Sulforaphane improves leptin responsiveness in high-fat high-sucrose diet-fed obese mice

Abstract

Diet-induced obesity (DIO) is commonly associated with hyperleptinemia and leptin resistance. Leptin acts centrally to inhibit food intake and increase energy expenditure, thereby preventing body weight gain. Resistance to the biological effects of leptin represents a major obstacle in utilizing exogenously administered leptin as a treatment option for obesity. Of importance, recent studies demonstrate that naturally occurring compounds improve leptin sensitivity in DIO mice, as revealed by anorectic and body weight-lowering effects. To date, the role of sulforaphane (SFN, an isothiocyanate derived from cruciferous vegetables) on leptin responsiveness has not been examined, in spite of its known beneficial effects toward lowering body weight gain in DIO. In the present study, we determined the extent to which SFN regulates leptin responsiveness in high-fat high-sucrose (HFHS) diet-fed obese mice. SFN treatment (0.5 mg/kg/day, s.c.) for 23 days in HFHS-fed mice improved the responsiveness to intraperitoneally-injected leptin by promoting significant decreases in cumulative food intake and body weight gain. A single leptin injection (2 mg/kg; i.p.) resulted in significant decreases in food intake at 24 h and 38 h time points. In addition, a triple leptin injection (1 mg/kg/day, 3 days; i.p.) led to significant decreases in food intake at 14 h, 24 h, 38 h, 48 h, and 62 h time points. Furthermore, single and triple leptin injections prevented body weight gain at 38 h and 62 h time points, respectively. The present findings suggest that intervention with SFN, a naturally occurring isothiocyanate, has the potential to improve leptin responsiveness in DIO.

Keywords: Diet-induced obesity; Food intake; Leptin responsiveness; Sulforaphane; Weight gain.

Fig. 1.

Experimental protocol to determine leptin responsiveness to food intake and body weight in high-fat high-sucrose (HFHS) diet-fed obese mice before and after sulforaphane (SFN) treatment. Prior to SFN treatment, leptin responsiveness test was performed using a single-injection protocol (on day 19) in HFHS-fed mice versus control (CON) group. After the start of SFN treatment (on day 35), leptin responsiveness tests were carried out using a single-injection protocol (on day 50) and a triple-injection protocol (once daily for 3 consecutive days; on days 54–56).

Fig. 2.

Effects of HFHS diet on leptin responsiveness as assessed by a single leptin injection. After the start of respective diets, control group (CON) and HFHS diet-fed mice were fasted for 9 h during daytime (on day 19) followed by injection with leptin (2 mg/kg,i.p.). As additional controls, saline was injected (10 ml/kg,i.p.) in the respective treatment groups. Subsequently, cumulative food intake was determined at three different time points (14 h, 24 h, and 38 h; upper panels). In addition, body weight gain was determined at the 38 h time point (lower panels). The data shown are the means ± S.E.M (n values for different groups are indicated within the bar graphs and beside the linear graphs). Statistical significance between saline- and leptin-injected mice of the same group was determined using repeated measures two-way ANOVA followed by Bonferroni test (upper panel) or unpaired student t test (lower panel). ^# P < 0.05, ^## P < 0.01, ^### P < 0.001; and NS, non-significant compared with saline-injected mice from the same group.

Fig. 3.

Effects of HFHS diet on leptin responsiveness as assessed by single and triple leptin injection(s) under vehicle-treated or SFN-treated conditions. Mice were maintained on CON diet or HFHS diet without or with SFN treatment, as illustrated in Fig. 1. A) All four groups of mice were fasted for 9 h during daytime (on day 50) followed by a single injection with leptin (2 mg/kg,i.p.). As controls, saline was injected (10 ml/kg,i.p.) in the respective treatment groups. Subsequently, cumulative food intake was determined at three different time points (14 h, 24 h, and 38 h; left panels). Body weight gain was determined at the 38 h time point (right panels). B) On day 54, the test was repeated with triple leptin injections (1 mg/kg, i.p.; once a day for 3 days with a 24 h time interval between injections). Cumulative food intake was determined at five time points (14 h, 24 h, 38 h, 48 h, and 62 h from the first injection; left panels). Body weight gain was determined at the 62 h time point from the first injection (right panels). The data shown are the means ± S.E.M. (n values for the different groups are indicated at bases of bars and beside the linear graphs). Statistical significance between saline- and leptin-injected mice of the same group was determined using repeated measures two-way ANOVA followed by Bonferroni test (left panels) or unpaired student t test (right panels). ^# P < 0.05, ^## P < 0.01, ^### P < 0.001; and NS, non-significant compared with saline-injected mice from the same group.

Fig. 3.

Effects of HFHS diet on leptin responsiveness as assessed by single and triple leptin injection(s) under vehicle-treated or SFN-treated conditions. Mice were maintained on CON diet or HFHS diet without or with SFN treatment, as illustrated in Fig. 1. A) All four groups of mice were fasted for 9 h during daytime (on day 50) followed by a single injection with leptin (2 mg/kg,i.p.). As controls, saline was injected (10 ml/kg,i.p.) in the respective treatment groups. Subsequently, cumulative food intake was determined at three different time points (14 h, 24 h, and 38 h; left panels). Body weight gain was determined at the 38 h time point (right panels). B) On day 54, the test was repeated with triple leptin injections (1 mg/kg, i.p.; once a day for 3 days with a 24 h time interval between injections). Cumulative food intake was determined at five time points (14 h, 24 h, 38 h, 48 h, and 62 h from the first injection; left panels). Body weight gain was determined at the 62 h time point from the first injection (right panels). The data shown are the means ± S.E.M. (n values for the different groups are indicated at bases of bars and beside the linear graphs). Statistical significance between saline- and leptin-injected mice of the same group was determined using repeated measures two-way ANOVA followed by Bonferroni test (left panels) or unpaired student t test (right panels). ^# P < 0.05, ^## P < 0.01, ^### P < 0.001; and NS, non-significant compared with saline-injected mice from the same group.

Fig. 4.

Effects of HFHS diet on caloric intake during leptin responsiveness under vehicle-treated or SFN-treated conditions. Mice were maintained on CON diet or HFHS diet without or with SFN treatment, as illustrated in Fig. 1. A-B) All four groups of mice were fasted for 9 h during daytime (on days 19 and 50, respectively) followed by a single injection with saline (10 ml/kg, i.p.) in the four groups. Subsequently, total caloric intake after 38 h was determined using the following formulas: 1 g CON diet = 3.91 Kcal; and 1 g HFHS diet = 4.68 Kcal. C-D) On day 54, the test was repeated with triple saline or leptin injections injections (10 ml/kg and 1 mg/kg, respectively, i.p.; once a day for 3 days). Subsequently, total caloric intake after 62 h from the first injection for saline-injected mice of 4 groups (C) or caloric intake for the first 14 h after each injection for leptin-injected mice of 4 groups (D) were similarly calculated. The data shown are the means ± S.E.M. (n values for the different groups are indicated within the bar graphs). Statistical analysis was carried out using unpaired student t-test (for total caloric intake of leptin responsiveness test performed on day 19) and regular two-way ANOVA followed by Bonferroni multiple comparisons test (for total food intake of leptin responsiveness test performed on days 50 and 54). ^## P < 0.01 and ^### P < 0.001 compared with saline-injected mice from the CON or CON-vehicle groups (A and C). ** P < 0.01 compared with saline-injected mice from the HFHS-vehicle group (B). ^### P < 0.001 compared with the food intake 14 h after the previous leptin injection (D).