LPL mRNA Research Articles

MicroRNA-186 promotes macrophage lipid accumulation and secretion of pro-inflammatory cytokines by targeting cystathionine γ-lyase in THP-1 macrophages

Background and aimsSeveral studies suggest that cardiomyocyte-enriched miR-186 is involved in cardiac injury and myocardial infarction, and also plays an important role in atherosclerotic diseases, but the underlying mechanism is unknown. Cystathionine-γ-lyase (CSE) is the predominant enzyme to produce H2S in the cardiovascular system. Here, miR-186 was identified to bind to the 3′UTR of CSE. In this study, we aimed at exploring whether miR-186 affects lipid accumulation and secretion of pro-inflammatory cytokines by targeting CSE and its underlying mechanism in human THP-1 macrophages and peripheral blood monocyte-derived macrophages (PBMDM). PBMDM just as a control group for the comparison with the THP-1 macrophages. MethodsMiR-186 target genes, CSE 3′UTR sequence and free energy were predicted and analyzed by bioinformatics analyses and dual-luciferase reporter assays. The expression of CSE mRNA and protein were measured by real-time quantitative PCR and western blot analyses. The lipid accumulation in THP-1 macrophages was detected by high performance liquid chromatography (HPLC). The effects of miR-186 on secretion of IL-6, IL-1β and TNF-α were examined by ELISA. Endogenous H2S was detected by spectrophotometry. Using small interfering RNA (siRNA) approach to decrease the expression of CSE protein and mRNA. ResultsWe found that miR-186 directly inhibited CSE protein and mRNA expression through targeting CSE 3′UTR by bioinformatics analyses and dual-luciferase reporter assays. HPLC assays showed that miR-186 increased the lipid accumulation in human THP-1 macrophages. We also showed that miR-186 enhanced secretion of pro-inflammatory cytokines in human THP-1 macrophages. Using siRNA approach, we found that CSE siRNA could inhibit the miR-186 inhibitor-induced decrease in the expression of LPL protein and mRNA in human THP-1 macrophages, which was accompanied a decrease in the level of H2S. ConclusionsMicroRNA-186 promotes macrophage lipid accumulation and pro-inflammatory cytokine secretion by targeting cystathionine γ-lyase in THP-1 macrophages.

Runx2-Modified Adipose-Derived Stem Cells Promote Tendon Graft Integration in Anterior Cruciate Ligament Reconstruction.

Runx2 is a powerful osteo-inductive factor and adipose-derived stem cells (ADSCs) are multipotent. However, it is unknown whether Runx2-overexpressing ADSCs (Runx2-ADSCs) could promote anterior cruciate ligament (ACL) reconstruction. We evaluated the effect of Runx2-ADSCs on ACL reconstruction in vitro and in vivo. mRNA expressions of osteocalcin (OCN), bone sialoprotein (BSP) and collagen I (COLI) increased over time in Runx2-ADSCs. Runx2 overexpression inhibited LPL and PPARγ mRNA expressions. Runx2 induced alkaline phosphatase activity markedly. In nude mice injected with Runx2-ADSCs, promoted bone formation was detected by X-rays 8 weeks after injection. The healing of tendon-to-bone in a rabbit model of ACL reconstruction treated with Runx2-ADSCs, fibrin glue only and an RNAi targeting Runx2, was evaluated with CT 3D reconstruction, histological analysis and biomechanical methods. CT showed a greater degree of new bone formation around the bone tunnel in the group treated with Runx2-ADSCs compared with the fibrin glue group and RNAi Runx2 group. Histology showed that treatment with Runx2-ADSCs led to a rapid and significant increase at the tendon-to-bone compared with the control groups. Biomechanical tests demonstrated higher tendon pullout strength in the Runx2-ADSCs group at early time points. The healing of the attachment in ACL reconstruction was enhanced by Runx2-ADSCs.

Expression of Lipid Metabolism-Associated Genes in Male and Female White Feather Chicken.

Differential lipid metabolic requirements of sexually-mature males and females may influence the regulation of lipid metabolism-associated genes and hence the content of adipose tissue. We measured the expression of eight lipid metabolism-associated genes (fatty acid synthase, FASN; acylglycerol- 3- phosphate O-acyltransferase 9, AGPAT9; peroxisomal proliferator-activated receptor γ, PPARγ; lipoprotein lipase, LPL; carnitine palmitoyl transferase 1 A, CPT1A; carnitine palmitoyl transferase 1 B, CPT1B; acyl-COA dehydrogenase long chain, ACADL; monoglyceride lipase, MGL) in eight tissues (hypothalamus, HYP; liver; heart; pectoralis major muscle, PM; gastrocnemius muscle, GAS; abdominal fat, AF; clavicular fat, CF; subcutaneous fat, SF) of five male and five female white feather chickens using real time PCR at 217 d (when the females were at peak egg production). There were no difference between sexes, nor were there sex by tissue interactions for CPT1A and MGL. In both cases expression was greater for liver than the other tissues. When interactions of sex by tissue were significant, the FASN mRNA abundance in HYP, liver, and PM was greater for females than males. There was no sexual dimorphism for any tissue for PPARγ. Overall values were greater for adipose depots than HYP and liver with muscles intermediate for AGPAT9. LPL mRNA abundance in PM and AF was greater for females than males, with the pattern reversed for heart and SF. CPT1B mRNA abundance in GAS and CF was greater for females than males, with the relationship reversed for liver. ACADL mRNA abundance in HYP, liver, and GAS was greater for females than males, and lower in PM than males. The results demonstrated that expression of lipid metablism–associated genes varies among sexes in mature chickens depending on the gene and the tissue.

Abstract 13566: A Novel New Chemical Entity, Gemcabene, Shows Significant Lipid Regulation in PPAR-α Knock-out Mice, Supporting a Mechanism Independent of PPAR-α

Background: In vitro and cell-based assays with a lipid lowering agent, gemcabene (Gem), ruled out a direct activation of PPAR-α, although some PPAR-α target genes were modulated in vivo. Therefore, PPAR-α may not be required for the in vivo lipid lowering efficacy of Gem. Experiment & Results: WT and PPAR-α KO mice were administered Gem (0.3%), Wy14643 (0.1%), a reference PPAR-α agonist, and a clinical lipid regulator, Gemfibrozil (0.3%) as diet admixtures for 8 days. PPAR-α KO mice exhibited a phenotype consistent with absence of PPAR-α, such as increased plasma triglycerides (TG), VLDL-C, LDL-C, HDL-C, and apoCIII. Hepatic apoCIII mRNA (negatively regulated by PPAR-α) showed upregulation in PPAR-α KO mice compared to WT. In WT mice, there was no change in liver to body weight ratio in the Gem-treated group, but a marked increase (212%) in the Wy14643-treated-group and marginal increase (22%) in gemfibrozil-treated mice. In WT, hepatomegaly was observed only in the Wy14643-treated group, which was associated with peroxisome proliferation, and a 20-fold increase in the PPAR-α target gene, acyl CoA oxidase (ACO). Absence of hepatomegaly in the Wy14643-treated PPAR-α KO mice and no change in ACO mRNA supported PPAR-α mediated effect of Wy14643. In WT, Gem and gemfibrozil showed a 3-fold increase in ACO mRNA. Gem lowered VLDL-C (-47%), LDL-C (-22%), and TG (-46%) in PPAR-α KO mice, suggesting a PPAR-α independent effect on circulating lipids. Wy14643 and Gem significantly reduced apoCIII by -39% and -24% in WT, and -20% and -16% in PPAR-α KO mice, respectively. ApoCIII mRNA was reduced -12% (NS) in Gem-treated and significantly by -46% in Wy14643-treated WT-mice. In KO mice, apoCIII mRNA was reduced by -8% (NS) by Gem and significantly by -20% by Wy14643. ApoCIII mRNA is a direct target of PPAR-α while plasma apoCIII level is influenced by several factors. Wy14643 induced hepatic LPL mRNA in WT, but not in PPAR-α KO mice. Interestingly, hepatic PPARγ expression showed increased level in the PPARα KO mice, suggesting the regulation of the two isoforms may be interdependent. Conclusions: Taken together, these studies demonstrate that the lipid lowering efficacy of Gem occurs in a PPAR-α -independent manner in vivo.

Xenobiotic-contaminated diets affect hepatic lipid metabolism: Implications for liver steatosis in Sparus aurata juveniles

The metabolic effects induced by feed contaminated with a lower or a higher concentration of -nonylpnenol (NP), 4-tert-octylphenol (t-OP) or bisphenol A (BPA), three environmental endocrine disruptors, were assessed in juvenile sea bream liver. Histological analysis demonstrated that all these three xenobiotics induced hepatic lipid accumulation and steatosis. These findings prompted analysis of the expression of the major molecules involved in lipid metabolism: peroxisome proliferator activated receptors (which is encoded by ppars), fatty acid synthase (encoded by fas), lipoprotein lipase (encoded by lpl) and hormone-sensitive lipase (encoded by hsl). The enzymes encoded by ppars and fas are in fact responsible for lipid accumulation, whereas lpl- and hsl- encoded proteins play a pivotal role in fat mobilization. The three xenobiotics modulated ppar mRNA expression: pparα mRNA expression was induced by the higher dose of each contaminant; pparβ mRNA expression was upregulated by the lower doses and in BPA2 fish ppary mRNA overexpression was induced by all pollutants. These data agreed with the lipid accumulation profiles documented by histology. Fas mRNA levels were modulated by the two NP doses and the higher BPA concentration. Lpl mRNA was significantly upregulated in all experimental groups except for BPA1 fish while hsl mRNA was significantly downregulated in all groups except for t-OP2 and BPA1 fish. The plasma concentrations of cortisol, the primary stress biomarker, were correlated with the levels of pepck mRNA level. This gene encodes phosphoenolpyruvate carboxykinase which is one of the key enzymes of gluconeogenesis. Pepck mRNA was significantly overexpressed in fish exposed to NP2 and both t-OP doses. Finally, the genes encoding cyclooxygenase 2 (cox2) and 5-lipoxygenase (5 lox), the products of which are involved in the inflammatory response, transcriptions were significantly upregulated in NP and BPA fish, whereas they were unchanged in t-OP specimens. The present findings suggest that dietary xenobiotic contamination can give rise to metabolic disorders also in fish and highlight the potential for their vertical transfer through the trophic levels and ultimately to humans.

Offspring subcutaneous adipose markers are sensitive to the timing of maternal gestational weight gain.

BackgroundExcessive maternal weight gain during pregnancy impacts on offspring health. This study focused on the timing of maternal gestational weight gain, using a porcine model with mothers of normal pre-pregnancy weight.MethodsTrial design ensured the trajectory of maternal gestational weight gain differed across treatments in early, mid and late gestation. Diet composition did not differ. On day 25 gestation, sows were assigned to one of five treatments: Control sows received a standard gestation diet of 2.3 kg/day (30 MJ DE/day) from early to late gestation (day 25–110 gestation). E sows received 4.6 kg food/day in early gestation (day 25–50 gestation). M sows doubled their food intake in mid gestation (day 50–80 gestation). EM sows doubled their food intake during both early and mid gestation (day 25–80 gestation). L sows consumed 3.5 kg food/day in late gestation (day 80–110 gestation). Offspring body weight and food intake levels were measured from birth to adolescence. Markers of lipid metabolism, hypertrophy and inflammation were investigated in subcutaneous adipose tissue of adolescent offspring.ResultsThe trajectory of gestational weight gain differed across treatments. However total gestational weight gain did not differ except for EM sows who were the heaviest and fattest mothers at parturition. Offspring birth weight did not differ across treatments. Subcutaneous adipose tissue from EM offspring differed significantly from controls, with elevated mRNA levels of lipogenic (CD36, ACACB and LPL), nutrient transporters (FABP4 and GLUT4), lipolysis (HSL and ATGL), adipocyte size (MEST) and inflammation (PAI-1) indicators. The subcutaneous adipose depot from L offspring exhibited elevated levels of CD36, ACACB, LPL, GLUT4 and FABP4 mRNA transcripts compared to control offspring.ConclusionsIncreasing gestational weight gain in early gestation had the greatest impact on offspring postnatal growth rate. Increasing maternal food allowance in late gestation appeared to shift the offspring adipocyte focus towards accumulation of fat. Mothers who gained the most weight during gestation (EM mothers) gave birth to offspring whose subcutaneous adipose tissue, at adolescence, appeared hyperactive compared to controls. This study concluded that mothers, who gained more than the recommended weight gain in mid and late gestation, put their offspring adipose tissue at risk of dysfunction.

The effect of long term under- and over-feeding on the expression of genes related to lipid metabolism in the mammary tissue of goats

Fat synthesis is under the control of a large number of genes whose nutritional regulation is still poorly documented. In this study, we examined the effect of long term under- and over-feeding on the expression of genes (acetyl-CoA carboxylase: ACACA, fatty acid synthetase: FASN, lipoprotein lipase: LPL, stearoyl-Co A desaturase 1: SCD1, peroxisome proliferator activated receptor G2: PPARG2, sterol regulatory element binding protein-1c: SREBP-1c and hormone sensitive lipase HSL) related to FA metabolism in goat mammary tissue (MT). Twenty four lactating goats were divided into three homogenous sub-groups and fed the same ratio in quantities covering 70% (underfeeding), 100% (control) and 130% (overfeeding) of their energy and crude protein requirements. The results showed a significant reduction on mRNA of ACACA, FASN, LPL, SCD1 and HSL in the MT of underfed goats, compared with the respective overfed. A numerical decrease on the mRNA level of PPARG2 and SREBP-1c in the MT of the underfed goats, compared with the respective controls and the overfed was also observed. In conclusion, the negative, compared to positive, energy balance in goats down regulates the ACACA, FASN, LPL, SCD1 and HSL expressions in their MT which indicates that the decrease in nutrient availability may lead to lower rates of lipid synthesis.

The effect of long term under- and over-feeding on the expression of genes related to lipid metabolism in mammary tissue of sheep.

Milk fatty acid (FA) synthesis by the mammary gland involves expression of a large number of genes whose nutritional regulation remains poorly defined. In this study, we examined the effect of long-term under- and over-feeding on the expression of genes (acetyl Co A carboxylase, ACC; fatty acid synthetase, FAS; lipoprotein lipase, LPL; stearoyl Co A desaturase, SCD; peroxisome proliferator activated receptor γ2, PPARγ2; sterol regulatory element binding protein-1, SREBP-1c; and hormone sensitive lipase, HSL) related to FA metabolism in sheep mammary tissue (MT). Twenty-four lactating sheep were divided into three homogenous sub-groups and fed the same ration in quantities covering 70% (underfeeding), 100% (control) and 130% (overfeeding) of their energy and crude protein requirements. The results showed a significant reduction of mRNA of ACC, FAS, LPL and SCD in the MT of underfed sheep, and a significant increase on the mRNA of LPL and SREBP-1c in the MT of overfed compared with the control respectively. In conclusion, the negative, compared to positive, energy balance in sheep down-regulates ACC, FAS, LPL, SCD, SREBP-1c and PPARγ2 expression in their MT which indicates that the decrease in nutrient availability may lead to lower rates of lipid synthesis.

Dietary Calcium Deficiency and Excess Both Impact Bone Development and Mesenchymal Stem Cell Lineage Priming in Neonatal Piglets

Background: Optimizing calcium nutrition to maximize bone accretion during growth to prevent fragility fractures later in life has spurred greater interest in calcium nutrition in neonates.Objective: The aim of this study was to determine the effect of dietary calcium, from deficiency through excess, on bone growth, and the in vivo and in vitro behavior of mesenchymal stem cells (MSCs) in neonatal pigs.Methods: Twenty-four male and female piglets (24 ± 6 h old) were fed either a calcium-deficient [Ca-D; 0.6% Ca on a dry matter (DM) basis], a calcium-adequate diet (Ca-A; 0.9% Ca on a DM basis), or a calcium-excessive diet (Ca-E; 1.3% Ca on a DM basis) for 14 d to assess the impact of dietary calcium on calcium homeostasis and on the behavior of MSCs.Results: Growth rate was not affected by the Ca-E diet, although bone ash content was 16% higher (P < 0.05) and urinary calcium excretion was 5-fold higher, when normalized to creatinine, compared with the Ca-A group at trial completion. Serum parathyroid hormone (PTH) concentrations were elevated (P < 0.05) in Ca-D piglets in comparison with other groups at both 7 and 14 d. In vivo proliferation of MSCs was 30% higher (P < 0.05) in Ca-E piglets than the other groups. MSCs from both Ca-D– and Ca-E–fed piglets had greater adipogenic potential based on increased gene expression (P < 0.05) of peroxisome proliferator-activated receptor γ (Pparg) and adipocyte fatty acid–binding protein (Ap2) than MSCs from Ca-A piglets. Interestingly, only MSCs from Ca-E–fed piglets had greater (P < 0.05) gene expression of lipoprotein lipase (Lpl) during adipocytic differentiation than those from Ca-A piglets. To assess alterations in lineage allocation and priming, the most and least osteogenic (O+ and O−, respectively) and adipogenic (A+ and A−, respectively) colonies from each MSC isolation were selected on the basis of functional staining. The O+ colonies from Ca-D piglets expressed lower (P < 0.05) levels of osteocalcin (OC) mRNA than did those from other groups, whereas the O− colonies from Ca-E piglets expressed higher (P < 0.05) levels of mRNA of Pparg, Ap2, and Lpl than did those from other groups.Conclusions: Neonatal calcium deficiency appears to reduce the osteogenic priming of MSCs while enlarging a subpopulation of potentially adipogenic cells, and excess dietary calcium appears to allow greater multipotency of MSCs. These programming alterations of MSCs could have long-term consequences for bone health.

Gestational stage affects amniotic epithelial cells phenotype, methylation status, immunomodulatory and stemness properties.

Stem cells isolated from amniotic epithelium (AECs) have shown great potential in cell-based regenerative therapies. Because of their fetal origin, these cells exhibit elevated proliferation rates and plasticity, as well as, immune tolerance and anti-inflammatory properties. These inherent attitudes make AECs well-suited for both allogenic and xenogenic cellular transplants in animal models. Since in human only at term amnion is easily obtainable after childbirth, limited information are so far available concerning the phenotypic and functional difference between AECs isolated from early and late amnia. To this regard, the sheep animal model offers an undoubted advantage in allowing the easy collection of both types of AECs in large quantity. The aim of this study was to determine the effect of gestational age on ovine AECs (oAECs) phenotype, immunomodulatory properties, global DNA methylation status and pluripotent differentiation ability towards mesodermic and ectodermic lineages. The immunomodulatory property of oAECs in inhibiting lymphocyte proliferation was mainly unaffected by gestational age. Conversely, gestation considerably affected the expression of surface markers, as well the expression and localization of pluripotency markers. In detail, with progression of gestation the mRNA expression of NANOG and SOX2 markers was reduced, while the ones of TERT and OCT4A was unaltered; but at the end of gestation NANOG, SOX2 and TERT proteins mainly localized outside the nuclear compartment. Regarding the differentiation ability, LPL (adipogenic-specific gene) mRNA content significantly increased in oAECs isolated from early amnia, while OCN (osteogenic-specific gene) and NEFM (neurogenic-specific gene) mRNA content significantly increased in oAECs isolated from late amnia, suggesting that gestational stage affected cell plasticity. Finally, the degree of global DNA methylation increased with gestational age. All these results indicate that gestational age is a key factor capable of influencing morphological and functional properties of oAECs, and thus probably affecting the outcome of cell transplantation therapies.

Combinatorial regulation of lipoprotein lipase by microRNAs during mouse adipogenesis

MicroRNAs (miRNAs) regulate gene expression directly through base pairing to their targets or indirectly through participating in multi-scale regulatory networks. Often miRNAs take part in feed-forward motifs where a miRNA and a transcription factor act on shared targets to achieve accurate regulation of processes such as cell differentiation. Here we show that the expression levels of miR-27a and miR-29a inversely correlate with the mRNA levels of lipoprotein lipase (Lpl), their predicted combinatorial target, and its key transcriptional regulator peroxisome proliferator-activated receptor gamma (Pparg) during 3T3-L1 adipocyte differentiation. More importantly, we show that Lpl, a key lipogenic enzyme, can be negatively regulated by the two miRNA families in a combinatorial fashion on the mRNA and functional level in maturing adipocytes. This regulation is mediated through the Lpl 3′UTR as confirmed by reporter gene assays. In addition, a small mathematical model captures the dynamics of this feed-forward motif and predicts the changes in Lpl mRNA levels upon network perturbations. The obtained results might offer an explanation to the dysregulation of LPL in diabetic conditions and could be extended to quantitative modeling of regulation of other metabolic genes under similar regulatory network motifs.

Effects of small interfering RNA-mediated hepatic glucagon receptor inhibition on lipid metabolism in db/db mice

Hepatic glucose overproduction is a major characteristic of type 2 diabetes. Because glucagon is a key regulator for glucose homeostasis, antagonizing the glucagon receptor (GCGR) is a possible therapeutic strategy for the treatment of diabetes mellitus. To study the effect of hepatic GCGR inhibition on the regulation of lipid metabolism, we generated siRNA-mediated GCGR knockdown (si-GCGR) in the db/db mouse. The hepatic knockdown of GCGR markedly reduced plasma glucose levels; however, total plasma cholesterol was increased. The detailed lipid analysis showed an increase in the LDL fraction, and no change in VLDL HDL fractions. Further studies showed that the increase in LDL was the result of over-expression of hepatic lipogenic genes and elevated de novo lipid synthesis. Inhibition of hepatic glucagon signaling via siRNA-mediated GCGR knockdown had an effect on both glucose and lipid metabolism in db/db mice.

Downregulated Lipid Metabolism in Differentiated Murine Adipocytes by Procyanidins from Defatted Grape Seed Meal

Recent reports have proposed possible anti-obesity mechanisms for antioxidants involving increased energy expenditure, pre-adipocyte differentiation and proliferation, decreased lipogenesis and increased lipolysis, and fat oxidation. The aim of this study was to examine and to confirm the anti-obesity effect of the oligomeric and polymeric procyanidin fractions from defatted grape seeds. The lipid metabolism-related mRNA level in the mouse preadipocytes, 3T3-L1 cells, was determined to evaluate the anti-obesity effect of the phenolic fractions from a grape seed meal. Lipid accumulation was reduced by 19% of the control level by the procyanidin fraction originating from the grape seed meal. Emerging from the effect of the treatment on HSL and LPL mRNA expression, lipolytic enzyme activity was not involved in the anti-obesity effects of CPE and FPP from the defatted grape seed meal. We tested and confirmed in this study the effect of the biological activities of oligomeric and polymeric procyanidins from the defatted grape seed meal. It is suggested from the results of this brief study that further studies would be desirable to focus on the anti-obesity effect of the purified extracts of a defatted grape seed meal.

Effect of a five-week high-fat diet on serum-lipid composition and LPL mRNA expression in rat skeletal muscles

Effect of a five-week high-fat diet on serum-lipid composition and LPL mRNA expression in rat skeletal muscles

DNA methylation variations at CETP and LPL gene promoter loci: New molecular biomarkers associated with blood lipid profile variability

BackgroundRecent findings suggest that DNA methylation, a well-known epigenetic mechanism, is involved in high-density lipoprotein cholesterol (HDL-C) metabolism and increased cardiovascular disease risk. The aim of this study was thus to assess whether DNA methylation within key genes of lipoprotein metabolism is associated with blood lipid profile variability. Methods and resultsNinety-eight untreated familial hypercholesterolaemia patients (61 men and 37 women) were recruited for leucocyte DNA methylation analyses at the LDLR, CETP, LCAT and LPL gene promoter loci using bisulfite pyrosequencing. LPL DNA methylation was correlated with HDL-C (r = 0.22; p = 0.031) and HDL particle size (r = 0.47, p = 0.013). In both sex, CETP DNA methylation was negatively associated with low-density lipoprotein cholesterol levels (r < −0.32; p < 0.05). In men, CETP DNA methylation was associated with HDL-C (r = −0.36; p = 0.006), HDL-triglyceride levels (r = 0.59; p < 0.001) and HDL particle size (r = −0.44, p = 0.019). In visceral adipose tissue from 30 men with severe obesity, the associations between LPL DNA methylation, HDL-C (r = −0.40; p = 0.03) and LPL mRNA levels (r = −0.61, p < 0.001) were confirmed. ConclusionCETP and LPL DNA methylation levels are associated with blood lipid profile, suggesting that further studies of epipolymorphisms should most certainly contribute to a better understanding of the molecular bases of dyslipidemia.

Korean pine nut oil decreases the amount of white adipose tissue by affecting lipid metabolism in C57BL/6 mice

Korean pine nut oil (PNO) has been reported to reduce weight gain and the amount of white adipose tissue. Effects of PNO on lipid metabolism were investigated to understand the mechanisms of decrease in amount of white adipose tissue. Five‐wk‐old C57BL/6 mice were fed control diets containing 10% kcal fat from PNO or soybean oil (SBO) (PC or SC) or high‐fat diets containing 35% kcal fat from lard and 10% kcal fat from PNO or SBO (PHF or SHF) for 12 weeks. Expression levels of jejunal Cd36, Ifabp, Dgat2, and Apoa4, and epididymal Lpl, Plin1, Ucp2, Adrb3, Pparg, Ppargc1a, and Ppard were determined by Real‐time PCR. There was no difference in daily food intake between SBO‐ and PNO‐fed mice. Overall, PNO‐fed mice gained less weight (P = 0.014) and had less white adipose tissue (P = 0.004). PC and PHF groups had significantly less amount of white adipose tissue compared with SC group (30% less, P = 0.046) and SHF group (18% less, P = 0.033), respectively. Overall, PNO downregulated the mRNA expression of Cd36 (P = 0.025) and Lpl (P = 0.020). PC group had significantly lower Lpl mRNA expression (0.62‐fold, P = 0.035) than SC group. Overall, PNO tended to downregulate the Apoa4 mRNA expression (P = 0.068) and upregulate Adrb3 mRNA expression (P = 0.081). These results indicate that PNO is less efficiently absorbed, utilized, and stored in the body than SBO, which leads to less accumulation of fat mass in C57BL/6 mice.Supported by the Korea Research Foundation grant funded by the Korea government (KRF‐2010–0024878)

Molecular Cloning and Transcript Expression of Genes Encoding Two Types of Lipoprotein Lipase in the Ovary of Cutthroat Trout,Oncorhynchus clarki

Large amounts of neutral lipids (NLs) are stored as lipid droplets in the ooplasm of fish oocytes, providing an essential energy resource for developing embryos and larvae. However, little is known about the origin of such lipids or about mechanisms underlying their uptake and accumulation in oocytes. We have proposed a model for this lipidation of teleost oocytes, as follows: very low density lipoprotein (Vldl) is metabolized by lipoprotein lipase (Lpl) outside and/or inside of the oocyte and the resulting fatty acids (FAs) are then utilized for de novo biosynthesis of NLs. As a first step toward verification of this model, cDNAs for genes encoding two types of Lpl, lpl and lpl2, were cloned from the ovary of cutthroat trout, Oncorhynchus clarki. Examination of Lpl polypeptide sequences deduced from the cDNAs revealed features similar to LPLs/Lpls in other species, including several conserved structural and functional domains. Both types of lpl mRNA were highly expressed in lipid storage tissues (e.g., adipose tissue, muscle, and ovary) and were predominantly expressed in the granulosa cells of ovarian follicles. Ovarian lpl1 mRNA levels showed a remarkable peak in April (early oocyte lipid droplet stage) and then decreased to low values sustained until November (mid-vitellogenesis), after which time a small peak in lpl1 gene expression was observed in December (late vitellogenesis). The mRNA levels of lpl2 also were elevated in April and were highest in June (late lipid droplet stage), but did not show other pronounced changes. These results suggest that, in the cutthroat trout, Vldl is metabolized by the action of Lpls in the granulosa cell layer to generate free FAs for uptake and biosynthesis of neutral lipids by growing oocytes.

A Novel Role of NF-YA Transcription Factor in in Vitro Adipocyte Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

AbstractAbstract 1250The CCAAT-binding transcription factor NF-Y, consists of three subunits, NF-YA, NF-YB and NF-YC, all necessary for DNA binding. NF-YA is characterized as the regulatory subunit of the complex because of its differential expression during cell cycle. NF-YA appears to have a complex role in hematopoietic stem cell (HSC) biology. So far, however, there are no available data on the role of NF-Y in mesenchymal stem cell (MSC) biology. The aim of this study is the evaluation of NF-YA expression levels during the ex vivo expansion of human bone marrow (BM)-MSCs and the assessment of its possible role for the BM-MSC differentiation process towards the osteoblastic and adipocytic lineages.BM-MSCs were isolated from posterior iliac crest aspirates of hematologically healthy individuals undergoing orthopedic surgery after written informed consent. BM-MSCs were ex vivo expanded until passage 10 (P10). Total RNA was isolated from BM-MSCs at P2 and P10 for the evaluation of NF-YA mRNA levels. P3 BM-MSCs’ ex vivo differentiation into adipocytes and osteoblasts was induced using the appropriate culture media. Mineralization was evidenced via Alizarin Red and Von Kosa staining and lipid droplets were revealed with Oil Red O staining. Total RNA and proteins were isolated from undifferentiated and differentiated BM-MSCs at various time-points. Protein levels of NF-YA were immunodetected by Western blot. The relative gene expression of NF-YA, as well as that of lineage-specific markers was evaluated by real-time PCR. All PCR results are expressed as 2−ΔCt. Adipocytic and osteoblastic differentiation of BM-MSCs was also induced following knockdown of NF-YA expression after BM-MSC transduction with lentiviral particles carrying either shRNA against NF-YA or nonsense shRNA. Total RNA was isolated from transduced BM-MSCs at several time-points during differentiation.A statistically significant (p=0.04) reduction in the mRNA levels of NF-YA was found in P10, as compared to P2 BM-MSC cultures (n=12).During adipogenic differentiation, NF-YA significantly increased (p=0.0478) at day 16, as compared to the onset of differentiation induction (day 0), (n=14). Similarly, protein levels of NF-YA also increased during adipogenesis Furthermore, NF-YA expression levels strongly correlated with the mRNA levels of adipogenesis-associated genes PPARG (r=0.74, p<0.0001), C/EBPA (r=0.76, p<0.0001) and LPL (r=0.74, p<0.0001). In contrast, during osteogenic differentiation no statistically significant change was detected in NF-YA mRNA or protein expression. (n=9). In order to evaluate if NF-YA is functionally related to adipogenic and osteogenenic differentiation of BM-MSCs, NF-YA knockdown was performed and adipocytic as well as osteoblastic differentiations were induced. Throughout adipogenesis, the relative mRNA levels of NF-YA in shNF-YA-transduced BM-MSCs were reduced to 51.03% compared with control (non-transduced) cells. At day 21 of adipogenesis, a reduction of 31.22%, 70.06% and 74.3% was also observed in PPARG, C/EBPA and LPL mRNA levels, respectively. In addition, Oil Red O staining indicated a lack of lipid droplets in shNF-YA-transduced BM-MSCs compared with control cells. During osteogenesis, NF-YA mRNA levels in shNF-YA-transduced BM-MSCs were reduced to 52.37% compared to control cells. At day 21 of differentiation the mRNA levels of the osteogenesis- associated genes DLX5, RUNX2, OSX and ALP remained unaffected in shNF-YA-transduced cells compared to control cells. Furthermore, Alizarin Red and Von Kossa staining did not reveal any significant difference between shNF-YA-transduced BM-MSCs and control cells.In conclusion we have shown for the first time, that BM-MSCs display decreased NF-YA mRNA levels after prolonged ex vivo expansion, a finding that might be associated with MSCs' senescence and/or loss of stemness., We have also shown that NF-YA does not have any major effect on BM-MSCs' osteogenic differentiation but displays a significant role in their adipogenic differentiation as was shown by (a) the increased NF-YA mRNA and protein levels during the ex vivo adipogenesis, (b) the positive correlation between the NF-YA mRNA expression levels and the adipocyte-associated genes PPARG, C/EBPA and LPL and (c) the impaired adipogenic differentiation following NF-YA knock down. Collectively our results imply a novel role for NF-YA in BM-MSCs' biology Disclosures:No relevant conflicts of interest to declare.

Comparative analysis of mesenchymal stem cells from adult mouse adipose, muscle, and fetal muscle

Recently, increasing evidence supports that adult stem cells are the part of a natural system for tissue growth and repair. This study focused on the differences of mesenchymal stem cells from adult adipose (ADSCs), skeletal muscle (MDSCs) and fetal muscle (FMSCs) in biological characteristics, which is the key to cell therapy success. Stem cell antigen 1 (Sca-1) expression of MDSCs and FMSCs at passage 3 was two times more than that at passage 1 (P<0.0001). After 28-day myogenic induction, higher expression levels of skeletal muscle-specific genes were observed in MDSCs than FMSCs (P<0.01), and the lowest expression levels were demonstrated in ADSCs among three cells (P<0.01). Besides, M-Cad and MyHC expressions in ADSCs were not detected by immunofluorescence or real-time quantitative PCR. Furthermore, after 14days adipogenic induction, PPARγ2, LPL and aP2 mRNA expressions were higher in ADSCs vs. MDSCs (P<0.01). Besides, MSCs from adult or fetal muscle expressed higher OCN and OPN than ADSCs after 28days osteogenic induction (P<0.01). Taken together, our results suggested that cell source and developmental stage had great impacts on biological properties of mesenchymal stem cells, and proper consideration of all the issues is necessary.

Effects of reproductive stage and 11-ketotestosterone on LPL mRNA levels in the ovary of the shortfinned eel

To understand the dynamics of lipid uptake into the ovary and the potential role that lipoprotein lipase plays in this event, changes in LPL transcript abundance during oogenesis were measured in both wild-caught and pituitary homogenate-induced artificially maturing eels. Also, the effects of 11-ketotestosterone (11-KT) on LPL mRNA levels were investigated in vivo and in vitro. Normalized ovarian LPL transcript abundance increased as oogenesis advanced, and it rose particularly rapidly during midvitellogenesis, corresponding to pronounced increases in ovarian lipid deposits and LPL activity. Furthermore, LPL mRNA levels were dramatically increased following 11-KT treatment in vivo, findings that were reinforced as trends in ovarian tissue incubated in vitro. Ovarian LPL appears to be directly involved in the uptake of lipids into the eel ovary, an involvement that appears to be controlled, at least in part, by the androgen 11-KT.