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Comparative Study
. 2022 Dec 22;17(12):e0271651.
doi: 10.1371/journal.pone.0271651. eCollection 2022.

Metabolic differences and differentially expressed genes between C57BL/6J and C57BL/6N mice substrains

Shino Nemoto  1 , Tetsuya Kubota  1   2   3   4   5 , Hiroshi Ohno  1   6   7
Affiliations
Comparative Study

Metabolic differences and differentially expressed genes between C57BL/6J and C57BL/6N mice substrains

Shino Nemoto et al. PLoS One. .

Abstract

C57BL/6J (B6J) and C57BL/6N (B6N) mice are the most frequently used substrains in C57BL/6 (B6) inbred mice, serving as physiological models for in vivo studies and as background strains to build transgenic mice. However, the differences in metabolic phenotypes between B6J and B6N mice are not coherent, and genotypic differences in metabolically important tissues have not been well studied. The phenotypic differences between B6J and B6N substrains have often been attributed to the role of the nicotinamide nucleotide transhydrogenase (Nnt) gene, whereby B6J has a spontaneous missense mutation of Nnt. Nevertheless, phenotypic differences between the two cannot be explained by Nnt mutations alone, especially in metabolic traits. Therefore, we aimed to investigate the genetic cause of the phenotypic differences between B6J and B6N mice. Determining consistent genetic differences across multiple tissues involved in metabolic traits such as subcutaneous and visceral white adipose tissues, brown adipose tissue, skeletal muscle, liver, hypothalamus, and hippocampus, may help explain phenotypic differences in metabolism between the two substrains. We report candidate genes along with comparative data on body weight, tissue weight, blood components involved in metabolism, and energy balance of B6J and B6N mice. Insulin degrading enzyme, adenylosuccinate synthase 2, and ectonucleotide triphosphate diphosphohydrolase 4 were highly expressed in B6J mice compared with those in B6N mice, and Nnt, WD repeat and FYVE domain containing 1, and dynein light chain Tctex-type 1 were less expressed in B6J mice compared with those in B6N mice in all seven tissues. Considering the extremely wide use of both substrains and their critical importance in generating transgenic and knock-out models, these findings guide future research across several interrelated fields.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Difference in body weight between B6J and B6N mice on ND or HF.
The body weight of male B6J and B6N mice on ND or HF was monitored up to 38 weeks of age (n = 5 per group). Data are presented as mean ± SEM. Asterisks (*) denote significant differences (* p<0.05, ** p<0.01) between B6J and B6N mice at each time point in the same food group. The lines and the equation were based on regression analysis (log-ratio transformed). ND, normal diet; HF, high-fat diet; open circles (blue), ND-fed B6J; open circles (red), ND-fed B6N; filled squares (blue), HF-fed B6J; filled squares (red), HF-fed B6N.
Fig 2
Fig 2. Tissue weights in B6J and B6N mice on ND or HF.
Relative tissue weights were calculated by organ weight/body weight (body mass percentage). (A) iWAT, inguinal white adipose tissue; (B) eWAT, epididymal white adipose tissue; (C) BAT, brown adipose tissue; (D) Muscle, skeletal muscle; and (E) Liver. Values are means + SEM (n = 5) for B6J group (blue color) and B6N group (red color). Two-way ANOVA indicated a significant interaction between substrain and diet for iWAT (p<0.01; F = 11.96; Df = 16) and eWAT (p <0.0001; F = 47.48; Df = 16). There was a significant effect of strain on muscle (p<0.001; F = 18.46; Df = 16) independent of diet, and significant effect of diet on muscle (p<0.001; F = 25.63; Df = 16) and on liver (p<0.0001; F = 27.80; Df = 16) independent of strain. Individual means were compared within groups using unpaired Student’s t-test. Asterisks (*) and daggers (†) indicate significant differences between B6J and B6N substrain groups, and ND and HF diet groups, respectively (** p<0.01 and **** p<0.0001, † p <0.05, †† p <0.01, ††† p <0.001, and †††† p<0.0001). J, B6J substrain; N, B6N substrain; ND, normal diet; HF, high-fat diet; open circles, ND-fed B6J; filled circles, ND-fed B6N; open squares, HF-fed B6J; filled squares, HF-fed B6N.
Fig 3
Fig 3. Differences in plasma leptin, adiponectin, and leptin to adiponectin ratio in B6J and B6N mice on ND or HF.
(A) Leptin, (B) Adiponectin, (C) Leptin/Adiponectin. Bars are representative of means + SEM (n = 5) for B6J group (blue color) and B6N groups (red color). Two-way ANOVA indicated a significant effect of strain on leptin (p <0.01; F = 8.78; Df = 16) and adiponectin (p<0.05; F = 5.97; Df = 16) independent of diet, and a significant effect of diet on leptin (p <0.01; F = 9.29; Df = 16) and on leptin/adiponectin (p <0.01; F = 11.40; Df = 16) independent of strain. Individual means were compared within groups using unpaired Student’s t-test. Asterisks (*) and daggers (†) indicate significant differences between B6J and B6N substrain groups and ND and HF diet groups, respectively (*** p <0.001, † p <0.05, and †† p<0.01). J, B6J substrain; N, B6N substrain; ND, normal diet, HF, high-fat diet; open circles, ND-fed B6J; filled circles, ND-fed B6N; open squares, HF-fed B6J; filled squares, HF-fed B6N.
Fig 4
Fig 4. Differences in metabolic parameters in the plasma of B6J and B6N mice on ND or HF.
(A) Insulin, (B) FFA, non-esterified fatty acids:, (C) T-Cho, total cholesterol;, (D) HDL, high-density lipoprotein cholesterol;, (E) ALT, alanine aminotransferase;, (F) AST, aspartate aminotransferase;, (G) LDH, lactate dehydrogenase;, (H) Glucose, and (I) TG, triglyceride. Bars are representative of means + SEM (n = 5), for B6J group (blue color) and B6N group (red color). Two-way ANOVA indicated a significant interaction between substrain and diet for TG (p<0.05; F = 5.89; Df = 15). There was a significant effect of strain on insulin (p<0.05; F = 6.87; Df = 16) and FFA (p<0.05; F = 4.67; Df = 15) independent of diet, and a significant effect of diet on T-Cho (p<0.0001; F = 27.50; Df = 16), HDL (p<0.001; F = 18.66; Df = 16), ALT (p<0.01; F = 11.83; Df = 16), AST (p <0.01; F = 10.74; Df = 16), LDH (p <0.001; F = 19.75; Df = 16), and TG (p<0.001; F = 22.85; Df = 15) independent of strain. Individual means were compared within groups using unpaired Student’s t-test. Asterisks (*) and daggers (†) indicate significant differences between B6J and B6N substrain groups and ND and HF diet groups, respectively (* p<0.05, † p<0.05, †† p<0.01, and ††† p<0.001). J, B6J substrain; N, B6N substrain; ND, normal diet, HF, high-fat diet; open circles, ND-fed B6J; filled circles, ND-fed B6N; open squares, HF-fed B6J; filled squares, HF-fed B6N.
Fig 5
Fig 5. Differences in metabolic assessments between B6J and B6N mice.
(A) Energy expenditure (B) Carbohydrate consumption (C) Fat consumption (D) Activity, and (E) Food intake. Values are means + SEM (n = 5, experimented in duplicates), and asterisks (*) indicate significant differences (* p<0.05, ** p<0.01, and *** p<0.001, unpaired Student’s t-test). Open circles, ND-fed B6J; filled circles, ND-fed B6N; open squares, HF-fed B6J; filled squares, HF-fed B6N.
Fig 6
Fig 6. Expression levels of DEGs between B6J and B6N on ND or HF.
The gene name is indicated at the top of each plot, and the y-axis represents the normalized signal values. (A) Ide, insulin degrading enzyme; (B) Adss2, adenylosuccinate synthase 2; (C) Entpd4, ectonucleotide triphosphate diphosphohydrolase 4; (D) Nnt, nicotinamide nucleotide transhydrogenase; (E) Wdfy1, WD repeat and FYVE domain containing 1; (F) Dynlt1, dynein light chain Tctex-type1. Boxes are representative of means ± SEM (n = 5) for the B6J (blue color) and B6N (red color) groups. Three-way ANOVA (Df = 110 for all analysis) indicated a significant interaction between substrain, diet, and tissue for Adss2 (p<0.05; F = 2.59) and Entpd4 (p<0.01; F = 3.24); a significant interaction between substrain and diet for Wdfy1 (p<0.001; F = 13.96); a significant interaction between diet and tissue for Ide (p<0.05; F = 2.71), Entpd4 (p<0.0001; F = 10.38), Nnt (p<0.001; F = 4.32), and Dynlt1 (p<0.0001; F = 9.51) and a significant interaction between strain and tissue for Adss2 (p<0.0001; F = 10.90), Entpd4 (p<0.0001; F = 5.49), Nnt (p<0.0001; F = 6.75), Wdfy1 (p<0.001; F = 4.34), and Dynlt1 (p<0.05; F = 2.24). There was a significant effect of strain on Ide, Adss2, Entpd4, Nnt, Wdfy1, and Dynlt1 (p<0.0001; F = 554, 453, 723, 289, 821, and 406, respectively) independent of diet and tissue, and a significant effect of diet on Ide (p<0.01; F = 10.40), Entpd4 (p<0.05; F = 6.56), Wdfy1 (p<0.01; F = 8.73), and Dynlt1 (p<0.0001; F = 17.78) independent of strain and tissue. There was a significant effect of tissue on Ide (p<0.05; F = 2.39), Adss2 (p<0.05; F = 2.53), Entpd4 (p<0.0001; F = 8.65), Nnt (p<0.05; F = 2.31), Wdfy1 (p<0.05; F = 2.54), and Dynlt1 (p<0.05; F = 2.21) independent of strain and diet. Daggers (†) indicate significant differences between ND and HF groups († p<0.05, †† p<0.01, and ††† p<0.001, Tukey’s post hoc test). ND, normal diet; HF, high-fat diet; open blue boxes, ND-fed B6J; open red boxes, ND-fed B6N; filled blue boxes, HF-fed B6J; filled red boxes, HF-fed B6N.
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This study was supported by grants from the JSPS KAKENHI Grant Number JP19K11734 (https://www.jsps.go.jp/j-grantsinaid/) to S.N. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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