2.2. Animals, Lieber-DeCarli liquid diets, and Experimental protocol

Type 2 diabetic male db/db homozygous mice (8 to 9 weeks of age; BKS.Cg-Dock7^m +/+ Lepr^db/J; no. 000642) and age-matched nondiabetic lean db/m heterozygous mice (Dock7^m Lepr^db) were purchased from Jackson Laboratory (Bar Harbor, ME). Unlike db/m mouse, the db/db mouse that is deficient in leptin receptor exhibits the characteristic features of T2D including hyperglycemia, hyperinsulinemia, insulin resistance, and obesity [28, 29]. Upon arrival, db/db and db/m mice were maintained in a humidity- and temperature-controlled animal room with a 12:12-h dark-light cycle. The mice had free access to water and food (regular chow diet) ad libitum. All animal experiments were performed in accordance with the Charlie Norwood Veterans Affairs Medical Center Institutional Animal Care and Use Committee guidelines and were approved by the committee.

In the present study, db/db and db/m mice were started on the liquid diets 7 days after arrival at the animal facility. Lieber-DeCarli`82, Shake and Pour, control liquid diet (Product No: F1259SP) and Lieber-DeCarli`82, Shake and Pour, ethanol liquid diet (Product No: F1258SP) were purchased from Bio-Serv (Frenchtown, NJ). Control and ethanol liquid diets were prepared fresh daily according to manufacturer’s instructions. Ethanol liquid diet (EtOH LD) consisted of 36% ethanol-derived calories, 36% fat-derived calories, 13% carbohydrate-derived calories, and 15% protein-derived calories. In control liquid diet (control LD), EtOH- derived calories were substituted by equivalent calories from carbohydrates, and the calories derived from all other components were found to be similar to EtOH LD.

Recent studies have shown that in normal chow diet-fed lean mice, short-term [18] or long-term [30] EtOH feeding alone does not result in significant development of steatosis and/or accumulation of triglycerides in the liver. In a different study, short-term EtOH feeding alone on a normal chow diet for 4 days does not induce hepatic steatosis in lean control mice and also does not appear to exaggerate macrovesicular steatosis in ob/ob mice (leptin gene-deficient obese phenotype) [31]. As noted in the Introduction, short-term EtOH-high fat diet feeding in mice has recently been used as a model of binge drinking [16–18]. For instance, EtOH feeding (6% v/v) in concomitance with high fat diet for 2 days has been shown to induce steatosis and inflammatory genes in mouse liver [16, 17]. In the present study, we employed a short-term EtOH-high fat diet feeding protocol for up to 9 days to determine its effects on systemic βOHB levels and the associated changes in glycemic control in db/db versus db/m mice. Due to the glucose-lowering effects of EtOH (as shown in Fig. 4B) and the potential hypoglycemic risk, we employed the 9-day feeding protocol in the present study.

Effects of ethanol on the systemic concentrations of βOHB and glucose in type 2 diabetic db/db mice versus nondiabetic db/m mice. βOHB (A) and glucose (B) concentrations. ^# p < 0.05 compared with db/db on control LD and * p < 0.05 compared with db/m on control LD; n = 7 mice/group on days 1, 3, 7 and 9; n = 4 mice/group on day 5.

As illustrated in Fig. 1, db/db mice and db/m mice were acclimatized with 1:1 mix of ethanol liquid diet and control liquid diet for 2 days (n = 7 mice/group), respectively. Subsequently, the respective mice were maintained on 5% (w/v) Lieber-DeCarli EtOH LD or an isocaloric control LD for 9 days. Body weights of mice were measured every other day from day 1 through day 9. Caloric intakes of liquid diets were measured following the same pattern using the equation; 1 mL = 1 kcal, as shown in Fig. 1.

Schematic of the experimental protocol to study the effects of EtOH in type 2 diabetic db/db mice versus nondiabetic db/m mice. Mice were initially acclimatized with liquid diets according to the study group, as described. After acclimatization, mice were fed Lieber-DeCarli ethanol liquid diet (EtOH LD, 5% w/v) or control liquid diet (control LD) for 9 days. βOHB, β-hydroxybutyrate; hmgcs2, 3-hydroxy-3-methylglutaryl-CoA synthase 2; IPGTT, intraperitoneal glucose tolerance test; GS, glycogen synthase; GSK-3β, glycogen synthase kinase-3β; H&E, haematoxylin and eosin; ORO, Oil Red O; TG, triglyceride; FA, fatty acid.