Glucocorticoids are among the most
potent and widely used immunosuppressant drugs available but have many
detrimental side effects; however, only limited data are available on
intervention studies to prevent these life-long side effects from occurring.
The purpose of this study was to test an experimental rat model mimicking the
bone loss and hyperglycemia associated with glucocorticoid administration. A
five-week treatment was compared to a control group. The bone loss in the
glucocorticoid-treated rats was found to be significant in only a brief
treatment; the serum parameters may reflect that the gradual onset of diabetes
was beginning. This study has provided a model of bone loss associated with
the glucocorticoid (prednisolone) administered that can be used to test
interventions to inhibit the adverse effects of glucocorticoids.
On September 21, 1948, compound E (cortisone) became the first
glucocorticoid to be administered to a patient with rheumatoid arthritis.
Since that time the anti-inflammatory and immunosuppressive actions of
glucocorticoids have benefited patients suffering from lupus and chronic
asthma, along with organ transplant recipients (Avioli 1984, Frauman 1996).
Unfortunately, glucocorticoid therapy does have several undesirable
side effects. These may include
central obesity, hypertension, impaired wound healing, increased infection
rates, and impaired growth in children (Frauman 1996).
However, of particular
interest and concern are the detrimental effects of glucocorticoids on bone
and on glucose levels. Research
has indicated that the use of glucocorticoids results in osteopenia (bone
loss) and hyperglycemia, which can eventually lead to osteoporosis and
diabetes mellitus (Naghavi et al 1975, Ravina et al 1999, Wimalawansa et al
Current osteoporosis trends
indicate that 10 million individuals in the United States already have
osteoporosis, and more than 18 million are at risk for developing osteoporosis
(National Osteoporosis Foundation 2001).
In addition, recent reports from the American Diabetes Association
state that approximately 15.7 million people or 5.9% of the United States
population currently suffer from diabetes mellitus (American Diabetes
Glucocorticoids have been shown to accelerate bone loss,
leading to osteopenia and osteoporosis and also to impair glucose tolerance,
leading to or worsening diabetes mellitus
(Wimalawansa et al 1997, Ravina et al 1999). Glucocorticoids are among the most potent and widely used
immunosuppressant drugs available (Frauman 1996) and due to the side effects,
are considered an enormous problem in clinical practice today (Wimalawansa et
al 1997). However, only limited
data are available on intervention studies to prevent these life-long side
effects from occurring. The
purpose of this study was to test an experimental rat model mimicking the bone
loss and hyperglycemia associated with glucocorticoid (prednisolone)
administration. This model will
be utilized in testing interventions to ameliorate the adverse side effects of
Materials and Methods
Animals and Diet
Twenty, six-month old, female Sprague-Dawley (Harlan-Teklad,
Indianapolis) rats were randomly assigned to groups of ten fed either 100 mg
prednisolone/kg diet or control diet with no added prednisolone.
Prednisolone and dosages were chosen based on Lingren’s and
colleagues’ model that concluded prednisolone- induced osteopenia occurs in
rats in doses of 100, 50, or 20 mg per kg of diet.
To ensure prednisolone-induced osteopenia 100mg per kg diet was chosen.
The rats were individually
housed in an environmentally controlled laboratory at the institution’s
Laboratory Animal Resource Center (LAR).
Rats were maintained on 12:12 light/dark cycles and allowed free access
to distilled water throughout the duration of the study.
Guidelines for the ethical care and treatment of animals established by
the Animal Care and Use Committee at the institution were followed.
Rats consumed their assigned diet for five weeks and had free access to
deionized water. During this
time, rats were weighed once a week. Throughout
the duration of the study, one rat from the prednisolone group, and one rat
from the control group died before completing the entire five weeks.
five weeks, the rats were placed in metabolic cages twelve hours before
necropsy where urine was collected. At the time of necropsy, fasting blood
glucose concentrations were measured using a Bayer Dex Glucometer Diabetes
Care System (Albertson's Pharmacy) on a drop of blood from the tip of the
tail. the animals then received an oral glucose load (1g/kg body weight) and
after two hours, their glucose level was again measured. animals were then
anesthetized intraperitoneally with ketamine (100 mg/kg body weight) and
sylazine (5 mg/kg body weight) and whole body scans were performed by Dual
Energy X-ray absorptiometry (DEXA) (Hologic QDR 4500 A, Waltham, MA>). The
animals were then exsanguinated from the abdominal aorta. Blood was allowed to
clot, centrifuged, serum aliquots frozen at -20ºC until analysis. Liver,
spleen, and kidneys were removed and weighed. Femur, tibia, third, fourth, and
fifth lumbar vertebra were collected from the animals, cleaned of adhering
tissues, and frozen at -20ºC for further analysis.
Bone mineral area and bone mineral content were performed by Dual
Energy X-ray Absorptiometry (DEXA) and analyzed with the small animal software
provided by the manufacturer.
Serum Clinical Chemistry
Insulin values were assayed with a rat insulin RIA Kit (Linco Cat. #
RI-13K, St. Charles, Mo.) and 125Iodine was measured on a gamma
counter. Glucose and fructosamine
were analyzed using a commercially available kit from Roche Diagnostics (Sommerville,
NJ). These tests were performed
using the Cobas-Fara II Clinical Analyzer (Roche Diagnostics, Sommerville,
Data analysis of means and standard errors of the mean were calculated
using SAS (version 8.0, SAS Institute, Cary, NC).
The generalized linear model procedure was used for analysis of
variance. The significance level
was set at p<0.05.
During the five-week treatment, the glucocorticoid group had a significant
loss in body weight compared to the control group.
The bone mineral area (Figure 1) and bone mineral content (Figure 2) of
the glucocorticoid group were significantly lower in comparison to their
basefline values. This was not observed in the control group. Apparently the
bones of animals in the glucocorticoid-treated group decreased in size as well
as in total mineral content.
There were no significant differences found between the two groups for
the blood parameters measured. The
blood glucose concentrations did not indicate that diabetes mellitus had been
induced in the rats. However,
even during this relatively short experiment, the glucocorticoid treated group
tended to have increase in their insulin (Figure 3).
Elevated insulin occurs prior to the onset of overt Type II diabetes in
humans. Fructosamine had
increased by eight percent, which was not significant (Figure 4). Fructosamine is a measure of glucose control over the
previous two to three weeks and higher fructosamine values reflect a history
of higher circulating glucose. Because
fructosamine changes gradually a longer study might be required to detect
glucose changes that could be very important over a lifetime.
Glucocorticoids are among the
most potent and widely used immunosuppressant drugs available but have many
detrimental side effects. However,
only limited data are available on intervention studies to prevent these
life-long side effects from occurring. Therefore,
an effective rat model would aid in the effort to reduce negative treatment
effects in humans associated with glucocorticoid treatment.
The bone loss in the
glucocorticoid-treated rats was found to be significant in only a brief
five-week period. In addition,
the serum parameters may reflect that the gradual onset of diabetes was
beginning. This study has
provided a model of bone loss associated with the glucocorticoid (prednisolone)
administered that can be used to test interventions to inhibit the adverse
effects of glucocorticoids.
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