Human intermittent fasting

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Hi All,

It seemed to be significant that:

" This experiment is the first in humans to show that intermittent fasting

increases

insulin mediated glucose uptake rates "

and:

" intermittent fasting and physical training may increase insulin action via

different mechanisms "

and the men were not diabetic, overweight or obese.

Nils Halberg, Morten Henriksen, Nathalie Soderhamn, Bente Stallknecht, Thorkil

Ploug, Schjerling, and Flemming Dela

The effect of intermittent fasting and re-feeding on insulin action in healthy

men

J Appl Physiol (July 28, 2005). doi:10.1152/japplphysiol.00683.2005 [Abstract]

[PDF]

Insulin resistance is currently a major health problem. This may be because of a

marked decrease in daily physical activity during the last decades combined with

constant food abundance. This lifestyle collide with our genome, which was most

likely selected in the late Palaeolithic era (50.000 - 10.000 BC) by criteria

that

favoured survival in an environment that was characterized by fluctuations

between

periods of feast and famine. The theory of thrifty genes states that these

fluctuations are required for an optimal metabolic function. We mimicked the

fluctuations in 8 healthy young males (25.0±0.1 yrs (mean±SE); BMI: 25.7±0.4 kg

..

m-2) by subjecting them to intermittent fasting every second day for 20 h for 15

days. Euglycemic hyperinsulinemic (40 mU . min-1 . m-2) clamps were performed

before

and after the intervention period. The subjects maintained body weight (86.4±2.3

kg;

coefficient of variation: 0.8±0.1%). Plasma FFA and ß-hydroxybutyrate

concentrations

were 347±18 µM and 0.06±0.02 mM, respectively, after an overnight fast, but

increased (p < 0.05) to 423±86 µM and 0.10±0.04 mM after 20 h fasting confirming

that the subjects were fasting. Insulin mediated whole body glucose uptake rates

increased from 6.3±0.6 to 7.3±0.3 mg . kg-1 . min-1 (p=0.03) and the

insulin-induced

inhibition of adipose tissue lipolysis was more prominent after than before the

intervention (p=0.05). After the 20 h fasting periods plasma adiponectin was

increased compared to the basal levels before and after the intervention

(5922±991

vs. 3860±784 ng . ml-1, p=0.02). This experiment is the first in humans to show

that

intermittent fasting increases insulin mediated glucose uptake rates and the

findings are compatible with the thrifty gene concept.

INTRODUCTION

Our genome was probably selected during the Late-Palaeolithic era (50.000 –

10.000

BC),

during a time man existed as hunter-gatherers (6). At that time there were no

guaranties in

finding food, resulting in intermixed periods of feast and famine. In addition,

physical activity

had to be a part of our ancestor’s daily living as forage and the hunt for food

must

have been

done through physical activity (15). Cycling between feast and famine, and thus

oscillations

in energy stores, as well as between exercise and rest have been characteristic

in

the Late-Palaeolithic

era and might have driven the selection of genes involved in the regulation of

metabolism (30).

Thus, our genotype selected centuries ago to favour an environment with

oscillations

in

energy stores, still exists with few if any changes. The modern sedentary

lifestyle

common in

the westernized countries are characterized by constant high food availability

and

low

physical activity, and it has led to an imbalance between our genotype and the

environment in

which we live today. This may predispose our potential “thrifty” genes to

miss-express

metabolic proteins manifesting in chronic diseases (e.g. type 2 diabetes) in the

industrialized

part of the world.

It is well known that physical training increases insulin action (10). The

molecular

events

leading to an exercise mediated increase in insulin action are not fully

characterized. In

addition, energy usage during each exercise bout in the training regimen with

subsequent

eating creates oscillations in energy stores. These oscillations are probably

not as

massive as

the oscillations seen between periods of feast and famine for the

Late-Paleolithic

people, but

some similarities might exist and we speculated if exercise induced oscillations

in

energy

stores could be mimicked by intermittent fasting. This study was undertaken to

test

the

hypothesis that 14 days of intermittent fasting/re-feeding improves insulin

stimulated glucse

disposal.

.... Experimental procedure

The subjects were examined on 2 occasions; before and after 14 days of fasting

every

second

day for 20 hours giving seven fasting periods. Each fasting period started at

22.00

and ended

at 18.00 the following day (for protocol see figure 1). During the fasting

periods

the subjects

were allowed to drink water and were instructed to maintain habitual activities.

.... RESULTS

Weight, body composition and indices of physical activity.

The body weight was maintained stable throughout the experiment (86.4 ± 2.3 kg,

0.8

± 0.1%

coefficient of variation) and % body fat was also unchanged before compared with

after the

fasting intervention (Table I).

Table I. Subject characteristics

......................

------Before the intervention After the intervention

......................

Age (y) 25±1 -

Body weight (kg) 87.1±2.3 86.2±2.4

BMI (kg · m -2 ) 25.7±0.4 25.5±0.3

% Body fat 20.1±0.8 20.4±1.1

Fasting plasma glucose (mmol·L -1 ) 5.0±0.1 5.1±0.1

Fasting insulin (pmol·L -1 ) 34±5 38±7

...........................

Data are mean±SE.

No significant differences were seen.

The level of habitual daily physical activity did not decrease during fasting

days.

Thus, the

average heart rate during daytime was not different during fasting (79 ± 3

min-1)

compared

with non-fasting days (80 ± 3 min-1).

Whole body glucose metabolism

Plasma glucose concentration during both clamps were kept constant (Fig. 2),

with a

coefficient of variance of 4.4% ± 1.3% mmol·L-1 during the last hour of the

clamps.

The glucose infusion rate was significantly increased during the last 30 min

(from

6.3 ± 0.6 to

7.3 ± 0.3 mg · min-1·kg-1) after the fasting intervention compared to before,

respectively

(p=0.03) (Fig. 2).

Glycerol metabolism in adipose tissue

There was no effect of intermittent fasting in either the adipose tissue blood

flow

(2.4 ± 0.5

vs. 2.9 ± 0.7 ml · 100 g-1· min-1 at basal and 2.6 ± 0.5 vs. 3.1 ± 0.5 ml · 100

g-1·

min-1 at the

insulin stimulated state) or the absolute interstitial glycerol concentrations

(Fig.

3A) during

the clamps. However, the interstitial glycerol concentrations decreased

exponentially with the

insulin infusion (R2 = 0.96 before and R2 = 0.99 after the fasting intervention)

and

the negative

slope of the curves were larger after the fasting intervention compared to

before

(p=0.05)

(Fig. 3B). This indicates that insulin had an enhanced inhibitory effect on

lipolysis after

intermittent fasting compared with before.

Substrates and metabolites

Fasting (8 h) plasma glucose concentrations were similar before (5.0 ± 0.1 mM)

and

after (5.1

± 0.1 mM) the intermittent fasting period. After 20 h fasting, i.e. days 4, 6,

and

10, plasma

glucose concentrations were lower (4.6 ± 0.1, 4.6 ± 0.1, and 4.7 ± 0.1 mM,

respectively)

compared with the shorter fasting periods (8 h) (p<0.05).

Fasting (8 h) plasma ß-hydroxybutyrate, FFA, and glycerol concentrations were

similar

before and after the intermittent fasting period, and all decreased (p<0.05)

with

insulin

infusion (Fig. 4). After 20 h fasting, i.e. days 4, 6, and 10, plasma FFA and

glycerol

concentrations were increased compared with the shorter fasting periods (p<0.05)

whereas the

increase in ß-hydroxybutyrate did not attain statistical significance (p=0.07)

(Fig.

4).

Hormones

Fasting (8 h) plasma insulin concentrations were similar before (33 ± 5 pM) and

after (38 ± 7

pM) the intermittent fasting period and concentrations increased (p<0.05) with

insulin

infusion (to 439 ± 63 and 404 ± 18 pM, respectively). After 20 h fasting, i.e.

days

4, 6, and

10, plasma insulin concentrations were unchanged (24 ± 4, 24 ± 5, and 16 ± 4

pmol/L)

compared with the shorter fasting period (Fig. 4).

Plasma adiponectin concentrations did not change with insulin infusion, and was

similar on

the two clamp days (Fig. 5). However, after 20 h fasting (day 6, 10 and 14) a

37%

increase

was seen compared with the shorter fasting days (p=0.02).

Plasma leptin concentrations were similar on the two clamp days and did not

change

with

insulin infusion (Fig. 5). However, after the 20 h fasting days (day 6, 10 and

14)

plasma

leptin concentrations decreased compared with the shorter fasting days (P=0.02)

(Fig. 5).

No significant differences were observed in either TNF-alpha or IL-6

concentrations

during this

study (Fig. 5).

Muscle triglyceride, glycogen, GLUT-4, and PGC-1ß mRNA

No overall changes were observed in concentrations of IMTG (p=0.11), glycogen

(p=0.26) or

in mRNA content of PGC-1alpha (p=0.18) when measured before and after each clamp

and

after

fasting on day 10 (Fig. 6 and 7). However, with insulin stimulation (data from

both

clamps

are included) we observed a significant decrease (p=0.04) in the IMTG

concentration.

Furthermore, total muscle GLUT 4 protein content did not change with the fasting

intervention (p=0.66) (Fig. 7).

Respiratory exchange ratios (RER)

RER were similar at basal (after 8 h fasting) on the two clamp days. With

insulin

stimulation

RER increased at both occasions (Fig. 8). No differences were observed in RER

values

between the overnight and the 20 h fasted state (Fig. 8).

DISCUSSION

In the present study we have used a very simple intervention protocol with the

aim

of

mimicking the perturbations in energy stores that is inherent in a physical

active

lifestyle with

regular exercise sessions. In a wider perspective we have tried to unravel the

significance of

genes that may be responsible for an evolutionary selection process, i.e. the

thrifty genes. In

this context the used intervention seems inevitably small. Nevertheless, by

subjecting healthy

males to cycles of feast and famine we did change the metabolic status to the

better, implying

that the mismatch between our ancient genotype and the living of the westernized

individual

of today became smaller. To our knowledge this is the first study in humans

where an

increased insulin action on whole body glucose uptake and adipose tissue

lipolysis

has been

obtained by means of intermittent fasting. This result is in accordance with

previously

reported in rodents (2; 32). In these studies, fasting every second day

increased

the insulin

sensitivity ~7 fold according to the homeostatic model assessment (2) and

decreased

the

incident of diabetes (32).

Prolonged fasting for 72 h with minimal physical activity has previously been

shown

to

increase IMTG levels in humans (46). With the present fasting protocol and

maintenance of

habitual daily physical activity in the fasting periods, we had expected to

detect a

decrease in

IMTG content in the skeletal muscle. The fact that this was not seen and that

muscle

glycogen

content was unchanged, could suggest that skeletal muscle is not immediately

involved in

recognition of acute energy oscillations. There is no doubt however, that

fasting

for 20 h

while maintaining normal daily physical activity must cause a temporary negative

energy

balance larger than normally experienced in a daily basis. This is also

indicated by

our finding

of decreased plasma glucose concentrations after 20 hr fasting. We did not have

the

possibility to estimate the hepatic glycogen stores, but from animal studies

(17),

we must infer

that liver glycogen probably also decreased considerably during the 20 h fasting

periods. It

has previously been suggested that usage of muscle energy depots during fasting

would be an

evolutionary disadvantage, as it would lessen the capacity of physical

performance

and hence

the ability to provide food (i.e. to hunt and gather) during periods of fasting

(6;

45). The

present findings support this view.

In contrast to the findings in skeletal muscle the adipose tissue responded to

the

changes in

energy balance as intermittent fasting changed the plasma concentrations of the

adipocyte

specific hormones leptin and adiponectin. However, as we did not measure the

energy

stores

in the adipose tissue during the intervention (e.g. by fat cell size) we cannot

determine

whether the change in adipokine release is merely a secondary response to

intermittent fasting

or if the adipose tissue is an active recognizer of energy oscillations.

Blood sampling for measurement of adipokines at basal levels before and after

the

fasting

intervention was performed at 10.00 whereas the three samples on days 6, 10 and

14

were

taken at 17.00. The amount of circulating adiponectin is constant or slightly

decreased during

daytime (20). Hence, the boosts of 37% we observed after each fasting period are

not

due to

nocturnal variation. Because the plasma adiponectin concentration is positively

correlated to

insulin sensitivity in humans (8; 23; 29) and adiponectin administration in

rodents

increases

insulin action (9; 38; 48) it seems likely that our finding of increases in

circulating

adiponectin after each fasting period would be able to exert an insulin

sensitizing

effect.

Skeletal muscle content of GLUT-4 protein after the overnight fast did not

differ

before and

after the fasting intervention. Future studies will have to determine if the

insulin

signalling,

e.g. phosphorylation of the insulin receptor substrate, is influenced by

fluctuations in energy

stores and thereby account for the increase insulin action as measured by the

clamp

method

reported herein.

Since 36 h passed between the last fasting period and the last clamp, it seems

most

likely that

the potential insulin sensitizing effects of adiponectin was due to adiponectin

induced changes

in gene expression. This could in turn be mediated through an AMPK activation

that

further

activates several transcription factors including myocyte enhancing factor (MEF)

that

increases GLUT-4 expression (24; 27). Another possibility is that the

adiponectin

boosts

peroxisome proliferated-activated receptor- gamma (PPAR-gamma) expression as

seen in

3T3-L1

adipocytes (1). And as PPAR-gamma induces adiponectin expression (16) it can be

speculated that

fasting starts a positive feedback loop which results in increased levels of

both

circulating

adiponectin and PPAR-gamma. Both are known to increase the insulin sensitivity.

A considerable increase in plasma FFA concentrations (5-fold) may raise the

amount

of

circulating adiponectin slightly (43) and glucocorticoids positively regulates

adiponectin gene

expression (21). FFA and glucocorticoid increases during fasting, but in

previous

studies no

effect of fasting on circulating adiponectin was seen (19; 49). Apart from

differences in

increases of FFA and glucocorticoids, different analysis methods used (RIA vs.

ELISA

(present study)) may recognize different isoforms of adiponectin and thereby

account

for the

discrepancy.

Leptin exhibit nocturnal differences with a peak during night (24.00-8.00)

whereas

there is no

difference between 10.00 and 17.00; if anything plasma leptin concentrations are

slightly

higher at 10.00 (40). In accordance with previous findings (19; 41; 49) we found

a

decrease in

circulating leptin after 8 h to 20 h of fasting. This decrease most likely

reflects

a state of

energy deficiency, and is probably not involved in the increased insulin action

we

have found

the present study.

The mechanism by which physical training increases whole-body insulin

sensitivity is

not

known in detail. It has previously been shown that in muscle the effect is

mediated

via local

contraction dependent mechanisms (11-13), and this could include

exercise-induced

oscillations in local energy stores. However, the insulin sensitizing effect of

exercise and

intermittent fasting may not exert their effects via the same pathway. While the

local effect of

exercise is well proven (there is no transfer of training induced increase in

insulin sensitivity

to non-trained muscle) it is less likely that the effects of intermittent

fasting is

a local, muscle

phenomenon. Thus, even though we were not able to detect changes in muscle and

TG

content after 20 h fasting, the intervention may still have exerted the effects

via

oscillations in

other energy stores (e.g. in adipose tissue or liver). The finding of decreased

leptin

concentrations corresponding to the intermittent fasting verifies that adipocyte

metabolism

was influenced by the intervention.

We did not find an effect of intermittent fasting on muscle PGC-1alpha mRNA

levels.

In contrast,

PGC-1alpha mRNA increases with acute exercise (34; 47) and is suggested to be

involved in the

enhancement of insulin mediated glucose uptake following exercise training (28;

39).

Thus,

PGC-1alpha may represent a step at which the insulin enhancement actions of

exercise

training

and intermittent fasting diverge.

Whole body insulin mediated glucose uptake was estimated by the euglycemic

hyperinsulinemic clamp technique. Even though this method is a standard for

measuring

insulin action, day to day coefficient of variation has been reported to vary

between 2.4% and

15% (4; 36; 42). Part of the observed effect of the intervention may therefore

be

due to

biological and instrumental variation.

It is important to note that in the present study, the subjects maintained their

body weight

throughout the intervention period, and % body fat did not change with

intermittent

fasting.

Thus, in contrast to previous studies using alternate day fasting (22), the

subjects

in the

present study kept their body weight by following the dietary instructions of

eating

abundantly every other day. It is well known that insulin sensitivity can be

influenced by

long-term profound changes of macronutrients in the diet. However, as the

subjects

were

instructed to maintain their usual diet habits (although increasing the amount

of

food) it is

unlikely that eventual minor changes in the macronutrient mix during eight

non-consecutive

days (i.e. the non-fasting days) would influence insulin sensitivity.

Furthermore, the increased insulin action after the intervention was not the

result

of the last

fasting period because from the last fasting period until the beginning of the

overnight fast the

subjects were allowed to eat for 30 h during which they consumed at least 250 g

of

carbohydrates. Muscle glycogen was not different between the pre and post

intervention

clamps, testifying that carbohydrate loading was sufficient prior to each clamp

experiment.

In keeping with previous findings (3) we observed a decrease in IMTG with

insulin

stimulation. At first glance this seems counterintuitive. However, during

insulin

stimulation

the FFA supply to the skeletal muscle decreases dramatically and since some

skeletal

muscle

FFA oxidation is still present (RER values of 0.90 ± 0.04 before and 0.86 ± 0.02

after the

fasting intervention) it seems arguable that FFA is provided by the IMTG pool,

which

accordingly will decrease.

In conclusion, the findings that intermittent fasting increases insulin

sensitivity

on the whole

body level as well as in adipose tissue, support the view that cycles of feast

and

famine are

important as an initiator of “thrifty genes” leading to improvements in

metabolic

function (6).

We suggest that a fasting induced increase in circulating adiponectin is at

least

partly

responsible for this finding, The change in adiponectin, together with changes

in

plasma

leptin with fasting underlines the important role of the adipose tissue in

recognizing the

oscillation in energy stores. Finally, the data indicate that intermittent

fasting

and physical training may increase insulin action via different mechanisms as

muscle

energy stores did not change with the present fasting intervention.

Al Pater, PhD; email: old542000@...

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