Formation Of Large Lipid Droplets Research Articles

Systematic characterization of all Toxoplasma gondii TBC domain-containing proteins identifies an essential regulator of Rab2 in the secretory pathway.

Toxoplasma gondii resides in its intracellular niche by employing a series of specialized secretory organelles that play roles in invasion, host cell manipulation, and parasite replication. Rab GTPases are major regulators of the parasite's secretory traffic that function as nucleotide-dependent molecular switches to control vesicle trafficking. While many of the Rab proteins have been characterized in T. gondii, precisely how these Rabs are regulated remains poorly understood. To better understand the parasite's secretory traffic, we investigated the entire family of Tre2-Bub2-Cdc16 (TBC) domain-containing proteins, which are known to be involved in vesicle fusion and secretory protein trafficking. We first determined the localization of all 18 TBC domain-containing proteins to discrete regions of the secretory pathway or other vesicles in the parasite. Second, we use an auxin-inducible degron approach to demonstrate that the protozoan-specific TgTBC9 protein, which localizes to the endoplasmic reticulum (ER), is essential for parasite survival. Knockdown of TgTBC9 results in parasite growth arrest and affects the organization of the ER and mitochondrial morphology. TgTBC9 knockdown also results in the formation of large lipid droplets (LDs) and multi-membranous structures surrounded by ER membranes, further indicating a disruption of ER functions. We show that the conserved dual-finger active site in the TBC domain of the protein is critical for its GTPase-activating protein (GAP) function and that the Plasmodium falciparum orthologue of TgTBC9 can rescue the lethal knockdown. We additionally show by immunoprecipitation and yeast 2 hybrid analyses that TgTBC9 preferentially binds Rab2, indicating that the TBC9-Rab2 pair controls ER morphology and vesicular trafficking in the parasite. Together, these studies identify the first essential TBC protein described in any protozoan and provide new insight into intracellular vesicle trafficking in T. gondii.

Cadmium facilitates the formation of large lipid droplets via PLCβ2-DAG-DGKε-PA signal pathway in Leydig cells

Cadmium (Cd) exposure damages the reproductive system. Lipid droplets (LDs) play an important role in steroid-producing cells to provide raw material for steroid hormone. We have found that the LDs of Leydig cells exposed to Cd are bigger than those of normal cells, but the effects on steroidogenesis and its underlying mechanism remains unclear. Using Isobaric tag for relative and absolute quantitation (iTARQ) proteomics, phosphodiesterase beta-2 (PLCβ2) was identified as the most significantly up-regulated protein in immature Leydig cells (ILCs) and adult Leydig cells (ALCs) derived from male rats exposed to maternal Cd. Consistent with high expression of PLCβ2, the size of LDs was increased in Leydig cells exposed to Cd, accompanied by reduction in cholesterol and progesterone (P4) levels. However, the high PLCβ2 did not result in high diacylglycerol (DAG) level, because Cd exposure up-regulated diacylglycerol kinases ε (DGKε) to promote the conversion from DAG to phosphatidic acid (PA). Exogenous PA, which was consistent with the intracellular PA concentration induced by Cd, facilitated the formation of large LDs in R2C cells, followed by reduced P4 level in the culture medium. When PLCβ2 expression was knocked down, the increased DGKε caused by Cd was reversed, and then the PA level was decreased to normal. As results, large LDs returned to normal size, and the level of total cholesterol was improved to restore steroidogenesis. The accumulation of PA regulated by PLCβ2-DAG-DGKε signal pathway is responsible for the formation of large LDs and insufficient steroid hormone synthesis in Leydig cells exposed to Cd. These data highlight that LD is an important target organelle for Cd-induced steroid hormone deficiency in males.

The rhythmic coupling of Egr-1 and Cidea regulates age-related metabolic dysfunction in the liver of male mice

The liver lipid metabolism of older individuals canbecome impaired and the circadian rhythm of genes involved in lipid metabolism is also disturbed. Although the link between metabolism and circadian rhythms is already recognized, how these processes are decoupled in liver during aging is still largely unknown. Here, we show that the circadian rhythm for the transcription factor Egr-1 expression is shifted forward with age in male mice. Egr-1 deletion accelerates liver age-related metabolic dysfunction, which associates with increased triglyceride accumulation, disruption of the opposite rhythmic coupling of Egr-1 and Cidea (Cell Death Inducing DFFA Like Effector A) at the transcriptional level and large lipid droplet formation. Importantly, adjustment of the central clock with light via a 4-hour forward shift in 6-month-old mice, leads to recovery the rhythm shift of Egr-1 during aging and largely ameliorated liver metabolic dysfunction. All our collected data suggest that liver Egr-1 might integrate the central and peripheral rhythms and regulate metabolic homeostasis in the liver.

DGAT1 protects against lipid induced-hepatic lipotoxicity in grass carp (Ctenopharyngodon idellus)

Farmed fish often suffers from fatty liver, which is characterized by the aberrant lipid accumulation. Lipid droplet is an intracellular organelle to store neutral lipid, and the formation of excessive lipid droplets in hepatocytes is associated with fatty liver disease. However, in the present study, we found that 2-week high fat (lipid 8%) intake have no adverse effect on grass carp hepatopancreas compared with control group (lipid 4%) accompanied by the lipid accumulation, indicating that the formation of lipid droplets may be a protection mechanism in response to exogenous fatty acids in farmed fish. Then, highly selective small-molecule inhibitors of DGAT (diacylglycerol acyltransferase) and oleic acid were combined to verify this hypothesis in grass carp hepatocytes. We found that inhibition of DGAT1 markedly abolished formation of lipid droplets and had a strong effect on TG (triglyceride) levels, while inhibition of DGAT2 decreased the formation of large lipid droplets and had no effect on TG levels, suggesting that DGAT1 is responsible for lipid droplet formation but DGAT2 only promotes the fusion of lipid droplet produced by DGAT1 in grass carp hepatocytes. Moreover, inhibition of DGAT1 not DGAT2 attenuated expression of lipogenic genes, which may contribute to the differential effects of DGAT1 and DGAT2 on TG synthesis. We demonstrate that inactivation of DGAT1 not DGAT2 set a metabolic switch towards fat oxidation that increased the content of reactive oxygen species, thereby leading to hepatic lipotoxicity via inducing endoplasmic reticulum stress/apotosis and inflammation. Taken together, DGAT1-dependent lipid droplet biogenesis is the cellular protective response to exogenous fatty acids in the development of hepatic steatosis. The results will help us to understand the physiological significance of lipid droplet in fish hepatocytes and the mechanisms of progressive development of farmed fish fatty liver

Nematotoxicity of a Cyt-like protein toxin from Conidiobolus obscurus (Entomophthoromycotina) on the pine wood nematode Bursaphelenchus xylophilus.

The pine wood nematode Bursaphelenchus xylophilus is a destructive pest on Pinus trees and lacks effective control measures. The present study identified a novel nematotoxic cytolytic (Cyt)-like protein originating from the entomopathogenic fungus Conidiobolus obscurus. The protein was successfully purified using heterologous expression in Escherichia coli and affinity chromatography. N-hydroxysuccinimide-rhodamine-labeled Cyt-like protein was used to establish the route of toxin uptake, and revealed that the toxin can enter the nematode via the stylet. In bioassays, the purified protein had high nematicide activity against B. xylophilus, with a median lethal concentration at 24 h of 15.8 and 29.4 μg mL-1 for juveniles and adults, respectively. Compared with the deionized water control, fecundity, thrashing, and egg hatching were significantly reduced by 97%, 98%, and 83%, respectively, with 40 μg mL-1 Cyt-like protein at 24-36 h. Staining with Oil-Red-O showed a decrease in large lipid droplet formation in the protein-treated adult nematodes. The Cyt-like protein toxin possesses high nematicide activity against B. xylophilus with effects on nematode vitality and fecundity. The potential exists to use the Cyt-like protein for the control of B. xylophilus.

Ezetimibe impairs transcellular lipid trafficking and induces large lipid droplet formation in intestinal absorptive epithelial cells

Ezetimibe inhibits Niemann-Pick C1-like 1 (NPC1L1) protein, which mediates intracellular cholesterol trafficking from the brush border membrane to the endoplasmic reticulum, where chylomicron assembly takes place in enterocytes or in the intestinal absorptive epithelial cells. Cholesterol is a minor lipid constituent of chylomicrons; however, whether or not a shortage of cholesterol attenuates chylomicron assembly is unknown. The aim of this study was to examine the effect of ezetimibe, a potent NPC1L1 inhibitor, on trans-epithelial lipid transport, and chylomicron assembly and secretion in enterocytes. Caco-2 cells, an absorptive epithelial model, grown onto culture inserts were given lipid micelles from the apical side, and chylomicron-like triacylglycerol-rich lipoprotein secreted basolaterally were analyzed after a 24-h incubation period in the presence of ezetimibe up to 50 μM. The secretion of lipoprotein and apolipoprotein B48 were reduced by adding ezetimibe (30% and 34%, respectively). Although ezetimibe allowed the cells to take up cholesterol normally, the esterification was abolished. Meanwhile, oleic acid esterification was unaffected. Moreover, ezetimibe activated sterol regulatory element-binding protein 2 by approximately 1.5-fold. These results suggest that ezetimibe limited cellular cholesterol mobilization required for lipoprotein assembly. In such conditions, large lipid droplet formation in Caco-2 cells and the enterocytes of mice were induced, implying that unprocessed triacylglycerol was sheltered in these compartments. Although ezetimibe did not reduce the post-prandial lipid surge appreciably in triolein-infused mice, the results of the present study indicated that pharmacological actions of ezetimibe may participate in a novel regulatory mechanism for the efficient chylomicron assembly and secretion.

Altered adipocyte differentiation and unbalanced autophagy in type 2 Familial Partial Lipodystrophy: an in vitro and in vivo study of adipose tissue browning

Type-2 Familial Partial Lipodystrophy is caused by LMNA mutations. Patients gradually lose subcutaneous fat from the limbs, while they accumulate adipose tissue in the face and neck. Several studies have demonstrated that autophagy is involved in the regulation of adipocyte differentiation and the maintenance of the balance between white and brown adipose tissue. We identified deregulation of autophagy in laminopathic preadipocytes before induction of differentiation. Moreover, in differentiating white adipocyte precursors, we observed impairment of large lipid droplet formation, altered regulation of adipose tissue genes, and expression of the brown adipose tissue marker UCP1. Conversely, in lipodystrophic brown adipocyte precursors induced to differentiate, we noticed activation of autophagy, formation of enlarged lipid droplets typical of white adipocytes, and dysregulation of brown adipose tissue genes. In agreement with these in vitro results indicating conversion of FPLD2 brown preadipocytes toward the white lineage, adipose tissue from FPLD2 patient neck, an area of brown adipogenesis, showed a white phenotype reminiscent of its brown origin. Moreover, in vivo morpho-functional evaluation of fat depots in the neck area of three FPLD2 patients by PET/CT analysis with cold stimulation showed the absence of brown adipose tissue activity. These findings highlight a new pathogenetic mechanism leading to improper fat distribution in lamin A-linked lipodystrophies and show that both impaired white adipocyte turnover and failure of adipose tissue browning contribute to disease.

Mapping and Profiling Lipid Distribution in a 3D Model of Breast Cancer Progression.

Aberrant lipid accumulation and marked changes in cellular lipid profiles are related to breast cancer metabolism and disease progression. In vitro, these phenomena are primarily studied using cells cultured in monolayers (2D). Here, we employ multicellular spheroids, generated using the MCF10A cell line series of increasing malignancy potential, to better recapitulate the 3D microenvironmental conditions that cells experience in vivo. Breast cancer cell lipid compositions were assessed in 2D and 3D culture models as a function of malignancy using liquid chromatography coupled with mass spectrometry. Further, the spatial distribution of lipids was examined using Raman chemical imaging and lipid staining. We show that with changes in the cellular microenvironment when moving from 2D to 3D cell cultures, total lipid amounts decrease significantly, while the ratio of acylglycerols to membrane lipids increases. This ratio increase could be associated with the formation of large lipid droplets (>10 μm) that are spatially evident throughout the spheroids but absent in 2D cultures. Additionally, we found a significant difference in lipid profiles between the more and less malignant spheroids, including changes that support de novo sphingolipid production and a reduction in ether-linked lipid fractions in the invasive spheroids. These differences in lipid profiles as a function of cell malignancy and microenvironment highlight the importance of coupled spatial and lipidomic studies to better understand the connections between lipid metabolism and cancer.

Differential Roles of CIDE Proteins in Promoting Lipid Droplet Fusion and Growth in Subpopulations of Hepatocytes

Liver plays a major regulatory role in whole-body lipid metabolism. Disruption of the hepatic lipid metabolism could lead to the initiation and progression of several metabolic disorders. The accumulation of fat in the form of lipid droplets (LDs) is an early pathophysiological feature of altered liver metabolism that is linked to insulin resistance and the potential progression of severe liver diseases, such as liver steatosis, liver cirrhosis and hepatocellular carcinoma. CIDE proteins including Cidea, Cideb and Cidec (also called Fsp27) play important roles in lipid metabolism. Cidea and Cidec are predominantly expressed in adipocytes, while Cideb is specifically expressed in the liver of wild-type mice. Cidea and Cidec are upregulated in the steatotic liver. Overexpression of Cidea/Cidec in mouse liver resulted in increased hepatic lipid accumulation and the formation of large LDs. Cidea expression in hepatocytes was specifically induced by saturated fatty acids, and such induction was reduced when SREBP1c was knocked down. Next, we find that CIDE proteins are all localized to LD-LD contact sites (LDCSs) and promote lipid transfer, LD fusion and growth in hepatocytes. We have identified two types of hepatocytes, one with small LDs (small LD-containing hepatocytes, SLHs) and one with large LDs (large LD-containing hepatocytes, LLHs) in the liver. Cideb is localized to LDCSs and promotes lipid exchange and LD fusion in both SLHs and LLHs, whereas Cidea and Cidec are specifically localized to the LDCSs and promote lipid exchange and LD fusion in LLHs. Fasting or High-fat-diet dramatically induces the expression of Cidea/Cidec and increases the percentage of LLHs in the liver. Knocking down Cidea or Cidec significantly reduced lipid storage in the livers of obese animals. Our data reveal that CIDE proteins play differential roles in promoting LD fusion and lipid storage; Cideb promotes lipid storage under normal diet conditions, while Cidea and Cidec are responsible for liver steatosis under fasting and obese conditions. Support or Funding Information This work was supported by grants from the National Basic Research Program, the National Natural Science Foundation of China and the China Postdoctoral Science Foundation. Proposed model of CIDEs mediated lipid droplets fusion in hepatocytes. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

LRRK2 mediated Rab8a phosphorylation promotes lipid storage

BackgroundSeveral mutations in leucine rich repeat kinase 2 (LRRK2) gene have been associated with pathogenesis of Parkinson’s disease (PD), a neurodegenerative disorder marked by resting tremors, and rigidity, leading to Postural instability. It has been revealed that mutations that lead to an increase of kinase activity of LRRK2 protein are significantly associated with PD pathogenesis. Recent studies have shown that some Rab GTPases, especially Rab8, serve as substrates of LRRK2 and undergo phosphorylation in its switch II domain upon interaction. Current study was performed in order to find out the effects of the phosphorylation of Rab8 and its mutants on lipid metabolism and lipid droplets growth.MethodsThe phosphorylation status of Rab8a was checked by phos-tag gel. Point mutant construct were generated to investigate the function of Rab8a. 3T3L1 cells were transfected with indicated plasmids and the lipid droplets were stained with Bodipy. Fluorescent microscopy experiments were performed to examine the sizes of lipid droplets. The interactions between Rab8a and Optineurin were determined by immunoprecipitation and western blot.ResultsOur assays demonstrated that Rab8a was phosphorylated by mutated LRRK2 that exhibits high kinase activity. Phosphorylation of Rab8a on amino acid residue T72 promoted the formation of large lipid droplets. T72D mutant of Rab8a had higher activity to promote the formation of large lipid droplets compared with wild type Rab8a, with increase in average diameter of lipid droplets from 2.10 μm to 2.46 μm. Moreover, phosphorylation of Rab8a weakened the interaction with its effector Optineurin.ConclusionsY1699C mutated LRRK2 was able to phosphorylate Rab8a and phosphorylation of Rab8a on site 72 plays important role in the fusion and enlargement of lipid droplets. Taken together, our study suggests an indirect relationship between enhanced lipid storage capacity and PD pathogenesis.

Lipid Metabolism in Mammary Epithelial Cells-A Comparison of Common In vitro Models

One of the main cellular functions of mammary epithelial cells (MEC) is the vast production of lipids to provide the new born with the energy and bioactive molecules that are essential for its development and survival. Milk lipids are secreted in a structure termed milk fat globule (MFG) which buds from the MEC, enveloped with the cellular bilayer membrane. MFG membrane composition changes as a function of lactation stage, nutrition and MFG size. In addition, MFG size differs at different stages of lactation and in response to nutritional treatments. Therefore, it is hypothesized that the MEC membrane changes according to metabolic and hormonal signals in the animal's body, and that these changes are associated with milk lipid production and secretion and MFG size. The aim of this work was to find a suitable model for studying lipogenic activity and its relation to membrane composition in the mammary gland. Two in vitro models were compared: mammary gland tissue culture ('explants') and primary culture of MEC. Prolactin treatment increased fat concentration in the explants. In addition, conjugated linoleic acid decreased fat content, but only in the presence of prolactin. Despite these interesting results, the model was not reproducible. Cells of the primary MEC culture showed cytokeratin-18 mRNA and protein expression, which validated the culture's epithelial content. In addition, this model was prolactin-sensitive, as reflected by induction of α-lactalbumin expression in response to prolactin administration. Oleic acid induced the formation of large lipid droplets, and methionine addition to the oleic acid treatment further increased lipid droplet size. In conclusion, primary culture of bovine MEC was found as an appropriate model for studying lipogenic activity with a focus on membrane composition and lipid droplet size in the mammary gland.

Cell death-inducing DNA fragmentation factor A-like effector A and fat-specific protein 27β coordinately control lipid droplet size in brown adipocytes

Adipose tissue stores neutral lipids and is a major metabolic organ involved in regulating whole-body energy homeostasis. Triacylglycerol is stored as unilocular large lipid droplets (LDs) in white adipocytes and as multilocular small LDs in brown adipocytes. Proteins of the cell death-inducing DNA fragmentation factor A-like effector (Cide) family include CideA, CideB, and fat-specific protein of 27 (FSP27). Of these, FSP27 has been shown to play a crucial role in the formation of unilocular large LDs in white adipocytes. However, the mechanisms by which brown adipocytes store small and multilocular LDs remain unclear. An FSP27 isoform, FSP27β, was recently identified. We herein report that CideA and FSP27β are mainly expressed in brown adipose tissue and that FSP27β overexpression inhibits CideA-induced LD enlargements in a dose-dependent manner in COS cells. Furthermore, RNAi-mediated FSP27β depletion resulted in enlarged LDs in HB2 adipocytes, which possess the characteristics of brown adipocytes. Brown adipocytes in FSP27-knock-out mice that express CideA, but not FSP27β, had larger and fewer LDs. Moreover, we confirmed that FSP27β and CideA form a complex in brown adipose tissue. Our results suggest that FSP27β negatively regulates CideA-promoted enlargement of LD size in brown adipocytes. FSP27β appears to be responsible for the formation of small and multilocular LDs in brown adipose tissue, a morphology facilitating free fatty acid transport to mitochondria adjacent to LDs for oxidation in brown adipocytes.

MTORC1 signalling mediates PI3K-dependent large lipid droplet accumulation in Drosophila ovarian nurse cells.

ABSTRACTInsulin and insulin-like growth factor signalling (IIS), which is primarily mediated by the PI3-kinase (PI3K)/PTEN/Akt kinase signalling cassette, is a highly evolutionarily conserved pathway involved in co-ordinating growth, development, ageing and nutrient homeostasis with dietary intake. It controls transcriptional regulators, in addition to promoting signalling by mechanistic target of rapamycin (mTOR) complex 1 (mTORC1), which stimulates biosynthesis of proteins and other macromolecules, and drives organismal growth. Previous studies in nutrient-storing germline nurse cells of the Drosophila ovary showed that a cytoplasmic pool of activated phosphorylated Akt (pAkt) controlled by Pten, an antagonist of IIS, cell-autonomously regulates accumulation of large lipid droplets in these cells at late stages of oogenesis. Here, we show that the large lipid droplet phenotype induced by Pten mutation is strongly suppressed when mTor function is removed. Furthermore, nurse cells lacking either Tsc1 or Tsc2, which negatively regulate mTORC1 activity, also accumulate large lipid droplets via a mechanism involving Rheb, the downstream G-protein target of TSC2, which positively regulates mTORC1. We conclude that elevated IIS/mTORC1 signalling is both necessary and sufficient to induce large lipid droplet formation in late-stage nurse cells, suggesting roles for this pathway in aspects of lipid droplet biogenesis, in addition to control of lipid metabolism.

Saturated fatty acid in the phospholipid monolayer contributes to the formation of large lipid droplets

The degree of saturation of fatty acid chains in the bilayer membrane structure is known to control membrane fluidity and packing density. However, the significance of fatty acid composition in the monolayers of lipid droplets (LDs) has not been elucidated. In this study, we noted a relationship between the size of LDs and the fatty acid composition of the monolayer. To obtain large LDs, we generated NIH3T3 cells overexpressing fat-specific protein 27 (FSP27). This induced the fusion of LDs, resulting in larger LDs in FSP27-overexpressing cells compared with LDs in control cells. Moreover, the lipid extracts of LDs from FSP27-overexpressing cells reconstituted large-droplet emulsions in vitro, implying that the lipid properties of LDs might affect the size of LDs. FSP27-overexpressing cells had more saturated fatty acids in the phospholipid monolayer of the LDs compared with control cells. To further investigate the effects of the degree of phospholipid unsaturation on the size of LDs, we synthesized artificial emulsions of a lipid mixed with distearoylphosphatidylcholine (DSPC, diC18:0-PC) and with dioleoylphosphatidylcholine (DOPC, diC18:1n-9-PC) and compared the sizes of the resulting LDs. The emulsions prepared from saturated PC had larger droplets than those prepared from unsaturated PC. Our results suggest that saturated fatty acid chains in phospholipid monolayers might establish the form and/or stability of large LDs.

Adipogenesis, lipogenesis and lipolysis is stimulated by mild but not severe hypoxia in 3T3-L1 cells

In-vitro investigation of the effects of hypoxia is limited by physical laws of gas diffusion and cellular O2 consumption, making prolonged exposures to stable O2 concentrations impossible. Using a gas-permeable cultureware, chronic effects of mild and severe hypoxia on triglyceride accumulation, lipid droplet size distribution, spontaneous lipolysis and gene expression of adipocyte-specific markers were assessed. 3T3-L1 cells were differentiated under 20%, 4% or 1% O2 using a gas-permeable cultureware. Triglyceride accumulation, expression of genes characteristic for advanced adipocyte differentiation and involvement of key lipogenesis enzymes were assessed after exposures. Lipogenesis increased by 375% under mild hypoxia, but dropped by 43% in severe hypoxia. Mild, but not severe, hypoxia increased formation of large lipid droplets 6.4 fold and strongly induced gene expression of adipocyte-specific markers. Spontaneous lipolysis increased by 488% in mild, but only by 135% in severe hypoxia. Inhibition of ATP-dependent citrate lyase suppressed hypoxia-induced lipogenesis by 81% and 85%. Activation of HIF inhibited lipogenesis by 59%. Mild, but not severe, hypoxia stimulates lipolysis and promotes adipocyte differentiation, probably through excess of acetyl-CoA originating from tricarboxylic acid cycle independently of HIF activation.

Large lipid droplets in leaf epidermis of angiosperms

Methods of light, scanning and transmission electron microscopy were used to study the lower epidermis of the leaves in evergreen Acokanthera oblongifolia (Apocynaceae), Exbucklandia populnea (Hamamelidaceae), Ternstroemia gymnanthera (Theaceae), and Viburnum suspensum (Caprifoliaceae). All these species show similar leaf anatomy. Our results together with those of earlier studies show that large lipid droplets (LDs) can regularly be found in leaf epidermis of some flowering plants. Their diameter varies in studied species from 3 to 11μm. Large lipid droplets are typical for cells of stomatal complexes and can have irregular shape. They also occur in some ordinary cells. The formation of large LDs is accompanied by the presence of thick cuticle covering ordinary and subsidiary cells, prominent cuticular outer ledges and rims on stomata, waxy plugs in the outer stomatal cavities (in A. oblongifolia), and abundant plastoglobules in plastids. All these indicate that intensive lipid synthesis occurs in leaf epidermis. The stomatal complexes are the place with the highest occurrence of the largest LDs. They are formed in subsidiary cells, which lie beneath the stomata. In these cells plastids and mitochondria are always present. The former contain lipid inclusions. We demonstrated that breakdown of LDs takes place in dying leaves of V. suspensum. This might indicate mobilization of lipids from the globules into the LDs. Based on these new data we suggest that the LDs found in cells of stomatal complexes play important role in maintaining normal function of stomata.

Perilipin-related protein regulates lipid metabolism in C. elegans.

Perilipins are lipid droplet surface proteins that contribute to fat metabolism by controlling the access of lipids to lipolytic enzymes. Perilipins have been identified in organisms as diverse as metazoa, fungi, and amoebas but strikingly not in nematodes. Here we identify the protein encoded by the W01A8.1 gene in Caenorhabditis elegans as the closest homologue and likely orthologue of metazoan perilipin. We demonstrate that nematode W01A8.1 is a cytoplasmic protein residing on lipid droplets similarly as human perilipins 1 and 2. Downregulation or elimination of W01A8.1 affects the appearance of lipid droplets resulting in the formation of large lipid droplets localized around the dividing nucleus during the early zygotic divisions. Visualization of lipid containing structures by CARS microscopy in vivo showed that lipid-containing structures become gradually enlarged during oogenesis and relocate during the first zygotic division around the dividing nucleus. In mutant embryos, the lipid containing structures show defective intracellular distribution in subsequent embryonic divisions and become gradually smaller during further development. In contrast to embryos, lipid-containing structures in enterocytes and in epidermal cells of adult animals are smaller in mutants than in wild type animals. Our results demonstrate the existence of a perilipin-related regulation of fat metabolism in nematodes and provide new possibilities for functional studies of lipid metabolism.

Roles of Acyl-CoA:Diacylglycerol Acyltransferases 1 and 2 in Triacylglycerol Synthesis and Secretion in Primary Hepatocytes.

Very low-density lipoprotein assembly and secretion are regulated by the availability of triacylglycerol. Although compelling evidence indicates that the majority of triacylglycerol in very low-density lipoprotein is derived from re-esterification of lipolytic products released by endoplasmic reticulum-associated lipases, little is known about roles of acyl-CoA:diacylglycerol acyltransferases (DGATs) in this process. We aimed to investigate the contribution of DGAT1 and DGAT2 in lipid metabolism and lipoprotein secretion in primary mouse and human hepatocytes. We used highly selective small-molecule inhibitors of DGAT1 and DGAT2, and we tracked storage and secretion of lipids synthesized de novo from [(3)H]acetic acid and from exogenously supplied [(3)H]oleic acid. Inactivation of individual DGAT activity did not affect incorporation of either radiolabeled precursor into intracellular triacylglycerol, whereas combined inactivation of both DGATs severely attenuated triacylglycerol synthesis. However, inhibition of DGAT2 augmented fatty acid oxidation, whereas inhibition of DGAT1 increased triacylglycerol secretion, suggesting preferential channeling of separate DGAT-derived triacylglycerol pools to distinct metabolic pathways. Inactivation of DGAT2 impaired cytosolic lipid droplet expansion, whereas DGAT1 inactivation promoted large lipid droplet formation. Moreover, inactivation of DGAT2 attenuated expression of lipogenic genes. Finally, triacylglycerol secretion was significantly reduced on DGAT2 inhibition without altering extracellular apolipoprotein B levels. Our data suggest that DGAT1 and DGAT2 can compensate for each other to synthesize triacylglycerol, but triacylglycerol synthesized by DGAT1 is preferentially channeled to oxidation, whereas DGAT2 synthesizes triacylglycerol destined for very low-density lipoprotein assembly.

Ipragliflozin, an SGLT2 inhibitor, exhibits a prophylactic effect on hepatic steatosis and fibrosis induced by choline-deficient l-amino acid-defined diet in rats

Ipragliflozin is a selective sodium glucose cotransporter 2 (SGLT2) inhibitor that increases urinary glucose excretion by inhibiting renal glucose reabsorption and thereby causes a subsequent antihyperglycemic effect. As nonalcoholic fatty liver disease (NAFLD), including nonalcoholic steatohepatitis (NASH), is closely linked to metabolic diseases such as obesity and diabetes, we investigated the effect of ipragliflozin on NAFLD in rats fed a choline-deficient l-amino acid-defined (CDAA) diet. Five weeks after starting the CDAA diet, rats exhibited hepatic triglyceride (TG) accumulation, fibrosis, and mild inflammation. Repeated oral administration of ipragliflozin (3mg/g, once daily for 5 weeks) prevented both hepatic TG accumulation (188 vs.290 mg/g tissue vehicle-treated group; P<0.001) and large lipid droplet formation. Further, ipragliflozin exerted a prophylactic effect on liver fibrosis, as indicated by a marked decrease in hydroxyproline content and fibrosis score. Pioglitazone, which is known to be effective on hepatic fibrosis in CDAA diet-fed rats as well as NASH patients with type 2 diabetes mellitus (T2DM), also exerted a mild prophylactic effect on fibrosis, but not on hepatic TG accumulation or inflammation. In conclusion, ipragliflozin prevented hepatic TG accumulation and fibrosis in CDAA-diet rats. These findings suggest the therapeutic potential of ipragliflozin for patients with NAFLD.

English

Surgical removal of intact hydatid cyst is the most effective treatment for hydatid disease. Recurrence of hydatid cyst is mainly due to dissemination of protoscolices (PSCs) rich fluid during the surgical operation. Therefore, preoperative instillation of a scolicidal agent into the cyst is a common practice with adverse side effects of the used drugs. All-trans retinoic acid (atRA) is the physiological mediator of most of the functions of vitamin A, particularly as a cellular differentiation and apoptosis regulating factor. We hypothesized that instillation of atRA could provide an alternative safe scolicidal approach. We tested the scolicidal effects and ultrastructural changes imposed by atRA on human hydatid cyst PSCs in vitro. Freshly isolated hydatid cyst PSCs were subjected to atRA (at 16.7, 1.67, 0.167 &micro;M and 16.7 nM/L). Changes in protoscolices viability (0.1% eosin exclusion) and morphology (scanning and transmission electron microscopy; SEM and TEM) were investigated. Dose-dependent PSCs death within few minutes to 7 days of exposure to atRA was observed. SEM demonstrated ultrastructural damages including rosteller disorganization, loss of hooks and distortion of hooks morphology. TEM revealed loss of the integrity of the internal tissues of PSCs, an increased vacuolization, formation of large lipid droplets in the distal cytoplasm and aberrant, rounded abnormally large sized mitochondria. atRA is a promising alternative to the available synthetic and chemical scolicidal agents. However, in vivo scolicidal activities of atRA and the possible side effects necessitate further studies. Key words: Hydatid cyst, protoscolices, retinoic acid, ultrastructure, scolicidal activity. &nbsp