Perilipin Protein Research Articles

Protein-protein interactions regulate the activity of Adipose Triglyceride Lipase in intracellular lipolysis

Carefully regulated lipid homeostastis generates an optimal physiological, non-toxic environment. Imbalances in lipid metabolism lead to obesity and are associated with type-2 diabetes, hepatic steatosis, hypertension and cardiovascular disease. Mammals store energy in lipid droplets predominantly in white adipose tissue. This energy reservoir builds up during periods of energy excess and is mobilized during energy deprivation. Triacylglycerols (TAGs) are unable to cross cell membranes for cell nutrition; they have to be cleaved before further transportation within the body. Lipolysis describes the cleavage of TAG and is carried out with the help of lipases. Adipose triglyceride lipase (ATGL) catalyzes the first step of intracellular lipolysis to mobilize TAG stores. In this minireview, we set the focus on molecular mechanism and interfaces behind co-activation and inhibition of ATGL, namely via its regulation by the co-activating protein CGI-58, the inhibitory proteins G0S2 and HILPDA, as well as the regulatory effect of fatty acid binding proteins and the perilipin protein family.

Perilipin5 protects against lipotoxicity and alleviates endoplasmic reticulum stress in pancreatic \u03b2-cells

BackgroundChronic exposure of pancreatic β-cells to excess free fatty acids is thought to contribute to type 2 diabetes pathogenesis in obesity by impairing β-cell function and even leading to apoptosis. In β-cells, lipid droplet-associated protein perilipin 5 (PLIN5) has been shown to enhance insulin secretion by regulating intracellular lipid metabolism; the roles of PLIN5 in response to lipotoxicity remain poorly understood.MethodsINS-1 β-cells were transfected with PLIN5-overexpression adenovirus (Ad-PLIN5) and treated with palmitate. C57BL/6 J male mice were fed with high fat diet and tail intravenous injected with adeno-associated virus overexpressing PLIN5 (AAV-PLIN5) in β-cells.ResultsOur data showed that palmitate and PPAR agonists including WY14643 (PPARα), GW501516 (PPARβ/δ), rosiglitazone (PPARγ) in vitro all induced PLIN5 expression in INS-1 cells. Under palmitate overload, although upregulating PLIN5 promoted lipid droplet storage, it alleviated lipotoxicity in INS-1 β-cells with improved cell viability, cell apoptosis and β-cell function. The protection role of PLIN5 in β-cell function observed in cell experiments were further verified in in vivo study indicated by mitigated glucose intolerance in high fat diet fed mice with β-cell-specific overexpression of PLIN5. Mechanistic experiments revealed that enhanced FAO induced by elevation of PLIN5, followed by decreased ER stress may be a major mechanism responsible for alleviation of lipotoxicity observed in the present study.ConclusionsOur finding substantiated the important role of PLIN5 in protection against lipotoxicity in β-cells.

Perilipin 5 deficiency promotes atherosclerosis progression through accelerating inflammation, apoptosis, and oxidative stress.

Excessive plasma triglyceride(TG) and cholesterol levels promote the progression of several prevalent cardiovascular risk factors, including atherosclerosis, which is a leading death cause. Perilipin 5 (Plin5), an important perilipin protein, is abundant in tissues with very active lipid catabolism and is involved in the regulation of oxidative stress. Although inflammation and oxidative stress play a critical role in atherosclerosis development, the underlying mechanisms are complex and not completely understood. In the present study, we demonstrated the role of Plin5 in high-fat-diet-induced atherosclerosis in apolipoprotein E null (ApoE-/- ) mice. Our results suggested that Plin5 expressions increased in the artery tissues of ApoE-/- mice. ApoE/Plin5 double knockout (ApoE-/- Plin5-/- ) exacerbated severer atherogenesis, accompanied with significantly disturbed plasma metabolic profiles, such as elevated TG, total cholesterol, and low-density lipoprotein cholesterol levels and reduced high-density lipoprotein cholesterol contents. ApoE-/- Plin5-/- exhibited a higher number of inflammatory monocytes and neutrophils, as well as overexpression of cytokines and chemokines linked with an inflammatory response. Consistently, the IκBα/nuclear factor kappa B pathway was strongly activated in ApoE-/- Plin5-/- . Notably, apoptosis was dramatically induced by ApoE-/- Plin5-/- , as evidenced by increased cleavage of Caspase-3 and Poly (ADP-ribose) polymerase-2. In addition, ApoE-/- Plin5-/- contributed to oxidative stress generation in the aortic tissues, which was linked with the activation of phosphatidylinositol 3-kinase/protein kinase B and mitogen-activated protein kinases pathways. In vitro, oxidized low-density lipoprotein (ox-LDL) increased Plin5 expression in RAW264.7 cells. Its knockdown enhanced inflammation, apoptosis, oxidative stress, and lipid accumulation, while promotion of Plin5 markedly reduced all the effects induced by ox-LDL in cells. These studies strongly supported that Plin5 could be a new regulator against atherosclerosis, providing new insights on therapeutic solutions.

Acute Carbohydrate Restriction During Recovery From Prolonged Exercise Enhances Intramuscular Triglyceride Resynthesis

Intramuscular triglyceride (IMTG) is an important substrate during moderate-intensity exercise, but providing a high carbohydrate (CHO) diet following exercise impairs IMTG resynthesis. Restricting CHO intake in the post-exercise period may augment the adaptive response to exercise, but whether this strategy enhances IMTG resynthesis is not known. Furthermore, because the lipid droplet (LD)-associated perilipin (PLIN) proteins promote IMTG storage, their distribution and interaction with LD may determine their role in post-exercise IMTG resynthesis. PURPOSE: To determine the effect of acutely restricting CHO during recovery from prolonged exercise on IMTG resynthesis and PLIN protein dynamics. METHODS: 14 male triathletes (27±1 y, 66.5±1.3 ml.kg-1.min-1) completed 4 h of cycling at ~56% VO2max. In the initial 4 h period following exercise 7 participants consumed a high CHO diet whereas 7 participants consumed only water. For the remaining recovery period (20 h) all participants received the same CHO-rich diet. Muscle samples collected pre and post-exercise, and 4 and 24 h post-exercise were analysed using confocal immunofluorescence microscopy to determine muscle fibre type-specific IMTG content and PLIN localisation to LDs. RESULTS: Exercise reduced IMTG content in type I fibres (-53%, P<0.05), with LD both associated and not associated with PLIN2, PLIN3, and PLIN5 being used. During recovery, IMTG content tended to increase in type I fibres after 4 h in the water condition only (+63%, P=0.076), and at 24 h IMTG content was similar to pre-exercise levels (P=0.987). Despite no change in PLIN protein content, the number of LD with PLIN2 or PLIN3 associated tended to increase during recovery in type I fibres only (P=0.08), and to a similar extent between conditions, whereas the number of LD with PLIN5 associated only increased in the CHO condition (P=0.002). CONCLUSION: Acute restriction of CHO during recovery from prolonged exercise has a tendency to initially enhance IMTG resynthesis, although a CHO-rich diet does not impair overall IMTG resynthesis 24 h later. A redistribution of PLIN proteins appears to support post-exercise IMTG resynthesis, but the predominant PLIN isoform supporting post-exercise IMTG storage may be dependent on post-exercise CHO availability.

Perilipin 1 Expression Differentiates Liposarcoma from Other Types of Soft Tissue Sarcoma

Lipid droplets, a morphologic feature of adipocytic tumors, are strongly regulated by associated proteins of the perilipin/PAT (perilipin, adipophilin, and tail-interacting protein of 47 kD) family. So far, the use of perilipins as markers for differential diagnosis of soft tissue tumors has only been studied in a few cases. The aim of this study was to investigate the expression of perilipins in 478 human soft tissue tumors and 60 respective normal tissues. Perilipin 1 was immunohistochemically positive in all studied cases of well-differentiated liposarcomas, >90% of myxoid round cell liposarcomas, and >70% of pleomorphic liposarcomas, whereas only the differentiated components of dedifferentiated liposarcomas were immunohistochemically positive for perilipin 1. All other types of soft tissue sarcomas were negative for perilipin 1. Perilipin 2 was more prominent in dedifferentiated and pleomorphic liposarcomas and nearly all other high-grade sarcomas. In well-differentiated liposarcomas, lipomas, or normal adipose tissue, perilipin 2 was virtually absent. In addition, long-term stimulation of adipogenesis in the liposarcoma cell line LiSa-2 restored perilipin 1 expression, as exhibited in the source tumor. Furthermore, knockdown of perilipin 2 or perilipin 3 in LiSa-2 cells influenced lipid droplet number and size as well as cell vitality. In summary, perilipin 1 is a promising marker for the differential diagnosis of liposarcomas from other soft tissue sarcomas, whereas perilipin 2 correlates negatively with tumor grade and may be therapeutically useful.

Plin5 deficiency promotes atherosclerosis progression through accelerating inflammation, apoptosis and oxidative stress.

Excessive plasma triglyceride and cholesterol levels promote the progression of several prevalent cardiovascular risk factors, including atherosclerosis, which is a leading death cause. Perilipin 5 (Plin5), an important perilipin protein, is abundant in tissues with very active lipid catabolism, and is involved in the regulation of oxidative stress. Although, in?ammation and oxidative stress play a critical role in atherosclerosis development, the underlying mechanisms are complex and not completely understood. In the present study, we demonstrated the role of Plin5 in high-fat-diet-induced atherosclerosis in apolipoprotein E null (ApoE-/- ) mice. Our results suggested that Plin5 expressions increased in the artery tissues of ApoE-/- mice. ApoE/Plin5 double knockout (ApoE-/- Plin5-/- ) exacerbated severer atherogenesis, accompanied with significantly disturbed plasma metabolic profiles, such as elevated triglyceride (TG), total cholesterol (TC) and low-density lipoprotein cholesterol (LDLC) levels and reduced high-density lipoprotein cholesterol (HDLC) contents. ApoE-/- Plin5-/- exhibited higher number of inflammatory monocytes and neutrophils, as well as over-expression of cytokines and chemokines linked with inflammatory response. Consistently, IκBα/nuclear factor kappa B (NF-κB) pathway was strongly activated in ApoE-/- Plin5-/- . Notably, apoptosis was dramatically induced by ApoE-/- Plin5-/- , as evidenced by increased cleavage of Caspase-3 and Poly (ADP-ribose) polymerase-2 (PARP-2). In addition, ApoE-/- Plin5-/- contributed to oxidative stress generation in the aortic tissues, which was linked with the activation of phosphatidylinositol 3-kinase /protein kinase B (PI3K/AKT) and mitogen-activated protein kinases (MAPKs) pathways. In vitro, oxidized low-density lipoprotein (oxLDL) increased Plin5 expression in RAW264.7 cells. Its knockdown enhanced inflammation, apoptosis, oxidative stress and lipid accumulation, while promotion of Plin5 markedly reduced all the effects induced by ox-LDL in cells. These studies strongly supported that Plin5 could be a new regulator against atherosclerosis, providing new insights on therapeutic solutions. This article is protected by copyright. All rights reserved.

Characterization and Structure Determination of the Perilipin 5 N‐Terminal Domain

Many countries, including the United States, have been afflicted with a group of lipid‐related diseases including type II diabetes, obesity and non‐alcoholic fatty liver disease. The commonality behind these disorders is lipid storage and lipid storage is mediated in part by the perilipin proteins. The perilipin family are a group of five conserved genes that differ with respect to size of the proteins they encode, tissue expression and transcriptional regulation. The only member of the perilipin family to have significant protein structural data collected is perilipin 3, and this is only on the carboxyl terminus. The C‐terminus of perilipin 5 has a high sequence homology to that of perilipin 3. Less is known about the amino terminus. The central focus of this study is to characterize and determine the structure of the N‐terminal domain of perilipin 5. The amino terminal 104 amino acids of perilipin 5 were expressed with a 6‐His tag in E. coli. Optimization of the expression of the protein fragment was ascertained using an immuno‐dot blot that contained cell samples treated with various ITPG concentrations and temperatures. Isolation and purification of the protein will then be performed using nickel affinity chromatography for further structural analysis. Secondary and tertiary structures of the N‐terminal fragment were predicted using Phyre2 and RaptorX. These computer models predicted a predominantly alpha helical structure with the presence of two 4‐helix bundles. Overall, determination of the N‐terminal structure for the perilipin 5 protein will increase our understanding of the perilipin family and their functions.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Adipocyte lipolysis affects Perilipin 5 and cristae organization at the cardiac lipid droplet-mitochondrial interface

This study investigated the effects of elevated fatty acid (FA) supply from adipose tissue on the ultrastructure of cardiac lipid droplets (LDs) and the expression and organization of LD scaffold proteins perilipin-2 (PLIN2) and perilipin-5 (PLIN5). Stimulation of adipocyte lipolysis by fasting (24 h) or β3-adrenergic receptor activation by CL316, 243 (CL) increased cardiac triacylglycerol (TAG) levels and LD size, whereas CL treatment also increased LD number. LDs were tightly associated with mitochondria, which was maintained during LD expansion. Electron tomography (ET) studies revealed continuity of LD and smooth endoplasmic reticulum (SER), suggesting interconnections among LDs. Under fed ad libitum conditions, the cristae of mitochondria that apposed LD were mostly organized perpendicularly to the tangent of the LD surface. Fasting significantly reduced, whereas CL treatment greatly increased, the perpendicular alignment of mitochondrial cristae. Fasting and CL treatment strongly upregulated PLIN5 protein and PLIN2 to a lesser extent. Immunofluorescence and immuno-electron microscopy demonstrated strong targeting of PLIN5 to the cardiac LD-mitochondrial interface, but not to the mitochondrial matrix. CL treatment augmented PLIN5 targeting to the LD-mitochondrial interface, whereas PLIN2 was not significantly affected. Together, our results support the concept that the interface between LD and cardiac mitochondria represents an organized and dynamic “metabolic synapse” that is highly responsive to FA trafficking.

Unveiling the function and regulation control of the DUF3129 family proteins in fungal infection of hosts.

Many prokaryotic and eukaryotic proteins contain domains of unknown function (DUFs). A DUF3129 family of proteins is widely encoded in the genomes of fungal pathogens. A few studies in plant and insect pathogens indicated that the DUF3129 genes are required for fungal penetration of host cuticles with an unclear mechanism. We found that a varied number of DUF3129 proteins is present in different fungal species and the proteins are evolutionarily diverged from each other at the inter- and intra-specific levels. By using the insect pathogenic fungus Metarhizium robertsii as a model, we performed experiments and found that the seven DUF3129 proteins encoded by this fungus are localized to cellular lipid droplets (LDs). Individual deletion of these genes did not affect fungal formation of the infection structure appressoria and the accumulation of LDs in fungal conidia. When compared with the wild-type (WT) strain, insect bioassays revealed that the virulence of most null mutants were significantly impaired during topical infection but not during injection of insects. Carbon starvation and the subsequent Western blot analysis indicated that the LD-specific perilipin protein was completely degraded in the WT cells whereas varied levels of perilipin could be detected in the mutant cells, which signified that depletion of LD content was delayed in mutant cells, and DUF3129 proteins are therefore involved in LD degradation. We also provided biochemical evidence that these DUF3129 genes are transcriptionally regulated by a yeast Ste12-like transcription factor. The findings of this study not only unveil the function of DUF3129 proteins but also better understand the diverse mechanism of fungus-host interactions. This article is part of the theme issue 'Biotic signalling sheds light on smart pest management'.

N-terminal acetylation and the N-end rule pathway control degradation of the lipid droplet protein PLIN2

Perilipin 2 (PLIN2) is a major lipid droplet (LD)-associated protein that regulates intracellular lipid homeostasis and LD formation. Under lipid-deprived conditions, the LD-unbound (free) form of PLIN2 is eliminated in the cytosol by an as yet unknown ubiquitin (Ub)-proteasome pathway that is associated with the N-terminal or near N-terminal residues of the protein. Here, using HeLa, HEK293T, and HepG2 human cell lines, cycloheximide chase, in vivo ubiquitylation, split-Ub yeast two-hybrid, and chemical cross-linking-based reciprocal co-immunoprecipitation assays, we found that TEB4 (MARCH6), an E3 Ub ligase and recognition component of the Ac/N-end rule pathway, directly targets the N-terminal acetyl moiety of Nα-terminally acetylated PLIN2 for its polyubiquitylation and degradation by the 26S proteasome. We also show that the TEB4-mediated Ac/N-end rule pathway reduces intracellular LD accumulation by degrading PLIN2. Collectively, these findings identify PLIN2 as a substrate of the Ac/N-end rule pathway and indicate a previously unappreciated role of the Ac/N-end rule pathway in LD metabolism.

Vitamin D produces a perilipin 2-dependent increase in mitochondrial function in C2C12 myotubes

Vitamin D has been connected with increased intramyocellular lipid (IMCL) and has also been shown to increase mitochondrial function and insulin sensitivity. Evidence suggests that perilipin 2 (PLIN2), a perilipin protein upregulated with calcitriol treatment, may be integral to managing increased IMCL capacity and lipid oxidation in skeletal muscle. Therefore, we hypothesized that PLIN2 is required for vitamin D induced IMCL accumulation and increased mitochondrial oxidative function. To address this hypothesis, we treated C2C12 myotubes with 100 nM calcitriol (the active form of vitamin D) and/or PLIN2 siRNA in a four group design and analyzed markers of IMCL accumulation and metabolism using qRT-PCR, cytochemistry, and oxygen consumption assay. Expression of PLIN2, but not PLIN3 or PLIN5 mRNA was increased with calcitriol, and PLIN2 induction was prevented with siRNA knockdown without compensation by other perilipins. PLIN2 knockdown did not appear to prevent lipid accumulation. Calcitriol treatment increased mRNA expression of triglyceride synthesizing genes DGAT1 and DGAT2 and also lipolytic genes ATGL and CGI-58. PLIN2 knockdown decreased the expression of CGI-58 and CPT1, and was required for calcitriol-induced upregulation of DGAT2. Calcitriol increased oxygen consumption rate while PLIN2 knockdown decreased oxygen consumption rate. PLIN2 was required for a calcitriol-induced increase in oxygen consumption driven by mitochondrial complex II. We conclude that calcitriol increases mitochondrial function in myotubes and that this increase is at least in part mediated by PLIN2.

PL-017 High-fat overfeeding increases intramuscular triglyceride content and perilipin protein expression in human skeletal muscle

Objective High-fat high-calorie diets can induce whole body insulin resistance (IR) whilst increasing stores of intramuscular triglyceride (IMTG) contained within lipid droplets (LD). Perilipin (PLIN) proteins assist in IMTG storage. Synaptosomal-associated protein (SNAP23) may support LD growth and also direct IMTG-derived fatty acids (FA) to mitochondria for β-oxidation. The objectives of this study were: 1) to test the hypothesis that 7 days of high-fat overfeeding increases IMTG content to prevent lipid induced muscle IR and 2) identify changes in PLINs, SNAP23 and mitochondria content and colocalisation of PLINs with LD, and SNAP23 with LD and mitochondria.&#x0D; Methods Muscle biopsies were obtained from thevastus lateralisof thirteen healthy individuals (age: 23±1years, BMI: 24.4±0.7kg.m-2) before (0min) and during (30min) an oral glucose tolerance test (OGTT), pre and post 7-days consuming a high-fat (65% energy) high-calorie (+50% kcal) diet. IMTG, PLIN2, PLIN3, PLIN5, SNAP23 and mitochondria content were measured using (semi)-quantitative confocal immunofluorescence microscopy. PLIN2, PLIN3 and PLIN5 colocalisation to LD was measured using object-based colocalisation analyses. Pearson’s correlation coefficient quantified colocalisation between SNAP23 and plasma membrane (PM), mitochondria and LD. Phosphorylation of intermediates of the muscle insulin-signalling cascade (Akt and AS160) were measured at 0 and 30 min of the OGTT before and after the dietary intervention.&#x0D; Results Following overfeeding phosphorylation of Akt and AS160 in muscle was not impaired during the OGTT, however Matsuda index of whole-body insulin sensitivity decreased (-23%; P &lt; 0.01). IMTG content increased in type I fibres (+100%; P &lt; 0.001) due to both an increase in LD number (+43%; P &lt; 0.001) and size (+44%; P&lt; 0.001). Of the PLINs investigated, only PLIN3 content increased (+50%;P &lt; 0.01) exclusively in type I fibres. PLIN2-associated LD increased (+80%; P &lt; 0.01) in type I fibres only, whereas PLIN3 and PLIN5-associated LD were unaltered. SNAP23 and mitochondria content did not change, nor did the colocalisation of SNAP23 with the PM, mitochondria or LD.&#x0D; Conclusions Our data confirm the hypothesis that following high-fat overfeeding IMTG stores increased whilst activation of key muscle insulin signalling components were maintained. The increase in IMTG stores is likely supported by the concurrent increase in total PLIN3 content and a redistribution of existing stores of PLIN2 to the expanded LD pool in type I fibres. To confirm if increased IMTG storage protects muscle from IR future research should determine whether meal-derived FAs are directed to IMTG rather than ceramides and diacylglycerol.

MRT, Functioning with NURF Complex, Regulates Lipid Droplet Size

Lipid droplets (LDs) are highly dynamic organelles that store neutral lipids. Through a gene overexpression screen in the Drosophila larval fat body, we have identified that MRT, an Myb/switching-defective protein 3 (Swi3), Adaptor 2 (Ada2), Nuclear receptor co-repressor (N-CoR), Transcription factor (TF)IIIB (SANT)-like DNA-binding domain-containing protein, regulates LD size and lipid storage. MRT directly interacts with, and is functionally dependent on, the PZG and NURF chromatin-remodeling complex components. MRT binds to the promoter of plin1, the gene encoding the LD-resident protein perilipin, and inhibits the transcription of plin1. Invitro LD coalescence assays suggest that mrt overexpression or loss of plin1 function facilitates LD coalescence. Our findings suggest that MRT functions together with chromatin-remodeling factors to regulate LD size, likely through the transcriptional repression of plin1.

The lipid-droplet associated protein perilipin 2 (PLIN2) plays a central role in lipid accumulation and cholesterol efflux via effects on LXR signaling in human macrophages

Aim: Cholesterol efflux is crucial in removing excess cholesterol from macrophage foam cells and is of importance in reducing atherosclerosis. Macrophages store lipids in lipid droplets (LDs) and perilipin 2 (PLIN2) is the major LD-associated protein. Thus we hypothesized that alterations of PLIN2 function affect macrophage lipid storage and can redirect intracellular cholesterol towards efflux pathways.

Impact of skeletal muscle IL-6 on regulation of liver and adipose tissue metabolism during fasting.

The liver and adipose tissue are important tissues in whole-body metabolic regulation during fasting. Interleukin 6 (IL-6) is a cytokine shown to be secreted from contracting muscle in humans and suggested to signal to the liver and adipose tissue. Furthermore, skeletal muscle IL-6 has been proposed to play a role during fasting. Therefore the aim of the present study was to investigate the role of skeletal muscle IL-6 in the regulation of substrate production in the liver and adipose tissue during fasting. Male skeletal muscle-specific IL-6 knockout (IL-6 MKO) mice and littermate floxed (control) mice fasted for 6 or 18h (6h fasting or 18h fasting) with corresponding fed control groups (6h fed or 18h fed) and liver and adipose tissue were quickly obtained. Plasma β-hydroxybutyrate increased and hepatic glucose, lactate and glycogen decreased with fasting. In addition, fasting increased phosphoenolpyruvate carboxykinase protein and phosphorylation of pyruvate dehydrogenase (PDH) in the liver as well as hormone-sensitive lipase (HSL)Ser660 and HSLSer563 phosphorylation, PDH phosphorylation, adipose triglyceride lipase phosphorylation and perilipin phosphorylation and protein content in adipose tissue without any effect of lack of skeletal muscle IL-6. In conclusion, fasting induced regulation of enzymes in adipose tissue lipolysis and glyceroneogenesis as well as regulation of hepatic gluconeogenic capacity and hepatic substrate utilization in mice. However, skeletal muscle IL-6 was not required for these fasting-induced effects, but had minor effects on markers of lipolysis and glyceroneogenesis in adipose tissue as well as markers of hepatic gluconeogenesis in the fed state.

LLC tumor cells-derivated factors reduces adipogenesis in co-culture system

Cancer cachexia (CC) is a multifactorial syndrome with an unknown etiology. The primary symptom is the progressive reduction of the body weight. Recently, down-regulation of adipogenic and lipogenic genes were demonstrated to be early affected during cachexia progression in adipose tissue (AT), resulting in AT remodeling. Thus, this study aimed to evaluate in a co-culture system the influence of the Lewis Lung Carcinoma (LLC) tumor cells (c/c-LLC) in an established pre-adipocyte cell line 3T3-L1 adipogenic capacity. c/c-LLC in the presence of 3T3-L1 caused a reduction in lipids accumulation, suggesting that secretory tumor cells products may affect adipogenesis. Interestingly, a very early (day 2) down-regulation of proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (C/EBPα), followed by late genes (day 4 and 8), adiponectin, perilipin, and fatty acid-binding protein 4 (FABP4). Caspase-3 expression was increased on the last day of cell differentiation; it occurred in the expression of pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α). Overall, our results suggest that LLC secretory products impair adipocyte differentiation in a co-culture system and increased apoptosis. In summary, our study has shown the inhibition of the adipogenic process in the 3T3-L1 co-culture system with LLC cells.

The Expression Pattern of PLIN2 in Differentiated Adipocytes from Qinchuan Cattle Analysis of Its Protein Structure and Interaction with CGI-58.

PLIN2 (Perilipin-2) is a protein that can anchor on the membrane of lipid droplets (LDs), playing a vital role in the early formation of LDs and in the regulation of LD metabolism in many types of cells. However, little research has been conducted in cattle adipocytes. In the present study, we found that the expression of PLIN2 mRNA peaks at Day 2 during cattle adipocyte differentiation (p < 0.01), but PLIN2 protein levels maintain high abundance until Day 4 and then decrease sharply. We first built an interaction model using PyMOL. The results of a pull-down assay indicated that bovine PLIN2 and CGI-58 (ABHD5, α/β hydrolase domain-containing protein 5) had an interaction relationship. Furthermore, Bimolecular Fluorescence Complementation-Flow Cytometry (BiFC-FC) was used to explore the function of the PLIN2-CGI-58 interaction. Interestingly, we found that different combined models had different levels of fluorescence intensity; specifically, PLIN2-VN173+CGI-58-VC155 expressed in bovine adipocytes exhibited the highest level of fluorescence intensity. Our findings elucidate the PLIN2 expression pattern in cattle adipocytes, the protein structure and the function of protein–protein interactions (PPI) as well as highlight the characteristics of bovine PLIN2 during the early formation and accumulation of lipid droplets.

Overexpression of PLIN2 is a prognostic marker and attenuates tumor progression in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common renal malignancy in adults, the incidence of which continues to increase. The lipid droplet protein perilipin 2 (PLIN2), which was originally considered an RNA transcript, is markedly expressed during adipocyte differentiation. In addition, it has been observed to be elevated in numerous types of cancer, including ccRCC; however, its essential function remains unclear in ccRCC. The present study examined the expression of PLIN2 in ccRCC, and aimed to determine the association between PLIN2 expression and patient survival. The present study mined the transcriptional, clinicopathological and survival data of PLIN2 in patients with ccRCC through The Cancer Genome Atlas. The expression levels of PLIN2 were also detected in human ccRCC tissues and cell lines by western blotting and immunohistochemistry, and its biological role was identified by functional analysis. The results demonstrated that PLIN2 was predominantly elevated in RCC tissues and cells. In addition, the expression levels of PLIN2 were significantly associated with various clinicopathological factors, and high PLIN2 expression was associated with a good prognosis. The results of a multivariate analysis demonstrated that high PLIN2 expression was an independent prognostic indicator of overall survival (hazard ratio, 0.586; P=0.001). Furthermore, PLIN2 knockdown promoted proliferation of ccRCC cells, and enhanced cell invasion and migration. These results suggested that PLIN2 may be considered a novel prognostic factor in ccRCC and a specific diagnostic indicator for patients with ccRCC. Furthermore, it could be a potential novel target for the clinical treatment of ccRCC.

Critical role of mTOR, PPARγ and PPARδ signaling in regulating early pregnancy decidual function, embryo viability and feto-placental growth.

What are the consequences of inhibiting mTOR, the mechanistic target of rapamycin (mTOR), and the peroxisome proliferator activated receptor gamma (PPARγ) and PPARδ pathways in the early post-implantation period on decidual function, embryo viability and feto-placental growth in the rat? mTOR inhibition from Days 7 to 9 of pregnancy in rats caused decidual PPARγ and PPARδ upregulation on Day 9 of pregnancy and resulted in embryo resorption by Day 14 of pregnancy. PPARγ and PPARδ inhibition differentially affected decidual mTOR signaling and levels of target proteins relevant to lipid histotrophic nutrition and led to reduced feto-placental weights on Day 14 of pregnancy. Although mTOR, PPARγ and PPARδ are nutrient sensors important during implantation, the role of these signaling pathways in decidual function and how they interact in the early post-implantation period are unknown. Perilipin 2 (PLIN2) and fatty acid binding protein 4 (FABP4), two adipogenic proteins involved in lipid histotrophic nutrition, are targets of mTOR and PPAR signaling pathways in a variety of tissues. Rapamycin (mTOR inhibitor, 0.75 mg/kg, sc), T0070907 (PPARγ inhibitor, 0.001 mg/kg, sc), GSK0660 (PPARδ inhibitor, 0.1 mg/kg, sc) or vehicle was injected daily to pregnant rats from Days 7 to 9 of pregnancy and the studies were performed on Day 9 of pregnancy (n = 7 per group) or Day 14 of pregnancy (n = 7 per group). On Day 9 of pregnancy, rat decidua were collected and prepared for western blot and immunohistochemical studies. On Day 14 of pregnancy, the resorption rate, number of viable fetuses, crown-rump length and placental and decidual weights were determined. Inhibition of mTOR in the early post-implantation period led to a reduction in FABP4 protein levels, an increase in PLIN2 levels and an upregulation of PPARγ and PPARδ in 9-day-pregnant rat decidua. Most embryos were viable on Day 9 of pregnancy but had resorbed by Day 14 of pregnancy. This denotes a key function of mTOR in the post-implantation period and suggests that activation of PPAR signaling was insufficient to compensate for impaired nutritional/survival signaling induced by mTOR inhibition. Inhibition of PPARγ signaling resulted in decreased decidual PLIN2 and FABP4 protein expression as well as in inhibition of decidual mTOR signaling in Day 9 of pregnancy. This treatment also reduced feto-placental growth on Day 14 of pregnancy, revealing the relevance of PPARγ signaling in sustaining post-implantation growth. Moreover, following inhibition of PPARδ, PLIN2 levels were decreased and mTOR complex 1 and 2 signaling was altered in decidua on Day 9 of pregnancy. On Day 14 of pregnancy, PPARδ inhibition caused reduced feto-placental weight, increased decidual weight and increased resorption rate, suggesting a key role of PPARδ in sustaining post-implantation development. Not applicable. This is an in vivo animal study and the relevance of the results for humans remains to be established. The early post-implantation period is a critical window of development and changes in the intrauterine environment may cause embryo resorption and lead to placental and fetal growth restriction. mTOR, PPARγ and PPARδ signaling are decidual nutrient sensors with extensive cross-talk that regulates adipogenic proteins involved in histotrophic nutrition and important for embryo viability and early placental and fetal development and growth. Funding was provided by the Agencia Nacional de Promoción Científica y Tecnológica de Argentina (PICT 2014-411 and PICT 2015-0130), and by the International Cooperation (Grants CONICET-NIH-2014 and CONICET-NIH-2017) to A.J. and T.J. The authors have no conflicts of interest.

Alterations in placental long chain polyunsaturated fatty acid metabolism in human intrauterine growth restriction.

Fatty acids (FA) are critical for fetal brain development and are transferred across the placenta by membrane-bound FA transport proteins (FATP), translocases (FAT/CD36), and cytosolic binding proteins (FABP). The cytosolic protein perilipin-2 aids in neutral lipid storage within lipid droplets. Decreased placental nutrient transport is believed to contribute to intrauterine growth restriction (IUGR); however, IUGR placental lipid transport and metabolism are poorly understood. We hypothesized that protein expression of FATPs, FABPs, and perilipin-2 in human placenta is decreased and placental lipid content and incorporation into lipid classes are reduced in IUGR. Placental tissue of idiopathic IUGR (n=25) and gestational age-matched, appropriately grown for gestational age (AGA) fetuses (n=19) was collected. We determined protein expression of FABP4 and perilipin-2 in placental homogenate and FATPs (2, 4, 6, CD36) in syncytiotrophoblast microvillous plasma membrane (MVM) by Western blot. Lipid droplet area (Oil Red O stain) and cellular FA content (GC/MS) were measured in chorionic villous tissue. MVM expression of FATP6 and CD36 was significantly increased in IUGR. The concentrations of seven n-6 and n-3 species long chain polyunsaturated FAs (LCPUFA) were significantly increased in the triglyceride fraction in IUGR vs AGA placenta. In summary, MVM FATP6 and CD36 protein expression is increased and LCPUFA are preferentially routed toward cellular storage in TG in the IUGR placenta, possibly to protect against oxidative stress associated with cellular FA accumulation. We speculate that these changes may be caused by impaired efflux of FA across the fetal-facing syncytiotrophoblast basal plasma membrane in IUGR placenta.