The role of the mitochondrial protein VDAC1 in inflammatory bowel disease: a potential therapeutic target.

Recent studies have implicated mitochondrial dysfunction as a trigger of inflammatory bowel diseases, including Crohn's disease (CD) and ulcerative colitis (UC). We have investigated the role of the mitochondria gate-keeper protein, the voltage-dependent-anion channel 1 (VDAC1), in gastrointestinal inflammation and tested the effects of the newly developed VDAC1-interacting molecules, VBIT-4 and VBIT-12, on UC induced by dextran sulfate sodium (DSS) or trinitrobenzene sulphonic acid (TNBS) in mice. VDAC1, which controls metabolism, lipids transport, apoptosis, and inflammasome activation, is overexpressed in the colon of CD and UC patients and DSS-treated mice. VBIT-12 treatment of cultured colon cells inhibited the DSS-induced VDAC1 overexpression, oligomerization, and apoptosis. In the DSS-treated mice, VBIT-12 suppressed weight loss, diarrhea, rectal bleeding, pro-inflammatory cytokine production, crypt and epithelial cell damage, and focal inflammation. VBIT-12 also inhibited the infiltration of inflammatory cells, apoptosis, mtDNA release, and activation of caspase-1 and NRLP3 inflammasome to reduce the inflammatory response. The levels of the ATP-gated P2X7-Ca2+/K+ channel and ER-IP3R-Ca2+ channel, and of the mitochondrial anti-viral protein (MAVS), mediating NLRP3 inflammasome assembly and activation, were highly increased in DSS-treated mice, but not when VBIT-12 treated. We conclude that UC may be promoted by VDAC1-overexpression and may therefore be amenable to treatment with novel VDAC1-interacting molecules. This VDAC1-based strategy exploits a completely new target for UC treatment and opens a new avenue for treating other inflammatory/autoimmune diseases.

A Mitochondrial VDAC1-Based Peptide Greatly Suppresses Steatosis and NASH-Associated Pathologies in a Mouse Model.

Non-alcoholic steatosis and non-alcoholic steatohepatitis (NASH) are liver pathologies characterized by severe metabolic alterations due to fat accumulation that lead to liver damage, inflammation, and fibrosis. We demonstrate that the voltage-dependent anion channel 1 (VDAC1)-based peptide R-Tf-D-LP4 arrested steatosis and NASH progression, as produced by a high-fat diet (HFD-32) in a mouse model, and reversed liver pathology to a normal-like state. VDAC1, a multi-functional mitochondrial protein, regulates cellular metabolic and energetic functions and apoptosis and interacts with many proteins. R-Tf-D-LP4 treatment eliminated hepatocyte ballooning degeneration, inflammation, and liver fibrosis associated with steatosis, NASH, and hepatocarcinoma, and it restored liver pathology-associated enzyme and glucose levels. Peptide treatment affected carbohydrate and lipid metabolism, increasing the expression of enzymes and factors associated with fatty acid transport to mitochondria, enhancing ß-oxidation and thermogenic processes, yet decreasing the expression of enzymes and regulators of fatty acid synthesis. The VDAC1-based peptide thus offers a promising therapeutic approach for steatosis and NASH.

VDAC1 and the TSPO: Expression, Interactions, and Associated Functions in Health and Disease States.

The translocator protein (TSPO), located at the outer mitochondrial membrane (OMM), serves multiple functions and contributes to numerous processes, including cholesterol import, mitochondrial metabolism, apoptosis, cell proliferation, Ca2+ signaling, oxidative stress, and inflammation. TSPO forms a complex with the voltage-dependent anion channel (VDAC), a protein that mediates the flux of ions, including Ca2+, nucleotides, and metabolites across the OMM, controls metabolism and apoptosis and interacts with many proteins. This review focuses on the two OMM proteins TSPO and VDAC1, addressing their structural interaction and associated functions. TSPO appears to be involved in the generation of reactive oxygen species, proposed to represent the link between TSPO activation and VDAC, thus playing a role in apoptotic cell death. In addition, expression of the two proteins in healthy brains and diseased states is considered, as is the relationship between TSPO and VDAC1 expression. Both proteins are over-expressed in in brains from Alzheimer's disease patients. Finally, TSPO expression levels were proposed as a biomarker of some neuropathological settings, while TSPO-interacting ligands have been considered as a potential basis for drug development.

Mitochondrial VDAC1 Silencing in Urethane-Induced Lung Cancer Inhibits Tumor Growth and Alters Cancer Oncogenic Properties.

Alterations in cellular metabolism are vital for cancer cell growth and motility. Here, we focused on metabolic reprogramming and changes in tumor hallmarks in lung cancer by silencing the expression of the mitochondrial gatekeeper VDAC1. To better mimic the clinical situation of lung cancer, we induced lung cancer in A/J mice using the carcinogen urethane and examined the effectiveness of si-m/hVDAC1-B encapsulated in PLGA-PEI nanoparticles. si-m/hVDAC1-B, given intravenously, induced metabolism reprogramming and inhibited tumor growth as monitored using MRI. Mice treated with non-targeted (NT) PLGA-PEI-si-NT showed many large size tumors in the lungs, while in PLGA-PEI-si-m/hVDAC-B-treated mice, lung tumor number and area were markedly decreased. Immunofluorescence staining showed decreased expression of VDAC1 and metabolism-related proteins and altered expression of cancer stem cell markers. Morphological analysis showed two types of tumors differing in their morphology; cell size and organization within the tumor. Based on specific markers, the two tumor types were identified as small cell (SCLC) and non-small cell (NSCLC) lung cancer. These two types of tumors were found only in control tumors, suggesting that PLGA-PEI-si-m/hVDAC1-B also targeted SCLC. Indeed, using a xenograft mouse model of human-derived SCLC H69 cells, si-m/hVDAC1-B inhibited tumor growth and reduced the expression of VDAC1 and energy- and metabolism-related enzymes, and of cancer stem cells in the established xenograft. Additionally, intravenous treatment of urethane-induced lung cancer mice with the VDAC1-based peptide, Retro-Tf-D-LP4, showed inhibition of tumor growth, and decreased expression levels of metabolism- and cancer stem cells-related proteins. Thus, silencing VDAC1 targeting both NSCLC and SCLC points to si-VDAC1 as a possible therapeutic tool to treat these lung cancer types. This is important as target NSCLC tumors undergo transformation to SCLC.

Hepatic GRK2 is dispensable for glucose homeostasis and other key metabolic parameters in mice.

OBJECTIVE: G-protein-coupled receptor (GPCR) kinases (GRKs) abrogate GPCR signaling by promoting receptor desensitization and internalization. Accumulating evidence suggests that GRK2 represents an important regulator of GPCR-mediated effects on systemic glucose metabolism, obesity, and insulin resistance. Despite the key role of the liver in maintaining euglycemia, the potential metabolic relevance of hepatic GRK2 has yet to be examined. Thus, the goal of this study was to explore the potential role of hepatic GRK2 in maintaining glucose homeostasis and other key metabolic functions. METHODS: To address this question, we generated mice that showed a ∼90% reduction in GRK2 protein expression selectively in hepatocytes (Hep-GRK2-KO mice) and subjected these mice, together with their control littermates, to systematic metabolic phenotyping studies. RESULTS: We found that Hep-GRK2-KO mice maintained on regular chow did not differ significantly from their control littermates in glycemia, glucose tolerance, insulin sensitivity, in vivo gluconeogenesis, and glucagon-induced hyperglycemia. We obtained similar findings when we analyzed Hep-GRK2-KO mice and control littermates consuming an obesogenic high-fat diet. Likewise, plasma levels of insulin, glucagon, free fatty acids, and ketone bodies remained unaffected by the lack of hepatocyte GRK2. The same was true when we examined the expression levels of key genes regulating hepatic glucose and fatty acid metabolism. CONCLUSION: In summary, our data suggest that hepatocyte GRK2 is dispensable for systemic glucose homeostasis and other key metabolic functions in both lean and obese mice. This finding suggests that drug development efforts aimed at inhibiting GRK2 to improve impaired glucose homeostasis and insulin sensitivity need to focus on other metabolically important tissues.

Correction: Pittala et al. The VDAC1-based R-Tf-D-LP4 Peptide as a Potential Treatment for Diabetes Mellitus. Cells 2020, 9, 481.

In the original publication [...].

Targeting the overexpressed mitochondrial protein VDAC1 in a mouse model of Alzheimer's disease protects against mitochondrial dysfunction and mitigates brain pathology.

BACKGROUND: Alzheimer's disease (AD) exhibits mitochondrial dysfunctions associated with dysregulated metabolism, brain inflammation, synaptic loss, and neuronal cell death. As a key protein serving as the mitochondrial gatekeeper, the voltage-dependent anion channel-1 (VDAC1) that controls metabolism and Ca2+ homeostasis is positioned at a convergence point for various cell survival and death signals. Here, we targeted VDAC1 with VBIT-4, a newly developed inhibitor of VDAC1 that prevents its pro-apoptotic activity, and mitochondria dysfunction. METHODS: To address the multiple pathways involved in AD, neuronal cultures and a 5 × FAD mouse model of AD were treated with VBIT-4. We addressed multiple topics related to the disease and its molecular mechanisms using immunoblotting, immunofluorescence, q-RT-PCR, 3-D structural analysis and several behavioral tests. RESULTS: In neuronal cultures, amyloid-beta (Aß)-induced VDAC1 and p53 overexpression and apoptotic cell death were prevented by VBIT-4. Using an AD-like 5 × FAD mouse model, we showed that VDAC1 was overexpressed in neurons surrounding Aß plaques, but not in astrocytes and microglia, and this was associated with neuronal cell death. VBIT-4 prevented the associated pathophysiological changes including neuronal cell death, neuroinflammation, and neuro-metabolic dysfunctions. VBIT-4 also switched astrocytes and microglia from being pro-inflammatory/neurotoxic to neuroprotective phenotype. Moreover, VBIT-4 prevented cognitive decline in the 5 × FAD mice as evaluated using several behavioral assessments of cognitive function. Interestingly, VBIT-4 protected against AD pathology, with no significant change in phosphorylated Tau and only a slight decrease in Aß-plaque load. CONCLUSIONS: The study suggests that mitochondrial dysfunction with its gatekeeper VDAC1 is a promising target for AD therapeutic intervention, and VBIT-4 is a promising drug candidate for AD treatment.

VDAC1 in the diseased myocardium and the effect of VDAC1-interacting compound on atrial fibrosis induced by hyperaldosteronism.

The voltage-dependent anion channel 1 (VDAC1) is a key player in mitochondrial function. VDAC1 serves as a gatekeeper mediating the fluxes of ions, nucleotides, and other metabolites across the outer mitochondrial membrane, as well as the release of apoptogenic proteins initiating apoptotic cell death. VBIT-4, a VDAC1 oligomerization inhibitor, was recently shown to prevent mitochondrial dysfunction and apoptosis, as validated in mouse models of lupus and type-2 diabetes. In the present study, we explored the expression of VDAC1 in the diseased myocardium of humans and rats. In addition, we evaluated the effect of VBIT-4 treatment on the atrial structural and electrical remodeling of rats exposed to excessive aldosterone levels. Immunohistochemical analysis of commercially available human cardiac tissues revealed marked overexpression of VDAC1 in post-myocardial infarction patients, as well as in patients with chronic ventricular dilatation\dysfunction. In agreement, rats exposed to myocardial infarction or to excessive aldosterone had a marked increase of VDAC1 in both ventricular and atrial tissues. Immunofluorescence staining indicated a punctuated appearance typical for mitochondrial-localized VDAC1. Finally, VBIT-4 treatment attenuated the atrial fibrotic load of rats exposed to excessive aldosterone without a notable effect on the susceptibility to atrial fibrillation episodes induced by burst pacing. Our results indicate that VDAC1 overexpression is associated with myocardial abnormalities in common pathological settings. Our data also indicate that inhibition of the VDAC1 can reduce excessive fibrosis in the atrial myocardium, a finding which may have important therapeutic implications. The exact mechanism\s of this beneficial effect need further studies.

The VDAC1-based R-Tf-D-LP4 Peptide as a Potential Treatment for Diabetes Mellitus.

Diabetes mellitus is a metabolic disorder approaching epidemic proportions. Non-alcoholic fatty liver disease (NAFLD) regularly coexists with metabolic disorders, including type 2 diabetes, obesity, and cardiovascular disease. Recently, we demonstrated that the voltage-dependent anion channel 1 (VDAC1) is involved in NAFLD. VDAC1 is an outer mitochondria membrane protein that serves as a mitochondrial gatekeeper, controlling metabolic and energy homeostasis, as well as crosstalk between the mitochondria and the rest of the cell. It is also involved in mitochondria-mediated apoptosis. Here, we demonstrate that the VDAC1-based peptide, R-Tf-D-LP4, affects several parameters of a NAFLD mouse model in which administration of streptozotocin (STZ) and high-fat diet 32 (STZ/HFD-32) led to both type 2 diabetes (T2D) and NAFLD phenotypes. We focused on diabetes, showing that R-Tf-D-LP4 peptide treatment of STZ/HFD-32 fed mice restored the elevated blood glucose back to close to normal levels, and increased the number and average size of islets and their insulin content as compared to untreated controls. Similar results were obtained when staining the islets for glucose transporter type 2. In addition, the R-Tf-D-LP4 peptide decreased the elevated glucose levels in a mouse displaying obese, diabetic, and metabolic symptoms due to a mutation in the obese (ob) gene. To explore the cause of the peptide-induced improvement in the endocrine pancreas phenotype, we analyzed the expression levels of the proliferation marker, Ki-67, and found it to be increased in the islets of STZ/HFD-32 fed mice treated with the R-Tf-D-LP4 peptide. Moreover, peptide treatment of STZ/HFD-32 fed mice caused an increase in the expression of ß-cell maturation and differentiation PDX1 transcription factor that enhances the expression of the insulin-encoding gene, and is essential for islet development, function, proliferation, and maintenance of glucose homeostasis in the pancreas. This increase occurred mainly in the ß-cells, suggesting that the source of their increased number after R-Tf-D-LP4 peptide treatment was most likely due to ß-cell proliferation. These results suggest that the VDAC1-based R-Tf-D-LP4 peptide has potential as a treatment for diabetes.

Rewiring of Cancer Cell Metabolism by Mitochondrial VDAC1 Depletion Results in Time-Dependent Tumor Reprogramming: Glioblastoma as a Proof of Concept.

Reprograming of the metabolism of cancer cells is an event recognized as a hallmark of the disease. The mitochondrial gatekeeper, voltage-dependent anion channel 1 (VDAC1), mediates transport of metabolites and ions in and out of mitochondria, and is involved in mitochondria-mediated apoptosis. Here, we compared the effects of reducing hVDAC1 expression in a glioblastoma xenograft using human-specific si-RNA (si-hVDAC1) for a short (19 days) and a long term (40 days). Tumors underwent reprograming, reflected in rewired metabolism, eradication of cancer stem cells (CSCs) and differentiation. Short- and long-term treatments of the tumors with si-hVDAC1 similarly reduced the expression of metabolism-related enzymes, and translocator protein (TSPO) and CSCs markers. In contrast, differentiation into cells expressing astrocyte or neuronal markers was noted only after a long period during which the tumor cells were hVDAC1-depleted. This suggests that tumor cell differentiation is a prolonged process that precedes metabolic reprograming and the "disappearance" of CSCs. Tumor proteomics analysis revealing global changes in the expression levels of proteins associated with signaling, synthesis and degradation of proteins, DNA structure and replication and epigenetic changes, all of which were highly altered after a long period of si-hVDAC1 tumor treatment. The depletion of hVDAC1 greatly reduced the levels of the multifunctional translocator protein TSPO, which is overexpressed in both the mitochondria and the nucleus of the tumor. The results thus show that VDAC1 depletion-mediated cancer cell metabolic reprograming involves a chain of events occurring in a sequential manner leading to a reversal of the unique properties of the tumor, indicative of the interplay between metabolism and oncogenic signaling networks.

Targeting Liver Cancer and Associated Pathologies in Mice with a Mitochondrial VDAC1-Based Peptide.

Hepatocellular carcinoma (HCC) is the third most lethal cancer worldwide. Despite progress in identifying risk factors, the incidence of HCC is increasing. Moreover, therapeutic options are limited and survival is poor. Therefore, alternative and innovative therapeutic strategies are urgently required. R-Tf-D-LP4, a cell-penetrating peptide derived from the mitochondrial multifunctional protein the voltage-dependent anion channel (VDAC1), is identified here as a highly effective liver cancer treatment. Recently, we demonstrated that R-Tf-D-LP4 induced apoptosis and inhibited tumor growth in mouse models. We now demonstrate that R-Tf-D-LP4 induced apoptosis in cancer liver-derived cell lines and inhibited tumor growth in three different liver cancer mouse models. These included diethylnitrosamine (DEN)-induced HCC, metabolically high-fat diet-induced HCC, and using a subcutaneous HepG2 cell xenograft model. Intravenous injection of the peptide into tumor-carrying DEN-treated mice resulted in dose-dependent inhibition of tumor growth up to complete tumor elimination. TUNEL staining of liver sections demonstrated peptide-induced apoptosis. Hematoxylin/eosin and Sirius red staining of liver sections showed decreased fibrotic formation. Immunohistochemical staining demonstrated reduced numbers of α-SMA-expressing cells in R-Tf-D-LP4-treated mouse livers. Additionally, macrophage presence in liver tissue was reduced in R-Tf-D-LP4-treated mice. Liver sections from DEN-treated mice showed steatohepatic pathology, reflected as fatty liver, inflammation, ballooning degeneration, and fibrosis; all were eliminated upon peptide treatment. Peptide treatment also inhibited tumor development in a nonalcoholic steatohepatitis-hepatocellular carcinoma mouse model induced by HFD. In HepG2 subcutaneous tumor xenografts, R-Tf-D-LP4 inhibited tumor growth. CONCLUSION: These results show that the VDAC1-based peptide R-Tf-D-LP4 has multiple effects on liver cancer cells, leading to impairment of cell energy and metabolism homeostasis, induction of apoptosis, and elimination of liver cancer-associated processes, and thus represents a promising therapeutic approach for liver cancer.

The prelude on novel receptor and ligand targets involved in the treatment of diabetes mellitus.

Metabolic disorders are a group of disorders, due to the disruption of the normal metabolic process at a cellular level. Diabetes Mellitus and Tyrosinaemia are the majorly reported metabolic disorders. Among them, Diabetes Mellitus is a one of the leading metabolic syndrome, affecting 5 to 7 % of the population worldwide and mainly characterised by elevated levels of glucose and is associated with two types of physiological event disturbances such as impaired insulin secretion and insulin resistance. Up to now, various treatment strategies are like insulin, alphaglucosidase inhibitors, biguanides, incretins were being followed. Concurrently, various novel therapeutic strategies are required to advance the therapy of Diabetes mellitus. For the last few decades, there has been an extensive research in understanding the metabolic pathways involved in Diabetes Mellitus at the cellular level and having the profound knowledge on cell-growth, cell-cycle, and apoptosis at a molecular level provides new targets for the treatment of Diabetes Mellitus. Receptor signalling has been involved in these mechanisms, to translate the information coming from outside. To understand the various receptors involved in these pathways, we must have a sound knowledge on receptors and ligands involved in it. This review mainly summarises the receptors and ligands which are involved the Diabetes Mellitus. Finally, researchers have to develop the alternative chemical moieties that retain their affinity to receptors and efficacy. Diabetes Mellitus being a metabolic disorder due to the glucose surfeit, demands the need for regular exercise along with dietary changes.