARα and the response was nil in serum from post-partum cows. The addition of LPL to the serum increased > 1.5-fold PPAR activation.Conclusion: Our results support dose-dependent activation of PPAR by circulating NEFA in bovine, specifically δand γ isotypes. Data also support the possibility of increasing PPAR activation by dietary FA;however, this nutrigenomics approach maybe only effective in pre-partum but not post-partum cows.

Rapid Decrease and Subsequent Increase in the Serum Triglycerides Accompanied by CD36 Transcript Increase in an Acute Stress Mice Model

In this article, we report the changes in serum triglyceride (TG) levels that occurred during repeated tail blood sampling using a mouse restrainer. We used three groups of mice, namely, “PBS-restrained” “PBS-unrestrained” and “mock-restrained”. The mice in the PBS-restrained and PBS-unrestrained groups were intraperitoneally (i.p.) injected with 100 mL PBS and tail blood sampling was performed at 1, 5, 8, 24, and 48 h after i.p. injection. For the mock-restrained group, no i.p. injection was performed whereas the subsequent tail blood sampling was similarly performed. During the tail blood sampling, the mice of the two “restrained” groups were placed inside the restrainer designed from an open-ended 50 mL conical tube. The blood from the mice in the PBS-unrestrained group mice was sampled from the tail held by the operator’s hands while being allowed to move on a stage. Strikingly, in all of the three groups, the serum TG level initially decreased to remarkably low levels (approximately 30 mg/dL) after several blood samplings were performed over 8 h. This decrease was followed by a 2 - 3-fold increase in the levels relative to that in the control mice in the subsequent 24 - 48 h time period. We concluded that the acute stress associated with blood sampling caused alterations in TG levels. Serum levels of free fatty acid showed only modest changes. Changes in TG levels were not associated with serum corticosterone levels but with a dramatic increase in CD36 transcript levels in the liver. The relevance of this finding to the previously reported release of lipoprotein lipase (LPL) from white fatty tissue into the plasma during acute stress is also discussed.

Effects of dietary sodium butyrate on growth,digestive enzymes,body composition and nutrient retention-related gene expression of juvenile yellow catfish (Pelteobagrus fulvidraco)

An 8-week feeding trial was conducted to evaluate the effects of sodium butyrate(SB)on growth,digestive enzymes,body composition and nutrient retention-related gene expression of juvenile yellow catfish(Pel-teobagrus fulvidraco).Five isonitmgenous and isolipidic diets(420 g/kg protein and 90 g/kg lipid)were formulated to contain 0(control),250,500,1,000 or 2,000 mg/kg SB.Triplicate groups of 40 fish(BW=1.26±0.01 g)per tank(300-L cylindrical fiberglass tanks)for each diet were fed to apparent satiation twice daily.Stomach,hepatopancreas and intestine samples were obtained for digestive enzymes activities analyses.A real-time quantitative PCR analysis was performed to determine the relative expression of target of rapa-mycin(TOR)and lipoprotein lipase(LPL)in the hepatopancreas and intestine.Fish fed the diets supplemented with SB at 500 and 1,000 mg/kg showed significantly higher specific growth rate and significantly lower feed conversion ratio compared to the control(P<0.05).Dietary SB inclusion did not alter activities of intestinal amylase,creatine kinase and sodium-potassium adenosine triphosphatase(Na+/K+-ATPase),but increased activities of hepatic trypsin,stomachic lipase,intestinal lipase,alkaline phosphatase and y-glutamyl trans-peptidase for fish fed 1,000 mg/kg SB compared to the control(P<0.05).Intestine length index,intestine somatic index,fold height and muscular thickness of distal intestine were significantly higher in 1,000 mg/kg SB groups compared to the control(P<0.05).Significantly higher levels of whole-body crude protein,ash,calcium,phosphorus,nutrition retention and relative mRNA of intestinal TOR were observed in 1,000 mg/kg SB group(P<0.05).Whole-body lipid content and hepatopancreas LPL mRNA expression in 2,000 mg/kg SB gmup were significantly higher than the control(P<0.05).Relative mRNA levels of intestinal LPL and hepatopancreas TOR were significantly higher in the 500 mg/kg SB group compared to those in other groups(P<0.05).The increased growth performance,digestive enzymes and nutrient retention in fish fed the diets supplemented with SB at 500 and 1,000 mg/kg suggests that SB can be a desirable growth promoter as an antibiotic alternative in diets.

Delayed Postprandial Hypertriglyceridemia,Slow Gastrointestinal Transit and Possible Links Between Them in Alloxan-Induced Diabetic Mice

Both postprandial hypertriglyceridemia and diabetic gastroparesis are common dysfunctions af-fecting diabetes mellitus; however, whether diabetic gastroparesis has an influence on postprandial hypertriglyceridemia still remains undetermined. Delayed postprandial hypertriglyceridemia, diabetic gastroparesis, and the possible links between them were investigated using alloxan-induced diabetic mice. After the oral administration of olive oil, delayed and exaggerated postprandial hypertriglyceridemia and diabetic gastroparesis were markedly presented in alloxan-induced diabetic mice. Domperidone shortened the time of triglycerides (TG) peak levels in diabetic mice. After intraperitoneal and intraduodenal administration of olive oil, no delay of TG peak levels occurred in diabetic mice. Simultaneously, serum post-heparin lipoprotein lipase activities significantly decreased just at the time of prolonged and elevated TG peak levels resulting from diabetic gastroparesis, and further deteriorated postprandial hypertriglyceridemia in diabetic mice. The results indicate that diabetic gastroparesis can be one of the important reasons for delayed and exaggerated postprandial hypertriglyceridemia in diabetes mellitus.

A peroxisome proliferator response elements regulatory system in xenopus oocytes and its application

Background Peroxisome proliferator-activated receptor-gamma （PPARγ） is a kind of ligand-activated transcription factors binding to peroxisome proliferator response element （PPRE）, a specific recognition site. It is thought to play a critical role in glucose and lipid metabolism and in inflammation control. The aim of this study was to establish a new cellular model for the quick screening of lipid-lowering drugs, which may be effective as PPAR-γ ligands on the PPRE-mediated pathway regulatory system. Methods Two plasmids were constructed： pXOE-PPARγ, in which the human PPARγ gene was in the downstream of TF Ⅲ A gene promoter, and pLXRN-PPRE-d2EGFP, in which the enhanced green fluorescent protein （EGFP） gene was subcloned into PPRE. The xenopus oocytes were injected with these two plamids, and consequently treated with prostaglandin E1, pioglitazone, and different kinds of lipid-lowering drugs. After 3 days, the oocytes were observed under a fluorescence microscope. To confirm the drug action,we injected pXOE- PPARγ plasmid into the oocytes, which then treated with prostaglandin E1 and Hawthorn flavonoids. The mass of expressed lipoprotein lipase （LPL） in the cells was determined by enzyme labeling linked immunosorbent assay （ELISA）. Results The expression of EGFP was only induced by prostagalandin E1, pioglitazone, Hawthorn flavonoids. A concentration-response relationship was seen between expressed EGFP and Hawthorn flawonids. The levels of LPL in both Hawthorn flaworvoids groups and PPARγ ligand prostagalandin E1 group injected with pXOE-PPARγ plamid increased significantly （ P 〈 0. 001 ） compared with controls, and a cocentration-response relationship was observed between LPL mass and Hawthorn flavonoids. Conclusions It is possible to establish a PPRE regulatory EGFP reporter system in xenopus oocytes to monitor the activity of PPARγ ligand. Hawthorn flavonoids can in increase the expression of gene downsteam of PPRE by effect on the PPRE pathway regulatory system.