Introduction

The obesity and obesity-related diseases, in particular, type 2 diabetes (T2D) have become the major medical challenges to all the nations [1]. In United States, over 78.6 million adults are obese and approximately 29.1 million people have T2D [2, 3]. T2D is a progressive disease characterized by insulin resistance and disrupted glucose and lipid metabolism. Insulin resistance, which precedes the development of T2D, is attributed to the major cause of T2D and other obesity-related metabolic disorders including fatty liver diseases, dyslipidemia, and cardiovascular diseases [4]. However, medications available nowadays are not effective in correcting the underlying cause of insulin resistance and bear inherent limitations [5, 6]. Therefore, anti-diabetic pharmacotherapies which target the cause of insulin resistance are preferable to improve T2D treatment.

The association between obesity and T2D has been recognized for decades. Several mechanisms have been proposed that ectopic lipid accumulation in liver and muscle plays causal role in insulin resistance and T2D [7,8,9,10,11]. Recently, a new class of compounds, mitochondrial uncouplers, was found to be promising as anti-obese and anti-diabetic pharmacotherapy with a novel mechanism [12, 13]. Mitochondrial uncoupling dissipates the energy from mitochondrial oxidation as heat without effectively producing ATP [14, 15]. The resultant cellular energy inefficiency in turn increases lipid catabolism and then reduces intracellular lipid loads [16, 17], which improves insulin sensitivity and obesity [18, 19].

Niclosamide is an anthelmintic drug approved by United States Food and Drug Administration [20]. Previous study showed that NEN, the ethanolamine salt of niclosamide, is a moderate hepatic mitochondrial uncoupler for treating obesity and T2D in animal models [13]. A more recent research showed that niclosamide itself also has effect on obesity [21]. Oral administration of NEN in mice significantly improved obesity, hyperglycemia, insulin resistance and hepatic steatosis symptoms by reducing cellular energy efficiency and increasing lipid oxidation in liver [13]. Niclosamide piperazine (NPP) is the other niclosamide salt form with known safety profile [22]. NPP has higher water solubility than niclosamide free base but lower water solubility than NEN. The higher water solubility of the salt formulation usually leads to the higher oral bioavailability, hence potency [23]. However, increased bioavailability may also enhance toxicity. For clinical drug development, it is important to identify the best salt formulation with proven efficacy and lower toxicity. In the current study, we determined the efficacy of NPP, as the other salt formulation of niclosamide on preventing the development of obesity and diabetic symptoms in high-fat diet (HFD)-induced obese/diabetic mice.

Materials and methods

Reagent

Niclosamide piperazine salt [NPP, 5-chloro-salicyl-(2-chloro-4-nitro) anilide piperazine salt] was purchased from BOC Sciences (Shirley, NY); niclosamide ethanolamine salt [NEN, niclosamide 5-chloro-salicyl-(2-chloro-4-nitro) anilide 2-aminoethanol salt] from 2A PharmaChem (Lisle, IL).

Measurement of mitochondrial membrane potential

NIH-3T3 cells were plated in 6-well plate and cultured in DMEM medium supplemented with 10% (vol/vol) fetal bovine serum, 100 U/mL of penicillin, 100 µg/mL of streptomycin and 0.29 mg/mL of l-glutamine at 37 °C in a humidified atmosphere of 5% CO2. For treatment, NPP or NEN was dissolved in DMSO as stock solutions, and then was added into medium to reach final desired concentrations by 1:1000 dilutions. Cells were incubated with vehicle (DMSO), NPP or NEN at indicated final concentration for 2 h and then immediately stained with tetramethylrhodamine, ethyl ester (TMRE) at final concentration of 100 nM for 10 min followed by PBS wash. Afterwards cells were subjected to florescence microscope for the determination of membrane potential indicated by florescence strength.

Mitochondrial respiration measurement

Mitochondrial respiration rate was evaluated by measuring oxygen consumption rate (OCR) under mitochondrial stress condition using Seahorse XF24e Extracellular Flux Analyzer (Seahorse Bioscience Inc., MA). One day before seahorse study, human liver carcinoma HepG2 cells were plated into XF24 microplate at the density of 50,000 cells/well in complete minimum essential medium, containing 10% (vol/vol) fetal bovine serum, 100 U/mL of penicillin, 100 µg/mL of streptomycin and 0.29 mg/mL of l-glutamine. The plate was placed at 37 °C in a humidified atmosphere of 5% CO2 for overnight. On the day of assay, cells were washed twice with fresh complete DMEM medium and the volume of each well was brought up to 525 μL. Cells were then placed in a CO2 free incubator for 1 h before the OCR assay. The OCR profile was measured at four consecutive stages, including baseline, injection of part A (2.5 μM oligomycin), injection of port B (2.5 μM NEN or 2.5 μM NPP), and injection of port C (2.5 μM antimycin and 2.5 μM rotenone). For each stage, the OCR was measured for 2–3 times; and for each measurement, there is a 2 min mix followed by a 2 min wait time to restore oxygen retention and a 3 min measure. The seahorse data was interpreted by Wave 2.3 software.

Animal and treatment

Four-week-old male C57BL/6J mice were purchased from Jackson Laboratory (Bar Harbor, ME) and shipped to the vivarium of Rutgers-Robert Wood Johnson Medical School. All the experimental protocols were approved by Institutional Animal Care and Use Committees (IACUCs). Mice were maintained in temperature/humidity-controlled room with 12 h light/dark cycle. Starting at age of 5 weeks, mice were randomized into two groups with matched body weights (n = 9), and were fed either HFD (60% fat calories; Research Diet, New Brunswick, NJ) or HFD containing 2000 ppm. NPP (mixed and formulated by Research Diet using NPP supplied by the Jin lab), respectively, for 11 weeks. Mice were allowed free access to water and food throughout the experiment period. Body weight was measured weekly, and food intake was recorded daily.

Blood glucose and plasma insulin measurement

Glucose level was determined by tail vein bleed from 16 h-fasted mice with a glucometer (OneTouch UltraSmart blood glucose monitoring system, Lifescan, Milpitas, CA). For measuring plasma insulin, mice were fasted 5 h before experiment. Blood samples were then collected and subjected to ultra-sensitive mouse insulin ELISA kit (Crystal Chem Inc., Downers Grove, IL).

Glucose tolerance assay and insulin sensitivity assay

Mice were fasted 16 h before glucose tolerance test. Glucose load was given by intraperitoneal injection of 20% d-glucose (Sigma Aldrich, St. Louis, MO) at a dosage of 2 g/kg body weight. Blood glucose levels were measured initially and at designated time points after administration (15, 30, 60, 90, and 120 min).

For insulin sensitivity assay, 5 h-fasted mice were intraperitoneally injected with recombinant human insulin (Eli Lilly, Indianapolis, IN) at a dosage of 0.75 U/kg body weight. Blood glucose concentrations were measured at the same time points indicated in glucose tolerance assay.

Histology of liver

At the end of treatment, mice were killed by decapitation. Liver tissues were dissected and fixed with 10% neutral buffered formalin (Surgipath Medical Industries Inc., Richmond, IL). Tissue sections were then prepared and stained with hematoxylin and eosin (H&E). Images were captured by Nikon Eclipse Ni-U imaging system.

Statistics

Data are presented as the mean ± standard deviation (SD). One-way ANOVA was used for all of the statistical comparisons among groups. Statistical significance is denoted.

Results

NPP uncouples mitochondria in mammalian cells

The structures of NPP and NEN are listed in Fig. 1a, b, respectively. NPP is another well-documented active salt form of niclosamide that has been used in veterinary medicine to control tapeworm infections through uncoupling mitochondria of the parasites [24]. To determine if NPP has mitochondrial uncoupling activity, we first analyzed mitochondrial membrane potential in two mammalian cell lines upon treatment with NPP. In cultured cells, the mouse fibroblast cell line NIH-3T3 and human hepatic cell line HepG2, NPP decreased mitochondrial membrane potential starting at the concentration of 500 nM and reached 50% reduction of membrane potential over concentration of 1 µM, which is comparable to that of NEN (Fig. 1c). The hallmark of mitochondrial uncoupling is the ability to increase mitochondrial oxygen consumption in the presence of a mitochondrial ATP synthase inhibitor such as oligomycin. We did oxygen consumption studies with Seahorse instrument in HepG2 cells. As shown in Fig. 1e, similar to NEN, NPP treatment stimulates mitochondrial oxygen consumption in the presence of oligomycin, indicating that NPP has mitochondrial uncoupling activity.

Fig. 1
figure 1

NPP uncouples mitochondria in mammalian cells. a Structures of 5-chloro-salicyl-(2-chloro-4-nitro) anilide piperazine salt (NPP), and b 5-chloro-salicyl-(2-chloro-4-nitro) anilide 2-aminoethanol salt (NEN). Effect of NPP and NEN on mitochondrial membrane potential determined by TMRE staining in NIH-3T3 cells (c) and in HepG2 cells (d). Cells were treated with vehicle (DMSO), NPP or NEN alone at indicated final concentration for 2 h. Cells were then stained with TMRE (100 nM) for 15 min to detect mitochondrial membrane potential. After washed twice with PBS, cells were observed under microscope. Scale bars 200 μm. e oxygen consumption rate (OCR) profile of HepG2 cells treated with 2.5 μM NEN or 2.5 μM NPP. Data are presented as mean value ± SD (n = 3)

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NPP-reduced diet-induced body weight gain

We further evaluated the efficacy of NPP on metabolism in animals. Long-term high-fat diet (HFD) feeding induces obesity, hyperglycemia, insulin resistance and hepatic steatosis in C57BL/6 mice. We either fed the C57/BL/6 mice HFD alone for 8 weeks to induce metabolic symptoms, or HFD containing 2000 ppm. NPP (125 mg/kg/day) during the same period of time. We found that despite the similar average initial body weight (Fig. 2a), mice treated with NPP exhibited slower rate of gaining body weight than the control group fed HFD alone, and ended with 25% less body weight gain after 8 weeks of treatment. The less body weight gain of NPP-treated mice was not caused by the less food intake, as shown in Fig. 2b.

Fig. 2
figure 2

Chronic oral treatment of NPP reduced body weight gain in high-fat diet (HFD)-induced obese/diabetic mice. a Body weights, b daily food intake of mice fed HFD (60% fat calories) or HFD containing 2000 ppm. NPP for 11 weeks. Data are mean value ± SD (n = 9). *p < 0.05, **p < 0.01, ***p < 0.001

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NPP-improved glycemic control and insulin resistance

We further determined the effects of NPP on glycemic control and insulin sensitivity. After 4-week HFD feeding, fasting blood glucose level of the control mice increased to ~122 mg/dL (Fig. 3a) which reaches prediabetic range [25], while the age-matched mice treated with NPP showed significantly lower blood glucose levels which are still within the normal range [25]. At the end of 8-week treatment, control mice fed HFD alone developed hyperglycemia to ~158 mg/dL and hyperinsulinemia to ~2.18 ng/mL, which reaches diabetic level [25]. Comparatively, NPP treatment significantly reduced blood glucose level and plasma insulin by 22 and 61%, respectively (Fig. 3b). To further investigate the effects of NPP on insulin resistance, intraperitoneal glucose tolerance assay and insulin resistance assay were performed. Consistent with the better glycemic control in NPP treatment group, mice treated with NPP showed significantly improved glucose tolerance and better insulin sensitivity in response to glucose and insulin load, respectively (Fig. 3c, d).

Fig. 3
figure 3

Chronic oral treatment of NPP-improved glycemic control and insulin resistance in high-fat diet (HFD)-induced obese/diabetic mice. a Fasting blood glucose levels of mice fed HFD (60% calories) or HFD containing 2000 ppm. NPP measured at week 4 and 8 of treatment; b plasma insulin level measured at week 9; c glucose tolerance assay and d insulin sensitivity assay performed at week 10 and 11, respectively. Data are mean value ± SD (n = 9). *p < 0.05, **p < 0.01, ***p < 0.001

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NPP prevented diet-induced hepatic steatosis

To determine whether NPP impacts hepatic lipid metabolism, we performed hepatic histology study. As shown in Fig. 4, the hepatocytes of control mice fed HFD alone had significant vacuolation composed of massive lipid droplets, which indicates severe hepatic steatosis. In contrast, fewer intracellular lipid droplets and regular shape of hepatocytes were found in the NPP-treated mice under same HFD feeding conditions. The histology results suggested that, similar to its analog NEN, NPP treatment prevented the HFD-induced hepatic lipid accumulation in mice.

Fig. 4
figure 4

Chronic oral treatment of NPP-reduced hepatic lipid accumulation. Representative pictures of H&E stained liver tissues from mice fed high-fat diet (HFD) alone or HFD containing 2000 ppm. NPP for 11 Weeks. The white areas in hepatocytes are cells with high lipid content. Scale bar 50 µM

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Discussion

Niclosamide is practically insoluble in water compared to its two salt forms, NEN and NPP [20]. For oral administration, it is mostly likely that NEN and NPP are more bioaccessible than niclosamide free base due to their higher water solubility, which gives rise to higher oral bioavailability and anti-diabetic efficacy of NEN and NPP. Thus, compared to niclosamide, the two salt forms exhibit higher potential for therapeutics development.

Previous study demonstrated that NEN is efficacious in correcting obesity, insulin resistance and diabetic symptoms [13]. The water solubility of NPP is between NEN and niclosamide. The current study addressed the question whether NPP could serve as an alternative to NEN as drug candidate for future clinical development. We demonstrated that NPP is effective in improving HFD-induced obesity, hyperglycemia, insulin resistance and hepatic steatosis at a dose similar to NEN in mice. The result provided evidence that NPP has comparable efficacy for treating obesity and T2D as NEN. Further studies, particularly the comparison in toxicity profile between NPP and NEN, will determine which salt form has a better therapeutic window. This would help us select a better lead compound for the development of a therapeutic for the treatment of obesity and T2D.

Conclusions

In brief, we found that NPP, a salt form of niclosamide, is efficacious in reducing HFD-induced obesity and improving HFD-induced diabetic symptoms and hepatic steatosis in mice. Its anti-obesity and anti-diabetic effect, together with our previous studies [13], confirmed the effectiveness of mitochondria uncoupling for the prevention or treatment of obesity and T2D. Moreover, based on the result from the current animal study, NPP may hold the promise to become an alternative to NEN as a drug lead for treating obesity and T2D. Future pre-clinical studies are required to compare the therapeutic window of the two drugs.