Inflammation In 3T3-L1 Adipocytes Research Articles

Cimifugin inhibits adipogenesis and TNF-α-induced insulin resistance in 3T3-L1 cells.

To investigate the effects of cimifugin on adipogenesis and tumor necrosis factor (TNF-α)-induced insulin resistance (IR) and inflammation in 3T3-L1 adipocytes. 3T3-L1 adipocytes were treated with 3-isobutyl-1-methyl-xanthine, dexamethasone, and insulin or cimifugin and then Oil Red O staining and intracellular triglyceride content detection were performed to assess adipogenesis. Subsequently, after cimifugin treatment, TNF-α was used to induce IR and inflammation. The results showed that cimifugin reduced intracellular lipids accumulation of 3T3-L1 adipocytes. Cimifugin improved IR of 3T3-L1 adipocytes induced by TNF-α, as reflected in decreased adiponectin, GLUT-4, and IRS-1 mRNA and protein expression. Moreover, cimifugin reduced TNF-α-induced pro-inflammatory factors production and phospho-P65 expression, and MAPK pathway activation in the 3T3-L1 adipocytes. These findings suggested that cimifugin might be useful for the prevention and therapy of obesity-related IR and inflammation.

Rutin attenuates tumor necrosis factor-α-induced inflammation and initiates fat browning in 3T3-L1 adipocytes: Potential therapeutic implications for anti-obesity therapy

Obesity is linked with a pathological state of inflammation, leading to the development and aggravation of metabolic diseases. The adipose tissue secretes adipokines and cytokines like tumor necrosis factor alpha (TNF-α) which are implicated in adipocyte dysfunction, exacerbation of inflammation, and insulin resistance. Rutin, a flavonoid that occurs naturally in plants such as Fagopyrum tataricum and Aspalathus linearis, has been found to have a variety of bioactive properties. However, experimental evidence on the therapeutic effects of rutin against a broad spectrum of obesity-associated complications remains to be explored. Hence, the potential therapeutic effects of rutin against TNF-α-induced inflammation in 3T3-L1 adipocytes were examined. The experimental design involved exposing differentiated adipocytes to tumor necrosis factor (TNF)-α before treatment with rutin (10 μM), in comparison to positive controls CL 316,243 (1 μM), and isoproterenol (10 μM) for 6 hours. The findings demonstrated that exposing these adipocytes to TNF-α was linked with enhanced markers of inflammation, increased lipolysis, and impaired lipid metabolism. Interestingly, treatment with rutin was effective in decreasing pro-inflammatory cytokines like TNF-α and IL-6. Rutin reversed adipocyte dysfunction by restoring intracellular lipid accumulation and preventing TNF-α-induced lipolysis. Importantly, rutin also potentially induced browning of 3T3-L1 adipocytes, as seen with increased mRNA expression of PR domain containing 16 (Prdm16) and protein levels of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC1α). These findings demonstrate the potential of rutin to block inflammation and reduce adipocyte dysfunction linked to obesity, in part by modulating molecular mechanisms involving adipogenesis, lipid metabolism, and fat browning. The current study is not without limitations, which include the use of only one cell line to test the efficacy of rutin, while in vivo or in human participants are required to confirm these findings. This could be essential to understand the potential therapeutic ramifications of rutin, including how they might influence the creation of novel anti-obesity treatments.

Anti-inflammatory efficacy of brown seaweed (Padina tetrastromatica) in 3T3-L1 adipocytes and low-dose LPS induced inflammation in C57BL6 mice

Chronic systemic inflammation is a central indicator of numerous diseases. Regulation of inflammation in adipose and liver is essential in mitigating obesity. SIRT1 is known to modulate inflammation and metabolic pathways. Brown algae Padina tetrastromatica possesses anti-oxidant and anti-inflammatory properties. However, the synergistic effects of P. tetrastromatica bioactives Fucoxanthin (Fx) and Total lipids (TL) to inhibit inflammation in SIRT1 silenced 3T3-L1 adipocytes remain unknown. Furthermore, the role of anti-inflammatory food supplements in mitigating systemic inflammation stays elusive. Therefore, here we studied the effects of P.tetrastromatica bioactives on cytokine production and activation of cell signals in SIRT1 silenced and LPS-induced inflammation in 3T3-L1 adipocytes and mice models. Preliminary in silico screening of Fx and its metabolites with target inflammatory markers was performed. The effects of Fx and TL on adipogenesis and reactive oxygen species (ROS) in silenced cells were detected by Oil Red O and DCFH-DA assay. Cytokine production and protein expression of NF-κB, COX-2, IL-10 and SIRT1 were determined by ELISA and western blotting. Fx and TL significantly inhibited adipogenesis, ROS, and Nitric oxide (NO), pro-inflammatory cytokine production (IL-6, IL-1ß, MCP-1, TNF-α) in SIRT1 silenced and LPS-challenged inflamed cells. Inflammatory marker (NF-κB, COX-2) expression was downregulated in Fx and TL treated cells compared to LPS and SIRT1 silenced cells indicating their modulating mechanistic ability to mitigate inflammation. Whereas, the expression of anti-inflammatory cytokine IL-10, adiponectin and NAD+ were upregulated significantly in Fx and TL treated cells compared to silenced and LPS insulted cells. These results were further validated by feeding seaweed and barley blended anti-inflammatory food (AIF) to LPS-treated mice. Results showed that AIF supplementation inhibited ROS generation, and pro-inflammatory cytokine, COX-2 and NF-κB expression. Whereas, feeding AIF upregulated adiponectin, NAD+, IL-10, and SIRT1 levels. Results suggest potential therapeutic food applications of P. tetrastromatica to manage inflammation and obesity.

Lpcat3 deficiency promotes palmitic acid-induced 3T3-L1 mature adipocyte inflammation through enhanced ROS generation.

Phosphatidylcholines (PCs) are major phospholipids in the mammalian cell membrane. Structural remodeling of PCs is associated with many biological processes. Lysophosphatidylcholine acyltransferase 3 (Lpcat3), which catalyzes the incorporation of polyunsaturated fatty acyl chains into the sn-2 site of PCs, plays an important role in maintaining plasma membrane fluidity. Adipose tissue is one of the main distribution organs of Lpcat3, while the relationship between Lpcat3 and adipose tissue dysfunction during overexpansion remains unknown. In this study, we reveal that both polyunsaturated PC content and Lpcat3 expression are increased in abdominal adipose tissues of high-fat diet-fed mice when compared with chow-diet-fed mice, indicating that Lpcat3 is involved in adipose tissue overexpansion and dysfunction. Our experiments in 3T3-L1 adipocytes show that inhibition of Lpcat3 does not change triglyceride accumulation but increases palmitic acid-induced inflammation and lipolysis. Conversely, Lpcat3 overexpression exhibits anti-inflammatory and anti-lipolytic effects. Furthermore, mechanistic studies demonstrate that Lpcat3 deficiency promotes reactive oxygen species (ROS) generation by increasing NOX enzyme activity by facilitating the translocation of NOX4 to lipid rafts, thereby aggregating 3T3-L1 adipocyte inflammation induced by palmitic acid. Moreover, overexpression of Lpcat3 exhibits the opposite effects. These findings suggest that Lpcat3 protects adipocytes from inflammation during adipose tissue overexpansion by reducing ROS generation. In conclusion, our study demonstrates that Lpcat3 deficiency promotes palmitic acid-induced inflammation in 3T3-L1 adipocytes by enhancing ROS generation.

Comparing the effects of tumor necrosis factor alpha, lipopolysaccharide and palmitic acid on lipid metabolism and inflammation in murine 3T3-L1 adipocytes

AimsThis study aimed to develop a model of dysregulated lipid metabolism and inflammation by treating 3T3-L1 adipocytes with tumor necrosis factor alpha (TNFα), lipopolysaccharide (LPS), and palmitic acid (PA) individually or in combination to assess their effects and mechanism of action. Main methodsDifferentiated 3T3-L1 adipocytes were treated with TNFα (10 ng/mL), LPS (100 ng/mL), and PA (0.75 mM) individually or in combination for 24 h. Lipolysis, lipid content, inflammation, and the expression of lipid metabolism and inflammation genes were assessed by glycerol release quantification, Oil Red O staining, enzyme-linked immunosorbent assays, and quantitative reverse transcription-polymerase chain reaction, respectively. Key findingsExposure of 3T3-L1 adipocytes to TNFα stimulated lipolysis, reduced lipid accumulation, decreased adiponectin (ADIPOQ) secretion, and increased secretion of pro-inflammatory adipokines, monocyte chemoattractant protein 1 (MCP-1), interleukin 6 (IL-6), and interleukin 1 beta (IL-1β). These changes were accompanied by decreased expression of lipid metabolism genes, increased expression of pro-inflammatory genes (MCP-1 and IL-6), and decreased expression of the anti-inflammatory gene, ADIPOQ. Exposure to LPS and PA, alone or in combination did not affect these parameters, while co-treatment with TNFα, LPS, and PA enhanced lipolysis and decreased ADIPOQ secretion compared to TNFα treatment. SignificanceDysregulation of lipid metabolism and inflammation in 3T3-L1 adipocytes is attributed to TNFα rather than LPS and PA. We propose that exposing 3T3-L1 adipocytes to TNFα presents a suitable in vitro model of adipocyte dysfunction that closely resembles the complexity of obesity in vivo.

Bovine α-lactalbumin-derived peptides attenuate TNF-α-induced insulin resistance and inflammation in 3T3-L1 adipocytes through inhibiting JNK and NF-κB signaling.

Bioactive peptides in bovine α-lactalbumin were isolated and identified, and the effects and mechanisms of peptide KILDK on insulin resistance in 3T3-L1 adipocytes were investigated. Mature 3T3-L1 adipocytes were stimulated with TNF-α to induce insulin resistance. Bovine α-lactalbumin hydrolysates (α-LAH) were subjected to stimulated gastrointestinal digestion and Caco-2 absorption, and GD-α-LAH and CA-α-LAH were obtained. Our results demonstrated that α-LAH, GD-α-LAH, and CA-α-LAH increased glucose uptake, enhanced Akt phosphorylation (Ser473), and decreased IRS-1 phosphorylation (Ser307) in insulin resistant 3T3-L1 adipocytes. Gel filtration chromatography and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI MS/MS) were used to separate and identify bioactive peptides. The identified peptide KILDK attenuated insulin resistance in 3T3-L1 adipocytes, which was attributed to the suppression of JNK phosphorylation (Thr183/Tyr185). Moreover, KILDK downregulated pro-inflammatory genes through blocking NF-κB signaling. Our findings suggested that bovine α-LAH might be a potential ingredient against insulin resistance.

Characterization of milk and soy phospholipid liposomes and inflammation in 3T3-L1 adipocytes

Milk phospholipids (PL) are valuable dairy components that appear to impart human health benefits, including improved cognitive function in infants and adults. The commercial food industry uses primarily plant-based sources of PL, such as soy lecithin. However, it remains unclear whether different compositions of PL from different dietary sources, such as milk, convey the same benefits. We hypothesized that PL derived from bovine milk or soy have differing physiological effects in terms of inflammation due to their differences in composition. The objectives of this study were to characterize milk and soy liposomes by their physicochemical properties and composition and to evaluate their effects in vitro by means of inflammatory gene expression analyses. Milk and soy phospholipid large unilamellar vesicles (MPL-LUV and SPL-LUV, respectively) prepared using thin-film hydration coupled with extrusion were similar in terms of structure, size, and stability; however, they differed significantly in composition. The 3T3-L1 adipocytes were selected for this work because adipocytes are the main site of uptake, synthesis, modification, and breakdown of lipids and are important inflammatory mediators in mammalian systems. In this work, these cells exposed to both liposome varieties showed high biocompatibility and low cytotoxicity up to concentrations of 0.5 mg/mL as measured by colorimetric MTT and lactate dehydrogenase assays. Furthermore, SPL-LUV showed trends toward stimulating inflammation compared with MPL-LUV as measured by expression of 2 proinflammatory cytokines, monocyte chemoattractant protein-1 (MCP-1) and IL-6. Expression of MCP-1 significantly increased 1.82-fold relative to the control upon SPL-LUV treatment, with similar trends for IL-6 (increased 1.59-fold). The MPL-LUV showed relatively no change in cytokine expression. The results obtained in this work suggest that the methodology used to prepare LUV and the composition and proportion of milk PL are important in measuring cell physiology changes and inflammatory status in mammalian cells.

Protein Digests and Pure Peptides from Chia Seed Prevented Adipogenesis and Inflammation by Inhibiting PPARγ and NF-κB Pathways in 3T3L-1 Adipocytes.

The objective was to evaluate the mechanisms of digested total proteins (DTP), albumin, glutelin, and pure peptides from chia seed (Salvia hispanica L.) to prevent adipogenesis and its associated inflammation in 3T3-L1 adipocytes. Preadipocytes (3T3-L1) were treated during differentiation with either DTP or digested albumin or glutelin (1 mg/mL) or pure peptides NSPGPHDVALDQ and RMVLPEYELLYE (100 µM). Differentiated adipocytes also received DTP, digested albumin or glutelin (1 mg/mL), before (prevention) or after (inhibition) induced inflammation by addition of conditioned medium (CM) from inflamed macrophages. All treatments prevented adipogenesis, reducing more than 50% the expression of PPARγ and to a lesser extent lipoprotein lipase (LPL), fatty acid synthase (FAS), sterol regulatory element-binding protein 1 (SREBP1), lipase activity and triglycerides. Inflammation induced by CM was reduced mainly during prevention, while DTP decreased expression of NF-κB (−48.4%), inducible nitric oxide synthase (iNOS) (−46.2%) and COX-2 (−64.5%), p < 0.05. Secretions of nitric oxide, PGE2 and TNFα were reduced by all treatments, p < 0.05. DTP reduced expressions of iNOS (−52.1%) and COX-2 (−66.4%). Furthermore, digested samples and pure peptides prevented adipogenesis by modulating PPARγ and additionally, preventing and even inhibiting inflammation in adipocytes by inhibition of PPARγ and NF-κB expression. These results highlight the effectiveness of digested total proteins and peptides from chia seed against adipogenesis complications in vitro.

Dynamic folding modulation generates FGF21 variant against diabetes.

Fibroblast growth factor 21 (FGF21) is a regulator of glucose and lipid metabolism. It has been widely considered as a promising candidate for the treatment of type 2 diabetes mellitus (T2DM) and other related metabolic disorders. However, lack of structural and dynamic information has limited FGF21-based drug development. Here, using nuclear magnetic resonance (NMR) spectroscopy, we determine the structure of FGF21 and find that its non-canonical flexible β-trefoil conformation affects the folding of β2-β3 hairpin and further overall protein stability. To modulate folding dynamics, we designed an FGF21-FGF19 chimera, FGF21SS . As expected, FGF21SS shows better thermostability without inducing hepatocyte proliferation. Functional characterization of FGF21SS shows its better insulin sensitivity, reduced inflammation in 3T3-L1 adipocytes, and lower blood glucose and insulin levels in ob/ob mice compared with wild type. Our dynamics-based rational design provides a promising approach for FGF21-based therapeutic development against T2DM.

꾸지뽕 열매 발효식초의 LPS에 의해 유도된 3T3-L1 세포 염증반응 억제 효과

Cudrania tricuspidata fruit has anti-inflammatory and anti-obesity effects. The present study aimed to determine the effect of C. tricuspidata fruits vinegar (CFV) on lipopolysaccharide (LPS)-induced adipocytes inflammation in 3T3-L1 adipocytes. The differentiation of 3T3-L1 adipocytes was induced by dexamethasone (DEX), 3-isobutyl-1-methylxanthine (IBMX) and insulin. Differentiated 3T3-L1 adipocytes were incubated with CFV followed by LPS treatment. The secretion of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) were measured using ELISA. The expression of toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), tumor necrosis factor receptor-associated factor 6 (TRAF6) and nuclear factor kappa B (NF-κB) were measured by western blotting. The results showed that CFV dose-dependently inhibited lipid droplet generation and LPS-induced TNF-α, IL-6 and MCP-1 production in the adipocytes. CFV inhibited NF-κB activation, and the LPS-induced expression of TLR4, MyD88, and TRAF6. These results indicated that CFV inhibits the LPS-induced inflammatory response in adipocytes by inhibiting TLR4 signaling, including NF-κB pathways.

Abstract 353: High Density Lipoprotein Regulates Tumor Necrosis Factor Alpha and Jun Kinase Signaling Pathway in Adipocytes

Background: High-density lipoproteins (HDL) is presumed to exhibit cardiovascular protection, since HDL mediate the reverse transport of cholesterol and exhibit strong anti-inflammatory potential. Previous studies have found that HDL can reduce the level of IL-8 secreted by the inflammatory adipocytes, as well as affect the expression of adiponectin and tumor necrosis factor-alpha (TNF-α) under the inflammatory condition in adipocytes. These studies indicate that HDL is closely related to the inflammatory response of adipose tissue, yet its mechanism has not been fully understood. Secretory leucocyte protease inhibitor (SLPI), a nonglycosylated single-chain polypeptide protein, may play an important role in diet-induced obesity and adipocyte inflammation. Purpose: To study the role of SLPI in HDL anti-inflammatory action in the adipocytes, and explore the mechanism of the action. Methods: 3T3-L1 Adipocytes were induced to differentiation and maturation by culturing. Acute inflammation in adipocytes was induced by LPS (100 ng/ml, 6h), and then the adipocytes were pretreated with HDL and/or SLPI-siRNA. The protein and mRNA levels of SLPI, TNF-α and JNK were examined by Western blotting and qRT-PCR assay, respectively. In addition, the levels of inflammatory factors were measured by ELISA. Results: HDL up-regulated SLPI expression, down-regulated TNF-α expression, and inhibited the secretion of inflammatory factors such as IL-1, IL-6, IL-8 and MCP-1 in 3T3-L1 Adipocytes pre-treated by LPS. Silencing of SLPI by its siRNA effectively abolished the anti-inflammatory effect of HDL. JNK inhibitor (SP600125) partially restored the anti-inflammatory effects of HDL decreased by SLPI knockdown and decreased the secretion of inflammatory factors. Conclusions: These data suggest that HDL up-regulate the expression of SLPI and inhibit the inflammation in 3T3-L1 Adipocytes pre-treated by LPS. The anti-inflammatory effect of HDL, including inhibition of the secretion of inflammatory factors via TNF-α/JNK signaling pathway, probably viat up-regulating the SLPI expression. Key words: high-density lipoprotein, Inflammation, Adipocytes

Protein Digests and Pure Peptides from Chia Seed (Salvia hispanica L) Prevented Adipogenesis and Its Associated Inflammation by Inhibition of PPAR-γ and NF-κB Pathways

Abstract Objectives To evaluate the mechanism of action of the effect of digested total proteins (DTP), albumin, and glutelin from chia seed to prevent and inhibit inflammation in 3T3-L1 adipocytes. Methods Preadipocytes (3T3-L1) were differentiated into mature adipocytes and received DTP, digested albumin, or glutelin (1 mg/ml) from chia seed together with conditioned medium (CM) from inflamed macrophages during 48 h (prevention), or the digested samples were added after 48 h of CM-stimulated inflammation (inhibition). ROS and cytokines secretion (IL-10, IL-12, IL-6, MCP-1, PGE2, and TNF-α) were analyzed by ELISA and lipid accumulation by oil red-O staining, triglyceride content, lipase activity and expression of proteins (PPAR-γ, SREBP1, FAS, LPL, COX-2, iNOS, and NF-κB) by western-blot. The interactions of peptides with FAS, MAGL, and PPAR-γ were evaluated in silico. Data analyses were performed in triplicate from two independent experiments by ANOVA and post-hoc of Tukey (P &amp;lt; 0.05). Results DTP and digested albumin and glutelin in both, prevention and inhibition of induced inflammation, decreased the expression of PPAR-γ more than 50% (P &amp;lt; 0.05). Digested samples reduced TNF-α secretion, ROS production, lipid accumulation, iNOS and COX-2 expression, especially glutelin (−71, −53, −15, −75, and -85%, respectively) on prevention of inflammation. Albumin digest inhibited NO and PGE2 (−42 and 64%, respectively). DTP reduced the lipase activity (−13%), triglyceride content (−27%), and NF-κB expression (−48%) (P &amp;lt; 0.05). On inhibition of inflammation, only SREBP-1 expression was reduced more than 33% (P &amp;lt; 0.05) by every digested protein. DTP was effective to reduce LPL (-32%), FAS (−29%), iNOS (−52%), and COX-2 (−66%) expression, as well as NO secretion (−15%), and triglyceride content (−18%) (P &amp;lt; 0.05). Peptides TGPSPTAGPPAPGGGTH and YLGAHPGTAN, from digested albumin, showed the highest interaction with PPAR-γ (−9.1 kcal/mol) and MAGL (−7.8 kcal/mol), respectively. Peptide APSPPVLGPP from DTP, showed the highest interaction with FAS (−9.8 kcal/mol). Conclusions Digested samples from chia seed were effective in preventing and even inhibiting inflammation in adipocytes by inhibition of PPAR-γ and NF-κB pathways which highlight the effectiveness these digested proteins against obesity complications. Funding Sources CNPq and CAPES (Brazil), and NIFA HATCH (USA).

Tst gene mediates protection against palmitate-induced inflammation in 3T3-L1 adipocytes

Obesity is a disease characterized by chronic inflammation and increases the risk of comorbidities such as type 2 diabetes mellitus, non-alcoholic fatty liver disease and cardiovascular dysfunction. In obese individuals, elevated levels of plasma circulating saturated fatty acids (e.g. palmitate) contribute to subclinical chronic inflammation. Genetic variants play a major role in determining susceptibility or resistance to obesity. Some recently described variant in genetically Lean mice that leads to upregulated expression selectively in adipose tissue of the nuclear encoded mitochondrial gene thiosulfate sulfurtransferase, Tst, has been causally linked to reduced adiposity and insulin sensitisation. Here we show that macrophages derived from the high-TST expressing Lean line of mice have a reduced inflammatory response to TLR-ligand activation than those from the low-TST expressing Fat line of mice. Furthermore, using the mouse clonal 3T3-L1 adipocytes we describe a positive effect of TST upregulation via its substrates, sodium thiosulfate (STS) and the garlic compound diallyl disulfide (DADS), on palmitate-induced inflammation resulting in significant reduction of inflammatory cytokines and inhibition of reactive oxygen species. Additionally, using an IL1β-induced insulin resistance 3T3-L1 cell model, upregulation of TST associated with antidiabetic effects. Therefore, we demonstrate that TST-mediated suppression of the inflammatory response protects in vitro cell model of white adipocytes from insulin resistance-induced inflammation. These results encourage the development of potent medicines in the future that specifically target TST in the fat tissue, which could lead to new ways of treating diabetes associated with obesity.

Ellagic Acid and Urolithins A and B Differentially Regulate Fat Accumulation and Inflammation in 3T3-L1 Adipocytes While Not Affecting Adipogenesis and Insulin Sensitivity.

Ellagic acid (EA) is a component of ellagitannins, present in crops such as pecans, walnuts, and many berries, which metabolized by the gut microbiota forms urolithins A, B, C, or D. In this study, ellagic acid, as well as urolithins A and B, were tested on 3T3-L1 preadipocytes for differentiation and lipid accumulation. In addition, inflammation was studied in mature adipocytes challenged with lipopolysaccharide (LPS). Results indicated that EA and urolithins A and B did not affect differentiation (adipogenesis) and only EA and urolithin A attenuated lipid accumulation (lipogenesis), which seemed to be through gene regulation of glucose transporter type 4 (GLUT4) and adiponectin. On the other hand, gene expression of cytokines and proteins associated with the inflammation process indicate that urolithins and EA differentially inhibit tumor necrosis factor alpha (TNFα), inducible nitric oxide synthase (iNOS), interleukin 6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1). Urolithins A and B were found to reduce nuclear levels of phosphorylated nuclear factor κB (p-NF-κB), whereas all treatments showed expression of nuclear phosphorylated protein kinase B (p-AKT) in challenged LPS cells when treated with insulin, indicating the fact that adipocytes remained insulin sensitive. In general, urolithin A is a compound able to reduce lipid accumulation, without affecting the protein expression of peroxisome proliferator-activated receptor-γ (PPARγ), CCAAT/enhancer binding protein-α (c/EBPα), and PPARα, whereas EA and urolithin B were found to enhance PPARγ and c/EBPα protein expressions as well as fatty acid (FA) oxidation, and differentially affected lipid accumulation.

Effect of saccharin on inflammation in 3T3-L1 adipocytes and the related mechanism.

BACKGROUND/OBJECTIVESExcessive intake of simple sugars induces obesity and increases the risk of inflammation. Thus, interest in alternative sweeteners as a sugar substitute is increasing. The purpose of this study was to determine the effect of saccharin on inflammation in 3T3-L1 adipocytes.MATERIALS/METHODS3T3-L1 preadipocytes were differentiated into adipocytes. The adipocytes were treated with saccharin (0, 50, 100, and 200 µg/mL) for 24 h. Inflammation was induced by exposure of treated adipocytes to lipopolysaccharide (LPS) for 18 h and cell proliferation was measured. The concentration of nitric oxide (NO) was measured by using Griess reagent. Protein expressions of nuclear factor kappa B (NF-κB) and inhibitor κB (IκB) were determined by western blot analysis. The mRNA expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin 1β (IL-1β), interleukin 6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor-α (TNF-α) were determined by real-time PCR.RESULTSCompared with the control group, the amount of NO and the mRNA expression of iNOS in the LPS-treated group were increased by about 17.6% and 46.9%, respectively, (P < 0.05), and those parameter levels were significantly decreased by saccharin treatment (P < 0.05). Protein expression of NF-κB was decreased and that of IκB was increased by saccharin treatment (P < 0.05). Saccharin decreased the mRNA expression of COX-2 and the inflammation cytokines (IL-1β, IL-6, MCP-1, and TNF-α) (P < 0.05).CONCLUSIONSThe results of this study suggest that saccharin can inhibit LPS-induced inflammatory responses in 3T3-L1 adipocytes via the NF-κB pathway.

Pentraxin 3 deficiency exacerbates lipopolysaccharide-induced inflammation in adipose tissue

Background/objectivesPentraxin 3 (PTX3) has been characterized as a soluble and multifunctional pattern recognition protein in the regulation of innate immune response. However, little is known about its role in adipose tissue inflammation and obesity. Herein, we investigated the role of PTX3 in the regulation of lipopolysaccharide (LPS)-induced inflammation in adipocytes and adipose tissue, as well as high-fat diet (HFD)-induced metabolic inflammation in obesity.MethodsPtx3 knockdown 3T3-L1 Cells were generated using shRNA for Ptx3 gene and treated with different inflammatory stimuli. For the in vivo studies, Ptx3 knockout mice were treated with 0.3 mg/kg of LPS for 6 h. Adipose tissues were collected for gene and protein expression by qPCR and western blotting, respectively. Ptx3 knockout mice were fed with HFD for 12 week since 6 week of age.ResultsWe observed that the expression of PTX3 in adipose tissue and serum PTX3 were markedly increased in response to LPS administration. Knocking down Ptx3 in 3T3-L1 cells reduced adipogenesis and caused a more profound and sustained upregulation of proinflammatory gene expression and signaling pathway activation during LPS-stimulated inflammation in 3T3-L1 adipocytes. In vivo studies showed that PTX3 deficiency significantly exacerbated the LPS-induced upregulation of inflammatory genes and downregulation of adipogeneic genes in visceral and subcutaneous adipose tissue of mice. Accordingly, LPS stimulation elicited increased activation of nuclear factor-κB (NF-κB) and p44/42 MAPK (Erk1/2) signaling pathways in visceral and subcutaneous adipose tissue. The expression of PTX3 in adipose tissue was also induced by HFD, and PTX3 deficiency led to the upregulation of proinflammatory genes in visceral adipose tissue of HFD-induced obese mice.ConclusionsOur results suggest a protective role of PTX3 in LPS- and HFD-induced sustained inflammation in adipose tissue.

Catechin attenuates TNF-α induced inflammatory response via AMPK-SIRT1 pathway in 3T3-L1 adipocytes

Chronic inflammation is a fundamental symptom of many diseases. Catechin possesses anti-oxidant and anti-inflammatory properties. However, the mechanism of catechin to prevent inflammation in 3T3-L1 adipocytes caused by TNF-α remains unknown. Therefore, the effects of catechin on the gene expression of cytokines and the activation of cell signals in TNF-α induced 3T3-L1 adipocytes were investigated. The effects of catechin on adipogenesis and cell viability were detected by Oil Red O staining and CCK-8 assay, respectively. The genes expression of cytokines was determined by real-time RT-PCR. The expression of NF-κB, AMPK, FOXO3a and SIRT1 on translation level was determined by western blotting analysis. The results demonstrated that catechin significantly enhanced adipogenesis and cell viability. catechin inhibited the gene expression of pro-inflammatory cytokines including IL-1α, IL-1β, IL-6, IL-12p35, and inflammatory enzymes including iNOS and COX-2, but enhanced the gene expression of anti-inflammatory cytokines including IL-4 and IL-10. Catechin also inhibited the activation of NF-κB, AMPK, FOXO3a and SIRT1, but increased the phosphorylation level of the above factors. All these results indicated that as a potential therapeutic strategy catechin has the ability of attenuating inflammatory response triggered by TNF-α through signaling cascades involved in inflammation and cytokines.

Inhibitory effects of lingonberry (Vaccinium vitis-idaea L.) fruit extract on obesity-induced inflammation in 3T3-L1 adipocytes and RAW 264.7 macrophages

Abstract In this study, the lingonberry fruit extract (LGBE) has been investigated for the ability to suppress the inflammatory response and mitigate oxidative stress in activated 3T3-L1 adipocytes and RAW 264.7 macrophages. The obtained results showed that LGBE significantly reduced IL-6, MCP-1 and IL-1β production, and counteracted the decrease in adiponectin and IL-10 expression in TNF-α-induced adipocytes. Moreover, LGBE exhibited a high anti-inflammatory potential in the macrophage cell culture by down-regulation the expression of pro-inflammatory mediators (IL-6, TNF-α, IL-1β, MCP-1, COX-2, iNOS). Furthermore, LGBE decreased intracellular ROS generation in inflamed adipocytes by enhancing expression of antioxidant defense enzymes (SOD, catalase, GPx) and inhibiting an oxidant enzyme (NADPH oxidase 4), which is one of the primary sources of ROS. In conclusion, we have demonstrated that lingonberry fruit may reduce adipose tissue inflammation and support immune cell homeostasis, and thus can be considered a natural tool for inflammation control.

Effect of epigallocatechin-3-gallate on oxidative stress and inflammation in 3T3-L1 adipocytes

Effect of epigallocatechin-3-gallate on oxidative stress and inflammation in 3T3-L1 adipocytes

Nod1-mediated lipolysis promotes diacylglycerol accumulation and successive inflammation via PKCδ-IRAK axis in adipocytes

Chronic inflammation contributes to obesity mediated metabolic disturbances, including insulin resistance. Obesity is associated with altered microbial load in metabolic tissues that can contribute to metabolic inflammation. Different bacterial components such as, LPS, peptidoglycans have been shown to underpin metabolic disturbances through interaction with host innate immune receptors. Activation of Nucleotide-binding oligomerization domain-containing protein 1 (Nod1) with specific peptidoglycan moieties promotes insulin resistance, inflammation and lipolysis in adipocytes. However, it was not clear how Nod1-mediated lipolysis and inflammation is linked. Here, we tested if Nod1-mediated lipolysis caused accumulation of lipid intermediates and promoted cell autonomous inflammation in adipocytes. We showed that Nod1-mediated lipolysis caused accumulation of diacylglycerol (DAG) and activation of PKCδ in 3T3-L1 adipocytes, which was prevented with a Nod1 inhibitor. Nod1-activated PKCδ caused downstream stimulation of IRAK1/4 and was associated with increased expression of proinflammatory cytokines such as, IL-1β, IL-18, IL-6, TNFα and MCP-1. Pharmacological inhibition or siRNA mediated knockdown of IRAK1/4 attenuated Nod1-mediated activation of NF-κB, JNK, and the expression of proinflammatory cytokines. These results reveal that Nod1-mediated lipolysis promoted accumulation of DAG, which engaged PKCδ and IRAK1/4 to augment inflammation in 3T3-L1 adipocytes.