Impact of glucose-dependent insulinotropic peptide on age-induced bone loss

Abstract

GIP is an important hormonal link between nutrition and bone formation. We show for the first time that BMSCs express functional GIP receptors, that expression decreases with aging, and that elevations in GIP can prevent age-associated bone loss.

Introduction: We previously showed that C57BL/6 mice lose bone mass as they age, particularly between 18 and 24 mo of age. The mechanisms involved in this age-dependent induced bone loss are probably multifactorial, but adequate nutrition and nutritional signals seem to be important. Glucose-dependent insulinotropic peptide (GIP) is an enteric hormone whose receptors are present in osteoblasts, and GIP is known to stimulate osteoblastic activity in vitro. In vivo, GIP-overexpressing C57BL/6 transgenic (GIP Tg(+)) mice have increased bone mass compared with controls. Bone histomorphometric data suggest that GIP increases osteoblast number, possibly by preventing osteoblastic apoptosis. However, potential GIP effects on osteoblastic precursors, bone marrow stromal cells (BMSCs), had not previously been examined. In addition, effects of GIP on age-induced bone loss were not known.

Materials and methods: Changes in BMD, biomechanics, biomarkers of bone turnover, and bone histology were assessed in C57BL/6 GIP Tg(+) versus Tg(-) (littermate) mice between the ages of 1 and 24 mo of age. In addition, age-related changes in GIP receptor (GIPR) expression and GIP effects on differentiation of BMSCs were also assessed as potential causal factors in aging-induced bone loss.

Results: We report that bone mass and bone strength in GIP Tg(+) mice did not drop in a similar age-dependent fashion as in controls. In addition, biomarker measurements showed that GIP Tg(+) mice had increased osteoblastic activity compared with wildtype control mice. Finally, we report for the first time that BMSCs express GIPR, that the expression decreases in an age-dependent manner, and that stimulation of BMSCs with GIP led to increased osteoblastic differentiation.

Conclusions: Our data show that elevated GIP levels prevent age-related loss of bone mass and bone strength and suggest that age-related decreases in GIP receptor expression in BMSCs may play a pathophysiological role in this bone loss. We conclude that elevations in GIP may be an effective countermeasure to age-induced bone loss.

FIG. 1

Body weight and length are not different between Tg⁻ and Tg⁺ mice. Tg⁻ and Tg⁺ mice at 1, 3, 6, 9, 16, and 24 mo of age (n = 12/time point) had body weight and length measured longitudinally. There was no statistically significant difference between the body weights (A, ———) or lengths (A, - - -) between Tg⁻ and Tg⁺ mice. In percent body fat measurements (B), there was a significant difference between the Tg⁻ and Tg⁺ mice at 3 and 9 mo of age (^a p < 0.05; ^b p < 0.01).

FIG. 2

High GIP levels increased osteocalcin levels. Serum GIP levels in Tg⁺ and Tg⁻ mice were measured at 4, 12, and 24 mo of age (A, n = 12 mice/time point). GIP levels in the Tg⁺ mice were much higher than those in the Tg⁻ mice; the levels dropped with mouse age but still remained significantly higher than those of Tg⁻ mice (*p < 0.001). Osteocalcin, a serum marker for bone formation (B), and Pyd, a marker for bone breakdown (C), were measured (n = 12 mice/time point) to assess bone turnover. Tg⁺ mice had significantly higher osteocalcin levels than Tg⁻ mice at 6, 16, and 24 mo of age (^a p < 0.05; ^b p < 0.01). Pyd levels from Tg⁻ mice were significantly higher than those of Tg⁺ mice only at 6 mo of age (^b p < 0.01).

FIG. 3

Tg⁺ mice have higher bone mass than Tg⁻ mice. Total BMD (A) was measured in Tg⁺ and Tg⁻ mice at 1, 3, 6, 9, 16, and 24 mo of age (n = 12 mice/time point). Starting at 6 mo, Tg⁺ mice had significantly higher BMD than Tg⁻ mice. BMD was also measured at various sites including spine (B) and femur (C). There was no significant difference between Tg⁺ and Tg⁻ spinal BMDs at any age (n = 12 mice/time point). In contrast, femoral BMDs were significantly different between Tg⁺ and Tg⁻ mice starting at 6 mo of age, with the difference widening over time (^a p < 0.05; ^b p < 0.01).

FIG. 4

Multiple biomechanical measurements of Tg⁺ mice at 24 mo of age were significantly higher than those of Tg⁻ mice. At 12 mo of age, there were no significant differences between Tg⁻ and Tg⁺ mice in either ultimate force at break (A), ultimate stress (B), ultimate displacement (C), ultimate strain at break (D), work to failure at break (E), or modulus of toughness at break (F). However, at 24 mo of age, Tg⁺ mice had significantly higher ultimate force, displacement, strain, work to failure, and modulus of toughness measurements compared with Tg⁻ mice (n = 10 mice/time point; ^a p < 0.05; ^b p < 0.01).

FIG. 5

Osteoblast number is increased in Tg⁺ mice versus Tg⁻ mice at 24 mo of age. Micrographs shown are transverse sections of the distal femur from Tg⁺ and Tg⁻ mice showing increased osteoblastic number in the Tg⁺ (B) vs. Tg⁻ mice (A). These figures are representative sections from bones of four different mice in each group.

FIG. 6

GIPR expression decreases in an age-dependent manner and GIP stimulates BMSC differentiation into osteoblasts. (A) BMSCs were examined for GIP receptor transcript expression by real-time PCR. GIPR copy number normalized to 1 at 6 mo showed a decrease to 0.65 at 18 mo and 0.18 at 24 mo. Shown are means ± SE from three separate experiments, *p < 0.01; **p < 0.001. (B) BMSCs from 18-mo-old C57BL/6 mice were treated with osteogenic induction media (OS). Some of the cells were also stimulated with 1 nM GIP. Alkaline phosphatase staining was performed at day 7 after initiation of the treatment (n = 3). (C) BMSCs purified from 18-mo-old C57BL/6 mice were treated to induce bone nodule formation. Positively stained bone nodules were counted and expressed as means ± SE (*p < 0.01; n = 3).