PATHOGENESIS/PATHOPHYSIOLOGY

ALCOHOL-INDUCED ADIPOGENESIS IN BONE MARROW AND 3T3 CELLS

Yisheng Wang; Quanjun Cui; Gary Balian; Gwo-Jaw Wang
Orthopaedic Research Laboratory; Department of Orthopaedic Surgery; University of Virginia, School of Medicine, Box 860374,
Charlottesville, VA 22908, Telephone: 804-924-1716, Fax: 804-924-1691, yw4n@Virginia.edu

Abstract:

Introduction: The patients population suffering from osteonecrosis (ON) has been increasing. In no-traumatic ON, the incidence of the steroid-induced ON is the highest, while alcohol-induced ON is second. The mechanism of action of alcohol on bone and marrow cells ahs not been defined. In this study, we investigated the effect of alcohol administration on rabbits in vivo and on marrow stromal cells (MSCs), D1, a cloned osteoprogenitor cell from bone marrow and 3T3 cells in vitro. Our experiments are directed towards understanding the role of alcohol on the development of a fatty marrow and ensuing osteonecrosis and that of adipogenesis in cells, as well as the changes in gene expression by bone marrow mesenchymal cells during adipogenesis and osteogenesis in vitro.

Materials and Methods: MSCs, D1 cells and 3T3 cells were maintained in culture. The MSCs were treated with 0.03, 0.09, and 0.15 M ethanol daily for 21 days. D1 and 3T3 cells were treated with 0.03, 0.06, 0.09, 0.15, and 0.21 M ethanol daily for 14 and 21 days. The effect of ethanol on adipogenesis was examined by determining the accumulation of triglyceride vesicles by phase contrast microscopy and by staining with Sudan IV. Osteogenesis and adipogenesis in D1 cells were measured using northern blot hybridization to detect 422 (aP2) and osteocalcin mRNA's.

A rabbit model was established to study osteonecrosis in vivo (approved by our institutional animal review board). New Zealand white rabbits were divided into two groups: Group A (n=40) received (10 ml/Kg glucose solution. In ten animals from each group, serum lipid peroxides (LPO), superoxide dismutase (SOD), triglyceride (TG) and cholesterol (CHO) were determined at 1, 2, 3 and 6 months after treatment. The femoral heads and livers were removed and examined histologically after staining with H & E and Sudan IV, and by electron microscopy.

Results: The cells were separately treated with increasing concentrations of ethanol for 14 days in MSCs, 3 days in D1 and 3T3 cells. The cells accumulated triglyceride vesicles, which were small initially and increased in size with time. The number of adipocytes in culture increased when the cells were treated at the higher concentrations of ethanol.

Northern blot hybridization with osteocalcin cDNA and 422 (aP2) cDNA in D1 cells showed that treatment with 0.15 M ethanol decreased expression of osteocalcin mRNA by 85% compared with control cells that were not treated with ethanol. 422 (aP2) mRNA did not change in the D1 cells treated with 0.15 M ethanol for 14 days.

Rabbits treated with ethanol showed significantly increased serum LPO, TG, CHO, and reduced SOD activity. At 6 months, LPO and SOD activity in Group A was 3.81 µmol/ml and 28.84 Nu/ml respectively, in Group B they were 2.52 µmol/ml and 31.68 Nu/ml. TG and CHO in Group A was 5.87 mmol/L and 2.54 mmol/L respectively, and that in Group B was 1.35 mmol/L and 1.64 mmol/L.

After ethanol treatment, fatty changes in the liver and bone marrow of Group A were found histologically. Fat cell hypertrophy and proliferation, and diminished hematopoiesis in the subchondral bone area of the femoral head was observed throughout the experiment. The percentage of empty osteocyte lacunae was increased and trabeculae became thinner and sparse at 2, 3 and 6 months. The femoral head became loose, and could be easily cut in group A at 6 months. By contrast, no abnormal changes were detected in the livers and femoral heads of animals in Group B.

Discussion: Results from our study show that alcohol can induce adipogenesis in bone marrow in vivo, and in cells in vitro. Analysis of gene expression demonstrated diminished expression of osteocalcin in cells treated with ethanol. Decreased osteogenic properties and increased adipogenesis were detected in cells treated with ethanol. Our study indicates that the rabbit is a suitable animal model in which to study osteonecrosis and alcohol-induced adipogenesis in bone marrow stroma. Systemic changes in lipid levels may be a direct result of treatment treatment with ethanol and may contribute to alcohol-related osteonecrosis. Inhibition of osteocalcin gene expression without an increase in 422 (aP2) mRNA suggests that adipogenesis i.e. increased triglycerides and vesicles, is brought about by the action of ethanol downstream in the fatty acid metabolism pathway.