Estrogen Deficiency Exacerbates Type 1 Diabetes-Induced Bone TNF-α Expression and Osteoporosis in Female Mice

Abstract

Estrogen deficiency after menopause is associated with rapid bone loss, osteoporosis, and increased fracture risk. Type 1 diabetes (T1D), characterized by hypoinsulinemia and hyperglycemia, is also associated with bone loss and increased fracture risk. With better treatment options, T1D patients are living longer; therefore, the number of patients having both T1D and estrogen deficiency is increasing. Little is known about the mechanistic impact of T1D in conjunction with estrogen deficiency on bone physiology and density. To investigate this, 11-week-old mice were ovariectomized (OVX), and T1D was induced by multiple low-dose streptozotocin injection. Microcomputed tomographic analysis indicated a marked reduction in trabecular bone volume fraction (BVF) in T1D-OVX mice (~82%) that was far greater than the reductions (~50%) in BVF in either the OVX and T1D groups. Osteoblast markers, number, and activity were significantly decreased in T1D-OVX mice, to a greater extent than either T1D or OVX mice. Correspondingly, marrow adiposity was significantly increased in T1D-OVX mouse bone. Bone expression analyses revealed that tumor necrosis factor (TNF)-α levels were highest in T1D-OVX mice and correlated with bone loss, and osteoblast and osteocyte death. In vitro studies indicate that estrogen deficiency and high glucose enhance TNF-α expression in response to inflammatory signals. Taken together, T1D combined with estrogen deficiency has a major effect on bone inflammation, which contributes to suppressed bone formation and osteoporosis. Understanding the mechanisms/effects of estrogen deficiency in the presence of T1D on bone health is essential for fracture prevention in this patient population.

Figure 1.

Estrogen deficiency exacerbates type 1 diabetes–induced trabecular bone loss. Eleven-week-old females underwent, sham or OVX operations. At 12 weeks of age, T1D (diabetes) was induced by streptozotocin injection, whereas controls were injected with saline. At 16 weeks of age, bones were imaged by microcomputed tomography. (a) Distal femur trabecular BVF. (c) L3-vertebral trabecular BVF. (b and d) Representative isosurface images of distal femoral and vertebral trabecular bone, respectively. Values represent mean ± standard error (n = 10 for intact control group; n = 9 for intact T1D group; n = 12 for both OVX control and T1D group). *P < 0.05 with respect to intact control; #P < 0.05 with respect to OVX control; ^P < 0.05 with respect to T1D intact.

Figure 2.

Type 1 diabetic, estrogen-deficient mice display lowest osteoblast marker levels. (a–c) Gene expression of osteoblast bone markers measured in RNA extracted from whole tibia (RUNX2, Osterix, and osteocalcin). (d) Osteocalcin protein levels in serum. (e) Osteoblast number per millimeter bone surface as determined histomorphometrically. (f) Calculated total osteoblast number per trabecular bone surface of the distal femur metaphyseal region. (g) Representative images (×40 magnification) of osteoblasts. Arrows indicate examples of osteoblasts that were quantified. Values represent mean ± standard error (n = 10 for intact control group; n = 9 for intact T1D group; n = 12 for both OVX control and T1D group). *P < 0.05 with respect to intact control; #P < 0.05 with respect to OVX control; ^P < 0.05 with respect to T1D intact. HPRT, hypoxanthine guanine phosphoribosyl transferase.

Figure 3.

T1D-OVX condition decreases dynamic markers of bone formation in both the vertebrae and humeri. (a and b) Calculated vertebral MAR and BFR. (c and d) Calculated humerus MAR and BFR. (e) Representative fluorescent microscopy images of calcein pulses located near the bone surface. Values represent mean ± standard error (n = 10 for intact control group; n = 9 for intact T1D group; n = 12 for both OVX control and T1D group). *P < 0.05 with respect to intact control; #P < 0.05 with respect to OVX control; ^P < 0.05 with respect to T1D intact.

Figure 4.

T1D-OVX mice display altered osteoclast markers. (a) TRAP expression levels in RNA extracted from whole tibias. (b) TRAP5b levels in mouse serum. (c) RANKL (an activator of osteoclastogenesis) expression relative to osteoprotegrin (OPG) (an inhibitor of osteoclastogenesis) messenger RNA levels. (d) Percentage of osteoclast surface per millimeter bone surface. (e) Calculated OC/OB. (f) Representative images (×40 magnification) of TRAP-stained osteoclasts, with arrows indicating examples of osteoclasts quantified. Values represent mean ± standard error (n = 10 for intact control group; n = 9 for intact T1D group; n = 12 for both OVX control and T1D group). *P < 0.05 with respect to intact control; #P < 0.05 with respect to OVX control; ^P < 0.05 with respect to T1D intact. HPRT, hypoxanthine guanine phosphoribosyl transferase.

Figure 5.

Bone marrow adiposity is increased in estrogen deficient diabetic mice. (a) Percentage of adipocyte area to total bone marrow area. (b) Representative marrow images (×40 magnification) displaying adipocyte differences. White circles are adipocytes. (c) Fatty acid binding protein 4 (FABP4) expression levels measured in RNA extracted from femur bone marrow. Values represent mean ± standard error (n = 10 for intact control group; n = 9 for intact T1D group; n = 12 for both OVX control and T1D group). *P < 0.05 with respect to intact control; #P < 0.05 with respect to OVX control; ^P < 0.05 with respect to T1D intact. HPRT, hypoxanthine guanine phosphoribosyl transferase.

Figure 6.

Bone TNF-α expression is increased by T1D-OVX conditions in vivo and in vitro and correlates to BVF and osteoblast numbers. (a) TNF-α RNA levels measured from whole tibia. (b) Ratio of the messenger RNA levels of TNF-α to interleukin (IL)-10. (c and d) Pearson correlation analyses of TNF-α RNA levels relative to (c) femoral BVF and (d) osteoblast number per square millimeter bone surface. Values represent mean ± standard error (n = 10 for intact control group; n = 9 for intact T1D group; n = 12 for both OVX control and T1D group). (e) Quantification of TNF-α protein in the femoral bone marrow. Below the graph are representative images (×40 magnification) of quantified immunohistochemical stain in the bone marrow. Values represent mean ± standard error (n = 8 for intact control group; n = 7 for intact T1D group; n = 6 for both OVX control and T1D group). *P < 0.05 with respect to intact control; #P < 0.05 with respect to OVX control; ^P < 0.05 with respect to T1D intact. (f) After 5 days in culture, MC3T3-E1 osteoblasts were grown with or without estrogen. After 24 hours, cells were treated with (c) vehicle, TNF-α (10 ng/mL) or glucose (30 mM) + TNF-α (10 ng/mL). RNA was extracted 24 hours later for TNF-α RNA expression analyses. Values represent mean ± standard error (5 separate experiments per group). *P < 0.05. HPRT, hypoxanthine guanine phosphoribosyl transferase; OB, osteoblast.

Figure 7.

Osteoblast and osteocyte death is increased in estrogen-deficient, T1D mice. (a) Ratio of RNA expression of proapoptotic marker, BAX, to the antiapoptotic marker, Bcl-2, from whole tibia. (b) Number of osteocytes per square millimeter of cortical bone in the distal femur metaphyseal region. (c) Representative images of TUNEL-stained osteocytes (×20 magnification) with arrows indicating examples of quantified osteocytes. (d) Percentage of TUNEL-stained osteocytes. (f) Percentage of TUNEL-stained osteoblasts. (e and g) Pearson correlation analyses of TUNEL-stained osteocytes and osteoblasts with TNF-α staining. Values represent the mean ± standard error (n = 10 for intact control group; n = 9 for intact T1D group; n = 12 for both OVX control and T1D group). *P < 0.05 with respect to intact control; #P < 0.05 with respect to OVX control; ^P < 0.05 with respect to T1D intact.