A novel chemical uncoupler ameliorates obesity and related phenotypes in mice with diet-induced obesity by modulating energy expenditure and food intake
- 2.3k Downloads
Decreasing mitochondrial coupling efficiency has been shown to be an effective therapy for obesity and related metabolic symptoms. Here we identified a novel mitochondrial uncoupler that promoted uncoupled respiration in a cell type-specific manner and investigated its effects on modulation of energy metabolism in vivo and in vitro.
We screened a collection of mitochondrial membrane potential depolarising compounds for a novel chemical uncoupler on isolated skeletal muscle mitochondria using a channel oxygen system. The effect on respiration of metabolic cells (L6 myotubes, 3T3-L1 adipocytes and rat primary hepatocytes) was examined and metabolic pathways sensitive to cellular ATP content were also evaluated. The chronic metabolic effects were investigated in high-fat diet-induced obese mice and standard diet-fed (SD) lean mice.
The novel uncoupler, CZ5, promoted uncoupled respiration in a cell type-specific manner. It stimulated fuel oxidation in L6 myotubes and reduced lipid accumulation in 3T3-L1 adipocytes but did not affect gluconeogenesis or the triacylglycerol content in hepatocytes. The administration of CZ5 to SD mice increased energy expenditure (EE) but did not affect body weight or adiposity. Chronic studies in mice on high-fat diet showed that CZ5 reduced body weight and improved glucose and lipid metabolism via both increased EE and suppressed energy intake. The reduced adiposity was associated with the restoration of expression of key metabolic genes in visceral adipose tissue.
This work demonstrates that a cell type-specific mitochondrial chemical uncoupler may have therapeutic potential for treating high-fat diet-induced metabolic diseases.
KeywordsCell type-specific Chemical uncoupler Metabolic diseases Obesity
Mitochondrial membrane potential
AMP-activated protein kinase
Carbonyl cyanide m-chlorophenylhydrazone
Fatty acid oxidation
Food and Drug Administration
Inhibitor of κB kinase β
White adipose tissue
The increasing obesity epidemic, primarily driven by overnutrition and a sedentary lifestyle, has become a serious worldwide public health concern. Obesity is one of the most important pathogenic factors causing type 2 diabetes mellitus and other metabolic disorders . Thermodynamically, therapeutic approaches to correct this energy imbalance should ameliorate obesity. However, lifestyle modification via diet and exercise or pharmacological interventions to reduce energy intake (EI) are inadequate and there has been limited approval for anti-obesity drugs in the past decade [2, 3, 4].
Mitochondria are the powerhouse of the cell, coupling the majority of energy derived from fuel substrates to ATP turnover and uncoupling less in the form of heat. In humans and rodents with obesity and related type 2 diabetes, fuel overload in the mitochondrial respiratory system and impaired mitochondrial oxidative capacity have been observed in energy-using organs and energy storage organs, such as skeletal muscle (SKM) [5, 6, 7] and white adipose tissue (WAT) [8, 9]. Increasing cellular energy expenditure (EE) by decreasing mitochondrial coupling efficiency has been proposed to be an appealing therapeutic alternative [10, 11]. Experiments using transgenic mice overexpressing uncoupling proteins (UCPs) in metabolic tissues showed that locally uncoupling oxidative phosphorylation (OXPHOS) could combat obesity and improve glucose homeostasis to varying extents [12, 13, 14]. Yet it remains to be verified how these UCPs are regulated by physiological ligands . Pharmacological evidence for the effectiveness of uncoupling includes the potent anti-obesity effect observed with 2,4-dinitrophenol (DNP), a chemical uncoupler with a non-specific effect widely used in the 1930s . However, severe side effects, such as uncontrolled hyperthermia at toxic doses, prevented the further development of other anti-obesity uncoupling agents largely due to their narrow therapeutic window .
In the present study, we screened compounds that depolarise mitochondrial membrane potential and identified a novel chemical uncoupler, CZ5, which potently uncouples respiration selectively in myocytes and adipocytes. The chronic administration of CZ5 induced beneficial metabolic effects in high-fat diet-fed (HFD) mice, not only by increasing EE but also by restricting EI.
L6 myoblasts and HepG2 cells were cultured in DMEM containing 10% FBS. For differentiation of L6 myoblasts, the concentration of FBS was decreased to 2%. The culture and differentiation of 3T3-L1 cells was conducted as described previously . Rat hepatocytes were isolated using Selgen’s two-step perfusion method  and maintained in DMEM.
Mitochondrial membrane potential assay
This assay was based on a previous report . Briefly, L6 myotubes were treated with compounds for 40 min before being stained with 0.1% JC-1 for another 20 min. The ratio of red to green fluorescence reflects the mitochondrial membrane potential (Δψm).
Measurement of respiration in isolated mitochondria and intact cells
Mitochondria were isolated from rat liver and cells as previously described . Respiration measurements were conducted using a Clark-type oxygen electrode (Strathkelvin Instruments, Motherwell, UK) as described . The procedure of respiration measurement is described in electronic supplementary material (ESM) Methods.
2-Deoxyglucose uptake was measured as described previously .
Measurement of glucose and fatty acid oxidation
The effects of treatments on these oxidative metabolisms in differentiated L6 myotubes were traced with respective isotope-labelled substrate. Full details are provided in ESM Methods.
Glucose production assay
After overnight attachment, glucose production was measured in primary hepatocytes following a 3 h pre-treatment. Cells were washed three times with phosphate-buffered saline and incubated in the gluconeogenic medium (glucose and phenol red-free DMEM containing 20 mmol/l sodium lactate and 2 mmol/l sodium pyruvate) for 3 h. The glucose concentration of the medium was measured with a colorimetric glucose assay kit (Fudan-Zhangjiang, Shanghai, China).
Oil red O staining and determination of triacylglycerol content
Cell staining with oil red O and triacylglycerols were determined in cell lysates using a colorimetric assay as described previously .
Sulforhodamine B cytotoxicity assay
L6 myotubes, rat hepatocytes and 3T3-L1 fibroblasts were treated with CZ5 followed by SRB assay as previously described .
All animal experiments were approved by the Animal Care and Use Committee of the Shanghai Institute of Materia Medica (Shanghai, China). Six-week-old male C57BL/6J mice (Shanghai SLAC Laboratory Animal Co., Shanghai, China) were housed in a temperature-controlled room (22 ± 2°C) with a light/dark cycle of 12 h. For chronic treatment, mice were fed high-fat diets (60% calories from fat; Research Diets, New Brunswick, NJ, USA) or standard diets ad libitum. At 14 weeks of age, mice were randomly assigned to treatment groups. For the study of chronic effect on standard diet-fed (SD) mice, either vehicle (0.5% methylcellulose, MC) or CZ5 (30 mg kg−1 day−1) was orally administered for 25 days. The metabolic effects were investigated (see ESM Methods). Hepatic and muscular triacylglycerol content was measured following a Folch extraction .
Measurement of adenine nucleotide, glutathione and glutathione disulfide levels
As described in ESM Methods, adenine nucleotide concentrations in cell or tissue extracts were determined by HPLC and reduced and oxidised glutathione levels in tissue extracts were measured using an enzymatic recycling method.
Quantitative RT-PCR for RNA, mitochondrial DNA (mtDNA) and genomic DNA copy number
Total proteins were prepared in RIPA buffer (50 mmol/l Tris-HCl, pH 8.0, 150 mmol/l NaCl, 1% NP-40, 1 mmol/l Na3VO4, 1 mmol/l phenylmethanesulfonyl fluoride, 1 mmol/l dithiothreitol, 1 mmol/l EDTA and 1 mmol/l EGTA) containing complete protease inhibitors (Roche, Basel, Switzerland). Protein (20 μg per sample) was electrophoresed through SDS-PAGE after boiling for 5 min in SDS loading buffer. The antibodies for AMPK, phospho-AMPK (Thr172), total ACC, phospho-ACC (Ser79), AS160, phospho-AS160 (Thr642), IKKβ and phospho-IKKα(Ser176/180)/IKKβ(Ser177/181) were purchased from Cell Signaling Technology (Beverly, MA, USA).
Acute toxicity study
Overnight-fasted C57BL/6J male mice weighing 20–25 g each were gavaged orally with vehicle or CZ5 and thereafter observed continuously for the first 4 h and then at 6 h intervals for the following 48 h and once daily for the following 7 days, to observe any death or changes in general behaviour and other physiological activities.
Results represented means ± SEM. All the in vitro experiments were conducted at least three times. Differences between two groups were examined using the unpaired two-tailed Student’s t test. The EE data in mice were assessed by analysis of covariance (ANCOVA) with body mass as a covariate. p < 0.05 was regarded as significant.
Identification of a novel cell type-specific chemical uncoupler, CZ5
Selective metabolic effects induced by CZ5 in myotubes and adipocytes
In response to low ATP/ADP ratios, cells generally tend to decrease anabolism to maintain energy homeostasis. To fulfil their important role in storing energy, adipocytes have the unique capabilities of adipogenesis and lipogenesis, two pathways dependent on cellular energy supply. We treated white adipocytes with CZ5 during differentiation (Fig. 2d, e) and after differentiation (Fig. 2f, g). CZ5 caused a dramatic reduction in lipid droplet accumulation compared with DMSO-treated adipocytes displaying normal differentiation (Fig. 2d). In fully differentiated adipocytes, CZ5-treatment also reduced intracellular triacylglycerols (Fig. 2g).
CZ5 had a minimal effect on the energy-sensitive metabolic pathway of gluconeogenesis and lipid accumulation in hepatocytes (Fig. 2h, i), even though there was some degree of uncoupled respiration in HepG2 hepatoma cells at the same working concentrations used in other cell types (far lower than CCCP) (Fig. 2j). These findings are consistent with the lack of a significant or weak uncoupling effect and an unchanged ADP/ATP ratio in hepatocytes.
The barely detectable cytotoxicity excluded the possibility that cell death interfered with the evaluation of metabolic effects (ESM Fig. 1).
CZ5 promoted EE but not weight loss in SD mice
Chronic treatment with CZ5 in HFD mice for 5 weeks produced an anti-obesity effect
CZ5 affects glucose and lipid homeostasis
Metabolic variables of SD mice (n = 6) and vehicle-treated, CZ5-treated or pair-fed HFD mice (n = 8 mice per group)
Plasma cholesterol (mmol/l)
2.30 ± 0.08***
4.79 ± 0.09
4.20 ± 0.25*
4.58 ± 0.21
0.98 ± 0.07**
1.64 ± 0.15
1.24 ± 0.08*
1.38 ± 0.13
Plasma triacylglycerols (mmol/l)
0.59 ± 0.03*
0.80 ± 0.07
0.76 ± 0.05
0.73 ± 0.05
Plasma NEFA (mmol/l)
0.67 ± 0.04
0.72 ± 0.05
0.68 ± 0.05
0.60 ± 0.02*
Plasma lactic acid (mmol/l)
5.75 ± 0.45
5.58 ± 0.45
4.71 ± 0.22
5.18 ± 0.26
Plasma leptin (ng/ml)
2.25 ± 0.24***
41.03 ± 7.00
8.58 ± 2.15***,†††
33.67 ± 5.47
Muscle triacylglycerols (μmol/g)
11.88 ± 2.51***
69.80 ± 9.59
23.61 ± 4.46***,††
53.63 ± 6.60
Liver triacylglycerols (μmol/g)
10.45 ± 0.52***
45.90 ± 6.95
47.58 ± 6.17
46.25 ± 4.17
Chronic effects of CZ5 in metabolic tissues of HFD mice
Mitochondrial uncoupling has been shown to reduce the production of reactive oxygen species , so to determine whether CZ5 affects oxidative stress levels we examined cellular glutathione status. CZ5 enhanced the GSH:GSSG ratio significantly in WAT and had a tendency (p = 0.07) to do so in SKM, which resulted from more than halved GSSG (Fig. 6d, e). These findings indicated that CZ5 reduced oxidative stress.
To understand the molecular basis of the markedly decreased white fat mass, we found that the expression levels of master transcription regulators of adipogenesis (Pparg and Srebf1) and mitochondrial biogenesis (Pgc-1α [also known as Ppargc1a]) and their respective downstream targets genes involved in lipid metabolism (e.g. Fasn, Glut4 [also known as Slc2a4], Fabp4, Atgl [also known as Pnpla2]) and mitochondrial OXPHOS (e.g. Cycs and Cox5b) were downregulated in mice fed high-fat diets compared with those fed standard diets (Fig. 6f), consistent with previous reports [8, 29, 30, 31]. Treatment with CZ5 restored the impaired expression of these genes, with the exception of Fasn mRNA, which was further decreased. However, CZ5 treatment did not significantly affect mtDNA copy number (ESM Fig. 5). Ppara plays a key role in the transcriptional control of genes involved in cellular fatty acid oxidation (FAO). Although the expression of Ppara was decreased in the epididymal fat of HFD mice, not all of the detected target genes (Acox1 and Cpt1b) were downregulated in comparison with SD mice. CZ5 not only upregulated Ppara mRNA but also induced its target genes. Obesity-induced chronic inflammation in adipose tissue has been suggested to be critical for the development of insulin resistance [32, 33]. HFD significantly increased the mRNA expression of Tnf and Il6, two key cytokines for obesity-induced insulin resistance in WAT, whereas the induction was largely reduced in CZ5-treated mice. Ucp3 is known to mediate FAO specifically in SKM. In contrast to the effects on FAO gene expression in WAT, high-fat diets increased the expression levels of Cpt1b and Ucp3 in SKM. CZ5 further promoted the expression of these two genes (Fig. 6g). For the majority of genes detected, their expression in the HFD-PF group was not substantially different from that in the obese controls (data not shown).
Uncoupling of oxidation from phosphorylation in mitochondria is an effective way to treat obesity mainly via augmenting EE . However, it is undesirable for a chemical uncoupler to cause widespread disturbance in ATP production in multiple organs . The capacity to reduce coupling efficiency selectively in muscle or fat may allow a chemical uncoupler to be used in obesity treatment . SKM, as a large organ and a major site of facultative thermogenesis, contributes significantly to resting EE (REE) . WAT is a major site of energy storage and does not directly contribute a large amount to REE, but has the capacity to indirectly influence whole-body metabolic efficiency via a range of secreted regulatory factors (adipokines) [8, 36, 37]. It has been shown in tissue-specific gain-of-function studies in mice that overexpression of UCPs in SKM increased REE and reduced body weight, accompanied by hyperphagia [14, 38], whereas UCP overexpression in WAT did not significantly raise REE and reduced body weight only in mice on high-fat diets .
CZ5 displayed selective potent uncoupling effects on myocytes and adipocytes compared with the uniform effects of the classic chemical uncoupler CCCP, which also uncoupled respiration in hepatocytes. Consistent with these findings a previous study reported suppression of hepatic gluconeogenesis with CCCP , while CZ5 did not affect glucose output in hepatocytes. The present study demonstrated that upregulation of EE may be responsible for CZ5-induced weight loss, by lowering fat mass and improving lipid metabolism in HFD mice. Consistent with the cell type-specific uncoupling effects of CZ5 in vitro, we found that in vivo markers of energy status, as well as triacylglycerol content, was altered in WAT and SKM. However, CZ5 had no remarkable effects on body weight and adiposity in SD mice, as the enhanced EI was able to compensate for the increased EE. These therapeutic effects induced by CZ5 appear to mimic those produced by overexpression of UCPs in SKM and WAT [12, 13, 14], which are in contrast to those effects reported for obesity treatment with non-specific uncouplers in humans or rodents [16, 17].
WAT is known to play an important role in the maintenance of whole-body glucose homeostasis through the sequestering of excess triacylglycerols and fatty acids. The increased expression of key adipogenic transcriptional factors and their downstream target genes involved in adipogenesis and lipogenesis indicated that CZ5 treatment restored the ‘de-differentiation’ induced by high-fat diets. The simultaneous increase in mitochondrial biogenesis regulator Pgc-1α may co-activate these adipogenic transcriptional factors . The induction of Pgc-1α expression may be a compensatory consequence of increased intracellular calcium concentrations levels, secondary to a compromised energy status induced by CZ5 . Combined with the upregulated expression of lipid oxidation genes, it can be speculated that CZ5 may enhance oxidative metabolism in WAT, leading to loss of white fat mass. We investigated in vivo whether changes in the expression of genes involved in metabolism were at least partially mediated by cell-autonomous effects in adipocytes. Consistent with the action of positive uncouplers described in a previous report , CZ5 enhanced Pgc-1α expression and reduced Leptin expression in adipocytes via incubation during differentiation (ESM Fig. 6).
Another interesting result is related to appetite regulation. In the chronic study in HFD mice, CZ5-treated obese mice exhibited suppression of food intake from the onset of therapy, but did not show any behavioural abnormalities related to compound-induced toxicity. There was also no obvious alteration in plasma markers of liver and kidney toxicity (ESM Table 2). The initial pharmacokinetic study showed that the plasma-to-brain concentration ratio was nearly 100 at the time of peak plasma concentration after a single oral dose (ESM Fig. 4), suggesting a limited blood–brain barrier permeability to CZ5. High-fat diet-induced obese rodents are known to harbour blunted central leptin sensitivity in comparison with SD rodents [44, 45]. Overexpression of UCP1 in epididymal fat after the development of obesity led to decreased food intake and reversed leptin resistance . In agreement, we observed that plasma leptin level and leptin mRNA expression in epididymal fat tissues were markedly decreased, though white fat mass was not significantly altered in obese mice treated by CZ5 for 2 weeks (ESM Fig. 7a–c). A leptin tolerance test showed that the acute anorexigenic effect of leptin was more potent in CZ5-treated obese mice, while no difference was observed between the control and pair-fed group (ESM Fig. 7d). These results suggest that increased leptin sensitivity might be one of the causes of the anorexic effect induced by CZ5, rather than the anorexia being a secondary effect of food restriction. The basal phosphorylation of the AMPK direct substrate acetyl-CoA carboxylase (ACC), downstream of leptin’s action in the hypothalamus [47, 48], was significantly decreased in CZ5-treated mice, despite phosphorylation levels of AMPK remaining unaltered (ESM Fig. 7e). This suggests the inhibition of hypothalamic AMPK activity may, in part, mediate the CZ5-induced reduction in EI. Furthermore, previous studies reported that overnutrition can induce leptin-insensitive hyperphagia through activation of inhibitor of κB kinase β (IKKβ)/nuclear factor-κB (NF-κB) . We found that hypothalamic phosphorylation of IKKβ was reduced after CZ5 treatment (ESM Fig. 7f). CZ5 treatment also weakened the expression of the Socs3 gene, another common inhibitor for leptin signalling (ESM Fig. 7g) . Meanwhile, chronic treatment of SD mice led to hyperphagia, which mimics the effects of overexpression of UCPs in SKM . This suggests that mitochondrial uncoupling in muscle or adipose tissue can regulate appetite in different ways, which might relate to leptin sensitivity and body composition.
The in vitro cell type-specific effects might allow CZ5 to have a milder uncoupling effect than that observed with the non-specific uncoupler DNP at the whole-body level. Examination for potential toxic cardiac effects after 5 weeks of administration of CZ5 to SD mice at a dose of 30 mg kg−1 day−1 showed that there were no obvious alterations of echocardiographic variables (ESM Table 3). In a small-scale acute toxicity study, all of the mice survived without any signs of compound-induced toxicity after a single oral dose 50-fold greater than that administered in the chronic study (ESM Table 4). Although the potential systemic toxicity of CZ5 requires a more complete evaluation, we initially conclude that CZ5 is a relatively safe chemical uncoupler, at least in rodent models.
Regarding the mechanism of the cell type-specific uncoupling effects, we found that discrepancies among uncoupling capacities in the three types of isolated mitochondria may partially contribute to the unusual characteristic of this compound. In isolated mitochondria from L6 myotubes and 3T3-L1 adipocytes, CZ5 stimulated the lowest and the maximal uncoupling respiration at lower concentrations than in isolated liver mitochondria (ESM Fig. 8). It is possible that CZ5 uncouples OXPHOS via its chemical properties, namely lipophilicity and weak acidity. Additionally CZ5 may also interact with certain proteins that mediate proton leak and are expressed specifically in myocytes and adipocytes . However, neither GDP nor carboxyatractyloside was able to attenuate the uncoupling effects of CZ5 (data not shown), suggesting no involvement of UCPs or the adenine nucleotide translocase in these effects. Although we favour the aforementioned explanation, the possibility of cell type-specific membrane permeability to CZ5 contributing to its various metabolic effects in the three cell types cannot be excluded.
Overall, we discovered a novel cell type-specific chemical uncoupler that has potential as an anti-obesity therapy by increasing EE and suppressing EI. Our findings provide a proof of concept for increasing mitochondrial uncoupling selectively in SKM and WAT as a novel approach for the treatment of obesity and type 2 diabetes.
We thank C.-L. Tang, L. Sheng and J. Zhang from the National Center for Drug Screening for their assistance in the animal experiments. We appreciate the support given by S.-L. Huang and Y. Leng at the Shanghai Institute of Materia Medica in the animal EE study and by J.-G. Jia and Y.-Z. Zou at the Shanghai Institute of Cardiovascular Disease in the assessment of cardiac safety study. We also appreciate F.-F. Guo from the Shanghai Institute for Nutritional Sciences and J.-M. Ye from the Royal Melbourne Institute of Technology (RMIT) university for helpful discussion and manuscript editing.
This work was supported by the National Natural Science Foundation of China (81001463, 81125023 and 81273566), the National Science and Technology Major Projects for Major New Drugs Innovation and Development (2012ZX09304011, 2012ZX09301-001-004 and 2013ZX09507002), National Program on Key Basic Research Project (2012CB524906 and G1998051104), Chinese Academy of Science ‘Strategic Leader in Science and Technology Projects’ (XDA01040303) and Shanghai Commission of Science and Technology (11DZ2292200). This work was also supported by the National Health and Medical Research Council of Australia and an Australian Research Council Future Fellowship to N. Turner.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
Y-YF, MZ, NT, L-NZ, T-CD, MG, SJL, J-YL, F-JN and JL all contributed to the conception and design of the study, and contributed to acquisition, analysis and interpretation of data. All the authors participated in drafting and revising the article and all have approved the final version of the article.
- 22.Turner N, Li JY, Gosby A et al (2008) Berberine and its more biologically available derivative, dihydroberberine, inhibit mitochondrial respiratory complex I: a mechanism for the action of berberine to activate AMP-activated protein kinase and improve insulin action. Diabetes 57:1414–1418CrossRefPubMedGoogle Scholar