Decreased Levels of Proapoptotic Factors and Increased Key Regulators of Mitochondrial Biogenesis Constitute New Potential Beneficial Features of Long-lived Growth Hormone Receptor Gene–Disrupted Mice

Abstract

Decreased somatotrophic signaling is among the most important mechanisms associated with extended longevity. Mice homozygous for the targeted disruption of the growth hormone (GH) receptor gene (GH receptor knockout; GHRKO) are obese and dwarf, are characterized by a reduced weight and body size, undetectable levels of GH receptor, high concentration of serum GH, and greatly reduced plasma levels of insulin and insulin-like growth factor-I, and are remarkably long lived. Recent results suggest new features of GHRKO mice that may positively affect longevity—decreased levels of proapoptotic factors and increased levels of key regulators of mitochondrial biogenesis. The alterations in levels of the proapoptotic factors and key regulators of mitochondrial biogenesis were not further improved by two other potential life-extending interventions—calorie restriction and visceral fat removal. This may attribute the primary role to GH resistance in the regulation of apoptosis and mitochondrial biogenesis in GHRKO mice in terms of increased life span.

DECREASES in insulin/insulin-like growth factor-I (IGF-I)-like signaling result in extended longevity in flies (1,2) and worms (3,4), and similarly, decreases in progrowth signaling result in life-span extension in yeast (reviewed by Fontana and coworkers (5)) (6). Furthermore, disruption of the growth hormone (GH)–IGF-I signaling can extend longevity in mice (7). One of the genetic interventions that leads to prolonged life span in mice is targeted disruption of the GH receptor/GH-binding protein gene (Ghr/bp gene) (8). Mice homozygous for this mutation are known as GH receptor/GH-binding protein knockout (GH receptor knockout [GHRKO]; Ghr/bp −/−) mice or “Laron mice” (8). These GH-resistant/insensitive dwarf mice live longer than their wild-type siblings and are characterized by reduced weight in spite of an obese phenotype, reduced body size, undetectable level of GH receptor, high level of serum GH, greatly reduced plasma levels of insulin and IGF-I, improved insulin sensitivity, reduced oxidative damage, and improved oxidative stress resistance (8–18). Furthermore, GHRKO mice have lower incidence and delayed onset of fatal neoplastic diseases (19). These mutant mice are also characterized by reduced inflammatory cytokines and beneficial changes in apolipoprotein levels (20). Moreover, GHRKO mice have decreased thyroid follicle size compared with normal mice (21), which may explain mild thyroid hypofunction observed in these mice. Comprehensive characteristics of these mice have been recently reviewed by List and coworkers (22). Importantly, as noticed previously, GHRKO mice constitute a mammalian model for human Laron syndrome (8). Therefore, the results of the studies on GHRKO mice may broaden our knowledge about that endocrine disorder.

Calorie restriction (CR) and surgical visceral fat removal (VFR) are other (beside GHRKO) potential life-extending interventions. The former has been shown to delay aging and increase life span (23), and the latter has been reported to improve insulin signaling in normal mice and rats and extend longevity in rats (24–26).

Apoptosis is a process in which cells play an active role in their own death (“cell suicide”) and it is the most common form of programmed eukaryotic cell death. Thus, apoptosis seems to be a crucial process which may contribute to the life-span regulation.

Biogenesis of mitochondria is another process which is indispensable for proper cell viability (27) and could be assumed to be essential for longevity regulation. Generally, it is the process by which new mitochondria are formed. Defects in mitochondrial biogenesis lead to the development of various age-related diseases (28).

Therefore, the role of apoptosis-related factors and key regulators of mitochondrial biogenesis in life-span regulation in long-lived GHRKO mice seems to be very relevant and interesting. For example, downregulation of myocyte apoptosis is linked with preservation of muscle integrity and thus myonuclear apoptosis may become an appropriate target for therapeutic interventions against sarcopenia (29).

This review focuses on changes in the examined factors in three types of tissues in dwarf GHRKO mice, particularly affected by the process of aging, namely skeletal muscles, hearts, and kidneys. Additionally, it reviews the possible role of other potential life-extending interventions, namely CR and VFR in the life-span regulation in mice with GH resistance.

Apoptosis

Apoptosis, or programmed cell death, is a normal component of the development and health of multicellular organisms, playing an essential role in many physiological processes, including the embryonic growth of tissues and organs (30). Disruption of this process can lead to numerous pathological conditions, such as neurodegenerative disorders, cancer, and autoimmune diseases (reviewed by Favaloro and coworkers (31)).

Two central apoptotic signaling pathways are known: intrinsic (mitochondrial) and extrinsic. The intrinsic pathway (including p53, bax, cytochrome c, and caspase-9) is initiated by various factors within the cell, such as free radicals, ionizing radiation, DNA damage, hypoxia, or chemotherapeutic drugs (32–34). The extrinsic (death receptor) pathway (including caspase-8) is responsible, among others, for elimination of unnecessary cells during development and education of immune system (33). Previous studies have shown decreased leukocyte caspase-3 gene expression as well as increased antiapoptotic bcl-2 expression in Ames dwarf mice (35). In contrast, Kennedy and coworkers (36) have shown increased apoptosis in liver of these dwarf mice. Therefore, the entire role of apoptosis in (patho)physiology and in life-span regulation in these long-lived animals still remains not fully clear and requires further analysis.

Caspases

Caspase-3.—

Caspase-3 is the main effector (executioner) caspase, responsible for inducing apoptosis and destroying cells. It has been previously shown that absence of caspase-3 protected against denervation-induced skeletal muscle atrophy in mice (37). Moreover, Russell and coworkers (38) demonstrated that high glucose caused an increased activity of caspase-3 in mice, resulting in muscle atrophy. Recently published studies have shown decreased levels of caspase-3 gene expression and protein in skeletal muscles of long-lived GHRKO mice compared with normal mice (39) (Tables 1 and 2). Therefore, these observations seem to be consistent, by analogy, with normal or low glucose level observed in dwarf GHRKO mice.

Table 1.

Effects of GHRKO Genetic Intervention on the Expression of the Apoptosis-Related Genes in Skeletal Muscles, Hearts, and Kidneys (39,47 )

	Skeletal Muscle	Heart	Kidney
Caspase-3	↓	—	↓
Caspase-9	↓	—	↓
bax	↓	—	↓
Smac/DIABLO	↓	—	—
Caspase-8	—	—	—
bcl-2	—	—	—
p53	—	—	—
cyc1	—	↑	↑

Note: “—” denotes that the level did not change.

Table 2.

Effects of GHRKO Genetic Intervention on the Level of the Apoptosis-Related Proteins in Skeletal Muscles and Kidneys (39,46)

	Skeletal Muscle	Kidney
Caspase-3	↓	↓
Caspase-9	↓	—
bax	↓	↓
Smac/DIABLO	↓	↓
Caspase-8	—	↓
bcl-2	↑	↑
bad	—	↓
pbad	↑	↑
Apaf-1	↓	↓
pp53	—	↓
cyc	—	—

Note: “—” denotes that the level did not change.

Renal caspase-3 expression level was increased in ischemic injury (40,41) and in mice with polycystic kidney disease (42). Moreover, insulin-resistant type 2 diabetic db/db mice have increased renal caspase-3 activity (43). Also, high glucose in vitro increased caspase-3 activity in MK4 embryonic metanephric mesenchymal cell line (44). Interestingly, the double mutants produced by crossing mice with caspase-3 gene deletion (casp3 −/−) and mice harboring the congenital polycystic kidney (cpk) mutation lived longer than control cpk animals (45). Gesing and coworkers (46,47) have recently demonstrated decreased mRNA and protein levels of caspase-3 in the kidneys of highly insulin-sensitive GHRKO mice (Tables 1 and 2). Therefore, one could hypothesize that decreased renal as well as skeletal muscle caspase-3 gene and protein levels may be considered as potentially beneficial for GHRKO mice in terms of extended longevity of these animals.

Caspase-9.—

It is known that caspase-9 activates the effector caspase-3. Mishra and coworkers (48) demonstrated that high glucose increased caspase-9 activity in human mesangial cells. Moreover, reduced caspase-9 mRNA level in the kidney of GHRKO compared with normal mice (47) (Table 1) and decreased caspase-9 gene expression and protein levels in skeletal muscles of GHRKO mice have been shown (39) (Tables 1 and 2). Thus, one could speculate that decreases in the levels of the examined caspase may benefit these mutants in the context of their increased life span.

Caspase-8.—

Caspase-8 is an initiator caspase, indispensable for induction of the extrinsic apoptotic signaling pathway mediated through death receptors (49). Caspase-8 inhibitors prevented cardiomyocyte death, cardiac dilation, and contractile dysfunction in murine model (50). On the other hand, Ghosh and coworkers (43) showed increased renal caspase-8 activity in diabetic db/db mice. In contrast, in GHRKO mice, which are characterized by improved insulin sensitivity and low insulin, decreased caspase-8 protein level in kidneys in comparison with normal animals has been reported (46) (Table 2). This decrease may be considered as potentially beneficial for GHRKO mice, in the context of their extended longevity.

Other Apoptosis-Related Factors

Bax.—

Bax (bcl-2-associated X protein) is one of proapoptotic bcl-2 family proteins (51). Upregulation of bax expression by aging has been previously demonstrated (52). A cardiac tolerance of bax knockout (−/−) mice to ischemia/reperfusion injury was improved compared with heterozygous (bax +/−) and wild-type (bax +/+) animals (53). Interestingly, β-catenin/Wnt signaling pathway may promote survival of renal epithelial cells after metabolic stress and this beneficial effect seems to be associated with an inhibition of bax expression (54). Similarly, Zhou and coworkers (55) have shown that β-catenin activation by Wnt1 in vitro protected tubular epithelial cells from apoptosis and decreased bax expression as well. In contrast, an increase of bax expression was observed in renal cortex of diabetic db/db mice (56) and also in kidneys of congenital polycystic kidney disease mice (42). Therefore, the results of the study in which the authors (39,46,47) have demonstrated that GH resistance leads, in highly insulin-sensitive GHRKO mice with low insulin level, to decreased bax gene expressions and protein levels in the kidney as well as in skeletal muscles (Tables 1 and 2), may be regarded as beneficial for GHRKO mice in terms of extended longevity of these animals.

Smac/DIABLO.—

Smac/DIABLO (second mitochondria-derived activator of caspase/direct inhibitor of apoptosis protein [IAP] binding protein with low pI) is a mitochondrial protein that, after being released from mitochondria into the cytosol, potentiates apoptosis, possibly by neutralizing the IAP family members and disrupting their ability to inactivate the caspase enzymes (57,58). Taking it into account, it was interesting to assess Smac/DIABLO gene and protein levels in GHRKO mice—animals with greatly prolonged longevity. It has been shown that gene expression in skeletal muscles and protein level of this factor in skeletal muscles and kidneys in GHRKO mice is decreased compared with normal mice (39,46) (Tables 1 and 2). One could speculate that these alterations may be beneficial for extended life span of GHRKO knockouts.

Bad and phospho-bad (Ser112).—

Bad is one of proapoptotic proteins. Phosphorylation at Ser112 (phospho-bad; p-bad) results in the binding of bad to 14-3-3 proteins, and through inhibition of bad binding to antiapoptotic bcl-2 (59) it may prevent caspase cascade activation. The results of previous published studies showed decreased renal bad protein as well as increased p-bad protein levels in the kidney and skeletal muscles of GHRKO mice compared with normal ones (39,46) (Table 2). With regard to the roles of bad and p-bad playing in living organisms, one could speculate that the previously mentioned alterations in the respective examined proteins may benefit GHRKO animals in the context of increased life span of these dwarf mice.

Apaf-1 and cytochrome c.—

The disrupted ratio between pro- and antiapoptotic proteins results in cytochrome c release from mitochondria into the cytosol. The released cytochrome c forms a multiprotein complex with procaspase-9 and Apaf-1 (apoptotic protease-activating factor-1), called the apoptosome (60–63). The formation of apoptosome leads subsequently to activation of caspase-9 and caspase cascade and finally to activation of caspase-3. Therefore, in order to broaden the knowledge about apoptosis regulation, it is of interest to assess Apaf-1 levels in GHR-disrupted mice. As was reported previously, levels of various proapoptotic factors are reduced in these mice. Also in that case, it turned out that the protein levels of the examined factor in skeletal muscle and kidney of GHRKO are decreased compared with normal mice (39,46) (Table 2). These alterations seem to be beneficial for GHRKO mice and may contribute to the extended longevity of these animals. Another important component of the apoptosome is cytochrome c. Previously, Miyazawa and coworkers (64) showed increased level of cytochrome c in kidney of 24-month-old C57BL/6J mice compared with 2-month-old C57BL/6J mice. Intriguingly, Gesing and coworkers (39,47) have found an increased cytochrome c1 (cyc1) gene expressions in the kidneys and hearts of young long-lived GHRKO mutants (Table 1). That result seems to be unexpected on the basis of the previously mentioned observations. However, it is worth mentioning that cytochrome c release may occur without loss of membrane potential, which normally precipitates opening of the permeability transition pores (65). Furthermore, cytochrome c is also involved in the electron transport chain. Thus, the entire interpretation of the data on cytochrome c expression is complicated by its multiple roles in physiological processes.

p53 and phospho (pp53) (Ser15).—

p53 protein is a tumor suppressor as well as an important factor which activates the intrinsic apoptotic pathway. p53, apart from its well-known role as a “guardian of the genome,” is said to be a mediator of senescence and a contributor to aging (66,67). Shizukuda and coworkers (68) demonstrated that targeted disruption of p53 attenuated doxorubicin-induced cardiac toxicity in mice. Interestingly, p53 inhibitors may be a potentially therapeutic treatment for patients after myocardial infarction (69). Nakamura and coworkers (70) have shown that cardiac expression of p53 is increased in diabetic mice, whereas in p53-deficient mice the cardiac abnormalities (including damaged myocytes due to reactive oxygen species) were prevented. Moreover, cisplatin-induced nephrotoxicity was abrogated in p53-deficient mice (71). Also, deacetylation of p53 by SIRT1 may lead to attenuation of cisplatin-induced kidney injury (72). On the other hand, p53 promoted renal injury in acute aristolochic acid-induced nephropathy (73). DNA damage induces phosphorylation of p53 at Ser15, leading to reduced interaction of p53 with its negative regulator—oncoprotein MDM2 (74).

On the basis of the previously mentioned observations, it was of interest to assess p53 level in GHRKO mice which are characterized by delayed aging. Intriguingly, no changes in p53 gene expressions in skeletal muscles, hearts and kidneys between GHRKO and normal mice were demonstrated (39,47) (Table 1). However, what seems to be more important, the same authors demonstrated a decrease of renal phospho-p53 (p-p53) protein level in GHRKO dwarfs (46) (Table 2). As was noticed previously, p-p53 level may reflect DNA damage. Therefore, one could hypothesize that decreased level of p-p53 protein in long-lived GHRKO mice may be considered as another beneficial effect for GHRKO dwarfs in the context of extended longevity of these animals.

Bcl-2.—

Bcl-2 is one of the main antiapoptotic bcl-2 family proteins (51), preventing permeabilization of the outer mitochondrial membrane by inhibiting the action of the proapoptotic proteins Bax and/or Bak (75). Mice deficient for bcl-2 gene have congenital renal hypoplasia, develop polycystic kidney disease and renal failure (76,77). Moreover, Ortiz and coworkers (56) demonstrated that high glucose decreased the expression of the bcl-2 in vitro and the expression of this gene in the renal cortex of diabetic db/db mice. Therefore, the assessment of bcl-2 in insulin-sensitive GHRKO mice seemed to be very interesting. It has been demonstrated that bcl-2 protein levels in skeletal muscles and kidneys of GHRKO mice are increased compared with normal mice (39,46) (Table 2). Therefore, with regard to known antiapoptotic action of bcl-2, one could speculate that an increase of bcl-2 protein levels may be potentially beneficial for GHRKO mice and their increased life span.

Mitochondrial Biogenesis

The biogenesis of mitochondria, the process by which new mitochondria are formed, is essential for proper cell viability (27). Mitochondria are complex essential eukaryotic organelles which play a crucial role in energy homeostasis and metabolism. They generate adenosine triphosphate (ATP), the cellular energy carrier, and contain essential enzymes indispensable for various biochemical processes, including lipid, cholesterol, steroid, heme, and nucleotide synthesis. Mitochondria are also important regulators of apoptosis (via cytochrome c that constitutes one of the elements of apoptosome). Disruption of mitochondrial biogenesis leads to impaired oxidative stress resistance, maintenance of energy production, and metabolism regulation and thus may result in the development of various degenerative and metabolic diseases that are often age associated (eg, type 2 diabetes) (reviewed by Joseph and coworkers) (78). On the other hand, increased mitochondrial biogenesis may prevent aging (79). Numerous key regulators of mitochondrial biogenesis are known, including peroxisome proliferator-activated receptor γ (PPARγ) coactivator 1α (PGC-1α), adenosine monophosphate (AMP)-activated protein kinase (AMPK), sirtuins, endothelial nitric oxide synthase (eNOS), nuclear respiratory factors (NRFs), mitochondrial transcription factor A (TFAM), and mitofusin-2 (MFN-2). Importantly, as far, the mitochondrial biogenesis has not been yet analyzed in long-lived GHRKO mice.

PGC-1α

PPARγ coactivator 1α (PGC-1α) is the key regulator (“master regulator”) of mitochondrial biogenesis. PGC-1α plays an essential role in the activation and control of mitochondrial biogenesis through, among others, coordination of actions of multiple transcription factors (80). It has been reported that decreased PGC-1α level may lead to lipid accumulation in skeletal muscles and finally to insulin resistance, obesity, and diabetes (81). Moreover, PGC-1α–null mice demonstrated reduced mitochondrial function and reduced thermogenic capacity (82). Interestingly, Boström and coworkers (83) have recently reported that PGC-1α is involved in expression of irisin—newly described myokine that contributes to brown fat-like development of white adipose tissue. Taking into account these important observations, it was of interest to assess PGC-1α gene expressions and protein levels in GHRKO mice which are characterized, as noticed earlier, by delayed aging and extended longevity. Increased PGC-1α gene expression in skeletal muscles and kidneys as well as increased renal level of PGC-1α protein in GHRKO mice has been reported (84,85) (Tables 3 and 4). Therefore, the above-mentioned data obtained in GHRKO mutants may be regarded as potentially beneficial for extended longevity in these animals. Furthermore, one should emphasize that a role of telomere-p53–PGC-1α mitochondrial/metabolic axis has been recently recognized as an important factor which may contribute to the aging regulation (eg, in the heart) (86). The role of PGC-1 coactivators during aging in heart and skeletal muscles has been recently reviewed (79).

Table 3.

Effects of GHRKO Genetic Intervention on the Gene Expression of Key Regulators of Mitochondrial Biogenesis in Skeletal Muscles, Hearts, and Kidneys (84)

	Skeletal Muscle	Heart	Kidney
PGC-1α	↑	—	↑
AMPK	—	↑	↑
SIRT-1	—	↑	—
SIRT-3	—	↑	↑
eNOS	—	↑	↑
NRF-1	↓	—	—
TFAM	↓	—	↓
MFN-2	—	↑	↑

Note: “—” denotes that the level did not change. AMPK = adenosine monophosphate-activated protein kinase; SIRT = sirtuin; eNOS = endothelial nitric oxide synthase; NRF = nuclear respiratory factor; PGC-1 = PPAR coactivator 1; TFAM = mitochondrial transcription factor A; MFN = mitofusin.

Table 4.

Effects of GHRKO Genetic Intervention on the Protein Level of Key Regulators of Mitochondrial Biogenesis in Skeletal Muscles and Kidneys (85)

	Skeletal Muscle	Kidney
PGC-1α	—	↑
AMPKα	—	↑
Phospho-AMPKα	—	↑
SIRT-3	—	↑
eNOS	—	↑
Phospho-eNOS	—	↑
NRF-1	—	—
MFN-2	↑	↑

Note: “—” denotes that the level did not change. AMPK = adenosine monophosphate-activated protein kinase; PGC-1 = PPAR coactivator 1; SIRT = sirtuin; eNOS = endothelial nitric oxide synthase; NRF = nuclear respiratory factor; MFN = mitofusin.

AMPK and Phospho-AMPKα (Thr172)

AMPK, playing a role of a cellular energy sensor, is activated by an increase in intracellular AMP:ATP ratio (87). The phosphorylation of a threonine residue (Thr172) within the kinase domain of the α-catalytic subunit is required for AMPK activation. Activated AMPK phosphorylates PGC-1α and increases PGC-1α activity (88). Moreover, AMPK may play an important role in the regulation of white adipose tissue metabolism (reviewed by Ceddia (89)). Importantly, activation of AMPK in the heart seems to be necessary for cardiomyocyte protection and survival during ischemia (90,91). An increase of AMPK expression may also protect cardiomyocytes from hypertrophy induced by angiotensin II (92). AMPK activation also protects cardiac function and structure in diabetic mice (93). On the other hand, cardiac AMPK-deficient mice showed severe left ventricular contractile dysfunction with increased apoptosis and necrosis (94). Moreover, deficiency in AMPK may exacerbate obesity-induced hypertrophy and contractile dysfunction in the heart (95). Therefore, it was of interest to examine a gene expressions and protein levels of that factor in long-lived mice with the targeted disruption of the GH receptor/GH-binding protein gene. Interestingly, AMPK mRNA level was increased in hearts and kidneys of long-lived GHRKO mice (84) (Table 3). Moreover, these mutants were characterized by increased renal levels of AMPK and phospho-AMPKα proteins (85) (Table 4). It may be consistent with the results of the study performed by Lieberthal and coworkers (96), showing beneficial role of AMPK in protecting kidney proximal tubular cells subjected to metabolic stress. Moreover, AMPK may contribute to slowing renal cystogenesis (97). On the basis of the earlier noticed observations, one could hypothesize that the alterations in the examined factors may contribute to the extended life span in GHRKO knockouts.

SIRT1

Sirtuin-1 (SIRT1) belongs to sirtuin (NAD⁺-dependent deacetylases) family, evolutionarily conserved enzymes found in all eukaryotic organisms. The sirtuins are involved in numerous biological processes, including DNA repair and the maintenance of chromosome stability. The role of sirtuins as potentially novel pharmacological targets for improving healthspan has been recently reviewed (98). SIRT1 playing the key role among sirtuins stimulates mitochondrial biogenesis via PGC-1α deacetylation. Due to activation in states of nutrient deprivation, including fasting and calorie restriction (99), SIRT1 is sometimes referred to as a nutrient deprivation sensor. Moreover, SIRT1 plays a role in sustaining normal immune function and in delaying the onset of autoimmune disease (100,101). Interestingly, transgenic mice with cardiac-specific overexpression of SIRT1 demonstrated delayed aging and protection against oxidative stress in the heart (102) and against myocardial ischemia/reperfusion (103). Vinciguerra and coworkers (104) have recently reported that IGF-I propeptide (mIGF-I) may protect the heart from oxidative stress via SIRT1/JNK activity. In contrast, deletion of SIRT1 results in embryonic lethality, causes chromosome abnormalities, and leads to impaired DNA damage repair (105). However, unexpectedly, high SIRT1 may cause dilated cardiomyopathy in mice (106). For all these reasons, it was of interest to examine SIRT1 level in long-lived GHRKO mice which are characterized by delayed aging. Particularly, the result of SIRT1 level assessment in heart would seem to be very important, taking into account the data from the studies performed by Hsu and coworkers (102,103). It turned out that a cardiac SIRT1 mRNA level was increased in these animals compared with normal mice (84) (Table 3). Therefore, in the context of the previously mentioned observations, this alteration may benefit these long-lived mutants and seems to be important in terms of extended longevity of GHRKO dwarfs.

SIRT3

Sirtuin-3 (SIRT3) is another member of the sirtuin family. SIRT3 may regulate mitochondrial metabolism, adaptive thermogenesis, energy homeostasis, and apoptosis (107,108) and additionally may play a relevant role in metabolic syndrome development (109). SIRT3 may also protect mouse heart by blocking the cardiac response to some hypertrophic stimuli (110). The beneficial role of SIRT3 in heart has been discussed by Liu and coworkers (111). Taking into account these observations, it was of interest to assess SIRT3 gene expression and protein level in GHRKO mice—experimental model of long-lived animals. We have shown increased SIRT3 mRNA levels in the hearts of GHRKO mice compared with normal animals (84) (Table 3). It can be assumed to be beneficial for these mutants and increased life span of these animals. Interestingly, high levels of SIRT3 expression were detected in various metabolically active tissues, including kidney, brown fat, liver, and brain (112). These observations seem to be consistent with the results of the studies demonstrating the increase of renal SIRT3 gene expression and protein level in GHRKO mice (84,85) (Tables 3 and 4). It appears reasonable to assume that also this alteration may benefit GHRKO mutants concerning the extended longevity of these mice.

SIRT6

Sirtuin-6 (SIRT6) constitutes one more member of the sirtuin family and plays a relevant role in the maintenance of genomic stability. It has been shown that SIRT6 knockout mice are characterized by impaired base excision repair and exhibit a premature aging phenotype (113). Moreover, telomere dysfunction was observed in SIRT6-deficient cells (114). Interestingly, SIRT6 may stimulate double-strand break repair in mammalian cells subjected to oxidative stress (115). Van Meter and coworkers (116) have recently shown that SIRT6 overexpression is selectively toxic to cancer cell lines but not to normal cells. It should also be stressed that SIRT6 overexpression in mice protects against impaired glucose tolerance and fat accumulation, both induced by high-fat diet (HFD) (117). Therefore, SIRT6 seems to play a protective role against the metabolic consequences of diet-induced obesity (117).

One should strongly emphasize that the most striking findings concerning SIRT6 have been recently published by Kanfi and coworkers (118). It turned out that SIRT6 is the only sirtuin so far that has been shown to positively affect life span. Namely, male, but not female, transgenic mice overexpressing SIRT6 live significantly longer compared with wild-type animals (118). Thus, SIRT6 may constitute a relevant regulator of longevity (see the role of sirtuins in mammalian aging; eg, reviewed in (119)). It is worth adding that male SIRT6-transgenic mice are characterized by lower serum concentration of IGF-I (118). Taking into account all mentioned observations and remembering about similar beneficial features of long-lived GHRKO mice (eg, decreased IGF-I level), one could hypothesize that SIRT6 level may be increased in these mutants. Further analysis is required to determine a level of this sirtuin in GH receptor gene–disrupted mice.

eNOS and Phospho-eNOS (Ser1177)

eNOS generates nitric oxide (NO) that activates mitochondrial biogenesis through the transcriptional activation of PGC-1α (120,121). Disruption of eNOS gene may lead to various pathological conditions. Yu and coworkers (122) showed that eNOS knockout mice [eNOS (−/−)] had defects in arteriogenesis after ischemia. Generally, eNOS (−/−) mice are characterized by hypertension, increased heart rate variability, impaired angiogenesis, age-dependent left ventricle hypertrophy, cardiac valve abnormalities, and insulin resistance (123) with fasting hyperinsulinemia and hyperlipidemia (124). Furthermore, eNOS knockout mice may have various renal dysfunctions and injuries (125–127). In contrast, prevention of the development of renovascular hypertension was observed in eNOS overexpressed C57BL/6 mice (128). Furthermore, appropriate eNOS activity may contribute to retardation of diabetic nephropathy progression in type 2 diabetic db/db mice (129). Interestingly, eNOS is required for beneficial effects of adiponectin (130). In the context of the mentioned observations, eNOS mRNA levels and protein levels of eNOS and its phosphorylated active form, phospho-eNOS, in insulin sensitive GHRKO mice have been assessed. The studies showed an increase of eNOS gene expression in the heart and kidneys of GHRKO mice as well as increase in renal eNOS and phospho-eNOS protein levels in these knockout mice (84,85) (Tables 3 and 4). Therefore, on the basis of the previously mentioned observations, one could conclude that these alterations may benefit GHRKO mice in terms of extended longevity of these animals.

NRF-1 and TFAM

NRF-1 is a nuclear DNA–binding factor which regulates transcription of numerous genes, including oxidative phosphorylation–related genes. Interestingly, PGC-1α induces transcription of NRF-1; subsequently, NRF-1 regulates expression of TFAM and plays an important role in nervous system and muscle development (131,132).

TFAM is another DNA–binding protein. Its main role relies on the coordination of nuclear and mitochondrial genomes both of which containing genes necessary for mitochondrial biogenesis. The protein in question is also known to be essential for the maintenance of mitochondrial DNA (133). It has been previously shown that mice with disrupted TFAM gene develop dilated cardiomyopathy and atrioventricular heart conduction blocks and die at a very young age (134). Furthermore, TFAM protein level was decreased in oxidative muscles in diabetic db/db mice (135). In contrast, TFAM overexpression ameliorates the cardiac dysfunctions caused by myocardial infarction (136,137).

In the context of the previously mentioned observations, the assessment of NRF-1 and TFAM expression levels in GHRKO mice has been performed. Intriguingly, the decrease of NRF-1 and TFAM mRNA levels in the skeletal muscles of GHRKO mice was reported, even though PGC-1α gene expression was increased in this tissue in GHRKO mutants (84) (Table 3). Moreover, the decrease of TFAM mRNA level in GHRKO’s kidneys was demonstrated, although the renal expression of PGC-1α was increased in these knockout mice (84) (Tables 3 and 4). Importantly, Pesce and coworkers (138) demonstrated the increase of TFAM amount as well as reduction in mitochondrial DNA (mtDNA) content in aged rat soleus skeletal muscle. The authors suggested that TFAM increase may be a cell compensatory response to decrease of mtDNA observed during aging (138). Therefore, the results of the studies, showing the decrease of skeletal muscle and renal TFAM gene expressions (84) (Table 3), may reflect potentially unaltered mtDNA content in GHRKO mice (although not assessed in these animals) and may be regarded as beneficial for long-lived GHRKO mice in the context of their increased life span.

MFN-2

MFN-2 is one of the factors playing an essential role in mitochondrial fusion and maintenance of the mitochondrial network architecture (139,140). Both of these processes are involved in the regulation of mitochondrial activity and biogenesis. Interestingly, decreased expression of MFN-2 may be linked to metabolic disturbances resulting in obesity and type 2 diabetes development (139,141). It is known that PGC-1α stimulates MFN-2 mRNA and protein expression in muscle cells (142). With regard to an important role of MFN-2 in the function of mitochondria, the gene expression and protein level of that factor in insulin-sensitive GHRKO mice are assessed. The results of the studies demonstrated the increased MFN-2 protein level in the skeletal muscles in GHRKO mice, even though no changes in PGC-1α protein were detected in this tissue (85) (Table 4). However, there are studies showing increased MFN-2 protein in PGC-1α knockout mice, demonstrating that level of MFN-2 does not have to depend on the levels of PGC-1α (143).

Decreased MFN-2 expression has been detected in hypertrophied cardiomyocytes (144). Furthermore, renal epithelial cell injury was exacerbated in MFN-2-deficient mice (145). Gesing and coworkers (84) have shown an increase of MFN-2 gene expression in the heart and kidneys in GHRKO mice (Table 3). Furthermore, MFN-2 protein level was increased in the kidneys of these long-lived animals compared with normal mice (85) (Table 4). Remembering about an essential role of MFN-2 in the regulation of mitochondrial function, one could hypothesize that the mentioned alterations in MFN-2 gene expressions and protein levels in various tissues of GHRKO mice may be considered beneficial for these animals in terms of their extended longevity.

Calorie Restriction

Calorie restriction (CR) is a well-known experimental intervention that delays aging and increases life span (23). Importantly, CR leads to protection of genome integrity and chromatin structure; CR may also increase DNA repair activity and reverse the decrease of this process in aged mice (146). Other effects of CR have been recently reviewed by Mercken and coworkers (147). Calorie restriction is also considered to affect numerous factors involved in the regulation of mitochondriogenesis (including AMPK, eNOS, sirtuins, and PGC-1α), leading to the increase of activity of this process in animals and humans (148–151). The characteristics of genetically normal mice subjected to CR resemble certain phenotypic features of GHRKO mice (such as reduced body size, reduced plasma insulin and IGF-I levels, and improved insulin sensitivity). Similarly, longevity of normal mice subjected to CR resembles that of GHRKO mice fed ad libitum (152). Interestingly, CR failed to further modify the alterations in insulin signaling in the livers of GHRKO mice compared with those of normal mice (153). CR also did not affect the insulin-signaling cascade in the heart of GHRKO mice (154). However, it should be emphasized that in the previously mentioned experiments, the authors performed 30% CR. Therefore, with regard to lack of effects of 30% CR on the examined parameters, it was of interest to perform more intensive CR (namely 40% CR). The gradually introduced 40% CR means that calorie-restricted mice received 90% of daily food consumption of ad libitum animals of the same genotype for 1 week, then 80% for the second week, 70% for the third week, and maintaining 60% for the rest of study (CR intervention lasted 6 months). Food consumption of ad libitum animals was monitored throughout the study, and the CR animals were fed daily 60% of the average amount of food consumed daily by ad libitum animals during the preceding week. It appeared reasonable to assume that increased intensity (40%) of CR might affect the examined parameters. The similar 40% CR has been recently used also in other studies in mice (155). However, the altered levels of proapoptotic factors and of key regulators of mitochondrial biogenesis, which may be, as it is hypothesized in that review, potentially beneficial for GHRKO mice in terms of the increased life span, did not improve further with CR (39,84,85). This appears to be consistent with the results of the study performed by Miller and coworkers (156), showing lack of differences in mitochondrial protein synthesis between ad libitum-fed B6D2F1 mice and calorie-restricted B6D2F1 mice. Also, Hancock and coworkers (157) demonstrated that 30% calorie restriction did not induce an increase in mitochondria in heart, brain, liver, adipose tissue, or skeletal muscle in male Wistar rats, as assessed by measurements of various mitochondrial proteins, including cytochrome c, citrate synthase, and cytochrome c oxidase subunit IV. Furthermore, recently published data show that neither PGC-1α in skeletal muscle nor muscle mitochondrial biogenesis are necessary for the short-term metabolic effects of CR (158). These data may support the hypothesis concerning potentially common mechanisms of CR and GH resistance (due to GH receptor knockout) contributing to extended longevity in GHRKO mice and suggesting that mechanisms linking GH resistance and CR to aging must overlap.

Visceral Fat Removal

Adipose tissue produces numerous proinflammatory cytokines, including tumor necrosis factor α, interleukin-6, and interleukin-1β which may cause the alterations in insulin sensitivity leading to the insulin resistance (159–161). Furthermore, adipose tissue also produces other factors, including adiponectin, a hormone which is known to improve insulin sensitivity, exert antiinflammatory effects, and promote cell survival. Interestingly, the long-living GHRKO mice have been shown to have increased plasma levels of adiponectin (162,163). For all these reasons, adipose tissue, and visceral fat particularly, is considered to have important role in aging regulation (164). Therefore, surgical removal of visceral adipose tissue can be expected to improve insulin signaling and potentially have beneficial effects for aging delay. Actually, surgical VFR has been reported to improve glucose tolerance in rats (165), as well as insulin signaling in normal mice and rats and extend longevity in rats (24–26). Thus, this procedure mimics some of the effects of CR. However, unexpectedly, surgical removal of omental fat did not improve insulin sensitivity in obese patients (166). In the context of these interesting observations, it was of interest to examine effects of VFR on gene expressions and protein levels of proapoptotic factors and key regulators of mitochondrial biogenesis in GHRKO mice. Unexpectedly, in contrast to the beneficial effects of VFR in genetically normal animals, this surgical intervention paradoxically promoted insulin resistance in GHRKO mice (47). These data are consistent with the results of experiments performed by Masternak and coworkers (163), confirming that intriguing effect of VFR. Importantly, VFR, similar to CR, did not improve the potentially beneficial effects of altered levels of proapoptotic factors and of key regulators of mitochondrial biogenesis in GHRKO mice (46,47,84,85). These results seem to be consistent with the observations showing lack of effects of CR on levels of the examined factors (39,84,85). Therefore, one could hypothesize that processes related to VFR may be involved in the overlapping of mechanisms linking GH resistance and CR to aging.

Summary

The long-lived GHRKO mice, generated as a result of the disruption of the GHR gene, are characterized by numerous endocrine abnormalities (GH resistance, improved insulin sensitivity, greatly reduced levels of insulin and IGF-I, etc.) (22). This review focuses on newly described unknown features of GHRKO mice (ie, decreased levels of proapoptotic factors and increased levels of key regulators of mitochondrial biogenesis). Therefore, one could speculate that these mechanisms may contribute to the extended longevity in GHRKO mice. As has been mentioned, the levels of key regulators of mitochondrial biogenesis were increased in GHRKO mice. Thus, one could hypothesize that mitochondrial biogenesis may also be increased in these animals, given that cytochrome c oxidase activity (regarded as a measure of mitochondrial content) was increased in skeletal muscles of GHRKO mice (84). Nevertheless, more data are required for comprehensive assessment of the process of mitochondrial biogenesis in long-lived GH receptor gene–disrupted mice. Furthermore, one should emphasize that both mitochondrial biogenesis and apoptosis are dynamic processes and thus it seems that the further analysis of these processes, regarding their dynamic nature, may support evaluation of steady state levels of the apoptosis-related factors and key regulators of mitochondrial biogenesis. When dealing with animal models, it is difficult to obtain more than a steady-state measurement.

Importantly, the alterations in gene expression and proteins levels of the proapoptotic factors and key regulators of mitochondrial biogenesis, as presented in current review, were not improved further by two other additional potential life-extending interventions—CR and VFR. This may attribute the primary role to GH receptor knockout in the regulation of apoptosis and mitochondrial biogenesis in GHRKO mice in terms of increased life span. Moreover, one could speculate that differences in the examined processes in the long-lived animals may be, at least, more apparent under stressful conditions such as HFD. Therefore, further studies are required to broaden our knowledge on other potential mechanisms which may regulate a life span in this unique animal experimental model as well as in other species, including humans. Furthermore, development of novel pharmacological interventions, which may delay the aging processes leading to an increased health span, will have increasing medical and public health significance (167–169).

Conclusions

• Besides well-known characteristics, decreased gene expressions and protein levels of proapoptotic factors in the skeletal muscles and kidneys and increased gene expressions of key regulators of mitochondrial biogenesis in the kidneys, hearts, and skeletal muscles as well as increased renal protein levels of these regulators may be regarded as newly revealed beneficial features of GHRKO mice.

• These new results may help explain the extended longevity of the GHRKO mice.

• No effects of CR or VFR—two other potential life-extending interventions, on the levels of the previously mentioned factors suggest the primary role of GH receptor knockout as an intervention strategy in the regulation of apoptosis and mitochondrial biogenesis in terms of increased life span.