Voltage-dependent Anion Channel Protein Research Articles

DNA methylation and histone deacetylation regulating insulin sensitivity due to chronic cold exposure

In this study, we investigated the causal relationship between chronic cold exposure and insulin resistance and the mechanisms of how DNA methylation and histone deacetylation regulate cold-reduced insulin resistance. 46 adult male mice from postnatal day 90–180 were randomly assigned to control group and cold-exposure group. Mice in cold-exposure group were placed at temperature from -1 to 4 °C for 30 days to mimic chronic cold environment. Then, fasting blood glucose, blood insulin level and insulin resistance index were measured with enzymatic methods. Immunofluorescent labeling was carried out to visualize the insulin receptor substrate 2 (IRS2), Obese receptor (Ob-R, a leptin receptor), voltage-dependent anion channel protein 1 (VDAC1), cytochrome C (cytC), 5-methylcytosine (5-mC) positive cells in hippocampal CA1 area. Furthermore, the expressions of some proteins mentioned above were detected with Western blot. The results showed: ① Chronic cold exposure could reduce the insulin resistance index (P < 0.01) and increase the number of IRS2 positive cells and Ob-R positive cells in hippocampus (P < 0.01). ② The expressions of mitochondrial energy-relative proteins, VDAC1 and cytC, were higher in cold-exposure group than in control group with both immunohistochemical staining and Western blot (P < 0.01). ③ Chronic cold exposure increased DNA methylation and histone deacetylation in the pyramidal cells of CA1 area and led to an increase in the expression of histone deacetylase 1 (HDAC1) and DNA methylation relative enzymes (P < 0.01). In conclusion, chronic cold exposure can improve insulin sensitivity, with the involvement of DNA methylation, histone deacetylation and the regulation of mitochondrial energy metabolism. These epigenetic modifications probably form the basic mechanism of cold-reduced insulin resistance.

Itraconazole, an Oral Antifungal Drug, Is Active in Chemotherapy Resistant B-Cell Non-Hodgkin Lymphoma and Enhances the Anti-Tumor Activity of Chemotherapy Agents

Background: Relapsed/refractory diffuse large B-cell lymphoma (DLBCL) patients previously treated with rituximab-based therapy have poor clinical outcome, according to the results from collaborative trial in relapsed aggressive lymphoma (CORAL) study. It stresses the need to identify and/or optimize novel targeted agents. To better understand the molecular mechanisms underlining the acquired resistance to rituximab, we generated and characterized several rituximab-resistant DLBCL cell lines (RRCLs). Itraconazole, an oral antifungal agent, was reported had novel anticancer activity in basal cell carcinoma, non-small cell lung cancer and prostate cancer. In our current work, we define and characterize the anticancer activity of itraconazole in preclinical rituximab-sensitive or -resistant lymphoma models.Methods: A panel of rituximab-sensitive (RSCL) and rituximab-resistant (RRCL) cell lines were exposed to escalating doses of itraconazole (0-20μM) for 24, 48 and 72h. Changes in cell viability and cell cycle distribution were evaluated using the Presto Blue assay and flow cytometry respectively. IC50 was calculated by Graphpad Prism6 software. Loss of mitochondrial membrane potential (Δψm) following itraconazole exposure was assessed by DiOC6 and flow cytometry. Subsequently lymphoma cells were exposed to itraconazole or vehicle and various chemotherapy agents such as doxorubicin (1µM), dexamethasone (1µM), cDDP (20μg/ml), bortezomib (20nM), carfilzomib (20nM) or MLN2238 (20nM) for 48 hours. Coefficient of synergy was calculated using the CalcuSyn software. Changes in hexokinase II (HKII), Voltage dependent anion channel protein (VDAC), LC3 and BCL-xL expression levels were determined by western blotting after exposure cells to itraconazole. VDAC-HKII interactions following in vitro exposure to itraconazole were determined by immunoprecipitation of VDAC and probing for HKII in RSCL and RRCLs.Result:Itraconazole consistently showed potent, specific, dose-and time- dependent inhibition of all our sensitive and resistant lymphoma cell lines. In vitro exposure cells to itraconazole resulted in a loss of mitochondrial membrane potential and caused G2 cell cycle arrest. Itraconazole significantly had a synergistic anti-tumor effect combined with various chemotherapeutic agents, including doxorubicin, dexamethasone, cisplatin and different generations of proteasome inhibitors (bortezomib, carfilzomib or ixazomib) in both RSCL and RRCL. Western blot and immunoprecipitation studies demonstrated that following exposure to itraconazole, HKII bound less to mitochondrial specific protein VDAC. Complete silencing of HKII (using HKII siRNA interference) resulted in a rescue of loss in the mitochondrial membrane potential induced by intraconazole.Conclusion: Taking together, our data suggest that itraconazole had a potent anti-tumor activity against rituximab-sensitive or resistant pre-clinical models. The disruption of HKII from mitochondria following itraconazole exposure may contribute to lower the mitochondrial membrane potential and enhance the chemotherapeutic efficacy. Our finding highlights itraconazole as a potential therapeutic agent in the treatment of B-cell malignancies, and strongly supports clinical translation of its use. DisclosuresNo relevant conflicts of interest to declare.

JQ1, a c-MYC Inhibitor Targets Hexokinase II and Enhances the Cytotoxic Effects of Chemotherapeutic Agents in Diffuse Large B-Cell Lymphoma (DLBCL)

Background: Results from thecollaborative trial in relapsed aggressive lymphoma (CORAL) study demonstrated a poor clinical among patients treated with second-line chemo-immunotherapy in the post-rituximab era, stressing the need to identify and/or optimize novel targeted agents. C-myc expression was found correlate with a poor clinical outcome in patients with newly diagnosed or relapsed/refractory DLBCL. Previously, we found that JQ1 overcomes rituximab-chemotherapy resistance in lymphoma pre-clinical models. JQ1 exhibited synergistic activity when combined with chemotherapy agents. We also demonstrated that hexokinase II was highly expressed in rituximab resistant cell lines (RRCL), and its expression was associated with a deregulation in the glucose metabolism and an increase in the apoptotic threshold leading to chemotherapy resistance. In our current work, we evaluated molecular studies dissecting the cellular pathways affected by JQ1.Methods: A panel of rituximab-sensitive (RSCL) and RRCL cell lines were exposed to escalating doses of JQ1 (0-8μM) for 24, 48 and 72h. Changes in cell viability and cell cycle distribution were evaluated using the Presto Blue assay and flow cytometry respectively. IC50 was calculated by Graphpad Prism 6 software. Loss of mitochondrial membrane potential (Δψm) following JQ1 exposure was assessed by DiOC6 staining. Changes in c-MYC, HKII, HKI, Voltage dependent anion channel protein (VDAC) and p21 expression levels were determined by western blotting. HKII was silencing using siRNA interference in RRCL (Raji-4RH) and changes in HKII, HKI, VDAC, c-MYC and p21 protein expression was determined. HKII knockdown or scramble Raji-4RH cells were exposed to different chemotherapy agents and JQ1; cell viability and mitochondrial membrane potential was accessed.Result:JQ1 induced a dose-and time- dependent cell death in cell lines. In vitro exposure cells to JQ1 resulted in a loss of mitochondrial potential and induced G1 cell cycle arrest. In vitro exposure to JQ1 decreased c-MYC and HKII expression levels and induced of p21 expression. Complete silencing of HKII re-sensitized cell to chemotherapy (doxorubicin, vincristine and bortezomib). On the other hand, HKII knockout abrogated JQ1 anti-tumor activity.Conclusion: Our data suggests that JQ1 had anti-tumor activity against rituximab-sensitive or resistant pre-clinical models. The inhibition of HKII following JQ1 exposure may contribute to lower the mitochondrial membrane potential and enhance the chemotherapeutic effect. Our finding highlights a better understanding in the molecular events triggered by JQ1 as a potential role in the treatment of B-cell malignancies. DisclosuresNo relevant conflicts of interest to declare.

Insights into the Response of Soybean Mitochondrial Proteins to Various Sizes of Aluminum Oxide Nanoparticles under Flooding Stress

Rapid developments in nanotechnology have led to the increasing use of nanoparticles (NPs) in the agricultural sector. For possible interactions between NPs and crops under flooding stress to be investigated, the molecular mechanisms in soybeans affected by exposure to various sizes of Al2O3 NPs were analyzed using a proteomic technique. In plants exposed to 30-60 nm Al2O3 NPs, the length of the root including hypocotyl was increased, and proteins related to glycolysis were suppressed. Exposure to 30-60 nm Al2O3 NPs mediated the scavenging activity of cells by regulating the ascorbate/glutathione pathway. Hierarchical clustering analysis indicated that ribosomal proteins were also increased upon exposure to flooding-stressed plants with 30-60 nm Al2O3 NPs. Mitochondrion was the target organelle of Al2O3 NPs under flooding-stress conditions. Mitochondrial proteomic analysis revealed that the abundance of voltage-dependent anion channel protein was increased upon exposure to flooding-stressed soybeans with 135 nm Al2O3 NPs, indicating the permeability of the mitochondrial membrane was increased. Furthermore, isocitrate dehydrogenase was increased upon exposure of plants to 5 nm Al2O3 NPs under flooding conditions. These results suggest that Al2O3 NPs of various sizes affect mitochondrial proteins under flooding stress by regulating membrane permeability and tricarboxylic acid cycle activity.

Novel Compounds Targeting the Mitochondrial Protein VDAC1 Inhibit Apoptosis and Protect against Mitochondrial Dysfunction

Apoptosis is thought to play a critical role in several pathological processes, such as neurodegenerative diseases (i.e. Parkinson's and Alzheimer's diseases) and various cardiovascular diseases. Despite the fact that apoptotic mechanisms are well defined, there is still no substantial therapeutic strategy to stop or even slow this process. Thus, there is an unmet need for therapeutic agents that are able to block or slow apoptosis in neurodegenerative and cardiovascular diseases. The outer mitochondrial membrane protein voltage-dependent anion channel 1 (VDAC1) is a convergence point for a variety of cell survival and death signals, including apoptosis. Recently, we demonstrated that VDAC1 oligomerization is involved in mitochondrion-mediated apoptosis. Thus, VDAC1 oligomerization represents a prime target for agents designed to modulate apoptosis. Here, high-throughput compound screening and medicinal chemistry were employed to develop compounds that directly interact with VDAC1 and prevent VDAC1 oligomerization, concomitant with an inhibition of apoptosis as induced by various means and in various cell lines. The compounds protected against apoptosis-associated mitochondrial dysfunction, restoring dissipated mitochondrial membrane potential, and thus cell energy and metabolism, decreasing reactive oxidative species production, and preventing detachment of hexokinase bound to mitochondria and disruption of intracellular Ca2+ levels. Thus, this study describes novel drug candidates with a defined mechanism of action that involves inhibition of VDAC1 oligomerization, apoptosis, and mitochondrial dysfunction. The compounds VBIT-3 and VBIT-4 offer a therapeutic strategy for treating different diseases associated with enhanced apoptosis and point to VDAC1 as a promising target for therapeutic intervention.

Characterization of a novel β-barrel protein (AtOM47) from the mitochondrial outer membrane of Arabidopsis thaliana.

In plant cells, mitochondria are major providers of energy and building blocks for growth and development as well as abiotic and biotic stress responses. They are encircled by two lipid membranes containing proteins that control mitochondrial function through the import of macromolecules and metabolites. Characterization of a novel β-barrel protein, OUTER MEMBRANE PROTEIN 47 (OM47), unique to the green lineage and related to the voltage-dependent anion channel (VDAC) protein family, showed that OM47 can complement a VDAC mutant in yeast. Mutation of OM47 in Arabidopsis thaliana by T-DNA insertion had no effect on the import of proteins, such as the β-barrel proteins translocase of the outer membrane 40 (TOM40) or sorting and assembly machinery 50 (SAM50), into mitochondria. Molecular and physiological analyses revealed a delay in chlorophyll breakdown, higher levels of starch, and a delay in the induction of senescence marker genes in the mutant lines. While there was a reduction of >90% in OM47 protein in mitochondria isolated from 3-week-old om47 mutants, in mitochondria isolated from 8-week-old plants OM47 levels were similar to that of the wild type. This recovery was achieved by an up-regulation of OM47 transcript abundance in the mutants. Combined, these results highlight a role in leaf senescence for this plant-specific β-barrel protein, probably mediating the recovery and recycling of chloroplast breakdown products by transporting metabolic intermediates into and out of mitochondria.

A Single Talent Immunogenic Membrane Antigen and Novel Prognostic Predictor: voltage-dependent anion channel 1 (VDAC1) in Pancreatic Cancer.

Immunogenic membrane antigens associated with multiple biological functions of human cancer cells, have significant value in molecule diagnosis and targeted therapy. Here we screened immunogenic membrane antigens in pancreatic cancer by immunobloting IgG purified from sera of 66 pancreatic cancer patients with membrane proteins separated from two-dimensional PAGE of human pancreatic cancer cell line SWl990, and identified voltage-dependent anion channel 1 (VDAC1) as one of the potential immunogenic membrane antigens. Further studies focusing on VDAC1 demonstrated that VDAC1 mRNA and protein were significantly expressed in the tested pancreatic cancer cell lines. VDAC1 silencing with RNAi significantly decreased cell growth, invasion and migration in the pancreatic cancer cell line Capan-1. Additionally, VDAC1 expression was upregulated in pancreatic cancer tissue compared with normal pancreas samples and patients with low VDAC1 expression had a significantly greater median survival compared to those with high expression (27.0 months vs. 17.8 months, P = 0.039). In multivariable analysis, VDAC1 staining was an independent prognostic factor for survival [(Hazard-Ratio) HR = 1.544, 95% CI = 0.794–3.0, P = 0.021]. These results demonstrated that VDAC1 may be a candidate immunogenic membrane antigen for pancreatic cancer, a potential independent prognostic marker, and an ideal drug target.

Cigarette smoke effects on TSPO and VDAC expression in a cellular lung cancer model.

As redox iron and copper ions are found in lung pleural fluid and parenchyma, we aimed to examine the effect of cigarette smoke (CS) alone and the combined effects of CS and redox metals, iron and copper ions, containing medium (saliva), on epithelial H1299 lung cancer cells. We also examined the expression levels of the anticarcinogenic and proapoptotic 18 kDa translocator protein (TSPO) and its closely associated protein voltage-dependent anion channel (VDAC). H1299 cells were subjected to western blot analysis using anti-TSPO and anti-VDAC antibodies. With the former, the 18 kDa band appeared as expected and a 72 kDa band also appeared. It may be assumed that in H1299 lung cancer cells, an additional form of TSPO protein appears as a four-unit tetrameric complex, which is affected by CS exposure. A significant decrease in the expression level of the 72 kDa protein occurred following only 60 min of CS exposure, whereas VDAC protein levels were increased following only 30 min of CS exposure. These results, together with our previous related studies, suggest a comprehensive two-arm novel paradigm for lung cancer induced by CS, and mediated by an altered TSPO protein, possibly resulting from both the 72 kDa TSPO degradation and redox metal ion-induced enhancement of free radical attack. We suggest that both of the most important proapoptotic and anticancer proteins, p53 and TSPO, are damaged by CS, paving the way for lung cancer initiation and progression.

Mild Alkalization Acutely Triggers the Warburg Effect by Enhancing Hexokinase Activity via Voltage-Dependent Anion Channel Binding.

To fully understand the glycolytic behavior of cancer cells, it is important to recognize how it is linked to pH dynamics. Here, we evaluated the acute effects of mild acidification and alkalization on cancer cell glucose uptake and glycolytic flux and investigated the role of hexokinase (HK). Cancer cells exposed to buffers with graded pH were measured for 18F-fluorodeoxyglucose (FDG) uptake, lactate production and HK activity. Subcellular localization of HK protein was assessed by western blots and confocal microscopy. The interior of T47D breast cancer cells was mildly alkalized to pH 7.5 by a buffer pH of 7.8, and this was accompanied by rapid increases of FDG uptake and lactate extrusion. This shift toward glycolytic flux led to the prompt recovery of a reversed pH gradient. In contrast, mild acidification rapidly reduced cellular FDG uptake and lactate production. Mild acidification decreased and mild alkalization increased mitochondrial HK translocation and enzyme activity. Cells transfected with specific siRNA against HK-1, HK-2 and voltage-dependent anion channel (VDAC)1 displayed significant attenuation of pH-induced changes in FDG uptake. Confocal microscopy showed increased co-localization of HK-1 and HK-2 with VDAC1 by alkaline treatment. In isolated mitochondria, acidic pH increased and alkaline pH decreased release of free HK-1 and HK-2 from the mitochondrial pellet into the supernatant. Furthermore, experiments using purified proteins showed that alkaline pH promoted co-immunoprecipitation of HK with VDAC protein. These findings demonstrate that mild alkalization is sufficient to acutely trigger cancer cell glycolytic flux through enhanced activity of HK by promoting its mitochondrial translocation and VDAC binding. This process might serve as a mechanism through which cancer cells trigger the Warburg effect to maintain a dysregulated pH.

Proteome Analysis of the Cerebellum Tissue in Chronically Alcohol-Fed Rats

Background: Cerebellar degeneration is one of the most common effects of chronic alcohol exposure, and Purkinje cells are the main targets of alcohol-induced cerebellum neuropathology, but the underlying mechanism remains unclear. Methods: Eight rats were fed for 8 weeks with a nutritionally adequate liquid diet containing either ethanol as 36% of the total caloric content or an isocaloric control diet. Rat cerebellum homogenates were subjected to agarose two-dimensional electrophoresis (2-DE), and the protein expression profiles in chronically alcohol-fed rats and the pair-fed controls were compared. The observed changes in the protein expression levels were confirmed using immunoblotting analysis. Results: Three protein spots changed significantly in intensity according to 2-DE. Based on immunoblotting analysis, low expression levels of microtubule-associated protein-2 (MAP2) and the overexpression of voltagedependent anion channel protein 1 (VDAC1) were observed in the cerebellum of alcohol-fed rats. The expression levels of both proteins did not change in other parts of the brain. Conclusions: Low expression levels of MAP2 and overexpression of VDAC1 were detected using proteome analysis of the cerebellum tissue from chronically alcohol-fed rats. Changes in the expression of these proteins may be related to cerebellar degeneration following chronic alcohol consumption.

Antifungal drug itraconazole targets VDAC1 to modulate the AMPK/mTOR signaling axis in endothelial cells

Itraconazole, a clinically used antifungal drug, was found to possess potent antiangiogenic and anticancer activity that is unique among the azole antifungals. Previous mechanistic studies have shown that itraconazole inhibits the mechanistic target of rapamycin (mTOR) signaling pathway, which is known to be a critical regulator of endothelial cell function and angiogenesis. However, the molecular target of itraconazole that mediates this activity has remained unknown. Here we identify the major target of itraconazole in endothelial cells as the mitochondrial protein voltage-dependent anion channel 1 (VDAC1), which regulates mitochondrial metabolism by controlling the passage of ions and small metabolites through the outer mitochondrial membrane. VDAC1 knockdown profoundly inhibits mTOR activity and cell proliferation in human umbilical vein cells (HUVEC), uncovering a previously unknown connection between VDAC1 and mTOR. Inhibition of VDAC1 by itraconazole disrupts mitochondrial metabolism, leading to an increase in the cellular AMP:ATP ratio and activation of the AMP-activated protein kinase (AMPK), an upstream regulator of mTOR. VDAC1-knockout cells are resistant to AMPK activation and mTOR inhibition by itraconazole, demonstrating that VDAC1 is the mediator of this activity. In addition, another known VDAC-targeting compound, erastin, also activates AMPK and inhibits mTOR and proliferation in HUVEC. VDAC1 thus represents a novel upstream regulator of mTOR signaling in endothelial cells and a promising target for the development of angiogenesis inhibitors.

CREB1-driven expression of miR-320a promotes mitophagy by down-regulating VDAC1 expression during serum starvation in cervical cancer cells.

The altered expression of miRNAs in response to stresses contributes to cancer pathogenesis. However, little is known regarding the mechanism by which cellular stresses drive alterations in miRNA expression. Here, we found that serum starvation enhanced mitophagy by downregulating the mitophagy-associated protein voltage-dependent anion channel 1 (VDAC1) and by inducing the expression of miR-320a and the transcription factor cAMP responsive element binding protein 1(CREB1). Furthermore, we cloned the promoter of miR-320a and identified the core promoter of miR-320a in the upstream -16 to -130 region of pre-miR-320a. Moreover, CREB1 was found to bind to the promoter of miR-320a to activate its expression and to induce mitophagy during serum starvation. Collectively, our results reveal a new mechanism underlying serum starvation-induced mitophagy in which serum starvation induces CREB1 expression, in turn activating miR-320a expression, which then down-regulates VDAC1 expression to facilitate mitophagy. These findings may provide new insights into cancer cell survival in response to environmental stresses.

Tubulin Tail Sequences and Post-translational Modifications Regulate Closure of Mitochondrial Voltage-dependent Anion Channel (VDAC)

It was previously shown that tubulin dimer interaction with the mitochondrial outer membrane protein voltage-dependent anion channel (VDAC) blocks traffic through the channel and reduces oxidative metabolism and that this requires the unstructured anionic C-terminal tail peptides found on both α- and β-tubulin subunits. It was unclear whether the α- and β-tubulin tails contribute equally to VDAC blockade and what effects might be due to sequence variations in these tail peptides or to tubulin post-translational modifications, which mostly occur on the tails. The nature of the contribution of the tubulin body beyond acting as an anchor for the tails had not been clarified either. Here we present peptide-protein chimeras to address these questions. These constructs allow us to easily combine a tail peptide with different proteins or combine different tail peptides with a particular protein. The results show that a single tail grafted to an inert protein is sufficient to produce channel closure similar to that observed with tubulin. We show that the β-tail is more than an order of magnitude more potent than the α-tail and that the lower α-tail activity is largely due to the presence of a terminal tyrosine. Detyrosination activates the α-tail, and activation is reversed by the removal of the glutamic acid penultimate to the tyrosine. Nitration of tyrosine reverses the tyrosine inhibition of binding and even induces prolonged VDAC closures. Our results demonstrate that small changes in sequence or post-translational modification of the unstructured tails of tubulin result in substantial changes in VDAC closure.

Motifs of VDAC2 required for mitochondrial Bak import and tBid-induced apoptosis

Voltage-dependent anion channel (VDAC) proteins are major components of the outer mitochondrial membrane. VDAC has three isoforms with >70% sequence similarity and redundant roles in metabolite and ion transport. However, only Vdac2(-/-) (V2(-/-)) mice are embryonic lethal, indicating a unique and fundamental function of VDAC2 (V2). Recently, a specific V2 requirement was demonstrated for mitochondrial Bak import and truncated Bid (tBid)-induced apoptosis. To determine the relevant domain(s) of V2 involved, VDAC1 (V1) and V2 chimeric constructs were created and used to rescue V2(-/-) fibroblasts. Surprisingly, the commonly cited V2-specific N-terminal extension and cysteines were found to be dispensable for Bak import and high tBid sensitivity. In gain-of-function studies, V2 (123-179) was the minimal sequence sufficient to render V1 competent to support Bak insertion. Furthermore, in loss-of-function experiments, T168 and D170 were identified as critical residues. These motifs are conserved in zebrafish V2 (zfV2) that also rescued V2-deficient fibroblasts. Because high-resolution structures of zfV2 and mammalian V1 have become available, we could superimpose these structures and recognized that the critical V2-specific residues help to create a distinctive open "pocket" on the cytoplasmic surface that could facilitate Bak recruitment.

VDAC3 gating is activated by suppression of disulfide-bond formation between the N-terminal region and the bottom of the pore

The voltage-dependent anion channels (VDACs), VDAC1, VDAC2, and VDAC3, are pore-forming proteins that control metabolite flux between mitochondria and cytoplasm. VDAC1 and VDAC2 have voltage-dependent gating activity, whereas VDAC3 is thought to have weak activity. The aim of this study was to analyze the channel properties of all three human VDAC isoforms and to clarify the channel function of VDAC3. Bacterially expressed recombinant human VDAC proteins were reconstituted into artificial planar lipid bilayers and their gating activities were evaluated. VDAC1 and VDAC2 had typical voltage-dependent gating activity, whereas the gating of VDAC3 was weak, as reported. However, gating of VDAC3 was evoked by dithiothreitol (DTT) and S-nitrosoglutathione (GSNO), which are thought to suppress disulfide-bond formation. Several cysteine mutants of VDAC3 also exhibited typical voltage-gating. Our results indicate that channel gating was induced by reduction of a disulfide-bond linking the N-terminal region to the bottom of the pore. Thus, channel gating of VDAC3 might be controlled by redox sensing under physiological conditions.

Dynamics of enhanced mitochondrial respiration in female compared with male rat cerebral arteries.

Mitochondrial respiration has never been directly examined in intact cerebral arteries. We tested the hypothesis that mitochondrial energetics of large cerebral arteries ex vivo are sex dependent. The Seahorse XFe24 analyzer was used to examine mitochondrial respiration in isolated cerebral arteries from adult male and female Sprague-Dawley rats. We examined the role of nitric oxide (NO) on mitochondrial respiration under basal conditions, using N(ω)-nitro-l-arginine methyl ester, and following pharmacological challenge using diazoxide (DZ), and also determined levels of mitochondrial and nonmitochondrial proteins using Western blot, and vascular diameter responses to DZ. The components of mitochondrial respiration including basal respiration, ATP production, proton leak, maximal respiration, and spare respiratory capacity were elevated in females compared with males, but increased in both male and female arteries in the presence of the NOS inhibitor. Although acute DZ treatment had little effect on mitochondrial respiration of male arteries, it decreased the respiration in female arteries. Levels of mitochondrial proteins in Complexes I-V and the voltage-dependent anion channel protein were elevated in female compared with male cerebral arteries. The DZ-induced vasodilation was greater in females than in males. Our findings show that substantial sex differences in mitochondrial respiratory dynamics exist in large cerebral arteries and may provide the mechanistic basis for observations that the female cerebral vasculature is more adaptable after injury.

Abstract 383: 17 beta-Estradiol Enhances Mitochondrial Function of Cerebral Arteries From Ovariectomized Female Rats

Previous studies have provided indirect evidence that circulating sex hormones alter the function of the cerebral circulation perhaps via effects on mitochondrial dynamics. However, effects of estrogen on mitochondrial respiration have never been directly examined. We have previously observed a difference in the mitochondrial function of cerebral arteries from male and female rats but the exact mechanisms are not clear. We tested the hypothesis that mitochondrial respiration in isolated cerebral arteries from ovariectomized (OVX) Sprague Dawley rats treated with a 21 d release, 0.5 mg of 17 β-estradiol pellet (OVX+E) was enhanced compared with arteries from placebo treated OVX rats. The Seahorse Bioscience XFe24 system was used to measure mitochondrial oxygen consumption rate (pM/min/μg protein) in cerebral arteries. Western blot was used to investigate the arterial expression of proteins. Radioimmunoassay was used to measure serum estradiol level. Treatment with 17 β-estradiol resulted in a higher serum estradiol level (146.9±18.16 pg/ml) and uterus weight (0.15±0.0058 g) in the OVX+E compared with the OVX (14.7±1.2 pg/ml, 0.07±0.003, respectively; p&lt;0.05). The components of mitochondrial respiration in pM/min/μg protein including basal respiration (147±9), ATP production (44±4), proton leak (102±7), and maximal respiration (212±13) were elevated in OVX+E compared with OVX (105±13, 21±4, 50±6, 138±10, respectively; p&lt;0.05). Expression of the mitochondrial DNA encoded Complexes I and III, the nuclear DNA encoded Complexes II, IV, V, and the voltage-dependent anion channel protein were similar in all groups. However, the ratios of phosphorylated endothelial and neuronal nitric oxide (NO) synthase normalized to total protein were significantly (p&lt;0.05) elevated in the OVX+E (1.43±0.06, 1.15±0.2, respectively) compared with OVX group (0.92±0.06, 0.53±0.19, respectively). Our findings provide direct evidence for sex-specific differences in mitochondrial function on freshly isolated cerebral arteries. Thus, estradiol replacement enhances the efficacy of the oxidative phosphorylation resulting in an increased mitochondrial respiration which is not due to increased mitochondrial protein expression but may be due to enhanced NO.

Daily heat stress treatment rescues denervation-activated mitochondrial clearance and atrophy in skeletal muscle.

Traumatic nerve injury or nerve disease leads to denervation and severe muscle atrophy. Recent evidence shows that mitochondrial loss could be a key mediator of skeletal muscle atrophy. Here, we show that daily heat stress treatment rescues denervation-induced loss of mitochondria and concomitant muscle atrophy. We also found that denervation-activated autophagy-dependent mitochondrial clearance (mitophagy) was suppressed by daily heat stress treatment. The molecular basis of this observation is explained by our results showing that heat stress treatment attenuates the increase of key proteins that regulate the tagging step for mitochondrial clearance and the intermediate step of autophagosome formation in denervated muscle. These findings contribute to the better understanding of mitochondrial quality control in denervated muscle from a translational perspective and provide a mechanism behind the attenuation of muscle wasting by heat stress. Traumatic nerve injury or motor neuron disease leads to denervation and severe muscle atrophy. Recent evidence indicates that loss of mitochondria and the related reduction in oxidative capacity could be key mediators of skeletal muscle atrophy. As our previous study showed that heat stress increased the numbers of mitochondria in skeletal muscle, we evaluated whether heat stress treatment could have a beneficial impact on denervation-induced loss of mitochondria and subsequent muscle atrophy. Here, we report that daily heat stress treatment (mice placed in a chamber with a hot environment; 40°C, 30minday(-1) , for 7days) rescues the following parameters: (i)muscle atrophy (decreased gastrocnemius muscle mass); (ii)loss of mitochondrial content (decreased levels of ubiquinol-cytochrome c reductase core proteinII, cytochrome c oxidase subunitsI and IV and voltage-dependent anion channel protein); and (iii)reduction in oxidative capacity (reduced maximal activities of citrate synthase and 3-hydroxyacyl-CoA dehydrogenase) in denervated muscle (produced by unilateral sciatic nerve transection). In order to gain a better understanding of the above mitochondrial adaptations, we also examined the effects of heat stress on autophagy-dependent mitochondrial clearance (mitophagy). Daily heat stress normalized denervation-activated induction of mitophagy (increased mitochondrial microtubule-associated protein 1A/1B-light chain3-II (LC3-II) with and without blocker of autophagosome clearance). The molecular basis of this observation was explained by the results that heat stress attenuated the denervation-induced increase in key proteins that regulate the following steps: (i)the tagging step of mitochondrial clearance (increased mitochondrial Parkin, ubiquitin-conjugated, P62/sequestosome 1 (P62/SQSTM1)); and (ii)the elongation step of autophagosome formation (increased Atg5-Atg12 conjugate and Atg16L). Overall, our results contribute to the better understanding of mitochondrial quality control and the mechanisms behind the attenuation of muscle wasting by heat stress in denervated skeletal muscle.

Sex‐differences in Mitochondrial Respiration in Rat Cerebral Arteries

We determined whether sex-differences exist in mitochondrial oxygen consumption rate (OCR) in cerebral arteries from male and female Sprague Dawley rats. The Seahorse XFe24 analyzer was used to explore mitochondrial OCR under basal conditions and after pharmacological challenge using the mitochondria-specific drug, diazoxide (DZ). Western blotting was used to determine levels of mitochondrial and non-mitochondrial proteins. The different components of mitochondrial respiration: including basal respiration (97 ± 15 pM/min/μg protein), ATP production (33 ± 5 pM/min/μg protein), proton leak (64 ± 11 pM/min/μg protein), maximal respiration (147 ± 22 pM/min/μg protein), and spare capacity (50 ± 8 pM/min/μg protein) were significantly elevated (p < 0.05) in female compared with male arteries (36 ± 9 pM/min/μg protein; 15 ± 4 pM/min/μg protein; 21 ± 5 pM/min/μg protein; 63 ± 12 pM/min/μg protein; 26 ± 7 pM/min/μg protein; respectively). Acute treatment with DZ had little effect on OCR of male arteries but decreased ATP production (19 ± 3 pM/min/μg), maximal respiration (78 ± 14 pM/min/μg protein), and spare respiratory capacity (26 ± 9 pM/min/μg protein) in female arteries to near male levels. The mitochondrial Complex proteins, the voltage-dependent anion channel protein, and phosphorylated endothelial and neuronal nitric oxide synthase levels were significantly elevated in female compared with male arteries. Our findings provide evidence for substantial sex-differences in mitochondrial respiratory dynamics in large cerebral arteries and may provide the mechanistic basis for greater adaptability and resistance to injury of the female cerebral vasculature. This work was supported by NIH grants HL-077731, HL-030260, HL-065380, and HL-093554.

Acetylproteomic Analysis Reveals Functional Implications of Lysine Acetylation in Human Spermatozoa (sperm)

Male infertility is a medical condition that has been on the rise globally. Lysine acetylation of human sperm, an essential posttranslational modification involved in the etiology of sperm abnormality, is not fully understood. Therefore, we first generated a qualified pan-anti-acetyllysine monoclonal antibody to characterize the global lysine acetylation of uncapacitated normal human sperm with a proteomics approach. With high enrichment ratios that were up to 31%, 973 lysine-acetylated sites that matched to 456 human sperm proteins, including 671 novel lysine acetylation sites and 205 novel lysine-acetylated proteins, were identified. These proteins exhibited conserved motifs XXXKYXXX, XXXKFXXX, and XXXKHXXX, were annotated to function in multiple metabolic processes, and were localized predominantly in the mitochondrion and cytoplasmic fractions. Between the uncapacitated and capacitated sperm, different acetylation profiles in regard to functional proteins involved in sperm capacitation, sperm-egg recognition, sperm-egg plasma fusion, and fertilization were observed, indicating that acetylation of functional proteins may be required during sperm capacitation. Bioinformatics analysis revealed association of acetylated proteins with diseases and drugs. Novel acetylation of voltage-dependent anion channel proteins was also found. With clinical sperm samples, we observed differed lysine acetyltransferases and lysine deacetylases expression between normal sperm and abnormal sperm of asthenospermia or necrospermia. Furthermore, with sperm samples impaired by epigallocatechin gallate to mimic asthenospermia, we observed that inhibition of sperm motility was partly through the blockade of voltage-dependent anion channel 2 Lys-74 acetylation combined with reduced ATP levels and mitochondrial membrane potential. Taken together, we obtained a qualified pan-anti-acetyllysine monoclonal antibody, analyzed the acetylproteome of uncapacitated human sperm, and revealed associations between functional protein acetylation and sperm functions.