Calorie restriction delays disease onset and mortality in monkeys

New study (Colman et al, 2009) in this week's issue of Science on the effect of calorie restriction over a 20 year period in rhesus macaques. Researchers at the Wisconsin National Primate Research Center examined monkeys under 30% CR and controls matched for baseline intake, age, and weight. According to the abstract:

Caloric restriction (CR), without malnutrition, delays aging and extends life span in diverse species; however, its effect on resistance to illness and mortality in primates has not been clearly established. We report findings of a 20-year longitudinal adult-onset CR study in rhesus monkeys aimed at filling this critical gap in aging research. In a population of rhesus macaques maintained at the Wisconsin National Primate Research Center, moderate CR lowered the incidence of aging-related deaths. At the time point reported, 50% of control fed animals survived as compared with 80% of the CR animals. Furthermore, CR delayed the onset of age-associated pathologies. Specifically, CR reduced the incidence of diabetes, cancer, cardiovascular disease, and brain atrophy. These data demonstrate that CR slows aging in a primate species.

Their general findings indicate a clear quantifiable effect of CR in reducing both mortality and age-related morbidities (cancer, cardiovascular disease, metabolic derangements) as well as age-associated loss of gray matter:

Food for thought, though confounding issues such as frequency of eating rather than total amount of caloric intake are not directly addressed.

Fasting and flying

As seen in the science newswire: new sleep research from Cliff Saper's lab at Harvard suggests a possible mechanism to moderate the effects of jetlag: fast before you fly, and eat (early in the day) when you get to your destination. They examined which parts of the circadian circuitry in the brain controls light-entrainable versus food-entrainable circadian rhythmicity, focusing on the hypothalamus:



Using knockout mice carrying a deletion of the clock gene Bma1, which lack circadian rhythmicity, they restored Bma1 function to specific hypothalamic nuclei using viral vector injection and show that different brain regions control light (suprachiasmatic nuclei) and food (dorsomedial hypothalamic nucleus) entrainable rhythmicity.

Figure 2 from Fuller et al. 2008. Mice heterozygous for Bma1 show normal food entrainment (A), seen as a rise in body temperature before feeding (gray bar), as well as light entrainment, seen as elevated body temperature during the presumptive dark cycle (12-24h). Both types of rhythmicity are abolished in -/- mice (B), but reintroduction of Bma1 to the DMH restores the preprandial increase in body temperature (C).

The general idea is that fasting induces a state where food consumption is prioritized, causing the body to entrain to periods of food availability. In the authors' words:

For a small mammal, finding food on a daily basis is a critical mission. Even a few days of starvation, a common threat in natural environments, may result in death. Hence, it is adaptive for animals to have a secondary "master clock" that can allow the animal to switch its behavioral patterns rapidly after a period of starvation to maximize the opportunity of finding food sources at the same time on following days.

In actuality, the fact that circadian rhythms are entrainable by food availability was already known (see Krieger 1974; more recently Gooley et al 2006 implicated the dorsomedial hypothalamic nucleus in the process), so the idea that fasting can prevent jetlag is newer to the news media than to the field. However, the positive identification of specific brain region control suggests mechanistic sources for the separability of different kinds of circadian entrainment.

Fasting and your body temperature

Fasting slows down your metabolism in order to reduce energy expenditure (Wang et al., 2004), which may be one of the reasons that animals live longer on dietary regimens such as caloric restriction or intermittent fasting (Liu & Walford, 1972; Rikke & Johnson, 2004; Salerian & Saleri, 2006). Under these regimens metabolic slowing manifests itself in a number of ways, usually a reduction in daily activity and a lower body temperature (Rikke et al., 2003; Severinsen & Munch, 1999). Lane and colleagues (1996) showed that caloric restriction in rhesus monkeys results in a rapid reduction in body temperature, and that the reduction endures for the duration of caloric restriction (in this case years).Humans under caloric restriction also exhibit reductions, albeit smaller, in body temperature. Heilbronn and colleagues (2006) randomized subjects into 1 of 4 groups for 6 months: (1) control (weight maintenance diet); (2) calorie restriction (25% calorie restriction of baseline energy requirements); (3) calorie restriction with exercise (12.5% calorie restriction plus 12.5% increase in energy expenditure by structured exercise); and (4) very low-calorie diet (very low-calorie diet [890 kcal/d] until 15% reduction in body weight, followed by a weight maintenance diet).
Group (3) is particularly interesting, showing that even relatively mild caloric restriction (a degree that can easily occur while intermittently fasting) decreases body temperature. So reducing body temperature is a common response to dietary restrictions, and together with decreases in physical activity are the largest contributors to reducing energy expenditures (reductions in basal metabolism occur, but apparently contribute relatively little).

Evidence suggests that the severity of the dietary restriction dictates the degree of temperature reduction. Rats subjected to total starvation versus caloric restriction (75%, which is quite severe) experienced a significantly greater reduction in body temperature for the duration of restriction, with temperatures in both groups normalizing once ad libitum feeding was reinstated (Severinsen & Munch, 1999).
I haven't found any primate data on body temperature under intermittent fasting, but mice exit torpor, which is a reduction in body temperature during inactive parts of the diurnal cycle, when intermittently fed double and triple rations (Rikke et al., 2003; see also Wan et al., 2003 for body temperature reductions under a more conventional alternate day fasting regimen). Presumably, part of this is due to the caloric expenditure associated with digesting a large meal.

The data also suggest, at least in this mouse strain, that when torpor is re-entered during the fast between meals, that the decrease in body temperature is particularly dramatic.

It's tempting to infer that caloric restriction and/or intermittent fasting cause reductions in body temperature which in turn causes the prolonged lifespans associated with both these dietary regimens. However, a recent experiment using genetically-engineered mice clearly shows that this cannot be the whole story. Conti and colleagues (2006) created transgenic mice where the preoptic area of the hypothalamus, which regulates body temperature, was warmer than normal. This tricked the brain into reducing body temperature by roughly half a degree Celsius, within the range attainable by dietary restrictions or meal patterning. These mice lived 10-20% longer than wild-type mice (in the figure black=wild-type, red=mutant).
The increase in lifespan occurred despite the fact that the transgenic mice ate as much as the normal mice, and actually weighed slightly more. These results indicate that a reduction in body temperature can lengthen lifespan independently of caloric restriction or intermittent fasting. It will be interesting to see whether calorically restricting or intermittently fasting these transgenic mice will lead to even longer lifespans.

I suspect that in practice the wide variation in fasting regimens results in wide variation in the magnitude of body temperature reductions. These factors probably interact with variations in the degree to which individual metabolisms react to dietary restrictions and meal patterning. I would venture to guess based on the evidence that people subjecting themselves to more severe caloric restriction or longer fasts (more severe caloric restriction on a local time scale) will experience the greatest reductions in body temperature. It's unclear to me whether this translates to impaired cold tolerance, although some first-hand reports suggest that it might.

Intermittent fasting or caloric restriction?

Intermittent fasting and caloric restriction are associated with many of the same benefits (Mattson, 2005). In trying to sort out the differences between these protocols, people will often treat IF and CR as if they are completely different things. It's not clear to me that they are entirely dissociable. I've previously mentioned that it is common for intermittently fasted rodents to eat slightly less than ad libitum controls. This varies from study to study and with the duration of dietary restriction (as does the degree of bodyweight difference between IF and control animals). The data in the figure below are from rats (Leveille, 1972) and mice (Anson et al., 2003), respectively.

It's worth noting that the C57BL/6 strain of mice (right panel) are intermediate (relative to other mouse strains) in terms of how much less IF mice eat than controls (Goodrick et al., 1990). And while Anson et al. (2003) claim a dissociation between IF and CR, the truth is that their animals were calorically restricted for much of the study. The question in my mind is whether both dietary protocols tap into the same underlying mechanism? Or can IF and CR interact to produce better than predicted results? The problem is that IF involves some CR and CR involves some IF. The following quote is from Leveille's paper, and is worth reproducing. Note that the term "meal-fed" refers to an intermittent diet where access to food is provided for only 2 hours each day and "nibbling" refers to ad libitum food access.

The results of the longevity study (experiment A) are difficult to interpret. Although the meal-fed animals had a significantly longer life-span than the nibbling rats the difference cannot be ascribed to meal-eating, per se. In addition to the metabolic differences between the meal-fed and nibbling rats there existed a significant difference in body weight and presumably in body fat. Thus these results can be interpreted as supportive of the classic experiments of McCay et al. (21, 22) showing that rats whose food intake was restricted had a significantly longer life-span. However, it is important to note that in McCay's studies as well as other similar studies (23,24) the restricted animals were very likely "meal-fed." It has recently been noted that restricting rats to 80% of ad libitum intake quickly results in a shift in eating pattern in which the animals ingest their daily food allowance within a 2-hour period (25). Thus one cannot determine unequivocally whether the increase in life span is due to the reduced food intake or to the meal pattern.

Masoro (2004) makes a similar point, and while his earlier study (Masoro et al., 1995) attempts to tease apart IF and CR, even he admits that the evidence does not conclusively eliminate the role of meal patterning. It seems that studying the meal patterning of individual animals within days may help to clarify this issue. This may also bear on the confusing results from human studies, since meal patterning is often not reported and maybe not even recorded.

Intermittent fasting facilitates learning

Several very interesting papers on the physiological changes that may underlie the benefits of intermittent fasting have been published recently. In rodents, intermittent fasting (typically implemented as alternate day fasting) results in numerous physiological changes that are correlated with disease reduction, increased stress resistance, improved insulin sensitivity and upregulation of neuroprotective trophic factors (reviewed by Mattson, 2005; Varady & Hellerstein, 2007). While many IF studies have used relatively short dietary interventions, a recent study by Fontán-Lozano and colleagues (2007) highlights the intriguing possibility that long-term intermittent fasting can lead to improved cognitive function. In a nice series of experiments, these authors demonstrate that fasted mice exhibit improved learning and memory compared to controls (fed ad libitum), and they go on to provide evidence for the underlying mechanism for this cognitive improvement.

Fontán-Lozano et al. mice placed on an alternate day fasting (ADF) regimen for 6-8 months (starting post-natal week 9, the average lifespan of a mouse is about 2 years). Mice on an ADF diet will typically eat more food on their feed days to compensate for the fasting day, and after some adjustment period will eat a bit less than twice as much food on their feed day as a normal mouse would on an typical diet. So in addition to fasting every other day, there is usually some mild caloric restriction involved. It's well-documented that ADF mice live longer than controls.



Survival distributions (n=40) of male C57BL/6J mice fed ad libitum (left curve) or every other day (ADF, right shifted curve). Souce: Talan & Ingram, 1985




ADF mice will also typically weigh less than control mice (Anson et al., 2003), although the ADF mice in Fontán-Lozano et al.'s experiments weighed the same as control mice. This might be due to the longer time spent on the ADF diet, although there are mouse strain differences in addition to diet duration differences (Goodrick et al., 1990). Fontán-Lozano et al. used a simple battery of behavioral tests to assess the learning and memory capacities of ADF mice. They found that compared to control mice, ADF mice learned faster (in a motor learning task, operant food reward task, and Pavlovian eyeblink conditioning task) and were better able to discriminate novel from familiar objects from briefer presentations.

Subsequent physiological experiments revealed that ADF mice exhibited increased theta-band activity in the hippocampus. Explorations of synaptic plasticity revealed that ADF mice exhibited enhanced paired-pulse facilitation at the CA3-CA1 synapse. Moreover, LTP could be elicited at the Schaffer's collateral–CA1 synapse using fewer high-frequency stimulations. These results are interesting since it is a well-accepted hypothesis that learning and memory are based on modifications of synaptic strength among neurons, an idea that goes at least back to Donald Hebb (1949).

A possible mechanism for the observed potentiation of synaptic plasticity is suggested by the observation that ADF mice also show changes in NMDA receptor subunit composition; this glutamate receptor is critical for many types of synaptic plasticity, and various isoforms exist which are composed of different protein subunits. It's known that the relative expression of NR2B subunits change over the course of a rodent's life; the fraction of NR2B subunits declines (and NR2A increases) in adulthood (Moyner et al., 1994). Fontán-Lozano et al. noted increased expression of NR2B NDMAR subunits in the hippocampus and perirhinal cortex of ADF mice.

Photomicrographs and immunohistochemical analyses of the NR2B expression pattern in the hippocampus of IFD (ADF) and control (ad libitum, AL) mice. The graphs represent the densitometric analysis of NR2B expression in the different areas of hippocampus (n = 5 animals per group in all tests). s luc, Stratum lucidum; mol, molecular layer; l mol, lacunosum moleculare layer; Molec, molecular layer; Lac mol, lacunosum moleculare layer; O.D., optical density. ***p ≤ 0.001. Source: Fontán-Lozano et al., 2007

Remarkably, the behavioral improvements as well as the synaptic enhancements seen in ADF mice are returned to control levels when these mice are administered a NR2B antagonist, which strongly suggests that the relative increase in NR2B subunit expression is responsible for arresting the cognitive decline that accompanies natural aging. This is reminiscent of the genetically-engineered smart mice created by Joe Tsien's group (Tang et al., 1999).

The causal mechanisms underlying the changes in NR2B expression are still unknown. Interestingly, exercise increases NR2B subunit expression in the hippocampus (Farmer et al., 2004), and there is some evidence that ADF can increase basal levels of activity (Carlson & Hoelzel, 1946). Whether there is a link between fasting-induced increases in activity and NR2B subunit expression is an important question for future research.

One thing to keep in mind is that enhanced synaptic plasticity may not necessarily be a good thing. Presumably, there was some selective pressure that led to downregulating NR2B subunit expression with age. It's not clear what this might be, but it may turn out that increased NR2B subunit expression is actually maladaptive once we figure out what the selective pressure was (is, it may still be acting?).