Lipid Droplet Area Research Articles

YC-1 inhibits lipid droplet accumulation and induces lipolysis in lipid-laden RAW264.7 macrophages

Macrophage foam cells play a central role in the initiation and progression of atherosclerosis. The present study investigates the effect of YC-1, a synthetic benzylindazole compound, on lipid metabolism in macrophage-derived foam cells. Foam cell formation was induced by incubating cultured RAW264.7 macrophages with oleic acid (OA) or cobalt chloride (CoCl 2) (a hypoxia mimetic). Morphological observation showed several Nile red-positive lipid droplets in the cytoplasm of OA-treated RAW264.7 macrophages. Pretreatment with 60 μMYC-1 could inhibit lipid accumulation. Co-incubation with OA and YC-1 also attenuated lipid accumulation due to the decrease in surface area of lipid droplets. Post-treatment with YC-1 induced lipolysis and free fatty acid release in lipid-laden macrophages in both dose- and time-dependent manners. In addition, ODQ, a soluble guanynyl cyclase (sGC) inhibitor, could not reverse the inhibitory effect of YC-1 on lipid accumulation, or block the lipolytic effect of YC-1 in lipid-laden macrophages. Neither another nitric oxide-independent sGC activator (BAY 41-2272) nor dibutyryl cGMP (db-cGMP) could mimic the effects of YC-1. Both intracellular sGC activity and the cGMP level remained unchanged following YC-1 incubation. These results suggested that the mechanism of YC-1-mediated lipid metabolism was via an sGC/cGMP-independent signal transduction pathway in RAW264.7 macrophages. In addition, YC-1 significantly attenuated CoCl 2-induced lipid droplets accumulation. Our results demonstrated that YC-1 could mediate lipid metabolism in macrophage and reduce foam cell formation, and it could be regarded as a potential drug for the prevention or therapy of atherosclerosis.

Octyl gallate induces hepatic steatosis in HepG2 cells through the regulation of SREBP-1c and PPAR-gamma gene expression.

Octyl gallate (OG) is an antioxidant commonly used in food, although there is no definition of its acceptable daily intake. There are reports in vitro and in vivo showing that food additives and drugs can alter lipid metabolism. Lipid droplet accumulation in hepatic cells is one of the main findings in the unregulated lipid metabolism and is strongly related to the development of nonalcoholic fatty liver disease (NAFLD). In this study, we investigated the effects of OG on lipid metabolism in the hepatocellular carcinoma cell line (HepG2). The results have shown, for the first time, that treatment with OG increased the overall amount of lipids, the triglyceride concentration, the lipid droplet area, and SREBP-1c and PPAR-γ gene expression. Taken together, the findings indicate that OG induces lipid droplet accumulation in HepG2 cells through the regulation of SREBP-1c and PPAR-γ gene expression without involving mTOR/S6K1 and may contribute to NAFLD when used as a food additive.

DHA Modulates Immune Response and Mitochondrial Function of Atlantic Salmon Adipocytes after LPS Treatment.

Adipocytes play a central role in overall energy homeostasis and are important contributors to the immune system. Fatty acids (FAs) act as signaling molecules capable to modulate adipocyte metabolism and functions. To identify the effects of two commonly used FAs in Atlantic salmon diets, primary adipocytes were cultured in the presence of oleic (OA) or docosahexaenoic (DHA) acid. DHA decreased adipocyte lipid droplet number and area compared to OA. The increase in lipid load in OA treated adipocytes was paralleled by an increase in iNOS activity and mitochondrial SOD2-GFP activity, which was probably directed to counteract increase in oxidative stress. Under lipopolysaccharide (LPS)-induced inflammation, DHA had a greater anti-inflammatory effect than OA, as evidenced by the higher SOD2 activity and the transcriptional regulation of antioxidant enzymes and pro- and anti-inflammatory markers. In addition, DHA maintained a healthy mitochondrial structure under induced inflammation while OA led to elongated mitochondria with a thin thread like structures in adipocytes exposed to LPS. Overall, DHA possess anti-inflammatory properties and protects Atlantic salmon against oxidative stress and limits lipid deposition. Furthermore, DHA plays a key role in protecting mitochondria shape and function.

HDL Receptor SR-B1 Deficiency Increased Inflammatory Dyslipidemia and Adipocyte Hypertrophy and Attenuated the Hepatic Steatosis in Murine Diet-Induced Obesity

Abstract Objectives We tested whether the scavenger receptor, class B type 1 (SR-B1), a physiologically relevant HDL receptor, modulates the metabolic and inflammatory phenotype of obese mice, accompanied by changes in adipocyte and hepatocyte lipid deposits. Methods Male SR-B1 knock-out (KO) mice and wild-type (WT) littermates were fed for 12 weeks with a high-fat diet (HFD, n = 12 per group) to induce obesity. Animals were euthanized after overnight food deprivation. Blood was obtained and adipose tissue and liver were removed. Plasma or frozen tissues were used for biochemical analyses or assessed by light microscopy and immunohistochemistry. Comparisons between WT and KO mice fed with HFD were performed and differences were considered statistically significant when P < 0.05. Results Compared to obese WT, obese HFD-fed SR-B1 KO mice showed increased plasma total cholesterol (p < 0.0001) and triglycerides (TG) (p < 0.01) as well as tumor necrosis factor-α (TNF-α) (p < 0.0001) levels. Also, these animals exhibited white fat with larger adipocytes (p < 0.0001) and increased macrophage-based crown-like structure formation (p < 0.01), compared to HFD-fed WT mice, revealing a more inflammatory adipose tissue. These changes in adipose tissue were associated with reduced hepatic steatosis: reduced hepatocyte lipid droplet area (p < 0.0001) and decreased liver TG content (p < 0.0001). In addition, obese SR-B1-deficient mice showed reduced hepatic peroxisome proliferator-activated receptor-γ (PPAR-γ) expression, more efficient hepatic fatty acid β-oxidation and increased very-low-density lipoprotein (VLDL)-TG secretion compared to obese HFD-fed WT mice Furthermore, liver fatty acid (FA) composition in obese SR-B1-deficient mice revealed a reduction in monounsaturated (MUFA) (p < 0.01), but an increase in polyunsaturated (PUFA) (p < 0.05), fatty acid content, compared to obese WT mice. Conclusions Our findings demonstrate that SR-B1 expression modulates high fat feeding-associated inflammatory dyslipidemia, adipocyte hypertrophy, and hepatic steatosis; key processes underlying the pathogenesis of highly prevalent chronic diseases, such as obesity and non-alcoholic fatty liver disease (NAFLD). Funding Sources FONDECYT 1,150,399, FONDECYT 1,180,525.

MP51-15 PERIPROSTATIC FAT INDUCES CENTROSOMAL AMPLIFICATION IN THE PROSTATE CANCER MICROENVIRONMENT: A MICROFLUIDIC APPROACH

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology II (MP51)1 Apr 2020MP51-15 PERIPROSTATIC FAT INDUCES CENTROSOMAL AMPLIFICATION IN THE PROSTATE CANCER MICROENVIRONMENT: A MICROFLUIDIC APPROACH Max Greenberg, Victoria Gil, Philip Fitchev, Francesca Nardi, Evanston, Deepak Daroach, Omar Franco, Simon Hayward, Renee Vickman, Xiaojing Su, Tristan Nicholson, Ashleigh Theberge, and Susan Crawford* Max GreenbergMax Greenberg More articles by this author , Victoria GilVictoria Gil More articles by this author , Philip FitchevPhilip Fitchev More articles by this author , Francesca Nardi, EvanstonFrancesca Nardi, Evanston More articles by this author , Deepak DaroachDeepak Daroach More articles by this author , Omar FrancoOmar Franco More articles by this author , Simon HaywardSimon Hayward More articles by this author , Renee VickmanRenee Vickman More articles by this author , Xiaojing SuXiaojing Su More articles by this author , Tristan NicholsonTristan Nicholson More articles by this author , Ashleigh ThebergeAshleigh Theberge More articles by this author , and Susan Crawford*Susan Crawford* More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000913.015AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Periprostatic fat (PPF) encases the prostate and is an understudied visceral fat depot and a stromal component of the prostate tumor microenvironment (TME). Visceral obesity is associated with ectopic lipid accumulation in non-adipocytes and tumor progression; however, the mechanisms responsible for aberrant adipocyte-tumor cell crosstalk are not clear. The PPF stores neutral lipid in the form of lipid droplets (LDs) and secretes a wide array of adipokines and cytokines. In human cancer-associated fibroblasts (CAFs), we recently found that increased lipid content promoted centrosomal and microtubule-organizing center (MTOC) amplification as well as a proliferative phenotype. Studying the TME has been hampered by limited experimental systems. We used an innovative multicellular open microfluidic platform, the Monorail, to study metabolic reprogramming and LD trafficking in tri-cultured human adipocytes, prostate tumor cells, and primary cancer-associated fibroblasts (CAFs). METHODS: Primary PPF derived from surgical prostatectomy specimens were co-cultured with prostate cancer and 3T3-L1 cell lines, and primary CAFs. The cells were cultured in a polystyrene open microfluidic device where only 22µL of medium is required per well. Low temperature-sensitive agarose gel was used to partition cells into various wells. The cells were analyzed using immunofluorescence stains (pericentrin and metalloproteinases), BODIPY and Oil-Red-O was used for neutral lipid. LD velocity was analyzed using live cell imaging. RESULTS: We found that the co-culture of prostate cancer cells with adipocytes (3T3-L1 or primary PPF) resulted in an increased LD area, and the addition of CAFs elevated both LD density and area in the tumor cells. Immunofluorescent studies for MTOCs revealed exposure of CAFs or adipocyte cells to cancer cells in the Monorail promoted amplification in centrosomes/MTOCs to two or more per cell. Live-cell imaging demonstrated that exposure to adipocytes caused the LD velocity to increase in cancer cells, and metalloproteinases were located on the surface of the LDs. CONCLUSIONS: The Monorail is an open microfluidic platform that simulates the complex TME. The device allows testing of primary human samples where cell number is limited. Adipocytes promote lipid reprogramming and MTOC amplification in both prostate cancer cells and CAFs. In the setting of obesity, higher intracytoplasmic lipid accumulation and centrosomal amplification are potential mechanisms responsible for accelerated prostate tumor growth and progression. Source of Funding: This work has been supported by the Rob Brooks Fund for Precision Prostate Cancer Care © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e769-e770 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Max Greenberg More articles by this author Victoria Gil More articles by this author Philip Fitchev More articles by this author Francesca Nardi, Evanston More articles by this author Deepak Daroach More articles by this author Omar Franco More articles by this author Simon Hayward More articles by this author Renee Vickman More articles by this author Xiaojing Su More articles by this author Tristan Nicholson More articles by this author Ashleigh Theberge More articles by this author Susan Crawford* More articles by this author Expand All Advertisement PDF downloadLoading ...

Ginsenoside Rg3 Induces Browning of 3T3-L1 Adipocytes by Activating AMPK Signaling.

Ginsenoside Rg3, one of the major components in Panax ginseng, has been reported to possess several therapeutic effects including anti-obesity properties. However, its effect on the browning of mature white adipocytes as well as the underlying mechanism remains poorly understood. In this study, we suggested a novel role of Rg3 in the browning of mature 3T3-L1 adipocytes by upregulating browning-related gene expression. The browning effects of Rg3 on differentiated 3T3-L1 adipocytes were evaluated by analyzing browning-related markers using quantitative PCR, immunoblotting, and immunostaining. In addition, the size and sum area of lipid droplets in differentiated 3T3-L1 adipocytes were measured using Oil-Red-O staining. In mature 3T3-L1 adipocytes, Rg3 dose-dependently induced the expression of browning-related genes such as Ucp1, Prdm16, Pgc1α, Cidea, and Dio2. Moreover, Rg3 induced the expression of beige fat-specific genes (CD137 and TMEM26) and lipid metabolism-associated genes (FASN, SREBP1, and MCAD), which indicated the activation of lipid metabolism by Rg3. We also demonstrated that activation of 5’ adenosine monophosphate-activated protein kinase (AMPK) is required for Rg3-mediated up-regulation of browning gene expression. Moreover, Rg3 inhibited the accumulation of lipid droplets and reduced the droplet size in mature 3T3-L1 adipocytes. Taken together, this study identifies a novel role of Rg3 in browning of white adipocytes, as well as suggesting a potential mechanism of an anti-obesity effect of Panax ginseng.

180 Glucose versus fatty acids: Different energy supplies for the bovine oocyte

Studies show that optimal concentrations of fatty acids (FA) and glucose are crucial for proper oocyte development, maturation, and further embryo quality support (Sutton McDowall et al. 2010 Reproduction 139, 685-95, https://doi.org/10.1530/REP-09-0345; Sutton McDowall et al. 2014 Theriogenology 82, 95-103, https://doi.org/10.1016/j.theriogenology.2014.03.011). A balance between metabolism of FA and glucose in oocytes needs to be maintained; however, it is not clear what the preferable pathway of energy production is and how it affects the oocyte. The aim of the experiment was to selectively block pathways of either glucose or FA metabolism during IVM of bovine oocytes to reveal changes within oocyte lipid droplets (LD) under crucial changes in energy metabolism. Cumulus-oocyte complexes were matured under standard conditions (Warzych et al. 2007Mol. Reprod. Dev. 74, 280-289; https://doi.org/10.1002/mrd.20610) without FA and glucose supplementation. The experimental groups were (1) control (IVM in basic medium), (2) group with inhibited glucose metabolism [supplementation with 1.5 µM iodoacetate (IO, inhibitor of glycolysis) and 150 µM dehydroepiandrosterone (DHEA, inhibitor of pentose phosphate pathway)], and (3) group with inhibited FA metabolism (150 µM etomoxir supplementation, ETO). Oocytes after 24h of IVM were stained with boron-dipyrromethene (BODIPY) 493/503 dye (lipid droplets) and 4′,6-diamidino-2-phenylindole (DAPI; chromatin) and analysed using a confocal microscope (LSM 880 AiryScan FAST; Zeiss). Obtained data were analysed using the Kruskal-Wallis test. The MII rate decreased to 60.5% for IO+DHEA (P<0.05) and to 78% for the ETO group compared with the control (83.8%). The average LD area (% of total oocyte area) significantly decreased in both experimental groups (IO+DHEA 6.72±2.5, ETO 6.28±3.2; P<0.01) compared with control (8.7±3.6). Total lipid content (intensity of the fluorescence), was significantly lower in experimental groups (IO+DHEA: 1.17×106±3×105, ETO: 7.1×104±2×104 vs. control: 1.68×106±4.6×105; P<0.01). With regard to the total number of LD, only in the IO+DHEA group were significantly fewer LD noted (987±343; P<0.05), whereas the ETO group did not differ significantly (1146±414) compared with control (1148±357). The ETO group had significantly lower total lipid content (P<0.01) and higher total LD number (P<0.05) compared with IO+DHEA. The area of lipid droplets did not differ between the experimental groups. These results show that blocking glucose metabolism strongly affects the nuclear maturation process of the oocyte, limiting the number of oocytes that reach the MII stage. A significant decrease in total lipid content in the ETO group may suggest strong utilisation of lipids during the maturation process, when de novo synthesis of lipids is blocked. Accumulation of lipids in LD before maturation allows this process to be passed even under the applied FA metabolism inhibitory conditions. In contrast, inhibition of glucose metabolism negatively affects oocyte development strictly after inhibition conditions, indicating the greater significance of glucose for proper oocyte maturation. Further studies are being conducted to investigate the effect of inhibitory systems on lipids in bovine cumulus cells and further embryos. Funding for this study was provided by National Science Centre, Poland (project no 2017/27/B/NZ9/00904).

76 Extracellular vesicles from oviduct and uterus in sequential culture improve the quality of bovine embryos produced invitro

Extracellular vesicles (EVs) are released by cells and transport cargo that affect functions of other cells. Oviductal fluid (OF) and uterine fluid (UF) have been shown to improve quality of embryos during invitro culture (Hamdi et al. 2017 Reprod. Fertil. Dev. 30, 935-945) which may be due to their content of EV (Lopera-Vásquez et al. 2017 Reproduction 153, 461-470). Thus, the aim of this study was to evaluate the effect of EVs from OF and UF on a sequential invitro culture system on the development and quality of bovine embryos. Zygotes were cultured in synthetic oviduct fluid (SOF) supplemented with 3mgmL−1 bovine serum albumin (BSA; n=1228) or 5% EV-depleted fetal calf serum (dFCS, n=1261) in the presence (BSAEV, n=1265 and dFCSEV, n=1253) or absence of 3×105 EVmL−1 from OF (Day 1 to Day 4) and UF (Day 4 to Day 9), mimicking invivo conditions. The EVs pooled from 5 oviducts (early luteal phase) and 5 uterine horns (middle luteal phase) from slaughtered heifers were isolated by a Size Exclusion Chromatography kit (Hansa BioMed). The EV size and concentration were assessed by the nanotracking analysis system and morphology by transmission electron microscopy. Embryo development was recorded on Days 7/9. Day 7/8 blastocysts were assessed for quality by staining with (a) Hoechst 33342 (10 µgmL−1, 30min) for total cell number, (b) Bodipy 493/503 (20 µgmL−1, 1h) for lipid content (lipid droplet area in µm2), and (c) for survival rate after vitrification/warming. Data were analysed by one-way ANOVA and Tukey test. The EV concentration was 2.97 and 7.98×1010 particlesmL−1, and mode size 137.2 and 151.2nm for OF and UF, respectively. Transmission electron microscopy confirmed EV presence and size, showing typical cup-shaped morphology. Blastocyst yield was lower (P<0.05) on Day 7 in the BSA groups (BSA: 15.7±1.9 and BSAEV: 15.2.4%) compared with serum groups (dFCS: 28.1±2.6 and dFCSEV: 30.1±2.9%) irrespective of EV supplementation; however, these differences were compensated at Days 8 and 9 (range: 30.0±3.2-40.8±3.9%). The EVs increased (P<0.05) blastocyst total cell number in dFCSEV (152.6±2.9) and BSAEV (140.5±1.5) compared with dFCS (117.9±2.0) and BSA groups (122.4±1.1). However, lipid content was decreased (P<0.05) in the presence of EVs only in dFCSEV (0.231±0.05µm2) compared with BSA (0.393±0.03µm2) and BSAEV (0.379±0.03µm2) groups. The dFCS did not differ from any group (0.371±0.05µm2; P>0.05). Blastocyst survival after vitrification/warming was high in all groups up to 72h (range: 80.0±3.8-100%; P>0.05). In conclusion, mimicking physiological conditions using EV from OF and UF during invitro culture does not affect development but improves embryo quality by increasing blastocyst total cell numbers and decreasing lipid contents. These results provide evidence of the association of the reproductive tract environment and developing embryo, confirming embryo-maternal communication. Funding was provided by MINECO-Spain AGL2015-70140-R; Y. N. Cajas, SENESCYT-Ecuador; C. L. V. Leal, FAPESP-Brazil 2017/20339-3.

Comparison of the Effects of Browning-Inducing Capsaicin on Two Murine Adipocyte Models.

The increasing prevalence of obesity and its associated comorbidities has gained attention in developing effective treatments and strategies that promote energy expenditure and the conversion of fat from a white to a brite phenotype. Capsaicin, bioactive component of chili peppers and a transient receptor potential channel vanilloid 1 (TRPV1) agonist, has been known to stimulate the process of thermogenesis. In this study, the effects of capsaicin were assessed on two murine cellular models by quantifying the dynamic of lipid droplets (LDs) and the expression of genes involved in adipocyte browning. Present findings demonstrated that treatment with norepinephrine or capsaicin combined with norepinephrine on 3T3-L1 cells and X9 cells significantly promoted the reduction of LDs area surface and size. The transcription of browning related genes such as uncoupling protein 1 (Ucp1), T-box transcription factor 1 (Tbx1), PR domain containing 16 (Prdm16), peroxisome proliferator-activated receptor γ coactivator 1α (Ppargc1a) and cell death-inducing DNA fragmentation factor A-like effector A (Cidea) was up-regulated by chronic capsaicin treatment on differentiated 3T3-L1 cells. Instead, X9 cells were significantly responsive only to the treatment with norepinephrine, used as positive control.

Involvement of G-Protein-Coupled Receptor 40 in the Inhibitory Effects of Docosahexaenoic Acid on SREBP1-Mediated Lipogenic Enzyme Expression in Primary Hepatocytes.

Nonalcoholic fatty liver disease is a frequent liver malady, which can progress to cirrhosis, the end-stage liver disease if proper treatment is not applied. Omega-3 fatty acids, such as docosahexaenoic acid (DHA) and eicosapentaenoic acid, have been clinically proven to lower serum triglyceride levels. Various physiological activities of omega-3 fatty acids are due to their agonistic actions on G-protein-coupled receptor 40 (GPR40) and GPR120. Lipid droplets (LD) accumulation in hepatocytes confirmed that DHA treatment reduced the number of larger ( >10 μm2) LDs, as well as the total area of LDs. Moreover, DHA lowered protein and mRNA expression levels of lipogenic enzymes such as fatty acid synthase (FAS), acetyl-CoA carboxylase and stearoyl-CoA desaturase-1 (SCD-1) in primary hepatocytes incubated with liver X receptor (LXR) agonist T0901317 or high glucose and insulin. DHA also decreased protein expression of nuclear and precursor sterol response-element binding protein (SREBP)-1, a key lipogenesis transcription factor. We further found that exposure of murine primary hepatocytes to DHA for 12 h increased GPR40 and GPR120 mRNA levels. Specific agonists (Compound A for GPR120 and AMG-1638 for GPR40), hepatocytes from GPR120 knock-out mice and GPR40 selective antagonist (GW1100) were used to assess whether DHA’s antilipogenic effects are mediated through GPR120 or GPR40. Compound A did not decrease SREBP-1 and FAS protein expression in hepatocytes exposed to T0901317 or high glucose with insulin. Moreover, DHA downregulated lipogenesis enzyme expression in GPR120-null hepatocytes. In contrast, AMG-1638 lowered SREBP-1 and SCD-1 protein levels. Additionally, GW1100, a GPR40 antagonist, reversed the antilipogenic effects of DHA. Collectively, our data demonstrate that DHA downregulates the expression SREBP-1-mediated lipogenic enzymes via GPR40 in primary hepatocytes.

Branched chain amino acids are associated with the heterogeneity of the area of lipid droplets in hepatocytes of patients with non-alcoholic fatty liver disease.

Macrovesicular steatosis around the central vein (zone 3) is one of the pathological features of non-alcoholic fatty liver disease or steatohepatitis (NAFLD/NASH). The aim of this study is to elucidate precisely the association between the area of lipid droplets (LDs) and the plasma metabolic parameters in patients with NAFLD/NASH. Eighty patients with NAFLD/NASH diagnosed by needle biopsy were enrolled. The LDs around zone 3 were counted automatically by image processing software, the total area of LDs (TLDs), the maximum area of LDs (MAXLDs), the average area of LDs (AVELDs) and the heterogeneity by the coefficient of variation (CV [%]) were quantified. The correlations between these values and plasma metabolic parameters were analyzed. We evaluated the association between branched chain amino acids (BCAAs) and the heterogeneity of LDs in hepatocytes in vitro and in vivo. The MAXLDs was significantly correlated with more metabolic parameters than AVELDs and TLDs. The level of BCAAs was independently associated with the CV among the metabolic parameters. In early stage NAFLD, aspartate and alanine aminotransferase were significantly higher in the high CV group than in the low CV group. The high concentration of BCAAs increased the CV of LDs in hepatocytes accompanied by the expression of phosphor-p70 S6 kinase and sterol regulatory element-binding protein 1 in vitro. A high BCAA diet induced high heterogeneity of LDs around zone 3 in ob/ob mice. The levels of BCAAs were associated with the LD heterogeneity of hepatocytes around zone 3 in patients with NAFLD/NASH.

Hepatic lipid metabolism and oxidative stress responses of grass carp (Ctenopharyngodon idella) fed diets of two different lipid levels against Aeromonas hydrophila infection

The present study was conducted to investigate the effect of Aeromonas hydrophila infection and different lipid level diets on the performance, hepatic lipid metabolism, and oxidative stress in grass carp (Ctenopharyngodon idella). To this end, grass carp (50.73 ± 3.17 g) were divided into 4 groups: control group, A. hydrophila group, high-fat diet group, high-fat diet + A. hydrophila group for 8 weeks. Compared with the control group, there was no significant difference in hepatic lipid content in the A. hydrophila group for 8 weeks, but it increased significantly in 4 weeks. The results showed that A. hydrophila infection and feeding with basal diet may have influenced hepatic lipid deposition in a time-dependent manner. The expression of lipid decomposition-related gene adipose triacylglyceride lipase (ATGL) and lipoprotein lipase (LPL) were reduced at 4 weeks, and lipid synthesis-related gene fatty acid synthase (FAS), acetyl-CoA carboxylase α (ACCα), and stearoyl-CoA desaturase (SCD) were suppressed at 8 weeks, which reduced the lipid metabolism of the liver. Compared with the high-fat diet group, the hepatic lipid content was significantly increased in high-fat diet + A. hydrophila group for 8 weeks, indicating that A. hydrophila infection aggravated hepatic lipid deposition induced by high-fat diet, the expression of ACCα and FAS were significantly up-regulated, and lipid decomposition-related genes (ATGL, LPL, CPT1) were not significantly changed, indicating that A. hydrophila infection of grass carp fed with a high-fat diet could increase liver lipid synthesis. Oil red O staining and the relative area of lipid droplets further support this finding. Moreover, in both groups, A. hydrophila infection enhanced serum aspartate transaminase (AST), which was found to cause liver injury. Total antioxidant capacity (T-AOC) was reduced and malondialdehyde (MDA) content was increased in the liver after infection of A. hydrophila in both basal diet and high-fat diet groups, indicating that A. hydrophila infection weakened the antioxidant defense ability of the liver, which led to the occurrence of oxidative stress and oxidative damage to the liver. Our results indicated that dysregulation of hepatic lipid metabolism and damage of the antioxidant system in grass carp could be caused by challenge to a certain dose of A. hydrophila, and hepatic lipid deposition induced by A. hydrophila infection was closely related to dietary lipid content.

Fat-specific protein 27α inhibits autophagy-dependent lipid droplet breakdown in white adipocytes.

Aims/IntroductionFat‐specific protein 27 (FSP27) α is the major isoform of FSP27 in white adipose tissue (WAT), and is essential for large unilocular lipid droplet (LD) formation in white adipocytes. In contrast, FSP27β is abundantly expressed in brown adipose tissue (BAT), and plays an important role in small multilocular LD formation. In FSP27 KO mice in which FSP27α and β are both depleted, WAT is characterized by multilocular LD formation, and by increased mitochondrial abundance and energy expenditure, whereas BAT conversely manifests large oligolocular LDs and reduced energy expenditure.Materials and MethodsWe investigated the effects of autophagy in WAT and BAT of wild type (WT) and FSP27 knockout (KO) mice. In addition, we examined the effects of FSP27α and FSP27β to the induction of autophagy in COS cells.ResultsFood deprivation induced autophagy in BAT of WT mice, as well as in WAT of FSP27 KO mice, suggesting that enhanced autophagy is characteristic of adipocytes with small multilocular LDs. Pharmacological inhibition of autophagy attenuated the fasting‐induced loss of LD area in adipocytes with small multilocular LDs (BAT of WT mice and WAT of FSP27 KO mice), without affecting that in adipocytes with large unilocular or oligolocular LDs (WAT of WT mice or in BAT of FSP27 KO mice). Overexpression of FSP27α inhibited autophagy induction by serum deprivation in COS cells, whereas that of FSP27β had no such effect.ConclusionsThe present results thus showed that FSP27α inhibits autophagy and might thereby contribute to the energy‐storage function of WAT.

Vitronectin deficiency attenuates hepatic fibrosis in a non-alcoholic steatohepatitis-induced mouse model.

Vitronectin (VN), an extracellular matrix protein, is a promising immune biomarker of non-alcoholic steatohepatitis (NASH); however, its precise function remains unclear. This study investigated how VN deficiency contributes to the development of NASH. Towards this aim, wild-type (WT) and VN-/- mice were fed with a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) for 6 and 10weeks to induce NASH, and the livers were isolated. In WT mice fed with CDAHFD for 6 and 10weeks, the expression of Vn mRNA and protein was up-regulated compared with that in mice fed with the MF control diet, indicating that VN is regulated in NASH condition. VN-/- mice showed decreased picrosirius red staining in the liver area and Col1a2 mRNA expression levels, compared with WT mice, indicating that the severity of hepatic fibrosis is attenuated in the CDAHFD-fed VN-/- mice. In addition, VN deficiency did not affect the area of lipid droplets in haematoxylin-eosin staining and the mRNA expression levels of fatty acid synthases, Srebp, Acc and Fas in the CDAHFD-fed mice. Moreover, VN deficiency decreased the inflammation score and the mRNA expression levels of Cd11b and F4/80, macrophage markers, as well as Tnf-α and Il-1β, inflammatory cytokines in the CDAHFD-fed mice. Furthermore, VN deficiency decreased the protein and mRNA expression levels of α-smooth muscle actin in the CDAHFD-fed mice, suggesting that VN deficiency inhibits the activation of hepatic stellate cells (HSCs). Our findings indicate that VN contributes to the development of fibrosis in the NASH model mice via modulation of the inflammatory reaction and activation of HSCs.

ВПЛИВ ВВЕДЕННЯ МЕЛАТОНІНУ В РІЗНИЙ ЧАС ДОБИ НА БУРУ ЖИРОВУ ТКАНИНУ ЩУРІВ ІЗ ВИСОКОКАЛОРІЙНИМ ОЖИРІННЯМ

The aim of our study was to determine morpho-functional state (area of nucleus, brown adipocytes and also area and number of lipid droplets in each cells, general optical density of tissue) of brown adipose tissue in rats with high-calorie (high fat) dietinduced obesity after melatonin administration in different time of the day (morning and evening). Melatonin was administered daily by gavage for 7 weeks in dose 30 mg/kg either 1 h after lights-on (ZT01) or 1 h before lights-off (ZT11) rats with high-calorie diet (HCD). Besides morphometric parameters as well were measured related visceral fat weight and related brown adipose tissue mass. Rats with HCD had huge changes in brown adipocytes morphology, which summarized in become resembles of classical white adipocytes: grown lipid droplets and cells area, but goes down lipid droplets number and optical density of brown adipose tissue. In general brown adipose tissue with above mentioned characteristic from HCD rats lose their ability to conduct strongly thermoproduction function. After melatonin used in rats with HCD arise leveling of pathological changes, which associated with consumption of HCD. Namely, in groups HCD ZT01 and HCD ZT11 we obtain decreased cells and lipid droplets area, increased lipid droplets number and optical density of brown adipose tissue, in relation to group HCD. Therese received changes has evidence about functionally active brown adipose tissue state, which can also dissipate of exceed energy (lipids – triacylglycerols) amount into whole organism during heat production for avoid to its storage in white adipose tissue and in outside adipose tissue. In addition, evening administration of melatonin (group HCD ZT11) demonstrate more activated state of brown adipose tissueand also related visceral weight gain less, than morning(group HCD ZT01). In conclusions, melatonin influence on morpho-functional state brown adipose tissue in rats with HCD, moreover evening administration can use for obesity therapy via its strong action on activate brown adipocytes.

Zingiber officinale formulation reduces hepatic injury and weight gain in rats fed an unhealthy diet.

This study investigated the ability of formulation containing Zingiber officinale (ginger) to reverse health changes promoted by unhealthy diet in Wistar rats. Five compounds from the gingerol family and three from the shogaol family were identified in the chromatographic analyzes of the extract. The animals were fed a combination of unhealthy foods, the cafeteria diet, which promoted increases in body weight, hepatocyte nucleus area, total hepatocyte area and liver fat accumulation, as well as reduced hepatic glutathione S-transferase concentration, compared to the control group, which received commercial chow. The treatment with ginger improved all these results, highlighting the reduction of 10% of body weight and 66% of the total area of lipid droplets deposited, compared to the group that received the cafeteria diet. Ginger treatments also attenuated lipid peroxidation, with a mean reduction of 41% in malondialdehyde levels and a mean increase of 222% in glutathione S-transferase activity in the liver. The cafeteria diet and ginger extract did not promote significant changes in glycemic and lipid profile, liver weight and liver enzymes compared to the control group. We suggest that ginger can have beneficial effects on health complications associated with unhealthy diet, such as excessive adiposity, oxidative stress and hepatic injury.

Altered Lipid Metabolism Impairs Skeletal Muscle Force in Young Rats Submitted to a Short-Term High-Fat Diet.

Obesity and ensuing disorders are increasingly prevalent in young populations. Prolonged exposure to high-fat diets (HFD) and excessive lipid accumulation were recently suggested to impair skeletal muscle functions in rodents. We aimed to determine the effects of a short-term HFD on skeletal muscle function in young rats. Young male Wistar rats (100–125 g) were fed HFD or a regular chow diet (RCD) for 14 days. Specific force, resistance to fatigue and recovery were tested in extensor digitorum longus (EDL; glycolytic) and soleus (SOL; oxidative) muscles using an ex vivo muscle contractility system. Muscle fiber typing and insulin signaling were analyzed while intramyocellular lipid droplets (LD) were characterized. Expression of key markers of lipid metabolism was also measured. Weight gain was similar for both groups. Specific force was decreased in SOL, but not in EDL of HFD rats. Muscle resistance to fatigue and force recovery were not altered in response to the diets. Similarly, muscle fiber type distribution and insulin signaling were not influenced by HFD. On the other hand, percent area and average size of intramyocellular LDs were significantly increased in the SOL of HFD rats. These effects were consistent with the increased expression of several mediators of lipid metabolism in the SOL muscle. A short-term HFD impairs specific force and alters lipid metabolism in SOL, but not EDL muscles of young rats. This indicates the importance of clarifying the early mechanisms through which lipid metabolism affects skeletal muscle functions in response to obesogenic diets in young populations.

Dehydroepiandrosterone reduced lipid droplet accumulation via inhibiting cell proliferation and improving mitochondrial function in primary chicken hepatocytes.

Dehydroepiandrosterone (DHEA) possesses fat-reducing effect, while little information is available on whether DHEA regulates cell proliferation and mitochondrial function, which would, in turn, affect lipid droplet accumulation in the broiler. In the present study, the lipid droplet accumulation, cell proliferation, cell cycle and mitochondrial membrane potential were analysis in primary chicken hepatocytes after DHEA treated. The results showed that total area and counts of lipid droplets were significantly decreased in hepatocytes treated with DHEA. The cell viability was significantly increased, while cell proliferation was significantly inhibited in a dose dependent manner in primary chicken hepatocytes after DHEA treated. DHEA treatment significantly increased the cell population in S phase and decreased the population in G2/M in primary chicken hepatocytes. Meanwhile, the cyclin A and cyclin-dependent kinases 2 (CDK2) mRNA abundance were significantly decreased in hepatocytes after DHEA treated. No significant differences were observed in the number of mitochondria, while the mitochondrial membrane permeability and succinate dehydrogenase (SDH) activity were significantly increased in hepatocytes after DHEA treated. In conclusion, our results demonstrated that DHEA reduced lipid droplet accumulation by inhibiting hepatocytes proliferation and enhancing mitochondrial function in primary chicken hepatocytes.

Abstract 581: Identification on Anti-Atherosclerotic Mechanism of SGLT-2 Inhibitor in Diabetic Rabbit Model

Aim: The sodium-glucose cotransporter 2 inhibitor (SGLT-2i) suggested a possible anti-atherosclerotic and cardioprotective effects beyond glucose lowering effect. However, further study is needed to prove underlying mechanisms of improve on cardiovascular outcomes by SGLT-2i. Therefore, we investigated possible underlying anti-atherosclerotic effect of SGLT-2i ‘Dapagliflozin’ with diabetic rabbit model by inducing atherosclerosis. Methods: Rabbit divided into two groups (each group/n=10); DA: Diabetic atherosclerosis, DAD: Diabetic atherosclerosis + Dapagliflozin. Dapagliflozin was given for a total of 8 weeks. Atherosclerotic plaques were induced with a high cholesterol (HC) diet and balloon inflation. In vivo intravascular imaging and histological assessment was performed. Results: From the histologic evaluation, atheromatous plaque and lipid accumulation were significantly less developed in the Diabetic atherosclerosis + Dapagliflozin group compared to the Diabetic atherosclerosis group. Significantly less macrophage infiltration and inflammatory proteins expression level was observed in the Diabetic atherosclerosis + Dapagliflozin group. The mRNA and protein level of pro-inflammatory markers such as HMGB1, TNF-α, iNOS, and RAGE were significantly lower in the Diabetic atherosclerosis + Dapagliflozin group. The polarization of M1 macrophages was also significantly lower in the Dapagliflozin treated group. The fibronectin and type IV collagen were less observed in the glomerulus of kidney in the Dapagliflozin treated group. The lipid droplet area and size in the liver of Dapagliflozin treated group was smaller and fewer than those in the non-treated group. Conclusion: These results suggest that dapagliflozin may prevent an acceleration atherosclerosis by reducing a vascular inflammation and modifying the macrophage polarization in diabetic animal model. This preliminary clinical observation demonstrates the therapeutic potential of dapagliflozin in diabetic patients with cardiovascular disease.

Skeletal muscle phosphatidylcholine and phosphatidylethanolamine respond to exercise and influence insulin sensitivity in men

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) composition in skeletal muscle have been linked to insulin sensitivity. We evaluated the relationships between skeletal muscle PC:PE, physical exercise and insulin sensitivity. We performed lipidomics and measured PC and PE in m. vastus lateralis biopsies obtained from 13 normoglycemic normal weight men and 13 dysglycemic overweight men at rest, immediately after 45 min of cycling at 70% maximum oxygen uptake, and 2 h post-exercise, before as well as after 12 weeks of combined endurance- and strength-exercise intervention. Insulin sensitivity was monitored by euglycemic-hyperinsulinemic clamp. RNA-sequencing was performed on biopsies, and mitochondria and lipid droplets were quantified on electron microscopic images. Exercise intervention for 12 w enhanced insulin sensitivity by 33%, skeletal muscle levels of PC by 21%, PE by 42%, and reduced PC:PE by 16%. One bicycle session reduced PC:PE by 5%. PC:PE correlated negatively with insulin sensitivity (β = −1.6, P < 0.001), percent area of mitochondria (ρ = −0.52, P = 0.035), and lipid droplet area (ρ = 0.55, P = 0.017) on EM pictures, and negatively with oxidative phosphorylation and mTOR based on RNA-sequencing. In conclusion, PC and PE contents of skeletal muscle respond to exercise, and PC:PE is inversely related to insulin sensitivity.