ERp46 binds to AdipoR1, but not AdipoR2, and modulates adiponectin signalling

The pleiotropic effects of the insulin-sensitizing adipokine adiponectin are mediated, at least in part, by two seven-transmembrane domain receptors AdipoR1 and AdipoR2. Recent reports indicate a role for AdipoR-binding proteins, namely APPL1, RACK1 and CK2β, in proximal signal transduction events. Here we demonstrate that endoplasmic reticulum protein 46 (ERp46) interacts specifically with AdipoR1 and provide evidence that ERp46 modulates adiponectin signalling. Co-immunoprecipitation followed by mass spectrometry identified ERp46 as an AdipoR1-, but not AdipoR2-, interacting protein. Analysis of truncated constructs and GST-fusion proteins revealed the interaction was mediated by the cytoplasmic, N-terminal residues (1-70) of AdipoR1. Indirect immunofluorescence microscopy and subcellular fractionation studies demonstrated that ERp46 was present in the ER and the plasma membrane (PM). Transient knockdown of ERp46 increased the levels of AdipoR1, and AdipoR2, at the PM and this correlated with increased adiponectin-stimulated phosphorylation of AMPK. In contrast, adiponectin-stimulated phosphorylation of p38MAPK was reduced following ERp46 knockdown. Collectively these results establish ERp46 as the first AdipoR1-specific interacting protein and suggest a role for ERp46 in adiponectin receptor biology and adiponectin signalling.     

Picroside III,an Active Ingredient of Picrorhiza scrophulariiflora,Ameliorates Experimental Colitis by Protecting Intestinal Barrier Integrity

This study aims to explore the protective effects of Picroside III, an active ingredient of Picrorhiza scrophulariiflora, on the intestinal epithelial barrier in tumor necrosis factor-α (TNF-α) induced Caco-2 cells and dextran sulfate sodium (DSS) induced colitis in mice. Results show that Picroside III significantly alleviated clinical signs of colitis including body weight loss, disease activity index increase, colon shortening, and colon tissue damage. It also increased claudin-3, ZO-1 and occludin expressions and decreased claudin-2 expression in the colon tissues of mice with colitis. In vitro, Picroside III also significantly promoted wound healing, decreased the permeability of cell monolayer, upregulated the expressions of claudin-3, ZO-1 and occludin and downregulated the expression of claudin-2 in TNF-α treated Caco-2 cells. Mechanism studies show that Picroside III significantly promoted AMP-activated protein kinase (AMPK) phosphorylation in vitro and in vivo, and blockade with AMPK could significantly attenuate the upregulation of Picroside III in ZO-1 and occludin expressions and the downregulation of claudin-2 expression in TNF-α treated Caco-2 cells. In conclusion, this study demonstrates that Picroside III attenuated DSS-induced colitis by promoting colonic mucosal wound healing and epithelial barrier function recovery via the activation of AMPK.     

Ginsenoside Rg1 protects H9c2 cells against nutritional stress-induced injury via aldolase /AMPK/PINK1 signalling

Insufficient nutrients supply will greatly affect the function of cardiac myocytes. The adaptive responses of cardiac myocytes to nutritional stress are not fully known. Ginsenoside Rg1 is one of the most pharmacologically active components in Panax Ginseng and possesses protective effects on cardiomyocyte. Here, we investigate the effects of ginsenoside Rg1 on H9c2 cells which were subjected to nutritional stress. Nutritional stress-induced by glucose deprivation strongly induced cell death and this response was inhibited by ginsenoside Rg1. Importantly, glucose deprivation decreased intracellular ATP levels and mitochondrial membrane potential. Ginsenoside Rg1 rescued ATP levels and mitochondrial membrane potential in nutrient-starved cells. For molecular mechanisms, ginsenoside Rg1 increased the expressions of PTEN-induced kinase 1 (PINK1) and p-AMPK in glucose deprivation treated H9c2 cells. Reducing the expression of aldolase in H9c2 cells inhibited ginsenoside Rg1′s actions on PINK1 and p-AMPK. Further, the nutritional stress mice were used to verify the mechanisms obtained in vitro. Ginsenoside Rg1 increased the expressions of aldolase, p-AMPK, and PINK1 in starved mice heart. Taken together, our results reveal that ginsenoside Rg1 limits nutritional stress-induced H9c2 cells injury by regulating the aldolase /AMP-activated protein kinase/PINK1 pathway.     

Autophagy induction by xanthoangelol exhibits anti‐metastatic activities in hepatocellular carcinoma

Xanthoangelol (XAG), a prenylated chalcone isolated from the Japanese herb Angelica keiskei Koidzumi, has been reported to exhibit antineoplastic properties. However, the specific anti‐tumor activity of XAG in human hepatocellular carcinoma (HCC), and the relevant mechanisms are not known. Herein, we evaluated the effect of XAG against HCC in vitro and in vivo. Although XAG treatment did not significantly reduce the viability of the Hep3B and Huh7 cell lines, it suppressed cell migration, invasion, and EMT. This anti‐metastatic effect of XAG was due to induction of autophagy, because treatment with the autophagy inhibitor 3‐methyadenine (3‐MA) or knockdown of the pro‐autophagy Beclin‐1 effectively abrogated the XAG‐induced suppression of metastasis. Mechanistically, XAG induced autophagy via activation of the AMPK/mTOR signaling pathway, and XAG treatment dramatically increased the expression of p‐AMPK while decreasing p‐mTOR expression. In addition, blocking AMPK/mTOR axis with compound C abrogated the autophagy‐mediated inhibition of metastasis. The murine model of HCC metastasis also showed that XAG effectively reduced the number of metastatic pulmonary nodules. Taken together, our results revealed that autophagy via the activation of AMPK/mTOR pathway is essential for the anti‐metastatic effect of XAG against HCC. These findings not only contribute to our understanding of the anti‐tumor activity of XAG but also provide a basis for its clinical application in HCC. Before this study, evidence of XAG on HCC was purely anecdotal; present study provides the first comprehensive assessments of XAG on HCC metastasis and investigates its underlying mechanism. Results suggest that XAG exerts anti‐metastatic properties against HCC through inducing autophagy which is mediated by the activation of AMPK/mTOR signaling pathway. This research extends our knowledge about the antineoplastic properties of XAG and suggests that induction autophagy may represent future treatment strategies for metastatic HCC.     

TIPE1‐mediated autophagy suppression promotes nasopharyngeal carcinoma cell proliferation via the AMPK/mTOR signalling pathway

Recent studies have shown that tumour necrosis factor‐α–induced protein 8 like‐1(TIPE1) plays distinct roles in different cancers. TIPE1 inhibits tumour proliferation and metastasis in a variety of tumours but acts as an oncogene in cervical cancer. The role of TIPE1 in nasopharyngeal carcinoma (NPC) remains unknown. Interestingly, TIPE1 expression was remarkably increased in NPC tissue samples compared to adjacent normal nasopharyngeal epithelial tissue samples in our study. TIPE1 expression was positively correlated with that of the proliferation marker Ki67 and negatively correlated with patient lifespan. In vitro, TIPE1 inhibited autophagy and induced cell proliferation in TIPE1‐overexpressing CNE‐1 and CNE‐2Z cells. In addition, knocking down TIPE1 expression promoted autophagy and decreased proliferation, whereas overexpressing TIPE1 increased the levels of pmTOR, pS6 and P62 and decreased the level of pAMPK and the LC3B. Furthermore, the decrease in autophagy was remarkably rescued in TIPE1‐overexpressing CNE‐1 and CNE‐2Z cells treated with the AMPK activator AICAR. In addition, TIPE1 promoted tumour growth in BALB/c nude mice. Taken together, results indicate that TIPE1 promotes NPC progression by inhibiting autophagy and inducing cell proliferation via the AMPK/mTOR signalling pathway. Thus, TIPE1 could potentially be used as a valuable diagnostic and prognostic biomarker for NPC.     

Exosomes derived from umbilical cord mesenchymal stem cells alleviate viral myocarditis through activating AMPK/mTOR‐mediated autophagy flux pathway

Human umbilical cord mesenchymal stem cell‐derived exosomes (hucMSC‐exosomes) have been implicated as a novel therapeutic approach for tissue injury repair and regeneration, but the effects of hucMSC‐exosomes on coxsackievirus B3 (CVB3)‐induced myocarditis remain unknown. The object of the present study is to investigate whether hucMSC‐exosomes have therapeutic effects on CVB3‐induced myocarditis (VMC). HucMSC‐exosomes were identified using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and Western blot. The purified hucMSC‐exosomes tagged with PKH26 were tail intravenously injected into VMC model mice in vivo and used to administrate CVB3‐infected human cardiomyocytes (HCMs) in vitro, respectively. The effects of hucMSC‐exosomes on myocardial pathology injury, proinflammatory cytokines and cardiac function were evaluated through haematoxylin and eosin (H&E) staining, quantitative polymerase chain reaction (qPCR) and Doppler echocardiography. The anti‐apoptosis role and potential mechanism of hucMSC‐exosomes were explored using TUNEL staining, flow cytometry, immunohistochemistry, Ad‐mRFP‐GFP‐LC3 transduction and Western blot. In vivo results showed that hucMSC‐exosomes (50 μg iv) significantly alleviated myocardium injury, shrank the production of proinflammatory cytokines and improved cardiac function. Moreover, in vitro data showed that hucMSC‐exosomes (50 μg/mL) inhibited the apoptosis of CVB3‐infected HCM through increasing pAMPK/AMPK ratio and up‐regulating autophagy proteins LC3II/I, BECLIN‐1 and anti‐apoptosis protein BCL‐2 as well as decreasing pmTOR/mTOR ratio, promoting the degradation of autophagy flux protein P62 and down‐regulating apoptosis protein BAX. In conclusion, hucMSC‐exosomes could alleviate CVB3‐induced myocarditis via activating AMPK/mTOR‐mediated autophagy flux pathway to attenuate cardiomyocyte apoptosis, which will be benefit for MSC‐exosome therapy of myocarditis in the future.     

有氧运动对糖尿病大鼠PPARα信号通路的影响及其与PPARγ关系*

目的: 探讨4周有氧运动对糖尿病大鼠血糖血脂的改善作用及其与PPARα和PPARγ的调控关系。方法: 6周龄雄性SD大鼠8周高脂饮食喂养后一次性腹腔注射链脲佐菌素以建立糖尿病模型大鼠。除普通膳食对照组(Con)外,建模成功的糖尿病大鼠随机分成糖尿病对照组(DM)、糖尿病运动组(TDM)、糖尿病运动加PPARγ激动剂吡格列酮组(TDP)和糖尿病运动加PPARγ抑制剂GW9662组(TDG),每组8只。TDP组和TDG组大鼠在运动前分别补充吡格列酮和GW9662,TDM、TDP和TDG组大鼠进行4周中等强度递增负荷跑台运动(第1周15 m/min,30 min;第2周15 m/min,60 min;第3周20 m/min,60 min;第4周20 m/min,90 min),每周运动6 d,每天1次。运动期间所有大鼠给予普通饲料。最后一次运动结束后36 h,大鼠麻醉、取血,然后处死大鼠、收集肝和腓肠肌。检测空腹血糖血脂指标(血糖、血胰岛素和血脂四项)。Western blot方法检测肝和腓肠肌PPARα、PPARα上游分子腺苷酸活化蛋白激酶(AMPK)和下游分子肉碱棕榈酰转移酶-1(CPT1)的蛋白水平。结果: ①与Con组大鼠比较,DM大鼠FBG(＞11.1 mmol/L)和血清TC、TG、LDL水平显著升高(P均＜0.01),表明DM造模成功。②与DM大鼠比较,TDM大鼠血糖血脂改善的同时,肝和腓肠肌PPARα、AMPK和CPT1的蛋白水平均显著升高(P均＜0.01)。③与TDM大鼠比较,TDG大鼠肝和腓肠肌的PPARα和CPT1,以及肝AMPK的蛋白水平无显著变化(仅腓肠肌AMPK显著降低(P＜0.05));而TDP大鼠的肝和腓肠肌PPARα、AMPK和CPT1蛋白水平均显著提高(P均＜0.01)。结论: 有氧运动对DM大鼠血糖血脂的改善作用-与运动激活肝和腓肠肌的AMPK-PPARα-CPT1通路有关。运动对DM大鼠PPARα通路的激活与PPARγ无关,但PPARγ激活可进一步增强运动对AMPK-PPARα-CPT1蛋白水平上调的作用。     

Development of a potent and orally active activator of adenosine monophosphate-activated protein kinase (AMPK), ASP4132, as a clinical candidate for the treatment of human cancer

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) plays a key role in maintaining cellular metabolism. AMP or adenosine diphosphate (ADP) levels rise during metabolic stress, such as during nutrient starvation, hypoxia and muscle contraction, and bind to AMPK to induce activity. Recently, activation of AMPK has been considered an attractive therapeutic strategy in the field of human oncology. Structural optimization of lead compound 2, a new type of AMPK activator with potent AMPK activation activity and attractive selective growth inhibition against human cancer cells, improved aqueous solubility, metabolic stability and animal pharmacokinetics (PK) and culminated in the identification of (5-{1-[(6-methoxypyridin-3-yl)methyl]piperidin-4-yl}-1H-benzimidazol-2-yl)(4-{[4-(trifluoromethyl)phenyl]methyl}piperazin-1-yl)methanone ditosylate, ASP4132 (28). Studies on ASP4132 had advanced to clinical trials for the treatment of cancer.