PAT Family Research Articles

Characterization of a splice variant of perilipin 5

Lipid storage provides energy stores for the body in times of increased oxidative requirements, such as fasting or exercise, but when the balance of lipid storage and mobilization is disrupted human health suffers. Implications for increased lipid storage include risk factors for developing type II diabetes mellitus and non‐alcoholic fatty liver disease. The perilipin family of proteins associate with the surface of lipid droplets and are essential to regulation of stored triacylglycerols. Perilipin 5 functions to maintain a balance between the storage of triacylglycerols in lipid droplets or their mobilization when energetically required. Several members of the perilipin gene family exhibit differential splicing, most notably perilipin 1 but reports exist of alternative messages for shortened forms of perilipins 2 and 3. We have identified a shortened form of perilipin 5 we have termed perilipin 5b. Western blots indicate that the protein, of approximately 35 kDa, is present in C2C12 cultured myoblasts and in heart and liver tissue from fed and fasted mice. Analysis of murine genomic DNA sequences reveals stop codons found in the introns between exons 6 and 7 and 7 and 8, either of which could give rise to a protein of approximately 35 kDa, indicating the likelihood of the observed protein being a splice variant, rather than a post‐translationally modified protein. Using RT‐PCR we have identified multiple amplicons suggesting that perilipin‐5b contains a read through of intron 6 of the murine perilipin 5 gene. Mining transcriptome databases reveals several transcripts with retained introns. Analysis of the amino acid sequence of perilipin 5b indicates that this truncation would lack the reported C‐terminal domain essential for interactions with ATGL and CGI‐58 but would retain the PKA site found at Ser155. The function of perilipin 5b is yet unknown, but the fact that it is found primarily in heart tissue seems to suggest that it plays a role in the oxidation of the lipid droplet. These data are consistent with what is currently known about Perilipin‐5 and the other PAT family proteins. Determination of perilipin 5b function will give a more accurate picture of the overall mechanism of lipid storage and metabolism.

T181. PROTEIN ACYL-THIOESTERASE ENZYME ACTIVITY IN THE POSTMORTEM DORSOLATERAL PREFRONTAL CORTEX IN SCHIZOPHRENIA

BackgroundThe role protein trafficking and localization is a recent target of investigation in schizophrenia pathophysiology. An important mediator of protein trafficking is S-acylation, also known as S-palmitoylation, which is the reversible attachment of long chain fatty acids to cysteine residues. S-acylation is a dynamic post-translational modification that modulates hydrophobicity of proteins, regulating their membrane association and subcellular localization. Notably, we have previously reported a proteome-wide decrease in S-acylated protein levels in the dorsolateral prefrontal cortex (DLPFC) of subjects with schizophrenia. One potential mechanism of decreased S-acylation is increased removal of acyl groups from proteins by protein acyl-thioesterase enzymes (PATs). Here we describe the optimization of an assay to measure the activity of the PAT family of enzymes in human postmortem cortical tissue and use the assay to address our hypothesis that PAT activity is increased in the DLPFC of subjects with schizophrenia.MethodsTo determine PAT activity, tissue homogenate was incubated with 4-methylumbelliferyl-6-thio-palmitate-β-D-glucopyranoside (4MU-Gluc-Palm) and 1U of exogenous β-glucosidase (to hydrolyze the 4MU-Gluc reaction intermediary). Released 4MU was excited at 360 ± 40 nm and fluorescent emission was measured, per minute, at 460 ± 40 nm. To determine the relationship between initial reaction rate and amount of enzyme, the initial reaction rate using 300 µM 4MU-Gluc-Palm was measured in homogenate containing 1 – 10 µg of total protein from the DLPFC of a subject with no history of psychiatric illness. The PAT activity of DLPFC homogenate boiled for 30 min and total protein homogenate from lymphocytes were measured as negative and positive control reactions, respectively.To estimate the maximum reaction rate (Vmax) and the concentration of 4MU-Gluc-Palm which achieved ½ Vmax (Km; a measure of enzyme-substrate affinity) the initial reaction rate was calculated in the presence of 0 – 200 µM 4MU-Gluc-Palm and the Michaelis-Menten equation was fit to plots of concentration vs. initial rate. Reactions were performed on 2.5 µg total protein homogenate from the DLPFC of 24 subjects with schizophrenia and 24 non-psychiatrically ill subjects.ResultsA fluorescent signal, which increases with time to a plateau upon substrate depletion, is detectable in total protein homogenate from DLPFC and lymphocytes, but not boiled DLPFC homogenate. In the DLPFC the initial reaction rate is linear with total protein amount [r2 = .99; p = .007], demonstrating that the reaction is sensitive to varying amounts of enzyme in a 10-fold range. When compared between schizophrenia and control subjects, neither Vmax [t(46) = 0.756; p = .45] nor Km [t(46) = 0.780; p = .44] were statistically significantly different.DiscussionHere we have demonstrated that PAT activity is measurable in human cortical tissue homogenate. Additionally, we have found no difference in the Vmax or Km of the combined PAT enzyme group in schizophrenia, providing no evidence to support our hypothesis that total PAT activity is increased in subjects with schizophrenia. This suggests that the proteome-wide decrease in S-acylated proteins in schizophrenia is caused by another mechanism, possibly increased expression or function of one or more of the specific PATs, leading to substrate specific changes in S-acylation, or a decrease in activity the acyl protein transferase enzymes, which attach acyl groups to proteins.

Characterization of a Splice Variant of Perilipin 5

Lipid storage provides energy stores for the body in times of increased oxidative requirements, such as fasting or exercise, but when the balance of lipid storage and mobilization is disrupted human health suffers. Implications for increased lipid storage include risk factors for developing type II diabetes mellitus and non‐alcoholic fatty liver disease. The perilipin family of proteins associate with the surface of lipid droplets and are essential to regulation of stored triacylglycerols. Perilipin 5 functions to maintain a balance between the storage of triacylglycerols in lipid droplets or their mobilization when energetically required. Several members of the perilipin gene family exhibit differential splicing, most notably perilipin 1 but reports exist of alternative messages for shortened forms of perilipins 2 and 3. We have identified a shortened form of perilipin 5 we have termed perilipin 5b. Western blots indicate that the protein, of approximately 35 kDa, is present in C2C12 cultured myoblasts and in heart and liver tissue from fed and fasted mice. Analysis of murine genomic DNA sequences reveals stop codons found in the introns between exons 6 and 7 and 7 and 8, either of which could give rise to a protein of approximately 35 kDa, indicating the likelihood of the observed protein being a splice variant, rather than a post‐translationally modified protein. Using RT‐PCR we have identified multiple amplicons suggesting that perilipin‐5b contains a read through of intron 6 of the murine perilipin 5 gene. Analysis of the amino acid sequence of perilipin 5b indicates that this truncation would lack the reported C‐terminal domain essential for interactions with ATGL and CGI‐58 but would retain the PKA site found at Ser155. The function of perilipin 5b is yet unknown, but the fact that it is found primarily in heart tissue seems to suggest that it plays a role in the oxidation of the lipid droplet. These data are consistent with what is currently known about Perilipin‐5 and the other PAT family proteins. Determination of perilipin 5b function will give a more accurate picture of the overall mechanism of lipid storage and metabolism.

Changes in liver perilipin gene expression during an oral lipid load in obese OLETF rats

Non‐alcoholic fatty liver disease (NAFLD) is one of the most prevalent liver maladies in the US. NAFLD is associated with obesity, hyperlipidemia, hypertension, insulin resistance, and diabetes; all traits linked to metabolic syndrome, affecting 20% of the US population. Inappropriate hepatic lipid accumulation is associated with metabolic syndrome. Intracellular lipid storage is regulated by lipid droplets (LDs) that are active components of the cell. LDs have surface proteins of which the most studied belong to the PAT family (Perilipin1–5), which participate in storage and lipid metabolism. To evaluate the effect that a high‐lipid load on the expression and dynamics of hepatic perilipins during early onset T2D, an oral fat tolerance test (OFTT) was performed in 28 wk‐old rats (n=5–6/group): (1) healthy, strain control LETO, and (2) obese, diabetic OLETF. Liver and blood were collected before (T0) and after (T1, T2, T4, and T8 hours) the lipid challenge. An acute increase in serum TG was observed at T2 (p= < 0.0001) and was maintained over the next 6 hours in OLETF whereas in LETO it decreased after the T2 mark. The mRNA expression of Plin2 increased 2.5‐fold in OLETF at T2 compared to LETO, and increased 2.5‐fold at T8 in LETO compared to baseline. Plin3 remained higher (p= 0.0077) in LETO compared to OLETF at T8. Plin4 did not change significantly in response to the lipid challenge between groups. Plin5 increased 1.8‐fold in LETO at T8 compared to baseline. These changes help explain the contributions of LDs to the regulation of hepatic lipids during both normal and diabetic conditions in response to a high lipid challenge similar to the consumption of a high‐fat meal. As Plin2 and 3 have been described as structural LD‐associated proteins, being present mostly in nascent LD, suggesting that a high fat load induces an acute induction of hepatocyte lipid storage. The increase in Plin5 suggests that lipid metabolism pathways are being activated, as Plin5 has been related with mitochondrial FA metabolism. The differential expression patterns of perilipins in response to an acute lipid challenge illustrates the timing of the cellular changes during the development of hepatic steatosis, providing useful insights into future development of treatment by targeting the PAT family of proteins.Support or Funding InformationThis work was supported by NIH's MHRT program.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Различие влияния липополисахарида E. coli , олеиновой кислоты и этомоксира на экспрессию белка ADRP в эпителиальных клетках

Intracellularly neutral lipids are deposited in lipid bodies (LB) – special organelles composed of triglyceride or cholesterol esters core surrounded by a phospholipid monolayer. LBs play a central role in cellular lipid metabolism. The biogenesis of LB and consumption of neutral lipids are controlled by proteins of the PAT family, which are expressed on the surface of LB and regulate lipids storage and degradation providing the interaction of LB with mitochondria and access of lipases to their substrates. The ADRP (adipose differentiation-related protein), a member of the PAT family, is a quantitatively major LB surface protein expressed in many cell types. The goal of the present work was to study the involvement of ADRP in the formation of LB and intracellular TAG accumulation under the action of different stimuli –lipopolysaccharide (LPS), exogenous oleic acid and etomoxir, an inhibitor of carnitine-palmitoyltransferase 1. The experiments were performed on epithelial cells isolated from the mucosal surface of the frog urinary bladder. Incubation of the cells for 21 hours with E. coli LPS (25 μg / ml), oleic acid (50 μM) and etomoxir (100 μM) led to a significant increase in the number and size of LBs and an increase in intracellular TAG accumulation. Despite the fact that all the substances used effectively stimulated LB growth, only incubation with LPS was accompanied by a sharp increase in the expression of ADRP protein, whereas in the presence of etomoxir or oleic acid the expression of this protein did not change. The obtained results indicate that the molecular mechanisms underlying the increase in LB formation and TAG accumulation under the action of different stimuli in the same cell type can be differed in relation to the involvement of LB surface proteins.

FAM134B promotes adipogenesis by increasing vesicular activity in porcine and 3T3-L1 adipocytes.

Family with sequence similarity 134, Member B (FAM134B), is a cis-Golgi transmembrane protein that is known to be necessary for the long-term survival of nociceptive and autonomic ganglion neurons. Recent work has shown that FAM134B plays a pivotal role in autophagy-mediated turnover of endoplasmic reticulum (ER) membranes, tumor inhibition and lipid homeostasis. In this study, we provide mechanistic links between FAM134B and ARF-related protein 1 (ARFRP1) and further show that FAM134B resides in the Golgi apparatus. Here, we found that FAM134B increased lipid accumulation in adipocytes. Transport vehicle number and ADP-ribosylation factor (ARF) family gene expression were also increased by FAM134B overexpression, suggesting that vesicular transport activity enhanced lipid accumulation. ARF-related protein 1 (ARFRP1) is a GTPase that promotes protein trafficking. We show that FAM134B regulates the expression of ARFRP1, and the knockdown of ARFRP1 abolishes enhancement on lipid accumulation caused by FAM134B. In addition, FAM134B upregulates the PAT family protein (PAT), which associates with the lipid droplets (LDs) surface and promotes lipolysis by recruiting adipocyte triglyceride lipase (ATGL). These findings indicate that FAM134B promotes lipid accumulation and adipogenic differentiation by increasing vesicle transport activity in the Golgi apparatus and inhibiting the lipolysis of LDs.

PATs and SNATs: Amino Acid Sensors in Disguise.

Solute Carriers (SLCs) are involved in the transport of substances across lipid bilayers, including nutrients like amino acids. Amino acids increase the activity of the microenvironmental sensor mechanistic Target of Rapamycin Complex 1 (mTORC1) to promote cellular growth and anabolic processes. They can be brought in to cells by a wide range of SLCs including the closely related Proton-assisted Amino acid Transporter (PAT or SLC36) and Sodium-coupled Neutral Amino acid Transporter (SNAT or SLC38) families. More than a decade ago, the first evidence emerged that members of the PAT family can act as amino acid-stimulated receptors, or so-called “transceptors,” connecting amino acids to mTORC1 activation. Since then, further studies in human cell models have suggested that other PAT and SNAT family members, which share significant homology within their transmembrane domains, can act as transceptors. A paradigm shift has also led to the PATs and SNATs at the surface of multiple intracellular compartments being linked to the recruitment and activation of different pools of mTORC1. Much focus has been on late endosomes and lysosomes as mTORC1 regulatory hubs, but more recently a Golgi-localized PAT was shown to be required for mTORC1 activation. PATs and SNATs can also traffic between the cell surface and intracellular compartments, with regulation of this movement providing a means of controlling their mTORC1 regulatory activity. These emerging features of PAT and SNAT amino acid sensors, including the transceptor mechanism, have implications for the pharmacological inhibition of mTORC1 and new therapeutic interventions.

Regulation of TG accumulation and lipid droplet morphology by the novel TLDP1 in Aurantiochytrium limacinum F26-b

Thraustochytrids are marine single-cell protists that produce large amounts of PUFAs, such as DHA. They accumulate PUFAs in lipid droplets (LDs), mainly as constituent(s) of triacylglycerol (TG). We identified a novel protein in the LD fraction of Aurantiochytrium limacinum F26-b using 2D-difference gel electrophoresis. The protein clustered with orthologs of thraustochytrids; however, the cluster was evolutionally different from known PAT family proteins or plant LD protein; thus, we named it thraustochytrid-specific LD protein 1 (TLDP1). TLDP1 surrounded LDs when expressed as a GFP-tagged form. Disruption of the tldp1 gene decreased the content of TG and number of LDs per cell; however, irregular and unusually large LDs were generated in tldp1-deficient mutants. Although the level of TG synthesis was unchanged by the disruption of tldp1, the level of TG degradation was higher in tldp1-deficient mutants than in the WT. These phenotypic abnormalities in tldp1-deficient mutants were restored by the expression of tldp1. These results indicate that TLDP1 is a thraustochytrid-specific LD protein and regulates the TG accumulation and LD morphology in A. limacinum F26-b.

Perilipin 1 is a highly specific marker for adipocytic differentiation in sarcomas with intermediate sensitivity.

Liposarcomas are the most common soft tissue sarcomas of adults. The identification of lipoblastic cells in soft tissue sarcomas is mandatory for the diagnosis of most subtypes of liposarcomas but may be difficult in conventional histology. The present study focuses on the expression and possible diagnostic impact of two PAT family proteins, perilipin 1/perilipin and perilipin 2/adipophilin in human liposarcomas. Eighty-seven cases of liposarcomas and 30 cases of non-lipomatous sarcomas were investigated immunohistochemically for perilipin 1 and 2 using entire tissue sections. Statistical analyses were performed using appropriate tests. Most liposarcomas and non-lipomatous sarcomas displayed positivity for perilipin 2. In contrast, while more than two-thirds of liposarcomas presented perilipin 1 positivity, all non-lipomatous sarcomas studied were negative for this marker, with statistical significance (p<0.001). Perilipin 1 expression increased with adipocytic differentiation of liposarcoma subtypes showing statistical significance (p<0.001). Non-lipomatous sarcomas demonstrated variable expression levels of perilipin 2. The expression level of perilipin 2 appeared to be correlated with tumor cell degeneration, e.g., through hypoxia. Perilipin 2 is not well suitable for distinction between liposarcomas and non-lipomatous sarcomas. However, perilipin 1 appeared to be a highly specific marker for liposarcoma and adipocytic differentiation in sarcomas with intermediate sensitivity.

Genomics and expression analysis of DHHC-cysteine-rich domain S-acyl transferase protein family in apple

S-acylation is one of a group of lipid modifications that occurs on eukaryotic proteins, mediated by DHHC-CRD-containing proteins, which plays an important role in regulating the membrane association, trafficking and function of target proteins. Although genome-wide identification of PAT family has been carried out in yeast, mice, humans and Arabidopsis, little is known about apple PAT genes. In this study, a total of 33 putative apple PAT proteins, containing DHHC-CRD by domain analysis, have been identified, and were classified into three groups according to the phylogenetic analysis of PAT proteins in apple and Arabidopsis. More complex TMDs in the most MdPATs revealed the PM location of the gene family. Gene structure, gene chromosomal location and paralogs analysis of MdPAT genes within the apple genome demonstrated that tandem and segmental duplications, as well as whole genome duplications, have likely contributed to the expansion and evolution of the PAT gene family in apple. According to the microarray and expressed sequence tag (ESTs) analysis, the different expression patterns indicate that they may play different roles during fruit development and rootstock-scion interactions process. Moreover, PATs were performed expression profile analyses in different tissues, indicating that the PATs are involved in various aspects of physiological and developmental processes of apple. To our knowledge, this is the first report of a genome-wide analysis of the apple PAT gene family, and this genomic analysis of apple DHHC-CRD PAT genes provides the first step towards a functional study of this gene family in apple.

Structural Characterization of the Lipid Storage Droplet Protein Perilipin 5

The ability of eukaryotic cells to maintain a reservoir of neutral lipids is critical in order maintain the plasma membrane, assist in the production of hormones, and to act as a source of energy if needed. Neutral lipids are stored within a structure known as a lipid droplet coated by members of the PAT family of lipid droplet proteins embedded within its lipid monolayer. The PAT family is comprised of perilipins 1–5 which have been shown to play a roles in regulating lipid droplet metabolism and the droplet's interaction with lipases. This work seeks to analyze a member of the PAT family, Perilipin 5, using proteolytic mapping in order to determine its domain topology which may provide clues to its function. We hypothesized that delipidation of cellular extracts using either detergents or lipases would alter the conformation of perilipin 5. This would alter the proteolytic cleavage patterns, indicating independently interacting domains of the protein and cleavage sites that were protected from proteases by being buried in lipid. Cellular lysates of Chinese Hamster Ovary cell lines expressing perilipin 5 were used as a source of protein. Following proteolysis, samples were analyzed by western blotting. Triton‐X‐100 treated samples had more cleavage sites than controls or Tween 20 treated samples. Collectively, these data show at least two major and four minor cleavage products resulting from proteolysis and suggest that perilipin 5 has at least two distinct domains.Support or Funding InformationThe Bert and Jane Horn endowed fund of Otterbein University.

Commentary: Heart Fat Infiltration in Subjects With and Without Coronary Artery Disease.

Commentary: Heart Fat Infiltration in Subjects With and Without Coronary Artery Disease.

Perilipin2 plays a positive role in adipocytes during lipolysis by escaping proteasomal degradation.

Perilipin2 (Plin2), also known as adipose differentiation-related protein (ADRP), or adipophilin, is a member of the PAT family involved in lipid droplet (LD) formation in the liver and peripheral tissues. Although Plin2 was originally identified as a highly expressed gene in adipocytes, its physiological role in mature adipocytes is largely unknown. In this report, we investigated the regulation of Plin2 expression and its function in differentiated adipocytes of mouse embryonic fibroblasts (MEFs). Plin2 mRNA levels increased during adipocyte differentiation whereas protein levels did not. Plin2 was degraded through the ubiquitin-proteasome pathway but was inhibited by lipolytic inducers. Furthermore, lentiviral-mediated Plin2 knockdown attenuated lipolysis in differentiated MEFs in a time-dependent manner. Oleic acid-induced LD formation enhanced Plin2 protein stability when it was localized to LDs. Furthermore, a mutational analysis revealed that the ubiquitination and degradation of Plin2 required both the second and third alanine in the N-terminal region. These results suggest that Plin2 is degraded in the cytosol in its N-terminal amino acid sequence-dependent manner and instead becomes stable when localized on LDs. Our findings highlight the relationship between protein stability and a previously unnoticed function of Plin2 during lipolysis in adipocytes.

Role of immunohistochemistry in the diagnosis of sebaceous carcinoma: a clinicopathologic and immunohistochemical study.

Sebaceous carcinoma (SC) is a relatively uncommon malignant epithelial neoplasm with a predilection for the periocular region. The diagnosis of SC can be difficult to make at initial presentation, as it can clinically and histopathologically resemble other common benign and malignant epithelial lesions. A diagnosis of SC is made by confirmation of sebaceous differentiation of neoplastic cells, which can often be accomplished by conventional microscopic findings; however, its recognition may be sometimes difficult and requires ancillary studies such as immunohistochemistry (IHC). Many studies have evaluated the role of IHC as a potential technique to differentiate SC from its mimics; however, most of these studies have used a limited panel of antibodies with variable results. The aim of this study was to determine the efficacy of IHC in the diagnosis of SC and to provide some guidelines for interpretation in the diagnosis of these neoplasms. We studied 27 cases of SC with a broad panel of IHC markers using a tissue microarray technique. We also studied 21 control cases of basal cell carcinoma (BCC) and 22 control cases of squamous cell carcinoma (SCC). Representative tissue cores were taken and processed from each case, and the tissue microarrays were stained by standard methods using antibodies to EMA, CK7, Ber-EP4, Factor XIIIA, androgen receptor, p53, adipophilin, progesterone receptor membrane component 1 (PGRMC1), squalene synthase (SQS), and alpha/beta hydrolase domain-containing protein 5 (ABHD5). Our studies show that EMA was expressed in all cases of SC, CK7 was expressed in 24 of 27 cases, Ber-EP4 was expressed in 7 of 27 cases, Factor XIIIA was negative in all cases, androgen receptor was expressed in 9 of 27 cases, P53 was expressed in 12 of 27 cases, adipophilin was expressed in all cases, PGRMC1 was expressed in 22 of 27 cases, SQS was expressed in 11 of 27 cases, and ABHD5 was expressed in 9 of 27 cases. EMA was negative in all cases of BCC, CK7 was expressed in 6 of 21 cases, Ber-EP4 was expressed in 21 of 21 cases, Factor XIIIA was negative in all cases, androgen receptor was expressed in 3 of 21 cases, P53 was expressed in 4 of 21 cases, adipophilin, PGRMC1, SQS, and ABHD5 were negative in all cases of BCC. Similarly, EMA was expressed in 16 of 22 cases of SCC, CK7 was expressed in 2 of 22 cases, Ber-EP4, Factor XIIIA, and androgen receptor were negative in all cases, P53 was expressed in 3 of 22 cases, adipophilin, PGRMC1, SQS, and ABHD5 were negative in all cases of SCC. Our study indicates that adipophilin represents a sensitive and reliable marker for the diagnosis of SC and can be of help in separating this tumor from some of its mimics. Additionally, inclusion of various epithelial markers in the panel will be of help if adequately used. Other antibodies against the PAT family of lipid droplet-associated proteins including PGRMC1, SQS, and ABHD5 were not as sensitive as adipophilin for identifying sebaceous differentiation and may therefore not be as useful for differential diagnosis as adipophilin.

Lipid droplet-associated proteins. Importance in steatosis, steatohepatitis and hepatocarcinogenesis

Hepatocellular steatosis constitutes the most frequent liver disease in western countries and may progress to steatohepatitis, liver cirrhosis and hepatocellular carcinoma (HCC). The lipid droplet (LD)-associated proteins perilipin, adipophilin, TIP47 ("tail interacting protein of 47 kDa"), S3-12 and myocardial LD protein (MLDP), so-called perilipins 1-5 (PAT family) govern formation, maintenance and degradation of LDs. A lack of perilipin in mice inhibits obesity and a lack of adipophilin or TIP47 inhibit the development of fatty liver disease. In long-term cell culture models as well as in liver biopsies of patients with different acute and chronic liver diseases, LD-associated proteins are sequentially recruited to LDs and regulated via peroxisome proliferator-activated receptor (PPAR) α and PPARγ as well as posttranscriptionally via alternative splicing, LD fusion and lipolysis. Whereas TIP47 and MLDP coat small newly formed LDs in acute microvesicular steatosis, adipophilin constitutes a robust general marker for LDs in many different cell types. Perilipin is important for the long-term storage of lipids in macrovesicular steatosis and controls lipolysis via hormone-dependent phosphorylation. During malignant transformation, increased formation of small LDs and overexpression of adipophilin, TIP47 and MLDP are detected, possibly as the expression of an altered tumor metabolism analogous to a Warburg effect. Adipophilin correlates positively with the proliferation rate of HCC cells. Cultured cells with downregulation of TIP47 or adipophilin via small interfering RNA (siRNA) or small hairpin RNA (shRNA) show less but larger LDs with reduced neutral fat content.

A Study of Lipid Interactions for the exchangeable PAT protein, Perilipin 3

Perilipin 3 (TIP 47) is a ubiquitously expressed PAT family protein carrying a C-terminal amphipathic α-helix bundle. In this work we focus on the mechanism of lipid binding by perilipin 3. Perilipin 3 associates with growing lipid droplets (LDs) and not with cellular membranes in vivo but the mechanism behind this specificity is not clear. We used several N-terminal truncation mutants as well as the full length perilipin 3 protein to investigate its lipid binding properties. Lipid monolayers at the air – buffer interface were used to study the lipid binding properties of all these constructs. Since monolayers are incredibly sensitive to surface active contaminants we had to purify large quantities of recombinant protein in order to carry out this work. The insertion of perilipin 3 into lipid monolayers was monitored by the increase in surface pressure of the lipid monolayer after injecting the proteins beneath a (phospho-)lipid monolayer. By systematically varying the lipid composition of the lipid monolayer we address the type of interactions responsible for perilipin 3 – LD interaction. This work addresses the types of interactions responsible for perilipin 3 – lipid insertion and also of the role of amphipathic α-helix bundles in lipid binding. We provide a model that describes the interactions responsible for perilipin 3 – LD interaction that help explain what sets this protein apart from other phospholipid membrane binding proteins.

PPARG modulated lipid accumulation in dairy GMEC via regulation of ADRP gene.

Peroxisome proliferator-activated receptor-gamma (PPARG) is considered to be a central regulator of lipid metabolism in mammary cells. Adipose differentiation-related protein (ADRP), a member of PAT family (Perilipin, ADRP, TIP47 family), plays a key role in lipid accumulation in mammary gland. It has been found that PPARG significantly promoted lipid storage in goat mammary epithelial cells (GMEC), which was abolished after knockdown of ADRP gene. The results of real-time PCR, Western blot and luciferase reporter assay for goat ADRP promoter showed that ligand-activated PPARG up-regulated the ADRP gene expression and activity of ADRP promoter. Moreover, PPARG directly interacted with a PPRE (PPAR response element) spanning at -1003 to -990 on ADRP promoter. In this study, to our knowledge, we are the first to verify the function of PPARG on lipid storage on cellular level of goat mammary gland and our results revealed a novel pathway that PPARG may regulate lipid accumulation by controlling the expression of ADRP gene.

CB-12 * LIPID DROPLET ACCUMULATION IN VON HIPPEL-LINDAU CNS HEMANGIOBLASTOMAS IS A MANIFESTATION OF HIF OVEREXPRESSION

CNS hemangioblastomas (HBs) are a major manifestation of the Von Hippel-Lindau (VHL) inherited tumor syndrome. Loss of functional VHL protein results in constitutive upregulation of hypoxia-inducible factor (HIF) and its downstream targets (e.g. VEGF) in affected cell types. The HB tumor cell exhibits a characteristic histologic lipid-laden foamy appearance, the biological basis of which has not been examined. Lipid accumulation in cells is an organized process and involves specific proteins that assist in assembling newly synthesized lipids into protein-covered droplets. The most studied lipid droplet proteins belong to the PAT family, named for the first three members: perilipin, adipose differentiation-related protein, and 47-kDa tail-interacting protein, now referred to as perilipin (PLIN) 1, 2, and 3, respectively. Of these, only PLIN2 is a HIF target gene, consistent with our observation that PLIN2 in glioblastoma occurs only focally in perinecrotic/hypoxic areas. We hypothesized that PLIN2 would be strongly upregulated in HB tumor cells. Twenty-two VHL-associated hemangioblastomas were examined immunohistochemically for PLIN1, -2, and -3. PLIN1 was absent and PLIN3 was observed only occasionally in small focal patches. However, all 22 HBs stained positively and robustly for PLIN2, which clearly outlined the lipid droplets in almost all HB tumor cells. In many cases, these droplets had enlarged and appeared to occupy the entire cytoplasm of the tumor cell. Double immunofluorescence indicated PLIN2 presence in the tumor/stromal, but not endothelial, cell compartment of HBs, another observation consistent with PLIN2 upregulation via HIF. The widespread, intense, and selective expression of PLIN2 in HBs suggests that HIF, via PLIN2 overexpression, promotes the extensive lipid droplet accumulation characteristic of these tumors. The extent to which this distinctive upregulation of PLIN2 has ramifications for understanding the biology of these tumors, or might be exploited for diagnostic or therapeutic purposes, remains to be determined.

Expression of PLIN2 and PLIN3 during oocyte maturation and early embryo development in cattle

In vitro-produced embryos store high lipid content in cytoplasmic lipid droplets (LD), and reduction or removal of LD has been demonstrated to improve freeze-thaw viability. The Perilipin Adipophilin Tail-interacting Protein of 47 kD (PAT) family of proteins is involved in the formation and regulation of LD in many cell types, but their presence has not been addressed either in cattle oocytes or preimplantation embryos. Therefore, this study aimed to detect the expression of PAT family transcripts (Perilipin-2 [PLIN2] and Perilipin-3 [PLIN3]) in immature and in vitro-matured (IVM) oocytes, and in in vitro-produced embryos at the stages of two to four cells, eight to 16 cells, morulae (MO), and blastocyst (BL). The expression of PLIN3 was downregulated in response to IVM, and PLIN2 was comparatively more expressed than PLIN3 in IVM oocytes (P < 0.001). During the early stages of embryo development, PLIN2 expression reached its peak at the MO stage (P < 0.001) and decreased again at the BL stage. In contrast, PLIN3 was expressed in low levels during the earliest stages of development, slightly upregulated at the MO stage (P < 0.05), and greatly increased its expression at the BL stage (15-fold; P < 0.001). PLIN3 was comparatively more expressed than PLIN2 during embryo culture in most stages analyzed (P < 0.05), except in eight- to 16-cell embryos. These results indicate that PLIN2 might be involved in the maintenance of lipid stocks necessary to support embryo development after fertilization of IVM oocytes. Also, we hypothesize that PLIN3 is the main PAT protein responsible for stabilization of LD formed in consequence of the acute lipid load seen during embryo development. We confirmed the presence of both PLIN2 and PLIN3 proteins in BL at Day 7 using immunocytochemistry: these PAT proteins colocalized with LD stained with BODIPY. PLIN3 seemed to be more ubiquitously spread out in the cytoplasm than PLIN2, consistent with the pattern seen in adipocytes. These findings suggest that both elderly (bigger) and newly formed (smaller) LD, positive for PLIN2 and PLIN3 respectively, coexist in blastocysts. To our knowledge this is the first report showing that transcripts of the PAT family are present in cattle oocytes and embryos.

Function of comparative gene identification-58 (CGI-58) on lipid metabolism in animals

There are some proteins on the surfaces of lipid droplets (LDs) in the cells which could regulate the lipogenesis and lipolysis, and play an important role in lipid metabolism of the whole body. Comparative gene identification-58 (CGI-58), distributing predominantly on the surface of LDs, which is a member of the α/β-hydrolase folding family and an activator of adipose triglyceride lipase (ATGL) and acyl-CoA-dependent lysophosphatidic acid acyltransfer (LPAAT). CGI-58 promotes lipolysis by combining with perilipin which is a member of the PAT family and ATGL. At the same time, the function of CGI-58 to activate ATGL is influenced by protein-protein interaction with the members of the perilipin family. This paper reviews the CGI-58 function in lipid metabolism of animals considering the hot topics.