Change in Bone Functions in Rats with Insulin Resistance Induced by High-fat Diet Consumption and Testosterone Deprivation for 12 Weeks

Abstract

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder associated with insulin resistant condition. We previously demonstrated that an obese-insulin resistant condition induced by long-term high-fat diet (HFD) consumption markedly impaired osteoblastic function leading to osteoporosis in the jaw bone. Previous studies also showed that testosterone deprivation lead to the development of osteoporosis. However, the effects of combined obesity and testosterone deprivation on bone funtion have not been investigated. In the present study, we hypothesized that testosterone deprivation impaired osteoblastic insulin signaling, decreased osteoblast survival, reduces bone density, and that obesity aggravated those deleterious effects in testosterone-deprived animals. Twenty four male rats were divided into sham-operated and orchiectomized groups (n=12/group). A week after the surgery, the rats in each group were divided into 2 subgroups and fed with either normal diet (ND; 19.7% energy from fat) or high fat diet (HFD;59.3% energy from fat) for 12 weeks. At the end of week 12, blood samples were collected from all rats [sham-operated ND rats (NDS), orchiectomized ND rats (NDO), sham-operated HFD rats (HFS) and orchiectomized HFD rats (HFO)] to determine the levels of glucose, insulin, cholesterol, testosterone, HOMA index and osteocalcin levels. The tibias were extracted to determine bone mass using microcomputed tomography. Furthermore, osteoblasts were isolated from rat tibiae in each subgroup, and were used to determine (1) osteoblast characteristic using alkaline phosphatase and Alizarin red staining, (2) osteoblastic cell proliferation using alamarBlue cell viability assay and proliferation protein marker, Cyclin D1, (3) cellular apoptosis using TUNEL assay, and (4) osteoblastic insulin signaling by measuring insulin-mediated insulin receptor phosphorylation (IR-p) and Akt phosphorylation (Akt-p). We found that only HFS and HFO groups were developed peripheral insulin resistance as indicated by increased HOMA index (26.6 ± 3.0, 25.2 ± 4.1, respectively), compared to that of NDS and NDO groups, (14.6 ± 1.7, 17.2 ± 3.2, respectively). The testosterone levels of NDO, HFS and HFO groups were significantly decreased compared to that of NDS group (0.07 ± 0.01, 0.46 ± 0.01, 0.15 ± 0.06 and 0.75 ± 0.2 ng/dl, respectively). Similar to testosterone levels, the trabecular bone density was significantly decreased in NDO, HFS and HFO groups (126.9 ± 11.1, 140.9 ± 15.3, 155.8 ± 10 mg/cm3, respectively), compared to that in the NDS group (203.4 ± 14.4 mg/cm3). NDO, HFS and HFO osteoblasts also demonstrated the decreased cell proliferation, decreased Cyclin D1 expression and increased cell apoptosis. Moreover, the impairment of osteoblastic insulin signaling isolated from NDO, HFS and HFO groups was demonstrated as indicated by the decreased IR-p and Akt-p. No differences were found among NDO, HFS and HFO rats. These findings indicate that either testosterone deprivation or obesity could impair osteoblastic insulin signaling, leading to the development of osteoporosis. However, obesity does not aggravate those deleterious effects of the testosterone-deprived condition on bone.