PPARγ mRNA Expression Research Articles

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.

Synthesis, in vitro, in silico and in vivo hypoglycemic and lipid-lowering effects of 4-benzyloxy-5-benzylidene-1,3-thiazolidine-2,4-diones mediated by dual PPAR α/γ modulation

In current work, we prepared a series of nine 4-benzyloxy-5-benzylidene-1,3-thiazolidine-2,4-diones using a two-step pathway. Compounds 1–9 were tested in vitro using a set of three proteins recognized as important targets in diabetes and related diseases: PPARα, PPARγ, and GLUT-4. Compounds 1–3, 5, and 7 showed significant increases in the mRNA expression of PPARγ and GLUT-4, whereas compounds 1–3 did it over PPARα. Compounds 1–3 were identified as a dual PPAR α/γ modulators and were selected for evaluating the in vivo antidiabetic action at 100 mg/kg dose, being orally actives and decreasing blood glucose concentration in a hyperglycemic mice model, as well as reducing the triacylglycerides levels in normolipidemic rats. Docking and molecular dynamics studies were conducted to clarify the dual effect and binding mode of compounds 1–3 on both PPARs. Compounds 2 and 3 exhibited robust in vitro and in vivo efficacy and could be considered dual PPAR modulators with antidiabetic and antidyslipidemic effects.

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.

Effects of Nigella sativa supplementation on blood concentration and mRNA expression of TNF-α, PPAR-γ and adiponectin, as major adipogenesis-related markers, in obese and overweight women: a crossover, randomised-controlled trial.

Adipocyte expansion through adipogenesis can offset the adverse metabolic effects of obesity. Nigella sativa (NS) (black seed) oil is shown to have therapeutic features in the management of obesity. NS oil might have beneficial changes in obese populations through mediating serum levels of adipogenesis-related parameters and relative transcriptional gene-diet interactions (nutrigenomics), though no previous studies assessed this mechanism in overweight/obese participants. This study assessed the effects of NS oil supplements on blood concentration and mRNA expression levels of TNF-α, PPAR-γ and serum adiponectin and expression of AdipoR1, as major adipogenesis and obesity-related parameters, in overweight/obese women using a cross-over design. Eligible women were randomised to receive either NS oil supplements (2000 mg/d) or placebo. Two periods of interventions (8 weeks in each) were cross-changed by a 4-week washout period. An individualised diet plan without calorie deficits was given to participants to match their energy/macronutrient intakes. The Pkcross procedure and intention-to-treat analysis were performed using Stata. Cohen's d(d) was estimated to measure the magnitude of the effects. Forty-six participants were included. NS oil capsules reduced transcription levels ((d = -2·31), P < 0·001) and blood concentrations of TNF-α ((d = -0·29), P < 0·001). AdipoR1 expression (d = 2·24, P < 0·001) and serum adiponectin (d = 0·88, P < 0·001) showed a significant augmentation with a medium-high effect size, as did gene expression (d = 0·69, P < 0·001) and serum levels of PPAR-γ (d = 0·97, P < 0·001). There was a moderate but significant decrease in body weight (d = 0·6, P < 0·001). The present beneficial findings would provide strong information for future nutrigenomics/clinical trial studies assessing the role of NS in the management of obesity and other comorbidities.

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.

Inhibition of palmitic acid induced adipogenesis by natural polyphenols in 3T3-L1 adipocytes.

Dietary free fatty acids induce preadipocyte differentiation in the presence of a hormonal cocktail in 3T3-L1 adipocytes. Plant polyphenols are curb adipocyte differentiation and protect from metabolic stress. In the present study, we examined the effects of the saturated fatty acid, palmitic acid (PA) in presence of flavonoids, chrysin (CR) and hesperidin (HD) and phenolic acid, syringic acid (SYA) and sinapic acid (SIA). Adipocytes were incubated for 10 d with 100μmol of PA along with 10-100µmol CR/HD and 100-1000µmol SYA/SIA. PA induced clonal expansion of preadipocytes, differentiation and oxidative stress in 3T3-L1 cells following 10 d of differentiation. Adipocytes treated with PA exhibited an increase of 300% in clonal population, 110% lipid and 172% reactive oxygen species accumulation. But treatment with CR, HD, SYA and SIA in the presence of PA concentration-dependent effect was observed. Concentrations of CR/HD and SYA/SIA inhibited PA-induced mRNA expression of PPARγ, C/EBPα, SREBP-1c, FAS and NOX4. Moreover, CR, HD, SYA and SIA did not exhibit toxicity in Drosophila DNA. In summary, these results suggest that dietary fatty acids act directly on adipocytes and addition of CR, HD, SYA and SIA resulted in reduction of PA-induced negative effects on 3T3-L1 adipocytes. HIGHLIGHTS: • Palmitic acid, the common dietary free fatty acid, is known to induce adipogenesis in 3T3-L1 adipocytes. • Treatment of differentiating adipocytes with flavonoids and phenolic acids reduced palmitic acid-induced clonal expansion of preadipocytes. • Phytocompounds reduced lipid accumulation and triglyceride production as well as ROS accumulation. • Thus, the phytocompounds showed effective anti-adipogenic activity even in palmitic acid challenged environment in adipocytes.

Ameliorative Effect of Ocimum forskolei Benth on Diabetic, Apoptotic, and Adipogenic Biomarkers of Diabetic Rats and 3T3-L1 Fibroblasts Assisted by In Silico Approach.

Diabetes mellitus (DM) is a complicated condition that is accompanied by a plethora of metabolic symptoms, including disturbed serum glucose and lipid profiles. Several herbs are reputed as traditional medicine to improve DM. The current study was designed to explore the chemical composition and possible ameliorative effects of Ocimum forskolei on blood glucose and lipid profile in high-fat diet/streptozotocin-induced diabetic rats and in 3T3-L1 cell lines as a first report of its bioactivity. Histopathological study of pancreatic and adipose tissues was performed in control and treatment groups, along with quantification of glucose and lipid profiles and the assessment of NF-κB, cleaved caspase-3, BAX, and BCL2 markers in rat pancreatic tissue. Glucose uptake, adipogenic markers, DGAT1, CEBP/α, and PPARγ levels were evaluated in the 3T3-L1 cell line. Hesperidin was isolated from total methanol extract (TME). TME and hesperidin significantly controlled the glucose and lipid profile in DM rats. Glibenclamide was used as a positive control. Histopathological assessment showed that TME and hesperidin averted necrosis and infiltration in pancreatic tissues, and led to a substantial improvement in the cellular structure of adipose tissue. TME and hesperidin distinctly diminished the mRNA and protein expression of NF-κB, cleaved caspase-3, and BAX, and increased BCL2 expression (reflecting its protective and antiapoptotic actions). Interestingly, TME and hesperidin reduced glucose uptake and oxidative lipid accumulation in the 3T3-L1 cell line. TME and hesperidin reduced DGAT1, CEBP/α, and PPARγ mRNA and protein expression in 3T3-L1 cells. Moreover, docking studies supported the results via deep interaction of hesperidin with the tested biomarkers. Taken together, the current study demonstrates Ocimum forskolei and hesperidin as possible candidates for treating diabetes mellitus.

Peroxisome proliferator-activated receptor expression in the canine endometrium with cystic endometrial hyperplasia-pyometra complex.

The most common uterine diseases in bitches occurring during diestrus are cystic endometrial hyperplasia (CEH) and pyometra. These diseases can coexist as CEH-pyometra complex (CEH-P). Their pathogenesis has not been fully explained. Peroxisome proliferator-activated receptors (PPARs) are important factors regulating mammalian reproductive function and inflammatory processes. Although there is a lack of data concerning the expression of PPARs in the canine endometrium during CEH and CEH-P, we hypothesized that they might be involved in the development of pathological disorders of the canine endometrium. Therefore, the current study was conducted to evaluate and compare PPARα, PPARδ and PPARγ mRNA expression using quantitative real-time PCR (qPCR) and their immunolocalization using immunohistochemistry (IHC) staining in the endometrium of clinically healthy bitches (control group; n=8) and those with CEH (n=8) or CEH-P (n=8). For quantification, the arithmetic means of all intensities of immunostaining from the cells were measured with the optical density. PPARα, PPARδ and PPARγ were detected in the luminal epithelium, glandular epithelium and stromal cells. The mRNA transcription of PPARα was higher in the CEH group than in the control group (p<.05). Additionally, the mRNA expression and immunostaining intensities of PPARδ and PPARγ in the endometrium in the CEH-P group were downregulated relative to those in the control group (p<.05). Moreover, the serum progesterone concentration measured by direct radioimmunoassay was decreased in the CEH-P group compared to the control group (p<.001) and CEH group (p<.05). The obtained results indicate that PPARs are present in the canine endometrium and that their mRNA profile and intensity levels change under pathological conditions such as CEH and CEH-P. This finding may suggest a correlation between changes in the PPAR expression profile and hormonal disturbances, as well as the potential involvement of PPARs in signal transduction during inflammatory processes occurring in the endometrium during CEH-P. These results pave the way to further research into the role of PPARs in the pathogenesis of CEH and CEH-P in female dogs.

Efficacy of aqueous extract of flower of Edgeworthia gardneri (Wall.) Meisn on glucose and lipid metabolism in KK/Upj-Ay/J mice.

To observe the effects of the flower of Edgeworthia gardneri (Wall.) Meisn (EWM) on glucose and lipid metabolism in KK/upj-Ay/J (KKAy) mice and investigate the possible mechanism of EWM in the liver of KKAy mice by transcriptome analysis. Forty KKAy mice were fed a high-sugar and high-fat diet for 3 weeks to establish the animal model of metabolic syndrome. After 5 weeks of continuous administration of EWM, serum high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides (TG), total cholesterol (TC), and free fatty acids (FFA) were detected by radioimmunoassay. Serum fasting insulin (Fins) and adiponectin levels were measured by enzyme-linked immunosorbent assay. Liver tissue fixed with paraformaldehyde was stained with hematoxylin-eosin and oil red O. Transcriptome analysis was used to evaluate the liver tissue. The expressions of lipoprotein lipase (LPL), peroxisome proliferator-activated receptor-γ (PPARγ), adenosine 5'-monophosphate-activated protein kinase (AMPK), sterol regulatory element binding protein-1c (SREBP-1c), and fatty acid synthase (Fas) mRNA and protein in liver tissue were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. EWM slightly reduced FBG and Fins in KKAy mice. Furthermore, EWM was able to downregulate serum LDL, TG, TC, and FFA and upregulate the expression of serum HDL and adiponectin. Transcriptome analysis revealed the following differential pathways: the peroxisome proliferator-activated receptor (PPAR) signaling pathway and the AMPK signaling pathway. RT-PCR and western blot analysis detected the associated genes and proteins. In addition, EWM was able to upregulate the expression of AMPK and downregulate the expression of PPARγ, SREBP1c, and Fas mRNA and protein and upregulate the expression of LPL mRNA. EWM can alleviate lipid metabolism disorders and to some extent improve glucose metabolism disorders in KKAy mice. These effects may be related to regulating PPARγ/LPL and activating the AMPK/SREBP1c/Fas pathway.

Co-Administration of Vitamin E and Atorvastatin Improves Insulin Sensitivity and Peroxisome Proliferator-Activated Receptor-γ Expression in Type 2 Diabetic Patients: A Randomized Double-Blind Clinical Trial.

Background: Negative effects of statins on glucose metabolism have been reported. The present study aimed to investigate the effects of co-administration of vitamin E and atorvastatin on glycemic control in hyperlipidemic patients with type 2 diabetes mellitus (T2DM).Methods: A randomized double-blind clinical trial was conducted at Vali-e-Asr Teaching Hospital (Zanjan, Iran) from July 2017 to March 2018. A total of 30 T2DM female patients were allocated to two groups, namely atorvastatin with placebo (n=15) and atorvastatin with vitamin E (n=15). The patients received daily 20 mg atorvastatin and 400 IU vitamin E or placebo for 12 weeks. Anthropometric and biochemical measures were recorded pre- and post-intervention. Peroxisome proliferator-activated receptor-γ (PPAR-γ) expression was measured in peripheral blood mononuclear cells (PBMCs). Independent sample t test and paired t test were used to analyze between- and within-group variables, respectively. The analysis of covariance (ANCOVA) was used to adjust the effect of baseline variables on the outcomes. P<0.05 was considered statistically significant.Results: After baseline adjustment, there was a significant improvement in homeostatic model assessment for insulin resistance (HOMA-IR) (P=0.04) and serum insulin (P<0.001) in the atorvastatin with vitamin E group compared to the atorvastatin with the placebo group. In addition, co-administration of vitamin E with atorvastatin significantly upregulated PPAR-γ expression (OR=5.4, P=0.04) in the PBMCs of T2DM patients.Conclusion: Co-administration of vitamin E and atorvastatin reduced insulin resistance and improved PPAR-γ mRNA expression. Further studies are required to substantiate our findings.Trial registration number: IRCT 20170918036256N

Modulatory effects of pioglitazone as a ligand for the peroxisome proliferator-activated receptor on semen quality and fertility potential of broiler breeder roosters

Fertility potential in roosters is a crucial topic in broiler breeder reproduction which is thought to be associated with age. This study aims to investigate effects of 2 levels of pioglitazone (PIO) supplementation on peroxisome proliferator-activated receptor gamma (PPAR-γ) expression, semen quality, and fertility parameters of aged broiler breeder roosters. The efficacy of PIO was divided into 2 sections: receptor-dependent and receptor-independent. Expression of PPAR-γ mRNA and protein was assessed in sperm to monitor receptor-dependent actions. Sperm motility, velocity parameters, viability, mitochondrial activity, and apoptosis were assessed for the receptor-independent actions. Broiler breeder roosters were randomly assigned to 3 groups: 1) control received a basal diet (CTRL); 2) PIO-5 received a basal diet supplemented with 5 mg PIO/bird/day, and 3) PIO-10 received a basal diet supplemented with 10 mg PIO/bird/day. In addition, semen samples were collected from 24 Ross broiler breeder roosters at 30, 43, and 53 wk of age. Effects of PIO were significant in terms of total motility, straight-line velocity, mitochondrial activity, and apoptosis (P ≤ 0.05). Total motility, straight-line velocity and mitochondrial activity improved in both PIO groups (P ≤ 0.05) along with a significant reduction in early and late apoptosis in the PIO groups (P ≤ 0.05). Pioglitazone addition affected total motility, mitochondrial activity, early apoptosis and late apoptosis in a linearly and quadratically manner (P < 0.05). PPAR-γ mRNA and protein expression were not significantly upregulated by the different doses of PIO (P > 0.05). Similarly, fertility performance was not significantly changed in the PIO groups (P > 0.05). Moreover, PIO improved mitochondrial activity and decreased the apoptosis rate in the sperm of aged broiler breeder roosters. These improvements were associated with the receptor-independent actions of PIO and the mechanism of action of PIO did not appear to be affected by the PPAR-γ receptor in broiler breeder roosters.

Washing Lipoaspirate Improves Fat Graft Survival in Nude Mice

The optimal fat processing technique of fat grafting has not been determined. We have proved the importance of washing lipoaspirate to remove blood, but the necessity of washing when there is no obvious bleeding during liposuction is not clear. The purpose of this study is to further investigate the effect of washing on fat graft survival and the underlying mechanisms, from the perspective of inflammation, oxidative stress and apoptosis. To exclude the influence of blood, de-erythrocyte infranatant (dEI) isolated from lipoaspirate was obtained. Purified fat processed by cotton pad filtration mixed with dEIs after sedimentation (sedimentation group), washing (washing group) or phosphate buffer solution (control group) was transplanted to nude mice subcutaneously. Samples were harvested at 1 day and 1, 3, 8 weeks after transplantation. Volume and weight retention, histologic examination, immunostaining of perilipin-1, CD31, CD45 and Ly6g, mRNA expression of PPAR-γ, C/EBPα, VEGF, bFGF, IL-6, IL10, TNF-α, TGF-β, Bax and Bcl-2, and protein contents of 8-iso-PGF2α, IL-6, IL10, TNF-α and TGF-β were all compared among groups. After transplantation, volume and weight retention, histologic scores, viable adipocytes and vascularization were all improved in the washing group, with increased expression of adipogenic and angiogenic genes. Compared with the sedimentation group, the washing group had milder inflammation, lower levels of oxidative stress and apoptosis. Washing lipoaspirate to eliminate mixed components can improve fat graft survival and promote adipogenesis and angiogenesis, possibly by relieving inflammation, reducing oxidative stress injury and inhibiting apoptosis. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of 47 these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors http://www.springer.com/00266 .

MiRNA-27a mediates insulin resistance in 3T3-L1 cells through the PPARγ.

The biological actions of insulin have been originated by activation of membrane receptors, which trigger a diversity of signaling pathways in facilitating their biological activities. Insulin homeostasis functions in promoting metabolism balance and promotes cell growth and proliferation. If these mechanisms are reformed, this could lead to insulin resistance as a result of defective insulin signaling triggered by mutations in receptors or effector molecules located downstream or by abnormal posttranslational modifications. The purpose of this is to preliminarily investigate the mechanism of miRNA-27a-mediating insulin resistance in 3T3-L1 cells. Insulin resistance in 3T3-L1 adipocytes as a cell model was induced by tumor necrosis factor-alpha (TNF-α) and the miRNA-27a expression in 3T3-L1 adipocytes had been experiential. The regulation of peroxisome proliferator-activated receptor-gamma (PPARγ) mRNA by miRNA-27a had been studied by reverse transcription receptor polymerase chain reaction (RT-PCR). MiRNA-27a was up-regulated in 3T3-L1 cells, miRNA-27a mimics reserved expression of PPARγ mRNA, and miRNA-27a inhibitors up-regulated the expression of PPARγ mRNA. The insulin resistance in 3T3-L1 cells mediated by miRNA-27a may be achieved by targeting PPARγ.

STAT3 Is the Master Regulator for the Forming of 3D Spheroids of 3T3-L1 Preadipocytes

To elucidate the currently unknown mechanisms responsible for the diverse biological aspects between two-dimensional (2D) and three-dimensional (3D) cultured 3T3-L1 preadipocytes, RNA-sequencing analyses were performed. During a 7-day culture period, 2D- and 3D-cultured 3T3-L1 cells were subjected to lipid staining by BODIPY, qPCR for adipogenesis related genes, including peroxisome proliferator-activated receptor γ (Pparγ), CCAAT/enhancer-binding protein alpha (Cebpa), Ap2 (fatty acid-binding protein 4; Fabp4), leptin, and AdipoQ (adiponectin), and RNA-sequencing analysis. Differentially expressed genes (DEGs) were detected by next-generation RNA sequencing (RNA-seq) and validated by a quantitative reverse transcription–polymerase chain reaction (qRT–PCR). Bioinformatic analyses were performed on DEGs using a Gene Ontology (GO) enrichment analysis and an Ingenuity Pathway Analysis (IPA). Significant spontaneous adipogenesis was observed in 3D 3T3-L1 spheroids, but not in 2D-cultured cells. The mRNA expression of Pparγ, Cebpa, and Ap2 among the five genes tested were significantly higher in 3D spheroids than in 2D-cultured cells, thus providing support for this conclusion. RNA analysis demonstrated that a total of 826 upregulated and 725 downregulated genes were identified as DEGs. GO enrichment analysis and IPA found 50 possible upstream regulators, and among these, 6 regulators—transforming growth factor β1 (TGFβ1), signal transducer and activator of transcription 3 (STAT3), interleukin 6 (IL6), angiotensinogen (AGT), FOS, and MYC—were, in fact, significantly upregulated. Further analyses of these regulators by causal networks of the top 14 predicted diseases and functions networks (IPA network score indicated more than 30), suggesting that STAT3 was the most critical upstream regulator. The findings presented herein suggest that STAT3 has a critical role in regulating the unique biological properties of 3D spheroids that are produced from 3T3-L1 preadipocytes.

Different sites of actions make different responses to thiazolidinediones between mouse and rat models of fatty liver

Therapeutic approach for NAFLD is limited and there are no approved drugs. Pioglitazone (PGZ), a thiazolidinedione (TZD) that acts via peroxisome proliferator activated receptor gamma (PPARγ) is the only agent that has shown consistent benefit and efficacy in clinical trials. However, the mechanism of its therapeutic effect on NAFLD remains unclear. The poor understanding may be due to problems with mouse, a species most used for animal experiments. TZDs exacerbate fatty liver in mouse models while they improve it in rat models like in human patients. Therefore, we compared the effects of TZDs including PGZ and rosiglitazone (RGZ) in ob/ob mice and Lepmkyo/Lepmkyo rats, models of leptin-deficient obesity, and A-ZIP/F-1 mice and seipin knockout (SKO) rats, models of generalized lipodystrophy. Pparg mRNA expression was markedly upregulated in fatty livers of mouse models while it was unchanged in rat models. TZDs exacerbated fatty liver in ob/ob and A-ZIP/F-1 mice, improved it in Lepmkyo/Lepmkyo rats and showed no effect in SKO rats. Gene expression analyses of Pparg and its target gene, Fsp27 revealed that PPARγ in the adipose tissue is the exclusive therapeutic target of TZDs in rats but PPARγ in the liver in addition to the adipose tissue is also a major site of actions for TZDs in mice. Although the response to TZDs in mice is the complete opposite of that in human patients, no report has pointed out the problem with TZD studies using mouse models so far. The present study might provide useful suggestions in research on TZDs.

Protective mechanisms of telmisartan against hepatic ischemia/reperfusion injury in rats may involve PPARγ-induced TLR4/NF-κB suppression.

Hepatic ischemia-reperfusion (I/R) is an important cause of liver damage in many clinical situations. Toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) is an inflammatory pathway activated in hepatic I/R injury. Telmisartan, a selective angiotensin II type 1 receptor antagonist and peroxisome proliferator-activated receptor-gamma (PPARγ) partial agonist, can inhibit the expression of pro-inflammatory cytokines. The present work investigated the possible protective effect of telmisartan against hepatic I/R injury and explored its possible mechanisms in rats. Rats were divided into four equal groups: sham-operated control, telmisartan-treated sham-operated control, I/R untreated, and I/R telmisartan-treated groups. Hepatic injury was evaluated biochemically by serum activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and histopathological examination. Hepatic oxidative stress biomarkers, myeloperoxidase level, PPARγ and TLR4 mRNA expression, and NF-κB and active caspase 3 immunoexpression were determined. The study showed that telmisartan attenuated hepatic I/R, as evidenced by decreased serum ALT and AST activities and confirmed by improvement of the histopathological changes. The protective effect of telmisartan was associated with modulation of oxidative stress parameters, myeloperoxidase level, PPARγ and TLR4 mRNA expression, and NF-κB and caspase 3 immunoexpression. Taken together, the current study showed that telmisartan could protect the rat liver from I/R injury. This hepatoprotective effect was attributed to, at least in part, increase in PPARγ expression and suppression of TLR4/NF-κB pathway.

Dermal White Adipose Tissue (dWAT) Is Regulated by Foxn1 and Hif-1α during the Early Phase of Skin Wound Healing.

Dermal white adipose tissue (dWAT) is involved in the maintenance of skin homeostasis. However, the studies concerning its molecular regulation are limited. In the present paper, we ask whether the introduction of two transcription factors, Foxn1 and Hif-1α, into the post-wounded skin of Foxn1−/− mice regulates dWAT during wound healing (days 3 and 6). We have chosen lentivirus vectors (LVs) as a tool to deliver Foxn1 and Hif-1α into the post-wounded skin. We documented that combinations of both transgenes reduces the number, size and diameter of dermal adipocytes at the wound bed area. The qRT-PCR analysis of pro-adipogenic genes, revealed that LV-Hif-1α alone, or combined with LV-Foxn1, increases the mRNA expression of Pparγ, Glut 4 and Fasn at post-wounding day 6. However, the most spectacular stimulatory effect of Foxn1 and/or Hif-1α was observed for Igf2, the growth factor participating in adipogenic signal transduction. Our data also shows that Foxn1/Hif-1α, at post-wounding day 3, reduces levels of CD68 and MIP-1γ mRNA expression and the percentage of CD68 positive cells in the wound site. In conclusion, the present data are the first to document that Foxn1 and Hif-1α cooperatively (1) regulate dWAT during the proliferative phase of skin wound healing through the Igf2 signaling pathway, and (2) reduce the macrophages content in the wound site.

Inhibitory Effect of Lactobacillus plantarum KFY02 Fermented Lemon Juice on Obesity and Intestinal Regulation in Mice Fed with High-fat Diet

The purpose of this study was to observe the fat reducing effect of lemon juice fermented by Lactobacillus plantarum KFY02 and its regulation on intestinal flora. This study induced obesity in mice through the establishment of high-fat diet, the experimental mice were divided into normal group, model group, Lactobacillus plantarum KFY02 group, unfermented lemon juice group and fermented lemon juice group, and biochemical kits, section observation and quantitative PCR were used to detect the related indexes of mouse serum and tissue, and the composition of intestinal microorganisms was observed by detecting the expression of microorganisms in feces. The results showed that Lactobacillus plantarum KFY02, unfermented lemon juice and fermented lemon juice could effectively reduce the body weight of obese mice, reduce the organ index of liver and epididymal adipose tissue in obese mice, and reduce the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AKP), total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C) and raise high density lipoprotein cholesterol (HDL-C) in serum and liver tissue of obese mice, and also could increase the contents of total cholesterol (TC), triglyceride (TG) and total protein (TP) in mouse feces. H&E pathological observation showed that Lactobacillus plantarum KFY02, unfermented lemon juice and fermented lemon juice could also reduce liver tissue lesions and adipocyte enlargement and accumulation caused by obesity. qPCR results showed that Lactobacillus plantarum KFY02, unfermented lemon juice and fermented lemon juice could up-regulate PPAR-α, CPT-1, LPL, CYP7A1 and down-regulate C/EBPα, PPAR-γ mRNA expression in liver tissue of obese mice induced by high fat diet, meanwhile, they could also up-regulate the expression of Bacteroidetes, Lactobacillus sp., Bifidobacterium sp. and down-regulate the expression of Firmicutes. These results showed that Lactobacillus plantarum KFY02, unfermented lemon juice and fermented lemon juice could reduce fat in mice, and the effect of fermented lemon juice was the best. The fat reduction effect of fermented lemon juice on mice was directly related to its regulation of intestinal function and microbial composition. It could be seen that lemon juice fermented by Lactobacillus plantarum KFY02 had good lipid-lowering and intestinal regulation in obese mice.

Effect of Metformin and Simvastatin in Inhibiting Proadipogenic Transcription Factors.

Obesity is a multifactorial chronic disease characterized by the excessive accumulation of fat in adipose tissue driven by hypertrophy and hyperplasia of adipocytes through adipogenesis. Adipogenesis plays a key role in the development of obesity and related metabolic disorders, which makes it potential target for the therapeutic approach to obesity. An increasing number of studies confirm the pleiotropic action of the combined treatment with metformin and statins, suggesting their anti-hypertensive, anti-inflammatory, and anti-adipogenic effect. The aim of this study was to analyze the effect of different doses of metformin (MET) and simvastatin (SIM) on the expression of key transcription factors of adipogenesis. Mouse 3T3-L1 preadipocytes were induced to differentiation in adipogenic medium with sustained MET and SIM treatment to assess the effect on adipogenesis. Nine days after initiating adipogenesis, the cells were prepared for further experiments, including Oil Red O staining, RT-PCR, Western blotting, and immunocytochemistry. Treating the cells with the combination of MET and SIM slightly reduced the intensity of Oil Red O staining compared with the control group, and down-regulated mRNA and protein expression of PPARγ, C/EBPα, and SREBP-1C. In conclusion, the inhibitory effect of MET and SIM on adipocyte differentiation, as indicated by decreased lipid accumulation, appears to be mediated through the down-regulation of adipogenic transcription factors, peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding pro-tein α (C/EBPα), and sterol regulatory element-binding protein 1 (SREBP-1C).