Affect Lipid Accumulation Research Articles

MYOSTATIN COORDINATING THE PROLIFERATION AND DIFFERENTIATION OF ADIPOSE AND SKELETAL MUSCLE CELLS AND ENERGY METABOLISM BALANCE BASED ON GENE CHIP TECHNOLOGY

ABSTRACT Myoblasts fuse into multinucleated muscle fibers to form and promote the growth of skeletal muscle. In order to analyze the role of myostatin (MSTN) in body fat, skeletal muscle cell proliferation and differentiation and energy metabolism, this study will use the antisense RNA technology of gene chip technology to study it. The results showed that the MSTN gene regulated the growth and proliferation of myoblasts and affected the development of skeletal muscle by affecting the expression of Cdc42, bnip2, p38 and other genes; knockout or overexpression of the MSTN gene would lead to a trend of fat-related genes from fat synthesis to fat decomposition; after the MSTN gene was knocked down, the expression levels of cpti-b, PPARG and other genes in the cells were corresponding after MSTN overexpression, the relative expression of the PPARG gene decreased. It is suggested that the knockout or overexpression of MSTN may affect lipid accumulation, and cpti-b and PPARG may directly regulate lipid level. It is hoped that this experiment can provide a reference for the study of MSTN effect on fat deposition.

Royal Jelly Proteins Inhibit Macrophage Proliferation: Interactions with Native- and Oxidized-Low Density Lipoprotein.

Macrophage proliferation is known to correlate with macrophage accumulation in atherosclerotic plaque, and therefore its inhibition and secondary reduction of plaque inflammation may have therapeutic beneficial effects on atherosclerosis. Recently, we reported that a peptide corresponding to positions 41-51 of royalisin (which consists of 51 amino acid residues), a potent antibacterial protein contained in royal jelly (RJ), can specifically bind to oxidized LDL (Ox-LDL), a major components of atherosclerotic lesions. Here, we investigated the interaction of RJ proteins including royalisin with LDL and Ox-LDL. Measurement of LDL oxidation bythe production ofthiobarbituric acid reactive substances and conjugated dienes, and by electrophoretic mobility on polyacrylamide gel electrophoresis showed that RJ proteins including royalisin and the degradation products of major RJ protein (MRJP) 1 and MRJP3 can induce oxidation of LDL and Ox-LDL. Surface plasmon resonance experiments showed that these RJ proteins can exhibit much higher binding affinity to LDL than Ox-LDL (the equilibrium dissociation constant, KD = 8.35 and 49.65μg proteins/mL for LDL and Ox-LDL, respectively). Experiments using cultured mouse J774A.1 macrophage cells proved that these RJ proteins can inhibit macrophage proliferation markedly and concentration-dependently, regardless of the absence or presence of LDL and Ox-LDL, but hardly affect lipid accumulation in macrophages. These results suggest that RJ proteins including royalisin and degradation products of MRJP1/MRJP3 may have therapeutic beneficial effects on atherosclerosis owing to the reduction of plaque inflammation. Further studies of these RJ proteins may lead to the discovery of novel anti-atherosclerotic drugs.

Alpha-Tocopherol supplementation reduces inflammation and apoptosis in high cholesterol mediated nonalcoholic steatohepatitis.

Inflammation and apoptosis signaling are crucial steps in the progression from nonalcoholic fatty liver disease (NAFLD) to nonalcoholic steatohepatitis (NASH). Alpha-tocopherol, the most active form of vitamin E, is an important modulator of signaling mechanisms, but its involvement to cholesterol-induced NASH pathogenesis remains poorly defined. Herein we have reported a novel effect of α-tocopherol in the transition from hepatic steatosis to NASH. High cholesterol diet alone (without α-tocopherol) in rabbits elevated NASH development as indicated by increased inflammatory response, apoptotic activity and liver fibrosis. Such elevation results from induction of signaling mechanisms since the expressions of IL1β, phospho c-Jun/c-Jun ratio, JNK, caspase 9, CHOP and Bax were increased, and recruitment of macrophage, α-smooth muscle actin (α-SMA) and COL1A1 into the liver tissue were induced. Alpha-tocopherol supplementation inhibited inflammatory response, apoptosis and fibrosis development without affecting lipid accumulation in high cholesterol-induced NASH. Specifically, α-tocopherol lowered the inflammatory level as observed by reduced macrophage infiltration and JNK/c-Jun signaling. Lower inflammatory status co-occurred with the reduction of CHOP and Bax expressions as well as fibrosis-related COL1A1 and α-SMA levels. Taken together, α-tocopherol supplementation inhibits cholesterol-induced NASH development by lowering JNK/c-Jun/inflammation axis in addition to JNK/CHOP/apoptosis signaling, which might contribute to resistance against NAFLD/NASH transition.

Comprehensive evaluation of the metabolic effects of porcine CRTC3 overexpression on subcutaneous adipocytes with metabolomic and transcriptomic analyses

BackgroundMeat quality is largely driven by fat deposition, which is regulated by several genes and signaling pathways. The cyclic adenosine monophosphate (cAMP) -regulated transcriptional coactivator 3 (CRTC3) is a coactivator of cAMP response element binding protein (CREB) that mediates the function of protein kinase A (PKA) signaling pathway and is involved in various biological processes including lipid and energy metabolism. However, the effects of CRTC3 on the metabolome and transcriptome of porcine subcutaneous adipocytes have not been studied yet. Here, we tested whether porcine CRTC3 expression would be related to fat deposition in Heigai pigs (a local fatty breed in China) and Duroc×Landrace×Yorkshire (DLY, a lean breed) pigs in vivo. The effects of adenovirus-induced CRTC3 overexpression on the metabolomic and transcriptomic profiles of subcutaneous adipocytes were also determined in vitro by performing mass spectrometry-based metabolomics combined with RNA sequencing (RNA-seq).ResultsPorcine CRTC3 expression is associated with fat deposition in vivo. In addition, CRTC3 overexpression increased lipid accumulation and the expression of mature adipocyte-related genes in cultured porcine subcutaneous adipocytes. According to the metabolomic analysis, CRTC3 overexpression induced significant changes in adipocyte lipid, amino acid and nucleotide metabolites in vitro. The RNA-seq analysis suggested that CRTC3 overexpression alters the expression of genes and pathways involved in adipogenesis, fatty acid metabolism and glycerophospholipid metabolism in vitro.ConclusionsWe identified significant alterations in the metabolite composition and the expression of genes and pathways involved in lipid metabolism in CRTC3-overexpressing adipocytes. Our results suggest that CRTC3 might play an important regulatory role in lipid metabolism and thus affects lipid accumulation in porcine subcutaneous adipocytes.

Lipid Droplets Accumulation during Hepatitis C Virus Infection in Cell-Culture Varies among Genotype 1-3 Strains and Does Not Correlate with Virus Replication.

Liver steatosis is a common complication of chronic hepatitis C virus (HCV) infection, which can result in accelerated liver fibrosis development, especially in patients infected with genotype 3a. The precise mechanisms of HCV-induced liver steatosis remain unclear, but it is often posited that increased intracellular lipid accumulation is the underlying cause of steatosis. To study experimentally how HCV infection in human liver derived cells by different genotypes and subtypes might affect lipid accumulation, we performed detailed cytofluorimetric and microscopy analyses of intracellular lipid droplets (LDs) in relation to the viral Core and to cell endoplasmic reticulum proteins. Following culture infection with HCV genotype 1a, 2a, 2b, 2c, and 3a strains, we found variable levels of intracellular LDs accumulation, associated to the infecting strain rather than to the specific genotype. Although two genotype 3a strains showed high levels of lipid accumulation, as previously observed, some strains of other genotypes displayed a similar phenotype. Moreover, the analyses of LDs size, number, and shape indicated that the apparent increase in lipid accumulation is due to an increase in the overall number rather than in the size of droplets. Finally, differences in total lipid content across genotypes did not correlate to differences in Core distribution nor Core levels. In conclusion, our study provides a quantitative in-depth analysis of the effect of HCV infection on LDs accumulation in cell-culture.

Bisphenol A induces hepatic triglyceride level in adult male rare minnow Gobiocypris rarus

Bisphenol A (BPA), an endocrine disruptor, exist in almost all waters. In the present study, we expose adult male Gobiocypris rarus rare minnow to 15 μg/L BPA to study the effect BPA on fish hepatic lipid metabolism. Following 1, 3 and 5 weeks exposure, the liver tissue of rare minnow was separated. The change of the hepatic morphology, hepatosomatic index, lipid composition and expression of lipid metabolism related genes were analyzed through paraffin section, oil red O staining, lipidomic analysis, and quantitative real-time PCR. BPA can cause significant hepatic lipid deposition in male rare minnow, leading to an increase in triglyceride (TG) level (1.84–22.87-fold), but it is also accompanied by a decrease in diglyceride level (1.67–4.78-fold). The expression of lipid metabolism related genes showed that BPA exposure can up-regulate TG synthesis related genes expression, and down-regulate TG degradation genes expression. Expression of TG transport related genes were also disrupted by BPA. It suggests that BPA can up-regulate rare minnow hepatic TG level through multi-path, and ultimately lead to lipid accumulation in the liver. The results of the present study enrich the mechanisms of environmental endocrine disruptors affecting lipid accumulation in fish.

Maturation system affects lipid accumulation in bovine oocytes.

This study evaluated the effects of three maturation systems, namely invitro (MatV) and invivo (MatS) systems, as well as intrafollicular transfer of immature oocytes (IFIOT; MatT), on the accumulation of lipid droplets in bovine oocytes. Lipids were evaluated using confocal microscopy and transmission electron microscopy. The expression of genes related to lipid metabolism, namely acyl-CoA synthetase short chain family member 2 (ACSS2), ELOVL fatty acid elongase 1 (ELOVL1) and fatty acid binding protein 3 (FABP3), was quantified by quantitative polymerase chain reaction. The mean (±s.d.) area occupied by lipids in immature oocytes (13±2%) was similar to those matured invivo (MatS, 16±2%; MatT, 12±2%). However, there was a significant increase in lipids in oocytes in the MatV group (24±2%) compared with all other groups (P<0.001). In the ultrastructural evaluations, MatV oocytes also showed the highest lipid content. The expression of ELOVL1 and FABP3 was similar in the MatS and IFIOT groups. However, transcript levels of ACSS2 were lower in IFIOT than MatV oocytes. These results indicate, for the first time, that oocytes matured by IFIOT are similar to those matured invivo with regard to lipid accumulation, which indicates better quality than those matured invitro.

Exogenous and Endogenous Serine Deficiency Exacerbates Hepatic Lipid Accumulation.

Serine is involved in the regulation of hepatic lipid metabolism. However, whether exogenous or endogenous serine deficiency affects lipid accumulation in the liver and related mechanisms is unclear. Here, we investigated the effects of serine deficiency on hepatic fat accumulation in mice fed a serine-deficient diet or in mice supplemented with the D-3-phosphoglycerate dehydrogenase (PHGDH) inhibitor NCT-503. Both treatments produced an increase in body weight and liver weight and higher triglyceride content in the liver. Both treatments also exacerbated hepatic inflammatory responses and oxidative stress. Importantly, NCT-503 supplementation significantly inhibited PHGDH activity and decreased the serine content in the liver. Dietary serine deficiency significantly affected the colonic microbiota, characterized by a decreased ratio of Firmicutes/Bacteroidetes and decreased proportion of Bifidobacterium. Dietary serine deficiency additionally resulted in significantly decreased colonic and serum acetate and butyrate levels. The collective results indicate that NCT-503 supplementation may contribute to overaccumulation of hepatic lipid, by causing hepatic serine deficiency, while dietary serine deficiency may produce similar outcomes by affecting the gut-microbiota-liver axis.

3'-Hydroxydaidzein Improves Obesity Through the Induced Browning of Beige Adipose and Modulation of Gut Microbiota in Mice with Obesity Induced by a High-Fat Diet.

Obesity is an important health issue nowadays. 3'-Hydroxydaidzein (OHD) is a metabolite of daidzein (DAI) that can be found in fermented soybean products, such as miso. DAI has been known to affect lipid accumulation, but the effect of OHD on lipid accumulation still needs to be investigated. In this study, we investigated the effects of OHD on mice with obesity induced by a high-fat diet (HFD). The results showed that mice treated with 0.1% OHD (HOHD) significantly reduced their body weight and inguinal fat without altering their food intake compared with the HFD group. The HOHD and DAI groups' hyperlipidemia were alleviated through decreased serum triacylglycerols and total cholesterol levels. The adipocyte sizes in inguinal fat were significantly smaller in the HOHD and DAI groups compared with the HFD group. Both the HOHD and DAI groups had increased PRDM16, C/EBP β, p-p38, SIRT1, PGC1 α, and UCP1 protein expression in their inguinal adipose tissue compared with the HFD group. Moreover, the OHD and DAI groups had significantly lower amounts of Lachnospira and GCA_900066225 compared with the HFD group. Collectively, OHD can ameliorate HFD-induced obesity in mice by stimulating the browning of the white adipose tissue and modulating gut microbiota.

Effects of β-glucan Rich Barley Flour on Glucose and Lipid Metabolism in the Ileum, Liver, and Adipose Tissues of High-Fat Diet Induced-Obesity Model Male Mice Analyzed by DNA Microarray.

We evaluated whether intake of β-glucan-rich barley flour affects expression levels of genes related to glucose and lipid metabolism in the ileum, liver, and adipose tissues of mice fed a high-fat diet. C57BL/6J male mice were fed a high-fat diet supplemented with high β-glucan barley, for 92 days. We measured the expression levels of genes involved in glucose and lipid metabolism in the ileum, liver, and adipose tissues using DNA microarray and q-PCR. The concentration of short-chain fatty acids (SCFAs) in the cecum was analyzed by GC/MS. The metabolic syndrome indices were improved by barley flour intake. Microarray analysis showed that the expression of genes related to steroid synthesis was consistently decreased in the liver and adipose tissues. The expression of genes involved in glucose metabolism did not change in these organs. In liver, a negative correlation was showed between some SCFAs and the expression levels of mRNA related to lipid synthesis and degradation. Barley flour affects lipid metabolism at the gene expression levels in both liver and adipose tissues. We suggest that SCFAs are associated with changes in the expression levels of genes related to lipid metabolism in the liver and adipose tissues, which affect lipid accumulation.

The native acyltransferase-coding genes DGA1 and DGA2 affect lipid accumulation in Blastobotrys raffinosifermentans differently when overexpressed.

Blastobotrys raffinosifermentans is an ascomycetous yeast with biotechnological applications, recently shown to be an oleaginous yeast accumulating lipids under nitrogen limitation. Diacylglycerol acyltransferases (DGATs) act in the lipid storage pathway, in the last step of triacylglycerol biosynthesis. Two DGAT families are widespread in eukaryotes. We first checked that B. raffinosifermentans strain LS3 possessed both types of DGAT, and we then overexpressed the native DGAT-encoding genes, DGA1 and DGA2, separately or together. DGA2 (from the DGAT1 family) overexpression was sufficient to increase lipid content significantly in LS3, to up to 26.5% of dry cell weight (DCW), 1.6 times the lipid content of the parental strain (16.90% of DCW) in glucose medium under nitrogen limitation. By contrast, DGA1 (of the DGAT2 type) overexpression led to a large increase (up to 140-fold) in the amount of the corresponding transcript, but had no effect on overall lipid content relative to the parental strain. Analysis of the expression of the native genes over time in the parental strain revealed that DGA2 transcript levels quadrupled between 8 and 24h in the N-limited lipogenic medium, whereas DGA1 transcript levels remained stable. This survey highlights the predominant role of the DGAT1 family in lipid accumulation and demonstrates the suitability of B. raffinosifermentans for engineering for lipid production.

Inhibitory effects of Porphyra dentata extract on 3T3-L1 adipocyte differentiation.

This study was aimed to investigate the inhibitory effects of Porphyra dentata (P. dentata) extract on the adipogenesis of 3T3-L1 cells and evaluate its anti-obesity effect. The proliferation of 3T3-L1 cells and differentiation of adipocytes under treatment of P. dentata extract was examined by measuring the cell viability using alamarBlue assay and lipid droplets by Oil Red O staining. Results showed that P. dentata extract has no cytotoxicity effect and lipid droplets formation decreased in a concentration-dependent manner in 3T3-L1 cells. It has been confirmed that transcription factors affecting lipid accumulation and anti-adipogenic effects during cell differentiation are linked to P. dentata extract. We observed that P. dentata shows lowering the mRNA expression of peroxisome proliferator-activated receptor γ2 (PPARγ2), CCAAT/enhancer binding protein α (C/EBPα) that adipogenesis-associated key transcription factors and inhibiting adipogenesis in the early stages of differentiation. Treating the cells with P. dentata did not only suppressed PPARγ2 and C/EBPα but also significantly decreased the mRNA expression of adiponectin, Leptin, fatty acid synthase, adipocyte protein 2, and Acetyl-coA carboxylase 1. Overall, the P. dentata extract demonstrated inhibitory property in adipogenesis, which has a potential effect in anti-obesity in 3T3-L1 cells.

Gilthead seabream (Sparus aurata) in vitro adipogenesis and its endocrine regulation by leptin, ghrelin, and insulin

Leptin, ghrelin, and insulin influence lipid metabolism and thus can directly affect adipose tissue characteristics, modulating the organoleptic quality of aquaculture fish. The present study explored gilthead seabream (Sparus aurata) cultured preadipocytes development, and the regulation of adipogenesis by those three hormones. Preadipocytes presented a fibroblast-like phenotype during the proliferation phase that changed to round-shaped with an enlarged cytoplasm filled with lipid droplets after complete differentiation, confirming the characteristics of mature adipocytes. peroxisome proliferator-activated receptor-γ (pparγ) expression was higher at the beginning of the culture, while fatty acid synthase and 3-hydroxyacyl-CoA dehydrogenase gradually increased with cell maturation. The expression of lipoprotein lipase-like, lysosomal acid lipase (lipa), fatty acid translocase/cluster of differentiation-36 (cd36), and leptin receptor (lepr) were not affected during cell culture development; and undetectable expression levels were observed for leptin. Concerning regulation, leptin inhibited lipid accumulation significantly reducing pparγ and cd36 gene expression, both in early differentiating and mature adipocytes, while ghrelin decreased the expression of pparγ in the early differentiating phase but did not reduce intracellular lipid content significantly. Additional insulin past the onset of adipogenesis did not affect lipid accumulation either. In conclusion, at present culture conditions leptin has an anti-adipogenic function in differentiating preadipocytes of gilthead seabream and continues exerting this role in mature adipocytes, while ghrelin and insulin do not seem to influence adipogenesis progression. A better understanding of leptin, ghrelin, and insulin impact on the adipogenic process could help in the prevention of fat accumulation, improving aquaculture fish production and quality.

Ellagic Acid and Urolithins A and B Differentially Regulate Fat Accumulation and Inflammation in 3T3-L1 Adipocytes While Not Affecting Adipogenesis and Insulin Sensitivity.

Ellagic acid (EA) is a component of ellagitannins, present in crops such as pecans, walnuts, and many berries, which metabolized by the gut microbiota forms urolithins A, B, C, or D. In this study, ellagic acid, as well as urolithins A and B, were tested on 3T3-L1 preadipocytes for differentiation and lipid accumulation. In addition, inflammation was studied in mature adipocytes challenged with lipopolysaccharide (LPS). Results indicated that EA and urolithins A and B did not affect differentiation (adipogenesis) and only EA and urolithin A attenuated lipid accumulation (lipogenesis), which seemed to be through gene regulation of glucose transporter type 4 (GLUT4) and adiponectin. On the other hand, gene expression of cytokines and proteins associated with the inflammation process indicate that urolithins and EA differentially inhibit tumor necrosis factor alpha (TNFα), inducible nitric oxide synthase (iNOS), interleukin 6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1). Urolithins A and B were found to reduce nuclear levels of phosphorylated nuclear factor κB (p-NF-κB), whereas all treatments showed expression of nuclear phosphorylated protein kinase B (p-AKT) in challenged LPS cells when treated with insulin, indicating the fact that adipocytes remained insulin sensitive. In general, urolithin A is a compound able to reduce lipid accumulation, without affecting the protein expression of peroxisome proliferator-activated receptor-γ (PPARγ), CCAAT/enhancer binding protein-α (c/EBPα), and PPARα, whereas EA and urolithin B were found to enhance PPARγ and c/EBPα protein expressions as well as fatty acid (FA) oxidation, and differentially affected lipid accumulation.

Effect of sequential recycling of spent media wastewater on docosahexaenoic acid production by newly isolated strain Aurantiochytrium sp. ICTFD5

The main aim of this work was the optimization of recycled spent media wastewater (SMW) concentration for high biomass production of Aurantiochytrium sp. ICTFD5. Further, optimization for growth patterns and lipid accumulation capacity with three subsequent recycling runs was also performed. The biomass production after 96 h fermentation for recycling with 50% SMW was; 21.3 ± 1.5, 19.1 ± 1.3, 19 ± 1.2, and 23 ± 1.2 g/L for the first, second, third recycle runs, and control respectively. All the recycle runs were carried out with the same media and cultivation conditions. Subsequent recycling affected lipid accumulation, and it was decreased by ~4 to 9% compared to the control. The compositional shift of fatty acids was observed with sequential recycle runs, changing more towards saturated fatty acids content, suggesting it to be a new potential source for biodiesel feedstock.

Effects of Nitrogen Source on the Lipid Accumulation in Phaeodactylum Tricornutum

Nitrogen source has an important influence on the growth and lipid synthesis of Phaeodactylum tricornutum. It affects lipid accumulation through global metabolic regulation. However, little research has been done on the correlation between other metabolites and lipid metabolism during the synthesis of Phaeodactylum tricornutum. In this study, we studied the effect of different nitrogen sources on lipid and other major metabolic pathways in Phaeodactylum tricornutum. We not only optimized the nutritional condition, but also revealed the competitive pathway affecting the lipid synthesis in Phaeodactylum tricornutum. The results showed that urea had the most significant effect on the growth and lipid accumulation of Phaeodactylum tricornutum. The highest lipid content could be obtained in the Modified Basal Medium (MBM) with a ratio of glycine 0.7 g/L, yeast extract 5 g/L, urea 0.6 g/L, glucose 20 g/L and the total lipid yield of the optimized group was 202, 118.9, 129.4 and 53.3% higher than the basic medium group from day 2-8. Through the analysis of metabolites, it was found that protein synthesis during the growth of Phaeodactylum tricornutum was the main competitive pathway affecting its biomass and lipid accumulation. This laid a theoretical and practical foundation for the regulation of lipid accumulation in Phaeodactylum tricornutum in the future.

Bisphenols B, E, F, and S and 4-cumylphenol induce lipid accumulation in mouse adipocytes similarly to bisphenol A.

Bisphenol A (BPA) has been widely reported to exert endocrine disrupting effects, including the induction of adipogenesis in cultured preadipocytes and intact animals. Because of the potential harm to human health, BPA is being substituted by structurally related bisphenols. Whether or not such BPA analogues are safe substitutes, however, remains largely unknown. Here, we investigated the potential of bisphenol B (BPB), bisphenol E (BPE), bisphenol F (BPF), bisphenol S (BPS), and 4-cumylphenol (4-CP) to affect lipid and hormone levels in 3 T3-L1 cells. We found that BPB, BPE, BPF, BPS, and 4-CP all affected lipid accumulation and leptin levels to the same extent and potencies as BPA. Based on these and other results, we conclude that these BPA analogues and 4-CP most likely will elicit similar effects on adipocytes as BPA. Using them to substitute BPA in products should be done with caution.

188 Improvement of bovine oocyte maturation invitro through cytokine supplementation

Oocyte competence is one of the key factors determining the proportion of embryos that develop to the blastocyst stage. There is vast evidence that IVM oocytes exhibit less developmental potential than their invivo counterparts. Here, we tested whether supplementation of three cytokines [FGF2 (40 ngmL−1), LIF (20ngmL−1), and IGF1 (20 ngmL−1), termed FLI] improved oocyte maturation, and as a consequence, preimplantation development of bovine embryos invitro. In the first experiment, cumulus-oocyte complexes (COCs) were collected from abattoir-derived ovaries and placed in maturation medium,±FLI, for 18 to 22h. At the end of maturation, COCs were fertilized with sperm from a single Holstein bull known to have high fertility. After an 18- to 20-h fertilization period, putative zygotes were cultured in synthetic oviductal fluid for 8 days. The number of embryos that underwent at least one cellular division (cleavage) and the number of embryos that developed to the blastocyst stage was recorded on Days 3 and 8 after insemination, respectively. The COCs supplemented with FLI (n=554) and controls (n=534) were evaluated across 5 replicates. There was no difference in the cleavage rate (P&amp;gt;0.05) between the two treatments. Development to the blastocyst stage was higher (P=0.05) for FLI-treated COCs (34.9%±1.96) than for the control group (23.9%±1.96). In a second experiment, COCs (n=204) supplemented±FLI were collected and fixed at 6, 12, 18, and 24h after placement in maturation medium. The number of transzonal projections in the COCs was determined by localization of actin filaments by using confocal microscopy. Data were analysed by ANOVA using the GLM procedure of SAS software (version 9.4; SAS Institute Inc.). The model included treatment, time, and the interaction of treatment×time as fixed effects. There was no difference (P&amp;gt;0.05) in the number of transzonal projections at 6h (166.3±8.6 vs. 143.9±8.8) and 12h (107.8±8.4 vs. 128.3±7.7) between FLI-treated and control COCs. However, FLI-treated COCs had fewer (P&amp;lt;0.05) transzonal projections at 18h (67.9±7.8 vs. 100.1±7.7) and 24h (56.4±7.2 vs. 80.6±7.4) compared with the controls. There was a significant treatment×time interaction (P=0.006). In a third experiment, we tested whether the timing of transzonal projection disassociation affected lipid accumulation in the embryo. Blastocysts (n=59) on Day 8 produced from COCs matured±FLI were collected and lipid content was determined by using Nile Red staining. There was no difference (P&amp;gt;0.05) in lipid content between treatments. Thus, supplementation of maturation medium with FLI accelerates the disassociation of transzonal projections in COCs and improves subsequent embryonic development to the blastocyst stage while having no detectable effect on lipid content. Further research is necessary to understand how these cytokines modulate IVM of bovine oocytes. This project was supported by Food for the 21st Century and the Clifton Murphy scholarship fund.

200 Maturation method affects lipid accumulation in bovine oocytes

Invitro maturation is a key step in invitro embryo production, since its success will depend on the availability of good quality oocytes. Previous studies have shown that it is during this stage that the greatest accumulation of lipid droplets occurs, which is reflected in the amount of lipid present in embryos produced invitro. However, this is not observed when maturation is performed invivo. Therefore, we hypothesised that lipid accumulation would be avoided if oocyte maturation were carried out in ovarian follicles following intrafollicular transfer of immature oocytes (IFIOT). We compared lipid accumulation in oocytes matured invitro, invivo, and by IFIOT. A total of 90 Nellore heifers were distributed in 3 experimental groups: donors of immature oocytes (D-IMA), ovulators of IFIOT oocytes (D-OV), and superstimulated donors of invivo-matured oocytes (D-FSH). All animals rotated through all groups during the experiment. To obtain immature oocytes, the D-IMA were submitted to ovum pickup (OPU), in which aspiration medium was supplemented with 500μM 3-isobutyl-1-methylxanthine (IBMX), and, after selection, part of the oocytes were cultured invitro for 22h (MatF) and part were used for IFIOT (MatT). To perform MatT, the D-OV had their ovulation synchronized by a progesterone and benzoate oestradiol protocol, in which 30h after the implant removal, the IFIOT was performed on the dominant follicle. The D-FSH oocytes were stimulated with 80mg of FSH (Folltropin; Vetoquinol) over 4 days, every 12h, in decreasing doses. At the same time that the immature oocytes were placed in MatF and IFIOT, ovulation was induced with the gonadotrophin releasing hormone (GnRH) analogue (50µg of lecirelin) in D-OV and D-FSH groups. After 22h, matured oocytes were either removed from culture (MatF) or recovered from follicles by OPU (MatT and MatS). From the recovered oocytes of all groups, only those with a polar body were used for lipid droplet evaluation. To quantify lipid accumulation, denuded oocytes were fixed and stained with boron-dipyrromethene (Bodipy) 493/503 (20 µgmL−1) and evaluated by confocal microscopy. Captured images were evaluated in the ImageJ program (National Institutes of Health), and lipid content was determined by calculating the ratio of the area of the lipid droplets to total oocyte area. Data were analysed by ANOVA with statistical significance set at P&amp;lt;0.05. A total of 95 oocytes were evaluated: 25 immature (CT), 24 invitro (MatF), 30 invivo (MatS), and 16 invivo (MatT). The mean area containing lipid droplets in immature oocytes (14%±0.9) was similar (P&amp;gt;0.05) to that observed in both invivo maturation systems (MatS=17.26%±0.8 and MatT=14.11%±0.9). However, in the MatF oocytes, lipid content (24.34%±1) increased during maturation and was higher than in the other groups (P&amp;lt;0.05). We showed for the first time that oocytes matured by IFIOT are similar to those invivo matured with regard to lipid content, which may imply their superior quality over those matured invitro. This new maturation method opens new possibilities for biotechnologies that need to use mature oocytes, such invitro embryo production, oocyte and embryo cryopreservation, cloning, and transgenesis. This study was supported by FAP-DF and Capes.

Lithium chloride promotes lipid accumulation through increased reactive oxygen species generation

LiCl is widely prescribed for bipolar disorder but adversely associated with a higher incidence of increased body weight. Here, we investigated effects and underlying mechanisms of LiCl on lipid accumulation. LiCl induced dose-dependent lipid accumulation in HepG2 and RAW264.7 cells under normal as well as high glucose conditions. LiCl exposure additionally promoted lipid accumulation in livers of zebrafish. SB216763, a specific GSK-3β inhibitor, did not affect lipid accumulation in HepG2 cells. Expression of key lipogenic enzymes, such as FAS and aP2, as well as SR-B1 were increased in RAW264.7 cells. LiCl enhanced FAS, ACC and SCD-1 mRNA levels while suppressing CPT-1 in HepG2 cells. LiCl stimulated DNA binding activities of SREBP-1c and ChREBP. LiCl activated AMPK phosphorylation but the AMPK inhibitor, AICAR, did not suppress LiCl-induced lipid accumulation in RAW264.7. LiCl, but not SB216763, induced a significant increase in ROS in RAW264.7 and HepG2 cells. NOX activity was dose-dependently enhanced by LiCl. Furthermore, NOX-1, NOX-2 and DUOX-1 mRNA levels were upregulated at an early stage of LiCl stimulation. LiCl-induced lipid accumulation was suppressed by the antioxidant, NAC, and inhibitors of NOX, DPI and APO. Phosphorylation and transcriptional activity of CREB were enhanced by LiCl. The cell-permeable cAMP analog, di-butyryl cAMP, not only promoted lipid accumulation itself but also LiCl-induced lipid accumulation in RAW264.7 cells. H-89, a PKA inhibitor, suppressed CREB activation, lipid accumulation and NOX activity in RAW264.7 cells. Our results indicate that LiCl stimulates lipid accumulation in hepatocyte and macrophage cells potentially through increased PKA-dependent ROS production.