Phosphorylation Of PPARγ Research Articles

Cardioprotective effects of nanoparticles green formulated by Spinacia oleracea extract on isoproterenol-induced myocardial infarction in mice by the determination of PPAR-γ/NF-κB pathway

Abstract We developed a contemporary cardioprotective medication using silver nanoparticles (AgNPs) loaded with Spinacia oleracea to treat isoproterenol (ISO)-induced myocardial infarction in mice, focusing on the PPAR-γ/NF-κB pathway. The physicochemical techniques, such as Fourier-transform infrared spectroscopy, field emission scanning electron microscopy, ultraviolet–visible spectroscopy, and energy dispersive X-ray analysis, were employed to characterize the AgNPs. In the in vivo experiment, myocardial infarction was induced in mice by administering ISO subcutaneously at a dose of 40 mg/kg every 12 h for a total of three times. The mice were divided into five groups in a random manner: (1 and 2) ISO + AgNPs at varying doses (10 and 20 μg/mL) and time points; (3) ISO; and (4) control. Following the treatment, cardiac function was assessed through electrocardiogram, as well as biochemical and histochemical analyses. In the study, we examined the inflammatory reactions and cell death in human coronary artery endothelial cells exposed to lipopolysaccharide (LPS). The PPAR-γ/NF-κB activation by LPS and the resulting cytokine production were checked using real-time PCR and western blot techniques. The typical ST segment depression in myocardial infarction mice is significantly inhibited by the administration of AgNPs. Additionally, the treatment with AgNPs leads to a significant improvement in ventricular wall infarction, a decrease in mortality rate, and inhibition of myocardial injury marker levels. Furthermore, the application of AgNPs resulted in a decrease in the inflammatory environment within the hearts of mice with myocardial infarction. This effectively prevented the increase in TNF-α, IL-1β, and IL-6. The gene expression normalization of PPAR-γ/NF-κB/IκB-α/IKKα/β and PPAR-γ phosphorylation could potentially be linked to the advantageous impacts of AgNPs. In the context of an in vitro experiment, the administration of AgNPs demonstrated a notable decrease in cell death and inflammation cytokines expression inhibition. The myocardial infarction mice in the pre + post-ISO group appear to experience more noticeable cardioprotective effects from the treatment with AgNPs than those in the post-ISO group. Our research findings demonstrate that AgNPs possess cardioprotective efficacies in ISO-induced myocardial infarction. This beneficial effect may be attributed to the PPAR-γ activation and the NF-κB signaling inhibition. Consequently, our study presents a novel remedial approach for myocardial infarction treatment in clinical settings.

Molecular regulation of PPARγ/RXRα signaling by the novel cofactor ZFP407.

Cofactors interacting with PPARγ can regulate adipogenesis and adipocyte metabolism by modulating the transcriptional activity and selectivity of PPARγ signaling. ZFP407 was previously demonstrated to regulate PPARγ target genes such as GLUT4, and its overexpression improved glucose homeostasis in mice. Here, using a series of molecular assays, including protein-interaction studies, mutagenesis, and ChIP-seq, ZFP407 was found to interact with the PPARγ/RXRα protein complex in the nucleus of adipocytes. Consistent with this observation, ZFP407 ChIP-seq peaks significantly overlapped with PPARγ ChIP-seq peaks, with more than half of ZFP407 peaks overlapping with PPARγ peaks. Transcription factor binding motifs enriched in these overlapping sites included CTCF, RARα/RXRγ, TP73, and ELK1, which regulate cellular development and function within adipocytes. Site-directed mutagenesis of frequent PPARγ phosphorylation or SUMOylation sites did not prevent its regulation by ZFP407, while mutagenesis of ZFP407 domains potentially necessary for RXR and PPARγ binding abrogated any impact of ZFP407 on PPARγ activity. These data suggest that ZFP407 controls the activity of PPARγ, but does so independently of post-translational modifications, likely by direct binding, establishing ZFP407 as a newly identified PPARγ cofactor. In addition, ZFP407 ChIP-seq analyses identified regions that did not overlap with PPARγ peaks. These non-overlapping peaks were significantly enriched for the transcription factor binding motifs of TBX19, PAX8, HSF4, and ZKSCAN3, which may contribute to the PPARγ-independent functions of ZFP407 in adipocytes and other cell types.

Sulfonylureas exert antidiabetic action on adipocytes by inhibition of PPARγ serine 273 phosphorylation

ObjectiveSulfonylureas (SUs) are still among the mostly prescribed antidiabetic drugs with an established mode of action: release of insulin from pancreatic β-cells. In addition, effects of SUs on adipocytes by activation of the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) have been described, which might explain their insulin-sensitizing potential observed in patients. However, there is a discrepancy between the impact of SUs on antidiabetic action and their rather moderate in vitro effect on PPARγ transcriptional activity. Recent studies have shown that some PPARγ ligands can improve insulin sensitivity by blocking PPARγ Ser-273 phosphorylation without having full agonist activity. It is unknown if SUs elicit their antidiabetic effects on adipocytes by inhibition of PPARγ phosphorylation. Here, we investigated if binding of SUs to PPARγ can interfere with PPARγ Ser-273 phosphorylation and determined their antidiabetic actions in vitro in primary human white adipocytes and in vivo in high-fat diet (HFD) obese mice. MethodsPrimary human white preadipocytes were differentiated in the presence of glibenclamide, glimepiride and PPARγ ligands rosiglitazone and SR1664 to compare PPARγ Ser-273 phosphorylation, glucose uptake and adipokine expression. Transcriptional activity at PPARγ was determined by luciferase assays, quantification of PPARγ Ser-273 phosphorylation was determined by Western blotting and CDK5 kinase assays. In silico modelling was performed to gain insight into the binding characteristics of SUs to PPARγ. HFD mice were administered SUs and rosiglitazone for 6 days. PPARγ Ser-273 phosphorylation in white adipose tissue (WAT), body composition, glucose tolerance, adipocyte morphology and expression levels of genes involved in PPARγ activity in WAT and brown adipose tissue (BAT) were evaluated. ResultsSUs inhibit phosphorylation of PPARγ at Ser-273 in primary human white adipocytes and exhibit a positive antidiabetic expression profile, which is characterized by up regulation of insulin-sensitizing and down regulation of insulin resistance-inducing adipokines. We demonstrate that SUs directly bind to PPARγ by in silico modelling and inhibit phosphorylation in kinase assays to a similar extend as rosiglitazone and SR1664. In HFD mice SUs reduce PPARγ phosphorylation in WAT and have comparable effects on gene expression to rosiglitazone. In BAT SUs increase UCP1 expression and reduce lipid droplets sizes. ConclusionsOur findings indicate that a part of SUs extra-pancreatic effects on adipocytes in vitro and in vivo is probably mediated via their interference with PPARγ phosphorylation rather than via classical agonistic activity at clinical concentrations.

Canagliflozin alleviates pulmonary hypertension by activating PPARγ and inhibiting its S225 phosphorylation

Pulmonary hypertension (PH) is a progressive fatal disease with no cure. Canagliflozin (CANA), a novel medication for diabetes, has been found to have remarkable cardiovascular benefits. However, few studies have addressed the effect and pharmacological mechanism of CANA in the treatment of PH. Therefore, our study aimed to investigate the effect and pharmacological mechanism of CANA in treating PH. First, CANA suppressed increased pulmonary artery pressure, right ventricular hypertrophy, and vascular remodeling in both mouse and rat PH models. Network pharmacology, transcriptomics, and biological results suggested that CANA could ameliorate PH by suppressing excessive oxidative stress and pulmonary artery smooth muscle cell proliferation partially through the activation of PPARγ. Further studies demonstrated that CANA inhibited phosphorylation of PPARγ at Ser225 (a novel serine phosphorylation site in PPARγ), thereby promoting the nuclear translocation of PPARγ and increasing its ability to resist oxidative stress and proliferation. Taken together, our study not only highlighted the potential pharmacological effect of CANA on PH but also revealed that CANA-induced inhibition of PPARγ Ser225 phosphorylation increases its capacity to counteract oxidative stress and inhibits proliferation. These findings may stimulate further research and encourage future clinical trials exploring the therapeutic potential of CANA in PH treatment.

Bifidobacterium bifidum inhibits the TNF-α induced increase in intestinal epithelial tight junction permeability via a TLR-2/TLR-6 and PPAR-γ dependent inhibition of NF-κB p50/p65

Background: Bifidobacterium bifidum (BB) is one of the most widely used probiotic bacterial species and BB has been shown to have a protective effect against intestinal inflammation by an enhancement of the intestinal epithelial tight junction (TJ) barrier. Previous studies from our laboratory showed that a specific BB strain, referred to as BB1, caused a marked enhancement of intestinal epithelial TJ barrier, while strain BB4 had no effect. TNF-α contributes to the development of intestinal inflammation in inflammatory conditions of the gut, in part by increasing intestinal permeability and antigenic penetration. TNF-α induced increase in intestinal epithelial TJ permeability is mediated by an NF-κB p50/p65 activation of myosin light chain kinase (MLCK) gene and MLCK-induced opening of the intestinal TJ barrier. Peroxisome proliferator-activated receptor gamma (PPAR-γ) is an important nuclear receptor known to have anti-inflammatory activity by interfering with NF-κB activation. However, the protective effect of BB1 against TNF-α induced increase in intestinal TJ permeability remain unclear. Aims: The major purpose of this study was to delineate the protective effect of BB1 against the TNF-α induced increase in intestinal TJ permeability and to elucidate the intracellular mechanism involved. Methods: Filter-grown Caco-2 monolayers ( in vitro) and recycling intestinal perfusion of live mice ( in vivo) were the model systems used to assess intestinal TJ permeability. Results: 1) TNF-α caused an increase Caco-2 TJ permeability, as assed by Caco-2 trans-epithelial resistance (TER) and trans-epithelial flux of paracellular marker, FITC-Dextran 10kD. 2) Addition of BB1 (1x108 CFU/ml) to the apical membrane compartment inhibited the TNF-α increase in Caco-2 epithelial TJ permeability, while strain BB4 had no effect. 3) BB1 but not BB4, caused an increase in Caco-2 TLR-2 expression on the apical membrane surface, and siRNA induced knockdown of TLR-2 prevent the BB1 inhibition of TNF-α induced increase in Caco-2 TJ permeability. 4) TNF-α caused a rapid phosphorylation and degradation of Caco-2 IKK-α, as well as and phosphorylation and nuclear translocation of NF-κB p50/p65, indicating NF-κB activation. BB1, but not BB4, inhibited the NF-κB activation. 5) TNF-α activation of NF-κB was associated with an increase in MLCK gene (increase in MLCK promoter activity and mRNA transcription) and kinase activity. BB1, but not BB4, inhibited the TNF-α induced increase in MLCK gene and kinase activity. 6) BB1 but not BB4, caused a rapid PPAR-γ activation (PPAR-γ phosphorylation and nuclear translocation), and siRNA induced knockdown of PPAR-γ inhibited the TNF-α induced activation of NF-κB and increase in Caco-2 TJ permeability. 7) Intraperitoneal injection of TNF-α (5 mg) also caused time-dependent increase in mouse small intestinal permeability (IP) to FITC-Dextran 10kD. TNF-α caused a degradation of IkB-α in intestinal tissue and nuclear translocation of NF-kB in mouse enterocyte. 8) BB1, but not BB4, selectively activated PPAR-γ in mouse enterocytes and inhibited the TNF-α-induced activation of NF-κB, resulting in a decrease in mouse IP. 9) BB1 inhibition of TNF-α activation of mouse enterocyte NF-κB and increase in IP was prevented in mice with intestinal epithelial cell-specific knockout of PPAR-γ Villin-cre and TLR-2 Villin-cre mice. Conclusion: BB1 protects against the TNF-α induced increase in intestinal TJ permeability in a strain-specific manner. BB1 protects against the TNF-α induced increase in intestinal TJ permeability via a novel intracellular mechanism involving BB1/TLR-2 pathway activation of PPAR-γ, and PPAR-γ mediated inhibition of NF-κB p50/p65 and MLCK gene in enterocyte. This research project was supported by the National Institute of Diabetes and Digestive and Kidney Diseases R01-DK-121073-01. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

TAT38 and TAT38 mimics potently inhibit adipogenesis by repressing C/EBPα, PPARγ, Pi-PPARγ, and SREBP1 expression

Antiretroviral therapy-naive people living with HIV possess less fat than people without HIV. Previously, we found that HIV-1 transactivator of transcription (TAT) decreases fat in ob/ob mice. The TAT38 (a.a. 20–57) is important in the inhibition of adipogenesis and contains three functional domains: Cys-ZF domain (a.a. 20–35 TACTNCYCAKCCFQVC), core-domain (a.a. 36–46, FITKALGISYG), and protein transduction domain (PTD)(a.a. 47–57, RAKRRQRRR). Interestingly, the TAT38 region interacts with the Cyclin T1 of the P-TEFb complex, of which expression increases during adipogenesis. The X-ray crystallographic structure of the complex showed that the Cys-ZF and the core domain bind to the Cyclin T1 via hydrophobic interactions. To prepare TAT38 mimics with structural and functional similarities to TAT38, we replaced the core domain with a hydrophobic aliphatic amino acid (from carbon numbers 5 to 8). The TAT38 mimics with 6-hexanoic amino acid (TAT38 Ahx (C6)) and 7-heptanoic amino acid (TAT38 Ahp (C7)) inhibited adipogenesis of 3T3-L1 potently, reduced cellular triglyceride content, and decreased body weight of diet-induced obese (DIO) mice by 10.4–11 % in two weeks. The TAT38 and the TAT38 mimics potently repressed the adipogenic transcription factors genes, C/EBPα, PPARγ, and SREBP1. Also, they inhibit the phosphorylation of PPARγ. The TAT peptides may be promising candidates for development into a drug against obesity or diabetes.

Ginsenoside Rg1 promotes β‑amyloid peptide degradation through inhibition of the ERK/PPARγ phosphorylation pathway in an Alzheimer's disease neuronal model.

β-Amyloid peptide (Aβ) deposition in the brain is an important pathological change in Alzheimer's disease (AD). Insulin-degrading enzyme (IDE), which is regulated transcriptionally by peroxisome proliferator-activated receptor γ (PPARγ), is able to proteolyze Aβ. One of the members of the MAPK family, ERK, is able to mediate the phosphorylation of PPARγ at Ser112, thereby inhibiting its transcriptional activity. Ginsenoside Rg1 is one of the active ingredients in the natural medicine ginseng and has inhibitory effects on Aβ production. The present study was designed to investigate whether ginsenoside Rg1 is able to affect the regulation of PPARγ based on the expression of its target gene, IDE, and whether it is able to promote Aβ degradation via inhibition of the ERK/PPARγ phosphorylation pathway. In the present study, primary cultured rat hippocampal neurons were treated with Aβ1-42, ginsenoside Rg1 and the ERK inhibitor PD98059, and subsequently TUNEL staining was used to detect the level of neuronal apoptosis. ELISA was subsequently employed to detect the intra- and extracellular Aβ1-42 levels, immunofluorescence staining and western blotting were used to detect the translocation of ERK from the cytoplasm to the nucleus, immunofluorescence double staining was used to detect the co-expression of ERK and PPARγ, and finally, western blotting was used to detect the phosphorylation of PPARγ at Ser112 and IDE expression. The results demonstrated that ginsenoside Rg1 or PD98059 were able to inhibit primary cultured hippocampal neuron apoptosis induced by Aβ1-42 treatment, reduce the levels of intra- and extraneuronal Aβ1-42 and inhibit the translocation of ERK from the cytoplasm to the nucleus. Furthermore, administration of ginsenoside Rg1 or PD98059 resulted in attenuated co-expression of ERK and PPARγ, inhibition of phosphorylation of PPARγ at Ser112 mediated by ERK and an increase in IDE expression. In addition, the effects when PD98059 to inhibit ERK followed by treatment with ginsenoside Rg1 were found to be more pronounced than those when using PD98059 alone. In conclusion, ginsenoside Rg1 was demonstrated to exert neuroprotective effects on AD via inhibition of the ERK/PPARγ phosphorylation pathway, which led to an increase in IDE expression, the promotion of Aβ degradation and the decrease of neuronal apoptosis. These results could provide a theoretical basis for the clinical application of ginsenoside Rg1 in AD.

Inhibitory effect and mechanism of action of blue light irradiation on adipogenesis

Blue light can reach the epidermis, dermis, and hypodermis of the skin and affect the physiology of each cell type. However, thus far, most studies have focused on the effects of blue light on the epidermis and dermis; no studies have been conducted on the effects of blue light on the hypodermis. In this study, we attempted to elucidate the effects of blue light on the hypodermis. Specifically, we investigated the effects of blue light on 3T3-L1 adipogenesis and its mechanism of action. In this study, we found that blue light reduced lipid accumulation and GPDH activity, indicating its antiadipogenic properties. Furthermore, blue light inhibited the mRNA and protein levels of peroxisome proliferator-activated receptor γ (PPARγ) and its target genes, such as Fasn and FABP4, and the luciferase activity of the PPRE promoter, suggesting that blue light downregulates the expression of adipogenic genes, leading to suppression of 3T3-L1 adipogenesis. Furthermore, blue light increased the phosphorylation of PPARγ, indicating its anti-adipogenic effect through PPARγ degradation. Blue light upregulates the expression and phosphorylation of transient receptor potential vanilloid 1 (TRPV1). Furthermore, blue light restored calcium influx, which was attenuated by adipogenesis. These data suggest that TRPV1 is involved in the anti-adipogenic effects of blue light. Blue light induced the phosphorylation of AMPK, ACC, and MAPKs, whereas capsazepine, an antagonist of TRPV1, attenuated the effects of blue light. Capsazepine treatment also reduced the blue light-induced anti-adipogenic effects. These results indicate that TRPV1 operates upstream of AMPK or MAPKs in the blue light-induced anti-adipogenesis process, which is mediated by activating the TRPV1-AMPK/MAPK signaling pathways. Meanwhile, inhibition of AMPK and MAPKs reduced the phosphorylation level of PPARγ, indicating that AMPK/MAPK signaling contributes to the phosphorylation of PPARγ. These results suggest the possibility of using blue light as an antiadipogenic agent.

Krüppel-like factor 15 deficiency exacerbates osteoarthritis through reduced expression of peroxisome proliferator-activated receptor gamma signaling in mice

Krüppel-like zinc finger transcription factors (KLFs) play diverse roles in mammalian cell differentiation and development. In this study, we investigated the function of KLF15 in the progression of osteoarthritis (OA). 0Destabilization of the medial meniscus (DMM) surgery was performed in 10-week-old male wild-type control (WT) mice and cartilage-specific KLF15 knockout (KO) mice. Histological analysis, immunohistochemistry, and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling staining were performed. Morphological changes were measured using microcomputed tomography. Six mice from each group were analyzed (total number of mice analyzed: 60). In vitro, immunofluorescence, quantitative reverse transcription-polymerase chain reaction, and western blot analyses were performed. KLF15 KO DMM mice exhibited significant cartilage degradation compared to WT mice. According to the Osteoarthritis Research Society International cartilage OA-histopathology scoring system, the mean sum score in KLF15 KO mice was significantly higher than that in WT mice at 8 weeks after surgery. Immunohistochemistry results revealed KLF15 KO mice exhibited reducedperoxisome proliferator-activated receptor gamma (PPARγ) expression, increased pIKKα/β, a disintegrin-like and metalloproteinase with thrombospondin motifs (ADAMTS) 5, and Matrix metalloproteinases (MMP13) expression, and reduced Forkhead box O (FOXO1) and Light chain 3B (LC3B) expression. Inhibition of PPARγ phosphorylation accelerated the effects of interleukin (IL) 1β-treatment in both KLF15 KO and WT chondrocytes, and activation of PPARγ expression canceled the IL1β-induced catabolic effects. Our results indicated that the OA phenotype of KLF15 KO DMM mice was influenced by reduced PPARγ expression, including enhanced pIKKα/β, ADAMTS5, and MMP13 expression, reduced autophagy, and increased apoptosis. KLF15 regulation may constitute a possible therapeutic strategy for the treating OA.

Exploring antidiabetic potential of a polyherbal formulation Madhurakshak Activ: An in vitro and in silico study

Madhurakshak Activ (MA), a commercial polyherbal antidiabetic preparation is known to manage diabetes mellitus (DM) by reducing blood glucose levels. However, lacks systematic mechanistic evaluation for their molecular and cellular mode of actions. In the present study, hydro-alcoholic and aqueous extract of MA were evaluated for their effects on glucose adsorption, diffusion, amylolysis kinetics and transport across the yeast cells using in vitro techniques. Bioactive compounds identified from MA by LC-MS/MS were assessed for their binding potential against DPP-IV and PPARγ via an in silico approach. Our results revealed that the adsorption of glucose increased dose dependently (5 mM -100 mM). Both extracts exhibited linear glucose uptake into the yeast cells (5 mM - 25 mM), whereas glucose diffusion was directly proportional to time (30–180 min). Pharmacokinetic analysis revealed drug-like properties and low toxicity levels for all the selected compounds. Among the tested compounds, 6-hydroxyluteolin (−8.9 against DPP-IV and PPARγ) and glycyrrhetaldehyde (DPP-IV -9.7 and PPARγ −8.5) have exhibited higher binding affinity compared to the positive control. Therefore, the above compounds were further considered for molecular dynamics simulation which showed stability of the docked complexes. Hence, studied mode of actions might produce a concerted role of MA in increasing the rate of glucose absorption and uptake followed by the in silico studies which suggest that the compounds identified from MA may inhibit DPP-IV and PPARγ phosphorylation.

Biological Screening and Crystallographic Studies of Hydroxy γ-Lactone Derivatives to Investigate PPARγ Phosphorylation Inhibition.

PPARγ represents a key target for the treatment of type 2 diabetes and metabolic syndrome. To avoid serious adverse effects related to the PPARγ agonism profile of traditional antidiabetic drugs, a new opportunity is represented by the development of molecules acting as inhibitors of PPARγ phosphorylation by the cyclin-dependent kinase 5 (CDK5). Their mechanism of action is mediated by the stabilization of the PPARγ β-sheet containing Ser273 (Ser245 in PPARγ isoform 1 nomenclature). In this paper, we report the identification of new γ-hydroxy-lactone-based PPARγ binders from the screening of an in-house library. These compounds exhibit a non-agonist profile towards PPARγ, and one of them prevents Ser245 PPARγ phosphorylation by acting mainly on PPARγ stabilization and exerting a weak CDK5 inhibitory effect.

Phloretin and phlorizin mitigates inflammatory stress and alleviate adipose and hepatic insulin resistance by abrogating PPARγ S273-Cdk5 interaction in type 2 diabetic mice

AimsThe rising prevalence of type 2 diabetes mellitus (T2DM) and accompanying insulin resistance is alarming globally. Natural and synthetic agonists of PPARγ are potentially attractive candidates for diabetics and are known to efficiently reverse adipose and hepatic insulin resistance, but related side effects and escalating costs are the causes of concern. Therefore, targeting PPARγ with natural ligands is advantageous and promising approach for the better management of T2DM. The present research aimed to assess the antidiabetic potential of phenolics Phloretin (PTN) and Phlorizin (PZN) in type 2 diabetic mice. Main methodsIn silico docking was performed to check the effect of PTN and PZN on PPARγ S273-Cdk5 interactions. The docking results were further validated in preclinical settings by utilizing a mice model of high fat diet-induced T2DM. Key findingsComputational docking and further MD-simulation data revealed that PTN and PZN inhibited the activation of Cdk5, thereby blocking the phosphorylation of PPARγ. Our in vivo results further demonstrated that PTN and PZN administration significantly improved the secretory functions of adipocytes by increasing adiponectin and reducing inflammatory cytokine levels, which ultimately reduced the hyperglycaemic index. Additionally, combined treatment of PTN and PZN decreased in vivo adipocyte expansion and increased Glut4 expression in adipose tissues. Furthermore, PTN and PZN treatment reduced hepatic insulin resistance by modulating lipid metabolism and inflammatory markers. SignificanceIn summary, our findings strongly imply that PTN and PZN are candidates as nutraceuticals in the management of comorbidities related to diabetes and its complications.

Novel Peptides from Sea Cucumber Intestinal Enzyme Hydrolysates Promote Osteogenic Differentiation of Bone Mesenchymal Stem Cells via Phosphorylation of PPARγ at Serine 112.

Osteo-adipogenic differentiation imbalance of bone mesenchymal stem cells (BMSCs) has been linked to a variety of pathophysiological processes such as obesity and osteoporosis. Recent studies report that the phosphorylation of peroxisome proliferator-activated receptor gamma (PPARγ) Ser112 affects the fate decision of BMSCs. Novel peptides from the sea cucumber intestinal peptide (SCIP) have been proved to promote the growth of longitudinal bone. However, it is unclear the effect of SCIP on BMSCs differentiation fate. BMSCs in vitro and glucocorticoid induced mice are employed to investigate the effects of SCIP on osteo-adipogenic differentiation of BMSCs. In vitro results show that SCIP supplement significantly promotes the proliferation and osteogenic differentiation of BMSCs, upregulates the expression of osteogenic marker. In vivo results show that SCIP supplement ameliorates the osteo-adipogenic differentiation imbalance in glucocorticoid-treated mice, decreases bone marrow fat, and elevates bone mineral density. Mechanistically, SCIP supplement promotes and maintains the phosphorylation of PPARγ Ser112 through AMPK/ERK and TAZ signals, thereby inducing the osteogenic differentiation of BMSCs. Supplement with SCIP promotes BMSCs to differentiate into osteoblasts. These results suggest that SCIP has potential as a functional food to improve obesity and osteoporosis.

Long-term exposure to low-dose Di(2-ethylhexyl) phthalate aggravated high fat diet-induced obesity in female mice

The potential obesogenic roles of di(2-ethylhexyl) phthalate (DEHP) have attracted great attention. The current study aimed to evaluate the combined effects of chronic low-dose DEHP (0.05 mg/kg BW) and a high-fat diet (HFD) on obesity in female mice and explore the underlying mechanisms. We found that low-dose DEHP challenge for 29 weeks increased fat accumulation both in CD- and HFD-fed mice and significantly accelerated the weight gain without affecting food intake in HFD-fed mice. DEHP exposure reduced the energy metabolism, down-regulated the uncoupling protein 1 (UCP1) and total oxidative phosphorylation (OXPHOS) proteins expression in the brown adipose tissue, and up-regulated the PPARγ expression and its phosphorylation at Ser273 in white adipose tissue (WAT). Besides, the combination of DEHP and HFD drove the remodeling of gut microbiota of mice, characterized by the reduced richness and diversity and the elevated Firmicutes to Bacteroidetes (F/B) ratio. Short-chain fatty acids (SCFAs) analysis revealed that DEHP and HFD cotreatment led to a decrease in levels of acetic acid, butyric acid, and pentanoic acid. Interestingly, sodium butyrate (NaB) significantly inhibited the adipogenesis and lipid accumulation of NIH/3T3 mouse embryonic fibroblasts (PPARγ2 overexpression) and the PPARγ phosphorylation at Ser273 induced by DEHP or MEHP. These findings demonstrate that chronic low-dose DEHP challenge could prompt fat accumulation by increasing PPARγ phosphorylation at Ser273 and decreasing thermogenesis in BAT, which might be associated with the SCFAs reduction.

Selective PPARγ modulator diosmin improves insulin sensitivity and promotes browning of white fat

Peroxisome proliferator-activated receptor γ (PPARγ) is a master regulator of adipocyte differentiation, glucolipid metabolism, and inflammation. Thiazolidinediones are PPARγ full agonists with potent insulin-sensitizing effects, whereas their oral usage is restricted because of unwanted side effects, including obesity and cardiovascular risks. Here, via virtual screening, microscale thermophoresis analysis, and molecular confirmation, we demonstrate that diosmin, a natural compound of wide and long-term clinical use, is a selective PPARγ modulator that binds to PPARγ and blocks PPARγ phosphorylation with weak transcriptional activity. Local diosmin administration in subcutaneous fat (inguinal white adipose tissue [iWAT]) improved insulin sensitivity and attenuated obesity via enhancing browning of white fat and energy expenditure. Besides, diosmin ameliorated inflammation in WAT and liver and reduced hepatic steatosis. Of note, we determined that iWAT local administration of diosmin did not exhibit obvious side effects. Taken together, the present study demonstrated that iWAT local delivery of diosmin protected mice from diet-induced insulin resistance, obesity, and fatty liver by blocking PPARγ phosphorylation, without apparent side effects, making it a potential therapeutic agent for the treatment of metabolic diseases.

Suppression of Lipid Accumulation in the Differentiation of 3T3-L1 Preadipocytes and Human Adipose Stem Cells into Adipocytes by TAK-715, a Specific Inhibitor of p38 MAPK

Excessive preadipocyte differentiation is linked with obesity. Although previous studies have shown that p38 MAPK is associated with adipogenesis, the regulation of preadipocyte differentiation by TAK-715, an inhibitor of p38 mitogen-activated protein kinase (MAPK), remains unclear. Interestingly, TAK-715 at 10 μM vastly suppressed the accumulation of lipid and intracellular triglyceride (TG) content with no cytotoxicity during 3T3-L1 preadipocyte differentiation. On mechanistic levels, TAK-715 significantly decreased the expressions of the CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor gamma (PPAR-γ), fatty acid synthase (FAS), and perilipin A. Similarly, the phosphorylation of the signal transducer and activator of transcription-3 (STAT-3) in differentiating 3T3-L1 cells was also reduced with TAK-715 treatment. Moreover, TAK-715 significantly blocked the phosphorylation of activating transcription factor-2 (ATF-2), a p38 MAPK downstream molecule, during 3T3-L1 preadipocyte differentiation. Of importance, TAK-715 also markedly impeded the phosphorylation of p38 MAPK and suppressed lipid accumulation during the adipocyte differentiation of human adipose stem cells (hASCs). Concisely, this is the first report that TAK-715 (10 μM) has potent anti-adipogenic effects on the adipogenesis process of 3T3-L1 cells and hASCs through the regulation of the expression and phosphorylation of p38 MAPK, C/EBP-α, PPAR-γ, STAT-3, FAS, and perilipin A.

Protein Profiling of Breast Carcinomas Reveals Expression of Immune-Suppressive Factors and Signatures Relevant for Patient Outcome.

Simple SummaryBreast cancer treatment has improved substantially over the last decade. Still, the failure of treatment and therapy resistance are urgent problems. Here, we assessed cellular signaling within primary cancer tissue to evaluate the possibility of developing strategies that lead to better patient stratification and the development of personalized treatment options. By employing DigiWest technology, the expression and activation of the regulators of key signaling pathways in breast cancer tissue were monitored. A positive correlation between immune cell infiltration and event-free survival was detected. PPARγ activation showed a negative correlation with immune cell infiltration, suggesting a novel immune evasion mechanism.In cancer, the complex interplay between tumor cells and the tumor microenvironment results in the modulation of signaling processes. By assessing the expression of a multitude of proteins and protein variants in cancer tissue, wide-ranging information on signaling pathway activation and the status of the immunological landscape is obtainable and may provide viable information on the treatment response. Archived breast cancer tissues from a cohort of 84 patients (no adjuvant therapy) were analyzed by high-throughput Western blotting, and the expression of 150 proteins covering central cancer pathways and immune cell markers was examined. By assessing CD8α, CD11c, CD16 and CD68 expression, immune cell infiltration was determined and revealed a strong correlation between event-free patient survival and the infiltration of immune cells. The presence of tumor-infiltrating lymphocytes was linked to the pronounced activation of the Jak/Stat signaling pathway and apoptotic processes. The elevated phosphorylation of PPARγ (pS112) in non-immune-infiltrated tumors suggests a novel immune evasion mechanism in breast cancer characterized by increased PPARγ phosphorylation. Multiplexed immune cell marker assessment and the protein profiling of tumor tissue provide functional signaling data facilitating breast cancer patient stratification.

An integrated study of Shenling Baizhu San against hyperuricemia: Efficacy evaluation, core target identification and active component discovery

Ethnopharmacological relevanceShenling Baizhu San (SLBZ) is a famous Traditional Chinese Medicine (TCM) formula that strengthens the spleen for replenishing qi, removing dampness, and inducing diuresis to relieve diarrhea. Combining the TCM interpretation that dampness is a vital pathogenesis factor in hyperuricemia occurrence and development, SLBZ has excellent potential against hyperuricemia from the perspective of TCM theories. Aim of the studyThis study aimed to investigate the efficacy of SLBZ against hyperuricemia and its possible mechanism with emphasis on the active components and the core targets. Materials and methodsIn the present study, we employed meta-analysis and a hyperuricemia quail model to evaluate the uric acid-lowering effect of SLBZ. Bodyweight, serum uric acid, and excreta uric acid levels in quails were assessed. Subsequently, we analyzed the potential active components and core targets of SLBZ against hyperuricemia by network pharmacology and calculated their interaction using molecular docking. Furthermore, the hyperuricemia rats treated with interfering agents of core targets were established to determine the central role of selected targets in hyperuricemia progression. Besides, we isolated and characterized the primary renal tubular epithelial cells of quails to verify the active components and core targets of SLBZ against hyperuricemia. Western blotting was used to observe the expression of core targets treated with active components under the stimulation of interfering agents. ResultsData from meta-analysis and animal experiments showed that SLBZ could work effectively against hyperuricemia. Hyperuricemia quails treated with SLBZ displayed significantly reduced serum uric acid levels accompanied by increased excretion of uric acid. According to network pharmacology and molecular docking results, 34 potential active components and the core target peroxisome proliferator-activated receptor gamma (PPARγ) for SLBZ against hyperuricemia were identified. The decreased serum uric acid levels in hyperuricemia rats treated with rosiglitazone, an agonist of PPARγ, confirms the essential role of PPARγ in the pathological process of hyperuricemia. Moreover, we first successfully isolated and characterized the primary renal tubular epithelial cells of quails and observed enhanced phosphorylation of PPARγ at Ser273 in cells handled with high-level uric acid. Whereas, the enhanced expression of p-PPARγ Ser273 could be down-regulated by luteolin and naringenin, two active components of SLBZ against hyperuricemia. ConclusionIn summary, SLBZ is a promising anti-hyperuricemia agent, and luteolin and naringenin are the active components for SLBZ against hyperuricemia by down-regulating phosphorylation of PPARγ at Ser273.

Sorghum (<i>Sorghum bicolor</i>) Extract-Induced Adipogenesis Is Independent of PPARγ Ser273 Phosphorylation in 3T3-L1 Adipocytes

Previously we showed that the water-soluble fraction of sorghum extract (SE) improves adipogenesis in 3-isobutyl-1-methylxanthine (IBMX)/dexamethasone/insulin (MDI)/thiazolidinedione (TZD)-induced 3T3-L1 preadipocytes but downregulates genes related to peroxisome proliferator-activated receptor γ (PPARγ) and adipogenesis in both MDI- and MDI/TZD-induced 3T3-L1 adipocytes. In this study, we showed that SE treatment altered the accumulation of stained lipids in 3T3-L1 adipocytes induced by MDI/troglitazone (Tro). Immunoblot analyses indicated that SE treatment reduced adipocyte protein 2 (aP2) expression and induced peroxisome proliferator-activated receptor α (PPARα) protein expression in the presence of Tro in 3T3-L1 adipocytes. MDI/Tro treatment, but not MDI treatment, of 3T3-L1 cells induced PPARγ phosphorylation at Ser273. SE downregulated PPARγ expression in MDI-induced 3T3-L1 adipocytes and did not affect its phosphorylation at Ser273 in MDI- and MDI/Tro-induced 3T3-L1 adipocytes. Therefore, SE likely promotes adipogenesis and lipid metabolism in 3T3-L1 preadipocytes by cooperating with Tro independent of PPARγ Ser273 phosphorylation.

Discovery by Virtual Screening of an Inhibitor of CDK5-Mediated PPARγ Phosphorylation.

Thiazolidinedione PPARγ agonists such as rosiglitazone and pioglitazone are effective antidiabetic drugs, but side effects have limited their use. It has been posited that their positive antidiabetic effects are mainly mediated by the inhibition of the CDK5-mediated Ser273 phosphorylation of PPARγ, whereas the side effects are linked to classical PPARγ agonism. Thus compounds that inhibit PPARγ Ser273 phosphorylation but lack classical PPARγ agonism have been sought as safer antidiabetic therapies. Herein we report the discovery by virtual screening of 10, which is a potent PPARγ binder and in vitro inhibitor of the CDK5-mediated phosphorylation of PPARγ Ser273 and displays negligible PPARγ agonism in a reporter gene assay. The pharmacokinetic properties of 10 are compatible with oral dosing, enabling preclinical in vivo testing, and a 7 day treatment demonstrated an improvement in insulin sensitivity in the ob/ob diabetic mouse model.