Biochemical and Biophysical Research Communications 294 (2002) 798–805

BBRC
www.academicpress.com

Acute and chronic treatment of ob/ob and db/db mice with

AICAR decreases blood glucose concentrationsq
Amy E. Halseth,* Nancy J. Ensor, Tommi A. White, Stuart A. Ross, and Eric A. Gulve
Cardiovascular and Metabolic Diseases, Mail Zone T1G, 800 N. Lindbergh Boulevard, Pharmacia Corporation, St. Louis, MO 63167, USA
Received 13 May 2002

Abstract

The enzyme 50 AMP-activated protein kinase (AMPK) is activated by increases in intracellular AMP concentration through a
complex interaction of phosphorylation and allosteric regulation. Actions of AMPK elucidated thus far suggest that AMPK may be
a viable target for pharmacologic intervention in type II diabetes. Activation of AMPK is believed to mediate both the acute in-
crease in skeletal muscle glucose uptake during exercise, as well as the adaptive responses to chronic exercise such as regulation of
expression of components of the muscle glucose uptake system. In addition, AMPK is known to inhibit key enzymes involved in
lipid and cholesterol synthesis, suggesting that activation of this kinase may also ameliorate dyslipidemia. To investigate the effects
of AMPK activation in animal models of type II diabetes, db/db and ob/ob mice were administered 5-aminoimidazole-4-carbox-
amide 1-b-ribofuranoside (AICAR) subcutaneously either acutely (single injection) or twice per day for 8 days (chronic treatment).
Blood glucose was lowered transiently in both db/db and ob/ob mice by acute AICAR treatment, returning to basal levels 3 h after
AICAR administration. In response to chronic treatment, blood glucose (measured 18 h post-AICAR administration) was signif-
icantly decreased in both mouse models when compared to vehicle control groups, with morning blood glucose values on Day 8
being decreased 30–35% in both mouse models. Chronic AICAR administration also resulted in an elevation of total Glut4
concentration in skeletal muscle from ob/ob mice, but not db/db mice. In contrast to the beneficial effects on glucose metabolism,
AICAR treatment of db/db and ob/ob mice led to approximately a 2.5–3-fold increase in serum triglyceride levels compared to
vehicle-treated controls. These data suggest that pharmacological activation of AMPK may enhance glucose uptake in individuals
with type II diabetes, however, this benefit may be offset by the concomitant elevation in triglycerides. Ó 2002 Elsevier Science
(USA). All rights reserved.

Keywords: 50 AMP-activated kinase; Diabetes; Skeletal muscle; Triglycerides; Glut4

50 AMP-activated protein kinase (AMPK) has been
described as a cellular fuel gauge [10,29], increasing its
kinase activity in response to decreases in cellular energy
charge. This activation is mediated by an increase in
cellular 50 AMP, which increases AMPK activity
through a complex series of mechanisms, including al-
losteric activation of the enzyme itself, activation of an
upstream kinase, AMPK kinase (AMPKK), and inhi-
bition of an inactivating phosphatase, PP2C [10,29]. In
q
Abbreviations: 50 AMP-activated protein kinase (AMPK); 5-amino-
imidazole-4-carboxamide 1-b-ribofuranoside (AICAR); hexokinase
(HK); glucose transporter isotype 4 (Glut4); fructose-1,6-bisphospha-
tase (F1,6BPase); phosphoenolpyruvate carboxykinase (PEPCK).
*
Corresponding author. Fax: +1-314-694-8153.
E-mail address: amy.e.halseth@pharmacia.com (A.E. Halseth).
the simplest sense, the function of activated AMPK is
to decrease the rate of cellular processes that require
energy, while at the same time increasing the rate of
energy-generating processes to return the cellular energy
charge to a more replete state.
The effects of skeletal muscle AMPK activation sug-
gest that pharmacological activation of this kinase may
be beneficial in the treatment of type II diabetes, a
condition characterized in part by skeletal muscle insu-
lin resistance. In response to contraction, AMPK ac-
tivity is increased in skeletal muscle of non-diabetic rat
[15,30] and human [6,8,31]. This activation has been
postulated to account for the exercise-induced increase
in skeletal muscle glucose uptake. Supporting evidence
comes from studies demonstrating that pharmacological
activation of AMPK with the compound 5-aminoimi-

0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved.
PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 0 5 5 7 - 0
 A.E. Halseth et al. / Biochemical and Biophysical Research Communications 294 (2002) 798–805
dazole-4-carboxamide 1-b-ribofuranoside (AICAR) re-
sults in increased glucose uptake in isolated rat skeletal
muscle [7,11] and the perfused rat hindlimb [21]. Im-
portantly, the combination of AICAR and a maximal
contractile stimulus is not greater than that observed
with just the contraction protocol [11], suggesting that
AICAR and contraction utilize the same pathway to
increase in glucose uptake. Consistent with the idea that
activation of AMPK is able to increase skeletal muscle
glucose uptake, in vivo administration of AICAR to
normal rats increases 2-deoxyglucose accumulation in
skeletal muscle under basal insulin conditions [1].
Repeated pharmacological activation of AMPK with
AICAR appears to mimic the effects of chronic exercise
on key proteins involved in muscle glucose uptake,
suggesting that AMPK activation may be a key com-
ponent of exercise training-induced increases in muscle
gene expression. Daily injections of non-diabetic rats
with AICAR for 5 or 8 days have been shown to in-
crease skeletal muscle Glut4 and hexokinase expression
approximately 2-fold [3,14]. This effect can be replicated
by incubating rat epitrochlearis muscles in AICAR-
containing culture medium for 18 h [23]. These latter
findings support the concept that the regulation of
hexokinase and Glut4 expression is a direct consequence
of AMPK activation in muscle and is not secondary to
any AICAR-induced perturbations in whole body me-
tabolism.
In addition to the potential therapeutic effects of
AMPK activation on glucose metabolism, existing data
suggest that activation of AMPK may also have favor-
able effects on disorders of lipid metabolism. For ex-
ample, HMG-CoA reductase (the rate-limiting step for
cholesterol synthesis), acetyl-CoA carboxylases (key
regulators of fatty acid synthesis/oxidation), and sn-
glycerol 3-phosphate acyltransferase (an essential en-
zyme in glycerolipid synthesis) are all phosphorylated
and inhibited by activated AMPK [10,22]. These find-
ings suggest that pharmacological activation of AMPK
should decrease cholesterol synthesis, lipid synthesis,
and blood glucose levels, which might improve a num-
ber of metabolic abnormalities manifested in individuals
with type II diabetes [29].
Exercise is one of the first-line treatments for diabe-
tes. In contrast to the impaired ability of insulin to
stimulate glucose uptake, diabetic individuals as well as
diabetic animal models typically display normal in-
creases in muscle glucose uptake and Glut4 transloca-
tion during exercise [12,16]. We thus hypothesized that
the AMPK-mediated pathway for glucose uptake would
be intact, despite the extreme insulin resistance. We
evaluated whether pharmacological activation of
AMPK would decrease blood glucose levels in two
mouse models of insulin resistance and diabetes, ob/ob
(obese, severely insulin resistant, and not diabetic) and
db/db (obese, severely insulin resistant, and diabetic)
799
mice. AICAR is taken up by cells and phosphorylated,
resulting in ZMP, an AMP analog that is able to acti-
vate AMPK via a similar mechanism as AMP [27].
Results from these studies showed that AICAR de-
creased blood glucose levels in these two mouse models
both acutely (over 1–3 h) as well as with repeated
treatment (8 days). Alterations in parameters of lipid
metabolism were also observed, although the mecha-
nism for these changes is not readily apparent.
Materials and methods
Animals
Male ob/ob mice (C57BL/6J-Lepob ), weighing 55 g, were obtained
from The Jackson Laboratory (Bar Harbor, ME). Male and female db/
db mice (C57BL/KsJ-Leprdb ) were obtained from a colony maintained
at Charles River Laboratory. db/db Mice were from 54 to 64 days old
at initiation of experiments (37 g) and groups were balanced for age
and gender of mice. All mice were maintained on a 12:12 h light–dark
cycle, fed standard rodent diet ad libitum, and had unlimited access to
water. All procedures were pre-approved by the Institutional Animal
Care and Use Committee.
Experimental procedures
In vivo procedures. Acute studies were performed to determine the
magnitude of changes in blood glucose in response to subcutaneous
AICAR (Sigma) in these mouse models and to determine appropriate
doses for chronic dosing. db/db mice were given subcutaneous AICAR
in sterile saline at doses ranging from 0.25 to 0.5 mg/g, while ob/ob
mice were given between 0.375 and 0.5 mg/g. Blood glucose levels were
measured periodically (for up to 300 min) from tail nick samples using
a Glucometer Elite Monitor (Bayer Corporation).
In chronic experiments, mice received subcutaneous AICAR twice
daily (approximately 8:00 a.m. and 5:00 p.m.) for 8 days. Blood glu-
cose was measured each morning, prior to AICAR administration
(approximately 18 h after the preceding dose). On the eighth day of
dosing, mice were fasted for 5 h, prior to blood collection from the
retro-orbital sinus for measurement of serum cholesterol and trigly-
ceride concentrations by the Clinical Chemistry Laboratory at St.
Louis University. Mice were then killed with CO2 and liver and gas-
trocnemius/plantaris muscle samples were rapidly removed and frozen
in liquid N2 .
Muscle Glut1 and Glut4 measurements. Gastrocnemius/plantaris
samples were homogenized individually on ice in 1 ml of 20 mM Hepes,
250 mM sucrose, 1 mM EDTA, pH 7.4 (HES), containing complete
(Boehringer–Mannheim) protease inhibitor cocktail with a motor-
driven stirrer (Caframo) at 1200 rpm with a glass on glass homoge-
nizer/tissue grinder (Kontes). Homogenization was continued until the
suspension contained little, if any, particulate matter, which was typ-
ically after 15–25 up and down strokes. Protein concentrations were
determined by the method of Bradford.
Proteins were separated electrophoretically, transferred to 0.2 lm
PVDF membranes (NOVEX) in 25 mM Tris, 190 mM glycine, 20%
methanol, 0.005% SDS, blocked in 5% carnation non-fat milk in TBS,
and immunoblotted with antibodies (each at 5 lg/ml) against Glut4 or
Glut1 in 1% non-fat milk TBS. Rabbit polyclonal affinity-purified
antibodies were generated by BioSource/Quality Controlled Bio-
chemical through immunization of synthetic peptides conjugated via
cysteine residues at the amino-termini of each peptide. Antibodies were
generated against the carboxy-terminal 19 amino acids of rat/human
Glut4 (amino acids 491–509; acetyl-CEQEVKPSTELEYLGPDEND-
OH) and the carboxy-terminal 16 amino acids of rat/human Glut1
800 A.E. Halseth et al. / Biochemical and Biophysical Research Communications 294 (2002) 798–805

(amino acids 477–492; acetyl-CKTPEELFHPLGADSQV-OH). De-

tection was with horseradish peroxidase-conjugated secondary anti-
bodies followed by washing with TBS/0.3% Tween 20 and exposure to
the reagents for enhanced chemiluminescence (Pierce). Images were
scanned using a Molecular Dymanics Personal Densitometer SI and
quantified using ImageQuant 5.0.
Liver triglyceride and cholesterol measurements. Aliquots of liver
(150–200 mg) were homogenized using a Tissue Tearor (Biospec
Products) in chloroform/methanol (2:1) and filtered through Whatman
0.45 lM filters. One ml of Milli-Q water was added to the filtrate and
samples were vortexed vigorously and then centrifuged at 3000 rpm at
4 °C. The lower chloroform phase was transferred to a glass tube and
solvent was evaporated under nitrogen. Sample remaining in the tube
was suspended in 0.5% Triton X-100 by sonication. This solution was
then used for the measurement of triglycerides (GPO-Trinder Trigly-
ceride Kit, Sigma Diagnostics) or cholesterol (Cholesterol CII Enzy-
matic Kit, Wako), using sonicated 0.5% Triton X-100 as the diluent for
the standard curves.

Results and discussion

Acute experiments

Initial experiments were performed to determine
whether AICAR would acutely lower glucose in db/db
and ob/ob mice, two models of insulin resistance. In db/
db mice, 0.5 mg/g AICAR given subcutaneously resulted
in a decrease in fed blood glucose of approximately 75%,
with the lowest glucose values (116  13 mg/dl) recorded
90 min after AICAR administration (Fig. 1A). Lower
blood glucose concentrations were still evident even at
150 min after AICAR administration.
In the first set of acute experiments performed on ob/
ob mice, this same dose of AICAR (0.5 mg/g) was used.
However, in ob/ob mice, this dose of AICAR given to
mice in the fed state resulted in overt hypoglycemia,
reaching an average value of 55  6 mg/dl 90 min post-
AICAR (Fig. 1B). In addition, we observed a tendency
toward rebound hyperglycemia in these mice, with val-
ues reaching 318  69 mg/dl 240 min after AICAR ad-
ministration, although this was not significantly different
than the time 0 point in the AICAR-treated mice (two-
tailed, paired t test P ¼ 0:096). We therefore tested the
ability of a lower AICAR dose to decrease blood glu-
cose in ob/ob mice. At 0.375 mg/g, the 90-min blood
glucose value was 90  9 mg/dl. Thus, 0.375 mg/g, twice
per day, was selected as an appropriate dose in this
model for use in chronic experiments.
Whether the fall in blood glucose that resulted from
an acute dose of AICAR is due to increased AMPK
activity is unclear, as ZMP will alter the activity of not
only AMPK, but also of other AMPK-regulated en-
zymes. For example, fructose-1,6-bisphosphatase
(F1,6BP), a key enzyme in the gluconeogenic pathway,
is inhibited directly by ZMP itself [27], which one would
predict would lower blood glucose levels via inhibition
of gluconeogenesis. Previous studies performed in mice
suggested that the inhibition of F1,6BP has only a major
Fig. 1. Blood glucose concentrations following acute injection of
0.5 mg/g AICAR or an equal volume of saline (vehicle). Data are
presented as means  SEM. (A) In db/db mice, glucose concentrations
were statistically different between control and AICAR-treated mice
from the 60 min time point onward (P < 0:01–0.00001, as indicated by
the asterisk; n ¼ 3 control mice and n ¼ 6 AICAR-treated mice). (B) In
ob/ob mice, glucose concentrations were statistically different between
control and AICAR-treated mice at the 60, 90, and 120 min time points
(P < 0:00001), as indicated by the asterisk. Glucose values at the 240
and 300 min time points were not significantly different between con-
trol and AICAR-treated groups (P < 0:20) nor were they different
from the time 0 point in the AICAR-treated mice (paired t test
P ¼ 0:096; n ¼ 6 for both control and AICAR-treated groups).
effect on blood glucose concentration after an overnight
fast [26], a condition where gluconeogenesis is the major
contributor to the maintenance of blood glucose. A re-
cent study performed in the obese, insulin-resistant
Zucker rat demonstrated that AICAR infusion de-
creased endogenous glucose production by 80% [2],
although again, the contribution of AMPK activation
versus direct effects of AICAR cannot be separated.
Given that the gluconeogenic rate is elevated in the
Zucker rat as well as the insulin-resistant mouse models
utilized in the present experiments [5,17,20], we cannot
rule out a contribution of AICAR-mediated inhibition
of gluconeogenesis to the observed blood glucose low-
ering.
Chronic experiments
In chronic dosing experiments, mice were adminis-
tered AICAR subcutaneously twice per day at approx-
imately 8 a.m. and 5 p.m. for 8 days. In db/db mice, five
of the mice were initially dosed at 0.25 mg/g, twice per
day for the first 5 days of the 8 day experiment. Because
 A.E. Halseth et al. / Biochemical and Biophysical Research Communications 294 (2002) 798–805
only minor diminutions in blood glucose were observed
in the first few days of the experiment in db/db mice
treated with this dose of AICAR, the dose was then
doubled to 0.5 mg/g, twice per day, for the remaining 3
days of the experiment. The other eight db/db mice
treated with AICAR received the higher dose for the
duration of the study. By Day 8, there were no statis-
tically significant differences in blood glucose values of
AICAR-treated mice whether the mice had received the
high dose AICAR throughout the study (Day 8 glucose:
368  37 mg/dl), or only for the final three days (Day 8
glucose: 291  40 mg/dl; two-tailed P ¼ 0:23). There-
fore, data from all db/db chronic experiments are pre-
sented together. Shown in Fig. 2A are the average
morning blood glucose values throughout the study,
which differed significantly between groups starting on
Day 3 through the conclusion of the experiment (P
values ranged between 0.02 and 0.003 on individual
days), with the exception of Day 7 (P ¼ 0:09).
Importantly, the data in Fig. 2A represent the mini-
mal difference between groups. That is, the upper limit
of measurement of the blood glucose monitors used in
these experiments is 600 mg/dl; therefore, a value of
Fig. 2. (A) Morning blood glucose concentrations following chronic
injections of 0.25–0.5 mg/g AICAR or an equal volume of saline twice
daily in db/db mice. Blood glucose was measured, prior to the morning
injection of AICAR or saline. Blood glucose concentrations were
significanly lower in AICAR-treated db/db mice from Day 3 onward
(P values ranged between 0.02 and 0.003 on individual days), with the
exception of Day 7 (P ¼ 0:09). Data are presented as means  SEM;
n ¼ 12 control mice and n ¼ 13 AICAR-treated mice. (B) Percentage
of db/db mice with morning blood glucose concentrations exceeding
600 mg/dl.
801
600 mg/dl was used to calculate group averages under
these conditions, even though their actual blood glucose
values may have been considerably higher. Shown in
Fig. 2B are the percentages of mice in each group that
had blood glucose values equal to or greater than
600 mg/dl. Fig. 2B clearly demonstrates that the severity
of diabetes increased during the experimental period, as
is known to occur during the period when islets of db/db
mice begin to fail. More than half (6 out of 11) of the
vehicle-treated mice progressed to this severely diabetic
state. In contrast, none of the mice in the AICAR-
treated group progressed to this stage of diabetes.
Therefore, while AICAR treatment did not reverse the
diabetic state that was already present in db/db mice,
AICAR treatment was able to delay the progressive
worsening of diabetes in this model.
Differential effects on body weight were observed
between vehicle and AICAR-treated db/db mice over
the course of the experiment. While weights of the
groups were similar on Day 0, following the 8 days of
the experiment, the vehicle-treated mice weighed on
average approximately 3 g less than the mice treated
with AICAR (P < 0:01, Table 1). This differential body
weight response most likely reflects the more severe
hyperglycemia of the vehicle-treated mice, which will
lose more calories due to glucose loss in the urine.
Blood glucose values in ob/ob mice treated chroni-
cally with AICAR are shown in Fig. 3. In contrast to db/
db mice, ob/ob mice are severely insulin resistant, but
are normoglycemic or modestly hyperglycemic in the fed
state and do not display the rapid progression to overt
diabetes on a standard chow diet. Even in these mice,
however, chronic AICAR dosing resulted in a decrease
in morning blood glucose levels compared to vehicle
controls, reaching statistical significance at Day 3 (P
values of subsequent days all <0.01). In ob/ob mice, in
contrast to what was observed in the db/db mice, there
were no differences in body weight between AICAR-
and vehicle-treated mice on either Day 0 or Day 8
(Table 1). These data support the hypothesis that the
body weight differences in db/db mice result from al-
terations in blood glucose levels and therefore urinary
Table 1
Body weight in vehicle- and AICAR-treated db/db and ob/ob mice
Vehicle AICAR
db/db Mice
Body weight (g) Day 0 38:7  1:0 38:5  0:5
Day 8 37:1  1:2 40:5  0:6
ob/ob Mice
Body weight (g) Day 0 55:7  0:7 54:7  0:9
Day 8 57:8  0:7 57:3  0:9
Fat pad weight/body Day 8 3:3  0:1 3:2  0:1
weight (%)
*
Indicates significant difference (P < 0:01) compared to vehicle-
treated mice.
802 A.E. Halseth et al. / Biochemical and Biophysical Research Communications 294 (2002) 798–805
Fig. 3. Morning blood glucose concentrations following chronic in-
jections of 0.375 mg/g AICAR or an equal volume of saline twice daily
in ob/ob mice. Blood glucose was measured, prior to the morning in-
jection of AICAR or saline. Data are presented as means  SEM.
Blood glucose concentrations were significanly lower in AICAR-trea-
ted ob/ob mice from Day 3 onward (P < 0:01–0.00001).
glucose loss, since the blood glucose levels of ob/ob mice
throughout the experiment were below the renal
threshold above which all the glucose cannot be reab-
sorbed from kidney filtrate.
As discussed earlier, the acute lowering of blood
glucose levels in these mice could be due either to inhi-
bition of gluconeogenesis or to an increase in AMPK-
stimulated muscle glucose utilization. In chronic studies,
the mechanism is most likely due to the sustained ac-
tions of AMPK. In these chronic studies, morning blood
glucose measurements were performed approximately
18 h after the previous AICAR administration, while it
has been shown that hepatic ZMP levels and inhibition
of flux through F1,6BPase return to baseline 4 h after
administration in normal mice [26,27]. The mechanism
by which repeated AMPK activation is able to modulate
whole body glucose metabolism is postulated to be due
to its ability to upregulate key proteins that mediate
glucose uptake in skeletal muscle.
Previous work performed in normal rats has shown
that once daily injections of AICAR (1 mg/g, subcutane-
ous) increase the expression of genes such as skeletal
muscle Glut4, the insulin responsive glucose transporter
[3,14]. A similar finding has been reported to occur in
isolated skeletal muscles incubated with AICAR [23],
suggesting that this is a direct effect of activated AMPK in
muscle and not secondary to other alterations in the
metabolic state of the animal. Glut4 protein is also well
known to be upregulated from approximately 1.5- to 3-
fold with chronic exercise training [24] and the increased
expression is thought to account for at least part of the
increase in whole body insulin-sensitivity that accompa-
nies this intervention. To assess whether AICAR was
capable of increasing gene expression in skeletal muscle of
diabetic animals, gastrocnemius Glut4 levels were as-
sessed through Western blotting. Fig. 4 shows that
treatment of ob/ob mice with AICAR for 8 days signifi-
cantly increased gastrocnemius Glut4 by approximately
Fig. 4. Glut4 protein expression in gatrocnemius from control and
AICAR-treated ob/ob (A) and db/db (B) mice. Data are presented as
means  SEM. Glut4 concentration was significantly increased by
AICAR treatment only in ob/ob mice (P < 0:03).
50%, which is similar to the range typically seen with ex-
ercise training [24]. In contrast, no increase in Glut4
protein was detected in gastrocnemius muscles from AI-
CAR-treated db/db mice. Glut1 protein levels in gas-
trocnemius remained unchanged in AICAR-treated ob/
ob and db/db mice relative to controls (data not shown).
In these experiments, we did not measure the con-
tribution of increased muscle glucose uptake compared
to decreased hepatic glucose production to the decrease
in blood glucose levels. Based on the measured increase
in Glut4 expression and putative increases in other
proteins involved in glucose uptake and metabolism, we
believe that muscle did play a role in this decrease in the
AICAR-treated mice. We cannot exclude the possibility
that AICAR treatment decreases hepatic glucose pro-
duction with a longer duration of action than is seen for
the acute inhibition of F1,6BPase. For example, treat-
ment of liver cell lines with AICAR has been shown to
decrease expression of two key gluconeogenic genes,
phosphoenolpyruvate carboxykinase (PEPCK) and
glucose-6-phosphatase [19]. Whether this occurs in vivo,
and the time course of the onset and maintenance of this
putative effect, remains to be determined.
Because one activity of AMPK is the phosphoryla-
tion and inhibition of enzymes involved in cholesterol
and lipid synthesis, we next investigated whether serum
 A.E. Halseth et al. / Biochemical and Biophysical Research Communications 294 (2002) 798–805
Fig. 5. (A) Serum cholesterol concentration in db/db and ob/ob mice
treated with AICAR or an equal volume of saline for 8 days. Data are
presented as means  SEM. Cholesterol concentration was signifi-
cantly increased (P < 0:0001) in db/db mice following AICAR treat-
ment, as indicated by the asterisk. (B) Serum triglyceride concentration
in db/db and ob/ob mice treated with AICAR or an equal volume of
saline for 8 days. Data are presented as means  SEM. Triglyceride
concentration was significantly increased (P < 0:0001) in both ob/ob
and db/db mice following AICAR treatment, as indicated by the as-
terisks.
or hepatic cholesterol and triglyceride levels were altered
by 8 days of AICAR treatment. The effects seen on
cholesterol differed depending on the mouse strain in-
vestigated. In the db/db mice, 8 days of AICAR treat-
ment resulted in an 40% increase in total cholesterol
compared to the vehicle-treated animals (P < 0:0001),
while there were no differences observed between AI-
CAR- and vehicle-treated ob/ob mice (Fig. 5A). The
reason for the different response in the two mouse
strains is unknown. It is worth noting that serum cho-
lesterol concentration was substantially higher, prior to
initiation of AICAR treatment in ob/ob than db/db
mice, and AICAR treatment may not result in further
elevation of this parameter. In contrast to the relatively
modest changes observed in serum cholesterol in these
experiments, serum triglycerides were elevated 2.4- and
3-fold by AICAR treatment of db/db and ob/ob mice,
respectively (Fig. 5B). This finding conflicts with the
postulated role of AMPK to decrease lipid synthesis.
Acute AICAR infusion in Zucker rats causes plasma
triglycerides to decrease by 80% [2]. One possible ex-
planation for this discrepancy may be that AICAR
acutely decreases triglyceride and cholesterol synthesis
by inhibition of key enzymes in these pathways, but as
803
Fig. 6. Liver triglyceride concentration in db/db and ob/ob mice
treated with AICAR or an equal volume of saline for 8 days. Data are
presented as means  SEM. AICAR treatment significantly decreased
liver triglyceride concentration in db/db mice (P < 0:05), while there
was no significant difference in liver triglyceride concentration between
AICAR- and vehicle-treated ob/ob mice.
the effect of the administered AICAR wears off, there is
a rebound in the rate of lipid synthesis or a decrease in
the rate of triglyceride clearance. A second possibility
could be that this is a side effect of AICAR unrelated to
its activation of AMPK. Further studies utilizing more
selective activators of AMPK will be required to test this
hypothesis. In any case, this elevation of circulating
triglyceride levels is distinct from that seen in these
models in response to two insulin sensitizing paradigms,
exercise training [13] and treatment with thiazolinedi-
ones [4,9,28], which both decrease triglycerides. In con-
trast to the dramatic elevation in serum triglycerides
observed after treatment with AICAR, hepatic trigly-
ceride concentrations were slightly but significantly de-
creased by AICAR in db/db mice (P < 0:05) and
unchanged by AICAR treatment in ob/ob mice (Fig. 6).
This suggests that the increase in serum triglycerides is
not due to increased production by the liver, since in-
creased hepatic production is typically accompanied by
increased hepatic triglyceride storage.
It has recently been suggested that the mechanism of
action for the anti-diabetic agent, metformin, is through
activation of the AMPK pathway [32]. Metformin was
shown to activate AMPK in hepatocytes, increasing
fatty acid oxidation and decreasing lipogenic enzyme
activity. Additionally, when hepatocytes were incubated
with an AMPK inhibitor, the effects of metformin to
inhibit glucose production were attenuated [32]. The
studies described in the present paper do not test
the hypothesis that metformin may be working through
the AMPK pathway directly, but it is worth pointing
out the differences between the current results obtained
with AICAR and those observed following metformin
treatment. For example, treatment of ob/ob mice with
metformin for 4 weeks does not alter blood glucose or
plasma triglyceride concentrations and decreases food
intake, body weight, and epididymal fat pad weight [18],
none of which were observed in the present study in
response to AICAR. The differences in in vivo effects of
metformin and AICAR may relate to different phar-
804 A.E. Halseth et al. / Biochemical and Biophysical Research Communications 294 (2002) 798–805
macokinetic parameters between the compounds, or
interaction of either metformin or AICAR with other
pathways besides the AMPK pathway.
In summary, both chronic and acute administration
of AICAR to two rodent models of insulin resistance
and type II diabetes decrease blood glucose levels.
Further studies will be required to determine the relative
contributions of decreased hepatic glucose production
versus increased skeletal muscle glucose uptake in the
hours following administration of AICAR. The more
sustained decrease in blood glucose (persisting for at
least 18 h following the last AICAR dose with repeated
dosing) is likely due to alterations in skeletal muscle
gene expression mediated via AMPK activation. In-
creases in expression of the proteins responsible for
skeletal muscle glucose uptake, as evidenced by the in-
crease in Glut4 shown in ob/ob mice, likely play an
important role in this process. Furthermore, a decrease
in expression of gluconeogenic enzyme expression may
also contribute to the decreased blood glucose levels.
AICAR administration resulted in 2.5–3-fold increase
in serum triglycerides in the absence of an increase in
hepatic triglyceride concentration. Whether this is a
consequence of AMPK activation or results from other
actions of AICAR cannot be determined with currently
available experimental tools. This work is in agreement
with results of Zierath and co-workers [25] who showed
both acute and chronic reductions in blood glucose
levels in diabetic ob/ob mice. Furthermore, the work
herein extends these findings to db/db animals. Since
both ob/ob and db/db mice are predictive animal models
when testing compounds for type II diabetes, the work
herein provides proof of principle for activation of
AMPK as a therapeutic target. However, the dyslipi-
demia that is observed following treatment of mice with
AICAR suggests one negative aspect for this potential
therapeutic mechanism.
Acknowledgments
We thank B. Ganesh Bhat, Mary Burney, and Traci Bollinger for
their technical assistance. We are also grateful for the excellent animal
care provided by the Pharmacia Laboratory Animal Medicine and
Science Group.
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