Effects of Insulin Resistance Induced by Dexamethasone on Bone Mass in Ovariectomized Rats

Glucocorticoids therapy is the most common cause of secondary iatrogenic osteoporosis.The bone loss occurs predominantly due to a decrease in bone formation, although increased bone resorption also occurs. Insulin resistance is the key pathology in type 2 diabetes negatively influence bone remodeling and leads to reduced bone strength. Loss of sex steroids, particularly oestradiol, as in ovariectomized rats,leads to increased skeletal remodeling over and above the age-related increment, together with excessive osteoclast activity. In this study, ovariectomy DEX group has highly significant increase in relative cortical resorptioncompared to ovaiectomy and sham DEX groups, also ovariectomy and DEX group has highly significant decrease in bone thickness compared to ovariectomy and sham DEX groups. The consequent increase in remodeling activation increases the overall resorption rate without a compensatory increase in formation, leading to rapid bone loss.This negative effect on bone which is due to the glucocorticoid excess is also mediated by indirect mechanisms such as the calcium malabsorption and hypercalciuria. In response to the enhanced supply of calcium from the skeleton, PTH secretion tends to be diminished, thereby reducing vitamin D [1,25(OH)2 cholecalciferol] concentration with a consequent reduction in calcium absorption.


Background
An excess of cortisol, as seen in Cushing's syndrome or with clinical administration of glucocorticoids that is used to treat acute and chronic inflammatory diseases, leads to symptoms of abdominal obesity, hypertension, glucose intolerance or diabetes and dyslipidemia, all of which are also features of insulin resistance [1][2].
Glucocorticoids modify osteoblastic cells through inhibiting osteoblast maturation and reduce their synthetic capacity, thereby reducing the amount of bone formed during each remodeling cycle [7] Osteoporosis is a skeletal disease characterized by low bone mass and the structural deterioration of bone resulting in, increase susceptibility to fractures [8] .Imbalance between bone formation and bone resorption is the predominant mechanism behind osteoporosis [9].

Objectives
In this study we injected dexamethasone in rats to induce insulin resistance and investigated the insulin resistance effects on bone mass either alone or in addition to ovariectomy in ovariectomized rat models.

Animals
24 Wistar female rats 8-10 WEEKS (155 -170 g) were obtained from animal house of institute of ophthalmology, Giza, Egypt. Rats were housed individually, allowed to acclimatize the new environment, free access to water and food ad libitum, temperature (23 C) and exposure to 12 hours day/night cycle.
Rats underwent overictomy surgery as follow: Anaesthesia: Ketamine 40 mg/kg mixed with xylazine 20 mg/kg and injected intra peritoneal. The abdominal hair was shaved abdominal wall was sterilized and midline anterior abdominal was incised. Both ovaries were removed. The postoperative antibiotic was cephalosporine 20 mg/kg intramuscular, once/day for 3 days. The post operative analgesia was Na Diclofenac 2ml/rat twice/day for 3 days. The rats were allowed to 2 weeks recovery before any additional procedures. 14days,and were allowed to rest for 12 hours before the surgery.
At end of experiment, blood samples were taken through puncture of the aorta. Bones of tibia are immediately removed for biochemical and histological studies.

Drugs and Chemicals
Dexamethasone [DEX] sodium phosphate powder and cholesterol powder were obtained from Sigma-Aldrich Company, USA. Commercial cinnamon powder was prepared as a water extract at a concentration of 60 g/100 ml distilled H2O2 Biochemical studies: 1-Serum glucose and insulin.

Histopathological Examinations
Specimens taken and sectioning The animals were sacrificed by using chloroform inhalation and the femurs were carefully dissected and cleared from adjacent muscles, then immediately fixed in neutral buffered formaldehyde for 2 days. After fixation, the femurs were processed for preparation of decalcified specimens. Decalcification was performed byusing the chelating agent ethylene diamine tetra acetic acid in the form of its disodium salt (5.5 g ethylene diamine tetra acetic acid in 90 ml distilled water and 10 ml formaldehyde 37-40%). Decalcification was carried out for 4 weeks, during which time the decalcifying solution was changed every day. The decalcified specimens were dehydrated and processed to form paraffin blocks. Serial longitudinal sections, parallel to the long axis of the bones extending from one-third to one-half the thickness of the distal femur, were prepared. Then sections of 5 µm thickness were subjected to:

Bone Histomorphometric Studies
1-Outer cortical bone thickness: mean width of outer cortical bone. This was measured by drawing a perpendicular line from the periosteum to the endosteum [10] 2-Relative bone resorption eroded surface (%): percentage of compact bone surface with characteristic features of resorption, including surfaces with and without osteoclasts [11].

Length of eroded trabecular surface % Total length of trabecular surface
The cortical thickness was determined at five levels (0.5mm apart), starting 0.2mm below the lowest point of the growth plate. All bone histomorphometric measurements were taken, beginning at more than 1.2mm from the growth plate-metaphyseal junction to exclude the primary spongiosa [12].

Statistical Analysis
Data are expressed as mean ± S.E.M. Statistical evaluation was performed by one-way analysis of variance [ANOVA] followed by Turkey test for multiple comparisons. All analyses were performed with SPSS software package [version 18]. P ˂ 0.05 was selected as the criterion for statistical significance.

Results
Dexamethasone injection in sham operated group induced significant increase in blood glucose and insulin levels compared to sham control group. Also Dexamethasone injection in ovariectomized rats results in highly significant increase in glucose and insulin levels compared to sham control ,sham DEX and ovariectomized groups Ovariectomized groups showed significant increase in blood glucose and highly significant increase in insulin levels compared to sham control group and sham DEX groups Ovariectomy + DEX group showed very highly significant increase in glucose and insulin compared to sham sham control, Sham +DEX and ovariectomized groups    Dexamethasone injection in sham operated group has insignificant increase in both calcium and phosphorous level and also insignificant increase in alkaline phosphatase enzyme compared to sham control.
Ovariectomy has insignificant increase in both calcium and phosphorous level and very highly significant increase in alkaline phosphatase enzyme compared to sham control and sham DEX groups.
Ovariectomy + Dex has significant increase in both calcium and phosphorous levels and very highly significant increase in alkaline phosphatase enzyme compared to sham DEX groups and significant increase compared to ovariectomized group.  Histopathological results: Sham DEX group has insignificant decrease in bone trabeculae thickness compared to sham control group. Ovariectomy group has insignificant decrease in bone trabeculae thickness compared to Sham DEX and Sham control. Ovariectomy and DEX group has highly significant decrease in bone thickness compared to ovariectomy and sham DEX groups.  Sham DEX group has insignificant increase in relative cortical resporption % compared to sham control group Ovariectomy group has significant increase in relative cortical resorption % compared to sham DEX group.
Ovariectomy DEX group has highly significant increase in relative cortical resorption compared to ovaiectomy and sham DEX groups.

Discussion
The current study confirms and extends the findings of an inverse association between bone strength indices and insulin resistance, noted by [13]. These findings are also consistent with those from studies in adolescents [14], young adults [15] bone marrow transplant patients [16], and diabetics [17] that have found associations between greater insulin resistance and reduced bone mass. Physiologically low estradiol has an effect on the skeleton in elderly women possibly by reducing skeletal remodeling [18]. The presence of osteoporosis related to diabetes mellitus is less acknowledged.In patients with type 2 diabetes mellitus the risk of osteopenia is not clear [19]. There is evidence that a decreased bone formation is one major mechanism leading to reduced bone mass in diabetics.The association between diabetes mellitus and osteoporosis remains controversial [20].
This study demonstrated that a longitudinal section of rat femur compact bone from group IV (OVARIECTOMY + DEX) showed thinned out trabeculae with deep erosions In sham DEX group and Ovariectomy group the histological changes in the bone shoed only either eroded endostium or periosteum with no signs of subperiosteal new bone deposition In this study, Ovariectomy DEX group has highly significant increase in relative cortical resorption compared to ovaiectomy and sham DEX groups , also ovariectomy and DEX group has highly significant decrease in bone thickness compared to ovariectomy and sham DEX groups. Our findings are in contradistinction to several previous studies that have documented higher bone mineral density in T2DM [21][22] despite the increased risk of fractures in T2DM [23][24][25][26][27][28]. A deleterious role for insulin resistance on bone is also consistent with experiments in mouse models that suggest that osteoblasts are insulin target cells [29] .Mice deficient in osteoblast-specific insulin receptors have reduced expression of genes implicated in bone resorption (CathepsinK and Tcirg1), less acidification of bone extracellular matrix, significantly smaller resorption pits and markedly lower serum levels of bone resorption marker, CTx Further, it was recently noted that in Wistar rats with an obese, insulin resistant condition induced by a 12week high fat diet, there was significant impairment of osteoblastic insulin signaling and osteoblast proliferation, with increased osteoblastic apoptosis culminating in osteoporosis in the jaw bone, compared to baseline, measured using micro computerized tomography (CT) of mandibular bone. Previous studies have also noted the lower spine bone volume in T2DM subjects. A recent study found that bone cross-sectional area is also lower in T2DM, suggesting a deficit in periosteal apposition which is normally stimulated by skeletal loading. Taken together, these studies suggest that increased insulin resistance and/or hyperinsulinemia, may interfere with the usual anabolic response in bone to skeletal loading, so that bone strength relative to load is negatively affected. Our study confirms and extends the findings of an inverse association between bone strength indices and insulin resistance, noted by Ishii et al [31]

Conclusions
Hyperinsulinemia and insulin resistance induced by long term glucocorticoids therapy affects negatively bone mass either alone or combined with ovariectomy .Induced menopause with loss of the anabolic hormone oestrogen have more effect in decreasing bone mass, which may lead to increased bone fractures in menopausal women taking glucocorticoids therapy for long times .This needs further research to uncover the biological mechanisms by which insulin resistance induce by glucocorticoids could deleteriously affect bone health.