PPARγ mRNA Research Articles

RSL1D1 modulates cell senescence and proliferation via regulation of PPARγ mRNA stability

AimsIn this study, we will examine if RSL1D1 influences PPARγ expression and explore the underlying mechanism that RSL1D1 regulates PPARγ expression. Moreover, the significance of RSL1D1-PPARγ pathway in cell senescence and proliferation will also be determined. Main methodsOur main methods include western blotting, immunoprecipitation (IP), real-time PCR, RNA Immunoprecipitation (RIP), biotin-labeled RNA pull down assay, dual luciferase reporter gene assay, senescence-associated β-galactosidase staining, cell proliferation assay, colony formation assay, wound healing assay, blood biochemistry test and Oil red O staining. Key findingsBy analyzing gene chip results we find that the expression of RSL1D1 and PPARγ might be correlated. Then we show that RSL1D1 is a posttranscriptional regulator of PPARγ. RSL1D1 overexpression elevates, while RSL1D1 knockdown inhibits, PPARγ mRNA and protein expression levels. Mechanistically, we find that RSL1D1 directly interacts with the 3′-untranslated region of PPARγ mRNA, and then promotes its stability and increases PPARγ protein expression level. We further demonstrate that RSL1D1 modulates cellular senescence and cell proliferation partially via PPARγ-regulated downstream target genes such as PTEN/p27, NF-κB, GLUT4, and ACL. Moreover, we find that RSL1D1 regulates PPARγ expression and function in a HuR-dependent manner. Last, we show that RSL1D1 knockout in mouse adipose tissue shortens mouse lifespan and leads to hepatic damage which may impair liver damage repair function. SignificanceCollectively, our findings unveil a novel posttranscriptional regulation of PPARγ by RSL1D1 and uncover a critical role of RSL1D1-PPARγ-PPARγ downstream target genes in regulating cellular senescence and cell proliferation.

Abstract P3008: Endothelial Stearoyl-CoA Desaturase-1 Catalyzes Fatty Acid Metabolites To Mitigate Arterial Inflammation And Stiffness

Introduction: Vascular metabolic plasticity is intimately linked with endothelial homeostasis. Stearoyl-CoA desaturase-1 (SCD1), the enzyme that catalyzes the saturated long-chain fatty acids (FA) into monounsaturated FA (MUFA), is responsive to pulsatile shear stress (PSS), and is associated with cancer cell metabolism. We assessed whether exercise-mediated PSS activates PPARγ-SCD1 to mediate anti-inflammatory metabolites for vascular protection. Methods and Results: Human aortic endothelial cells (HAEC) were subjected to PSS (τ avg = 23±8 dyne/cm 2 at 1 Hz) for 4 hours, followed by metabolomic analysis. SCD1-mediated fatty acid metabolites, azelaic acid (AZA) and oleic acid (OA), were identified to mitigate NFκB luciferase activity and monocyte binding ( p < 0.05, n= 5, vs. static condition). PSS upregulated PPARγ and SCD1 mRNA expression, whereas knockdown of PPARγ using siRNA downregulated SCD1. Wild type mice (C57BL/6J) undergoing 2-wk voluntary exercise developed increased perinuclear SCD1 expression in the aortic endothelium ( p < 0.05, n=6, vs. sedentary controls), whereas SCD1 expression was reduced in PPARγ -dominant negative mice following exercise. LDLR -/- mice with endothelial-specific SCD1 deletion (SCD1 EC-KO : Cdh5 Cre , Scd1 f/f ) on 4-wk high-fat diet (HFD) developed significantly increased VCAM-1 and Oil Red O positive lesions in the aortic arch, whereas exercise or selective overexpression (OE) of SCD1 in endothelial cells by pAd5/F35/CD144 adenoviral vector mitigated the lesions ( p < 0.05, n=5, HFD + exercise or HFD+Ad-SCD1 vs. HFD). Arterial stiffness as measured by Pulse Wave Velocity of the left common carotid was elevated in LDLR -/- SCD1 EC-KO on HF diet, but was decreased with exercise or SCD1 OE. Conclusion: PPARγ-SCD1 signaling is flow-responsive to mitigate arterial inflammation and stiffness. Despite SCD1-associated cancer cell metabolism, endothelial-specific SCD1 confers vascular protection, consistent with the reported skeletal muscle-specific model. Thus, we demonstrate that exercise activates PPARγ-SCD1 and SCD1 catalyzes AZA and OA as biomarkers with therapeutic potential.

New 4-thiazolidinone-based molecules Les-2769 and Les-3266 as possible PPARγ modulators

Development of cancer drug-resistance is still an ongoing problem in the modern anticancer treatment. Therefore, there is a need to search for a new active substance, which may become a potential anticancer agent. 4-Thiazolidinones are well-described substances with cytotoxicity against cancer cells in vitro. Therefore, the aim of this study was to evaluate the effect of two 4-thiazolidinone-based derivatives (Les-2769 and Les-3266) on the PPARγ-dependent cytotoxicity in normal human skin fibroblasts (BJ) and squamous cell carcinoma (SCC-15) in vitro. The data obtained showed a cytotoxic effect of Les-2769 and Les-3266 used in micromolar concentrations on SCC-15 and BJ cells, manifesting by a decrease in the metabolic activity, an increase in the release of lactate dehydrogenase, and caspase-3 activity. The co-treatment of the cells with Les-3266 and an antagonist (GW9662) or an agonist (rosiglitazone) of the PPARγ receptor induced changes in the above-mentioned parameters in the BJ and SCC-15 cells, compared to the Les-3266 alone exposure; this was not found in the Les-2769-treated cells. The further analysis of the compounds indicated changes in the expression of the PPARγ, KI67, and NF-κB genes. Moreover, the tested compounds caused an increase in the level of PPARγ mRNA expression in a similar way to rosiglitazone in SCC-15, which may indicate the affinity of the compounds for PPARγ. Molecular docking is consistent with experimental in vitro data about the potential agonistic activity of Les-2769 and Les-3266 towards PPARγ receptors. Summarizing, the anticancer effect of both compounds was observed in the SCC-15 cells in vitro; moreover, the mechanism of action of Les-3266 in cells is mediated probably by interaction with the PPARγ receptor pathway, which needs in-depth study.

Evaluation of the expression pattern and diagnostic value of PPARγ in malignant and benign primary bone tumors

PurposeThe quantifiable description of PPARγ expression pattern beside mechanistic in-vitro evidence will provide insights into the involvement of this mediator in tumor pathogenesis. This study is focused on illuminating the PPARγ gene and protein expression pattern, its association with tumor deterioration and its diagnostic value in different types of primary bone tumors.MethodsThe expression pattern of PPARγ was investigated in the 180 bone tissues including 90 bone tumor tissues and 90 non-cancerous bone tissues. The local PPARγ expression level was assessed using real-time qRT-PCR and the PPARγ protein expression pattern was measured using immunohistochemistry. The correlation of PPARγ expression level with patients’ clinic-pathological features, also the value of the variables in predicting PPARγ expression level in tumors and the value of PPARγ to discriminate tumor subtypes were assessed.ResultsThe mean PPARγ mRNA expression was significantly higher in bone tumors compared to healthy bone tissues, also the malignant tumors including osteosarcoma and Ewing sarcoma had the elevated level of PPARγ mRNA compared to GCT tumors. Consistently, the protein expression of PPARγ in the tumor site was significantly higher in the bone tumors and malignant tumors compared to non-cancerous and benign tumors, respectively. The PPARγ protein could predict malignant tumor features including tumor grade, metastasis and recurrence significantly. Moreover, PPARγ could potentially discriminate the patients from the controls also malignant tumors from benign tumors with significant sensitivity and specificity.ConclusionsPPARγ might be involved in primary bone tumor pathogenesis and determining its molecular mechanism regarding bone cancer pathogenesis is of grave importance.

The preventive effect of low-dose aspirin in a PPAR-γ antagonist treated mouse model of preeclampsia

BackgroundPreeclampsia (PE) is one of the leading causes of maternal and perinatal mortality and morbidity. Low-dose aspirin (LDA) is the most widely used drug to prevent PE, but the recommended dose of LDA varies according to different guidelines. Peroxisome proliferator-activated receptor (PPAR)-γ is involved in the formation of the placenta during pregnancy and is expressed in women with severe PE. In the present study, Our purpose was to investigate whether aspirin intervention in preeclampsia was related to PPAR-γ.MethodsWe administered pregnant mice with PPAR-γ-specific antagonist(T0070907) 2 mg/kg/d at 8.5–12.5 days of pregnancy. Mice treated with T0070907 developed key features of preeclampsia. Two doses of LDA (10 mg/kg/d and 20 mg/kg/d) were administered to the mice with a PE phenotype for intervention.ResultsLDA effectively decreased the increase in blood pressure in mice caused by T0070907 and decreased urinary protein levels and the urinary protein/creatinine ratio. LDA also inhibited the overexpression of endoglin and IL-β treated by T0070907. In addition, LDA evidently increased the placental weight and alleviates the degree of placental lesions of placenta and kidney. LDA alleviated the inhibition of PPAR-γ mRNA expression. The beneficial effect of 20 mg LDA was significantly better than that of 10 mg.Conclusions(1) LDA has a preventive effect against PE treated by PPAR-γ antagonist. (2) The preventive effect of LDA against PE is dose-dependent.

Overexpression of DGAT2 Stimulates Lipid Droplet Formation and Triacylglycerol Accumulation in Bovine Satellite Cells.

Simple SummaryTriacylglycerols are the major component of intramuscular fat, and the final step in their biosynthesis is catalyzed by DGAT2. Bovine skeletal muscle satellite cells were infected with the overexpression adenovirus Ad-DGAT2 and interfering adenovirus sh-DGAT2. The results showed that overexpression of DGAT2 upregulated the expression of genes involved in lipid accumulation and adipogenesis and increased cellular triacylglycerol content. The differentially expressed genes were mainly enriched in the PPAR signaling, glycerolipid metabolism, fatty acid biosynthesis, and AMPK signaling pathways. These results highlight the important regulatory role of DGAT2 during adipogenic transdifferentiation of BSCs and the complexity of intramuscular adipogenesis, as well as providing a theoretical basis for producing high marbling content beef.Intramuscular fat (IMF) is closely related to the tenderness, juiciness, and flavor of beef, and is an important indicator for beef quality assessment internationally. The main components of skeletal intramuscular fat (IMF) are phospholipids and triacylglycerols (TAG), and the final step of TAG biosynthesis is catalyzed by diacylglycerol acyltransferase 2 (DGAT2). To explore the effect of DGAT2 on the differentiation of bovine muscle satellite cells (BSCs) and its role in the signaling pathway related to lipid metabolism, the adenovirus overexpression and interference vector of the DGAT2 gene was constructed in this study, and the overexpression adenovirus Ad-DGAT2 and interfering adenovirus sh-DGAT2 were used to infect BSCs. Overexpression of DGAT2 resulted in a significant increase in the contents of TAG and ADP, and the mRNA and protein expression levels of PPARγ, C/EBPα, and SREBF1 (p < 0.05). Interfering with the expression of DGAT2 reduced the intracellular TAG content and lipid droplet accumulation. Furthermore, the mRNA and protein expression levels of PPARγ, C/EBPα, and SREBF1 (p < 0.05) were significantly downregulated. Transcriptome sequencing showed that a total of 598 differentially expressed genes (DEGs) were screened in BSCs infected with Ad-DGAT2, and these DEGs included 292 upregulated genes and 306 downregulated genes. A total of 49 DEGs were screened in BSCs infected with sh-DGAT2, and these DEGs included 25 upregulated and 24 downregulated genes. KEGG enrichment analysis showed that the DEGs, after overexpression of DGAT2, were mainly enriched in the PPAR signaling pathway, and the fat digestion and absorption, glycerophospholipid metabolism, fatty acid biosynthesis, and AMPK signaling pathways. The DEGs obtained after interfering with DGAT2 were mainly enriched in the metabolic pathways, such as the PPAR signaling pathway and PI3K/AKT signaling pathway. In summary, our study demonstrated that the lipid droplet formation, TAG accumulation, and adipogenic gene expression in BSCs overexpressing DGAT2 were higher than those in the control cells. These results highlight the important role of DGAT2 in regulating BSCs during adipogenic transdifferentiation and underscore the complexity of intramuscular adipogenesis.

Breast Cancer-Stromal Interactions: Adipose-Derived Stromal/Stem Cell Age and Cancer Subtype Mediated Remodeling.

Adipose tissue is characterized as an endocrine organ that acts as a source of hormones and paracrine factors. In diseases such as cancer, endocrine and paracrine signals from adipose tissue contribute to cancer progression. Young individuals with estrogen receptor-alpha positive (ER-α+) breast cancer (BC) have an increased resistance to endocrine therapies, suggesting that alternative estrogen signaling is activated within these cells. Despite this, the effects of stromal age on the endocrine response in BC are not well defined. To identify differences between young and aged ER-α+ breast tumors, RNA sequencing data were obtained from The Cancer Genome Atlas. Analysis revealed enrichment of matrix and paracrine factors in young (≤40 years old) patients compared to aged (≥65 years old) tumor samples. Adipose-derived stromal/stem cells (ASCs) from noncancerous lipoaspirate of young and aged donors were evaluated for alterations in matrix production and paracrine secreted factors to determine if the tumor stroma could alter estrogen signaling. Young and aged ASCs demonstrated comparable proliferation, differentiation, and matrix production, but exhibited differences in the expression levels of inflammatory cytokines (Interferon gamma, interleukin [IL]-8, IL-10, Tumor necrosis factor alpha, IL-2, and IL-6). Conditioned media (CM)-based experiments showed that young ASC donor age elevated endocrine response in ER-α+ BC cell lines. MCF-7 ER-α+ BC cell line treated with secreted factors from young ASCs had enhanced ER-α regulated genes (PGR and SDF-1) compared to MCF-7 cells treated with aged ASC CM. Western blot analysis demonstrated increased activation levels of p-ER ser-167 in the MCF-7 cell line treated with young ASC secreted factors. To determine if ER-α+ BC cells heightened the cytokine release in ASCs, ASCs were stimulated with MCF-7-derived CM. Results demonstrated no change in growth factors or cytokines when treated with the ER-α+ secretome. In contrast to ER-α+ CM, the ER-α negative MDA-MB-231 derived CM demonstrated increased stimulation of pro-inflammatory cytokines in ASCs. While there was no observed change in the release of selected paracrine factors, MCF-7 cells did induce matrix production and a pro-adipogenic lineage commitment. The adipogenesis was evident by increased collagen content through Sirius Red/Fast Green Collagen stain, lipid accumulation evident by Oil Red O stain, and significantly increased expression in PPARγ mRNA expression. The data from this study provide evidence suggesting more of a subtype-dependent than an age-dependent difference in stromal response to BC, suggesting that this signaling is not heightened by reciprocal signals from ER-α+ BC cell lines. These results are important in understanding the mechanisms of estrogen signaling and the dynamic and reciprocal nature of cancer cell-stromal cell crosstalk that can lead to tumor heterogeneity and variance in response to therapy.

Sinapic Acid Attenuated Cardiac Remodeling After Myocardial Infarction by Promoting Macrophage M2 Polarization Through the PPARγ Pathway.

BackgroundMacrophage polarization is an important regulatory mechanism of ventricular remodeling. Studies have shown that sinapic acid (SA) exerts an anti-inflammatory effect. However, the effect of SA on macrophages is still unclear.ObjectivesThe purpose of the study was to investigate the role of SA in macrophage polarization and ventricular remodeling after myocardial infarction (MI).MethodsAn MI model was established by ligating the left coronary artery. The rats with MI were treated with SA for 1 or 4 weeks after MI. The effect of SA on bone marrow-derived macrophages (BMDMs) was also observed in vitro.ResultsCardiac systolic dysfunction was significantly improved after SA treatment. SA reduced MCP-1 and CCR2 expression and macrophage infiltration. SA decreased the levels of the inflammatory factors TNF-α, IL-1α, IL-1β, and iNOS and increased the levels of the M2 macrophage markers CD206, Arg-1, IL-10, Ym-1, Fizz-1, and TGF-β at 1 week after MI. SA significantly increased CD68+/CD206+ macrophage infiltration. Myocardial interstitial fibrosis and MMP-2 and MMP-9 levels were decreased, and the sympathetic nerve marker TH and nerve sprouting marker GAP43 were suppressed after SA treatment at 4 weeks after MI. The PPARγ level was notably upregulated after SA treatment. In vitro, SA also increased the expression of PPARγ mRNA in BMDMs and IL-4-treated BMDMs in a concentration-dependent manner. SA enhanced Arg1 and IL-10 expression in BMDMs, and the PPARγ antagonist GW9662 attenuated M2 macrophage marker expression.ConclusionsOur results demonstrated that SA attenuated structural and neural remodeling by promoting macrophage M2 polarization via PPARγ activation after MI.

Paternal diabetes programs sex-dependent placental alterations and fetal overgrowth.

The aim of this study was to evaluate the paternal programming of sex-dependent alterations in fetoplacental growth and placental lipid metabolism regulated by peroxisome proliferator-activated receptor (PPAR) target genes in F1 diabetic males born from F0 pregestational diabetic rats. F1 control and diabetic male rats were mated with control female rats. On day 21 of gestation, F2 male and female fetoplacental growth, placental lipid levels, and protein and mRNA levels of genes involved in lipid metabolism and transport were evaluated. Fetal but not placental weight was increased in the diabetic group. Triglyceride, cholesterol and free fatty acid levels were increased in placentas of male fetuses from the diabetic group. The mRNA levels of Pparα and Pparγ coactivator 1α (Pgc-1α) were increased only in placentas of male fetuses from the diabetic group. Protein levels of PPARα and PGC-1α were decreased only in placentas of male fetuses from the diabetic group. No differences were found in Pparγ mRNA and protein levels in placentas from the diabetic group. The mRNA levels of genes involved in lipid synthesis showed no differences between groups, whereas the mRNA levels of genes involved in lipid oxidation and transport were increased only in placentas of male fetuses from the diabetic group. In conclusion, paternal diabetes programs fetal overgrowth and sex-dependent effects on the regulation of lipid metabolism in the placenta, where only placentas of male fetuses show an increase in lipid accumulation and mRNA expression of enzymes involved in lipid oxidation and transport pathways.

Peroxisome Proliferator-Activated Receptor Gene Knockout Promotes Podocyte Injury in Diabetic Mice

Objective To investigate the effects of peroxisome proliferator-activated receptor (PPARγ) expression on renal podocyte in diabetic mice by conditionally knockout mouse PPARγ gene. Methods Wild-type C57BL mice and PPARγ gene knockout mice were used as research objects to establish the diabetic mouse model, which was divided into normal control group (NC group), normal glucose PPARγ gene knockout group (NK group), diabetic wild-type group (DM group), and diabetic PPARγ gene knockout group (DK group), with 8 mice in each group. After 16 weeks, the mice were sacrificed for renal tissue collection. Morphological changes of renal tissue were observed by HE and Masson staining, and ultrastructure of renal tissue was observed by transmission electron microscope. Protein expressions of PPARγ, podocin, nephrin, collagen IV, and fibronectin (FN) in renal tissues were detected by immunohistochemistry and Western blot, and mRNA changes of PPARγ, podocin, and nephrin in renal tissues were detected by qRT-PCR. Results Compared with the NC group, the protein and mRNA expressions of PPARγ, podocin, and nephrin decreased in the kidney tissue of mice in the DM group, while the protein expressions of collagen IV and FN increased. The expression of various proteins in kidney tissues of the DK group was consistent with that of the DM group, and the difference was more obvious. The expression of PPARγ protein and mRNA decreased in the NK group, while the expression of podocin, nephrin protein and mRNA, collagen IV, and FN protein showed no significant difference. Conclusion In diabetic renal tissue, the loss of PPARγ can aggravate podocellular damage and thus promote the occurrence of diabetic renal fibrosis. Increasing the expression of PPARγ may effectively relieve renal podocyte impairment in diabetic patients, which can be used for the treatment of diabetic nephropathy.

Curcuma zedoaria 50% methanol extracts increase adiponectin secretion by enhancing PPARγ mRNA expression in 3T3-L1 cells.

Curcuma zedoaria is a characteristic species of its genus that contains little to no curcuminoid. Here, we demonstrate that C. zedoaria extracts with 50% methanol increases adiponectin secretion into the media by enhancing PPARγ mRNA expression in 3T3-L1 cells. These results indicate that C. zedoaria may be useful for preventing/improving lifestyle-related diseases such as diabetes and atherosclerosis.

The transported active mulberry leaf phenolics inhibited adipogenesis through PPAR-γ and Leptin signaling pathway.

The effective components of mulberry leaf polyphenols (MLPs) should be absorbed and transported by the intestinal cells before regulating lipid metabolism. The Caco-2 intestinal epithelial cell and 3T3-L1 adipocytes were coupled to screen the effective components of MLPs that are being absorbed and transported by intestinal cells. The regulation and molecular mechanism by which the effective components affect adipogenesis were analyzed in this study. Among the 12 main components identified, five main compounds were well absorbed with Papp in the order of benzoic acid > chlorogenic acid > astragaloside > hyperoside > rutin. Chlorogenic acid and benzoic acid were mainly absorbed through passive diffusion, while rutin, astragaloside, and hyperoside were mainly by active transport, of which chlorogenic and rutin absorption were mediated by the efflux protein, P-glycoprotein (P-pg). Based on the transport volume of 2 mg/ml MLPs within 2 h, 25% of the maximum transported MLPs (TMLPs) was a safe concentration for 3 T3-L1 preadipocytes. Except for astragaloside, the other four components showed a significant inhibitory effect on lipid droplets, TG and TC, and chlorogenic acid and benzoic acid had the strongest effect. Additionally, we observed a synergistic effect as TMLPs were the most effective. We hypothesized that TMLPs, chlorogenic acid and benzoic acid suppressed adipogenesis and regulated lipid metabolism by inhibiting PPAR-γ, C/EBP-α, and FAS mRNA while promoting ADIPO and Leptin mRNA expression. PRACTICAL APPLICATIONS: The absorption and adipogenesis inhibition effect of mulberry leaf phenolics were evaluated in this study. The results provided guideline for the development of functional foods in regulating lipid metabolism.

The Role of Peroxisome Proliferator-Activated Receptors in PGF2α-Induced Luteolysis in the Bovine Corpus Luteum.

Simple SummaryThe corpus luteum (CL) is responsible for progesterone (P4) secretion. In the absence of pregnancy, luteolysis occurs, which leads to a reduction in P4 production, followed by the structural regression of the CL. In cows, prostaglandin F2α (PGF2α) is the main luteolytic factor. It is also an endogenous ligand for peroxisome proliferator-activated receptors (PPARs), which are important factors regulating mammalian reproductive function. However, the mechanisms of action of PPAR isoforms, i.e., PPARα, PPARδ and PPARγ, in the luteolytic pathways in cattle are still not fully understood. The aim of this in vitro study was to determine the expression of PPAR isoforms in the bovine CL throughout the estrous cycle, and their involvement in PGF2α-induced processes related to luteolysis. The obtained results indicate that the expression of PPARs changes in the bovine CL throughout the estrous cycle; moreover, PGF2α affects its expression. This study provides evidence that PPARγ, among all examined PPAR isoforms, could be involved in the regulation of PGF2α-induced luteolysis in cattle, and PPARs may affect CL regression at multiple sites. These results help to widen the knowledge of the mechanisms of luteal regression in the bovine CL.The participation of peroxisome proliferator-activated receptors (PPARs) in ovarian function in cattle is still not fully understood. The aim of this in vitro study was to determine: (i) the immunolocalization, mRNA expression and tissue concentration of PPARα, PPARδ and PPARγ in the bovine corpus luteum (CL) (n = 40) throughout the estrous cycle, and (ii) the involvement of PPAR in PGF2α-induced processes related to luteolysis. CL (n = 9) explants were cultured in the presence of PPAR antagonists (10−5 M) in combination with or without PGF2α receptor antagonist (10−5 M) and PGF2α (10−6 M). The mRNA and protein expression of PPARs was evaluated through qPCR, IHC, and ELISA, respectively. The results showed that PPAR mRNA and protein expression differed according to the luteal stages. PGF2α upregulated PPARδ and PPARγ mRNA expression in the bovine CL in vitro, whereas PPARγ increased the inhibitory effect of PGF2α by decreasing progesterone secretion and the mRNA expression of hydroxy-delta-5-steroid dehydrogenase, 3 β- and steroid delta-isomerase 1 (HSD3B1) in the CL explants; mRNA transcription of tumor necrosis factor α (TNFα) and inducible nitric oxide synthase (iNOS) was increased. The obtained results indicate that the mRNA and protein expression of PPARs changes in the bovine CL throughout the estrous cycle and under the influence of PGF2α. We suggest that isoform γ, among all examined PPARs, could be a factor involved in the regulation of PGF2α-induced processes related to luteolysis in the bovine CL. Further studies are needed to understand the role of PPAR in luteal regression in the CL of cattle.

4-Hydroxybenzoic Acid and β-Sitosterol from Cucurbita ficifolia Act as Insulin Secretagogues, Peroxisome Proliferator-Activated Receptor-Gamma Agonists, and Liver Glycogen Storage Promoters: In Vivo, In Vitro, and In Silico Studies.

Insulin secretion and GLUT4 expression are two critical events in glucose regulation. The receptors G-protein-coupled receptor 40 (GPR40) and peroxisome proliferator-activated receptor-gamma (PPARγ) modulate these processes, and they represent potential therapeutic targets for new antidiabetic agent's design. Cucurbita ficifolia fruit is used in traditional medicine for diabetes control. Previous studies demonstrated several effects: a hypoglycemic effect mediated by an insulin secretagogue action, antihyperglycemic effect, and promoting liver glycogen storage. Anti-inflammatory and antioxidant effects were also reported. Moreover, some of its phytochemicals have been described, including d-chiro-inositol. However, to understand these effects integrally, other active principles should be investigated. The aim was to perform a chemical fractionation guided by bioassay to isolate and identify other compounds from C. ficifolia fruit that explain its hypoglycemic action as insulin secretagogue, its antihyperglycemic effect by PPARγ activation, and on liver glycogen storage. Three different preparations of C. ficifolia were tested in vivo. Ethyl acetate fraction derived from aqueous extract showed antihyperglycemic effect in an oral glucose tolerance test and was further fractioned. The insulin secretagogue action was tested in RINm5F cells. For the PPARγ activation, C2C12 myocytes were treated with the fractions, and GLUT4 mRNA expression was measured. Chemical fractionation resulted in the isolation and identification of β-sitosterol and 4-hydroxybenzoic acid (4-HBA), which increased insulin secretion, GLUT4, PPARγ, and adiponectin mRNA expression, in addition to an increase in glycogen storage. 4-HBA exhibited an antihyperglycemic effect, while β-sitosterol showed hypoglycemic effect, confirming the wide antidiabetic related results we found in our in vitro models. An in silico study revealed that 4-HBA and β-sitosterol have potential as dual agonists on PPARγ and GPR40 receptors. Both compounds should be considered in the development of new antidiabetic drug development.

Dietary Micronutrients Reduce Insulin Resistance via Adipose Tissue Modulation in Mice Fed a High Fat Diet

ObjectivesObesity is associated with increased insulin resistance (IR) and white adipose tissue (WAT) dysregulation (e.g., decrease in PPAR-γ mRNA expression and impaired leptin sensitivity). Dietary vitamins A, B1, B6, and B12, selenium (Se) and zinc (Zn) have been shown to reduce IR in obesity in animals and humans, but mechanisms have not been defined. The objective of this study is to investigate the effects of selected micronutrients on adipose tissue metabolism and IR in a mouse model of diet-induced obesity (DIO). We hypothesize that these additions modulate genes regulating IR in WAT and improve leptin sensitivity.Methods28 DIO male mice were randomly assigned to two high fat (HF, 60 kcal % fat) diets with or without the inclusion of a mineral-vitamin mixture (MVM: 5 × vitamin A, B1, B6, B12, zinc, and 2 × selenium), respectively. Similarly, 28 lean mice were randomized into a low-fat diet (LF, 10 kcal % fat) with or without the MVM. Mice were fed diets ad libitum for 8 weeks. Bodyweight (BW), IR, serum glucose, fasting insulin, C-peptide, leptin, as well as mRNA expression of genes involved in insulin signaling and adipokine secretion were measured in epididymal white adipose tissue (eWAT).ResultsCompared to HF mice, HF-MVM mice exhibited reduced body weight gain over time (by 6%, P < 0.05), improved insulin sensitivity (P < 0.05), reduced fasting glucose (by 18%), insulin (by 45%), C-peptide concentrations (by 26%) and Homeostatic Model Assessment for Insulin Resistance (by 47%, HOMA-IR, P < 0.05 for all). Similarly, LF-MVM had reduced fasting glucose level (P < 0.05) compared to LF mice. As expected, serum leptin concentration (adjusted for a visceral fat mass) was 6-fold higher in HF mice compared to LF mice; HF-MVM mice had lower leptin level compared to the HF mice suggesting improved leptin sensitivity. Ppar-γ gene expression in eWAT was 77% lower in HF vs LF group, suggesting eWAT dysfunction and systemic IR in DIO mice; the addition of MVM to LF diet attenuated this effect.ConclusionsThis MVM mixture may reduce IR through the upregulation of the PPAR-γ system in WAT and improve leptin sensitivity in DIO. Understanding the mechanism of action of micronutrients in reducing IR in a highly relevant animal model will provide new avenues for identifying food component solutions to modify IR in humans.Funding SourcesSupport provided by Various Sponsors (unrestricted funds).

PPAR-γ Agonist Pioglitazone Restored Mouse Liver mRNA Expression of Clock Genes and Inflammation-Related Genes Disrupted by Reversed Feeding.

Introduction The master clock, which is located in the suprachiasmatic nucleus (SCN), harmonizes clock genes present in the liver to synchronize life rhythms and bioactivity with the surrounding environment. The reversed feeding disrupts the expression of clock genes in the liver. Recently, a novel role of PPAR-γ as a regulator in correlating circadian rhythm and metabolism was demonstrated. This study examined the influence of PPAR-γ agonist pioglitazone (PG) on the mRNA expression profile of principle clock genes and inflammation-related genes in the mouse liver disrupted by reverse feeding. Methods Mice were randomly assigned to daytime-feeding and nighttime-feeding groups. Mice in daytime-feeding groups received food from 7 AM to 7 PM, and mice in nighttime-feeding groups received food from 7 PM to 7 AM. PG was administered in the dose of 20 mg/kg per os as aqueous suspension 40 μl at 7 AM or 7 PM. Each group consisted of 12 animals. On day 8 of the feeding intervention, mice were sacrificed by cervical dislocation at noon (05 hours after light onset (HALO)) and midnight (HALO 17). Liver expressions of Bmal1, Clock, Rev-erb alpha, Cry1, Cry2, Per1, Per2, Cxcl5, Nrf2, and Ppar-γ were determined by quantitative reverse transcription PCR. Liver expression of PPAR-γ, pNF-κB, and IL-6 was determined by Western blotting. Glucose, ceruloplasmin, total cholesterol, triglyceride concentrations, and ALT and AST activities were measured in sera by photometric methods. The null hypothesis tested was that PG and the time of its administration have no influence on the clock gene expression impaired by reverse feeding. Results Administration of PG at 7 AM to nighttime-feeding mice did not reveal any influence on the expression of the clock or inflammation-related genes either at midnight or at noon. In the daytime-feeding group, PG intake at 7 PM led to an increase in Per2 and Rev-erb alpha mRNA at noon, an increase in Ppar-γ mRNA at midnight, and a decrease in Nfκb (p65) mRNA at noon. In general, PG administration at 7 PM slightly normalized the impaired expression of clock genes and increased anti-inflammatory potency impaired by reversed feeding. This pattern was supported by biochemical substrate levels—glucose, total cholesterol, ALT, and AST activities. The decrease in NF-κB led to the inhibition of serum ceruloplasmin levels as well as IL-6 in liver tissue. According to our data, PG intake at 7 PM exerts strong normalization of clock gene expression with a further increase in Nrf2 and, especially, Ppar-γ and PPAR-γ expression with inhibition of Nfκb and pNF-κB expression in daytime-feeding mice. These expression changes resulted in decreased hyperglycemia, hypercholesterolemia, ALT, and AST activities. Thus, PG had a potent chronopharmacological effect when administered at 7 PM to daytime-feeding mice. Conclusions Our study indicates that reversed feeding induced the disruption of mouse liver circadian expression pattern of clock genes accompanied by increasing Nfκb and pNF-κB and IL-6 expression and decreasing Nrf2 and PPAR-γ. Administration of PG restored the clock gene expression profile and decreased Nfκb, pNF-κB, and IL-6, as well as increased Nrf2, Ppar-γ, and PPAR-γ expression. PG intake at 7 PM was more effective than at 7 AM in reversed feeding mice.

Brown adipocytes promote epithelial mesenchymal transition of neuroblastoma cells by inducing PPAR-γ/UCP2 expression

ABSTRACT Neuroblastoma (NB) is an embryonic malignant tumour of the sympathetic nervous system, and current research shows that activation of brown adipose tissue accelerates cachexia in cancer patients. However, the interaction between brown adipose tissues and NB remains unclear. The study aimed to investigate the effect of brown adipocytes in the co-culture system on the proliferation and migration of NB cells. Brown adipocytes promoted the proliferation and migration of Neuro-2a, BE(2)-M17, and SH-SY5Y cells under the co-culture system, with an increase of the mRNA and protein levels of UCP2 and PPAR-γ in NB cells. The UCP2 inhibitor genipin or PPAR-γ inhibitor T0090709 inhibited the migration of NB cells induced by brown adipocytes. Genipin or siUCP2 upregulated the expression of E-cadherin, and downregulated the expression of N-cadherin and vimentin in NB cells. We suggest that under co-cultivation conditions, NB cells can activate brown adipocytes, which triggers changes in various genes and promotes the proliferation and migration of NB cells. The PPAR-γ/UCP2 pathway is involved in the migration of NB cells caused by brown adipocytes.

Epimedii folium Polysaccharide Ameliorated Glucose Metabolic Disorder in Type 2 Diabetic Mice by Regulating the SIRT1/PPARγ Signaling Pathway

Abstract: Background and Aim: Epimedii folium is a widely used herbal medicine with numerous medicinal effects. In this study, we aimed to investigate the effects of Epimedii folium polysaccharide (EFP) in db/db metabolic disorder mice model. Materials and Methods: Studies were carried out by the db/db mice with separately receiving EFP (100 mg/ kg, 400 mg/kg) and metformin (300 mg/kg/day) for 8 weeks. Results: In the present study, we show that EFP and metformin ameliorated metabolic disorders and decreased the levels of glucose metabolism parameters in a dose-dependent manner. Besides, the hepatorenal function was also improved with EFP and metformin. In addition, EFP and metformin also reduced the lipid vacuoles accumulated in the hepatocytes and alleviated the hepatocyte’s hypertrophy and nuclei shrunk. Moreover, EFP and metformin also reduced the activity level of MDA and increased the antioxidant enzymes levels, including CAT, SOD, and GSH-Px, as well as the mRNA and proteins expression of SIRT1 and PPARγ in the mice liver. Conclusion: Our data demonstrate that EFP ameliorated hyperglycemia mediated oxidative stress and promoted lipid metabolism in T2D mice, and was partially associated with activation of SIRT1/PPARγ pathway in db/db mice. Key words: Type 2 diabetes, Epimedii folium polysaccharide, Hyperglycemia, Oxidative stress, Lipid metabolism.

Study on the role of heat shock protein 90 (HSP90) gene in chicken preadipocytes proliferation and differentiation

In this study, we examined the effects of Heat Shock Protein 90 (HSP90) on adipocyte proliferation and differentiation in chickens. To achieve this, we constructed RNA interference (RNAi) vectors to target HSP90 and transfected the vectors into primary adipocytes. After transfection, oil red O staining was performed to determine the status of triglyceride accumulation in the cells, whereas the CCK-8 cell kit and 5-Ethynyl-2'-Deoxyuridine (EdU) assays were used to determine cell proliferation. Thereafter, the mRNA and protein expression levels of PPARγ, FAS, SREBP-1c, and HSP90 were determined, and the results showed that after the interference of HSP90, the mRNA and protein expression levels of HSP90 in the chicken adipocytes decreased significantly compared to the control and blank groups (p < 0.05). The decreased mRNA and protein expression of PPARγ, FAS, and SREBP-1c was related to adipocyte differentiation (p < 0.05). However, HSP90 interference had no effect on adipocyte proliferation (p > 0.05). Taken together, the results of this study showed that HSP90 influenced the expression of PPARγ and adipose-differentiation-related genes, thereby regulating triglyceride accumulation and adipocyte differentiation in chickens.

Abstract 442: Strenuous Exercise And Polyphenol Intake Differentially Modulate Lipoproteins

Objective: To investigate gene expression levels of ATP-binding cassette transporter A1 (ABCA1), proprotein convertase subtilisin/kexin type 9 (PCSK9), paraoxonase 1 (PON1), angiopoietin-like 3 (ANGPTL3), angiopoietin-like 4 (ANGPTL4), peroxisome proliferator-activated receptor gamma (PPARγ), apolipoprotein C-III (APO CIII), and apolipoprotein A-5 (APO A5) after 30 days of quercetin intake combined with exercise. Methods: Forty C57BL/6 mice were divided into four groups (n=10): no exercise and no quercetin (C); quercetin and no exercise (Q); exercise and no quercetin (E); and exercise combined with quercetin (EQ). All animals were fed a high-fat diet containing 1.5% cholesterol as part of a 42% fat diet without antioxidants. After 2 months the animals were sacrificed and liver tissues were analyzed for ABCA1, PCSK9, PON1, ANGPTL3, ANGPTL4, PPARγ, APO CIII, and APO A5 expression using qPCR and western blot. Results: After the treatment period ABCA1, PPARγ, APO A5, and ANGPTL4 mRNA expressions differentially altered with exercise and quercetin intake. The results also indicated that PCSK9 mRNA expression was up regulated in the combination group; however, this did not correlate with an increase in secreted protein. Finally, the level of PON1 protein expression was reduced in the exercise quercetin combination group compared to the control. Conclusions: Hepatocyte lipoprotein expression levels were influenced by quercetin consumption combined with exercise. The changes were not consistent with the perceived effects of the exercise polyphenols combination.