3 times per week feeding versus CR

[Guest guest]

Hi All,

This experiment has the Medline abstract:

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?

cmd=Retrieve & db=pubmed & dopt=Abstract & list_uids=15613681

http://tinyurl.com/4lmyh

Hsieh EA, Chai CM, Hellerstein MK.

Effects of Caloric Restriction on Cell Proliferation in Several

Tissues in

Mice: Role of Intermittent Feeding.

Am J Physiol Endocrinol Metab. 2004 Dec 21; [Epub ahead of

print]

PMID: 15613681 [PubMed - as supplied by publisher]

Excerpts from the available pdf are below.

Of note,

1. The definition of the different treatments of the mice is

highly complex.

2. The body weights were greatly affected by the use of

intermittent feeding versus daily feeding versus continuous

feeding, as illustrated by " Non-significant differences in

body weight between mice with the same

caloric intake but fed by different feeding

patterns may be due to the presence or absence

of food in the stomach during weighing " .

3. Searching the text for " lifespan " identifies many

shortcomings of the present study hinging on there being

no longevity data for the mice in these experiments, in

my opinion.

4. Experiments on cells grown in culture may not reflect

the results of experiments on pathology in the animal,

as for examining cell cultures for their proliferation rates

as a proxy for examinations in animals for cancer.

5. Examining the effect on cancer rates using intermittent

feeding may merit further studies, even in humans.

Introduction

Caloric restriction (CR) ... A range of 30-70%

extension of maximal lifespan has been achieved using variations on

CR regimens (45),

including both early- and adult-onset CR (44, 46). CR also exerts a

number of other

beneficial health effects, including reduced carcinogenesis, enhanced

insulin sensitivity,

and reduced cardiovascular disease risk (15). The inhibitory effect

of CR on

carcinogenesis is of particular interest, as CR effectively inhibits

spontaneous tumor

formation as well as neoplasias in knockout/transgenic models of

cancer and chemically-induced

tumorigenesis (18, 19, 45). ...

... Materials and Methods

Mice and CR regimens. For all studies, 7-week-old female C57BL/6J

mice

.... were fed a semi-purified AIN-93M diet ad libitum

.... Studies were then started at 8 weeks of age. 3 studies were

carried out.

Study #1: Time course of CR effects (Figure 1A). A 33% CR diet was

fed for varying

durations of time to the 3 treatment groups (n = 8 per group): 2

weeks CR (2W), 1 month

CR (1M) or 2 months CR (2M). 2W received a control diet for 6 weeks

prior to onset of

the CR diet, and 1M received a control diet for 1 month prior to

onset of the CR diet, so

that the ages of all 3 groups were matched at the end of the

experiment. Accordingly, all

3 groups were sacrificed at 16 weeks of age. Two different control

groups were also used

(n = 8 per group): ad libitum fed mice (AL) and mice fed 95% of ad

libitum intake (C95).

These animals were also sacrificed at 16 weeks of age. These control

groups represent the

different types of control groups that have commonly been used in CR

studies

demonstrating lifespan extension or reduced carcinogenesis (1, 37,

41, 44, 46). One

additional group of mice (n = 4) was placed on CR for a longer period

of time (3 months,

3M), also starting at 8 weeks of age. This group was sacrificed at 20

weeks of age.

During non-CR periods, the treatment groups were maintained on the

C95 diet regimen.

During CR periods, mice were fed 67% of C95 intake, or about 64% of

AL intake, as

previously described (37). The CR and C95 groups were fed 3 days a

week, such that 2-times

the daily allotment was given on Mondays and Wednesdays, and 3-times

the daily

allotment was given on Fridays, as has been commonly used in previous

CR studies (13,

37, 42, 44, 46). AL and C95 mice were fed a semi-purified AIN-93M

diet, while CR

mice were fed an enriched AIN-93M diet that contains 33% more

protein, minerals, and

vitamins per gram of diet (Bio-Serv). All mice were housed

individually. Food intake and

body weight were monitored weekly.

Study #2: Refeeding effects (Figure 1B). The time course of

response to refeeding was

also studied. Mice received a 33% CR diet for 1 month and were

subsequently given a

C95 diet (n = 8 per group) for either 2 weeks of refeeding (R2W) or 1

month of refeeding

(R1M). The CR diet for the R2W group started 2 weeks into the study

(10 weeks old)

while CR diet for R1M started immediately (8 weeks old), so that both

groups were

sacrificed at 16 weeks of age. One additional group of mice received

a 33% CR diet for 1

month and was refed for a longer period of time (2 months, n = 4)

(R2M). These mice

were sacrificed at 20 weeks of age. All mice were housed

individually. Food intake and

body weight were monitored weekly.

Study #3: Intermittency of feeding study. The role of intermittent

food intake was also

investigated. 3 groups of mice were put on a 33% CR diet,

administered via different

feeding protocols (n = 6 per group): intermittent feeding of 3 times

per week (CR-INT),

as described above (37); daily feeding (CR-DF); or continuous feeding

via an electronic

pellet dispenser (CR-PD). 3 other groups of mice were fed 95% of ad

libitum diet via the

same 3 feeding protocols (n = 6 per group): intermittent feeding of 3

times a week (95-INT);

daily feeding (95-DF); or continuous feeding (95-PD). The 95-INT, 95-

DF, and

95-PD groups were also compared with a group fed ad libitum (AL)

concurrently.

Intermittent feeding was as described above, with 2 times the daily

allotment given on

Mondays and Wednesdays, and 3 times the daily allotment given on

Fridays. Mice fed

daily were given their food allotment for each day, every morning.

The amount and type

of diet (33% enriched or standard AIN-93M) depended on whether the

mice were in the

CR groups (CR-INT, CR-DF) or the control groups (95-INT, 95-DF).

Continuously fed

mice were housed in cages containing an electronic pellet dispenser

that delivered a 45

mg pellet of AIN-93M diet (standard for 95-PD, 33% enriched for CR-

PD, Bio-Serv),

into the cage every 20 to 30 minutes, depending on the caloric

intake. All mice were

housed individually. Food intake and body weight were monitored

weekly. Mice were

sacrificed at 12 weeks of age, after 4 weeks of treatment.

...

Results

Study #1: Time course

Food intake and body weight.

On average, AL mice consumed 22 grams of food per

week. Therefore, C95 mice were fed 21 grams of food per week and CR

mice were fed

14 grams of food per week. The body weight of CR mice dropped

initially by as much as

30% but stabilized over time (Figure 2A). Mice then gained weight on

CR diets.

Time course. When compared to AL, proliferation of epidermal cells,

MECs, and T-cells

was significantly decreased in the CR groups at all time points

studied (Figures 3A-3C).

When compared to C95, in contrast, cell proliferation in all tissues

was not significantly

decreased until 1 month of CR, after which the response was again not

significant. At 1

month of CR, the time of greatest effect of CR, epidermal cell

proliferation was 61% of

that in AL mice and 76% of that in C95 mice. MEC proliferation was

only 11% of AL

and 29% of C95 mice values at 1 month, while T-cell proliferation was

41% of that in AL

mice and 57% of that in C95 mice.

Differences between C95 and AL control groups.

C95 mice exhibited statistically

significantly lower cell proliferation than AL mice in all tissues

examined (Figures 3A-3C).

After 2 months on respective diets, epidermal cell proliferation in

C95 mice was

81% of that in AL mice, MEC proliferation was 37%, and T-cell

proliferation was 71%.

Thus, CR exerted significant effects on proliferation of all 3 cell

types studied, but C95

also had a potent impact that appeared to account for at least part

of the CR effect.

Estrus cycle. Based on cell morphology analysis of vaginal cells

collected from 1M and

C95 mice, CR mice were anestrus (not cycling), while C95 mice were

actively cycling.

The marked reduction in MEC proliferation in the CR groups might

therefore in part be

explained by reduction in reproductive hormone levels (31), but the

substantial effect

observed in the C95 groups exclude this as the primary cause of

reduced MEC

proliferation.

Study #2: Refeeding

Food intake and body weight.

As in study #1, AL mice consumed about 22 grams of

food per week. During the CR phase, mice were therefore fed 14 grams

of food per week,

and during the refeeding phase, mice were fed 21 grams of food per

week. Refeeding

resulted in a rapid gain of lost weight (Figure 2B). Body weights of

CR mice had caught

up to the body weights of C95 mice by the end of the study, despite

the 1-month period

of CR.

Time course of refeeding effects.

When compared to the C95 control group, cell

proliferation in all tissues rebounded to a significantly higher rate

after 2 weeks of

refeeding, persisting through 1 month of refeeding but normalizing

after 2 months of

refeeding (Figures 4A-4C). When compared to the AL group, cell

proliferation in all

tissues was no longer significantly different after 2 weeks of

refeeding. Subsequent

comparisons revealed tissue-specific differences. After 1 month of

refeeding of the C95

diet, T-cell proliferation rate was statistically higher than AL

levels; this was normalized

after 2 months of refeeding. MEC proliferation was significantly

lower than AL levels

after 2 months of refeeding of C95 diet, consistent with the

observation that MEC

proliferation was lower in C95 mice than in AL mice (Figure 3B).

Study #3: Intermittency of feeding

Food intake and body weight.

Throughout this study, all groups of CR mice were fed

14 grams of food per week, and all groups of control mice were fed 21

grams of food per

week. All mice gained weight on their diets (Figure 2C). Non-

significant differences in

body weight between mice with the same caloric intake but fed by

different feeding

patterns may be due to the presence or absence of food in the stomach

during weighing.

Feeding intermittency effects among groups of CR mice.

In the three tissues studied,

intermittency of feeding (i.e. food given 3 times per week) had no

additional effect

compared to daily or continuous feeding on cell proliferation when CR

was present

(Figures 5A-5C).

Feeding intermittency effects among groups of control mice.

There was lower cell

proliferation in all tissues of the group fed intermittently at 95%

of ad libitum diet (95-INT)

compared to daily feeding (95-DF), continuous feeding (95-PD), or ad

libitum

feeding (AL), although not all comparisons were statistically

significant (Figures 6A-6C).

MEC proliferation was significantly lower in 95-INT than in 95PD

mice, while T-cell

proliferation was significantly lower in 95-INT mice compared to 95-

DF and 95-PD

mice. Epidermal and T-cell proliferation rates in AL were not

statistically different from

95-DF or 95-PD but were significantly greater than 95-INT. An

intermittent feeding

regimen (i.e. food given 3 times per week) therefore caused

significant reductions in cell

proliferation rates compared to isocaloric diets fed by more constant

patterns.

Discussion

We demonstrate here the application of a relatively simple method for

measuring cell

proliferation in multiple tissues in mice. By this technique, it is

clear that cell

proliferation rates in mice are extremely sensitive to changes in

caloric intake, whether

due to CR or feeding pattern.

Previous methods for measuring cell proliferation include cell-cycle

indices such as Ki67

or PCNA staining (28, 38). These techniques do not accurately reveal

rate of progression

through the cell cycle, however (16). Dynamic measurements, including

incorporation of

BrdU and 3 HdT, also have limitations. DNA incorporation of these

precursors occurs via

nucleoside salvage pathways and is dependent on a number of

variables, including

efficiency of cellular uptake, competition with extracellular

nucleosides, etc., which can

differ among cell types (34, 35). Labeled deoxyribonucleosides

released after cell death

may also be reincorporated into other cells (16). The stable isotope

labeling method used

here is safe, yields quantitative kinetic information, does not

depend on the

deoxyribonucleoside salvage pathway, and is not susceptible to

artifacts related to re-utilization

(16, 34, 35).

We show here that early-onset 33% CR in C57BL/6J mice, administered

by a commonly

used feeding regimen in this field (i.e. food given 3 times per week)

(13, 37, 42, 44, 46),

reduces proliferation of epidermal cells (keratinocytes), MECs, and

splenic T-cells. When

mice were refed after CR, cell proliferation rates were restored

within 2 weeks to values

equal to ad libitum fed controls, and some tissues became transiently

hyperproliferative

in comparison to 95% ad libitum fed controls. These data suggest that

the effects of CR

on cell proliferation are rapid and reversible. Whether or not these

effects on cell

proliferation are sustained over extended duration of CR cannot be

deduced from these

data.

The mediator(s) of the CR effect on cell proliferation in multiple

tissues remain

uncertain. IGF-1 has been hypothesized to mediate the decrease in

cell proliferation in

response to CR (18, 19). Serum IGF-1 levels have been consistently

reported to be

reduced in CR studies (4, 9, 14, 20), and exogenous replacement of

IGF-1 has been found

to negate the benefits against bladder cancer conferred by CR in p53-

deficient mice (9).

In addition, modulations in IGF-1 signaling have been correlated to

lifespan extension (3,

8, 11). We were unable to accurately compare IGF-1 levels between

groups due to

differences in fasting times prior to sacrifice. A priority for

future studies will be to

characterize the relationship between changes in cell proliferation

and concentrations of

potential mediators.

Our data demonstrate that an intermittent pattern of feeding,

resulting in periodic fasting,

contributes to the anti-proliferative effects of CR regimens, along

with caloric deficit. We

observed that a 5% decrease in total caloric intake, combined with an

intermittent feeding

pattern (food given 3 times per week), decreased cell proliferation

compared to mice fed

isocalorically but according to a more constant feeding pattern

(daily or continuously).

Intermittency of feeding did not appear to have an additive effect in

CR mice. In

particular, among mice receiving 95% of AL caloric intake,

intermittent feeding

decreased MEC and T-cell proliferation compared to continuously fed

mice.

Continuously and daily fed mice at 95% AL caloric intake also did not

have significantly

lower epidermal and T-cell proliferation compared to AL controls,

whereas intermittently

fed mice at 95% AL caloric intake did, ruling out an effect of the 5%

reduction in caloric

intake per se. Recently, intermittent feeding was found to impart

greater benefits than

daily feeding at a 40% level of CR (2). The intermittent feeding

model employed by

Anson et al. involved alternating ad libitum feeding and complete

food deprivation, every

other day. Although the mice compensated for food deprivation on the

days during which

they were fed, they were only able to attain a caloric intake of

about 90% of ad libitum

levels. Thus, their model, resulting in 10% CR with intermittent

feeding, is similar to our

C95 group, fed 5% CR intermittently. Anson et al. reported improved

insulin sensitivity

in this model, compared to a daily fed 40% CR model (2). Both studies

therefore suggest

that minimal CR in conjunction with intermittent feeding induces

health effects similar to

that from traditional, much more substantial CR.

Our data do not suggest, however, that the effects of substantial CR

can be completely

reproduced by intermittency of feeding. Although intermittent feeding

with 5% CR (95-INT)

resulted in lower cell proliferation than more continuous feeding at

the same caloric

level, it is worth noting that the degree of hypoproliferation is not

as pronounced as in

mice fed 33% CR, regardless of feeding intermittency. This result

suggests that

substantial CR still has a dominant effect over feeding

intermittency. Similarly, Lee et al.

have shown that mice fed intermittently on 41% CR have greater

lifespan extension and

lower tumor incidence than those fed intermittently on 15% CR as

controls (25).

and Halberg also investigated the role of intermittent feeding

and found that 25%

CR with 6 smaller meals versus 1 big meal a day both extended

lifespan to the same

extent in mice but resulted in a different circadian rhythm, such

that less frequent meals

resulted in lower core body temperature (36). This finding may be

significant, as CR-

induced torpor and cell proliferation are linked (22, 45), but cell

proliferation was not

measured in this study. The finding that 25% CR with increased

feeding intermittency did

not extend lifespan beyond daily feeding of 25% CR may suggest that

substantial CR

overcomes or masks any effect of intermittency on lifespan. This

interpretation is also

consistent with our data, as 33% CR groups had the same cell

proliferation rates, despite

different feeding intermittency patterns. There has yet to be a study

comparing lifespan

expectancy in animals with minimal CR using different feeding

patterns, however. Such a

study would be necessary to investigate the effect of intermittency

of feeding apart from

caloric deficit on lifespan extension.

The suggestion that intermittent feeding may produce benefits similar

to caloric

restriction is potentially of great interest to human applications.

While it may be

impractical to maintain humans on substantial calorically restricted

diets for their

lifetime, intermittent food deprivation may be feasible. If some of

the health benefits of

CR can be reproduced, including reduction in cancer promotion, this

might be a

therapeutic strategy worth pursuing. Human CR studies using the

techniques described

here (e.g. proliferation of skin cells and mammary epithelial cells

(17, 34)) can, in

principle, be performed to test this hypothesis.

Cheers, Alan Pater

[Guest guest]

Hi folks:

I don't know whether or not anyone took a close look at the study

posted below by Al.

They have not made it easy to figure out what they are saying, but

from my point of view the most interesting aspects appear to be the

following:

1. They were trying to determine the relative cancer-prevention

merits of ad-lib, 5% CR, and 36% CR, WITH DIFFERENT FREQUENCIES OF

FEEDING. I.E. ***FASTING*** or not fasting.

2. Cell proliferation is considered to be a decent measure of cancer

susceptibility - less proliferation, less cancer.

3. They measured cell proliferation of three types in mice:

epidermal cells; mammary epithelial cells; and T-cell, for various

degrees of CR and various different feeding frequencies.

4. Feeding frequencies tested were: ad-lib available all day; 95% of

ad-lib (5% CR) available steadily all day, once daily, and fed only

Monday/Wednesday/Friday; and 36% CR fed steadily all day, once daily,

and only Monday/Wednesday/Friday.

5. Those fed 5% CR had appreciably lower cell proliferation than

those fed ad-lib, and, in comparison with other feeding schedules,

ESPECIALLY SO if only fed Monday/Wednesday/Friday. In this study, at

5% CR, fasting appears to be decisively better than daily or

continuous feeding.

6. Those fed 36% less than ad-lib had less still cell proliferation

than those on 5% CR (of course). Of those on 36% CR, the ones fed

only three days a week had the least cell proliferation, but only

rather marginally better than 36% CR fed daily or continuously.

7. Compared with ad-lib, the reduction in cell proliferation for the

mice fed three times a week for 5% CR and 36% CR were:

Epidermal: 22% less; 33% less.

Mammary: 65% less; 87% less.

T-cells: 27% less; 60% less.

So, 36% CR is much better than 5% CR (not news); and three days a

week feeding is a lot better than being fed daily for 5% CR, but only

slightly better at 36% CR.

One final point ................ the above may only apply, of

course, if you are a female mouse ;; ^ )))

Rodney.

PS: If anyone thinks I got this summary, above, wrong please say so!

-------------------

--- In , " old542000 " <apater@m...>

wrote:

>

> Hi All,

>

> This experiment has the Medline abstract:

>

> http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?

> cmd=Retrieve & db=pubmed & dopt=Abstract & list_uids=15613681

>

> http://tinyurl.com/4lmyh

>

> Hsieh EA, Chai CM, Hellerstein MK.

> Effects of Caloric Restriction on Cell Proliferation in Several

> Tissues in

> Mice: Role of Intermittent Feeding.

> Am J Physiol Endocrinol Metab. 2004 Dec 21; [Epub ahead

of

> print]

> PMID: 15613681 [PubMed - as supplied by publisher]

>

> Excerpts from the available pdf are below.

>

> Of note,

>

> 1. The definition of the different treatments of the mice is

> highly complex.

>

> 2. The body weights were greatly affected by the use of

> intermittent feeding versus daily feeding versus continuous

> feeding, as illustrated by " Non-significant differences in

> body weight between mice with the same

> caloric intake but fed by different feeding

> patterns may be due to the presence or absence

> of food in the stomach during weighing " .

>

> 3. Searching the text for " lifespan " identifies many

> shortcomings of the present study hinging on there being

> no longevity data for the mice in these experiments, in

> my opinion.

>

> 4. Experiments on cells grown in culture may not reflect

> the results of experiments on pathology in the animal,

> as for examining cell cultures for their proliferation rates

> as a proxy for examinations in animals for cancer.

>

> 5. Examining the effect on cancer rates using intermittent

> feeding may merit further studies, even in humans.

>

> Introduction

> Caloric restriction (CR) ... A range of 30-70%

> extension of maximal lifespan has been achieved using variations on

> CR regimens (45),

> including both early- and adult-onset CR (44, 46). CR also exerts a

> number of other

> beneficial health effects, including reduced carcinogenesis,

enhanced

> insulin sensitivity,

> and reduced cardiovascular disease risk (15). The inhibitory effect

> of CR on

> carcinogenesis is of particular interest, as CR effectively

inhibits

> spontaneous tumor

> formation as well as neoplasias in knockout/transgenic models of

> cancer and chemically-induced

> tumorigenesis (18, 19, 45). ...

>

> ... Materials and Methods

> Mice and CR regimens. For all studies, 7-week-old female C57BL/6J

> mice

> ... were fed a semi-purified AIN-93M diet ad libitum

> ... Studies were then started at 8 weeks of age. 3 studies were

> carried out.

> Study #1: Time course of CR effects (Figure 1A). A 33% CR diet

was

> fed for varying

> durations of time to the 3 treatment groups (n = 8 per group): 2

> weeks CR (2W), 1 month

> CR (1M) or 2 months CR (2M). 2W received a control diet for 6 weeks

> prior to onset of

> the CR diet, and 1M received a control diet for 1 month prior to

> onset of the CR diet, so

> that the ages of all 3 groups were matched at the end of the

> experiment. Accordingly, all

> 3 groups were sacrificed at 16 weeks of age. Two different control

> groups were also used

> (n = 8 per group): ad libitum fed mice (AL) and mice fed 95% of ad

> libitum intake (C95).

> These animals were also sacrificed at 16 weeks of age. These

control

> groups represent the

> different types of control groups that have commonly been used in

CR

> studies

> demonstrating lifespan extension or reduced carcinogenesis (1, 37,

> 41, 44, 46). One

> additional group of mice (n = 4) was placed on CR for a longer

period

> of time (3 months,

> 3M), also starting at 8 weeks of age. This group was sacrificed at

20

> weeks of age.

> During non-CR periods, the treatment groups were maintained on the

> C95 diet regimen.

> During CR periods, mice were fed 67% of C95 intake, or about 64% of

> AL intake, as

> previously described (37). The CR and C95 groups were fed 3 days a

> week, such that 2-times

> the daily allotment was given on Mondays and Wednesdays, and 3-

times

> the daily

> allotment was given on Fridays, as has been commonly used in

previous

> CR studies (13,

> 37, 42, 44, 46). AL and C95 mice were fed a semi-purified AIN-93M

> diet, while CR

> mice were fed an enriched AIN-93M diet that contains 33% more

> protein, minerals, and

> vitamins per gram of diet (Bio-Serv). All mice were housed

> individually. Food intake and

> body weight were monitored weekly.

> Study #2: Refeeding effects (Figure 1B). The time course of

> response to refeeding was

> also studied. Mice received a 33% CR diet for 1 month and were

> subsequently given a

> C95 diet (n = 8 per group) for either 2 weeks of refeeding (R2W) or

1

> month of refeeding

> (R1M). The CR diet for the R2W group started 2 weeks into the study

> (10 weeks old)

> while CR diet for R1M started immediately (8 weeks old), so that

both

> groups were

> sacrificed at 16 weeks of age. One additional group of mice

received

> a 33% CR diet for 1

> month and was refed for a longer period of time (2 months, n = 4)

> (R2M). These mice

> were sacrificed at 20 weeks of age. All mice were housed

> individually. Food intake and

> body weight were monitored weekly.

> Study #3: Intermittency of feeding study. The role of

intermittent

> food intake was also

> investigated. 3 groups of mice were put on a 33% CR diet,

> administered via different

> feeding protocols (n = 6 per group): intermittent feeding of 3

times

> per week (CR-INT),

> as described above (37); daily feeding (CR-DF); or continuous

feeding

> via an electronic

> pellet dispenser (CR-PD). 3 other groups of mice were fed 95% of ad

> libitum diet via the

> same 3 feeding protocols (n = 6 per group): intermittent feeding of

3

> times a week (95-INT);

> daily feeding (95-DF); or continuous feeding (95-PD). The 95-INT,

95-

> DF, and

> 95-PD groups were also compared with a group fed ad libitum (AL)

> concurrently.

> Intermittent feeding was as described above, with 2 times the daily

> allotment given on

> Mondays and Wednesdays, and 3 times the daily allotment given on

> Fridays. Mice fed

> daily were given their food allotment for each day, every morning.

> The amount and type

> of diet (33% enriched or standard AIN-93M) depended on whether the

> mice were in the

> CR groups (CR-INT, CR-DF) or the control groups (95-INT, 95-DF).

> Continuously fed

> mice were housed in cages containing an electronic pellet dispenser

> that delivered a 45

> mg pellet of AIN-93M diet (standard for 95-PD, 33% enriched for CR-

> PD, Bio-Serv),

> into the cage every 20 to 30 minutes, depending on the caloric

> intake. All mice were

> housed individually. Food intake and body weight were monitored

> weekly. Mice were

> sacrificed at 12 weeks of age, after 4 weeks of treatment.

> ...

>

> Results

> Study #1: Time course

> Food intake and body weight.

> On average, AL mice consumed 22 grams of food per

> week. Therefore, C95 mice were fed 21 grams of food per week and CR

> mice were fed

> 14 grams of food per week. The body weight of CR mice dropped

> initially by as much as

> 30% but stabilized over time (Figure 2A). Mice then gained weight

on

> CR diets.

> Time course. When compared to AL, proliferation of epidermal cells,

> MECs, and T-cells

> was significantly decreased in the CR groups at all time points

> studied (Figures 3A-3C).

> When compared to C95, in contrast, cell proliferation in all

tissues

> was not significantly

> decreased until 1 month of CR, after which the response was again

not

> significant. At 1

> month of CR, the time of greatest effect of CR, epidermal cell

> proliferation was 61% of

> that in AL mice and 76% of that in C95 mice. MEC proliferation was

> only 11% of AL

> and 29% of C95 mice values at 1 month, while T-cell proliferation

was

> 41% of that in AL

> mice and 57% of that in C95 mice.

> Differences between C95 and AL control groups.

> C95 mice exhibited statistically

> significantly lower cell proliferation than AL mice in all tissues

> examined (Figures 3A-3C).

> After 2 months on respective diets, epidermal cell proliferation in

> C95 mice was

> 81% of that in AL mice, MEC proliferation was 37%, and T-cell

> proliferation was 71%.

> Thus, CR exerted significant effects on proliferation of all 3 cell

> types studied, but C95

> also had a potent impact that appeared to account for at least part

> of the CR effect.

> Estrus cycle. Based on cell morphology analysis of vaginal cells

> collected from 1M and

> C95 mice, CR mice were anestrus (not cycling), while C95 mice were

> actively cycling.

> The marked reduction in MEC proliferation in the CR groups might

> therefore in part be

> explained by reduction in reproductive hormone levels (31), but the

> substantial effect

> observed in the C95 groups exclude this as the primary cause of

> reduced MEC

> proliferation.

>

> Study #2: Refeeding

> Food intake and body weight.

> As in study #1, AL mice consumed about 22 grams of

> food per week. During the CR phase, mice were therefore fed 14

grams

> of food per week,

> and during the refeeding phase, mice were fed 21 grams of food per

> week. Refeeding

> resulted in a rapid gain of lost weight (Figure 2B). Body weights

of

> CR mice had caught

> up to the body weights of C95 mice by the end of the study, despite

> the 1-month period

> of CR.

> Time course of refeeding effects.

> When compared to the C95 control group, cell

> proliferation in all tissues rebounded to a significantly higher

rate

> after 2 weeks of

> refeeding, persisting through 1 month of refeeding but normalizing

> after 2 months of

> refeeding (Figures 4A-4C). When compared to the AL group, cell

> proliferation in all

> tissues was no longer significantly different after 2 weeks of

> refeeding. Subsequent

> comparisons revealed tissue-specific differences. After 1 month of

> refeeding of the C95

> diet, T-cell proliferation rate was statistically higher than AL

> levels; this was normalized

> after 2 months of refeeding. MEC proliferation was significantly

> lower than AL levels

> after 2 months of refeeding of C95 diet, consistent with the

> observation that MEC

> proliferation was lower in C95 mice than in AL mice (Figure 3B).

>

> Study #3: Intermittency of feeding

> Food intake and body weight.

> Throughout this study, all groups of CR mice were fed

> 14 grams of food per week, and all groups of control mice were fed

21

> grams of food per

> week. All mice gained weight on their diets (Figure 2C). Non-

> significant differences in

> body weight between mice with the same caloric intake but fed by

> different feeding

> patterns may be due to the presence or absence of food in the

stomach

> during weighing.

> Feeding intermittency effects among groups of CR mice.

> In the three tissues studied,

> intermittency of feeding (i.e. food given 3 times per week) had no

> additional effect

> compared to daily or continuous feeding on cell proliferation when

CR

> was present

> (Figures 5A-5C).

> Feeding intermittency effects among groups of control mice.

> There was lower cell

> proliferation in all tissues of the group fed intermittently at 95%

> of ad libitum diet (95-INT)

> compared to daily feeding (95-DF), continuous feeding (95-PD), or

ad

> libitum

> feeding (AL), although not all comparisons were statistically

> significant (Figures 6A-6C).

> MEC proliferation was significantly lower in 95-INT than in 95PD

> mice, while T-cell

> proliferation was significantly lower in 95-INT mice compared to 95-

> DF and 95-PD

> mice. Epidermal and T-cell proliferation rates in AL were not

> statistically different from

> 95-DF or 95-PD but were significantly greater than 95-INT. An

> intermittent feeding

> regimen (i.e. food given 3 times per week) therefore caused

> significant reductions in cell

> proliferation rates compared to isocaloric diets fed by more

constant

> patterns.

>

> Discussion

> We demonstrate here the application of a relatively simple method

for

> measuring cell

> proliferation in multiple tissues in mice. By this technique, it is

> clear that cell

> proliferation rates in mice are extremely sensitive to changes in

> caloric intake, whether

> due to CR or feeding pattern.

> Previous methods for measuring cell proliferation include cell-

cycle

> indices such as Ki67

> or PCNA staining (28, 38). These techniques do not accurately

reveal

> rate of progression

> through the cell cycle, however (16). Dynamic measurements,

including

> incorporation of

> BrdU and 3 HdT, also have limitations. DNA incorporation of these

> precursors occurs via

> nucleoside salvage pathways and is dependent on a number of

> variables, including

> efficiency of cellular uptake, competition with extracellular

> nucleosides, etc., which can

> differ among cell types (34, 35). Labeled deoxyribonucleosides

> released after cell death

> may also be reincorporated into other cells (16). The stable

isotope

> labeling method used

> here is safe, yields quantitative kinetic information, does not

> depend on the

> deoxyribonucleoside salvage pathway, and is not susceptible to

> artifacts related to re-utilization

> (16, 34, 35).

> We show here that early-onset 33% CR in C57BL/6J mice, administered

> by a commonly

> used feeding regimen in this field (i.e. food given 3 times per

week)

> (13, 37, 42, 44, 46),

> reduces proliferation of epidermal cells (keratinocytes), MECs, and

> splenic T-cells. When

> mice were refed after CR, cell proliferation rates were restored

> within 2 weeks to values

> equal to ad libitum fed controls, and some tissues became

transiently

> hyperproliferative

> in comparison to 95% ad libitum fed controls. These data suggest

that

> the effects of CR

> on cell proliferation are rapid and reversible. Whether or not

these

> effects on cell

> proliferation are sustained over extended duration of CR cannot be

> deduced from these

> data.

> The mediator(s) of the CR effect on cell proliferation in multiple

> tissues remain

> uncertain. IGF-1 has been hypothesized to mediate the decrease in

> cell proliferation in

> response to CR (18, 19). Serum IGF-1 levels have been consistently

> reported to be

> reduced in CR studies (4, 9, 14, 20), and exogenous replacement of

> IGF-1 has been found

> to negate the benefits against bladder cancer conferred by CR in

p53-

> deficient mice (9).

> In addition, modulations in IGF-1 signaling have been correlated to

> lifespan extension (3,

> 8, 11). We were unable to accurately compare IGF-1 levels between

> groups due to

> differences in fasting times prior to sacrifice. A priority for

> future studies will be to

> characterize the relationship between changes in cell proliferation

> and concentrations of

> potential mediators.

> Our data demonstrate that an intermittent pattern of feeding,

> resulting in periodic fasting,

> contributes to the anti-proliferative effects of CR regimens, along

> with caloric deficit. We

> observed that a 5% decrease in total caloric intake, combined with

an

> intermittent feeding

> pattern (food given 3 times per week), decreased cell proliferation

> compared to mice fed

> isocalorically but according to a more constant feeding pattern

> (daily or continuously).

> Intermittency of feeding did not appear to have an additive effect

in

> CR mice. In

> particular, among mice receiving 95% of AL caloric intake,

> intermittent feeding

> decreased MEC and T-cell proliferation compared to continuously fed

> mice.

> Continuously and daily fed mice at 95% AL caloric intake also did

not

> have significantly

> lower epidermal and T-cell proliferation compared to AL controls,

> whereas intermittently

> fed mice at 95% AL caloric intake did, ruling out an effect of the

5%

> reduction in caloric

> intake per se. Recently, intermittent feeding was found to impart

> greater benefits than

> daily feeding at a 40% level of CR (2). The intermittent feeding

> model employed by

> Anson et al. involved alternating ad libitum feeding and complete

> food deprivation, every

> other day. Although the mice compensated for food deprivation on

the

> days during which

> they were fed, they were only able to attain a caloric intake of

> about 90% of ad libitum

> levels. Thus, their model, resulting in 10% CR with intermittent

> feeding, is similar to our

> C95 group, fed 5% CR intermittently. Anson et al. reported improved

> insulin sensitivity

> in this model, compared to a daily fed 40% CR model (2). Both

studies

> therefore suggest

> that minimal CR in conjunction with intermittent feeding induces

> health effects similar to

> that from traditional, much more substantial CR.

> Our data do not suggest, however, that the effects of substantial

CR

> can be completely

> reproduced by intermittency of feeding. Although intermittent

feeding

> with 5% CR (95-INT)

> resulted in lower cell proliferation than more continuous feeding

at

> the same caloric

> level, it is worth noting that the degree of hypoproliferation is

not

> as pronounced as in

> mice fed 33% CR, regardless of feeding intermittency. This result

> suggests that

> substantial CR still has a dominant effect over feeding

> intermittency. Similarly, Lee et al.

> have shown that mice fed intermittently on 41% CR have greater

> lifespan extension and

> lower tumor incidence than those fed intermittently on 15% CR as

> controls (25).

> and Halberg also investigated the role of intermittent

feeding

> and found that 25%

> CR with 6 smaller meals versus 1 big meal a day both extended

> lifespan to the same

> extent in mice but resulted in a different circadian rhythm, such

> that less frequent meals

> resulted in lower core body temperature (36). This finding may be

> significant, as CR-

> induced torpor and cell proliferation are linked (22, 45), but cell

> proliferation was not

> measured in this study. The finding that 25% CR with increased

> feeding intermittency did

> not extend lifespan beyond daily feeding of 25% CR may suggest that

> substantial CR

> overcomes or masks any effect of intermittency on lifespan. This

> interpretation is also

> consistent with our data, as 33% CR groups had the same cell

> proliferation rates, despite

> different feeding intermittency patterns. There has yet to be a

study

> comparing lifespan

> expectancy in animals with minimal CR using different feeding

> patterns, however. Such a

> study would be necessary to investigate the effect of intermittency

> of feeding apart from

> caloric deficit on lifespan extension.

> The suggestion that intermittent feeding may produce benefits

similar

> to caloric

> restriction is potentially of great interest to human applications.

> While it may be

> impractical to maintain humans on substantial calorically

restricted

> diets for their

> lifetime, intermittent food deprivation may be feasible. If some of

> the health benefits of

> CR can be reproduced, including reduction in cancer promotion, this

> might be a

> therapeutic strategy worth pursuing. Human CR studies using the

> techniques described

> here (e.g. proliferation of skin cells and mammary epithelial cells

> (17, 34)) can, in

> principle, be performed to test this hypothesis.

>

>

> Cheers, Alan Pater