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Chromatin immunoprecipitation assays of the perilipin promoter in 3T3-L1 adipocytes. Chromatin immunoprecipitation assays were performed as described under " Experimental Procedures . " Soluble chromatin was immunoprecipitated with mouse IgG (lane 2), antibodies against mouse PPAR (lane 3), or acetyl-histone H3 (lane 4). Immunoprecipitates were analyzed by PCR with specific primers for the mouse perilipin or aP2 promoter. PCR was performed with total chromatin input (lane 1).
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Recent studies have shown that lipid droplets are covered with a proteinaceous coat, although the functions and identities of the component proteins have not yet been well elucidated. The first identified lipid droplet-specific proteins are the perilipins, a family of proteins coating the surfaces of lipid droplets of adipocytes. The generation of...
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... Different groups claim 3T3-L1 to be mature at different days after differentiation initiation. 1,15,16,18 According to our protocol the process of adipogenesis is fully activated after day 10 of cell culture/ day 6 of differentiation and, thereafter, 3T3-L1 cells are steadily accumulating lipids and increase the expression of marker genes. Further, Malekpour-Dehkordi et al. showed that prolonged incubation of 3T3-L1 adipocytes for additional 30 days leads to an even higher lipid accumulation into the cells. ...
3T3-L1 is a model cell line which can be differentiated from preadipocytes into mature adipocytes. Here, we present a protocol for changing gene expression in 3T3-L1 (pre)adipocytes using small interfering RNA (siRNA)-mediated knockdown. We describe steps to perform the knockdown of a certain gene prior to differentiation (day 4) to analyze the impact on adipogenesis. We then detail procedures for knockdown on day 8 of differentiation to study the role of a certain gene in mature adipocyte function.
For complete details on the use and execution of this protocol, please refer to Kaczmarek et al.¹
... Peroxisome proliferator-activated receptors (PPARs) are transcriptional factors that control the expression of genes that code for proteins involved in FA storage, glucose metabolism, and adipocyte differentiation [152], including PLINs, as described above [142,153,154]. Out of the three known PPAR isoforms (α, β/δ, and γ), PPARγ and PPARα have the most significant impact on the function of PLINs. ...
... Two specific mechanisms seem to underpin the enhancement of LD loading mediated by PPARγ. One is the upregulation of PLIN1 expression, which improves the capacity to restrict lipolytic enzymes from acting primarily on LDs in adipocytes [153]. The other involves an increase in PLIN2 expression, which bolsters the ability to block lipolytic enzymes' access to the LD core [61,[63][64][65][66][67]. ...
... These regulatory pathways exert unique influences on different PLINs [41,142,146,149,153,154]. Therefore, their impact on the coordination of PLIN functions and on the allocation of FAs between adipocytes and recipient cells may also be distinct. ...
Simple Summary
This review highlights the importance of perilipins in lipid metabolism and their potential as therapeutic targets for lipid-associated diseases like cancer and obesity. Perilipins are proteins found in lipid droplets that regulate lipase activity and play a crucial role in maintaining the balance between lipid synthesis and breakdown. Modulating perilipins could improve existing treatments or offer new therapeutic opportunities for addressing these diseases.
Abstract
Perilipins (PLINs) are the most abundant proteins in lipid droplets (LD). These LD-associated proteins are responsible for upgrading LD from inert lipid storage structures to fully functional organelles, fundamentally integrated in the lipid metabolism. There are five distinct perilipins (PLIN1–5), each with specific expression patterns and metabolic activation, but all capable of regulating the activity of lipases on LD. This plurality creates a complex orchestrated mechanism that is directly related to the healthy balance between lipogenesis and lipolysis. Given the essential role of PLINs in the modulation of the lipid metabolism, these proteins can become interesting targets for the treatment of lipid-associated diseases. Since reprogrammed lipid metabolism is a recognized cancer hallmark, and obesity is a known risk factor for cancer and other comorbidities, the modulation of PLINs could either improve existing treatments or create new opportunities for the treatment of these diseases. Even though PLINs have not been, so far, directly considered for pharmacological interventions, there are many established drugs that can modulate PLINs activity. Therefore, the aim of this study is to assess the involvement of PLINs in diseases related to lipid metabolism dysregulation and whether PLINs can be viewed as potential therapeutic targets for cancer and obesity.
... Perilipin knockout mice are lean with accelerated basal lipolysis and are proof against food regimen-caused weight problems; however, these mice increase glucose intolerance and insulin resistance greater without problems, probably because of expanded levels of serum FFAs [81]. further, perilipin has a peroxisome proliferator-activated receptor γ (PPAR γ) responsive element in its promoter region and is precipitated by using thiazolidinedione agonists of PPARY [82]. ...
Insulin, a peptide hormone synthesized and secreted by pancreatic β-cells, exerts a wide range of anabolic effects on multiple tissues. It plays a crucial role in maintaining complete-frame gas homeostasis by promoting carbohydrate, fat, and amino acid uptake and storage in skeletal muscle, adipose tissue, and the liver. Insulin stimulates glucose transportation and storage as glycogen in skeletal muscle, while simultaneously inhibiting glycogenolysis and gluconeogenesis in the liver. In adipocytes, it enhances glucose uptake, glycerol synthesis, and triglyceride formation while suppressing lipolysis. During fasting periods, decreased circulating insulin levels and increased secretion of counter-regulatory hormones lead to the breakdown of stored fuels, providing metabolic substrates for cellular energy. The dynamic changes in insulin levels during feeding and fasting states play a key role in fuel metabolism and blood glucose regulation. Additionally, insulin influences protein catabolism, translation, cell growth, differentiation, and survival through mitogenic and anti-apoptotic processes, resulting in a net synergistic effect on various biological pathways.
... Accordingly, alcoholinduced expression of Plin2, a direct PPARg transcriptional target, was decreased by the knockdown of PPARg ( Figure 7C). 53,54 Furthermore, PPARg knockdown reduced alcohol-induced lipid accumulation after 48 hours of treatment as measured by Oil Red O staining ( Figure 7D and E). Moreover, we established stable cell lines overexpressing either a control vector (beta-galactosidase, herein referred (F) Western blot analysis of liver tissue from NIAAA 10d þ 1b AlbCre and ABL1 KO pair-and alcohol-fed mice (n ¼ 2 mice per group). ...
Background and aims:
Alcohol-associated liver disease (ALD) represents a spectrum of alcohol use-related liver diseases. Outside of alcohol abstinence, there are currently no FDA-approved treatments for advanced ALD, necessitating a greater understanding of ALD pathogenesis and potential molecular targets for therapeutic intervention. The ABL-family proteins, including ABL1 and ABL2, are non-receptor tyrosine kinases that participate in a diverse set of cellular functions. We investigated the role of the ABL kinases in alcohol-associated liver disease.
Methods:
We utilized samples from patients with ALD compared to healthy controls to elucidate a clinical phenotype. We established strains of liver-specific Abl1 and Abl2 knockout mice and subjected them to the NIAAA acute-on-chronic alcohol feeding regimen. Murine samples were subjected to RNA-sequencing, AST, Oil Red O staining, H&E staining, western blotting, and qPCR to assess phenotypic changes following alcohol feeding. In vitro modeling in HepG2 cells as well as primary hepatocytes from C57BL6/J mice was used establish this mechanistic link of ALD pathogenesis.
Results:
We demonstrate that the ABL kinases are highly activated in ALD patient liver samples as well as in liver tissues from mice subjected to an alcohol feeding regimen. We found that the liver-specific knockout of Abl2, but not Abl1, attenuated alcohol-induced steatosis, liver injury, and inflammation. Subsequent RNA-sequencing and gene set enrichment analyses of mouse liver tissues revealed that, relative to wild-type alcohol-fed mice, Abl2 knockout alcohol-fed mice exhibited numerous pathway changes, including significantly decreased PPAR signaling. Further examination revealed that PPARγ, a previously identified regulator of ALD pathogenesis, was induced upon alcohol feeding in wild-type mice, but not in Abl2 knockout mice. In vitro analyses revealed that shRNA-mediated knockdown of ABL2 abolished the alcohol-induced accumulation of PPARγ as well as subsequent lipid accumulation. Conversely, forced overexpression of ABL2 resulted in increased PPARγ protein expression. Further, we demonstrated that the regulation of HIF1α by ABL2 is required for alcohol-induced PPARγ expression. Furthermore, treatment with ABL kinase inhibitors attenuated alcohol-induced PPARγ expression, lipid droplet formation and liver injury.
Conclusions:
Based on our current evidence, we propose that alcohol-induced-ABL2 activation promotes ALD through increasing HIF1α and the subsequent PPARγ expression, and ABL2 inhibition may serve as a promising target for the treatment of ALD.
... Thus, Plin1 may play a key role in adipogenic and lipid metabolic processes. Moreover, Plin1 expression may be specifically regulated by PPAR-γ, and activating PPAR-γ significantly increased Plin1 expression, and Plin1 may be a downstream target of PPAR-γ (Arimura et al., 2004). Our data exhibited that Plin1 was significantly raised after BPBP treatment. ...
Dihydrxytetraphenylmethane, also known as Bisphenol BP (BPBP), has been increasingly used in industrial production and more frequently detected in the environment as an alternative plasticizer of BPA. However, there are no reports about BPBP in food safety or its effects on cellular lipogenesis. The purpose of this research was to investigate the influence and potential mechanisms of BPBP on adipogenesis in 3T3-L1 cells. Cells were treated with 4 concentrations (0.01, 0.1, 1, and 10 μM) of BPBP and the results showed that treatment with at low concentrations (0.01 μM) promoted cell fat differentiation and triglyceride accumulation. RNA-seq data showed that a total of 370 differentially expressed genes between control and the low-dose BPBP-treated group were determined, including 227 upregulated genes and 143 downregulated genes. Some key genes related to adipo-cyte differentiation and adipogenesis were significantly enriched after BPBP treatment, including PPAR-γ, Adi-poq, Nr1h3 and Plin1. Pathway analyses suggest that the activation of PPAR-γ signaling pathway may be key for BPBP to promote adipocyte differentiation and fat accumulation. Our work provides evidence for the potential obesogenic effect of BPBP and may call for further research on the safety of the chemical in food products.
... Studies on mammals revealed that the transcription of the Plin1 gene is mainly regulated by PPARγ, the main regulator of adipogenesis. In mammals, PPARγ can regulate the transcription of the Plin1 gene by binding to the functional response element of PPARγ located in the 5′"flanking" region of the Plin1 gene (Arimura et al., 2004). In the basal or resting state, Plin1 protects lipids in LD from interacting with adipose triglyceride lipase (ATGL), hormonesensitive lipase (HSL), and binds to comparative gene identification-58, which is an activator of ATGL, to reduce ineffective lipolysis, thus maintaining intracellular TG levels. ...
Background: We determined the effects of Cuscutae semen ( Cuscuta chinensis Lam. or Cuscuta australis R. Br.)–Radix rehmanniae praeparata ( Rehjnannia glutinosa Libosch.) on the protein levels in testicular tissues of rats gavaged with tripterygium wilfordii multiglycosides (GTW) and elucidated the molecular mechanism underlying Cuscutae semen–Radix rehmanniae praeparata for relieving GTW-induced reproductive injury.
Methods: A total of 21 male Sprague–Dawley rats were randomly divided into the control group, model group, and Cuscutae semen–Radix rehmanniae praeparata group based on their body weights. The control group was given 10 mLkg ⁻¹ of 0.9% normal saline by gavage daily. The model group (GTW group) was administered with 12 mg kg ⁻¹ GTW by gavage daily. Cuscutae semen–Radix rehmanniae praeparata group (the TSZSDH group) was administered with 1.56 gkg ⁻¹ of Cuscutae semen–Radix rehmanniae praeparata granules daily according to their model group dosing. The serum levels of luteinizing hormone, follicle-stimulating hormone, estradiol, and testosterone were measured after 12 weeks of continuous gavage, and the pathological lesion of testicular tissues was observed. Differentially expressed proteins were evaluated by quantitative proteomics and verified by western blotting (WB) and Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR).
Results: Cuscutae semen–Radix rehmanniae praeparata can effectively relieve pathological lesions of GTW-induced testicular tissues. A total of 216 differentially expressed proteins were identified in the TSZSDH group and model group. High-throughput proteomics revealed that differentially expressed proteins are closely associated with the peroxisome proliferator-activated receptor (PPAR) signaling pathway, protein digestion and absorption, and protein glycan pathway in cancer. Cuscutae semen–Radix rehmanniae praeparata can significantly upregulate the protein expressions of Acsl1, Plin1, Dbil5, Plin4, Col12a1, Col1a1, Col5a3, Col1a2, Dcn, so as to play a protective role on testicular tissues. Acsl1, Plin1, and PPARγ on the PPAR signaling pathway were verified by WB and RT-qPCR experiments, which were found to be consistent with the results of proteomics analysis.
Conclusion: Cuscutae semen and Radix rehmanniae praeparata may regulate the PPAR signaling pathway mediated Acsl1, Plin1 and PPARγ to reduce the testicular tissue damage of male rats caused by GTW.
... C/EBPα is PPARγdependent, indicating that C/EBPα alone cannot induce adipogenesis in the absence of PPARγ [19]. Perilipin is a protein found in lipid droplets present on the surface of differentiated adipocytes, where it blocks access to lipases to regulate lipogenesis and promotes PPARγ expression [20]. Therefore, our results indicate that the expression of adipogenesis and lipogenesis-related proteins was suppressed by isoacteoside treatment (Fig. 5). ...
Isoacteoside is a caffeoyl phenylethanoide glycoside found in various plant parts, such as the flower of Magnolia denudata . In particular, magnolia has been studied for its anti-obesity, anticancer, and anti-inflammatory effects. However, isoacteoside has not been extensively studied, except for its anti-inflammatory effects. In this study, the anti-obesity effects of isoacteoside were investigated in 3T3-L1 mouse pre-adipocytes. Isoacteoside treatment did not induce cytotoxicity in 3T3-L1 cells up to a concentration of 1000 μM. The anti-obesity effects on 3T3-L1 cells were confirmed using oil red O staining. In addition, the expression of obesity-related proteins and genes, such as peroxisome proliferator-activated acceptor gamma ( PPARγ ), CCAAT/enhancer-binding protein alpha ( C/EBPα ), and perilipin ( PLIN1 ), was determined by western blotting and qRT-PCR assays to confirm the anti-obesity effects of isoacteoside. The results of this study suggest that isoacteoside, a natural substance isolated from plant extracts, is not highly toxic to normal cells, such as pre-adipocytes, and displays anti-obesity effects in vitro.
... PLIN1, a protein on the surface of lipid droplets, converts triglycerides into glycerol and free fatty acids through phosphorylation (Wen et al., 2011;Maurizi et al., 2017). In mice, PLIN1 is a downstream target gene of PPAR-γ (Ying et al., 2015;Maurizi et al., 2017), and its depletion can cause abnormal expression of some genes related to fat metabolism (CAAT/enhancer-binding proteins, sterol regulatory element-binding protein-1, FABP4) and impair the ability to break down fat (Naoto et al., 2004). FABP4 is an important long-chain fatty acid binding transporter (Coe and Bernlohr, 1998;Labbe et al., 2015). ...
Cold tolerance is an important trait for sheep raised at high altitudes. Muscle tissue, comprising 30–40% of the total body mass, produces heat during cold exposure. However, little is known about the genetic mechanisms of this tissue and its role in thermogenesis in lambs. We examined genes in skeletal muscle tissue in a cold-adapted sheep breed, Altay, and a cold-intolerant sheep breed, Hu, when exposed to low air temperature. Three ewe-lambs of each breed were maintained at −5°C and three ewe-lambs of each breed were maintained at 20°C. After cold exposure for 25 days, the longissimus dorsi of each lamb was collected, and transcriptome profiles were sequenced and analyzed. The results of RNA-seq showed that the average reads among the four groups were 11.0 Gbase. The genome mapping rate averaged 88.1% and the gene mapping rate averaged 82.5%. The analysis of differentially expressed genes (DEGs) indicated that the peroxisome proliferator-activated receptors (PPAR), cAMP, and calcium signaling pathways and muscle contraction in muscle tissue were linked to thermogenesis in cold-exposed lambs. Furthermore, PCK1 (phosphoenolpyruvate carboxykinase1) increased glyceroneogenesis in cold-exposed Altay lambs, and APOC3 (apolipoprotein C3), LPL (lipoprotein lipase), and FABP4 (fatty acid binding protein 4, adipocyte) were involved in the intake and transport of free fatty acids. In Hu sheep, cAMP biosynthesis from ATP hydrolysis was regulated by ADCY10 (adenylate cyclase) and ADORA2a (adenosine A2a receptor). Skeletal muscle contraction was regulated by MYL2 (myosin light chain 2). In conclusion, cold exposure altered the expression level of genes involved in heat production in muscle tissue. Some potential mechanisms were revealed, including calcium ion transport in the calcium signaling pathway, fatty acid metabolism in the PPAR signaling pathway, and cAMP biosynthesis in the cAMP signaling pathway. This study implied that skeletal muscle plays an important role in thermoregulation in lambs.
... In mature white adipocytes, various proteins are localized on the surface of LDs and control fusion, growth, fission, and lipolysis. Perilipin 1, a downstream target of PPARγ, is one of the major regulatory proteins involved in LD formation [11]. Perilipin 1 is localized on the surface of LDs and prevents lipase from binding to LDs for lipolysis. ...
Hypertrophy and hyperplasia of white adipocytes induce obesity, leading to diseases such as type 2 diabetes and hypertension, and even cancer. Hypertrophy of white adipocytes is attributed to the excessive storage of the energy form of triglycerides in lipid droplets (LDs). LDs are fat storage organelles that maintain whole-body energy homeostasis. It is important to understand the mechanism of LD formation for the development of obesity therapy; however, the regulatory mechanisms of LD size and formation are not fully understood. In this study, we demonstrated that the PPM family phosphatase PPM1D regulates LD formation. PPM1D specific inhibitor, SL-176 significantly decreased LD formation via two different pathways: dependent of and independent of adipocyte-differentiation processes. In the mature white adipocytes after differentiation, LD formation was found to be controlled by PPM1D via dephosphorylation of Ser511 of perilipin 1. We found that inhibition of PPM1D in mature white adipocytes significantly reduced the size of the LDs via dephosphorylation of Ser511 of perilipin 1 but did not change the lipolysis sensitivity and the total amount of lipid in cells. Collectively, the results of this study provide evidence that PPM1D plays an important role in LD formation in mature adipocytes.
... In contrast to the normal BMECs, the KLF6 knock-out cell line showed a markedly reduced expression of PPARA and their selected genes in terms of mRNA and protein, suggesting that these genes are involved in lipogenesis and cholesterol metabolism. Preliminary studies have demonstrated an important role for PPAR in controlling the transcriptional activity of several key adipocyte-related genes, including aP2, C/EBP, GLUT4, and perilipin in response to insulin [56][57][58][59][60][61][62]. Meanwhile, another study in the mitochondria reported that PPARA regulates the fatty acid in the muscle [63,64], and the liver regulates fatty acid by targeting the a-carnitine palmitoyl transferase I genes [65]. ...
MicroRNAs (miRNAs) are non-coding RNAs that regulate the expression of their target genes involved in many cellular functions at the post-transcriptional level. Previously, bta-miR-148a showed significantly high expression in bovine mammary epithelial cells (BMECs) of Chinese Holstein cows producing high milk fat compared to those with low milk fat content. Here, we investigated the role of bta-miR-148a through targeting Krüppel-like factor 6 (KLF6) and further analyzed the role of KLF6 in regulating fat metabolism through targeting PPARA, AMPK/mTOR/PPARG, and other fat marker genes in BMECs of Chinese Holstein. The bioinformatics analysis showed that the 3’ UTR of KLF6 mRNA possesses the binding sites for bta-miR-148a, which was further verified through dual-luciferase reporter assay. The BMECs were transfected with bta-miR-148a-mimic, inhibitor, and shNC, and the expression of KLF6 was found to be negatively regulated by bta-miR-148a. Moreover, the contents of triglyceride (TG), and cholesterol (CHO) in BMECs transfected with bta-miR-148a-mimic were significantly lower than the contents in BMECs transfected with bta-miR-148a-shNC. Meanwhile, the TG and CHO contents were significantly increased in BMECs transfected with bta-miR-148a-inhibitor than in BMECs transfected with bta-miR-148a-shNC. In addition, the TG and CHO contents were significantly decreased in BMECs upon the down-regulation of KLF6 through transfection with pb7sk-KLF6-siRNA1 compared to the control group. Contrarily, when KLF6 was overexpressed in BMECs through transfection with pBI-CMV3-KLF6, the TG and CHO contents were significantly increased compared to the control group. Whereas, the qPCR and Western blot evaluation of PPARA, AMPK/mTOR/PPARG, and other fat marker genes revealed that all of the genes were considerably down-regulated in the KLF6-KO-BMECs compared to the normal BMECs. Taking advantage of deploying new molecular markers and regulators for increasing the production of better-quality milk with tailored fat contents would be the hallmark in dairy sector. Hence, bta-miR-148a and KLF6 are potential candidates for increased milk synthesis and the production of valuable milk components in dairy cattle through marker-assisted selection in molecular breeding. Furthermore, this study hints at the extrapolation of a myriad of functions of other KLF family members in milk fat synthesis.
