*2.1. Animals*

All animal experiments were approved by the University of Alabama at Birmingham (IACUC-20467, approved on 06/16/2016) and Georgetown University (animal project #2017-0059, approved on 10/27/2017), and followed the Association for Research in Vision and Ophthalmology Statement for the Use of Animals. Six to eight-week-old C57BL/6J mice were fed either a low-fat diet (LFD) (10%kcal fat, 70%kcal carbohydrate, 20% protein), a Western diet (40% kcal fat, 43% kcal carbohydrate, 17%kcal protein), or a HFD (60% kcal fat, 20% kcal carbohydrate, 20% kcal protein) for up to 12 months. Diets were purchased from Research Diets, Inc, New Brunswick, NJ, USA. Full details of the composition of each diet is given in Supplementary Table S1.

### *2.2. Body Composition, Glucose Tolerance, and Insulin Sensitivity Testing*

The fat mass, lean mass, and water content of the animals were measured by magnetic resonance imaging using EchoMRI (Echo Medical Systems, LLC, Houston, TX, USA). For glucose and insulin tolerance tests, mice were fasted for 5-6 h, injected intraperitoneally with D-glucose at 1.5 g/kg of lean mass and tail bled for glucose and insulin measurements. Blood glucose and insulin levels were

measured 0, 15, 30, 45, 60, and 120 min after glucose administration. The insulin sensitivity index (ISI) was estimated using the Matsuda–Defronzo method [31].
