Attenuation of age-related metabolic dysfunction in mice with a targeted disruption of the Cbeta subunit of protein kinase A

Abstract

The cyclic adenosine monophosphate-dependent protein kinase A (PKA) pathway helps regulate both cell growth and division, and triglyceride storage and metabolism in response to nutrient status. Studies in yeast show that disruption of this pathway promotes longevity in a manner similar to caloric restriction. Because PKA is highly conserved, it can be studied in mammalian systems. This report describes the metabolic phenotype of mice lacking the PKA catalytic subunit Cbeta. We confirmed that Cbeta has high levels of expression in the brain but also showed moderate levels in liver. Cbeta-null animals had reduced basal PKA activity while appearing overtly normal when fed standard rodent chow. However, the absence of Cbeta protected mice from diet-induced obesity, steatosis, dyslipoproteinemia, and insulin resistance, without any differences in caloric intake or locomotor activity. These findings have relevant pharmacological implications because aging in mammals is characterized by metabolic decline associated with obesity, altered body fat distribution, and insulin resistance.

Figure 1.

Cα and Cβ protein levels in brain and liver. Immunoblots showed no detectable levels of Cβ in either total brain or in liver of Cβall^−/− mice. Cα levels were found to be unaltered by disruption of Cβ in these tissues. Each lane represents an individual mouse.

Figure 2.

Weight gain and adiposity of male and female wild-type and Cβall^−/− mice on regular and diabetogenic diets. (A and B) Body weights of males maintained on a regular (A) and diabetogenic (B) diet for 14–15 weeks. Mutants weighed about 10% less than wild-type littermates when maintained on a regular diet; this weight difference became more pronounced if mice were raised on a diabetogenic diet. (C and D) Body weights of female wild-type and Cβall^−/− mice fed either a regular (A) or a diabetogenic (B) diet for 14–15 weeks. There was no difference in body weight between genotypes if maintained on a regular diet. Mutants had a slower rate of weight gain than wild-type littermates if raised on a diabetogenic diet. (E) Quantitative nuclear magnetic resonance imaging body composition analysis. There was no difference in percent fat or percent lean mass between genotypes for male mice, regardless of diet. Female mutants showed increased slightly higher adiposity compared with wild types when on a regular diet but reduced adiposity when maintained on the diabetogenic diet. (F–I) Final fat pad percent body weights of male and female wild-type and Cβall^−/− mice on regular and diabetogenic diets. (F) No differences were seen in fat pad percent body weights between genotypes of males on a regular diet. (G) Fat distribution was different in mutant males raised on a diabetogenic diet compared with wild-type mice. Reproductive fat pads comprised a larger percentage of the body weight in mutants than wild-type mice, whereas retroperitoneal, mesenteric and brown adipose fat pads had lower percent body weights. (H) Borderline significant to no significant differences was seen in fat pad weights between genotypes of females on a regular diet. Inguinal fat pads had slightly higher percent body weights in the mutants. (I) All fat pads of female mutants on the diabetogenic diet were much smaller than those of their wild-type littermates. *p < .05, **p < .001, ***p < .0001, and b represents borderline significance. Error bars represent standard deviations. n = 7–10 mice per set.

Figure 3.

Cβall^−/− mutants are protected against fatty liver disease. (A) Wild-type (WT) mice fed the diabetogenic diet developed large livers, whereas livers from mutants remained the same size as those from mice on a regular diet. (B) Livers from WT mice fed a diabetogenic diet were large and pale in color compared with those of mutants. (C and D) Periodic acid/Schiff-stained sections of paraffin-embedded livers from male mice fed a diabetogenic diet. Livers from WT mice were full of fat-filled vacuoles, absent from livers of Cβall^−/− mice. (E) Correlation of percent fat content of livers from male mice maintained on a diabetogenic diet (measured with quantitative nuclear magnetic resonance imaging), with total liver weight. R² = .9762. *p < .05, **p < .001, ***p < .0001. Error bars represent standard deviations. n = 7–10 mice per set.

Figure 4.

Lipoproteins in male (left side) and female (right side) wild-type and Cβall^−/− mice on regular and diabetogenic diets. All mice on the diabetogenic diet experienced rises in serum HDL levels, but male mutants had significantly lower levels than wild types. Male wild-type mice, but not mutant mice, also showed increases in low-density lipoprotein (LDL) and very low–density lipoprotein (VLDL) when fed the diabetogenic diet. *p < .05, **p < .001, ***p < .0001. Error bars indicate standard deviations. n = 7–10 mice per set.

Figure 5.

Glucose homeostasis in male wild-type and Cβall^−/− mice on regular and diabetogenic diets. (A) Blood glucose levels of male mutants on the regular diet were frequently found to be lower than those of wild-type mice over the course of the study. Differences between genotypes were even greater when mice were fed the diabetogenic diet (B). (C) Serum insulin in both genotypes remained very low (barely detectable) over the course of the study when mice were fed the regular diet. (D) Serum insulin levels in wild-type mice fed the diabetogenic diet increased 10-fold by the end of the study but remained low in mutants. (E and F) Intraperitoneal glucose tolerance testings (IPGTTs) on mice fed regular (E) and diabetogenic (F) diets. There were no significant differences between genotypes of mice fed the regular diet in the ability to clear intraperitoneally injected glucose, but mutants were found to have better glucose disposal than wild types if fed the diabetogenic diet for 12 weeks. (G and H) Insulin resistance of mice fed regular (G) and diabetogenic (H) diets. Differences were only seen between genotypes if mice were fed the diabetogenic diet; mutants were found to be more sensitive to insulin than wild-type mice. *p < .05, **p < .001, ***p < .0001, and b represents borderline significance. Error bars represent standard deviations. n = 7–10 mice per set. For IPGTT and insulin resistance analyses, results were standardized by setting initial blood glucose levels at 100%.

Figure 6.

Glucose homeostasis in female wild-type and Cβall^−/− mice on regular and diabetogenic diets. (A) Blood glucose levels of female mutants on the regular diet were found to be similar to those of wild-type mice over the course of the study, but mutants were frequently found to have lower blood glucose levels when mice were fed the diabetogenic diet. (C and D) Insulin resistance of mice fed regular (C) and diabetogenic (D) diets. Mutant mice on both diets were found to be more insulin sensitive than wild-type mice when injected intraperitoneally with insulin. *p < .05, **p < .001, ***p < .0001, and b represents borderline significance. Error bars represent standard deviations. n = 7–10 mice per set. For IPGTT and insulin resistance analyses, results were standardized by setting initial blood glucose levels at 100%.