Calorie Restriction
The last paragraph of the below pdf-availed paper is:
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"In summary, both caloric restriction and protein restriction increase
maximum longevity (by different amounts) and both decrease the rate of
mitochondrial ROS production and oxidative damage to mtDNA in rats. In
contrast, neither carbohydrate nor lipid restriction seem to increase
maximum longevity and neither of them decrease mitochondrial ROS production
and oxidative damage to mtDNA. These results, taken together, indicate that
the decrease in mitochondrial ROS production and mtDNA oxidative damage
observed in caloric restriction is due to the reduced intake of proteins
that occurs during this dietary manipulation, and not to the decreased
ingestion of lipids or carbohydrates. This conclusion is also consistent
with the recent observation that methionine restriction (which also
increases maximum longevity) also decreases mitochondrial ROS generation and
oxidative damage to mtDNA." =====================================================
Sanz A, Gomez J, Caro P, Barja G.
Carbohydrate restriction does not change mitochondrial free radical generation and oxidative DNA damage. J Bioenerg Biomembr. 2006 Nov 30; [Epub ahead of print]
PMID: 17136610
Many previous investigations have consistently reported that caloric
restriction (40%), which increases maximum longevity, decreases
mitochondrial reactive species (ROS) generation and oxidative damage to
mitochondrial DNA (mtDNA) in laboratory rodents. These decreases take place
in rat liver after only seven weeks of caloric restriction. Moreover, it has
been found that seven weeks of 40% protein restriction, independently of
caloric restriction, also decrease these two parameters, whereas they are
not changed after seven weeks of 40% lipid restriction. This is interesting
since it is known that protein restriction can extend longevity in rodents,
whereas lipid restriction does not have such effect. However, before
concluding that the ameliorating effects of caloric restriction on
mitochondrial oxidative stress are due to restriction in protein intake,
studies on the third energetic component of the diet, carbohydrates, are
needed. In the present study, using semipurified diets, the carbohydrate
ingestion of male Wistar rats was decreased by 40% below controls without
changing the level of intake of the other dietary components. After seven
weeks of treatment the liver mitochondria of the carbohydrate restricted
animals did not show changes in the rate of mitochondrial ROS production,
mitochondrial oxygen consumption or percent free radical leak with any
substrate (complex I- or complex II-linked) studied. In agreement with this,
the levels of oxidative damage in hepatic mtDNA and nuclear DNA were not
modified in carbohydrate restricted animals. Oxidative damage in mtDNA was
one order of magnitude higher than that in nuclear DNA in both dietary
groups. These results, together with previous ones, discard lipids and
carbohydrates, and indicate that the lowered ingestion of dietary proteins
is responsible for the decrease in mitochondrial ROS production and
oxidative damage in mtDNA that occurs during caloric restriction.
Introduction
Caloric restriction (CR) slows down the aging rate and increases maximum longevity in laboratory rodents and other animals (Barger et al., 2003). Nevertheless, the basic mechanisms underlying the effects of CR on aging and longevity are still unclear. The oxygen free radical mitochondrial theory of aging is currently receiving considerable support both from comparative and caloric restriction studies (Beckman and Ames, 1998; Barja, 2004a; Ramsey et al., 2004). Many investigations have consistently found that CR decreases the rate of production of reactive oxygen species (ROS) at mitochondria and the steady-state level of oxidative damage to mitochondrial DNA (mtDNA) in rodent tissues (Gredilla and Barja, 2005). Low levels of these two characteristics are also constitutively exhibited by long-lived species when compared to short-lived ones (Barja, 2004b). These results offer a plausible mechanism by which CR could slow down the rate of aging, by decreasing oxidative damage and long-term accumulation of mutations in mitochondrial DNA (Barja, 2004a). However, it has not been clarified if the decreases in mitochondrial ROS production and oxidative DNA damage during CR are due to the reduction in calories themselves or are specifically related to the decreased ingestion of any of the three main energetic dietary components, proteins, lipids or carbohydrates.
Although the general consensus was reached in the last
decade that the life extension effect of CR is related to the reduced ingestion of calories themselves, recent studies and revision of old data suggest that variations in the main individual dietary components can also modulate longevity in rodents (Archer, 2003; Pamplona and Barja, 2006) and insects (Mair et al., 2005, Piper et al., 2005).We have thus initiated a series of studies to clarify the possible effect of restriction of those dietary components on mitochondrial oxidative stress. Two reports on the separate effects of protein and lipid restriction without changing the rest of the dietary components have been already published (Sanz et al., 2004, 2006a).
In the present study we investigate whether restriction of
the third energetic component of the diet, the carbohydrates, can be responsible for the two main effects of CR related to mitochondrial oxidative stress described above. In our dietary protocol carbohydrate ingestion is decreased while the intake of proteins, lipids and other dietary components ismaintained at the same level as in control animals. This avoids confusing the effects of carbohydrate restriction with those of increasing the percentage of other dietary components. In previous studies we have found that CR decreases mitochondrial ROS production and oxidative DNA damage in liver (Gredilla et al., 2001a), heart (Gredilla et al., 2001b), and brain (Sanz et al., 2005a) ofWistar rats. However, while detection of these decreases usually needs long-term restriction in other tissues (Gredilla and Barja, 2005), in the liver the effects are quicker and can be detected after only seven weeks CR (Gredilla et al., 2001a). We have thus selected this rat organ and implementation time for the present study of carbohydrate restriction because it allows performing the experiment (which needs the use of semipurified diets) in a much shorter time. The results obtained are discussed together with those previously obtained after seven weeks of protein (Sanz et al., 2004) or lipid (Sanz et al., 2006a) restriction also in the liver of Wistar rats, as well as with the available results on the effects on protein, lipid or carbohydrate restriction on animal longevity.
Materials and methods
A total number of 14 male Wistar rats of 250 g of body
weight were obtained from the Complutense University Animal Facility and were caged individually and maintained in a 12:12 (light:dark) cycle at 22±2?C. Control animals were fed ad libitum a semipurified diet prepared by MP Biomedicals (Irvine, CA, USA) based on the American Institute of Nutrition AIN-93G diet: 39.7486% cornstarch, dextrinized cornstarch 13.20%, sucrose 10.00%, soy protein 20.00%, soybean oil 7.00%, alphacel (non-nutritive bulk) 5%, mineral mix 3.5%, vitamin mix 1.0%, L-cystine 0.3%, choline bitartrate 0.25% and tert-butylhydroquinone 0.0014%. The diet given to the carbohydrate restricted animals was a modified AIN-93G diet. Its carbohydrate content was reduced while its content in proteins, in fats and in all the rest of its components was appropriately increased. Its composition
was: cornstarch 31.8%, dextrinized cornstarch 10.56%, sucrose 8%, soy protein 26.67%, soybean oil 9.33%, alphacel 6.91%, mineral mix 4.67%, vitamin mix 1.33%, L-cystine 0.4%, choline bitartrate 0.33%, and tert-butylhydroquinone 0.0019%. This diet was given each day to the carbohydrate restricted animals in an amount equal to 75% of the food eaten by the controls. The final result was that carbohydrate restricted animals ingested daily 40% less carbohydrates than the controls while the total amount of protein, fat and the rest of dietary components eaten was the same in control and carbohydrate restricted animals. After seven weeks of dietary treatment ...
Results
The body weight of the carbohydrate restricted animals was significantly lower than that of the ad libitum-fed rats after sevenweeks of treatment (Table 1). Theweight of the kidneys and spleen were also significantly smaller in carbohydrate restricted animals. These differences were no longer present when theweight of these tissueswas referred to body weight. The weight of heart, liver, and brain did not show significant differences between the two groups.
The rate of oxygen consumption of liver mitochondria
was measured without (State 4) and with (State 3) ADP
in the presence of complex I- (pyruvate/malate or glutamate/
malate) and complex II- (succinate) linked substrates.
The addition of ADP strongly increased the rate of oxygen consumption in all cases, indicating tight coupling of the mitochondrial preparations (Table 2). No significant differences in oxygen consumption were found between control and carbohydrate restricted groups with any substrate in either state 4 or 3.
The rate of H2O2 production of liver mitochondria was
measured in control and carbohydrate restricted rats using different combinations of substrates and inhibitors of the respiratory chain (Table 3). Addition of rotenone to pyruvate/ malate supplemented mitochondria strongly increased their rates of H2O2 generation both in ad libitum-fed and in carbohydrate restricted animals, and the same was observed with glutamate/malate in control animals. Antymicin A strongly stimulated the rate of H2O2 generation with succinate as substrate in both control and carbohydrate restricted rats. No significant differences in H2O2 production were found between control and carbohydrate-restricted rats with any substrate or substrate plus inhibitor combination (Table 3). The free radical leak of liver mitochondria did not show significant differences between both groups with any substrate (Table 4).
The level of 8-oxodG was significantly higher (7 to 9 fold
higher) in mtDNA than in nDNA in the liver of both control
and carbohydrate restricted animals (Fig. 1). Similarly
to what was observed for the rate of mitochondrial ROS generation, carbohydrate restriction did not significantly change the level of 8-oxodG in either mtDNA or nDNA.
Discussion
In this investigation the carbohydrate ingestion of male
Wistar rats was restricted by 40% during 7 weeks without recently found that methionine restriction without caloric restriction also decreases mitochondrial ROS production and oxidative damage to mtDNA in rat liver and heart (Sanz et al., 2006b), thus further supporting the possibility that protein and methionine restriction increases rodent longevity through decreases in mitochondrial oxidative stress.
Caloric and protein restriction share many common
effects in addition to life prolongation, including delays in puberty, decreases in growth rate, changes in metabolic rate, boosting of cell-mediated immunity, lowering of cholesterol levels, or decreases in preneoplastic lesions and tumours and lowering of protein oxidation (Youngman et al., 1992). Low protein diets also decelerate glomerulosclerosis in mice (Doi et al., 2001), delay the occurrence of chronic nephropathy and cardiomyopathy in rats (Maeda et al., 1985), and protect rat liver against exposure to toxic chemicals (Rodrigues et al., 1991). A lower (but significant) life extension effect in protein restriction than in CR would agree with the widely held notion that aging has multiple causes. CR could decrease aging rate through decreases in mitochondrial oxidative stress (due to protein restriction) as well as through other mechanisms like, e.g., lowering insulin/IGF-1 signalling (Richardson et al., 2004). This would be consistent with recent findings of a lack of relationship between insulin/IGF-1 signalling and mitochondrial ROS generation (Sanz et al., 2005b), which could be two independent mechanisms lowering aging rate in parallel during CR.
What are the effects of lipid restriction on longevity? Various investigations have discussed whether or not lipids are involved in CR effects, some supporting and some rejecting this idea based on different kinds of end point biochemical measurements and experiments (Masoro, 2000; Barzilai and Gabriely, 2001; Muurling et al., 2002). But very few studies have directly tested the effect of lipid restriction on longevity. An investigation in Fisher rats did not found changes in longevity after lipid or mineral restriction without CR (Iwasaki et al., 1988). Another long-term study performed also in Fisher 344 rats found increases in maximum and medium life span after 40% CR but not after 40% lipid restriction (Shimokawa et al., 1996). These and other investigations led to the conclusion that restriction of calories, but not of fats, slows down the primary aging process (Masoro, 1990). Thus, although available direct information is scarce and mainly limited to a particular rat strain, it seems safe to conclude that lipid restriction does not delay aging (Table 5). If that is indeed the case, it will fit well with the finding that lipid restriction without CR does not decrease mitochondrial ROS production and oxidative damage to mtDNA or nDNA (Sanz et al., 2006a).
Concerning carbohydrates, most of the available investigations have studied the effect on rodent survival after changing the kind of carbohydrate rather than restricting total carbohydrates in the diet. Although information is scarce, it seems that simple carbohydrates like sucrose, glucose or fructose shorten longevity compared to diets containing complex carbohydrates (Archer, 2003). Thus, it has been reported that carbohydrate given either as sucrose or as glucose, compared to starch, decreased the life span of rats (Dalderup and Viser, 1969) and mice (Mlekusch et al., 1996), whereas changing the source of complex carbohydrate from corn to rice did not modify maximum longevity in mice (Yamaki et al., 2005). However, the situation can be more complex since studies in Fisher 344 rats have found that a cornstarch compared to a sucrose-based diet increases both mean and maximum life span when the experiment is performed in ad libitum-fed animals, whereas sucrose is better for mean life span and cornstarch is better for maximum life span when the experiment is performed in CR animals (Murtagh-Mark et al., 1995). Concerning the total amount of carbohydrate in the diet the available information is even more limited and contradictory. One study reported an inverse relationship between total carbohydrate intake and life span in experimental animals (Ross, 1976). However, another study found that increasing the carbohydrate (dextrin) proportion in the diet of male Fisher 344 rats increased their 10th percentile survival by 41 days, a much smaller effect than that of CR which increased the 10th percentile survival in this experiment by 396 days (Khorakova et al., 1990). In any case, such effect would be incompatible with the possibility that carbohydrate restriction increases maximum longevity. Moreover, in several of the 16 studies described above in which protein restriction increased maximum longevity (reviewed in Pamplona and Barja, 2006) the decrease in dietary protein was compensated by corresponding increases in dietary carbohydrate. This is also strongly contradictory with the possibility that carbohydrate restriction increases longevity. Another study did not find changes in the longevity of shortlived autoimmune-prone mice after increasing total dietary carbohydrate under ad libitum feeding conditions (Kubo et al., 1987). Thus, it seems safe to conclude that most of the available information suggests that carbohydrate restriction does not increase rodent longevity (Table 5). This would agree again with the lack of decrease in mitochondrial ROS production and oxidative damage to mtDNA and nDNA observed in our study.
Last edited by Whoa182 : Thu, Dec-14-06 at 15:39.
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. It's been quite a while now. And you haven't let up for an instant. It really is a bit CR evangelical. Why do you bother? Every now and then somebody feels sorry for you and leaps to your defense. But usually even they (Woo being a prime example) just can't help noticing that CR is a very strange eating disorder. 
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