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 patients with relapsing-remitting multiple sclerosis (RRMS) and 14 healthy age-matched controls. Of 632 known metabolites detected, 60 were significantly altered in RRMS. Bioinformatics analysis identified an altered metabotype in patients with RRMS, represented by four changed metabolic pathways of glycerophospholipid, citrate cycle, sphingolipid, and pyruvate metabolism. Interestingly, the common upstream metabolic pathway feeding these four 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 antiinflammatory phenotype of monocytes/macrophage in the central nervous system. Our study provided a proof of principle for how a blood-based metabolomic approach using patient samples could lead to the identification of a therapeutic target for developing potential therapy.

Untargeted and temporal analysis of retinal lipidome in bacterial endophthalmitis.

Bacterial endophthalmitis is a blinding infectious disease typically acquired during ocular surgery. We previously reported significant alterations in retinal metabolism during Staphylococcus (S) aureus endophthalmitis. However, the changes in retinal lipid composition during endophthalmitis are unknown. Here, using a mouse model of S. aureus endophthalmitis and an untargeted lipidomic approach, we comprehensively analyzed temporal alterations in total lipids and oxylipin in retina. Our data showed a time-dependent increase in the levels of lipid classes, sphingolipids, glycerolipids, sterols, and non-esterified fatty acids, whereas levels of phospholipids decreased. Among lipid subclasses, phosphatidylcholine decreased over time. The oxylipin analysis revealed increased prostaglandin-E2, hydroxyeicosatetraenoic acids, docosahexaenoic acid, eicosapentaenoic acid, and α-linolenic acid. In-vitro studies using mouse bone marrow-derived macrophages showed increased lipid droplets and lipid-peroxide formation in response to S. aureus infection. Collectively, these findings suggest that S. aureus-infection alters the retinal lipid profile, which may contribute to the pathogenesis of bacterial endophthalmitis.

Oral administration of S-nitroso-L-glutathione (GSNO) provides anti-inflammatory and cytoprotective effects during ocular bacterial infections.

Bacterial endophthalmitis is a severe complication of eye surgeries that can lead to vision loss. Current treatment involves intravitreal antibiotic injections that control bacterial growth but not inflammation. To identify newer therapeutic targets to promote inflammation resolution in endophthalmitis, we recently employed an untargeted metabolomics approach. This led to the discovery that the levels of S-nitroso-L-glutathione (GSNO) were significantly reduced in an experimental murine Staphylococcus aureus (SA) endophthalmitis model. In this study, we tested the hypothesis whether GSNO supplementation via different routes (oral, intravitreal) provides protection during bacterial endophthalmitis. Our results show that prophylactic administration of GSNO via intravitreal injections ameliorated SA endophthalmitis. Therapeutically, oral administration of GSNO was found to be most effective in reducing intraocular inflammation and bacterial burden. Moreover, oral GSNO treatment synergized with intravitreal antibiotic injections in reducing the severity of endophthalmitis. Furthermore, in vitro experiments using cultured human retinal Muller glia and retinal pigment epithelial (RPE) cells showed that GSNO treatment reduced SA-induced inflammatory mediators and cell death. Notably, both in-vivo and ex-vivo data showed that GSNO strengthened the outer blood-retinal barrier during endophthalmitis. Collectively, our study demonstrates GSNO as a potential therapeutic agent for the treatment of intraocular infections due to its dual anti-inflammatory and cytoprotective properties.

Aldehyde dehydrogenase 2 augments adiponectin signaling in coronary angiogenesis in HFpEF associated with diabetes.

4-hydroxy-2-nonenal (4HNE), an oxidative stress byproduct, is elevated in diabetes which decreases coronary angiogenesis, and this was rescued by the 4HNE detoxifying enzyme, aldehyde dehydrogenase 2 (ALDH2). Adiponectin (APN), an adipocytokine, has pro-angiogenic properties and its loss of function is critical in diabetes and its complications. Coronary endothelial cell (CEC) damage is the initiating step of diabetes-mediated heart failure with preserved ejection fraction (HFpEF) pathogenesis. Thus, we hypothesize that ALDH2 restores 4HNE-induced downregulation of APN signaling in CECs and subsequent coronary angiogenesis in diabetic HFpEF. Treatment with disulfiram, an ALDH2 inhibitor, exacerbated 4HNE-mediated decreases in APN-induced increased coronary angiogenesis and APN-signaling cascades, whereas pretreatment with alda1, an ALDH2 activator, rescued the effect of 4HNE. We employed control mice (db/m), spontaneous type-2 diabetic mice (db/db), ALDH2*2 knock-in mutant mice with intrinsic low ALDH2 activity (AL), and diabetic mice with intrinsic low ALDH2 activity (AF) mice that were created by crossing db/db and AL mice to test our hypothesis in vivo. AF mice exhibited heart failure with preserved ejection fraction (HFpEF)/severe diastolic dysfunction at 6 months with a preserved systolic function compared with db/db mice as well as 3 months of their age. Decreased APN-mediated coronary angiogenesis, along with increased circulatory APN levels and decreased cardiac APN signaling (index of APN resistance) were higher in AF mice relative to db/db mice. Alda1 treatment improved APN-mediated angiogenesis in AF and db/db mice. In summary, 4HNE-induces APN resistance and a subsequent decrease in coronary angiogenesis in diabetic mouse heart which was rescued by ALDH2.

A UPLC-MS/MS Based Rapid, Sensitive, and Non-Enzymatic Methodology for Quantitation of Dietary Isoflavones in Biological Fluids.

Dietary isoflavones, a type of phytoestrogens, have gained importance owing to their health-promoting benefits. However, the beneficial effects of isoflavones are mediated by smaller metabolites produced with the help of gut bacteria that are known to metabolize these phytoestrogenic compounds into Daidzein and Genistein and biologically active molecules such as S-Equol. Identifying and measuring these phytoestrogens and their metabolites is an important step towards understanding the significance of diet and gut microbiota in human health and diseases. We have overcome the reported difficulties in quantitation of these isoflavones and developed a simplified, sensitive, non-enzymatic, and sulfatases-free extraction methodology. We have subsequently used this method to quantify these metabolites in the urine of mice using UPLC-MS/MS. The extraction and quantitation method was validated for precision, linearity, accuracy, recoveries, limit of detection (LOD), and limit of quantification (LOQ). Linear calibration curves for Daidzein, Genistein, and S-Equol were set up by performing linear regression analysis and checked using the correlation coefficient (r2 > 0.995). LOQs for Daidzein, Genistein, and S-Equol were 2, 4, and 2 ng/mL, respectively. This UPLC-MS/MS swift method is suitable for quantifying isoflavones and the microbial-derived metabolite S-Equol in mice urine and is particularly useful for large numbers of samples.

AMP-Activated Protein Kinase Restricts Zika Virus Replication in Endothelial Cells by Potentiating Innate Antiviral Responses and Inhibiting Glycolysis.

Viruses are known to perturb host cellular metabolism to enable their replication and spread. However, little is known about the interactions between Zika virus (ZIKV) infection and host metabolism. Using primary human retinal vascular endothelial cells and an established human endothelial cell line, we investigated the role of AMP-activated protein kinase (AMPK), a master regulator of energy metabolism, in response to ZIKV challenge. ZIKV infection caused a time-dependent reduction in the active phosphorylated state of AMPK and of its downstream target acetyl-CoA carboxylase. Pharmacological activation of AMPK using 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), metformin, and a specific AMPKα activator (GSK621) attenuated ZIKV replication. This activity was reversed by an AMPK inhibitor (compound C). Lentivirus-mediated knockdown of AMPK and the use of AMPKα-/- mouse embryonic fibroblasts provided further evidence that AMPK has an antiviral effect on ZIKV replication. Consistent with its antiviral effect, AMPK activation potentiated the expression of genes with antiviral properties (e.g., IFNs, OAS2, ISG15, and MX1) and inhibited inflammatory mediators (e.g., TNF-α and CCL5). Bioenergetic analysis showed that ZIKV infection evokes a glycolytic response, as evidenced by elevated extracellular acidification rate and increased expression of key glycolytic genes (GLUT1, HK2, TPI, and MCT4); activation of AMPK by AICAR treatment reduced this response. Consistent with this, 2-deoxyglucose, an inhibitor of glycolysis, augmented AMPK activity and attenuated ZIKV replication. Thus, our study demonstrates that the anti-ZIKV effect of AMPK signaling in endothelial cells is mediated by reduction of viral-induced glycolysis and enhanced innate antiviral responses.

An emerging potential of metabolomics in multiple sclerosis: a comprehensive overview.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the nervous system that primarily affects young adults. Although the exact etiology of the disease remains obscure, it is clear that alterations in the metabolome contribute to this process. As such, defining a reliable and disease-specific metabolome has tremendous potential as a diagnostic and therapeutic strategy for MS. Here, we provide an overview of studies aimed at identifying the role of metabolomics in MS. These offer new insights into disease pathophysiology and the contributions of metabolic pathways to this process, identify unique markers indicative of treatment responses, and demonstrate the therapeutic effects of drug-like metabolites in cellular and animal models of MS. By and large, the commonly perturbed pathways in MS and its preclinical model include lipid metabolism involving alpha-linoleic acid pathway, nucleotide metabolism, amino acid metabolism, tricarboxylic acid cycle, D-ornithine and D-arginine pathways with collective role in signaling and energy supply. The metabolomics studies suggest that metabolic profiling of MS patient samples may uncover biomarkers that will advance our understanding of disease pathogenesis and progression, reduce delays and mistakes in diagnosis, monitor the course of disease, and detect better drug targets, all of which will improve early therapeutic interventions and improve evaluation of response to these treatments.

Enhanced oxidative phosphorylation in NKT cells is essential for their survival and function.

Cellular metabolism and signaling pathways are key regulators to determine conventional T cell fate and function, but little is understood about the role of cell metabolism for natural killer T (NKT) cell survival, proliferation, and function. We found that NKT cells operate distinct metabolic programming from CD4 T cells. NKT cells are less efficient in glucose uptake than CD4 T cells with or without activation. Gene-expression data revealed that, in NKT cells, glucose is preferentially metabolized by the pentose phosphate pathway and mitochondria, as opposed to being converted into lactate. In fact, glucose is essential for the effector functions of NKT cells and a high lactate environment is detrimental for NKT cell survival and proliferation. Increased glucose uptake and IFN-γ expression in NKT cells is inversely correlated with bacterial loads in response to bacterial infection, further supporting the significance of glucose metabolism for NKT cell function. We also found that promyelocytic leukemia zinc finger seemed to play a role in regulating NKT cells' glucose metabolism. Overall, our study reveals that NKT cells use distinct arms of glucose metabolism for their survival and function.

Current and Future Biomarkers in Multiple Sclerosis.

Multiple sclerosis (MS) is a debilitating autoimmune disorder. Currently, there is a lack of effective treatment for the progressive form of MS, partly due to insensitive readout for neurodegeneration. The recent development of sensitive assays for neurofilament light chain (NfL) has made it a potential new biomarker in predicting MS disease activity and progression, providing an additional readout in clinical trials. However, NfL is elevated in other neurodegenerative disorders besides MS, and, furthermore, it is also confounded by age, body mass index (BMI), and blood volume. Additionally, there is considerable overlap in the range of serum NfL (sNfL) levels compared to healthy controls. These confounders demonstrate the limitations of using solely NfL as a marker to monitor disease activity in MS patients. Other blood and cerebrospinal fluid (CSF) biomarkers of axonal damage, neuronal damage, glial dysfunction, demyelination, and inflammation have been studied as actionable biomarkers for MS and have provided insight into the pathology underlying the disease process of MS. However, these other biomarkers may be plagued with similar issues as NfL. Using biomarkers of a bioinformatic approach that includes cellular studies, micro-RNAs (miRNAs), extracellular vesicles (EVs), metabolomics, metabolites and the microbiome may prove to be useful in developing a more comprehensive panel that addresses the limitations of using a single biomarker. Therefore, more research with recent technological and statistical approaches is needed to identify novel and useful diagnostic and prognostic biomarker tools in MS.

Aldehyde Dehydrogenase 2 Activator Augments the Beneficial Effects of Empagliflozin in Mice with Diabetes-Associated HFpEF.

To ameliorate diabetes mellitus-associated heart failure with preserved ejection fraction (HFpEF), we plan to lower diabetes-mediated oxidative stress-induced 4-hydroxy-2-nonenal (4HNE) accumulation by pharmacological agents that either decrease 4HNE generation or increase its detoxification.A cellular reactive carbonyl species (RCS), 4HNE, was significantly increased in diabetic hearts due to a diabetes-induced decrease in 4HNE detoxification by aldehyde dehydrogenase (ALDH) 2, a cardiac mitochondrial enzyme that metabolizes 4HNE. Therefore, hyperglycemia-induced 4HNE is critical for diabetes-mediated cardiotoxicity and we hypothesize that lowering 4HNE ameliorates diabetes-associated HFpEF. We fed a high-fat diet to ALDH2*2 mice, which have intrinsically low ALDH2 activity, to induce type-2 diabetes. After 4 months of diabetes, the mice exhibited features of HFpEF along with increased 4HNE adducts, and we treated them with vehicle, empagliflozin (EMP) (3 mg/kg/d) to reduce 4HNE and Alda-1 (10 mg/kg/d), and ALDH2 activator to enhance ALDH2 activity as well as a combination of EMP + Alda-1 (E + A), via subcutaneous osmotic pumps. After 2 months of treatments, cardiac function was assessed by conscious echocardiography before and after exercise stress. EMP + Alda-1 improved exercise tolerance, diastolic and systolic function, 4HNE detoxification and cardiac liver kinase B1 (LKB1)-AMP-activated protein kinase (AMPK) pathways in ALDH2*2 mice with diabetes-associated HFpEF. This combination was even more effective than EMP alone. Our data indicate that ALDH2 activation along with the treatment of hypoglycemic agents may be a salient strategy to alleviate diabetes-associated HFpEF.

Cytokine Storm and Failed Resolution in COVID-19: Taking a Cue from Multiple Sclerosis.

INTRODUCTION: Excessive inflammatory responses and failed resolution are major common causes of tissue injury and organ dysfunction in a variety of diseases, including multiple sclerosis (MS), diabetes, and most recently, COVID-19, despite the distinct pathoetiology of the diseases. The promotion of the natural process of inflammatory resolution has been long recognized to improve functional recovery and disease outcomes effectively. To mitigate the excessive inflammation in MS, scientific investigations identified a group of derivatives of omega fatty acids, known as specialized pro-resolving lipid mediators (SPM) that have been significantly effective in treating preclinical disease models of MS. METHODS: This chapter is based on our observations from MS. It is being increasingly deliberated that the ongoing COVID-19 infection induces severe cytokine storm that ultimately triggers rampant inflammation. The impact of infection and associated mortality is much higher in patients with co-morbid diseases. Also, reports suggest a better outcome in diabetic patients with reasonable glycemic control, which certainly hints towards a hidden role of anti-hyperglycemic drugs such as metformin in alleviating disease pathology through its anti-inflammatory feature. Notably, SPM and metformin share common therapeutic features in exerting a broad-spectrum anti-inflammatory activity in human patients with a superior safety profile. RESULTS: When there is an immediate need to encounter the fast-rampant infection of COVID-19 and control the viral-infection associated morbid inflammatory cytokine storm causing severe organ damage, SPM and metformin should be seriously considered as a potential adjunctive treatment. CONCLUSION: Given the fact that current treatment for COVID-19 is only supportive, global research is aimed at developing safe and effective therapeutic options that can result in a better clinical course in patients with comorbid conditions. Accordingly, taking a cue from our experiences in controlling robust inflammatory response in MS and diabetes by simultaneously inhibiting inflammatory process and stimulating its resolution, combinatorial therapy of metformin and SPM in COVID-19 holds significant promise in treating this global health crisis.

Glycolytic inhibitor 2-deoxyglucose suppresses inflammatory response in innate immune cells and experimental staphylococcal endophthalmitis.

Previously, we have shown that Staphylococcus (S) aureus induces a glycolytic response in retinal residential (microglia) and infiltrated cells (neutrophils and macrophages) during endophthalmitis. In this study, we sought to investigate the physiological role of glycolysis in bacterial endophthalmitis using a glycolytic inhibitor, 2-deoxyglucose (2DG). Our data showed that 2DG treatment attenuated the inflammatory responses of mouse bone marrow-derived macrophages (BMDM) and neutrophils (BMDN) when challenged with either live or heat-killed S. aureus (HKSA). Among the inflammatory mediators, 2DG caused a significant reduction in levels of cytokines (TNF-α, IL-1ß, IL-6) and chemokines (CXCL1 and CXCL2). Western blot analysis of 2DG treated cells showed downregulation of bacterial-induced MEK/ERK pathways. In vivo, intravitreal administration of 2DG both pre- and post-bacterial infection resulted in a significant reduction in intraocular inflammation in C57BL/6 mouse eyes and downregulation of ERK phosphorylation in retinal tissue. Collectively, our study demonstrates that 2DG attenuates inflammatory response in bacterial endophthalmitis and cultured innate immune cells via inhibition of ERK signaling. Thus glycolytic inhibitors in combination with antibiotics could mitigate inflammation-mediated tissue damage in ocular infections.

AMP-Activated Protein Kinase Suppresses Autoimmune Central Nervous System Disease by Regulating M1-Type Macrophage-Th17 Axis.

The AMP-activated protein kinase, AMPK, is an energy-sensing, metabolic switch implicated in various metabolic disorders; however, its role in inflammation is not well defined. We have previously shown that loss of AMPK exacerbates experimental autoimmune encephalomyelitis (EAE) disease severity. In this study, we investigated the mechanism through which AMPK modulates inflammatory disease like EAE. AMPKα1 knockout (α1KO) mice with EAE showed severe demyelination and inflammation in the brain and spinal cord compared with wild-type due to higher expression of proinflammatory Th17 cytokines, including IL-17, IL-23, and IL-1ß, impaired blood-brain barrier integrity, and increased infiltration of inflammatory cells in the CNS. Infiltrated CD4 cells in the brains and spinal cords of α1KO with EAE were significantly higher compared with wild-type EAE and were characterized as IL-17 (IL-17 and GM-CSF double-positive) CD4 cells. Increased inflammatory response in α1KO mice was due to polarization of macrophages (Mϕ) to proinflammatory M1 type phenotype (IL-10(low)IL-23/IL-1ß/IL-6(high)), and these M1 Mϕ showed stronger capacity to induce allogenic as well as Ag-specific (myelin oligodendrocyte glycoprotein [MOG]35-55) T cell response. Mϕ from α1KO mice also enhanced the encephalitogenic property of MOG35-55-primed CD4 T cells in B6 mice. The increased encephalitogenic MOG-restricted CD4(+) T cells were due to an autocrine effect of IL-1ß/IL-23-mediated induction of IL-6 production in α1KO Mϕ, which in turn induce IL-17 and GM-CSF production in CD4 cells. Collectively, our data indicate that AMPK controls the inflammatory disease by regulating the M1 phenotype-Th17 axis in an animal model of multiple sclerosis.

5-Aminoimidazole-4-carboxamide ribonucleoside-mediated adenosine monophosphate-activated protein kinase activation induces protective innate responses in bacterial endophthalmitis.

The retina is considered to be the most metabolically active tissue in the body. However, the link between energy metabolism and retinal inflammation, as incited by microbial infection such as endophthalmitis, remains unexplored. In this study, using a mouse model of Staphylococcus aureus (SA) endophthalmitis, we demonstrate that the activity (phosphorylation) of 5' adenosine monophosphate-activated protein kinase alpha (AMPKα), a cellular energy sensor and its endogenous substrate; acetyl-CoA carboxylase is down-regulated in the SA-infected retina. Intravitreal administration of an AMPK activator, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), restored AMPKα and acetyl-CoA carboxylase phosphorylation. AICAR treatment reduced both the bacterial burden and intraocular inflammation in SA-infected eyes by inhibiting NF-kB and MAP kinases (p38 and JNK) signalling. The anti-inflammatory effects of AICAR were diminished in eyes pretreated with AMPK inhibitor, Compound C. The bioenergetics (Seahorse) analysis of SA-infected microglia and bone marrow-derived macrophages revealed an increase in glycolysis, which was reinstated by AICAR treatment. AICAR also reduced the expression of SA-induced glycolytic genes, including hexokinase 2 and glucose transporter 1 in microglia, bone marrow-derived macrophages and the mouse retina. Interestingly, AICAR treatment enhanced the bacterial phagocytic and intracellular killing activities of cultured microglia, macrophages and neutrophils. Furthermore, AMPKα1 global knockout mice exhibited increased susceptibility towards SA endophthalmitis, as evidenced by increased inflammatory mediators and bacterial burden and reduced retinal function. Together, these findings provide the first evidence that AMPK activation promotes retinal innate defence in endophthalmitis by modulating energy metabolism and that it can be targeted therapeutically to treat ocular infections.

Synergistic effect of MEK inhibitor and metformin combination in low grade serous ovarian cancer.

OBJECTIVE: Low-grade serous ovarian cancer (LGSOC) constitutes 5-8% of epithelial ovarian cancers and is refractory to chemotherapy. We and others have shown metformin to cause significant growth inhibition in high-grade ovarian cancer both in vitro and in vivo. Here, we aimed to analyze if metformin was effective in inhibiting proliferation of LGSOC alone and in combination with MEK inhibitor. METHODS: Three LGSOC lines (VOA1056, VOA1312 and VOA5646) were treated with metformin, trametinib or 2-deoxyglucose (2DG) alone or in combination with metformin. Proliferation was measured by MTT assay over a period of four days. Protein expression was measured by western blotting. Seahorse Analyzer was used to measure effect of metformin on glycolysis and mitochondrial respiration. RESULTS: All LGSOC cell lines showed significant inhibition with metformin in a dose- and time-dependent manner. Trametinib significantly inhibited the growth of Ras mutated LGSOC lines (VOA1312 and VOA1056), while VOA5646 cells without RAS mutation did not show any response. Metformin and trametinib combination showed synergistic inhibition of RAS mutated VOA1312 and VOA1056 cells, but not for non-Ras mutated VOA5646 cells. Metformin and trametinib increased phosphorylated AMPK expression in LGSOC lines with combination showing stronger expression. Trametinib decreased 42/44 mitogen activated kinase phosphorylation in all cell lines, while metformin and combination had no significant effect. 2-DG significantly inhibited glycolysis in all LGSOC lines and combination with metformin showed synergistic inhibitory effect. CONCLUSIONS: Metformin alone or in combination with MEK and glycolytic inhibitors may be a potential therapy for LGSOC, a cancer that is indolent but chemo-resistant.

Discrete mechanisms of mTOR and cell cycle regulation by AMPK agonists independent of AMPK.

The multifunctional AMPK-activated protein kinase (AMPK) is an evolutionarily conserved energy sensor that plays an important role in cell proliferation, growth, and survival. It remains unclear whether AMPK functions as a tumor suppressor or a contextual oncogene. This is because although on one hand active AMPK inhibits mammalian target of rapamycin (mTOR) and lipogenesis--two crucial arms of cancer growth--AMPK also ensures viability by metabolic reprogramming in cancer cells. AMPK activation by two indirect AMPK agonists AICAR and metformin (now in over 50 clinical trials on cancer) has been correlated with reduced cancer cell proliferation and viability. Surprisingly, we found that compared with normal tissue, AMPK is constitutively activated in both human and mouse gliomas. Therefore, we questioned whether the antiproliferative actions of AICAR and metformin are AMPK independent. Both AMPK agonists inhibited proliferation, but through unique AMPK-independent mechanisms and both reduced tumor growth in vivo independent of AMPK. Importantly, A769662, a direct AMPK activator, had no effect on proliferation, uncoupling high AMPK activity from inhibition of proliferation. Metformin directly inhibited mTOR by enhancing PRAS40's association with RAPTOR, whereas AICAR blocked the cell cycle through proteasomal degradation of the G2M phosphatase cdc25c. Together, our results suggest that although AICAR and metformin are potent AMPK-independent antiproliferative agents, physiological AMPK activation in glioma may be a response mechanism to metabolic stress and anticancer agents.

Untargeted Plasma Metabolomics Identifies Endogenous Metabolite with Drug-like Properties in Chronic Animal Model of Multiple Sclerosis.

We performed untargeted metabolomics in plasma of B6 mice with experimental autoimmune encephalitis (EAE) at the chronic phase of the disease in search of an altered metabolic pathway(s). Of 324 metabolites measured, 100 metabolites that mapped to various pathways (mainly lipids) linked to mitochondrial function, inflammation, and membrane stability were observed to be significantly altered between EAE and control (p < 0.05, false discovery rate <0.10). Bioinformatics analysis revealed six metabolic pathways being impacted and altered in EAE, including α-linolenic acid and linoleic acid metabolism (PUFA). The metabolites of PUFAs, including ω-3 and ω-6 fatty acids, are commonly decreased in mouse models of multiple sclerosis (MS) and in patients with MS. Daily oral administration of resolvin D1, a downstream metabolite of ω-3, decreased disease progression by suppressing autoreactive T cells and inducing an M2 phenotype of monocytes/macrophages and resident brain microglial cells. This study provides a proof of principle for the application of metabolomics to identify an endogenous metabolite(s) possessing drug-like properties, which is assessed for therapy in preclinical mouse models of MS.

Metformin-induced mitochondrial function and ABCD2 up-regulation in X-linked adrenoleukodystrophy involves AMP-activated protein kinase.

X-linked adrenoleukodystrophy (X-ALD) is a progressive neurometabolic disease caused by mutations/deletions in the Abcd1 gene. Similar mutations/deletions in the Abcd1 gene often result in diagonally opposing phenotypes of mild adrenomyeloneuropathy and severe neuroinflammatory cerebral adrenoleukodystrophy (ALD), which suggests involvement of downstream modifier genes. We recently documented the first evidence of loss of AMP-activated protein kinase α1 (AMPKα1) in ALD patient-derived cells. Here, we report the novel loss of AMPKα1 in postmortem brain white matter of patients with ALD phenotype. Pharmacological activation of AMPK can rescue the mitochondrial dysfunction and inhibit the pro-inflammatory response. The FDA approved anti-diabetic drug Metformin, a well-known AMPK activator, induces mitochondrial biogenesis and is documented for its anti-inflammatory role. We observed a dose-dependent activation of AMPKα1 in metformin-treated X-ALD patient-derived fibroblasts. Metformin also induced mitochondrial oxidative phosphorylation and ATP levels in X-ALD patient-derived fibroblasts. Metformin treatment decreased very long chain fatty acid levels and pro-inflammatory cytokine gene expressions in X-ALD patient-derived cells. Abcd2 [adrenoleukodystrophy protein-related protein] levels were increased in metformin-treated X-ALD patient-derived fibroblasts and Abcd1-KO mice primary mixed glial cells. Abcd2 induction was AMPKα1-dependent since metformin failed to induce Abcd2 levels in AMPKα1-KO mice-derived primary mixed glial cells. In vivo metformin (100 mg/Kg) in drinking water for 60 days induced Abcd2 levels and mitochondrial oxidative phosphorylation protein levels in the brain and spinal cord of Abcd1-KO mice. Taken together, these results provide proof-of-principle for therapeutic potential of metformin as a useful strategy for correcting the metabolic and inflammatory derangements in X-ALD by targeting AMPK. There is no effective therapy for inherited peroxisomal disorder X-linked adrenoleukodystrophy (X-ALD). We document the therapeutic potential of FDA approved drug, Metformin, for X-ALD by targeting AMPK. Metformin induced peroxisomal Abcd2 levels in vitro and in vivo. Metformin lowered VLCFA levels, improved mitochondrial function and ameliorated inflammatory gene expression in X-ALD patient-derived cells. Metformin-induced Abcd2 levels were dependent on AMPKα1, a metabolic and anti-inflammatory gene, recently documented by our laboratory to play a putative role in X-ALD pathology. Read the Editorial Highlight for this article on page 10.

Folic acid tagged nanoceria as a novel therapeutic agent in ovarian cancer.

BACKGROUND: Nanomedicine is a very promising field and nanomedical drugs have recently been used as therapeutic agents against cancer. In a previous study, we showed that Nanoceria (NCe), nanoparticles of cerium oxide, significantly inhibited production of reactive oxygen species, cell migration and invasion of ovarian cancer cells in vitro, without affecting cell proliferation and significantly reduced tumor growth in an ovarian cancer xenograft nude model. Increased expression of folate receptor-α, an isoform of membrane-bound folate receptors, has been described in ovarian cancer. To enable NCe to specifically target ovarian cancer cells, we conjugated nanoceria to folic acid (NCe-FA). Our aim was to investigate the pre-clinical efficacy of NCe-FA alone and in combination with Cisplatin. METHODS: Ovarian cancer cell lines were treated with NCe or NCe-FA. Cell viability was assessed by MTT and colony forming units. In vivo studies were carried in A2780 generated mouse xenografts treated with 0.1 mg/Kg NCe, 0.1 mg/Kg; NCe-FA and cisplatinum, 4 mg/Kg by intra-peritoneal injections. Tumor weights and burden scores were determined. Immunohistochemistry and toxicity assays were used to evaluate treatment effects. RESULTS: We show that folic acid conjugation of NCe increased the cellular NCe internalization and inhibited cell proliferation. Mice treated with NCe-FA had a lower tumor burden compared to NCe, without any vital organ toxicity. Combination of NCe-FA with cisplatinum decreased the tumor burden more significantly. Moreover, NCe-FA was also effective in reducing proliferation and angiogenesis in the xenograft mouse model. CONCLUSION: Thus, specific targeting of ovarian cancer cells by NCe-FA holds great potential as an effective therapeutic alone or in combination with standard chemotherapy.