Prolonged Fasting Identifies Skeletal Muscle Mitochondrial Dysfunction as Consequence Rather Than Cause of Human Insulin Resistance

Abstract

Type 2 diabetes and insulin resistance have been associated with mitochondrial dysfunction, but it is debated whether this is a primary factor in the pathogenesis of the disease. To test the concept that mitochondrial dysfunction is secondary to the development of insulin resistance, we employed the unique model of prolonged fasting in humans. Prolonged fasting is a physiologic condition in which muscular insulin resistance develops in the presence of increased free fatty acid (FFA) levels, increased fat oxidation and low glucose and insulin levels. It is therefore anticipated that skeletal muscle mitochondrial function is maintained to accommodate increased fat oxidation unless factors secondary to insulin resistance exert negative effects on mitochondrial function.
While in a respiration chamber, twelve healthy males were subjected to a 60 h fast and a 60 h normal fed condition in a randomized crossover design. Afterward, insulin sensitivity was assessed using a hyperinsulinemic-euglycemic clamp, and mitochondrial function was quantified ex vivo in permeabilized muscle fibers using high-resolution respirometry.
Indeed, FFA levels were increased approximately ninefold after 60 h of fasting in healthy male subjects, leading to elevated intramuscular lipid levels and decreased muscular insulin sensitivity. Despite an increase in whole-body fat oxidation, we observed an overall reduction in both coupled state 3 respiration and maximally uncoupled respiration in permeabilized skeletal muscle fibers, which could not be explained by changes in mitochondrial density.
These findings confirm that the insulin-resistant state has secondary negative effects on mitochondrial function. Given the low insulin and glucose levels after prolonged fasting, hyperglycemia and insulin action per se can be excluded as underlying mechanisms, pointing toward elevated plasma FFA and/or intramuscular fat accumulation as possible causes for the observed reduction in mitochondrial capacity.

Full text

Prolonged Fasting Identifies Skeletal Muscle
Mitochondrial Dysfunction as Consequence Rather Than
Cause of Human Insulin Resistance
Joris Hoeks,
1
Noud A. van Herpen,
1,2
Marco Mensink,
3
Esther Moonen-Kornips,
1
Denis van Beurden,
1,2
Matthijs K.C. Hesselink,
4
and Patrick Schrauwen
1,2
OBJECTIVE—Type 2 diabetes and insulin resistance have been
associated with mitochondrial dysfunction, but it is debated
whether this is a primary factor in the pathogenesis of the
disease. To test the concept that mitochondrial dysfunction is
secondary to the development of insulin resistance, we employed
the unique model of prolonged fasting in humans. Prolonged
fasting is a physiologic condition in which muscular insulin
resistance develops in the presence of increased free fatty acid
(FFA) levels, increased fat oxidation and low glucose and insulin
levels. It is therefore anticipated that skeletal muscle mitochon-
drial function is maintained to accommodate increased fat oxi-
dation unless factors secondary to insulin resistance exert
negative effects on mitochondrial function.
RESEARCH DESIGN AND METHODS—While in a respiration
chamber, twelve healthy males were subjected to a 60 h fast and
a 60 h normal fed condition in a randomized crossover design.
Afterward, insulin sensitivity was assessed using a hyperinsuline-
mic-euglycemic clamp, and mitochondrial function was quanti-
fied ex vivo in permeabilized muscle fibers using high-resolution
respirometry.
RESULTS—Indeed, FFA levels were increased approximately
ninefold after 60 h of fasting in healthy male subjects, leading to
elevated intramuscular lipid levels and decreased muscular insu-
lin sensitivity. Despite an increase in whole-body fat oxidation,
we observed an overall reduction in both coupled state 3
respiration and maximally uncoupled respiration in permeabil-
ized skeletal muscle fibers, which could not be explained by
changes in mitochondrial density.
CONCLUSIONS—These findings confirm that the insulin-resis-
tant state has secondary negative effects on mitochondrial func-
tion. Given the low insulin and glucose levels after prolonged
fasting, hyperglycemia and insulin action per se can be excluded
as underlying mechanisms, pointing toward elevated plasma FFA
and/or intramuscular fat accumulation as possible causes for the
observed reduction in mitochondrial capacity. Diabetes 59:
2117–2125, 2010
Although the existence of mitochondrial abnor-
malities in patients with type 2 diabetes has
been extensively reported during the last de-
cade (1–5), there is no evidence that a reduced
mitochondrial function is a primary factor in the patho-
physiology of this disease. In fact, alternative theories
state that impaired mitochondrial capacity is secondary to
the insulin-resistant or diabetic state. In this context, it has
been shown that insulin can stimulate mitochondrial bio-
genesis and increases ATP synthesis in skeletal muscle
(6,7). A reduced insulin action in skeletal muscle, as
observed in type 2 diabetic patients, could therefore
contribute to the origin of mitochondrial dysfunction.
Additionally, the increased exposure of skeletal muscle
mitochondria to elevated levels of free fatty acids (FFA),
seen in insulin resistance and type 2 diabetes, has been
suggested to interfere with proper mitochondrial function.
Thus, Szendroedi et al. (8) showed that plasma FFA levels
negatively correlated with mitochondrial function mea-
sured by magnetic resonance spectroscopy. Furthermore,
we showed that the acute elevation of plasma FFA by lipid
infusion is accompanied by downregulation of the tran-
scriptional coactivator peroxisome proliferator-activated
receptor gamma coactivator-1␣(PGC1␣) and other genes
involved in mitochondrial metabolism (9). Moreover, it
was shown in a comparable study that short-term eleva-
tion of lipid availability reduces insulin-stimulated in-
crease in ATP synthase flux in skeletal muscle (10),
although this may mainly reflect an effect of muscular
insulin resistance on ATP flux.
Prolonged fasting (⬎48 h) in humans is accompanied by
a reduction in insulin sensitivity, elevated plasma FFA
levels, elevated intramuscular fat levels, but also an in-
crease in whole-body fat oxidative capacity (11,12). Fur-
thermore, prolonged fasting-induced insulin resistance is
not accompanied by hyperglycemia or hyperinsulinemia,
factors that have been suggested to cause mitochondrial
dysfunction in diabetes (7,13). In fact, prolonged fasting is
a physiologic condition in which insulin resistance devel-
ops to spare glucose for utilization by the brain, and
increased FFA levels are accompanied by increased fat
oxidation. It could therefore be anticipated that despite
the development of insulin resistance, mitochondrial func-
tion is maintained to accommodate increased fat oxidation
during prolonged fasting. Alternatively, if (lipid-induced)
insulin resistance or factors associated with the insulin-
resistant state indeed cause mitochondrial dysfunction,
we anticipate a reduction in mitochondrial function with
prolonged fasting. Therefore, we aim to test the concept
that mitochondrial dysfunction originates secondary to
From the
1
School for Nutrition, Toxicology and Metabolism, Department of
Human Biology, Maastricht University Medical Centre, Maastricht, the
Netherlands;
2
Top Institute Food and Nutrition, Wageningen, the Nether-
lands; the
3
Department of Human Nutrition, Wageningen University,
Wageningen, the Netherlands; and the
4
School for Nutrition, Toxicology and
Metabolism, Department of Human Movement Sciences, Maastricht Univer-
sity Medical Centre, Maastricht, the Netherlands.
Corresponding author: J. Hoeks, j.hoeks@hb.unimaas.nl.
Received 13 April 2010 and accepted 15 June 2010. Published ahead of print
at http://diabetes.diabetesjournals.org on 23 June 2010. DOI: 10.2337/db10-0519.
J.H. and N.A.v.H. contributed equally to this study.
The study has been registered at www.trialregister.nl with registration num-
ber NTR 2042.
© 2010 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit,
and the work is not altered. See http://creativecommons.org/licenses/by
-nc-nd/3.0/ for details.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
ORIGINAL ARTICLE
diabetes.diabetesjournals.org DIABETES, VOL. 59, SEPTEMBER 2010 2117

the development of insulin resistance by employing the
physiologic model of prolonged fasting-induced insulin
resistance.
RESEARCH DESIGN AND METHODS
Twelve healthy, lean, male volunteers who had no family history of diabetes
or any other endocrine disorder participated in this study (Table 1). None of
the subjects engaged in sports activities for more than 2 h per week. Body
composition (14) and maximal aerobic capacity (15) were measured as
described previously. The study protocol was reviewed and approved by the
Medical Ethical Committee of Maastricht University Medical Centre and all
subjects gave their written informed consent before participating in the study.
Experimental design. Subjects participated in two experimental trials: a
60 h fast and a 60 h normal fed condition, in a randomized crossover design
with a 2-week washout period. In the fast condition, subjects were fasted for
60 h (calorie-free drinks only), whereas in the second condition, subjects were
fed in energy balance (50 –35–15% of energy as carbohydrates, fat, and protein,
respectively). Before the start of each experimental period, a standardized
evening meal was provided. Subject stayed in a respiration chamber during
the entire 60 h to ensure compliance to the dietary regime and to allow the
measurement of 24 h substrate oxidation and energy expenditure (16). In the
respiration chamber, subjects followed an activity protocol as previously
described (17). During the intervention, blood samples were taken after 12, 36,
and 60 h after an overnight fast in case of the fed condition.
Hyperinsulinemic-euglycemic clamp. After leaving the respiration chamber
on the morning of the third day, a muscle biopsy was taken (18) and a
hyperinsulinemic-euglycemic clamp procedure (4) was performed. Insulin-
stimulated plasma glucose rate of disappearance (Rd), endogenous glucose
production (EGP), and nonoxidative glucose disposal (mainly reflecting
glycogen synthesis) were calculated as by Phielix et al. (4). Substrate
oxidation in the basal and the insulin-stimulated state was measured using
indirect calorimetry (Omnical, Maastricht, the Netherlands) and calculated
according to Frayn (19).
Muscle biopsy. After taking the muscle biopsy, a portion of the muscle tissue
was directly frozen in melting isopentane and stored at ⫺80°C until assayed.
Another portion (⬃30 mg) was immediately placed in ice cold preservation
medium (4).
Blood analyses. Plasma nonesterified fatty acids (Wako Nefa C test kit; Wako
Chemicals, Neuss, Germany) and glucose (hexokinase method; LaRoche,
Basel, Switzerland) were measured with enzymatic assays automated on a
Cobas Fara/Mira. Insulin concentration was determined using a radioimmu-
noassay (Linco Reseach, St. Charles, MO).
Intramuscular triacylglycerols. Fresh cryosections (5 ␮m) were stained for
intramuscular triacylglycerols (IMTG) by Oil Red O staining combined with
fibertyping and immunolabeling of the basal membrane marker laminin to
allow quantification of IMTG, as described previously (20,21).
Mitochondrial DNA copy number and citrate synthase activity. Mito-
chondrial DNA (mtDNA) copy number, the ratio of NADH dehydrogenase
subunit one (ND1) to lipoprotein lipase (LPL) (mtDNA/nuclear DNA) was
determined as described previously (4). Citrate synthase (CS) activity was
measured spectrophotometrically as described previously (22).
High resolution respirometry. Permeabilized skeletal muscle fibers were
immediately prepared from the muscle tissue collected in the preservation
medium, as described elsewhere (4,23). Subsequently, the permeabilized
muscle fibers (⬃2.5 mg wet weight) were analyzed for mitochondrial function
using an oxygraph (OROBOROS Instruments, Innsbruck, Austria), in essence
according to Phielix et al. (4). To prevent oxygen limitation, the respiration
chambers were hyperoxygenated up to ⬃500 ␮mol/l O
2
. Subsequently, two
different multisubstrate/inhibition protocols were used in which substrates
and inhibitors were added consecutively in saturating concentrations. State 2
respiration was measured after the addition of malate (4 mmol/l) plus
octanoyl-carnitine (50 ␮mol/l) or malate (4 mmol/l) plus glutamate (10
mmol/l). Subsequently, an excess of 2 mmol/l of ADP was added to determine
coupled (state 3) respiration. Coupled respiration was then maximized with
convergent electron input through Complex I and Complex II by adding
saturating concentrations of succinate (10 mmol/l). Finally, the chemical
uncoupler carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP)
was titrated or oligomycin (2 ␮g/ml) was added to evaluate the maximal
capacity of the electron transport chain and the respiration not coupled to
ATP synthesis (state 4o respiration), respectively. The integrity of the outer
mitochondrial membrane was assessed by the addition of cytochrome C (10
␮mol/l) upon maximal coupled respiration. All measurements were performed
in duplicate.
Western blotting. Oxidative phosphorylation (OXPHOS) protein levels and
mitochondrial uncoupling protein-3 (UCP3) content were measured in whole
muscle by Western blotting as described previously (24). UCP3 was expressed
as a ratio over the sum of OXPHOS complexes to correct for differences in
mitochondrial density.
Statistics. Data are reported as means ⫾SE. Statistical analyses were
performed using the statistical computer program SPSS 16.0 for Mac OS X.
Statistical comparisons between the two conditions (fed versus fast) were
performed using the paired Student ttest. Plasma FFA, glucose, and insulin
were compared over time by two-way repeated measures ANOVA for inves-
tigation of treatment and time (treatment*time) interactions. When the
interaction was significant, we performed post hoc testing to determine the
exact location of the difference. Differences were considered statistically
significant if P⬍0.05.
RESULTS
Plasma parameters. A significant treatment*time inter-
action (P⬍0.001) was observed for all plasma parameters
(Fig. 1). At the start of the intervention (t ⫽12 h, after an
overnight fast) plasma FFA levels (Fig. 1A) were similar
between both conditions (221 ⫾18 vs. 215 ⫾27 ␮mol/l,
respectively, P⫽0.82). Upon 60 h of fasting, plasma FFA
increased dramatically, up to 1,981 ⫾95 ␮mol/l vs. 387 ⫾
37 in the fed condition (P⬍0.001).
Plasma glucose values (Fig. 1B) were similar at baseline,
averaging 5.05 ⫾0.09 and 4.98 ⫾0.06 mmol/l in the fed and
the fasted state, respectively (P⫽0.28), and remained
unchanged throughout the fed condition. During fasting,
however, plasma glucose levels gradually decreased to
3.72 ⫾0.13 mmol/l at t ⫽60h(P⬍0.001).
Baseline plasma insulin levels (Fig. 1C) were similar in
both conditions (12.9 ⫾0.9 vs. 11.9 ⫾1.2 ␮U/ml in fed
versus fasted, respectively, P⫽0.25) and did not change in
the fed condition. In the fasted condition however, plasma
insulin levels were markedly reduced to 7.1 ⫾0.6 ␮U/ml at
t⫽36h(P⬍0.001) and were maintained at this lower
level (7.0 ␮U/ml ⫾0.74) at t ⫽60h(P⬍0.001).
Indirect calorimetry. Twenty-four hour energy expendi-
ture during the last 24 h of the 60 h intervention was
slightly but significantly reduced upon prolonged fasting
(10.88 ⫾0.33 vs. 10.30 ⫾0.30 MJ/day, in fed versus fasted,
respectively, P⫽0.02). The difference was mainly caused
by a reduction in diet-induced thermogenesis, and not
caused by a decrease in resting metabolic rate (data not
shown). Additionally, whole-body 24-h fat oxidation was
increased upon prolonged fasting as evidenced by a sig-
nificant reduction in 24-h respiratory exchange ratio
(RER) (0.91 ⫾0.009 vs. 0.77 ⫾0.003 in fed versus fasted,
respectively, P⬍0.001).
Insulin sensitivity. All subjects displayed a decrease in
glucose infusion rate upon 60 h of fasting (Fig. 2A). We
also calculated the insulin sensitivity index, an index that
takes into account the variation in insulin and glucose
levels during the clamp (25). Insulin sensitivity index was
reduced by ⬃45% upon 60 h of fasting, as compared with
the fed condition (Fig. 2B,P⬍0.001).
The reduction in whole-body insulin sensitivity was
mainly accounted for by a reduction in insulin-stimulated
TABLE 1
Subject characteristics
Parameter Mean ⫾SE
Age (years) 23.6 ⫾1.0
Body weight (kg) 78.5 ⫾2.5
Fat-free mass (kg) 65.9 ⫾1.8
Height (m) 1.86 ⫾0.02
BMI (kg/m
2
)22.6 ⫾0.5
Maximal aerobic capacity (ml O
2
/kg
FFM
/min) 57.5 ⫾1.5
PROLONGED FASTING AND MITOCHONDRIAL FUNCTION
2118 DIABETES, VOL. 59, SEPTEMBER 2010 diabetes.diabetesjournals.org

glucose disposal (⌬Rd, P⬍0.001, Table 2). The reduced
insulin-stimulated glucose disposal after fasting, mainly
reflecting muscle glucose uptake, was due to both reduced
insulin-stimulation of glucose oxidation and reduced non-
oxidative glucose disposal (Table 2). However, insulin-
stimulated glucose oxidation seemed to be more severely
suppressed by 60 h of fasting (Table 2). Also, baseline
endogenous glucose production was reduced after 60 h of
fasting. However, insulin-induced suppression of endoge-
nous glucose production, reflecting hepatic insulin sensi-
tivity, was only marginally affected by fasting and was
almost complete in both conditions (Table 2).
Metabolic flexibility. Metabolic flexibility was blunted in
the fasted condition when compared with the fed condi-
tion (Fig. 3A,P⬍0.001). Basal whole-body fat oxidation
was increased by 1.5-fold upon prolonged fasting (Fig. 3B,
P⬍0.001). During the glucose clamp, fat oxidation
significantly decreased in both conditions (Pⱕ0.001), but
the suppression was significantly less in the fasted condi-
tion (Fig. 3B,P⬍0.001). Basal carbohydrate oxidation
after fasting was only ⬃35% of the value obtained in the
fed situation (P⬍0.001), but increased in both conditions
during the glucose clamp (Fig. 3C). However, this insulin-
induced change in carbohydrate oxidation was blunted
upon fasting (P⬍0.001).
Intramuscular triacylglycerols. The mean intramuscu-
lar IMTG area fraction (Fig. 4), was ⬃2.7-fold higher after
60 h of fasting in comparison with the fed condition (P⫽
0.001). The increase in lipid accumulation was more
pronounced (⬃3.5 fold, P⬍0.001) in fibers identified as
slow, oxidative (type 1) fibers. Within type 2 muscle fibers,
IMTG levels increased by approximately twofold after 60 h
of fasting (P⫽0.015).
Mitochondrial function. Mitochondrial DNA copy num-
ber was similar in both the fed and the fasting condition (Fig.
5A,P⫽0.96). Also, OXPHOS protein levels (Complex I:
21.2 ⫾6.4 vs. 17.7 ⫾5.7 arbitrary units, P⫽0.50; Complex II:
48.7 ⫾14.6 vs. 48.3 ⫾16.2 AU, P⫽0.97; Complex III: 11.6 ⫾
1.5 vs. 11.9 ⫾1.1 AU, P⫽0.88; Complex IV: 92.8 ⫾7.0 vs.
93.7 ⫾9.1 AU, P⫽0.85; Complex V: 4.4 ⫾0.7 vs. 4.8 ⫾0.9,
P⫽0.66) and CS activity (76.2 ⫾7.3 vs. 70.2 ⫾6.6
␮mol/min/g protein, P⫽0.41) were similar in the fed versus
the fasted state, respectively, confirming an equal mitochon-
drial mass in both conditions.
Nonetheless, we adjusted the oxygen fluxes for individ-
ual differences in mtDNA copy number. However, similar
results were obtained without this correction (see supple-
mental Fig. 1 in the online appendix available at http://
diabetes.diabetesjournals.org). State 2 respiration (i.e.,
respiration in the presence of substrate alone) was not
different between conditions on any of the substrate
combinations studied (Fig. 5B). However, ADP-stimulated
(state 3) respiration on a lipid substrate (malate ⫹oc-
tanoyl-carnitine, MO) was significantly reduced upon fast-
ing, as compared with the fed situation (P⫽0.03, Fig. 5C).
Similarly, state 3 upon the Complex I substrates malate ⫹
glutamate (MG) was ⬃22% lower after fasting, although
this did not reach statistical significance (P⫽0.12, Fig.
5D).
Additionally, respiration upon parallel electron input to
both Complex I and II was reduced by ⬃20% upon
prolonged fasting. Thus, state 3 respiration upon malate ⫹
octanoyl-carnitine ⫹glutamate (MOG) was significantly
lower in the fasted state compared with the fed condition
(P⫽0.01, Fig. 5E). Similar differences were observed for
state 3 respiration upon malate ⫹octanoyl-carnitine ⫹
glutamate ⫹succinate (MOGS, P⫽0.02, Fig. 5E) and
malate ⫹glutamate ⫹succinate (MGS, P⫽0.04, Fig. 5E).
Maximal FCCP-induced uncoupled respiration, reflecting
the maximal capacity of the electron transport chain, was
also reduced by ⬃23% after 60 h of fasting (P⫽0.008, Fig.
5F). Finally, state 4o respiration (reflecting mitochondrial
proton leak) was similar between the fed and fasted state
(P⫽0.48, Fig. 5G). The average increase in oxygen
consumption upon cytochrome C was less than 10% (un-
Time (hours)
12 36 60
Plasma Glucose (mmol/L)
0
1
2
3
4
5
6
Time (hours)
12 36 60
Plasma FFA (mmol/L)
0.0
0.5
1.0
1.5
2.0
2.5
*
*
**
Time (hours)
12 36 60
Plasma Insulin (µU/ml)
0
2
4
6
8
10
12
14
16
**
A
B
C
FIG. 1. Plasma free fatty acids (A), plasma glucose (B), and plasma
insulin (C) levels after 12, 36, and 60 h of fasting. Open circles
represent the fed condition; closed circles represent the fasted condi-
tion. Values are mean ⴞSE. *P<0.05.
J. HOEKS AND ASSOCIATES
diabetes.diabetesjournals.org DIABETES, VOL. 59, SEPTEMBER 2010 2119

derscoring the viability of the muscle fibers) and similar in
both conditions (P⫽0.54).
Mitochondrial UCP3 did not change upon 60 h of fasting
in humans and averaged 2.21 ⫾0.38 vs. 2.20 ⫾0.41 AU in
the fed versus fasted condition (P⫽0.96).
DISCUSSION
In this study, we evaluated the effect of prolonged fasting
on skeletal muscle mitochondrial functional capacity in
humans to examine whether the mitochondrial dysfunc-
tion that is frequently reported in insulin resistance and
type 2 diabetes can be a consequence of lipid-induced
insulin resistance, rather than a cause. In contrast to the
hyperglycemia and hyperinsulinaemia accompanying
“energy excess”-induced insulin resistance (lipid infu-
sion, high-fat diets), prolonged fasting-induced insulin
resistance is associated with hypoglycemia and hypoin-
sulinemia. Moreover, prolonged fasting-induced lipid
accumulation and insulin resistance are considered to be a
functional physiologic response. Thus, reduced insulin
sensitivity saves carbohydrates for the central nervous
system, being obligate for glucose and not requiring insu-
lin for its uptake, whereas increased lipid availability at
the same time can serve as a direct available energy source
for the muscles and is paralleled by an enhanced fat
oxidative capacity (12). Therefore, we anticipated that
skeletal muscle mitochondrial function would not be
impaired in this model unless mitochondrial function is
impaired by factors that are secondary to the lipid-induced
insulin-resistant state. Intriguingly, we found that only 60 h
of fasting in humans was accompanied by an overall
reduction in skeletal muscle mitochondrial capacity,
which was not explained by changes in mitochondrial
density.
We assessed mitochondrial functional capacity in detail
(Fig. 5) by using a wide variety of substrates and substrate
combinations to determine the maximum ADP-stimulated
respiration (state 3) fueled by a lipid substrate, by Com-
plex I substrates, and upon parallel electron input into
Complex I (NADH) and II (FADH
2
). Interestingly, state 3
respiration was reduced by ⬃20% upon all substrates,
which reduces the possibility that the decline is caused by
substrate-specific alterations such as substrate uptake into
the mitochondria. Therefore, both a reduction in the
activity of the electron transport chain and the oxidative
phosphorylation system could underlie the reduced state 3
respiration. Using the chemical uncoupler FCCP, control
over respiration by the oxidative phosphorylation system
is bypassed; thus, FCCP-induced respiration reflects the
maximal capacity of the electron transport chain. Irrespec-
tive of the intervention, FCCP was able to enhance mito-
chondrial respiration considerably over state 3 values,
indicating that the electron transport chain is not rate-
limiting in state 3. However, FCCP-induced respiration in
Fed Fasted
Glucose Infusion Rate (ml/h)
40
60
80
100
120
140
160
180
200
220
240
SI index (ml min-1 kg-1/µU ml-1)
0
2
4
6
8
10
12
*
AB
FIG. 2. Assessment of insulin sensitivity by hyperinsulinemic-euglycemic clamping after 60 h of fasting. Arepresents the individual data for the
glucose infusion rate. Bdisplays the group results of the insulin sensitivity index, i.e., the glucose infusion rate corrected for body weight,
glucose, and plasma insulin levels during the clamp procedure. The white bar represents the fed condition whereas the black bar depicts the fasted
condition. Values are mean ⴞSE. *P<0.05. S
I
, insulin sensitivity index.
TABLE 2
Substrate kinetics
Fed Fasted
Rd Glucose (␮mol/kg/min)
Basal 11.3 ⫾0.6 7.6 ⫾0.4*
Clamp 38.0 ⫾2.8 18.8 ⫾0.9*
Delta 26.7 ⫾2.6 11.2 ⫾1.0*
EGP (␮mol/kg/min)
Basal 10.7 ⫾0.6 6.8 ⫾0.3*
Clamp ⫺1.0 ⫾0.4 0.7 ⫾0.4*
Delta absolute ⫺11.7 ⫾0.6 ⫺6.07 ⫾0.5*
Delta % 111.6 ⫾5.9 90.0 ⫾6.7*
CHO oxidation (␮mol/kg/min)
Basal 9.7 ⫾0.8 3.4 ⫾0.6*
Clamp 20.8 ⫾1.1 6.6 ⫾0.8*
Delta 11.1 ⫾0.9 3.4 ⫾0.7*
NOGD (␮mol/kg/min)
Basal 1.6 ⫾0.5 4.2 ⫾0.8*
Clamp 17.2 ⫾2.2 12.6 ⫾0.8*
Delta 15.6 ⫾2.4 8.2 ⫾1.1*
Lipid oxidation (␮mol/kg/min)
Basal 1.2 ⫾0.0 1.8 ⫾0.2*
Clamp 0.3 ⫾0.1 1.52 ⫾0.1*
Delta ⫺0.8 ⫾0.1 ⫺0.31 ⫾0.1*
Values are mean ⫾SE. *P⬍0.05. EGP, endogenous glucose
production; CHO, carbohydrate; NOGD, nonoxidative glucose
disposal.
PROLONGED FASTING AND MITOCHONDRIAL FUNCTION
2120 DIABETES, VOL. 59, SEPTEMBER 2010 diabetes.diabetesjournals.org

itself was reduced by ⬃23% upon prolonged fasting (Fig.
5F), indicating that a combined reduction of both the
capacity of the electron transport chain and oxidative
phosphorylation underlies the reduced mitochondrial ox-
idative capacity upon fasting.
The reduced mitochondrial capacity was not accounted
for by a reduction in mitochondrial density. Thus, mtDNA
copy number, CS activity, and OXPHOS protein levels
remained unaffected by fasting. Although this does not
exclude the possibility that prolonged exposure to high
FFA and/or insulin resistance may lead to a reduced
mitochondrial function as observed in type 2 diabetes via
reduced mitochondrial biogenesis (e.g., via PGC-1␣), this
finding indicates that fasting interferes with intrinsic mi-
tochondrial capacity in skeletal muscle. Interestingly, a
similar reduction in intrinsic mitochondrial capacity, with-
out differences in mitochondrial content, was recently
reported by us in type 2 diabetic patients and first degree
relatives when compared with BMI- and age-matched
obese control subjects (4). Thus, the reduction in mito-
chondrial function upon fasting mimics the situation as
observed in the diabetic state, and suggests that similar
(secondary) effects are involved in causing mitochondrial
dysfunction. However, it should be noted that fasting is
not a direct model for type 2 diabetes. Furthermore,
although the available data in the literature underpin the
notion that fasting-induced lipid accumulation is respon-
sible for reduced insulin sensitivity upon fasting, we
cannot exclude the possibility that the reduced mitochon-
drial function upon prolonged fasting triggers the insulin
resistance observed.
Resistance of skeletal muscle to insulin action per se
has been suggested to explain the reduction in mitochon-
drial functional capacity observed in diabetes. Thus, in
healthy individuals, it was shown thata7hinsulin infusion
increased mitochondrial protein synthesis, cytochrome C
oxidase (COX), and citrate synthase (CS) enzyme activi-
ties and ATP production (26). Moreover, it has been
reported that exposing human primary muscle cells to
insulin upregulates the expression of PGC-1␣(27). There-
fore, the reduction in insulin action in type 2 diabetes may
underlie the observed mitochondrial defects. In agreement
with this hypothesis, it was demonstrated that at low
doses of insulin (reflecting postabsorptive levels) the
skeletal mitochondrial ATP synthesis rate was not differ-
ent between diabetic patients and controls, indicating that
Basal Insulin-stimulated
Fat oxidation (µmol kg-1 min-1)
0.0
0.5
1.0
1.5
2.0
2.5
Basal Insulin-stimulated
Respiratory Exchange Ratio
0.70
0.75
0.80
0.85
0.90
0.95
1.00
*
*
A
Basal Insulin-stimulated
Carbohydrate oxidation (µmol kg-1 min-1)
0
5
10
15
20
25
*
*
B
C
*
*
FIG. 3. Indirect calorimetry results after 60 h of fasting in the basal
state and upon the hyperinsulinemic-euglycemic clamp. Ashows that
metabolic flexibility, defined as the change in respiratory exchange
ratio upon insulin stimulation, is blunted upon prolonged fasting. B
and Cdisplay whole-body lipid and carbohydrate oxidation, respec-
tively. White bars/circles represent the fed condition; black bars/circles
represent the fasted condition. Values are mean ⴞSE. *P<0.05.
Area fraction (%)
0
2
4
6
8
10
12
*
Tot a l Type 2Type 1
*
*
FIG. 4. Intramuscular triacylglycerols measured by Oil Red O staining
combined with an immunofluorescence staining against slow myosin
heavy chain (sMHC), to determine fibertyping. White bars represent
the fed condition; black bars represent the fasted condition. Values are
mean ⴞSE. *P<0.05.
J. HOEKS AND ASSOCIATES
diabetes.diabetesjournals.org DIABETES, VOL. 59, SEPTEMBER 2010 2121

State 3, fat oxidation
0
10
20
30
40
50
State 3, complex I+II
MOG MOGS MGS
0
20
40
60
80
100
120
140
Mitochondrial density
Mitochondrial DNA copy number (AU)
0
2000
4000
6000
8000
10000
A
State 2
MMOMG
O
2
flux ([pmol mg
-1
s
-1
] / mtDNA copy # *10
4
)
O
2
flux ([pmol mg
-1
s
-1
] / mtDNA copy # *10
4
)
O
2
flux ([pmol mg
-1
s
-1
] / mtDNA copy # *10
4
)
O
2
flux ([pmol mg
-1
s
-1
] / mtDNA copy # *10
4
)
O
2
flux ([pmol mg
-1
s
-1
] / mtDNA copy # *10
4
)
O
2
flux ([pmol mg
-1
s
-1
] / mtDNA copy # *10
4
)
0
2
4
6
8
10
12
14
B
CE
**
State uncoupled, complex I+II
0
20
40
60
80
100
120
140
160
180
200
220
F
*
State 4o, complex I+II
0
10
20
30
40
50
60
G
*
*
State 3, complex I
0
20
40
60
80
D
MO MG
MOGS MGS
FIG. 5. Evaluation of mitochondrial function. A: Mitochondrial density. B: Mitochondrial respiration upon substrates only (state 2). C:
ADP-stimulated respiration (state 3) upon a lipid substrate. D: State 3 respiration fueled by Complex I-linked substrates. E: State 3 respiration
upon parallel electron input into Complex I and II. F: Maximally uncoupled respiration upon FCCP. G: Mitochondrial respiration uncoupled from
ATP synthesis (state 4o). White bars represent the fed condition, black bars represent the fasted condition. Values are mean ⴞSE, nⴝ10. *P<
0.05. M, malate; O, octanoyl-carnitine; G, glutamate; S, succinate.
PROLONGED FASTING AND MITOCHONDRIAL FUNCTION
2122 DIABETES, VOL. 59, SEPTEMBER 2010 diabetes.diabetesjournals.org

there is no intrinsic muscle mitochondrial defect in type 2
diabetic patients (7). On the other hand, high (postpran-
dial) levels of insulin increased the mitochondrial ATP
production rate in nondiabetic subjects, whereas this
increase was absent in type 2 diabetic patients (7). Fur-
thermore, the lack of response in the diabetic patients was
accompanied by a reduced expression of PGC-1␣, CS, and
COX.
Besides reduced insulin action, the hyperglycemia asso-
ciated with insulin resistance and type 2 diabetes has also
been suggested to exert harmful effects on mitochondrial
functional capacity via induction of oxidative stress. In-
deed, hyperglycemia has been shown to increase mito-
chondrial ROS production in endothelial cells (28), as well
as in other cell types (29). In addition, it was reported that
severe hyperglycemia inhibited respiration in human skel-
etal muscle, which was restored upon insulin treatment
(13).
It should be noted that the insulin-resistant state after
prolonged fasting was accompanied by hypoinsulinemia
and hypoglycemia. Mitochondrial function was thus
assessed after exposure to low insulin and glucose
concentrations. It is therefore unlikely that the reduced
mitochondrial functional capacity observed here is
caused by hyperinsulinemia and/or hyperglycemia asso-
ciated with reduced insulin action. However, it remains
possible that chronic hyperinsulinemia and hyperglyce-
mia may negatively affect mitochondrial function in type
2 diabetes patients.
An alternative candidate to explain the observed reduc-
tion in mitochondrial capacity upon fasting is prolonged
exposure to elevated plasma FFA levels. This is under-
scored by previous findings in isolated mouse and human
skeletal muscle mitochondria showing a dosage-depen-
dent inhibition of ATP synthesis upon incubation with high
but physiologic levels of FFA metabolites (30). Further-
more, it was shown in mice that prolonged consumption of
a high-fat diet for the duration of 16 weeks reduced
mitochondrial function (31). Also in human in vivo studies,
negative associations between high fatty acid availability
and markers for mitochondrial function have been re-
ported. Thus, PGC-1␣expression (9) and the insulin-
stimulated increase in skeletal muscle ATP synthesis (10)
were reduced upon lipid infusion. Furthermore, it was
recently shown that mitochondrial membrane potential
was impaired upon short-term lipid infusion in healthy
individuals, although several other markers of mitochon-
drial function remained unaffected (32). Despite the re-
ported negative associations between mitochondrial
function and (plasma) FFA, there are also several lines of
evidence suggesting the opposite. Thus, raising plasma
FFA by high-fat feeding combined with daily heparin
injections for 4 weeks in rats increased skeletal muscle
mitochondrial biogenesis and mitochondrial enzymes in-
volved in fat oxidation, the citric acid cycle, and the
respiratory chain (33). Furthermore, we previously
showed that high-fat feeding for 8 weeks in rats resulted in
a twofold increase of PGC-1␣protein levels (34).
Despite the obvious species differences between these
studies, the explanation for the discrepancy in these
results remains unclear. Adding to the complexity is the
fact that several approaches (high-fat feeding and lipid
infusion combined with a hyperinsulinemic-euglycemic
clamp) to elevate plasma FFA levels are accompanied by
hyperinsulinemia and/or hyperglycemia and insulin resis-
tance, all factors that have also been suggested to interfere
with mitochondrial capacity (26,29). Finally, differences in
absolute levels of plasma FFA achieved in the different
studies may contribute to (part of) the variation.
Within the context of mitochondrial lipotoxicity, we and
others have previously postulated that mitochondrial
UCP3 may be involved in protecting mitochondria against
(lipid-induced) oxidative damage (35). Therefore, we de-
termined protein levels of UCP3 and found that UCP3
content was similar between the fed and the fasted con-
dition. This is a surprising finding since fasting has been
quite convincingly shown to increase UCP3 protein levels
in animal studies (36,37). Moreover, UCP3 mRNA levels
were also elevated after 15 h (⬃5-fold) and 40 h (⬃10-fold)
of fasting in humans (38). It should be noted, however, that
this is the first study to evaluate UCP3 protein content
upon prolonged fasting in humans.
The impressive increase in plasma FFA upon pro-
longed fasting is in line with previous findings in hu-
mans (12,39), although the absolute values achieved in
this study (⬃2.0 mol/l) are high. Also, the ⬃2.7-fold
increase in IMTG levels after 60 h of fasting is slightly
higher in comparison with previous reports (12,39). The
high plasma FFA and IMTG levels might be caused by
complete compliance to the fasting regimen in the present
study since, in contrast to other studies, the subjects
stayed in a respiration chamber throughout the whole
period.
The reduction in insulin-stimulated glucose uptake ob-
served in the present study confirms previous observa-
tions showing that prolonged fasting reduced glucose Rd,
which was accounted for by a reduction in both insulin-
stimulated glucose oxidation and nonoxidative glucose
disposal (40). In agreement with previous reports (40), we
also detected a decreased metabolic flexibility (i.e., the
ability to switch from predominantly fat oxidation to
glucose oxidation upon insulin stimulation) upon pro-
longed fasting (Fig. 3). However, not all studies show this
effect (41).
As anticipated, whole-body fat oxidation increased sig-
nificantly upon prolonged fasting. Therefore, the decrease
in mitochondrial capacity in skeletal muscle is counterin-
tuitive, especially since this decrease was substrate-inde-
pendent and also apparent upon a lipid substrate.
These results indicate that the reduced mitochondrial
capacity is secondary to the fatty acid surplus associated
with the insulin-resistant state. It is important to note
however, that the reduction in muscle mitochondrial ca-
pacity does not (yet) affect the capability of the body to
enhance fat oxidation. This is an important finding since it
has generally been assumed that a reduction in muscle
mitochondrial function will result in reductions in whole-
body fat oxidative capacity (3–5,42,43). Here we show that
this extrapolation may not be justified, although we cannot
exclude that the fasting-induced reduced mitochondrial
function in muscle may decrease muscle-specific fat oxi-
dation compensated by increased fat oxidation in other
organs, or may have an impact on the capacity to switch
from carbohydrate to fat oxidation (metabolic flexibility).
In conclusion, 60 h of fasting in humans lowered insulin-
stimulated glucose uptake down to ⬃50% along with drasti-
cally elevated plasma FFA and IMTG levels. This was
accompanied by an overall reduction in intrinsic mitochon-
drial functional capacity in skeletal muscle, despite a pro-
nounced increase in whole-body fat oxidation. Since
prolonged fasting is a physiologic condition in which in-
creased fat oxidation becomes very important, a reduced
J. HOEKS AND ASSOCIATES
diabetes.diabetesjournals.org DIABETES, VOL. 59, SEPTEMBER 2010 2123

mitochondrial function seems unbeneficial from a physio-
logic point of view. Our findings suggest that the elevated
plasma FFA and/or intramuscular lipid levels associated with
the insulin-resistant state are responsible for the secondary
negative effects on mitochondrial function.
ACKNOWLEDGMENTS
This study was funded by Top Institute Food and Nutri-
tion. J.H. was supported by a grant from the Netherlands
Organization for Scientific Research. M.K.C.H. is sup-
ported by a VIDI Research Grant for innovative research
from the Netherlands Organization for Scientific Research
(Grant 917.66.359). P.S. is supported by a VICI Research
Grant for innovative research from the Netherlands Orga-
nization for Scientific Research (Grant 918.96.618).
No potential conflicts of interest relevant to this article
were reported.
J.H. wrote the manuscript and researched data. N.A.v.H.
researched data and reviewed/edited the manuscript. M.M.
contributed to discussion and reviewed/edited the manu-
script. E.M.-K. and D.v.B. were involved in analysis of
material. M.K.C.H. reviewed/edited the manuscript. P.S.
edited the manuscript and contributed to discussion.
The authors thank Esther Phielix and Gert Schaart for
practical help and technical support.
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