Calorie restriction induces mitochondrial biogenesis and bioenergetic efficiency

Results and Discussion

To investigate whether the CR-dependent decrease of mitochondrial ROS production is reproduced in vitro, HeLa cells, FaO cells, and primary hepatocytes were incubated in the presence of serum from rats submitted to long-term CR (40% for 6–12 months) and compared with those incubated with serum from age-matched, ad libitum (AL)-fed rats (18). Results from this in vitro model of CR showed a clear decrease of intracellular fluorescence signals for ROS forms as determined by carboxy-2′,7′-dichlorodihydrofluorescein diacetate (cH₂DCFDA) and dihydroethidium (Het) (Fig. 1A). Thus, a significant decrease in ROS production in CR cells was observed when compared with cells grown in AL serum (Fig. 1B). The decrease of ROS production could not be attributed to changes in antioxidant enzyme expression as demonstrated by the levels of both catalase or Cu/Zn SOD in whole cell extracts (Fig. 1C). These data obtained by our in vitro system confirm the previous results showing decreased ROS production in mitochondria induced by in vivo CR experiments (6, 8, 20). Our results contradict the recent results of Lambert and Merry (16) showing that CR did not affect bioenergetics or ROS production in intact rat hepatocytes isolated from CR rats. This discrepancy may be caused by differences in the dietary regimen and methods used for ROS determination in both studies but also because we have treated cells in vitro with CR serum, whereas the hepatocytes in the Lambert and Merry study were isolated from CR rats and grown in Hanks’ balanced salt solution (HBSS).

Because the mitochondrial membrane potential (ΔΨm) is the central bioenergetic parameter controlling the generation of ROS (1), we examined the ΔΨm signal by using 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidalolylcarbocyanine iodide (JC1) in HeLa cells treated with either CR or AL sera (Fig. 1D). After 48 h, ratiometric analysis of JC1-aggregated forms vs. the free form demonstrates that a lower ΔΨm in these cells was confirmed by direct visualization by confocal microscopy. Then, incubation with CR serum seems to decrease ΔΨm in these cells. Our in vitro results confirmed previous in vivo data showing a CR-dependent decrease of ΔΨm in liver mitochondria caused by decreased mitochondrial respiration together with increased proton leak (20). It has been demonstrated that the probability of electron leakage to form superoxide increases at high ΔΨm (1, 21, 22). Our results show that CR slowed down both ΔΨm and ROS. Do these findings necessarily mean a lower rate of O₂ consumption or a lower ATP production in CR cells? Based on previous studies of basal metabolism of rodents measured as oxygen consumption, it is expected that short-term CR will reduce metabolic rate, but that in long-term measurements, the metabolic rate of CR animals will not differ significantly from that of AL controls because of changes in body composition (23, 24). In the current in vitro analysis of CR serum-treated cells and isolated hepatocytes, the levels of ATP (Fig. 1E) or the ATP/ADP ratio (Fig. 4, which is published as supporting information on the PNAS web site) were not significantly different compared with AL controls. Interestingly, we observed that oxygen consumption compared with AL conditions was reduced by 35–40% in a variety of cells grown in CR serum and in freshly isolated primary hepatocytes from 1-yr-old CR rats (Fig. 1F). Part of this decrease in oxygen consumption could be caused by the minor ROS production by mitochondria together with a lower respiration rate (15).

Thus, our results support the proposal that CR produces very efficient electron transport through the respiratory chain, that is, equivalent ATP production under conditions of lower oxygen consumption and ROS production. This change in mitochondrial efficiency could attenuate molecular and cellular damage resulting from oxidative stress and thus reduce the rate of aging at cellular and organismic levels (25, 26). Our results agree with previous findings in isolated mitochondria from liver from rats fed under CR conditions that show lower respiration and higher proton leak at the same time (15). This fact would explain the lower oxygen consumption and the decrease of ΔΨm at the same time. However, recent studies have reported the reduction of mitochondrial ROS production by the decrease of proton leak and a higher ΔΨm in CR mitochondria (16). Both studies are based on in vitro analysis of isolated mitochondria. Our cytometrical approach permits us to determine ΔΨm and ROS in the living cell environment without isolation procedures and buffer disturbances that could have impacted the conclusions drawn from past studies.

The next question to ask then is how does the cell increase this respiratory efficiency? We hypothesize that CR supports efficient respiration by increasing the number of low potential mitochondria (state 3). To investigate this hypothesis, we incubated HeLa cells, FaO cells, and rat primary hepatocytes in sera obtained from rats on either CR or AL diets and analyzed the cells for different mitochondrial markers after this exposure. HeLa cells incubated with CR serum showed a significant increase of mitotracker green FM (MTG) signal, indicating the increase of mitochondrial mass (Fig. 2A). This fact was confirmed by using the nonyl acridine orange (NaO) probe, which specifically binds to cardiolipin, a mitochondrial-specific phospholipid and is widely considered a mitochondrial mass marker (Fig. 2B). Immunolocalization of both cytochrome c and cytochrome c oxidase in CR sera-treated HeLa cells also confirmed the increase of mitochondria compared with AL-treated cells (Fig. 2B) and the specificity of MTG and NaO staining as determined by the mitochondrial staining pattern (Fig. 2B). Both FaO cells incubated with CR serum and primary hepatocytes isolated from rats fed under CR long term also showed a significant increase of cardiolipin-dependent NaO staining signal (Fig. 2C), indicating that this CR effect seems to be independent of cell type.

Citrate synthase activity is also considered a biomarker of mitochondrial mass and function. Reduced activity of this enzyme has been observed in livers from aged AL rats and is attenuated by CR (27). Activity of citrate synthase was increased significantly in CR sera-treated cells compared with AL conditions and in CR liver compared with AL tissue (Fig. 2D).

To confirm an actual increase in the number of mitochondria, we next conducted an EM analysis of mitochondria in liver tissue obtained from old AL and CR rats. We could detect a significant increase in the number of mitochondria in this tissue (Fig. 2E), which was quantified from EM micrographs as represented in Fig. 2F. Thus, based on a variety of assays to determine mitochondrial mass in different cell types, the results would support the hypothesis that CR increases the number of mitochondria per cell.

Taken together, these results support the conclusion that a larger amount of low-potential mitochondria would maintain a balanced respiratory chain activity fully capable of handling reduced oxygen consumption while maintaining ATP synthesis but with a lower ROS production. In fact, the mitochondrial protein density in liver, which can be interpreted as a marker of mitochondrial mass, is increased in CR rats without any change in the respiration rate (17). All of these results are in agreement with the fact that CR reduces body temperature in mammals (28), which may be caused by increased coupling of oxidative phosphorylation to ATP synthesis (29). The increase of CR-dependent low-potential mitochondria would explain these previously well established observations.

Having established that CR can up-regulate mitochondria and improve metabolic efficiency, questions emerge regarding what processes are involved in this up-regulation. Mitochondrial biogenesis is a highly regulated process operating through peroxisome proliferation-activated receptor (PPAR) coactivator 1 α (PGC-1α)-dependent nuclear respiratory factors (NRFs). Interestingly, recent reports suggest a role for Sir2 homolog (SIRT) in the regulation of PGC-1α during CR and fasting (13, 30, 31). We have previously shown an induction of SIRT1 in cells and tissues under CR and stress conditions (18, 19). To investigate how CR can induce a greater number of low-potential mitochondria, we determined the expression of mRNA of NRF1 and NRF2, which control the expression of nuclear genes that codify most of the subunits of mitochondrial complexes (32–34), and PPARα, another factor that would contribute to mitochondrial biogenesis by the activation of the fatty acid oxidation pathway (35). The expression of the respective mRNAs was determined by RT-PCR in HeLa cells incubated for 24 h with both AL and CR serum. Both NRF1 and NRF2βγ transcripts were significantly increased in CR serum-treated cells compared with AL serum-treated cells; whereas, NRF2α did not show any significant change of expression (Fig. 3A). PPARα was not induced by CR serum treatment in vitro (Fig. 3A), and these results agree with previous ones showing that this factor is not activated in rat kidney and liver by short-term CR treatment in vivo (36, 37). On the other hand, both induced factors, NRF1 and NRF2, are involved in mitochondrial biogenesis under conditions that promote increased respiration (34, 38). In both cases, NRF1 and NRF2 depend on PGC-1α transcriptional coactivator (33), which is up-regulated by both short- and long-term CR (36). In our hands, in vivo expression of PGC-1α and PPARα were elevated in livers from 1-year-old CR rats compared with AL-fed rats (Fig. 3B). In agreement with this finding we have shown previously an activation of PGC-1α and some of its downstream targets in liver of CR rats ranging from 12 to 24 months of age (36).

Several of the effects of CR serum on cultured cells have been linked to low levels of insulin. In fact, the resistance of cells to heat shock and the induction of SIRT1 expression in cells cultured in the presence of serum from rats fed under CR conditions were inhibited by the addition of insulin to the culture medium (18, 19). In our hands, the addition of insulin to CR-serum cultures also inhibited the induction of NRF1 and NRF2βγ mRNA (Fig. 3C), suggesting that the insulin-dependent pathway is involved in the regulation of the mitochondrial changes induced by CR serum.

In numerous experiments, the SIRT1 pathway has been shown to be involved in the antiaging mechanisms of CR, particularly in the protection against stress (13, 19). The CR mechanism identified in the current investigation differs from that induced in vitro in response to fasting and pyruvate wherein SIRT1 deacetylates PGC-1α and induces the expression of gluconeogenic genes but not mitochondrial genes (31).

The antiaging effect of CR in mammals is a multidimensional phenomenon that must be highly regulated (39). The in vitro model of CR has helped to clarify the SIRT1-dependent regulatory pathway that enhances stress resistance in a wide variety of organisms (18, 19). Another basic aspect of the antiaging effects of CR is a decrease of endogenous oxidative stress production mainly in mitochondria, which would prevent the accumulation of damage (6, 8, 40). Our current study demonstrates that this effect can be caused by a PGC-1α-dependent mechanism that increases mitochondrial mass. These mitochondria show a low-membrane potential, under lower oxygen consumption without diminishing ATP production, and generate lower levels of ROS, thus attenuating damage caused by endogenous oxidative stress and maintaining a balanced respiratory chain. PGC-1α or SIRT1 are critical to regulate this response. As evidence for their role, we disrupted expression of PGC-1α or SIRT1 by using small inhibitory RNA and observed an attenuation of decreased oxygen consumption in CR serum-treated cells (Fig. 5, which is published as supporting information on the PNAS web site).

These in vitro results provide strong evidence to support how CR might exert its antiaging effects in vivo (29). We propose then that CR activates diverse regulatory pathways that greatly enhance stress resistance via the SIRT1 pathway (19) and markedly improve bioenergetics through the activation of the PGC-1α pathway.

Methods

Supplementary Material