Acta Physiol 2007, 191, 205–216

Effects of a 3-day fast on regional lipid and glucose

metabolism in human skeletal muscle and adipose tissue

J. Gjedsted,1,2 L. C. Gormsen,2,3 S. Nielsen,2,3 O. Schmitz,3,4 C. B. Djurhuus,2 S. Keiding,5

H. Ørskov,2 E. Tønnesen1 and N. Møller2,3
1 Department of Anaesthesia and Intensive Care Medicine, Aarhus University Hospital, Noerrebrogade, Aarhus, Denmark
2 Medical Research Laboratories, Clinical Institute, Aarhus University, Aarhus, Denmark
3 Medical Department M (Endocrinology and Diabetes), Aarhus University Hospital, Noerrebrogade, Aarhus, Denmark
4 Institute of Clinical Pharmacology, Aarhus University, Aarhus, Denmark
5 Department of Medicine V (Hepatology and PET centre), Aarhus University Hospital, Noerrebrogade, Aarhus, Denmark

Received 5 February 2007, Abstract

revision requested 19 April 2007,
revision received 2 May 2007,
accepted 25 May 2007
Correspondence: J. Gjedsted,
Medical Research Laboratories,
Aarhus University Hospital,
Noerrebrogade, DK-8000 Aarhus
C, Denmark. E-mail:
jakobgjedsted@dadlnet.dk
Aim: Fasting is characterized by increased whole body lipolysis and lipid
oxidation, decreased glucose oxidation and insulin resistance. To identify the
regional sources and underlying mechanisms, we studied 10 healthy male
volunteers post-absorptively and after 72 h of fasting.
Methods: Each study comprised a 3-h basal period and a 3-h hyperinsulin-
aemic euglycaemic clamp and we used a combination of leg and forearm
arteriovenous techniques, upper and lower body microdialysis and glucose
and palmitate tracers.
Results: In the basal state, plasma levels, fluxes and oxidation rates of free
fatty acids all roughly doubled after fasting. Palmitate fluxes across the
forearm and leg also increased by two to threefold and interstitial leg muscle
glycerol concentrations doubled. Subcutaneous femoral glycerol concentra-
tions and blood flows were unaltered, but abdominal subcutaneous blood
flow increased by 50% in the presence of unchanged glycerol concentrations,
indicating stimulated abdominal lipolysis. During the clamp, we observed
whole body insulin resistance and glucose uptake across the leg and forearm
decreased by 60%.
Conclusion: Our data show that fasting induces insulin resistance in upper
and lower body muscles and suggest that increased lipolysis, is primarily due
to the activation of lipolysis in muscle-associated fat (in the leg) and in upper
body subcutaneous fat, whereas peripheral subcutaneous fat is spared.
Keywords A/V balance technique, fasting, metabolism, microdialysis.

In modern western societies fasting or semi-fasting is subsequently on an appropriately regulated release of

mainly relevant to obese patients on weight loss
regimens and to anorectic patients with chronic
diseases. Fasting has, however, always been an inevi-
table component of human life throughout evolution
and successful adaptation to fasting has been a
prerequisite for survival of the human species. As
depots of carbohydrate and protein are limited,
successful adaptation to fasting firstly depends on a
high ability to store surplus calories as fat and
free fatty acids (FFA) from adipose tissue. Classic
studies by Cahill et al. (1966, 1970) have established
that during fasting FFA and ketone bodies become the
dominant fuels. It is well established that fasting
induces overall insulin resistance (Newman & Bro-
dows 1983, Mansell & Macdonald 1990, Mansell
et al. 1990, Duska et al. 2005). Studies have indicated
that this is caused by a decreased ability of insulin to
stimulate peripheral glucose disposal. In contrast to

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Journal compilation  2007 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2007.01740.x 205
Fasting glucose and lipid metabolism Æ J Gjedsted et al. Acta Physiol 2007, 191, 205–216

this it seems that the effects of insulin on endogenous
glucose production (EGP) may be either enhanced or
unaltered (Jensen et al. 1988, Castillo et al. 1991).
One study has shown that insulin stimulated forearm
glucose uptake is decreased during fasting (Mansell &
Macdonald 1990).
Other studies have shown that after 84 h of fasting,
whole body lipolysis, assessed by palmitate turnover,
doubles from 1.4 to 2.7 lmol kg)1 min)1 (Jensen et al.
1987), in part due to decreased insulin and increased
adrenaline levels (Wolfe et al. 1987). Progressive
fasting is also characterized by increasing lipid oxida-
tion and decreasing glucose oxidation (Klein et al.
1993).
As regards the regional sources of lipolysis during
fasting, studies utilizing arteriovenous balance com-
bined with isotope dilution techniques have suggested
that regional FFA release rates across the splanchnic
bed and leg rise in the same relative proportions as
those previously observed in the overnight post-
absorptive state (Jensen et al. 2001, Nielsen et al.
2004). Another study has suggested that in the leg
increased skeletal muscle lipolysis (assessed by micro-
dialysis), rather than subcutaneous lipolysis, may be
important during caloric restriction in obese subjects
(Hagstrom-Toft et al. 2001). It has also been shown
that early starvation (14–20 h) is associated with an
increase of lipolysis (assessed by increased FFA efflux)
in abdominal subcutaneous tissue (Samra et al. 1996)
and that weight loss in obesity particularly reduces
abdominal obesity (Ross et al. 2000). Although these
observations have been made in different subjects,
studied under different conditions with different
methods, it may therefore be hypothesized that the
increase in whole body lipolysis during fasting is
caused by a combination of (i) increased peripheral
lipolysis, in particular in skeletal muscle and (ii)
increased abdominal/central lipolysis, including subcu-
taneous adipose tissue.
The present protocol was accordingly designed to
define mechanisms underlying the effects of fasting on
basal and insulin-stimulated lipid and glucose metabo-
lism at the whole body level and regionally in upper and
lower body subcutaneous adipose tissue and muscle and
more specifically to test whether skeletal muscle and
abdominal subcutaneous tissue are predominant regio-
nal sources of lipolysis. For this purpose, we employed a
combination of isotope dilution with labelled palmitate
and glucose, arteriovenous balance techniques across
the forearm and leg and microdialysis in subcutaneous
adipose tissue and in muscle.
Materials, methods and study design
We used [3-3H] glucose (Lægemiddelstyrelsen, Isotop
Apoteket, Denmark) and albumine bound [9,10-3H]
palmitate (Lægemiddelstyrelsen). The chemical, isoto-
pic and optical purity of the isotopes were tested before
use. Solutions were prepared under sterile conditions
and were shown to be free of bacteria and pyrogens
before use.
Indocyanine green (ICG – Cardiogreen) was pur-
chased from Hyson, Wescott and Dunning, Baltimore,
MD, USA.
The study was approved by the local Ethics Commit-
tee and all participants gave written informed consent
before participating.
Ten healthy, lean, male volunteers were included in
the study (Table 1). All had normal fasting glucose
values and no further examinations of glucose tolerance
were performed. The volunteers were investigated
twice, during post-absorptive conditions (12 h fast)
and after 72 h of fast [Breakfast allowed on day 1
(08:00 hours) and admitted on day 4]. The volunteers

Table 1 Volunteer characteristics

Basal Clamp
postabs Fasting P-value postabs Fasting P-value

Age (years) 25  3.4

Weight (kg) 83.6  1.87 80.8  2.1 P < 0.01
BMI (kg m)2) 24.5  0.36 23.7  0.4 P < 0.01
Leg blood flow (mL min)1) 438  67 658  93 P < 0.01 553  78 541  93 P = 0.5
Arm blood flow (mL 100 mL)1 · min) 2.3  0.2 3.5  0.2 P < 0.01 3.6  0.4 4.3  0.5 P < 0.05
Abd.Xenon133 wash-out slope (·10)5) )6.8  0.8 )10.9  1.5 P < 0.05 )3.8  0.8 )5.5  0.7 P = 0.9
Fem.Xenon133 wash-out slope (·10)5) )5.4  0.6 )7.0  1.0 P = 0.18 )4.5  0.6 )6.9  0.9 P = 0.07
Energy expenditure (kcal day)1) 1945  36 1999  69 P = 0.4 2033  37 2077  38 P = 0.4
RQ 0.84  0.01 0.77  0.001 P < 0.01 0.88  0.01 0.78  0.01 P < 0.01

BMI, body mass index; Leg blood flow, calculated from dye dilution (ICG) data; Arm blood flow, calculated from plethysmography;
Abd.Xenon133 and Fem.Xenon133, slope from the xenon133 wash-out curve; Energy expenditure, calculated from calorimetry; RQ,
respiratory quotient: calculated from indirect calorimetry; P-values, postabsorptive vs. fasting.

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206 Journal compilation  2007 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2007.01740.x
Acta Physiol 2007, 191, 205–216
were allowed to drink tap water and nothing else
throughout the fasting period. Physical exercise was not
allowed 2 days before and throughout the fasting
periods. The fasting took place at home, but a 24-h
telephone number to contact the responsible physician
were available if necessary. The subjects were instructed
that any caloric ingestion would be detectable in the
blood samples. The study days were separated by at
least 1 month to allow tracer wash-out. The volunteers
were admitted to The Medical Research Laboratories at
07:30 hours, and remained in bed throughout the day.
The experiments were performed under thermo neutral
conditions (21–23 C).
The study days started with a 180-min basal period,
henceforth referred to as ‘basal’, followed by a 180-min
hyperinsulinaemic euglycaemic clamp referred to as
‘clamp’ (Fig 1).
At t = 0 primed constant infusion of [3-3H] glucose
was started, and at t = 180 min, insulin (Insulin Act-
rapid; Novo-Nordisk, Copenhagen, Denmark) was
infused intravenously at a constant rate of
0.6 mU kg)1 min)1 for 180 min (time, 180–360 min).
Plasma glucose was clamped at 5 mmol L)1 (DeFronzo
et al. 1979). During the clamp, plasma glucose were
measured in duplicate every 10 min on a Beckman
Analyzer (Beckman Instruments, Palo Alto, CA, USA).
During the clamp period (180–360 min) amino acids
were infused (2 mL min)1, Vamin 14 gN L)1, Frese-
nius Kabi) to avoid a decrease in amino acid levels
(Fig 1) and subsequent changes in insulin sensitivity
(Flakoll et al. 1992). Urine was sampled during the
basal and during the clamp period to estimate urea
excretion. Tritiated palmitate was infused from 120–
180 min and again from 300–360 min. Unless other-
wise specified blood samples were obtained from the
femoral artery during the last 30 min of the basal and
the clamp periods.
Figure 1 Study flowsheet. On both study
days, Palm.: Tritiated Palmitate, Glucose:
Tritiated glucose.
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Journal compilation  2007 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2007.01740.x
J Gjedsted et al. Æ Fasting glucose and lipid metabolism
The forearm model
Intravenous catheters (Viggo, Helsingborg, Sweden)
were placed in an antecubital vein for infusions, and
in a retrograde manner in the contralateral antecubital
vein for deep venous samples, arterial blood was
sampled from the femoral artery (Moller et al. 1989).
Before deep venous sampling the wrist cuff were inflated
to a supra systolic pressure. Preceding the blood
sampling, forearm blood flow was determined by
venous occlusion plethysmography.
The leg model
Under local anaesthesia (lidocain 10 mg mL)1, ‘SAD’)
the left femoral artery and vein were cannulated
percutaneously with double lumen 4 fr. Catheters (BD
CareflowTM, Becton Dickinson, Singapore) placed with
the arterial catheter tip in the cranial direction,
approximately at the level of the inguinal ligament and
the venous catheter tip in the caudal direction with the
tip distal to the merge of the great saphenous vein into
the femoral vein. The double lumen catheters allowed
simultaneous blood sampling and infusion in the arterial
catheter with the proximal lumen used for sampling and
the distal lumen for ICG infusion. The exact location
of the catheter tips were visualized by ultrasound
(vingmed vivid 5). Blood sampling for ICG measuring
in quadruplicate allowed blood flow to be determined by
dye dilution technique (Indocyanine green). Due to
difficulties in placing femoral catheters, two volunteers
were only examined with forearm technique.
Microdialysis
Microdialysis fibres (CMA 60 microdialysis catheter;
CMA, Stockholm, Sweden) were placed in the right
207
Fasting glucose and lipid metabolism Æ J Gjedsted et al.
abdominal and femoral subcutaneous adipose tissue.
Prior to the insertion of the muscular catheters,
lidocaine (Lidokain, 10 mg mL)1, ‘SAD’) was injected
superficial to the fascia of the lateral vastus muscle
14 cm above the patella and 20 cm below the tibial
plateau in the medial head of the gastrocnemius muscle.
Correct placement of the microdialysis catheters in the
muscles was confirmed by the presence of muscle
twitches during insertion. Immediately after placement,
fibres were perfused at a rate of 1 lL min)1 (CMA-107
perfusion pump; CMA) with Ringer chloride (T1;
CMA; Na+ 147 mmol L)1; K+ 1.4 mmol L)1; Ca2+
2.3 mmol L)1; Cl– 156 mmol L)1, pH 6; osmolality,
290 mosmol kg)1). A small amount of [3H] glycerol
was added to measure the relative recovery by an
internal reference method (Gravholt et al. 1999). Per-
fusion fluid samples were collected every 30 min from
t = 0 to t = 360 min. An automated spectrophotometric
kinetic enzymatic analyzer (CMA 600; CMA, Solna,
Sweden) was used for duplicate measurements of
glycerol in the microdialysate. Changes in interstitial
glycerol concentration were used as an index of lipolysis
(Jansson et al. 1990). Microdialysis data are presented
as average interstitial glycerol concentrations during the
last 60 min of each period.
Regional blood flow measurements
Blood flow of subcutaneous adipose tissues in the
abdominal and femoral regions was measured by the
local 133Xe wash-out method (Larsen et al. 1966). In
short, 3.7 MBq (0.1 mL) 133Xe was injected subcuta-
neously in the abdomen and leg. The decay curve of
133
Xe was continuously measured starting 30 min after
injection using an NaI detector (Mediscint; Oakfield
Instruments, Workingham, Berkshire, UK) as previously
described (Christiansen et al. 2005).
Blood flow of the left leg was determined using
continuous infusion of ICG. ICG was infused in the
femoral artery in an upstream manner to ensure rapid
and complete mixture of the dye in the vessel. Samples
from the femoral artery and vein were taken in
quadruplicate at the end of each period for measure-
ments of ICG (Jorfeldt & Rutberg 1990, Ott et al.
1993) and haematocrit. Correction of non-steady state
was <10% of the infusion dose, and mean arterial and
venous concentrations were used for calculations and
blood flow was calculated. Forearm blood flow was
determined by venous occlusion plethysmography, pre-
ceding every blood sample.
Glucose tracer
At t = 0, a bolus dose (15 lCi) of [3-3H] glucose was
injected, followed by a constant-rate infusion
208 Journal compilation  2007 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2007.01740.x
Acta Physiol 2007, 191, 205–216
(0.15 lCi min)1) throughout the experiment. To min-
imize rapid dilution of the labelled glucose pool with
unlabelled glucose, [3-3H] glucose was added to the
glucose infused during the clamp (Finegood et al. 1987,
Hother-Nielsen et al. 1992).
The specific activity (SA) of tritiated glucose was
measured as previously described (Moller et al. 1990).
Rates of appearance (Ra) and disappearance (Rd) of
glucose were calculated using Steele’s equation for
non-steady state, and a pool fraction of 0.65 was
used. Endogenous glucose production during the
clamp was calculated by subtracting the rate of
glucose infusion (M value) from glucose Ra, deter-
mined isotopically (Moller et al. 1990, Hother-Nielsen
et al. 1992).
Palmitate tracer
Infusion of [9,10-3H] palmitate (0.3 lCi min)1) was
started at 120 and 300 min and maintained for 1 h.
Blood samples for measurements of palmitate concen-
tration and SA were drawn before the infusion and after
40, 50 and 60 min of the infusion period. Systemic
palmitate flux were calculated using the [9,10-3H]
palmitate infusion rate divided by the steady-state
palmitate SA. Forearm and leg palmitate balances
(uptake and release) were estimated using blood flow
and specific activities (SA) from arterial and venous
samples and calculated as previously described (Nielsen
et al. 2004). Forearm and leg palmitate balances were
not determined during the clamp period due to insuf-
ficient sample material.
Analyses
We used a two-site immunoassay ELISA (DAKO,
Glostrup, Denmark) (Andersen et al. 1993) to measure
serum insulin. A double monoclonal immunofluoromet-
ric assay (Delfia; Perkin-Elmer, Turku, Finland) was
used to measure serum GH, while plasma glucagon
(Orskov et al. 1968) was measured by radioimmunoas-
say (Immunoclear, Stillwater, MN, USA). Serum corti-
sol was measured with a solid-phase time-resolved
fluoroimmunoassay (Delfia; Perkin-Elmer). Plasma
adrenaline and noradrenaline concentrations were
determined by electrochemical detection after high-
pressure liquid chromatography (Eriksson & Persson
1982). Serum FFA were determined by a colorimetric
method employing a commercial kit (Wako Chemicals,
Neuss, Germany). Blood samples were deproteinized
with perchloric acid for determination of, glycerol,
3-hydroxybutyrate (3-OHB), and lactate by an auto-
mated fluorometric method (Lloyd et al. 1978). Plasma
ICG was determined by spectrophotometric analysis
(Ott et al. 1993). Whole blood concentration of amino
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Acta Physiol 2007, 191, 205–216 J Gjedsted et al. Æ Fasting glucose and lipid metabolism
acids determined by HPLC technique (Jones & Gilligan
1983). Plasma palmitate concentration and SA were
determined by HPLC (Miles et al. 1987) by use of
[2H31] palmitate as internal standard (Jensen et al.
1988). Blood and urine urea was measured by the
urease–Berthelot method (Fawcett & Scott 1960).
Respiratory exchange rates (RQ) and total energy
expenditure (EE) was calculated from indirect calorim-
etry (Deltatrac; Datex Instrumentarium, Helsinki,
Finland) and urea excretion. Oxidation rates of glucose
and of lipids were calculated based on non-protein EE
and RQ (Frayn 1983).

Statistics
All results are given as mean  standard error. All
comparisons are basal post-absorptive vs. fasting and
clamp post-absorptive vs. fasting unless otherwise
specified. Paired t-tests were applied, and P-values
<0.05 were considered statistically significant.
Results
The characteristics of the volunteers are given in
Table 1. Fasting resulted in an average weight loss of
2.7 kg. Specific activities of glucose- and palmitate
tracers were stable during the sampling periods (data
not shown).
Basal
Energy expenditure was unaltered whereas RQ signif-
icantly declined during fasting (Table 1, Fig. 2). Limb
blood flow increased significantly by around 50%
(P < 0.01) after fasting in both forearm and leg
Figure 2 Calorimetry data. Calorimetry was performed at the
end of each period, urine collection were performed during
each period. PA, post absorptive.
(Table 1). Fasting increased abdominal subcutaneous
blood flow by 60% (xenon wash-out curve slopes 10.9
vs. 6.8, P < 0.05), whereas femoral subcutaneous blood
flow tended to increase (xenon wash-out curve slopes
7.0 vs. 5.4, P = 0.18).
Insulin levels decreased significantly during fasting
(Table 2) whereas the counter regulatory hormones
GH, glucagon, adrenaline and noradrenaline all
increased. We did not detect any increase in cortisol.
Baseline plasma-glucose and circulating levels of
alanine decreased, whereas glycerol FFA, 3-hydroxybu-
tyrate significantly increased (Table 2). Lactate and
total amino acid levels were unaltered (Table 2).
Fasting caused a decrease in EGP and a 75% decrease
in glucose oxidation (Table 3, Fig. 2). Non-oxidative
glucose disposal was unaltered.

Table 2 Hormone and metabolite levels

Basal postabs. Fasting P-value Clamp postabs. Fasting P-value

Insulin (pmol L)1) 43.0  6.0 28.9  4.8 P < 0.01 294  21.2 279  18.5 P = 0.16
Glucagon (pg mL)1) 33.5  4 71.6  12 P < 0.01 69.3  11.4 92.7  20.6 P = 0.09
GH (ng mL)1) 1.1  0.5 2.9  0.7 P < 0.05 3.2  1.2 1.1  0.3 P = 0.07
Cortisol (nmol L)1) 346  25.6 340  42.5 P = 0.9 234  14.0 238  26.7 P = 0.9
Adrenaline (pg mL)1) 27.4  3 57.3  11.1 P < 0.05 35.3  3.3 43.8  7.4 P = 0.1
Noradrenaline (pg mL)1) 142  11 228  29 P < 0.05 187  24 147  12 P = 0.14
Glucose (mmol L)1) 5.2  0.1 3.8  0.2 P < 0.01 5.0  0.04 5.1  0.1 P = 0.2
Glycerol (lmol L)1) 45.6  6.65 76.3  10.2 P < 0.01 18.9  4.5 30.6  2.1 P < 0.05
FFA (lmol L)1) 584  50 1304  80 P < 0.01 61.5  9.4 358  34.9 P < 0.01
3-OHB (lmol L)1) 70.2  27 2963  420 P < 0.01 7.4  1.2 274  53.6 P < 0.01
Alanine (lmol L)1) 585  165 398  32 P < 0.01 894  98 623  42 P < 0.05
Lactate (lmol L)1) 371  70 352  45 P = 0.78 406  55.2 278  21.9 P < 0.05
Total amino acid (lmol L)1) 4953  911 5024  401 P = 0.87 6914  337 6206  489 P = 0.18

Blood samples were drawn from femoral artery in triplicate during the last 30 min of each period (basal and clamp). GH, growth
hormone; FFA, free fatty acids; 3-OHB, 3-hydroxybutyrate. P-values, postabsorptive vs. fasting (paired t-test).

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Journal compilation  2007 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2007.01740.x 209
Fasting glucose and lipid metabolism Æ J Gjedsted et al. Acta Physiol 2007, 191, 205–216

Table 3 Whole body glucose metabolism

Basal Clamp
postabs. Fasting P-value post abs. Fasting P-value
)1 )1
M-value (mg kg min ) 4.8  0.5 2.3  0.3 P < 0.01
EGP (mg kg)1 min)1) 2.0  0.2 1.3  0.04 P < 0.01 0.2  0.1 0.3  0.1 P = 0.4
GlucoseRd (mg kg)1 min)1) 2.1  0.5 1.4  0.05 P < 0.01 5.2  0.3 2.5  0.2 P < 0.01
GlucoseOX (mg kg)1 min)1) 1.5  0.2 0.5  0.2 P < 0.01 1.9  0.2 0.6  0.1 P < 0.01
GlucoseNonOx (mg kg)1 min)1) 0.5  0.1 0.9  0.2 P = 0.57 3.4  0.4 1.9  0.3 P < 0.01

M-value, glucose infusion rate per kilogram bodyweight; EGP, endogenous glucose production; GlucoseRd, glucose rate of disap-
pearance; GlucoseOX, glucose oxidation rate; GlucoseNonOx, non-oxidative glucose disposal. All mean values of last 30 min of the
basal and clamp period. P-values, postabsorptive vs. fasting.

In parallel with FFA, palmitate concentrations dou-
bled after fasting (P < 0.01), palmitate flux increased
from 236 to 390 lmol min)1 (P < 0.01) and lipid
oxidation rates increased (Table 4). Regional limb
palmitate rates of appearance and rates of disappear-
ance all increased two to threefold (P < 0.05). The
fractional extraction of palmitate were unaltered in the
forearm (0.35  0.05 vs. 0.33  0.4, P = 0.7) and in
the leg (0.36  0.05 vs. 0.25  0.02, P = 0.1).
There were no detectable alterations in net arterio-
venous exchange of glucose, FFA, glycerol or 3-hydrox-
ybutyrate across the leg or the arm (Table 5).
Microdialysis showed a significant increase in intersti-
tial muscle glycerol levels, from the post-absorptive
state to the fasting state – in m. gastrocnemius (97  10
lmol L)1 vs. 163  18 lmol L)1) and m. vastus later-
alis (69  7 lmol L)1 vs. 149  6 lmol L)1) – but not
in abdominal (332  43 lmol L)1 vs. 368  30 lmol
L)1) or femoral (332  59 lmol L)1 vs. 298  29
lmol L)1) subcutaneous adipose tissue (Fig. 3).
Clamp
Insulin, GH, glucagon, cortisol, adrenaline and
noradrenaline concentrations were comparable during
HE-clamp steady-state (Table 2). Alanine and lactate
concentrations were significantly lower during fasting,
but levels of total amino acids were comparable
(Table 2). EE was also unaltered but the respiratory
quotients remained decreased during fasting (Table 1).
Plasma glucose levels were clamped at 5 mmol L)1
and were not significantly different (Table 2). During
fasting limb A/V clamp differences of glucose (Table 5)
and M-values (Table 3) were more than halved. Both
oxidative and non-oxidative glucose disposal rates
decreased, however, EGP was not significantly different
in the two situations (Table 3).
Glycerol, FFA and 3-hydroxybutyrate levels remained
significantly higher in the fasting state (Table 2). Palm-
itate concentrations, whole body palmitate fluxes and
lipid oxidation rates were all significantly increased
(Table 4). Net leg release (Table 5) of FFA and
3-hydroxybutyrate were significantly increased, and
limb release of glycerol also tended to increase. Net
forearm release of glycerol increased.
During the clamp, microdialysis showed that glycerol
levels after fasting were significantly higher in the vastus
lateralis muscle (30.73  4.97 lmol L)1 vs. 74.69 
7.49 lmol L)1), but not in the gastrocnemius muscle
(73.53  17.13 lmol L)1 vs. 86.94  11.96 lmol L)1),

Table 4 Whole body and regional lipid metabolism

Basal postabs Fasting P-value Clamp postabs Fasting P-value

Palmitate (lmol L)1) 165  13.5 329  14.5 P < 0.01 18.6  2.3 92.1  9.2 P < 0.01
Palmitate flux (lmol min)1) 236  19 390  24 P < 0.01 43.3  3.3 142  10.5 P < 0.01
LipidOX (mg kg)1 min)1) 0.7  0.1 1.1  0.01 P < 0.05 0.4  0.01 1.0  0.01 P < 0.01
Arm palmitateRd (nmol/min · 100 mL)1) 65  7.1 207  39 P < 0.01 * *
Leg palmitateRd (lmol/min · leg)1) 11.5  1.3 30.0  5.3 P < 0.05 * *
Arm palmitateRa (nmol/min · 100 mL)1) 73  14 207  56 P < 0.05 * *
Leg palmitateRa (lmol/min · leg)1) 10  1.8 31.4  3.6 P < 0.05 * *

LipidOX, lipid oxidation rate calculated from indirect calorimetry; Arm palmitateRd, palmitate rate of disappearance in forearm
calculated from tracer fractional uptake; Leg palmitateRd, palmitate rate of disappearance in leg calculated from tracer fractional
uptake; Arm palmitateRa, palmitate rate of appearance in forearm; Leg palmitateRa, palmitate rate of appearance in leg. *Palmitate
tracer balances were not measured during the clamp period due to lack of sample material. P-values, postabsorptive vs. fasting.

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Acta Physiol 2007, 191, 205–216 J Gjedsted et al. Æ Fasting glucose and lipid metabolism
Table 5 Net arterio-venous metabolite differences (arterial minus venous)

Basal Clamp
postabs. Fasting P-value post abs. Fasting P-value
)1
Glucose (mmol L )
Arm 0.1  0.1 0.2  0.1 P = 0.07 0.5  0.1 0.2  0.1 P < 0.05
Leg )0.01  0.02 )0.02  0.02 P = 0.64 0.73  0.2 0.27  0.1 P < 0.05
Glycerol (lmol L)1)
Arm )14  5.8 )140.14 P = 0.99 )1.5  2.5 )9.8  2.9 P < 0.05
Leg 2.6  4.6 )4.6  9.2 P = 0.5 )4.1  14.0 )15.7  5 P = 0.09
FFA (lmol L)1)
Arm )20.7  50.4 )56.4  103 P = 0.6 2.4  5.3 )8.6  21.3 P = 0.63
Leg 12.4  17.3 )80.6  54.6 P = 0.2 )4.6  5.4 )77.1  28.7 P < 0.05
3-OHB (lmol L)1)
Arm 12  11.9 309  291 P = 0.33 )0.6  0.9 )109  44 P < 0.05
Leg 12.9  1 )14.2  117.6 P = 0.82 0.02  1.7 )81.9  30.7 P < 0.05
Alanine (lmol L)1)
Arm )74  19 )40  11 P = 0.19 0  9.5 )33.5  19.5 P = 0.21
Leg )30.4  13.6 )99.2  17.2 P < 0.05 )1.7  21.5 )5.4  13.9 P < 0.9
Lactate (lmol L)1)
Arm )118  53 )57  34 P = 0.33 )31  20 )103  41 P = 0.21
Leg )57.9  38 )68.8  35.5 P = 0.79 )22.9  31.5 )53.1  23.3 P = 0.45

Mean values of samples drawn in triplicate during the last 30 min of each period. FFA, free fatty acids; 3-OHB, 3-hydroxybutyrate.
P-values, postabsorptive vs. fasting.

Figure 3 Microdialysis data. Glycerol

levels in adipose tissue (abdominal and
femoral) and skeletal muscle (gastrocne-
mial and lateral vastus muscles),
*P < 0.05 (postabsorptive vs. fasting);
NS, not significant.

nor in the femoral (204.8  49.98 lmol L)1 vs. The limb blood flow within the study days responded
178.91  19.44 lmol L)1) or the abdominal (171.91  different to the hyperinsulinaemic clamp as the leg
18.81 lmol L)1 vs. 254.64  39.89 lmol L)1) subcu- blood flow both in the post-absorptive and the fasting
taneous adipose tissue. state were unaltered whereas the forearm blood flow in

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Journal compilation  2007 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2007.01740.x 211
Fasting glucose and lipid metabolism Æ J Gjedsted et al.
the post-absorptive state increased by 50% (P < 0.01)
and in the fasting state by 20% (P < 0.05).
Discussion
Our study was designed to address the issues of whether
and how upper and lower body subcutaneous fat and
muscle contribute to lipolysis and whether muscles in
the arm and in the leg contribute to insulin resistance
after 72 h of fasting. In accordance with previous
studies, we found substantially increased whole body
lipolysis, as evidenced by two- and 40-fold increased
concentrations of FFA and 3-hydroxybutyrate,
increased palmitate concentrations and fluxes and
increased lipid oxidation rates during fasting. We also
saw two to threefold increases in regional leg and
forearm palmitate fluxes, in the presence of increased
muscle glycerol and unchanged subcutaneous fat glyc-
erol concentrations, suggesting that lipolysis in muscle
are stimulated to a larger extent than lipolysis in
subcutaneous fat. During the glucose clamp, glucose
uptakes in the leg and the forearm were substantially
decreased. These data add new evidence that fasting is
associated with insulin resistance in both upper and
lower body muscles and that lipolysis in non-subcuta-
neous lower body fat compartments, such as muscle, is
significantly activated during fasting. At the same time
our finding that subcutaneous abdominal glycerol
concentrations are unchanged in the presence of a
50% increase in local blood flow suggests that upper
body subcutaneous lipolysis is stimulated.
Ever since the seminal studies of Benedict (1915) and
of Cahill et al. (1966), Cahill (1970), it has been clear
that fasting leads to increased lipolysis and lipid
oxidation. The sources of FFA, however, remain
uncertain. One study by Jensen et al. (2001), utilizing
hepatic vein catheterization and dilution with labelled
palmitate and glycerol, reported a small splanchic FFA
release of around 100 lmol min)1 or 15% of whole
body flux after a 60 h fast, which considering the
methodology may be an underestimate. At the same
time, a five to sixfold dilution of labelled glycerol across
the splanchnic bed was observed (Jensen et al. 2001). A
high femoral release of glycerol was also observed and it
was assessed that each leg contributed a total of around
15% of whole body FFA release. Another study utilizing
venous cannulation of subcutaneous abdominal fat after
early (14–20 h) starvation reported a 30% increase in
FFA release (Samra et al. 1996). Some considerations,
however, have to be taken into account. Fasting is a
progressive process and it is likely that there may be
regional differences between FFA sources after early
14–20 h of fasting (Samra et al. 1996) and after 72 h of
fasting. In the study by Jensen et al. (2001), splanchnic
and leg lipolysis each only contributed around
212 Journal compilation  2007 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2007.01740.x
Acta Physiol 2007, 191, 205–216
100 lmol min)1 (corresponding to below 40%) of a
total whole body FFA flux of 740 lmol min)1, so the
remaining fraction by inference must have originated
from upper body non-splanchnic adipose tissue. That
study did not allow a direct comparison to basal state
conditions, but the findings were comparable with
studies by the same group in non-fasted healthy, lean
participants in the basal post-absorptive state, in whom
80–90% of whole body lipolysis was estimated to occur
in non-splanchnic upper body fat (Nielsen et al. 2004).
As noted by the authors it is, however, conceivable that
the splanchnic model employed due to simultaneous
mesenterial and omental release of FFA and subsequent
hepatic uptake underestimates splanchnic FFA release
(Jensen et al. 2001). That notion, which in our view is
supported by the five to sixfold dilution of glycerol in
the same study (Jensen et al. 2001), together with our
current data, by inference, support, that the splanchnic
bed contributes substantially to the increased lipolysis
during fasting.
In our study, we also found that during fasting each
leg contributed around 10% of whole body FFA/
palmitate flux. In this context, it should be noted that
the regional leg and forearm palmitate fluxes include
contributions from all tissue compartments in the limbs,
including both subcutaneous and intramuscular lipoly-
sis. Somewhat surprisingly we did not see any indices of
increased lipolysis in subcutaneous femoral fat. Com-
pared with the overnight post-absorptive state, adipose
tissue glycerol concentration and local blood flow was
unchanged. The observations that local leg palmitate
rate of appearance was increased threefold together
with a doubling of interstitial muscle glycerol concen-
trations and a 50% increase in leg blood flow clearly
suggest that lipolysis in muscle is increased during
fasting. The notion of an increase in muscle lipolysis is
further supported by the fact that the vastus lateralis
and the gastrocnemicus muscles exhibited very similar
glycerol patterns. The mechanisms behind this process
could be intramyocellular lipolysis or more likely
intermyocellular lipolysis, lipolysis in intermuscular
adipose tissue or lipoprotein lipase (LPL) activated
lipolysis of circulating triacylglycerols. Recent studies
have demonstrated the existence of significant inter-
muscular fat depots, amounting to around 1 kg (Galla-
gher et al. 2005) and have also shown that skeletal
muscle LPL activity increases, whereas adipose tissue
LPL activity decreases during fasting (Ruge et al. 2005).
The fact that net arterio-venous differences of glycerol
across muscle remained unaltered, despite two to
threefold increased interstitial glycerol concentrations
and limb palmitate fluxes, may in part be explained by
simultaneous uptake and release of glycerol in skeletal
muscle (Guo & Jensen 1999, van Hall et al. 2002), in
part by the observed increase in limb blood flow and in
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Acta Physiol 2007, 191, 205–216
part by increased activity of the newly discovered
adipose triglyceride lipase (ATGL), which hydrolyses
triacylglycerol to diacylglycerol without any subsequent
glycerol release (Zimmermann et al. 2004).
Bone marrow adipose tissue, which reacts to the
lipolytic actions of growth hormone (Gevers et al.
2002) is another potential source of leg lipolysis.
Regulation of lipolysis is complex and appears to be
different in muscle and adipose tissue (Enoksson et al.
1998) and the low levels of insulin and high levels of
catecholamines in the fasting state could potentially
activate lipolysis in skeletal muscle more than in
subcutaneous tissue (Navegantes et al. 2003). A study
with caloric restriction in obese women (Hagstrom-Toft
et al. 2001) suggests that lipolytic insulin sensitivity is
preserved in adipose tissue, but not in skeletal muscle
during caloric restriction and muscle lipolysis, therefore
may become increasingly important during caloric
restriction. Our data show that this also appears to be
the case during strict fasting.
These events do, however, not explain dramatic
increase in whole body lipolysis, which occurs during
fasting. If one assumes that local glycerol release
increases in a curvilinear fashion with increasing blood
flow (Coppack et al. 2005) the combination of a 50%
increase in local abdominal subcutaneous blood flow
and unchanged interstitial glycerol concentrations is
compatible with a 50% increase in upper body subcu-
taneous lipolysis, which explains the major part of the
increase in whole body lipolysis. Again it is very
possible that other upper body sources, such as intra-
muscular, intermuscular and splanchnic fat compart-
ments, may contribute. It has for instance been shown
that during weight loss in obese subjects, there is a
preferential loss of visceral fat (Leenen et al. 1992) and
that this loss of visceral fat is more pronounced in men
than in women (Wirth & Steinmetz 1998).
Glucose metabolism is fundamentally affected by
fasting. A decline in plasma glucose is seen in the early
phases, and after 72 h of fasting, the plasma glucose
stabilizes just below 4 mmol L)1. A decrease in EGP,
similar to the one we observed, is seen after 72 h of
fasting both in obese and in normal weight (Bjorkman &
Eriksson 1985, Rothman et al. 1991, Hellerstein et al.
1997) subjects. Several investigators also have described
a significant decrease in insulin sensitivity with IVGTT
(Cahill et al. 1966) and euglycaemic clamp techniques
(Bjorkman & Eriksson 1985). In our study, muscle
glucose uptake significantly decreases by 60–70% in
both arm and leg during a HE-clamp after a 72-h fast
(Table 3), thus explicitly showing that skeletal muscle
sensitivity to insulin is grossly impaired during fasting.
These findings are in accordance with a previous study
showing decreased insulin stimulated forearm glucose
uptake during fasting (Mansell & Macdonald 1990).
 2007 The Authors
Journal compilation  2007 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2007.01740.x
J Gjedsted et al. Æ Fasting glucose and lipid metabolism
Another interesting observation was that lipid and
glucose oxidation rates remained virtually unchanged
during the clamp, demonstrating that the majority of
infused glucose was used for non-oxidative restoration
of carbohydrate stores. It should also be noted that
during the clamp forearm and leg glycerol and FFA
releases were increased indicating resistance of lipolysis
to insulin in these two compartments.
As with most studies, these experiments have limita-
tions. The participants fasted at home without any strict
supervision. On the other hand even at a hospital it is
impossible to ensure strict adherence to fasting and our
metabolic results show that compliance to the fasting
protocol has been very good. We measured interstitial
glycerol concentrations with microdialysis, which esti-
mates flux-generating concentrations of a variety of
compounds across a diminutive dialysis membrane and
permits assessment of changes in interstitial concentra-
tions of these compounds in various tissues (Fernqvist
et al. 1988, Millet et al. 1998, Rosdahl et al. 1998).
True (or quasi-true) equilibrium across the membrane is
only accomplished with very low flow rates, as used in
the present study (Rosdahl et al. 1998, Djurhuus et al.
2002). Under these circumstances, any increase in
glycerol concentrations in the perfusate may be seen as
a reflection of increased regional lipolysis, provided that
local blood flow and glycerol clearance are not altered. It
should thus be considered that any change in regional
blood flow could alter the flux generating concentration
gradients across the dialysis catheter. It is generally
assumed that local glycerol concentrations decreases and
local glycerol release increases in strict parallel with local
blood flow and a recent study has shown that a tight
correlation exists between adipose tissue blood flow and
capillary glycerol permeability (Coppack et al. 2005). In
the present study, we found increased adipose tissue
abdominal and leg and forearm blood flow together with
unchanged femoral adipose tissue blood flow. Never-
theless our findings of increased muscle glycerol con-
centrations should be interpreted cautiously, as the leg
blood flow measured represents the whole limb and not
solely muscle and as the increase in muscle glycerol to
some extent may be due to spill-over from circulating
glycerol. On the other hand increased total leg blood
flow in the presence of unchanged subcutaneous blood
flow indicates that muscle blood flow was increased,
further supporting that muscle lipolysis was increased.
In this study, we have given many data as simple a/v
differences rather than uptake or release rates and we
have not extrapolated from regional data to whole body
data. The main reason for this approach is that regional
blood flow is highly variable and fluctuating (Butler
1987) and that extrapolation to the whole body level is
based on many assumptions and may not always be
appropriate.
213
Fasting glucose and lipid metabolism Æ J Gjedsted et al.
In conclusion, our data show that the increased
lipolysis seen during fasting is accompanied by two to
threefold increase in regional leg and forearm palmitate
fluxes together with increased muscle glycerol and
unchanged subcutaneous fat glycerol concentrations in
the leg, suggesting that lipolysis in muscle are stimulated
in preference to lipolysis in subcutaneous lower body
fat. Our finding of a 50% increase in subcutaneous
abdominal blood flow in combination with unaltered
glycerol concentrations strongly suggest that upper
body subcutaneous fat lipolysis is markedly stimulated
during fasting. During the glucose clamp glucose
uptakes in the leg and the forearm were substantially
decreased. These data add new evidence that fasting is
associated with insulin resistance in upper and lower
body muscles and suggest that lipolysis in non-subcu-
taneous lower body fat compartments, such as muscle,
and in abdominal subcutaneous fat is significantly
activated during fasting. Our data support the hypoth-
esis that during fasting and caloric restriction lipolysis is
predominantly activated in skeletal muscle and subcu-
taneous (and visceral) abdominal adipose tissue,
whereas peripheral adipose tissue is spared.
Conflict of interest
There is no conflict of interest.
We thank Annette Mengel for excellent expert technical
assistance. Roche Diagnostics kindly donated the microdialysis
catheters. The study received financial support from the FOOD
study group.
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