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High glucose-6-phosphatase activity in osteoblasts in the metaphysis of femur of growing rats.

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THE ANATOMICAL RECORD 220:252-257 (1988)
High GIucose-6-Phosphatase Activity in Osteoblasts
in the Metaphysis of Femur of Growing Rats
HIROHIKO TOKUNAGA, SHINSUKE KANAMURA, JUN WATANABE,
KAZUO KANAI, AND MINORU SAKAIDA
Department of Anatomy and Orthopaedic Surgery, Kansai Medical University,
Fumizonecho 1, Moriguchi, Osaka, 570 Japan
ABSTRACT
Glucose-6-phosphatase (G6Pase) activity was examined cytochemically in the metaphysis of femurs of 3- and 7-day-old rats. G6Pase and hexokinase
activities were also examined biochemically in the femur and tibia of 3-day-old
animals. The reaction product for G6Pase activity was seen in the endoplasmic
reticulum and nuclear envelope of all cell types composing the metaphysis. The
amount of the reaction product was abundant in osteoblasts, moderate in osteocytes,
and moderate to scarce in osteoclasts and capillary endothelial cells. Biochemical
G6Pase activity in the bones was higher than that in the brain, submandibular
gland, or pancreas of the animals. Hexokinase activity in the bones was not different
from that in the submandibular gland, pancreas, or kidney. The activity ratio of
G6Pase and hexokinase in the bones (0.603) was greater than that in the submandibular gland, pancreas, or brain and smaller than that in the kidney. Possible
physiological significances of the higher G6Pase activity in osteoblasts are discussed.
were fixed with 2% glutaraldehyde containing 0.1 M
sodium cacodylate (pH 7.4) at 4°C for 1 hr and washed
in 0.1 M sodium cacodylate (pH 7.4) containing 0.2 M
ethylenediamine tetraacetate (EDTA) and 8% sucrose
for 1 h r at 4°C. EDTA was used for decalcification and
inhibiting alkaline phosphatase activity. The fixed slices
were sectioned at 30 pm with a freezing microtome and
washed in the buffer containing 0.2 M EDTA for 1h r at
4°C. The sections were preincubated in 0.25 M sucrose
containing 10 mM levamisole for 15 min and incubated
in a reaction medium (3.7 mM G6P, 3.6 mM lead nitrate,
30 mM sodium cacodylate, 230 mM sucrose, and 10 mM
levamisole, pH 6.7; Kanai et al., 1983, 1986; Shugyo et
al., 1986; Watanabe et al., 1983, 1986) a t room temperature for 1h r with a change of the medium. Levamisole
was used to inhibit alkaline phosphatase activity (van
Belle, 1972). The sections were postfixed in 1%buffered
osmium tetroxide (pH 7.4) for 2 h r a t 4"C, dehydrated,
and embedded in Spurr. Thin sections were stained with
uranyl acetate and lead citrate and examined in a JEM
100-S electron microscope.
To ascertain whether the reaction product is due to
G6Pase activity, control experiments (Tice and Barrnett,
1962; Kanamura, 1971a,b, 1975b) were carried out as
follows: The glutaraldehyde-fixed sections were incubated in the reaction medium lacking G6P; sections
were incubated in 0.1 acetate buffer (pH 5.0) for 30 min
at 37°C before incubation in the reaction medium; sections were incubated in a reaction medium containing
MATERIALS AND METHODS
a n equimolar amount of /3-glycerophosphate in place of
One hundred seventy-two male Wistar rats, 3 and 7 G6P; or sections were preincubated in 0.25 M sucrose
days old, were used. The animals were killed by
decapitation.
The osteoblast is a functionally active cell bearing
well-developed endoplasmic reticulum and participating
in the synthesis and secretion of the organic components
of the bone matrix. Generally, glucose-6-phosphatase
(G6Pase; E.C. 3.1.3.9; D-glucose-6-phosphate phosphohydrolase) activity is shown to be high in functionally
active cells, in which the endoplasmic reticulum is well
developed, such as hepatocytes (Tice and Barmett, 1962;
Ericsson, 1966; Kanamura, 1971a,b; Leskes et al., 1971)
and jejunal epithelial cells (Hugon et al., 1970, 1971).
The osteoblast is also supposed to provide storage sites
for calcium and phosphate, which are used in the calcification process (Matthews and Martin, 1971; Lehninger, 1977; Brighton and Hunt, 1974,1978; Boskey, 1981).
In vivo, G6Pase hydrolyzes glucose-6-phosphate (G6P)to
produce glucose and phosphate (Arion et al., 1972); the
enzyme is related to phosphate production. For these
reasons, it is of interest to examine whether G6Pase
activity is present in osteoblasts.
However, there have been no data in the literature on
G6Pase activity in osteoblasts. In the present study,
therefore, we examined in the metaphysis of femurs of
3- and 7-day-old rats whether G6Pase activity is high in
osteoblasts and whether the high activity in osteoblasts
decreases with the development into osteocytes that are
not concerned with the bone formation. Further, biochemical G6Pase and hexokinase activities were measured in the femur and tibia.
Cytochemical Methods
Metaphyses of the distal femurs were quickly removed
and sliced at about 1 mm with razor blades. The slices
0 1988 ALAN R. LISS, INC.
Received July 23, 1987; accepted August 31, 1987.
GGPASEIN OSTEOBLASTS
253
containing 10 mM NaF for 15 min and then incubated nate was mixed with an equal volume of 12N HCl, and
in the reaction medium containing an equimolar amount the pellet was dissolved in 2 ml of 12N HC1. Calcium in
of NaF.
an aliquot (0.1 ml) of the mixture or the solution was
measured by the Calcium C-test (Wako Pure Chemical).
Biochemical Methods
All biochemical data were subjected to statistical analBone shafts of bilateral femurs and tibias of ten ani- ysis (analysis of variance and Student’s t-test).
mals were used in one experiment, and five experiments
RESULTS
were done within an age group. Bone marrows were
Cytochemical Results
eliminated from bone shafts. The bone shafts were dissected into small pieces and homogenized in 20 mM TrisIn spite of the washing in the buffer containing 0.2 M
HC1 buffer (pH 7.6) containing 50 mM KC1,2 mMMgC12, EDTA, the preservation of the fine structures of cells
and 0.25 M sucrose in a Potter-Elvehjem homogenizer appeared generally satisfactory. The reaction product
at 4,000 revlmin for 2 min at 4°C. The homogenate was €or G6Pase activity was present in the endoplasmic recentrifuged at 3,OOOg for 10 min at 4°C. The 3,OOOg ticulum and nuclear envelope in all cell types composing
supernate was used for the measurement of G6Pase and the metaphysis of both 3- and 7-day-old animals: osteohexokinase activities (3-day-oldanimals).
blasts, osteocytes, osteoclasts, capillary endothelial cells,
Microsomal fractions were prepared according to the and fibroblasts. However, the amount of reaction prodmethod of Hogeboom et al. (1948). The 3,OOOg supernate uct was abundant in osteoblasts (Fig. l), decreased to
was saved, and the pellet was again homogenized and moderate with development into the typical osteocyte
centrifuged as above. The first and second supernates (Fig. 2), and moderate to scarce in osteoclasts (Fig. 41,
were combined and centrifuged at 8,OOOg €or 10 min at fibroblasts, and endothelial cells.
4°C. The supernate was then centrifuged at 12,OOOgfor
Deposition of final product was sometimes observed in
15 rnin at 4°C. The supernate was further centrifuged lysosomes and in the Golgi apparatus of osteocytes and
at 105,OOOg for 60 rnin at 4°C. The resultant pellet was osteoclasts (Fig. 2); on the plasma membrane facing the
washed with 0.15 M KC1, resuspended, and centrifuged free surface and in lysosomes and the Golgi apparatus
again at 105,OOOg for 60 rnin at 4°C. The pellet was of osteoblasts (Fig. 1);and on the plasma membrane of
suspended in 2 ml of 0.25 M sucrose and was used for endothelial cells and fibroblasts. Mitochondria in all cell
assay of G6Pase activity (3-and 7-day-oldanimals).
types showed no reaction product. No reaction product
For comparing levels of G6Pase and hexokinase activ- was seen in matrix vesicles.
ities in the bones with other organs, the 3,OOOg superOmission of G6P from the incubation medium resulted
nates were also made from the brain, submandibular in a complete absence of the reaction product. Immergland, pancreas, and kidney of 3-day-oldanimals.
sion of the fixed sections in 0.1 M acetate buffer (pH 5.0)
G6Pase activity was assayed according to the method before incubation in the reaction medium or use of 0of Leskes et al. (1971). An aliquot (0.1 ml) of the 3,OOOg glycerophosphate in place of G6P in the reaction mesupernate or microsomal fraction was incubated with 1 dium caused a loss of the reaction product except in
ml of the medium (30 mM G6P, 30 mM sodium cacodyl- lysosomes and in the Golgi apparatus and on the plasma
ate, pH 6.7). Levamisole (5 mM) was added to the reac- membrane (Fig. 3). Preincubation and incubation of the
tion medium to inhibit alkaline phosphatase activity. fixed sections with NaF abolished the total reaction, but
Incubation was done at 37°C for 30 min. The inorganic the final product was still visible on the plasma memphosphorus released was determined by a Phosphor C- brane and sometimes in the Golgi apparatus. These
test (Wako Pure Chemical, Osaka, Japan). Protein was results indicate that the reaction product in the endodetermined according to the method of Lowry et al. plasmic reticulum and nuclear envelope is due to G6Pase
(1951).The enzyme activity was expressed as nanomoles activity, but the deposition of final product in lysosomes
and in the Golgi apparatus and on the plasma memof G6P used per minute per milligram of protein.
Hexokinase activity was assayed according to the brane is probably related to acid or alkaline phosphatase
method of Joshi and Jagannathan (1966). An aliquot (0.1 activity.
ml) of the 3,OOOg supernate was mixed with 2.8 ml of
the medium containing 15 mM glucose, 10 mM MgC12,
Biochemical Results
40 mM Tris-HC1 buffer, 1.4 IU glucose-6-phosphate deG6Pase activity in the 3,OOOg supernate from the bones
hydrogenase, 0.01 mM EDTA and 0.3 mM 0-nicotinamide adenine dinucleotide phosphate (NADP), pH 7.4. of 3-day-old animals was 9.37 & 1.90 (nmol G6P used
Then, 0.1 ml of 3.3 mM adenosine triphosphate (ATP) per min per mg of protein; mean f S.D. for five experiwas added to the mixture, and the formation of NADP- ments), and this value was higher than values of the
reduced form was estimated at 30°C from the second to brain, submandibular gland, and pancreas, although it
the tenth minute after adding ATP. The enzyme activity was lower than the value of the kidney (Table 1).The
was expressed as nanomoles of G6P formed per minute activity in the microsomal fraction was 30.13 k 7.99 in
3-day-oldanimals and 36.73 k 3.17 in 7-day-oldanimals.
per milligram of protein.
Hexokinase activity (11.3 f 3.77 nmol G6P formed per
As an index showing whether an organ is inclined to
uptake glucose or to release glucose, the ratio of G6Pase min per mg of protein; mean f S.D. for five experiactivity and hexokinase activity at 37°C was calculated. ments) in the bones of 3-day-old animals was not differHexokinase activity was converted into the value at ent from values in the submandibular gland, pancreas,
and kidney, although it was lower than the value in the
37°C by the method of Long (1952).
To estimate the contamination of the bone matrix in brain.
The activity ratio of G6Pase and hexokinase was 0.603.
the 3,OOOg supernate, calcium was assayed in the 3,OOOg
supernate and 3,OOOg pellet. One milliliter of the super- This value was greater than that of the submandibular
Figs. 1-4. Cytochemical demonstration of GGPase activity in the
cells of the metaphyses of femurs of 3- and 7-day-old rats. Sections (30
pm) cut from glutaraldehyde-fixed tissues and washed in the buffer
containing 0.2 M EDTA were incubated for 1 hr in a medium modified
from that of Wachstein and Meisel (1956). x 16,000.
Fig. 1. A osteoblast from a 7-day-old animal. An abundant amount of
the reaction product for GGPase activity is seen in the nuclear envelope
and well-developed endoplasmic reticulum. The reaction product in
lysosomes and on the plasma membrane facing the free surface (the
upper side) is probably related to acid or alkaline phosphatase activity.
Fig. 2. Two osteocytes from a 3-day-old (a)and a 7-day-old(a) animal.
Note the decrease in the amount of reaction product for G6Pase activity in the endoplasmic reticulum. In addition, the endoplasmic reticulum decreases markedly in amount. The reaction product in lysosomes
is probably related to acid phosphatase activity.
255
G ~ P A SIN
E OSTEOBLASTS
TABLE 1. G6Pase and hexokinase activities in 3,OOOg supernates from the bone
and other organs of 3-day-old rats*
Brain
Bone
Submandibular
gland
Pancreas
Kidney
G6Pase
(37°C)
Hexokinase
(30°C)
Hexokinase
(37°C)'
G6PaseI
hexokinase
at
37°C
3.6 & 0.72
9.4 & 1.90
3.9 ?C 1.75
21.9 f 4.30
11.3 & 3.77
8.7 f 3.00
30.0 & 5.90
15.5 + 5.17
11.9 f 4.11
0.120
0.603
0.328
4.2 & 0.53
52.4 & 4.64
8.4 1.22
17.4 2 6.04
+
11.5 & 1.67
23.8 8.28
0.368
2.201
+
*Values are means f S.D. for five experiments (ten animals/experiment). Activities of G6Pase
and hexokinase are nmol G6P usedmidmg protein and nmol G6P formedmidmg protein,
respectively.
'The rate of G6P formed at 37°C was calculated by the method of Long (1952).
Fig. 3. Portion of an osteoblast from a 7-day-old animal. The fixed
section was incubated in the reaction medium containing an equimolar
amount of 0-glycerophosphate in place of G6P. Note disappearance of
Fig. 4. An osteoclast from a 7-day-old animal. A moderate amount of
the reaction product in the endoplasmic reticulum and nuclear envelope. The reaction product in the Golgi apparatus and a lysosome is the reaction product for G6Pase activity is seen in the endoplasmic
reticulum and nuclear envelope.
probably due to acid phosphatase activity.
clear envelope of osteoblasts in the metaphyses of femurs
of 3- and 7-day-old rats. Further, biochemical G6Pase
activities in the femur and tibia (except bone marrow)
were higher than values in the brain, submandibular
gland, and pancreas of the animals. The higher G6Pase
activity in osteoblasts is probably related to their
functions.
G6Pase has a wide spectrum of hydrolytic and synthetic activities (Nordlie, 1972). However, hydrolysis of
DISCUSSION
G6P is probably the sole function of this enzyme in vivo
As revealed in the present cytochemical results, an (Arion et al., 1972). The role of the enzyme in the liver
abundant amount of reaction product for G6Pase activ- and kidney is to release glucose into blood by hydrolyzity was present in the endoplasmic reticulum and nu- ing G6P produced via gluconeogenesis and glycogeno-
gland or pancreas, much greater than that of the brain,
and smaller than that of the kidney (Table 1).
Calcium content in the bones was 6.10 k 2.48 pglmg
of wet tissue (mean _+ S.D. for five experiments). Percentages of calcium recovered in the 3,OOOg pellet and
3,OOOg supernate were 94.7 + 1.7 and 5.3 k 1.7, respectively. Therefore, contamination of the bone matrix in
the 3,OOOg supernate was negligible.
256
H. TOKUNAGA ET AL.
lysis (Krebs, 1963; Nordlie, 1972). We postulated that
the role of relatively high activity in epididymal principal cells or in the seminal epithelium is to supply glucose into the epididymal fluid or fructose into the
seminal fluid (Kanai et al., 1981, 1983, 1986) and that
the role of the increased activity in skeletal muscle cells
of starved mice is to release glucose into the blood (Hirose et al., 1986; Sakaida et al., 1987). However, the role
of this enzyme in other various cell types containing low
or moderate activities is unknown, although we supposed a role of regulation of G6P concentration in the
cells, hydrolyzing if there is any excess (Kanamura,
1975a; Watanabe et al., 1983, 1986).
Osteoblasts probably consume a large amount of glucose from the blood for the synthesis of organic components of bone matrix. In the present results, hexokinase
activity in the bone was not different from that in the
submandibular gland or pancreas, which is considered
to be functionally active and to use glucose from the
blood abundantly (Martin, 1967; Hokin, 1967). Therefore, it is probable that G6P is steadily produced by the
hexokinase activity from the blood glucose in osteoblasts. The higher G6Pase activity possibly hydrolyzes
G6P thus produced, if there is any excess. Thus, a role
of the higher G6Pase in osteoblasts is possibly to regulate the intracellular concentration of G6P.
Phosphate that is thus produced in the well-developed
endoplasmic reticulum of osteoblasts may be abundant.
Some of such phosphate may be used for new calcification in the bone; the phosphate may diffuse freely to
new mineralization sites to increase regional phosphate
levels. As to the mechanism of how calcium-phosphate
particles in mitochondria of chondrocytes can influence
the formation of large amounts of a solid phase of calcium-phosphate in the extracellular tissue spaces,
Glimcher (1976) and Landis and Glimcher (1982) postulated as follows: Calcium and phosphate of mitochondrial particles may be released and then dissolved in the
extracellular fluid and the resulting increases in ion
concentrations produce a more metastable solution of
calcium and phosphate, a situation that facilitates the
heterogenous nucleation of solid phase calcium-phosphate particles by major connective tissue components
such as collagen. A similar mechanism is supposed for
the participation of calcium and phosphate in the matrix
vesicle for tissue calcification (Anderson, 1976). The
phosphate released by G6Pase activity from osteoblasts
may also serve for the calcification by a similar mechanism. However, the mechanism is postulated for the
initial step of calcification without any existing mineralization front, while considerable mineralization front,
which can serve as a nucleator of new crystals (Glimcher,
19761, already exists in the bone used in the present
study. Thus, the present results suggest that the endoplasmic reticulum in osteoblasts may play a role of phosphate production for new calcification in the bone.
During the process of development into osteocytes,
G6Pase activity decreased to a moderate level with a
decline in the amount of the endoplasmic reticulum.
Thus, GGPase activity decreases with the decline in
functional activity of the cell. The production of phosphate also probably becomes lower in osteocytes, which
are not concerned with the bone formation, than in
osteoblasts.
The activity ratio of G6Pase and hexokinase was 0.120
in the brain, and it was 2.201 in the kidney. The values
are consistent with the facts that the brain is a n organ
consuming much glucose (Sokoloff, 1960) and that the
kidney releases glucose into the blood (Nordlie, 1972).
The ratios (0.328 and 0.368) of the submandibular gland
and pancreas possibly show that these organs neither
consume much glucose nor release glucose into the blood.
However, the ratio in the bone was 0.603. This suggests
that the cells of the bone are more inclined to “release
glucose and phosphate” than the cells of the submandibular gland and pancreas.
G6Pase activity was moderate in osteocytes and moderate to scarce in osteoclasts and endothelial cells. The
role of the activity in these cells is also possibly to
regulate the intracellular concentration of G6P, as has
been postulated in various cell types containing low or
moderate activities (Kanamura, 1975a; Watanabe et al.,
1983, 1986).
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