LEAP2 attenuates the ability of alcohol to induce locomotor stimulation, rewarding memory, and release of dopamine in NAcS in male mice.
There was an overall effect on locomotor activity in male mice treated with LEAP2 and alcohol (F3,30=6.17, P = 0.0013; Fig. 1A). LEAP2 blocked (P = 0.0223, n = 8) the alcohol-induced locomotor stimulation (P = 0.0071, n = 11) in male mice. This is further evident as the activity between vehicle (n = 7) and LEAP2-alcohol-treated mice are similar (P = 0.8742). LEAP2 did not influence the activity per se (P = 0.9973, n = 8). In total 8 mice were excluded due to misplaced infusion sites. Next, LEAP2’s effect on the memory of alcohol reward was evaluated using the mCPP paradigm. As expected, LEAP2 attenuated the memory of alcohol reward (Fig. 1B, n = 7) compared to vehicle (n = 8, t13 = 3,31, P = 0.0057). One mouse was excluded due to a misplaced infusion site.
Further support for LEAP2’s ability to attenuate the stimulatory and rewarding properties of alcohol is provided by the in vivo microdialysis studies in which LEAP2 suppresses the ability of alcohol to cause a release of dopamine (Fig. 1C). Supportively, there was an overall effect of treatment (F1,15=14.70, P = 0.0016), time (F12,180=2.89, P = 0.0011), and interaction (F12,180=2.91, P = 0.0011). The dopamine levels were lower in LEAP2-alcohol-treated mice (n = 7) compared to those treated with vehicle-alcohol (n = 8) at 20 (P < 0.01), 40–60 (P < 0.05), and 140–160 (P < 0.05) minutes. Three mice were excluded due to misplaced infusion sites/probe placements.
LEAP2 reduces alcohol intake in male and female rats.
Additional evidence for the role of LEAP2 in alcohol responses was provided by alcohol-drinking experiments in male (Fig. 2, n = 6) and female rats (Fig. 3, n = 9), where each rat got both treatments. Four males and three females were excluded due to infusion misplacements.
Compared to vehicle, LEAP2 reduced the alcohol intake at the 2-hour time point in male rats (t5 = 3.36, P = 0.0202, Fig. 2A), exposed to alcohol for 6 weeks before treatment (4.3±0.5 g/kg). However, LEAP2 did not influence alcohol intake at 4- (t5 = 0.19, P = 0.8579, Fig. 2B) or 24-hour (t5 = 0.00, P = 0.9966, Fig. 2C) timepoints. Additionally, the food intake was lowered after LEAP2 at the 2-hour timepoint (t5 = 3.05, P = 0.0286, Fig. 2D). Similar to alcohol, LEAP2 did not alter food intake at the 4- (t5 = 1.26, P = 0.2641, Fig. 2E) or 24- (t5 = 1.83, P = 0.1275, Fig. 2F) hour timepoints or alter the body weight change (Fig. 2J, t5 = 0.05, P = 0.9603). Moreover, LEAP2 did not alter the water intake at any investigated timepoints (2-hour, Fig. 2G, t5 = 0.39, P = 0.7099; 4-hour, Fig. 2H, t5 = 0.90, P = 0.4075; 24-hour, Fig. 2I, t5 = 0.64, P = 0.5523), the preference for alcohol (2-hour, t5 = 0.60, P = 0.5719; 4-hour, t5 = 0.73, P = 0.5005; 24-hour, t5 = 0.35, P = 0.7409) or total fluid intake (2-hour, t5 = 0.2.04, P = 0.0971; 4-hour, t5 = 1.01, P = 0.3609; 24-hour, t5 = 0.67, P = 0.5307, Supplementary Fig. 1A-F).
Circulating LEAP2 levels could only be reliably detected in males (n = 23, 1.06±0.02 ng/ml, Fig. 4). While the above data revealed that central LEAP2 lowers alcohol intake in male rats, circulating LEAP2 did not correlate to alcohol drinking in a separate cohort of male rats. Specifically, the LEAP2 levels in serum were similar in high (> 3.5 g/kg, n = 12, 1.05±0.02 ng/ml) and low (< 3.5 g/kg, n = 11, 1.08±0.04 ng/ml) alcohol-consuming rats (t21 = 0.70, P = 0.493, Fig. 4A). Further, there was no correlation between alcohol consumption and circulating LEAP2 levels (p = 0.960, Fig. 4B). One serum sample from male rats was excluded due to contamination.
While LEAP2 did not alter the alcohol intake at the 2-hour timepoint (t8 = 0.65, P = 0.5359, Fig. 3A) in female rats under the same conditions (5.9±0.6 g/kg), it reduced it at the 4-hour timepoint (t8 = 3.36, P = 0.0100, Fig. 3B). Additionally, LEAP2 did not influence alcohol intake at the 24-hour (t8 = 1.11, P = 0.3011, Fig. 3C) timepoint. Further, LEAP2 did not change the food intake at the 2- (t8 = 0.40, P = 0.6994, Fig. 3D), 4- (t8 = 1.06, P = 0.3308, Fig. 3E) or 24- (t8 = 1.10, P = 0.3052, Fig. 3F) hour timepoints. Neither did LEAP2 influence the water intake at any investigated timepoints (2-hour, Fig. 3G, t8 = 0.98, P = 0.3561; 4-hour, Fig. 3H, t8 = 1.41, P = 0.1970; 24-hour, Fig. 3I, t8 = 0.36, P = 0.7316). However, LEAP2 reduced the body weight change (Fig. 3J, t8 = 2.58, P = 0.0325). LEAP2 treatment did not alter the preference for alcohol (2-hour, t8 = 1.54, P = 0.1620; 4-hour, t8 = 0.45, P = 0.6656; 24-hour, t8 = 0.58, P = 0.5802) or total fluid intake (2-hour, t8 = 0.84, P = 0.4242; 4-hour, t8 = 2.16, P = 0.0627; 24-hour, t8 = 0.77, P = 0.4637, Supplementary Fig. 2G-L).
In a separate set of alcohol-consuming female rats, LEAP2 was measured in serum. However, LEAP2 was not detected in serum from female rats due to concentrations below the detection limit.
In male rats, central LEAP2 infusion lowered the total caloric intake at the 2-hour time point (t5 = 3.13, P = 0.0259; Supplementary Fig. 3A), while it did not influence it at 4- or 24-hour time points (t5 = 1.09, P = 0.3260 and t5 = 1.28, P = 0.2571; Supplementary Fig. 3B-C). In female rats, the caloric intake is similar in rats treated with vehicle or LEAP2 (t8 = 0.41, P = 0.6897; t8 = 1.36, P = 0.2123; t8 = 1.35, P = 0.2131; Supplementary Fig. 3D-F).