Clinical diabetes is associated with disturbances in carnitine metabolism during Diabetes was induced with streptozotocin (STZ) at the dose of 50 mg/kg …
Metabolic Response to Exercise
PLASMA FREE AND ESTERIFIED CARNITINE LEVELS IN THE STREPTOZOTOCIN DIABETIC
RAT AFTER A SINGLE BOUT OF EXERCISE
TOM L BRODERICK1, ANDR? NADEAU2
1Department of Physiology, Midwestern University, Glendale, USA
2Diabetes Research Unit, Laval University Medical Center, Ste-Foy, Canada
ABSTRACT
Broderick TL, Nadeau A Plasma free and esterified carnitine levels in the
streptozotocin rat after a single bout of exercise JEPonline 2006;93:17-
23 Clinical diabetes is associated with disturbances in carnitine
metabolism during exercise In type 1 diabetic patients, the typical
reduction in plasma free carnitine FC is absent during an acute session
of exercise To establish an experimental model of study, male Wistar rats
were divided into a control n8 and a diabetic n8 group Diabetes was
induced with streptozotocin STZ at the dose of 50 mg/kg body weight One
week following the induction of diabetes, exercise was performed on a
treadmill at 22 m/min for 60 min at an incline of 8 Arterial blood was
collected at rest and immediately following exercise in previously
cannulated rats In
control rats, compared to rest, FC levels decreased
significantly after 60 min of exercise In diabetic rats, on the other
hand, the decrease in FC levels was not observed after exercise Plasma
esterified carnitine levels increased in both groups post-exercise, but
this increase was non significant from resting levels Our results indicate
that the plasma FC response at the end of acute exercise in the STZ-model
of diabetes is consistent with the observations in type 1 diabetic
patients This model may thus be suited to examine the disturbances in
carnitine metabolism during exercise typically reported in diabetic
patients
Key Words: Exercise, Metabolism, Diabetes, Insulin, Glucose
INTRODUCTION
Carnitine -hydroxy–trimethylaminobutyric acid is an essential cofactor
in the transfer and oxidation of fatty acids in the mitochondria of highly
oxidative tissues 1 The role of carnitine in fatty acid metabolism
becomes increasingly important during exercise, as fatty acids are a major
source of energy for contracting muscle 2 As the demand for fatty acids
increases, the requirement for carnitine in working muscle also increases
and its use is reflected by a reduction in free
carnitine FC in the
plasma Also reflecting this state is a heightened acylcarnitine production
from fatty acid esterification with subsequent appearance of esterified
carnitine EC in the plasma 2-4 The association between carnitine and
fatty acid metabolism is supported by the observation that hyperinsulinemia
in exercising humans, achieved by intravenous infusion, suppresses fatty
acid metabolism and attenuates the changes in both FC and EC 5
The effects of exercise on plasma carnitine metabolism have also been
reported in patients with diabetes mellitus 6,7 Since this condition is
associated with alterations in whole body substrate utilization 8,
disturbances in carnitine metabolism are particularly evident In type 1
diabetic patients, we have shown that the typical reduction in plasma FC
was not observed 6, whereas in type 2 diabetic patients, the increase in
EC was attenuated during exercise 7 Although these changes remain
unexplained, a greater reliance on glucose as energy substrate and defects
in fatty acid metabolism, frequent in type 1 and type 2, respectively, may
account for these changes 6,8,9 Part of the difficulty in explaining
these disturbances in carnitine
metabolism during exercise also stems from
the lack of an appropriate experimental model to investigate the impact of
diabetes
Indeed, studies intended to investigate from a mechanistic approach the
changes in carnitine metabolism that occur in diabetes must consider an
experimental model The streptozotocin STZ-induced diabetic rat is
undoubtedly the most widely used model and has provided valuable
information on the diabetic state 10 An attractive feature of this
model is that the severity of diabetes can be tailored by adjusting the
dose of STZ and if necessary implementing a diet rich in fat to produce a
state of insulin resistance 11 By manipulating these variables in
rodents, it is therefore possible to induce either type 1 or type 2-
diabetes In the present study, we chose to induce an insulinopenic state
in rats that closely resembles type 1 diabetes and determined whether a
plasma carnitine profile can be obtained during acute exercise, and whether
this response closely resembles that previously observed in the type 1
diabetic patient 6
METHODS
Animals And Induction Of Diabetes
The animals used in this study were cared for according to the
recommendations in the
Canadian Council on Animal Cares Guide to the Care
and Use of Experimental Animals Male Wistar rats n16 weighing 195 g
were used for this study Each animal was individually housed at 23 C
under standard lighting 0500-1900 hours and allowed free access to rat
chow and tap water Diabetes was induced in ether-anesthetized rats n8
by a single penile vein injection of STZ at the dose of 50 mg/kg dissolved
in citrate buffer, pH 45 STZ is selectively toxic to the insulin-
producing ?-cells of the pancreatic islets Destruction of the cells that
regulate blood glucose levels results in hyperglycemia, the hallmark of
diabetes Control rats n8 received an equivalent amount of citrate
buffer
One week later, after an overnight fast, diabetes was confirmed by the
level of glucose assessed in tail-blood with Dextrostix strips read with a
Glucometer Ames Division, Miles Laboratories, Rexdale, Ontario, Canada
With the concentration of STZ used, all rats developed severe diabetes with
plasma glucose levels ranging between 19 and 28 mmol/L
Exercise Protocol
The animals were first familiarized to the treadmill for 1 week by exposure
to daily 10 min runs Thereafter, rats were anaesthetized and the
left
carotid artery was cannulated 12 to later obtain blood samples in
unrestrained animals Two- to three days later, after an overnight fast,
exercise was performed on motor-driven treadmill Quinton Instruments,
model 42-15, Seattle, Washington, USA by both control and diabetic
animals The duration of the exercise bout was 60 min and the treadmill
speed was set at 22 m/min with a grade set at 8, corresponding to an
estimated oxygen cost of 58 ml/kg/min 13 Arterial blood samples were
obtained at rest and immediately after exercise for later measurement of
plasma glucose, insulin, and carnitine The blood was quickly centrifuged
after the exercise sessions at 3,000 rpm and the plasma separated and
frozen at -80C until analysis
Assay Methods
Plasma glucose was determined spectrophotometrically by an enzymatic method
using hexokinase and glucose-6-phosphate dehydrogenase 14 Plasma
insulin was determined by radioimmunoassay with rat insulin as standard and
polyethylene glycol separation 15 Plasma carnitine was measured by the
radioenzymatic assay described by McGarry and Foster 16 using carnitine
acetyltransferase and [14C] acetyl CoA All assays were performed at the
end of the
study
Statistical Analyses
Statistical analysis was performed using unpaired t-tests for comparisons
between group means Comparison of plasma values before and after exercise
in each group was determined using paired t-tests All values are reported
as mean SD
RESULTS
Figure 1A shows plasma glucose levels at rest and following 60 min of
exercise in control and diabetic rats Confirming the diabetic state,
glucose levels were significantly increased in diabetic rats compared to
control rats Sixty min of exercise was associated with a significant
decrease in glucose levels in both groups of rats However, at the end of
exercise, glucose levels remained significantly higher in diabetic rats
As expected, following STZ treatment, pancreatic function was altered in
diabetic rats Indeed, as illustrated in Figure 1B, plasma insulin levels
before exercise were significantly lower in diabetic rats compared to
control rats After exercise, plasma insulin levels were decreased and
remained significantly lower in control rats compared to diabetic rats In
diabetic rats, however, the decrease in insulin levels was not observed
The effect of acute exercise on plasma FC levels is
depicted in figure 2A
FC levels were significantly higher in control rats compared to diabetic
rats Exercise was associated with a significant decrease in FC in the
control rats, whereas in diabetic rats, FC levels remained unchanged from
resting values
As shown in Figure 2B, the levels of plasma EC at rest were similar in
control rats and diabetic rats After 60 min of exercise, plasma EC levels
were significantly higher in control rats compared to diabetic rats
DISCUSSION
The present study is the first to make use of a chemically-induced form of
diabetes in the rat to measure the changes in plasma carnitine during acute
exercise Using the STZ-model of diabetes, measurements of plasma FC and EC
were made before and immediately after 60 min of exercise Our results
demonstrate that close similarities exist in the plasma FC profile to
exercise between the STZ-rat and type 1 diabetic patient The level of
plasma FC levels was decreased after exercise in the control rat, whereas
they remained unchanged in the STZ-rat, a pattern that clearly mimics the
response observed in both the non-diabetic and type 1 diabetic patient,
respectively Based on these observations, the
STZ-model of diabetes may
be pertinent to investigate the metabolic disturbances in carnitine
metabolism during exercise seen in human type 1 diabetes That this model
is suited to study carnitine metabolism is further supported by the
findings that the changes in both plasma glucose and insulin in the STZ-
model are similar to the responses reported in the type 1 diabetic patient
6
Since we have succeeded in developing a model for the study of plasma
carnitine metabolism in an exercise setting using the diabetic rat, at this
point, we can only speculate on the similarity in the FC response between
the STZ-rat and type 1 diabetic patient Of interest, though care must be
taken in this interpretation, is the role of insulin on fatty acid
metabolism The suppressive role of insulin on adipose tissue lipolysis has
been suggested to explain the FC response in type 1 diabetic patients
5,6 In diabetic patients receiving exogenous insulin, absorption from
injection sites results in over-insulinization and inhibition of lipolysis
17,18 This limits the availability of free fatty acids resulting in a
preferential use of glucose as energy source 19-21 An elevated
respiratory exchange ratio
observed in hyperinsulinemic type 1 diabetic
patients is consistent with the preferential utilization of carbohydrates
as an energy substrate during exercise 6 A hyperinsulinemic state
induced by an intravenous infusion of insulin in the non-diabetic patient
is also associated with a limited blood level of free fatty acids and
reduced FC response during exercise 5
In the present study, however, diabetic rats were not hyperinsulinemic Pre-
exercise insulin levels were lower in diabetic rats compared to control
rats and remained unchanged after exercise, a profile that would inevitably
be associated with an elevated blood level of free fatty acids 22 Under
these conditions, the FC response in diabetic rats is clearly not
consistent with the role of insulin in suppressing free fatty acid
mobilization An intriguing question then arises in what explains the
altered FC response in the diabetic rat? One possibility is that the
hyperglycemic state of diabetic animals is associated with a preferential
use of glucose Indeed, glucose uptake and oxidation by exercising muscle
are stimulated under conditions of hyperglycemia 20,21
While more studies are required to delineate the effects of
STZ on
carnitine metabolism during exercise, it should be clear that the
mechanisms explaining the FC response based solely substrate oxidation and
plasma changes are assumptions This is because 99 of total carnitine is
located in the intracellular pool and the relation between carnitine and
its function in fatty acid flux is highly sensitive to the mitochondrial
metabolic state 1 During exercise, the rate of fatty acid oxidation in
muscle is related to the concentration of fatty acids into the cytoplasm to
which the mitochondria is exposed rather than the concentration of free
fatty acids in the plasma As the availability of fatty acids in the plasma
is reduced with hyperinsulinemia, it remains unknown whether this would
limit the involvement of carnitine in fatty acid oxidation Clearly, future
studies are warranted to elucidate this possibility
CONCLUSIONS
In conclusion, using the STZ-model of diabetes, we were able to demonstrate
that acute exercise presents a plasma carnitine pattern that is similar to
that reported in the type 1 diabetic patient The STZ-diabetic rat model
seems appropriate for studies requiring extensive tissue sampling not
possible in humans to
elucidate the disturbances in carnitine metabolism
ACKNOWLEDGEMENTS
The authors wish to thank Rachèle Duchesne, Marie Martin and Stéfane
Charest for their skilled technical assistance This study was funded by
the Canadian Diabetes Association AN
Address for correspondence: Tom L Broderick, PhD, Department or
Physiology, Midwestern University, Glendale, Arizona, USA, 85308, ph: 623-
572-3664; fax: 623-572-3664; Email: tbrode@midwesternedu
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A
B
Figure 2 Values are expressed as mean SD for 8 rats in each group
P005, compared to control rats; , P005, compared to rest inXZ\ z
| E KLM respective group
A
B
Figure 1 Values are shown as mean SD for 8 rats in each group for A
plasma glucose and B plasma insulin P005, compared to control rats;
, P005, compared to rest in respective group
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