Two protocols for the detection of oleaginous bacteria using Oil Red O.

The selection of oleaginous bacteria, potentially applicable to biotechnological approaches, is usually carried out by different expensive and time-consuming techniques. In this study, we used Oil Red O (ORO) as an useful dye for staining of neutral lipids (triacylglycerols and wax esters) on thin-layer chromatography plates. ORO could detect minimal quantities of both compounds (detection limit, 0.0025 mg of tripalmitin or 0.005 mg of cetylpalmitate). In addition, we developed a specific, rapid, and inexpensive screening methodology to detect triacylglycerol-accumulating microorganisms grown on the agar plate. This staining methodology detected 9/13 strains with a triacylglycerol content higher than 20% by cellular dry weight. ORO did not stain polyhydroxyalkanoates-producing bacteria. The four oleaginous strains not detected by this screening methodology exhibited a mucoid morphology of their colonies. Apparently, an extracellular polymeric substance produced by these strains hampered the entry of the lipophilic dye into cells. The utilization of the developed screening methodology would allow selecting of oleaginous bacteria in a simpler and faster way than techniques usually used nowadays, based on unspecific staining protocols and spectrophotometric or chromatographic methods. Furthermore, the use of ORO as a staining reagent would easily characterize the neutral lipids accumulated by microorganisms as reserve compounds. KEY POINTS: • Oil Red O staining is specific for triacylglycerols • Oil Red O staining is useful to detect oleaginous bacteria • Fast and inexpensive staining to isolate oleaginous bacteria from the environment.

Effect of four hydroxy fatty acids on lipid accumulation in the 3T3-L1 cells: A comparative study.

Notwithstanding the several investigations of the hydroxy fatty acids (hFAs)' physiological functions, studies focusing on their anti-obesity effects are limited. This study investigated the anti-obesity effects of four hFAs, 10-hydroxy stearic acid (10-hSA), 12-hydroxy stearic acid (12-hSA), 9,12-hydroxy stearic acid (9,12-dhSA), and 12-hydroxy oleic acid (12-hOA), on the 3T3-L1 cells. All hFAs suppressed lipid accumulation, with 10-hSA and 12-hOA exhibiting the strongest suppression, followed by 12-hSA and 9, 12-hSA. This trend was similar to that observed for the glycerol-3-phosphate dehydrogenase (GPDH) activity degree. Contrastingly, only 9,12-dhSA suppressed cell viability. The mRNA levels of HK1 and Aldoa were markedly suppressed by 10-hSA and 12-hSA compared to the control. Additionally, mRNA expression of Gyk was considerably suppressed by 12-hSA. Thus, all hFAs suppressed lipid accumulation by suppressing GPDH activity, although their molecular mechanisms were different. These findings will aid the application of hFAs in the food and medical industries.

Lipoprotein glomerulopathy with markedly increased arterial stiffness successfully treated with a combination of fenofibrate and losartan: a case report.

BACKGROUND: Lipoprotein glomerulopathy (LPG) is a apolipoprotein E (ApoE)-related glomerular disease and has been associated with type III hyperlipidemia. Without appropriate treatment, chronic kidney disease (CKD) caused by LPG progresses, and approximately half of the patients develop end-stage kidney disease within 1-27 years of disease onset. However, few studies have highlighted the clinical course of cardiovascular diseases (CVDs) in patients with LPG. Herein, we report the first case of LPG in which the CVD risk was assessed using arterial stiffness. CASE PRESENTATION: A 32-year-old Japanese man was referred to our hospital due to persistent proteinuria. Kidney biopsy showed markedly dilated capillary lumens containing pale-stained thrombi, which stained positively with Oil Red O. Electron microscopy revealed the presence of thrombi in the capillary lumen with low electron density and vacuoles of various sizes in part of the thrombi. Toluidine blue and Sudan IV stains were used to stain the thin sections of Epon-embedded tissue samples for electron microscopy. Sudan IV-positive droplets were observed in the capillary lumens, vascular walls, and cytoplasm of tubular cells. Increased serum ApoE concentration was observed. Liquid chromatography-tandem mass spectrometry of laser-microdissected glomeruli from paraffin sections revealed an increase in ApoE. Direct deoxyribonucleic acid sequencing of ApoE revealed a heterozygous ApoE Sendai mutation (Arg145Pro). The patient was finally diagnosed with LPG with heterozygosity for ApoE-Sendai mutation (Arg145Pro). Notably, at the time of diagnosis, he had markedly increased arterial stiffness for his age. Arterial stiffness was measured using brachial-ankle pulse wave velocity (baPWV), which was equivalent to that of a 56-year-old man. After three months of treatment with fenofibrate and losartan, a significant reduction in proteinuria was achieved along with an improvement in baPWV. Furthermore, these effects were maintained despite the lack of decrease in serum ApoE levels. CONCLUSION: Herein, we report the case of a patient with LPG with markedly increased arterial stiffness at the time of diagnosis, in whom combination therapy with fenofibrate and losartan successfully improved proteinuria and arterial stiffness. To the best of our knowledge, this is the first case report of LPG in which CVD risk was assessed using arterial stiffness.

Differential Lipid Accumulation on HepG2 Cells Triggered by Palmitic and Linoleic Fatty Acids Exposure.

Lipid metabolism pathways such as ß-oxidation, lipolysis and, lipogenesis, are mainly associated with normal liver function. However, steatosis is a growing pathology caused by the accumulation of lipids in hepatic cells due to increased lipogenesis, dysregulated lipid metabolism, and/or reduced lipolysis. Accordingly, this investigation hypothesizes a selective in vitro accumulation of palmitic and linoleic fatty acids on hepatocytes. After assessing the metabolic inhibition, apoptotic effect, and reactive oxygen species (ROS) generation by linoleic (LA) and palmitic (PA) fatty acids, HepG2 cells were exposed to different ratios of LA and PA to study the lipid accumulation using the lipophilic dye Oil Red O. Lipidomic studies were also carried out after lipid isolation. Results revealed that LA was highly accumulated and induced ROS production when compared to PA. Lipid profile modifications were observed after LA:PA 1:1 (v/v) exposure, which led to a four-fold increase in triglycerides (TGs) (mainly in linoleic acid-containing species), as well as a increase in cholesterol and polyunsaturated fatty acids (PUFA) content when compared to the control cells. The present work highlights the importance of balancing both PA and LA fatty acids concentrations in HepG2 cells to maintain normal levels of free fatty acids (FFAs), cholesterol, and TGs and to minimize some of the observed in vitro effects (i.e., apoptosis, ROS generation and lipid accumulation) caused by these fatty acids.

Germinated Rice Seeds Improved Resveratrol Production to Suppress Adipogenic and Inflammatory Molecules in 3T3-L1 Adipocytes.

Obesity is a major risk factor for a variety of diseases and contributes to chronic inflammation. Resveratrol is a naturally occurring antioxidant that can reduce adipogenesis. In this study, the antiadipogenic and anti-inflammatory activities of resveratrol-enriched rice were investigated in 3T3-L1 adipocyte cells. Cotreatment of dexamethasone and isobutylmethylxanthin upregulated adipogenic transcription factors and signaling pathways. Subsequent treatment of adipocytes with rice seed extracts suppressed the differentiation of 3T3-L1 by downregulating adipogenic transcription factors (peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α) and signaling pathways (extracellular signal-regulated kinase 1/2 and protein kinase B Akt), this was especially observed in cells treated with germinated resveratrol-enriched rice seed extract (DJ526_5). DJ526_5 treatment also markedly reduced lipid accumulation in the cells and expression of adipogenic genes. Lipopolysaccharide (LPS)-induced inflammatory cytokines (prostaglandin-endoperoxide synthase 2 (COX-2), tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6) decreased in cells treated with DJ526_5. Collectively, DJ526_5 exerts antiadipogenic effects by suppressing the expression of adipogenesis transcription factors. Moreover, DJ526_5 ameliorates anti-inflammatory effects in 3T3-L1 adipocytes by inhibiting the activation of phosphorylation NF-κB p65 and ERK ½ (MAPK). These results highlight the potential of resveratrol-enriched rice as an alternative obesity-reducing and anti-inflammatory agent.

In Vitro Assessment of Anti-Adipogenic and Anti-Inflammatory Properties of Black Cumin (Nigella sativa L.) Seeds Extract on 3T3-L1 Adipocytes and Raw264.7 Macrophages.

Background and Objectives: This study evaluated the in vitro anti-adipogenic and anti-inflammatory properties of black cumin (Nigella sativa L.) seed extract (BCS extract) as a potential candidate for developing herbal formulations targeting metabolic disorders. Materials and Methods: We evaluated the BCS extract by assessing its 2,2-diphenyl-1-picrohydrazyl (DPPH) radical scavenging activity, levels of prostaglandin E2 (PGE2) and nitric oxide (NO), and mRNA expression levels of key pro-inflammatory mediators. We also quantified the phosphorylation of nuclear factor kappa light chain enhancer of activated B cells (NF-κB) and mitogen-activated protein kinases (MAPK) signaling molecules. To assess anti-adipogenic effects, we used differentiated 3T3-L1 cells and BCS extract in doses from 10 to 100 µg/mL. We also determined mRNA levels of key adipogenic genes, including peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein α (C/BEPα), adipocyte protein 2 (aP2), lipoprotein lipase (LPL), fatty acid synthase (FAS), and sterol-regulated element-binding protein 1c (SREBP-1c) using real-time quantitative polymerase chain reaction (qPCR). Results: This study showed a concentration-dependent DPPH radical scavenging activity and no toxicity at concentrations up to 30 µg/mL in Raw264.7 cells. BCS extract showed an IC50 of 328.77 ± 20.52 µg/mL. Notably, pre-treatment with BCS extract (30 µg/mL) significantly enhanced cell viability in lipopolysaccharide (LPS)-treated Raw264.7 cells. BCS extract treatment effectively inhibited LPS-induced production of PGE2 and NO, as well as the expression of monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), inducible NO synthase (iNOS), interleukin (IL)-1ß and IL-6, possibly by limiting the phosphorylation of p38, p65, inhibitory κBα (I-κBα), and c-Jun N-terminal kinase (JNK). It also significantly attenuated lipid accumulation and key adipogenic genes in 3T3-L1 cells. Conclusions: This study highlights the in vitro anti-adipogenic and anti-inflammatory potential of BCS extract, underscoring its potential as a promising candidate for managing metabolic disorders.

Isolation of phenolic acids and tannin acids from Mangifera indica L. kernels as inhibitors of lipid accumulation in 3T3-L1 cells.

Mango (Mangifera indica L.) kernels are usually discarded as waste, but they contain many pharmacological properties and bioactivities. In this study, we isolated antiobesity agents from mango kernels that inhibit intracellular lipid formation in 3T3-L1 adipocytes. Two phenolic acids, ethyl gallate and ethyl digallate, and 2 tannin acids, 1,2,3,4,6-penta-O-galloyl-ß-d-glucose (PGG) and 3-O-digalloyl-1,2,4,6-tetra-O-ß-d-glucose (HGG), were identified from mango kernels and were found to be suppressed lipid accumulation as evidenced by Oil Red O staining. Furthermore, ethyl digallate, PGG, and HGG significantly downregulated the mRNA expression of adipogenic transcription factors such as C/EBPα and PPARγ. However, ethyl gallate did not affect the expression of these transcription factors. Our findings reveal the presence of antiobesity compounds in mango kernels, implying its therapeutic role against obesity.

A Real-Time Detection Device for the Rapid Quantification of Skin Casual Sebum Using the Oil Red O Staining Method.

The human skin sebum suggests that it (along with other epidermal surface lipids) plays a role in skin barrier formation, the moderation of cutaneous inflammation, and antimicrobial defense. Various methods have been developed for collecting and measuring skin sebum. We tested methods of detection using "color intensity", by staining the skin casual sebum. This process was conducted in three steps; first, the selection of materials for sebum collection; second, staining the collected sebum; third, the development of a device that can measure the level of stained sebum. A plastic film was used to effectively collect sebum that increased with the replacement time of the sebum. In addition, the collected sebum was stained with Oil Red O (ORO) and checked with RGB; as a result, the R2 value was higher than 0.9. It was also confirmed that the correlation value was higher than 0.9 in the comparison result with Sebumeter®, which is a common standard technology. Finally, it was confirmed that the R2 value was higher than 0.9 in the detection value using the sensor. In conclusion, we have proven the proof of concept (PoC) for this method, and we would like to introduce an effective sebum measurement method that differs from the existing method.

Vaping-Associated Lung Injury: A Review.

Since commercial development in 2003, the usage of modern electronic cigarette (e-cigarette) continues to increase amongst people who have never smoked, ex-smokers who have switched to e-cigarettes, and dual-users of both conventional cigarettes and e-cigarettes. With such an increase in use, knowledge of the irritative, toxic and potential carcinogenic effects on the lungs is increasing. This review article will discuss the background of e-cigarettes, vaping devices and explore their popularity. We will further summarise the available literature describing the mechanism of lung injury caused by e-cigarette or vaping use.

FABP5 Is a Sensitive Marker for Lipid-Rich Macrophages in the Luminal Side of Atherosclerotic Lesions.

Lipid-rich macrophages in atherosclerotic lesions are thought to be derived from myeloid and vascular smooth muscle cells. A series of studies with genetic and pharmacological inhibition of fatty acid binding protein 4 (FABP4) and FABP5 and bone marrow transplant experiments with FABP4/5 deficient cells in mice have demonstrated that these play an important role in the development of atherosclerosis. However, it is still uncertain about the differential cell-type specificity and distribution between FABP4- and FABP5-expressing cells in early- and late-stage atherosclerotic lesions. In this study, we first explored spatial distribution of FABP4/5 in atherosclerotic lesions in apolipoprotein E deficient (ApoE-/-) mice. FABP4 was only marginally detected in early and advanced lesions, whereas FABP5 was abundantly expressed in these lesions. In advanced lesions, the FABP5-positive area was mostly restricted to the foam cell layer adjacent to the lumen above collagen and elastic fibers with a high signal/noise ratio. Oil red O (ORO) staining revealed that FABP5-positive cells were lipid-rich in early and advanced lesions. Together, most of lipid-rich FABP5-positive cells reside adjacent to the lumen above collagen and elastic fibers. We next studied involvement of FABP5 in lesion formation of atherosclerosis using ApoE-/- FABP5-/- mice. However, deletion of FABP5 did not affect the development of atherosclerosis. These findings, along with previous reports, suggest a novel notion that FABP5 is a sensitive marker for bone marrow-derived lipid-rich macrophages in the luminal side of atherosclerotic lesions, although its functional significance remains elusive.

Divergence exists in the subcellular distribution of intramuscular triglyceride in human skeletal muscle dependent on the choice of lipid dye.

Despite over 50 years of research, a comprehensive understanding of how intramuscular triglyceride (IMTG) is stored in skeletal muscle and its contribution as a fuel during exercise is lacking. Immunohistochemical techniques provide information on IMTG content and lipid droplet (LD) morphology on a fibre type and subcellular-specific basis, and the lipid dye Oil Red O (ORO) is commonly used to achieve this. BODIPY 493/503 (BODIPY) is an alternative lipid dye with lower background staining and narrower emission spectra. Here we provide the first quantitative comparison of BODIPY and ORO for investigating exercise-induced changes in IMTG content and LD morphology on a fibre type and subcellular-specific basis. Estimates of IMTG content were greater when using BODIPY, which was predominantly due to BODIPY detecting a larger number of LDs, compared to ORO. The subcellular distribution of intramuscular lipid was also dependent on the lipid dye used; ORO detects a greater proportion of IMTG in the periphery (5 µm below cell membrane) of the fibre, whereas IMTG content was higher in the central region using BODIPY. In response to 60 min moderate-intensity cycling exercise, IMTG content was reduced in both the peripheral (- 24%) and central region (- 29%) of type I fibres (P < 0.05) using BODIPY, whereas using ORO, IMTG content was only reduced in the peripheral region of type I fibres (- 31%; P < 0.05). As well as highlighting some methodological considerations herein, our investigation demonstrates that important differences exist between BODIPY and ORO for detecting and quantifying IMTG on a fibre type and subcellular-specific basis.

Histological validation of adipogenic differentiation potential of ASC on collagen-based 2D scaffolds.

Determination of the adipogenic potential and behavior of adipose-derived mesenchymal stem/stromal cells (ASCs) is particularly relevant for their potential clinical application in regenerative medicine, especially when regeneration is supported by biomaterials or scaffolds. Scaffolds need to be able to induce tissue repair and limit undesired adipogenic differentiation. Depending on the scaffold employed, determination of cell behavior may be hindered by material interference with staining, which will limit either cells identification or dye quantification. Collagen is a promising biomaterial in regenerative medicine, however, histological analysis of cells cultured on collagen-based scaffolds is challenging. Here we describe a new histological method based on iron hematoxylin combined with Oil red O (ORO) staining, for the determination of the adipogenic differentiation of ASCs cultivated on a collagen-based 2D scaffold. ASCs were seeded on collagen films or plastic, differentiated into adipocytes for 14 days, and then stained with either ORO or iron hematoxylin and ORO combined. The collagen films avidly absorbed the ORO dye; conventional staining and quantification by dye extraction failed to discriminate between differentiated and undifferentiated cells on the films. On the contrary, the iron hematoxylin-ORO combination provided a quantitative and more reliable determination of adipocytes based on single cell count. This method is particularly recommended for determining the adipogenic differentiation potential of ASCs and other cell types grown on highly absorptive materials that need to be validated for their potential use in bioengineering and regenerative medicine.

Tectorigenin attenuates diabetic nephropathy by improving vascular endothelium dysfunction through activating AdipoR1/2 pathway.

Diabetic nephropathy (DN), a kind of microvascular complication, is a primary cause of end-stage renal disease worldwide. However, therapeutic drugs for DN treatment are still in lack. The glomerular endothelium is essential to maintain selective permeability of glomerular filtration barrier and glomerular vasculature function. Growing evidences show that endothelial dysfunction or injury is the initial stage of vascular damage in DN, which can be induced by hyperglycemia, lipotoxicity, and inflammation. Therefore, to improve the function of vascular endothelium in kidney is a key point for treatment of DN. As a plant isoflavone, tectorigenin (TEC) has attracted considerable attention due to its anti-proliferative and anti-inflammatory functions. However, whether TEC could inhibit the DN development remains unknown. In this study, we examined the effects of TEC on DN development in db/db mice, a type of genetic defect diabetic mice that can spontaneously develop into severe renal dysfunction. Intriguingly, TEC treatment restored diabetes-induced glucose and lipid metabolic disorder; and improved the deterioration of renal function, particularly the renal endothelium function in db/db mice. Additionally, TEC inhibited the renal inflammation via reducing macrophages infiltration and M1 polarization. Moreover, TEC inhibited lipopolysaccharide (LPS)-induced endothelial injury and M1 polarization in vitro. Mechanistically, TEC partially restored the reduction in expression of adiponectin receptor 1/2 (AdipoR1/2), pi-LKB1, pi-AMPKα, and PPARα in vitro and in vivo. Noteworthy, these beneficial pharmacological activities mediated by TEC were significantly attenuated after AdipoR1/2 knockdown by siRNA, indicating that AdipoR1/2 plays a critical role in protection against DN. Collectively, these results suggested that TEC have a potently effect for retarding type 2 diabetes-associated DN.

9-PAHSA Prevents Mitochondrial Dysfunction and Increases the Viability of Steatotic Hepatocytes.

Nonalcoholic fatty liver disease (NAFLD) is quickly becoming the most common liver disease worldwide. Within the NAFLD spectrum, patients with nonalcoholic steatohepatitis (NASH) are at the highest risk of developing cirrhosis and disease progression to hepatocellular carcinoma. To date, therapeutic options for NASH patients have been ineffective, and therefore, new options are urgently needed. Hence, a model system to develop new therapeutic interventions is needed. Here, we introduce two new in vitro models of steatosis induction in HepG2 cells and primary murine hepatocytes. We used a recently discovered novel class of bioactive anti-inflammatory lipids called branched fatty acid esters of hydroxyl fatty acids. Among these bioactive lipids, palmitic-acid-9-hydroxy-stearic-acid (9-PAHSA) is the most promising as a representative nondrug therapy based on dietary supplements or nutritional modifications. In this study, we show a therapeutic effect of 9-PAHSA on lipotoxicity in steatotic primary hepatocytes and HepG2 cells. This could be shown be increased viability and decreased steatosis. Furthermore, we could demonstrate a preventive effect in HepG2 cells. The outcome of 9-PAHSA administration is both preventative and therapeutically effective for hepatocytes with limited damage. In conclusion, bioactive lipids like 9-PAHSA offer new hope for prevention or treatment in patients with fatty liver and steatosis.

The significance of lipid accumulation in breast carcinoma cells through perilipin 2 and its clinicopathological significance.

Both systemic and intratumoral lipid metabolism have been recently reported to play pivotal roles in both tumor development and progression in various human malignancies including breast cancer. However, its details have remained largely unknown in breast cancer patients. Therefore, in this study, we focused on perilipin 2, which is involved in constituting the intracellular lipid composition. Perilipin 2 was first immunolocalized in 105 cases of breast cancer. The status of perilipin 2 immunoreactivity was significantly positively associated with histological grade, Ki-67 labeling index and HER2 status and negatively with estrogen receptor status of these patients. Subsequent in vitro study also revealed that its mRNA expression in triple negative breast carcinoma cells was higher than cells of other subtypes. We then examined the correlation between perilipin 2 immunoreactivity and intracellular lipid droplet evaluated by Oil-red O stating in 13 cases of breast carcinoma tissues. A significantly positive correlation was detected between the status of perilipin 2 and Oil-red O staining. These findings above did indicate that perilipin 2 could represent the status of intracellular lipid droplets in surgical pathology specimens of breast cancer and perilipin 2 was also associated with its more aggressive biological phenotypes.

Crude microcystins extracted from Microcystis aeruginosa exert anti-obesity effects by downregulating angiogenesis and adipogenesis related signaling molecules in HUVEC and 3 T3-L1 cells.

BACKGROUND: Obesity is a risk factor for many diseases including diabetes, cancer, arthritis, and cardiovascular diseases. Angiogenesis nourishes adipose tissues and contributes to obesity; it can be prevented by suppressing the expression of associated signaling molecules. Natural products have garnered attention owing to their safety and efficacy in treating several diseases, including obesity. METHODS: Crude Microcystins were extracted from the blooming Microcystis aeruginosa under stress conditions, by ultrasonication following by solvent extraction. The microcystin extract was evaluated for its potential of inhibiting angiogenesis and adipogenesis. The antiangiogenic activity of the microcystins extract was investigated using human umbilical vein endothelial cells (HUVECs), and its anti-obesity activity was determined in vitro by quantification of the accumulated lipids in mouse 3 T3-L1 cells via Oil Red O staining method. RESULTS: The microcystin extract suppressed HUVECs proliferation and tubes formation in Matrigel in a dose-dependent manner. RT-PCR analysis revealed the downregulation of the mRNA expression of angiogenesis-related signaling molecules, such as PI3K, ß-catenin, vascular endothelial growth factor receptor-2 (VEGFR-2), vascular endothelial-cadherin, Akt1, and NF-κB. Additionally, it inhibited the differentiation of premature 3 T3 cells and lipid accumulation in a dose-dependent manner. It suppressed adipogenesis and lipogenesis by reducing the expression level of peroxisome proliferator-activated receptor γ, CCAAT/enhancer binding protein α, and sterol regulatory element-binding protein. CONCLUSIONS: Crude microcystin exerts anti-angiogenic and anti-obesity effects due to the inhibitory effects on the genes expression of associated signaling molecules and transcriptional factors.

Live cell imaging and analysis of lipid droplets biogenesis in hepatatis C virus infected cells.

Lipid droplets (LDs) are regulated neutral lipid storage organelles having a central role in numerous cellular processes as well as in various pathologies such as metabolic disorders, immune responses and during pathogen infection. Due to the growing significance of LDs, extensive efforts are made to study the mechanism and the dynamics of their formation and life history and how are these diverted or modified by pathogens. Real-time visualization of lipid droplet biogenesis can assist in clarifying these and other important issues and may have implications towards understanding the pathogenesis of the associated diseases. Typically, LDs are post-experimentally stained using lipophilic dyes and are visualized under a microscope. Alternatively, overexpression of LD-associated proteins or immunofluorescence analyses are used to identify and follow LDs. These experimental approaches only examine a single end point of the experiment and cannot answer questions regarding LD dynamics. Here, we describe a simple and novel experimental setting that allows real-time fluorescence staining and detection of LDs in cultured living as well as infected cells. This method is quick and simple and is not restricted to a specific dye or cell line. Using this system, the biogenesis of LDs and their growth is demonstrated in cells infected with hepatitis C virus (HCV), confirming the strength of this method and the wide range of its applications.

Microsomal triglyceride transfer protein in the ectoparasitic crustacean salmon louse (Lepeophtheirus salmonis).

The salmon louse, Lepeophtheirus salmonis, is an endemic ectoparasite on salmonid fish that is challenging for the salmon farming industry and wild fish. Salmon lice produce high numbers of offspring, necessitating sequestration of large amounts of lipids into growing oocytes as a major energy source for larvae, most probably mediated by lipoproteins. The microsomal triglyceride transfer protein (MTP) is essential for the assembly of lipoproteins. Salmon lice have three L. salmonis MTP (LsMTP) transcript variants encoding two different protein isoforms, which are predicted to contain three ß-sheets (N, C, and A) and a central helical domain, similar to MTPs from other species. In adult females, the LsMTPs are differently transcribed in the sub-cuticular tissues, the intestine, the ovary, and in the mature eggs. RNA interference-mediated knockdown of LsMTP in mature females gave offspring with significantly fewer neutral lipids in their yolk and only 10-30% survival. The present study suggests the importance of LsMTP in reproduction and lipid metabolism in adult female L. salmonis, a possible metabolic bottleneck that could be exploited for the development of new anti-parasitic treatment methods.

Exploration of Underlying Mechanism of Anti-adipogenic Activity of Sulfuretin.

Sulfuretin is a natural flavonoid found in the plant Rhus verniciflua STOKES. The plant has been traditionally used as medicinal agent for antiviral, cathartic, diaphoretic, anti-rheumatic and sedative activities in East Asia. In this study we isolated and identified sulfuretin from R. verniciflua and investigated its anti-adipogenic activity against 3T3-L1 preadipocytes cells. We evaluated the effects of sulfuretin on the adipogenic transcription factors like peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), fatty acid synthase (FAS), Fabp4, adiponectin and zinc fingerprint protein (Zfp) 521 by gene expression (real-time QPCR) and Western blot analysis. Sulfuretin treatment at Day 0 and 2 showed significant reduction of lipid production in 3T3-L1 cells in concentration dependent manner. Gene expression analysis (real-time PCR) revealed that sulfuretin inhibited the both major adipogenic factors (C/EBPα, C/EBPß and PPARγ) and minor adipogenic factors (sterol regulatory element-binding protein (SREBP1c), adiponectin, FAS, Fabp4, Zfp423, and Ebf1). Western blot analysis showed the increased expression of ß-catenin and suppression of PPARγ after sulfuretin treatment. Overall, sulfuretin is a natural flavonoid having potent anti-adipogenic activity through the suppression of major adipogenic factors C/EBPα, C/EBPß and PPARγ, which initiate adipogenesis.

Diversity of Lipid Distribution in Fish Skeletal Muscle.

Adipose tissue is a lipid storage organ characterized by the pronounced accumulation of adipocytes. Although adipose tissues are found in various parts of the vertebrate body, it is unclear whether these tissues have a common ancestral origin or have evolved in several phylogenetic lineages by independent adipocyte accumulation events. To gain insight into the evolutionary history of vertebrate adipose tissues, we determined the distribution of adipocytes by oil red O staining in skeletal muscle of 10 teleost species spanning eight orders: Tetraodontiformes, Pleuronectiformes, Spariformes, Salmoniformes, Clupeiformes, Beloniformes, Osmeriformes, and Cypriniformes. Accumulation of adipocytes in the myoseptum was observed in many species, including red seabream, rainbow trout, Pacific herring, Pacific saury, zebrafish and giant danio. We also found some order-, species-, and swimming mode-specific distribution patterns of adipocytes: 1) almost complete absence of intramuscular adipocytes in the order Tetraodontiformes (torafugu and spotted green pufferfish), 2) clear adipocyte accumulation in the inclinator muscles of fin in Japanese flounder, 3) a large intramuscular adipose tissue at the root of the dorsal fin in ayu, and 4) thick lipid layers consisting of subcutaneous adipose tissue and red muscle lipids in pelagic migratory fish (Pacific herring and Pacific saury). Of note, Pacific herring and Pacific saury are phylogenetically distinct species sharing a similar niche and swimming mode, suggesting that their analogous adipocyte/lipid distribution patterns are the consequence of convergent evolution. The potentially heterogeneous origin of adipose tissues has significant implications for the interpretation of their functional diversity.