BAM15

Mitochondrial uncoupling attenuates sarcopenic obesity by enhancing skeletal muscle mitophagy and quality control.

Sarcopenic obesity is a highly prevalent disease with poor survival and ineffective medical interventions. Mitochondrial dysfunction is purported to be central in the pathogenesis of sarcopenic obesity by impairing both organelle biogenesis and quality control. We have previously identified that a mitochondrial-targeted furazano[3,4-b]pyrazine named BAM15 is orally available and selectively lowers respiratory coupling efficiency and protects against diet-induced obesity in mice. Here, we tested the hypothesis that mitochondrial uncoupling simultaneously attenuates loss of muscle function and weight gain in a mouse model of sarcopenic obesity. Eighty-week-old male C57BL/6J mice with obesity were randomized to 10&#xa0;weeks of high fat diet (CTRL) or BAM15 (BAM15; 0.1% w/w in high fat diet) treatment. Body weight and food intake were measured weekly. Body composition, muscle function, energy expenditure, locomotor activity, and glucose tolerance were determined after treatment. Skeletal muscle was harvested and evaluated for histology, gene expression, protein signalling, and mitochondrial structure and function. BAM15 decreased body weight (54.0&#xa0;&#xb1;&#xa0;2.0 vs. 42.3&#xa0;&#xb1;&#xa0;1.3&#xa0;g, P&#xa0;<&#xa0;0.001) which was attributable to increased energy expenditure (10.1&#xa0;&#xb1;&#xa0;0.1 vs. 11.3&#xa0;&#xb1;&#xa0;0.4&#xa0;kcal/day, P&#xa0;<&#xa0;0.001). BAM15 increased muscle mass (52.7&#xa0;&#xb1;&#xa0;0.4 vs. 59.4&#xa0;&#xb1;&#xa0;1.0%, P&#xa0;<&#xa0;0.001), strength (91.1&#xa0;&#xb1;&#xa0;1.3 vs. 124.9&#xa0;&#xb1;&#xa0;1.2&#xa0;g, P&#xa0;<&#xa0;0.0001), and locomotor activity (347.0&#xa0;&#xb1;&#xa0;14.4 vs. 432.7&#xa0;&#xb1;&#xa0;32.0&#xa0;m, P&#xa0;<&#xa0;0.001). Improvements in physical function were mediated in part by reductions in skeletal muscle inflammation (interleukin 6 and gp130, both P&#xa0;<&#xa0;0.05), enhanced mitochondrial function, and improved endoplasmic reticulum homeostasis. Specifically, BAM15 activated mitochondrial quality control (PINK1-ubiquitin binding and LC3II, P&#xa0;<&#xa0;0.01), increased mitochondrial activity (citrate synthase and complex II activity, all P&#xa0;<&#xa0;0.05), restricted endoplasmic reticulum (ER) misfolding (decreased oligomer A11 insoluble/soluble ratio, P&#xa0;<&#xa0;0.0001) while limiting ER stress (decreased PERK signalling, P&#xa0;<&#xa0;0.0001), apoptotic signalling (decreased cytochrome C release and Caspase-3/9 activation, all P&#xa0;<&#xa0;0.001), and muscle protein degradation (decreased 14-kDa actin fragment insoluble/soluble ratio, P&#xa0;<&#xa0;0.001). Mitochondrial uncoupling by agents such as BAM15 may mitigate age-related decline in muscle mass and function by molecular and cellular bioenergetic adaptations that confer protection against sarcopenic obesity.

BAM15 treats mouse sepsis and kidney injury, linking mortality, mitochondrial DNA, tubule damage, and neutrophils.

Sepsis pathogenesis is complex and heterogeneous; hence, a precision-medicine strategy is needed. Acute kidney injury (AKI) following sepsis portends higher mortality. Overproduction of mitochondrial ROS (mtROS) is a potential mediator of sepsis and sepsis-induced AKI. BAM15, a chemical uncoupler, dissipates mitochondrial proton gradients without generating mtROS. We injected BAM15 into mice at 0, 6, or 12 hours after cecal ligation and puncture (CLP), and these mice were treated with fluids and antibiotics. BAM15 reduced mortality, even after 12 hours, when mice were ill, and BAM15 reduced kidney damage and splenic apoptosis. Serial plasma and urinary mitochondrial DNA (mtDNA) levels increased after CLP and decreased after BAM15 administration (at 0 or 6 hours). In vitro septic serum proportionately increased mtROS overproduction and mtDNA release from kidney tubule cells, which BAM15 prevented. BAM15 decreased neutrophil apoptosis and mtDNA release; neutrophil depletion counteracted BAM15 benefits. Further, mtDNA injection in vivo replicated inflammation and kidney injury, which was prevented by BAM15. A large dose of exogenous mtDNA reversed protection by BAM15. We conclude that BAM15 is an effective preventive and therapeutic candidate in experimental sepsis and that BAM15 and mtDNA, a potential drug-companion diagnostic/drug-efficacy pair for clinical sepsis, are mechanistically linked via mtROS.

Low-Dose Sorafenib Acts as a Mitochondrial Uncoupler and Ameliorates Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is becoming one of the leading causes of hepatocellular carcinoma (HCC). Sorafenib is the only first-line therapy for advanced HCC despite its serious adverse effects. Here, we report that at an equivalent of approximately one-tenth the clinical dose for HCC, sorafenib treatment effectively prevents the progression of NASH in both mice and monkeys without any observed significant adverse events. Mechanistically, sorafenib's benefit in NASH is independent of its canonical kinase targets in HCC, but involves the induction of mild mitochondrial uncoupling and subsequent activation of AMP-activated protein kinase (AMPK). Collectively, our findings demonstrate a previously unappreciated therapeutic effect and signaling mechanism of low-dose sorafenib treatment in NASH. We envision that this new therapeutic strategy for NASH has the potential to translate into a beneficial anti-NASH therapy with fewer adverse events than is observed in the drug's current use in HCC.

The magic bullet: Niclosamide.

The term 'magic bullet' is a scientific concept proposed by the German Nobel laureate Paul Ehrlich in 1907, describing a medicine that could specifically and efficiently target a disease without harming the body. Oncologists have been looking for a magic bullet for cancer therapy ever since. However, the current therapies for cancers-including chemotherapy, radiation therapy, hormone therapy, and targeted therapy-pose either pan-cytotoxicity or only single-target efficacy, precluding their ability to function as a magic bullet. Intriguingly, niclosamide, an FDA-approved drug for treating tapeworm infections with an excellent safety profile, displays broad anti-cancer activity in a variety of contexts. In particular, niclosamide inhibits multiple oncogenic pathways such as Wnt/&#x3b2;-catenin, Ras, Stat3, Notch, E2F-Myc, NF-&#x3ba;B, and mTOR and activates tumor suppressor signaling pathways such as p53, PP2A, and AMPK. Moreover, niclosamide potentially improves immunotherapy by modulating pathways such as PD-1/PDL-1. We recently discovered that niclosamide ethanolamine (NEN) reprograms cellular metabolism through its uncoupler function, consequently remodeling the cellular epigenetic landscape to promote differentiation. Inspired by the promising results from the pre-clinical studies, several clinical trials are ongoing to assess the therapeutic effect of niclosamide in cancer patients. This current review summarizes the functions, mechanism of action, and potential applications of niclosamide in cancer therapy as a magic bullet.

Beneficial effects of MGL-3196 and BAM15 combination in a mouse model of fatty liver disease.

Metabolic dysfunction-associated steatohepatitis (MASH) is a metabolic disorder with limited treatment options. The thyroid hormone receptor (THR)-&#x3b2; agonist resmetirom/MGL-3196 (MGL) increases liver fat oxidation and has been approved for treating adult MASH. However, over 60% of patients receiving MGL treatment do not achieve MASH resolution. Therefore, we investigated the potential for combination therapy of MGL with the mitochondrial uncoupler BAM15 to improve fatty liver disease outcomes in the GAN mouse model of MASH. C57BL/6J male mice were fed GAN diet for 38&#x2009;weeks before stratification and randomization to treatments including MGL, BAM15, MGL&#x2009;+&#x2009;BAM15, or no drug control for 8&#x2009;weeks. Treatments were admixed in diet and mice were pair-fed to control for drug intake. Treatment effectiveness was assessed by body weight, body composition, energy expenditure, glucose tolerance, tissue lipid content, and histological analyses. MGL&#x2009;+&#x2009;BAM15 treatment resulted in better efficacy versus GAN control mice than either monotherapy in the context of energy expenditure, liver fat loss, glucose control, and fatty liver disease activity score. Improvements in ALT, liver mass, and plasma cholesterol were primarily driven by MGL, while improvements in body fat were primarily driven by BAM15. No treatments altered liver fibrosis. MGL&#x2009;+&#x2009;BAM15 treatment had overall better efficacy to improve metabolic outcomes in mice fed GAN diet than either monotherapy alone. These data warrant further investigation into combination therapies of THR-&#x3b2; agonists and mitochondrial uncouplers for the potential treatment of disorders related to fatty liver, obesity, and insulin resistance.

BAM15 as a mitochondrial uncoupler: a promising therapeutic agent for diverse diseases.

Subcellular organelles dysfunction is implicated in various diseases, including metabolic diseases, neurodegenerative diseases, cancer, and cardiovascular diseases. BAM15, a selective mitochondrial uncoupler, has emerged as a promising therapeutic agent due to its ability to enhance mitochondrial respiration and metabolic flexibility. By disrupting the coupling between electron transport and ATP synthesis, BAM15 dissipates the proton gradient, leading to increased mitochondrial respiration and energy expenditure. This review provides a comprehensive overview of BAM15, including its mechanism of action and potential therapeutic applications in diverse disease contexts. BAM15 has shown promise in obesity by increasing energy expenditure and reducing fat accumulation. In diabetes, it improves glycemic control and reverses insulin resistance. Additionally, BAM15 has potential in non-alcoholic fatty liver disease, sepsis, and cardiovascular diseases by mitigating oxidative stress, modulating inflammatory responses, and promoting cardioprotection. The safety profile of BAM15 is encouraging, with minimal adverse effects and remarkable tolerability. However, challenges such as its high lipophilicity and the need for alternative delivery methods need to be addressed. Further research is necessary to fully understand the therapeutic potential of BAM15 and optimize its application in clinical settings.

Head-to-head comparison of BAM15, semaglutide, rosiglitazone, NEN, and calorie restriction on metabolic physiology in female db/db mice.

Metabolic disorders such as type 2 diabetes, fatty liver disease, hyperlipidemia, and obesity commonly co-occur but clinical treatment options do not effectively target all disorders. Calorie restriction, semaglutide, rosiglitazone, and mitochondrial uncouplers have all demonstrated efficacy against one or more obesity-related metabolic disorders, but it currently remains unclear which therapeutic strategy best targets the combination of hyperglycaemia, liver fat, hypertriglyceridemia, and adiposity. Herein we performed a head-to-head comparison of 5 treatment interventions in the female db/db mouse model of severe metabolic disease. Treatments included &#x223c;60&#xa0;% calorie restriction (CR), semaglutide, rosiglitazone, BAM15, and niclosamide ethanolamine (NEN). Results showed that BAM15 and CR improved body weight and liver steatosis to levels superior to semaglutide, NEN, and rosiglitazone, while BAM15, semaglutide, and rosiglitazone improved glucose tolerance better than CR and NEN. BAM15, CR, semaglutide, and rosiglitazone all had efficacy against hypertriglyceridaemia. These data provide a comprehensive head-to-head comparison of several key treatment strategies for metabolic disease and highlight the efficacy of mitochondrial uncoupling to correct multiple facets of the metabolic disease milieu in female db/db mice.

The new mitochondrial uncoupler BAM15 induces ROS production for treatment of acute myeloid leukemia.

Acute myeloid leukemia (AML) is a malignant proliferative disease of myeloid hematopoietic origin and cannot be treated appropriately at present. This is due to the fact that leukemia cells are not sensitive to some of the traditional chemotherapy drugs. Or some chemotherapeutic drugs are too toxic to normal cells, affecting their wide clinical application. In this study, we identified BAM15 as a novel mitochondrial uncoupling agent by screening a library of small molecule compounds that inhibit AML cell activity. BAM15 significantly inhibited proliferation and promoted apoptosis in AML cells while at the same time being less cytotoxic to normal cells. The mechanism may be related to the disturbance of the ROS production balance. In vivo investigations revealed that BAM15 effectively suppressed AML progression and prolonged the survival time of mice. In addition, we found that BAM15 can be used in combination with cytarabine to enhance its anti-cancer activity and inhibit the activity of primary cells in AML. Therefore, we identified BAM15 as a potential drug candidate for the treatment of AML.

Mitochondrial uncoupler BAM15 reverses diet-induced obesity and insulin resistance in mice.

Obesity is a health problem affecting more than 40% of US adults and 13% of the global population. Anti-obesity treatments including diet, exercise, surgery and pharmacotherapies have so far failed to reverse obesity incidence. Herein, we target obesity with a pharmacotherapeutic approach that decreases caloric efficiency by mitochondrial uncoupling. We show that a recently identified mitochondrial uncoupler BAM15 is orally bioavailable, increases nutrient oxidation, and decreases body fat mass without altering food intake, lean body mass, body temperature, or biochemical and haematological markers of toxicity. BAM15 decreases hepatic fat, decreases inflammatory lipids, and has strong antioxidant effects. Hyperinsulinemic-euglycemic clamp studies show that BAM15 improves insulin sensitivity in multiple tissue types. Collectively, these data demonstrate that pharmacologic mitochondrial uncoupling with BAM15 has powerful anti-obesity and insulin sensitizing effects without compromising lean mass or affecting food intake.

Targeting Mitochondrial Dysfunction to Prevent Endothelial Dysfunction and Atherosclerosis in Diabetes: Focus on the Novel Uncoupler BAM15.

Diabetes mellitus is a chronic metabolic disorder characterized by persistent hyperglycemia, leading to endothelial dysfunction and accelerated atherosclerosis. Mitochondrial dysfunction, oxidative stress, and dysregulated lipid metabolism contribute to endothelial cell (EC) injury, promoting plaque formation and increasing cardiovascular disease risk. Current lipid-lowering therapies have limited effectiveness in restoring endothelial function, highlighting the need for novel strategies. Mitochondrial uncoupling has emerged as a promising approach, with BAM15-a newly identified mitochondrial uncoupler-showing potential therapeutic benefits. BAM15 enhances fatty acid oxidation (FAO), reduces reactive oxygen species, and protects ECs from hyperglycemia-induced apoptosis. Unlike conventional uncouplers, BAM15 demonstrates improved tolerability and efficacy without severe off-target effects. It restores mitochondrial function, improves endothelial survival, and supports metabolic homeostasis under hyperglycemic conditions. This review uniquely integrates emerging evidence on mitochondrial dysfunction, endothelial metabolism, and FAO to highlight the novel role of BAM15 in restoring vascular function in diabetes. We provide the first focused synthesis of BAM15's mechanistic impact on EC bioenergetics and position it within the broader landscape of mitochondrial-targeted therapies for diabetic vascular complications. Further research is needed to elucidate the molecular mechanism through which BAM15 modulates EC metabolism and to evaluate its long-term vascular effects in diabetic models.

BAM15 inhibits endothelial pyroptosis via the NLRP3/ASC/caspase-1 pathway to alleviate atherosclerosis.

Atherosclerosis (AS) is a chronic inflammatory disease contributing to major cardiovascular events. This study aimed to investigate the effects of BAM15, a mitochondrial uncoupler, on regulating the NLRP3/ASC/caspase-1 signaling pathway to suppress endothelial cell pyroptosis and mitigate AS. AS was induced in ApoE-/- mice through a high-fat diet (HFD), and the therapeutic effects of BAM15 (5&#xa0;mg/kg/day, s. c.) were evaluated. Histological analyses, including HE staining and oil red O staining, were used to assess aortic pathology and lipid deposition. Serum inflammatory cytokines (IL-1&#x3b2;, IL-18) were quantified by ELISA. Mouse primary aortic endothelial cells (MAECs) were treated with oxidized low-density lipoprotein (ox-LDL) to simulate AS condition in vitro. Mitochondrial reactive oxygen species (mtROS) expression and oxidized (ox)-mtDNA content were detected by Mitosox staining and ELISA, respectively. Western blot was used to assess the expression of pyroptosis-related proteins, including GSDMD-NT, NLRP3, ASC, and cleaved-caspase-1. BAM15 reduced atherosclerotic plaque formation, lipid deposition, and inflammation, and diminished mtROS expression and ox-mtDNA content in the AS mouse models. In both in vivo and in vitro experiments, BAM15 markedly inhibited the activation of the NLRP3 inflammasome, leading to reduced pyroptosis in endothelial cells. Activation of the NLRP3/ASC/caspase-1 signaling pathway by Nigericin partially reversed the protective effects of BAM15, underscoring the pivotal role of NLRP3 inflammasome inhibition in endothelial pyroptosis suppression. BAM15 effectively inhibits endothelial cell pyroptosis by reducing mtROS production and ox-mtDNA release to suppress the NLRP3/ASC/caspase-1 signaling pathway, thereby alleviating AS in both in vivo and in vitro models.

Mitochondrial uncoupler BAM15 enhances the function of CD7CAR-TCD7- cells and reduces the release of cytokines for the therapy of T-cell malignancies.

Traditional therapies for relapsed/refractory T-cell malignancies, such as T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphomas, have limited efficacy. Chimeric antigen receptor T-cell (CAR-T) therapy has shown potential in treating hematologic malignancies, but challenges such as tumor immune evasion, CAR-T resistance, and cytokine release syndrome (CRS) hinder its clinical application. In this study, we generated CD7CAR-TCD7- cells by modifying naturally occurring CD7 negative T cells from human peripheral blood with a novel CD7 targeted CAR construct. The cytotoxic efficacy of CD7CAR-TCD7- cells against CD7 positive T cell malignancies was assessed through in vitro experiments and xenograft mouse models. To address CAR-T therapy limitations, we identified BAM15, a mitochondrial uncoupling agent, from a small molecule library. BAM15 enhanced the cytotoxic function of CD7CAR-TCD7- cells in a concentration-dependent manner while reducing cytokine release profile in both cellular assays and xenograft models. Notably, at low concentrations (2.5&#x3bc;M, 1&#xa0;&#x3bc;M and 0.5&#xa0;&#x3bc;M), BAM15 improved antitumor efficacy at suboptimal effector-to-target ratios (E:T&#xa0;=&#xa0;1:1 and 1:2), reduced inflammatory cytokines like IL-6 and TNF&#x3b1;, and alleviated inflammatory cell infiltration in lung and liver. This study confirms the feasibility of constructing CD7CAR-TCD7- cells from CD7- T cells and first reveals the synergistic effects of BAM15 on CD7CAR-TCD7- cells, for overcoming dose limitations and CRS of CAR-T therapy, and providing a novel strategy for T-cell malignancies.

Neuroblastoma Differentiation: The Untapped Potential of Mitochondrial Uncouplers.

While the goal of most anticancer treatments is to kill cancer cells, some therapies halt cancer progression by inducing cancer cell differentiation. For example, retinoic acid induces neuroblastoma cell differentiation in vitro and is used as maintenance therapy for children with high-risk neuroblastoma. A new study by Jiang and colleagues has revealed the mitochondrial uncoupler niclosamide ethanolamine (NEN) induces neuroblastoma cell differentiation in vitro and slows neuroblastoma tumor growth in vivo. Mitochondrial uncoupler molecules alter cell metabolism by forcing cells to "burn" more nutrients, resulting in a switch from anabolic to catabolic metabolism. NEN-induced neuroblastoma cell differentiation was associated with disruption of Warburg metabolism, epigenetic remodeling, and downregulation of key oncogenic drivers of neuroblastoma development, including MYCN. NEN is currently used as an antiparasitic worm treatment and is safe to use in children but has poor pharmacokinetic properties. However, derivatives of NEN and structurally distinct uncouplers that have improved pharmacokinetic properties are in development. Results of this study ignite the idea that mitochondrial uncouplers could be used as differentiating agents and expand the pharmacotherapy toolkit to treat cancer, including neuroblastoma. See related article by Jiang et al., p. 181.

Mitochondrial Respiration-Dependent ANT2-UCP2 Interaction.

Adenine nucleotide translocases (ANTs) and uncoupling proteins (UCPs) are known to facilitate proton leak across the inner mitochondrial membrane. However, it remains to be unravelled whether UCP2/3 contribute to significant amount of proton leak in vivo. Reports are indicative of UCP2 dependent proton-coupled efflux of C4 metabolites from the mitochondrial matrix. Previous studies have suggested that UCP2/3 knockdown (KD) contributes to increased ANT-dependent proton leak. Here we investigated the hypothesis that interaction exists between the UCP2 and ANT2 proteins, and that such interaction is regulated by the cellular metabolic demand. Protein-protein interaction was evaluated using reciprocal co-immunoprecipitation and in situ proximity ligation assay. KD of ANT2 and UCP2 was performed by siRNA in human embryonic kidney cells 293A (HEK293A) cells. Mitochondrial and cellular respiration was measured by high-resolution respirometry. ANT2-UCP2 interaction was demonstrated, and this was dependent on cellular metabolism. Inhibition of ATP synthase promoted ANT2-UCP2 interaction whereas high cellular respiration, induced by adding the mitochondrial uncoupler FCCP, prevented interaction. UCP2 KD contributed to increased carboxyatractyloside (CATR) sensitive proton leak, whereas ANT2 and UCP2 double KD reduced CATR sensitive proton leak, compared to UCP2 KD. Furthermore, proton leak was reduced in double KD compared to UCP2 KD. In conclusion, our results show that there is an interaction between ANT2-UCP2, which appears to be dynamically regulated by mitochondrial respiratory activity. This may have implications in the regulation of mitochondrial efficiency or cellular substrate utilization as increased activity of UCP2 may promote a switch from glucose to fatty acid metabolism.

Anilinopyrazines as potential mitochondrial uncouplers.

Mitochondrial protonophores transport protons through the mitochondrial inner membrane into the matrix to uncouple nutrient oxidation from ATP production thereby decreasing the proton motive force. Mitochondrial uncouplers have beneficial effects of decrease reactive oxygen species generation and have the potential for treating diseases such as obesity, neurodegenerative diseases, non-alcoholic fatty liver disease (NAFLD), diabetes, and many others. In this study, we report the structure-activity relationship profile of the pyrazine scaffold bearing substituted aniline rings. Our work indicates that a trifluoromethyl group is best at the para position while the trifluoromethoxy group is preferred in the meta position of the aniline rings of 2,3-substituted pyrazines. As proton transport and cycling requires the formation of a negative charge that has to traverse the mitochondrial membrane, a stabilizing internal hydrogen bond is a key feature for efficient mitochondrial uncoupling activity.

Diverse actions of 15 structurally unrelated mitochondrial uncouplers in cells and mice.

Mitochondrial uncouplers are used as chemical tools to study mitochondrial function in vitro and in vivo, and some molecules are in development for the treatment of metabolic diseases. One problem in the field is that any molecule that increases proton transport into the mitochondrial matrix independent of ATP production can be classified as an uncoupler regardless of off-target activities. Therefore, there are dozens of classes of molecules that exhibit a wide spectrum of phenotypes. Herein we directly compared 15 mitochondrial uncouplers side-by-side in a well-defined cell system to better understand their in vitro dose response profiles and the top molecules with suitable pharmacology and safety profiles were compared in db/db mice. Fifteen mitochondrial uncouplers were characterised in vitro in CHO-K1 cells. The top five candidates were selected for further characterisation in male db/db mice based on their in vitro dose response and/or tolerability. We tested two doses of each mitochondrial uncoupler in mice and benchmarked their efficacy to a lifestyle intervention of 35% calorie restriction as well as to lean db/+ metabolically healthy mice. Eleven groups of mice were fed ad libitum either; 1) chow (control), 2) chow with 0.15% BAM15 (w/w), 3) chow with 0.2% BAM15 (w/w), 4) chow with 0.1% NEN (w/w), 5) chow with 0.25% NEN (w/w), 6) chow with 0.01% OPC-163493 (w/w), 7) chow with 0.02% OPC-63493 (w/w), 8) chow with 0.015% ES9 (w/w), 9) chow with 0.03% ES9 (w/w), 10) chow with 0.2% NTZ (w/w), and 11) chow with 0.4% NTZ (w/w). Another group of mice was fed chow to receive &#x223c;65% of the average daily food intake of control mice as a model of calorie restriction (CR). Mice were metabolically phenotyped over 4 weeks of treatment with assessment of key readouts including body weight, HbA1c, blood glucose and glucose tolerance tests. At termination, key tissues were collected and plasma was analysed for markers of toxicity. Few mitochondrial uncouplers behaved similarly in vitro, with 11 molecules impairing maximal mitochondrial capacity. In vivo, BAM15 dose-dependently improved body weight and metabolic parameters in db/db mice, with 0.2% BAM15 treatment yielding statistically significant improvements in body weight, fat pad weight, glucose tolerance, blood glucose, HbA1c, liver weight and triglyceride content. The next-best treatment was 0.03% ES9 which significantly improved glucose tolerance, blood glucose levels, and HbA1c, but increased body weight, liver size and steatosis relative to db/db controls. Mitochondrial uncouplers BAM15 and ES9 had the greatest dose tolerance range in vitro, while BAM15 had the best overall effects on body weight, glucose control and liver steatosis in db/db mice. This study reveals diverse phenotypes across 15 classes of mitochondrial uncouplers and underscores the need for rigorous evaluation to identify molecules that drive stable mitochondrial respiration without unwanted mitochondrial inhibition or off-target effects. Ultimately, mitochondrial uncouplers should not be generalized and each uncoupler molecule needs to be considered by its own actions in well-defined experimental conditions.

Mitochondrial uncoupler BAM15 ameliorates liver lipid metabolism disorders by activating the AMPK pathway.

N5,N6-bis(2-Fluorophenyl)-[1,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine (BAM15) is a recently identified mitochondrial uncoupler with antitumor, anti-inflammatory, antioxidant and antiobesity properties. Although it has been shown that BAM15 has a high targeting ability to the liver, its capacity to improve liver metabolic disorders and the underlying mechanisms are not well understood. This study examined how BAM15 works in high-fat-diet (HFD) induced obese mice. Our results showed that compared with 2,4-Dinitrophenol (DNP) and carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), BAM15 has a higher binding capacity and stronger activity in mediating proton uncoupling, and effectively promoted mitochondrial fusion, division, autophagy, and the tricarboxylic acid cycle. BAM15 improved hepatic lipid metabolism disorders by enhancing mitochondrial autophagy through activation of the 5'-AMP-activated protein kinase (AMPK) pathway. This indicates that BAM15 could be used to treat liver lipid metabolism issues and offers a solid theoretical foundation for managing lipid-related diseases.

Self-assembly drug-albumin nanocomposites for nonalcoholic fatty liver disease treatment.

Nonalcoholic fatty liver disease (NAFLD) is a chronic metabolic liver disease closely related to obesity, which has become a global health problem. However, current pharmacological therapies for NAFLD are limited by potential side effects, low effectiveness and poor aqueous solubility. Herein, we designed functionalized drug-albumin nanocomposites (BAM15@BSA NPs), which were prepared by self-assembly of the anti-obesity small-molecule drug (BAM15) and bovine serum albumin (BSA), for treatment of NAFLD. The proposed BAM15@BSA NPs not only improve aqueous solubility and half-life of BAM15 but also exhibit hepatic-targeted capacity and an increased therapeutic efficacy. In vitro experiments revealed that BAM15@BSA NPs possessed excellent biocompatibility, and improved resistance to adipogenesis and reduced lipid accumulation in human hepatocellular carcinoma cells. In vivo, BAM15@BSA NPs showed liver targeting ability and powerful anti-obesity effects without altering body temperature or affecting food intake, and could effectively alleviate hepatic steatosis and improve therapeutic efficacy for NAFLD treatment. The above findings demonstrated that BAM15@BSA NPs potentially served as a safe and effective drug for NAFLD treatment.

Mitochondrial Uncoupling Induces Epigenome Remodeling and Promotes Differentiation in Neuroblastoma.

The Warburg effect is the major metabolic hallmark of cancer. According to Warburg himself, the consequence of the Warburg effect is cell dedifferentiation. Therefore, reversing the Warburg effect might be an approach to restore cell differentiation in cancer. In this study, we used a mitochondrial uncoupler, niclosamide ethanolamine (NEN), to activate mitochondrial respiration, which induced neural differentiation in neuroblastoma cells. NEN treatment increased the NAD+/NADH and pyruvate/lactate ratios and also the &#x3b1;-ketoglutarate/2-hydroxyglutarate (2-HG) ratio. Consequently, NEN treatment induced promoter CpG island demethylation and epigenetic landscape remodeling, activating the neural differentiation program. In addition, NEN treatment upregulated p53 but downregulated N-Myc and &#x3b2;-catenin signaling in neuroblastoma cells. Importantly, even under hypoxia, NEN treatment remained effective in inhibiting 2-HG generation, promoting DNA demethylation, and suppressing hypoxia-inducible factor signaling. Dietary NEN intervention reduced tumor growth rate, 2-HG levels, and expression of N-Myc and &#x3b2;-catenin in tumors in an orthotopic neuroblastoma mouse model. Integrative analysis indicated that NEN treatment upregulated favorable prognosis genes and downregulated unfavorable prognosis genes, which were defined using multiple neuroblastoma patient datasets. Altogether, these results suggest that mitochondrial uncoupling is an effective metabolic and epigenetic therapy for reversing the Warburg effect and inducing differentiation in neuroblastoma. Targeting cancer metabolism using the mitochondrial uncoupler niclosamide ethanolamine leads to methylome reprogramming and differentiation in neuroblastoma, providing a therapeutic opportunity to reverse the Warburg effect and suppress tumor growth. See related commentary by Byrne and Bell, p.167.

Temporal Analysis of Protein Ubiquitylation and Phosphorylation During Parkin-Dependent Mitophagy.

Mitophagy, the selective degradation of mitochondria by autophagy, affects defective mitochondria following damage or stress. At the onset of mitophagy, parkin ubiquitylates proteins on the mitochondrial outer membrane. While the role of parkin at the onset of mitophagy is well understood, less is known about its activity during later stages in the process. Here, we used HeLa cells expressing catalytically active or inactive parkin to perform temporal analysis of the proteome, ubiquitylome, and phosphoproteome during 18&#xa0;h after induction of mitophagy by mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazine. Abundance profiles of proteins downregulated in parkin-dependent manner revealed a stepwise and "outside-in" directed degradation of mitochondrial subcompartments. While ubiquitylation of mitochondrial outer membrane proteins was enriched among early parkin-dependent targets, numerous mitochondrial inner membrane, matrix, and cytosolic proteins were also found ubiquitylated at later stages of mitophagy. Phosphoproteome analysis revealed a possible crosstalk between phosphorylation and ubiquitylation during mitophagy on key parkin targets, such as voltage-dependent anion channel 2.

Restricting bioenergetic efficiency enhances longevity and mitochondrial redox capacity in Drosophila melanogaster.

Mitochondria are essential for survival and as such, impairments in organelle homeostasis significantly accelerate age-related morbidity and mortality. Here, we determined the contribution of bioenergetic efficiency to life span and health span in Drosophila melanogaster utilizing the mitochondrial uncoupler BAM15. Life span was determined in flies fed a normal diet (ND) or high fat diet (HFD) supplemented with vehicle or BAM15. Locomotor function was determined by negative geotaxis assay in middle-aged flies fed vehicle or BAM15 under ND or HFD conditions. Redox capacity (high-resolution respirometry/fluorometry), citrate synthase (enzyme activity), mtDNA content (qPCR), gene expression (qPCR), and protein expression (western blot) were assessed in flight muscle homogenates of middle-aged flies fed vehicle or BAM15 ND. The molar ratio of H2O2 and O2 (H2O2:O2) in a defined respiratory state was calculated as a measure of redox balance. BAM15 extended life span by 9% on ND and 25% on HFD and improved locomotor activity by 125% on ND and 53% on HFD. Additionally, BAM15 enhanced oxidative phosphorylation capacity supported by pyruvate + malate, proline, and glycerol 3-phosphate. Concurrently, BAM15 enhanced the mitochondrial H2O2 production rate, reverse electron flow from mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) to Complex I, mGPDH, and Complex I without altering the H2O2:O2 ratio. BAM15 upregulated transcriptional signatures associated with mitochondrial function and fitness as well as antioxidant defense. BAM15-mediated restriction of bioenergetic efficiency prolongs life span and health span in Drosophila fed a ND or HFD. Improvements in life span and health span in ND were supported by synergistic enhancement of muscular redox capacity.

Organelle-Targeted BODIPY Photocages: Visible-Light-Mediated Subcellular Photorelease.

Photocaging facilitates non-invasive and precise spatio-temporal control over the release of biologically relevant small- and macro-molecules using light. However, sub-cellular organelles are dispersed in cells in a manner that renders selective light-irradiation of a complete organelle impractical. Organelle-specific photocages could provide a powerful method for releasing bioactive molecules in sub-cellular locations. Herein, we report a general post-synthetic method for the chemical functionalization and further conjugation of meso-methyl BODIPY photocages and the synthesis of endoplasmic reticulum (ER)-, lysosome-, and mitochondria-targeted derivatives. We also demonstrate that 2,4-dinitrophenol, a mitochondrial uncoupler, and puromycin, a protein biosynthesis inhibitor, can be selectively photoreleased in mitochondria and ER, respectively, in live cells by using visible light. Additionally, photocaging is shown to lead to higher efficacy of the released molecules, probably owing to a localized and abrupt release.

Mitochondrial uncoupler BAM15 inhibits artery constriction and potently activates AMPK in vascular smooth muscle cells.

Our previous studies found that mitochondrial uncouplers CCCP and niclosamide inhibited artery constriction and the mechanism involved AMPK activation in vascular smooth muscle cells. BAM15 is a novel type of mitochondrial uncoupler. The aim of the present study is to identify the vasoactivity of BAM15 and characterize the BAM15-induced AMPK activation in vascular smooth muscle cells (A10 cells). BAM15 relaxed phenylephrine (PE)-induced constricted rat mesenteric arteries with intact and denuded endothelium. Pretreatment with BAM15 inhibited PE-induced constriction of rat mesenteric arteries with intact and denuded endothelium. BAM15, CCCP, and niclosamide had the comparable IC50 value of vasorelaxation in PE-induced constriction of rat mesenteric arteries. BAM15 was less cytotoxic in A10 cells compared with CCCP and niclosamide. BAM15 depolarized mitochondrial membrane potential, induced mitochondrial fission, increased mitochondrial ROS production, and increased mitochondrial oxygen consumption rate in A10 cells. BAM15 potently activated AMPK in A10 cells and the efficacy of BAM15 was stronger than that of CCCP, niclosamide, and AMPK positive activators metformin and AICAR. In conclusion, BAM15 activates AMPK in vascular smooth muscle cells with higher potency than that of CCCP, niclosamide and the known AMPK activators metformin and AICAR. The present work indicates that BAM15 is a potent AMPK activator.

BAM15 Relieves Neurodegeneration in Aged Caenorhabditis elegans and Extends Lifespan.

BAM15 was recently screened as a protonophore uncoupler specifically for the mitochondrial membrane but not the plasma membrane. It is equally as potent as FCCP, but less toxic. Previously, mitochondrial uncoupling via DNP alleviates neurodegeneration in the nematode Caenorhabditis elegans during aging. Therefore, we investigated whether BAM15 uncouplers could phenotypically and functionally reduce neuronal defects in aged nematodes. We observed green fluorescence protein-tagged mechanosensory neurons and performed touch and chemotaxis assays during aging. Wild-type animals treated with both 50 &#xb5;M BAM15 and 10 &#xb5;M DNP showed reduced mechanosensory neuronal defects during aging, which correlates with the maintenance of touch responses and short-term memory during aging. Uncoupler mutant ucp-4 also responded the same way as the wild-type, reducing neurodegeneration in 50 &#xb5;M BAM15 and 10 &#xb5;M DNP-treated animals compared to the DMSO control. These results suggest that 50 &#xb5;M BAM15 alleviates neurodegeneration phenotypically and functionally in C. elegans during aging, potentially through mitochondrial uncoupling. In accordance with the preserved neuronal shape and function in aged C. elegans, 50 &#xb5;M BAM15 extended the mean lifespan of both wild-type and ucp-4 mutants.

A New Mitochondrial Uncoupler Improves Metabolic Homeostasis in Mice.

BAM15-mediated mitochondrial uncoupling protects against obesity and improves glycemic control.

Obesity is a leading cause of preventable death worldwide. Despite this, current strategies for the treatment of obesity remain ineffective at achieving long-term weight control. This is due, in part, to difficulties in identifying tolerable and efficacious small molecules or biologics capable of regulating systemic nutrient homeostasis. Here, we demonstrate that BAM15, a mitochondrially targeted small molecule protonophore, stimulates energy expenditure and glucose and lipid metabolism to protect against diet-induced obesity. Exposure to BAM15 in&#xa0;vitro enhanced mitochondrial respiratory kinetics, improved insulin action, and stimulated nutrient uptake by sustained activation of AMPK. C57BL/6J mice treated with BAM15 were resistant to weight gain. Furthermore, BAM15-treated mice exhibited improved body composition and glycemic control independent of weight loss, effects attributable to drug targeting of lipid-rich tissues. We provide the first phenotypic characterization and demonstration of pre-clinical efficacy for BAM15 as a pharmacological approach for the treatment of obesity and related diseases.

Niclosamide ethanolamine inhibits artery constriction.

We previously demonstrated that the typical mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) inhibited artery constriction, but CCCP was used only as a pharmacological tool. Niclosamide is an anthelmintic drug approved by FDA. Niclosamide ethanolamine (NEN) is a salt form of niclosamide and has been demonstrated to uncouple mitochondrial oxidative phosphorylation. The aim of the present study was to elucidate the vasoactivity of NEN and the potential mechanisms. Isometric tension of rat mesenteric artery and thoracic aorta was recorded by using multi-wire myograph system. The protein levels were measured by using western blot techniques. Niclosamide ethanolamine (NEN) treatment relaxed phenylephrine (PE)- and high K+ (KPSS)-induced constriction, and pre-treatment with NEN inhibited PE- and KPSS-induced constriction of rat mesenteric arteries. In rat thoracic aorta, NEN also showed antagonism against PE- and KPSS-induced constriction. NEN induced increase of cellular ADP/ATP ratio in vascular smooth muscle cells (A10) and activated AMP-activated protein kinase (AMPK) in A10 cells and rat thoracic aorta. NEN-induced aorta relaxation was attenuated in AMPK&#x3b1;1 knockout (-/-) mice. SERCA inhibitors cyclopiazonic acid and thapsigargin, but not KATP channel blockers glibenclamide and 5-hydroxydecanoic acid, attenuated NEN-induced vasorelaxation in rat mesenteric arteries. NEN treatment increased cytosolic [Ca2+]i and depolarized mitochondrial membrane potential in vascular smooth muscle cells (A10). Niclosamide in non-salt form showed the similar vasoactivity as NEN in rat mesenteric arteries. Niclosamide ethanolamine inhibits artery constriction, indicating that it would be promising to be developed as an anti-hypertensive drug or it would induce vasodilation-related side effects when absorbed in vivo.

Mitochondrial NAD+ Controls Nuclear ARTD1-Induced ADP-Ribosylation.

In addition to its role as an electron transporter, mitochondrial nicotinamide adenine dinucleotide (NAD+) is an important co-factor for enzymatic reactions, including ADP-ribosylation. Although mitochondria harbor the most intra-cellular NAD+, mitochondrial ADP-ribosylation remains poorly understood. Here we provide evidence for mitochondrial ADP-ribosylation, which was identified using various methodologies including immunofluorescence, western blot, and mass spectrometry. We show that mitochondrial ADP-ribosylation reversibly increases in response to respiratory chain inhibition. Conversely, H2O2-induced oxidative stress reciprocally induces nuclear and reduces mitochondrial ADP-ribosylation. Elevated mitochondrial ADP-ribosylation, in turn, dampens H2O2-triggered nuclear ADP-ribosylation and increases MMS-induced ARTD1 chromatin retention. Interestingly, co-treatment of cells with the mitochondrial uncoupler FCCP decreases PARP inhibitor efficacy. Together, our results suggest that mitochondrial ADP-ribosylation is a dynamic cellular process that impacts nuclear ADP-ribosylation and provide evidence for a NAD+-mediated mitochondrial-nuclear crosstalk.

Mitochondrial uncoupler BAM15 attenuates cryopreservation-induced damage in human sperm by stabilizing mitochondrial homeostasis.

Human sperm cryopreservation is essential for sperm banking and assisted reproduction, yet freeze-thaw stress promotes oxidative injury that reduces motility and damages the acrosome and nuclear DNA. Here, we tested whether the mitochondrial uncoupler BAM15 improves post-thaw human sperm quality and examined mechanisms linked to mitochondrial homeostasis. Ejaculates were cryopreserved using a standard protocol supplemented with graded concentrations of BAM15. After thawing, total and progressive motility and viability were assessed. Flow cytometry quantified the DNA fragmentation index and the proportion of high DNA stainability cells. Mitochondrial membrane potential, intracellular reactive oxygen species, and lipid peroxidation were measured to evaluate mitochondrial function and oxidative status. Ultrastructural preservation of the acrosome, plasma membrane, midpiece mitochondria, and flagellar axoneme was examined by transmission electron microscopy. Compared with untreated controls, BAM15 increased total and progressive motility and improved viability. BAM15 reduced DNA fragmentation and decreased high DNA stainability, indicating enhanced genomic integrity. Consistently, BAM15 improved mitochondrial membrane potential while suppressing intracellular reactive oxygen species and lipid peroxidation, supporting attenuation of freeze-thaw oxidative damage. Transmission electron microscopy further revealed more continuous acrosomal and plasma membranes, fewer swollen or vacuolated midpiece mitochondria, and improved preservation of axonemal architecture. Collectively, these findings identify BAM15 as a promising cryopreservation supplement that stabilizes mitochondrial homeostasis and improves the functional and structural quality of human sperm after thawing.

Tumor metabolome remolded by low dose mitochondrial uncoupler elicites robust CD8+ T cell response.

Tumor cells balance ATP production and carbon skeleton synthesis by flexibly altering catabolic pathways to sustain their significant growth advantage. Uncouplers have shown potential for tumor suppression by converting chemical energy from catabolism into heat. However, their use may be limited due to indiscriminate metabolic interference in both tumor and normal cells, as well as the uncertainty surrounding their effects on the immune microenvironment. Herein, we found that low-dose uncoupler BAM15 promoted AMPK, AKT signaling, and the TCA cycle without increasing cell proliferation or inducing cell death in vitro, suggesting an increase in futile cycling. Intratumoral injection of 50&#x2009;ng/mL BAM15 accelerated catabolic processes while inhibiting anabolic pathways, resulting in a metabolomic reshaping with increased levels of linoleic acid, C5DC, and others. These changes were shown to enhance tumor-killing effects by T cells. To reduce side effects on normal tissues and improve tumor retention, BAM15 was targeted for delivery by loading it into TCVs. This TCV-BAM15 treatment significantly increased CD8+ T cell counts and granzyme B levels. Our findings highlight a previously unrecognized therapeutic effect and signaling mechanism of low-dose BAM15 treatment in tumors. We propose that this novel strategy holds promise as a tumor immunity therapy with fewer adverse effects compared to free uncoupling drugs at high concentrations.

Preservation of Autophagy May Be a Mechanism Behind Healthy Aging.

Autophagy is intricately linked with protective cellular processes, including mitochondrial function, proteostasis, and cellular senescence. Animal studies have indicated that autophagy becomes dysfunctional with aging and may contribute to T cell immunosenescence. In humans, it remains unclear whether autophagy is impaired in CD4+ T cells as people age. To answer this question, we examined basal and inducible autophagic activity in a series of experiments comparing CD4+ T cells from younger (23-35&#x2009;years old) and older (67-93&#x2009;years old) healthy donors. We used immunofluorescence to detect LC3 (a marker of autophagosomes and autolysosomes) and LAMP2 (a marker of endolysosomes) in conjunction with bafilomycin A1 (which inhibits the acidification of lysosomes) and CCCP (a mitochondrial uncoupler) to manipulate autophagic flux. We found a significantly higher autophagy flux in CD4+ T cells from older compared to younger donors and a higher number of LC3+ compartments among older donors. Since the overall amount of autophagosomes degraded was comparable between the two groups, we concluded that autophagosome biogenesis was reduced in the older group. Rather than a decline, our findings in healthy older donors point toward a compensatory enhancement of human CD4+ T cell autophagy with age, which may be a mechanism behind healthy aging.

Mitochondrial uncoupler triclosan induces vasorelaxation of rat arteries.

Our previous studies found that mitochondrial uncouplers induced vasodilation. Triclosan, the broad spectrum antibacterial agent, is the active ingredient in soaps and toothpastes. It was reported that triclosan induced mitochondrial uncoupling, so we aim to investigate the effects of triclosan on vascular function of rat mesenteric arteries and aorta. The isometric tension of rat mesenteric artery and thoracic aorta was recorded by multi-wire myograph system. The cytosolic [Ca2+]i, mitochondrial reactive oxygen species (mitoROS), and mitochondrial membrane potential of smooth muscle cells (A10 cells) were measured using laser scanning confocal microscopy. Triclosan treatment relaxed phenylephrine (PE)- and high K+ (KPSS)-induced constriction, and pre-treatment with triclosan inhibited PE- and KPSS-induced constriction of rat mesenteric arteries. In rat thoracic aorta, triclosan also relaxed PE- and KPSS-induced constriction. Triclosan induces vasorelaxation without involving KATP channel activation in smooth muscle cells of arteries. Triclosan treatment increased cytosolic [Ca2+]i, mitochondrial ROS production and depolarized mitochondrial membrane potential in A10 cells. In conclusion, triclosan induces mitochondrial uncoupling in vascular smooth muscle cells and relaxes the constricted rat mesenteric arteries and aorta of rats. The present results suggest that triclosan would indicate vasodilation effect if absorbed excessively in vivo.

BAM15 attenuates transportation-induced apoptosis in iPS-differentiated retinal tissue.

BAM15 is a novel mitochondrial protonophore uncoupler capable of protecting mammals from acute renal ischemic-reperfusion injury and cold-induced microtubule damage. The purpose of our study was to investigate the effect of BAM15 on apoptosis during 5-day transportation of human-induced pluripotent stem (hiPS)-differentiated retinal tissue. Retinal tissues of 30&#x2009;days and 60&#x2009;days were transported with or without BAM15 for 5&#x2009;days in the laboratory or by real express. Immunofluorescence staining of apoptosis marker cleaved caspase3, proliferation marker Ki67, and neural axon marker NEFL was performed. And expression of apoptotic-related factors p53, NFkappaB, and TNF-a was detected by real-time PCR. Also, location of ganglion cells, photoreceptor cells, amacrine cells, and precursors of neuronal cell types in retinal tissue was stained by immunofluorescence after transportation. Furthermore, cell viability was assessed by CCK8 assay. Results showed transportation remarkably intensified expression of apoptotic factor cleaved caspase3, p53, NFkappaB, and TNF-a, which could be reduced by supplement of BAM15. In addition, neurons were severely injured after transportation, with axons manifesting disrupted and tortuous by staining NEFL. And the addition of BAM15 in transportation was able to protect neuronal structure and increase cell viability without affecting subtypes cells location of retinal tissue. BAM15 might be used as a protective reagent on apoptosis during transporting retinal tissues, holding great potential in research and clinical applications.

Artemisinin synergizes with CCCP in autophagic cell death induction via ER stress in uveal melanoma.

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults, and it is associated with a poor prognosis due to the lack of effective targeted therapies. Artemisinin (ARS), a widely used antimalarial drug, has demonstrated anti-tumor effects in several types of cancer, including UM. However, these effects are primarily observed at high concentrations, which restricts its broader use. Our study reveals mitochondrial uncoupler agents as synergistic partners with DHA in treating UM, both in vitro and in vivo. Specifically, the combination of DHA and CCCP, a representative mitochondrial uncoupler, induced endoplasmic reticulum (ER) stress by enhancing the binding of ATF4 and CHOP to the SESN2 promoter region. This ER stress subsequently activated autophagic cell death, augmenting UM cell eradication. Our findings suggest that combining DHA with mitochondrial protonophore uncouplers inhibits UM proliferation, underscoring the therapeutic promise of this approach for UM treatment.

Targeting negative energy balance with calorie restriction and mitochondrial uncoupling in db/db mice.

Calorie restriction is a first-line treatment for overweight individuals with metabolic impairments. However, few patients can adhere to long-term calorie restriction. An alternative approach to calorie restriction that also causes negative energy balance is mitochondrial uncoupling, which decreases the amount of energy that can be extracted from food. Herein we compare the metabolic effects of calorie restriction with the mitochondrial uncoupler BAM15 in the db/db mouse model of severe hyperglycemia, obesity, hypertriglyceridemia, and fatty liver. Male db/db mice were treated with &#x223c;50% calorie restriction, BAM15 at two doses of 0.1% and 0.2% (w/w) admixed in diet, or 0.2% BAM15 with time-restricted feeding from 5 weeks of age. Mice were metabolically phenotyped over 4 weeks with assessment of key readouts including body weight, glucose tolerance, and liver steatosis. At termination, liver tissues were analysed by metabolomics and qPCR. Calorie restriction and high-dose 0.2% BAM15 decreased body weight to a similar extent, but mice treated with BAM15 had far better improvement in glucose control. High-dose BAM15 treatment completely normalized fasting glucose and glucose tolerance to levels similar to lean db/+ control mice. Low-dose 0.1% BAM15 did not affect body mass but partially improved glucose tolerance to a similar degree as 50% calorie restriction. Both calorie restriction and high-dose BAM15 significantly improved hyperglucagonemia and liver and serum triglyceride levels. Combining high-dose BAM15 with time-restricted feeding to match the time that calorie restricted mice were fed resulted in the best metabolic phenotype most similar to lean db/+ controls. BAM15-mediated improvements in glucose control were associated with decreased glucagon levels and decreased expression of enzymes involved in hepatic gluconeogenesis. BAM15 and calorie restriction treatments improved most metabolic disease phenotypes in db/db mice. However, mice fed BAM15 had superior effects on glucose control compared to the calorie restricted group that consumed half as much food. Submaximal dosing with BAM15 demonstrated that its beneficial effects on glucose control are independent of weight loss. These data highlight the potential for mitochondrial uncoupler pharmacotherapies in the treatment of metabolic disease.

Beneficial effects of simultaneously targeting calorie intake and calorie efficiency in diet-induced obese mice.

Semaglutide is an anti-diabetes and weight loss drug that decreases food intake, slows gastric emptying, and increases insulin secretion. Patients begin treatment with low-dose semaglutide and increase dosage over time as efficacy plateaus. With increasing dosage, there is also greater incidence of gastrointestinal side effects. One reason for the plateau in semaglutide efficacy despite continued low food intake is due to compensatory actions whereby the body becomes more metabolically efficient to defend against further weight loss. Mitochondrial uncoupler drugs decrease metabolic efficiency, therefore we sought to investigate the combination therapy of semaglutide with the mitochondrial uncoupler BAM15 in diet-induced obese mice. Mice were fed high-fat western diet (WD) and stratified into six treatment groups including WD control, BAM15, low-dose semaglutide without or with BAM15, and high-dose semaglutide without or with BAM15. Combining BAM15 with either semaglutide dose decreased body fat and liver triglycerides, which was not achieved by any monotherapy, while high-dose semaglutide with BAM15 had the greatest effect on glucose homeostasis. This study demonstrates a novel approach to improve weight loss without loss of lean mass and improve glucose control by simultaneously targeting energy intake and energy efficiency. Such a combination may decrease the need for semaglutide dose escalation and hence minimize potential gastrointestinal side effects.

Ammonia sensitive SLC4A11 mitochondrial uncoupling reduces glutamine induced oxidative stress.

SLC4A11 is a NH3 sensitive membrane transporter with H+ channel-like properties that facilitates Glutamine catabolism in Human and Mouse corneal endothelium (CE). Loss of SLC4A11 activity induces oxidative stress and cell death, resulting in Congenital Hereditary Endothelial Dystrophy (CHED) with corneal edema and vision loss. However, the mechanism by which SLC4A11 prevents ROS production and protects CE is unknown. Here we demonstrate that SLC4A11 is localized to the inner mitochondrial membrane of CE and SLC4A11 transfected PS120 fibroblasts, where it acts as an NH3-sensitive mitochondrial uncoupler that enhances glutamine-dependent oxygen consumption, electron transport chain activity, and ATP levels by suppressing damaging Reactive Oxygen Species (ROS) production. In the presence of glutamine, Slc4a11-/- (KO) mouse CE generate significantly greater mitochondrial superoxide, a greater proportion of damaged depolarized mitochondria, and more apoptotic cells than WT. KO CE can be rescued by MitoQ, reducing NH3 production by GLS1 inhibition or dimethyl &#x3b1;Ketoglutarate supplementation, or by BAM15 mitochondrial uncoupling. Slc4a11 KO mouse corneal edema can be partially reversed by &#x3b1;Ketoglutarate eye drops. Moreover, we demonstrate that this role for SLC4A11 is not specific to CE cells, as SLC4A11 knockdown in glutamine-addicted colon carcinoma cells reduced glutamine catabolism, increased ROS production, and inhibited cell proliferation. Overall, our studies reveal a unique metabolic mechanism that reduces mitochondrial oxidative stress while promoting glutamine catabolism.

Breast cancer growth and proliferation is suppressed by the mitochondrial targeted furazano[3,4-b]pyrazine BAM15.

Enhanced metabolic plasticity and diversification of energy production is a hallmark of highly proliferative breast cancers. This contributes to poor pharmacotherapy efficacy, recurrence, and metastases. We have previously identified a mitochondrial-targeted furazano[3,4-b]pyrazine named BAM15 that selectively reduces bioenergetic coupling efficiency and is orally available. Here, we evaluated the antineoplastic properties of uncoupling oxidative phosphorylation from ATP production in breast cancer using BAM15. The anticancer effects of BAM15 were evaluated in human triple-negative MDA-MB-231 and murine luminal B, ER&#x3b1;-negative EO771 cells as well as in an orthotopic allograft model of highly proliferative mammary cancer in mice fed a standard or high fat diet (HFD). Untargeted transcriptomic profiling of MDA-MB-231 cells was conducted after 16-h exposure to BAM15. Additionally, oxidative phosphorylation and electron transfer capacity was determined in permeabilized cells and excised tumor homogenates after treatment with BAM15. BAM15 increased proton leak and over time, diminished cell proliferation, migration, and ATP production in both MDA-MB-231 and EO771 cells. Additionally, BAM15 decreased mitochondrial membrane potential, while inducing apoptosis and reactive oxygen species accumulation in MDA-MB-231 and EO771 cells. Untargeted transcriptomic profiling of MDA-MB-231 cells further revealed inhibition of signatures associated with cell survival and energy production by BAM15. In lean mice, BAM15 lowered body weight independent of food intake and slowed tumor progression compared to vehicle-treated controls. In HFD mice, BAM15 reduced tumor growth relative to vehicle and calorie-restricted weight-matched controls mediated in part by impaired cell proliferation, mitochondrial respiratory function, and ATP production. LC-MS/MS profiling of plasma and tissues from BAM15-treated animals revealed distribution of BAM15 in adipose, liver, and tumor tissue with low abundance in skeletal muscle. Collectively, these data indicate that mitochondrial uncoupling may be an effective strategy to limit proliferation of aggressive forms of breast cancer. More broadly, these findings highlight the metabolic vulnerabilities of highly proliferative breast cancers which may be leveraged in overcoming poor responsiveness to existing therapies.

Sepsis Encephalopathy Is Partly Mediated by miR370-3p-Induced Mitochondrial Injury but Attenuated by BAM15 in Cecal Ligation and Puncture Sepsis Male Mice.

BAM15 (a mitochondrial uncoupling agent) was tested on cecal ligation and puncture (CLP) sepsis mice with in vitro experiments. BAM15 attenuated sepsis as indicated by survival, organ histology (kidneys and livers), spleen apoptosis (activated caspase 3), brain injury (SHIRPA score, serum s100&#x3b2;, serum miR370-3p, brain miR370-3p, brain TNF-&#x3b1;, and apoptosis), systemic inflammation (cytokines, cell-free DNA, endotoxemia, and bacteremia), and blood-brain barrier (BBB) damage (Evan's blue dye and the presence of green fluorescent E. coli in brain after an oral administration). In parallel, brain miR arrays demonstrated miR370-3p at 24 h but not 120 h post-CLP, which was correlated with metabolic pathways. Either lipopolysaccharide (LPS) or TNF-&#x3b1; upregulated miR370-3p in PC12 (neuron cells). An activation by sepsis factors (LPS, TNF-&#x3b1;, or miR370-3p transfection) damaged mitochondria (fluorescent color staining) and reduced cell ATP, possibly through profound mitochondrial activity (extracellular flux analysis) that was attenuated by BAM15. In bone-marrow-derived macrophages, LPS caused mitochondrial injury, decreased cell ATP, enhanced glycolysis activity (extracellular flux analysis), and induced pro-inflammatory macrophages (iNOS and IL-1&#x3b2;) which were neutralized by BAM15. In conclusion, BAM15 attenuated sepsis through decreased mitochondrial damage, reduced neuronal miR370-3p upregulation, and induced anti-inflammatory macrophages. BAM15 is proposed to be used as an adjuvant therapy against sepsis hyperinflammation.

Restoring mitochondrial quantity and quality to reverse the Warburg effect and drive neuroblastoma differentiation.

Reduced mitochondrial quality and quantity in tumors is associated with dedifferentiation and increased malignancy. However, it remains unclear how to restore mitochondrial quantity and quality in tumors and whether mitochondrial restoration can drive tumor differentiation. Our study shows that restoring mitochondrial function using retinoic acid (RA) to boost mitochondrial biogenesis and a mitochondrial uncoupler to enhance respiration synergistically drives neuroblastoma differentiation and inhibits proliferation. U-13C-glucose/glutamine isotope tracing revealed a metabolic shift from the pentose phosphate pathway to oxidative phosphorylation, accelerating the tricarboxylic acid cycle and switching substrate preference from glutamine to glucose. These effects were abolished by electron transport chain (ETC) inhibitors or in &#x3c1;0 cells lacking mitochondrial DNA, emphasizing the necessity of mitochondrial function for differentiation. Dietary RA and uncoupler treatment promoted tumor differentiation in an orthotopic neuroblastoma xenograft model, evidenced by neuropil production and Schwann cell recruitment. Single-cell RNA sequencing of xenografts revealed that this strategy effectively eliminated the stem cell population, promoted differentiation, and increased mitochondrial gene signatures along the differentiation trajectory, potentially improving patient outcomes. Collectively, our findings establish a mitochondria-centric therapeutic strategy for inducing tumor differentiation, suggesting that maintaining/driving differentiation in tumor requires not only ATP production but also continuous ATP consumption and sustained ETC activity.