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. 2009 Jun 18;4(6):e5963.
doi: 10.1371/journal.pone.0005963.

Disruption of protein kinase A in mice enhances healthy aging

Linda C Enns  1 , John F Morton, Piper R Treuting, Mary J Emond, Norman S Wolf, Dao-Fu Dai, G S McKnight, Peter S Rabinovitch, Warren C Ladiges
Affiliations

Disruption of protein kinase A in mice enhances healthy aging

Linda C Enns et al. PLoS One. .

Erratum in

  • PLoS One. 2010;5(2) doi: 10.1371/annotation/c7cad2dc-1eca-487e-89ae-151a22d8a0b4. Dai, Dao-Fu [added]

Abstract

Mutations that cause a reduction in protein kinase A (PKA) activity have been shown to extend lifespan in yeast. Loss of function of mammalian RIIbeta, a regulatory subunit of PKA expressed in brain and adipose tissue, results in mice that are lean and insulin sensitive. It was therefore hypothesized that RIIB null (RIIbeta(-/-)) mice would express anti-aging phenotypes. We conducted lifespan studies using 40 mutant and 40 wild type (WT) littermates of equal gender numbers and found that both the median and maximum lifespans were significantly increased in mutant males compared to WT littermates. The median lifespan was increased from 884 days to 1005 days (p = 0.006 as determined by the log rank test) and the 80% lifespan (defined here as 80% deaths) was increased from 941 days to 1073 days (p = 0.004 as determined by the Wang-Allison test). There was no difference in either median or 80% lifespan in female genotypes. WT mice of both genders became increasingly obese with age, while mutant mice maintained their lean phenotype into old age. Adiposity was found to correlate with lifespan for males only. 50% of male mice between 30 and 35 g, corresponding to about 5% body fat, for either genotype lived over 1000 days. No male mouse outside of this weight range achieved this lifespan. During their last month of life, WT mice began losing weight (a total of 8% and 15% of body weight was lost for males and females, respectively), but RIIbeta(-/-) male mice maintained their lean body mass to end of life. This attenuation of decline was not seen in female mutant mice. Old male mutant mice were insulin sensitive throughout their life. Both genders showed modestly lower blood glucose levels in old mutants compared to WT. Male mutants were also resistant to age-induced fatty liver. Pathological assessment of tissues from end of life male mutant mice showed a decrease in tumor incidence, decreased severity of renal lesions, and a trend towards a decrease in age-related cardiac pathology. These findings help establish the highly conserved nature of PKA and suggest that disruption of PKA affects physiological mechanisms known to be associated with healthy aging.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. A. Disruption of the RIIβ subunit of Protein Kinase A increases lifespan in male mice on the C57BL/6 background.
A. Both the median and maximum lifespans were significantly increased in mutant males compared to WT littermates. The median lifespan was increased from 884 days to 1005 days (p = 0.006 as determined by the log rank test) and the maximum lifespan (defined here as 80% deaths) was increased from 941 days to 1073 days (p = 0.004 as determined by the Wang-Allison test). B. There was no difference in either median or maximum lifespan in female genotypes.
Figure 2
Figure 2. RIIβ−/− mice are resistant to age-related obesity.
A,B. RIIβ−/− mice maintain lower body weights than WT littermates. Body weights for RIIβ−/− and WT littermates were measured weekly beginning at 19 to 24 months of age. Measurements were started with 15 mice per gender per genotype. Measurements taken in the last month of each individual mouse's life were not included. Each point represents a mean. Error bars represent standard deviations. N≤15 (sample size decreased with time). b = borderline significance; *P<0.05; **P<0.001; ***P<0.0001 (as determined by Student's t-test). C,D. Body weight and body fat are directly proportional. Body weight and percentage body fat were measured in both old (18 month old) males and females (8 WT and 9 RIIβ−/− males; 3 heterozygous RIIβ+/− and 2 RIIβ−/− females) and found to be directly proportional for both genders (R2 = 0.9774 and 0.6182, respectively). E. Old RIIβ−/− mice are resistant to age-induced increases in serum leptin. Serum leptin for young (2 month) and old (16 month) male, RIIB−/− and WT littermates (5 WT and 4 RIIβ−/−; and 8 WT and 9 RIIβ−/−, respectively). Data presents as means. Error bars represent standard deviations. **P<0.001.
Figure 3
Figure 3. Adiposity correlates with lifespan for males only.
A, C. When lifespan of WT mice was plotted against their maximal body weight, a correlation was found for males, but not females (N = 13 and 15, and R2 = 0.4795 and 0.0369, respectively). Males with the longest lifespans (over 1000 days) had body weights between 30 and 35 g. B. Most (82%) of the RIIβ−/− male mice examined had body weights between 30 and 35 g. 50% of these mice lived over 1000 days, and the 3 mice that fell outside of this weight range had shortened lifespans of under 700 days (Total N = 17). D. Of the 20 female RIIβ−/− mice measured, only 4 lived to 1000 days or higher. All 5 mice with maximal body weights below 24 g had shortened lifespans equal to or under 800 days, as did the one mouse above 35 g, but lifespans for mice falling between these two body weights were variable.
Figure 4
Figure 4. Old RIIβ−/− male mice have attenuated age-related fatty livers.
A. Old (18 month old) WT mice developed livers that were twice the size of, and paler in color than livers from young mice. B,C. Livers from 18 month-old RIIβ−/− littermates were smaller, darker in color, and 25% lower in weight than those from their WT littermates. N = 5 (WT) and 6 (RIIβ−/−). D. Liver weight correlated directly with % fat content (R2 = 0.7064) N = 4 (WT) and 6 (RIIβ−/−). For both C and D: Numbers represent means. Error bars represent standard deviations. P's determined by Student's t-test.
Figure 5
Figure 5. Both young and old RIIβ−/− male mice are insulin sensitive compared to WT, and RIIβ−/− males are resistant to age-induced hyperinsulinemia.
A. RIIβ−/− blood glucose levels were slightly lower than WT for both old males and females, but both genotypes of both genders fell into an acceptable range. N = 15. B. Male WT mice experienced over 4-fold increases in serum insulin levels with age, while RIIβ−/− littermates maintained low serum insulin levels between 2 and 16 months of age. 2 month-old mice: N = 5 (WT) and 4 (RIIβ−/−). 16 month-old mice: N = 8 (WT) and 9 (RIIβ−/−). C,D. Blood glucose levels of both 2 to 5 month (C) and 18 month (D) RIIβ−/− males dropped more rapidly and further in response to an inter-peritoneal insulin injection. N = 6; points represent means. Results were standardized by setting initial blood glucose levels at 100%. Data for all figures: Numbers represent means, and error bars represent standard deviations. **P<0.001; *P<0.05 and b represents borderline significance (as determined by Student's t-test).
Figure 6
Figure 6. Old RIIβ−/− male mice have attenuated cardiac dysfunction.
A. RIIβ−/− mice were significantly protected from age-related LV hypertrophy, shown by lower LV mass normalized to tibia length in 24 month old WT and RIIβ−/− mice. B. These RIIβ−/− mice also had superior Ea/Aa ratios and myocardial performance indices (MPI) compared to WT littermates. N = 6. Numbers represent means. Error bars represent standard deviations. P's determined by Student's t-test.
Figure 7
Figure 7. RIIB−/− male mice do not lose weight during aging.
Weights of individuals were compared between the first body weight measurements at 19 to 24 months of age and the final measurements taken during the last month of life. WT mice showed an average loss of several grams during senescence (A,B) while male RIIβ−/− littermates maintained their body weight (A). This phenomenon was not seen in RIIβ−/− females (B). Data presented as means. Error bars represent standard deviations. N = 15. *P<0.05 (as determined by Student's t-test).
Figure 8
Figure 8. Old RIIβ−/− male mice show attenuated renal dysfunction, tumor incidence, and a trend towards attenuated cardiac pathology.
A. Old RIIβ−/− male mice showed attenuated tubular degeneration and interstitial and pelvic inflammation compared to wild-type littermates although renal lesions in both genotypes was relatively mild. N = 6. Numbers represent means. Error bars represent standard deviations. *P<0.05 (determined by Student's t-test). B. Hematoxylin and eosin-stained section of kidneys from WT and RIIβ−/− male mice. The WT mouse had mild perivascular and interstitial lymphohistiocytic accumulations (severity score 2) and mild tubular changes including attenuated epithelium and intralumenal proteinaceous material (arrow). This mouse also had marked neutrophilic pyleitis that likely contributed to moribundity. In contrast, the RIIβ−/− mouse had minimal interstitial inflammation (severity score 1) surrounding a small caliper tangentially sectioned vessel (arrows). Original magnification for both images, 20X. C. Spleens from 100% of WT mice but only 40% of RIIβ−/− mice examined at end of life, presented with lesions. N = 6. D. Hearts from old (18 month) RIIβ−/− male mice tended towards better scores than WT for 4 of 6 types of lesions graded, although differences were not significant. IR = interstitial fibrosis, cytomeg = cytomegaly, inflamm = inflammation, arteriosc = arteriosclerosis. N = 5 (WT) and 6 (RIIβ−/−). Numbers represent means. Error bars represent standard deviations. Numbers above columns represent probability values, determined by Student's t-test.
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