Mitochondrial Hexokinase II Research Articles

Natural product triptolide induces GSDME-mediated pyroptosis in head and neck cancer through suppressing mitochondrial hexokinase-\u0399\u0399

BackgroundPyroptosis is a lytic cell death form executed by gasdermins family proteins. Induction of tumor pyroptosis promotes anti-tumor immunity and is a potential cancer treatment strategy. Triptolide (TPL) is a natural product isolated from the traditional Chinese herb which possesses potent anti-tumor activity in human cancers. However, its role in pyroptosis remains to be elucidated.MethodsCell survival was measured by colony formation assay. Cell apoptosis was determined by Annexin V assay. Pyroptosis was evaluated by morphological features and release of interleukin 1β and lactate dehydrogenase A (LDHA). Immunofluorescence staining was employed to measure subcellular localization of proteins. Tumorigenicity was assessed by a xenograft tumor model. Expression levels of mRNAs or proteins were determined by qPCR or western blot assay, respectively.ResultsTriptolide eliminates head and neck cancer cells through inducing gasdermin E (GSDME) mediated pyroptosis. Silencing GSDME attenuates the cytotoxicity of TPL against cancer cells. TPL treatment suppresses expression of c-myc and mitochondrial hexokinase II (HK-II) in cancer cells, leading to activation of the BAD/BAX-caspase 3 cascade and cleavage of GSDME by active caspase 3. Silencing HK-II sensitizes cancer cells to TPL induced pyroptosis, whereas enforced expression of HK-II prevents TPL induced pyroptosis. Mechanistically, HK-II prevents mitochondrial translocation of BAD, BAX proteins and activation of caspase 3, thus attenuating cleavage of GSDME and pyroptosis upon TPL treatment. Furthermore, TPL treatment suppresses NRF2/SLC7A11 (also known as xCT) axis and induces reactive oxygen species (ROS) accumulation, regardless of the status of GSDME. Combination of TPL with erastin, an inhibitor of SLC7A11, exerts robust synergistic effect in suppression of tumor survival in vitro and in a nude mice model.ConclusionsThis study not only provides a new paradigm of TPL in cancer therapy, but also highlights a crucial role of mitochondrial HK-II in linking glucose metabolism with pyroptosis.

Regulation of Parkin-dependent mitophagy by Bcl-2-associated athanogene (BAG) family members.

Regulation of Parkin-dependent mitophagy by Bcl-2-associated athanogene (BAG) family members.

Flavonoid GL-V9 induces apoptosis and inhibits glycolysis of breast cancer via disrupting GSK-3β-modulated mitochondrial binding of HKII

Energy metabolism plays important roles in the growth and survival of cancer cells. Here, we find a newly synthesized flavonoid named GL-V9, which inhibits glycolysis and induces apoptosis of human breast cancer cell lines, and investigate the underlying mechanism. Results show that hexokinase II (HKII) plays important roles in the anticancer effects of GL-V9. GL-V9 not only downregulates the expression of HKII in MDA-MB-231 and MCF-7 cells, but also induces dissociation of HKII from voltage-dependent anion channel (VDAC) in mitochondria, resulting in glycolytic inhibition and mitochondrial-mediated apoptosis. The dissociation of mitochondrial HKII is attributed to GSK-3β-induced phosphorylation of mitochondrial VDAC. Our in vivo experiments also show that GL-V9 significantly inhibits the growth of human breast cancer due to activation of GSK-3β and inactivation of AKT. Thus, GL-V9 induces cytotoxicity in breast cancer cells via disrupting the mitochondrial binding of HKII. Our works demonstrate the significance of metabolic regulators in cancer growth and offer a fresh insight into the molecular basis for the development of GL-V9 as a candidate for breast carcinoma treatment.

Hexokinase II promotes the Warburg effect by phosphorylating alpha subunit of pyruvate dehydrogenase.

ObjectiveTumor cells rely heavily on glycolysis regardless of oxygen tension, a phenomenon called the Warburg effect. Hexokinase II (HKII) catalyzes the first irreversible step of glycolysis and is often overexpressed in tumor cells. Mitochondrial HKII couples glycolysis and oxidative phosphorylation while maintaining mitochondrial membrane integrity. In this study, we investigated the role of HKII in promoting the Warburg effect in cancer cells.MethodsHKII-mediated phosphorylation of the alpha subunit of pyruvate dehydrogenase (PDHA1) was tested in HEK293T cells and clear cell renal cell carcinoma (ccRCC) specimens using gene knockdown, western blotting, immunohistochemistry, and immunofluorescence.ResultsIt was determined that HKII could not only transform glucose into glucose-6-phosphate, but also transfer the phosphate group of ATP onto PDHA1. In addition, it was found that HKII increased the phosphorylation of Ser293 on PDHA1, decreasing pyruvate dehydrogenase (PDH) complex activity and thus rerouting the metabolic pathway and promoting the Warburg effect. The overexpression of HKII correlated with the phosphorylation of PDHA1 and disease progression in ccRCC.ConclusionsThe data presented here suggest that HKII is an important biomarker in the evaluation and treatment of cancer.

Astragaloside IV reduces neuronal apoptosis and parthanatos in ischemic injury by preserving mitochondrial hexokinase-II

Cerebral ischemia induces neuronal cell death in different ways and mitochondrial dysfunction is an important cause. Astragaloside IV (AIV) is a natural saponin abandent in Astragalus membranaceus and this study aims to find if AIV protects neuronal survival via preserving mitochondrial hexokinase-II (HK-II). Glutamate stimulation induced HK-II dissociation from mitochondria and impaired mitochondrial function, indicated by the opening of the mitochondrial permeability transition pore, the collapse of mitochondrial membrane potential and reduced mitochondrial oxygen consumption ratio in neurons. Accompanied with apoptosis, oxidative DNA damage, PAR formation and nuclear translocation of apoptosis inducing factor (AIF) indicated the presence of parthanatos. AIV activated Akt and protected mitochondrial HK-II via promoting the binding of Akt to HK-II and protected hexokinase activity with improved glycolysis. As a consequence of preserved mitochondrial HK-II, AIV reduced the release of pro-apoptotic proteins and AIF, resultantly protected neurons from apoptosis and parthanatos. Moreover, the neuroprotective effects of AIV were also reproduced in mice subjected to middle cerebral artery occlusion to support the findings in vitro. Together, these results showed that glutamate excitotoxicity impaired mitochondrial HK-II and simultaneously induced apoptosis and parthanatos owing to mitochondrial dysfunction. AIV activated Akt to promote HK-II binding to mitochondria, and the structural and functional integrity of mitochondria contributed to protecting neurons from apoptosis and DNA damage. These findings address the important role of mitochondrial HK-II in neuronal protection.

Melatonin improves neurological outcomes and preserves hippocampal mitochondrial function in a rat model of cardiac arrest.

Cerebral injury after cardiac arrest (CA)/cardiopulmonary resuscitation (CPR) has been implicated in the poor prognosis of CA survivors. This study was designed to evaluate the impact of melatonin on postresuscitation neurological outcomes and to explore the underlying mechanism. Sprague-Dawley rats were randomly assigned to four groups: sham group, CPR group, melatonin pretreatment group (Pre-M) and posttreatment group (Post-M). For the last 2 groups, daily melatonin gavage was performed for 12 consecutive days before or 24 hours after rat survival from CA/CPR. No statistical differences were observed in heart rate (HR), mean arterial blood pressure (MAP), and end-tidal carbon dioxide (ETCO2) at baseline and after restoration of spontaneous circulation (ROSC) among groups. However, melatonin pretreatment or posttreatment significantly improved neurological deficit score and memory and spatial learning ability after CA/CPR. Further studies demonstrated that the complex I- and complex-II supported mitochondrial respiration were greatly increased under melatonin treatment. In addition, melatonin treatment preserved the mitochondrial-binding hexokinase II (HKII) and ATP levels and suppressed the upregulated protein lysine acetylation in hippocampus after CA/CPR. In conclusion, using a rat asphyxial CA model we have demonstrated that treatment with melatonin either before or after CA/CPR provides a promising neuroprotective effect, and this protection was mediated by increasing mitochondrial HKII expression, suppressing protein acetylation and improving mitochondrial function in hippocampus.

Astragaloside IV Reduces Neuronal Apoptosis and Parthanatos in Ischemic Injury by Preserving Mitochondrial Hexokinase-II

Background: Cerebral ischemia induces neuronal cell death in different ways and mitochondrial dysfunction is an important cause. Astragaloside IV (AIV) is a natural saponin abandent in Astragalus membranaceus and this study aims to find if AIV protects neuronal survival via preserving mitochondrial hexokinase-II (HK-II). Methods: The primary cortical neuron were prepared to observe the effects of AIV on neuroprotection with focus on the regulation of mitochondrial HK-II via Akt activation. Meanwhile, we also observed the protection in mice MCAO model. Results: Glutamate stimulation induced HK-II dissociation from mitochondria and impaired mitochondrial function, indicated by the opening of the mitochondrial permeability transition pore and the collapse of mitochondrial membrane potential. Accompanied with apoptosis, oxidative DNA damage, PAR formation and nuclear translocation of apoptosis inducing factor (AIF) indicated the presence of parthanatos. AIV activated Akt and protected mitochondrial HK-II via promoting the binding of Akt to HK-II. As a consequence of preserved mitochondrial HK-II, AIV reduced the release of pro-apoptotic proteins and AIF, resultantly protected neurons from apoptosis and parthanatos. Moreover, the neuroprotective effects of AIV were also reproduced in mice subjected to middle cerebral artery occlusion to support the findings in vitro. Interpretation: Glutamate excitotoxicity impaired mitochondrial HK-II and simultaneously induced apoptosis and parthanatos owing to mitochondrial dysfunction. AIV activated Akt to promote HK-II binding to mitochondria, and the structural and functional integrity of mitochondria contributed to protecting neurons from apoptosis and DNA damage. These findings address the important role of mitochondrial HK-II in neuronal protection. Funding:This study is funded by National Natural Science Foundation of China (Grant No. 81703738) and Foundation of The Science and Technology Department of Henan Province (Grant No. 182102310169) Declaration of Interest: The authors declare that they have no competing interests. Ethical Approval: The animal care and all experimental procedures were approved by Animal Ethics Committee of China Pharmaceutical University.

Mangiferin protects mitochondrial function by preserving mitochondrial hexokinase-II in vessel endothelial cells

Hexokinase-II (HK-II) confers protection against cell death and this study was designed to investigate the effect of mangiferin on the regulation of mitochondrial HK-II. In vessel endothelial cells, saturated fatty acid palmitate (PA) stimulation induced HK-II detachment from mitochondria due to cellular acidification. Mangiferin reduced lactate accumulation by improving pyruvate dehydrogenase activity, promoted Akt translocation to HK-II and prevented HK-II detachment from mitochondria. Knockdown of Akt2 diminished the protective effect of mangiferin on mitochondrial HK-II, confirming the role of Akt in the regulation of HK-II. Mangiferin prevented mitochondrial permeability transition pore opening, restored mitochondrial membrane potential and thereby protected cell from apoptosis. In high-fat diet fed mice, oral administration of mangiferin induced Akt phosphorylation, increased HK-II binding to mitochondria and resultantly protected vessel endothelial function, demonstrating its protective effect on endothelial integrity in vivo. This finding provided a novel strategy for the protection of mitochondrial function in the endothelium.

Acute detachment of hexokinase II from mitochondria modestly increases oxygen consumption of the intact mouse heart

ObjectiveCardiac hexokinase II (HKII) can translocate between cytosol and mitochondria and change its cellular expression with pathologies such as ischemia–reperfusion, diabetes and heart failure. The cardiac metabolic consequences of these changes are unknown. Here we measured energy substrate utilization in cytosol and mitochondria using stabile isotopes and oxygen consumption of the intact perfused heart for 1) an acute decrease in mitochondrial HKII (mtHKII), and 2) a chronic decrease in total cellular HKII. Methods/resultsWe first examined effects of 200nM TAT (Trans-Activator of Transcription)-HKII peptide treatment, which was previously shown to acutely decrease mtHKII by ~30%. In Langendorff-perfused hearts TAT-HKII resulted in a modest, but significant, increased oxygen consumption, while cardiac performance was unchanged. At the metabolic level, there was a nonsignificant (p=0.076) ~40% decrease in glucose contribution to pyruvate and lactate formation through glycolysis and to mitochondrial citrate synthase flux (6.6±1.1 vs. 11.2±2.2%), and an 35% increase in tissue pyruvate (27±2 vs. 20±2pmol/mg; p=0.033). Secondly, we compared WT and HKII+/− hearts (50% chronic decrease in total HKII). RNA sequencing revealed no differential gene expression between WT and HKII+/− hearts indicating an absence of metabolic reprogramming at the transcriptional level. Langendorff-perfused hearts showed no significant differences in glycolysis (0.34±0.03μmol/min), glucose contribution to citrate synthase flux (35±2.3%), palmitate contribution to citrate synthase flux (20±1.1%), oxygen consumption or mechanical performance between WT and HKII+/− hearts. ConclusionsThese results indicate that acute albeit not chronic changes in mitochondrial HKII modestly affect cardiac oxygen consumption and energy substrate metabolism.

Ginsenoside Rg5 increases cardiomyocyte resistance to ischemic injury through regulation of mitochondrial hexokinase-II and dynamin-related protein 1

Hexokinase-II (HK-II) and dynamin-related protein 1 (Drp1) regulate mitochondrial function differently. This study was designed to investigate the cardioprotective effect of ginsenoside Rg5 (Rg5) with emphasis on the regulation of mitochondrial HK-II and Drp1. Saturated acid palmitate (PA) stimulation increased lactate accumulation and induced cellular acidification by impairing the activity of pyruvate dehydrogenase (PDH) in cardiomyocytes, leading to HK-II dissociation from mitochondria. Rg5 improved PDH activity and prevented cellular acidification by combating fatty-acid oxidation, contributing to protecting mitochondrial HK-II. HK-II binding to mitochondria prevented mitochondrial Drp1 recruitment, whereas Drp1 activation decreased the content of mitochondrial HK-II, demonstrating the reciprocal control for binding to mitochondria. Rg5 promoted Akt translocation to mitochondria and increased HK-II binding to mitochondria while coordinately suppressing Drp1 recruitment and mitochondrial fission. Akt inhibitor triciribine or knockdown of Akt with small interfering RNA diminished the effects of Rg5, indicating that Rg5 inhibited Drp1 activation and promoted HK-II mitochondrial binding through Akt activation. Rg5 prevented the opening of mitochondrial permeability transition pore and increased ATP production, resultantly increasing cardiomyocyte resistance to hypoxia/reoxygenation injury. Meanwhile, Rg5 prevented cell apoptosis with increased HK-II binding and reduced Drp1 recruitment to mitochondria in isoproterenol-induced ischemic heart of mice. Taken together, these findings not only established a previously unrecognized role of ginsenosides in cardioprotection but also suggest that mitochondrial HK-II binding and Drp1 recruitment could be targeted therapeutically to prevent ischemic injury in the heart.

Suppression of miR-181a attenuates H2O2-induced death of mesenchymal stem cells by maintaining hexokinase II expression.

BackgroundLow survival rate of transplanted cells compromises the efficacy of cell therapy. Hexokinase II (HKII) is known to have anti-apoptotic activity through its interaction with mitochondria. The objective was to identify miRNAs targeting HKII and investigate whether miRNA-mediated modulation of HKII could improve the survival of mesenchymal stem cells (MSCs) exposed to H2O2. The expression of HKII in MSCs exposed to H2O2 was evaluated, and HKII-targeting miRNA was screened based on miRNA-target prediction databases. The effect of H2O2 on the expression of the selected HKII-targeting miRNA was examined and the effect of modulation of the selected HKII-targeting miRNA using anti-miRNA on H2O2-induced apoptosis of MSC was evaluated.ResultsH2O2 (600 μM) induced cell death of MSCs and decreased mitochondrial HKII expression. We have identified miR-181a as a HKII-targeting miRNA and H2O2 increased the expression of miR-181a in MSCs. Delivery of anti-miR-181a, which neutralizes endogenous miR-181a, significantly attenuated H2O2-induced decrease of HKII expression and disruption of mitochondrial membrane potential, improving the survival of MSCs exposed to H2O2.ConclusionsThese findings suggest that H2O2-induced up-regulation of miR-181a contributes to the cell death of MSCs by down-regulating HKII. Neutralizing miR-181a can be an effective way to prime MSCs for transplantation into ischemic tissues.

Curcumin inhibits aerobic glycolysis and induces mitochondrial-mediated apoptosis through hexokinase II in human colorectal cancer cells in vitro.

Curcumin, the major pigment of the dietary spice turmeric, has the potential for chemoprevention by promotion of apoptosis. Here, we investigated the molecular mechanisms of curcumin in glycolytic inhibition and apoptotic induction in human colorectal cancer HCT116 and HT29 cells. On the one hand, curcumin downregulated the expression and activity of hexokinase II (HKII) in HCT116 and HT29 cells in a concentration-dependent manner, but had little effect on the other key glycolytic enzymes (PFK, PGM, and LDH). On the other, curcumin induced dissociation of HKII from the mitochondria, resulting in mitochondrial-mediated apoptosis. Furthermore, the phosphorylation of mitochondrial HKII through AKT was responsible for the curcumin-induced dissociation of HKII, which was different from the mechanism of HKII inhibitor 3-BrPA. These results have important implications for the metabolism reprogramming effect and the susceptibility to curcumin-induced mitochondrial cytotoxicity through the regulation of HKII, and provide a molecular basis for the development of naturally compounds as novel anticancer agents for colorectal carcinoma.

Abstract 16870: Dissociation of Mitochondrial HK-II Triggers Mitochondria Specific Autophagy

Introduction: There is emerging evidence that the metabolic pathway interplays with the survival pathway to preserve cellular homeostasis. Hexokinases (HKs) catalyze the first step of glucose metabolism and hexokinase-II (HK-II) is the predominant isoform in the heart. Our recent study revealed that HK-II positively regulates general autophagy in the absence of glucose. Mitochondrial HK-II (mitoHK-II) is regulated by Akt and provides cardioprotection while it is decreased in the ischemic heart. Hypothesis: We tested the hypothesis that mitoHK-II dissociation triggers mitochondria specific autophagy (mitophagy). Results: As previously reported, mitoHK-II levels were decreased by ~40% in the perfused mouse heart subjected to global ischemia and in neonatal rat ventricular myocytes (NRVMs) subjected to simulated ischemia. To assess the role of mitoHK-II dissociation, mitoHK-II dissociating peptide (15NG) was expressed in NRVMs. MitoHK-II was decreased by 40% in NRVMs expressing 15NG which was accompanied with Parkin translocation to mitochondria and ubiquitination of mitochondrial proteins. This response was attenuated by Parkin knockdown and reversed by the recovery of mitoHK-II by co-expression of HK-II but not by that of mitochondria binding deficient mutant. 15NG expression did not induce mitochondrial membrane depolarization nor PINK1 stabilization at mitochondria, suggesting that the effects of mitoHK-II dissociation is not dependent on the previously established mitochondria depolarization/PINK1 pathway. This was confirmed by the experiments using PINK1 siRNA. Modest dissociation of mitoHK-II (by 20%) did not induce mitophagic responses but remarkably enhanced FCCP induced mitophagy, indicating that these two pathways are synergetic. We will be analyzing 15NG transgenic mice generated in our lab to determine the mitophagic role of mitoHK-II dissociation in vivo. Conclusions: These results suggest that mitoHK-II dissociation can regulate Parkin dependent mitophagy, in conjunction with depolarization dependent mechanisms and that HK-II could confer cardioprotection by switching the cell from an energy production to an energy conservation mode under ischemia.

Akt Phosphorylates HK-II at Thr-473 and Increases Mitochondrial HK-II Association to Protect Cardiomyocytes

Hexokinase II (HK-II) is an enzyme that catalyzes the first step in glycolysis and localizes not only in the cytosol but also at mitochondria. Akt, activated by insulin-like growth factor 1 (IGF-1) treatment in neonatal rat ventricular myocytes, translocates to mitochondria and increases mitochondrial HK-II binding. Expression of an HK-II-dissociating peptide diminished IGF-1-induced increases in mitochondrial HK-II as well as protection against hydrogen peroxide treatment, suggesting an important role of mitochondrial HK-II in IGF-1/Akt-mediated protection. We hypothesized, on the basis of an Akt phosphorylation consensus sequence present in HK-II, that Thr-473 is the target of Akt kinase activity. Indeed, recombinant kinase-active Akt robustly phosphorylates WT HK-II, but not Thr-473 mutants. Phosphomimetic (T473D)HK-II, but not non-phosphorylatable (T473A)HK-II, constitutively increased mitochondrial binding compared with WT HK-II and concomitantly confers greater protection against hydrogen peroxide. Glucose 6-phosphate (G-6P), a product of the catalytic activity of HK-II, is well known to dissociate HK-II from mitochondria. Addition of G-6P to isolated mitochondria dose-dependently dissociates WT HK-II, and this response is inhibited significantly in mitochondria isolated from cardiomyocytes expressing T473D HK-II. Pretreatment with IGF-1 also inhibits G-6P-induced overexpressed or endogenous HK-II dissociation, and this response was blocked by Akt inhibition. These results show that Akt phosphorylation of HK-II at Thr-473 is responsible for the Akt-mediated increase in HK-II binding to mitochondria. This increase is, at least in part, due to the decreased sensitivity to G-6P-induced dissociation. Thus, phosphorylation-mediated regulation of mitochondrial HK-II would be a critical component of the protective effect of Akt.

Cerulenin-induced apoptosis is mediated by disrupting the interaction between AIF and hexokinase II

Fatty acid synthase (FASN) is a key enzyme that plays a critical role in numerous metabolic functions by catalyzing the synthesis for long-chain fatty acids. FASN is highly expressed in various human cancers. This preferential expression makes FASN an attractive target for anticancer therapy. Hexokinase II (HKII) is overexpressed in most cancer cells, and it generally localizes to the outer mitochondrial membrane. Recent studies have demonstrated the protective role of mitochondrial HKII in preservation of mitochondrial integrity. The association of hexokinase with mitochondria has emerged as a powerful mechanism in protecting numerous cell types against cell death. We performed this study to examine the mechanism underlying apoptosis induced by cerulenin and with specific focus on its effect on HKII in ZR-75-1 human breast cancer cells. Additionally, we sought to elucidate whether inhibition of the PI3K/Akt pathway can potentiate the anticancer effect of cerulenin. Here, we showed that cerulenin disrupts the physical association between HKII and AIF, leading to eventual cell death. In addition, LY294002, a PI3K/Akt inhibitor, sensitized ZR-75-1 breast cancer cells to cerulenin-induced apoptosis. Collectively, cerulenin induces apoptosis via disrupting the interaction between AIF and HKII and inhibition of PI3K sensitizes cells to cerulenin-induced apoptosis in ZR-75-1 cells.

PHLPP-1 Negatively Regulates Akt Activity and Survival in the Heart

The recently discovered PHLPP-1 (PH domain leucine-rich repeat protein phosphatase-1) selectively dephosphorylates Akt at Ser473 and terminates Akt signaling in cancer cells. The regulatory role of PHLPP-1 in the heart has not been considered. To test the hypothesis that blockade/inhibition of PHLPP-1 could constitute a novel way to enhance Akt signals and provide cardioprotection. PHLPP-1 is expressed in neonatal rat ventricular myocytes (NRVMs) and in adult mouse ventricular myocytes (AMVMs). PHLPP-1 knockdown by small interfering RNA significantly enhances phosphorylation of Akt (p-Akt) at Ser473, but not at Thr308, in NRVMs stimulated with leukemia inhibitory factor (LIF). The increased phosphorylation is accompanied by greater Akt catalytic activity. PHLPP-1 knockdown enhances LIF-mediated cardioprotection against doxorubicin and also protects cardiomyocytes against H(2)O(2). Direct Akt effects at mitochondria have been implicated in cardioprotection and mitochondria/cytosol fractionation revealed a significant enrichment of PHLPP-1 at mitochondria. The ability of PHLPP-1 knockdown to potentiate LIF-mediated increases in p-Akt at mitochondria and an accompanying increase in mitochondrial hexokinase-II was demonstrated. We generated PHLPP-1 knockout (KO) mice and demonstrate that AMVMs isolated from KO mice show potentiated p-Akt at Ser473 in response to agonists. When isolated perfused hearts are subjected to ischemia/reperfusion, p-Akt in whole-heart homogenates and in the mitochondrial fraction is significantly increased. Additionally in PHLPP-1 KO hearts, the increase in p-Akt elicited by ischemia/reperfusion is potentiated and, concomitantly, infarct size is significantly reduced. These results implicate PHLPP-1 as an endogenous negative regulator of Akt activity and cell survival in the heart.

Mitochondrial Hexokinase II Promotes Neuronal Survival and Acts Downstream of Glycogen Synthase Kinase-3

Mitochondrial alterations are detected in most neurodegenerative disorders and may contribute to the dysfunction and demise of neuronal cells. Because glycogen synthase kinase-3 (GSK-3) is considered to be a critical factor in regulating neuronal cell survival and death, we studied the effects of modulating GSK-3 activity in cultured neurons treated with the mitochondrial inhibitor, rotenone. Interestingly, chronic inhibition of GSK-3 protects against rotenone-induced apoptosis in cultured neuronal cells. In an attempt to elucidate the molecular mechanisms underlying this neuroprotection, we demonstrated that chronic inhibition of GSK-3 reprograms the metabolism of neuronal cells, leading to an enhancement of glycolysis. This effect was accompanied by the induction and accumulation of hexokinase II (HKII) in the mitochondria. Interfering with either the mitochondrial binding of HKII or HKII expression significantly diminished the neuroprotection evoked by GSK-3 inhibition, and importantly, HKII overexpression is sufficient to protect against rotenone-induced cell death. Thus, mitochondrial HKII is a promoter of neuronal survival under the regulation of GSK-3. Furthermore, the neuroprotective effect of HKII may be relevant to neurodegenerative diseases in which glucose hypometabolism and mitochondrial dysfunction are prominent features.

Akt mediates mitochondrial protection in cardiomyocytes through phosphorylation of mitochondrial hexokinase-II

Akt activation supports survival of cardiomyocytes against ischemia/reperfusion, which induces cell death through opening of the mitochondrial permeability transition pore (PT-pore). Mitochondrial depolarization induced by treatment of cardiomyocytes with H(2)O(2) is prevented by activation of Akt with leukemia inhibitory factor (LIF). This protective effect is observed even when cardiomyocytes treated with LIF are permeabilized and mitochondrial depolarization is elicited by elevating Ca(2+). Cell fractionation studies demonstrate that LIF treatment increases both total and phosphorylated Akt in the mitochondrial fraction. Furthermore, the association of Akt with HK-II is increased by LIF. HK-II contains consensus sequences for phosphorylation by Akt and LIF treatment induces PI3K- and Akt-dependent HK-II phosphorylation. Addition of recombinant kinase-active Akt to isolated adult mouse heart mitochondria stimulates phosphorylation of HK-II and concomitantly inhibits the ability of Ca(2+) to induce cytochrome c release. This protection is prevented when HK-II is dissociated from mitochondria by incubation with glucose 6-phosphate or HK-II-dissociating peptide. Finally LIF increases HK-II association with mitochondria and dissociation of HK-II from mitochondria attenuates the protective effect of LIF on H(2)O(2)-induced mitochondrial depolarization in cardiomyocytes. We conclude that Akt has a direct effect at the level of the mitochondrion, which is mediated via phosphorylation of HK-II and results in protection of mitochondria against oxidant or Ca(2+)-stimulated PT-pore opening.

Akt mediates cardioprotection through phosphorylation of mitochondrial hexokinase-II

Akt activation supports survival of many cell types including cardiomyocytes. Apoptotic pathways activated in cardiomyocytes in response to cellular stresses involve opening of the mitochondrial permeability transition pore (PT-pore) and can be inhibited by Akt activation. Mitochondrial depolarization induced by H2O2 is prevented by activation of Akt with leukemia inhibitory factor (LIF). This protective effect of LIF is still observed when cells treated with LIF are permeabilized to remove soluble second messengers and mitochondrial depolarization elicited by elevating Ca2+.