Expression Of PLIN2 Research Articles

Perilipin 5 Reduces Oxidative Damage Associated With Lipotoxicity by Activating the PI3K/ERK-Mediated Nrf2-ARE Signaling Pathway in INS-1 Pancreatic β-Cells.

Oxidative stress induced by free fatty acid overload in pancreatic β-cells is a potential contributory factor to dysfunction of insulin secretion and apoptotic cell death. Perilipin 5 (Plin5) has been reported to ameliorate oxidative stress-mediated damage in non-insulin-secreting tissues. We tested the hypothesis that Plin5 plays a similar role in pancreatic β-cells, which are extremely sensitive to oxidative stress. Here, our in vitro data showed that Plin5-mediated alleviation of palmitate-triggered apoptosis involves the mitochondrial pathway. And the protective role of Plin5 on β-cells was partially dependent on its modulation in oxidative stress. Upregulation of Plin5 in INS-1 cells decreased reactive oxygen species production, enhanced cellular glutathione levels, and induced expression of antioxidant enzymes glutamate-cysteine ligase catalytic subunit and heme oxygenase-1. However, knocking out of Plin5 abolished all of these beneficial effects. Furthermore, by using the O2− scavenger MnTMPyP, we verified that altering Plin5 expression impacted lipotoxic cell death partially via modulating oxidative stress. Mechanistic experiments revealed that Plin5 induced Nrf2-ARE system, a master regulator in the cellular adaptive response to oxidative stress, by activating PI3K/Akt and ERK signal pathways, contributing to the increase of antioxidant defense and consequently improving β-cell function and survival in the presence of lipotoxic oxidative stress. Overall, our findings indicate that Plin5 abrogates oxidative damage in INS-1 β-cells during lipotoxic stress partially through the enhancement of antioxidant defense involving the PI3K/Akt and ERK mediated Nrf2-ARE system.

Maternal diets enriched in olive oil regulate lipid metabolism and levels of PPARs and their coactivators in the fetal liver in a rat model of gestational diabetes mellitus

In a rat model of gestational diabetes mellitus (GDM) programmed in the offspring of neonatal streptozotocin-induced (nSTZ) diabetic rats, lipids are accumulated in the fetal liver in a sex-dependent way. Here, we evaluated whether maternal diets enriched in olive oil in rats that will develop GDM ameliorate lipid metabolic impairments in the fetal livers. Pregnant offspring of control and nSTZ diabetic rats (F0) were fed a 6% olive oil-supplemented diet throughout the F1 gestation. We evaluated maternal metabolic parameters as well as lipid content, expression of lipid metabolizing enzymes and protein expression of PLIN2, PPARs and PPAR coactivators in the fetal livers. The offspring of nSTZ diabetic rats developed GDM regardless of the maternal treatment. Hypertriglyceridemia in GDM rats was prevented by the olive oil-enriched maternal treatment. In the livers of male fetuses of GDM rats, the maternal olive oil-supplemented diet prevented lipid overaccumulation and prevented the increase in PPARγ and PPARδ levels. In the livers of female fetuses of GDM rats, the maternal olive oil supplementation prevented the increase in PPARδ levels and the reduction in PGC1α levels, but did not prevent the reduced lipid content. Control and GDM rats showed a reduction of lipid metabolic enzymes in the fetal livers, which was associated with reduced levels of the PPAR coactivators PGC-1α and SRC-1 in males and of SRC-1 in females. These results suggest powerful effects of a maternal olive oil-supplemented diet in the fetal liver, possibly providing benefits in the fetuses and offspring from GDM rats.

The Expression of Perilipin Family Proteins can be used as Diagnostic Markers of Liposarcoma and to Differentiate Subtypes.

Objective: Liposarcoma is a mesenchymal malignant tumor characterized by adipocyte differentiation which is divided into four subtypes with different prognosis. Accurate histopathological diagnosis is essential for precise treatment. Perilipins, including PLIN1, PLIN2, PLIN3, PLIN4, PLIN5, is a family of lipid droplet-associated proteins that participate in lipid metabolism regulation. The role that perilipins play in sarcomas is not clear. This study aims to assess perilipins expression in subtypes of liposarcoma and various non-lipomatous sarcomas.Methods: A large set of 245 soft tissue sarcoma paraffin-embedded samples including 66 liposarcomas and 179 non-lipomatous sarcomas were collected for tissue microarray and immunohistochemistry to assess perilipins expression.Results: PLIN1 expression was shown in most liposarcomas (41/66) and was absent in non-lipomatous sarcomas (0/179). PLIN4 expression was shown in some liposarcomas (21/66) and was almost negative in non-lipomatous sarcomas (2/179). PLIN1 and PLIN4 expressions in liposarcoma were higher (both P<0.001) than those in non-lipomatous sarcoma. Both PLIN1 and PLIN4 also had a significant difference in liposarcoma subtypes (both P<0.001). PLIN2, PLIN3 and PLIN5 were widely expressed in liposarcomas, rhabdomyosarcomas, leiomyosarcomas, dermatofibrosarcoma protuberans, undifferentiated sarcomas, fibrosarcomas, Ewing's sarcomas and epithelioid sarcomas. PLIN2, PLIN3 and PLIN5 expressions were significantly different among non-lipomatous sarcoma (all P<0.01).Except for PLIN3, the expression of the other four perilipin members in liposarcoma was pairwise related.Conclusions: PLIN1 and PLIN4 can be used as diagnostic markers of liposarcoma and to differentiate liposarcoma subtypes. The combined application of whole perilipin family immunohistochemistry may help to distinguish differently differentiated sarcomas.

Effects of (−)-epicatechin on the time course of the expression of perilipins in a diet-induced model of nonalcoholic steatohepatitis

The existing treatments for nonalcoholic steatohepatitis (NASH) are not completely effective. The need for new alternatives without adverse effects and low cost, such as the flavonoid (−)-epicatechin (EC), which has beneficial effects on lipid metabolism and cardiovascular diseases, arises. The objective of this work was to analyze EC effects in the NASH induced by a Paigen-type diet (PD). Mice were administered with (1) normal chow and water, (2) PD + fructose 30% and (3) PD + fructose 30% + EC (1 mg/kg) per gavage during 9 weeks. At the end of each treatment, serum was collected for analysis of the biochemical profile and liver enzymes. The liver was collected for microscopic analysis and for the evaluation of the relative expression of Plin2, Plin3, CD36, adiponectin and UCP2. Results showed that EC reduced weight gain and decreased triglyceride (TG), low-density lipoprotein cholesterol, TG/high-density lipoprotein and the activity of liver enzymes (alanine aminotransferase and alkaline phosphatase), suggesting lower liver damage. The microscopic analysis showed less “balloonization” of the hepatocyte, small drops of lipids, less accumulation of collagen and infiltration of inflammatory cells as compared to nontreated group. Finally, a decrease in the expression of Plin 2 was observed. While CD36 decreased, adiponectin and UCP2 increased. In conclusion, EC improves the biochemical profile, the microscopic characteristics and protein expression. Therefore, it may be a possible therapeutic approach for NASH since it prevents the progression of the hepatic and metabolic damage induced by high-fat diets.

Plin5/p-Plin5 Guards Diabetic CMECs by Regulating FFAs Metabolism Bidirectionally.

Background Hyper-free fatty acidemia (HFFA) impairs cardiac capillaries, as well as type 2 diabetes mellitus (T2DM). Perilipin 5 (Plin5) maintains metabolic balance of free fatty acids (FFAs) in high oxidative tissues via the states of nonphosphorylation and phosphorylation. However, when facing to T2DM-HFFA, Plin5's role in cardiac microvascular endothelial cells (CMECs) is not defined. Methods In mice of WT or Plin5−/−, T2DM models were rendered by high-fat diet combined with intraperitoneal injection of streptozocin. CMECs isolated from left ventricles were incubated with high glucose (HG) and high FFAs (HFFAs). Plin5 phosphorylation was stimulated by isoproterenol. Plin5 expression was knocked down by small interfering RNA (siRNA). We determined cardiac function by small animal ultrasound, apoptotic rate by flow cytometry, microvessel quantity by immunohistochemistry, microvascular integrity by scanning electron microscopy, intracellular FFAs by spectrophotometry, lipid droplets (LDs) by Nile red staining, mRNAs by quantitative real-time polymerase chain reaction, proteins by western blots, nitric oxide (NO) and reactive oxygen species (ROS) by fluorescent dye staining and enzyme-linked immunosorbent assay kits. Results In CMECs, HFFAs aggravated cell injury induced by HG and activated Plin5 expression. In mice with T2DM-HFFA, Plin5 deficiency reduced number of cardiac capillaries, worsened structural incompleteness, and enhanced diastolic dysfunction. Moreover, in CMECs treated with HG-HFFAs, both ablation and phosphorylation of Plin5 reduced LDs content, increased intracellular FFAs, stimulated mitochondrial β-oxidation, added ROS generation, and reduced the expression and activity of endothelial nitric oxide synthase (eNOS), eventually leading to increased apoptotic rate and decreased NO content, all of which were reversed by N-acetyl-L-cysteine. Conclusion Plin5 preserves lipid balance and cell survival in diabetic CMECs by regulating FFAs metabolism bidirectionally via the states of nonphosphorylation and phosphorylation.

High-fat diet leads to increased ovarian lipid deposition even in the absence of obesity

We have previously shown that high-fat diet (HFD) feeding leads to depletion of the ovarian reserve, subfertility, and altered expression of key ovarian genes, irrespective of the development of obesity1,2. It has been proposed that HFD-induced ovarian dysfunction is due to increased lipid accumulation in the ovary3,4, however, it is currently unknown if there is increased HFD-induced lipid accumulation in the ovary in the absence of obesity. We hypothesized that HFD exposure would increase ovarian lipid deposition regardless of the induction of obesity. Prospective laboratory animal study. 5-week-old C57BL/6J mice were fed either a 60% HFD or standard chow (N = 15) for 10 weeks. After 10 weeks body weights were determined. Lean and fat mass was measured by quantitative MRI. HFD mice were divided into HFD-lean (HFLn, N=10) and HFD-obese (HFOb, N=9) groups based on body weight: mice < 26 g were considered HFLn and mice ³ 30 g were considered HFOb. Ovaries were collected, one for qRT-PCR analysis of the lipid droplet protein plin2 and the other for immunohistochemical analysis of lipid droplets via anti-plin2 staining. A one-way ANOVA (Tukey’s post hoc) was used for statistical analysis. After 10 weeks of diet HFOb mice weighed more and had a higher percentage of body fat (33.2 ± 0.9 g, 37.2 ± 1.2%, respectively) than both HFLn mice (23.5 ± 0.6g, 17.7 ± 1.5%, respectively) and chow controls (21.6 ± 0.3g, 11.2 ± 0.6%, respectively) (p<0.0001). Ovarian expression of plin2 was dramatically increased after HFD, with a 5-fold increase observed in HFLn mice and a 4.7-fold increase observed in HFOb mice (p = 0.01) compared to chow-fed controls. Immunohistochemical analysis of plin2 in the ovary confirmed our qRT-PCR data showing dramatically increased levels of plin2 in the ovaries of both the HFLn and HFOb mice. In particular, increased lipid deposition was observed in the corpora lutea, ovarian stroma, and outside of the theca cells of large follicles, and to a far lesser extent in the granulosa cells. HFD exposure leads to increased ovarian lipid deposition regardless of the development of obesity in mice. This phenomenon may be responsible for the ovarian dysfunction and reproductive defects observed with HFD exposure, even in the absence of obesity. The presence of increased lipid droplets in the ovaries of lean mice fed a HFD provides further evidence that even if a lean phenotype is maintained, consumption of HFDs will still negatively impact the ovary. More research is needed to determine the exact mechanism by which excessive ovarian lipid accumulation is responsible for the HFD-induced reproductive dysfunction.

The relationship between the expression of PLIN3 and PLIN5 protein flowing endurance training in streptozotocin rats

Background and aims: Intermuscular lipolysis disorder plays an important role in insulin resistance and diabetes mellitus and perilipin PLIN5 and PLIN3 are the key proteins in regulating muscle cellular lipolysis. Therefore, the purpose of this study was to examine the relationship between the expression of PLIN3 and PLIN5 protein following endurance training in streptozotocin (STZ) rats. Methods: A number of 24 male Wistar rats were randomly divided into low endurance training group (n = 8), high-intensity training group (n = 8), and control group (n = 8). Diabetes was induced in every rat by STZ injection. Three days after injection, the blood samples were taken from the cut tip of the tails of the mice and animals with blood glucose greater than 300 mg/dL were considered diabetic. The training program included eight weeks of aerobic training at different intensities. Training in high- and low-intensity groups included 22-25 and 5-8 m/min of training. Finally, one-way analysis of variance (ANOVA) and correlation was used to determine the significance of the differences between variables, followed by utilizing Tukey’s post-hoc test for significance. Results: The comparison between the groups by ANOVA showed significant differences in PLIN3 (P=0.0006) and PLIN5 (P=0.012). The results of Tukey post hoc test also demonstrated a statistical difference between the mean values of diabetic control group and high-intensity endurance group regarding PLIN3 (P=0.01) and PLIN5 (P=0.009), but no significant increase was observed in the lowintensity exercise group as compared to the control group (PLIN3, P=0.067 &amp; PLIN5, P=0.44). As regards insulin resistance, there was a significant difference among the three groups (P=0.0001). Eventually, the result of the correlation between PLIN3 and PLIN5 showed similar enhancement by increasing the intensity (P=0.0026). Conclusion: According to research results, high-intensity endurance training increased the expression of PLIN3 and PLIN5 in diabetic specimens and PLIN3 and PLIN5 followed a similar increase pattern in high-intensity training

Targeting PLIN2/PLIN5-PPARγ: Sulforaphane Disturbs the Maturation of Lipid Droplets.

The effects of sulforaphane (SFN) on the maturation of lipid droplets (LDs)-the storage units for free fatty acids and sterols as triacylglycerides (TAG) and cholesterol esters (CE)-are far from being understood, despite the fact that SFN is known to be beneficial for ameliorating lipid metabolism disorders. High-fat-intake models are established in both HHL-5 hepatocytes and rodents. The numbers and sizes of LDs are decreased by SFN. The accumulation of lipid core components (TAG & CE) is reduced and the expression of their key synthetases, acyl-coenzyme A: diacylglycerol acyltransferases 2 (DGAT2) and acyl-coenzyme A: cholesterol acyltransferases 1 (ACAT1), is also inhibited. Moreover, SFN decreases LD-associated protein PLIN2 and PLIN5 expression, but not that of PLIN1 and PLIN3, both in vivo and in vitro. Furthermore, over-expression of peroxisome proliferator-activated receptor gamma (PPARγ) induces the accumulation of TAG and the up-regulation of PLIN2 and PLIN5, which are not reversed by SFN. These results suggest that PPARγ may be a target of SFN in lipid metabolism. SFN disturbs LD maturation by inhibiting the formation of the neutral lipid core and decreases PLIN2 and PLIN5 via down-regulation of PPARγ.

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.

Oleic acid-induced perilipin 5 expression and lipid droplets formation are regulated by the PI3K/PPARα pathway in HepG2 cells

Perilipin 5 (Plin5), a member of the PAT (Perilipin, ADRP, and Tip47) protein family, has been implicated in the regulation of cellular neutral lipid accumulation in nonalcoholic fatty liver diseases. However, the underlying regulatory mechanisms of Plin5 are not clear. The goal of the present study was to explore the mechanism of oleic acid (OA)-induced Plin5 expression in HepG2 cells. We found that the expression of Plin5 was increased during OA-induced lipid droplets formation in a dose- and time-dependent manner. During this process of OA-stimulated lipid droplets formation, peroxisome proliferator-activated receptor alpha (PPARα) was also upregulated. When PPARα activation was blocked by GW6471, OA-induced Plin5 expression and lipid droplets formation were effectively ablated. We further found that the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 was able to downregulate both PPARα and Plin5 expression and lipid droplets formation. Thus, we concluded that PI3K may, at least in part, act upstream of PPARα to regulate Plin5 expression and lipid droplets formation in HepG2 cells.

Perilipin 2 and lipid droplets provide reciprocal stabilization

The lipid droplet (LD)-associated protein adipose differentiation-related protein (ADRP or PLIN2) is required for the formation and stability of the LD organelle, whereas its biological roles are still obscure. Herein, we show that PLIN2 is the most abundant protein on the lipid droplets (LDs) of mouse myoblast cell line C2C12. Both the expression of PLIN2 and the accumulation of LDs were up-regulated in a time- and dose-dependent manner when the cells were treated with oleate (OA). The protein level of PLIN2 was positively correlated with the formation of LDs, suggesting that LDs stabilize PLIN2. Furthermore, knocking out PLIN2 in C2C12 cells led to enlarged LDs and higher triacylglycerol hydrolysis activity. The isolated PLIN2 null LDs became closely contact with mitochondria and other cellular organelles. Additionally, mitochondrial activity was suppressed by OA in PLIN2 null cells. Our results reveal the pivotal roles of PLIN2 in governing LD dynamics and their relationship to mitochondria, and suggest a reciprocal stabilization between PLIN2 and LDs.

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.

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= &lt; 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.

Transcriptome analysis after PPARγ activation in human meibomian gland epithelial cells (hMGEC)

PurposePPARγ plays a critical role in the maturation of immortalized human meibomian gland epithelial cells (hMGEC). To further understand the molecular changes associated with meibocyte differentiation, we analyzed transcriptome profiles from hMGEC after PPARγ activation. MethodsThree sets of cultivated hMGEC with or without exposure to PPARγ agonist, rosiglitazone were used for RNA-seq analysis. RNA was isolated and processed to generate 6 libraries. The libraries were then sequenced and mapped to the human reference genome, and the expression results were gathered as reads per length of transcript in kilobases per million mapped reads (RPKM) values. Differential gene expression analyses were performed using DESeq2 and NOISeq. Gene ontology enrichment analysis (GOEA) was performed on gene sets that were upregulated or downregulated after rosiglitazone treatment. Five genes were selected for validation and differential expression was confirmed using quantitative PCR. The Differential expression of CK5 was evaluated using Western blotting. ResultsExpression data indicated that about 58,000 genes are expressed in hMGEC. DESeq2 and NOISeq indicated that 296 and 3436 genes were upregulated and 258 and 3592 genes were down regulated after rosiglitazone treatment, respectively. Of genes showing significant differences > 2 fold, GOEA indicated that cellular and metabolic processes were highly represented. Expression of ANGPTL4, PLIN2, SQSTM1, and DDIT3 were significantly upregulated and HHIP was downregulated by rosiglitazone. CK5 was downregulated by rosiglitazone. ConclusionsThe RNA-seq data suggested that PPARγ activation induced alterations in cell differentiation and metabolic process and affected multiple signaling pathways such as PPAR, autophagy, WNT, and Hedgehog.

LIPG-promoted lipid storage mediates adaptation to oxidative stress in breast cancer.

Endothelial lipase (LIPG) is a cell surface associated lipase that displays phospholipase A1 activity towards phosphatidylcholine present in high‐density lipoproteins (HDL). LIPG was recently reported to be expressed in breast cancer and to support proliferation, tumourigenicity and metastasis. Here we show that severe oxidative stress leading to AMPK activation triggers LIPG upregulation, resulting in intracellular lipid droplet accumulation in breast cancer cells, which supports survival. Neutralizing oxidative stress abrogated LIPG upregulation and the concomitant lipid storage. In human breast cancer, high LIPG expression was observed in a limited subset of tumours and was significantly associated with shorter metastasis‐free survival in node‐negative, untreated patients. Moreover, expression of PLIN2 and TXNRD1 in these tumours indicated a link to lipid storage and oxidative stress. Altogether, our findings reveal a previously unrecognized role for LIPG in enabling oxidative stress‐induced lipid droplet accumulation in tumour cells that protects against oxidative stress, and thus supports tumour progression.

Type 2 diabetes is associated with suppression of autophagy and lipid accumulation in β-cells.

Both type 2 diabetes (T2D) and obesity are characterized by excessive hyperlipidaemia and subsequent lipid droplet (LD) accumulation in adipose tissue. To investigate whether LDs also accumulate in β‐cells of T2D patients, we assessed the expression of PLIN2, a LD‐associated protein, in non‐diabetic (ND) and T2D pancreata. We observed an up‐regulation of PLIN2 mRNA and protein in β‐cells of T2D patients, along with significant changes in the expression of lipid metabolism, apoptosis and oxidative stress genes. The increased LD buildup in T2D β‐cells was accompanied by inhibition of nuclear translocation of TFEB, a master regulator of autophagy and by down‐regulation of lysosomal biomarker LAMP2. To investigate whether LD accumulation and autophagy were influenced by diabetic conditions, we used rat INS‐1 cells to model the effects of hyperglycaemia and hyperlipidaemia on autophagy and metabolic gene expression. Consistent with human tissue, both LD formation and PLIN2 expression were enhanced in INS‐1 cells under hyperglycaemia, whereas TFEB activation and autophagy gene expression were significantly reduced. Collectively, these results suggest that lipid clearance and overall homeostasis is markedly disrupted in β‐cells under hyperglycaemic conditions and interventions ameliorating lipid clearance could be beneficial in reducing functional impairments in islets caused by glucolipotoxicity.

Perilipin 5 Deletion in Hepatocytes Remodels Lipid Metabolism and Causes Hepatic Insulin Resistance in Mice.

Defects in hepatic lipid metabolism cause nonalcoholic fatty liver disease and insulin resistance, and these pathologies are closely linked. Regulation of lipid droplet metabolism is central to the control of intracellular fatty acid fluxes, and perilipin 5 (PLIN5) is important in this process. We examined the role of PLIN5 on hepatic lipid metabolism and systemic glycemic control using liver-specific Plin5-deficient mice (Plin5LKO ). Hepatocytes isolated from Plin5LKO mice exhibited marked changes in lipid metabolism characterized by decreased fatty acid uptake and storage, decreased fatty acid oxidation that was associated with reduced contact between lipid droplets and mitochondria, and reduced triglyceride secretion. With consumption of a high-fat diet, Plin5LKO mice accumulated intrahepatic triglyceride, without significant changes in inflammation, ceramide or diglyceride contents, endoplasmic reticulum stress, or autophagy. Instead, livers of Plin5LKO mice exhibited activation of c-Jun N-terminal kinase, impaired insulin signal transduction, and insulin resistance, which impaired systemic insulin action and glycemic control. Re-expression of Plin5 in the livers of Plin5LKO mice reversed these effects. Together, we show that Plin5 is an important modulator of intrahepatic lipid metabolism and suggest that the increased Plin5 expression that occurs with overnutrition may play an important role in preventing hepatic insulin resistance.

Plin5/p-Plin5 Guards Diabetic CMECs by Regulating FFAs Metabolism Bidirectionally

Background: Hyper-free fatty academia (HFFA) impairs cardiac capillaries, as well as type 2 diabetes mellitus (T2DM). Perilipin 5 (Plin5) maintains metabolic balance of free fatty acids (FFAs) in high oxidative tissues via the states of non-phosphorylation and phosphorylation. However, when facing to T2DM-HFFA, Plin5's role in cardiac microvascular endothelial cells (CMECs) is not defined. Methods: In mice of WT or Plin5-/-, T2DM models were rendered by high-fat diet combined with intraperitoneal injection of streptozocin. CMECs isolated from left ventricles were incubated with high glucose (HG) and high FFAs (HFFAs). Plin5 phosphorylation was stimulated by isoproterenol. Plin5 expression was knocked down by siRNA. We determined cardiac function by small animal ultrasound, apoptotic rate by flow cytometry, microvessel quantity by immunohistochemistry, microvascular integrity by scanning electron microscopy, intracellular FFAs by spectrophotometry, lipid droplets (LDs) by nile red staining, mRNAs by quantitative real time polymerase chain reaction, proteins by western blots, nitric oxide (NO) and reactive oxygen species (ROS) by fluorescent dye staining and enzyme-linked immunosorbent assay kits. Results: In CMECs, HFFAs aggravated cell injury induced by HG and activated Plin5 expression. In mice with T2DM-HFFA, Plin5 deficiency reduced number of cardiac capillaries, worsened structural incompleteness and enhanced diastolic dysfunction. Moreover, in CMECs treated with HG-HFFAs, both ablation and phosphorylation of Plin5 reduced LDs content, increased intracellular FFAs, stimulated mitochondrial β-oxidation and added ROS generation, reduced the expression and activity of endothelial nitric oxide synthase (eNOS), eventually leading to increased apoptotic rate and decreased NO content, all of which were reversed by acetylcysteine. Conclusion: Plin5 preserves lipid balance and cell survival in diabetic CMECs by regualting FFAs metabolism bidirectionally via the states of non-phosphorylation and phosphorylation. Funding Statement: This work was supported by grants from Science Fund for Distinguished Young Scholars of Sichuan Province (No.2017JQ0012), National Science Funds of China (No.81500208), China Postdoctoral Science Foundation (No.2017M613429). Declaration of Interests: The authors declare: No competing financial interests exist. Ethics Approval Statement: All experiments were conducted in adherence with National Institutes of Health Guidelines on the Use of Laboratory Animals, and were approved by Institutional Animal Care and Use Committee of Chengdu Military General Hospital.

Chronic Lymphocytic Leukaemia Relies on Lipid Scavenging and Synthesis As an Energy Source

Dysregulation of cancer cell bioenergetics is one of the hallmarks of cancer. The Warburg effect is one such documented change. However, glucose metabolism is not universally increased in cancer cells. Uptake of radiolabelled glucose in chronic lymphocytic leukaemia (CLL) fails as a marker of proliferation and the underlying reason is not well elucidated [Conte et al, 2014]. Using proteomic and comprehensive lipid analyses, the preferred metabolic pathways of CLL cells have been identified.We have previously shown by proteomic analysis that circulating peripheral blood CLL cells demonstrate a significant increase in the expression of proteins pivotal in endogenous lipid synthesis pathways compared to B-cells from healthy individuals. These include fatty acid synthase (+3.84 fold), farnesyl diphosphate synthase (+2.17 fold), ATP-citrate lyase (+4.63 fold), citrate synthase (+2.45 fold) and acetyl-CoA acetyltransferase 1 (+5.84 fold). Conversely, analysis of the proliferation centres in CLL lymph nodes reveals increased expression of proteins involved in beta-oxidation, such as hydroxyacyl-CoA dehydrogenase (+2.02 fold). Additionally CLL cells shows a large increase in the levels of phospholipids found associated with lipid droplets compared to healthy B-cells, including phosphatidylinositol and phosphatidylethanolamine.Our current studies have expanded these observations in an attempt to determine if CLL cells have a preponderance for endogenous synthesis of lipids or exogenous uptake. We performed a comprehensive analysis of CLL cells from the bone marrow and peripheral blood using a variety of techniques. Transmission electron microscopy (TEM) images demonstrate striking morphological differences between normal B-cells and CLL cells with the inclusion of lipid droplets in the cytoplasm of CLL cells (Figure 1A) which was most evident in peripheral CLL samples. These were confirmed to be lipid droplets by the specific marker Bodipy 493/503 on flow cytometry (Fig. 1B) and confocal microscopy imaging with ReZolveL1 which targets neutral lipids (Fig. 1C). These showed a substantially higher level of lipid droplet staining in CLL cells compared to normal B-cells. Additionally there was higher expression of CD36 in CLL cells which is the receptor for exogenous lipid uptake into cells (Fig 1D).TEM analysis also revealed a high number of lysosomes in CLL cells, which was confirmed with lysotracker imaging on confocal microscopy (Fig. 1E). Lysosomes are now emerging as key players in lipid transport and biogenesis [Thelen and Zoncu, 2017]. Lipid droplets may be broken down by a process called autophagy mediated lipid degradation, or lipophagy, which relies on autophagosomes delivering lipid droplets to lysosomes. Autophagosomes were observed in several further bone marrow samples (Fig. 1F). The bone marrow samples also showed evidence of ‘condensed mitochondria’, indicative of high rates of beta-oxidation [Rossignol et al, 2004].qPCR was then carried out on 90 genes involved in lipid metabolism. These results revealed significant differences between healthy B-cells and CLL cells. All CLL samples analysed demonstrated high expression of PLIN1, which protects lipid droplets from degradation, and low expression of APOC1, which prevents fatty acid uptake. From these observations we propose that peripheral CLL cells scavenge lipids from the periphery, mainly by uptake via the CD36 receptor, and increased endogenous lipid synthesis pathways.These results underpin a model for metabolism and enhanced survival for CLL cells in the hypoxic and potentially nutrient poor bone marrow/lymph node microenvironments. Peripheral CLL cells scavenge lipids; these excess lipids, which are toxic to the cell, are stored in lipid droplets which are protected from degradation by a high expression of PLIN proteins. Once these cells circulate back to the bone marrow and lymph nodes to proliferate, the lipid droplets are degraded, likely by lipophagy and neutral lipolysis which frees fatty acids to be used in beta oxidation in the mitochondria to sustain cell proliferation in the bone marrow and lymph node compartments. Our results begin to unravel CLL bioenergetics and dysregulation of cellular metabolism that occurs in this disease. We are now investigating whether the manipulation of these pathways, particularly lipophagy, may represent a novel therapeutic approach in CLL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Reactive Oxygen Species Induces Lipid Droplet Accumulation in HepG2 Cells by Increasing Perilipin 2 Expression.

Non-alcoholic fatty liver disease (NAFLD) has become the world’s most common liver disease. The disease can develop liver fibrosis or even carcinomas from the initial hepatic steatosis, and this process is influenced by many factors. Reactive oxygen species (ROS), as potent oxidants in cells, have been reported previously to play an important role in the development of NAFLD progression via promoting neutral lipid accumulation. Here, we found that ROS can promote lipid droplet formation in hepatocytes by promoting perilipin2 (PLIN2) expression. First, we used different concentrations of hydrogen peroxide to treat HepG2 cells and found that the number of lipid droplets in the cells increased, however also that this effect was dose-independent. Then, the mRNA level of several lipid droplet-associated genes was detected with hydrogen peroxide treatment and the expression of PLIN2, PLIN5, and FSP27 genes was significantly up-regulated (p < 0.05). We overexpressed PLIN2 in HepG2 cells and found that the lipid droplets in the cells were markedly increased. Interference with PLIN2 inhibits ROS-induced lipid droplet formation, revealing that PLIN2 is a critical factor in this process. We subsequently analyzed the regulatory pathway and protein interaction network that is involved in PLIN2 and found that PLIN2 can regulate intracellular lipid metabolism through the PPARα/RXRA and CREB/CREBBP signaling pathways. The majority of the data indicated the correlation between hydrogen peroxide-induced PLIN2 and lipid droplet upregulation. In conclusion, ROS up-regulates the expression of PLIN2 in hepatocytes, whereas PLIN2 promotes the formation of lipid droplets resulting in lipid accumulation in liver tissues.