How stra(i)nge are your controls? A comparative analysis of metabolic phenotypes in commonly used C57 substrains

In recent years, the use of insufficiently characterised control subjects has been a contributing factor to increasing irreproducibility in different areas of biomedical research including neuroscience and metabolism. There is now a growing awareness of phenotypic differences between the metabolic profiles of C57BL/6 substrains which are commonly used as control animals. We here investigated baseline metabolic characteristics such as glucose regulation, fasted serum insulin levels and hepatic insulin signalling in five different C57BL/6 sub-strains (N, J, JOla, JRcc) of both sexes, obtained from two commercial vendors Charles River Laboratories (Crl) and Envigo (Env). Our results indicated systematic and tissue-specific differences between substrains, modulated by both vendor and sex in all parameters investigated, not necessarily mediated by the presence of the Nnt mutation. Not only were there differences between 6J and 6N as expected, all three 6J sub-strains exhibited different profiles, even from the same breeder. Two distinct metabolic profiles were identified, one in which low insulin levels resulted in impaired glucose clearance (6JCrl; both sexes) and the other, where sustained elevations in fasted basal insulin levels led to glucose intolerance (male 6JRccEnv). Further, 6JRccEnv displayed sex differences in both glucose clearance and hepatic insulin signalling markers. In comparison, the two 6N substrains of either sex, irrespective of vendor, did not exhibit considerable differences, with 6NCrl animals presenting a good choice as a healthy baseline control for many types of experiments. Overall, our data emphasise the importance of selecting and characterising control subjects regarding background, sex, and supplier to ensure proper experimental outcomes in biomedical research.