Intrinsic Activity of C57BL/6 Substrains Associates with High-Fat Diet-Induced Mechanical Sensitivity in Mice

Abstract

Pain is significantly impacted by the increasing epidemic of obesity and the metabolic syndrome. Our understanding of how these features impact pain is only beginning to be developed. Herein, we have investigated how small genetic differences among C57BL/6 mice from 2 different commercial vendors lead to important differences in the development of high-fat diet-induced mechanical sensitivity. Two substrains of C57BL/6 mice from Jackson Laboratories (Bar Harbor, ME; C57BL/6J and C57BL/6NIH), as well as C57BL/6 from Charles Rivers Laboratories (Wilmington, MA; C57BL/6CR) were placed on high-fat diets and analyzed for changes in metabolic features influenced by high-fat diet and obesity, as well as measures of pain-related behaviors. All 3 substrains responded to the high-fat diet; however, C57BL/6CR mice had the highest weights, fat mass, and impaired glucose tolerance of the 3 substrains. In addition, the C57BL/6CR mice were the only strain to develop significant mechanical sensitivity over the course of 8 weeks. Importantly, the C57BL/6J mice were protected from mechanical sensitivity, which may be based on increased physical activity compared with the other 2 substrains. These findings suggest that activity may play a powerful role in protecting metabolic changes associated with a high-fat diet and that these may also be protective in pain-associated changes as a result of a high-fat diet. These findings also emphasize the importance of selection and transparency in choosing C57BL/6 substrains in pain-related research. PERSPECTIVE: Obesity and the metabolic syndrome play an important role in pain. This study identifies key differences in the response to a high-fat diet among substrains of C57BL/6 mice and differences in intrinsic physical activity that may influence pain sensitivity. The results emphasize physical activity as a powerful modulator of obesity-related pain sensitivity.

Keywords: C57BL/6; Pain; diet; insulin resistance; metabolism; obesity.

Figure 1. All C57BL/6 Mice Display Increased Body Weight, Fat Mass, and Glucose When Fed a High-Fat Diet

A) All high fat-fed groups had increased bodyweight relative to control-fed groups beginning at 3 weeks (n=10 for all groups). B) Beginning after 1 week on a high fat diet all groups displayed an increase in fat mass. C) All high fat groups display slightly elevated blood glucose beginning 1 week after the start of a high fat diet. D) After 7 weeks of a high fat diet all groups display an increase in body weight, with C57BL/6J animals displaying the smallest increase in weight. E) Following 7 weeks of a high fat diet C57BL/6CR animals have the greatest increase in fat mass. F) After 7 weeks of a high fat diet all groups display mildly elevated blood glucose. All data presented as mean ± SEM * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001 CF: Control-fed HF: High fat-fed

Figure 2. C57BL/6CR and C57BL/6NJ Mice Develop Glucose Intolerance When Fed a High Fat Diet

A) High fat-fed C57BL/6CR animals display significantly reduced glucose tolerance following 6 weeks on a high fat diet (n=6 for all groups). B) High fat-fed C57BL/6J mice showed no change in glucose tolerance following 6 weeks of a high fat diet (n=6 for all groups). C) High fat-fed C57BL/6NJ mice display slightly reduced glucose tolerance 6 weeks on a high fat diet (n=6 on all groups). D) Fasting insulin was increased in all groups on a high fat diet, with C57BL/6CR mice having the highest levels of hyperinsulinemia (n=10 for all groups). All data presented as mean ± SEM * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001 CF: Control-fed HF: High fat-fed

Figure 3. C57BL/6 Sub-strains Have Different Intrinsic Activity and Energy Expenditure on a High Fat Diet

A) Control-fed and high fat-fed C57BL/6J mice display significantly greater activity during dark cycle than C57BL/6CR or C57BL/6NJ mice (n=8 for all groups). B) No sub-strain or diet group displayed an alteration in energy intake during active dark cycle time. C) All high fat-fed substrains displayed a reduced respiratory quotient associated with increased fat fuel utilization during their active dark cycle. D) Control-fed C57BL/6CR showed reduced energy expenditure relative to high fat-fed C57BL/6CR and control-fed C57BL/6J and C57BL/6NJ animals. All data presented as mean ± SEM * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001 CF: Control-fed HF: High fat-fed

Figure 4. High Fat-Fed C57BL/6CR mice Develop Mechanical Allodynia

A) Following 7 weeks of a high fat diet C57BL/6CR mice have increased mechanical sensitivity relative to control-fed mice (n=10 for all groups). B) C57BL/6J mice had no changes in mechanical sensitivity after 7 weeks of a high fat diet. C) High fat-fed C57BL/6NJ mice did not develop significant alterations in mechanical sensitivity after 7 weeks. D) No diet group or substrain showed a significant change in thermal sensitivity across 7 weeks when fed a high fat diet. All data presented as mean ± SEM * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001 CF: Control-fed HF: High fat-fed

Figure 5. C57BL/6CR Animals Display Heighted Chemogenic Sensitivity When Fed a High Fat Diet

A) High fat-fed C57BL/6CR mice were the only group to display increased nociceptive behaviors phase 1 as a response to formalin injection. Control-fed C57BL/6J mice also showed an increased response in phase 1 as compared to C57BL/6CR mice (n=15 for all groups). B) No sub-strain or diet group displayed changes in phase 2 responses following formalin injection. C) Control-fed vs. high fat C57BL/6CR mice D) Control-fed vs. high fat-fed C57BL/6J mice E) Control-fed vs. high fat-fed C57BL/6NJ mice. All data presented as mean ± SEM * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001 CF: Control-fed HF: High fat-fed

Figure 6. Epidermal Fiber Density Was Not Altered by a High Fat Diet

A) Sections of the plantar surface of the hind paw stained with PGP9.5 to identify sensory axons innervating the epidermis B) No substrain displayed differences in their IENFD when fed a high fat diet. On the control diet, C57BL/6NJ mice did have an increased IENFD relative to C57BL/6CR and C57BL/6J mice (n=8 for all groups) C) IENF levels in C57BL/6CR mice following 7 weeks of diet D) IENF levels in C57BL/6J mice following 7 weeks of diet E) IENF levels in C57BL/6NJ mice following 7 weeks of diet. All data presented as mean ± SEM * p<0.05 CF: Control-fed HF: High fat-fed