Inhibitory Effect of Tangeretin and Cardamonin on Human Intestinal SGLT1 Activity In Vitro and Blood Glucose Levels in Mice In Vivo

Abstract

Rapid postprandial blood glucose elevation can cause lifestyle-related diseases, such as type II diabetes. The absorption of food-derived glucose is primarily mediated by sodium/glucose cotransporter 1 (SGLT1). Moderate SGLT1 inhibition can help attenuate postprandial blood glucose elevation and prevent lifestyle-related diseases. In this study, we established a CHO cell line stably expressing human SGLT1 and examined the effects of phytochemicals on SGLT1 activity. Among the 50 phytochemicals assessed, tangeretin and cardamonin inhibited SGLT1 activity. Tangeretin and cardamonin did not affect the uptake of L-leucine, L-glutamate, and glycyl-sarcosine. Tangeretin, but not cardamonin, inhibited fructose uptake, suggesting that the inhibitory effect of tangeretin was specific to the monosaccharide transporter, whereas that of cardamonin was specific to SGLT1. Kinetic analysis suggested that the suppression of SGLT1 activity by tangeretin was associated with a reduction in V_max and an increase in K_m, whereas suppression by cardamonin was associated with a reduction in V_max and no change in K_m. Oral glucose tolerance tests in mice showed that tangeretin and cardamonin significantly suppressed the rapid increase in blood glucose levels. In conclusion, tangeretin and cardamonin were shown to inhibit SGLT1 activity in vitro and lower blood glucose level in vivo.

Keywords: SGLT1; cardamonin; intestinal epithelial cell; tangeretin; transporter.

Figure 1

Glucose uptake in stable hSGLT1-expressing Chinese Hamster Ovary (CHO) cells in the absence or presence of sodium ions (A). (B) Sodium-dependent glucose uptake in stable hSGLT1-expressing CHO cells. Glucose uptake in the absence or presence of sodium ions was measured (A). Sodium-dependent glucose uptake was calculated by subtracting the glucose uptake in the presence of sodium ions and in the absence of sodium ions. Each value represents the mean ± standard error of mean (n = 3).

Figure 2

Effect of the phytochemicals on glucose uptake in stable hSGLT1-expressing CHO cells. Glucose uptake was measured in the absence or presence of the phytochemicals (1 μM). The values are expressed in terms of mean ± standard error of mean (n = 3); * p < 0.05 vs. the control value (Student’s t-test).

Figure 3

Concentration-dependent inhibition of glucose uptake by tangeretin (A) and cardamonin (B) in stably hSGLT1-expressed CHO cells. Glucose uptake was measured in the absence or presence of 0–50 μM tangeretin (A) and cardamonin (B). The values are the mean ± SE (n = 3), and the values indicated by different characters are significantly different from each other (Tukey’s test; p < 0.05).

Figure 4

Effect of tangeretin (A) and cardamonin (B) on leucine and glutamic acid uptake in stable hSGLT1-expressing CHO cells. The uptake of glucose, L-leucine, and L-glutamic acid uptake was measured in the absence or presence of 10 μM tangeretin (A) or cardamonin (B). The values are expressed in terms of mean ± standard error of mean (n = 3); * p < 0.05 vs. the control value (Student’s t-test).

Figure 5

Effect of tangeretin (A) and cardamonin (B) on fructose and glycyl-sarcosine uptake in Caco-2 cells. Fructose and glycyl-sarcosine uptake were measured in the absence or presence of 10 μM tangeretin (A) or cardamonin (B). The values are expressed in terms of mean ± standard error of mean (n = 3); * p < 0.05 vs. the control value (Student’s t-test).

Figure 6

Effect of tangeretin-related compounds on glucose uptake in stable hSGLT1-expressing CHO cells. Glucose uptake was measured in the absence or presence of 1 μM tangeretin, nobiletin, and sinensetin (A). The values are expressed in terms of mean ± standard error of mean (SE) (n = 3), and the values indicated by the different characters are significantly different from each other (Tukey’s test; p < 0.05). Sodium-dependent glucose uptake was assessed by subtracting glucose uptake in the presence of sodium ions and in the absence of sodium ions, further, in the absence or presence of tangeretin-related compounds (B). Each value represents mean ± SE (n = 3).

Figure 7

Effect of cardamonin-related compounds on glucose uptake in stable hSGLT1-expressing CHO cells. Glucose uptake was measured in the absence or presence of 10 μM cardamonin and its related compound, respectively (A). The values represent the mean ± standard error of mean (SE) (n = 3), and the values indicated by different characters are significantly different from each other (Tukey’s test; p < 0.05). Sodium-dependent glucose uptake was assessed by subtracting glucose uptake in the presence of sodium ions and in the absence of sodium ions, and further, in the absence or presence of cardamonin-related compounds (B). Each value represents the mean ± SE (n = 3).

Figure 8

Kinetic analysis of sodium-dependent glucose uptake in the absence (A) or presence of tangeretin (B) and cardamonin (C). Sodium-dependent glucose uptake was measured after the administration of 0–0.4 mM glucose without (A) or along with 10 μM tangeretin (B) or cardamonin (C). The values are expressed in terms of mean ± standard error of mean (n = 3). Lineweaver–Burk plots were constructed to calculate the Vmax and Km values for sodium-dependent glucose uptake in the absence or presence of 10 μM tangeretin or cardamonin.

Figure 9

Effect of tangeretin on plasma glucose levels after oral glucose administration in ICR mice. ICR mice fasted overnight were orally administered 1 g glucose/kg body weight (BW) (20% glucose solution) along with or without 250 mg or 400 mg tangeretin/kg BW, and blood was drawn at 0, 30, 60, and 120 min. The plasma glucose concentration was measured (A), and the AUC under each experimental condition was calculated based on the values obtained (B). The values are expressed in terms of mean ± standard deviation of mean (n = 6); (A) * p < 0.05 vs. the control value (Dunnett’s test). (B) The values indicated by the different characters are significantly different from each other (Tukey’s test; p < 0.05).

Figure 10

Effect of cardamonin on plasma glucose levels after oral glucose administration in ICR mice. ICR mice fasted overnight were orally administered 1 g glucose/kg body weight (BW) (20% glucose solution) along with or without 250 mg or 400 mg cardamonin/kg BW, and blood was drawn at 0, 30, 60, and 120 min. The plasma glucose concentration was measured (A), and the AUC under each experimental condition was calculated based on the values obtained (B). The values are expressed in terms of mean ± standard deviation of mean (n = 6); (A) * p < 0.05 vs. the control value (Dunnett’s test). (B) The values indicated by the different characters are significantly different from each other (Tukey’s test; p < 0.05).