Anat Cell Biol.  2013 Dec;46(4):254-261. 10.5115/acb.2013.46.4.254.

Ascorbic acid insufficiency induces the severe defect on bone formation via the down-regulation of osteocalcin production

• Kim W
• Bae S
• Kim H
• Kim Y
• Choi J
• Lim SY
• Lee HJ
• Lee J
• Choi J
• Jang M
• Lee KE
• Chung SG
• Hwang YI
• Kang JS
• Lee WJ

• KMID: 2046766
• DOI: http://doi.org/10.5115/acb.2013.46.4.254

Abstract

The L-gulono-gamma-lactone oxidase gene (Gulo) encodes an essential enzyme in the synthesis of ascorbic acid from glucose. On the basis of previous findings of bone abnormalities in Gulo-/- mice under conditions of ascorbic acid insufficiency, we investigated the effect of ascorbic acid insufficiency on factors related to bone metabolism in Gulo-/- mice. Four groups of mice were raised for 4 weeks under differing conditions of ascorbic acid insufficiency, namely, wild type; ascorbic acid-sufficient Gulo-/- mice, 3-week ascorbic acid-insufficient Gulo-/- mice, and 4-week ascorbic acid-insufficient Gulo-/- mice. Four weeks of ascorbic acid insufficiency resulted in significant weight loss in Gulo-/- mice. Interestingly, average plasma osteocalcin levels were significantly decreased in Gulo-/- mice after 3 weeks of ascorbic acid insufficiency. In addition, the tibia weight in ascorbic acid-sufficient Gulo-/- mice was significantly higher than that in the other three groups. Moreover, significant decreases in trabecular bone volume near to the growth plate, as well as in trabecular bone attachment to the growth plate, were evident in 3- or 4-week ascorbic acid-insufficient Gulo-/-. In summary, ascorbic acid insufficiency in Gulo-/- mice results in severe defects in normal bone formation, which are closely related to a decrease in plasma osteocalcin levels.

Keyword

L-Gulonolactone oxidase; Ascorbic acid; Osteogenesis; Osteocalcin

MeSH Terms

Animals
Ascorbic Acid*
Down-Regulation*
Glucose
Growth Plate
L-Gulonolactone Oxidase
Metabolism
Mice
Osteocalcin*
Osteogenesis*
Plasma
Tibia
Weight Loss
Ascorbic Acid
Glucose
L-Gulonolactone Oxidase
Osteocalcin

Figure

• 

  Fig. 1 Mice weight at baseline and at the conclusion of the experiment. The weight of the wild-type (WT), ascorbic acid-sufficient Gulo-/- mice (Gulo-/-+AsA), and ascorbic acid-insufficient Gulo-/- mice (Gulo-/-) was measured at baseline and after 3 or 4 weeks of ascorbic acid withdrawal. (A) No significant differences in weight were observed among the groups at baseline. (B) Weight was significantly decreased in Gulo-/- mice after 4 weeks of ascorbic acid insufficiency compared with ascorbic acid-sufficient (-/-) mice. Data are presented as the means±SD, and each group included 5 animals. **P<0.01.

• 

  Fig. 2 Plasma levels of calcium and phosphorus after ascorbic acid withdrawal. Plasma levels of calcium (A) and phosphorus (B) were measured in 3-week (3w Gulo-/-) and 4-week (4w Gulo-/-) ascorbic acid-insufficient Gulo-/- mice. No significant differences were observed among the groups. The data are presented as the means±SD, and each group included 5 animals. WT, wild-type.

• 

  Fig. 3 The plasma levels of alkaline phosphatase and osteocalcin after ascorbic acid withdrawal. Plasma levels of alkaline phosphatase (A) and osteocalcin (B) were measured in 3-week (3w Gulo-/-) and 4-week (4w Gulo-/-) ascorbic acid-insufficient Gulo-/- mice. (A) The plasma level of alkaline phosphatase was not significantly different among the groups. (B) The plasma level of osteocalcin was significantly lower in 3w Gulo-/- mice than in wild-type (WT) and ascorbic acid-sufficient Gulo-/- mice (Gulo-/-+AsA). Osteocalcin was undetectable in the plasma of 4w Gulo-/- mice. Data are presented as the means±SD, ND, not detected, each group included 5 animals. **P<0.01.

• 

  Fig. 4 Tibia dry weight and the ratio of the tibia weight to the final body weight at the end of the experiment. Tibia dry weight was measured in 3-week (3w Gulo-/-) and 4-week (4w Gulo-/-) ascorbic acid-insufficient Gulo-/- mice. Tibia dry weight (A) and ratio of the tibia weight to the final body weight (B) was significantly higher in the ascorbic acid-sufficient Gulo-/- mice (Gulo-/-+AsA) than in the other three groups. Data are shown as the means±SD, each group included 5 animals. *P<0.05, **P<0.01.

• 

  Fig. 5 The proximal metaphysis and growth plate of the proximal tibia after ascorbic acid withdrawal. Mouse tibias from 3-week (3w Gulo-/-) and 4-week (4w Gulo-/-) ascorbic acid-insufficient Gulo-/- mice were harvested, fixed, decalcified, sectioned at 4 µm and stained with H&E. Compared with the wild-type (WT) (A, E) and ascorbic acid-sufficient Gulo-/- mice (Gulo-/-+AsA) (B, F), 3w Gulo-/- (C, G), 4w Gulo-/- (D, H) mice had decreased trabecular bone volume near the growth plate, and decreased trabecular bone attachment to the growth plate. Scale bars=400 µm (A-D), 100 µm (E-H).

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