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Fasted Plasma Chemistry Metabolic Panel (week 15) [Mice were fed on High Fat Diet (21.4% crude fat content, Western RD, 829100, Special Diets Services) from 4 weeks of age]: Mice are fasted overnight (maximum 16 hours) then terminally anaesthetised with ketamine/xylazine, after which blood is collected into Lithium/Heparin coated tubes via the retro-orbital sinus. The resulting plasma is analysed for glucose, triglycerides, cholesterol, high density lipoproteins, low density lipoproteins, non-esterified free fatty acids (NEFAC) and glycerol on an Olympus AU400. For a subset of genes, fructosamine is analysed using the AU400 and plasma insulin concentration is measured by ELISA and/or Mesoscale Discovery (MSD) array technology.

Plasma Chemistry panel (week 14) (Mice were fed on Mouse Breeder Diet (5021, Labdiet) from weaning): Non-fasted mice are terminally anaesthetised with ketamine/xylazine and blood is collected into Lithium/Heparin coated tubes via the retro-orbital sinus. Plasma is analysed for the following parameters on an Olympus AU400:

Indirect Calorimetry (week 12, male mice prioritised) [Mice were fed on High Fat Diet (21.4% crude fat content, Western RD, 829100, Special Diets Services) from 4 weeks of age]: Respiratory metabolic function is assessed using the LabMaster system (TSE-systems). Mice are housed individually in calorimetry cages for a period of approximately 21 hours including a 5 hour light phase acclimatisation period. This is followed consecutively by a 12 hour dark (19:30 - 07:30) and minimum 3 hour light phase. Cumulative food intake, activity (beam breaks), volume of oxygen consumed and volume of carbon dioxide produced are all measured. From this data the respiratory exchange ratio and energy expenditure are derived. Water bottles are weighed before and after the experiment to assess water consumption.

ipGTT (week 13) [Mice were fed on High Fat Diet (21.4% crude fat content, Western RD, 829100, Special Diets Services) from 4 weeks of age]: Mice are fasted overnight (maximum of 16 hours) before the glucose tolerance test. A fasting blood sample is taken before a bolus of glucose is administered by intra-peritoneal injection. Blood samples are tested for glucose concentration (Accu-Check Aviva, Roche) at 15, 30, 60 and 120 minutes following the glucose administration, and data presented as plasma glucose concentration.

Core Temperature (week 14) [Mice were fed on High Fat Diet (21.4% crude fat content, Western RD, 829100, Special Diets Services) from 4 weeks of age]: Rectal core body temperature is measured using a TH-5 thermometer with a RET-3 probe (Viking Medical).