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J Appl Physiol 99: 2128 –2136, 2005.
First published July 28, 2005; doi:10.1152/japplphysiol.00683.2005.
Halberg, Nils, Morten Henriksen, Nathalie Söderhamn, Bente availability and low physical activity, and it has led to an
Stallknecht, Thorkil Ploug, Peter Schjerling, and Flemming Dela. imbalance between our genotype and the environment in which
Effect of intermittent fasting and refeeding on insulin action in healthy we live today. This may predispose our potential “thrifty”
men. J Appl Physiol 99: 2128 –2136, 2005. First published July 28, genes to misexpress metabolic proteins, manifesting in chronic
2005; doi:10.1152/japplphysiol.00683.2005.—Insulin resistance is
diseases (e.g., Type 2 diabetes) in the industrialized part of the
currently a major health problem. This may be because of a marked
decrease in daily physical activity during recent decades combined world.
with constant food abundance. This lifestyle collides with our ge- It is well known that physical training increases insulin
nome, which was most likely selected in the late Paleolithic era action (10). The molecular events leading to an exercise-
Address for reprint requests and other correspondence: N. Halberg, Dept. of The costs of publication of this article were defrayed in part by the payment
Medical Physiology, The Panum Institute, Blegdamsvej 3, DK-2200 Copen- of page charges. The article must therefore be hereby marked “advertisement”
hagen N, Denmark (e-mail: nilsh@mfi.ku.dk). in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
2128 8750-7587/05 $8.00 Copyright © 2005 the American Physiological Society http://www. jap.org
METABOLIC EFFECTS OF INTERMITTENT FASTING 2129
Table 1. Subject characteristics plasma, and saline. Each clamp started with a 2-ml insulin infusate
bolus followed by a constant infusion (40 mU 䡠 min⫺1 䡠 m⫺2) for 120
Before the After the min. Plasma glucose concentrations were maintained at a preexperi-
Intervention Intervention mental level by frequent analysis of arterialized blood samples (ABL-
Age, yr 25⫾1 system 700, Radiometer) with subsequent adjustments of the glucose
Body weight, kg 87.1⫾2.3 86.2⫾2.4 infusion rate.
BMI, kg/m2 25.7⫾0.4 25.5⫾0.3 Blood flow. Subcutaneous blood flow was determined by the
%Body fat 20.1⫾0.8 20.4⫾1.1 standard local 133Xe washout method (5, 26) in the abdominal
Fasting plasma glucose, mmol/l 5.0⫾0.1 5.1⫾0.1 subcutaneous adipose tissue in close proximity to the microdialysis
Fasting insulin, pmol/l 34⫾5 38⫾7 catheter. The tissue-blood partition coefficient was set to 10 (5).
Data are means ⫾ SE. BMI, body mass index. No significant differences Muscle biopsies. Muscle biopsies were obtained from the middle
were seen. portion of the vastus lateralis before and in the end of each clamp
experiment. After administration of local anesthesia, an incision of 10
mm was made and the biopsy was taken (Bergström needle method
catheter (18-gauge, Becton Dickinson, Helsingborg, Sweden) was modified to apply suction). In addition, smaller biopsies were obtained
inserted in the medial cubital vein for infusion of glucose and insulin, from the mid portion of vastus lateralis after the fourth fasting period
and one catheter (18-gauge, Becton Dickinson) was inserted in a (i.e., day 10). The biopsy was then obtained with a Tru-core I biopsy
superficial hand vein in the retrograde direction. The hand was then
needle and instrument (Medical Device Technology, Gainesville, FL).
placed in a heating pad for sampling of arterialized blood. After basal
Muscle biopsies were quickly cleaned from visible blood and
Fig. 1. Experimental protocol. Eight men were subjected to fasting (marked with bars) every second day for a total of 7 fasting periods. Before and after this
intervention euglycemic clamps and microdialysis were performed. Blood samples and expiratory gas measurements were performed after the fasting on the days
marked with black bars (days 6, 10, and 14). Additionally, after the fasting on day 10 a muscle biopsy was taken for measurement of glycogen and IMTG.
blocked in 1% defatted milk powder plus 5% BSA in TS buffer [10 the intermittent fasting. When a significant main effect was observed,
mM Tris (pH 7.4), 150 mM NaCl], incubated for 90 and 60 min with the Student-Newman-Keuls test was used post hoc. In comparison of
primary and horseradish peroxidase-labeled secondary antibodies, a single parameter before, during, and after the experiment, a one-way
respectively, and diluted in blocking solution. Antigen-antibody com- ANOVA for repeated measures was used. Comparison of a single
plexes were visualized and quantitated by a LAS 3000 imaging parameter before and after the fasting intervention was performed
system (Fuji Film). Monoclonal antibody F-27 was used for detec- with Student’s paired t-test. The SigmaStat version 2.03 software
tion of GLUT4 (35). Signals were normalized against amount of package was used for all statistical analysis. P ⬍ 0.05 was considered
desmin by reprobing the polyvinylidene difluoride membrane with a statistically significant in two-tailed testing.
monoclonal antibody against desmin (DakoCytomation, Glostrup,
Denmark). RESULTS
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 in-
sulin infusion and were similar on the 2 clamp days (Fig. 5).
However, after 20-h fasting (days 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 2 clamp
days and did not change with insulin infusion (Fig. 5). How-
ever, after the 20-h fasting days (days 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-␣ or
IL-6 concentrations during this study (Fig. 5).
concentration. Furthermore, total muscle GLUT4 protein con- sevenfold according to the homeostatic model assessment (2)
tent did not change with the fasting intervention (P ⫽ 0.66) and decreased the incidence of diabetes (32).
(Fig. 7). Prolonged fasting for 72 h with minimal physical activity
has previously been shown to increase IMTG levels in humans
Respiratory Exchange Ratios (46). With the present fasting protocol and maintenance of
Respiratory exchange ratios (RER) were similar at basal habitual daily physical activity in the fasting periods, we had
(after 8-h fasting) on the 2 clamp days. With insulin stimula- expected to detect a decrease in IMTG content in the skeletal
tion RER increased at both occasions (Fig. 8). No differences muscle. The fact that this was not seen and that muscle
were observed in RER values between the overnight and the glycogen content was unchanged could suggest that skeletal
20-h fasted state (Fig. 8). muscle is not immediately involved in recognition of acute
energy oscillations. There is no doubt, however, that fasting for
DISCUSSION 20 h while maintaining normal daily physical activity must
In the present study we have used a very simple intervention cause a temporary negative energy balance larger than nor-
protocol with the aim of mimicking the perturbations in energy mally experienced in a daily basis. This is also indicated by our
stores that are inherent in a physical active lifestyle with finding of decreased plasma glucose concentrations after 20-h
regular exercise sessions. In a wider perspective we have tried fasting. We did not have the possibility to estimate the hepatic
to unravel the significance of genes that may be responsible for glycogen stores, but from animal studies (17) we must infer
an evolutionary selection process, i.e., the thrifty genes. In this that liver glycogen probably also decreased considerably dur-
context the used intervention seems inevitably small. Never- ing the 20-h fasting periods. It has previously been suggested
theless, by subjecting healthy men to cycles of feast and famine that usage of muscle energy depots during fasting would be an
we did change the metabolic status to the better, implying that evolutionary disadvantage, because it would lessen the capac-
the mismatch between our ancient genotype and the lifestyle of ity for physical performance and hence the ability to provide
the westernized individual of today became smaller. To our food (i.e., to hunt and gather) during periods of fasting (6, 45).
knowledge this is the first study in humans in which an The present findings support this view.
increased insulin action on whole body glucose uptake and In contrast to the findings in skeletal muscle, the adipose
adipose tissue lipolysis has been obtained by means of inter- tissue responded to the changes in energy balance as intermit-
mittent fasting. This result is in accordance with previously tent fasting changed the plasma concentrations of the adipo-
reported in rodents (2, 32). In these studies, fasting every cyte-specific hormones leptin and adiponectin. However, be-
second day increased the insulin sensitivity approximately cause we did not measure the energy stores in the adipose
J Appl Physiol • VOL 99 • DECEMBER 2005 • www.jap.org
METABOLIC EFFECTS OF INTERMITTENT FASTING 2133
tissue during the intervention (e.g., by fat cell size), we cannot several transcription factors including myocyte enhancing fac-
determine whether the change in adipokine release is merely a tor that increases GLUT4 expression (24, 27). Another possi-
secondary response to intermittent fasting or whether the adi- bility is that the adiponectin boosts peroxisome proliferated-
pose tissue is an active recognizer of energy oscillations. activated receptor-␥ (PPAR-␥) expression as seen in 3T3-L1
Blood sampling for measurement of adipokines at basal adipocytes (1). In addition, because PPAR-␥ induces adiponec-
levels before and after the fasting intervention was performed tin expression (16), it can be speculated that fasting starts a
at 1000 whereas the three samples on days 6, 10, and 14 were positive feedback loop that results in increased levels of both
taken at 1700. The amount of circulating adiponectin is con- circulating adiponectin and PPAR-␥. Both are known to in-
stant or slightly decreased during daytime (20). Hence, the crease the insulin sensitivity.
boosts of 37% we observed after each fasting period are not A considerable increase in plasma FFA concentrations (5-
due to nocturnal variation. Because the plasma adiponectin fold) may raise the amount of circulating adiponectin slightly
concentration is positively correlated to insulin sensitivity in (43), and glucocorticoids positively regulate adiponectin gene
humans (8, 23, 29) and adiponectin administration in rodents
expression (21). FFA and glucocorticoid increase during fast-
increases insulin action (9, 38, 48), it seems likely that our
ing, but in previous studies no effect of fasting on circulating
finding of increases in circulating adiponectin after each fasting
adiponectin was seen (19, 49). Apart from differences in
period would be able to exert an insulin-sensitizing effect.
Skeletal muscle content of GLUT4 protein after the over- increases of FFA and glucocorticoids, different analysis meth-
night fast did not differ before and after the fasting interven- ods used [RIA vs. ELISA (present study)] may recognize
tion. Future studies will have to determine whether the insulin different isoforms of adiponectin and thereby account for the
signaling, e.g., phosphorylation of the insulin receptor sub- discrepancy.
strate, is influenced by fluctuations in energy stores and thereby Leptin exhibits nocturnal differences with a peak during the
accounts for the increase insulin action as measured by the night (2400 – 0800), whereas there is no difference between
clamp method reported herein. 1000 and 1700; if anything, plasma leptin concentrations are
Because 36 h passed between the last fasting period and the slightly higher at 1000 (40). In accordance with previous
last clamp, it seems most likely that the potential insulin- findings (19, 41, 49), we found a decrease in circulating leptin
sensitizing effects of adiponectin were due to adiponectin- after 8 –20 h of fasting. This decrease most likely reflects a
induced changes in gene expression. This could in turn be state of energy deficiency and is probably not involved in the
mediated through an AMPK activation that further activates increased insulin action we have found in the present study.
J Appl Physiol • VOL 99 • DECEMBER 2005 • www.jap.org
2134 METABOLIC EFFECTS OF INTERMITTENT FASTING
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