Qun Zhu

Resveratrol Attenuates Renal Hypertrophy in Early-Stage Diabetes by Activating AMPK

Background: Recent studies suggest the involvement of the adenosine monophosphate-activated serine/threonine protein kinase (AMPK) pathway in the pathogenesis of diabetic nephropathy (DN). Resveratrol, an agent that activates AMPK, may have the potential to protect against the development of DN. This study was designed to investigate the therapeutic effects of resveratrol on renal hypertrophy in early-stage diabetes and the underlying mechanisms. Method: Molecular and structural changes involved in the pathogenesis of DN were tested in a rat model of early-stage diabetes. Renal mesangial cells (RMCs) were cultured in media containing different concentrations of glucose with or without resveratrol. Cellular DNA synthesis was assayed by measuring 3H-thymidine incorporation. The phosphorylation status of AMPK, eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), and phospho- ribosomal protein S6 (S6) was analyzed by Western blot. Results: Resveratrol reduced plasma creatinine and urinary albumin excretion and attenuated renal hypertrophy without affecting blood glucose levels. Moreover, resveratrol activated AMPK and inhibited phosphorylation of 4E-BP1 and S6 in diabetic rat kidneys. In vitro, resveratrol blocked high glucose-induced dephosphorylation of AMPK and phosphorylation of 4E-BP1 and S6 and strongly inhibited both the DNA synthesis and proliferation of RMCs. Conclusion: These findings suggest the possibility that resveratrol exerts antiproliferative, antihypertrophic effects by activating AMPK and reducing 4E-BP1 and S6 phosphorylation, thus suppressing the development and progression of DN.

Heme oxygenase-1 enhances autophagy in podocytes as a protective mechanism against high glucose-induced apoptosis.

Injury and loss of podocytes play vital roles in diabetic nephropathy progression. Emerging evidence suggests autophagy, which is induced by multiple stressors including hyperglycemia, plays a protective role. Meanwhile, heme oxygenase-1 (HO-1) possesses powerful anti-apoptotic properties. Therefore, we investigated the impact of autophagy on podocyte apoptosis under diabetic conditions and its association with HO-1. Mouse podocytes were cultured in vitro; apoptosis was detected by flow cytometry. Transmission electron microscopy and biochemical autophagic flux assays were used to measure the autophagy markers microtubule-associated protein 1 light chain 3-II (LC3-II) and beclin-1. LC3-II and beclin-1 expression peaked 12-24h after exposing podocytes to high glucose. Inhibition of autophagy with 3-methyladenine or Beclin-1 siRNAs or Atg 5 siRNAs sensitized cells to apoptosis, suggesting autophagy is a survival mechanism. HO-1 inactivation inhibited autophagy, which aggravated podocyte injury in vitro. Hemin-induced autophagy also protected podocytes from hyperglycemia in vitro and was abrogated by HO-1 siRNA. Adenosine monophosphate-activated protein kinase phosphorylation was higher in hemin-treated and lower in HO-1 siRNA-treated podocytes. Suppression of AMPK activity reversed HO-1-mediated Beclin-1 upregulation and autophagy, indicating HO-1-mediated autophagy is AMPK dependent. These findings suggest HO-1 induction and regulation of autophagy are potential therapeutic targets for diabetic nephropathy.

Chronic activation of neutral ceramidase protects β-cells against cytokine-induced apoptosis

AbstractAim:To investigate the activity and expression of neutral ceramidase (N-CDase) in the insulin-secreting cell line INS-1 and its role in the cellular response to cytokines.Methods:HPLC, Western blotting, and quantitative real-time PCR were performed to detect the activity and expression of N-CDase in INS-1 cells treated with a cytokine mixture (5 ng/mL interleukin-1β, 10 ng/mL TNF-α, and 50 ng/mL interferon-γ). The expression and activity of N-CDase in the INS-1 cells were specifically inhibited using N-CDase-siRNA transfection. Annexin V-fluorescein-isothiocyanate/propidium iodide flow cytometry was used to assess apoptosis in the INS-1 cells.Results:The INS-1 cells exhibited some basal N-CDase activity, and cytokines induced a time-dependent delay in the activation of N-CDase. As a result, the activation of N-CDase was first detectable at 8 h after stimulation. It peaked at 16 h and remained elevated at 24 h. Cytokines also upregulated the mRNA and protein expression of N-CDase in the INS-1 cells. Furthermore, when N-CDase activity was inhibited by RNA interference, cytokine-induced apoptosis in the INS-1 cells was markedly increased.Conclusion:The N-CDase pathway is active in INS-1 cells, and the chronic activation of N-CDase is involved in the pathological response of β-cells to cytokines, potentially providing protection against cytokine toxicity.

Acute activation of acid ceramidase affects cytokine-induced cytotoxicity in rat islet β-cells

Ceramidase hydrolyzes ceramide and produces sphingosine as a substrate of sphingosine kinase (SPHK), which transforms sphingosine to sphingosine‐1‐phosphate. It has been reported that cytokines elicit SPHK activation in rat β‐cells. As a sphingosine provider, ceramidase should also be activated. In our previous work, we showed that the increase in mRNA and protein levels in cytokine‐treated INS‐1 rat β‐cells resulted in chronic activation of neutral ceramidase. Here we found that acid ceramidase (AC) is activated by cytokines at an early stage via tyrosine phosphorylation. In addition, basal AC activity was first detected in INS‐1 cells and isolated rat islets, and cytokine‐induced cell growth was significantly repressed when AC was pharmacologically inhibited.

Low‐dose cytokine‐induced neutral ceramidase secretion from INS‐1 cells via exosomes and its anti‐apoptotic effect

It has been reported that the effect of inflammatory cytokines on β‐cell destruction in type 1 diabetes is concentration‐dependent. However, the underlying mechanisms remain unclear. In the present study, we found that a high concentration of cytokines promoted apoptosis in the rat β‐cell line INS‐1, whereas a low concentration of cytokines had no effect. We also found that cytokines at a low concentration stimulated neutral ceramidase (NCDase) release via exosomes from INS‐1 cells, whereas cytokines at a high concentration inhibited NCDase release. Furthermore, the results showed that the NCDase‐containing exosomes isolated from the culture medium of INS‐1 cells treated with cytokines at a low concentration inhibited apoptosis induced by a high concentration of cytokines. Finally, the results also showed that the protective action of NCDase in the exosomes on apoptosis was mediated by the generation of sphingosine 1‐phosphate (S1P) and its interaction with S1P receptor 2. Taken together, these findings revealed a novel NCDase‐S1P‐phosphate‐S1P receptor 2‐dependent mechanism by which a low level of inflammatory cytokines protects pancreatic β‐cells from apoptosis induced by a high level of inflammatory cytokines.

Tauroursodeoxycholate, a chemical chaperone, prevents palmitate-induced apoptosis in pancreatic β-cells by reducing ER stress.

BACKGROUND Free fatty acids (FFA) can have deleterious effects on β-cells and promote type 2 diabetes, a process known as lipotoxicity. Recently, the induction of endoplasmic reticulum (ER) stress is one mechanism proposed to contribute to the detrimental effects of FFA on β-cells. Tauroursodeoxycholic acid (TUDCA) has been reported to show cytoprotective effects by alleviating ER stress induced by some cytotoxic stimuli. The aim of this study was to investigate the effects of TUDCA on FFA (palmitate)-induced apoptosis and ER stress in rat islet β-cells. METHODS The rat pancreatic β-cell line INS-1 was cultured with palmitate (0.5 mM), or cultured togther with TUDCA (100 μM), Annexin V-fluorescein-isothiocyanate/propidium iodide flow cytometry was used to assess apoptosis in INS-1 cells. Cell viability was evaluated with MTT reduction conversion assay. The expressions of ER stress marker GRP78, ER stress-associated pro-apoptotic effectors CHOP and ATF4 were detected by Western blotting. RESULTS TUDCA significantly reduced palmitate-induced cell apoptosis and growth inhibition in INS-1 cells. TUDCA also attenuated palmitate-induced expressions of GRP78, CHOP and ATF4 in INS-1 cells. CONCLUSIONS Our results thus suggested that TUDCA could protect INS-1 cells from palmitate-induced injury, which might be due to the amelioration of ER stress and blocking the ATF4/CHOP signaling pathway.

Neutral ceramidase activity inhibition is involved in palmitate-induced apoptosis in INS-1 cells

Neutral ceramidase (NCDase) is a class of ceramidases, a key enzyme in ceramide degradation. Recently, it was observed that NCDase activity was suppressed by saturated fatty acids to increase ceramide content in rat muscle. However, little is known about its changes in activity and roles in palmitate (Palm)-induced lipotoxicity in pancreatic β cells. Here, we demonstrated that Palm treatment significantly down-regulated NCDase activity, mRNA and protein levels in rat INS-1 cells. In addition, Palm caused a significant accumulation of ceramide, while SPH level remained unchanged, suggesting that inhibition of NCDase activity led to no change of SPH level after treatment with Palm for 24 h. Furthermore, NCDase overexpression significantly reduced Palm-induced apoptosis in INS-1 cells. Conversely, NCDase siRNA knockdown markedly exacerbated Palm-induced apoptosis. In conclusion, Palm treatment suppressed the activity of NCDase and down-regulated its mRNA and protein expression. Furthermore, NCDase inhibition was involved in Palm-induced apoptosis by blocking ceramide degradation in INS-1 cells.

Neutral Ceramidase Secreted Via Exosome Protects Against Palmitate-Induced Apoptosis in INS-1 Cells

Aims: To investigate the effects of neutral ceramidase (NCDase) packaged in exosomes that are secreted from β-cells on free fatty acid (FFA)-induced β-cells apoptosis and its role in regulation of sphingolipid-mediated signaling pathway. Methods: HPLC and Western blotting were performed to determine NCDase activity and expression. Annexin V-fluorescein-isothiocyanate/propidium iodide flow cytometry was used to assess apoptosis. Electrospray ionization tandem mass spectrometry was used for ceramide (Cer), sphingosine-1-phosphate (S1P), and sphingosine (SPH) determination. Results: INS-1 cells over-expressed NCDase secreted active NCDase via exosomes. Exosomes isolated from the cultured medium of INS-1 cells that oxpressed NCDase could ameliorate palmitate-induced apoptosis. Furthermore, the results showed that exosome-derived NCDase treatment reduced intracellular Cer/S1P ratio. Conclusions: β-cell secreted active NCDase via exosome, the exosome-packaged-NCDase protected β-cells from FFA-induced apoptosis through regulating sphingolipid metabolites and it might be a potential treatment for β-cell lipotoxicity and type 2 diabetes.