The growth hormone receptor gene-disrupted mouse fails to respond to an intermittent fasting diet

Abstract

The interaction of longevity-conferring genes with longevity-conferring diets is poorly understood. The growth hormone receptor gene-disrupted (GHR-KO) mouse is long lived; and this longevity is not responsive to 30% caloric restriction, in contrast to wild-type animals from the same strain. To determine whether this may have been limited to a particular level of dietary restriction, we subjected GHR-KO mice to a different dietary restriction regimen, an intermittent fasting diet. The intermittent fasting diet increased the survivorship and improved insulin sensitivity of normal males, but failed to affect either parameter in GHR-KO mice. From the results of two paradigms of dietary restriction, we postulate that GHR-KO mice would be resistant to any manner of dietary restriction; potentially due to their inability to further enhance insulin sensitivity. Insulin sensitivity may be a mechanism and/or a marker of the lifespan extending potential of an intervention.

Figure 1. Gender-specific Effects of Intermittent Fasting (IF) on Body Weight (BW) and Insulin Sensitivity

GHR-KO mice and their littermate controls (derived from 129/Ola founders, provided by J. J. Kopchick [Ohio University, Athens, OH], and outbred to Balb/c, C57Bl/6J and C3HJ strains) were bred in a closed colony, housed under standard conditions (12-hr. light/12-hr. dark cycling and 20–23°C), and fed Lab Diet Formula 5001 (23% protein, 4.5% fat, 6% fiber) (Nestle Purina, St. Louis, MO). All animals were fed AL for the first 8–10 weeks of age. Thereafter, the mice were either fed AL every day (AL group) or every other day (IF group). Mice were weighed in the morning after a feeding day, approximately 16–20 hrs. after the IF group had been fed. Animal protocols were approved by the Animal Care and Use Committee of Southern Illinois University. A. Body weight trajectory for male mice shows IF reduces BW for only littermate control mice. B. Body weight trajectory for female mice shows IF reduces BW solely for GHR-KO mice. Unpaired Student’s t-test (Microsoft Excel, Redmond, WA) was employed to compare food consumption data. Body weight-gain data was contrasted with Repeated-Measures Analysis of Variance (ANOVA) (SPSS, Chicago, IL). Cohorts of mice not allocated for the lifespan assay, but similarly subjected to IF for ten months, were tested for insulin tolerance (ITT). After AL access to food overnight, food was removed, 0.75 international units (I.U.)/kg body weight of porcine insulin (Sigma-Aldrich, St. Louis, MO) was injected intra-peritoneal (IP) and tail-blood glucose was determined with a glucometer (Lifescan; Johnson & Johnson, New Brunswick, NJ) at 0, 15, 30, and 60 min. C. Insulin Tolerance Test for male mice depicts amelioration of insulin resistance of the littermate control mouse by IF. D. ITT for females shows IF has no effect on insulin sensitivity for either phenotype. Repeated-Measures ANOVA was used for comparisons of data from the ITT (SPSS, Chicago, IL). Graphs were generated with Excel (Microsoft, Redmond, WA). All measures of central tendency are arithmetic means, and all depictions of variation (error bars) represent standard deviations.

Figure 1. Gender-specific Effects of Intermittent Fasting (IF) on Body Weight (BW) and Insulin Sensitivity

GHR-KO mice and their littermate controls (derived from 129/Ola founders, provided by J. J. Kopchick [Ohio University, Athens, OH], and outbred to Balb/c, C57Bl/6J and C3HJ strains) were bred in a closed colony, housed under standard conditions (12-hr. light/12-hr. dark cycling and 20–23°C), and fed Lab Diet Formula 5001 (23% protein, 4.5% fat, 6% fiber) (Nestle Purina, St. Louis, MO). All animals were fed AL for the first 8–10 weeks of age. Thereafter, the mice were either fed AL every day (AL group) or every other day (IF group). Mice were weighed in the morning after a feeding day, approximately 16–20 hrs. after the IF group had been fed. Animal protocols were approved by the Animal Care and Use Committee of Southern Illinois University. A. Body weight trajectory for male mice shows IF reduces BW for only littermate control mice. B. Body weight trajectory for female mice shows IF reduces BW solely for GHR-KO mice. Unpaired Student’s t-test (Microsoft Excel, Redmond, WA) was employed to compare food consumption data. Body weight-gain data was contrasted with Repeated-Measures Analysis of Variance (ANOVA) (SPSS, Chicago, IL). Cohorts of mice not allocated for the lifespan assay, but similarly subjected to IF for ten months, were tested for insulin tolerance (ITT). After AL access to food overnight, food was removed, 0.75 international units (I.U.)/kg body weight of porcine insulin (Sigma-Aldrich, St. Louis, MO) was injected intra-peritoneal (IP) and tail-blood glucose was determined with a glucometer (Lifescan; Johnson & Johnson, New Brunswick, NJ) at 0, 15, 30, and 60 min. C. Insulin Tolerance Test for male mice depicts amelioration of insulin resistance of the littermate control mouse by IF. D. ITT for females shows IF has no effect on insulin sensitivity for either phenotype. Repeated-Measures ANOVA was used for comparisons of data from the ITT (SPSS, Chicago, IL). Graphs were generated with Excel (Microsoft, Redmond, WA). All measures of central tendency are arithmetic means, and all depictions of variation (error bars) represent standard deviations.

Figure 1. Gender-specific Effects of Intermittent Fasting (IF) on Body Weight (BW) and Insulin Sensitivity

GHR-KO mice and their littermate controls (derived from 129/Ola founders, provided by J. J. Kopchick [Ohio University, Athens, OH], and outbred to Balb/c, C57Bl/6J and C3HJ strains) were bred in a closed colony, housed under standard conditions (12-hr. light/12-hr. dark cycling and 20–23°C), and fed Lab Diet Formula 5001 (23% protein, 4.5% fat, 6% fiber) (Nestle Purina, St. Louis, MO). All animals were fed AL for the first 8–10 weeks of age. Thereafter, the mice were either fed AL every day (AL group) or every other day (IF group). Mice were weighed in the morning after a feeding day, approximately 16–20 hrs. after the IF group had been fed. Animal protocols were approved by the Animal Care and Use Committee of Southern Illinois University. A. Body weight trajectory for male mice shows IF reduces BW for only littermate control mice. B. Body weight trajectory for female mice shows IF reduces BW solely for GHR-KO mice. Unpaired Student’s t-test (Microsoft Excel, Redmond, WA) was employed to compare food consumption data. Body weight-gain data was contrasted with Repeated-Measures Analysis of Variance (ANOVA) (SPSS, Chicago, IL). Cohorts of mice not allocated for the lifespan assay, but similarly subjected to IF for ten months, were tested for insulin tolerance (ITT). After AL access to food overnight, food was removed, 0.75 international units (I.U.)/kg body weight of porcine insulin (Sigma-Aldrich, St. Louis, MO) was injected intra-peritoneal (IP) and tail-blood glucose was determined with a glucometer (Lifescan; Johnson & Johnson, New Brunswick, NJ) at 0, 15, 30, and 60 min. C. Insulin Tolerance Test for male mice depicts amelioration of insulin resistance of the littermate control mouse by IF. D. ITT for females shows IF has no effect on insulin sensitivity for either phenotype. Repeated-Measures ANOVA was used for comparisons of data from the ITT (SPSS, Chicago, IL). Graphs were generated with Excel (Microsoft, Redmond, WA). All measures of central tendency are arithmetic means, and all depictions of variation (error bars) represent standard deviations.

Figure 1. Gender-specific Effects of Intermittent Fasting (IF) on Body Weight (BW) and Insulin Sensitivity

GHR-KO mice and their littermate controls (derived from 129/Ola founders, provided by J. J. Kopchick [Ohio University, Athens, OH], and outbred to Balb/c, C57Bl/6J and C3HJ strains) were bred in a closed colony, housed under standard conditions (12-hr. light/12-hr. dark cycling and 20–23°C), and fed Lab Diet Formula 5001 (23% protein, 4.5% fat, 6% fiber) (Nestle Purina, St. Louis, MO). All animals were fed AL for the first 8–10 weeks of age. Thereafter, the mice were either fed AL every day (AL group) or every other day (IF group). Mice were weighed in the morning after a feeding day, approximately 16–20 hrs. after the IF group had been fed. Animal protocols were approved by the Animal Care and Use Committee of Southern Illinois University. A. Body weight trajectory for male mice shows IF reduces BW for only littermate control mice. B. Body weight trajectory for female mice shows IF reduces BW solely for GHR-KO mice. Unpaired Student’s t-test (Microsoft Excel, Redmond, WA) was employed to compare food consumption data. Body weight-gain data was contrasted with Repeated-Measures Analysis of Variance (ANOVA) (SPSS, Chicago, IL). Cohorts of mice not allocated for the lifespan assay, but similarly subjected to IF for ten months, were tested for insulin tolerance (ITT). After AL access to food overnight, food was removed, 0.75 international units (I.U.)/kg body weight of porcine insulin (Sigma-Aldrich, St. Louis, MO) was injected intra-peritoneal (IP) and tail-blood glucose was determined with a glucometer (Lifescan; Johnson & Johnson, New Brunswick, NJ) at 0, 15, 30, and 60 min. C. Insulin Tolerance Test for male mice depicts amelioration of insulin resistance of the littermate control mouse by IF. D. ITT for females shows IF has no effect on insulin sensitivity for either phenotype. Repeated-Measures ANOVA was used for comparisons of data from the ITT (SPSS, Chicago, IL). Graphs were generated with Excel (Microsoft, Redmond, WA). All measures of central tendency are arithmetic means, and all depictions of variation (error bars) represent standard deviations.

Figure 2. Sexual Dimorphism of Effect of Intermittent Fasting (IF) on Survivorship

For the survivorship assay, all animals were fed AL for the first 8–10 weeks of age. Henceforth, the mice were either fed AL every day (AL group) or every other day (IF group). Mice found either moribund or with a neoplasm approximately 10% of their total body volume were euthanized, and the date of euthanasia was recorded as the date of death. A. IF confers longevity to male littermates. B. For females, IF has no effect on either phenotype. Log-rank (Mantel-Cox) test analysis was utilized to compare the overall survivorship data with GraphPad Prism 5.01 (GraphPad Software Inc., La Jolla, CA); and maximal lifespan, of the 90th percentile of the appropriate control population, was analyzed with quantile regression exploiting an exact unconditional (Score Statistic) test (http://www.stat.ncsu.edu/exact/); for the latter, a binomial model was used, the hypothesis was two-sided, 99.9000% confidence interval assigned, and the test statistic employed was Fisher's Exact-Boschloo Test Statistic. Graphs were generated with Excel (Microsoft, Redmond, WA). All measures of central tendency are arithmetic means, and all depictions of variation (error bars) represent standard deviations. Mice physically lost from the study, euthanized due to senescence-independent matters, or included in a supplemental cohort in the survivorship assay account for the discrepancies in the sample sizes (within subgroup) between the body weight data and the survivorship data.

Figure 2. Sexual Dimorphism of Effect of Intermittent Fasting (IF) on Survivorship

For the survivorship assay, all animals were fed AL for the first 8–10 weeks of age. Henceforth, the mice were either fed AL every day (AL group) or every other day (IF group). Mice found either moribund or with a neoplasm approximately 10% of their total body volume were euthanized, and the date of euthanasia was recorded as the date of death. A. IF confers longevity to male littermates. B. For females, IF has no effect on either phenotype. Log-rank (Mantel-Cox) test analysis was utilized to compare the overall survivorship data with GraphPad Prism 5.01 (GraphPad Software Inc., La Jolla, CA); and maximal lifespan, of the 90th percentile of the appropriate control population, was analyzed with quantile regression exploiting an exact unconditional (Score Statistic) test (http://www.stat.ncsu.edu/exact/); for the latter, a binomial model was used, the hypothesis was two-sided, 99.9000% confidence interval assigned, and the test statistic employed was Fisher's Exact-Boschloo Test Statistic. Graphs were generated with Excel (Microsoft, Redmond, WA). All measures of central tendency are arithmetic means, and all depictions of variation (error bars) represent standard deviations. Mice physically lost from the study, euthanized due to senescence-independent matters, or included in a supplemental cohort in the survivorship assay account for the discrepancies in the sample sizes (within subgroup) between the body weight data and the survivorship data.