Pairing Binge Drinking and a High-Fat Diet in Adolescence Modulates the Inflammatory Effects of Subsequent Alcohol Consumption in Mice

Abstract

Alcohol binge drinking (BD) and poor nutritional habits are two frequent behaviors among many adolescents that alter gut microbiota in a pro-inflammatory direction. Dysbiotic changes in the gut microbiome are observed after alcohol and high-fat diet (HFD) consumption, even before obesity onset. In this study, we investigate the neuroinflammatory response of adolescent BD when combined with a continuous or intermittent HFD and its effects on adult ethanol consumption by using a self-administration (SA) paradigm in mice. The inflammatory biomarkers IL-6 and CX3CL1 were measured in the striatum 24 h after BD, 3 weeks later and after the ethanol (EtOH) SA. Adolescent BD increased alcohol consumption in the oral SA and caused a greater motivation to seek the substance. Likewise, mice with intermittent access to HFD exhibited higher EtOH consumption, while the opposite effect was found in mice with continuous HFD access. Biochemical analyses showed that after BD and three weeks later, striatal levels of IL-6 and CX3CL1 were increased. In addition, in saline-treated mice, CX3CL1 was increased after continuous access to HFD. After oral SA procedure, striatal IL-6 was increased only in animals exposed to BD and HFD. In addition, striatal CX3CL1 levels were increased in all BD- and HFD-exposed groups. Overall, our findings show that adolescent BD and intermittent HFD increase adult alcohol intake and point to neuroinflammation as an important mechanism modulating this interaction.

Keywords: alcohol; binge; binge drinking; cytokines; high-fat diet; inflammation; microbiota.

Figure 1

Body weight of mice over the procedure. Mean (± SEM) amount measured animals’ body weight. (A) No differences in body weight between the standard diet (SD) and high-fat diet binge (HFDb) groups were detected; (B) Significant differences between continuous high-fat diet (HFDc) groups and SD groups. * p < 0.05; ** p < 0.01.

Figure 2

(A) Binge sessions. Caloric intake (kcal) of high-fat diet (HFD) in the 2-h high-fat binge-eating sessions that took place on Monday, Wednesday and Friday in the high-fat diet binge (HFDb) groups. The mean (±SEM) amount of kcal consumed in 2 h of access to high-fat food in every binge session * p < 0.05; *** p < 0.001 significant difference with respect to the first and second binge sessions. (B) Weekly energy consumption per cage (four mice) across the procedure. Mean (±SEM) 24 h energy consumption (kcal) of mice (standard chow shown by open bars and HFD by solid bars) *** p < 0.001 significant difference of both continuous high-fat diet (HFDc) groups with respect to the standard diet (SD) groups and HFDb groups.

Figure 3

Intermittent ethanol intake during adolescence and high-fat diet (HFD) increase IL-6 levels in the striatum. The columns represent the mean and the vertical lines ± SEM of concentration levels of IL-6 (pg/mg protein) (A) after ethanol binge drinking (BD) during adolescence, (B) 3 weeks after the last ethanol injection and (C) after the end of the oral self-administration (SA) procedure in the groups with food deprivation and (D) in the groups without food deprivation *** p < 0.001, ** p < 0.01 with respect to the corresponding non-ethanol-treated groups; +++ p < 0.001 with respect to the corresponding standard diet fed group.

Figure 4

Intermittent ethanol intake during adolescence and continuous high-fat diet (HFD) increase CX3CL1 levels in the striatum. The columns represent the mean and the vertical lines ± SEM of concentration levels of CX3CL1 (ng/mg protein) (A) after ethanol BD during adolescence, (B) 3 weeks after the last ethanol injection and (C) after the end of the oral self-administration (SA) procedure in the groups with food deprivation and (D) in the groups without food deprivation *** p < 0.001, * p < 0.05 with respect to the corresponding non-ethanol-treated groups; +++ p < 0.001, ++ p < 0.01 with respect to the corresponding standard diet fed group. # p < 0.05 with respect to the corresponding binge HFD group.

Figure 5

Effects of intermittent high-fat diet (HFD) bingeing on effective responses and oral ethanol EtOH self-administration in OF1 mice. Data are presented as mean (± SEM): (A) g/kg of 6% EtOH consumption and (B) the number of effective responses during FR1 and FR3. The columns represent means and the vertical lines ± SEM of (C) breaking point values during PR and (D) amount of 6% EtOH consumption during the PR session. * p < 0.05; ** p < 0.01; *** p < 0.001 values that are significantly different from animals that received BD compared to the saline groups. + p < 0.05; +++ p < 0.001 significant differences in the HFDb-E compared to the HFD-S group. @ p < 0.05 significant differences in SD-E group with respect to SD-S. # p < 0.05 significant differences in the HFDb group between Day 1 compared to Day 2, 3 and 4.

Figure 6

Effects of continuous access to high-fat diet (HFD) on effective responses and oral ethanol EtOH self-administration in OF1 mice. Data are presented as mean (± SEM): (A) g/kg of 6% EtOH consumption (B) the number of effective responses during FR1 and FR3. The columns represent means and the vertical lines ± SEM of (C) breaking point values during PR and (D) amount of 6% EtOH consumption during the PR session * p < 0.05 significant differences in the groups that received BD (SD-E and HFD-E) compared to the saline groups (SD-S and HFDc-S). + p < 0.05 significant differences between SD-E with respect to SD-S. # p < 0.05 significant differences between the BD-treated groups and the saline-treated groups in Day 9. ## p < 0.01 significant differences between the HFDc groups (HFDc-S and HFDc-E) compared to the SD groups (SD-S and SD-E).

Figure 7

Schematic representation of experimental design. OF1 adolescent mice arrived in the laboratory at PND 21. At PND 26 all animals changed their feeding schedules according to the experimental condition (1) Standard diet (SD), (2) High-fat diet binge (HFDb) or (3) High-fat diet continuous access (HFDc). From PND 29 to 41 mice underwent the (1.25% ethanol) binge drinking procedure (BD) (SD-E, HFDb-E and HFDc-E) or were treated with saline (SD-S, HFDb-S, HFDc-S). After reaching adulthood (PND 61), mice started the self-administration (SA) procedure. Tissue sampling was performed at three different timepoints: after BD, three weeks after BD and after the SA procedure.