Endrin potentiates early-stage adipogenesis in 3T3-L1 cells by activating the mammalian target of rapamycin.

Obesogens are a type of endocrine-disrupting chemicals (EDCs) that disrupt the human endocrine system, resulting in obesity and metabolic disease. Several obesogens, including bisphenol A, tolylfluanid, and some pesticides, have been identified and studied previously; however, the underlying molecular mechanisms by which obesogens interfere with adipogenesis and induce insulin resistance in adipocyte remain unknown. This study aims to determine which type of chemical is the most potent obesogen and to investigate its effect on adipogenesis-related gene expressions. 3T3-L1, a pre adipocyte cell line, was differentiated into mature adipocytes with either vehicle or various obesogens, including bisphenol A, tolylfluanid, and endrin, as well as corticosterone, at the same dose. Subsequently, intracellular and secreted triglyceride levels were measured, and the expression of genes and proteins involved in adipogenesis and lipogenesis was investigated. We found that endrin was the most potent regulator of adipogenic differentiation, as compared to tolylfluanid, bisphenol A, and corticosterone. Endrin increased intracellular and secreted triglyceride levels and enhanced the expression of adipogenic transcription factors as well as the terminal differentiation marker in a dose-dependent manner. During the early stages of differentiation, endrin enhanced mammalian target of rapamycin (mTOR) activity, which was suppressed by the pharmacological blockade of the protein kinase B-mTOR pathway, with repressed adipogenic differentiation. However, endrin did not change the expression levels of the downstream members of the mTOR signaling pathway or proteins related to lipolysis in response to insulin. Thus, we suggest that endrin potentiates early-stage adipogenic differentiation by activating the mTOR pathway.

Inhibition of the PI3K-AKT-mTOR pathway suppresses the adipocyte-mediated proliferation and migration of breast cancer cells.

Objective: Although it is well known that adipocyte significantly affects breast cancer progression, its mechanism has not been fully understood. Here, we analyzed the effect of adipocytes on breast cancer progression including cell proliferation and migration. Materials and Methods: We treated the conditioned media obtained from mouse 3T3-L1-derived or human adipose tissue-derived mesenchymal stem cells (hAMSC)-derived adipocytes to breast cancer cells, MCF-7 and MDA-MB-231. And then, cells viability and proliferation were analyzed using MTT assays and colony forming assays, respectively. Also mRNA expression of inflammatory cytokines and proteins expression in main signal pathway were analyzed by RT-qPCR and immunoblotting, respectively. Results: Adipocyte-derived conditioned media increased the proliferation and migration of MCF-7 and MDA-MB-231 cells while little effects in a human normal immortalized mammary epithelial cell line MCF10A. In addition, adipocyte-derived conditioned media induced phosphorylation of AKT and mTOR and upregulated the expression of target genes of the PI3K-AKT-mTOR pathway including IL6, IL1ß, IL1α and TNFα in breast cancer cells. Furthermore, BEZ235 a dual inhibitor of PI3K and mTOR significantly decreased the adipocyte-mediated the proliferation and migration of breast cancer cells. Conclusion: Adipocyte-derived conditioned media enhance the proliferation and migration of breast cancer cells through the PI3K-AKT-mTOR pathway, supporting the importance of heterotypic interactions between breast cancer cells and adipocytes in the tumor microenvironment.

Inhibitory Effects of Roseoside and Icariside E4 Isolated from a Natural Product Mixture (No-ap) on the Expression of Angiotensin II Receptor 1 and Oxidative Stress in Angiotensin II-Stimulated H9C2 Cells.

Hypertension is a major risk factor for the development of cardiovascular diseases. This study aimed to elucidate whether the natural product mixture No-ap (NA) containing Pine densiflora, Annona muricate, and Monordica charantia, or its single components have inhibitory effects on hypertension-related molecules in Angiotensin II (Ang II)-stimulated H9C2 cells. Individual functional components were isolated and purified from NA using various columns and solvents, and then their structures were analyzed using ESI⁻MS, ¹H-NMR, and 13H-NMR spectra. H9C2 cells were stimulated with 300 nM Ang II for 7 h. NA, telmisartan, ginsenoside, roseoside (Roseo), icariside E4 (IE4), or a combination of two components (Roseo and IE4) were administered to the cells 1 h before Ang II stimulation. The expression and activity of hypertension-related molecules or oxidative molecules were determined using RT-PCR, western blot, and ELISA. Ang II stimulation increased the expression of Ang II receptor 1 (AT1), tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), tumor growth factor-ß (TGF-ß) mRNA, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and the levels of hydrogen peroxide (H2O2) and superoxide anion (•O2-) and reduced anti-oxidant enzyme activity. NA significantly improved the expression or activities of all hypertension-related molecules altered in Ang II-stimulated cells. Roseo or IE4 pretreatment either decreased or increased the expression or activities of all hypertension-related molecules similar to NA, but to a lesser extent. The pretreatment with a combination of Roseo and IE4 (1:1) either decreased or increased the expression of all hypertension-related molecules, compared to each single component, revealing a synergistic action of the two compounds. Thus, the combination of single components could exert promising anti-hypertensive effects similar to NA, which should be examined in future animal and clinical studies.

Inhibition of Osteoarthritis-Related Molecules by Isomucronulatol 7-O-ß-d-glucoside and Ecliptasaponin A in IL-1ß-Stimulated Chondrosarcoma Cell Model.

Osteoarthritis (OA) is the common form of arthritis and is characterized by disability and cartilage degradation. Although natural product extracts have been reported to have anti-osteoarthritic effects, the potential bioactivity of Ryupunghwan (RPH), a traditional Korean medicinal botanical formula that contains Astragalus membranaceus, Turnera diffusa, Achyranthes bidentata, Angelica gigas, Eclipta prostrata, Eucommia ulmoides, and Ilex paraguariensis, is not known well. Therefore, the inhibitory effects of single compounds isolated from RPH on the OA-related molecules were investigated using IL-1ß-stimulated chondrosarcoma SW1353 (SW1353) cell model. Two bioactive compounds, isomucronulatol 7-O-ß-d-glucoside (IMG) and ecliptasaponin A (ES) were isolated and purified from RPH using column chromatography, and then the structures were analyzed using ESI-MS, ¹H-NMR, and 13C-NMR spectrum. The expression or amount of matrix metalloproteinase 13 (MMP13), COX1/2, TNF-α, IL-1ß or p65 was determined by RT-PCR, Western blot, and enzyme-linked immunosorbent assay (ELISA). RPH pretreatment reduced the expression and amounts of MMP13, and the expression of collagen II, COX1/2, TNF-α, IL-1ß or p65, which were increased in IL-1ß-stimulated SW1353 cells. IMG reduced the expression of all OA-related molecules, but the observed inhibitory effect was less than that of RPH extract. The other single compound ES showed the reduced expression of all OA-related molecules, and the effect was stronger than that in IMG (approximately 100 fold). Combination pretreatment of both single components remarkably reduced the expression of MMP13, compared to each single component. These synergic effects may provide potential molecular modes of action for the anti-osteoarthritic effects of RPH observed in clinical and animal studies.

Chronic HMGCR/HMG-CoA reductase inhibitor treatment contributes to dysglycemia by upregulating hepatic gluconeogenesis through autophagy induction.

Statins (HMGCR/HMG-CoA reductase [3-hydroxy-3-methylglutaryl-CoA reductase] inhibitors) are widely used to lower blood cholesterol levels but have been shown to increase the risk of type 2 diabetes mellitus. However, the molecular mechanism underlying diabetogenic effects remains to be elucidated. Here we show that statins significantly increase the expression of key gluconeogenic enzymes (such as G6PC [glucose-6-phosphatase] and PCK1 (phosphoenolpyruvate carboxykinase 1 [soluble]) in vitro and in vivo and promote hepatic glucose output. Statin treatment activates autophagic flux in HepG2 cells. Acute suppression of autophagy with lysosome inhibitors in statin treated HepG2 cells reduced gluconeogenic enzymes expression and glucose output. Importantly, the ability of statins to increase gluconeogenesis was impaired when ATG7 was deficient and BECN1 was absent, suggesting that autophagy plays a critical role in the diabetogenic effects of statins. Moreover autophagic vacuoles and gluconeogenic genes expression in the liver of diet-induced obese mice were increased by statins, ultimately leading to elevated hepatic glucose production, hyperglycemia, and insulin resistance. Together, these data demonstrate that chronic statin therapy results in insulin resistance through the activation of hepatic gluconeogenesis, which is tightly coupled to hepatic autophagy. These data further contribute to a better understanding of the diabetogenic effects of stains in the context of insulin resistance.

Oligonol suppresses lipid accumulation and improves insulin resistance in a palmitate-induced in HepG2 hepatocytes as a cellular steatosis model.

BACKGROUND: Oligonol is a low molecular weight form of polyphenol polymers derived from lychee fruits. Several studies suggest that Oligonol has an anti-obesity effect. Since obesity is tightly associated with insulin resistance, we investigated a possible remission effect of Oligonol on lipid accumulation and insulin resistance in human hepatic HepG2 cells. METHODS: HepG2 cells were treated with palmitate for 24 h to induce cellular hepatic steatosis and insulin resistance. The cells were then treated with Oligonol at subtoxic concentrations and examined for lipid metabolism, cytokine production, and insulin signaling using quantitative RT-PCR and western blot analysis. RESULTS: Oligonol treatment reversed the palmitate-induced intracellular lipid accumulation, down regulated the expression of lipogenic genes, and up-regulated genes for fatty acid degradation. Oligonol restored insulin sensitivity, as was determined by the phosphorylation states of IRS-1. Oligonol also inhibited STAT3-SOCS3 signaling and increased AMPK phosphorylation in HepG2 cells. CONCLUSION: Oligonol treatment improved palmitate-induced cellular steatosis and insulin resistance in HepG2 cells with concomitant reduction of inflammatory cytokines and decrease in STAT3-SOCS3 and AMPK-mTOR pathways. Oligonol may have beneficial effects in lipid metabolism and insulin resistance in the liver.

Inhibition of Adipogenesis by Oligonol through Akt-mTOR Inhibition in 3T3-L1 Adipocytes.

Polyphenols have recently become an important focus of study in obesity research. Oligonol is an oligomerized polyphenol, typically comprised of catechin-type polyphenols from a variety of fruits, which has been found to exhibit better bioavailability and bioreactivity than natural polyphenol compounds. Here, we demonstrated that Oligonol inhibits 3T3-L1 adipocyte differentiation by reducing adipogenic gene expression. During adipogenesis, Oligonol downregulated the mRNA levels of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding proteins α (C/EBPα), and δ (C/EBPδ) in a dose-dependent manner and the expression of genes involved in lipid biosynthesis. The antiadipogenic effect of Oligonol appears to originate from its ability to inhibit the Akt and mammalian target of rapamycin (mTOR) signaling pathway by diminishing the phosphorylation of ribosomal protein S6 kinase (p70S6K), a downstream target of mTOR and forkhead box protein O1 (Foxo1). These results suggest that Oligonol may be a potent regulator of obesity by repressing major adipogenic genes through inhibition of the Akt signaling pathway, which induces the inhibition of lipid accumulation, ultimately inhibiting adipogenesis.

Visceral adiposity is associated with SIRT1 expression in peripheral blood mononuclear cells: a pilot study.

Sirtuin1 (SIRT1) is activated during calorie restriction and appears to be related to energy balance through glucose or lipid metabolism and insulin signaling. These findings suggest that SIRT1 may play a role in the pathophysiology of visceral obesity. Therefore, we investigated the relationship between SIRT1 gene expression in circulating peripheral blood mononuclear cells (PBMCs) and abdominal visceral adiposity as measured by computed tomography. We recruited 43 men and women without history of diabetes or cardiovascular disease Biomarkers of metabolic disease and body composition by computed tomography were assessed. SIRT1 gene expression was determined using isolated PBMCs. SIRT1 expression levels negatively correlated with body mass index, waist circumference, abdominal visceral fat area, and homeostasis model of assessment of insulin resistance (HOMA-IR) and positively correlated with adiponectin levels. Results of step-wise multiple regression analysis revealed that abdominal visceral fat area and HOMA-IR were independently associated with SIRT1 expression. The significant association between abdominal visceral fat accumulation and SIRT1 gene expression in PBMCs suggests that SIRT1 may be a new therapeutic target for the prevention of disease related to obesity, especially visceral obesity.

CD4+ TH1 cells generated by Ii-PADRE DNA at prime phase are important to induce effectors and memory CD8+ T cells.

The requirement for CD4 T cells in priming and maintaining cytotoxic T-lymphocyte responses presents a long-standing paradox in cellular immunology. In this study, we used sequential coadministration of a DNA vaccine encoding an invariant (Ii) chain in which the class II-associated Ii-peptide region is replaced with CD4 T-helper epitope, PADRE [Pan human leukocyte antigen-DR reactive epitope (Ii-PADRE)] or Bcl-xL with a DNA vaccine encoding Sig/E7/LAMP-1 to verify the role of CD4 T cells for the generation of effectors and memory E7-specific CD8 T-cell immune responses. Sequential vaccination, with Ii-PADRE+Sig/E7/LAMP-1 priming followed by Bcl-xL+Sig/E7/LAMP-1 boosting led to generation of E7-specific CD8 T cells, and was nearly equivalent in effect to coadministration with Ii-PADRE+Sig/E7/LAMP-1 or Bcl-xL+Sig/E7/LAMP-1 at both prime and boost. The mice vaccinated with the Ii-PADRE+Sig/E7/LAMP-1 prime-Bcl-xL+Sig/E7/LAMP-1 boost regimen exhibited better long-term E7-specific immune responses and tumor prevention effects in vivo than the mice vaccinated with the reverse sequential coadministration. After CD4 T-cell depletion, mice primed with Ii-PADRE+Sig/E7/LAMP-1 generated low numbers of E7-specific CD8 T cells and suppressed long-term memory CD8 T-cell response regardless of the sequence or combination of DNA vaccines administered. Mice primed with Bcl-xL+Sig/E7/LAMP-1 only suppressed long-term memory CD8 T-cell response after depletion of CD4 T cells before priming. Our findings suggest that activated CD4 T cells at prime phase are important to generate the antigen-specific CD8 T-cell immune responses and CD4 T cells, which are naive or activated, play a role to maintain the long-term memory responses.

Doxorubicin enhances CD4(+) T-cell immune responses by inducing expression of CD40 ligand and 4-1BB.

Chemotherapy agents have adverse immunotherapeutic effects secondary to inhibition of hematopoietic stem cell proliferation, particularly in committed lymphoblast. Certain anti-cancer drugs have immuno-modulatory properties, although mechanisms are still not fully understood. In the current study, we explored the effects of doxorubicin on peripheral blood CD4(+) and CD8(+) T-cell responses pre- and post-stimulation. Doxorubicin treatment alone had no effects on peripheral blood T lymphocytes and regulatory T-cells in vivo and in vitro. However, CD4(+) T-cells were resistant to doxorubicin and demonstrated more robust proliferation than CD8(+) T-cells after doxorubicin pre-treatment. CD40 ligand and 4-1BB expression on the surface of CD4(+) T-cells were increased after TCR-ligation activation; however, expression on CD8(+) T-cells was not increased. Dendritic cells cultured in the presence of activated CD4(+) T-cells pre-treated with doxorubicin had greater survival rates than those cultured with activated CD8(+) T-cells. Doxorubicin pre-treatment followed by anti-CD3epsilon+anti-4-1BB activation led to proliferation of CD4(+) T-cells and no proliferative effects on CD8(+) T-cells. Our results collectively suggest that doxorubicin pre-treatment in cancer patients may be a more effective way to enhance anti-cancer immune responses by increased antigen-specific CD4(+) Th1 immune responses.