The question addressed in this study was whether modified ADF regimens, which allow some caloric intake on the restricted day, can reproduce global reductions in cell proliferation rates observed with true ADF and classic CR regimens. Our results document, for the first time, that a modified ADF regimen, which allowed for the consumption of 15% of energy needs on the fast day, produced essentially identical reductions in cell proliferation rates as true ADF. Moreover, both true ADF and modified ADF regimens produced similar reductions as classic 25% CR for all cell types monitored (epidermal cells, splenic T cells, and mammary epithelial cells). Interestingly, the modified ADF regimen, which allowed for consumption of 25% of energy needs on the calorie-restricted day, did not result in such beneficial modulations. These results suggest that a dose-response relationship of a nonlinear type exists between the extent of calorie intake on the restricted day of an ADF regimen and the signals responsible for reducing global cell proliferation and that this dose-response relationship may have a thresh-hold character.
We investigated some of the circulating factors that might act as signals to reduce cell proliferation. ADF-100% and CR-25%, but not modified ADF regimens, reduced circulating IGF-1 levels, consistent with the proposed role of IGF-1 in the hypoproliferative response induced by CR (23
). The observation that equivalent reductions in cell proliferation were observed in the ADF-85% regimen without any reduction in IGF-1 levels, however, calls into question the uniqueness of IGF-1 in this regard. It is important to note that these antiproliferative effects in both the true (ADF-100%) and modified ADF (ADF-85%) groups occurred here in the absence of weight loss, suggesting that these outcomes occurred as a result of an energy intake pattern rather than a net negative energy balance. We have previously reported (6
) that ADF-100% regimens result in weight loss, due to an inability of the hyperphagic response to compensate fully for the complete absence of caloric intake on the fast day. It has not previously been shown, however, that any regimen that allows energy intake on the restricted day and no change in body weight could reproduce the effects of CR or true ADF.
Cell proliferation plays a central role in the promotional phase of carcinogenesis (5
). We demonstrate here that modified ADF regimens work just as well as daily CR to reduce cell proliferation rates in a variety of tissue types of both epithelial (mammary and skin) and mesenchymal (lymphocyte) origin. Although the ability of classic CR to reduce cell turnover is well documented (24
), very few studies (6
) have examined the effect of true and modified ADF regimens on this indicator of cancer risk. In our previous study (6
), we showed that modified ADF regimens that allow consumption of 50 and 75% of baseline energy needs on the restricted day, alternating with ad libitum
feeding on the feed day, had very little effect on proliferation rates of prostate epithelial or splenic T cells, whereas the group undergoing a complete 24-h fast (ADF-100%) exhibited markedly reduced proliferation rates (6
). This led us to test the hypothesis that a reduction in caloric intake on the fast day by >50 but <100% could produce beneficial effects. The results here indicate that 15% of energy needs, but not 25%, can be consumed on the restricted day and achieve similar reductions in cell proliferation rates as daily CR and true ADF. It will be important to confirm in longer term carcinogenesis studies that the reductions in global cell proliferation rates shown here result in reduced cancer rates. If this is confirmed, and data can be extrapolated to ADF in humans, it is possible that subjects need not undergo a complete 24 h fast but may instead consume a portion of energy needs on the restricted day and still achieve benefits such as a reduction in cancer risk. If the results with modified ADF regimens shown here in rodents can be confirmed by studies in man, the feasibility of ADF strategies would be considerably enhanced, as true ADF regimens are generally not well tolerated in human subjects (29
These antiproliferative effects occurred in the absence of weight loss in the ADF groups. Based on the hyperphagic response observed on the ad libitum feeding day, the ADF groups were able to maintain and gain body weight by compensating on the feed day for the degree of energy restriction experienced on the fast day. More specifically, the mice in the ADF-75%, ADF-85%, and ADF-100% groups ate 60, 70, and 85% more food on the feed days, respectively, than control mice. Reduced activity levels or resting energy expenditure may also contribute but was not evaluated here. The lack of a requirement for weight loss or changes in body fat mass with ADF regimens makes this approach to dietary restriction potentially attractive for normal weight and mildly overweight subjects. Individuals in higher weight classes might be more appropriate for daily CR, as this diet regimen generally results in weight loss.
Evidence has linked circulating IGF-1 concentrations to risk for cancer. Prospective studies (31
) in humans indicate that individuals with higher plasma IGF-1 concentrations have an increased risk of colon, lung, breast, and prostate cancer. These findings are supported by animal data, which indicate that IGF-1 functions as a mitogen and an antiapoptotic survival factor (34
). In the present study, we show that ADF-100% decreases circulating IGF-1 concentrations to an equal extent as daily 25% CR. These reductions in IGF-1 concentrations showed significant, although modest, correlations with epidermal and mammary epithelial cell proliferation rates. Similar associations between ADF-induced decreases in IGF-1 levels and reduced cell turnover rates were observed previously (6
). Classic CR has also been shown to reduce plasma IGF-1 levels while decreasing tumorigenesis (12
). Interestingly, the decreases in cell proliferation rates observed in the ADF-85% group were not accompanied by significant reductions in IGF-1 levels. Accordingly, the relation between circulating IGF-1 concentrations and the anti-proliferative effects of modified ADF regimens remains uncertain.
Plasma levels of ghrelin have been shown to increase in response to fasting (35
) and prolonged CR (36
). During periods of starvation, ghrelin is thought to promote the resumption of feeding by activating hypothalamic neurons (37
). Ghrelin has also been shown to be a potent stimulator of GH release (39
). These actions are dependent on the octanoyl modification of the third serine residue of ghrelin, which is the form of ghrelin active at the GHSR (39
). Accordingly, we quantified the circulating levels of oct
ghrelin (active ghrelin) as well as des
ghrelin after an 8 h fast in all animals. We found that ADF-100%, but not modified ADF, increased plasma levels of oct
ghrelin, relative to controls. Interestingly, this effect on oct
ghrelin did not occur in the CR group. These preliminary findings suggest that true ADF is a more potent stimulator of ghrelin release than CR during periods of fasting. It may be speculated that the cyclic intake of food in the ADF-100% animals induced patterned release of ghrelin by the stomach in this group. It will be of interest in future studies to measure diurnal changes in ghrelin levels after 24 h of feasting alternated with 24 h fasting.
In summary, our data demonstrate that a modified ADF regimen, consisting of ad libitum feeding on the feed day alternated with the consumption of 15% of energy needs on the calorie-restricted day, decreases global cell proliferation rates to a similar extent as true ADF and daily CR. Over the course of the study, body weight within the ADF groups remained stable, indicating that these antipromotional effects were mediated by signals other than weight reduction or body fat stores. These effects may be mediated in part by decreases in circulating levels of IGF-1, although IGF-1 levels were not reduced in the modified ADF group. Since allowing some food intake on the calorie-restricted day could increase subject compliance and tolerability of ADF regimens, these findings may have practical implications. Direct confirmation of these observations in human subjects will be necessary before the implications for human health or dietary practices can be judged.