D12492 diet

Physiological fuel values were calculated from modified Atwater factors (3.5 kcal/g carbohydrate; 3.5 kcal/g protein; 8.5 kcal/g fat). The high fat diet was prepared from purified food-grade reagents according to a commercial formulation (D12492 diet, Research Diets Inc., New Brunswick, NJ, USA). It had a macronutrient weight content of 26.3% carbohydrate (19.2% kcal), 26.2% protein (19% kcal), and 34.9% fat (61.8% kcal) and a physiological fuel value of 4.80 kcal/g. Carbohydrate sources were maltodextrin and sucrose (64.5 and 35.5%, respectively), protein sources were casein and L-cystine (98.5 and 1.5%, respectively), while lipid sources were lard and soybean oil (90.7 and 9.3%, respectively). The diet also contained cellulose (64.6 g/kg), calcium carbonate (7.1 g/kg), dicalcium phosphate (16.8 g/kg), potassium citrate (21.3 g/kg), and choline bitartrate (2.6 g/kg), as well as mineral (12.9 g/kg) and vitamin (12.9 g/kg) mixes. The regular chow diet (Charles River Rodent Diet # 5075, Cargill Animal Nutrition, MN, USA) had a macronutrient weight content of 55.2% carbohydrate (65.6% kcal), 18% protein (21.4% kcal), and 4.5% fat (13% kcal) and a physiological fuel value of 2.89 kcal/g.

The high fat diet (HFD) was prepared from purified food-grade reagents according to a commercial formulation (D12492 diet, Research Diets Inc., New Brunswick, NJ, United States) as previously reported (Andrich et al., 2018 (link)). It had a physiological fuel value (calculated using a modified Atwater factor) of 20.09 kJ/g (4.80 kcal/g) and a macronutrient weight content of 26.3% carbohydrate (19.2% kJl), 26.2% protein (19% kJ) and 34.9% fat (61.8% kJ). The regular chow diet (Charles River Rodent Diet # 5075, Cargill Animal Nutrition, United States) had a physiological fuel value of 12.09 kJ/g (2.89 kcal/g) and a macronutrient weight content of 55.2% carbohydrate (65.6% kJ), 18% protein (21.4% kJ) and 4.5% fat (13% kJ).

Animal use and care were in accordance with institutional and National Institutes of Health guidelines. DIO C57BL/6 mice and lean littermates were purchased from Taconic (Taconic Farms Inc, Germantown, NY) for all studies. The study protocol was approved by the Brigham and Women’s Hospital (BWH) Institutional Animal Care and Use Committee (IACUC) animal protocol (animal protocol 2016N000371). Obesity was induced by feeding animals ad libitum with a high-fat diet (HFD) that provides 60% of total energy as fat (D12492 diet, Research Diets Inc, New Brunswick, NJ) starting at 6 weeks of age for a duration of 12 weeks. Wistar rats used for intracerebral administration of recombinant MCH were group-housed and fed D12492 for 12 weeks (60% k/cal diet). Rats subsequently underwent cranial surgery placing a resealable canula in the lateral cerebral ventricle. All animals were maintained in specific pathogen-free conditions at the BWH animal facility in accordance with federal, state, and institutional guidelines. Animals were maintained on 12 hr light, 12 hr dark cycle in facilities with an ambient temperature of 19–22°C and 40–60% humidity and were allowed free access to water and standard chow. Euthanasia was performed by cervical dislocation following anesthesia with isoflurane (Patterson Veterinary, Devens, MA).

The animal experiments and animal care were performed in compliance with and were approved by the Institutional Animal Care and Use Committees of the Massachusetts General Hospital, Boston, MA (PHS Assurance Number D16-00361). Diet-induced obese (DIO) male Sprague Dawley rats (Charles River Laboratories, Wilmington, MA, USA) were used for all studies. Obesity was induced by feeding the animals ad libitum with a high-fat diet (HFD), which provides 60% of total energy as fat, 20% as carbohydrate, and 20% as protein (D12492 diet, Research Diets Inc., New Brunswick, NJ, USA). At the time of surgery, DIO rats weighed 675 ± 25 g. Animals were individually housed and were maintained on 12-h light, 12-h dark cycle (lights on at 0700 h) in facilities with an ambient temperature of 19–22 °C and 40–60% humidity. For each experimental group and each time point, five animals were used.

Male C57BL/6J mice were purchased from Japan SLC (Shizuoka, Japan), housed in a conventional animal room at 24°C, and maintained under a 12 h light/dark cycle. Gpr84^−/− mice with a C57BL/6J background were generated using the CRISPR/Cas9 system. Mice were acclimated to the CLEA Rodent Diet (CE-2, CLEA Japan, Inc., Tokyo, Japan) for 1 week prior to treatment. Seven-week-old mice were placed on an MCT diet or modified D12492 diet (60% kcal fat, Research Diets Inc., New Brunswick, NJ, USA) for 5 weeks. These diets were formulated as either lard or MCT (C8:0 triglyceride, C10:0 triglyceride, or C12:0 triglyceride, Nisshin OilliO Group, Ltd., Tokyo, Japan), or based on the D12492 diet (Research Diets Inc.) supplemented with or without 5% decanoate (C10:0, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The composition of the diets is provided in Supplementary Tables 1, 2. Body weight was measured once per week for the duration of the experiment. All mice were sacrificed under deep isoflurane induced anesthesia. All experimental procedures involving mice were performed according to protocols approved by the Committee on the Ethics of Animal Experiments of the Kyoto University Animal Experimentation Committee (Lif-K21020) and the Tokyo University of Agriculture and Technology (permit number: 28–87). All efforts were made to minimize suffering.

Animals (n = 48) were fed a high-calorie diet (D12492 diet; Research Diets, Inc., USA) for 3 weeks followed by the injection of two lower doses of streptozotocin (STZ) (30 mg/kg intraperitoneally) which was administered at weekly intervals. [12 (link)]. Three days after the last injection, tail vein blood glucose was measured after fasting for 5 h using a blood glucose meter (Accu-Chek Performa; Roche Diagnostics, USA). Rats having blood glucose > 15 mmol/liter were considered diabetic and were used for our study [12 (link)]. Insulin resistance in diabetic animals was further confirmed through an insulin tolerance test [12 (link)]. The high-fat diet was continued throughout the experimental period in the diabetic group of animals. The blood glucose concentrations and body weight were monitored fortnightly.