Abstract
The oxidative phosphorylation system1 in mammalian mitochondria plays a key role in transducing energy from ingested nutrients2. Mitochondrial metabolism is dynamic and can be reprogrammed to support both catabolic and anabolic reactions, depending on physiological demands or disease states. Rewiring of mitochondrial metabolism is intricately linked to metabolic diseases and promotes tumour growth3,4,5. Here, we demonstrate that oral treatment with an inhibitor of mitochondrial transcription (IMT)6 shifts whole-animal metabolism towards fatty acid oxidation, which, in turn, leads to rapid normalization of body weight, reversal of hepatosteatosis and restoration of normal glucose tolerance in male mice on a high-fat diet. Paradoxically, the IMT treatment causes a severe reduction of oxidative phosphorylation capacity concomitant with marked upregulation of fatty acid oxidation in the liver, as determined by proteomics and metabolomics analyses. The IMT treatment leads to a marked reduction of complex I, the main dehydrogenase feeding electrons into the ubiquinone (Q) pool, whereas the levels of electron transfer flavoprotein dehydrogenase and other dehydrogenases connected to the Q pool are increased. This rewiring of metabolism caused by reduced mtDNA expression in the liver provides a principle for drug treatment of obesity and obesity-related pathology.
Main
The first attempts to target mitochondria to treat obesity were reported in the 1930s when more than 100,000 individuals were treated with the uncoupler dinitrophenol (DNP)7,8,9. Although this treatment increased the metabolic rate and reduced obesity, serious side effects prevented DNP from becoming an established treatment7,8. Metformin provides an alternate way to inhibit oxidative phosphorylation (OXPHOS) and this mild complex I inhibitor is widely used as an anti-diabetic medication and also protects against cancer10,11,12,13,14,15. The possible connection between beneficial metabolic effects and anti-cancer activity of drugs targeting mitochondria prompted us to investigate whether inhibitor of mitochondrial transcription (IMT) treatment, which is known to impair tumour metabolism and growth in mouse models6, also may have beneficial metabolic effects. Treatment of tumour cell lines with IMT induces a dose-dependent impairment of OXPHOS and cellular metabolic starvation, with progressively reduced levels of a range of critical metabolites and eventually cell death6. Despite the drastic effects on metabolism in cancer cell lines and cancer xenografts, treatment of whole animals is well tolerated6. We therefore decided to test the hypothesis that IMT treatment aiming to moderately impair the OXPHOS capacity in whole animals may induce beneficial metabolic effects in healthy and metabolically challenged mice.
Male C57BL/6N mice at the age of 4 weeks were randomly chosen to be fed a chow diet or high-fat diet (HFD) for 8 weeks. Thereafter, the two groups were subdivided for oral treatment (gavage) with either IMT (LDC4857, 30 mg kg−1) or vehicle for 4 weeks while continuing the respective diets (Fig. 1a). The IMT compound used in this study was developed within an optimization programme based on the structurally closely related IMT1B compound. IMT treatment of mice on HFD causes a rapid marked reduction of body weight after 1 week, with a cumulative weight loss of ~7 g after 4 weeks (Fig. 1b). Measurements of body composition with non-invasive magnetic resonance imaging (EchoMRI-100) after 4 weeks of IMT treatment showed markedly reduced fat mass without any change of lean mass (Fig. 1c). Haematoxylin and eosin (H&E) staining of tissue sections of epididymal white adipose tissue (eWAT) showed that HFD results in large lipid-filled adipocytes and that IMT treatment leads to a drastic decrease in adipocyte size (Extended Data Fig. 1a).
We next assessed whole-body energy homoeostasis in mice on chow diet or HFD treated with vehicle or the IMT compound using the Oxymax/Comprehensive Lab Animal Monitoring System (CLAMS). The four groups of mice were subjected to five continuous days of CLAMS analysis during the fourth week of gavage treatment with vehicle or IMT compound. The first 3 days were used to acclimate the animals to the CLAMS system, followed by measurements during the fourth day. Day five included a 12-h period of fasting followed by 6 h of refeeding. Notably, IMT treatment did not alter food intake (Fig. 1d) or physical activity (Extended Data Fig. 1b). We found that IMT treatment did not increase the lipid content in faeces (Fig. 1e) or the total diurnal lipid excretion in faeces (Extended Data Fig. 1c,d). We performed bomb calorimetry and found a higher energy content in faeces in mice on a chow diet in comparison with mice on HFD, consistent with results from other studies16,17. IMT treatment did not additionally alter the total energy content in faeces (Fig. 1f). These analyses of faeces thus exclude that drug-induced malabsorption explains the weight loss.
IMT-treated mice on HFD showed enhanced oxygen consumption during both the light and dark cycle (Extended Data Fig. 2a). Regression-based analysis of covariance (ANCOVA)18,19 with either total mass or lean mass as a covariate did not clearly link increased energy expenditure to IMT treatment (Extended Data Fig. 2b,c). Although these results indicate that IMT treatment may not exert its effect through increasing energy expenditure, subtle differences in energy expenditure can be hard to detect by indirect calorimetry despite having a profound long-term impact on body weight18,20. We therefore proceeded to assess the respiratory exchange ratio (RER) as this parameter needs no normalization to body weight or body composition. Mice on a standard chow diet had a RER of ~0.9–1.1, whereas it was decreased to ~0.8 on HFD (Fig. 1g and Extended Data Fig. 2d), as expected. Upon refeeding after fasting, IMT treatment resulted in a lower RER in comparison with vehicle treatment, regardless of the diet (Fig. 1g and Extended Data Fig. 2d), consistent with drug-induced activation of fat metabolism. These data provide evidence that IMT treatment reverses HFD-induced obesity by promoting metabolism of fat at the organismal level.
We found normal fasting blood glucose levels accompanied by markedly increased fasting serum insulin levels (Extended Data Fig. 3a,b) and pathological intraperitoneal glucose tolerance tests (ipGTT; Extended Data Fig. 3c–e) with an increased peak concentration of serum insulin (Extended Data Fig. 3e) in mice on HFD, consistent with a pre-diabetic state and insulin resistance. Glucose homoeostasis was markedly improved when mice on HFD were treated with an IMT compound for 4 weeks; the fasting blood glucose was reduced (Extended Data Fig. 3a), serum insulin levels were decreased (Extended Data Fig. 3b) and the ipGTT responses were normalized (Extended Data Fig. 3c–e). IMT treatment leads to reduced circulating insulin levels (Extended Data Fig. 3b), but ex vivo glucose-stimulated insulin secretion (GSIS) assays showed that IMT treatment did not impair insulin secretion or insulin biosynthesis in isolated pancreatic islets (Extended Data Fig. 3f,g). The reduced circulating insulin levels and normalized glucose homoeostasis in IMT-treated mice on HFD are, thus, probably explained by increased insulin sensitivity.
We observed a large macrovesicular steatosis in the liver of mice on HFD (Fig. 2a). Notably, IMT treatment markedly reduced hepatosteatosis (Fig. 2a), leading to a decreased lipid content in the liver (Fig. 2b) and reduced liver weight (Fig. 2c). We performed lipidomics and found a large accumulation of diglycerides and triglycerides in the liver of mice on HFD, which was reversed by 4 weeks of IMT treatment (Fig. 2d). In contrast, the phospholipid and sphingolipid levels in the liver were mainly affected by the diet and not markedly impacted by IMT treatment (Extended Data Fig. 4). IMT treatment of mice on HFD was accompanied by an improvement of the liver function, as demonstrated by decreased aminotransferase activities in the serum (Fig. 2e,f). The serum albumin levels were normal in all investigated groups (Fig. 2g). Taken together these data show that IMT treatment can reverse diet-induced hepatosteatosis and normalize liver function….
