D12450ji

Six-week-old male C57BL/6NTac mice (Taconic Biosciences, Rensselaer, NY) were fed a high-fat diet (60% fat, derived from lard and soybean oil; D12492, Research Diets, New Brunswick, NJ) alone are hereafter referred to as the DIO mice or a standard diet (10% fat, derived from lard and soybean oil; D12450Ji, Research Diets) alone from here onwards are referred to as the lean mice until 18 and 29 weeks of age. Mice were then euthanized by CO₂ inhalation and were necropsied. Serum was collected for clinical chemistry and tissues were collected for histopathological examination. The liver was weighed at necropsy and the organ weights relative to body weights were calculated. For glucose tolerance test, DIO and lean mice at 19 weeks of age were injected intraperitoneally with D-glucose (Sigma-Aldrich, St. Louis, MO) at the dose of 2 g/kg after 16 h fasting, and euthanized by CO₂ inhalation at 0 (control), 30, 60, and 120 min. Serum was collected for analyses of glucose, insulin, and leptin as previously described^{23 (link)}. All experiments and methods in this study were performed in accordance with relevant guidelines and regulations and were approved by the Animal Care and Use Committee at National Institute of Environmental Health Science (code number: 2018-0002).

Animal studies were conducted according to the National Institutes of Health guide for the care and use of Laboratory animals. Maine Medical Center Research Institute approved all experimental procedures. Snca^fl/fl (B6(Cg)-Sncatm1.2Vlb/J strain^{24 (link)}, stock #028559) mice were crossed to Prrx1Cre mice (B6.Cg-Tg(Prrx1-cre)1Cjt/J^{13 (link)}, stock# 005584), both purchased from The Jackson Laboratory (Bar Harbor, ME). Prrx1Cre;Snca^fl/fl and littermate controls were sham operated or ovariectomized (OVX) at 8 weeks old and sacrificed at 20 weeks old. Independently, eight-week old female and male Prrx1Cre;Snca^fl/fl mice and littermate controls were randomly assigned treatment with high fat (60% fat) (D12492i, Research Diets, Inc.) or low fat (10% fat) diet (D12450Ji, Research Diets, Inc.) for 15 weeks.

Eight-week-old female C57BL/6J (Jackson stock number: 000664) mice were fed with either obesogenic high fat diet (HFD, D12492i – 60% kcal derived from fat) or low fat diet (LFD, D12450Ji- 10% kcal derived from fat, Research Diets Inc., New Brunswick, NJ) for seventeen weeks at Jackson labs (Figure S1A). Mice were shipped to the University of Tennessee Health Science Center at 25 weeks of age. Animal studies were performed with approval and in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) at the University of Tennessee Health Science Center and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All animals were housed in a temperature-controlled facility with a 12-h light/dark cycle and ad libitium access to food and water. Weights are provided in Figure S1. Mice were never singly housed. Mice were randomized to IgG2a or anti-PD-1 interventions.