How stra(i)nge are your controls? A comparative analysis of metabolic phenotypes in commonly used C57BL/6 substrains

Abstract

In recent years, insufficiently characterised controls have been a contributing factor to irreproducibility in biomedical research including neuroscience and metabolism. There is now a growing awareness of phenotypic differences between the 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 substrains (N, J, JOla, JRcc) of both sexes, obtained from two commercial vendors, Charles River Laboratories (Crl) and Envigo (Env). Our results indicate systematic and tissue-specific differences between substrains, affected by both vendor and sex, in all parameters investigated, and not necessarily mediated by the presence of the NntC57BL/6J mutation. Not only were there differences between 6J and 6N as expected, all three 6J substrains 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.

Fig 1. Body weight and GTT profiles of C57 substrains of both sexes.

Body weight (A) of male and female animals belonging to five C57 substrains i.e., 6JCrl (black with shaded pattern), 6JOlaEnv (blue), 6JRccEnv (maroon), 6NCrl (green with shaded pattern), 6NEnv (purple). Blood glucose levels (mmol/L) measured every 15 minutes (left) and total glycaemic excursion measured as total area under curve (right) during the GTT for females (B) and male (C) C57 substrains. All data sets visualised as scatter plots with mean ± SD with significances for displayed on graph as: ** = p<0.01, * = p<0.05 (Tukey’s post-hoc test), p<0.09 in italics for marginal significances (see text for statistics). Key for substrains displayed at bottom of figure with Crl animals (J/N) having shaded pattern to distinguish from Env.

Fig 2. Basal fasted glucose and serum insulin levels in C57 substrains of both sexes.

Basal glucose and serum insulin levels after a 5hr fast in female (A, C respectively) and male (B, D respectively) animals belonging to five C57 substrains as described above. All data sets visualised as scatter plots (mean ± SD); significances: **** = p<0.0001, ** = p<0.01, * = p<0.05 (Tukey’s post-hoc test), p<0.09 in italics for marginal significances (see main text for statistics). Key to substrains: bottom of figure with Crl animals (J/N) having shaded pattern to distinguish from Env.

Fig 3. Hepatic insulin signalling markers in C57 substrains of both sexes.

Representative images of western blots probed with antibodies for the detection of phosphorylated (p-) or total (t-) AKT, rpS6 and GSK3β in liver of female (A) and male (D) C57 substrains with molecular weight in kDA. Fold increase in protein levels of p-, t-, p/t ratio of: AKT in female (B) and male (F); rpS6 in female (C) and male (G); GSK3β in female (D) and male (H) C57 substrains, relative to 6JCrl group within each sex displayed. All data sets visualised as scatter plots with mean ± SD; significances: **** = p<0.0001 vs all other groups, ** = p<0.01, * = p<0.05 (Tukey’s post-hoc test), p<0.09 in italics for marginal significances. Key:6JCrl (black), 6JOlaEnv (blue), 6JRccEnv (maroon), 6NCrl (green), 6NEnv (purple) with Crl animals having shaded pattern inside to distinguish from Env.

Fig 4. Correlation matrices between GTT glycemic excursion, serum insulin levels and insulin signalling markers in male 6J substrains.

Heat plots depict Z-score converted (overall mean) correlation matrices with positive (blue) and negative (red) correlations based on Pearson’s r correlational coefficient, for male 6JCrl (A) 6JOlaEnv (B), 6JRccEnv (C) animals. Upper and lower triangle represent mirror images with GTT indicating area under curve or total glycemic excursion during GTT, insulin indicating basal fasted serum insulin levels followed by hepatic insulin signalling markers as mentioned above. For further detail, see Results.