03-PX-Anabolic-FSH

J Bone Miner Metab (2000) 18:2–8
Review article
© Springer-Verlag 2000
The pancreas: A storehouse of protein anabolic factors
and bone/calcium metabolism-regulating factors
Yoshito Takaoka 1 and Toshiyuki Yoneda 2
1
Emeritus Professor of Nagasaki University, Nagasaki, Japan
2
Department of Biochemistry, Osaka University Faculty of Dentistry, 1-8 Yamadaoka, Suita 565-0871, Japan
Key words: pancreas, salivary glands, protein anabolism,
bone/calcium metabolism, elastase
Classical pancreas extract
Salivary glands/pancreas compensation theory
In 1947, Y. Takaoka, then in the Third Department
of Internal Medicine, Tokyo University School of
Medicine, noticed for the first time that many diabetic
patients manifested hypertrophy of their parotid glands.
Of interest, there was a positive correlation between the
extent of hypertrophy in these patients and their general
physical condition [1–3] (Fig. 1). Although several
earlier reports had described the occurrence of parotid
gland hypertrophy in some endocrine diseases [3–5], the
significance of this observation in light of the pathophysiology
of diabetes was unclear. Later, Takaoka
confirmed that 126 of 150 (80%) diabetic patients had
parotid gland hypertrophy [3], and the weight of the
gland determined at autopsy was greater in diabetic
patients (11 patients; average, 21.2g) than normal
individuals (100 subjects; average, 17.2g) [2,3].
Subsequently, Takaoka demonstrated that intravenous
administration of salivary gland extracts from
alloxan-induced diabetic dogs caused more than 20%
decrease in blood sugar levels in rabbits in a similar
manner to insulin. In contrast, extracts from the liver
of diabetic dogs or salivary glands from healthy dogs
did not show such an effect [2,4]. These clinical and
experimental results collectively led Takaoka to propose
that the salivary glands (parotid glands) possess
compensatory functions for hypofunction of the pancreas
[2,3].
Offprint requests to: T. Yoneda
Received: June 19, 1999
Consistent with these observations, malnourished
Japanese and German POWs who were released from
prisons in Siberia also demonstrated parotid gland
hypertrophy [3,5,6]. Likewise, Takizawa and Murata [7]
also found at autopsy that Japanese civilian prisoners
who died of malnutrition had hypertrophy in the anabolic
organs such as pancreas and parotid glands and
atrophy in catabolic organs including pituitary, thyroid,
and adrenal glands. From these observations, they
hypothesized that these apparent organ changes under
malnutrition conditions might reflect reactions of the
human body to survive. Consequently, Takaoka [8]
postulated that salivary glands may play some role in
protein metabolism as well as in glucose metabolism.
This assumption was supported by the finding that
sialoadenectomy caused a profound decrease in serum
A/G ratio in dogs and rabbits. The result suggests that
the salivary glands may control serum A/G ratio in an
endocrine fashion.
Parotin and protein anabolism
Preceding these studies by Takaoka, Drs. Tomosaburo
and his brother Akira Ogata, who were both Emeritus
Professors of Tokyo University, had been studying the
physiological role of the parotid glands. After enormous
effort, they succeeded in purifying a protein from
bovine parotid gland extracts to homogeneity using the
Tiselius method and named it parotin [9]. Parotin
showed an ability to regenerate the periodontal tissues
of sialodectomized dogs. They also demonstrated that
parotin caused hypocalcemia in rabbits and promoted
the formation of bone and cartilage in dogs [10]. From
these findings, Ogata proposed the hypothesis that the
parotid gland is an endocrine organ [11].
Based on the idea of an endocrine role for the salivary
glands in the regulation of protein metabolism,
Takaoka examined the activity of highly purified
parotin. He found that it not only decreased ionized

Y. Takaoka and T. Yoneda: Pancreas: a storehouse of factors 3
Fig. 1. Hypertrophy of the parotid
glands in diabetic patients. Left, 60-
year-old woman; right, 68-year-old
man [6]
blood calcium, total amino acids, and total protein but
also increased blood A/G ratio and leukocyte number
in rabbits. These findings suggest that parotin had an
action equivalent to a growth hormone. From these
results together with other data that are not described
here, Takaoka suggested that the primary physiological
action of parotin might be associated with stimulation
of protein anabolism and, possibly, oxidative phosphorylation
[12].
Because parotin was shown to decrease urinary creatine
in rabbits, an attempt was made to administer
parotin to patients with progressive muscular dystrophy
(PMD) that was characterized by creatinuria. Although
parotin was not effective in patients with Duchenne and
limb girdle-type PMD (the first and second most severe
types of PMD, respectively), it unexpectedly and dramatically
cured one patient with refractory myasthenia
gravis (MG) and later three MG patients who also
exhibited creatinuria [3,13].
Pancreas extract (PX)
According to his parotid gland–pancreas compensation
theory [2], Takaoka began in 1952 to attempt to isolate
a more potent protein anabolic hormone-like factor
than parotin from pancreas (hereinafter called PX, for
pancreas extract) [6,14,15] for treating PMD. Since
then, 46 scientists have collaborated in this project
(until 1998), innovated diverse techniques to purify PX,
and made invaluable contributions to advance this
study. In the early stages of PX purification, PX activity
was monitored for its capacity to decrease ionized blood
calcium, blood urea nitrogen (BUN), total protein, and
amino acids and to increase white blood cell number in
rabbits. These time-consuming and cumbersome studies
were primarily conducted by Takaoka, then professor
and Chairman of the First Department of Internal
Medicine at Nagasaki University Schools of Medicine,
and his collaborators in the Third Department of
Internal Medicine at Tokyo University School of
Medicine [1,2,8]. Initial problems in PX purification
were overcome after they introduced acetone powder
of porcine pancreas for purification. The PX prepared
using this method showed several intriguing effects. In
1964, an inpatient with severe MG (a 26-year-old
woman) at Nagasaki University Hospital who had been
unable to move for almost 4 years was cautiously given
intramuscular injections of PX that had powerful activity
to decrease BUN and Ca 2 . The patient exhibited
marked improvement in her performance in response to
PX within a week [16] and became able to return to her
home only 1 month later. Being encouraged by this
case, six additional MG patients were treated with PX
and all showed remarkable recovery from their disease.
Furthermore, a patient (a 37-year-old woman) with
facioscapulohumeral (FSH) PMD who volunteered
to be treated with PX also demonstrated notable responses
to PX (Fig. 2) [17–19]. In this case, it was noted
that the patient not only became able to raise her legs
but also gained weight. In fact, Takaoka noticed that the

4 Y. Takaoka and T. Yoneda: Pancreas: a storehouse of factors
Fig. 2. Effects of pancreas extract
(PX) on facioscapulohumeral
progressive muscular dystrophy
(FSH PMD) patient (37 years
old). Left upper panel, 7 months
after treatment. Left lower panel,
before treatment. Right upper
panel, 10 months after treatment.
Right lower panel, 8 months after
treatment. Note that the patient
became able to raise her legs and
also significantly gained weight
after PX treatment [18]
Fig. 3. Effects of PX on forced swimming
test. Mice (ddy, male, 20–30 g)
were treated with either NaCl or PX
(3mg/kg, ip, 90 min before test). Mice
administered with PX could swim
significantly longer than control mice.
Numbers in parentheses indicate number
of mice studied [14]
majority of patients who were treated with PX showed
an increase in their appetite.
The calcium-lowering action of PX was another
beneficial effect. In one case of synovial sarcoma of the
knee joint associated with hypercalcemia, PX decreased
calcium in a manner similar to salmon calcitonin [6].
This biological property of PX suggested that PX might
play a role in regulating bone/calcium metabolism.
Consistent with these clinical results, PX was shown
to prolong the survival of dystrophic mice [17], extend
forced swimming time in mice (Fig. 3) [14], and increase
RNA synthesis in cultured cells [20]. Histochemical
study also revealed an increased RNA synthesis around
the nuclei of skeletal muscles in rats [18].
In 1967, a new technique of parotin purification from
bovine parotid glands was established. This parotin was
named MP parotin. Of interest, immunohistochemical
studies using 125 I-labeled anti-MP-parotin antibodies
revealed much stronger expression of the protein in
Langerhans’ islets in the pancreas than in the parotid
gland, suggesting the presence of an MP-patotin-like
substance in pancreas [21]. Takaoka, therefore, applied
this new technique for PX purification and observed
that the PX prepared using this technique had more
consistent and potent effects on serum ionized calcium
than PX prepared by the old technique. Takaoka then
began to collaborate with Suzuki and Yoneda in the
Department of Biochemistry at Osaka University

Y. Takaoka and T. Yoneda: Pancreas: a storehouse of factors 5
Faculty of Dentistry early in 1981. They decided to
focus on the calcium-lowering action of PX based on
this observation and established an in vitro bioassay
that assesses the capacity of PX to inhibit parathyroid
hormone- (PTH-) stimulated bone resorption in organ
cultures of 45 Ca-labeled fetal rat long bones [22]. Introduction
of this assay markedly facilitated purifying the
calcium-lowering activity of PX. Moreover, Kikkawa
independently found that this newly prepared PX
promoted migration of corneal epithelial cells in a
similar manner to epidermal growth factor in vitro
(unpublished observation).
New generation PX
Potential role of pancreas in bone/calcium metabolism
At the present time, it is widely recognized that bone,
intestine, and kidney are the organs that maintain calcium
homeostasis by regulating bone/calcium metabolism
in cooperation with PTH, 1,25-dihydroxyvitamin
D 3 (1,25-D 3 ), and calcitonin (CT). However, the observations
described earlier suggest that the pancreas
could be another organ that influences bone/calcium
metabolism. There are several lines of evidence suggesting
that pancreas may be involved in the regulation
of bone/calcium metabolism [23–29]. With this background
information, we attempted to identify a molecule
that plays a role in the regulation of bone/calcium
metabolism in the pancreas.
Effects of PX on blood calcium levels and
bone resorption
We showed that systemic administration of PX that
was prepared according to the methods described by
Takaoka et al. [2] reproducibly decreased blood ionized
calcium levels in normal rabbit and mice. PX also
inhibited bone resorption, which was stimulated by
PTH, interleukin-1α, and 1,25-D 3 [30] in organ cultures
of fetal rat long bones. Inhibition of bone resorption
by PX was caused by inhibition of osteoclast formation
[30]. These data confirmed Takaoka’s previous
data.
Partial purification of PX by anion exchange, followed
by size exclusion column chromatography
Acetone powder of porcine pancreas was processed
through a DE-52 anion exchange column and subsequently
Sephacryl S-200 HR columns. Fractions eluted
between 25 and 43 KDa demonstrated an activity that
promoted DNA synthesis in MG-63 human osteoblastic
osteosarcoma cells and inhibited 1,25-D 3 -stimulated
bone resorption. This partially purified PX was approximately
200 times more potent than crude PX in hypocalcemic
activity in normal mice.
Effects of partially purified PX on cancer-associated
hypercalcemia
PX was tested in nude mice bearing a human squamous
cell carcinoma (MH-85) [31] that causes hypercalcemia
with no bone metastases and cachexia with anorexia
and also increases osteoclastic bone resorption [32].
In these tumor-bearing nude mice, PX prevented the
progression of hypercalcemia and inhibited osteoclastic
bone resorption [33]. Moreover, PX inhibited
osteoclast-like cell formation and bone resorption that
had been stimulated by conditioned medium of MH-85
cells. Interestingly, PX increased food intake, decreased
weight loss, and prevented the development of cachexia
[33]. Eventually, PX profoundly prolonged survival of
MH-85-tumor-bearing nude mice [33]. Takaoka also
found that PX prolonged the survival of irradiated mice
(unpublished observation). Thus, PX may potentially
be a beneficial agent for hypercalcemia and cachexia
associated with malignancy.
Purification of PX to homogeneity
PX was purified to homogeneity by successive steps
including anion exchange chromatography on DE-52,
isoelectric focusing on Miniphor, and reverse-phase
HPLC on a C 18 column. The purified material was
eluted as a single protein peak at 50% acetonitrile in
0.1% trifluoroacetic acid on the C 18 column and showed
promotion of DNA synthesis in MG-63 cells and inhibition
of 1,25-D 3 -stumulated bone resorption. Silver
staining on SDS-PAGE demonstrated that this protein
peak migrated as a single band at approximately
28 KDa. Amino acid sequence analysis of the 28-KDa
protein revealed 32 amino acids from the N-terminus
that had 92% homology with human elastase III B [34]
as previously reported [35].
Effects of recombinant human elastase IIIB (rhEIIIB)
on bone resorption and blood calcium levels
RhEIIIB markedly inhibited 1,25-D 3 -stimulated bone
resorption and pit formation by isolated osteoclasts.
These effects were blocked by polyclonal antibodies to
rhEIIIB. RhEIIIB also decreased blood ionized calcium
levels, which were increased by local administration
of interleukin-1 in normal mice [34]. These results
clearly demonstrate that the bone/calcium metabolismregulating
activity of PX is accounted for by elastase
IIIB, and thus, PX is distinct from known bone/calciumregulating
hormones and cytokines such as CT, amylin,
glucagon, and transforming growth factor-β.

6 Y. Takaoka and T. Yoneda: Pancreas: a storehouse of factors
Table 1. Effects of pancreas extract (PX) on bone/calcium
metabolism
In vitro
Stimulates proliferation and differentiation of
osteoblast-like cells
Increases bone nodule formation
Inhibits osteoclastic bone resorption
Decreases osteoclast formation
In vivo
Decreases serum calcium in normal mice
Inhibits bone resorption
Stimulates bone formation
In tumor-bearing mice with hypercalcemia:
Reverses calcium levels
Promotes appetite
Decrease loss of body weight
Prolongs survival
Effects of rhEIIIB on bone formation in vitro and in
vivo
We have recently found that repeated subcutaneous
injections of rhEIIIB on mouse calvariae induced new
bone formation and that treatment of fetal rat calvarial
cells with rhEIIIB increased bone nodule formation in
vitro (manuscript in preparation).
The effects of PX on bone/calcium metabolism are
summarized in Table 1.
in turn modulates the proliferation, differentiation, and
function of osteoblasts and osteoclasts.
Some elastases, such as endogenous vascular elastase
[44], are reported to have a homology with adipsin, of
which expression is correlated with the differentiation of
adipocytes [45]. It is notable that adipocytes differentiate
from mesenchymal stem cells, which also differentiate
into osteoblasts [46]. Thus, adipsin might be involved in
the regulation of differentiation of osteoblasts as well as
adipocytes. If this is the case, elastase might also play a
role in osteoblastic differentiation as well.
Recently, a unique cell-surface receptor that mediates
responses of platelets to a serine protease thrombin
has been identified [47]. This receptor, which has seven
transmembrane-spanning structures, is activated by
proteolytic cleavage of the N-terminus at its tethered
end by thrombin. It thus is called protease-activated
receptor. Of note, expression of thrombin receptors has
recently been found in bone [48]. Moreover, thrombin is
shown to stimulate bone resorption and elicit a variety
of cellular responses in osteoblasts [47]. Because the
osteotropic activity of PX is at least in part accounted
for by elastase IIIB, which also belongs to the serine
protease family, it is plausible to speculate that osteoblasts
and osteoclasts might possess receptors that
are activated by elastase IIIB. PX might exhibit its
biological effects through these receptors.
Mechanisms of EIIIB effects on bone/calcium
metabolism
Mechanisms by which EIIIB affects bone/calcium metabolism
are unknown at the present time. The enzyme
catalytic activity may be essential for exertion of its
inhibitory effect on bone resorption because protease
inhibitors markedly impaired bone resorption-inhibiting
activity (unpublished observation). On the other
hand, Tomomura et al. [36–38], who independently
identified a calcium-lowering molecule named caldecrin
that is homologous to elastase IV in rat pancreas, reported
that a protease inhibitor (PMSF) did not diminish
the activity. Thus, the requirement of enzyme
catalytic activity of elastase for osteotropic activity is yet
to be elucidated.
Elastase-like serine proteases have recently been
found to generate soluble mature form of transforming
growth factor-α (TGF-α) by cleavage of two alanine/
valine sequences located at positions 38–39 and 88–89 in
membrane-bound pro-TGF-α (39–41). Furthermore, it
has also been described that elastase releases extracellular
matrix-bound TGF-β 1 [42] and basic fibroblast
growth factor [43]. Thus, elastases may cleave or release
membrane-anchored or matrix-bound cytokines and
growth factors and increase the local concentrations of
these molecules in the bone microenvironment, which
Concluding summary
In this review article, we have described diverse effects
of pancreatic factor PX, including long-standing large
bodies of work by Takaoka, who has been characterizing
PX over the last half-century. Although the precise
molecular mechanisms by which PX shows these diverse
effects are unclear, it is likely that PX has protein
anabolic effects that evidently cured some patients with
PMD and MG. Whatever the mechanisms are, these
data of Takaoka strongly suggest that the pancreas is
the organ that participates in the regulation of protein
metabolism. His data also suggest that the pancreas
contains yet unknown additional protein anabolic factors
that are involved in controlling BUN, total protein,
and white blood cell count. Further studies are required
for identifying these factors.
Very little has been studied about the role of the
pancreas in the regulation of bone and calcium metabolism
to date. Progress of state-of-the-art techniques for
protein purification and molecular biology has enabled
us to demonstrate that pancreas produces a molecule
that modulates bone/calcium metabolism. This molecule
turns out to be elastase IIIB, which belongs to the
serine protease family. These results suggest that the
pancreas as well as bone, kidney, and intestine also

Y. Takaoka and T. Yoneda: Pancreas: a storehouse of factors 7
plays a role in regulating bone/calcium metabolism. It is
expected that the study of PX deepens our insights into
bone biology and calcium metabolism and, it is hoped,
will lead to the development of alternative interventions
for the treatment of metabolic bone diseases such
as osteoporosis.
The pancreas is, therefore, a storehouse of a variety
of previously unappreciated molecules that regulate not
only protein metabolism but also bone/calcium metabolism.
Pursuit of further molecular characterization of
the pancreas is eagerly awaited.
Acknowledgments. The authors are very grateful to all
the collaborators who devoted their invaluable efforts
to PX study during the past 50 years. We greatly regret
that not all the names of these researchers can be listed
here. We wish to note that the PX study could not have
been accomplished without these people. The authors
also thank Miss Mie Masuda for her excellent secretarial
assistance.
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