Long-term low-dose rapamycin effects on sugar metabolism and immunity in aging-prone and normal mice

Aging is a natural process characterized by the gradual accumulation of cellular defects, which progressively impair proliferative capacity of cells and disrupt essential functions required for organismal survival (1). During the aging process, defective cells that are not eliminated often transition into a senescent phenotype, marked by irreversible cell cycle arrest while maintaining metabolic activity (2, 3). Over time, an increasing number of cells adopt this senescent phenotype, accumulating in tissues and organs, thereby impairing homeostasis and regeneration (4). This accumulation arises not only from reduced proliferative capacity but also from the secretion of senescence-associated secretory phenotype (SASP) inflammatory factors, such as IL-1β, IL-6, and TNF-α. Together, these factors contribute to a state of chronic, low-grade inflammation known as inflammaging, which is implicated in the pathogenesis of common age-related conditions such as cardiovascular disease and diabetes (5 –7). To maintain homeostasis, cells rely on various repair mechanisms, with autophagy, a cellular degradation process regulated by the mTOR (or mechanistic target of rapamycin) receptor playing a crucial role (1, 8). Specifically, mitophagy, the autophagic clearance of mitochondria, is essential to preserve mitochondrial function during aging, ensuring the bioenergetic homeostasis necessary for optimal cellular function (1, 9, 10). The accumulation of dysfunctional mitochondria is a hallmark of aging (10 –12). In fact, mitochondrial homeostasis is vital not only for cellular metabolism but also for overall health. Mutations in mitochondrial DNA are associated with a range of adverse phenotypes, including myopathies, neuropathies, diabetes, premature aging, and shortened lifespan. Mitochondrial dysfunction is a common feature of aging, and mTOR downregulation promotes mitochondrial biogenesis and maintenance, thereby contributing to longevity (12).

Aging also fosters a vicious cycle between inflammation and immunosenescence. Increased levels of inflammatory mediators reduce adaptive immune responses, further promoting immunosenescence. As a result, the innate immune cells intensify their functions, releasing more pro-inflammatory mediators, which in turn sustain the low-grade inflammation typical of aging (13).

The discovery that mTOR inhibition can alleviate various aspects of aging and extend lifespan across multiple species has fueled scientific interest in developing mTOR inhibitors to potentially enhance human longevity (1, 8). Rapamycin (RAPA), a well-studied mTOR inhibitor isolated from Streptomyces strains, has shown promise as an immunosuppressant and autophagy inducer. RAPA induces a nutrient-starvation response, enabling cells to conserve resources, which delays the onset of cellular senescence and extends lifespan (2, 9, 14). In aged mice, RAPA treatment doubles B lymphocyte counts in bone marrow, enhancing viral infection response and vaccination efficacy (15). Furthermore, RAPA treatment increases the levels of regulatory T (Treg) cells in the spleen and thymus without impairing their function in vitro and/or in vivo, though it does suppress the CD4⁺ T cell response (16).

Despite its considerable potential, extended use of RAPA can lead to clinical complications, including glucose metabolism disruption, insulin resistance, gonadal atrophy, and cataract formation (15, 17, 18). Additionally, RAPA also improves mitochondrial homeostasis, enhancing metabolic stress adaptation during replicative aging in vitro and in vivo. This adaptation encompasses increased mitochondrial carbon oxidation, maintaining ATP levels and redox balance for bioenergetic requirements of cellular maintenance and proliferation (2). RAPA, by inhibiting mTORC1, indirectly activates the PINK1/Parkin pathway and other mitophagy-related proteins such as BNIP3 and NIX, promoting the recognition and removal of dysfunctional mitochondria and thereby supporting mitochondrial quality Control (19 –21).

Replicative senescence often manifests as a metabolic shift from glycolysis to oxidative phosphorylation, despite increased mitochondrial mass and activity markers, likely due to lysosomal pH elevation from proton pump failure, impairing cellular organelle clearance. The buildup of dysfunctional mitochondria in senescent cells amplifies the production of reactive oxygen species (ROS), leading to cellular damage, with mTOR being central to these metabolic changes. Consequently, inhibition of mTOR by RAPA mitigates metabolic stress and delays the onset of senescence (1).

Negative mTOR regulation, via caloric restriction or RAPA inhibition, has been shown to extend lifespan across a wide range of species, including fungi (22), nematodes (23), arthropods (24), rodents (25), and non-human primates (26). This longevity effect is primarily attributed to induction of autophagy, a vital process that rejuvenates cells (27), maintains protein and mitochondrial homeostasis (22, 28), ensures genetic stability (29), and downregulates inflammasome activity (2, 30). These findings underscore the evolutionary significance of mTOR as a longevity regulator (1).

Aging represents a significant and growing public health concern, underscoring the urgent need for innovative therapeutic strategies aimed at enhancing the well-being of older populations. While mTOR inhibition via RAPA has shown promising anti-aging benefits, its long-term application is hindered by adverse effects on immune function and glucose homeostasis. To address this critical gap, our study evaluates the effects of intermittent low-dose RAPA administration on glucose metabolism, immune modulation, and mitochondrial function in Senescence-Accelerated Mouse Prone 8 (SAMP8) and Senescence-Accelerated Mouse Resistant 1 (SAMR1) mouse models. This approach seeks to maximize the therapeutic potential of RAPA while mitigating its systemic side effects, advancing the development of safer anti-aging interventions.

The SAMP mouse strain is a valuable model for aging research due to its characteristics of accelerated senescence and a shorter lifespan (average of 9.7 months for SAMP8 versus 16.3 months for SAMR1) (36). The SAMP8 strain mimics many hallmarks of human aging, such as mitochondrial dysfunction and chronic inflammation, which are particularly relevant to this study's focus on RAPA's impact on these processes. In contrast, SAMR1 mice serve as a robust baseline for distinguishing aging-independent outcomes (37).

Aging profoundly affects the immune system, leading to immunosenescence, which in turn increases vulnerability to infections, autoimmune diseases, and cancer. Enhancing immune cell function could improve an individual's resilience to both internal and external threats, potentially contributing to a longer lifespan. However, using RAPA as an anti-aging treatment presents a challenge due to its immunosuppressive properties, which inhibit cell proliferation by blocking mTOR. In their study, Arriola Apelo et al. (38) found that intermittent administration of RAPA at 2 mg/kg every five days minimized adverse effects. Based on these results, we employed a similar regimen to investigate whether low-dose, intermittent treatment could ameliorate RAPA-induced immune and metabolic alterations.

Phenotypes of immunosenescence include thymic atrophy, reduced numbers of naïve T cells, increased numbers of memory T cells, and a reversal of the CD4/CD8 ratio, all of which directly influence immune responses in aged individuals (13). In the study by Neff et al. (2013), C57BL/6 mice received encapsulated RAPA at 14 ppm (approximately 2.24 mg/kg/day), and analysis of peripheral blood lymphocyte subpopulations in young and aged animals showed that the frequency of CD4⁺ T lymphocytes declined with age in both Control and RAPA-treated groups. Importantly, RAPA treatment did not reverse this age-associated decline in CD4⁺ T cells (39). This pattern closely parallels our findings in the accelerated aging SAMP8 mice, where a reduction in CD4⁺ lymphocytes was observed in both the thymus and spleen compared to the SAMR1 strain. Similarly, RAPA treatment at a low dose was not able to prevent or restore this loss. These results underscore the importance of examining RAPA's effects on both primary and secondary lymphoid organs, as well as on in vitro lymphocyte proliferation, to better understand its influence on immune aging.

Comparative studies evaluating the effects of RAPA across primary and secondary lymphoid organs highlight distinct tissue-specific responses. For example, Luo et al. (1994) demonstrated that BALB/c and DBA/2 mice treated intraperitoneally with 2 mg/kg RAPA for 14 days developed significant thymic atrophy and decreased thymocyte proliferation, yet spleen mass and cellularity remained unaffected (40). Similarly, Tian et al. (2004) found that a regimen of 1.5 mg/kg RAPA i.p. for 14 days in Wistar rats accelerated apoptosis in thymic CD4⁺CD8⁺ thymocytes but did not decrease splenic cell numbers, although it increased the proportion of CD4⁺CD25⁺ T cells, suggesting functional but not quantitative alterations (41). Lu et al. (2015) further demonstrated that only higher chronic doses of RAPA (1–3 mg/kg/day) reduced cellularity in the thymic and spleen of young rats, whereas lower doses had milder effects (42). In our previous work, we showed that 2 months of low-dose RAPA treatment induced more rapid thymic involution in SAMP8 mice, which exhibited significantly lower thymic cellularity than SAMR1. Importantly, RAPA treatment did not promote thymic atrophy in these animals. In contrast, in the spleen, SAMP8 mice displayed increased CD4⁺ and decreased CD8⁺ splenic T cells, while SAMR1 mice showed the opposite effect (31). However, these alterations were not sustained during chronic exposure, as observed in the present study.

Qu et al. (2007) reported that 1.5 mg/kg/day of RAPA i.p. for 14 days in C57BL/6 mice increased the proportion of splenic CD4⁺CD25⁺FoxP3⁺ Treg cells, despite a reduction in total lymphocyte numbers, indicating differential sensitivity of Treg cells to RAPA (16). In our own study, we observed a reduction in Treg cell percentages in SAMP8 RAPA animals, while SAMR1 RAPA animals showed decreased thymic CD3⁺ cellularity and diminished in vitro lymphoproliferative capacity. These observations align with the partial resistance of Treg cells to RAPA reported by Wang et al. (2011), who used 1.25 mg/kg daily or 3 mg/kg on alternate days in C57BL/6 mice (43), as well as with the broader variability seen in long-term regimens, such as Hurez et al. (2015) using 2.24 mg/kg/day encapsulated RAPA for 12 months (44).

It is important to note that RAPA administration may impact gut microbiota, as shown in recent studies. Hurez et al. (2015) demonstrated that RAPA can modify microbial composition, which may in turn influence immune function, given the bidirectional nature of the gut–immune axis (44). Although our study focused on direct effects of RAPA on glucose metabolism and immune cells, we recognize that the absence of microbiota monitoring is a limitation. Future investigations incorporating gut microbiota analysis will be crucial to fully elucidate the mechanisms underlying the systemic effects of RAPA.

In our study, we observed a reduction in the proportion of FoxP3⁺ Tregs in SAMP8 mice treated with RAPA. Although previous reports (41) indicate that RAPA may accelerate apoptosis in CD4⁺CD8⁺ thymocytes, our methodology, by flow cytometry with LIVE/DEAD™ viability staining, did not allow us to determine whether the observed decrease in FoxP3⁺ cells reflects increased apoptosis, suppressed proliferation, or both.

Beyond quantitative changes, the decrease of CD3⁺ thymocytes and altered CD4/CD8 ratios observed here have broader functional implications in the context of immunosenescence. Thymic atrophy and reduced naïve T cell output are hallmarks of aging, contributing to diminished repertoire diversity and impaired responses to novel antigens (45). Our findings suggest that intermittent RAPA, despite its mitochondrial benefits, may accentuate some of these age-associated immune limitations. This raises important translational considerations, as any clinical use of RAPA in elderly populations would need to balance its anti-aging benefits with potential risks of reduced immune competence.

A study by Aoki et al. (1995) (46) analyzed SAMP8 mice at 9 months of age, which is the same age as the animals in our study at the time of analysis. In their study, SAMP8 mice exhibited increased IFN-γ and decreased IL-2, IL-4, and IL-5 compared to the background AKR/J strain. This was attributed to immunosenescence in SAMP8 mice, resulting in an inflammatory profile and reduced IL-4 production associated with poor antibody responses. While our results differed from Aoki et al. (1995) for most cytokines, both studies consistently showed a significant decrease in IL-4 in SAMP8, reinforcing its association with immune aging and poor antibody responses in this strain. Collectively, these findings underscore that the effects of RAPA on immune aging are shaped by protocol, dosage, animal strain, and underlying immune phenotype.

In addition to differences in inflammatory profiles, aging-related metabolic changes, such as altered glucose uptake, offer further insights into the physiological distinctions between SAMP8 and SAMR1 strains. A crucial aspect of in vivo glucose metabolism is cellular responsiveness to insulin, which is frequently impaired with advancing age. Notably, prolonged RAPA treatment has been associated with the development of insulin resistance and hyperglycemia (39). To address these concerns, we adopted the intermittent dosing regimen proposed by Arriola Apelo et al. (2016) (38), in which RAPA is administered every five days, a strategy shown to effectively mitigate both glucose-related and immunosuppressive side effects. Indeed, in our results, no difference in glucose metabolism was observed following RAPA treatment, except for reduced glucose uptake in the bladder of SAMP8 RAPA animals. The apparent reduction in bladder ¹⁸F-FDG signal in SAMP8 animals treated with RAPA likely reflects urinary excretion rather than metabolic uptake, as the bladder does not actively metabolize glucose. This secondary observation may be linked to renal physiology or urine output and should not be overinterpreted as an effect on systemic glucose metabolism. Furthermore, our results demonstrate that SAMP8 Control animals exhibit a higher basal glucose demand compared to SAMR1 Control, likely attributable to oxidative stress, which can activate compensatory glucose uptake pathways even under low-glucose conditions. This is consistent with the ex vivo findings of Barquissau et al. (2017) (47), who reported increased glucose uptake in muscle from SAMP8 and SAMR1 mice under overnight fasting. Our current study extends these observations to the in vivo context.

Consistent with prior evidence of a chronic low-grade inflammatory state in SAMP8 mice, which is associated with increased oxidative stress and metabolic reprogramming (48, 49), the elevated basal glucose uptake we observed may reflect compensatory glucose transport and mitochondrial substrate use rather than enhanced adaptive immune activity. This supports the concept of inflammaging, where metabolic and immune alterations are partially uncoupled.

Finally, the bioenergetic profile analysis of liver mitochondria revealed no statistical differences in mitochondrial respiration between strains, contrary to expectations that accelerated aging drives oxidative stress and energy production inefficiency (50). This finding can be explained by biological factors: all animals were approximately 9 months old at the end of the experiment, an age at which SAMP8 mice may not yet display overt mitochondrial dysfunction in the liver (51). Moreover, SAMP8 is primarily a model of sarcopenia, and it is well recognized that different organs age at different rates. The liver, in particular, has a high regenerative capacity and cellular turnover, which may delay or attenuate mitochondrial impairment compared with more aging-sensitive tissues such as skeletal muscle (52, 53). However, RAPA improved ATP synthesis in state 3 of mitochondrial respiratory in treated animals from both strains. Similar beneficial effects of RAPA have been observed in skeletal muscle (54), cardiac muscle (55), and even at low doses (0.8 mg/kg) in the muscle and brain of mice (56). This improvement can be attributed to RAPA's role in promoting mitophagy, a crucial process for mitochondrial quality control, a mechanism we did not directly investigate in this study. Furthermore, our results showed that RAPA treatment enhanced respiratory control in SAMR1 animals, indicating improved mitochondrial efficiency in oxygen utilization. This finding aligns with previous research, such as that by Johnson et al. (2013) in a mouse model of Leigh syndrome treated with RAPA (57).

Although our data demonstrate improved mitochondrial bioenergetics and distinct strain-specific immune outcomes, the mechanistic basis of these effects remains to be fully elucidated. Our interpretation that RAPA promotes mitophagy is hypothesis-driven and relies on indirect evidence from the literature, as we did not measure canonical markers of mitophagy such as PINK1/Parkin activation, LC3-II turnover, or mitochondrial DNA integrity. Likewise, the strain-specific immune responses observed, such as reduced FoxP3⁺ Tregs in SAMP8 and decreased CD3⁺ thymocytes in SAMR1, may reflect differences in basal mTORC activity, IL-2 signaling, or epigenetic regulation that were not assessed here. Future investigations integrating direct mitophagy assays, detailed cytokine profiling, and epigenetic analyses will be critical to define the molecular drivers of these RAPA effects and to refine therapeutic strategies based on mTOR modulation.

These results demonstrate that RAPA treatment exerts distinct effects between the strains, impacting not only the immune system but also mitochondrial bioenergetics. These differences may be attributable to a relatively preserved mitochondrial function and metabolic integrity in SAMR1 animals, which experience normal aging and likely exhibit less age-associated cellular damage compared to SAMP8. SAMR1 mice showed a more pronounced immunosuppressive response and greater mitochondrial adaptability to RAPA, which may be due to a higher baseline capacity for bioenergetic and metabolic regulation. This enhanced responsiveness could reflect more efficient mitochondrial dynamics, energy production, and cellular signaling pathways that are possibly maintained in SAMR1 but compromised in SAMP8 as a result of accelerated aging. These findings underscore the need for further research to clarify the mechanisms underlying these strain-specific responses and to explore the broader implications of RAPA treatment in modulating mitochondrial bioenergetics, immune function, and aging-related processes.

While our study provides novel insights into the long-term effects of intermittent low-dose RAPA, several limitations should be acknowledged. On the immune side, analyses were restricted to lymphocyte subsets, proliferative responses, and a limited cytokine panel, without in vivo tests of immune competence (e.g., infection or vaccination models) or functional characterization of specific subsets such as the suppressive capacity of FoxP3⁺ Tregs, antigen-specific proliferation, cytotoxic activity, or effector cytokine production at the single-cell level. On the metabolic side, although ¹⁸F-FDG PET enabled monitoring of tissue glucose handling, we did not perform insulin or glucose tolerance tests, indirect calorimetry, or detailed metabolic flux analyses needed to fully define the adaptive nature of RAPA's effects. Furthermore, although SAMP8 mice are widely used as a model of accelerated aging, we did not include direct measures of lifespan, healthspan, or age-related pathologies, and immunosenescence was inferred primarily from changes in lymphocyte subsets and cytokine production without validation against canonical senescence markers or broader SASP components. In addition, baseline molecular differences in mTOR signaling, mitochondrial function, or immune homeostasis were not directly assessed, and the absence of significant differences in liver mitochondrial respiration between strains likely reflects the true metabolic status of 9-month-old animals, when SAMP8 typically exhibit only moderate hepatic mitochondrial impairment and the liver's high regenerative capacity may delay dysfunction, rather than methodological artifacts. Taken together, these limitations indicate that our findings provide indirect but meaningful evidence of age-associated immune and metabolic modulation. Future studies combining functional immune assays, comprehensive senescence and SASP marker profiling, and longitudinal aging outcomes, as well as testing combinatorial strategies with immunomodulatory adjuvants or metabolic regulators, will be critical to validate and expand the translational implications of chronic intermittent low-dose RAPA.

The findings presented here confirm the potential of RAPA as a modulator of aging-related processes when administered chronically at intermittent low doses, as evidenced by improvements in mitochondrial function and energy metabolism observed across different strains. However, the immunosuppressive effects of RAPA, including reductions in Treg percentages and total CD3⁺ T lymphocytes in SAMP8 and SAMR1, respectively, highlight the challenge of balancing efficacy and safety in therapeutic applications. These results lay the groundwork for refining RAPA-based interventions by focusing on the optimization of dosing regimens to effectively target specific hallmarks of aging while minimizing systemic effects. Further research is essential to tailor these interventions, improve the therapeutic index, and ensure more precise modulation of the aging process.