Obesity affects more than 40 percent of adults in the United States and 13 percent of the global population.
With obesity comes a variety of other interconnected diseases including cardiovascular disease, diabetes, and fatty liver disease, which makes the disease one of the most difficult – and most crucial – to treat.
“Obesity is the biggest health problem in the United States. But, it is hard for people to lose weight and keep it off; being on a diet can be so difficult. So, a pharmacological approach, or a drug, could help out and would be beneficial for all of society,” said Webster Santos, professor of chemistry and the Cliff and Agnes Lilly Faculty Fellow of Drug Discovery in the College of Science at Virginia Tech.
Santos and his colleagues have recently identified a small mitochondrial uncoupler, named BAM15, that decreases the body fat mass of mice without affecting food intake and muscle mass or increasing body temperature.
Additionally, the molecule decreases insulin resistance and has beneficial effects on oxidative stress and inflammation.
The findings, published in Nature Communications on May 14, 2020, hold promise for future treatment and prevention of obesity, diabetes, and especially nonalcoholic steatohepatitis (NASH), a type of fatty liver disease that is characterized by inflammation and fat accumulation in the liver.
In the next few years, the condition is expected to become the leading cause of liver transplants in the United States.
The mitochondria are commonly referred to as the powerhouses of the cell. The organelle generates ATP, a molecule that serves as the energy currency of the cell, which powers body movement and other biological processes that help our body to function properly.
In order to make ATP, nutrients need to be burned and a proton motive force (PMF) needs to be established within the mitochondria.
The PMF is generated from a proton gradient, where there is a higher concentration of protons outside of the inner membrane and a lower concentration of protons in the matrix, or the space within the inner membrane.
The cell creates ATP whenever protons pass through an enzyme called ATP synthase, which is embedded in the membrane. Hence, nutrient oxidation, or nutrient burning, is coupled to ATP synthesis.
“So anything that decreases the PMF has the potential to increase respiration. Mitochondrial uncouplers are small molecules that go to the mitochondria to help the cells respire more.
Effectively, they change metabolism in the cell so that we burn more calories without doing any exercise,” said Santos, an affiliated member of the Fralin Life Sciences Institute and the Virginia Tech Center for Drug Discovery.
Mitochondrial uncouplers transport protons into the matrix by bypassing ATP synthase, which throws off the PMF. To reestablish the gradient, protons must be exported out of the mitochondrial matrix. As a result, the cell begins to burn fuel at higher than necessary levels.
Knowing that these molecules can change a cell’s metabolism, researchers wanted to be sure that the drug was reaching its desired targets and that it was, above all, safe. Through a series of mouse studies, the researchers found that BAM15 is neither toxic, even at high doses, nor does it affect the satiety center in the brain, which tells our body if we are hungry or full.
In the past, many anti-fat drugs would tell your body to stop eating.
But as a result, patients would rebound and eat more. In the BAM15 mouse studies, animals ate the same amount as the control group – and they still lost fat mass.
Another side effect of previous mitochondrial uncouplers was increased body temperature. Using a rectal probe, researchers measured the body temperature of mice who were fed BAM15. They found no change in body temperature.
But one issue arises concerning the half-life of BAM15. The half-life, or the length of time that a drug is still effective, is relatively short in the mouse model. For oral dosing in humans, the optimal half-life is much longer.
Even as BAM15 has some serious potential in mouse models, the drug won’t necessarily be successful in humans – at least not this same exact molecule.
“We are essentially looking for roughly the same type of molecule, but it needs to stay in the body for longer to have an effect. We are tweaking the chemical structure of the compound. So far, we have made several hundred molecules related to this,” said Santos.
The ultimate goal of the Santos lab is to transition the anti-fat treatment from animal models to a treatment for NASH in humans. The lab has used their better compounds in animal models of NASH, which have been proven to be effective as anti-NASH compounds in mice.
Working alongside Santos is Kyle Hoehn, an assistant professor of pharmacology from the University of Virginia and an associate professor of biotechnology and biomolecular sciences at the University of New South Wales in Australia.
Hoehn is a metabolic physiology expert who is in charge of conducting the animal studies. Santos and Hoehn have been collaborating for several years now and they even founded a biotech company together.
Co-founded by Santos and Hoehn in 2017, Continuum Biosciences aims to improve the ways in which our bodies burn fuel and fight back against our bodies ability to store excess nutrients as we age.
These promising NASH treatment compounds are licensed by their company and are patented by Virginia Tech.
The company is looking to use mitochondrial uncouplers for more than just obesity and NASH. The molecules also have a unique anti-oxygen effect that can minimize the accumulation of reactive oxygen species, or oxidative stress, in our bodies, which ultimately results in neurodegeneration and aging.
“If you just minimize aging, you could minimize the risk of Alzheimer’s disease and Parkinson’s disease. All of these reactive oxygen species-related or inflammation-related diseases could benefit from mitochondrial uncouplers. So, we could see this heading that way,” said Santos.
Discussion
Obesity is a complex disorder that is difficult to overcome. Obesity is influenced by genetic and environmental factors that include basal metabolic rate, satiety, hormone balance, physical activity, and food choices2,33. Weight loss is difficult to sustain in part because each person is individually programmed to defend a physiological set-point for body weight and initial weight loss is counteracted by feedback mechanisms that lower metabolic rate and/or increase hunger34,35,36,37.
Calorie restriction is frequently used as an anti-obesity treatment, but it can have unwanted effects including depression and withdrawal symptoms particularly in patients with food addiction35,38.
Importantly, interventions such as mitochondrial uncoupling can cause weight loss without affecting food intake. Pharmacologically decreasing metabolic efficiency by mitochondrial uncoupling represents a potential therapeutic intervention that may surmount genetic and environmental causes of obesity by overcoming the feedback mechanisms that defend body weight.
For example, by definition, mitochondrial uncoupling causes metabolic inefficiency to directly override the body’s attempt to improve metabolic efficiency or lower metabolic rate. Mitochondrial uncoupling also does not cause complications associated with either undereating or overeating, indicating that it does not trigger the adaptive responses caused by calorie restriction or treatment with the SGLT2 inhibitor canagliflozin37. The lack of compensatory food intake in mice fed BAM15 is consistent with human data showing that people taking DNP did not report increased hunger22.
The lack of altered food intake is psychologically important because the mesolimbic reward center in the brain is intimately connected with food consumption39,40; therefore, targeting metabolic efficiency with mitochondrial uncoupling may not be associated with the same psychological risk profile as dieting or using pharmacological agents that target satiety.
In this study, BAM15 demonstrated a direct on-target mechanism to lower metabolic efficiency that resulted in loss of body fat without altering activity or food intake. Mice fed BAM15 had lower RER values than control animals concomitant with elevated plasma NEFA levels suggesting that release of adipose-derived NEFAs fuel fatty acid oxidation in other tissues.
We show direct evidence that BAM15 treatment increases oxygen consumption and 14C-palmitate oxidation in liver tissue ex vivo and decreases hepatic TG and NEFA in mice. These data demonstrate a direct effect of BAM15 to increase nutrient metabolism in the liver with lesser effects evidenced in skeletal muscle and fat.
BAM15 treatment had no effect on fat-free lean mass, no impact on body temperature, and no effect on clinical biochemistry or haematology indicators of tissue damage. These rigorous assessments show that BAM15 safely reversed obesity and insulin resistance without observed adverse effects. The only serum parameters that differed statistically after chronic BAM15 treatment were lower triglyceride and higher BUN levels.
The average BUN level in WD + BAM15-fed mice was 35 mg/dL, which is not considered elevated as it is within the normal physiological range for C57BL/6 mice41. Furthermore, creatinine levels were normal and acute oral gavage of BAM15 did not alter BUN levels.
Metabolomic analysis of liver tissue from WD + BAM15-fed mice showed that there was no major destabilisation across the metabolic profile as few metabolites changed more than twofold. The strongest signatures were antioxidant effects, evidenced by an increased GSH:GSSG ratio, decreased bioactive oxidised lipids (e.g. 4-HNE), and decreased pro-inflammatory lipid mediators in the form of eicosanoids and docosanoids.
The antioxidant effect was expected because mild mitochondrial uncoupling has antioxidant effects that decrease superoxide production from the electron transport chain42,43. Metabolomics data identified changes in amino acid metabolism that may be related to increased amino acid oxidation or anaplerosis into the TCA cycle.
Increased anaplerosis is an expected phenotype because it counterbalances the increase in fat oxidation to prevent outstripping the TCA cycle44. Mice treated with BAM15 had normal ATP levels and there was no indication of energy stress via AMPK signaling compared to control mice, thereby indicating that nutrient flux through mitochondria in BAM15-treated liver was sufficient to maintain normal physiology without energetic stress.
In the WD reversal study, mice fed WD for 4 weeks had a threefold increase in fat mass. Groups of mice were stratified and randomized to WD with or without 0.1% w/w BAM15. Within 3 weeks of treatment, BAM15 completely reversed diet-induced glucose intolerance and hyperinsulinemia.
Hyperinsulinemic-euglycemic clamp experiments performed after 6 weeks of treatment showed that BAM15 improved insulin sensitivity to levels similar to chow mice evidenced by normalization of glucose infusion rate and improvements in muscle glucose clearance and suppression of adipose NEFA production.
WD-fed mice have impaired insulin-mediated suppression of hepatic glucose output compared to chow-fed mice; however, mice fed WD containing BAM15 have an intermediate phenotype between chow and WD whereby they are statistically no different from either chow or WD groups.
When considering this collectively with the biochemical data, BAM15 appears to have a stronger phenotype for fat oxidation over glucose metabolism that may underlie the anti-obesity effects. Cardiac muscle glucose uptake was not statistically different for any group.
Recent years have seen a resurgence of interest in mitochondrial uncouplers as human medicines. For example, the FDA recently granted IND approval for DNP to re-enter the clinic as a potential therapeutic for patients with Huntington’s disease45, and niclosamide and nitazoxanide are FDA-approved anti-parasitic drugs that were discovered to have mitochondrial uncoupling activity. Nitazoxanide is not known to have anti-obesity effects, but it is currently in phase 2 clinical trials for NASH-induced fibrosis46.
Niclosamide has been studied in obese mouse models where it slowed not only fat mass gain but also decreased lean mass gain when administered in a diet-induced obesity prevention model47. Niclosamide unexpectedly increased body weight when fed to db/db mice47.
In contrast to niclosamide, BAM15 treatment in the obesity prevention model decreased fat mass below that of normal chow-fed mice without decreasing lean mass thereby revealing a remarkably high specificity for BAM15 to target fat loss.
The preservation of lean mass during weight loss is highly desired and distinguishes BAM15 from other uncouplers. Another mitochondrial uncoupler OPC-163493 recently discovered by Otsuka Pharmaceuticals had strong anti-diabetes effects in multiple rodent models, but did not impact adiposity29. Finally, controlled-release DNP and the methyl ether prodrug version of DNP both failed to alter adiposity25,26.
In summary, BAM15 represents a rare mitochondrial uncoupler that prevents and reverses obesity without affecting food intake or lean mass. One limitation of BAM15 is low aqueous solubility, but this property did not affect oral bioavailability and indeed low aqueous solubility is an important parameter that enables BAM15 to penetrate membranes and enter mitochondria.
Another limitation of BAM15 is a 1.7 h half-life and future directions will investigate formulation strategies to improve exposure. Collectively, the data presented herein supports further development of BAM15 as a potential therapeutic for obesity and metabolic diseases.
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More information: Stephanie J. Alexopoulos et al. Mitochondrial uncoupler BAM15 reverses diet-induced obesity and insulin resistance in mice, Nature Communications (2020). DOI: 10.1038/s41467-020-16298-2
