Expression of key regulators of mitochondrial biogenesis in growth hormone receptor knockout (GHRKO) mice is enhanced but is not further improved by other potential life-extending interventions

Abstract

Mitochondrial biogenesis is essential for cell viability. Growth hormone receptor knockout (GHRKO), calorie restriction, and surgical visceral fat removal constitute experimental interventions to delay aging and increase life span. We examined the expression of known regulators of mitochondriogenesis: peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α), adenosine monophosphate (AMP)-activated protein kinase (AMPK), sirtuin-1 (SIRT-1) and sirtuin-3 (SIRT-3), endothelial nitric oxide synthase (eNOS), nuclear respiratory factor-1, mitochondrial transcription factor A (TFAM), and mitofusin-2 (MFN-2) in the skeletal muscles and hearts of control and calorie-restricted female GHRKO mice and in the kidneys of male GHRKOs after visceral fat removal or sham surgery. Expression of PGC-1α in skeletal muscles, AMPK, SIRT-1, SIRT-3, eNOS, and MFN-2 in the heart and PGC-1α, AMPK, SIRT-3, eNOS, and MFN-2 in kidneys was increased in GHRKO mice but was not affected by calorie restriction or visceral fat removal. GHRKO mice have increased expression of key regulators of mitochondriogenesis, which is not improved further by calorie restriction or visceral fat removal.

Figure 1.

PGC-1α messenger RNA (mRNA) expression in skeletal muscle (A), heart (B), or kidney (C) of normal (N) and growth hormone receptor/–binding protein knockout (GHRKO; KO) mice fed ad libitum (AL), subjected to 40% calorie restriction (CR; A and B—Experiment 1), sham operated (sham), or subjected to visceral fat removal (VFR; C—Experiment 2). The data from real-time polymerase chain reaction were normalized by the housekeeping gene β2-microglobulin (B2M) and expressed as the relative expression. Values are means ± SEM. a and b—values that do not share the same letter in the superscript are statistically significant (p < .05). *p = .034 vs N mice (the significance for genotype) and **p < .001 vs N mice (the significance for genotype).

Figure 2.

AMPK messenger RNA (mRNA) expression in skeletal muscle (A), heart (B), or kidney (C) of normal (N) and growth hormone receptor/–binding protein knockout (GHRKO; KO) mice fed ad libitum (AL), subjected to 40% calorie restriction (CR; A and B—Experiment 1), sham operated (sham), or subjected to visceral fat removal (VFR; C—Experiment 2). The data from real-time polymerase chain reaction were normalized by the housekeeping gene β2-microglobulin (B2M) and expressed as the relative expression. Values are means ± SEM. a and b—values that do not share the same letter in the superscript are statistically significant (p < .05). *p = .001 vs N mice (the significance for genotype) and **p = .011 vs N mice (the significance for genotype).

Figure 3.

SIRT-1 messenger RNA (mRNA) expression in skeletal muscle (A), heart (B), or kidney (C) of normal (N) and growth hormone receptor/–binding protein knockout (GHRKO; KO) mice fed ad libitum (AL), subjected to 40% calorie restriction (CR; A and B—Experiment 1), sham operated (sham), or subjected to visceral fat removal (VFR; C—Experiment 2). The data from real-time polymerase chain reaction were normalized by the housekeeping gene β2-microglobulin (B2M) and expressed as the relative expression. Values are means ± SEM. a—values that share the same letter in the superscript are not statistically significant. *p =.037 vs N mice (the significance for genotype).

Figure 4.

SIRT-3 messenger RNA (mRNA) expression in skeletal muscle (A), heart (B), or kidney (C) of normal (N) and growth hormone receptor/–binding protein knockout (GHRKO; KO) mice fed ad libitum (AL), subjected to 40% calorie restriction (CR; A and B—Experiment 1), sham operated (sham), or subjected to visceral fat removal (VFR; C—Experiment 2). The data from real-time polymerase chain reaction were normalized by the housekeeping gene β2-microglobulin (B2M) and expressed as the relative expression. Values are means ± SEM. a and b—values that do not share the same letter in the superscript are statistically significant (p < .05). *p = .011 vs N mice (the significance for genotype) and **p < .001 vs N mice (the significance for genotype).

Figure 5.

Endothelial nitric oxide synthase (eNOS) messenger RNA (mRNA) expression in skeletal muscle (A), heart (B), or kidney (C) of normal (N) and growth hormone receptor/–binding protein knockout (GHRKO; KO) mice fed ad libitum (AL), subjected to 40% calorie restriction (CR; A and B—Experiment 1), sham operated (sham), or subjected to visceral fat removal (VFR; C—Experiment 2). The data from real-time polymerase chain reaction were normalized by the housekeeping gene β2-microglobulin (B2M) and expressed as the relative expression. Values are means ± SEM. a and b—values that do not share the same letter in the superscript are statistically significant (p < .05). *p = .024 vs N mice (the significance for genotype) and **p < .001 vs N mice (the significance for genotype).

Figure 6.

Nuclear respiratory factor-1 (NRF-1) messenger RNA (mRNA) expression in skeletal muscle (A), heart (B), or kidney (C) of normal (N) and growth hormone receptor/–binding protein knockout (GHRKO; KO) mice fed ad libitum (AL), subjected to 40% calorie restriction (CR; A and B—Experiment 1), sham operated (sham), or subjected to visceral fat removal (VFR; C—Experiment 2). The data from real-time polymerase chain reaction were normalized by the housekeeping gene β2-microglobulin (B2M) and expressed as the relative expression. Values are means ± SEM. a and b—values that do not share the same letter in the superscript are statistically significant (p < .05). *p = .011 vs N mice (the significance for genotype).

Figure 7.

TFAM messenger RNA (mRNA) expression in skeletal muscle (A), heart (B), or kidney (C) of normal (N) and growth hormone receptor/–binding protein knockout (GHRKO; KO) mice fed ad libitum (AL), subjected to 40% calorie restriction (CR; A and B—Experiment 1), sham operated (sham), or subjected to visceral fat removal (VFR; C—Experiment 2). The data from real-time polymerase chain reaction were normalized by the housekeeping gene β2-microglobulin (B2M) and expressed as the relative expression. Values are means ± SEM. a and b—values that do not share the same letter in the superscript are statistically significant (p < .05). *p = .021 vs N mice (the significance for genotype) and **p = .012 vs N mice (the significance for genotype).

Figure 8.

MFN-2 messenger RNA (mRNA) expression in skeletal muscle (A), heart (B), or kidney (C) of normal (N) and growth hormone receptor/–binding protein knockout (GHRKO; KO) mice fed ad libitum (AL), subjected to 40% calorie restriction (CR; A and B—Experiment 1), sham operated (sham), or subjected to visceral fat removal (VFR; C—Experiment 2). The data from real-time polymerase chain reaction were normalized by the housekeeping gene β2-microglobulin (B2M) and expressed as the relative expression. Values are means ± SEM. a and b—values that do not share the same letter in the superscript are statistically significant (p < .05). *p = .028 vs N mice (the significance for genotype) and **p < .001 vs N mice (the significance for genotype).

Figure 9.

COXIV messenger RNA (mRNA) expression in skeletal muscle (A) and heart (B) of normal (N) and growth hormone receptor/–binding protein knockout (GHRKO; KO) mice fed ad libitum (AL) or subjected to 40% calorie restriction (CR; Experiment 1). The data from real-time polymerase chain reaction were normalized by the housekeeping gene β2-microglobulin (B2M) and expressed as the relative expression. Values are means ± SEM. a and b—values that do not share the same letter in the superscript are statistically significant (p < .05). *p = .048 vs AL mice (the significance for diet intervention) and **p < .001 vs N mice (the significance for genotype).

Figure 10.

COXIV protein level in skeletal muscle (A) and kidney (B) of normal (N) and growth hormone receptor/–binding protein knockout (GHRKO; KO) mice fed ad libitum (AL), subjected to 40% calorie restriction (CR; A—Experiment 1), sham operated (sham), or subjected to visceral fat removal (VFR; B—Experiment 2). The protein level was expressed as the arbitrary unit (AU) per square millimeter. Values are means ± SEM. a—values that share the same letter in the superscript are not statistically significant. *p = .019 vs AL mice (the significance for diet intervention).

Figure 11.

COX activity in skeletal muscle (A), heart (B), and kidney (C) of normal (N) and growth hormone receptor/–binding protein knockout (GHRKO) mice. The COX activity was expressed as the units (U) per gram of tissue. Values are means ± SEM. a and b—values that do not share the same letter in the superscript are statistically significant (p < .05). *p = .007.

Figure 12.

NAMPT protein level in skeletal muscle (A) and kidney (B) of normal (N) and growth hormone receptor/–binding protein knockout (GHRKO; KO) mice fed ad libitum (AL), subjected to 40% calorie restriction (CR; A—Experiment 1), sham operated (sham), or subjected to visceral fat removal (VFR; B—Experiment 2). The protein level was expressed as the arbitrary unit (AU) per square millimeter. Values are means ± SEM. a–c—values that do not share the same letter in the superscript are statistically significant (p < .05).