healthmedicinet

Animal care

All procedures adopted and experiments undertaken were approved by the University
of Tasmania Animal Ethics Committee and performed in accordance with the Australian
Code of Practice for the Care and Use of Animals for Scientific Purposes—2004, 7th
Edition. Male Hooded Wistar rats weighing 243 ± 1 g at the time of the experiments
were obtained from the University of Tasmania Animal House (Hobart, Australia). Animals
were raised on a commercial diet (Pivot, Launceston, Australia) containing 21.4% protein,
4.6% lipid, 68% carbohydrate and 6% crude fiber with added vitamins and minerals together
with water ad libitum and were housed at 21 ± 1°C on a 12:12 h light:dark cycle.

Surgery

Complete details are as described previously 1]. Briefly, rats were anesthetized and cannulas surgically implanted into a carotid
artery for arterial sampling and measurement of blood pressure, and into both jugular
veins for continuous administration of anesthetic and other intravenous infusions.
Animals were allowed to spontaneously breathe room air through a tracheostomy tube.
Animals were maintained at 37°C under anesthesia for the duration of the experiment
using a continual infusion of sodium pentobarbital (0.6 mg/kg/min) via the left jugular
cannula. Once the surgery was completed, a period of equilibration of approximately
60 min was allowed so that cardiovascular parameters could become stable and constant.

Experimental protocols

All animals underwent one of the three following protocols in Figure 1.

Figure 1. Animals were subject to a 2 h euglycemic clamp of 3 mU/kg/min insulin or saline infusion
with or without AICAR. Arterial blood samples (droplets) were taken for glucose and lactate analysis. A bolus injection (i.v.) of AICAR (20 mg/kg)
is indicated by pointing hand and followed by AICAR infusion (3.75 mg/min/kg). Other bolus infusions are indicated
by pointing hand. In protocol A, arterial and venous blood samples were taken (indicated as A–V) for
plasma glucose determination as well as arterial insulin and AICAR determination.
The gastrocnemius group of muscles was freeze clamped at 120 min for the determination
of AICAR and ZMP content as well as R’g determination. In protocol B, microbubble
infusion (40 µL/min) and periods where ultrasound measurement of MvV and MvP were
made are shown by CEU. Muscle contraction (field stimulation, 2 Hz, 0.1 ms, 30–50 V)
is indicated by EX. In protocol C L-NAME (NOS inhibitor) was infused 15 min prior
to and during AICAR infusion.

Procotol A: metabolic and hemodynamic actions of AICAR

Rats were randomly assigned into four experimental groups: (1) saline, n = 33 (2)
AICAR (3.75 mg/min/kg, Sigma-Aldrich., St. Louis, MO, USA), n = 16 (3) insulin (3 mU/min/kg,
Human R Eli Lilly, Indianapolis, IN, USA), n = 39 or (4) insulin + AICAR (3 mU/min/kg
and 3.75 mg/min/kg respectively), n = 18.

AICAR administration was initiated by an intravenous bolus injection (20 mg/kg) at
t = 60 min followed by a constant infusion (3.75 mg/min/kg, via the jugular vein)
for the remainder of the experiment. Hyperinsulinemic euglycemic clamps (3 mU/min/kg)
were performed in the insulin and insulin + AICAR groups to assess insulin sensitivity.
Insulin infusions commenced at t = 0 min and continued until the end of the experiment.
In the experiments where rats received insulin, basal blood glucose concentration
was maintained (euglycemia) by co-infusion of variable rates of glucose (30% w/v solution)
over the course of the experiment.

Epigastric vessels were ligated, and an ultrasonic flow probe (Transonic Systems,
VB series 0.5 mm) was positioned around the femoral artery of the right leg just distal
to the rectus abdominis muscle. Data for femoral artery blood flow (FBF), heart rate
(HR), and blood pressure (BP) were sampled using WINDAQ data acquisition software
(DATAQ Instruments).

Blood and plasma samples were collected from the carotid artery (A) and femoral vein
(V) for determination of blood and plasma glucose, and blood lactate concentration
at the end of the experiment. Hindleg glucose uptake was calculated from the arterio-venous
difference (A–V) multiplied by the FBF (the Fick Principle) and expressed as µmol/min.
The calf muscles (gastrocnemius, soleus and plantaris) were removed from the right
leg and freeze clamped in situ at the conclusion of the experiment and stored at ?80°C
for later determination of AICAR and 5-Aminoimidazole-4-carboxamide-1-?-D-ribofuranosyl 5?-monophosphate (ZMP) content. Both of these metabolites were measured
because AICAR is taken up by cells and converted to its active form ZMP.

Muscle glucose uptake was also assessed using isotopic tracers. A intravenous bolus
dose (50 µCi, 0.2 ml) of 2-deoxy-D-[1-
¹⁴
C]glucose (2-DG), specific activity 1.85-2.29 GBq/mmol; Amersham Life Science, Castle
Hill, NSW, Australia) was given 45 min before the end of the experiment. The plasma
specific activity of 2-DG was determined from plasma samples (25µL) collected at 5,
10, 15, 30 and 45 min after administration of the 2-DG bolus. At the conclusion of
the experiment, individual muscles [soleus, plantaris (Plant), gastrocnemius red (GR),
gastrocnemius white (GW), extensor digitorum longus (EDL) and tibialis (Tib)] from
the lower left hindlimb were rapidly removed, frozen in liquid nitrogen and stored
at ?80°C until assayed for 2-DG radioactivity and the determination of muscle specific
glucose uptake as described previously 18].

Effect of fasting on AICAR-mediated metabolic actions

A sub-set of insulin + AICAR experiments were performed using a modified version of
protocol A, to assess effect of fasting on AICAR-mediated metabolic actions. These
experiments were carried out in order to determine the effect of AICAR on insulin-mediated
metabolic responses with different degrees of liver glycogen content (i.e. prolonged
fasting depletes liver glycogen). All rats underwent insulin + AICAR (3 mU/min/kg
and 3.75 mg/min/kg, respectively) infusion. Rats were randomly assigned into two experimental
groups: (1) overnight fast (which represents abstaining from food for 16 h), n = 12
or (2) prolonged fast (which represents abstaining from food for 40 h), n = 16. Whole
body GIR and muscle specific glucose uptake (by isotopic tracer) was performed as
described above.

Protocol B: muscle microvascular actions of AICAR

The effect of AICAR on microvascular perfusion in skeletal muscle was assessed. These
measures could not be conducted in Protocol A because microbubbles interfere with
the Doppler signal from the Transonic flow probe used to measure FBF.

Rats were randomly assigned into four experimental groups: (1) saline, n = 6 (2) AICAR
(3.75 mg/min/kg, Sigma-Aldrich., St. Louis, MO, USA), n = 6 (3) insulin (3 mU/min/kg,
Human R Eli Lilly, Indianapolis, IN, USA), n = 6 or (4) insulin + AICAR (3 mU/min/kg
and 3.75 mg/min/kg respectively), n = 6.

AICAR, insulin or saline infusion was identical to the procedure described in protocol
A. Muscle microvascular perfusion was assessed by contrast-enhanced ultrasound (CEU)
as described previously 2].

CEU imaging occurred at baseline (t = 0 min) and at the end of the experiment (t = 120 min)
to assess microvascular responses to saline, AICAR, insulin and Insulin + AICAR. Contraction
is a well-accepted stimulus for maximal microvascular recruitment. At the end of the
experiment, an incision was made through the skin at the lateral side of the hip.
Electrodes were attached to the muscle of the hindleg and the Achilles tendon. Twitch
contraction was performed with 0.1 ms pulses of 30–50 V. Microvascular responses to
contraction were assessed by CEU at the time indicated (Figure 1b).

CEU imaging

The adductor magnus and semimembranosus muscles of the left hind limb were imaged
in short axis with a linear array transducer (L7-4), secured in position for the duration
of the experiment, connected to an ultrasound system (HDI-5000, Ultrasound, Phillips
Ultrasound). The acoustic focus was set at the mid-muscle level and gain settings
were optimised and held constant throughout the experiment. Albumin microbubbles (Optisonâ„¢,
GE Healthcare) were diluted 1:5 with perfluoropropane gassed saline and infused via
the jugular vein at 40 µL/min for the duration of the data acquisition. The acoustic
signal that is generated from the microbubbles exposed to ultrasound is proportional
to the concentration of microbubbles within the volume of tissue being imaged. All
microbubbles within the ultrasound beam are simultaneously imaged and destroyed in
response to a single pulse of high-energy ultrasound. As the time between successive
pulses is prolonged, the beam becomes progressively replenished with microbubbles
(refer to Figure in 2]). The beam will eventually become fully replenished with microbubbles, and further
increases in the time between each ultrasound pulse will not affect the microbubble
signal in tissue. Intermittent imaging was performed using pulsing intervals (PIs)
ranging from 0.5 to 15 s to allow incremental microvascular replenishment with microbubbles
between each pulse until the volume within the beam was completely refilled. Several
frames were obtained at each PI. Data was analysed using QLABâ„¢ Software (Version 6.0,
Phillips Ultrasound, Bothwell). The ultrasound intensity in decibels within the region
of interest (semimembranous and adductor magnus) were converted to acoustic intensity
and after background subtraction using 0.5 s ultrasound images (to eliminate signal
from larger blood vessels and muscle per se), a pulsing interval (time) versus acoustic-intensity
curve was plotted. This allowed calculation of microvascular volume (MvV) as well
as an index of microvascular perfusion (MvP) according to the equation y = A (1 ? e
^??(t?0.5)
) where y is the acoustic intensity at a given pulsing interval, A = MvV, and Ax? = MvP
2], 29].

Protocol C: role of NOS on AICAR-mediated microvascular actions

We have previously demonstrated that insulin 4], but not contraction 30], stimulate microvascular recruitment in muscle via NOS-dependent pathway. We wanted
to determine whether AICAR-mediated microvascular recruitment was similar to insulin
or contraction. The femoral artery, femoral vein and nerve were all carefully separated.
In one leg (test leg), the epigastric artery was cannulated for infusion of N
^?
–L-nitro-arginine-methyl ester (L-NAME, Sigma-Aldrich, St. Louis, MO, USA) to achieve 10 ?M, and in the contralateral
leg (control leg), the epigastric artery was ligated. Rats were randomly assigned
into two groups as follows; (1) AICAR (3.75 mg/min/g), n = 9, or (2) AICAR + local
L-NAME, n = 9 (Figure 1c). We have previously demonstrated that this dose of L-NAME supresses insulin-mediated microvascular recruitment while avoiding systemic
effects on blood pressure and heart rate 18]. Microvascular responses were assessed by CEU at baseline (t = 0 min) and after AICAR
or AICAR + L-NAME infusion. Similar to Protocol B, all animals underwent a bout of acute contraction
to assess maximal microvascular recruitment.

Analytical methods

Blood glucose, plasma glucose and blood lactate concentrations were determined using
a glucose analyzer (Yellow Springs Instruments, Model 2300 Stat plus). Plasma insulin
concentrations were determined by ELISA (Mercodia, Uppsala, Sweden) from arterial
plasma samples taken at beginning and the conclusion of the experiment. AICAR and
ZMP concentrations were determined from perchloric acid treated plasma or homogenized
muscle samples, that were centrifuged for 10 min and the supernatant analysed using
reverse-phase HPLC as generally used to resolve nucleoside mixtures 25], 31].

Statistical analysis

All data are presented as mean ± SE. Repeated-measures two-way ANOVA was used to determine
if there were differences between treatment groups over the time course of the experiment,
or one-way ANOVA was used for single point measurements. When a significant difference
(p  0.05) was found, pair wise comparisons by the Student–Newman–Keuls test was used
to determine treatment differences. Comparisons between the treatments for plasma
AICAR values were determined by an unpaired t test. All tests were performed using SigmaStatâ„¢ (Systat Software, Inc., San Jose,
CA, USA).