TRIB3 promotes the progression of renal cell carcinoma by upregulating the lipid droplet-associated protein PLIN2.

Abnormal lipid metabolism and lipid accumulation are characteristic hallmarks of renal cell carcinoma (RCC). While there is prior evidence closely linking such lipid accumulation within RCC cells and consequent tumorigenesis, the mechanisms underlying this process remain incompletely understood. In this study, a series of bioinformatics analyses were initially performed by screening RCC databases and gene sets, ultimately leading to the identification of TRIB3 as an oncogene that functions as a central regulator of lipid metabolism. TRIB3 overexpression was observed in both RCC patient tumor tissues and cell lines, and this upregulation was correlated with a worse RCC patient prognosis. When TRIB3 was knocked down, this resulted in a reduction in lipid accumulation and the consequent induction of endoplasmic reticulum (ER) stress-related apoptotic cell death. At the molecular level, interactions between TRIB3 and PLIN2 were found to abrogate TEB4-mediated PLIN2 ubiquitination and consequent degradation, thus maintaining higher PLIN2 expression levels. This simultaneously helps facilitate the accumulation of lipids while preserving ER homeostasis, thus driving accelerated RCC tumor progression. This TRIB3-PLIN2 axis thus represents a promising new target for efforts to treat RCC.

Phosphoproteomics Revealed Differentially Expressed Sites and Function of the Bovine Milk Fat Globule Membrane in Colostrum and Mature Milk.

One type of large and intricate post-translational modification of milk proteins that has significant biological implications is phosphorylation. The characterization of phosphoproteins found in the bovine milk fat globule membrane (MFGM) is still mostly unknown. Here, label-free phosphoproteomics was used to identify 94 phosphorylation sites from 54 MFGM phosphoproteins in bovine colostrum (BC) and 136 phosphorylation sites from 91 MFGM phosphoproteins in bovine mature milk (BM). αs1-Casein and ß-casein were the most phosphorylated proteins in bovine colostrum. In bovine mature milk, perilipin-2 was the protein with the greatest number of phosphorylation sites. The results show that bovine colostrum MFGM phosphoproteins were mainly involved in immune function, whereas bovine mature MFGM phosphoproteins were mainly involved in metabolic function. Plasminogen and osteopontin were the most strongly interacting proteins in colostrum, whereas perilipin-2 was the most strongly interacting protein in bovine mature milk. This work demonstrates the unique alterations in the phosphorylation manner of the bovine MFGM protein during lactation and further expands our knowledge of the site characteristics of bovine MFGM phosphoproteins. This result confirms the value of MFGM as a reference ingredient for infant formula during different stages.

Arginine (315) is required for the PLIN2-CGI-58 interface and plays a functional role in regulating nascent LDs formation in bovine adipocytes.

Perilipin-2 (PLIN2) can anchor to lipid droplets (LDs) and play a crucial role in regulating nascent LDs formation. Bimolecular fluorescence complementation (BiFC) and flow cytometry were examined to verify the PLIN2-CGI-58 interaction efficiency in bovine adipocytes. GST-Pulldown assay was used to detect the key site arginine315 function in PLIN2-CGI-58 interaction. Experiments were also examined to research these mutations function of PLIN2 in LDs formation during adipocytes differentiation, LDs were measured after staining by BODIPY, lipogenesis-related genes were also detected. Results showed that Leucine (L371A, L311A) and glycine (G369A, G376A) mutations reduced interaction efficiencies. Serine (S367A) mutations enhanced the interaction efficiency. Arginine (R315A) mutations resulted in loss of fluorescence in the cytoplasm and disrupted the interaction with CGI-58, as verified by pulldown assay. R315W mutations resulted in a significant increase in the number of LDs compared with wild-type (WT) PLIN2 or the R315A mutations. Lipogenesis-related genes were either up- or downregulated when mutated PLIN2 interacted with CGI-58. Arginine315 in PLIN2 is required for the PLIN2-CGI-58 interface and could regulate nascent LD formation and lipogenesis. This study is the first to study amino acids on the PLIN2 interface during interaction with CGI-58 in bovine and highlight the role played by PLIN2 in the regulation of bovine adipocyte lipogenesis.

Plin2 inhibits autophagy via activating AKT/mTOR pathway in non-small cell lung cancer.

Perilipin 2 (Plin2) is known to be dysregulated in several human malignancies, which facilitates cancer progression. Recent studies have found that the abnormal expression of Plin2 is associated with poor prognosis of non-small cell lung cancer (NSCLC). However, the specific role of Plin2 and its underlying mechanism remain unclear. This study revealed that Plin2 expression was low in NSCLC tissues, and its relatively higher expression indicated larger tumor size and poorer prognosis. In vitro experiments proved that Plin2 promoted NSCLC cellular proliferation and inhibited autophagy by activating the AKT/mTOR pathway. Meanwhile, treatment with the AKT phosphorylation promoter or inhibitor neutralized the influence of Plin2 depletion or over-expression on proliferation and autophagy, respectively. In vivo study showed that Plin2 stimulated subcutaneous tumorigenesis of NSCLC cells in nude mice. Collectively, this study clarified the carcinogenic role of Plin2 and its molecular mechanism in NSCLC progression, which may facilitate a targeted therapy in the future.

Nazo, the Drosophila homolog of the NBIA-mutated protein-c19orf12, is required for triglyceride homeostasis.

Lipid dyshomeostasis has been implicated in a variety of diseases ranging from obesity to neurodegenerative disorders such as Neurodegeneration with Brain Iron Accumulation (NBIA). Here, we uncover the physiological role of Nazo, the Drosophila melanogaster homolog of the NBIA-mutated protein-c19orf12, whose function has been elusive. Ablation of Drosophila c19orf12 homologs leads to dysregulation of multiple lipid metabolism genes. nazo mutants exhibit markedly reduced gut lipid droplet and whole-body triglyceride contents. Consequently, they are sensitive to starvation and oxidative stress. Nazo is required for maintaining normal levels of Perilipin-2, an inhibitor of the lipase-Brummer. Concurrent knockdown of Brummer or overexpression of Perilipin-2 rescues the nazo phenotype, suggesting that this defect, at least in part, may arise from diminished Perilipin-2 on lipid droplets leading to aberrant Brummer-mediated lipolysis. Our findings potentially provide novel insights into the role of c19orf12 as a possible link between lipid dyshomeostasis and neurodegeneration, particularly in the context of NBIA.

Perilipin 1: a systematic review on its functions on lipid metabolism and atherosclerosis in mice and humans.

The function of perilipin 1 in human metabolism was recently highlighted by the description of PLIN1 variants associated with various pathologies. These include severe familial partial lipodystrophy and early onset acute coronary syndrome. Additionally, certain variants have been reported to have a protective effect on cardiovascular diseases. The role of this protein remains controversial in mice and variant interpretation in humans is still conflicting. This literature review has two primary objectives (i) to clarify the function of the PLIN1 gene in lipid metabolism and atherosclerosis by examining functional studies performed in cells (adipocytes) and mice and (ii) to understand the impact of PLIN1 variants identified in humans based on the variant's location within the protein and the type of variant (missense or frameshift). To achieve these objectives, we conducted an extensive analysis of the relevant literature on perilipin 1, its function in cellular models and mice, and the consequences of its mutations in humans. We also utilized bioinformatics tools and consulted the Human Genetics Cardiovascular Disease Knowledge Portal to enhance the pathogenicity assessment of PLIN1 missense variants.

Lysine tRNA fragments and miR-194-5p co-regulate hepatic steatosis via ß-Klotho and perilipin 2.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) involves hepatic accumulation of intracellular lipid droplets via incompletely understood processes. Here, we report distinct and cooperative NAFLD roles of LysTTT-5'tRF transfer RNA fragments and microRNA miR-194-5p. METHODS: Combined use of diet induced obese mice with human-derived oleic acid-exposed Hep G2 cells revealed new NAFLD roles of LysTTT-5'tRF and miR-194-5p. RESULTS: Unlike lean animals, dietary-induced NAFLD mice showed concurrent hepatic decrease of both LysTTT-5'tRF and miR-194-5p levels, which were restored following miR-132 antisense oligonucleotide treatment which suppresses hepatic steatosis. Moreover, exposing human-derived Hep G2 cells to oleic acid for 7 days co-suppressed miR-194-5p and LysTTT-5'tRF levels while increasing lipid accumulation. Inversely, transfecting fattened cells with a synthetic LysTTT-5'tRF mimic elevated mRNA levels of the metabolic regulator ß-Klotho while decreasing triglyceride amounts by 30% within 24 h. In contradistinction, antisense suppression of miR-194-5p induced accumulation of its novel target, the NAFLD-implicated lipid droplet-coating PLIN2 protein. Further, two out of 15 steatosis-alleviating screened drug-repurposing compounds, Danazol and Latanoprost, elevated miR-194-5p or LysTTT-5'tRF levels. CONCLUSION: Our findings highlight the different yet complementary roles of miR-194-5p and LysTTT-5'tRF and offer new insights into the complex roles of small non-coding RNAs and the multiple pathways involved in NAFLD pathogenesis.

Binding of perilipin 3 to membranes containing diacylglycerol is mediated by conserved residues within its PAT domain.

Perilipins (PLINs) constitute an evolutionarily conserved family of proteins that specifically associate with the surface of lipid droplets (LDs). These proteins function in LD biogenesis and lipolysis and help to stabilize the surface of LDs. PLINs are typically composed of three different protein domains. They share an N-terminal PAT domain of unknown structure and function, a central region containing 11-mer repeats that form amphipathic helices, and a C-terminal domain that adopts a 4-helix bundle structure. How exactly these three distinct domains contribute to PLIN function remains to be determined. Here, we show that the N-terminal PAT domain of PLIN3 binds diacylglycerol (DAG), the precursor to triacylglycerol, a major storage lipid of LDs. PLIN3 and its PAT domain alone bind liposomes with micromolar affinity and PLIN3 binds artificial LDs containing low concentrations of DAG with nanomolar affinity. The PAT domain of PLIN3 is predicted to adopt an amphipathic triangular shaped structure. In silico ligand docking indicates that DAG binds to one of the highly curved regions within this domain. A conserved aspartic acid residue in the PAT domain, E86, is predicted to interact with DAG, and we found that its substitution abrogates high affinity binding of DAG as well as DAG-stimulated association with liposome and artificial LDs. These results indicate that the PAT domain of PLINs harbor specific lipid-binding properties that are important for targeting these proteins to the surface of LDs and to ER membrane domains enriched in DAG to promote LD formation.

Altered hepatic lipid droplet morphology and lipid metabolism in fasted Plin2-null mice.

Perilipin 2 (Plin2) binds to the surface of hepatic lipid droplets (LDs) with expression levels that correlate with triacylglyceride (TAG) content. We investigated if Plin2 is important for hepatic LD storage in fasted or high-fat diet-induced obese Plin2+/+ and Plin2-/- mice. Plin2-/- mice had comparable body weights, metabolic phenotype, glucose tolerance, and circulating TAG and total cholesterol levels compared with Plin2+/+ mice, regardless of the dietary regime. Both fasted and high-fat fed Plin2-/- mice stored reduced levels of hepatic TAG compared with Plin2+/+ mice. Fasted Plin2-/- mice stored fewer but larger hepatic LDs compared with Plin2+/+ mice. Detailed hepatic lipid analysis showed substantial reductions in accumulated TAG species in fasted Plin2-/- mice compared with Plin2+/+ mice, whereas cholesteryl esters and phosphatidylcholines were increased. RNA-Seq revealed minor differences in hepatic gene expression between fed Plin2+/+ and Plin2-/- mice, in contrast to marked differences in gene expression between fasted Plin2+/+ and Plin2-/- mice. Our findings demonstrate that Plin2 is required to regulate hepatic LD size and storage of neutral lipid species in the fasted state, while its role in obesity-induced steatosis is less clear.

Perilipin 2-positive mononuclear phagocytes accumulate in the diabetic retina and promote PPARγ-dependent vasodegeneration.

Type 2 diabetes mellitus (T2DM), characterized by hyperglycemia and dyslipidemia, leads to nonproliferative diabetic retinopathy (NPDR). NPDR is associated with blood-retina barrier disruption, plasma exudates, microvascular degeneration, elevated inflammatory cytokine levels, and monocyte (Mo) infiltration. Whether and how the diabetes-associated changes in plasma lipid and carbohydrate levels modify Mo differentiation remains unknown. Here, we show that mononuclear phagocytes (MPs) in areas of vascular leakage in DR donor retinas expressed perilipin 2 (PLIN2), a marker of intracellular lipid load. Strong upregulation of PLIN2 was also observed when healthy donor Mos were treated with plasma from patients with T2DM or with palmitate concentrations typical of those found in T2DM plasma, but not under high-glucose conditions. PLIN2 expression correlated with the expression of other key genes involved in lipid metabolism (ACADVL, PDK4) and the DR biomarkers ANGPTL4 and CXCL8. Mechanistically, we show that lipid-exposed MPs induced capillary degeneration in ex vivo explants that was inhibited by pharmaceutical inhibition of PPARγ signaling. Our study reveals a mechanism linking dyslipidemia-induced MP polarization to the increased inflammatory cytokine levels and microvascular degeneration that characterize NPDR. This study provides comprehensive insights into the glycemia-independent activation of Mos in T2DM and identifies MP PPARγ as a target for inhibition of lipid-activated MPs in DR.

The combination of the low immunohistochemical expression of peroxiredoxin 4 and perilipin 2 predicts longer survival in pancreatic ductal adenocarcinoma with peroxiredoxin 4 possibly playing a main role.

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with poor prognosis. Therefore, indicators that can be used for the early prediction of the prognosis of PDAC are needed. Peroxiredoxin (PRDX) 4 is a secretion-type antioxidant enzyme located in the cytoplasmic endoplasmic reticulum. Recent studies have reported that it is closely related to the development and prognosis of many types of cancer. Perilipin (PLIN) 2 is a lipid droplet coating protein. The high expression of PLIN2 is known to be an indicator of some types of cancer and oxidative stress management. It is highly suggestive of the interplay between PRDX4 and PLIN2 to some degree. In this study, we collected 101 patients' clinical data and paraffin-embedded specimens with PDAC and analyzed them with immunohistochemical staining of PRDX4 and PLIN2. We found that the low expression of PRDX4 predicts longer survival and a better clinical condition in PDAC patients. Moreover, when the low expression of PRDX4 is combined with the low expression of PLIN2, the 3-year survival is significantly improved. Univariate and multivariate Cox proportional hazard analyses showed that the PRDX4 expression in PDAC was an independent prognostic factor for survival. Taken together, between PRDX4 and PLIN2, PRDX4 plays a main role in prognosis and has the potential to become a clinical prognostic indicator of PDAC.

Ceramide synthase 6 (CerS6) is upregulated in alcohol-associated liver disease and exhibits sex-based differences in the regulation of energy homeostasis and lipid droplet accumulation.

OBJECTIVE: Alcohol-associated liver disease (ALD) is the leading cause of liver-related mortality worldwide. Current strategies to manage ALD focus largely on advanced stage disease, however, metabolic changes such as glucose intolerance are apparent at the earliest stage of alcoholic steatosis and increase the risk of disease progression. Ceramides impair insulin signaling and accumulate in ALD, and metabolic pathways involving ceramide synthase 6 (CerS6) are perturbed in ALD during hepatic steatosis. In this study, we aimed to investigate the role of CerS6 in ALD development and the relevance of CerS6 to human ALD. METHODS: C57BL/6 WT and CerS6 KO mice of both sexes were fed either a Lieber-DeCarli control (CON) or 15% ethanol (EtOH) diet for six weeks. In vivo metabolic tests including glucose and insulin tolerance tests (GTT and ITT) and energy expenditure were performed. The mice were euthanized, and serum and liver lipids and liver histology were examined. For in vitro studies, CerS6 was deleted in human hepatocytes, VL17A and cells were incubated with EtOH and/or C16:0-ceramides. RNAseq analysis was performed in livers from mice and human patients with different stages of ALD and diseased controls. RESULTS: After six weeks on an EtOH diet, CerS6 KO mice had reduced body weight, food intake, and %fat mass compared to WT mice. Energy expenditure increased in both male and female KO mice, however, was only statistically significant in male mice. In response to EtOH, WT mice developed mild hepatic steatosis, while steatosis was ameliorated in KO mice as determined by H&E and ORO staining. KO mice showed significantly decreased long-chain ceramide species, especially C16:0-ceramides, in the serum and liver tissues compared to WT mice. CerS6 deletion decreased serum TG and NEFA only in male not female mice. CerS6 deletion improved glucose tolerance and insulin resistance in EtOH-fed mice of both sexes. RNAseq analysis revealed that 74 genes are significantly upregulated and 66 genes are downregulated by CerS6 deletion in EtOH-fed male mice, with key network pathways including TG biosynthetic process, positive regulation of lipid localization, and fat cell differentiation. Similar to RNAseq results, absence of CerS6 significantly decreased mRNA expression of lipid droplet associated proteins in EtOH-fed mice. In vitro, EtOH stimulation significantly increased PLIN2 protein expression in VL17A cells while CerS6 deletion inhibited EtOH-mediated PLIN2 upregulation. C16:0-ceramide treatment significantly increased PLIN2 protein expression compared to CON. Notably, progression of ALD in humans was associated with increased hepatic CerS6 expression. CONCLUSIONS: Our findings demonstrate that CerS6 deletion improves glucose homeostasis in alcohol-fed mice and exhibits sex-based differences in the attenuation of EtOH-induced weight gain and hepatic steatosis. Additionally, we unveil that CerS6 plays a major role as a regulator of lipid droplet biogenesis in alcohol-induced intra-hepatic lipid droplet formation, identifying it as a putative target for early ALD management.

Methacrylated Gelatin as a Scaffold for Mechanically Isolated Stromal Vascular Fraction for Cutaneous Wound Repair.

Mechanically processed stromal vascular fraction (mSVF) is a highly interesting cell source for regenerative purposes, including wound healing, and a practical alternative to enzymatically isolated SVF. In the clinical context, SVF benefits from scaffolds that facilitate viability and other cellular properties. In the present work, the feasibility of methacrylated gelatin (GelMA), a stiffness-tunable, light-inducible hydrogel with high biocompatibility is investigated as a scaffold for SVF in an in vitro setting. Lipoaspirates from elective surgical procedures were collected and processed to mSVF and mixed with GelMA precursor solutions. Non-encapsulated mSVF served as a control. Viability was measured over 21 days. Secreted basic fibroblast growth factor (bFGF) levels were measured on days 1, 7 and 21 by ELISA. IHC was performed to detect VEGF-A, perilipin-2, and CD73 expression on days 7 and 21. The impact of GelMA-mSVF on human dermal fibroblasts was measured in a co-culture assay by the same viability assay. The viability of cultured GelMA-mSVF was significantly higher after 21 days (p < 0.01) when compared to mSVF alone. Also, GelMA-mSVF secreted stable levels of bFGF over 21 days. While VEGF-A was primarily expressed on day 21, perilipin-2 and CD73-positive cells were observed on days 7 and 21. Finally, GelMA-mSVF significantly improved fibroblast viability as compared with GelMA alone (p < 0.01). GelMA may be a promising scaffold for mSVF as it maintains cell viability and proliferation with the release of growth factors while facilitating adipogenic differentiation, stromal cell marker expression and fibroblast proliferation.

Parallel CRISPR-Cas9 screens identify mechanisms of PLIN2 and lipid droplet regulation.

Despite the key roles of perilipin-2 (PLIN2) in governing lipid droplet (LD) metabolism, the mechanisms that regulate PLIN2 levels remain incompletely understood. Here, we leverage a set of genome-edited human PLIN2 reporter cell lines in a series of CRISPR-Cas9 loss-of-function screens, identifying genetic modifiers that influence PLIN2 expression and post-translational stability under different metabolic conditions and in different cell types. These regulators include canonical genes that control lipid metabolism as well as genes involved in ubiquitination, transcription, and mitochondrial function. We further demonstrate a role for the E3 ligase MARCH6 in regulating triacylglycerol biosynthesis, thereby influencing LD abundance and PLIN2 stability. Finally, our CRISPR screens and several published screens provide the foundation for CRISPRlipid (http://crisprlipid.org), an online data commons for lipid-related functional genomics data. Our study identifies mechanisms of PLIN2 and LD regulation and provides an extensive resource for the exploration of LD biology and lipid metabolism.

Alcoholic Setdb1 suppression promotes hepatosteatosis in mice by strengthening Plin2.

BACKGROUND AND AIMS: Hepatosteatosis is one of the early features of alcoholic liver disease (ALD) and pharmaceutical or genetic interfering of the development of hepatosteatosis will efficiently alleviate the progression of ALD. Currently, the role of histone methyltransferase Setdb1 in ALD is not yet well understood. METHOD: Lieber-De Carli diet mice model and NIAAA mice model were constructed to confirm the expression of Setdb1. The hepatocyte-specific Setdb1-knockout (Setdb1-HKO) mice was established to determine the effects of Setdb1 in vivo. Adenovirus-Setdb1 were produced to rescue the hepatic steatosis in both Setdb1-HKO and Lieber-De Carli mice. The enrichment of H3k9me3 in the upstream sequence of Plin2 and the chaperone-mediated autophagy (CMA) of Plin2 were identified by ChIP and co-IP. Dual-luciferase reporter assay was used to detect the interaction of Setdb1 3'UTR and miR216b-5p in AML12 or HEK 293 T cells. RESULTS: We found that Setdb1 was downregulated in the liver of alcohol-fed mice. Setdb1 knockdown promoted lipid accumulation in AML12 hepatocytes. Meanwhile, hepatocyte-specific Setdb1-knockout (Setdb1-HKO) mice exhibited significant lipid accumulation in the liver. Overexpression of Setdb1 was performed with an adenoviral vector through tail vein injection, which ameliorated hepatosteatosis in both Setdb1-HKO and alcoholic diet-fed mice. Mechanistically, downregulated Setdb1 promoted the mRNA expression of Plin2 by desuppressing H3K9me3-mediated chromatin silencing in its upstream sequence. Pin2 acts as a critical membrane surface-associated protein to maintain lipid droplet stability and inhibit lipase degradation. The downregulation of Setdb1 also maintained the stability of Plin2 protein through inhibiting Plin2-recruited chaperone-mediated autophagy (CMA). To explore the reasons for Setdb1 suppression in ALD, we found that upregulated miR-216b-5p bound to the 3'UTR of Setdb1 mRNA, disturbed its mRNA stability, and eventually aggravated hepatic steatosis. CONCLUSIONS: Setdb1 suppression plays an important role in the progression of alcoholic hepatosteatosis via elevating the expression of Plin2 mRNA and maintaining the stability of Plin2 protein. Targeting hepatic Setdb1 might be a promising diagnostic or therapeutic strategy for ALD.

Liposarcoma of the pleural cavity.

Liposarcoma rarely occurs in the pleura or thoracic cavity, and few reports appear in the literature. We hypothesized that combining clinicopathologic, immunohistochemical, and fluorescence in situ hybridization methods would allow definite diagnoses. Using formalin-fixed, paraffin-embedded blocks, we examined 6 atypical lipomatous tumor/well-differentiated liposarcomas (ALT/WDLPS), 5 dedifferentiated liposarcomas (DDLPSs), 2 pleomorphic liposarcomas, and 1 myxoid liposarcoma (MLPS). We used the Kaplan-Meier method and the Wilcoxon test for survival analysis for prognostic factor evaluation. Histologically, ALT/WDLPS was composed of a relatively mature adipocytic proliferation, accompanied by some lipoblasts. DDLPS exhibited round-to-oval tumor cells with a high nucleus-to-cytoplasm ratio that had proliferated in nests, accompanied in case 10 by some giant cells but no fatty cells. The pleomorphic type contained a varying proportion of pleomorphic lipoblasts. MLPS displayed uniform round- to oval-shaped cells and small signet-ring lipoblasts in a myxoid stroma. Immunohistochemically, 11 (79%), 11 (79%), and 10 (71%) of 14 cases were positive for S-100, p16, and CDK4, respectively. Six of the 14 cases (43%) were positive for MDM2 and adipophilin. One case of ALT/WDLPS and 3 cases of DDLPS exhibited MDM2 amplification by fluorescence in situ hybridization (Vysis LSI MDM2 SpectrumGreen Probe plus Vysis CEP 12 SpectrumOrange probe). ALT/WDLPS was the most favorable type for survival, while adipophilin tended to be a negative prognostic factor for pleural liposarcoma. For a firm diagnosis of liposarcoma in the pleura, immunohistochemistry for CDK4, MDM2, and adipophilin together with MDM2 gene amplification by fluorescence in situ hybridization may be an important diagnostic tool.

Inhibition of perilipin 2 attenuates cerebral ischemia/reperfusion injury by blocking NLRP3 inflammasome activation both in vivo and in vitro.

Cerebral ischemia/reperfusion (CI/R) usually causes neuroinflammation within the central nervous system, further prompting irreversible cerebral dysfunction. Perilipin 2 (Plin2), a lipid droplet protein, has been reported to exacerbate the pathological process in different diseases, including inflammatory responses. However, the role and mechanism of Plin2 in CI/R injury are unclear. In this study, the rat models of transient middle cerebral artery occlusion followed by reperfusion (tMCAO/R) were established to mimic I/R injury, and we found that Plin2 was highly expressed in the ischemic penumbra of tMCAO/R rats. The siRNA-mediated knockdown of Plin2 significantly decreased neurological deficit scores and reduced infarct areas in rats induced by I/R. Detailed investigation showed that Plin2 deficiency alleviated inflammation of tMCAO/R rats as evidenced by reduced secretion of proinflammatory factors and the blockade of NLR family pyrin domain containing 3 (NLRP3) inflammasome activation. In vitro experiments showed that Plin2 expression was upregulated in mouse microglia subjected to oxygen-glucose deprivation/reoxygenation (OGD/R). Plin2 knockdown inhibited OGD/R-induced microglia activation and the accumulation of inflammation-related factors. Taken together, this study demonstrates that lipid droplet protein Plin2 contributes to the pathologic process of CI/R damage by impacting inflammatory response and NLRP3 inflammasome activation. Thus, Plin2 may provide a new therapeutic direction for CI/R injury.

Aflatoxin B1 exposure triggers hepatic lipotoxicity via p53 and perilipin 2 interaction-mediated mitochondria-lipid droplet contacts: An in vitro and in vivo assessment.

Aflatoxin B1 (AFB1) is one of the most toxic mycotoxins widely found in food contaminants, and its target organ is the liver. It poses a major food security and public health threat worldwide. However, the lipotoxicity mechanism of AFB1 exposure-induced liver injury remains unclear and requires further elucidation. Herein, we investigated the potential hepatic lipotoxicity of AFB1 exposure using in vitro and in vivo models to assess the public health hazards of high dietary AFB1 exposure. We demonstrated that low-dose of AFB1 (1.25 µM for 48 h, about one-fifth of the IC50 in HepG2 and HepaRG cells, IC50 are 5.995 µM and 5.266 µM, respectively) exposure significantly induced hepatic lipotoxicity, including abnormal lipid droplets (LDs) growth, mitochondria-LDs contacts increase, lipophagy disruption, and lipid accumulation. Mechanistically, we showed that AFB1 exposure promoted the mitochondrial p53 (mito-p53) and LDs-associated protein perilipin 2 (PLIN2) interaction-mediated mitochondria-LDs contacts, resulting in lipid accumulation in hepatocytes. Mito-p53-targeted inhibition, knockdown of PLIN2, and rapamycin application efficiently promoted the lysosome-dependent lipophagy and alleviated the hepatic lipotoxicity and liver injury induced by AFB1 exposure. Overall, our study found that mito-p53 and PLIN2 interaction mediates three organelles-mitochondria, LDs, and lysosomal networks to regulate lipid homeostasis in AFB1 exposure-induced hepatotoxicity, revealing how this unique trio of organelles works together and provides a novel insight into the targeted intervention in inter-organelle lipid sensing and trafficking for alleviating hazardous materials-induced hepatic lipotoxicity.

Effects of Long-Term Physical Activity and BCAA Availability on the Subcellular Associations between Intramyocellular Lipids, Perilipins and PGC-1α.

Cellular skeletal muscle lipid metabolism is of paramount importance for metabolic health, specifically through its connection to branched-chain amino acids (BCAA) metabolism and through its modulation by exercise. In this study, we aimed at better understanding intramyocellular lipids (IMCL) and their related key proteins in response to physical activity and BCAA deprivation. By means of confocal microscopy, we examined IMCL and the lipid droplet coating proteins PLIN2 and PLIN5 in human twin pairs discordant for physical activity. Additionally, in order to study IMCLs, PLINs and their association to peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in cytosolic and nuclear pools, we mimicked exercise-induced contractions in C2C12 myotubes by electrical pulse stimulation (EPS), with or without BCAA deprivation. The life-long physically active twins displayed an increased IMCL signal in type I fibers when compared to their inactive twin pair. Moreover, the inactive twins showed a decreased association between PLIN2 and IMCL. Similarly, in the C2C12 cell line, PLIN2 dissociated from IMCL when myotubes were deprived of BCAA, especially when contracting. In addition, in myotubes, EPS led to an increase in nuclear PLIN5 signal and its associations with IMCL and PGC-1α. This study demonstrates how physical activity and BCAA availability affects IMCL and their associated proteins, providing further and novel evidence for the link between the BCAA, energy and lipid metabolisms.

Novel prognostication biomarker adipophilin reveals a metabolic shift in uveal melanoma and new therapeutic opportunities.

Metastatic uveal melanoma remains incurable at present. We previously demonstrated that loss of BAP1 gene expression in tumour cells triggers molecular mechanisms of immunosuppression in the tumour microenvironment (TME) of metastatic uveal melanoma. Adipophilin is a structural protein of lipid droplets involved in fat storage within mammalian cells, and its expression has been identified in uveal melanoma. We comprehensively evaluated adipophilin expression at the RNA (PLIN2) and protein levels of 80 patients of the GDC-TCGA-UM study and in a local cohort of 43 primary uveal melanoma samples respectively. PLIN2 expression is a survival prognosticator biomarker in uveal melanoma. Loss of adipophilin expression is significantly associated with monosomy 3 status and nuclear BAP1 losses in uveal melanoma tumours. Integrative transcriptomic and secretome studies show a relationship between transient loss of adipophilin expression and increased levels of tumour-associated macrophages and hypoxia genes, suggesting PLIN2-dependent changes in oxygen and lipid metabolism in the TME of low and high-metastatic risk uveal melanoma. We designed four adipophilin-based multigene signatures for uveal melanoma prognostication using a transcriptomic and secretome survival-functional network approach. Adipophilin-based multigene signatures were validated in BAP1-positive and BAP1-negative uveal melanoma cell lines using a next-generation RNA sequencing approach. We identified existing small molecules, mostly adrenergic, retinoid, and glucocorticoid receptor agonists, MEK, and RAF inhibitors, with the potential to reverse this multigene signature expression in uveal melanoma. Some of these molecules were able to impact tumour cell viability, and carvedilol, an adrenergic receptor antagonist, restored PLIN2 levels, mimicking the expression of normoxia/lipid storage signatures and reversing the expression of hypoxia/lipolysis signatures in co-cultures of uveal melanoma cells with human macrophages. These findings open up a new research line for understanding the lipid metabolic regulation of immune responses, with implications for therapeutic innovation in uveal melanoma. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.