Animal model

Our study was carried out by the Department of Pediatrics of the University Hospital of Cologne. The study was approved by the appropriate governmental authority (Institutional protocol number of the animal welfare application: AZ 8.87–50.10.37.09.292, Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen, Germany). All animal procedures were performed in accordance with the German Animal Welfare Law. Animal care and use was performed by qualified individuals, supervised by a veterinarian. The manuscript complies with the Animals in Research: Reporting In Vivo Experiments (ARRIVE) guidelines⁵².

Mice (C57BL/6N) were bred and held at the animal facility of the Department of Pharmacology of the University Hospital of Cologne (Cologne, Germany). Mice were housed in a room maintained at 22 ± 2 °C, exposed to humidity of 50–60% and a 12/12-h light/dark cycle. All breeding colonies were kept in individually ventilated cages (IVCs, Blue line cages type II long, Tecniplast, Italy). Three-week-old female mice were randomly assigned to a voluntary wheel running group (Maternal-INT, n = 9) or a control sedentary group (Maternal- CO_, n = 35) and were fed a regular standard laboratory chow diet (#R/M-H Ssniff, Germany; containing 412 g/kg carbohydrates, 190 g/kg protein, and 33 g/kg fat; total metabolizable energy 3220 kcal/kg, 9% of total metabolizable energy from fat) for 9–10 weeks during preconception, gestation and lactation period. While both groups (Maternal-CO and Maternal-INT) received the same standard diet, the running intervention group (Maternal-INT) had continuous access to a running wheel in their home cage during gestation (Fig. 1A). The running wheel was available from gestational day (G) 0 until G18, starting with mating. Running wheels were equipped with tachometers measuring distance (km), average speed (km/h) and time (h:m) as described before¹¹. Male breeders were chow fed and had limited access to the running wheel during the mating process for 48 h. Body weight of the dams was monitored daily from G0 to G18. Blood samples for serum analysis were collected via submandibular puncture at gestational day G16. In order to minimize stress-induced side effects, dams were not fasted. Postpartum the body weight of both offspring groups (CO vs. INT, named after maternal conditions) was monitored daily starting immediately after birth at postnatal day (P) 1 (P1) up to P21. On P3, litter size was randomly adjusted to six for each litter. A subset of offspring was sacrificed at P21 and non-fasted blood samples were collected via intracardial puncture for further analyses. The remaining animals of CO and INT offspring continued on a standard diet until P70 (Fig. 1A). From P71 to P112, one-half of the CO- and INT-offspring was subjected to an obesogenic high-fat diet (#C1057 modified Altromin, Germany; containing 269 g/kg carbohydrates, 208 g/kg protein, and 351 g/kg fat; total metabolizable energy 5,237, 60% of metabolizable energy from fat), while the other half continued on standard chow. Consequently, four groups with distinct condition resulted from P70 to P112, termed as follows: CO, CO-HFD, INT and INT-HFD (Fig. 1A). After six week of HFD feeding, at P112, all animals were sacrificed and processed as described before^11,14. All offspring were sacrificed via CO2-inhalation. Food intake measurement was performed in offspring at P70 when offspring were still on a standard diet and at P112 in offspring on a high fat diet (CO-HFD and INT-HFD, Supplementary Fig. ²F,G). From P21 to P112, animals were weighed weekly. For the duration of the whole study, all mice had free access to the experimental diets and water. To exclude gender influences, all studies were performed using male offspring only^6,8,50. 1–2 pups per litter were studied at each time point.