The Resistance of Cortical Bone Tissue to Failure under Cyclic Loading is Reduced with Alendronate

Bisphosphonates are the most prescribed preventative treatment for osteoporosis. However, their long-term use has recently been associated with atypical fractures of cortical bone in patients who present with low-energy induced breaks of unclear pathophysiology. The effects of bisphosphonates on the mechanical properties of cortical bone have been exclusively studied under simple, monotonic, quasi-static loading. This study examined the cyclic fatigue properties of bisphosphonate-treated cortical bone at a level in which tissue damage initiates and is accumulated prior to frank fracture in low-energy situations. Physiologically relevant, dynamic, 4-point bending applied to beams (1.5 mm × 0.5 mm × 10 mm) machined from dog rib (n=12/group) demonstrated mechanical failure and micro-architectural features that were dependent on drug dose (3 groups: 0, 0.2, 1.0 mg/kg/day; Alendronate [ALN] for 3 years) with cortical bone tissue elastic modulus (initial cycles of loading) reduced by 21% (p<0.001) and fatigue life (number of cycles to failure) reduced in a stress-life approach by greater than 3-fold with ALN1.0 (p<0.05). While not affecting the number of osteons, ALN treatment reduced other features associated with bone remodeling, such as the size of osteons (−14%, ALN1.0: 10.5±1.8, VEH: 12.2±1.6, ×103 µm2; p<0.01) and the density of osteocyte lacunae (−20%; ALN1.0: 11.4±3.3, VEH: 14.3±3.6, ×102 #/mm2; p<0.05). Furthermore, the osteocyte lacunar density was directly proportional to initial elastic modulus when the groups were pooled (R=0.54, p<0.01). These findings suggest that the structural components normally contributing to healthy cortical bone tissue are altered by high-dose ALN treatment and contribute to reduced mechanical properties under cyclic loading conditions

Growth Hormone Protects Against Ovariectomy-Induced Bone Loss in States of Low Circulating Insulin-like Growth Factor (IGF-1)*

Early after estrogen loss in postmenopausal women and ovariectomy (OVX) of animals, accelerated endosteal bone resorption leads to marrow expansion of long bone shafts that reduce mechanical integrity. Both growth hormone (GH) and insulin-like growth factor (IGF-1) are potent regulators of bone remodeling processes. To investigate the role of the GH/IGF-1 axis with estrogen deficiency, we used the liver IGF-1-deficient (LID) mouse. Contrary to deficits in controls, OVX of LID mice resulted in maintenance of cortical bone mechanical integrity primarily owing to an enhanced periosteal expansion affect on cross-sectional structure (total area and cortical width). The serum balance in LID that favors GH over IGF-1 diminished the effects of ablated ovarian function on numbers of osteoclast precursors in the marrow and viability of osteocytes within the cortical matrix and led to less endosteal resorption in addition to greater periosteal bone formation. Interactions between estrogen and the GH/IGF-1 system as related to bone remodeling provide a pathway to minimize degeneration of bone tissue structure and osteoporotic fracture. © 2010 American Society for Bone and Mineral Researc

Chronic High Fructose Intake Reduces Serum 1,25 (OH)2D-3 Levels in Calcium-Sufficient Rodents

Excessive fructose consumption inhibits adaptive increases in intestinal Ca2+ transport in lactating and weanling rats with increased Ca2+ requirements by preventing the increase in serum levels of 1,25(OH)(2)D-3. Here we tested the hypothesis that chronic fructose intake decreases 1,25(OH)(2)D-3 levels independent of increases in Ca2+ requirements. Adult mice fed for five wk a high glucose-low Ca2+ diet displayed expected compensatory increases in intestinal and renal Ca2+ transporter expression and activity, in renal CYP27B1 (coding for 1 alpha-hydroxylase) expression as well as in serum 1,25(OH)(2)D-3 levels, compared with mice fed isocaloric glucose- or fructose-normal Ca2+ diets. Replacing glucose with fructose prevented these increases in Ca2+ transporter, CYP27B1, and 1,25(OH)(2)D-3 levels induced by a low Ca2+ diet. In adult mice fed for three mo a normal Ca2+ diet, renal expression of CYP27B1 and of CYP24A1 (24-hydroxylase) decreased and increased, respectively, when the carbohydrate source was fructose instead of glucose or starch. Intestinal and renal Ca2+ transporter activity and expression did not vary with dietary carbohydrate. To determine the time course of fructose effects, a high fructose or glucose diet with normal Ca2+ levels was fed to adult rats for three mo. Serum levels of 1,25(OH)(2)D-3 decreased and of FGF23 increased significantly over time. Renal expression of CYP27B1 and serum levels of 1,25(OH)(2)D-3 still decreased in fructose-compared to those in glucose-fed rats after three mo. Serum parathyroid hormone, Ca2+ and phosphate levels were normal and independent of dietary sugar as well as time of feeding. Thus, chronically high fructose intakes can decrease serum levels of 1,25(OH)(2)D-3 in adult rodents experiencing no Ca2+ stress and fed sufficient levels of dietary Ca2+. This finding is highly significant because fructose constitutes a substantial portion of the average diet of Americans already deficient in vitamin D

Expression of 1,25(OH)₂D₃ metabolizing enzymes and of Ca²⁺transporters in the kidney of mice fed for three mo a high fructose, starch, or glucose diet containing normal Ca²⁺ levels.

<p>(A) Marked effects of excessive fructose intake on mRNA expression levels of renal 1,25(OH)₂D₃ metabolic enzymes, CYP27B1 and CYP24A1. (B) mRNA expression levels of renal TRPV6, CaBP28k, CaBP9k and PMCA1. All expression data were analyzed by real-time PCR using <i>EF1</i>α as a reference and normalized relative to levels seen in mice fed glucose diet. Data are means ± SEM (<i>n</i> = 5 per group). Differences (<i>P</i><0.05) among means are indicated by differences in superscript letters, as analyzed by 1-way ANOVA LSD. Thus, within a gene of interest, bars with superscript “a” are > bars with “b”. Chronic consumption of high fructose levels has dramatic effects on mRNA expression of renal 1,25(OH)₂D₃ metabolizing enzymes but not on Ca²⁺ transporter mRNA expression.</p

Fructose-fed mice demonstrate an inhibition of compensation for intestinal Ca²⁺transport rate and transporter expression induced by dietary Ca²⁺-deficiency.

<p>(A) Ca²⁺ transport was measured using everted duodenal sacs from mice fed diets containing either 43% glucose or fructose, in combination with either normal or low Ca²⁺. Active transepithelial Ca²⁺ transport from the luminal to the basolateral compartment was expressed as a ratio of the final quantity of ⁴⁵Ca²⁺ inside/outside of the everted gut sacs. (B) mRNA expression levels of intestinal Ca²⁺ (TRPV6, CaBP9k, PMCA1) transporters. All expression data were analyzed by real-time PCR using <i>EF1</i>α as a reference and then normalized to levels in mice fed a normal Ca²⁺-glucose diet (Nor Ca²⁺-G). Data are means ± SEM (<i>n</i> = 6 per group). Differences (<i>P</i><0.05) among means are indicated by differences in superscript letters, as analyzed by 1-way ANOVA LSD. Thus, within a gene of interest, bars with superscript “a” are > bars with “b” which in turn are > bars with “c”. Dietary fructose inhibits compensatory increases in Ca²⁺ transporter activity and expression induced by dietary Ca²⁺-deficiency.</p

Effect of chronic fructose feeding on renal expression of 1,25(OH)₂D₃ metabolizing enzymes and of Ca²⁺transporters.

<p>Rats were fed for three mo a normal Ca²⁺ diet containing either 43% glucose or fructose. (<b>A</b>) mRNA expression level of renal 1,25(OH)₂D₃ metabolic enzymes, CYP27B1 and CYP24A1 (<b>B</b>) The protein abundance of CYP27B1 and CYP24B1 using β-actin as a reference. (<b>C</b>) mRNA expression levels of renal TRPV6, CaBP28k, CaBP9k and PMCA1. All expression data were analyzed by real-time PCR using <i>EF1</i>α as a reference and normalized relative to levels seen in rat fed glucose diet. Data are means ± SEM (<i>n</i> = 6 per group). Differences (<i>P</i><0.05) between means are indicated by asterisks. Chronic consumption of high fructose diets reduces mRNA and protein expression of CYP27B1.</p

Fructose inhibits compensatory increases in renal expression of the 1,25(OH)₂D₃ metabolizing enzyme CYP27B1 induced by dietary Ca²⁺deficiency in mice.

<p>(A) mRNA expression levels of renal CYP27B1 (1α-hydroxylase) and CYP24A1 (24-hydroxylase). (B) The protein abundance of CYP27B1 and CYP24A1, using β-actin as a reference. (C) mRNA expression levels of renal TRPV6, CaBP28k, CaBP9k and PMCA1. Nor  =  normal; G  =  glucose; F  =  fructose. All mRNA expression level data were analyzed by real-time PCR using <i>EF1</i>α as a reference and normalized to levels in mice fed glucose and normal Ca²⁺ diet. Data are means ± SEM (<i>n</i> = 5–6 per group). Differences (<i>P</i><0.05) among means are indicated by differences in superscript letters, as analyzed by 1-way ANOVA LSD. Thus, within a gene of interest, bars with superscript “a” are > bars with “b” which in turn > bars with “c”. Dietary fructose inhibits compensatory increases in renal expression of Ca²⁺ transporters and 1,25(OH)₂D₃ metabolizing enzymes induced by dietary Ca²⁺-deficiency.</p

Effects of age on expression of intestinal Ca²⁺ transporters and of renal 1,25(OH)₂D₃ metabolic enzymes in mice.

<p>A comparison of the expression levels of Ca²⁺ transporters (<b>A</b>) and 1,25(OH)₂D₃ metabolic enzymes (<b>B</b>) in four mo old mice after three mo of feeding on normal Ca²⁺ diet containing 63% glucose (from study 2) and in two mo old mice fed a normal Ca²⁺ diet containing 43% glucose for five wk (from study 1). All expression data were analyzed by real-time PCR using <i>EF1</i>α as a reference and normalized relative to levels seen in four mo old mice. Data are means ± SEM (<i>n</i> = 5–6 per group). Differences (<i>P</i><0.05) between means are indicated by asterisks. Expression of intestinal Ca²⁺ transporters decreases with age.</p

Time course of the fructose-induced reduction in serum levels of 1,25-(OH)₂D₃ in adult rats fed for three mo a normal Ca²⁺ diet with either glucose or fructose.

<p>Glucose or Fructose = 43%. FGF  =  fibroblast growth factor, PTH  =  parathyroid hormone. <i>n</i> = 6 per group. Data are means ± SEM. Means with different superscript letters are significantly different from others in the same row (<i>P</i><0.05 by <i>posthoc</i> LSD test).</p

Chronic consumption of fructose has no significant effect on intestinal Ca²⁺ transport rate and transporter mRNA expression in mice fed for three mo a high fructose, starch, or glucose diet containing normal Ca²⁺ levels.

<p>(A) Active transduodenal Ca²⁺ transport from the luminal to the basolateral compartment was expressed as a ratio of the final quantity of (⁴⁵Ca²⁺ inside/⁴⁵Ca²⁺ outside) of the everted sacs of mice fed diets containing 63% fructose, glucose or starch. (B) mRNA expression levels of intestinal Ca²⁺ (TRPV6, CaBP9k, PMCA1) transporters. All expression data were analyzed by real-time PCR using <i>EF1</i>α as a reference and normalized relative to levels seen in mice fed glucose diet. Data are means ± SEM (<i>n</i> = 5 per group). Chronic consumption of high fructose levels has no significant effect on intestinal Ca²⁺ transport rate and transporter mRNA expression.</p