Effects of exercise training on adipogenesis of stromal-vascular fraction cells in rat epididymal white adipose tissue

Aim: Previous studies have shown that exercise training reduced white adipose tissue (WAT) mass compared to that in sedentary controls, and that the smaller mass contained fewer adipocytes. However, the effect of exercise training on adipogenesis is not completely clear. Therefore, we re-examined the effect of exercise training on adipocyte numbers in WAT and, if such an effect was found tested the adipogenic responses of stromal-vascular fraction (SVF) cells containing adipose tissue-derived stem cells (ADSC) in epididymal WAT from exercise-trained (TR) rats. Methods: Wistar male rats were divided into two groups: control (C) and TR. The TR rats were subjected to exercise on a treadmill for 9 weeks. SVF cells containing ADSC were separated from epididymal WAT by centrifugation. Expression of adipocyte differentiation-related genes and adipogenesis of SVF cells were examined. Results: In SVF cells of TR rats, the expression of peroxisome proliferator-activated receptor γ (PPARγ) and that of PPARγ target lipogenic genes was dramatically downregulated, whereas that of preadipocyte factor-1 gene was significantly upregulated. Lipid accumulation in SVF cells of TR rats after the induction of adipocyte differentiation was significantly suppressed in comparison with that of C rats. Moreover, increased expression of hypoxia-inducible factor-1α (HIF-1α) protein was observed in SVF cells of TR rats. Pre-treatment of YC-1, a potent HIF-1α inhibitor, in SVF cells of TR rats restored adipogenesis. Conclusion: These results suggest that exercise training suppresses the ability of SVF cells to differentiate into adipocytes, and that underlying mechanisms involve the upregulation of HIF-1α expression.     

PPARγ-dependent peptidoglycan recognition protein 3 (PGlyRP3) expression regulates proinflammatory cytokines by microbial and dietary fatty acids

PGlyRPs recognize bacterial peptidoglycan and function in antibacterial innate immunity. Focusing on the interference between nutrition and recognition pattern proteins, free fatty acids (FFA) of dietary and bacterial sources may exert their immunological response through modulating the expression level of the PGlyRPs in enterocytes. PGlyRP3 was the only PGlyRPs member expressed in Caco2 cells. In silico analysis showed that the promoter of PGlyRP3 has some PPRE regions that, as tested by EMSA, bind physically to the PPARγ-RXRα complex. PGlyRP3 gene expression was induced by PPARγ ligands including GW1929 and some FFA. Overexpression of PGlyRP3 in Caco2 cells down regulated the expression of the inflammatory cytokines IL-8, IL-12 and TNF-α, while its silencing increased the expression of these cytokines. FFA that induced the PGlyRP3 inhibited the tested cytokines. Silencing of PGlyRP3 gene caused the same FFA to increase the cytokine gene expression. A negative regulation of NF-κB pathway, including up-regulation of Iκβ-α and down regulation of NF-κB and COX-2, is involved in the anti-inflammatory effects of PGlyRP3. In conclusion, PPARγ mediates a modulation of PGlyRP3 gene expression, which is involved in inhibiting inflammation through negative regulation of NF-κB pathway.     

IFNγ‐responsiveness of endothelial cells leads to efficient angiostasis in tumours involving down‐regulation of Dll4

Although IFNγ is regarded as a key cytokine in angiostatic response, our poor understanding of its effective cellular target drastically limits its clinical trials against angiogenesis‐related disorders. Here, we investigated the effect of IFNγ on endothelial cells (ECs) and possible molecular mechanisms in angiostasis. By employing Tie2^IFNγR mice, in which IFNγR expression was reconstituted under the control of Tie2 promoter in IFNγR‐deficient mice, we found that the response of ECs to IFNγ was highly effective in inhibiting blood supply and retarding tumour growth. Interestingly, the expression of IFNγR on Tie2^? cells did not inhibit, but promoted tumour growth in control wild‐type mice. Mechanism studies showed that IFNγ reacting on ECs down‐regulated the delta‐like ligand 4 (Dll4)/Notch signalling pathway. Accordingly, overexpression of Dll4 in human ECs diminished the effect of IFNγ on ECs. This study demonstrates that the action of IFNγ on ECs, but not other cells, is highly effective for tumour angiostasis, which involves down‐regulating Dll4. It provides insights for EC‐targeted angiostatic therapy in treating angiogenesis‐associated disorders in the clinic. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Interferon Alpha Treatment of Patients with Impaired Interferon Gamma Signaling

Patients with deficiency in the interferon gamma receptor (IFN-γR) are unable to respond properly to IFN-γ and develop severe infections with nontuberculous mycobacteria (NTM). IFN-γ and IFN-α are known to signal through STAT1 and activate many downstream effector genes in common. Therefore, we added IFN-α for treatment of patients with disseminated mycobacterial disease in an effort to complement their IFN-γ signaling defect. We treated four patients with IFN-γR deficiency with adjunctive IFN-α therapy in addition to best available antimicrobial therapy, with or without IFN-γ, depending on the defect. During IFN-α treatment, ex vivo induction of IFN target genes was detected. In addition, IFN-α driven gene expression in patients’ cells and mycobacteria induced cytokine response were observed in vitro. Clinical responses varied in these patients. IFN-α therapy was associated with either improvement or stabilization of disease. In no case was disease exacerbated. In patients with profoundly impaired IFN-γ signaling who have refractory infections, IFN-α may have adjunctive anti-mycobacterial effects.     

Multiple roles of PPARα in brown adipose tissue under constitutive and cold conditions

Peroxisome proliferator‐activated receptor α (PPARα) is a member of the nuclear receptor family, regulating fatty acid degradation in many organs. Two‐dimensional SDS‐PAGE of brown adipose tissue (BAT) from PPARα‐null mice produced a higher‐density spot. Proteomic analysis indicated that the protein was pyruvate dehydrogenase β (PDHβ). To observe PDHβ regulation in BAT, the organ was stimulated by long‐term cold exposure, and the activities of associated enzymes were investigated. Histological and biochemical analyses of BAT showed a significant decrease in the triglyceride content in wild‐type mice and some degree of decrease in PPARα‐null mice on cold exposure. Analyses of molecules related to glucose metabolism showed that the expression of PDHβ is under PPARα‐specific regulation, and that glucose degradation ability may decrease on cold exposure. In contrast, analyses of molecules related to fatty acid metabolism showed that numerous PPARα/γ target molecules are induced on cold exposure, and that fatty acid degradation ability in wild‐type mice is markedly enhanced and also increases to same degree in PPARα‐null mice on cold exposure. Thus, this study proposes novel and multiple roles of PPARα in BAT.     

IFN-α cannot substitute lack of IFN-γ responsiveness in cells of an IFN-γR1 deficient patient

Patients with complete IFN-γR deficiency are unable to respond to IFN-γ and have impaired Th1-immunity and recurrent, severe infections with weakly virulent Mycobacteria. Since IFN-α and IFN-γ share signalling pathways, treatment with IFN-α has been proposed in complete IFN-γR deficiency. We stimulated cells from healthy controls and from a patient lacking IFN-γR1 with IFN-α and IFN-γ, to establish whether IFN-α would substitute for IFN-γ effects. IFN-α induced STAT1 phosphorylation in monocytes of the IFN-γR1(-/-) patient, but did not prime for LPS-induced IL-12p70, IL-12p40, IL-23 or TNF production. In control cells, IFN-α inhibited the priming effect of IFN-γ on LPS-induced pro-inflammatory cytokine release. Finally, IFN-γ but not IFN-α induced killing of M. smegmatis in cultured macrophages. In conclusion, no evidence was found to support the use of IFN-α in IFN-γR-deficient patients as intervention against mycobacterial infection; on the contrary, treatment of individuals with IFN-α may even adversely affect host defence against Mycobacteria.     

Detection of Fcγ receptors on human endothelial cells stimulated with cytokines tumour necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ)

This investigation was conducted to detect Fcγ receptors (FcγR) on cytokine-stimulated human endothelial cells (EC) by measuring anti-FcγR MoAb binding with an ELISA. TNF-α and IFN-γ significantly increased the expression of FcγR type II (FcγRII) and type III (FcγRIII) on aortic EC. Simultaneous treatment with both cytokines had a synergistic effect and pretreatment of EC with IFN-γ augmented the effect of TNF-α. The greatest effect was the increase (up to four-to-six-fold) in expression of FcγRII found by the simultaneous treatment of aortic EC with both cytokines. The receptors were expressed on the cell surface and showed receptor capping after incubation at 37°C. This study showed that the inflammatory cytokines TNF-α and IFN-γ enhanced low-affinity FcγR expression on human EC in vitro. The expression of FcγR may contribute to the specific localization of circulating immune complexes on blood vessels in areas of vasculitis.     

过氧化物酶增殖物活化受体γ调节胰腺癌生长部分依赖于NF-κB和AP-1

目的：探讨过氧化物酶增殖物活化受体γ（PPARγ）在人胰腺癌生长中的调节作用，以及核因子-κB（NF-κB）和活化蛋白（AP-1）在该过程的变化，旨在进一步揭示PPARγ抑制胰腺癌生长的机制。 方法： 培养细胞经PPARγ配体15-脱氧-前列腺素J2（15d-PGJ2）、RXRα配体9-顺式-维甲酸（9-cis-RA）及其联合作用后，用MTT法测定细胞活力，并评价药物的抗增殖效果；用TransAMTM方法检测其对SW1990细胞中核因子-κB （NF- κB） p65活性蛋白表达的影响；用逆转录聚合酶链式反应（RT-PCR）检测其对SW1990细胞活化蛋白-1（AP-1）表达的调节作用。 结果： 15d-PGJ2和9-cis-RA及其联合应用对胰腺癌细胞的增殖均具有抑制作用，且呈剂量依赖性。9-cis-RA对15d-PGJ2抑制胰腺癌细胞增殖具有协同效应。TransAMTM检测显示，15d-PGJ2、9-cis-RA及其联合作用干预组NF- κB p65活性蛋白含量均表现为先降后升的趋势，但都未达到对照组的水平。RT-PCR揭示随着15d-PGJ2、9-cis-RA及其联合作用浓度的提高，c-jun mRNA表达水平均呈现先增高后降低的趋势；在15d-PGJ2或9-cis-RA单独干预组中，c-fos mRNA表达水平逐渐减弱；而在两者联合作用组中表现为逐渐增强的趋势。 结论： PPARγ的活化在体外对胰腺癌的生长呈负调节作用。RXRα的激活可协同增强PPARγ激动剂的抗增殖作用。其机制可能是其通过下调NF-κB和AP-1表达而实现。     

Role of PPARγ in COX-2 Activation in Mycobacterial Pulmonary Inflammation

Preliminary studies show that intranasal (i.n.) administration of BCG in mice induces M1 activation of alveolar macrophages (M?) that increase TNF-α production and cyclooxygenase-2 (COX-2) expression but reduce constitutive peroxisome proliferator-activated receptor gamma (PPARγ) expression. However, COX-2 is catalytically inactive for prostaglandin E(2) release, unlike COX-2 that is active in M1 activation in vitro by BCG. In this study, we determined the role of PPARγ for BCG-induced M1 activation in vivo and in vitro. We found that treatment of mice with GW9662, a PPARγ antagonist, prior to i.n. BCG, partially restored PPARγ expression, and decreased TNF-α production and COX-2 expression. But COX-2 was still inactive. The decreased effects on TNF-α and COX-2 were also observed when alveolar M? were treated in vitro with GW9662/BCG, but COX-2 was still active. Our results indicate that PPARγ upregulates M1 activation of alveolar M?, but inactive COX-2 formation is independent of PPARγ in mycobacterial pulmonary inflammation.     

IL-6 regulates exercise and training-induced adaptations in subcutaneous adipose tissue in mice

Aim: The aim of this study was to test the hypothesis that IL‐6 regulates exercise‐induced gene responses in subcutaneous adipose tissue in mice. Methods: Four‐month‐old male IL‐6 whole body knockout (KO) mice and C57B wild‐type (WT) mice performed 1 h of treadmill exercise, where subcutaneous adipose tissue (AT) was removed either immediately after, 4 h or 10 h after exercise as well as from mice not running acutely. Moreover, AT was sampled at resting conditions after 5 weeks of exercise training. Results: AT leptin mRNA decreased immediately after a single running exercise bout in both genotypes and returned to baseline within 10 h of recovery in IL‐6 KO mice, but not WT mice. Leptin mRNA content decreased in WT and increased in IL‐6 KO mice with training, but without significant alterations in leptin protein. Acute exercise induced a decrease in the AT TNFα mRNA content in WT, but not in IL‐6‐KO mice, while training lowered resting levels of TNFα mRNA in both genotypes. In addition, an exercise‐induced decline in AT PPARγ mRNA content was absent in IL‐6 KO mice and in line training increased PPARγ mRNA only in IL‐6 KO mice. Conclusion: The present findings indicate a role of IL‐6 in regulating exercise‐ and training‐induced leptin and PPARγ expression in adipose tissue. In addition, while IL‐6 is required for TNF‐α mRNA reduction in response to acute exercise, IL‐6 does not appear to be mandatory for anti‐inflammatory effects of exercise training in adipose tissue.     

腺病毒介导的mPPARγ1基因转染抑制IFN-γ诱导ECV304细胞galectin-9基因和蛋白表达

目的：观察腺病毒介导的mPPARγ1转染抑制IFN-γ诱导ECV304细胞galectin-9基因和蛋白表达。方法：构建表达小鼠PPARγ1基因的复制缺陷型腺病毒表达载体；将融合80%的ECV304细胞给予不同刺激量（1×104 U/L、5×104 U/L、1×105 U/L和2×105 U/L）的IFN-γ干预；将IFN-γ（1×105 U/L）预刺激并孵育 24 h 的ECV304细胞分成对照组（C）、PPARγ基因过度表达组(P)、PPARγ活化剂曲格列酮干预组(T)以及PPARγ基因过度表达和曲格列酮共刺激组(PT)进行干预，观察不同剂量IFN-γ对ECV304细胞galectin-9基因和蛋白表达的作用，以及PPARγ基因过度表达和/或活化对上述作用的影响。结果：正常ECV304细胞galectin-9基因表达弱。IFNγ孵育24 h后， ECV304细胞galectin-9基因和蛋白表达增加，且galectin-9表达与IFN-γ具有量效关系。PPARγ1基因转染抑制IFN-γ诱导galectin-9基因/蛋白表达，曲格列酮对上述作用无影响；PPARγ1基因转染和曲格列酮共刺激抑制IFN-γ诱导galectin-9基因/蛋白表达与单一PPARγ1基因转染效应相似。正常ECV304细胞PPARγ表达量低，而PPARγ基因过表达和活化不影响内源性PPARγ基因表达。结论：PPARγ1基因转染抑制IFN-γ诱导ECV304细胞galectin-9基因/蛋白表达可能是PPARγ基因发挥免疫调控作用的一个重要机制。     

Resveratrol prevents TNFα-induced suppression of adiponectin expression via PPARγ activation in 3T3-L1 adipocytes

Tumor necrosis factor α (TNFα) is an adipokine, whose increase is known to suppress the expression and secretion of adiponectin in adipocytes. Resveratrol has been ever reported to recover the suppression of adiponectin by TNFα, but the underlying mechanism remains poorly understood. In this study, we validated the roles of resveratrol in the inhibition of the adiponectin by TNFα in 3T3-L1 cells. Exposure to TNFα for 24 h inhibited adiponectin synthesis and secretion, but the inhibitions were partially recovered by resveratrol treatment in 3T3-L1 adipocytes. Furthermore, we found that resveratrol improved the expression of adiponectin by the increase of PPARγ DNA-binding activity. Our results suggest that resveratrol may attenuate the inhibition of adiponectin expression by TNFα via activation of PPARγ, thereby possibly improving insulin resistance. However, significant preventive effects of resveratrol were only observed when it was administrated before TNFα increase, limiting its use as preventive strategy for insulin resistance.     

Macrophage PPARγ and impaired wound healing in type 2 diabetes

Macrophages undergo a transition from pro‐inflammatory to healing‐associated phenotypes that is critical for efficient wound healing. However, the regulation of this transition during normal and impaired healing remains to be elucidated. In our studies, the switch in macrophage phenotypes during skin wound healing was associated with up‐regulation of the peroxisome proliferator‐activated receptor (PPAR)γ and its downstream targets, along with increased mitochondrial content. In the setting of diabetes, up‐regulation of PPARγ activity was impaired by sustained expression of IL‐1β in both mouse and human wounds. In addition, experiments with myeloid‐specific PPARγ knockout mice indicated that loss of PPARγ in macrophages is sufficient to prolong wound inflammation and delay healing. Furthermore, PPARγ agonists promoted a healing‐associated macrophage phenotype both in vitro and in vivo, even in the diabetic wound environment. Importantly, topical administration of PPARγ agonists improved healing in diabetic mice, suggesting an appealing strategy for down‐regulating inflammation and improving the healing of chronic wounds. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Increasing proliferation of murine adipose tissue-derived mesenchymal stem cells by TNF-α plus IFN-γ

The objective of the current study was to assess the effects of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) along with their simultaneous application on proliferation and pluripotency genes of murine adipose tissue-derived mesenchymal stem cells (AT-MSCs). The proliferation, doubling time (DT), colony-forming unit–fibroblast (CFU-F), pluripotency genes expression, and proliferation-related immunomodulatory markers of MSCs were analyzed upon activation with TNF-α (10?ng/ml), IFN-γ (10?ng/ml) and both TNF-α and IFN-γ (5?ng/ml?+?5?ng/ml). Pluripotency genes including Oct-4, Sox-2, and Nanog as well as proliferation-associated immunomodulatory cytokines such as insulin-like growth factor 1 (IGF-1) and transforming growth factor-β (TGF-β) expression were evaluated using real-time PCR. Surface expression of Qa2 (HLA-G) was analyzed by flow cytometry. Pretreatment of MSCs with TNF-α plus IFN-γ led to significantly increased proliferation, DT and CFU-F as well as expression of pluripotency genes in AT-MSCs (p < 0.01). MSCs expressed more IGF-1, TGF-β, and Qa2 upon activation with TNF-α plus IFN-γ and IFN-γ. MSCs expressed significantly decreased amounts of TGF-β and Qa2 in presence of TNF-α. TNF-α combined with IFN-γ may be improved the proliferation of AT-MSCs. Conversely, expanded MSCs pointed out low levels of the immunomodulatory marker, s especially Qa2 in the presence of TNF-α. In conclusion, we showed that TNF-α together with IFN-γ increased the proliferation of MSCs and slightly enhanced the expression of pluripotency genes.