論文要旨・審査の要旨

学位論文の内容の要旨
PRIVATANANUPUNT Jutiporn
論文提出者氏名
論文審査担当者
主査森山啓司
副査柴田俊一
横山三紀
Expression of glucose-dependent insulinotropic polypeptide
論
文
題
目
and its receptor in the rat major salivary gland
(論文の内容の要旨)
Introduction
Saliva plays a variety of functions which are essential for the maintenance of health and quality of life.
Components of saliva are synthesized by three pairs of major salivary glands: parotid gland (PG),
submandibular gland (SMG) and sublingual gland (SLG), as well as numerous minor salivary glands.
Salivary glands are generally classified as exocrine glands; however evidence over the past few decades
suggests that it may also possess endocrine secretory capability. Because a large number of
physiologically active proteins and peptides are secreted by the salivary gland, more detail regarding its
secretion and function is required.
Glucose-dependent insulinotropic polypeptide (GIP) is one of incretin hormones secreted mainly from
duodenum, less found in stomach, lens, brain and pancreas, and it has been recently reported the GIP
expression in SMG. In opposite to the ligands; GIP receptor (GIPR) is expressed throughout the body.
Aside from its remarkable role in potentiating insulin secretion, the effects of GIP on the facilitation of
nutrient uptake into adipose tissue and bone, stimulation of bone formation, stimulation of lipogenesis,
and promotion of β-cell and hippocampal progenitor cell proliferation have been demonstrated.
Nevertheless, the action of GIP secreted in the SMG is still not understood. Further, nothing is known
about the GIP expression in other types of salivary glands. Thus, we focused on GIP gene expression in
the rat PG and SLG, as well as the distribution of GIP and GIPR proteins in the rat major salivary
glands. In addition, the levels of GIP gene expression levels in the rat SMG at different ages were
quantitatively compared.
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Materials and methods
Animal and tissue preparation
Eight-week-old male Wistar rats, weighing 230–260g each, were deeply anesthetized then one side of
PG, SMG and SLG was used for RNA isolation, while the other side was immediately immersed in 10%
formalin neutral buffer solution at 4°C overnight and embedded in paraffin. To observe the alteration of
GIP expression during different growth periods, the SMG of 8-, 9- and 11-week-old male Wistar rats
(n=6 for each group) were collected and used for quantitative analysis.
Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted from the PG, SMG and SLG tissues using RNA STAT-60 reagent. Briefly, 5
μg of total RNA was converted to cDNA using SuperScript III Reverse Transcriptase. PCR was
performed using GIP-specific primers (forward 5’-ACC ACG AGG CCC AAG GTA TG-3’; reverse
5’-CAG AGA CTT TGG ACC AGG GCA-3’) amplifying a 400 bp GIP cDNA fragment. The PCR
conditions were as follows: 94°C for 10 min, then 35 cycles of 94°C for 45 s, 64°C for 45 s, and 72°C
for 1 min, followed by 72°C for 10 min. The amplified PCR products were analyzed by electrophoresis
on a 1.5% agarose gel and stained with ethidium bromide.
Immunohistochemistry
Immunostaining was performed using the three-step streptavidin-biotin-peroxidase method (Vectastain
Universal Quick Kit). Five-μm-thick sections were deparaffinized and unmasked the antigen in sodium
citrate buffer (pH 6.0). Then specimens were treated with a solution of 0.3% H2O2 in methanol for 20
min. The samples were blocked with blocking serum for 10 min followed by incubation overnight with
primary rabbit anti-GIP (YII-Y103-EX, 1:1000, Cosmo Bio) and anti-GIPR (LS-C138883, 1:50,
LSBio) antibodies in a humidified chamber at 4°C. The sections were then incubated with a
biotinylated universal secondary antibody for 10 min at room temperature, followed by incubation with
streptavidin peroxidase complex for 5 min. The immunoreactive sites were visualized with DAB and
counterstained with hematoxylin.
Real-time quantitative RT-PCR (qRT-PCR)
Reverse transcription was performed using 1 μg of total RNA using the high capacity RNA-to-cDNA
Kit. Next, 1 μl of cDNA along with a 20- l reaction of 2× PCR master mixture and gene-specific
Taqman assay purchased from Applied Biosystems (GIP: Rn00571500_m1 and GAPDH:
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Rn99999916_s1) were loaded into the plate. The reaction was carried out in a StepOne™ Real-Time
PCR detection system using the conditions as follow; 10 min at 95°C, and 45 cycles of 15 s at 95°C and
1 min at 60°C. The relative quantification (RQ) method, which represents the mean fold differences of
mRNA level compared with those of the 8-week-old rats, was used for analysis.
Statistical Analysis
Gene expression data were presented as mean ± standard error of the mean (S.E.M.) of RQ values.
Statistical analysis was performed using the Student’s t-test method at a 5% significance level using the
statistical software SPSS version 16.
Results
GIP gene expression in the rat PG, SMG, and SLG
Electrophoresis of the amplified products showed a single band of expected size corresponding to GIP
mRNA in all salivary glands; where the expression seemed to be higher in the SMG and SLG compared
with the PG.
Localization of GIP and GIPR in the rat PG, SMG, and SLG
GIP immunopositive staining was observed throughout the cytoplasmic region of the epithelial cells of
the striated ducts (SD) in all major salivary glands, where the immunostaining was more prominent in
the SMG and SLG than in the PG. Moreover, GIP immunopositive staining was also observed in the
secretory granules of the granular ducts (GD) and in the luminal portion of the intercalated duct (ID) of
the SMG. On the other hand, GIPR-positive immunostaining was found in the SD, excretory duct and
less intense staining in the ID of all glands, and also intensely found in the GD of the SMG. However,
neither GIP nor GIPR immunostaining was detected in the acini of any salivary tissues.
GIP gene expression in the rat SMG during the growth period
Relative to the GIP mRNA level in the SMG of 8-week-old rats (1.11±0.22), the expression was
significantly down-regulated 2–3-fold in the 9-week-old (0.48±0.10) and 11-week-old (0.30±0.08) rat
SMG (p<0.05), respectively. However, there was no significant difference in GIP mRNA expression
levels between the 9- and 11-week-old rats.
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Discussion
This is the first report of GIP gene expression in the rat PG and SLG, and the localization of GIP and
GIPR proteins in the rat major salivary glands. Previous report have shown the presence of GIPR
mRNA in human salivary tissues and in our study, we found positive immunostaining of GIPR in the
ducts of all major salivary glands, suggesting that GIP of salivary origin may act in an autocrine and/or
paracrine manner. Salivary ductal cells are known to play a role in modifying the saliva compositions
by absorbing sodium chloride and by adding proteins, growth factors, and potassium – thus it is likely
that salivary GIP may regulate any of these steps. GIP has been reported to reduce sodium resorption in
the isolated main duct of the rabbit mandibular salivary gland, it might be possible that GIP synthesized
within the salivary glands binds to the GIPR in the ducts and alters its electrolyte transport.
In addition, the specific GD in the rodent SMG contains many secretory granules with a variety of
biologically active polypeptides. The presence of GIPR in the GD of the SMG may be relevant to the
effect of salivary GIP in inducing protein secretion from these GD cells.
Although the secretion of saliva has been thought to be solely controlled by the autonomic nervous
system, studies in the past decade have also mentioned an influence of hormones on salivary
composition and metabolism. These new findings raise the possibility that salivary GIP might play a
role in the modification of saliva by altering the ductal transport properties or synthesis of secretory
proteins. GIP release is known to be stimulated by oral ingestion of carbohydrates and fats. While
eating, a number of sensory receptors are activated in response to food intake. Salivary GIP expression
may also be up-regulated in response to glucose and lipids in food that synergistically work with the
autonomic control system to modify the secretion of saliva.
The ductal phase of postnatal development of the rat SMG took place between 4 and 12 weeks of age,
and the proliferative activity of all cell types decreased progressively during that period. From our
study, rats during the late stage of SMG development were examined and found that GIP gene
expression decreased 2–3-fold between the 8- week-old to 9- and 11-week-old rats, respectively. It can
be concluded that GIP gene expression decreases when the SMG reaches the mature state and
proliferation declines. GIP is known to stimulate several cell proliferations; GIP originating from
salivary glands might therefore play a role in regulating ductal cell proliferation. However, this function
remains unclear in the absence of any supporting evidence.
In summary, this is the first study to indicate both GIP and GIPR gene expression and GIP histological
localization in the rat major salivary glands, as well as its variation in the rat SMG during the growth
period. These findings are crucial for a better understanding of the physiological function of the rat
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major salivary gland.
（和文による要約）
唾液は口腔内の健康および QOL の維持向上において極めて重要な役割を果たしている。唾液腺
は、生理活性をもつ数多くのタンパク質やペプチドを分泌するが、その詳細については不明な点が多
い。GIP (Glucose-dependent insulinotropic polypeptide)は主に十二指腸の K 細胞で分泌される消化
管ホルモンで、膵臓のβ細胞からのインスリン分泌を促進することから、glucagon-like peptide (GLP)
-1 と合わせてインクレチンと呼ばれている。口腔内における GIP については顎下腺での発現の報告は
あるものの、その詳細は全く知られていない。そこで我々は、ラット大唾液腺における GIP mRNA の発
現の有無と GIP および GIP 受容体の局在について明らかにすること、ならびに成長期ラットにおける
顎下腺 GIP mRNA の発現量の変化について詳細な検討を行うことを目的として実験を行った。
8 週齢雄性 Wistar ラット（体重 230～260ｇ、6 匹）を抱水クロラール深麻酔下にて屠殺後、大唾液腺
を摘出し、片側各唾液腺から total RNA を抽出し GIP および GIP 受容体の RT-PCR および GIP の定
量 PCR を行い、対側の各唾液腺は組織切片作製を目的に浸漬固定を行い通法に従ってパラフィン
伏埋の後 GIP および GIP 受容体の免疫染色を行った。GIP の遺伝子発現量（mRNA 量）は RQ 値の
平均値±SEM で表記した。統計学的検定にはステューデントの t 検定（p<0.05）を用いた。
RT-PCR の結果、ラット顎下腺、耳下腺、舌下腺において GIP mRNA の発現が認められ、顎下腺お
よび舌下腺は耳下腺に比べより発現が強く認められた。また免疫組織染色の結果、GIP および GIP
受容体は顎下腺、舌下腺、耳下腺の導管周囲の上皮組織の細胞質に特異的に発現が認められた。
GIP の定量 PCR の結果、9 週齢および 11 週齢ラット顎下腺での GIP 発現量は 8 週に比較して有意
に少なかった。
本研究により、ラット顎下腺、舌下腺、耳下腺での GIP および GIP 受容体の発現および局在が初め
て明らかになった。唾液腺は、一般的に外分泌腺として様々な生理活性物質を分泌するが、GIP が
唾液腺導管における輸送や分泌調整に対してある一定の役割を果たす可能性や、食物摂取後の糖
代謝において重要な機能を持つ可能性が考えられる。また顎下腺 GIP の発現量は、週齢によって変
化することが確認されたことから、GIP は唾液腺において成長因子としての機能をもつ可能性が考え
られる。
本研究によって、ラット唾液腺における GIP および GIP 受容体の発現とその局在、さらに増齢にともな
う GIP の発現量変化が明らかになった。これらの発見はラット唾液腺の生理機能解明の一助になると考
えられる。
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論文審査の要旨および担当者
報告番号
論文審査担当者
甲第 4611 号
主査
副査
森山啓司
柴田俊一
PRIVATANANUPUNT Jutiporn
横山三紀
Expression of glucose-dependent insulinotropic polypeptide
論文題目
and its receptor in the rat major salivary gland
（論文審査の要旨）
唾液は口腔内の健康および QOL の維持向上において極めて重要な役割を果たしている。唾液の主成分は
水であるが、それ以外にも唾液の機能に影響を与える生理活性をもつ、タンパク質やペプチドを始めとした
数多くの分泌物が含まれている。しかし、その組成物の分泌および機能については未だ不明な点も多い。
Glucose-dependent insulinotropic polypeptide（GIP）は主に上部小腸 K 細胞から分泌される消化管ホル
モンであり、glucagon-like peptide (GLP) -1 と合わせてインクレチンと呼ばれ、膵臓 β 細胞からのインスリ
ン分泌を促進する。GIP は、1971 年、J. C. Brown らが単離した胃酸分泌抑制作用をもつ消化管ホルモンで
Gastric inhibitory polypeptide と命名されていたが、後に膵臓 β 細胞のインスリン分泌促進、耐糖能改善作
用が確認された経緯をもち、栄養素を脂肪細胞に細胞に蓄積する作用や、骨芽細胞の機能を亢進させる作用
など膵臓以外の多様な臓器への作用が確認されている。しかし口腔領域ではラット顎下腺およびヒト唾液中
に GIP の発現に関する報告はあるものの、唾液腺における GIP についての詳細は不明である。
このような背景のもと、Jutiporn はラット唾液腺における GIP の発現と週齢変化を検討する目的で実験
を行った。方法としては、8、9、11 週齢雄性 Wistar ラットの大唾液腺を摘出し、片側各唾液腺から total RNA
を抽出し、対側の各唾液腺からは組織切片を作製した。抽出した total RNA から逆転写ポリメラーゼ連鎖反
応法（RT-PCR）によって GIP 遺伝子の発現を検出するとともに定量 RT-PCR を行い、作製したパラフィン
組織切片を用いて GIP および GIP 受容体の免疫組織化学染色を行い光学顕微鏡による観察を行った。得ら
れた結果の概要は以下のようなものである。
1. RT-PCR の結果、ラット耳下腺、顎下腺、舌下腺において GIP mRNA の発現が認められ、顎下腺および
舌下腺は耳下腺に比べより発現が強く認められた。
2. 免疫染色の結果、GIP および GIP 受容体は耳下腺、顎下腺、舌下腺の導管周囲の上皮組織に特異的に発
現が認められた。さらに、GIP の免疫染色では顎下腺および舌下腺は耳下腺に比べより発現が強く認め
られた。
3. GIP の定量 PCR の結果、9 週齢および 11 週齢ラット顎下腺での GIP 発現量は 8 週に比較して有意に少
なかった。
本研究により、ラット耳下腺、舌下腺での GIP 遺伝子発現が確認され、免疫組織学的手法によるラット顎
下腺、舌下腺、耳下腺での GIP および GIP 受容体の発現および局在が明らかになった。また、ラット顎下
腺 GIP 遺伝子は、週齢により発現量が変化していることが示唆された。これらの知見は、今後の唾液腺よ
び GIP の生理的機能を理解する上で極めて重要であると考えられる。
以上のように本論文は、唾液腺の GIP および GIP 受容体の発現および局在に着目し、また週齢による
GIP 遺伝子発現量の変化を初めて明らかにしたものであり、今後の歯学の基礎および臨床の各分野における
発展に寄与するところが極めて大きいと考えられる。したがって、本論文は博士（歯学）の学位を申請する
に十分値するものと認められた。
（１）

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