Surface Of Lipid Droplets Research Articles

CIDE Family-Mediated Unique Lipid Droplet Morphology in White Adipose Tissue and Brown Adipose Tissue Determines the Adipocyte Energy Metabolism.

White adipose tissue (WAT) stores energy as triacylglycerol in preparation for fasting state. In contrast, brown adipose tissue (BAT) consumes energy and produces heat in a cold environment. One of the major differences between these two adipose tissues is the morphology of the intracellular lipid droplet (LD), which is large and unilocular in WAT and small and multilocular in BAT. Although the fat-specific protein 27 alpha (FSP27α), belonging to the cell death-inducing DNA fragmentation factor A (DFFA)-like effector (Cide) family, was known to be indispensable for large unilocular LD formation in WAT, the mechanism that regulated small multilocular LD formation in BAT remained unknown. We recently uncovered that FSP27β, a novel isoform of FSP27 abundantly expressed in BAT, plays a crucial role in small multilocular LD formation by inhibiting the homodimerization of CideA in BAT. We speculate that unilocular LD formation is ideal for efficient lipid storage in WAT because lipolysis from the LD surface is restricted due to the minimum LD surface area. In addition, hydrolyzed free fatty acid (FFA) and glycerol can efficiently flow out into the circulation from the cell surface. In contrast, small multilocular LD formation is ideal for efficient intracellular lipolysis from the LD surface and the subsequent facilitation of FFA transport to mitochondria that are adjacent to LDs for β-oxidation in BAT. Thus, intracellular LD morphology is closely related to the functions and characteristics of adipose tissues. Given that the browning of adipose tissue leads to enhanced energy expenditure and the prevention of obesity, clarification of the mechanism with respect to intracellular LD formation is very meaningful.

COPI-TRAPPII activates Rab18 and regulates its lipid droplet association.

The transport protein particle (TRAPP) was initially identified as a vesicle tethering factor in yeast and as a guanine nucleotide exchange factor (GEF) for Ypt1/Rab1. In mammals, structures and functions of various TRAPP complexes are beginning to be understood. We found that mammalian TRAPPII was a GEF for both Rab18 and Rab1. Inactivation of TRAPPII-specific subunits by various methods including siRNA depletion and CRISPR-Cas9-mediated deletion reduced lipolysis and resulted in aberrantly large lipid droplets. Recruitment of Rab18 onto lipid droplet (LD) surface was defective in TRAPPII-deleted cells, but the localization of Rab1 on Golgi was not affected. COPI regulates LD homeostasis. We found that the previously documented interaction between TRAPPII and COPI was also required for the recruitment of Rab18 to the LD We hypothesize that the interaction between COPI and TRAPPII helps bring TRAPPII onto LD surface, and TRAPPII, in turn, activates Rab18 and recruits it on the LD surface to facilitate its functions in LD homeostasis.

The cytoskeletal protein septin 11 is associated with human obesity and is involved in adipocyte lipid storage and metabolism.

Septins are newly identified members of the cytoskeleton that have been proposed as biomarkers of a number of diseases. However, septins have not been characterised in adipose tissue and their relationship with obesity and insulin resistance remains unknown. Herein, we characterised a member of this family, septin 11 (SEPT11), in human adipose tissue and analysed its potential involvement in the regulation of adipocyte metabolism. Gene and protein expression levels of SEPT11 were analysed in human adipose tissue. SEPT11 distribution was evaluated by immunocytochemistry, electron microscopy and subcellular fractionation techniques. Glutathione S-transferase (GST) pull-down, immunoprecipitation and yeast two-hybrid screening were used to identify the SEPT11 interactome. Gene silencing was used to assess the role of SEPT11 in the regulation of insulin signalling and lipid metabolism in adipocytes. We demonstrate the expression of SEPT11 in human adipocytes and its upregulation in obese individuals, with SEPT11 mRNA content positively correlating with variables of insulin resistance in subcutaneous adipose tissue. SEPT11 content was regulated by lipogenic, lipolytic and proinflammatory stimuli in human adipocytes. SEPT11 associated with caveolae in mature adipocytes and interacted with both caveolin-1 and the intracellular fatty acid chaperone, fatty acid binding protein 5 (FABP5). Lipid loading of adipocytes caused the association of the three proteins with the surface of lipid droplets. SEPT11 silencing impaired insulin signalling and insulin-induced lipid accumulation in adipocytes. Our findings support a role for SEPT11 in lipid traffic and metabolism in adipocytes and open new avenues for research on the control of lipid storage in obesity and insulin resistance.

Fluorescence imaging of spatial location of lipids and proteins during digestion of protein-stabilized oil-in-water emulsions: A simulated gastrointestinal tract study.

A whey protein isolate-rhodamine B conjugate (WPI-RB) was synthesized to visualize changes in the location of a protein emulsifier in oil-in-water emulsions during digestion. An oil-soluble dye (Nile Red) was used to visualize changes in the lipid phase during digestion. Protein-labeled and lipid-labeled emulsions were passed through a simulated gastrointestinal tract consisting of mouth, stomach, and intestinal phases, and changes in protein and lipid location and morphology were monitored using confocal laser scanning microscopy. The proteins remained attached to the lipid droplet surfaces in the mouth and stomach, but formed large aggregates in the small intestine. The lipid droplets were highly flocculated in the mouth, highly coalesced in the stomach, and fully digested in the small intestine. Our results show that conjugation of fluorescence dyes to protein emulsifiers is useful for direct visualization of their location, as well as for understanding the behavior of food emulsions within the gastrointestinal tract.

MiR-148a and miR-30a limit HCV-dependent suppression of the lipid droplet protein, ADRP, in HCV infected cell models.

Hepatitis C Virus (HCV) promotes lipid droplet (LD) formation and perturbs the expression of the LD associated PAT proteins ADRP and TIP47, to promote its own lifecycle. HCV enhances TIP47 and suppresses ADRP by displacing it from LD surface in infected cell models. We have previously shown that suppression of TIP47 by miR-148a and miR-30a decreased intracellular LDs and HCV RNA. Thus, this study aimed at examining whether this microRNA-mediated suppression of HCV would limit HCV-dependent displacement of ADRP from LDs. ADRP expression was examined in 21 HCV-infected liver biopsies and 9 healthy donor liver tissues as well as in HCV-infected Huh7 cells using qRT-PCR. miR-148a and miR-30a expression was manipulated using specific oligos in JFH-1 infected, oleic acid treated cells, to study their impact on ADRP expression using qRT-PCR, and immunofluorescence microscopy. Intracellular HCV RNA was assessed using qRT-PCR. ADRP is down regulated in patients as well as HCVcc-JFH-I infected cell models. Forcing the expression of both miRNAs induced ADRP on the mRNA and protein levels. This study shows that HCV suppresses hepatic ADRP expression in infected patients and cell lines. Forcing the expression of miR-148a and miR-30a limits the suppressive effect of HCV on ADRP. J. Med. Virol. 89:653-659, 2017. © 2016 Wiley Periodicals, Inc.

Effect of Quercetin on Hepatitis C Virus Life Cycle: From Viral to Host Targets.

Quercetin is a natural flavonoid, which has been shown to have anti hepatitis C virus (HCV) properties. However, the exact mechanisms whereby quercetin impacts the HCV life cycle are not fully understood. We assessed the effect of quercetin on different steps of the HCV life cycle in Huh-7.5 cells and primary human hepatocytes (PHH) infected with HCVcc. In both cell types, quercetin significantly decreased i) the viral genome replication; ii) the production of infectious HCV particles and iii) the specific infectivity of the newly produced viral particles (by 85% and 92%, Huh7.5 and PHH respectively). In addition, when applied directly on HCV particles, quercetin reduced their infectivity by 65%, suggesting that it affects the virion integrity. Interestingly, the HCV-induced up-regulation of diacylglycerol acyltransferase (DGAT) and the typical localization of the HCV core protein to the surface of lipid droplets, known to be mediated by DGAT, were both prevented by quercetin. In conclusion, quercetin appears to have direct and host-mediated antiviral effects against HCV.

The Perilipins: Major Cytosolic Lipid Droplet-Associated Proteins and Their Roles in Cellular Lipid Storage, Mobilization, and Systemic Homeostasis.

The discovery by Dr. Constantine Londos of perilipin 1, the major scaffold protein at the surface of cytosolic lipid droplets in adipocytes, marked a fundamental conceptual change in the understanding of lipolytic regulation. Focus then shifted from the enzymatic activation of lipases to substrate accessibility, mediated by perilipin-dependent protein sequestration and recruitment. Consequently, the lipid droplet became recognized as a unique, metabolically active cellular organelle and its surface as the active site for novel protein-protein interactions. A new area of investigation emerged, centered on lipid droplets' biology and their role in energy homeostasis. The perilipin family is of ancient origin and has expanded to include five mammalian genes and a growing list of evolutionarily conserved members. Universally, the perilipins modulate cellular lipid storage. This review provides a summary that connects the perilipins to both cellular and whole-body homeostasis.

Spatial control of lipid droplet proteins by the ERAD ubiquitin ligase Doa10.

The endoplasmic reticulum (ER) plays a central role in the biogenesis of most membrane proteins. Among these are proteins localized to the surface of lipid droplets (LDs), fat storage organelles delimited by a phospholipid monolayer. The LD monolayer is often continuous with the membrane of the ER allowing certain membrane proteins to diffuse between the two organelles. In these connected organelles, how some proteins concentrate specifically at the surface of LDs is not known. Here, we show that the ERAD ubiquitin ligase Doa10 controls the levels of some LD proteins. Their degradation is dependent on the localization to the ER and appears independent of the folding state. Moreover, we show that by degrading the ER pool of these LD proteins, ERAD contributes to restrict their localization to LDs. The signals for LD targeting and Doa10‐mediated degradation overlap, indicating that these are competing events. This spatial control of protein localization is a novel function of ERAD that might contribute to generate functional diversity in a continuous membrane system.

Influence of methylcellulose on attributes of β-carotene fortified starch-based filled hydrogels: Optical, rheological, structural, digestibility, and bioaccessibility properties

There is considerable interest in controlling the gastrointestinal fate of nutraceuticals to improve their efficacy. In this study, the influence of methylcellulose (an indigestible polysaccharide) on lipid digestion and β-carotene bioaccessibility was determined. The carotenoids were encapsulated within lipid droplets that were then loaded into rice starch hydrogels containing different methylcellulose levels. Incorporation of 0 to 0.2% of methylcellulose had little impact on the dynamic shear rheology of the starch hydrogels, which may be important for formulating functional foods with desirable textural attributes. The microstructure, lipid digestion, and β-carotene bioaccessibility of the filled hydrogels were measured as the samples were passed through simulated oral, gastric, and small intestinal phases. The lipid digestion rate and carotenoid bioaccessibility decreased with increasing methylcellulose. This effect was attributed to the ability of the methylcellulose to inhibit molecular diffusion, promote droplet flocculation, or bind gastrointestinal components thereby inhibiting triacylglycerol hydrolysis at the lipid droplet surfaces. This information may be useful for rationally designing functional foods with improved nutritional benefits.

Biochemical characterization of human acyl coenzyme A: 2-monoacylglycerol acyltransferase-3 (MGAT3)

BackgroundMGAT3 catalyzes the synthesis of 1,2-diacylglycerol from 2-monoacylglycerol in an acyl CoA-dependent reaction. Although initially identified as an MGAT enzyme, MGAT3 is more closely related to DGAT2 than to MGAT1 and MGAT2. Furthermore, MGAT3 possesses both DGAT and MGAT activities, in vitro. MGAT3 is almost exclusively expressed in the small intestine in humans, suggesting that it has a role in dietary fat absorption. Although identified many years ago, little information is available regarding the contribution of MGAT3 to triacylglycerol biosynthesis. ResultsThis study confirmed the initial observations that MGAT3 possessed both MGAT and DGAT activities. When expressed in cells in culture, MGAT3 stimulated lipid droplet growth, but unlike DGAT2, does not become concentrated around the lipid droplet surface. We also characterized the MGAT activity of an MGAT3 mutant in which a conserved cysteine was changed to a tyrosine residue. Lastly, although they share significant sequence identity, MGAT3 is a much more stable protein than DGAT2, yet they are both polyubiquitinated and degraded through ER-associated degradation by the proteasome. ConclusionOur findings provide additional evidence that MGAT3 likely functions as a TG synthase in cells.

Pressure-induced conformational switch of an interfacial protein.

A special class of proteins adopts an inactive conformation in aqueous solution and activates at an interface (such as the surface of lipid droplet) by switching their conformations. Lipase, an essential enzyme for breaking down lipids, serves as a model system for studying such interfacial proteins. The underlying conformational switch of lipase induced by solvent condition is achieved through changing the status of the gated substrate-access channel. Interestingly, a lipase was also reported to exhibit pressure activation, which indicates it is drastically active at high hydrostatic pressure. To unravel the molecular mechanism of this unusual phenomenon, we examined the structural changes induced by high hydrostatic pressures (up to 1500 MPa) using molecular dynamics simulations. By monitoring the width of the access channel, we found that the protein undergoes a conformational transition and opens the access channel at high pressures (>100 MPa). Particularly, a disordered amphiphilic α5 region of the protein becomes ordered at high pressure. This positive correlation between the channel opening and α5 ordering is consistent with the early findings of the gating motion in the presence of a water-oil interface. Statistical analysis of the ensemble of conformations also reveals the essential collective motions of the protein and how these motions contribute to gating. Arguments are presented as to why heightened sensitivity to high-pressure perturbation can be a general feature of switchable interfacial proteins. Further mutations are also suggested to validate our observations. Proteins 2016; 84:820-827. © 2016 Wiley Periodicals, Inc.

Intracellular mechanisms underlying lipid accumulation (white opaque substance) in gastric epithelial neoplasms: A pilot study of expression profiles of lipid-metabolism-associated genes.

White opaque substance (WOS) is a novel endoscopic finding in gastric neoplasms, indicating the intracellular accumulation of lipid droplets (LDs). However, gastric lipid metabolism has not been extensively investigated, even in normal mucosa. We investigated the expression profiles of lipid-metabolism-associated genes in gastric neoplasms. Thirty-four patients with early gastric cancer or adenoma were enrolled in this study. Paired biopsy samples from tumor and adjacent non-tumor areas were obtained and analyzed by real-time polymerase chain reaction. Endoscopically resected specimens were evaluated histopathologically. Genes associated with β-oxidation (peroxisome proliferator-activated receptor α, carnitine palmitoyltransferase 1A, and hydroxyacyl-CoA dehydrogenase), lipoprotein excretion (apolipoprotein B, microsomal triglyceride transfer protein, and acyl-CoA:cholesterol acyltransferase 2), fatty acid transport (fatty acid-binding protein), construction of triglycerides in the endoplasmic reticulum (acyl-CoA:diacylglycerol acyltransferase 1), and LD degradation/lipolysis (comparative gene identification-58, adipose triglyceride lipase) were significantly downregulated in neoplasms compared with non-tumor areas. Pyruvate dehydrogenase lipoamide kinase isozyme 4 (negative regulator of glycolysis) and adipophilin (LD surface component) were also repressed. Conversely, expression levels of genes associated with de novo lipogenesis (sterol regulatory element-binding protein 1c, acyl-CoA:diacylglycerol acyltransferase 2) were significantly enhanced in neoplasms. There was no significant difference in gene expression levels between carcinomas and adenomas, or between WOS-positive and WOS-negative neoplasms. Gene expression profiles in neoplasms suggest a predominance of lipid storage (lipogenesis/LD formation) over consumption (β-oxidation/excretion/lipolysis). Lipid accumulation and WOS in gastric epithelial neoplasms may be caused by impaired mitochondrial oxidation, lipoprotein excretion, and LD degradation.

Targeting Fat: Mechanisms of Protein Localization to Lipid Droplets.

How proteins specifically localize to the phospholipid monolayer surface of lipid droplets (LDs) is being unraveled. We review here the major known pathways of protein targeting to LDs and suggest a classification framework based on the localization origin for the protein. Class I proteins often have a membrane-embedded, hydrophobic 'hairpin' motif, and access LDs from the endoplasmic reticulum (ER) either during LD formation or after formation via ER-LD membrane bridges. Class II proteins access the LD surface from the cytosol and bind through amphipathic helices or other hydrophobic domains. Other proteins require lipid modifications or protein-protein interactions to bind to LDs. We summarize knowledge for targeting and removal of the different classes, and highlight areas needing investigation.

Intestinal inflammation requires FOXO3 and prostaglandin E2-dependent lipogenesis and elevated lipid droplets.

Intestinal inflammation has been recently characterized by the dysregulation of lipids as metabolic and energy sources, revealing a novel feature of its pathophysiology. Because intracellular lipids, stored in dynamic lipid droplets (LDs), provide energy for cellular needs, we investigated whether they play a role in intestinal inflammation. In the inflamed intestine of mice, elevated LDs were found in colonic and infiltrating immune cells as shown by staining for the LD coat protein PLIN2 and for lipids with BODIPY. In colonic cells, TNF stimulated LD increases by receptor signaling rely on phosphatidylinositol 3-kinase activation. Downstream, TNF triggered a negative regulatory loop between LDs and the transcription factor FOXO3. This was shown in the colon of Foxo3-deficient mice, where elevation in PLIN2 and lipids were further facilitated by inflammation and were more prominent relative to wild-type, whereas, in colonic cells, inhibition of lipogenesis blocked the TNF-mediated loss of FOXO3. Furthermore, blockade of PGE2 synthesis abrogated TNF-stimulated increases in LDs and FOXO3 inactivation. We found in colonic tissue of Foxo3-deficient mice higher levels of cyclooxygenase-2, a mediator of prostaglandin E2 (PGE2) synthesis, supporting involvement of PGE2 in the LD-FOXO3 regulatory loop. Ultimately, TNF-stimulated lipogenesis leading to elevated LDs facilitated NF-κB-mediated increases in IL-8 protein, which is associated with the surface of LDs found in the lumina of the endoplasmic reticulum and Golgi apparatus. This novel immunometabolic mechanism of colonic inflammation involving elevated LDs could provide opportunities for new treatment options.

Conserved Amphipathic Helices Mediate Lipid Droplet Targeting of Perilipins 1–3

Perilipins (PLINs) play a key role in energy storage by orchestrating the activity of lipases on the surface of lipid droplets. Failure of this activity results in severe metabolic disease in humans. Unlike all other lipid droplet-associated proteins, PLINs localize almost exclusively to the phospholipid monolayer surrounding the droplet. To understand how they sense and associate with the unique topology of the droplet surface, we studied the localization of human PLINs in Saccharomyces cerevisiae, demonstrating that the targeting mechanism is highly conserved and that 11-mer repeat regions are sufficient for droplet targeting. Mutations designed to disrupt folding of this region into amphipathic helices (AHs) significantly decreased lipid droplet targeting in vivo and in vitro. Finally, we demonstrated a substantial increase in the helicity of this region in the presence of detergent micelles, which was prevented by an AH-disrupting missense mutation. We conclude that highly conserved 11-mer repeat regions of PLINs target lipid droplets by folding into AHs on the droplet surface, thus enabling PLINs to regulate the interface between the hydrophobic lipid core and its surrounding hydrophilic environment.

Lipid Droplet-Associated Proteins (LDAPs) Are Required for the Dynamic Regulation of Neutral Lipid Compartmentation in Plant Cells.

Eukaryotic cells compartmentalize neutral lipids into organelles called lipid droplets (LDs), and while much is known about the role of LDs in storing triacylglycerols in seeds, their biogenesis and function in nonseed tissues are poorly understood. Recently, we identified a class of plant-specific, lipid droplet-associated proteins (LDAPs) that are abundant components of LDs in nonseed cell types. Here, we characterized the three LDAPs in Arabidopsis (Arabidopsis thaliana) to gain insight to their targeting, assembly, and influence on LD function and dynamics. While all three LDAPs targeted specifically to the LD surface, truncation analysis of LDAP3 revealed that essentially the entire protein was required for LD localization. The association of LDAP3 with LDs was detergent sensitive, but the protein bound with similar affinity to synthetic liposomes of various phospholipid compositions, suggesting that other factors contributed to targeting specificity. Investigation of LD dynamics in leaves revealed that LD abundance was modulated during the diurnal cycle, and characterization of LDAP misexpression mutants indicated that all three LDAPs were important for this process. LD abundance was increased significantly during abiotic stress, and characterization of mutant lines revealed that LDAP1 and LDAP3 were required for the proper induction of LDs during heat and cold temperature stress, respectively. Furthermore, LDAP1 was required for proper neutral lipid compartmentalization and triacylglycerol degradation during postgerminative growth. Taken together, these studies reveal that LDAPs are required for the maintenance and regulation of LDs in plant cells and perform nonredundant functions in various physiological contexts, including stress response and postgerminative growth.

Depletion of Rab32 decreases intracellular lipid accumulation and induces lipolysis through enhancing ATGL expression in hepatocytes

Nonalcoholic fatty liver disease (NAFLD) is a disease caused by the accumulation of lipids in hepatocytes. To date, however, the pathogenesis of NAFLD is still unclear. Recent studies have shown that Rab GTPases, a major protein family in vesicle trafficking, are associated with intracellular lipid accumulation. Here, we show that Rab32, the only Rab GTPase located in mitochondria, participates in hepatic steatosis. Ablation of Rab32 can decrease intracellular lipid accumulation in hepatocytes (HepG2, L02). Further studying the possible mechanism, we found that knockdown of Rab32 can enhance lipolysis instead of lipogenesis via inducing the expression of adipose triglyceride lipase (ATGL), a key enzyme on the surface of lipid droplets which has been proved to be significant in controlling intracellular lipid accumulation. Co-immunoprecipitation shows that Rab32 and ATGL are not directly associated. These findings suggest that knockdown of Rab32 indirectly affects lipolysis through increasing the expression of ATGL. Taken together, our study reveals that Rab32 can participate in regulating intracellular lipid accumulation and that knockdown of Rab32 can decrease intracellular lipid accumulation in hepatocytes. We also demonstrated that ablation of Rab32 can induce intracellular lipolysis by enhancing the expression of ATGL.

AMPK-dependent phosphorylation of lipid droplet protein PLIN2 triggers its degradation by CMA

ABSTRACTLipids stored in lipid droplets are hydrolyzed via either cytosolic lipases or a selective form of macroautophagy known as lipophagy. We recently demonstrated that chaperone-mediated autophagy (CMA) is required for the initiation of lipolysis by either of these independent lipolytic pathways. CMA selectively degrades the lipid droplet proteins perilipins (PLIN) 2 and 3 from the lipid droplet surface, thus, facilitating the recruitment of cytosolic lipases and autophagy effector proteins to the lipid droplets. PLIN2 phosphorylation was observed upon induction of lipolysis, but the phosphorylating kinase and the relation of this phosphorylation with CMA of PLIN2 remained unknown. Here, we report that phosphorylation of PLIN2 is dependent on AMPK and occurs after the interaction of PLIN2 with the CMA chaperone HSPA8/Hsc70. Our results highlight a role for posttranslational modifications in priming proteins to be amenable for degradation by CMA.

Hemozoin is a product of heme detoxification in the gut of the most medically important species of the family Opisthorchiidae

Many species of trematodes such as Schistosoma spp., Fasciola hepatica and Echinostoma trivolvis are blood-feeding parasites. Nevertheless, there is no consensus on the feeding habits of the family Opisthorchiidae (Opisthorchis felineus, Opisthorchis viverrini and Clonorchis sinensis). Previously, histological studies of O. felineus and C. sinensis revealed some dark stained material in their gut lumen. In this study we conducted a comprehensive analysis of the gut contents of three members of the family Opisthorchiidae (O. felineus, O. viverrini and C. sinensis). Using transmission electron microscopy, we demonstrated for the first known time the presence of disintegrating blood cells in the gut of O. felineus as well as electron-dense crystals in the gut of O. felineus and C. sinensis. Electron energy loss spectroscopy revealed iron atoms in these crystals, and mass spectrometry of the purified pigment demonstrated the presence of heme. Fourier-transform infrared spectroscopy identified the signature peaks of the common iron–carboxylate bond characteristic in crystals isolated from O. felineus and C. sinensis. Scanning electron microscopy showed layered ovoid crystals of various sizes from 50nm to 2μm. Morphological, chemical and paramagnetic properties of these crystals were similar to those of hemozoin from Schistosoma mansoni. Crystal formation occurs on the surface of lipid droplets in O. felineus and C. sinensis guts. Our results suggest that the diet of O. felineus and C. sinensis includes blood. Detoxification of the free heme produced during the digestion proceeds via formation of insoluble crystals that contain iron and heme dimers, i.e. crystals of hemozoin. Furthermore, we believe that biocrystallisation of hemozoin takes place on the surface of the lipid droplets, similar to S. mansoni. Hemozoin was not detected in the closely related species O. viverrini.

Triglyceride Mobilization from Lipid Droplets Sustains the Anti-Steatotic Action of Iodothyronines in Cultured Rat Hepatocytes.

Adipose tissue, dietary lipids and de novo lipogenesis are sources of hepatic free fatty acids (FFAs) that are stored in lipid droplets (LDs) as triacylglycerols (TAGs). Destiny of TAGs stored in LDs is determined by LD proteomic equipment. When adipose triglyceride lipase (ATGL) localizes at LD surface the lipid mobilization is stimulated. In this work, an in vitro model of cultured rat hepatocytes mimicking a mild steatosis condition was used to investigate the direct lipid-lowering action of iodothyronines, by focusing, in particular, on LD-associated proteins, FFA oxidation and lipid secretion. Our results demonstrate that in “steatotic” hepatocytes iodothyronines reduced the lipid excess through the recruitment of ATGL on LD surface, and the modulation of the LD-associated proteins Rab18 and TIP47. As an effect of ATGL recruitment, iodothyronines stimulated the lipid mobilization from LDs then followed by the up-regulation of carnitine-palmitoyl-transferase (CPT1) expression and the stimulation of cytochrome-c oxidase (COX) activity that seems to indicate a stimulation of mitochondrial function. The lipid lowering action of iodothyronines did not depend on increased TAG secretion. On the basis of our data, ATGL could be indicated as an early mediator of the lipid-lowering action of iodothyronines able to channel hydrolyzed FFAs toward mitochondrial beta-oxidation rather than secretion.