Sucralose, a Non-nutritive Artificial Sweetener Exacerbates High Fat Diet-Induced Hepatic Steatosis Through Taste Receptor Type 1 Member 3

Abstract

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease globally, and it is strongly associated with obesity. To combat obesity, artificial sweeteners are often used to replace natural sugars, and sucralose is one of the most extensively used sweeteners. It was known that sucralose exerted effects on lipid metabolism dysregulation, and hepatic inflammation; however, the effects of sucralose on hepatic steatosis were still obscure. In this study, we found that supplements of sucralose enhanced high-fat-diet (HFD)-induced hepatic steatosis. In addition, treatment of sucralose increased reactive oxygen species (ROS) generation and induced endoplasmic reticulum (ER) stress in HepG2 cells. Pretreatment of ROS or ER stress inhibitors reversed the effects of sucralose on lipogenesis. Furthermore, pretreatment of taste receptor type 1 membrane 3 (T1R3) inhibitor or T1R3 knockdown reversed sucralose-induced lipogenesis in HepG2 cells. Taken together, sucralose might activate T1R3 to generate ROS and promote ER stress and lipogenesis, and further accelerate to the development of hepatic steatosis.

Keywords: artificial sweetener; endoplasmic reticulum stress; hepatic steatosis; high fat diet; sucralose.

FIGURE 1

Supplemen of sucralose in high-fat diet showed no significant effects on the body weight of mice. Eight-week-old C57BL/6 mice were fed with a chow diet (Chow), high-fat diet (HFD), or HFD supplemented with sucralose (HFSUC) for 12 weeks, and the body weight (A), food intake (B), and water intake (C) were measured using metabolic cages. At the end of the experiments, each group of the mice was sacrificed and the epididymal fat pads (eWAT) were removed and weighed (D). Data are mean ± SEM (n = 7–8 for each group of the mice) ^***p < 0.001 as compared with the Chow group.

FIGURE 2

Supplement of sucralose exacerbated high fat diet-induced hepatic steatosis in mice. Eight-week-old C57BL/6 mice were fed with a chow diet (Chow), high-fat diet (HFD), or HFD supplemented with sucralose (HFSUC) for 12 weeks. At the end of the experiments, each group of the mice was sacrificed and the liver tissues were removed and weighed (A). Hepatic triglyceride contents for each group of the mice were measured using a commercialized assay kit (B). In addition, the liver sections were stained with hematoxylin and eosin (100X) (C). Serum samples were collected for the determination of alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels (D). Data are mean ± SEM (n = 7–8 for each group of the mice) *p < 0.05, ^**p < 0.01, ^***p < 0.001 as compared with the Chow group or indicated groups.

FIGURE 3

Sucralose increased hepatic ER stress- and lipogenesis-related protein expressions in mice. Eight-week-old C57BL/6 mice were fed with a chow diet (Chow), high-fat diet (HFD), or HFD supplemented with sucralose (HFSUC) for 12 weeks. At the end of the experiments, each group of the mice was sacrificed and the liver tissues were removed. Western blots analysis was used to analyze the hepatic expressions of lipogenesis-related proteins, carbohydrate-response element-binding protein (ChREBP) (A), sterol regulatory element-binding protein 1 (SREBP1) (B), fatty acid synthase (FASN) (C), and acetyl-CoA carboxylase 1 (ACC1) (D), and ER stress-related proteins, IRE1α (E), and XBP1s (F). Data are mean ± SEM (n = 6 for each group) *p < 0.05, ^**p < 0.01, ^***p < 0.001 as compared with indicated groups.

FIGURE 4

Sucralose increased lipogenesis to facilitate lipid accumulation in HepG2 cells. HepG2 cells were pretreated with indicated doses of sucralose (SUC) for 24 h, and then treated with 0.5 mM oleic acid (OA) chelated with 2% bovine serum albumin (BSA) for another 24 h. Oil Red O staining was used to detect lipid accumulation (original magnification 200X) (A). In addition, the gene (B–F), and protein (G–K) expressions of carbohydrate-response element-binding protein (ChREBP), sterol regulatory element-binding protein 1 (SREBP1), fatty acid synthase (FASN), and acetyl-CoA carboxylase 1 (ACC1) were analyzed by real-time polymerase chain reaction and Western blots. Data are mean ± SEM (n = 6 for each group) *p < 0.05, ^**p < 0.01, ^***p < 0.001 as compared with control group.

FIGURE 5

Sucralose increased reactive oxygen species to induce endoplasmic reticulum stress in HepG2 cells. HepG2 cells were treated with indicated concentrations of sucralose (SUC) for 16 h to analyze the expressions of IRE1α (A) and XBP1s (B) by Western blots. The cells were pretreated with STF083010 (IRE1 inhibitor) at indicated doses for 30 min, and then treated with 10 mM sucralose for another 24 h for the determination of SREBP1 by Western blots (C). HepG2 cells were treated with 10 mM sucralose and the reactive oxygen species production was determined by DCFDA staining at indicated times. The fluorescence intensity was analyzed by ImageJ software (100X) (D). Cells were pretreated with N-acetylcysteine (NAC) at indicated doses for 30 min, and then treated with 10 mM sucralose for another 16 h to determine IRE1α (E) and XBP1s (F) expressions by Western blots. Data are the mean ± SEM (n = 6 for each group) *p < 0.05, ^**p < 0.01, ^***p < 0.001 as compared with indicated groups.

FIGURE 6

Sucralose induced reactive oxygen species generation and lipogenesis through T1R3. HepG2 cells were pretreated with gymnemic acid (GA) (A) or lactisole (LAC) (B) at indicated doses for 30 min, and then treated with 10 mM sucralose (SUC) for another 90 min. The reactive oxygen species production was determined by DCFDA staining, and the fluorescence intensity was analyzed by ImageJ software (original magnification 100X). HepG2 cells were pretreated with GA (C,D) or LAC (E,F) at indicated doses for 30 min, and then treated with 10 mM sucralose for another 24 h to determine XBP1s, and SREBP1 expressions by Western blots. In addition, the cells were harvested to determine the intracellular triglyceride contents using a commercialized assay kit (G). The cells were treated with 10 mM sucralose for 24 h to determine T1R2 (H) and T1R3 (I) expressions by Western blots. Data are mean ± SEM (n = 6 for each group) *p < 0.05, ^**p < 0.01, ^***p < 0.001 as compared with indicated groups.

FIGURE 7

Knockdown of T1R3 in HepG2 cells diminished the effects of sucralose on lipogenesis. Different clones of lentiviral vectors containing short hairpin RNA targeted to T1R3 (shT1R3) were used to evaluate the knockdown efficiency of T1R3 (A). HepG2 cells transfected with lentiviral vectors containing luciferase (LV^LUC) or short hairpin RNA targeted to T1R3 (shT1R3²¹) were treated with 10 mM sucralose (SUC) for 24 h to determine IRE1α (B), XBP1s (C), and SREBP1 expressions (D) by Western blots. In addition, HepG2 cells transfected with lentiviral particles were pretreated with 10 mM sucralose for 24 h, and then treated with 0.5 mM oleic acid (OA) for another 24 h. Cells were harvested and the intracellular triglyceride contents using a commercialized assay kit (E). ^**p < 0.01, ^***p < 0.001 as compared with LV^LUC group or indicated groups. N.S., no significance.