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Protein kinase A signaling as an anti-aging target
Ageing Research Reviews 9 (2010) 269–272
Contents lists available at ScienceDirect
Ageing Research Reviews journal homepage: www.elsevier.com/locate/arr
Review
Protein kinase A signaling as an anti-aging target Linda C. Enns, Warren Ladiges ∗ Department of Comparative Medicine, University of Washington, Seattle, WA 98195, USA
a r t i c l e
i n f o
Article history: Received 23 December 2009 Received in revised form 10 February 2010 Accepted 17 February 2010 Keywords: PKA signaling Aging Obesity
a b s t r a c t Protein kinase A (PKA) is a multi-unit protein kinase that mediates signal transduction of G-proteincoupled receptors through its activation by adenyl cyclase (AC)-mediated cAMP. The vital importance of PKA signaling to cellular function is reflected in the widespread expression of PKA subunit genes. As one of its many functions, PKA plays a key role in the regulation of metabolism and triglyceride storage. The PKA pathway has become of great interest to the study of aging, since mutations that cause a reduction in PKA signaling have been shown to extend lifespan in yeast, and to both delay the incidence and severity of age-related disease, and to promote leanness and longevity, in mice. There is increasing interest in the potential for the inhibition or redistribution of adiposity to attenuate aging, since obesity is associated with impaired function of most organ systems, and is a strong risk factor for shortened life span. Its association with coronary heart disease, hypertension, type 2 diabetes, cancer, sleep apnea and osteoarthritis is leading to its accession as a major cause of global ill health. Therefore, gene signaling pathways such as PKA that promote adiposity are potential inhibitory targets for aging intervention. Since numerous plant compounds have been found that both prevent adipogenesis and inhibit PKA signaling, a focused investigation into their effects on biological systems and the corresponding molecular mechanisms would be of high relevance to the discovery of novel and non-toxic compounds that promote healthy aging. © 2010 Elsevier Ireland Ltd. All rights reserved.
Protein kinase A (PKA) is a ubiquitous cellular multi-unit kinase that phosphorylates serine and threonine residues in response to adenyl cyclase (AC)-mediated cAMP (Niswender et al., 1975). The widespread expression of PKA subunit genes, coupled with the myriad of mechanisms by which cAMP is regulated within a cell, suggests that PKA signaling is one of extreme importance to cellular function. PKA is known to mediate signal transduction downstream of G-protein-coupled receptors and plays a key role in the regulation of metabolism and triglyceride storage. Genes in the PKA pathway are of interest based on loss of function lifespan studies in yeast, worms and flies. Loss of function of CYR1, an AC ortholog in yeast, was shown to extend lifespan (Longo, 2003), and reduced function of yeast TPK1, TPK2, and TPK3, homologs of both the mouse and human PKA catalytic subunits, has been shown to extend invertebrate lifespan (Lin et al., 2000). PKA activity has also been found to mediate age-related decline in flies (Yamazaki et al., 2007; Laviada et al., 1997). If findings in other species extend to mammals, a reduction in AC or PKA activity in the mouse might be expected to have similar lifespan extending or enhancing effects. In support of this idea, deletion of AC5 has been reported to delay cardiac aging and extend lifespan in mice (Yan et al., 2007). In more recent studies we have described increased lifespan and health benefits in mice lacking PKA subunit genes (Enns et al., 2009a,b), which will be the
∗ Corresponding author. Tel.: +1 206 685 3260; fax: +1 206 685 3006. E-mail address: [email protected] (W. Ladiges). 1568-1637/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.arr.2010.02.004
focus of this review. Anti-aging effects in divergent species indicate a conserved function for this pathway. The purpose of genetic studies in aging is to identify the entire set of genes involved in regulating aging in humans. One of the most powerful methods for determining the role of specific genes in an organism’s development or physiology is by generating genetic mutants. Genetic testing of humans is restricted to either epidemiological studies or cell culture systems. Invertebrate models are inexpensive and easy to make, but using them to identify potential genetic targets for human clinical treatment is only a first step. Ultimately, and before costly pharmaceutical development and subsequent clinical testing on primates and humans can progress, genetic findings need to be verified in a mammalian model. In this regard, the mouse is an ideal model organism (Ladiges et al., 2009). Mice are convenient model organisms because of their short reproductive cycles, their small size which makes them inexpensive to maintain in large numbers, and the fact that they are genetically easily manipulated and well understood (Enns et al., 2008). But more importantly, mice age similarly to humans. The frequency and type of cancer in both species can be similar, depending on the background strain (Balmain and Harris, 2000). Loss of tissue elasticity, immunological ability, muscle strength, sensory perception and reflexes occur in both species, as well as age-associated diseases such as osteoporosis, osteoarthritis, type II diabetes, cardiovascular disease, cataracts and neurodegenerative diseases (Kappeler and Epelbaum, 2005). Mice, like humans, also show an age-related decline in cognitive ability (Murphy et al., 2006a,b).
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L.C. Enns, W. Ladiges / Ageing Research Reviews 9 (2010) 269–272
Fig. 1. PKA consists of two regulatory subunits, which function to maintain two catalytic subunits in an inactive status. When the regulatory units are activated by cAMP, the catalytic units are released as active holoenzymes. C␣ phosphorylates cAMP-response element binding protein (CREB), and C phosphorylates c-REL, a subunit of nuclear factor B (NF-B).
1. Deletion of PKA RII enhances healthy aging in mice Murine PKA consists of four regulatory and two catalytic subunits, each with multiple isoforms (Fig. 1). PKA RII is one of the regulatory isoforms and is predominantly expressed in brown and white adipose tissue and in brain (McKnight et al., 1998; Cummings et al., 1996), tissues known to be important in the regulation of energy homeostasis. Nearly all PKA activity in adipose tissue and 50% of PKA activity in the striatum, hypothalamus, and cortex is attributed to the RII subunit (Cummings et al., 1996). Partial compensation for the loss of RII occurs in these tissues with up-regulation of the RI regulatory subunit (Amieux et al., 1997; Planas et al., 1999; Brandon et al., 1998). RII null mice are lean in comparison to their wild type littermates, have increased resting metabolic activity, body temperature, uncoupling protein 1 (UCP1) concentrations, and lipid hydrolysis (Cummings et al., 1996). Loss of the RII subunit has been shown to protect mice on a highfat, high-carbohydrate diet against weight gain, hyperinsulinemia, fatty livers and insulin resistance, reduce plasma levels of VLDL and LDL cholesterol, and to improve glucose dispersal (Schreyer et al., 2001). Lower serum glucose and cholesterol levels and improved insulin sensitivity are known to occur during caloric restriction (Sohal and Weindruch, 1999; Roth et al., 1999). Caloric restriction (CR) is the most widely studied model of longevity, and the lifespan extension from CR is thought to be mediated in part by the PKA pathway (Lin et al., 2002; Steffen et al., 2008; Wei et al., 2008). Mammalian RII is a PKA regulatory subunit, in contrast to the yeast TPK genes which encode PKA catalytic subunits. However, in mice, loss of RII results in a compensatory increase in the PKA regulatory subunit RI␣, which dissociates at a lower concentration of cAMP. This leads to an increased release and subsequent degradation of the catalytic subunits (Cummings et al., 1996; Amieux et al., 1997; Planas et al., 1999). In both organisms, therefore, these mutations lead to a down-regulation of PKA activity. Based on both the anti-obesity and anti-diabetic phenotypes of these mice, as well the data showing that disruption of PKA extends lifespan in yeast, we hypothesized that old RII null mice would be characterized by anti-aging phenotypes. We conducted a lifespan study and found that both median and maximum lifespans were significantly increased in RII null male mice (Enns et al., 2009a) thus fulfilling our prediction for delayed aging. It is of interest that
the lifespan of females was not affected by the mutation. The reason for the gender difference is not yet known but we now have preliminary data suggesting it may be related to differences in the role that metabolic perturbations play in longevity. We have shown that body composition, including body weight, percent body fat mass, and percent lean body mass, are associated with extended lifespan in PKA RII null male mice. Young RII null and WT littermates weigh about the same and have about the same amount of body fat and lean body mass, but with age, there is a striking difference in these parameters between the two genotypes. WT mice of both genders experience increased adiposity with age, and both RII null male and female mice are resistant to this age-related obesity. Both genotypes eat about the same amount, so the body composition differences cannot be attributed to differences in food intake. Importantly, higher body fat percentages in old mice correlate with shorter lifespans in males only, suggesting that body composition may be the physiological factor contributing to the gender-specific longevity phenotype of the mutant males. RII null males are also more insulin sensitive that WT littermates throughout their lifetime, and this may contribute to their longevity as well. We also saw a decreased incidence and severity of a number of age-related diseases in RII null mice. These included a decrease in incidence and severity of renal pathology, decreased cardiovascular lesions and a decrease in incidence and severity of lymphoreticular tumors. One of the more interesting attenuations we observed in the RII null mutants was in age-related cardiac dysfunction. PKA plays an important role in regulating cardiac contractility, acting downstream of -adrenergic receptors (ARs) to control calcium transients (Antos et al., 2001). PKA regulates calcium signaling in the heart by interacting with multiple substrates, and cardiac hypertrophy and dysfunction in several different animal models has been associated with the perturbed activity of a number of these downstream targets (Antos et al., 2001; Sipido and Eisner, 2005). Furthermore, disruption of AC5, an upstream regulator of PKA, was shown to preserve cardiac function against pressure overload in mice (Okumura et al., 2003). Using echocardiography, we found that RII null male mice at 24 months of age had decreased interventricular septal diameter and decreased left ventricular wall diameter compared to wild type littermates (Enns et al., 2009a). Left ventricular (LV) hypertrophy is caused by cardiac wall stress and is an independent predictive risk factor of cardiovascular morbidity and mortality in humans (Casale et al., 1986; Devereux, 1989). The delay in age-related progression of cardiac dysfunction suggests a possible connection with the findings that the absence of AC5, and hence disruption of PKA signaling, is protective of cardiac dysfunction. The fact that RII is not expressed, or expressed at very low levels, in cardiac tissue suggests that signaling from adipose tissue or the brain may be involved. It is also possible that the delayed cardiac function is a secondary effect due to lack of adiposity. Left ventricular mass has been found to be associated with obesity, even in the absence of hypertension (Crisostomo et al., 2001; Lauer et al., 1992). Our novel findings therefore confirm a role for PKA in mammalian longevity and suggest that disruption of PKA RII affects physiological mechanisms known to be associated with healthy aging in mammals.
2. PKA catalytic C subunit null mice are resistant to a high caloric diet PKA catalytic subunits are maintained in an inactive state by binding of a regulatory subunit. Each regulatory subunit binds one catalytic subunit. Binding of cAMP to the regulatory subunits causes their dissociation with the catalytic subunits, activating
L.C. Enns, W. Ladiges / Ageing Research Reviews 9 (2010) 269–272
them (Fig. 1). In general, C␣ is expressed ubiquitously in all tissues, while C shows a more restricted pattern of expression in the brain (Brandon et al., 1998), liver (Enns et al., 2009b) and hematopoietic cells (Genomics Institute of the Novartis Research Foundation (“GNF”) Gene Expression Database). In addition to having different tissue-specific expression patterns, the C␣ and C subunits of PKA are believed to have unique functions (Gamm et al., 1996), and are known to phosphorylate different downstream targets (Yu et al., 2004). In keeping with both the tissue and function-specific natures of these catalytic subunits, mutating one or the other in mice causes dramatically different effects. Knocking out C␣ in mice results in extremely detrimental phenotypes, including growth retardation, and sperm dysfunction (Skalhegg et al., 2002). In contrast, we have found that while C null animals appear overtly normal when fed standard rodent chow, they are protected from diet-induced obesity, steatosis, dyslipoproteinemia and insulin resistance, without any differences in caloric intake or locomotor activity (Enns et al., 2009b). The significant increase in body weight in wild type littermates maintained on a high caloric diet was shown by quantitative magnetic resonance (QMR) imaging to be due to an increase in fat mass. Both PKA C null males and females displayed obesity resistance. Generally, there was no difference in amount of food consumed by either genotype. When individual fat depots were weighed there was an attenuation in the development of visceral fat in PKA C null mice, of interest due to observations that accumulation of visceral fat is a high risk factor for age-related disease (Carr et al., 2004). The fat sparing effect was especially evident in the liver, showing that PKA C null mice are resistant to the hepatic steatosis-like condition associated with ingesting a high fat diet. Blood glucose was elevated in wild type littermates, but not PKA C null mice, as early as four weeks on the high fat diet, and a glucose tolerance test showed that PKA C null mice maintained their tolerance to glucose and sensitivity to insulin in contrast to wild type littermates. We have also found the PKA C mutant mice to be leptin sensitive (unpublished data). PKA C null mice display neither the nocturnal hyperactivity, nor the increased body temperature, UCP1 expression, and resting metabolic rate reported for RII null mice (preliminary unpublished data). These observations indicate that the PKA C null mice are metabolically different from PKA RII null mice, possibly due to differences in tissue distribution, subcellular localization, or function of the protein. More importantly, PKA C null mice also do not show the cognitive defects reported in RII null mice (Fischer et al., 2004; Enns et al., 2008). Our findings that PKA C null mice are resistant to diet-induced obesity and leptin resistance have relevant pharmacological implications, since aging in mammals is characterized by metabolic decline. While plant-based PKA inhibitors may show promise for the treatment of age-related metabolic syndrome in mammals, studies on mutations in different mouse PKA subunits clearly suggest that a C-specific PKA inhibitor is desirable in order to avoid the potentially toxic effects of C␣ inhibition. 3. PKA inhibitors are novel anti-aging compounds The design of novel kinase-specific inhibitors is a major scientific effort. Our mouse studies now provide the rationale for focusing some of this attention to the development of PKA inhibitors for preventing or delaying aging and age-related diseases. The cAMP-PKA signaling pathway, while well known for its role in the regulation of body weight and metabolism in response to nutrient status, is involved in numerous processes, including regulation of other kinases, intracellular calcium concentrations, and transcription of a number of different downstream targets (Murray, 2008; Taskén and Aandahl, 2004). Mammalian PKA is a tetrameric holoenzyme and its modulation of so many independent and different processes may be
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in part due to the existence of a number of different isomers for each of its 4 subunits (McKnight et al., 1998). The regulatory subunits are highly dynamic multi-domain proteins that interact with a variety of proteins in addition to serving as major receptors for cAMP. Inhibition of PKA RII is of interest for improving health span based on our mouse studies. However, it has also shown that cognitive function is compromised (Fischer et al., 2004) so stringent specificity would be required to avoid inhibitory activity in the brain. The beta catalytic subunit of PKA is also of interest as an anti-aging target based on both our published studies and unpublished observations on PKA C null mutant mice. As already mentioned, the complete absence of the C subunit gene results in no detectable deleterious affects in mice on the C57BL/6 background. Therefore the anti-obesity and cardiac sparing effects provide the rationale for the development of compounds that specifically inhibit PKA C signaling activity. Plant secondary metabolites (flavonoids, anthocyanins and polyphenols) have been the subject of increasing interest as potential agents for both disease prevention and therapy. Many plant-derived compounds, such as those found in pomegranate, grapeseed, apples and green tea, have been shown to have cancerpreventing properties and to protect against heart disease and artherosclerosis (Nakachi et al., 2000; Clifton, 2004). In addition, some have been found to target adipocyte function, resulting in suppression of adipogenesis in cell culture (Hsu and Yen, 2007). Extracts from green tea, crowberry, clove, cinnamon and pomegranate have been shown to be inhibitors of PKA activity (Moskaug et al., 2008). However, their specific mechanism of inhibition is not known so they could be inducing harmful effects by altering vital PKA signaling. A screening assay is therefore needed to distinguish those compounds that specifically inhibit PKA C and not other PKA subunit genes. The PKA pathway has become of great interest to the study of aging, since mutations in this pathway that cause a reduction in PKA activity have been shown to extend lifespan in yeast, and to promote leanness and longevity, and resistance to stress-induced cardiomyopathy in mice. There is increasing interest in the reduction and/or redistribution of adiposity in conjunction with delayed aging. Obesity is associated with impaired function of most organ systems, and is a strong risk factor for shortened life span (Fontaine et al., 2003). Its association with coronary heart disease, hypertension, type 2 diabetes, cancer, sleep apnea and osteoarthritis is leading to its accession as a leading cause of global ill health, ahead of under nutrition and infectious disease (Kopelman, 2000). Therefore, genes that promote adiposity through cellular signaling pathways are potential inhibitory targets for aging intervention. Since numerous plant compounds have been found that both prevent adipogenesis and inhibit PKA activity, a focused investigation into identifying and characterizing their biological effects and molecular mechanisms would be of high relevance to the development of novel and non-toxic compounds as a way to maintain good health with increasing age. Acknowledgement Supported in part by the Ellison Medical Foundation. References Amieux, P.S., Cummings, D.E., Motamed, K., Brandon, E.P., Wailes, L.A., 1997. Compensatory regulation of RI␣ protein levels in protein kinase A mutant mice. Journal of Biological Chemistry 272, 3993–3998. Antos, C.L., Frey, N., Marx, S.O., Reiken, S., Gaburjakova, M., Richardson, J.A., Marks, A.R., Olson, E.N., 2001. Dilated cardiomyopathy and sudden death resulting from constitutive activation of protein kinase A. Circulation Research 89, 997–1004. Balmain, A., Harris, C., 2000. Carcinogenesis in mouse and human cells: parallels and paradoxes. Carcinogenesis 21, 371–377.
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Contents lists available at ScienceDirect
Ageing Research Reviews journal homepage: www.elsevier.com/locate/arr
Review
Protein kinase A signaling as an anti-aging target Linda C. Enns, Warren Ladiges ∗ Department of Comparative Medicine, University of Washington, Seattle, WA 98195, USA
a r t i c l e
i n f o
Article history: Received 23 December 2009 Received in revised form 10 February 2010 Accepted 17 February 2010 Keywords: PKA signaling Aging Obesity
a b s t r a c t Protein kinase A (PKA) is a multi-unit protein kinase that mediates signal transduction of G-proteincoupled receptors through its activation by adenyl cyclase (AC)-mediated cAMP. The vital importance of PKA signaling to cellular function is reflected in the widespread expression of PKA subunit genes. As one of its many functions, PKA plays a key role in the regulation of metabolism and triglyceride storage. The PKA pathway has become of great interest to the study of aging, since mutations that cause a reduction in PKA signaling have been shown to extend lifespan in yeast, and to both delay the incidence and severity of age-related disease, and to promote leanness and longevity, in mice. There is increasing interest in the potential for the inhibition or redistribution of adiposity to attenuate aging, since obesity is associated with impaired function of most organ systems, and is a strong risk factor for shortened life span. Its association with coronary heart disease, hypertension, type 2 diabetes, cancer, sleep apnea and osteoarthritis is leading to its accession as a major cause of global ill health. Therefore, gene signaling pathways such as PKA that promote adiposity are potential inhibitory targets for aging intervention. Since numerous plant compounds have been found that both prevent adipogenesis and inhibit PKA signaling, a focused investigation into their effects on biological systems and the corresponding molecular mechanisms would be of high relevance to the discovery of novel and non-toxic compounds that promote healthy aging. © 2010 Elsevier Ireland Ltd. All rights reserved.
Protein kinase A (PKA) is a ubiquitous cellular multi-unit kinase that phosphorylates serine and threonine residues in response to adenyl cyclase (AC)-mediated cAMP (Niswender et al., 1975). The widespread expression of PKA subunit genes, coupled with the myriad of mechanisms by which cAMP is regulated within a cell, suggests that PKA signaling is one of extreme importance to cellular function. PKA is known to mediate signal transduction downstream of G-protein-coupled receptors and plays a key role in the regulation of metabolism and triglyceride storage. Genes in the PKA pathway are of interest based on loss of function lifespan studies in yeast, worms and flies. Loss of function of CYR1, an AC ortholog in yeast, was shown to extend lifespan (Longo, 2003), and reduced function of yeast TPK1, TPK2, and TPK3, homologs of both the mouse and human PKA catalytic subunits, has been shown to extend invertebrate lifespan (Lin et al., 2000). PKA activity has also been found to mediate age-related decline in flies (Yamazaki et al., 2007; Laviada et al., 1997). If findings in other species extend to mammals, a reduction in AC or PKA activity in the mouse might be expected to have similar lifespan extending or enhancing effects. In support of this idea, deletion of AC5 has been reported to delay cardiac aging and extend lifespan in mice (Yan et al., 2007). In more recent studies we have described increased lifespan and health benefits in mice lacking PKA subunit genes (Enns et al., 2009a,b), which will be the
∗ Corresponding author. Tel.: +1 206 685 3260; fax: +1 206 685 3006. E-mail address: [email protected] (W. Ladiges). 1568-1637/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.arr.2010.02.004
focus of this review. Anti-aging effects in divergent species indicate a conserved function for this pathway. The purpose of genetic studies in aging is to identify the entire set of genes involved in regulating aging in humans. One of the most powerful methods for determining the role of specific genes in an organism’s development or physiology is by generating genetic mutants. Genetic testing of humans is restricted to either epidemiological studies or cell culture systems. Invertebrate models are inexpensive and easy to make, but using them to identify potential genetic targets for human clinical treatment is only a first step. Ultimately, and before costly pharmaceutical development and subsequent clinical testing on primates and humans can progress, genetic findings need to be verified in a mammalian model. In this regard, the mouse is an ideal model organism (Ladiges et al., 2009). Mice are convenient model organisms because of their short reproductive cycles, their small size which makes them inexpensive to maintain in large numbers, and the fact that they are genetically easily manipulated and well understood (Enns et al., 2008). But more importantly, mice age similarly to humans. The frequency and type of cancer in both species can be similar, depending on the background strain (Balmain and Harris, 2000). Loss of tissue elasticity, immunological ability, muscle strength, sensory perception and reflexes occur in both species, as well as age-associated diseases such as osteoporosis, osteoarthritis, type II diabetes, cardiovascular disease, cataracts and neurodegenerative diseases (Kappeler and Epelbaum, 2005). Mice, like humans, also show an age-related decline in cognitive ability (Murphy et al., 2006a,b).
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Fig. 1. PKA consists of two regulatory subunits, which function to maintain two catalytic subunits in an inactive status. When the regulatory units are activated by cAMP, the catalytic units are released as active holoenzymes. C␣ phosphorylates cAMP-response element binding protein (CREB), and C phosphorylates c-REL, a subunit of nuclear factor B (NF-B).
1. Deletion of PKA RII enhances healthy aging in mice Murine PKA consists of four regulatory and two catalytic subunits, each with multiple isoforms (Fig. 1). PKA RII is one of the regulatory isoforms and is predominantly expressed in brown and white adipose tissue and in brain (McKnight et al., 1998; Cummings et al., 1996), tissues known to be important in the regulation of energy homeostasis. Nearly all PKA activity in adipose tissue and 50% of PKA activity in the striatum, hypothalamus, and cortex is attributed to the RII subunit (Cummings et al., 1996). Partial compensation for the loss of RII occurs in these tissues with up-regulation of the RI regulatory subunit (Amieux et al., 1997; Planas et al., 1999; Brandon et al., 1998). RII null mice are lean in comparison to their wild type littermates, have increased resting metabolic activity, body temperature, uncoupling protein 1 (UCP1) concentrations, and lipid hydrolysis (Cummings et al., 1996). Loss of the RII subunit has been shown to protect mice on a highfat, high-carbohydrate diet against weight gain, hyperinsulinemia, fatty livers and insulin resistance, reduce plasma levels of VLDL and LDL cholesterol, and to improve glucose dispersal (Schreyer et al., 2001). Lower serum glucose and cholesterol levels and improved insulin sensitivity are known to occur during caloric restriction (Sohal and Weindruch, 1999; Roth et al., 1999). Caloric restriction (CR) is the most widely studied model of longevity, and the lifespan extension from CR is thought to be mediated in part by the PKA pathway (Lin et al., 2002; Steffen et al., 2008; Wei et al., 2008). Mammalian RII is a PKA regulatory subunit, in contrast to the yeast TPK genes which encode PKA catalytic subunits. However, in mice, loss of RII results in a compensatory increase in the PKA regulatory subunit RI␣, which dissociates at a lower concentration of cAMP. This leads to an increased release and subsequent degradation of the catalytic subunits (Cummings et al., 1996; Amieux et al., 1997; Planas et al., 1999). In both organisms, therefore, these mutations lead to a down-regulation of PKA activity. Based on both the anti-obesity and anti-diabetic phenotypes of these mice, as well the data showing that disruption of PKA extends lifespan in yeast, we hypothesized that old RII null mice would be characterized by anti-aging phenotypes. We conducted a lifespan study and found that both median and maximum lifespans were significantly increased in RII null male mice (Enns et al., 2009a) thus fulfilling our prediction for delayed aging. It is of interest that
the lifespan of females was not affected by the mutation. The reason for the gender difference is not yet known but we now have preliminary data suggesting it may be related to differences in the role that metabolic perturbations play in longevity. We have shown that body composition, including body weight, percent body fat mass, and percent lean body mass, are associated with extended lifespan in PKA RII null male mice. Young RII null and WT littermates weigh about the same and have about the same amount of body fat and lean body mass, but with age, there is a striking difference in these parameters between the two genotypes. WT mice of both genders experience increased adiposity with age, and both RII null male and female mice are resistant to this age-related obesity. Both genotypes eat about the same amount, so the body composition differences cannot be attributed to differences in food intake. Importantly, higher body fat percentages in old mice correlate with shorter lifespans in males only, suggesting that body composition may be the physiological factor contributing to the gender-specific longevity phenotype of the mutant males. RII null males are also more insulin sensitive that WT littermates throughout their lifetime, and this may contribute to their longevity as well. We also saw a decreased incidence and severity of a number of age-related diseases in RII null mice. These included a decrease in incidence and severity of renal pathology, decreased cardiovascular lesions and a decrease in incidence and severity of lymphoreticular tumors. One of the more interesting attenuations we observed in the RII null mutants was in age-related cardiac dysfunction. PKA plays an important role in regulating cardiac contractility, acting downstream of -adrenergic receptors (ARs) to control calcium transients (Antos et al., 2001). PKA regulates calcium signaling in the heart by interacting with multiple substrates, and cardiac hypertrophy and dysfunction in several different animal models has been associated with the perturbed activity of a number of these downstream targets (Antos et al., 2001; Sipido and Eisner, 2005). Furthermore, disruption of AC5, an upstream regulator of PKA, was shown to preserve cardiac function against pressure overload in mice (Okumura et al., 2003). Using echocardiography, we found that RII null male mice at 24 months of age had decreased interventricular septal diameter and decreased left ventricular wall diameter compared to wild type littermates (Enns et al., 2009a). Left ventricular (LV) hypertrophy is caused by cardiac wall stress and is an independent predictive risk factor of cardiovascular morbidity and mortality in humans (Casale et al., 1986; Devereux, 1989). The delay in age-related progression of cardiac dysfunction suggests a possible connection with the findings that the absence of AC5, and hence disruption of PKA signaling, is protective of cardiac dysfunction. The fact that RII is not expressed, or expressed at very low levels, in cardiac tissue suggests that signaling from adipose tissue or the brain may be involved. It is also possible that the delayed cardiac function is a secondary effect due to lack of adiposity. Left ventricular mass has been found to be associated with obesity, even in the absence of hypertension (Crisostomo et al., 2001; Lauer et al., 1992). Our novel findings therefore confirm a role for PKA in mammalian longevity and suggest that disruption of PKA RII affects physiological mechanisms known to be associated with healthy aging in mammals.
2. PKA catalytic C subunit null mice are resistant to a high caloric diet PKA catalytic subunits are maintained in an inactive state by binding of a regulatory subunit. Each regulatory subunit binds one catalytic subunit. Binding of cAMP to the regulatory subunits causes their dissociation with the catalytic subunits, activating
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them (Fig. 1). In general, C␣ is expressed ubiquitously in all tissues, while C shows a more restricted pattern of expression in the brain (Brandon et al., 1998), liver (Enns et al., 2009b) and hematopoietic cells (Genomics Institute of the Novartis Research Foundation (“GNF”) Gene Expression Database). In addition to having different tissue-specific expression patterns, the C␣ and C subunits of PKA are believed to have unique functions (Gamm et al., 1996), and are known to phosphorylate different downstream targets (Yu et al., 2004). In keeping with both the tissue and function-specific natures of these catalytic subunits, mutating one or the other in mice causes dramatically different effects. Knocking out C␣ in mice results in extremely detrimental phenotypes, including growth retardation, and sperm dysfunction (Skalhegg et al., 2002). In contrast, we have found that while C null animals appear overtly normal when fed standard rodent chow, they are protected from diet-induced obesity, steatosis, dyslipoproteinemia and insulin resistance, without any differences in caloric intake or locomotor activity (Enns et al., 2009b). The significant increase in body weight in wild type littermates maintained on a high caloric diet was shown by quantitative magnetic resonance (QMR) imaging to be due to an increase in fat mass. Both PKA C null males and females displayed obesity resistance. Generally, there was no difference in amount of food consumed by either genotype. When individual fat depots were weighed there was an attenuation in the development of visceral fat in PKA C null mice, of interest due to observations that accumulation of visceral fat is a high risk factor for age-related disease (Carr et al., 2004). The fat sparing effect was especially evident in the liver, showing that PKA C null mice are resistant to the hepatic steatosis-like condition associated with ingesting a high fat diet. Blood glucose was elevated in wild type littermates, but not PKA C null mice, as early as four weeks on the high fat diet, and a glucose tolerance test showed that PKA C null mice maintained their tolerance to glucose and sensitivity to insulin in contrast to wild type littermates. We have also found the PKA C mutant mice to be leptin sensitive (unpublished data). PKA C null mice display neither the nocturnal hyperactivity, nor the increased body temperature, UCP1 expression, and resting metabolic rate reported for RII null mice (preliminary unpublished data). These observations indicate that the PKA C null mice are metabolically different from PKA RII null mice, possibly due to differences in tissue distribution, subcellular localization, or function of the protein. More importantly, PKA C null mice also do not show the cognitive defects reported in RII null mice (Fischer et al., 2004; Enns et al., 2008). Our findings that PKA C null mice are resistant to diet-induced obesity and leptin resistance have relevant pharmacological implications, since aging in mammals is characterized by metabolic decline. While plant-based PKA inhibitors may show promise for the treatment of age-related metabolic syndrome in mammals, studies on mutations in different mouse PKA subunits clearly suggest that a C-specific PKA inhibitor is desirable in order to avoid the potentially toxic effects of C␣ inhibition. 3. PKA inhibitors are novel anti-aging compounds The design of novel kinase-specific inhibitors is a major scientific effort. Our mouse studies now provide the rationale for focusing some of this attention to the development of PKA inhibitors for preventing or delaying aging and age-related diseases. The cAMP-PKA signaling pathway, while well known for its role in the regulation of body weight and metabolism in response to nutrient status, is involved in numerous processes, including regulation of other kinases, intracellular calcium concentrations, and transcription of a number of different downstream targets (Murray, 2008; Taskén and Aandahl, 2004). Mammalian PKA is a tetrameric holoenzyme and its modulation of so many independent and different processes may be
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in part due to the existence of a number of different isomers for each of its 4 subunits (McKnight et al., 1998). The regulatory subunits are highly dynamic multi-domain proteins that interact with a variety of proteins in addition to serving as major receptors for cAMP. Inhibition of PKA RII is of interest for improving health span based on our mouse studies. However, it has also shown that cognitive function is compromised (Fischer et al., 2004) so stringent specificity would be required to avoid inhibitory activity in the brain. The beta catalytic subunit of PKA is also of interest as an anti-aging target based on both our published studies and unpublished observations on PKA C null mutant mice. As already mentioned, the complete absence of the C subunit gene results in no detectable deleterious affects in mice on the C57BL/6 background. Therefore the anti-obesity and cardiac sparing effects provide the rationale for the development of compounds that specifically inhibit PKA C signaling activity. Plant secondary metabolites (flavonoids, anthocyanins and polyphenols) have been the subject of increasing interest as potential agents for both disease prevention and therapy. Many plant-derived compounds, such as those found in pomegranate, grapeseed, apples and green tea, have been shown to have cancerpreventing properties and to protect against heart disease and artherosclerosis (Nakachi et al., 2000; Clifton, 2004). In addition, some have been found to target adipocyte function, resulting in suppression of adipogenesis in cell culture (Hsu and Yen, 2007). Extracts from green tea, crowberry, clove, cinnamon and pomegranate have been shown to be inhibitors of PKA activity (Moskaug et al., 2008). However, their specific mechanism of inhibition is not known so they could be inducing harmful effects by altering vital PKA signaling. A screening assay is therefore needed to distinguish those compounds that specifically inhibit PKA C and not other PKA subunit genes. The PKA pathway has become of great interest to the study of aging, since mutations in this pathway that cause a reduction in PKA activity have been shown to extend lifespan in yeast, and to promote leanness and longevity, and resistance to stress-induced cardiomyopathy in mice. There is increasing interest in the reduction and/or redistribution of adiposity in conjunction with delayed aging. Obesity is associated with impaired function of most organ systems, and is a strong risk factor for shortened life span (Fontaine et al., 2003). Its association with coronary heart disease, hypertension, type 2 diabetes, cancer, sleep apnea and osteoarthritis is leading to its accession as a leading cause of global ill health, ahead of under nutrition and infectious disease (Kopelman, 2000). Therefore, genes that promote adiposity through cellular signaling pathways are potential inhibitory targets for aging intervention. Since numerous plant compounds have been found that both prevent adipogenesis and inhibit PKA activity, a focused investigation into identifying and characterizing their biological effects and molecular mechanisms would be of high relevance to the development of novel and non-toxic compounds as a way to maintain good health with increasing age. Acknowledgement Supported in part by the Ellison Medical Foundation. References Amieux, P.S., Cummings, D.E., Motamed, K., Brandon, E.P., Wailes, L.A., 1997. Compensatory regulation of RI␣ protein levels in protein kinase A mutant mice. Journal of Biological Chemistry 272, 3993–3998. Antos, C.L., Frey, N., Marx, S.O., Reiken, S., Gaburjakova, M., Richardson, J.A., Marks, A.R., Olson, E.N., 2001. Dilated cardiomyopathy and sudden death resulting from constitutive activation of protein kinase A. Circulation Research 89, 997–1004. Balmain, A., Harris, C., 2000. Carcinogenesis in mouse and human cells: parallels and paradoxes. Carcinogenesis 21, 371–377.
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