Mitochondrial Hexokinase Research Articles

VDAC as a voltage-dependent mitochondrial gatekeeper under physiological conditions

Mitochondria, composed of two membranes, play a key role in energy production in eukaryotic cells. The main function of the inner membrane is oxidative phosphorylation, while the mitochondrial outer membrane (MOM) seems to control the energy flux and exchange of various charged metabolites between mitochondria and the cytosol. Metabolites cross MOM via the various isoforms of voltage-dependent anion channel (VDAC). In turn, VDACs interact with some enzymes, other proteins and molecules, including drugs. This work aimed to analyze various literature experimental data related to targeting mitochondrial VDACs and VDAC-kinase complexes on the basis of the hypothesis of generation of the outer membrane potential (OMP) and OMP-dependent reprogramming of cell energy metabolism. Our previous model of the VDAC-hexokinase-linked generation of OMP was further complemented in this study with an additional regulation of the MOM permeability by the OMP-dependent docking of cytosolic proteins like tubulin to VDACs. Computational analysis of the model suggests that OMP changes might be involved in the mechanisms of apoptosis promotion through the so-called transient hyperpolarization of mitochondria. The high concordance of the performed computational estimations with many published experimental data allows concluding that OMP generation under physiological conditions is highly probable and VDAC might function as an OMP-dependent gatekeeper of mitochondria, controlling cell life and death. The proposed model of OMP generation allows understanding in more detail the mechanisms of cancer death resistance and anticancer action of various drugs and treatments influencing VDAC voltage-gating properties, VDAC content, mitochondrial hexokinase activity and VDAC-kinase interactions in MOM.

Dual-targeted nanoformulation with Janus structure for synergistic enhancement of sonodynamic therapy and chemotherapy

The accurate delivery of nanoparticles and organic small molecule drugs remains a serious challenge in nanoparticle-based tumor therapy. Dual-targeted therapy combining tumor cell targeting and organelle targeting is an effective solution. Here, an anticancer nanoformulation accurate delivery system was prepared using hyaluronic acid (HA) targeting CD44 receptors on the surface of tumor cells and IR780 iodine (IR780) targeting mitochondrial for delivery. The system is based on an ultra-small Janus structured inorganic sensitizer TiO2-x@NaGdF4 nanoparticles (TN NPs) prepared by one-step pyrolysis, further loaded with organic small molecule acoustic sensitizer IR780 and mitochondrial hexokinase II inhibitor lonidamine (LND), followed by encapsulation of HA. Ultra-small size nanoparticles exhibit strong tissue penetration, tumor inhibition and in vivo metabolism. Under ultrasound radiation, TN NPs and IR780 could produce a synergistic effect, effectively increased the efficiency of reactive oxygen species (ROS) production. Meanwhile, the released IR780 could smoothly target the mitochondria, and the ROS produced by IR780 can destroy the mitochondrial structure and disrupt the mitochondrial respiration. LND could inhibit the energy metabolism of tumor cells by reducing the activity of hexokinase II (HK II), which further accelerates the process of apoptosis. Furthermore, since the Janus structure allows the integration of multifunctional components into a single system, TN NPs can not only serve as an acoustic sensitizer to generate ROS, but the Gd element contained can also act as the nuclear magnetic resonance (MR) imaging contrast agent, suggesting that the nanoformulation can enable imaging-guided diagnosis and therapy. In conclusion, a new scheme to enhance sonodynamic therapy (SDT) and chemotherapy synergistically is proposed here based on ultra-small dual-targeted nanoformulation with Janus structure in the ultrasound radiation environment.

Detachment of Hexokinase II From Mitochondria Promotes Collateral Sensitivity in Multidrug Resistant Chronic Myeloid Leukemia Cells.

Chronic Myeloid Leukemia is a neoplastic disease characterized by the abnormal expansion of hematopoietic cells with compromised functions. Leukemic cells often display a multidrug resistance phenotype, enabling them to evade a number of structurally unrelated cytotoxic compounds. One of those mechanisms relies on the high expression of efflux transporters, such as the ABC proteins, whose activity depends on the hydrolysis of ATP to reduce intracellular drug accumulation. In the present work, we employed a well-known erythroleukemia cell line, K562, and a multidrug resistant derivative cell, FEPS, to evaluate how hexokinase II, a key regulator for the rate-limiting step glycolysis, contributes to the establishment of the multidrug resistance phenotype. We found that multidrug resistant cells primarily resort to glycolysis to generate ATP. Clotrimazole reduced the expression of mitochondrial hexokinase II, which destabilized bioenergetic parameters such as reactive oxygen species production, ATP, and glutathione levels on multidrug resistant cells. This impaired the activity of ABCC1, leading to increased drug accumulation and cell death. In summary, we propose that decoupling of hexokinase II from the mitochondria emerges as a promising strategy to generate collateral sensitivity and aid in the management of chronic myeloid leukemia in chemotherapy-refractory patients.

How Do Hexokinases Inhibit Receptor-Mediated Apoptosis?

Simple SummaryIn multicellular animals, cells autonomously respond to lethal stress by inducing cell death programs. The most common regulated cell death is apoptosis. Cells protect their neighbors from damage by their cell contents or infection through this process. Apoptosis can occur as a result of intrinsic stress or induced by surface receptors, for example, by immune cells. In most cases, receptor-mediated apoptosis also requires the intrinsic signaling pathway. Intrinsic apoptosis is controlled by proteins of the B-cell lymphoma 2 (BCL-2) family. Pro-apoptotic BCL-2 proteins are inhibited by retrotranslocation from the mitochondria into the cytosol until the cell commits to apoptosis. Increasingly, discoveries show that BCL-2 proteins are regulated by proteins that are not themselves members of the BCL-2 family. Here, we discuss the selective inhibition of the link between death receptors activation and intrinsic apoptosis by hexokinases. These enzymes funnel glucose into the cellular metabolism. Independently, hexokinases retrotranslocate BCL-2 proteins and thereby protect cells from receptor-mediated apoptosis.The regulated cell death apoptosis enables redundant or compromised cells in ontogeny and homeostasis to remove themselves receptor-dependent after extrinsic signaling or after internal stress by BCL-2 proteins on the outer mitochondrial membrane (OMM). Mitochondrial BCL-2 proteins are also often needed for receptor-mediated signaling in apoptosis. Then, the truncated BH3-only protein BID (tBID) blocks retrotranslocation of the pro-apoptotic BCL-2 proteins BAX and BAK from the mitochondria into the cytosol. BAX and BAK in turn permeabilize the OMM. Although the BCL-2 proteins are controlled by a complex regulatory network, a specific mechanism for the inhibition of tBID remained unknown. Curiously, it was suggested that hexokinases, which channel glucose into the metabolism, have an intriguing function in the regulation of apoptosis. Recent analysis of transient hexokinase interactions with BAX revealed its participation in the inhibition of BAX and also BAK by retrotranslocation from mitochondria to the cytosol. In contrast to general apoptosis inhibition by anti-apoptotic BCL-2 proteins, hexokinase I and hexokinase 2 specifically inhibit tBID and thus the mitochondrial apoptosis pathway in response to death receptor signaling. Mitochondrial hexokinase localization and BH3 binding of cytosolic hexokinase domains are prerequisites for protection against receptor-mediated cell death, whereas glucose metabolism is not. This mechanism protects cells from apoptosis induced by cytotoxic T cells.

Insulin-Induced Recurrent Hypoglycemia Up-Regulates Glucose Metabolism in the Brain Cortex of Chemically Induced Diabetic Rats.

Diabetes is a chronic metabolic disease that seriously compromises human well-being. Various studies highlight the importance of maintaining a sufficient glucose supply to the brain and subsequently safeguarding cerebral glucose metabolism. The goal of the present work is to clarify and disclose the metabolic alterations induced by recurrent hypoglycemia in the context of long-term hyperglycemia to further comprehend the effects beyond brain harm. To this end, chemically induced diabetic rats underwent a protocol of repeatedly insulin-induced hypoglycemic episodes. The activity of key enzymes of glycolysis, the pentose phosphate pathway and the Krebs cycle was measured by spectrophotometry in extracts or isolated mitochondria from brain cortical tissue. Western blot analysis was used to determine the protein content of glucose and monocarboxylate transporters, players in the insulin signaling pathway and mitochondrial biogenesis and dynamics. We observed that recurrent hypoglycemia up-regulates the activity of mitochondrial hexokinase and Krebs cycle enzymes (namely, pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase and succinate dehydrogenase) and the protein levels of mitochondrial transcription factor A (TFAM). Both insults increased the nuclear factor erythroid 2–related factor 2 (NRF2) protein content and induced divergent effects in mitochondrial dynamics. Insulin-signaling downstream pathways were found to be down-regulated, and glycogen synthase kinase 3 beta (GSK3β) was found to be activated through both decreased phosphorylation at Ser9 and increased phosphorylation at Y216. Interestingly, no changes in the levels of cAMP response element-binding protein (CREB), which plays a key role in neuronal plasticity and memory, were caused by hypoglycemia and/or hyperglycemia. These findings provide experimental evidence that recurrent hypoglycemia, in the context of chronic hyperglycemia, has the capacity to evoke coordinated adaptive responses in the brain cortex that will ultimately contribute to sustaining brain cell health.

Dopamine signaling impairs ROS modulation by mitochondrial hexokinase in human neural progenitor cells.

Dopamine signaling has numerous roles during brain development. In addition, alterations in dopamine signaling may be also involved in the pathophysiology of psychiatric disorders. Neurodevelopment is modulated in multiple steps by reactive oxygen species (ROS), byproducts of oxidative metabolism that are signaling factors involved in proliferation, differentiation, and migration. Hexokinase (HK), when associated with the mitochondria (mt-HK), is a potent modulator of the generation of mitochondrial ROS in the brain. In the present study, we investigated whether dopamine could affect both the activity and redox function of mt-HK in human neural progenitor cells (NPCs). We found that dopamine signaling via D1R decreases mt-HK activity and impairs ROS modulation, which is followed by an expressive release of H2O2 and impairment in calcium handling by the mitochondria. Nevertheless, mitochondrial respiration is not affected, suggesting specificity for dopamine on mt-HK function. In neural stem cells (NSCs) derived from induced-pluripotent stem cells (iPSCs) of schizophrenia patients, mt-HK is unable to decrease mitochondrial ROS, in contrast with NSCs derived from healthy individuals. Our data point to mitochondrial hexokinase as a novel target of dopaminergic signaling, as well as a redox modulator in human neural progenitor cells, which may be relevant to the pathophysiology of neurodevelopmental disorders such as schizophrenia.

Hexokinases inhibit death receptor–dependent apoptosis on the mitochondria

Death receptor-mediated apoptosis requires the mitochondrial apoptosis pathway in many mammalian cells. In response to death receptor signaling, the truncated BH3-only protein BID can activate the proapoptotic BCL-2 proteins BAX and BAK and trigger the permeabilization of the mitochondria. BAX and BAK are inhibited by prosurvival BCL-2 proteins through retrotranslocation from the mitochondria into the cytosol, but a specific resistance mechanism to truncated BID-dependent apoptosis is unknown. Here, we report that hexokinase 1 and hexokinase 2 inhibit the apoptosis activator truncated BID as well as the effectors BAX and BAK by retrotranslocation from the mitochondria into the cytosol. BCL-2 protein shuttling and protection from TRAIL- and FasL-induced cell death requires mitochondrial hexokinase localization and interactions with the BH3 motifs of BCL-2 proteins but not glucose phosphorylation. Together, our work establishes hexokinase-dependent retrotranslocation of truncated BID as a selective protective mechanism against death receptor-induced apoptosis on the mitochondria.

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.

Neuronal Pentraxin 1 Promotes Hypoxic-Ischemic Neuronal Injury by Impairing Mitochondrial Biogenesis via Interactions With Active Bax[6A7] and Mitochondrial Hexokinase II.

Mitochondrial dysfunction is a key mechanism of cell death in hypoxic-ischemic brain injury. Neuronal pentraxin 1 (NP1) has been shown to play crucial roles in mitochondria-mediated neuronal death. However, the underlying mechanism(s) of NP1-induced mitochondrial dysfunction in hypoxia-ischemia (HI) remains obscure. Here, we report that NP1 induction following HI and its subsequent localization to mitochondria, leads to disruption of key regulatory proteins for mitochondrial biogenesis. Brain mitochondrial DNA (mtDNA) content and mtDNA-encoded subunit I of complex IV (mtCOX-1) expression was increased post-HI, but not the nuclear DNA-encoded subunit of complex II (nSDH-A). Up-regulation of mitochondrial proteins COXIV and HSP60 further supported enhanced mtDNA function. NP1 interaction with active Bax (Bax6A7) was increased in the brain after HI and in oxygen-glucose deprivation (OGD)-induced neuronal cultures. Importantly, NP1 colocalized with mitochondrial hexokinase II (mtHKII) following OGD leading to HKII dissociation from mitochondria. Knockdown of NP1 or SB216763, a GSK-3 inhibitor, prevented OGD-induced mtHKII dissociation and cellular ATP decrease. NP1 also modulated the expression of mitochondrial transcription factor A (Tfam) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), regulators of mitochondrial biogenesis, following HI. Together, we reveal crucial roles of NP1 in mitochondrial biogenesis involving interactions with Bax[6A7] and mtHKII in HI brain injury.

ROCK2 Promotes Osteosarcoma Growth and Glycolysis by Up-Regulating HKII via Phospho-PI3K/AKT Signalling.

BackgroundOsteosarcoma (OS) is a malignant bone tumour that exhibits a high mortality. While tumours thrive in a state of malnutrition, the mechanism by which OS cells adapt to metabolic stress through metabolic reprogramming remains unclear.MethodsWe analysed the expression of ROCK2 in osteosarcoma tissues by RT-qPCR and Western blot. Cell proliferation were analysed using CCK8, EdU and colony formation assays. The level of cell glycolysis was detected by glucose-6 phosphate, glucose consumption, lactate production and ATP levels.ResultsHerein, our study showed that ROCK2 expression in OS tissues was higher than in adjacent tissues. Functional assays have demonstrated that ROCK2 contributes to the growth of OS cells by inducing aerobic glycolysis. The current study revealed that ROCK2 knockdown decreased the levels of mitochondrial hexokinase II (HKII). And also indicated that ROCK2 served as a key enzyme in glycolysis and that it served an important role in tumour growth. A significant positive correlation was identified between the mRNA and protein expressions of ROCK2 and HKII, further demonstrating that ROCK2-induced glycolysis and proliferation was dependent on HKII expression in OS cells. Mechanistically, ROCK2 promotes HKII expression by activating the phospho-PI3K/AKT signalling pathway.ConclusionTaken together, the results of the current study linked the two drivers of OS growth and aerobic glycolysis and identified a new mechanism of ROCK2 control in OS.

Metabolic Features of Mouse and Human Retinas: Rods versus Cones, Macula versus Periphery, Retina versus RPE.

SummaryPhotoreceptors, especially cones, which are enriched in the human macula, have high energy demands, making them vulnerable to metabolic stress. Metabolic dysfunction of photoreceptors and their supporting retinal pigment epithelium (RPE) is an important underlying cause of degenerative retinal diseases. However, how cones and the macula support their exorbitant metabolic demand and communicate with RPE is unclear. By profiling metabolite uptake and release and analyzing metabolic genes, we have found cone-rich retinas and human macula share specific metabolic features with upregulated pathways in pyruvate metabolism, mitochondrial TCA cycle, and lipid synthesis. Human neural retina and RPE have distinct but complementary metabolic features. Retinal metabolism centers on NADH production and neurotransmitter biosynthesis. The retina needs aspartate to sustain its aerobic glycolysis and mitochondrial metabolism. RPE metabolism is directed toward NADPH production and biosynthesis of acetyl-rich metabolites, serine, and others. RPE consumes multiple nutrients, including proline, to produce metabolites for the retina.

Knockout of VDAC1 in H9c2 Cells Promotes Oxidative Stress-Induced Cell Apoptosis through Decreased Mitochondrial Hexokinase II Binding and Enhanced Glycolytic Stress.

Background/Aims:The role of VDAC1, the most abundant mitochondrial outer membrane protein, in cell death depends on cell types and stimuli. Both silencing and upregulation of VDAC1 in various type of cancer cell lines can stimulate apoptosis. In contrast, in mouse embryonic stem (MES) cells and mouse embryonic fibroblasts (MEFs), the roles of VDAC1 knockout (VDAC1−/−) in apoptotic cell death are contradictory. The contribution and underlying mechanism of VDAC1−/− in oxidative stress-induced cell death in cardiac cells has not been established. We hypothesized that VDAC1 is an essential regulator of oxidative stress-induced cell death in H9c2 cells.Methods:We knocked out VDAC1 in this rat cardiomyoblast cell line with CRISPR-Cas9 genome editing technique to produce VDAC1−/− H9c2 cells, and determined if VDAC1 is critical in promoting cell death via oxidative stress induced by tert-butylhydroper-oxide (tBHP), an organic peroxide, or rotenone (ROT), an inhibitor of mitochondrial complex I by measuring cell viability with MTT assay, cell death with TUNEL stain and LDH release. The mitochondrial and glycolytic stress were examined by measuring O2 consumption rate (OCR) and extracellular acidification rate (ECAR) with a Seahorse XFp analyzer.Results:We found that under control conditions, VDAC1−/− did not affect H9c2 cell proliferation or mitochondrial respiration. However, compared to the wildtype (WT) cells, exposure to either tBHP or ROT enhanced the production of ROS, ECAR, and the proton (H+) production rate (PPR) from glycolysis, as well as promoted apoptotic cell death in VDAC1−/− H9c2 cells. VDAC1−/− H9c2 cells also exhibited markedly reduced mitochondria-bound hexokinase II (HKII) and Bax. Restoration of VDAC1 in VDAC1−/− H9c2 cells reinstated mitochondria-bound HKII and concomitantly decreased tBHP and ROT-induced ROS production and cell death. Interestingly, mitochondrial respiration remained the same after tBHP treatment in VDAC1−/− and WT H9c2 cells.Conclusion:Our results suggest that VDAC1−/− in H9c2 cells enhances oxidative stress-mediated cell apoptosis that is directly linked to the reduction of mitochondria-bound HKII and concomitantly associated with enhanced ROS production, ECAR, and PPR.

A Protocol to Study Mitochondrial Function in Human Neural Progenitors and iPSC-Derived Astrocytes.

Mitochondrial dysfunction is a central component in the pathophysiology of multiple neuropsychiatric and degenerative disorders. Evaluating mitochondrial function in human-derived neural cells can help characterize dysregulation in oxidative metabolism associated with the onset of brain disorders, and may also help define targeted therapies. Astrocytes play a number of different key roles in the brain, being implicated in neurogenesis, synaptogenesis, blood-brain-barrier permeability, and homeostasis, and, consequently, the malfunctioning of astrocytes is related to many neuropathologies. Here we describe protocols for generating induced pluripotent stem cell (iPSC)-derived astrocytes and evaluating multiple aspects of mitochondrial function. We use a high-resolution respirometry assay that measures real-time variations in mitochondrial oxygen flow, allowing the evaluation of cellular respiration in the context of an intact intracellular microenvironment, something not possible with permeabilized cells or isolated mitochondria, where the cellular microenvironment is disrupted. Given that an impairment in the mitochondrial regulation of intracellular calcium homeostasis is involved in many pathologic stresses, we also describe a protocol to evaluate mitochondrial calcium dynamics in human neural cells, by fluorimetry. Lastly, we outline a mitochondrial function assay that allows for the measurement of the enzymatic activity of mitochondrial hexokinase (mt-HK), an enzyme that is functionally coupled to oxidative phosphorylation and is involved in redox homeostasis, particularly in the brain. In all, these protocols allow a detailed characterization of mitochondrial function in human neural cells. High-resolution respirometry, calcium dynamics, and mt-HK activity assays provide data regarding the functional status of mitochondria, which may reflect mitochondrial stress or dysfunction. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation of iPSC-derived human astrocytes Basic Protocol 2: Measuring real-time oxygen flux in human iPSC-derived astrocytes using a high-resolution OROBOROS Oxygraph 2k (O2k) Basic Protocol 3: Measuring mitochondrial calcium dynamics fluorometrically in permeabilized human neural cells Basic Protocol 4: Measuring OXPHOS-dependent activity of mitochondrial hexokinase in permeabilized human neural cells using a spectrophotometer.

293-OR: Mitochondrial Hexokinase Dissociation Correlates with Obesity-Induced Inflammation and Regulates the Fate of Glucose

It has been shown that high fat diet (HFD) induced obesity triggers local immune cell infiltration leading to pro-inflammatory cytokine production and subsequently to a state of chronic low-grade inflammation in adipose and spleen tissues. However, the underlying mechanisms driving this chronic inflammation remains poorly defined. The product of hexokinase (HK) enzymes, glucose-6-phosphate, can be metabolized through glycolysis to produce ATP and pyruvate or alternative pathways, such as the pentose-phosphate-pathway (PPP) to generate anabolic intermediates. HK1 contains an N-terminal domain that permits mitochondrial binding, but its physiologic significance is unclear. We found that adipose and spleen tissues from HFD mice had increased cytosolic HK1, which correlated with an increase in inflammatory cytokine mRNA levels. To elucidate whether HK1 subcellular localization to the mitochondria affects the inflammatory response we generated mice lacking the HK1 mitochondrial binding domain, termed ΔE1HK1. These mice appear normal and show no change in body weight, Mendelian ratios, glucose tolerance or hexokinase activity across different tissues as compared to littermate controls. However, lipopolysaccharide challenge in these mice produced a hyper-inflammatory phenotype in vitro and in vivo. We performed steady-state and flux metabolomics in ΔE1HK1 mouse tissues and found increased glucose derived metabolites above the level of GAPDH, including PPP metabolites. Additionally, we found that GAPDH activity was attenuated in the ΔE1HK1 mice and may explain the elevated PPP flux upstream of GAPDH. Since PPP is vital for proper inflammatory cell activation, we treated cells from these mice with PPP inhibitors and found a reversal of the hyper-inflammatory phenotype observed in ΔE1HK1 mice. Our data suggests that mitochondrial dissociation of HK1 alters glucose utilization, which subsequently increases the pro-inflammatory response. Disclosure F. Keyhani Nejad: None. A. DeJesus: None. J.A. Geier: None. H. Ardehali: None.

Targeting mitochondrial hexokinases increases efficacy of histone deacetylase inhibitors in solid tumor models

Hexokinase 1 and 2 have been shown to inhibit Bak- and Bax-mediated apoptosis, leading us to combine the histone deacetylase inhibitor romidepsin with clotrimazole or bifonazole, two compounds that reportedly decrease mitochondrial localization of hexokinases. Cancer cell lines derived from breast, kidney, lung, colon or ovarian cancers were treated with a short-term exposure to 25 ng/ml romidepsin combined with either clotrimazole or bifonazole. The combination of romidepsin with 25 µM clotrimazole or bifonazole resulted in increased annexin staining compared to cells treated with any of the drugs alone. Cell death was caspase-mediated, as the pan-caspase inhibitor Q-VD-OPh was found to inhibit apoptosis induced by the combination. A549 lung cancer cells or HCT-116 cells deficient in Bak and Bax were also resistant to apoptosis with the combination implicating the intrinsic apoptotic pathway. We found that a 24 h treatment with clotrimazole or bifonazole decreased total hexokinase 2 expression, resulting in a 76% or 60% decrease, respectively, of mitochondrial expression of hexokinase 2. Mitochondrial hexokinase 1 levels increased 2-fold or less. Our work suggests that the combination of a short-term romidepsin treatment with bifonazole or clotrimazole leads to increased apoptosis, most likely due to decreased mitochondrial expression of hexokinase 2.

Glycogen synthase kinase-3β opens mitochondrial permeability transition pore through mitochondrial hexokinase II dissociation.

Accumulating evidence has revealed pivotal roles of glycogen synthase kinase-3β (GSK3β) inactivation on cardiac protection. Because the precise mechanisms of cardiac protection against ischemia/reperfusion (I/R) injury by GSK3β-inactivation remain elusive, we investigated the relationship between GSK3β-mediated mitochondrial hexokinase II (mitoHK-II; a downstream target of GSK3β) dissociation and mitochondrial permeability transition pore (mPTP) opening. In Langendorff-perfused hearts, GSK3β inactivation by SB216763 improved the left ventricular-developed pressure and retained mitoHK-II binding after I/R. In permeabilized myocytes, GSK3β depolarized mitochondrial membrane potential with accelerated mitochondrial calcein release (suggesting GSK3β-mediated mPTP opening) and decreased mitoHK-II bindings. GSK3β-mediated mPTP opening depended on mitoHK-II binding, i.e., it was accelerated by dissociation of mitoHK-II (dicyclohexylcarbodiimide) and attenuated by enhancement of mitoHK-II binding (dextran). However, inactivation of mitoHK-II by glucose-depletion or glucose-6-phosphate inhibited the GSK3β-mediated mPTP opening. We conclude that GSK3β-mediated mPTP opening may be involved in I/R injury and regulated by mitoHK-II binding and activity.

Empagliflozin effects on ischemic contracture and I/R injury in isolated mouse hearts perfused with or without insulin

IntroductionThe kidney‐targeted Empagliflozin (EMPA) has shown large unexpected cardiovascular benefits, which cannot be explained by general cardiovascular risk factors. We recently reported that EMPA has sodium hydrogen exchanger (NHE) inhibition properties in isolated cardiomyocytes. Classic NHE inhibitors such as Cariporide (CARI) delay ischemic contracture onset and reduce ischemia reperfusion (I/R) injury in the heart. Here we examined whether EMPA delays contracture onset and reduces I/R injury. Both normal‐ and low‐glycogen hearts were used, because the rate of glycogen depletion during ischemia affects contracture onset and IR injury.Methods/ResultsIsolated mouse hearts were perfused with and without 50 mU/L insulin, to create normal‐ and low‐glycogen hearts respectively, and subjected to 25 min I and 120 min R. 1μM EMPA, 10μM cariporide (CARI; positive control) or vehicle was administered prior to ischemia until 10 minutes post‐reperfusion. In normal‐glycogen hearts, EMPA and CARI did not change ischemic contracture (in seconds, vehicle 1056±56, EMPA 900±44, CARI 880±84) and IR injury (infarct size (%): vehicle 59±6, Empa 62±10, Cari 68±4), however in low‐glycogen hearts, both EMPA and CARI delayed ischemic contracture onset (vehicle 459±25, EMPA 559±22, CARI 588±31). Higher EMPA concentration further delayed contracture onset. Only CARI reduced I/R injury (from 51±6 to 34±5). The presence of insulin (same concentration used) did not alter NHE activity, nor the ability of CARI or EMPA to inhibit NHE, in isolated cardiomyocytes. The increased IR injury in normal‐glycogen hearts was associated with increased end‐ischemic lactate and glucose‐6‐phosphate accumulation, and consequently decreased mitochondrial hexokinase ll.ConclusionEMPA does not protect against I/R injury in isolated hearts, yet EMPA delayed ischemic contracture onset in low‐glycogen hearts, suggesting a direct cardiac, ATP preserving, effect of EMPA during ischemia. Cardiac glycogen content interferes with the effects of NHE inhibitors on I/R injury in isolated hearts. These data indicate that empagliflozin and cariporide may inhibit NHE in different manners.Support or Funding InformationNone.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mitochondria-Bound Hexokinase (mt-HK) Activity Differ in Cortical and Hypothalamic Synaptosomes: Differential Role of mt-HK in H2O2 Depuration.

Glucose and oxygen are vital for the brain, as these molecules provide energy and metabolic intermediates that are necessary for cell function. The glycolysis pathway and mitochondria play a pivotal role in cell energy metabolism, which is closely related to reactive oxygen species (ROS) production. Hexokinase (HK) is a key enzyme involved in glucose metabolism that modulates the level of brain mitochondrial ROS by recycling ADP for oxidative phosphorylation (OxPhos). Here, we hypothesize that the control of mitochondrial metabolism by hexokinase differs in distinct areas of the brain, such as the cortex and hypothalamus, in which ROS might function as signaling molecules. Thus, we investigated mitochondrial metabolism of synaptosomes derived from both brain regions. Cortical synaptosomes (CSy) show a predominance of glutamatergic synapses, while in the hypothalamic synaptosomes (HSy), the GABAergic synapses predominate. Significant differences of oxygen consumption and ROS production were related to higher mitochondrial complex II activity (succinate dehydrogenase-SDH) in CSy rather than to mitochondrial number. Mitochondrial HK (mt-HK) activity was higher in CSy than in HSy regardless the substrate added. Mitochondrial O2 consumption related to mt-HK activation by 2-deoxyglucose was also higher in CSy. In the presence of substrate for complex II, the activation of synaptosomal mt-HK promoted depuration of ROS in both HSy and CSy, while ROS depuration did not occur in HSy when substrate for complex I was used. The impact of the mt-HK inhibition by glucose-6-phosphate (G6P) was the same in synaptosomes from both areas. Together, the differences found between CSy and HSy indicate specific roles of mt-HK and SDH on the metabolism of each brain region, what probably depends on the main metabolic route that is used by the neurons.

Cyclophilin D ablation is associated with increased end-ischemic mitochondrial hexokinase activity

Both the absence of cyclophilin D (CypD) and the presence of mitochondrial bound hexokinase II (mtHKII) protect the heart against ischemia/reperfusion (I/R) injury. It is unknown whether CypD determines the amount of mtHKII in the heart. We examined whether CypD affects mtHK in normoxic, ischemic and preconditioned isolated mouse hearts. Wild type (WT) and CypD−/− mouse hearts were perfused with glucose only and subjected to 25 min ischemia and reperfusion. At baseline, cytosolic and mtHK was similar between hearts. CypD ablation protected against I/R injury and increased ischemic preconditioning (IPC) effects, without affecting end-ischemic mtHK. When hearts were perfused with glucose, glutamine, pyruvate and lactate, the preparation was more stable and CypD ablation−resulted in more protection that was associated with increased mtHK activity, leaving little room for additional protection by IPC. In conclusion, in glucose only-perfused hearts, deletion of CypD is not associated with end-ischemic mitochondrial-HK binding. In contrast, in the physiologically more relevant multiple-substrate perfusion model, deletion of CypD is associated with an increased mtHK activity, possibly explaining the increased protection against I/R injury.

Abstract 4040: Short-term romidepsin treatment combined with clotrimazole or bifonazole leads to decreased mitochondrial hexokinase 2 and apoptosis in cancer cells

Abstract In light of disappointing clinical trials in solid tumors, we have sought novel combinations to increase the efficacy of the histone deacetylase inhibitor (HDI) romidepsin. Hexokinase 2 (HK2) expression is increased in cancer cells and is postulated to prevent Bax-mediated cytochrome c release from mitochondria. Since romidepsin treatment induces apoptosis via the mitochondrial pathway, combining romidepsin with agents that detach mitochondrial HK2 might result in synergistic apoptotic effects. HCT-116 colon carcinoma cells were treated with 25 ng/ml romidepsin alone for 6h, or with romidepsin in the presence of 25 µM clotrimazole or bifonazole, compounds known to detach mitochondrial HK2. The medium was removed and cells were treated for an additional 42 h with or without clotrimazole or bifonazole. Cells were also treated with clotrimazole or bifonazole alone for 48 h. Apoptosis was then quantitated by annexin staining. While short-term romidepsin treatment alone induced modest annexin staining and 25 µM clotrimazole or bifonazole had little effect, the combination resulted in significant apoptosis. Similar effects were observed for A549 lung cancer cells, MDA-MB-231 breast cancer cells and 786-0 renal carcinoma cells. We also treated HCT-116 and A549 cells lacking Bak, Bax or both with the clotrimazole/romidepsin combination. Apoptosis was slightly increased in Bak-/- cells, reduced in Bax-/- cells and nearly completely abrogated in cells lacking both proteins compared to wild-type cells suggesting apoptosis occurred through the intrinsic pathway. The clotrimazole analog TRAM-34 was similarly active to clotrimazole. Protein expression was determined in cytoplasmic and mitochondrial fractions of cells treated with romidepsin, clotrimazole or both and we found a >60% decrease in mitochondrial HK2 in cells treated with the combination versus untreated cells. Our results suggest that combining romidepsin with a compound that leads to decreased mitochondrial hexokinase, such as clotrimazole or bifonazole, results in increased apoptosis. We postulate that cell death following combined romidepsin and clotrimazole treatment requires an intact intrinsic apoptotic pathway and that HK2 serves an anti-apoptotic function at the mitochondria. Citation Format: Robert W. Robey, Andrew J. McDonald, Hanna Kozlowski, Michael M. Gottesman, Susan E. Bates. Short-term romidepsin treatment combined with clotrimazole or bifonazole leads to decreased mitochondrial hexokinase 2 and apoptosis in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4040. doi:10.1158/1538-7445.AM2017-4040