Isopro rmh 3000

H67D knock-in mice^{5 (link)} and their control wild-type mice were kindly provided by Dr. James Connor (Penn State University, PA, USA). H67D mutation (C199G) in mice is homologous to H63D mutation in humans and recapitulates H63D-related hemochromatosis^{3 (link), 5 (link)}. All mice used for these studies were on the mixed background of C57BL/6 and 129Sv/J strains^{5 (link)}. Wild-type and H67D mutant mice (8-week-old; male and female) were fed facility chow (Prolab Isopro RMH 3000, LabDiet; 96 mg manganese and 380 mg iron per kg diet) and given water ad libitum. For olfactory exposure to Mn, mice were intranasally-instilled (0.08 mL/kg) with manganese chloride (MnCl₂) dissolved in double distilled water daily for 3 days. Four doses of MnCl₂ were selected for the study: 0 (saline only), 0.2, 1 and 5 mg/kg body weight. For the dietary iron overload mice, weanling wild-type mice (3–4 weeks old) were fed iron overload diet (10,000 mg iron/kg, as carbonyl iron; TD.09077, Harlan Teklad, Madison, WI, USA) or control diet (50 mg iron/kg, TD.07800, Harlan Teklad) for 4 weeks^{32 (link)–34 (link)}, and intranasally instilled with saline or 1 mg MnCl₂/kg daily for 3 days. We selected a dose of 1 mg/kg because it was the minimum dose required to show the difference in Mn accumulation in the brain between wild-type and H67D mutant mice.

Group housed male and female C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME) were delivered at 6–8 weeks of age and maintained on a 12-h reverse dark/light cycle. After 5–7 days habituation to the animal facility, mice were single housed in polycarbonate (GM500, Tecniplast, Italy) Plexiglas cages, given an additional 5–7 days to adjust to the housing conditions, and assigned to the water control or two-bottle choice (2BC) alcohol access group. Subjects were matched for age and weight (N = 98 males: N = 73 females). Unless otherwise specified, mice were given ad libitum access to water and Isopro RMH 3000 chow (LabDiet, St. Louis, MO). Behavioral experiments were conducted during the dark cycle and with the room lights off, and males and females were run separately. The Institutional Animal Care and Use Committee at UNC Chapel Hill approved all experimental procedures, which were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

Mice received standard feed (Prolab Isopro RMH 3000; LabDiet, St. Louis, MO) which provides 60% of calories from carbohydrates, 14% from fat, and 26% from protein sources. After significant [¹⁸F]FDG uptake was noted in the native heart, mice were trialed on a low-carbohydrate diet (Dyets, Bethlehem, PA) to determine if dietary changes could induce a ketogenic state and decrease active glucose transport into the myocardial cells. The diet provided a standard caloric density with only 5% of calories from carbohydrate sources, 60% from fat and 35% from protein. Animals were placed on the special feed 5 days prior to [¹⁸F]FDG injection and returned to a standard diet following each scan.