Ablation of Hepatic Production of the Acid-Labile Subunit in Bovine-GH Transgenic Mice: Effects on Organ and Skeletal Growth

Abstract

Growth hormone (GH) and insulinlike growth factor 1 (IGF-1) are anabolic hormones that facilitate somatic and skeletal growth and regulate metabolism via endocrine and autocrine/paracrine mechanisms. We hypothesized that excess tissue production of GH would protect skeletal growth and integrity in states of reduction in serum IGF-1 levels. To test our hypothesis, we used bovine GH (bGH) transgenic mice as a model of GH hypersecretion and ablated the liver-derived acid-labile subunit, which stabilizes IGF-1 complexes with IGF-binding protein-3 and -5 in circulation. We used a genetic approach to create bGH/als gene knockout (ALSKO) mice and small interfering RNA (siRNA) gene-silencing approach to reduce als or igf-1 gene expression. We found that in both models, decreased IGF-1 levels in serum were associated with decreased body and skeletal size of the bGH mice. Excess GH produced more robust bones but compromised mechanical properties in male mice. Excess GH production in tissues did not protect from trabecular bone loss in response to reductions in serum IGF-1 (in bGH/ALSKO or bGH mice treated with siRNAs). Reduced serum IGF-1 levels in the bGH mice did not alleviate the hyperinsulinemia and did not resolve liver or kidney pathologies that resulted from GH hypersecretion. We concluded that reduced serum IGF-1 levels decrease somatic and skeletal growth even in states of excess GH.

Figure 1.

Genetic ablation of ALS in bGH mice. Pictures of (a) male mice and (b) female mice used in the study. Body weights of (c) male and (d) female mice followed up from 4 to 16 weeks of age. Liver expression of the Igf-1, als, Igfbp3, Ghr, and Insr genes in (e) male and (f) female mice at 16 weeks of age. Serum IGF-1 levels in (g) male and (h) female mice at 16 weeks of age. Serum levels of IGFBP-3 in 16-week-old (i) male and (j) female mice. Western immunoblotting of serum ALS in (k) male and (l) female mice at 16 weeks of age. Data are presented as mean ± standard error of the mean. Significance is accepted at P < 0.05 [(a) = vs. controls; (b) = vs. ALSKO mice; (c) = vs. bGH mice].

Figure 2.

Microscopic effects of serum IGF-1 reductions on kidney and liver of bGH mice. (a) Livers from WT mice had expected background levels of hepatocellular glycogen. Livers from (b) bGH and (c) bGH/ALSKO mice, as well as from mice administered siRNA targeting (d) ALS, (e) IGF-1, or (f) ALS/IGF-1, had hepatocellular hypertrophy with no differences between groups. (b–f) Black arrowheads indicate single-cell necrosis, and white arrowheads indicate mitotic figures. (g) Kidneys from WT mice had thin capillary loops containing erythrocytes in glomerular capillaries. Kidneys from (h) bGH and (i) bGH/ALSKO mice, as well as (j) mice administered siRNA duplexes ALS, had membranoproliferative glomerulonephropathy characterized by increased glomerular mesangium and hypercellularity. Mice administered siRNA targeting (k) IGF-1 or (l) ALS/IGF-1 had slightly increased severity of glomerulonephropathy compared with that of bGH mice. (h–l) Black arrowheads indicate glomerular lesions, and white arrowheads indicate tubules with basophilic, often vacuolated, cytoplasm and occasional casts.

Figure 3.

Effects of genetic ablation of ALS in bGH mice on skeletal morphology and mechanical properties. Femurs dissected from 16-week-old mice were analyzed by microCT (WT males, n = 13; ALSKO males, n = 10; bGH males, n = 9; bGH/ALSKO males, n = 14; WT females, n = 11; ALSKO females, n = 6; bGH females, n = 13; bGH/ALSKO females, n = 13). (a, b) Three-dimensional images of cortical and trabecular volumes at the mid-diaphysis and distal metaphysis, respectively, from male and female mice. (c, d) Total cross-sectional area (T.Ar), (e, f) bone area, (g, h) cortical bone thickness, (i, j) relative cortical bone area (RCA; Ct.Ar/T.Ar × 100), and (k, l) polar MMI of cortical bone taken at the femur mid-diaphysis in male and female mice. (m, n) Cortical bone mineral density. Mechanical properties of the femur were determined using a three-point bending assay (WT males, n = 7; ALSKO males, n = 6; bGH males, n = 5; bGH/ALSKO males, n = 10; WT females, n = 6; ALSKO females, n = 6; bGH females, n = 8; bGH/ALSKO females, n = 10). (o, p) Bone strength (maximal force), (q, r) bone stiffness (work to fracture), and (s, t) bone toughness (yield stress). Data are presented as mean ± standard error of the mean. Significance is accepted at P < 0.05 [(a) = vs. control; (b) = vs. ALSKO; (c) = vs. bGH]. NS, not significant.

Figure 4.

Effects of genetic ablation of ALS in bGH mice on serum osteocalcin, CTX, in vivo osteoclastogenesis, and gene expression in bone. (a, b) Serum osteocalcin levels (n > 8 per group). (c, d) Serum CTX levels (n > 6 per group). (e, f) Primary osteoclast (OC) cultures (n > 3 samples per group). (g, h) Gene expression in cortical bone shells. (i) Cathepsin K−positive cells/bone surface (n = 3 per group). Data are presented as mean ± standard error of the mean. Significance is accepted at P < 0.05 [(a) = vs. control; (b) = vs. ALSKO; (c) = vs. bGH]. CTSK, cathepsin K; NS, not significant.

Figure 5.

siRNA approach to silence Igf-1 and als gene expression in bGH mice. Body weights of (a) male and (b) female mice followed up from 4 to 11 weeks of age. Liver expression of the Igf-1, als, Igfbp3, Ghr, and Insr genes in (c) male and (d) female mice at 11 weeks of age. Serum IGF-1 levels in (e) male and (f) female mice at 11 weeks of age. Serum IGFBP-3 levels in (g) male and (h) female mice at 11 weeks of age. Western immunoblotting of serum ALS in (i) male and (j) female mice at 11 weeks of age. Data are presented as mean ± standard error of the mean. Significance is accepted at P < 0.05 [(a) = vs. bGH-PBS; (b) = vs. bGH-siRNA-ALS; (c) = vs. bGH-siRNA-IGF-1].

Figure 6.

Effects of siRNA silencing of Igf-1 and als gene expression on skeletal morphology and mechanical properties. Femurs dissected from 11-week-old mice were analyzed by microCT (bGH-PBS males, n = 9; bGH-siRNA-ALS males, n = 5; bGH-siRNA-IGF-1 males, n = 5; bGH-siRNA-ALS/IGF-1 males, n = 5; bGH-PBS females, n = 13; bGH-siRNA-ALS females, n = 8; bGH-siRNA-IGF-1 females, n = 5; bGH-siRNA-ALS/IGF-1 females, n = 5). (a, b) Three-dimensional images from male and female mice of cortical and trabecular volumes at the mid-diaphysis and distal metaphysis, respectively. (c, d) Total cross-sectional area; (e, f) bone area; (g, h) cortical bone thickness; (i, j) relative cortical bone area (RCA; Ct.Ar/T.Ar × 100); and (k, l) polar MMI of cortical bone taken at the femur mid-diaphysis in male and female mice. (m, n) Cortical BMD. Mechanical properties of the femur were determined using a three-point bending assay (bGH-PBS males, n = 5; bGH-siRNA-ALS males, n = 5; bGH-siRNA-IGF-1 males, n = 5; bGH-siRNA-ALS/IGF-1 males, n = 5; bGH-PBS females, n = 8; bGH-siRNA-ALS females, n = 8; bGH-siRNA-IGF-1 females, n = 5; bGH-siRNA-ALS/IGF-1 females, n = 5). (o, p) Bone strength (maximal force), (q, r) bone toughness (work to fracture), and (s, t) bone stiffness (yield stress). Data are presented as mean ± standard error of the mean. Significance is accepted at P < 0.05 [(a) = vs. bGH; (b) = vs. bGH-SiRNA-ALS; (c) = vs. bGH-SiRNA-IGF-1]. NS, not significant.