Epoxyeicosatrienoic Acid Agonist Regulates Human Mesenchymal Stem Cell–Derived Adipocytes Through Activation of HO-1-pAKT Signaling and a Decrease in PPARγ

Department of Physiology and Pharmacology, University of Toledo College of Medicine, Toledo, Ohio 43614, USA.
Stem cells and development (Impact Factor: 3.73). 12/2010; 19(12):1863-73. DOI: 10.1089/scd.2010.0098
Source: PubMed


Human mesenchymal stem cells (MSCs) expressed substantial levels of CYP2J2, a major CYP450 involved in epoxyeicosatrienoic acid (EET) formation. MSCs synthesized significant levels of EETs (65.8 ± 5.8 pg/mg protein) and dihydroxyeicosatrienoic acids (DHETs) (15.83 ± 1.62 pg/mg protein), suggesting the presence of soluble epoxide hydrolase (sEH). The addition of an sEH inhibitor to MSC culture decreased adipogenesis. EETs decreased MSC-derived adipocytes in a concentration-dependent manner, 8,9- and 14,15-EET having the maximum reductive effect on adipogenesis. We examined the effect of 12-(3-hexylureido)dodec-8(Z)-enoic acid, an EET agonist, on MSC-derived adipocytes and demonstrated an increased number of healthy small adipocytes, attenuated fatty acid synthase (FAS) levels (P < 0.01), and reduced PPARγ, C/EBPα, FAS, and lipid accumulation (P < 0.05). These effects were accompanied by increased levels of heme oxygenase (HO)-1 and adiponectin (P < 0.05), and increased glucose uptake (P < 0.05). Inhibition of HO activity or AKT by tin mesoporphyrin (SnMP) and LY2940002, respectively, reversed EET-induced inhibition of adipogenesis, suggesting that activation of the HO-1-adiponectin axis underlies EET effect in MSCs. These findings indicate that EETs decrease MSC-derived adipocyte stem cell differentiation by upregulation of HO-1-adiponectin-AKT signaling and play essential roles in the regulation of adipocyte differentiation by inhibiting PPARγ, C/EBPα, and FAS and in stem cell development. These novel observations highlight the seminal role of arachidonic acid metabolism in MSCs and suggest that an EET agonist may have potential therapeutic use in the treatment of dyslipidemia, diabetes, and the metabolic syndrome.

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    • "A decrease in HO-1 expression results in increased insulin resistance and adiposity in Zucker rats and obese mice [34]. Additionally, induction of HO-1 in adipocyte cell culture is associated with increased adiponectin levels and decreased pro-inflammatory cytokines, TNFα and IL-6 [35,36]. "
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    ABSTRACT: Introduction Heme oxygenase (HO), a major cytoprotective enzyme, attenuates oxidative stress and obesity. The canonical Wnt signaling cascade plays a pivotal role in the regulation of adipogenesis. The present study examined the interplay between HO-1and the Wnt canonical pathway in the modulation of adipogenesis in mesenchymal stem cell (MSC)-derived adipocytes. Methods To verify the role of HO-1 in generating small healthy adipocytes, cobalt protoporphyrin (CoPP), inducer of HO-1, was used during adipocyte differentiation. Lipid accumulation was measured by Oil red O staining and lipid droplet size was measured by BODIPY staining. Results During adipogenesis in vitro, differentiating pre-adipocytes display transient increases in the expression of genes involved in canonical Wnt signaling cascade. Increased levels of HO-1 expression and HO activity resulted in elevated levels of β-catenin, pGSK3β, Wnt10b, Pref-1, and shh along with increased levels of adiponectin (P < 0.05). In addition, induction of HO-1 resulted in a reduction in C/EBPα, PPARγ, Peg-1/Mest, aP2, CD36 expression and lipid accumulation (P < 0.05). Suppression of HO-1 gene by siRNA decreased Wnt10b, pGSK3β and β-catenin expression, and increased lipid accumulation. The canonical Wnt responsive genes, IL-8 and SFRP1, were upregulated by CoPP and their expression was decreased by the concurrent administration of tin mesoporphyrin (SnMP), an inhibitor of HO activity. Furthermore, knockdown of Wnt10b gene expression by using siRNA showed increased lipid accumulation, and this effect was not decreased by concurrent treatment with CoPP. Also our results show that blocking the Wnt 10b antagonist, Dickkopf 1 (Dkk-1), by siRNA decreased lipid accumulation and this effect was further enhanced by concurrent administration of CoPP. Conclusions This is the first study to demonstrate that HO-1 acts upstream of canonical Wnt signaling cascade and decreases lipogenesis and adipocyte differentiation suggesting that the HO-1 mediated increase in Wnt10b can modulate the adipocyte phenotype by regulating the transcriptional factors that play a role in adipogenesis. This is evidenced by a decrease in lipid accumulation and inflammatory cytokine levels, increased adiponectin levels and elevation of the expression of genes of the canonical Wnt signaling cascade.
    Full-text · Article · Mar 2013 · Stem Cell Research & Therapy
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    • "Human MSCs and mouse pre-adipocyte cell line, 3T3L1, has been widely used for elucidating mechanisms involved in mammalian adipogenesis (Kim et al., 2010;MacDougald et al., 1995). These pre-adipocytes undergo a well-characterized process of differentiation upon induction with insulin, dexamethasone, and indomethacin (MacDougald et al., 1995). "
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    ABSTRACT: Patho-physiological conditions with high oxidative stress, such as conditions associated with increased denatured heme-proteins, are associated with enhanced adipogenic response. This effect predominantly manifests as adipocyte hypertrophy characterized by dysfunctional, pro-inflammatory adipocytes exhibiting reduced expression of anti-inflammatory hormone, adiponectin. To understand how increased levels of cellular heme, a pro-oxidant molecule, modulates adipogenesis; the following study was designed to evaluate effects of heme on adipogenesis in human mesenchymal stem cells (hMSCs) and mouse pre-adipocytes (3T3L1). Experiments were conducted in the absence and in the presence of a superoxide dismutase (SOD) mimetic (tempol, 100 µM). Heme (10 µM) increased (P<0.05) adipogenesis in hMSCs and mouse pre-adipocytes, where tempol alone (100 µmol/L) attenuated adipogenesis in these cells (P<0.05). Tempol also reversed heme-induced increase in adipogenesis in both hMSCs and mouse pre-adipocytes (P<0.05). In addition, heme exposed 3T3L1 exhibited reduced (P<0.05) expression of transcriptional regulator-sirtuin 1 (Sirt1), along with, increased (P<0.05) expression of adipogenic markers peroxisome proliferators-activated receptor-gamma (PPARγ), C/EBPα, and aP2. These effects of heme were rescued (P<0.05) in cells concurrently treated with heme and tempol (P<0.05) and prevented in cells over-expressing Sirt1. Taken together, our results indicate that heme-induced oxidative stress inhibits Sirt1, thus un-inhibiting adipogenic regulators such as PPARγ and C/EBPα; which in turn induce increased adipogenesis along with adipocyte hypertrophy in pre-adipocytes. Anti-oxidant induced offsetting of these effects of heme supports the role of heme-dependent oxidative stress in mediating such events.
    Full-text · Article · Jun 2012 · Journal of Cellular Biochemistry
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    • "EETs induce HO-1 expression and signalling cascade [107], including activation of AMP-activated kinase (AMPK) and pAKT, thus reducing adiposity and insulin resistance in animal model of obesity and diabetes. In addition, EETs decrease MSC-derived adipocyte stem cell differentiation by the upregulation of HO-1-adiponectin-AKT signalling, suggesting that EET agonist may have potential therapeutic role in the treatment of dyslipidemia, diabetes, and the metabolic syndrome [108]. The potential action of EETs as intracellular lipid signalling modulators of adipogenesis was further supported by the recent finding that the treatment with EET agonists inhibits adipogenesis and decreases the levels of inflammatory cytokines. "
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    ABSTRACT: Cardiovascular risk factors contribute to enhanced oxidative stress which leads to endothelial dysfunction. These events trigger platelet activation and their interaction with leukocytes and endothelial cells, thus contributing to the induction of chronic inflammatory processes at the vascular wall and to the development of atherosclerotic lesions and atherothrombosis. In this scenario, endogenous antioxidant pathways are induced to restrain the development of vascular disease. In the present paper, we will discuss the role of heme oxygenase (HO)-1 which is an enzyme of the heme catabolism and cleaves heme to form biliverdin and carbon monoxide (CO). Biliverdin is reduced enzymatically to the potent antioxidant bilirubin. Recent evidence supports the involvement of HO-1 in the antioxidant and antiinflammatory effect of cyclooxygenase(COX)-2-dependent prostacyclin in the vasculature. Moreover, the role of HO-1 in estrogen vasoprotection is emerging. Finally, possible strategies to develop novel therapeutics against cardiovascular disease by targeting the induction of HO-1 will be discussed.
    Full-text · Article · Feb 2012
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