1．開発の経緯 1982年，Charles River Canada Inc.より Long-Evans 系ラットを導入しクローズドコロニーで飼育繁殖していた中に，肥満を伴い多食，多飲，多尿，糖尿を呈するものの経過が極めて緩徐で，ヒトのインスリン非依存性糖尿病（Non Insulin Dependent Diabetes Mellitus ; NIDDM）の病態に類似したラットを発見した。この形質を保持する目的で糖尿と経口ブドウ糖負荷試験（Oral Glucose Tolerance Test ; OGTT）の成績を指標にして選抜交配を重ね，1991年，肥満を伴い高血糖を長期聞持続し糖尿病性腎症を併発するラット OLETF（Otsuka Long-Evans Tokushima Fatty）を作出した。また1983年に同じクローズドコロニーの Long-Evans 系ラットの中に多食，多飲，多尿，糖尿を呈し，急激な体重減少を伴って死亡するヒトのインスリン依存性糖尿病（Insulin Dependent Diabetes Mellitus ; IDDM）の病態に類似した糖尿病ラットを発見し，そこから糖尿を指標に選抜交配を重ね，糖尿病好発系の LETL ラット（Long-Evans Tokushima Lean）を1989年に近交化した。LETL ラットの病態の特徴は，生後8から15週齢にかけて突然多食，多飲，多尿，糖尿が発現し，インスリンで治療しない限り2週間以内で死亡するが，糖尿病発症にはTリンパ球減少症を必ずしも伴わないという点にある。さらに糖尿病を全く発症しないコントロールラットで，遺伝的にも近縁である LETO（Long-Evans Tokushima Otsuka）ラットを得た。 本論文においては OLETF ラットの病態を遺伝的，臨床病理学的および病理学的に解析し，本ラットの糖尿病モデル動物としての有用性について明らかにした。2．臨床経過とその病理組織像 OLETF 雄ラットは若週齢より肥満を呈し，25週齢で高インスリン血症，高血糖を示す糖尿病の発症がほぼ全例にみられた。膵ラ島では発病初期にB細胞が増生・肥大し，生後40週齢ごろより膵ラ島の萎縮が顕著となった。多食，多飲，多尿，体重減少，低インスリン血症などの症状がこの膵ラ島の疲弊・萎縮に伴って徐々に発現した。高インスリン血症から低インスリン血症に移行する病像は OLETF ラットの特徴でもあった。この病像と膵ラ島の病理組織学的変化とは非常に密接な関係があった。すなわち膵ラ島が肥大する増生期では血漿インスリン値が高く，線維化などによって膵ラ島の疲弊・萎縮が進行した病期には逆に低くなっていた。一方，OLETF 雌ラットは生後25週齢では糖尿病および耐糖能障害の発症は認められず，生後40週を過ぎると徐々に血漿グリコース値は上昇し，糖尿病の発症基準を満たすようになり，生後65週齢になると糖尿病が発症した。膵は生後40週齢ごろより雄と同様に膵ラ島の肥大，線維化による膵ラ島の分断・置換が種々の程度でほとんどの個体にみられたが，全く変化が認められない個体もあった。生後60週齢を過ぎる頃より雌では雄と同様に膵ラ島の大きさおよび数の減少を来していたが，概してその病変の拡がり，重症度とも雄に比して軽度であった。 OLETF ラットの膵に認められた膵ラ島の肥大，膵ラ島の線維化，外分泌部の膵炎，膵ラ島周囲炎および膵ラ島近傍のヘモジデリン沈着について OLETF ラットと LETO ラットならびに F344/Ducrj ラットとの交雑試験群の膵組織を用いて糖尿病発症との関連性についてロジスティックモデルに当てはめて解析したところ，膵ラ島の肥大と線維化が糖尿病発症に重要な要因であり，膵ラ島周囲炎，膵炎およびヘモジデリン沈着は糖尿病発症と直接的な関連性のないことが明らかとなった。3．糖尿病の発症率 OGTT の成績を基に以下の三つのタイプに分けた。1）糖尿病（Diabetes Mellitus ; DM）：血漿グルコースの頂値が300 mg/dl以上で，120分値が200 ㎎/dl以上のもの。2）耐糖能障害（Impaired Glucose Tolerance ; IGT）：血漿グルコースの頂値と120分値のどちらか一方が糖尿病の基準を満たしているもの。3）正常（Normal）：血漿グルコ一スの頂値と120分値の両者がともに糖尿病の基準を満たしていないもの。以上の診断基準によるF20世代以降における生後25週齢雄の糖尿病発症率は87.8%（341/388），耐糖能障害が7.2%（28/388），正常が4.8%（19/388）であった。 一方雄では生後25週齢では発症せず，生後65週齢で糖尿病発症率は33.5%（5/15），耐糖能障害が20.0%（3/15），正常が46.7%（7/15）であった。4．催糖尿病遺伝子の解析 OLETF ラットの糖尿病発症の遣伝解析をおこなう目的で OLETF ラットと LETO ラットならびに OLETF ラットと血縁関係のない F344/Ducrj ラットとの交雑試験をおこなった結果，催糖尿病遺伝子は雌雄ともに劣性で複数存在し，コントロールラットである LETO もそのいくつかを共有していた。さらにX染色体上にその一つが存在している可能性が示唆された。また OLETF ラットの糖尿病発症と RT1（ラットの主要組織適合遺伝子複合体）との関連はなかった。 現在，ヒトで NIDDM の発症に遺伝要因が強く関与していることは周知の事実であるが，まだ候補遺伝子を特定するに至っていない。今回 OLETF ラットのX染色体上に催糖尿病遺伝子の一つが存在する可能性が示唆され，このことが OLETF ラット固有の現象なのか，それともヒトの NIDDM と共通したことなのか非常に興味深いが，X染色体上の位置とその機能の検討は今後の課題である。5．糖尿病発症に関与する性ホルモン OLETF ラットの糖尿病は雄に多く発症をみるという性差が存在するが，この性差が OLETF ラットの糖尿病発症要因にどのように関わっているのか，精巣あるいは卵巣を摘出した群，さらにそれらの処置後テストステロンを投与した群を設けて調べた。その結果，精巣を摘出した雄では糖尿病や耐糖能障害の発現が抑制あるいは遅延する傾向が，卵巣を摘出した雌では逆に糖尿病が発現した。また，同様の処置をおこなった OLETF ラットにテストステロンを投与することにより，雌雄 OLETF ラットのどちらにも糖尿病が発症したが，OLETF 雄ラットにエストロジェンを投与すると糖尿病あるいは耐糖能障害どちらも発症しなかった。これらのことから老齢雌における糖尿病の発症には加齢に伴う卵巣からのエストロジェン分泌が減少し，相対的に精巣外からのテストステロン量が増加することで糖尿病が発症するのではないかと考えられた。また，雌雄の OLETF ラットにテストステロンを投与すると糖尿病の発症が認められたが，LETO ラットと F344/Ducrj ラットにテストステロンを投与しても糖尿病は発症しないこと，OLETF ラット，LETO ラット，F344/Ducrj ラットの血漿中のテストステロン濃度に差がみられなかったことなどから，テストステロンは催糖尿病遺伝子の存在下で作用し糖尿病発症に一翼をになっていると考えられた。6．糖尿病性腎症 OLETF ラットの糖尿病は緩徐な発症で，しかも長期間にわたって徐々に増悪する特徴をもっているため糖尿病性合併症がみられた。とくに腎症の指標である尿蛋白値は LETO ラットに比較して27週齢より顕著に上昇，47週齢では800 mg/dl以上となった。OLETF ラットの早期の腎糸球体病変として23週齢でメサンギウムの拡大と増殖がみられた。LETO ラットの腎糸球体と比較して腎糸球体の肥大も認めた。29週齢で fibrin cap を伴う滲出性病変が少数例みられ，55週齢になると多数認められるようになり，滲出性病変が腎糸球体全体に及びつつあるものもあった。その他，55週齢では結節性病変と腎糸球体の毛細血管が毛細血管瘤様に拡張した像も認めた。またヒトの糖尿病牲腎症と比較的特異性が高いと言われている capsular drop もみられた。96週齢ではほとんどの腎糸球体が萎縮硬化を示し，尿細管は萎縮消失したり，PAS 陽性の貯留物のうっ滞などを呈していた。また間質は拡大し炎症性細胞浸潤が伴う，いわゆる終末腎に近い像が認められた。 以上，OLETF ラットは肥満を伴い高インスリン血症から低インスリン血症に移行する特徴を有し，現在社会的問題となっている糖尿病性合併症，とくにヒトの腎症の特徴とされている腎糸球体の滲出性病変や結節性病変が出現したことから，糖尿病の成立機構の研究のみならず，糖尿病性合併症の予防や治療の研究に多いに貢献出来るモデル動物と期待される。さらに遺伝解析においても催糖尿病遺伝子の一つがX染色体上にあることが示唆されたことは今後の NIDDM の遺伝解析に貴重なデータを提供することとなるであろう。Abstract The characteristic features of OLETF rats are 1) late onset of hyperglycemia (after 18 weeks of age), 2) a chronic course of disease, 3) mild obesity, 4) clinical onset of diabetes mellitus (DM) mostly in males, 5) hereditary trait a); multiple recessive genes are involved in the induction of DM, b); rat MHC, RT1 has no diabetogenic effect, c); control strain, LETO appears to share some of diabetogenic genes with OLETF rats, d); female OLETF rats also carry diabetogenic genes, and e); one of the diabetogenic genes, designated as ODB-1, is transmitted linked with the X-chromosome of OLETF rats, however testosterone is an important factor involved in developing diabetes. 6) the changes of pancreatic islets can be classified into three stages: (1) an early stage (at less than 9 weeks of age) mild lymphocyte infiltration, (2) a hyperplastic stage (10-40 weeks of age); hyperplastic change and fibrosis in or around islets, (3) a final stage (at more than 40 weeks of age) showing atrophy of islets. 7) Diabetic nephropathy; after wk 50, glomeruli of OLETF rat enlarged, diffuse glomerulosclerosis, nodular glomerulosclerosis, the fibrin cap lesion, capsular drop lesion. These clinical and pathologic features of disease in OLETF rats resemble those of human NIDDM.Introduction We have reported a new inbred strain of Long-Evans Tokushima Lean (LETL) rats with IDDM without lymphopenia (1). This LETL strain was established in 1989 from an outbred colony of Long-Evans rats that had been purchased from Charles River Canada Inc. From the same colony of rats, we established another inbred strain of rats, Otsuka Long-Evans Tokushima Fatty (OLETF), that develops spontaneous persistent hyperglycemia (2). In this paper, we report here that pathophysiological characteristics and genetic profiles of the OLETF rats. The value of this strain as an animal model of human type 2 diabetes is discussed.Establishment of OLETF strain A spontaneously diabetic rat with polyuria, polydipsia and mild obesity was discovered in 1984 in an outbred colony of Long-Evans rats, which had been purchased from Charles River Canada (St.Constant, Quebec, Canada) in 1982. A strain of rats developed from this rat by selective breeding has since been maintained at the Tokushima Research Institute (Otsuka Pharmaceutical, Tokushima, Japan) and named Otsuka Long-Evans Tokushima Fatty (OLETF). Selective brother-sister matings were then started using males of more than 400g and apparently normal females at 9-10 weeks old. The males used for mating were examined by the OGTT at 25 weeks old, and normal glucose toleranced rats and their offspring were killed. The LETO line was obtained by different original matings from those for OLETF rats, but both strain originated from the same colony of Long-Evans rats. The LETO line has not shown the diabetic syndrome.Clinical features Male OLETF rats began to gain weight faster than LETO rats from 5 weeks and the difference gradually increased with age, being about 200g at 40 weeks of age. Then in the early stage of diabetes mellitus (DM) with glycosuria, the body weight decreased slightly, the decrease becoming more prominent with increase in severity of DM. After the onset of glycosuria at 40 weeks, water and food intakes and urine production increased gradually, and after persistent glycosuria for more than 10 months, the rats showed marked polyphagia, polydipsia and polyuria. However, they survived and death from diabetes was very rare.Incidence of DM The cumulative incidences of male rats with diabetic or IGT type were 86.0% (301/350) or 9.7% (34/350), respectively, after the 20th generation. However, incidence of DM in female OLETF was 33.3% at 65 wk-old.Biochemical analysis The plasma glucose level of male OLETF rats became higher than that of LETO rats from 18 weeks. The triglyceride level increased from 8 weeks. The plasma cholesterol level became slightly higher than that of control rats. At 30 weeks of age, the level of plasma glucose was twice that of controls, and the plasma triglyceride level was 5 times that of controls, whereas the plasma cholesterol level was elevated only slightly. At 40 weeks of age, the plasma cholesterol level was 4.73±O.68mM in OLETF rats and 2.71±O.15mM in LETO rats and the plasma triglyceride level was 6.45±2.26mM in OLETF rats and 1.31±O.35mM in LETO rats, both differences being highly significant (P<O.OO1).Oral Glucose Tolerance test (OGTT) The results of OGTTs at various ages are shown in Fig.1. After 24 weeks of age, elevation of plasma glucose in the OGTT became marked, indicating the development of DM. At 55 weeks, the peak values exceeded 400mg/dl. At 24 weeks, the basal value and the values of plasma IRI during the OGTT were increased in OLETF rats and these increases were more prominent at 55 weeks. When OLETF rats developed glycosuria, their plasma glucose levels increased, whereas their IRI values decreased. At 65 weeks of age, plasma glucose level elevated to over 450mg/dl, and the IRI value became less than 300pg/ml, and these rats were rescued by insulin injection and their body weights increased gradually. The plasma glucose and IRI responses of LETO rats in the OGTT were within the normal ranges throughout the term of observation.(Fig. 1)Hereditary trait The mode of inheritance of DM in OLETF rats was examined by genetic segregation studies using F1, F2 and backcross male progenies. The results shown in Table 1 and Fig.2 are summarized as follows: 1) no diabetic rat was found in reciprocal F1 hybrids of OLETF and F344/Ducrj rats, 2) approximately 46 % of (OLETF × LETO) F1 hybrids were diabetic, whereas in the reverse combination only 2 of 63 F1 hybrids (3.2%) were diabetic, 3) approximately 12 % of the (OLETF × F344) F2 hybrids were diabetic, whereas the approximately 29-35% of (OLETF × LETO) F2 and (LETO × OLETF) F2 rats were diabetic, 4) on backcross with OLETF rats, more than 60% of the progenies were diabetic, whereas those obtained by backcrosses with LETO or F344 developed no diabetes except those of (OLETF × LETO) F1 × LETO mating which showed a relatively high incidence of diabetes (21.5%). No association of diabetes with the RT1.A phenotype was observed (data not shown). These results clearly indicate that 1) more than two recessive genes are involved in the development of diabetes, 2) both male and female OLETF rats carry a set of diabetogenic genes, and 3) one of the diabetogenic genes designated as ODB-1, is transmitted linked with the X-chromosome of OLETF rats, and 4) LETO rats appear to share some diabetogenic genes with the OLETF strain.Histopathological finding Pancreas: Histopathologic changes were classified into three stages: 1) an early stage (at less than 9 weeks), 2) a hyperplastic stage (at 10-40 weeks) and 3) a final stage (at more than 40 weeks). An early stage, mild to moderate lymphocyte infiltration into or around almost all pancreatic islets and partially in the acinus area were seen in both male and female OLETF rats. However, at this stage, the islet structure was normal and insulin granules were clearly identified by immunohistochemical staining. After week 12, fibrosis of the islets became apparent. A characteristic feature is hyperplasia of pancreatic ducts in or around the islets. After week 20, fibrosis of the islets became prominent; the islets were either divided or completely replaced by fibrotic fibers. In the final stage, after 40 weeks, replacement of islets by connective tissues became more marked. After 70 weeks, the pancreas was extremely atrophic and the tissue was replaced by fatty and connective tissue. Both the number and size of islets were significantly decreased. Loss of the islets was seen in the final stage.(Table 1)(Fig. 2)Diabetic nephropathy The results of urinary protein levels at various ages are shown in Fig.3. Urinary protein levels in male OLETF rats began to increase more than LETO rats from 30 weeks and drastically increased thereafter, ultimately reached over 800 mg/day.(Fig. 3) The early histological change of the kidney was focal mesangial lesion with proliferation of mesangial cells at 23 weeks of age before the stage of clinical diabetic nephropathy. At 29 weeks of age, besides mesangial lesion, a few glomeruli showed the segmental lesion with PAS-positive deposits in the mesangium or capillary that seemed to be identical with the fibrin (hyalin) cap in the exudative lesion commonly observed in human diabetic glomerulopathy. After 50 week of age, the glomeruli showing the exudative lesion with prominent fibrin caps became numerous and more dominant finding than the mesangial lesion. Most of these lesions were characteristic in that they arose as the focal and segmental lesions in the early stage and showed the segmental obsolescence in the later stage. However, a few lesions of them advanced so much as to be the global exudative lesions. The similar deposits were often observed at inside of Bowmann\u27s capsule which had same finestructure with fibrin cap and were located between the basement membrane and parietal epithelial cells of the capsule. This was identical with the lesion called the capsular drop, one of characteristic findings for human diabetic glomerulopathy. On the other hand, the aneurysmal dilatation of intraglomerular vessels, another characteristic finding in glomerulus, was frequently observed. These dilated vessels were contoured with thickened wall of mesangial tissue. However, the vascular changes in the interstitium or vascular pole of the glomerulus such as the arteriolo-hyalinosis and -sclerosis were not seen. Through all stages, the severity of mesangial lesions increased with aging, some of which showed nodular expansion of the mesangial matrix. However, they were not identical with the typical nodular lesion described by Kimmelstiel-Wilson(3) because they were at most a minor proportion and, if present, lacked the consolidated appearance with acellular core. After 55 weeks of age, the segmental or global obsolescence (sclerosis) of glomeruli and involved numerous glomeruli so advanced ultimately as to be the end stage kidney at 96 weeks of age.References1. Kawano,K., Hirashima,T., Mori,S., Saitoh,Y., Kurosumi,M. and Natori,T. (1991) New inbred strain of Long-Evans Tokushima Lean rats with IDDM without lymphopenia. Diabetes 40, 1375-1381.2. Kawano,K., Hirashima,T., Mori,S., Kurosumi,M., Saitoh,Y. and Natori,T. (1991) Spontaneous long-term hyperglycemic rat with diabetic complications Otsuka Long-Evans Tokushima Fatty (OLETF) strain. Diabetes 41,1422-1428. 3. kimmelstiel P. Wilson C. (1936) Intercapillary lesions in the glomeruli of the kidney. Am J Pathol 12: 83-97博士(獣医学)麻布大
