The primary purpose of the present set of studies was to provide a direct comparison of the effects of the ACEi enalapril and the ARB losartan on body composition and physical performance when administered late in life to aged rats. To ensure that any observed change in body composition was not secondary to other physiological processes, we measured locomotor activity, body temperature, food intake, and glucose and insulin levels. In addition, we assessed overall tissue pathology, to ensure that the treatment itself did not promote conditions, such as increased tumorigenesis, that would also result in weight loss. To link changes in adiposity to improvements in skeletal muscle quality, we performed gene array analyses to generate hypotheses regarding which age-related signaling pathways might be altered with enalapril treatment. Based on these results, our primary follow-up pathway was mitochondria-mediated apoptosis of myocytes.
Our results indicate that enalapril treatment consistently attenuates age-related increases in adiposity relative to both placebo and losartan. The maximal effect was achieved after 3 months of treatment (between 24 and 27 months of age), at a dose of 40 mg/kg, and was observed in the absence of any changes in physical activity, body temperature, or food intake. These data are consistent with our previous findings and a larger developing literature demonstrating selective loss of body fat compartment with ACEi treatment in a variety of species, under various feeding regimens and across various time points in the life span (Weisinger et al. 2009a
; Santos et al. 2009
). However, one exception we note is that most studies, especially those in young animals, report decreased food intake. For example, enalapril administration (10 mg/kg) to young adult rats, fed either regular or a high-fat chow, was accompanied by a decrease in food intake (Santos et al. 2009
), although when adjusted for changing body weight, food intake is equivalent or increased. A similar finding has been reported in captopril-treated young mice maintained on a high-fat diet (Weisinger et al. 2009b
). One potential difference between our study and those using young rats and mice as subjects is that young rodents are highly leptin sensitive. Leptin is a primary modulator of ingestive behavior, and with age, the F344BN rat becomes leptin resistant (Zhang and Scarpace 2006
; Scarpace and Zhang 2009
). It is therefore likely that the large losses in fat mass observed in those studies using young animals resulted in decreased leptin levels, initiating an anorectic response. However, it is unlikely that this loss of fat was a direct action of enalapril on leptin signaling given that in the study of Santos et al. (2009
), young rats fed regular chow or a high-fat diet had a similar anorectic response to ICV leptin administration regardless of enalapril treatment. Our group has previously demonstrated that enalapril treatment in old rats did indeed result in a reduction in leptin levels commensurate with the loss of fat mass; however, this effect did not translate in any change in food intake (Carter et al. 2004
). In fact, in the present study, all animals decrease food intake between 24 and 27 months of age, even though fat mass is increasing. Therefore, enalapril most likely is working through other pathways which modulate fat loss (enhanced fatty acid oxidation, increased adiponectin levels, and improved insulin and peroxisome proliferator-activated receptor (PPAR)-γ signaling; Santos et al. 2009
; Weisinger et al. 2009a
) rather than through decreased food intake. Future studies in older animals are needed to address these mechanisms.
The weight loss observed was also not due to changes in pathology. In fact, quite the opposite occurred in that enalapril, at the highest dose, attenuated the age-related increase in tumor development relative to placebo- and losartan-treated animals. This finding is especially remarkable in light of a recent meta-analysis showing an increased risk for cancer associated with chronic ARB treatment (Sipahi et al. 2010
). However, lifespan was not an outcome in our studies given that enalapril administration which occurred was not administered over the entire life of these rats (only between 24 and 30 months of age). However, in a recent study, as part of the NIA Intervention Testing Program, 15 mg/kg enalapril administered to mice from middle age throughout the lifespan did not impact maximal life span (Harrison et al. 2009
). In contrast to these findings, others reported increased life span in laboratory rodents treated with enalapril. For example, Santos et al. showed that enalapril, administered for 26 months to Wistar rats, increased mean life span (Santos et al. 2009
). The effect on maximum life span was not ascertainable given that the study was censored at 26 months. In addition, Basso et al. found that inhibition of the renin–angiotensin system (RAS) via either enalapril (10 mg/kg) or losartan (30 mg/kg) increased life span in normotensive Wistar rats (Basso et al. 2007
). However, in this study, many animals were removed during the experiment for other experimental purposes, thereby changing the composition of the original population and making a conclusive statement regarding life span problematic. Increased longevity has been observed in ACEi-treated mice and rats fed high-fat diets, although these studies were also censored at particular ages (Weisinger et al. 2009b
). Hence, additional research is needed to establish the impact of pharmacological RAS inhibition on life span, taking into consideration the dose and duration of the intervention.
In the current study, losartan had no impact on body composition. One interpretation is that blockade of the actions of ANGII at the AT1 receptor has no anti-adiposity action. However, recent reports suggest that ARBs other than losartan, such as telmisartan, prevent weight gain in normally fed and high-fat-fed mice through PPAR/AMPK signaling pathways (Feng et al. 2010
; He et al. 2010
). Indeed, the same series of studies showed, using an in vitro preparation (3 T3-L1 preadipocytes), that only telmisartan activated PPAR- γ signaling when compared directly with losartan. Furthermore, PPAR- γ and PPAR-delta knockout mice do not respond with weight loss to telmisartan treatment. Most interestingly, mice fed telmisartan had improved exercise endurance, accompanied by a shift to increased slow-twitch muscle fiber profile (Feng et al. 2010
). Whether or not this effect of telmisartan is directly regulated at the AT1 receptor or some other direct action of this drug on alternative signaling pathways is yet unknown. However, there is emerging evidence that telmisartan operates as a partial PPAR-γ agonist and that it inhibits the proliferative capacity of some cells that lack ANGII receptors (Benson et al. 2004
). These data highlight the need for comparative effectiveness of ARBs in late-life intervention studies, using preclinical models.
Importantly, at 27 months of age, both enalapril and losartan attenuated the age-dependent decline in physical performance. However, in contrast to the enalapril group, in losartan-treated rats, this effect occurred in the absence of changes in body weight or composition. This finding suggests that both compounds might improve physical performance through a direct action on the skeletal muscle, independent of changes in body mass. Therefore, we performed gene array analyses in order to develop working hypotheses regarding potential molecular signaling pathways which might be altered with enalapril treatment, and apoptosis emerged as a leading candidate. This hypothesis is supported by previous studies showing that interventions reducing the severity of skeletal muscle apoptosis improve physical function (Song et al. 2006
; Marzetti et al. 2008a
). Apoptosis, a process of programmed cell death, is a highly conserved and tightly regulated systematic set of events resulting in cellular self-destruction without the induction of inflammation or damage to the surrounding tissue (Kerr et al. 1972
). Cysteine-aspartic proteases (caspases) are the executioner enzymes that carry out the dismantling of the cell and are normally present as inactive zymogens (procaspases) (Danial and Korsmeyer 2004
). Upon apoptotic stimuli, initiator caspases (i.e., caspase 8, caspase 9, caspase 12) are activated, subsequently leading to the activation of effector caspases (i.e., caspase 3, caspase 6, caspase 7) that perform the actual cellular degradation (Danial and Korsmeyer 2004
). Effector caspases can be activated through extrinsic and intrinsic pathways (Hengartner 2000
). The extrinsic apoptotic signaling is initiated by the activation of death receptors present on the cell surface, such as the Fas receptor and tumor necrosis factor receptor (Danial and Korsmeyer 2004
). Intrinsic pathways of caspase activation include several internal cellular stimuli mediated by the endoplasmic reticulum or the mitochondria (Danial and Korsmeyer 2004
). Mitochondria-mediated intrinsic signaling pathway of apoptosis is probably the more prominent means of programmed cell death and has been the subject of intense scientific scrutiny in the aging community (Jeong and Seol 2008
In the present study, decreases in DNA fragmentation were only observed in enalapril-treated rats, regardless of the dosage. Interestingly, the Bax-to-Bcl-2 ratio, a critical checkpoint in the apoptotic signaling, was lowered by both enalapril and losartan. Our data do not allow inferring the mechanisms whereby the decreased Bax/Bcl-2 induced by losartan did not translate into a mitigation of muscle apoptotic DNA fragmentation. One explanation for this finding may be that enalapril treatment, besides increasing mitochondrial Bcl-2 expression, was also able to promote Bcl-2 phosphorylation, which is required for its anti-apoptotic activity (Horiuchi et al. 1997
). Importantly, phosphorylation of Bcl-2 induced by mitogen-activated protein kinase (MAPK) is inhibited by the activation of ANGII type 2 (AT2) receptor (Horiuchi et al. 1997
). It is therefore conceivable that the reduced AT2 receptor signaling elicited by enalapril might have resulted in increased MAPK activation and, hence, enhanced Bcl-2 phosphorylation. On the other hand, the lack of AT2 receptor inhibition by losartan might have induced an incomplete anti-apoptotic response, with increased mitochondrial Bcl-2 levels not supported by adequate Bcl-2 phosphorylation. This possibility warrants further investigation.
In addition, although both enalapril doses were able to reduce the extent of apoptosis in the gastrocnemius muscle, only the highest dose decreased the activation of the mitochondrial caspase-dependent apoptotic pathway. Furthermore, neither enalapril dose affected the caspase-independent pathway. Based on our findings, it is therefore unclear how low-dose enalapril attenuated apoptosis. One possibility is that enalapril given at the lowest dose did indeed result in downregulation of mitochondrial caspase-dependent apoptotic signaling. However, changes in the expression levels of apoptogenic mediators might have been below the detection limit of our Western blot analysis. Alternatively, it may be hypothesized that low-dose enalapril was still able to attenuate the caspase catalytic activity, possibly via upregulation of caspase inhibitors (e.g., cIAPs and cFLIPs). The impact of caspase activity and caspase inhibitors should be addressed in future studies.
In conclusion, enalapril treatment, between 20 and 40 mg/kg, consistently lowers body weight in older animals, even when initiated late in life, with a maximal efficacy achieved after 3 months of treatment. This effect was not observed in losartan-treated animals, suggesting that blocking the AT1 receptor pathway does not mediate these benefits. Changes in body weight and composition appear dissociated from improvements in physical function and may reflect a differential impact of enalapril and losartan on muscle quality. These data suggest that attenuation of the severity of skeletal muscle apoptosis promoted by enalapril may represent a distinct mechanism through which this compound improves muscle strength/quality.