Male Wistar rats (n = 55, 260 ± 7 g) were obtained from a commercial breeder (Harlan, UK). All procedures were in accordance with Home Office (UK) guidelines under The Animals (Scientific Procedures) Act, 1986 and with institutional guidelines. Control rats were fed for three weeks on a standard chow diet (Harlan Laboratories), with an Atwater Fuel Energy of 3.0 kcal/g, comprising 66% calories from carbohydrate, 22% from protein and 12% from fat (Additional file 1: Table S1). To induce diabetes, rats were fed a high-fat diet (Special Diet Services) for three weeks, with an Atwater Fuel Energy of 5.3 kcal/g, comprising 60% calories from fat, 35% from protein and 5% from carbohydrate, according to a modification of the protocols of Reed et al. and Srinivasan et al.[18 (link),19 (link)]. On day 13, rats were fasted overnight and given a single intraperitoneal injection of streptozotocin (STZ in citrate buffer, pH 4) the following morning, and the high-fat diet feeding was continued for a further week (or chow diet for controls). Different doses of STZ (0, 15, 20, 25 and 30 mg/kg bodyweight w/w) in combination with high-fat diet, were investigated to determine the optimal dose to induce a type 2 diabetic phenotype with modified cardiac metabolism. We started our study with a dose of 30 mg/kg, to closely replicate that used by others [19 (link)], then included additional groups on lower doses of STZ until hyperglycaemia was no longer induced, mortality was not observed with any dose of STZ. After three weeks on their designated diet, rats in the fed state were terminally anaesthetised with sodium pentobarbital, hearts and livers were rapidly excised, freeze clamped and stored at −80°C for subsequent analysis. Following excision of the heart, blood was collected from the chest cavity, plasma separated and analysed for metabolites using a Pentra analyser (ABX, UK) and an insulin ELISA (Mercodia, Sweden). Both left and right epididymal fat pads were excised, trimmed and weighed, for assessment of adiposity.
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