Physiological and metabolic characteristics of novel double-mutant female mice with targeted disruption of both growth hormone-releasing hormone and growth hormone receptor

Abstract

Mice with disruptions of growth hormone-releasing hormone (GHRH) or growth hormone receptor (GHR) exhibit similar phenotypes of prolonged lifespan and delayed age-related diseases. However, these two models respond differently to calorie restriction indicating that they might carry different and/or independent mechanisms for improved longevity and healthspan. In order to elucidate these mechanisms, we generated GHRH and GHR double-knockout mice (D-KO). In the present study, we focused specifically on the characteristics of female D-KO mice. The D-KO mice have reduced body weight and enhanced insulin sensitivity compared to wild-type (WT) controls. Growth retardation in D-KO mice is accompanied by decreased GH expression in pituitary, decreased circulating IGF-1, increased high-molecular-weight (HMW) adiponectin, and leptin hormones compared to WT controls. Generalized linear model-based regression analysis, which controls for body weight differences between D-KO and WT groups, shows that D-KO mice have decreased lean mass, bone mineral density, and bone mineral content, but increased adiposity. Indirect calorimetry markers including oxygen consumption, carbon dioxide production, and energy expenditure were significantly lower in D-KO mice relative to the controls. In comparison with WT mice, the D-KO mice displayed reduced respiratory exchange ratio (RER) values only during the light cycle, suggesting a circadian-related metabolic shift toward fat utilization. Interestingly, to date survival data suggest extended lifespan in D-KO female mice.

Keywords: CRISPR; GHR; GHRH; indirect calorimetry; insulin sensitivity; metabolism.

FIGURE 1

GHR/GHBP knockout mice with CRISPR technology have altered body composition. The alignment of exon 4 (gray rectangle) reference sequence with electropherograms from wild‐type and mutant (28208) alleles (a). The translated sequence of single letter amino acids (maroon rectangle) shows the five amino acids inferred to be deleted in the mutant allele (orange rectangle) and position of a missense mutation at residue E44 (blue rectangle) in a young human patient. The CRISPR target site is indicated by the blue bar, with a red arrowhead representing the PAM (NGG). The red rectangle with dotted lines in the bottom electropherogram indicates the 15 bp deleted sequence. Body weights of WT and D‐KO mice from weaning to adulthood (b). Body composition parameters were measured by DXA. Body composition parameters were plotted on the y‐axis, and body weights are plotted on the x‐axis (c‐f). Relationship between body weight and BMD in WT and D‐KO mice (c). Relationship between body weight and BMC in WT and GHRH^‐/‐ mice (d). Relationship between body weight and lean mass in WT and D‐KO mice (e). Relationship between body weight and fat mass in WT and GHRH^‐/‐ mice (f). WT n = 12, D‐KO n = 11. The WT and D‐KO groups were statistically analyzed with ANCOVA method, which was used to calculate p values, shown on each panel

FIGURE 2

VO₂ recordings in D‐KO mice. VO₂ was measured by indirect calorimetry for 6 days in WT and D‐KO mice (a). 6 days of VO₂ data were averaged into a single day format (b). VO₂ recordings measured on light (c) and dark (d) cycles were averaged for individual animals. VO₂ is plotted on the y‐axis, and body weights are plotted on the x‐axis for light (e) and dark (f) cycles. WT n = 11, D‐KO n = 12. Each bar represents mean ±SEM. For panels c‐d, statistical analysis was performed with unpaired Student's t test with Welch's correction; ****p < 0.0001. For panels e and f, statistical significance was determined by ANCOVA

FIGURE 3

VCO₂ recordings in D‐KO mice. VCO₂ was measured by indirect calorimetry for 6 days in WT and D‐KO mice (a). 6 days of VCO₂ data were averaged into a single day format (b). VCO₂ recordings measured on light (c) and dark (d) cycles were averaged for individual animals. VCO₂ is plotted on the y‐axis, and body weights are plotted on the x‐axis for light (e) and dark (f) cycles. WT n = 11, D‐KO n = 12. Each bar represents mean ±SEM. For panels b‐d, statistical analysis was performed with unpaired Student's t test with Welch's correction; ****p < 0.0001. For panels e and f, statistical significance was determined by ANCOVA

FIGURE 4

Metabolic rate in D‐KO mice. Energy expenditure was calculated using respiratory parameters measure by indirect calorimetry for 6 days in WT and D‐KO mice (a). 6 days of energy expenditure data were averaged into a single day format (b). Energy expenditure values on light (c) and dark (d) cycles were averaged for individual animals. Energy expenditure values are plotted on the y‐axis, and body weights are plotted on the x‐axis for light (e) and dark (f) cycles. WT n = 11, D‐KO n = 12. Each bar represents mean ±SEM. For panels b‐d, statistical analysis was performed by unpaired Student's t test with Welch's correction; ****p < 0.0001. For panels e and f, statistical significance was determined by ANCOVA

FIGURE 5

Respiratory exchange ratio (VCO₂/VO₂) in D‐KO mice. RER values were calculated by dividing VCO₂ with VO₂. RER data, which were collected during a 6‐day period, were averaged into a single day format (a). Averaged RER values are shown as light (b) and dark (c) cycles for WT and D‐KO mice. Female WT n = 11, D‐KO n = 12. Each bar represents mean ±SEM. Statistical analysis was performed with unpaired Student's t test with Welch's correction; ns=not significant, a; *p < 0.05, b; **p < 0.01, c; ***p < 0.001, d; ****p < 0.0001

FIGURE 6

Insulin and glucose tolerance tests. D‐KO and WT mice were fasted overnight and injected with 1 g glucose per kg of body weight. Blood glucose levels of mice were measured during the following 2 hours (a). Area under the curve analysis is shown (c). D‐KO and WT mice were fasted for 4 hours and injected with 1 IU porcine insulin per kg of body weight. Blood glucose levels of mice were measured during the following 2 hours (b). Area under the curve analysis is shown (d). WT n = 13–14, D‐KO n = 10–11. Each bar represents mean ±SEM. Statistical analysis was performed with unpaired Student's t test with Welch's correction; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001