Energy metabolism adaptations and gene expression reprogramming in a cellular MAFLD model

Mitochondrial dysfunction plays a critical role in metabolic associated fatty liver disease (MAFLD). This study aims to characterize mitochondrial dysfunctions in a human MAFLD Huh7 cell model triggered by free fatty acid (FFA) (palmitate and oleate) overload for 24 hours. We investigate its impact on cellular energy metabolism and identify potential targets for MAFLD treatment. FFA-treated cells displayed an accumulation of lipid droplets and slightly decreased viability but no significant changes in mitochondrial superoxide levels. Bioenergetic analysis showed a shift to more respiration and less glycolytic fermentation. Comprehensive transcriptomics and proteomics analyses identified changes in the expression of genes prominently involved in fatty acid handling and metabolism. The expressions of seven genes were consistently and significantly (p<0.05) altered (4 upregulated and 3 downregulated genes) in both proteomics and transcriptomics. The FFA-treated Huh7 cell model is an appropriate in vitro model to study fatty acid metabolism and suitable to investigate the role of mitochondria, glycolysis, and multiple metabolic pathways in MAFLD. Our comprehensive analyses form a basis for drug discovery and screening using this model.

Venetoclax Reverses Metabolic Reprogramming Induced By S1P Modulator FTY720, Suppresses Oxidative Phosphorylation and Synergistically Targets Multiple Myeloma

Patients with multiple myeloma (MM) invariably relapse with chemotherapy-resistant disease, underscoring the need for new therapeutic modalities that bypass these resistance mechanisms. FTY720, also known as fingolimod, is an S1P modulator approved by the FDA to treat the relapsing form of multiple sclerosis. Previously we reported that FTY720 exhibits potent anti-myeloma effect in vitro and in vivo in disseminated xenograft model of MM (Beider et al., Clin Cancer Res 2017). Cytotoxic activity of FTY720 was associated with down-regulation of anti-apoptotic protein MCL-1 while not affecting BCL-2 levels. It is therefore conceivable that BCL-2 inhibition using BH3-mimetic venetoclax may improve responses to FTY720. Incubation of human MM cell lines (n=8) and primary MM cells (n=3) with venetoclax and FTY720 combination synergistically potentiated cell death (CI&lt;0.02), regardless of the MM cells t (11; 14) status. The robust apoptosis induced by venetoclax /FTY720 treatment was accompanied by cytochrome C release, activation of caspase-3 and extensive DNA damage, demonstrated by increased TUNEL staining and elevated levels of phosphorylated histone H2AX, respectively . These effects were associated with down-regulation of BCL-2 protein, stabilization of pro-apoptotic Bak protein, loss of mitochondrial membrane potential, ER stress induction, and inhibition of the AKT/mTOR signaling pathway. Furthermore, the venetoclax /FTY720 combination markedly induced mitochondrial calcium flux and mitochondrial ROS generation. Corresponding with mitochondrial destabilization, venetoclax/FTY720 combination promoted the release of apoptosis-inducing factor (AIF) from the mitochondria to the cytosol and subsequently increased AIF nuclear localization, suggesting its functional role in the execution phase of the apoptosis in response to the dual treatment. AIF is a mitochondrial oxidoreductase that contributes to cell death programs and participates in the assembly of the respiratory chain. Of note, single-agent treatment with FTY720 profoundly up-regulated mitochondrial AIF levels. Given the regulative role of AIF in mitochondrial bioenergetics, we could suggest that increased mitochondrial levels of AIF upon FTY720 exposure may support adaptive responses and promote MM survival upon mitochondrial stress. We thus investigated a possible effect of venetoclax and FTY720 separately or in combination on the metabolic activity of MM cells, observing distinct metabolic profiles of single versus combined exposures. FTY720 significantly suppressed glycolysis, down-regulating the transcript levels of the glycolytic enzymes HK2, PDK1, and LDHA. Glycolytic suppression may result in upregulation of mitochondrial content, which maintains cell survival. In accordance, increased mitochondrial activity was detected in FTY720-treated MM cells, detected by high uptake of MitoSpy Red, a dye that stains mitochondria in a membrane potential-dependent manner. To determine if the changes in the mitochondrial content also altered mitochondrial function, bioenergetic analysis was undertaken. FTY720-treated MM cells demonstrated increased levels of NDUFB8 and UQCRC2 (subunits of mitochondrial respiratory complexes I and III, respectively). Furthermore, FTY720 exposure up-regulated ATP production, suggesting an increase in tumor-protective oxidative phosphorylation (OXPHOS). In agreement, inhibition of mitochondrial electron transport chain using rotenone sensitized MM cells to FTY720, synergistically promoting cell death. Notably, co-treatment with venetoclax effectively reversed the metabolic changes mediated by FTY720, reducing mitochondrial mass, suppressing mitochondrial activity and strongly down-regulating the pathways related to OXPHOS. Furthermore, venetoclax/FTY720 combination significantly reduced glutathione (GSH) levels, therefore suppressing antioxidative cell responses. To conclude, we unveil venetoclax role in the metabolic regulation in MM cells. Venetoclax reverses metabolic reprogramming induced by FTY720, suppresses mitochondrial respiration, induces vigorous mitochondrial damage and preferentially targets MM cells in combination with FTY720. These findings may provide the scientific basis for a novel combinatorial anti-myeloma therapy. Figure 1 Figure 1. Disclosures Peled: Biokine Therapeutics Ltd: Current Employment; Gamida Cell: Research Funding.

Blood-based untargeted metabolomics in Relapsing-Remitting Multiple Sclerosis revealed the testable therapeutic target

Metabolic aberrations impact the pathogenesis of multiple sclerosis (MS) and possibly can provide clues for new treatment strategies. Using untargeted metabolomics, we measured serum metabolites from 35 relapsing-remitting patients and 14 healthy age-matched controls. Out of 632 known metabolites detected, 60 were significantly altered in relapsing-remitting MS (RRMS). Bioinformatics analysis identified an altered metabotype in RRMS patients, represented by 4 changed metabolic pathways of glycerophospholipid, citrate cycle, sphingolipid, and pyruvate metabolism. Interestingly, the common upstream metabolic pathway feeding these 4 pathways is the glycolysis pathway. Real-time bioenergetic analysis of the patient derived peripheral blood mononuclear cells, showed enhanced glycolysis, supporting the altered metabolic state of immune cells. Experimental autoimmune encephalomyelitis mice treated with the glycolytic inhibitor, 2-deoxy-D-glucose ameliorated the disease progression and inhibited the disease pathology significantly by promoting the anti-inflammatory phenotype of monocytes/macrophage in the central nervous system. Our study suggests that targeting glycolysis offers a potential target for MS.

Physiologically Relevant Concentrations of Dolutegravir, Emtricitabine, and Efavirenz Induce Distinct Metabolic Alterations in HeLa Epithelial and BV2 Microglial Cells

Microglia, the resident brain phagocytes, likely play a key role in human immunodeficiency virus (HIV) infection of the central nervous system (CNS) and subsequent neuropathogenesis; however, the nature of the infection-induced changes that yield damaging CNS effects and the stimuli that provoke microglial activation remains elusive, especially in the current era of using antiretroviral (ARV) drugs for ARV therapy (ART). Altered microglial metabolism can modulate cellular functionality and pathogenicity in neurological disease. While HIV infection itself alters brain energy metabolism, the effect of ARV drugs, particularly those currently used in treatment, on metabolism is understudied. Dolutegravir (DTG) and emtricitabine (FTC) combination, together with tenofovir (TAF or TDF), is one of the recommended first line treatments for HIV. Despite the relatively good tolerability and safety profile of FTC, a nucleoside reverse transcriptase inhibitor, and DTG, an integrase inhibitor, adverse side effects have been reported and highlight a need to understand off-target effects of these medications. We hypothesized that similar to previous ART regimen drugs, DTG and FTC side effects involve mitochondrial dysfunction. To increase detection of ARV-induced mitochondrial effects, highly glycolytic HeLa epithelial cells were forced to rely on oxidative phosphorylation by substituting galactose for glucose in the growth media. We assessed ATP levels, resazurin oxidation-reduction (REDOX), and mitochondrial membrane potential following 24-hour exposure (to approximate effects of one dose equivalent) to DTG, FTC, and efavirenz (EFV, a known mitotoxic ARV drug). Further, since microglia support productive HIV infection, act as latent HIV cellular reservoirs, and when dysfunctional likely contribute to HIV-associated neurocognitive disorders, the experiments were repeated using BV2 microglial cells. In HeLa cells, FTC decreased mitochondrial REDOX activity, while DTG, similar to EFV, impaired both mitochondrial ATP generation and REDOX activity. In contrast to HeLa cells, DTG increased cellular ATP generation and mitochondrial REDOX activity in BV2 cells. Bioenergetic analysis revealed that DTG, FTC, and EFV elevated BV2 cell mitochondrial respiration. DTG and FTC exposure induced distinct mitochondrial functional changes in HeLa and BV2 cells. These findings suggest cell type-specific metabolic changes may contribute to the toxic side effects of these ARV drugs.

Self-Regulation and Psychodynamics in Bioenergetic Analysis

This article presents ten theses containing theoretical considerations for a postgraduate curriculum as currently tested by the Polish Society for Bioenergetic Analysis. The bioenergetic notion of the self, of affect regulation and of mental defense are modified in order to allow for psychodynamic conflict-, structureand trauma-pathological concepts to be integrated into Bioenergetic Analysis. Among other advantages, this approach facilitates the work with structural disorders (narcissism, borderline personality disorder) in Bioenergetic Analysis.

A Traumatic Event

The article describes a work of Bioenergetic Analysis performed in the business sector. The therapists provided group therapy in a company context in a situation of serious grief (an employee had been killed in an occupational accident while several of his colleagues were present). Good results were achieved. Initially, the client requested individual psychological therapy for the thirty colleagues of the deceased man. This request was redefined to cover two approaches: immediate individual therapy (for those employees most affected) and group therapy. This paper describes the group therapy performed in the company environment. The paper also discusses some of the techniques used; the theoretical basis (Lowen’s Bioenergetic Analysis); incorporation of neurological advances on trauma (Porges’ Polyvagal Theory), trauma treatment (Berceli, Levine, Ogden, Rothschild and Van der Kolk); and the concepts that might be most helpful for anyone replicating this work in similar contexts.

Mitochondrial dysfunction underlying sporadic inclusion body myositis is ameliorated by the mitochondrial homing drug MA-5

Sporadic inclusion body myositis (sIBM) is the most common idiopathic inflammatory myopathy, and several reports have suggested that mitochondrial abnormalities are involved in its etiology. We recruited 9 sIBM patients and found significant histological changes and an elevation of growth differential factor 15 (GDF15), a marker of mitochondrial disease, strongly suggesting the involvement of mitochondrial dysfunction. Bioenergetic analysis of sIBM patient myoblasts revealed impaired mitochondrial function. Decreased ATP production, reduced mitochondrial size and reduced mitochondrial dynamics were also observed in sIBM myoblasts. Cell vulnerability to oxidative stress also suggested the existence of mitochondrial dysfunction. Mitochonic acid-5 (MA-5) increased the cellular ATP level, reduced mitochondrial ROS, and provided protection against sIBM myoblast death. MA-5 also improved the survival of sIBM skin fibroblasts as well as mitochondrial morphology and dynamics in these cells. The reduction in the gene expression levels of Opa1 and Drp1 was also reversed by MA-5, suggesting the modification of the fusion/fission process. These data suggest that MA-5 may provide an alternative therapeutic strategy for treating not only mitochondrial diseases but also sIBM.

NIMG-49. VALIDATION OF SIMULTANEOUS PH- AND O2-WEIGHTED MOLECULAR MRI USING 18F-FDG PET, IMMUNOHISTOCHEMISTRY, AND BIOENERGETICS

This study aimed to investigate the potential of a novel MRI metric related to aerobic glycolysis in patients with diffuse gliomas. All subjects (Study I–III; 7, 26, and 11 subjects, respectively) were scanned on 3-T systems and underwent pH-weighted amine chemical exchange saturation transfer spin-and-gradient-echo echoplanar imaging (CEST-SAGE-EPI) or CEST-EPI, and perfusion imaging. Relative oxygen extraction fraction (rOEF) was calculated by dividing hypoxia-sensitive R2’ by normalized relative cerebral blood volume (nrCBV); a novel metric that characterizes glycolytic status (aerobic glycolytic index, AGI) was calculated by dividing amine CEST contrast by rOEF. Patients in Study I were additionally scanned by 18F-fluorodeoxyglucose (FDG)-PET. Stereotactic image-guided biopsies were performed on patients in Study II and III, and samples were analyzed by immunohistochemistry (IHC) and extracellular flux bioenergetic analysis, respectively. Pairwise correlation between MR metrics and standardized uptake value of 18F-FDG, IHC metrics, or indices of cellular metabolism was calculated using Spearman’s correlation analysis. In Study I, AGI showed very strong significant correlation with 18F-FDG uptake in glioma (correlation coefficient ρ = 0.86, P =0.014). In Study II, AGI was significantly correlated with glucose transporter 3 (ρ = 0.71; P = 0.0041) and hexokinase 2 (ρ = 0.73; P = 0.0029) in IDH wild-type glioma, while it was significantly correlated with monocarboxylate transporter 1 (ρ = 0.59; P = 0.0094) in IDH mutant glioma. This result may reflect the different glycolytic statuses of these gliomas; specifically, the rate-limiting steps in glycolysis. In Study III, a strong significant correlation with cellular AGI derived from the bioenergetic analysis was found for AGI derived from MRI (ρ = 0.79, P = .036). In conclusion, AGI derived from MRI was correlated with FDG, IHC measurements, and cellular AGI. Future studies investigating the clinical utility of AGI in prediction and evaluation of treatment effects are warranted.