Niclosamide Ethanolamine Improves the Liver Damage and Lipid Abnormalities in Lysosomal Acid Lipase Deficient Mice

Lysosomal acid lipase deficiency (LAL-D) is an autosomal recessive genetic disease caused by the mutations of the LIPA gene which encodes LAL. Loss of LAL activity leads to massive accumulation of cholesterol ester and triglycerides in lysosome of cells in liver, spleen and other organs. Hepatomegaly, progressive liver impairment and lipid abnormalities are common presentations found in patients with LAL-D, which result in high morbidity and high premature mortality. Niclosamide is an anthelmintic drug approved by the US FDA with the mechanism of action that uncouples mitochondria of parasitic worms. Niclosamide ethanolamine (NEN), a salt form of niclsoamide, is a mild mitochondrial uncoupler primarily targeting liver and bears excellent safety profile. Previous study showed that NEN is effective to reduce hepatic steatosis and improve diabetic dyslipidemia symptoms by increasing lipid oxidation in mice. Here we investigated whether NEN can be used for treating liver damage and lipid abnormalities in LAL deficient (lal−/−) mice. Three-week-old female lal−/− mice were randomized into two groups (n=6) based on body weight which were fed AIN-93M diet or AIN-93M diet containing 2000ppm NEN, respectively, for 4 weeks. In the same time frame, age-matched lal+/+ mice were fed AIN-93M diet alone. The results showed that NEN-treated lal−/− mice presented significantly lower weight of liver, spleen and small intestine compared to the untreated counterparts (Figure 1). Consistently, markedly lower levels of plasma ALT and AST as well as lower hepatic lipid accumulation were found in NEN-treated lal−/− mice (Figure 2). In addition, NEN treatment results in lower levels of very low density lipoprotein (VLDL) and low density lipoprotein (LDL) cholesterol in lal−/− mice (Figure 3), which is accompanied with significant reduction in hepatic apolipoprotein B (ApoB) level (Figure 4). Consistent with lower hepatic apoB level, NEN significantly reduces the ApoB net secretion from HepG2 cells in a dose-dependent manner (Figure 4). Moreover, in a separate longevity study, long-term treatment of NEN reduces the mortality of lal−/− mice (Figure 5). Together, our data demonstrate that NEN is effective to reduce liver lipid accumulation and damage, and prolong lifespan in lal−/− mice. NEN holds the potential to be used as a therapy for the management of LAL-D. Support or Funding Information The work is supported by Mitobiopharm company. NEN treatment reduced hepatomegaly. 21-day-old female lal−/− mice were fed AIN-93M diet supplemented with (KO/NEN) or without (KO) 2000ppm NEN and matching lal+/+ (WT) were fed AIN-93M diet alone for 4 weeks. (a) Liver weight, (b) ratio of liver mass to body weight, (c) spleen weight, (d) ratio of spleen weight to body weight, (e) small intestine weight, (f) ratio of small intestine weight to body weight, (g) body weight, and (h) daily food intake. (□) P < 0.05, (□□) P < 0.01, and (□□□) P < 0.001 Oral treatment of NEN reduced liver damage and hepatic lipid content. (a) Plasma ALT, (b) plasma AST, (c) hepatic total cholesterol content (TC), (d) hepatic triglycerides content (TG) of female lal−/− mice fed AIN-93M diet or AIN-93M diet containing NEN for 4 weeks starting from 21 days of age. n = 6. (□) P < 0.05, (□□) P < 0.01, and (□□□) P < 0.001 denote significant difference between lal−/− mice with and without NEN treatment; all error bars, SD. Oral treatment of NEN improved plasma lipid profile. (a) Plasma triglycerides (TG), (b) plasma total cholesterol (TC), (c) plasma VLDL&LDL cholesterol (VLDL&LDL-C), and (d) plasma HDL-cholesterol (HDL-C) of female lal−/− mice fed AIN-93M diet or AIN-93M diet containing 2000ppm NEN for 4 weeks starting from 21 days of age. n = 6. (□) P < 0.05, (□□) P < 0.01, and (□□□) P < 0.001 denote significant difference between lal−/− mice with and without NEN treatment; all error bars, SD. NEN reduced apoB-100 secretion from liver and HepG2 cells. Immunoblot analyses of (a) plasma apoB-100, (b) hepatic apoB-100 and hepatic LDL-R in female lal−/− mice fed AIN-93M diet or AIN-93M diet containing 2000ppm NEN for 4 weeks starting from 21 days of age. (c) Immunoblot analyses of apoB-100 secreted from HepG2 cells treated with insulin (100ng/mL), 0.1μM NEN, 0.5μM NEN, or 1.0μM NEN in the absence or presence of 1.0g/L glucose for 24 hours. Long-term oral treatment of NEN reduced mortality of lal−/− mice. Starting from the age of 21 days, four lal−/− mice (2 male, 2 female) were fed AIN-93M diet and five lal−/− mice (1 male, 4 female) were fed AIN-93M diet containing 2000ppm NEN till the end of their natural lifespan. The survival curves of the two groups were compared by log-rank test. NEN treatment reduced hepatomegaly. 21-day-old female lal−/− mice were fed AIN-93M diet supplemented with (KO/NEN) or without (KO) 2000ppm NEN and matching lal+/+ (WT) were fed AIN-93M diet alone for 4 weeks. (a) Liver weight, (b) ratio of liver mass to body weight, (c) spleen weight, (d) ratio of spleen weight to body weight, (e) small intestine weight, (f) ratio of small intestine weight to body weight, (g) body weight, and (h) daily food intake. (□) P < 0.05, (□□) P < 0.01, and (□□□) P < 0.001 Oral treatment of NEN reduced liver damage and hepatic lipid content. (a) Plasma ALT, (b) plasma AST, (c) hepatic total cholesterol content (TC), (d) hepatic triglycerides content (TG) of female lal−/− mice fed AIN-93M diet or AIN-93M diet containing NEN for 4 weeks starting from 21 days of age. n = 6. (□) P < 0.05, (□□) P < 0.01, and (□□□) P < 0.001 denote significant difference between lal−/− mice with and without NEN treatment; all error bars, SD. Oral treatment of NEN improved plasma lipid profile. (a) Plasma triglycerides (TG), (b) plasma total cholesterol (TC), (c) plasma VLDL&LDL cholesterol (VLDL&LDL-C), and (d) plasma HDL-cholesterol (HDL-C) of female lal−/− mice fed AIN-93M diet or AIN-93M diet containing 2000ppm NEN for 4 weeks starting from 21 days of age. n = 6. (□) P < 0.05, (□□) P < 0.01, and (□□□) P < 0.001 denote significant difference between lal−/− mice with and without NEN treatment; all error bars, SD. NEN reduced apoB-100 secretion from liver and HepG2 cells. Immunoblot analyses of (a) plasma apoB-100, (b) hepatic apoB-100 and hepatic LDL-R in female lal−/− mice fed AIN-93M diet or AIN-93M diet containing 2000ppm NEN for 4 weeks starting from 21 days of age. (c) Immunoblot analyses of apoB-100 secreted from HepG2 cells treated with insulin (100ng/mL), 0.1μM NEN, 0.5μM NEN, or 1.0μM NEN in the absence or presence of 1.0g/L glucose for 24 hours. Long-term oral treatment of NEN reduced mortality of lal−/− mice. Starting from the age of 21 days, four lal−/− mice (2 male, 2 female) were fed AIN-93M diet and five lal−/− mice (1 male, 4 female) were fed AIN-93M diet containing 2000ppm NEN till the end of their natural lifespan. The survival curves of the two groups were compared by log-rank test.