Alpha-lipoic acid regulates the autophagy of vascular smooth muscle cells in diabetes by elevating hydrogen sulfide level

Dysfunctional vascular smooth muscle (VSM) plays a vital role in the process of atherosclerosis in patients with type 2 diabetes mellitus (T2DM). Alpha-lipoic acid (ALA) can prevent the altered VSM induced by diabetes. However, the precise mechanism underlying the beneficial effect of ALA is not well understood. This study aimed to determine whether ALA ameliorates VSM function by elevating hydrogen sulfide (H₂S) level in diabetes and whether this effect is associated with regulation of autophagy of VSM cells (VSMCs). We found decreased serum H₂S levels in Chinese patients and rats with type 2 diabetes mellitus (T2DM). ALA treatment could increase H₂S level, which reduced the autophagy-related index and activation of the 5′-monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway, thereby protecting vascular function in rats with T2DM. Propargylglycine (PPG), a cystathionine-γ-lyase inhibitor, could weaken the ALA effect. In cultured VSMCs, high glucose level also reduced H₂S level, upregulated the autophagy-related index and activated the AMPK/mTOR pathway, which were reversed by concomitant application of sodium hydrosulfide (NaHS, an H₂S donor) or ALA. The protective effect of NaHS or ALA was attenuated by rapamycin (an autophagy activator), 5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide (an AMPK activator) or PPG. In contrast, Compound C (an AMPK inhibitor) enhanced the effect of ALA or NaHS. ALA may have a protective effect on VSMCs in T2DM by elevating H₂S level and downregulating autophagy via the AMPK/mTOR pathway. This study provides a new target for addressing diabetic macroangiopathy.     

Hydrogen sulfide mitigates hyperglycemic remodeling via liver kinase B1-adenosine monophosphate-activated protein kinase signaling

Hyperglycemia (HG) reduces AMPK activation leading to impaired autophagy and matrix accumulation. Hydrogen sulfide (H₂S) treatment improves HG-induced renovascular remodeling however, its mechanism remains unclear. Activation of LKB1 by the formation of heterotrimeric complex with STRAD and MO25 is known to activate AMPK. We hypothesized that in HG; H₂S induces autophagy and modulates matrix synthesis through AMPK-dependent LKB1/STRAD/MO25 complex formation. To address this hypothesis, mouse glomerular endothelial cells were treated with normal and high glucose in the absence or presence of sodium hydrogen sulfide (NaHS), an H₂S donor. HG decreased the expression of H₂S regulating enzymes CBS and CSE, and autophagy markers Atg5, Atg7, Atg3 and LC3B/A ratio. HG increased galectin-3 and periostin, markers of matrix accumulation. Treatment with NaHS to HG cells increased LKB1/STRAD/MO25 formation and AMPK phosphorylation. Silencing the encoded genes confirmed complex formation under normoglycemia. H₂S-mediated AMPK activation in HG was associated with upregulation of autophagy and diminished matrix accumulation. We conclude that H₂S mitigates adverse remodeling in HG by induction of autophagy and regulation of matrix metabolism through LKB1/STRAD/MO25 dependent pathway.     

Toll‐like receptor signalling cross‐activates the autophagic pathway to restrict Salmonella Typhimurium growth in macrophages

It has been long recognised that activation of toll‐like receptors (TLRs) induces autophagy to restrict intracellular bacterial growth. However, the mechanisms of TLR‐induced autophagy are incompletely understood. Salmonella Typhimurium is an intracellular pathogen that causes food poisoning and gastroenteritis in humans. Whether TLR activation contributes to S. Typhimurium‐induced autophagy has not been investigated. Here, we report that S. Typhimurium and TLRs shared a common pathway to induce autophagy in macrophages. We first showed that S. Typhimurium‐induced autophagy in a RAW264.7 murine macrophage cell line was mediated by the AMP‐activated protein kinase (AMPK) through activation of the TGF‐β‐activated kinase (TAK1), a kinase activated by multiple TLRs. AMPK activation led to increased phosphorylation of Unc‐51‐like autophagy activating kinase (ULK1) at S317 and S555. ULK1 phosphorylation at these two sites in S. Typhimurium‐infected macrophages overrode the inhibitory effect of mTOR on ULK1 activity due to mTOR‐mediated ULK1 phosphorylation at S757. Lipopolysaccharide (LPS), flagellin, and CpG oligodeoxynucleotide, which activate TLR4, TLR5, and TLR9, respectively, increased TAK1 and AMPK phosphorylation and induced autophagy in RAW264.7 cells and in bone marrow‐derived macrophages. However, LPS was unable to induce TAK1 and AMPK phosphorylation and autophagy in TLR4‐deficient macrophages. TAK1 and AMPK‐specific inhibitors blocked S. Typhimurium‐induced autophagy and xenophagy and increased the bacterial growth in RAW264.7 cells. These observations collectively suggest that activation of the TAK1–AMPK axis through TLRs is essential for S. Typhimurium‐induced autophagy and that TLR signalling cross‐activates the autophagic pathway to clear intracellular bacteria.     

Activation of AMPK participates hydrogen sulfide-induced cyto-protective effect against dexamethasone in osteoblastic MC3T3-E1 cells

Long-time glucocorticoids (GCs) usage causes osteoporosis. In the present study, we explored the potential role of hydrogen sulfide (H₂S) against dexamethasone (Dex)-induced osteoblast cell damage, and focused on the underlying mechanisms. We showed that two H₂S-producing enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), were significantly downregulated in human osteonecrosis tissues as well as in Dex-treated osteoblastic MC3T3-E1 cells. H₂S donor NaHS as well as the CBS activator S-adenosyl-l-methionine (SAM) inhibited Dex-induced viability reduction, death and apoptosis in MC3T3-E1 cells. NaHS activated adenosine monophosphate (AMP)-activated protein kinase (AMPK) signaling, which participated its cyto-protective activity. AMPK inhibition by its inhibitor (compound C) or reduction by targeted-shRNA suppressed its pro-survival activity against Dex in MC3T3-E1 cells. Further, we found that NaHS inhibited Dex-mediated reactive oxygen species (ROS) production and ATP depletion. Such effects by NaHS were again inhibited by compound C and AMPKα1-shRNA. In summary, we show that H₂S inhibits Dex-induced osteoblast damage through activation of AMPK signaling. H₂S signaling might be further investigated as a novel target for anti-osteoporosis treatment.     

Hydrogen sulfide improves left ventricular function in smoking rats via regulation of apoptosis and autophagy

The present study was designed to investigate the protective effects of hydrogen sulfide (H₂S) against cigarette smoking-induced left ventricular dysfunction in rats. Left ventricular structure and function were assessed using two-dimensional echocardiography. Cardiomyocyte apoptosis was determined by Annexin V/PI and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining. Cardiac autophagy was evaluated by detection of autophagy-related protein expression and observation of autophagosomes. Our results indicated that administration of NaHS (a donor of H₂S) could protect against smoking-induced left ventricular systolic dysfunction. H₂S was found to exert anti-apoptotic effects in the myocardium of smoking rats by inhibiting JNK and P38 mitogen-activated protein kinases pathways and activating PI3K/Akt signaling. Moreover, H₂S could also reduce smoking-induced autophagic cell death via regulation of AMPK/mTOR signaling pathway. In conclusion, our study demonstrates that H₂S can improve left ventricular systolic function in smoking rats via regulation of apoptosis and autophagy.     

Endogenous Prostaglandins and Afferent Sensory Nerves in Gastroprotective Effect of Hydrogen Sulfide against Stress-Induced Gastric Lesions

Hydrogen sulfide (H₂S) plays an important role in human physiology, exerting vasodilatory, neuromodulatory and anti-inflammatory effects. H₂S has been implicated in the mechanism of gastrointestinal integrity but whether this gaseous mediator can affect hemorrhagic lesions induced by stress has been little elucidated. We studied the effect of the H₂S precursor L-cysteine, H₂S-donor NaHS, the H₂S synthesizing enzyme (CSE) activity inhibitor- D,L-propargylglycine (PAG) and the gastric H₂S production by CSE/CBS/3-MST activity in water immersion and restraint stress (WRS) ulcerogenesis and the accompanying changes in gastric blood flow (GBF). The role of endogenous prostaglandins (PGs) and sensory afferent nerves releasing calcitonin gene-related peptide (CGRP) in the mechanism of gastroprotection induced by H₂S was examined in capsaicin-denervated rats and those pretreated with capsazepine to inhibit activity of vanilloid receptors (VR-1). Rats were pretreated with vehicle, NaHS, the donor of H₂S and or L-cysteine, the H₂S precursor, with or without the concurrent treatment with 1) nonselective (indomethacin) and selective cyclooxygenase (COX)-1 (SC-560) or COX-2 (rofecoxib) inhibitors. The expression of mRNA and protein for COX-1 and COX-2 were analyzed in gastric mucosa pretreated with NaHS with or without PAG. Both NaHS and L-cysteine dose-dependently attenuated severity of WRS-induced gastric lesions and significantly increased GBF. These effects were significantly reduced by pretreatment with PAG and capsaicin denervation. NaHS increased gastric H₂S production via CSE/CBS but not 3-MST activity. Inhibition of COX-1 and COX-2 activity significantly diminished NaHS- and L-cysteine-induced protection and hyperemia. NaHS increased expression of COX-1, COX-2 mRNAs and proteins and raised CGRP mRNA expression. These effects of NaHS on COX-1 and COX-2 protein contents were reversed by PAG and capsaicin denervation. We conclude that H₂S exerts gastroprotection against WRS-induced gastric lesions by the mechanism involving enhancement in gastric microcirculation mediated by endogenous PGs, sensory afferent nerves releasing CGRP and the activation of VR-1 receptors.     

Activation of AMPK protects against hydrogen peroxide-induced osteoblast apoptosis through autophagy induction and NADPH maintenance: New implications for osteonecrosis treatment?

Elevated hydrogen peroxide (H₂O₂) causes osteoblast dysfunction and apoptosis, serving as an important contributor to the development of osteonecrosis. Here we aimed to understand the role of AMP-activated protein kinase (AMPK) in the process. We observed a high level of AMPK activation in surgery isolated patients' osteonecrosis tissues. In cultured osteoblastoma MG63 cells, H₂O₂ stimulation induced significant AMPK activation, oxidative stress, cell death and apoptosis. Inhibition of AMPK by its inhibitor (compound C) or by shRNA-mediated knockdown dramatically enhanced H₂O₂-induced MG63 cell apoptosis, while over-expression of AMPK in HEK-293 cells alleviated H₂O₂-induced cell damage. These results confirmed that H₂O₂-activated AMPK is pro-cell survival. We observed that H₂O₂ induced protective autophagy in MG63 cells, and AMPK-dependent Ulk1 activation and mTORC1 (mTOR complex 1) inactivation might involve autophagy activation. Further, AMPK activation inhibited H₂O₂-induced oxidative stress, probably through inhibiting NADPH (nicotinamide adenine dinucleotide phosphate) depletion, since more NADPH depletion and oxidative stress were induced by H₂O₂ in AMPK deficient MG63 cells. Finally, we observed a significant AMPK activation in H₂O₂-treated primary cultured and transformed (MC3T3-E1) osteoblasts, and AMPK inhibitor compound C enhanced death by H₂O₂ in these cells. Based on these results, we concluded that H₂O₂-induced AMPK activation is pro-survival and anti-apoptosis in osteoblasts. Autophagy induction and NADPH maintenance are involved in AMPK-mediated pro-survival effects. AMPK might represent a novel molecular target for osteonecrosis treatment.     

Starvation-induced autophagy is regulated by mitochondrial reactive oxygen species leading to AMPK activation

Starvation is the most extensively studied condition that induces autophagy. Previous studies have demonstrated that starvation-induced autophagy is regulated by reactive oxygen species (ROS) such as superoxide (O₂^?) but the source for ROS under starvation conditions and the downstream signaling pathways regulating autophagy are unclear. In this study, a cervical cancer HeLa cell line was generated that was deficient in mitochondrial electron transport chain (mETC) (HeLa ρ° cells). This resulted in endogenous levels of O₂^? being significantly reduced and failed to be induced under starvation of glucose, L-glutamine, pyruvate, and serum (GP) or of amino acids and serum (AA) compared to wild type (wt) HeLa cells. In contrast, H₂O₂ production failed to increase under GP starvation in both wild type and ρ° cells whereas it increased in wt cells but not in ρ° cells under AA starvation. GP or AA starvation induced autophagy was blocked in ρ° cells as determined by the amount of autophagosomes and autolysosomes. Autophagy is regulated by 5′ adenosine monophosphate-activated protein kinase (AMPK) activation and AMPK is activated under starvation conditions. We demonstrate that ρ° cells and HeLa cells over expressing manganese-superoxide dismutase 2 (SOD2) cells fail to activate AMPK activation following starvation. This indicates that mitochondrial ROS might regulate AMPK activation. In addition, inhibiting AMPK activation either by siRNA or compound C resulted in reduced autophagy during starvation. Using a ROS scavenger NAC, AMPK activation is reduced under starvation condition and mTOR signaling is increased. Taken together, mitochondria-generated ROS induces autophagy mediated by the AMPK pathway under starvation conditions.     

Activation of Autophagic Flux against Xenoestrogen Bisphenol-A-induced Hippocampal Neurodegeneration via AMP kinase (AMPK)/Mammalian Target of Rapamycin (mTOR) Pathways

The human health hazards related to persisting use of bisphenol-A (BPA) are well documented. BPA-induced neurotoxicity occurs with the generation of oxidative stress, neurodegeneration, and cognitive dysfunctions. However, the cellular and molecular mechanism(s) of the effects of BPA on autophagy and association with oxidative stress and apoptosis are still elusive. We observed that BPA exposure during the early postnatal period enhanced the expression and the levels of autophagy genes/proteins. BPA treatment in the presence of bafilomycin A₁ increased the levels of LC3-II and SQSTM1 and also potentiated GFP-LC3 puncta index in GFP-LC3-transfected hippocampal neural stem cell-derived neurons. BPA-induced generation of reactive oxygen species and apoptosis were mitigated by a pharmacological activator of autophagy (rapamycin). Pharmacological (wortmannin and bafilomycin A₁) and genetic (beclin siRNA) inhibition of autophagy aggravated BPA neurotoxicity. Activation of autophagy against BPA resulted in intracellular energy sensor AMP kinase (AMPK) activation, increased phosphorylation of raptor and acetyl-CoA carboxylase, and decreased phosphorylation of ULK1 (Ser-757), and silencing of AMPK exacerbated BPA neurotoxicity. Conversely, BPA exposure down-regulated the mammalian target of rapamycin (mTOR) pathway by phosphorylation of raptor as a transient cell''s compensatory mechanism to preserve cellular energy pool. Moreover, silencing of mTOR enhanced autophagy, which further alleviated BPA-induced reactive oxygen species generation and apoptosis. BPA-mediated neurotoxicity also resulted in mitochondrial loss, bioenergetic deficits, and increased PARKIN mitochondrial translocation, suggesting enhanced mitophagy. These results suggest implication of autophagy against BPA-mediated neurodegeneration through involvement of AMPK and mTOR pathways. Hence, autophagy, which arbitrates cell survival and demise during stress conditions, requires further assessment to be established as a biomarker of xenoestrogen exposure.     

Mechanistic insight of platelet apoptosis leading to non-surgical bleeding among heart failure patients supported by continuous-flow left ventricular assist devices

Vascular endothelial cells are highly sensitive to oxidative stress, and this is one of the mechanisms by which widespread endothelial dysfunction is induced in most cardiovascular diseases and disorders. However, how these cells can survive in oxidative stress environments remains unclear. Salidroside, a traditional Chinese medicine, has been shown to confer vascular protective effects. We aimed to understand the role of autophagy and its regulatory mechanisms by treating human umbilical vein endothelial cells (HUVECs) with salidroside under oxidative stress. HUVECs were treated with salidroside and exposed to hydrogen peroxide (H₂O₂). The results indicated that salidroside exerted cytoprotective effects in an H₂O₂-induced HUVEC injury model and suppressed H₂O₂-induced apoptosis of HUVECs. Pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, increased oxidative stress-induced HUVEC apoptosis, while the autophagy activator rapamycin induced anti-apoptosis effects in HUVECs. Salidroside increased autophagy and decreased apoptosis of HUVECs in a dose-dependent manner under oxidative stress. Moreover, 3-MA attenuated salidroside-induced HUVEC autophagy and promoted apoptosis, whereas rapamycin had no additional effects compared with salidroside alone. Salidroside upregulated AMPK phosphorylation but downregulated mTOR phosphorylation under oxidative stress; however, administration of compound C, an AMPK inhibitor, abrogated AMPK phosphorylation and increased mTOR phosphorylation and apoptosis compared with salidroside alone. These results suggest that autophagy is a protective mechanism in HUVECs under oxidative stress and that salidroside might promote autophagy through activation of the AMPK pathway and downregulation of mTOR pathway.     

Pimozide reduces toxic forms of tau in TauC3 mice via 5′ adenosine monophosphate‐activated protein kinase‐mediated autophagy

In neurodegenerative diseases like Alzheimer's disease (AD), tau is hyperphosphorylated and forms aggregates and neurofibrillary tangles in affected neurons. Autophagy is critical to clear the aggregates of disease‐associated proteins and is often altered in patients and animal models of AD. Because mechanistic target of rapamycin (mTOR) negatively regulates autophagy and is hyperactive in the brains of patients with AD, mTOR is an attractive therapeutic target for AD. However, pharmacological strategies to increase autophagy by targeting mTOR inhibition cause various side effects. Therefore, autophagy activation mediated by non‐mTOR pathways is a new option for autophagy‐based AD therapy. Here, we report that pimozide activates autophagy to rescue tau pathology in an AD model. Pimozide increased autophagic flux through the activation of the AMPK‐Unc‐51 like autophagy activating kinase 1 (ULK1) axis, but not of mTOR, in neuronal cells, and this function was independent of dopamine D2 receptor inhibition. Pimozide reduced levels of abnormally phosphorylated tau aggregates in neuronal cells. Further, daily intraperitoneal (i.p.) treatment of pimozide led to a recovery from memory deficits of TauC3 mice expressing a caspase‐cleaved form of tau. In the brains of these mice, we found increased phosphorylation of AMPK1 and ULK1, and reduced levels of the soluble oligomers and NP40‐insoluble aggregates of abnormally phosphorylated tau. Together, these results suggest that pimozide rescues memory impairments in TauC3 mice and reduces tau aggregates by increasing autophagic flux through the mTOR‐independent AMPK‐ULK1 axis.     

Hydrogen sulfide induces human colon cancer cell proliferation: Role of Akt,ERK and p21

H₂S (hydrogen sulfide), regarded as the third gaseous transmitter, is implicated in ulcerative colitis and colorectal cancers. The present study investigates the effects of H₂S on cell proliferation in human colon cancer HCT 116 cells and SW480 cells. We identified the two key enzymes, CBS and CSE, for H₂S synthesis in HCT 116 cells. An exogenously administered H₂S donor NaHS induced cell proliferation in a concentration‐dependent manner, with optimal proliferative concentration at 200 μmol/l. NaHS administration increased Akt and ERK phosphorylation. Blockade of Akt and ERK activation attenuated NaHS‐induced cell proliferation. Cell‐cycle analysis showed that NaHS treatment for 6 h decreased the proportion of cells in G₀–G₁ phase and increased the proportion of cells in S phase. Protein expressions of Cyclin D1 and PCNA (proliferating cell nuclear antigen) were not altered, but the cyclin‐dependent kinase inhibitor p21^Waf1/Cip1 was inhibited significantly by NaHS treatment. NaHS significantly reduced NO metabolite levels. In conclusion, NaHS induced human colon cancer cell proliferation. This effect might be mediated by the increase of Akt and ERK phosphorylation and the decrease of p21^Waf1/Cip1 expression and NO production. The results suggested a role for H₂S in human colonic cancer development.     

To die or to live: the dual role of poly(ADP-ribose) polymerase-1 in autophagy and necrosis under oxidative stress and DNA damage

Poly(ADP-ribose) polymerase-1 (PARP-1), activated by DNA strand breaks, participates in the DNA repair process physiologically. Excessive activation of PARP-1 mediates necrotic cell death under the status of oxidative stress and DNA damage. However, it remains elusive whether and how PARP-1 activation is involved in autophagy and what is the function of PARP-1-mediated autophagy under oxidative stress and DNA damage. We recently demonstrate that hydrogen peroxide (H₂O₂) induces autophagy through a novel autophagy signalling mechanism linking PARP-1 activation to the LKB1-AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) pathway. Furthermore, PARP-1-mediated autophagy plays a cytoprotective role in H₂O₂-induced necrotic cell death as suppression of autophagy greatly sensitizes H2O2-induced cell death. Our study thus identifies a novel function of PARP-1 in mediating autophagy and it appears that PAPR-1 possesses a dual role in modulating necrosis and autophagy under oxidative stress and DNA damage: on the one hand, overactivation of PARP-1 leads to ATP depletion and necrotic cell death; on the other hand, PARP-1 activation promotes autophagy via the LKB1-AMPK-mTOR pathway to enhance cell survival. The cellular decision of life or death depends on the balance between autophagy and necrosis mediated by these two distinct pathways.     

Hydrogen Sulfide Promotes Adipogenesis in 3T3L1 Cells

The effect of hydrogen sulfide (H₂S) on differentiation of 3T3L1-derived adipocytes was examined. Endogenous H₂S was increased after 3T3L1 differentiation. The expression of the H₂S-synthesising enzymes, cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST), was increased in a time-dependent manner during 3T3L1 differentiation. Expression of genes associated with adipogenesis related genes including fatty acid binding protein 4 (FABP4/aP2), a key regulator of this process, was increased by GYY4137 (a slow-releasing H₂S donor compound) and sodium hydrosulfide (NaHS, a classical H₂S donor) but not by ZYJ1122 or time-expired NaHS. Furthermore expression of these genes were reduced by aminooxyacetic acid (AOAA, CBS inhibitor), DL-propargylglycine (PAG, CSE inhibitor) as well as by CSE small interference RNA (siCSE) and siCBS. The size and number of lipid droplets in mature adipocytes was significantly increased by both GYY4137 and NaHS, which also impaired the ability of CL316,243 (β3-agonist) to promote lipolysis in these cells. In contrast, AOAA and PAG had the opposite effect. Taken together, we show that the H₂S-synthesising enzymes CBS, CSE and 3-MST are endogenously expressed during adipogenesis and that both endogenous and exogenous H₂S modulate adipogenesis and adipocyte maturation.     

Neuroprotective effects of hydrogen sulfide on Parkinson’s disease rat models

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by a progressive loss of dopaminergic neurons in the substantia nigra (SN). The present study was designed to examine the therapeutic effect of hydrogen sulfide (H₂S, a novel biological gas) on PD. The endogenous H₂S level was markedly reduced in the SN in a 6‐hydroxydopamine (6‐OHDA)‐induced PD rat model. Systemic administration of NaHS (an H₂S donor) dramatically reversed the progression of movement dysfunction, loss of tyrosine‐hydroxylase positive neurons in the SN and the elevated malondialdehyde level in injured striatum in the 6‐OHDA‐induced PD model. H₂S specifically inhibited 6‐OHDA evoked NADPH oxidase activation and oxygen consumption. Similarly, administration of NaHS also prevented the development of PD induced by rotenone. NaHS treatment inhibited microglial activation in the SN and accumulation of pro‐inflammatory factors (e.g. TNF‐α and nitric oxide) in the striatum via NF‐κB pathway. Moreover, significantly less neurotoxicity was found in neurons treated with the conditioned medium from microglia incubated with both NaHS and rotenone compared to that with rotenone only, suggesting that the therapeutic effect of NaHS was, at least partially, secondary to its suppression of microglial activation. In summary, we demonstrate for the first time that H₂S may serve as a neuroprotectant to treat and prevent neurotoxin‐induced neurodegeneration via multiple mechanisms including anti‐oxidative stress, anti‐inflammation and metabolic inhibition and therefore has potential therapeutic value for treatment of PD.     

Autophagy-dependent and -independent involvement of AMP-activated protein kinase in 6-hydroxydopamine toxicity to SH-SY5Y neuroblastoma cells

The role of the main intracellular energy sensor adenosine monophosphate (AMP)-activated protein kinase (AMPK) in the induction of autophagic response and cell death was investigated in SH-SY5Y human neuroblastoma cells exposed to the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA). The induction of autophagy in SH-SY5Y cells was demonstrated by acridine orange staining of intracellular acidic vesicles, the presence of autophagosome- and autophagolysosome-like vesicles confirmed by transmission electron microscopy, as well as by microtubule-associated protein 1 light-chain 3 (LC3) conversion and p62 degradation detected by immunoblotting. 6-OHDA induced phosphorylation of AMPK and its target Raptor, followed by the dephosphorylation of the major autophagy inhibitor mammalian target of rapamycin (mTOR) and its substrate p70S6 kinase (S6K). 6-OHDA treatment failed to suppress mTOR/S6K phosphorylation and to increase LC3 conversion, p62 degradation and cytoplasmatic acidification in neuroblastoma cells in which AMPK expression was downregulated by RNA interference. Transfection of SH-SY5Y cells with AMPK or LC3β shRNA, as well as treatment with pharmacological autophagy inhibitors suppressed, while mTOR inhibitor rapamycin potentiated 6-OHDA-induced oxidative stress and apoptotic cell death. 6-OHDA induced phosphorylation of p38 mitogen-activated protein (MAP) kinase in an AMPK-dependent manner, and pharmacological inhibition of p38 MAP kinase reduced neurotoxicity, but not AMPK activation and autophagy triggered by 6-OHDA. Finally, the antioxidant N-acetyl cysteine antagonized 6-OHDA-induced activation of AMPK, p38 and autophagy. These data suggest that oxidative stress-mediated AMPK/mTOR-dependent autophagy and AMPK/p38-dependent apoptosis could be valid therapeutic targets for neuroprotection.     

Hydrogen sulfide inhibits high glucose-induced matrix protein synthesis by activating AMP-activated protein kinase in renal epithelial cells

Hydrogen sulfide, a signaling gas, affects several cell functions. We hypothesized that hydrogen sulfide modulates high glucose (30 mm) stimulation of matrix protein synthesis in glomerular epithelial cells. High glucose stimulation of global protein synthesis, cellular hypertrophy, and matrix laminin and type IV collagen content was inhibited by sodium hydrosulfide (NaHS), an H(2)S donor. High glucose activation of mammalian target of rapamycin (mTOR) complex 1 (mTORC1), shown by phosphorylation of p70S6 kinase and 4E-BP1, was inhibited by NaHS. High glucose stimulated mTORC1 to promote key events in the initiation and elongation phases of mRNA translation: binding of eIF4A to eIF4G, reduction in PDCD4 expression and inhibition of its binding to eIF4A, eEF2 kinase phosphorylation, and dephosphorylation of eEF2; these events were inhibited by NaHS. The role of AMP-activated protein kinase (AMPK), an inhibitor of protein synthesis, was examined. NaHS dose-dependently stimulated AMPK phosphorylation and restored AMPK phosphorylation reduced by high glucose. Compound C, an AMPK inhibitor, abolished NaHS modulation of high glucose effect on events in mRNA translation as well as global and matrix protein synthesis. NaHS induction of AMPK phosphorylation was inhibited by siRNA for calmodulin kinase kinase β, but not LKB1, upstream kinases for AMPK; STO-609, a calmodulin kinase kinase β inhibitor, had the same effect. Renal cortical content of cystathionine β-synthase and cystathionine γ-lyase, hydrogen sulfide-generating enzymes, was significantly reduced in mice with type 1 diabetes or type 2 diabetes, coinciding with renal hypertrophy and matrix accumulation. Hydrogen sulfide is a newly identified modulator of protein synthesis in the kidney, and reduction in its generation may contribute to kidney injury in diabetes.     

Calcineurin suppresses AMPK-dependent cytoprotective autophagy in cardiomyocytes under oxidative stress

Calcineurin signalling plays a critical role in the pathogenesis of many cardiovascular diseases. Calcineurin has been proven to affect a series of signalling pathways and to exert a proapoptotic effect in cardiomyocytes. However, whether it is able to regulate autophagy remains largely unknown. Here, we report that prolonged oxidative stress-induced activation of calcineurin contributes to the attenuation of adaptive AMP-activated protein kinase (AMPK) signalling and inhibits autophagy in cardiomyocytes. Primary cardiomyocytes exhibited rapid formation of autophagosomes, microtubule-associated protein 1 light chain 3 (LC3) expression and phosphorylation of AMPK in response to hydrogen peroxide (H₂O₂) treatment. However, prolonged (12 h) H₂O₂ treatment attenuated these effects and was accompanied by a significant increase in calcineurin activity and apoptosis. Inhibition of calcineurin by FK506 restored AMPK function and LC3 expression, and decreased the extent of apoptosis caused by prolonged oxidative stress. In contrast, overexpression of the constitutively active form of calcineurin markedly attenuated the increase in LC3 induced by short-term (3 h) H₂O₂ treatment and sensitised cells to apoptosis. In addition, FK506 failed to induce autophagy and alleviate apoptosis in cardiomyocytes expressing a kinase-dead K45R AMPK mutant. Furthermore, inhibition of autophagy by 3-methylanine (3-MA) or by knockdown of the essential autophagy-related gene ATG7 abrogated the protective effect of FK506. These findings suggest a novel role of calcineurin in suppressing adaptive autophagy during oxidative stress by downregulating the AMPK signalling pathway. The results also provide insight into how altered calcineurin and autophagic signalling is integrated to control cell survival during oxidative stress and may guide strategies to prevent cardiac oxidative damage.     

β‐Guanidinopropionic acid extends the lifespan of Drosophila melanogaster via an AMP‐activated protein kinase‐dependent increase in autophagy

Previous studies have demonstrated that AMP‐activated protein kinase (AMPK) controls autophagy through the mammalian target of rapamycin (mTOR) and Unc‐51 like kinase 1 (ULK1/Atg1) signaling, which augments the quality of cellular housekeeping, and that β‐guanidinopropionic acid (β‐GPA), a creatine analog, leads to a chronic activation of AMPK. However, the relationship between β‐GPA and aging remains elusive. In this study, we hypothesized that feeding β‐GPA to adult Drosophila produces the lifespan extension via activation of AMPK‐dependent autophagy. It was found that dietary administration of β‐GPA at a concentration higher than 900 mm induced a significant extension of the lifespan of Drosophila melanogaster in repeated experiments. Furthermore, we found that Atg8 protein, the homolog of microtubule‐associated protein 1A/1B‐light chain 3 (LC3) and a biomarker of autophagy in Drosophila, was significantly upregulated by β‐GPA treatment, indicating that autophagic activity plays a role in the effect of β‐GPA. On the other hand, when the expression of Atg5 protein, an essential protein for autophagy, was reduced by RNA interference (RNAi), the effect of β‐GPA on lifespan extension was abolished. Moreover, we found that AMPK was also involved in this process. β‐GPA treatment significantly elevated the expression of phospho‐T172‐AMPK levels, while inhibition of AMPK by either AMPK‐RNAi or compound C significantly attenuated the expression of autophagy‐related proteins and lifespan extension in Drosophila. Taken together, our results suggest that β‐GPA can induce an extension of the lifespan of Drosophila via AMPK‐Atg1‐autophagy signaling pathway.     

Differential mechanisms underlying neuroprotection of hydrogen sulfide donors against oxidative stress

This study investigated whether slow-releasing organic hydrogen sulfide donors act through the same mechanisms as those of inorganic donors to protect neurons from oxidative stress. By inducing oxidative stress in a neuronal cell line HT22 with glutamate, we investigated the protective mechanisms of the organic donors: ADT-OH [5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione], the most widely used moiety for synthesizing slow-releasing hydrogen sulfide donors, and ADT, a methyl derivative of ADT-OH. The organic donors were more potent than the inorganic donor sodium hydrogensulfide (NaHS) in protecting HT22 cells against glutamate toxicity. Consistent with previous publications, NaHS partially restored glutamate-depleted glutathione (GSH) levels, protected HT22 from direct free radical damage induced by hydrogen peroxide (H₂O₂), and NaHS protection was abolished by a K_ATP channel blocker glibenclamide. However, neither ADT nor ADT-OH enhanced glutamate-depleted GSH levels or protected HT22 from H₂O₂-induced oxidative stress. Glibenclamide, which abolished NaHS neuroprotection against oxidative stress, did not block ADT and ADT-OH neuroprotection against glutamate-induced oxidative stress. Unexpectedly, we found that glutamate induced AMPK activation and that compound C, a well-established AMPK inhibitor, remarkably protected HT22 from glutamate-induced oxidative stress, suggesting that AMPK activation contributed to oxidative glutamate toxicity. Interestingly, all hydrogen sulfide donors, including NaHS, remarkably attenuated glutamate-induced AMPK activation. However, under oxidative glutamate toxicity, compound C only increased the viability of HT22 cells treated with NaHS, but did not further increase ADT and ADT-OH neuroprotection. Thus, suppressing AMPK activation likely contributed to ADT and ADT-OH neuroprotection. In conclusion, hydrogen sulfide donors acted through differential mechanisms to confer neuroprotection against oxidative toxicity and suppressing AMPK activation was a possible mechanism underlying neuroprotection of organic hydrogen sulfide donors against oxidative toxicity.