Breeze2

Mice were bled by cheek puncture on day-4 and day-10 of the dietary cycles #3 and #9 (see Supplementary Fig. 1a for timeline). All mice were fasted for 6 h (from 7:00 AM-1:00 PM) prior to blood collection. Fasting blood glucose was measured in fresh whole blood using a handheld instant glucose meter Bayer Breeze2 (Bayer, Mishawaka, IN). Additional blood was collected in 1.1 ml Z-Gel tubes (Sarstedt, Nümbrecht, Germany) and centrifuged 15 min at 4 °C at 18,500 × g to separate serum, which was stored at −80 °C. Serum insulin was measured using a mouse ultra-sensitive enzyme-linked immunosorbent assay (Catalog #90080; Crystal Chem, Downers Grove, IL). Circulating levels of 3-hydroxybutyrate (3-HB) and leptin were determined with commercially available kits according to the manufacturer’s instructions [3-HB: Catalog #700190; Cayman Chemicals, Ann Arbor, MI; leptin—Catalog #EZML—82 K; Millipore, Burlington, MA). Insulin resistance was calculated using the HOMA2 Calculator software available from the Oxford Centre for Diabetes, Endocrinology and Metabolism, Diabetes Trials Unit website (http://www.dtu.ox.ac.uk/). n = 7–17 per group.

During the 4th week of diet treatment, female mice were fasted for 3 h and blood glucose was recorded via tail nick using a standard glucose testing meter (Bayer Breeze2; Bayer HealthCare LLC, Tarrytown, NY, USA). Animals received a bolus of 2 g dextrose (Bimeda^®, Oakbrook Terrace, IL, USA) per kg body weight via oral gavage. Blood glucose readings were repeated from the same tail nick 15, 30, 60 and 120 min after glucose was administered. At 6 weeks and 3 months of age, offspring from all groups were fasted for 3 h and subjected to the same glucose tolerance testing protocol, except dextrose was delivered by intraperitoneal injection. At 8, 10 and 12 months of age, offspring were subjected to glucose tolerance testing with dextrose administered via oral gavage, but otherwise following the same procedure as described. At 15 months of age, offspring were fasted overnight (18 h) and underwent the intraperitoneal glucose tolerance testing procedure but were injected with 2.67 g dextrose/kg body weight. Varied fasting times, doses and alternate routes of delivery were used in order to attempt to elucidate any potential differences in glucose tolerance. Offspring glucose tolerance represents no more than one pup per litter per sex at each time point, though not necessarily all the same animals at every time point.