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ネコの抗インスリン自然自己抗体（IgG）に関する研究

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(1)Title. ネコの抗インスリン自然自己抗体（IgG）に関する研究( 本 文(Fulltext) ). Author(s). 髙島, 諭. Report No.(Doctoral Degree). 博士(獣医学) 甲第417号. Issue Date. 2014-03-13. Type. 博士論文. Version. ETD. URL. http://hdl.handle.net/20.500.12099/49040. ※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。.

(2) gT-ůG†X†ɯǵɯĻů«˺IgG˻,ː?ȥȯ. ˿˽˾̀Ń. ij˒ėĠėĠ˙ʶùǾéĠȥȯȪ ˺ij˒ėĠ˻ ˶ Ķ ʝ.

(3) țƻ ȍʗ‹ʇ‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰˾ ɟƘ‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰˿ ȵ1Ȳ     Ɍʋ‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰́     ƣƋ<0Ǝǐ‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰̂     Ʌƨ‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰̃     ɗĪ‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰17     įŶ‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰20 ȵ2Ȳ     Ɍʋ‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰21     ƣƋ<0Ǝǐ‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰23     Ʌƨ‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰31     ɗĪ‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰38 įŶ‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰42 ȵ3Ȳ     Ɍʋ‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰43     ƣƋ<0Ǝǐ‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰44     Ʌƨ‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰47     ɗĪ‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰53     įŶ‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰55 ɋŶ‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰56 ʞʬ‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰57 ŊȇƊǽ‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰‰58.

(4) ȍʗ‹ʇ.  ‰ IgG‰‰‰Immunoglobulin G˺ÅȐQ„p† G˻ ‰ ELISA‰‰‰Enzyme-linked immunosorbent assay˺ˆȿɅùÅȐüȠǧĢǐ˻ ‰ RTK‰‰‰Receptor tyrosine kinase˺õħ«^„V†NdŠ[˻ ‰ RIA‰‰‰Radioimmuneassay˺ƅĭÅȐǧĢǐ˻ ‰ HRP‰‰‰Horseradish peroxidase˺ʂǕ Ums‚KNV]Š[˻ ‰ SDS-PAGE‰‰‰SDS polyacrylamide gel electrophoresis˺SDS vEP‚ExcS‚ˡdžǔ äǐ˻ ‰ CBB‰‰‰Coomassie brilliant blue˺PŠwVŠpE†bp‚Š˻ ‰ PBS‰‰‰Phosphate-buffered saline solution˺†ˇɎɼˬĐLJ˻ ‰ BCS‰‰‰Body condition score˺uaFT†aFV†XTE˻ ‰ BSA‰‰‰Bovine serum albumin˺HVɹǡE‚px†˻ ‰ CV‰‰‰Coefficient of variation˺Ĕä³ƈ˻ ‰ HPLC‰‰‰High pressure liquid chromatography˺˵ʴǜ«P„wbQ€oFŠ˻ ‰ GLUT4‰‰‰Glucose transporter 4˺Q‚TŠXʫʱų« 4˻ ‰ HSBS‰‰‰HEPES salts buffer solution˺HEPES ĐɎɼǜ˻ ‰ [3H]-2-DOG‰‰‰[1,2-3H]-2-Deoxy-D-glucose ‰ TBS‰‰‰Tris-buffered saline solution˺bXɎɼˬĐLJ˻ ‰ IRS-1‰‰‰Insulin receptor substrate 1˺G†X†õħ«čʥ 1˻ ‰ IR≉‰Insulin receptor β subunit˺G†X†õħ«Up~e_b˻ ‰ EGF‰‰‰Epidermal growth factor˺ŒȚŧˍĄĞ˻. 1.

(5) ɟ Ƙ  ůG†X†ů«.˼ɯĻÅȐśɫĶDZ,ːʶ? 1 ċȻıȒ-lb, '˼ɫĶDZBʦȜſ Ș+îĄ)+?[39]6!˼ȏȭäǸ˺HV<0p\˻Ȉƥ-G†X†Ůǁ- ?lb;Gf-Ȼ ıȒŞɘ-ɹ‘=ƱÒ@?ůG†X†ů«[12, 43].˼ُG†X†-ãƨBǣ?[44]= ,˼G†X†Ůǁ,ː³+ůG†X†ů«ÒȀ?lb-G†X†ɯĻÅȐȓ¸ɔ[9](.˼ ů«)ɯĻÓǏG†X†)-Ʌù,<>G†X†-ãƨǣ?˼ȰȖȘ+Ʌù-ʊ˚,<>¨ ɹȻBŊʦ[4]‹ɰȘ,ůG†X†ů«.˼ȑŞ-ȒĄ)'ʕʟ@' ?  gT-ůG†X†ů«.˼ȏȭäǸ˺lb<0p\˻Ȉƥ-G†X†Ůǁ- ?ȻıȒŞɘ -ɹǡ=ƱÒ@!) 2000 Ń, Hoenig =[17],<>Ďý@' ? Hoenig =.˼G †X†Ůǁ-+ gT˺¼ň˼ȻıȒ<0ȻıȒ¢ĕ-Ȓdž˻=.˼β Ɂɠůî;G†X†, Ĭ?ɯĻů«ƱÒ@+ )B­'Ďý' ?-ĎýB:),'˼gT.ůG†X †ů«Bɯǵ,µƞ?).+ )ɗ =@'! 2010 Ń,+$'˼Nishii =[36].˼ɮŅ Ș,¼ň+gT-ɹǡ‘,ɯĻÓǏG†X†,ʈĀśBȨ IgG˺ůG†X† IgG˻ğĈ? )BĎý!ůG†X† IgG ɹǡ=ƱÒ@!gT,G†X†Ůǁ-+$!)=˼ Nishii =.˼ůG†X† IgG .ĕƥśG†X†,Ĭ'Ȇȅ@!ů«(.+˼ɯĻÓǏG†X †,ɅùɢBȨɯǵɯĻů«( ?)ɗ !  ɯĻů«.˼‹ɰȘ,ɯĻÅȐȑŞ-ȖȅʃĄ)+?)ȣ=@' ?ȅ«-ÅȐVX`y.˼ Ƣƥ˼Ɂɵ;HG‚X˼ɦȔɁɠ+*-ȏǸBʕʟ'@BŽ˚?!9,À˼ɯĻÅȐ ȑŞ(.ɯĻů«ɯĻ-Ɂɠ;Ƀɐ,Ĭ'ʺÞ+ÅȐòŚB˼@=B¿Ħ?)ȒĄ)+ ?[2] $5

(6) (˼¼ň+lb, ':ɹǡ\†kPʥ;ɁɠɽˤǸʥ˼ ? .ɁɠËǸʥ,òŚ ?ɯĻů«ɯǵ,Ȇȅ@?)Ďý@' ?[27]ɯǵɯĻů«.˼ĕ˃=-ǹ×+ůîÚ ǭBõ+':ɯǵ,Ȇȅ@?ů«[11, 27, 31, 32]( >˼ƍȅÆ-ɬĿɹ‘[31];˼ŧš-ɹ‘ =[11, 30] ȅǛBʳ'ƱÒ@?ɯǵɯĻů«.˼ɯĻůî,òŚ!†kȁÓæ-ƒ ǃ˛( Ž˚@?P„Š†ˀŰʙ,ȢȞ?˼ƽŁ+ÅȐȼ)7+@' ?[27]ɯǵɯĻů«.ɯĻÅ. 2.

(7) ȐȑŞ-ȒĄ)+?ɯĻů«).ȏ+>˼B †kȁÓæ-ŭÙB!ÅȐʛȸ[3, 23];ɖæ<0Ĕ ś!ɯĻůî-Ž˚[15]+*,<>ȅ«-ŜŁśBɊŷ?ÀBų

(8) )ɗ =@' ?[30]¼ň+ gT-ɹǡ=Ȗʄ@!ůG†X† IgG ,% '.˼-ȅȂȘŐß.ŽƔ( ?˼ɯĻG†X †,Ʌù?ɯǵɯĻů«)'˼gT-ȅÿǗä,ƞȇ+ÀBų$' ?öɢś ?  Nishii =[36]-Ďý, '˼ůG†X† IgG Bµƞ?¼ň+gT˼ɹȻT†b„Š‚-ȏŁ ( ?Ƴȳ+˵ɹȻ;¨ɹȻBȨ+ )Bɗť?)˼gT-ůG†X† IgG G†X†-¬ ȇ,<4ãƨ.˼lb;Gf, 'ȒĄśBŷ%ůG†X†ů«).ƢʥȘ,ȏ+?öɢś. ?gTBˢɤËQ‚TŠXʠɴʔ˴,<>ģ˴Ș,˵ɹȻ,ʖĮ?)˼G†X†-ÓǏ.˼l b[28, 42];Gf[29],Dž2'ŷɉƖˏˍ˼%•ĵśBȨ[24, 34, 37, 49]!9˼gT.ɹȻ¹ŒƓ ,Ĭ?G†X†ÓǏk\Š†lb;Gf).ȏ+?)ɗ =@' ?ȀƖdz(ƔȦ+ƭŵ.+ ˼ůG†X†ɯǵɯĻů«.˼gT-ǹŖȘ+G†X†ÓǏk\Š†Bɿ

(9) ŐßBų

(10) öɢś  ? -ȥȯ(.˼gT-ůG†X†ɯǵɯĻů«-ȅȂȘŐß-ʊƔBțȘ)'˼¢-ģ˴Bģ Ə!ȵ 1 Ȳ(.˼gT-ɹǫ‘, û6@?ůG†X† IgG ǯŇBǧĢ? ELISA B¬ʀ˼ @Bȇ 'gT-ɹǫǯŇBǧĢ!=,ɹǫůG†X† IgG ǯŇ)ś×;Ń˹˼ɜǨŇ˼ɹǫ t‚{†ǯŇȶ-øȭk€zŠ\Š)-ː³śBÓƧ!ȵ 2 Ȳ(.˼¼ň+gT-ɹǡ=Ó˟ !ůG†X† IgG )Č˰gTŧǷɣɝɁɠBȇ '˼ůG†X† IgG G†X†-’+¬ȇ(. ?Ȼ-ô>ʮ7ɢ)@,ːA? RTK VQd‚-Ǘśæ, ?ãƨBƱʍ!6!ȵ 3 Ȳ(.˼ȵ 2 Ȳ, 'ʕ9=@!ůG†X† IgG -‹˃-ÓȊG†X†-¬ȇBđŌ?ãƨ,˼ůG† X† IgG )G†X†,<?ĖÓĞʁù«Ŏŧː³?*

(11) BƱʍ!. 3.

(12) ȵ1Ȳ ELISA ,<?gT-ɹǫůG†X† IgG ǯŇ-ǧĢ. Ɍ ʋ  Nishii =[36].˼G†X†Ůǁ-+ ɮŅȘ,¼ň+gT 10 ˨-ɹǡ=G†X†,ʈĀśB Ȩ IgG BƱÒ!-ůG†X† IgG .˼ĕƥśG†X†,òŚ'Ȇȅ@! IgG (.+ )=˼ɯĻÓǏG†X†,ɅùɢBȨɯǵɯĻů«)ɗ =@!˼ůG†X† IgG -ƱÒ,°!gT. 10 ˨( >˼ 2'-¼ň+gTůG†X† IgG Bɯǵ,µƞ' ?) BʒƔ?!9,.˼<>ėʅƷ+¯ƈ,% 'Ȧʕ?Řʃ ?6!˼ůG†X† IgG )¶× -gT-ŷ%øȭk€zŠ\Š˺ś×˼Ń˹˼«Ʈ˼t‚{†ǯŇ<0ȑŞ˻)-ː³ś-ÓƧ.˼ gT,?ůG†X†ɯǵɯĻů«-ȅȂȘŐß-ʊƔ,)$'ˉʃ+şĎ)+?gT-ůG† X† IgG - ELISA -¬ʀ.˼Ė-ʔƋ-ãǿ< ǧĢ)ɹǫǯŇ)'-ĤʉȘ+ʓ²Böɢ, ?!9,Řʃ( ?  ɯǵɯĻů«.˼äǸ-ƽŁ+ȅÿǗäBɿâ?ÀBų

(13) )ɗ =@' ?[30]˼G†X†B ʕʟ?ɯǵů«,% '.˼lb[40];$ƿ˫[41], 'ɹǡ‘,ğĈ?)Ďý@' ? ,+ gT-ůG†X†ɯǵɯĻů«˼ɹ‘, 'ɯĻÓǏG†X†)Ʌù?)( G†X†-¬ȇBʛȸ' ?)£Ģ?+=˼gT-ɹǫůG†X† IgG ǯŇ-ǧĢ.˼ɹȻT †b„Š‚B.9)!G†X†-¬ȇBʓ²?!9,Ř˧-Ʊƫ˦ț)+?  ȵ 1 Ȳ(.˼gT-ɹǫůG†X† IgG ǯŇBǧĢ? ELISA B¬ʀ˼ǧĢȺŇBƱʍ?) ):,˼¼ň+gT-ɹǫǯŇBǧĢ˼=,ś×;Ń˹˼«ċ˼ɹǫt‚{†ǯŇȶ)-ː³BÓ Ƨ!á '˼ȑŞgT˺ȻıȒ˼ǏıĂ˼ËÓǏ˼ǚæĂȑŞȶ˻-ɹǫůG†X† IgG ǯŇB ǧĢ˼¼ňgT-ǧĢ¹)Džʪ!ǹ,˼ȻıȒ,ɓŞ!gT,% '.˼ɹǫůG†X† IgG -ʺŽʧ+ ,ǒț!¬ʀ! ELISA .˼qƒŠb-ćȝæůî,gT-ɫɭ=Ⱥʀ!G †X†Bȇ ?)(˼ɯĻÓǏG†X†,ʈĀśBȨ IgG BƱÒ(?<

(14) ,ĹĘ!. 4.

(15) ƣƋ<0Ǝǐ 1. @?  ELISA qƒŠb-ćȝæ,ȇ ?G†X†)'˼1 ˨-¼ň+ŧgT-ɫɭ=Ó˟Ⱥʀ!G †X†Bȇ !Ó˟Ⱥʀ,ȇ !ɫɭ.ƂÒőȜ#,-80°C (ÏɅ˼Ⱥʀ6(µğ!ɫɭɃɐ 10 g , ethanol/0.7 M HCl Bá ˼4°C (t{WdGY˺3: 1 v/v, 4 ml/g tissue˻ ˼ŦǮǜB 37°C ( 2 ƖˏƃŴ!ʽŗÓ˟ő˺4°C˼10000g, 20 Ó˻ ˼ŒǡB Fractgel EMD-SO3-650 column˺Merck, Darmstadt, Germany˻Bȇ 'GK†˜ƁP„wbQ€oFŠ,°!Resource RPC column˺GE Healthcare, Chalfont St. Giles, UK˻Bȇ !ʲȝ HPLC ,<> 0.1%bo‚K„˅ˇ(ǪÒ@? ÓȊBŔ! L€yɅùǸʥ.˼ 60%EZbeb‚Bûƞ?0.1%bo‚K„˅ˇȇ 'ǪÒ! G†X†Bûƞ?ÓȊ˺EZbeb‚ǯŇȽ 50%(ǪÒ˻.˼ÓȊʔƋ˺1.5 μl˻BdG„†z† pƒ†˺Trans-Blot Transfer Medium, Bio-Rad, Hercules, CA, USA˻Œ,Xv_b?c_bp„_ bÓƧ,<>Ȧʕ!z†pƒ†.p„_PJŠX˺ėƑƢ©ñʀɷ˼ė˓˻(p„_N†Q!ő ,˼mK^†ƶʟů€_bG†X†ů«˺Vj|O˼Ǣķ˼ɔ˲˻)):, 4°C (‹ƗòŚ˼Em W†ƶʟ HRP˺Thermo Fisher Scientific, Rochester, NY, USA˻Bá ' 30 Óˏˢɒ!G†X †)ů«-ʁù«. 4-chloro-1-naphthol (öʆæ!  ELISA ƶǩǸʥ)'¼ň+ŧgT-ɹǡ=EoFe`FȺʀ!ůG†X† IgG Bȇ !H VG†X†˺Sigma, St. Louis, MO, USA˻BćĢ!L_q†QS‚˺CNBr-activated Sepharose 4B, GE Healthcare˻Bȇ '˼žƵƛ,!$'ɹǡ‘-G†X†ɅùǸʥBÓ˟!Ó˟@ !G†X†ɅùǸʥBȦʕ?!9, 4%-20%Q€WJ†b SDS EP‚ExcS‚˺SDS-PAGE Gradient Gel, TEFCO, Tokyo, Japan˻Bȇ 'ˡdžǔä˼ɉ ' CBB ƪɲBƏ!ůG†X† IgG B=,Ⱦæ?!9, HiTrap Protein A HP column˺GE Healthcare˻Bȇ 'Ŕ=@!G†X †ɅùǸʥBȺʀ!IgG ǯŇ.üijƈ=NjĢ!˺280 nm ,?üÄŇ 1.4  IgG 1.0 g ,ȝō?˻ ELISA -Żźů«)'.˼ HRP ƶʟHUOůgT IgG Bȇ !˺R1308 HRP, Acris Antibodies GmbH, Herford, Germany˻ . 5.

(16) 2. 2K YE>4  ȵ 1 Ȳ(.84 ˨˺Ơïå˜ 6 ˨ïå˜ 38 ˨ƠˁĜ˞ 9 ˨ˁĜ˞ 31 ˨˻-¼ň+ŧGJgT Bȇ !@=-gT.˼ʨ«Ʊƫ˼ɹȁƈ˼ɹǜȅæĠ<0ıƱƫ,<>ɮŅȘ,¼ň( ? )BȦʕ!Ń˹. 1 = 13 ǀ-ȹĆ, >˼‘ę¹. 6 ǀ( $!9 ǃ˛ʓ² BCS . 2 = 9 ( >˼‘ę¹. 5 ( $!gT-ś×.˼˞˜<0ïå˼íĸƂÒ-ƞǴ<$'Ơïå˜˼ïå ˜˼ƠˁĜ˞˼ˁĜ˞,Ó˫'ÓƧ!6!ɹǫůG†X† IgG ǯŇ)ȑŞ)-ː³BÓƧ? !9,˼ij˒ėĠŚȇȅǸȪĠ˃˖IJäǸȒ˙<0˷ôėĠʭĠ˃˖IJäǸéȕZ†\Š,ƥ˙! 58 ˨˺Ơïå˜ 20 ˨˼ïå˜ 16 ˨˼ƠˁĜ˞ 8 ˨˼ˁĜ˞ 14 ˨˻-ȑŞgTBȇ !ɹǜƱƫ; ȊÁƱƫũʄ+*--gT-ȑŞ,ː?şĎ.˼ʑȕʎˋ<0˯ ’=-əô>ʛƫ+=0 ,ʨ«Ʊƫ,<>Ŕ!ȑŞ-Ëʐ.˼ǏıĂȑŞ 22 ˨˺ŤśɥɭȒ 18 ˨˼ıȤȓ 3 ˨˼ɩɡDZ 1 ˨˻ ˼ ËÓǏȑŞ 20 ˨˺ȻıȒ 18 ˨˼ȉǺɨƺɢ—ʷȓ 2 ˨˻ ˼ǚæĂȑŞ 6 ˨˺DZȓśɧȑŞ 3 ˨˼Ɂɵ śɧDZ 3 ˨˻ ˼-Ÿ˺ɦȔ+*˻10 ˨( $!ȻıȒgT 18 ˨˺Ơïå˜ 9 ˨˼ïå˜ 5 ˨˼Ơˁ Ĝ˞ 2 ˨˼ˁĜ˞ 2 ˨˻-

(17) # 3 ˨˺Ơïå˜ 2 ˨˼ƠˁĜ˞ 1 ˨˻.G†X†Ůǁ-+ ȓ¯(. $!ɹǜ. 12 ƖˏɈˬǺţ(˩ˢɤ=žô˼G†X†ĚǗB˔!9, EDTA-2Na )Eq „^e†Bû8^}Šp,žô!žɹő˼Ȝ#,ʽŗÓ˟˺4°C, 1000 g, 15 min˻ ˼-80°C (µğ ˼3 ˆƝ¢Ë,ǧĢ!-ȥȯ.˼ij˒ėĠģ˴äǸMGc€G†,ǎ$'ģƏ!˺ūʕȎ÷: 09044 <0 09075˻ . 3. YI=&#& IgG J: H8  96 HI‚wGP„qƒŠb˺NUNC MaxiSorp, Thermo Fisher Scientific, NY, USA˻,gTG†X †˺500 ng/well˻BÓǒ˼2 ƖˏĥǦ(ćȝæ!qƒŠbB PBS˺10 mM PBS containing 0.025% Tween20˻(ǖǘ!ő˼BSA 10 mg/ml˺A8022, Sigma˻Bû8 PBS ,<>ĥǦ( 18 Ɩ ˏp„_N†Q!ǖǘő˼ƶǩ<0ɹǫʔƋBHI‚,á ˼ĥǦ( 2 ƖˏČ˰!ǖǘő˼ PBS ( 50000 ·,ľˈ@! HRP ƶʟHUOůgT IgG Bá ˼=,ĥǦ( 2 ƖˏČ˰˼ǖǘ. 6.

(18) ő, 3,3',5,5'-tetramethylebenzidine˺Dako, Produktionsvej, Glostrup, Denmark˻Bû8čʥǪǜB á !40 ÓˏČ˰ő˼»Ƽǜ˺2N H2SO4˻Bá ˼wGP„qƒŠbŠ]Š˺Model 680, Bio-Rad, Hercules, USA˻,<>’Ǒˍ 450 nm ÝǑˍ 620 nm (üÄŇBǧĢ!  ELISA -ȺŇȦʕ-!9,˼ ľˈȜɍśʔ˴)Ǡáăóʔ˴BģƏ! ľˈȜɍśʔ˴, '.˼ ɮŅȘ,¼ň+ 2 ˨-gT=žô!ɹǫB PBS (ʶɉľˈ ˺ľˈDž; ʔƋ:PBS = 0: 1, 1: 1, 1: 2, 1: 4˻ ˼@ @ůG†X† IgG ǯŇBĢˊ!Ǡáăóʔ˴.˼ƐȣǯŇ-ůG†X† IgG BgT ɹǫʔƋ,Ǡá?)(ģƏ!E_ZGË CV .˵ǯŇ)¨ǯŇ- 2 ȭ˫-ɹǫʔƋBȇ !ú qƒŠb,<? 4 ă-ǧĢ=ȷÒ!E_ZGˏ CV . 2 ȭ˫-ɹǫʔƋBȇ !ȏ+?qƒŠ b,<? 4 ă-ǧĢ=ȷÒ!  -ȥȯ(.˼ɹǫʔƋ.žôő 3 ˆƝˏ-80°C (ÏɅ'=ůG†X IgG ǯŇBǧĢ!ů G†X† IgG -ÏɅ,<?ǯŇ-ĔæBȦʕ?!9,˼ 2 ¶«-ɹǫʔƋ,% 'žôȜő<0 3 ˆƝˏµğ!ő-ǧĢ¹BDžʪ!. 4. YIS IgG  &#&J: H8  ɹǫɋ IgG ǯŇ.ļʢ-U†cG_^ ELISA N_b˺Cat IgG ELISA Quantitation Set, Bethyl Laboratories, Montgomery, TX, USA˻Bȇ ' Campbell =[10]-Ǝǐ,!$'ǧĢ!ɹǫ G†X†ǯŇ. 0.1 = 3.6 ng/ml -ȹĆ(ɹǫǯŇǧĢöɢ+[33]ļʢ-U†cG_^gTȇ ELISA N_b˺gTG†X†ǧĢN_b˼ưljȅȪĠȥȯũ˼ƸǙ˼ȩěķ˻Bȇ 'ǧĢ!. 5. bW,$'\X[d  3 ˨-¼ň(ƽŁ«ċ˺BCS: 5/9˻-gTBȇ 'ˢɤËQ‚TŠXʠɴʔ˴BģƏ!ƜÖ-ɹǜ žôő,˼Q‚TŠXǪǜ˺50%, w/v, 1 g/kg «ˉ˻Bˢɤُ!žɹ.Q‚TŠXُő 10, 30, 60, 90, 120 Óő,ģƏ!ɹǫQ‚TŠXǯŇ.c€GRxXbŠǐ˺ĩĒc€GRy, Ʀ™˻(ǧĢ !. 7.

(19) 6. RZ.A  aŠ\.˼‘ę¹)ȹĆ(ɽʎ!ÏɅµğś-Ȧʕ-!9,˼žôȜő) 3 ‡ƝÏɅµğő, ?ǧĢ¹ÓĽ-ƽʅśBȦʕ!ő,łĊ¹BĬŚ- ? t-test (Džʪ!ś×)ɹǫůG†X † IgG ǯŇ)-ː³.˼aŠ\ÓĽ-ˣƽʅśBȦʕ!ő˼Kruskal-Wallis test Bȇ 'ÓƧ! ɹǫG†X† IgG ǯŇ)Ń˹˼BCS˼ɋ IgG ǯŇ<0G†X†ǯŇ-ȝːː³,% '.˼ Spearman’s correlation coefficient or by rank test ,<>ÓƧ!ˢɤËQ‚TŠXʠɴʔ˴,?˼ ɹǫQ‚TŠX˼G†X†<0ůG†X† IgG ǯŇ.˼Bartlett's test ,<>ÓƇ-DžʪB! Q‚TŠXُÛ)ُő,?ɹǫǯŇ,% '.˼single-factor ANOVA ) Dunnett test ,<> ÓƧ!¼ňgT)ȑŞgT,?ɹǫůG†X† IgG ǯŇ-Džʪ.˼):,ǧĢ¹ÓĽ-ƽʅ śȦʕ@+$!!9 Mann-Whitney’s U test ,<>– -‘ę¹BDžʪ˼p<0.05 BɇʌȘ, ƞš)!. 8.

(20) Ʌ ƨ 1. =&#& IgG H8L ELISA P:  Figure 1-1 ,.ůG†X† IgG BǧĢ? ELISA ,?ÊċȘ+ƶǩƙɍBȨƙɍ-Ȝɍ ś.˼33.8 ng/ml = 2160 ng/ml -ȹĆ(Ɋŷ@' !PBS (ʶɉľˈ@! 2 ȭ˫-ɹǫʔƋ Bȇ !ľˈȜɍśʔ˴-ăŀȜɍ˺Fig. 1-2˻.˼):,34îdzBʳ?Ȝɍ( $!ɹǫʔƋ, 3 ȭ˫-ƐȣˊůG†X† IgG BǠá!ďù-ăóǿ.˼@ @ 92.2%, 91.9%<0 92.0%)˵ $!E_ZGË CV .˼ɹǫǯŇ 799 ng/ml (. 4.0%66 ng/ml (. 3.7%( $!2 ȭ˫-ɹǫ ʔƋ-ǧĢ=NJ9=@!E_ZGˏ CV˺n=4˻.˼ɹǫǯŇ 799 ng/ml , ' 5.1%˼72 ng/ml ,  ' 6.9%"$! ɹǫʔƋ-ÏɅµğś.˼ µğÛ, 390±17˺łĊ¹±ƶǩʘĺ˻ µg/ml ) 209±7 µg/ml ( $!ǧĢ¹.˼̀ˆƝ-ÏɅµğő,. 403±15 µg/ml ) 203±11 µg/ml ( >˼ǧĢ¹-ˏ,ƞ šĺ.+$!. 2. YI=&#& IgG J: H8  +;/ 9eh)5hYI$!&J: `*  ɮŅȘ,¼ň+gT, '˼ɹǫůG†X† IgG ǯŇ.˼80 µg/ml˺0.5 µM˻= 1578 µg/ml ˺10.8 µM˻-ȹĆË, >˼‘ę¹. 221 µg/ml˺1.5 µM˻( $!˺Fig. 1-3˻ Table 1-1 .ś×, <$'Ó˫@!gT-ɹǫůG†X† IgG ǯŇBȨ˞˜<0ïå˼íĸƂÒ-ƞǴ, '˼ gT-ɹǫůG†X† IgG ǯŇ,ĺ.+$!6!ɹǫůG†X† IgG ǯŇ.˼Ń˹<0 BCS )ƞš-ȝːː³,+$!˺Ń˹: n=84, r=0.19, p=0.09; BCS: n=84, r=-0.01, p=0.76˻   ɹǫůG†X† IgG ǯŇBǧĢ!¼ň+gT-ɹǫ-ɋ IgG ǯŇ)G†X†ǯŇB Table 1-2 ,Ȩ ɹǫůG†X† IgG ǯŇ.˼ ɋ IgG ǯŇ)ƞš+ƽ-ȝːː³ʕ9=@! ˺Fig. 1-4; n=84, r=0.383, p<0.01˻ ˼G†X†ǯŇ).ƞš-ȝːː³ʕ9=@+$!˺n=84, r=-0.100, p=0.67˻ . 9.

(21) Absorbance 450-620 nm. 2.5 2 1.5 1 0.5 0 0. 500. 1000. 1500. 2000. 2500. Anti-insulin IgG (ng/ml). Anti-insulin IgG concentration ng/ml). Fig. 1-1. A typical standard curve of anti-insulin IgG concentration for the ELISA.. 800 y = 780.95x + 19.968 600. 400 y = 498.38x - 13.97 200. 0 0. 0.2. 0.4. 0.6. 0.8. 1. Dilution factor Fig. 1-2. The dilution curves of plasma anti-insulin IgG concentrations.. 10.

(22) Plasma anti-insulin IgG concentration (μg/ml) . 1600 1400 1200 1000 800 600 400 200 0 Fig. 1-3. The plasma anti-insulin IgG concentrations in 84 clinically healthy cats. The plasma anti-insulin IgG concentrations ranged from 80 to 1578 μg/ml, with a median concentration of 221 µg/ml (broken line).. Table 1-1. Plasma anti-insulin IgG concentrations in 84 clinically healthy cats categorized by gender. Plasma anti-insulin IgG concentration (g/ml) Variable Male Gender. Female. Category. n. Median. Minimun-Maximum. Intact. 6. 234. 161 - 908. Castrated. 38. 188. 80 - 831. Intact. 9. 397. 85 - 1094. Spayed. 31. 237. 86 - 1578. 11.

(23) Table 1-2. Plasma concentrations of anti-insulin IgG, total IgG and insulin in 84 healthy cats. Variable. Median. Minimun-Maximum. (µg/ml). 221. 80 - 1578. Total IgG. (mg/ml). 25.4. 11.9 - 59.3. Insulin. (ng/ml). 0.54. 0.27 - 2.3. Plasma anti-insulin IgG concentration (μg/ml). Anti-insulin IgG. n=84 r=0.383 p<0.01 y = 14.704x - 43.578. 1600. 1200. 800. 400. 0 0. 10. 20. 30. 40. 50. 60. Plasma total IgG concentration (mg/ml) Fig. 1-4. Correlation between plasma anti-insulin IgG and total IgG concentrations. Plasma anti-insulin IgG concentrations were correlated significantly (p<0.01) with total plasma IgG concentrations.. 12.

(24) 3. bW,$'\X[dYI$'h&#& =&#& IgG J: 73  ɹǫQ‚TŠXǯŇ.˼Q‚TŠXǪǜ-ˢɤËُ 10 Óő,ɶŒƓ˺p<0.01˻˼30 Óő ˺p<0.01˻ ˼60 Óő˺p<0.05˻6(.ُÛ-ǯŇ<>:ƞš,˵¹( $!˼-őŒ,ǣİ˼ 90 Ó(ُÛǯŇ,Ũ$!˺Fig. 1-5A˻ ɹǫG†X†ǯŇ.Q‚TŠXǪǜُ 60 Óő,ƜėǯŇ BȨ!˺ُÛǯŇ)Džʪ' p<0.01˻ ˼ɹǫůG†X† IgG ǯŇ. 120 Ó-ʉĪƖˏ‘ Ĕä+$!˺Fig. 1-5B˻ . 4. N<YI=&#& IgG J:  Figure 1-6 ,ȑŞgT,?ɹǫůG†X† IgG ǯŇB¼ň+gT-Ʌƨ)­'ȨǧĢ !ȑ޵ƞgT 58 ˨-ɹǫůG†X† IgG ǯŇ.80 µg/ml = 2749 µg/ml -ȹĆË, >˼‘ ę¹ 251 µg/ml .˼¼ň+gT-ɹǫǯŇ)ĺ+$!ȑŞgT-

(25) # 2 ¯, '¼ňgT-Ɯ ˵ǧĢ¹BėŒă?˵¹BȨ!˼@=.ŤśɥŽÈ)˵M†wQ„p†ɹȓB­Ȗ' ?¶«( $! $5

(26) ˼ȻıȒgT 18 ˨-ɹǫůG†X† IgG ǯŇ.˼80 µg/ml = 1428 µg/ml -ȹĆ(˼¼ň+gT-ǧĢ¹-ȹĆË,ÓĽ'>˼‘ę¹ 143 µg/ml .˼¼ň+gT-ɹǫǯŇ )ĺ+$!˺Fig. 1-7˻ ˼G†X†Ůǁ-+ ȻıȒȓ¯ 3 ˨-ɹǫůG†X† IgG ǯŇ.@ @ 86 µg/ml˼95 µg/ml˼84 µg/ml ( >˼¼ň+gT<0ȻıȒgT-Ɯ¨¹,ʯ ǯ Ň( $!. 13.

(27) Plasma glucose concentration (mg/dl). A. Glucose **. 400 300. **. 200. *. 100 0 0. 20. 40. 60. 80. 100. 120. Plasma insulin concentration (ng/ml). Time after admonistration of glucose (min). B. Insulin. 3. **. 2. 1. 0 0. 20. 40. 60. 80. 100. 120. Time after administration of glucose (min). C. Anti-insulin IgG. Plasma anti-insulin IgG concentration (μg/ml). 200 150 100 50 0 0. 20. 40. 60. 80. 100. 120. Time after administration of glucose (min). Fig. 1-5. The results of intravenous glucose tolerance test (1 g/kg) in three normal domestic cats. Data are expressed as means±SEMs (vertical bar). * and ** denote significant differences from pretreatment level at p<0.05 and p<0.01. A: Plasma glucose concentrations (■). B: Plasma insulin concentrations (▲). C: Plasma anti-insulin IgG concentrations (●). 14. 14.

(28) Plasma anti-insulin IgG concentration (μg/ml). 3000. 2000. 1000. 0 Clinical healthy (○). Diseased (●). Fig. 1-6. The plasma anti-insulin IgG concentrations in 84 clinically healthy cats (broken line shows median: 221 µg/ml) and 58 diseased cats (broken line shows median: 251 µg/ml). No difference between medians of both groups by Mann-Whitney’s U test.. 15.

(29) Plasma anti-insulin IgG concentration (µg/ml). 10000. 1000. Median Median. 100. 10 Clinical healthy. Diabetic. Fig. 1-7. The plasma anti-insulin IgG concentrations in 84 clinically healthy cats (box; 25th and 75th percentiles, whiskers; 25th percentile minus and 75th percentiles plus 1.5 times the interquartile range or the minimum and maximum, the median: 221 µg/ml) and 18 diabetic cats (15 closed and 3 opened circles indicate the data of cats with and without insulin treatment, respectively, median: 143 µg/ml). No difference between medians of both groups by Mann-Whitney’s U test.. 16.

(30) ɗ Ī  ȵ 1 Ȳ(.˼gT-ɹǫ‘-gTG†X†,ʈĀśBȨ IgG ǯŇBǧĢ? ELISA B¬ʀ˼ -ǧĢȺŇBƱʍ!-ǧĢȼ.˼Ȝɍś-˵ ƶǩȜɍBƞ˼¨ E_ZGË<0E_Z Gˏ CV BȨ!6!ľˈʔ˴;Ǡáăóʔ˴, '˼Âɱ+ɅƨBŔ!¬ʀ! ELISA ˼g T-ɹǫůG†X† IgG ǯŇ-ǧĢ, 'êÓ+śɢBȨ)Ȧʕ@!6!˼¬ʀ!g TůG†X† IgG - ELISA Bȇ '˼¼ň+gT 84 ˨-ɹǫǯŇBǧĢ˼2'-gT-ɹǫ =gT-G†X†,ʈĀśBȨ IgG BƱÒ!-Ʌƨ.˼¼ň+gTɹ‘,ɯĻG†X† Bůî)'ʕʟ? IgG Bɯǵů«)'µƞ?)! Nishii =-Ďý[36]BƄŷ!=,˼ ¼ň+gT-ɹǫůG†X† IgG ǯŇ 80 µg/ml = 1578 µg/ml -ȹĆ, >˼ ‘ę¹ 221 µg/ml ( ?)BȨ!Ʌƨ.˼œő˼gTůG†X†ɯǵɯĻů«-ȅȂȘŐßBʊƔ' Œ(ʣˉ+čȧaŠ\)+?  gT, 'ůG†X†ů«.˼Hoenig =[17],<>˼p\ȈƥG†X†<0p\ȈƥG†X †)Exhˇ˄Õ- 1 Lũ˺B30˻-7ȏ+$!lbȈƥG†X†[16]-ُǁ- ?gT=ƱÒ @' ?˼ȵ 1 Ȳ, '¬ʀ! ELISA ,<?G†X†Ůǁ-+ 84 ˨-¼ň+g T-ɹǫʔƋ-ǧĢ(.˼2'-ʔƋ=ůG†X† IgG BƱÒ!¬ʀ! ELISA .˼gT -ɫɭ=ŲÒ@!G†X†BŻź! IgG BůG†X† IgG )'ƱÒ' ?!9˼ǧĢ, °!84˨-¼ň+gTɯĻÓǏG†X†,ʈĀśBȨɯǵɯĻů«Bµƞ' ?),ˏʼ .+ Hoenig =[17]- RIA .˼®ȇ!ůG†X†ů«p\G†X†BŻź?ʏʌ( $ !p\G†X†.gTG†X†) A8A10 <0 A18 - 3 Lũ(Exhˇ˄Õȏ+$' ? [16]-!9 Hoenig =- RIA (.˼gTG†X†,ʈĀśBȨůG†X†ɯǵɯĻů«BƱ Ò(+$!öɢś ?-öɢś.˼Hoenig =- RIA , '˼p\<0lbȈƥ-G† X†-ُǁ-+ gT˺¼ň˼ȻıȒ<0ȻıȒ¢ĕ-Ȓdž˻-ɹǡʔƋ=ůG†X†ů« ƱÒ@' + Ʌƨ=ɾ =@?  gT-ůG†X†ɯǵɯĻů«G†X†-¬ȇBʛȸ' ?)£Ģ?)˼ɹ‘-ůG†X. 17.

(31) † IgG -Žʧ;ʺÞȻıȒ-Ȓţ,ː³?öɢś ?!9˼ȻıȒ 18 ȓ¯-ůG†X† IgG -ɹǫǯŇBǧĢ!˼ȻıȒgT-ůG†X† IgG ǯŇ.2'¼ň+gT-ǧĢ¹-ȹ ĆË( >˼¼ň+gT)ȻıȒgT-ůG†X† IgG ǯŇ,ɇʌȘ+ĺ.+$!ȻıȒ 18 ȓ¯ ,., G†X†Ǎȕ‘-¶« 15 ¯û6@!G†X†Ǎȕ‘-gT.˼ُG†X†,Ĭ? ů«BȆȅ?) ?[17]G†X†Ǎȕ‘-ȻıȒgT(.˼ɹǫ‘,gTG†X†,ʈĀś BȨ IgG )ُ@' ?G†X†ʀÜ,Ĭ?ů«ǟĈ?öɢś >˼ȵ 1 Ȳ(¬ʀ! ELISA , '˼Ǝ-ů«Bè×(+$!)ɗ =@!gT, '˼ȻıȒ)ůG†X† ɯǵɯĻů«-ː³BȂʊ?!9,.˼ُG†X†,òŚ'Ȇȅ@!ůG†X†ů«-ː BŽ˚?!9,˼G†X†Ůǁ-+ ȻıȒȓ¯,% 'ʓ²?Řʃ ?ȵ 1 Ȳ, '.˼̀¯-G†X†Ůǁ-+ ȻıȒgT-ɹǫůG†X† IgG ǯŇBǧĢ!˼84 µg/ml˼ 86 µg/ml˼95 µg/ml ( >˼@=.¼ň+gT-ǧĢ¹-ȹĆË˺80 µg/ml = 1578 µg/ml˻( $ !˼¼ň+gT-Ɯ¨,ʯ ǯŇ( $!-Ʌƨ.˼ȻıȒȓ¯A 3 ¯)İ+ !9ŽȦ Ģ(. ?˼G†X†-ƽŁ+¬ȇ,ůG†X†ɯǵɯĻů«ːA?öɢśBȨ' ? -),% '.˼=,ǧĢ¯ƈBđ;'˼Ʊʒ?Řʃ ?  Nishii =[35, 36].˼G†X†-ˌ6!.ˌBʕʟ? 2 ȭ˫-ů€_bG†X†{hP„Š d‚ů«Bȇ !gTG†X†ǧĢȇU†cG_^ ELISA -ˎȖʺȬ, '˼ǴÑɒ-ɹǫ(.Ž öɢ( $!G†X†ǯŇ-ǧĢ˼ɹǫBˇ-E‚TŠ‚Ñɒ,<>Q„p†B˚ï?)(ǧ Ģöɢ)+$!)BĎý' ?-”ģ.˼gTɹǫ‘-3)C*-G†X†,Q„p† Ʌù' ?)BȨā?6!ȵ 1 Ȳ, '˼¼ň+gT-ɹǫůG†X† IgG -{‚ǯŇ˺ȹ Ć: 0.5 µM-10.8 µM˻˼G†X†-{‚ǯŇ˺ȹĆ: 0.05 nM-0.9 nM˻Bʾ,Œă$' !”ģ.˼gT-ɹ‘,.G†X†ÓĞB2'Ʌù?)-öɢ+ˊ-ůG†X† IgG ÓĞğ Ĉ' ?)BȨā?gT-ůG†X† IgG )G†X†-ɹ‘ǯŇ-ː³)úƵ,˼t‚{ †-ʫʱ;µğ-ŐßBų$' ?ɹ‘-ɅùQ„p†[8]( ?ȉǺɨt‚{†ɅùQ„p†[19] ;T‚^TX`„GcɅùQ„p†[45]-ɹ‘ǯŇ.˼ø-Ʌù?t‚{†-ɹ‘ǯŇ,Dž'ɶ. 18.

(32) ˵ )ȣ=@' ?gT-ɹ‘, '.˼ɹ‘Bʹ˟' ?ůG†X† IgG ÓǏ @!G†X†)Ʌù'˼IgG-G†X†ʁù«)'ơƯBŕȃ' ?öɢśɗ =@!ȵ 1 Ȳ, '˼ɹǫůG†X† IgG ǯŇ.ɋ IgG ǯŇ)-ˏ,ƽ-ȝːː³ʕ9=@!˼ɹǫG† X†ǯŇ)-ˏ,.ʕ9=@+$!-).˼ɋ IgG ,.‹Ģ-ßù(ůG†X† IgG û 6@˼ɹ‘-ůG†X† IgG -ˊ.G†X†,ː³+˼ɋ IgG -đǣ,±ğ'Ĕä?öɢś BȨ!  $ƿ˫(.ɍɊɳɁɠ;ɣɝɁɠ, '˼ůG†X†ů«G†X†-õħ«1-Ʌù;G †X†-¬ȇ[18, 46]BđŌ?)Ďý@' ?6!ȻıȒ-Gf, '˼ůG†X†ů «˼ُ@!G†X†-ëǣƟ-ʼnˍ,ː³' ?öɢśĎý@' ?[12]Nishii =-ȥ ȯ[36]<0-ȥȯ-ȵ 1 Ȳ6(, '˼gT-ůG†X†ɯǵɯĻů«,ː?ȣʄ.ɹ‘, ?ğĈ-7( >˼ůG†X†ɯǵɯĻů«-ȅȂȘƺɢ˼ǹ,ɯĻÓǏG†X†-¬ȇ, ?ãƨBȂʊ?,.şĎêÓ(+ gT-ůG†X†ɯǵɯĻů«-ȅȂȘ+ŐßBʊƔ? !9,.˼¼ň+gT-ɹǡ=Ⱥʀ!ůG†X† IgG )gTȈƥ-G†X†ƶȘɃɐBȇ !. in vitro ģ˴,<>˼ůG†X†ɯǵɯĻů«-ğĈȅ«,Ĭ?G†X†-¬ȇ, ?ãƨB Ʊʍ?Řʃ ?. 19.

(33) į Ŷ  ȵ 1 Ȳ(.gTG†X†,ʈĀśBȨ IgG -ɹǫǯŇBǧĢöɢ+ ELISA B¬ʀ!ELISA Bȇ '˼G†X†Ůǁ-+ ¼ň+gT 84 ˨-ɹǫǯŇBǧĢ˼ǧĢ!2'-g TůG†X† IgG Bµƞ?)˼-ǯŇ 80 µg/ml = 1578 µg/ml -ȹĆ, >˼‘ę¹ 221 µg/ml ( ?)BȨ!-Ʌƨ.˼¼ň+gTɹ‘,ɯĻG†X†Bůî)'ʕʟ? IgG Bɯǵů«)'µƞ?)-ʒŵ)+? $5

(34) ˼gT-ɹǫůG†X† IgG ǯŇ.˼g T-ś×˼Ń˹˼«ċ<0ɹǫG†X†ǯŇ)-ː³+˼=,ȻıȒ+*-ȑȒ-gT, '˵ǯŇ ? .¨ǯŇBȨ).+$!¼ň+gT-ŷ%ůG†X†ɯǵɯĻů«.˼l b;Gf, 'ȑŞ-ȒĄ)+?ůG†X†ů«).ƢʥȘ,ȏ+?˼G†X†-ÀBʛȸ ?ɯǵɯĻů«)'ƽŁ+ȅÿǗä-Ɋŷ,ʡǽ?öɢś ?. 20.

(35) ȵ2Ȳ gT-ůG†X† IgG G†X†-¬ȇ, ?ãƨ. Ɍ ʋ  Nishii =[36],<$'˼¼ň+gTµƞ?)BȦʕ@!ůG†X† IgG .˼G†X†Ů ǁ-+ ¼ň+gT 10 ˨-ɹǡ=ƱÒ@!)=˼ĕƥśG†X†,òŚ'Ȇȅ@!ů «(.+˼ɯĻG†X†,ɅùɢBȨɯǵɯĻů«( ?)ɗ =@!6!ȵ 1 Ȳ, '˼ gTG†X†,ʈĀśBŷ% IgG BƱÒ? ELISA ,<>˼ǧĢ!G†X†Ůǁ-+ ¼ň +gT 84 ˨-ɹǫ2'=ůG†X† IgG ƱÒ!˼ -Ʌƨ.gTůG†X†ɯǵɯĻ ů«Bµƞ?)BʒƔ' ?  ȏȭäǸ˺HV˼p\˻Ȉƥ-G†X†ŮǁBŷ%lb-ȻıȒŞɘ(.˼ůG†X†ů« ُG†X†-õħ«Ʌù˃§,ȴùȘ,Ʌù?)(˼Ǎȕűůś-îĄ)+?)Ďý@ ' ?[6, 7, 44] $5

(36) (˼wHXȈƥ-Č˰ɁɠBȇ !ȥȯ, '˼{‚{_b,p\ȈƥG †X†Bُ'Ȇȅ!ůG†X†ů«˼G†X†-õħ«Ʌù) RTK VQd‚ǗśæB đŌ?)Ďý@' ?[18, 21, 46]G†X†Bʕʟ?ɯǵɯĻů«-ğĈ,% '.˼g T¢ĕ(:lb[40];wHX[41], 'Ďý@' ?˼G†X†-¬ȇ, ?ãƨ,ː?ş Ď.+ ¼ň+gT-ŷ%ůG†X† IgG .˼lb, 'ȑŞ-ȒĄ)+?ůG†X†ů«) .ƢʥȘ,ȏ+?)ɗ =@?˼‹ȅBʳġĢ'ɹ‘,ğĈ?)(ƽŁ+ȅÿǗäBɊŷ ?ÀBų

(37) ɯǵɯĻů«[30]( ?)Bɗť?)˼gT,)$'ƞȇ+ȅȂȘŐßBŷ%öɢś. ?gTȪäǸ.˼ȟśɚˬäǸ( >[5]˼DzLJæǸ(.+˼Exhˇ)ɣʥB‘ŗ)!ǹƞƬ˰¡ʞBŷ%-ǹƞ-Ƭ˰¡ʞ,ʿŚ?!9,˼ƔȦ+ƭŵ.+ ˼gTůG†X†ɯ ǵɯĻů«BØȇ' ?öɢśƀĢ@?  ȵ 2 Ȳ(.˼gT-ůG†X†ɯǵɯĻů«-ȅȂȘŐßBʊƔ?!9,˼gT-ůG†X† IgG G†X†-¬ȇ,ð4ãƨBʓ²!¼ň+gT-ɹǡ=G†X†,Ĭ?ʈĀśB. 21.

(38) č,Ó˟!ůG†X† IgG )gT-Č˰ŧǷɣɝɁɠBȇ '˼ůG†X† IgG -ğĈ˼G† X†Ǡá,<?ɁɠË1-Ȼô>ʮ7ɢ)@,ːA?õħ«VQd‚Ǘśæ, ?ãƨBƱʍ !. 22.

(39) ƣƋ<0Ǝǐ 1. YG =&#& IgG .a  G†X†Ůǁ-+ 8 ˨-¼ň+ŧgT=žô!ɹǜ=ɹǡBÓ˟˼ǟù!ǟùɹ ǡ‘,û6@? IgG B MelonTM Gel Chromatography Cartridge˺Thermo Fisher Scientific˻Bȇ ' Ó˟!L€yǪÒǜ‘- IgG ,.G†X†B.9)?sq^cɅù' ?!9˼ǪÒǜ 1 ml ,Ĭ 1N - HCl ǪǜB 150 μl á ?),<$' IgG )-sq^c-ɅùBʊ˼ʊ˟!s q^cB˘ĕǰʺ~e_b˺Vivaspin20-30K, GE Healthcare˻(˚ï!˘ĕǰʺ~e_b,ǂȋ ! IgG Bû8njǥ,.˼ÅȐQ„p†-ĔśBˁ?!9,˼0.01 M - PBS 20 ml Bá '‘Ā !‘Ā! IgG njǥ.˼=,˘ĕǰʺ~e_b(ǯɏ˼ɋ IgG ÓȊ)!  ɋ IgG ÓȊ.˼G†X†,Ĭ?ʈĀś,č& !EoFe`FP„wbQ€oFŠ,°?) ,<>ůG†X†IgG BÓ˟! EoFe`FL€y ˺HiTrap NHS-activated HP, GE Healthcare˻ ,ćȝæ?G†X†)'˼ÇŪąˠ+gTG†X†-¡A>,HVG†X†˺Alpha Diagnostic, San Antonio, TX, USA˻Bȇ !HVG†X†.gTG†X†)Exhˇ˄Õ A18 - 1 Lũȏ+?-7[16]( ?EoFe`FL€y-žƵƛ,œ ˼G†X†-ćȝæ)ɋ IgG ÓȊ-ǠáBģƏ˼ÓȊ-ǪÒ. HPLC˺TOSO HPLC system, TOSO, Tokyo, Japan˻,<>˼3 M NaCl, 1.5 M glycine buffer solution˺buffer A, pH 8.5˻) 0.05 M phosphate buffer solution˺buffer B, pH 7.2˻Bȇ !¢-ɍŎQ€WJ†bq„Q€y,œ$'ģƏ!; 0 Ó 25 Ó, 100% buffer A; 25 Ó 35 Ó, 100%-50% buffer A + 0%-50% buffer B; 35 Ó 40 Ó, 50% buffer A + 50% buffer B; 40 Ó 50 Ó, 50%-0% buffer A + 50%-100% buffer BǪÒʴŇ. 0.5 ml/Ó,ʏĢ˼ǪÒÓȊ-üÄŇ.ì Ǒˍ 280 nm ({e\Š!˺Fig. 2-1; UV-8010, TOSO˻   HPLC ,<?ǪÒÓȊ, '˼üÄŇ-ŒƓBȨÓȊ,.ůG†X† IgG û6@˼ǪÒƤ ¤B buffer A B 100%= 50%,˼buffer B B 0%= 50%,Ĕƚ' ʺȬ(ǪÒ@?ȵ 1 ÓȊ ) buffer A B 50%= 0%,˼buffer B B 50%= 100%,Ĕƚ' ʺȬ(ǪÒ@?ȵ 2 ÓȊB Ŕ!HPLC ,<>ÓȊ!ȵ 1 ÓȊ<0ȵ 2 ÓȊ-G†X†,Ĭ?ɅùʈĀś.˼ɽˤq€Y. 23.

(40) {†É˸Ŭɻ˺BIAcoreTM J, GE Healthcare˻,<>ȷÒ@!ɅùʴŇĢƈ˺ka˻<0ʊ˟ʴŇĢ ƈ˺kd˻= BIA evaluation˺ver. 3.1˻software˺GE healthcare˻Bȇ ' KD ¹˺KD = kd/ka˻) 'Ŕ!ȵ 1 ÓȊ<0ȵ 2 ÓȊ-G†X†,Ĭ?ɅùʈĀś.˼@ @˼1.6110-4 M˼2.00 10-5 M ( $!. 10. 20. 2nd fraction. KD: 1.61×10-4 M. KD: 2.00×10-5 M. 30. 40. 50. 3. 60. 2.25. 1.5. Glycine (M). 1st fraction. NaCl (M). 4.5. Absorbance at 280 nm. 0. The composition of elution buffer. Anti-insulin IgG. Pass fraction . 1.5. 0.75. 0. 0. 70. Time (min). Fig. 2-1. Representative elution pattern of anti-insulin IgG from sera of cats by affinity chromatography on immobilized bovine insulin. .  ɋ IgG ÓȊ)EoFe`FP„wbQ€oFŠ,?ȿʳ>ÓȊ<0ǪÒÓȊ( ?ȵ 1 ÓȊ) ȵ 2 ÓȊB 15%SDS EP‚ExcS‚˺SuperSep TM Ace; ĀÄȾɷ, ė˓˻,<?ˡdžǔä) CBB ƪɲ,°!ůG†X† IgG -ȵ 1 ÓȊ<0ȵ 2 ÓȊ,.˼EoFe`FP„wbQ€oFŠ, ?Żźůî)'ȇ !HVG†X†-ǟÇ+ )BȦ9!˺Fig. 2-2˻ ģ˴,.˼ůG †X† IgG )'ȵ 1 ÓȊ<0ȵ 2 ÓȊBȇ ! ȵ 1 ÓȊ<0ȵ 2 ÓȊ- IgG ǯŇ.˼ gT IgG ǧĢȇ ELISA˺Cat IgG ELISA Quantitation Set, Bethyl Laboratories, Montgomery, TX, USA˻Bȇ 'Ȧʕ!. 24.

(41) (kDa). 1. 2. 3. 4. 5. 6. 75 50 37 25 20. 15 10. ←Approximately 6 kDa. Fig. 2-2. SDS-PAGE results of purified IgG from sera of healthy domestic cats. SDS-PAGE show clear biphasic bands 25 and 50 kDa in size, which is consistent with immunoglobulin. Lane 1: Molecular weight marker. Lane 2: Purified whole IgG. Lane 3: Passed fraction. Lane 4: 1st fraction of affinity chromatography on immobilized bovine insulin. Lane 5: 2nd fraction of affinity chromatography on immobilized bovine insulin. Lane 6: Bovine insulin.. 25.

(42) 2. VTQU 6c.3  gTȈƥ-ɣɝÛ˳Ɂɠ˺Primary Normal Feline Preadipocytes, Zen-Bio, Research Triangle, NC, USA˻.˼ļʢ-Ȗɞ˼Óæ˼ɊŷČĉ˺Zen-Bio˻B®$'ŧǷČ˰!˼2'-Č˰ĹȬB˼37°C˼ 5% CO2 (ģƏ!ŧǷɣɝɁɠ.˼ģ˴Û,ɹǡ)G†X†-ŏ˥B˚ï?!9, 4 Ɩˏ˼ɹ ǡ)G†X†Bû6+ ɊŷČĉ(˭˱Č˰!. 3. 2-Deoxy-D-glucose uptake assay  gTŧǷɣɝɁɠ1-Ȼô>ʮ7ɢ-ʓ².˼Klip =[25]-Ǝǐ,œ$'ģƏ!Q‚TŠXbƒ ŠUŠ)'[3H]-2-DOG˺PerkinElmer, Boston, MA, USA˻Bȇ !Q‚TŠXbƒŠUŠ.˼0.1 mM [3H]-2-DOG ) 10.2 mM ˣƶʟ 2-DOG B PBS (ľˈ'˼0.2 μCi ,ʛƉ'®ȇ!6!˼ Ǡá?G†X†)'HVG†X†Bȇ !˭˱Č˰!ɣɝɁɠ.ȅȂƤ¤,ġĢ? !9,˼Q‚TŠXBû6+ 0.1% BSA ûƞ HSBS˺pH 7.4˻Bá ' 37°C˼5% CO2 ( 30 Óˏ ÛČ˰! -ő˼ Ɂɠ, HSBS (ɋˊ 100 μl ,ʛƉ!¢- 5 ȭ˫-ǪǜB¢-ǯŇ(Ǡá˼ =, 30 ÓˏČ˰!; 1) ǴÑȂ˼2) ÛČ˰,®ȇ! HSBS˺vehicle˻ ˼3) G†X† 10-7 M -ì ǻ˼4) ůG†X† IgG -ȵ 1 ÓȊ<0ȵ 2 ÓȊ 10-12 M, 10-11 M, 10-10 M -ìǻ5) G†X† 10-7 M )ůG†X† IgG -ȵ 1 ÓȊ<0ȵ 2 ÓȊ 10-12 M, 10-11 M, 10-10 M -ǟùǠá?G†X† -ʿȇǯŇ,.˼ “¾ʔ˴, 'ǴÑȂ-Ɂɠ-Ƚ 2.5 ·-[3H]-2-DOG ô>ʮ7ˊBʕ9! 10-7 M B žȇ!˺Fig. 2-3˻ bƒŠUŠǪǜBɁɠ,á ' 10 ÓˏČ˰!ő˼LjÍ! PBS B 1 ă !> 0.5 ml ȇ '˼4 ăǖǘ!-ő˼0.5 ml - NaOH (ɁɠBǪʊ'˼ÈˊBV†^ƒŠV†j GE‚,ȫ!=, 0.5 ml - HCl Bá 'ɁɠǪʊǜB‘Ā!ő,˼9 ml -ǜ«V†^ƒŠV †LP`‚˺Ultima Gold, PerkinElmer˻Bá '˼[3H]-2-DOG -ô>ʮ7ˊB radioactivity ) 'ǜ«V†^ƒŠV†LH†\Š˺Tri-Carb2900TR; Packerd, Meriden, CT, USA˻(ǧĢ!Ŕ =@!¹.˼BCA ǐ(ǧĢ!ɁɠǪʊǜ-\†kPʥǯŇ˺Pierce BCA Protein Assay Kit; Thermo Fisher Scientific˻(ɿƽ!Ʌƨ.ǴÑȂ-Ɂɠ-Ȼô>ʮ7ˊB 100%)!ȗÓǿ˺%˻(ɽȨ. 26.

(43) !ģ˴. 1 ʔ˴ɔ !> 3 ăɑ>ʰ˼Ʌƨ.łĊ¹ƶǩºĺ(Ȩ!. [3H]-2-DOG uptake percentage of non-treatment control. 300. 200. 100. 0 0. 12. 11. 10. 9. 8. 7. -log [Insulin] M. Fig. 2-3. Mean (+SD) percentages of the insulin-induced [1,2-3H]-2-Deoxy-D-glucose ([3H]-2-DOG) uptake in adipocytes derived from cats. Results (n=3) describe non-treatment control as 100%.. 4. VTQU#&_3%&&] C gTŧǷɣɝɁɠBȇ '˼G†X†<0ůG†X† IgG -ȵ 1 ÓȊ6!.ȵ 2 ÓȊ-ğĈ RTK VQd‚Ǘśæ, ?ãƨBʓ²?!9,˼†ˇæ^„V†\†kPʥ-ɄƖȘĔæBHJ X\†p„_b,<>ƱÒ!†ˇæ^„V†\†kPʥ-ƱÒ,.˼wHXů†ˇæ^„V† ů«˺p-Tyr (PY20): sc-508, Santa Cruz biotechnology, Santa Cruz, CA, USA˻Bȇ !ů†ˇæ ^„V†ů«.wHXȈƥ-ìP„Š†ů«-!9˼G†X†BǠá!gTɣɝɁɠ=ŲÒ! ɁɠǪʊǜ,?òŚśB“¾ʔ˴(Ȧʕ!G†X†BǠá!wHXȈƥ-Ɂɠ=ɄƖȘ ,ŲÒ!ɁɠǪʊǜ,ů†ˇæ^„V†ů«Bެ?)˼†ˇæ! IRS-1˺185 kDa˻ ˼IRβ ˺95 kDa˻ ˼Akt˺60 kDa˻- 3 ȭ-†ˇæ^„V†\†kPʥƱÒ@?[47]“¾ʔ˴, '˼ gTɣɝɁɠ=ŲÒ!ɁɠǪʊǜ,wHXů†ˇæ^„V†ů«Bެ?)˼Ɩˏ<0ǯŇ ±ğś,ƱÒ@!†ˇæ^„V†\†kPʥ.˼†ˇæ IRS-1 -7( $!˺Fig. 2-4˻ (˼ ƱÒ?†ˇæ^„V†\†kPʥ)'†ˇæ Akt Bá !ƱÒ,.˼Ser473 B†ˇæ!. 27.

(44) Akt BǹȏȘ,ƱÒ?HUOů†ˇæ Akt ů«˺Cell Signaling Technology, Beverly, MA, USA˻ Bȇ !ȇ !HUOů†ˇæ Akt ů«.gT-†ˇæ Akt ,Ĭ?˜ĺòŚśȦʕ@' +$!!9˼G†X† 10-7 M BǠá!gTŧǷɣɝɁɠ=ŲÒ!ɁɠǪʊǜ,?˜ĺò ŚśB“¾Ș,Ʊʍ˼Ɩˏ±ğś-òŚBȦʕ!˺Fig. 2-5˻ 6!˼iHXNŠn†Q\†kPʥ -ƱÒ,.˼ů β-actin ů«˺Cell Signaling Technology˻Bȇ !  ˭˱Č˰!ɣɝɁɠ,, ˭˱Č˰,ȇ !ɹǡ)G†X†Bû6+ ɊŷČĉ(ɋˊ 100 μl ,ʛ Ɖ!¢- 4 ȭ˫-ǪǜB¢-ǯŇ(Ǡá˼=, 0 Ó˼1 Ó˼3 Ó˼5 Ó˼15 Ó˼30 Ó˼60 Ó ˏČ˰!; 1˻˭˱Č˰,ȇ !ɊŷČĉ˺vehicle˻ ˼2˻G†X† 10-7 M -ìǻ˼3˻ůG†X† IgG -ȵ 1 ÓȊ6!.ȵ 2 ÓȊ 10-10 M -ìǻ˼4) G†X† 10-7 M )ůG†X† IgG -ȵ 1 ÓȊ 6!.ȵ 2 ÓȊ 10-10 M -ǟùČ˰ő-Ɂɠ.˼†ˇæˆȿ˕ĦÜ˺PhosSTOP; Roche Applied Science, Basel, Switzerland˻Ç>-Ɂɠ\†kPʥŲÒN_b˺Complete Lysis-M; Roche˻,<>Ǫ ʊ!Ǫʊ!Ɂɠ.˼SDS-PAGE ȇU†q‚j_oDŠ˺0.125 M Tris-HCl, 4.3% SDS, 30% glycerol, 10% 2-mercaptoethanol, 0.01% bromophenol blue [pH 6.8]; TX{jGK, Ʀ™˻)ȶˊǟù ˼2 ÓˏuG‚!ő,˼10% SDS vEP‚ExcS‚˺SuperSepTM Ace; ĀÄȾɷ˻,<>ˡ džǔä!Ó˟!\†kPʥ.˼eb„Z‚„ŠXɪ˺AmershamTM HybondTM –ECL; GE healthcare˻, 80 V˼3 Ɩˏʳˡ-Ƥ¤(ʩÌ!ʩÌő-eb„Z‚„ŠXɪ. 2%HVɹǡE‚p x†Bá ! 0.2% Tween 20-TBS (p„N†Q˺4, overnight˻!0.2% Tween 20-TBS (ǖǘ ő˼Can Get SignalTM ˺ƦǕɀ, Ʀ™˻(ľˈ!‹ƻů«˺ů†ˇæ^„V†ů«, 1 : 200, ů† ˇæ Akt ů«, 1 : 3000, ů β-actin ů«, 1 : 1000˻(òŚ!˺ĥǦ˼1 Ɩˏ˻ ‹ƻů«òŚő˼0.2% Tween 20-TBS (eb„Z‚„ŠXɪBǖǘ˼Can Get SignalTM ( 4000 ·,ľˈ! HRP ƶʟ• ƻů«˺anti-mouse or rabbit HRP conjugate, GE Healthcare˻(òŚ!eb„Z‚„ŠXɪ. 0.2% Tween 20-TBS (ǖǘ!ő˼ECL system˺Western Lightning ECL Pro; PerkinElmer˻(ů«òŚ BƱÒ!ƱÒ!VQd‚ŌŇ. ChemiDoc ˺Bio-Rad, Hercules, CA, USA˻(ƈ¹æ˼β-actin -ƱÒˊ(ɿƽ!ő˼Ʌƨ.Č˰Ɩˏ 0 Ó-Ɂɠ,?ƱÒˊB 100%)!ȗÓǿ˺%˻(ɽȨ. 28.

(45) !ģ˴. 1 ʔ˴ɔ !> 3 ăɑ>ʰ˼Ʌƨ.łĊ¹ƶǩºĺ(Ȩ!. 5. RZ.A  gTɣɝɁɠ1-G†X†˼ůG†X† IgG ÓȊ-ȵ 1 ÓȊ<0ȵ 2 ÓȊ-ìǻǠá,<?Ɂ ɠ1-[3H]-2-DOG ô>ʮ7ˊ.˼øģ˴ɔ-aŠ\-ȶÓƇśB Bartlett ƱĢ,<>Ȧʕ!ő,˼ vehicle Ǡá,<?ô>ʮ7ˊ)-ĺB‹Ã˄ɒÓƇÓƧ) Dunnett ǐ,<>Džʪ!6!˼ůG† X† IgG -ȵ 1 ÓȊ6!.ȵ 2 ÓȊ-ğĈ,<?G†X†Ǡáő-Ɂɠ-[3H]-2-DOG ô>ʮ7ˊ .˼øģ˴ɔ-aŠ\-ȶÓƇśB Bartlett ƱĢ,<>Ȧʕ!ő,˼G†X†ìǻǠá,<?ô> ʮ7ˊ)-ĺB‹Ã˄ɒÓƇÓƧ) Dunnett ǐ,<>Džʪ!=,˼Ʊʍ!ůG†X† IgG ȵ 1 ÓȊ<0ȵ 2 ÓȊ, 'ǠáǯŇ˺10-12 M, 10-11 M, 10-10 M˻ˏ-[3H]-2-DOG ô>ʮ7ˊ-ĺ .˼‹Ã˄ɒÓƇÓƧ) Tukey-Kramer ǐ,<>@ @Džʪ!Ɂɠ1-ůG†X† IgG -ȵ 1 ÓȊ6!.ȵ 2 ÓȊ-gTɣɝɁɠ1-ìǻǠá,<?†ˇæ^„V†\†kPʥ˺†ˇæ IRS-1 <0†ˇæ Akt˻-ƱÒˊ.˼Ǡáő-ɄʺƖˏ˺Ó˻DŽ, F ƱĢ,<>aŠ\-ȶÓƇśBȦʕ !ő,˼ vehicle Ǡá,<?ƱÒˊ)-ĺB Student - t-ƱĢ,<>Džʪ!6!˼ůG†X† IgG -ȵ 1 ÓȊ6!.ȵ 2 ÓȊ-ğĈ,<?G†X†Ǡáő-Ɂɠ,?†ˇæ^„V†\†k Pʥ-ƱÒˊ.˼Ǡáő-ɄʺƖˏDŽ, F ƱĢ,<>aŠ\-ȶÓƇśBȦʕ!ő,˼G†X†ì ǻǠá,<?ƱÒˊ)-ĺB Student - t-ƱĢ,<>Džʪ!p<0.05 BɇʌȘ,ƞš)!. 29.

(46) B) Insulin dose dependent responses. A) Time dependent responses after addition of insulin 10-7 M. (kDa) 250. 0. 3. (Incubation time; 3 min) 10-9 10-8 10-7 (M). 30 (min). 10. ← Phospho-IRS-1. 150. (185 kDa). 100 75. 50 β-actin (45 kDa). Fig. 2-4. Effect of insulin on tyrosine–phosphorylated proteins in adipocytes derived from cats. Matured adipocytes were treated with bovine insulin for indicated times (A) and indicated concentrations (B). Cells were lysed, and phosphorylated proteins were detected with immunoblotting with anti-phosphotyrosine antiboby (PY20; mouse monoclonal antibody designed to specially detect phosphorylated tyrosine residues). Although PY20 can detect three major phosphoproteins (phospho-IRS-1, -IRβ and -Akt) observed after insulin treatment in 3T3-L1 adipocytes derived from mouse, only phosphorylated IRS-1 are detectable on adipocytes derived from cats.. 0. 1. 3. 5. 10. 20. 30. 60. 90. 120. (min). Phospho-Akt (60 kDa). β-actin (45 kDa). Fig. 2-5. Effect of insulin 10-7 M on phosphorylated Akt and β-actin in adipocytes derived from cats. Matured adipocytes were treated with bovine insulin for indicated times. Cells were lysed, and phosphorylated Akt and β-actin were detected with immunoblotting with anti-phosphorylated Akt antibody and anti-β-actin antibody. Antiphosphorylated Akt antibody detected a time dependent response of phosphorylated Akt in insulin-treated cells.. 30.

(47) Ʌƨ 1i =&#& IgG &#&F0VTQU [1,2-3H]-2-Deoxy-D-glucose 4 ^

(48) (1B  Vehicle )'ȇ ! HSBS -Ǡá,<?gTɣɝɁɠ1-[3H]-2-DOG -ô>ʮ7ˊ.˼ǴÑȂ-Ɂ ɠ,?ô>ʮ7ˊ˺100%˻)34úȶ˺105.46.6%˻( >˼vehicle Ǡá,<?ô>ʮ7ˊđá .+$!G†X† 10-7 M -ìǻǠá,<?Ɂɠ1- [3H]-2-DOG -ô>ʮ7ˊ.˼vehicle Ǡá ,<?ô>ʮ7ˊ,Dž2'ɶđá!˺p<0.01, Fig. 2-6A˻  $5

(49) ˼ȵ 1 ÓȊ<0ȵ 2 ÓȊìǻǠá,<?Ɂɠ1- [3H]-2-DOG -ô>ʮ7ˊ.˼Ʊʍ!ǯŇ˺10-12 M, 10-11 M <0 10-10 M˻ , ' vehicle Ǡá,<?ô>ʮ7ˊ)úȶ(˼Ȼô>ʮ7ˊBđá+$!˺Fig. 2-6A˻ G† X†Ǡá,<?Ɂɠ1-[3H]-2-DOG -ô>ʮ7ˊ.˼ȵ 1 ÓȊB):,Ǡá?)(ɶđá !˺p<0.01˻˼đáˊ.ȇˊ±ğś(.+$!˺Fig. 2-6B˻ ˼G†X†Ǡá,<? Ɂɠ 1-[3H]-2-DOG -ô>ʮ7ˊ.˼ȵ 2 ÓȊB):,Ǡá':˼Ʊʍ!2'-ǯŇ(˼G†X† ìǻǠá,<?ô>ʮ7ˊ)úȶ( $!˺Fig. 2-6B˻ . 31.

(50) 300. p<0.01. 200. 100. 0. Vehicle. -12 10Low-affinity 10-11 IgG 10-10 M. Insulin 10-7 M I. N. Nontreatment control. V. [3H]- 2-DOG uptake percentage of non-treatment control. A) Insulin alone and anti-insulin IgG fractions alone . Anti-insulin IgG 1st fraction. -12 10-10 M 10High-affinity 10-11 IgG. Anti-insulin IgG 2nd fraction. B) Insulin with or without anti-insulin IgG fractions. *. [3H]- 2-DOG uptake percentage of non-treatment control. **. *. 400. ††. †† ††. 300. 200. 100. 0 Insulin Insulin 10-7 M. 10-11 IgG 10-12 10-10 M Low-affinity Anti-insulin IgG 1st fraction + Insulin 10-7 M. 10-10 M 10-12 10-11 IgG High-affinity Anti-insulin IgG 2nd fraction + Insulin 10-7 M. Fig. 2-6. Mean (±SD) percentages of [1,2-3H]-2-Deoxy-D-glucose ([3H]-2-DOG) uptake in feline adipocytes. Results (n=3) describe non-treatment control as 100%. †† denote significant differences from insulin alone at p<0.01. * and ** denote significant differences from other concentration at p<0.05 and p<0.01 respectively. A) Insulin alone and anti-insulin IgG fractions alone. B) Insulin with or without anti-insulin IgG fractions. . 32.

(51) 2. =&#& IgG &#&F0VTQU RTK $D;3(1B  Vehicle )'ȇ ! HSBS -gTɣɝɁɠ1-Ǡá,<?†ˇæ IRS-1 -ƱÒˊ.˼Ǡá= 60 ÓˏĔæ+$!˺Fig. 2-7 A˻ ɣɝɁɠ1-G†X†-ìǻǠá,<?†ˇæ IRS-1 -ƱÒˊ .˼˾Ó= 3 Ó, ' vehicle Ǡá,<?ƱÒˊ<>:ƞš,Ė$!˺p<0.05˻˼5 Ó¢˗, 'úƵ,Ĕæ!˺Fig. 2-7 B˻ ůG†X† IgG -ȵ 1 ÓȊ<0ȵ 2 ÓȊ-ɣɝɁɠ1-ìǻǠ á,<?†ˇæ IRS-1 -ƱÒˊ.˼60 ÓˏBʳ'˼vehicle Ǡá,<?ƱÒˊ)úƵ,Ĕæ! ˺Fig.2-7C <0 D˻  $5

(52) ˼ɣɝɁɠ1-G†X†Ǡá,<?†ˇæ IRS-1 -ƱÒˊ.˼ȵ 1 ÓȊB):,Ǡá?)˼5 Óő,Ɯė,ʻ'˼@¢˗˼Œ,ǣİ!˼Ǡá= 30 Ó6(G †X†ìǻǠá,<?ƱÒˊ<>ƞš,Ė$!˺Fig. 2-8B˻ 6!˼ɣɝɁɠ,G†X†)ȵ 2 Ó ȊB):,Ǡá?)˼†ˇæ IRS-1 -ƱÒˊ.˼60 ÓˏBʳ'˼G†X†ìǻǠá,<?ƱÒ ˊ)úƵ,Ĕæ!(Fig. 2-8C)  Vehicle )'ȇ ! HSBS -gTɣɝɁɠ1-Ǡá,<?†ˇæ Akt -ƱÒˊ.˼Ǡá= 60 ÓˏĔæ+$!˺Fig. 2-9A˻ ɣɝɁɠ1-G†X†-ìǻǠá,<?†ˇæ Akt -ƱÒˊ.˼ 5 Ó(Ɯė,ʻ!ő˼Œ,¨!˼Ǡá= 60 ÓˏBʳ'˼vehicle Ǡá,<?ƱÒˊ<> ƞš,Ė$!˺Fig. 2-9B˻ ůG†X† IgG -ȵ 1 ÓȊ-ɣɝɁɠ1-ìǻǠá,<?†ˇæ Akt -ƱÒˊ.˼15 Ó˺p<0.05˻) 30 Ó˺p<0.01˻, '˼vehicle Ǡá,<?ƱÒˊ<>@ @Ė $! (Fig. 2-9C) 6!˼ ȵ 2 ÓȊ-ɣɝɁɠ1-ìǻǠá,<?†ˇæ Akt -ƱÒˊ.˼ 5Ó ˺p<0.05˻ ) 15 Ó˺p<0.01˻, '˼vehicle Ǡá,<?ƱÒˊ<>@ @Ė$!˺Fig. 2-9D˻  $5

(53) ˼ ɣɝɁɠ1-G†X†Ǡá,<?†ˇæ Akt -ƱÒˊ.˼ȵ 1 ÓȊB):,Ǡá?)˼1 ÓőƱÒˊ.G†X†ìǻǠá<>:ƞš,İ+$!˺p<0.01˻˼-őđá˼5 Óő,Ɯė,ʻ !˺Fig. 2-10B˻ ˼ɣɝɁɠ1-G†X†ìǻǠá,<?ƱÒˊǣİĝ9! 15 Ó¢˗˼ G†X†)ȵ 1 ÓȊ-Ǡá,<?†ˇæ Akt -ƱÒˊ.Ǡá= 30 Óő6(G†X†ìǻǠá <>ƞš,Ė$!˺p<0.05˻ 6!˼ɣɝɁɠ,G†X†)ȵ 2 ÓȊB):,Ǡá?)˼†ˇæ Akt -ƱÒˊ. 60 ÓˏBʳ'˼G†X†ìǻǠá)úƵ,Ĕæ!˺Fig. 2-10C˻ . 33.

(54) A) Vehicle (○). 1. 3. 5. 15. 30. 60. 1. 3. 5. 15. 30. 60. 0. Time (min) (○). Phosphorylated IRS-1 percentage of non-treatment cells. B) Insulin alone (●) and vehicle alone (○) 400. Time (min) § §. 0. (●). 300 200 100 0 0. 15. 30. 45. 60. Time (min). Phosphorylated IRS-1 percentage of non-treatment cells. C) 1st fraction alone () and vehicle alone (○) 400. Time (min). 300. (). 0. 1. 3. 5. 15. 30. 60. 200 100 0 0. 15. 30. 45. 60. Time (min). Phosphorylated IRS-1 percentage of non-treatment cells. D) 2nd fraction alone () and vehicle alone (○) 400. Time (min) (). 300. 0. 1. 3. 5. 15. 30. 60. 200 100 0 0. 15. 30. 45. 60. Time (min ). Fig. 2-7. Mean (±SD) percent changes and representative western blots of phosphorylated IRS-1 in feline adipocytes. Results (n=3) describe time 0 as 100%. § denote significant differences from vehicle at p<0.05. A) Vehicle alone (○). B) Insulin 10-7 M alone (●) and vehicle alone (○). C) 1st fraction 10-10 M alone () and vehicle alone (○). D) 2nd fraction 10-10 M alone () and vehicle alone (○).. 34.

(55) A) Insulin (●) Time (min). 0. 1. 3. 5. 15. 30. 60. 3. 5. 15. 30. 60. (●). Phosphorylated IRS-1 percentage of non-treatment cells. B) 1st fraction + insulin (▲) and insulin (●) Time (min). 1,500. * *. **. 0. 1. (▲). 1,000. **. *. 500. 0 0. 15. 30. 45. 60. Time (min). Phosphorylated IRS-1 percentage of non-treatment cells. C) 2nd fraction + insulin () and insulin(●) 1,500. Time (min). 0. 1. 3. 5. 15. 30. 60. () 1,000. 500. 0 0. 15. 30. 45. 60. Time (min ). Fig. 2-8. Mean (±SD) percent changes and representative western blots of phosphorylated IRS-1 in feline adipocytes. Results (n=3) describe time 0 as 100%. * and ** denote significant differences from insulin alone at p<0.05 and p<0.01 respectively.  A) Insulin 10-7 M alone (●). B) 1st fraction 10-10 M + insulin 10-7 M (▲) and insulin 10-7 M alone (●). C) 2nd fraction 10-10 M + insulin 10-7 M () and insulin 10-7 M alone (●).. 35.

(56) A) Vehicle (○) Time (min) (○). 0. 1. 3. 5. 15. 30. 60. 0. 1. 3. 5. 15. 30. 60. Phosphorylated Akt percentage of non-treatment cells. B) Insulin alone (●) and vehicle alone (○) Time (min). 2000 1500. §§. 1000 500. (●). §§ §§. §§. 15. 30. §§. §§. 0 0. 45. 60. Time (min). Phosphorylated Akt percentage of non-treatment cells. C) 1st fraction alone () and vehicle alone (○) 0. Time (min) (). 2000. 1. 3. 5. 15. 30. 60. 1500 1000 500. §. §. 15. 30. 0 0. 45. 60. Time (min). Phosphorylated Akt percentage of non-treatment cells. D) 2nd fraction alone () and vehicle alone (○) 0. Time (min). 2000. 1. 3. 5. 15. 30. 60. (). 1500 1000 500. §. §§. 0 0. 15. 30. 45. 60. Time (min). Fig. 2-9. Mean (±SD) percent changes and representative western blots of phosphorylated Akt in feline adipocytes. Results (n=3) describe time 0 as 100%. § and §§ denote significant differences from vehicle at p<0.05 and p<0.01 respectively. A) Vehicle alone (○). B) Insulin 10-7 M alone (●) and vehicle alone (○). C) 1st fraction 10-10 M alone () and vehicle alone (○). D) 2nd fraction 10-10 M alone () and vehicle alone (○). 36.

(57) A) Insulin alone (●) Time (min) (●). 0. 1. 3. 5. 15. 30. 60. 5. 15. 30. 60. B) 1st fraction group + insulin (▲) and insulin alone (●). Phosphorylated Akt percentage of non-treatment cells. Time (min) 2000. 0. 1. 3. (▲). 1500. *. *. 15. 30. 1000 500. ** 0 0. 45. 60. Time (min). Phosphorylated Akt percentage of non-treatment cells. C) 2nd fraction + insulin () and insulin alone (●) Time (min). 2000. 0. 1. 3. 5. 15. 30. 60. () 1500 1000 500 0 0. 15. 30. 45. 60. Time (min). Fig. 2-10. Mean (±SD) percent changes and representative western blots of phosphorylated Akt in feline adipocytes. Results (n=3) describe time 0 as 100%. * and ** denote significant differences from insulin alone at p<0.05 and p<0.01 respectively. A) Insulin 10-7 M alone (●). B) 1st fraction 10-10 M + insulin 10-7 M (▲) and insulin 10-7 M alone (●). C) 2nd fraction 10-10 M + insulin 10-7 M () and insulin 10-7 M alone (●).. 37.

(58) ɗ Ī  ȵ 2 Ȳ(., ¼ň+gT-ɹǡ=EoFe`FP„wbQ€oFŠ) HPLC ,<>G†X†,Ĭ ?ɅùʈĀś˺KD ¹˻-¨ ȵ 1 ÓȊ)˼˵ ȵ 2 ÓȊ,Ó˟!ůG†X† IgG Bȇ '˼gT -ůG†X†ɯǵɯĻů«-ğĈG†X†-¬ȇ, ?ãƨBƱʍ!ůG†X† IgG ȵ 1 ÓȊ.˼G†X†)):,gTŧǷɣɝɁɠ,Ǡá?)˼G†X†ìǻ(Ǡá!Ɩ<>Ɂ ɠ1-Ȼô>ʮ7ˊđá˼6!@,ːA? RTK VQd‚( ?†ˇæ IRS-1 )†ˇæ Akt -ƱÒˊ:đá!ůG†X† IgG -ȵ 1 ÓȊ-ğĈ,<>ɁɠË1-Ȼ-ô>ʮ7ɢđŌ! ȂȈ.˼G†X†-õħ«Ʌùő,? RTK VQd‚Ǘśæ-đŌ,):+$!îŎʥɪɽˤ1GLUT4 translocation ´ʷ-Ʌƨ[14, 20]( ?)ƀĪ@! $5

(59) (˼ůG†X† IgG -ȵ 2 ÓȊ.˼G†X†)):,Ɂɠ,Ǡá':˼G†X†ìǻ(Ǡá!))úȶ-ɁɠË1-Ȼ ô>ʮ7) RTK VQd‚ǗśæBȨ!gT-ůG†X†ɯǵɯĻů«,G†X†-¬ȇBđŌ ?ÓȊ)˼đŌ+ ÓȊ ?)Ɣ=)+$!  gT-ůG†X†ɯǵɯĻů«)G†X†-Ŏŧ?ůîů«ʁù«*-<

(60) +žɃ7(G† X†-¬ȇ,ãƨBð4-.ŽƔ( ?Nishii =,<?gT-ůG†X† IgG ,ː?ȥȯ [36], '˼¼ň+gT-ɹǡ=Ó˟@!ůG†X† IgG ĖP„Š†ś<0ĖòŚś(. $!ɅƨBɗť?)˼gT-ůG†X† IgG .˼G†X†ÓĞŒ,ʁƈğĈ?ůî˃§,úƖ ,Ʌù?)(ĖÓĞʁù«BŎŧ?öɢś ?˺Fig. 2-11˻ wHX-ɛɪ;ɍɊɳɁɠBȇ !ȥȯ[18, 46], '˼p\G†X†Bůî)'{‚{_b,Ȇȅ!ůG†X†ů«˺ɯǵ ɯĻů«(.+ ˻˼-ğĈ, 'G†X†-¬ȇ)õħ«ɅùɢBđŌ?)Ďý @' ?-ãƨ.˼G†X†)ůG†X†ů«-Ŏŧ?ĖÓĞʁù«-ʖĮ?õħ«Ð˝ ,ʦĄ?)ƀĢ@' ?6! 3T3 ɍɊɳɁɠBȇ !ȥȯ[46], '˼EGF ,ʈĀśBȨů EGF ů« EGF )ʁù«BŎŧ?)(˼EGF -ãƨBđŌ?)Ďý@' ?ȵ 2 Ȳ, ?gT-ůG†X† IgG -ȵ 1 ÓȊ,<?G†X†-¬ȇđŌãƨ,.˼IgG )G†X†, <?ĖÓĞʁù«Ŏŧː˼G†X†õħ«Ð˝-ʖĮ+*Bʳ'˼RTK VQd‚-ǗśæB. 38.

(61) Ō9˼ɁɠË1-Ȼô>ʮ7ɢBđŌ!öɢś ?. Insulin. Anti-insulin IgG 1st fraction. Fig. 2-11. The presumed multivalent complex formed of insulin and 1st fraction of antiinsulin IgG. Anti-insulin IgGs are bound with insulin as polyvalent antigen by binding to multiple antigenic sites at a time. .  gT-ůG†X† IgG -ȵ 1 ÓȊ)ȵ 2 ÓȊ,?G†X†-¬ȇ, ?ãƨ-ʼ .˼ @ @-ÓȊBƴŧ? IgG G†X†)Ŏŧ?ůîů«ʁù«-ƴʵȘǹŖ,ʦĄ?öɢś. ?‹ɰȘ+ůîů«ʁù«-ƴʵNjĢ,.˼ůî-ů«ɅùJnbŠq-§ɒ;ÓĽĨŇ˼Ʌùʈ Āś˼ů«-öäȹĆ˺segmental flexibility˻;Ʌùà˺avidity˻+*ː³?)ɗ =@' ?[38] -

(62) #˼gT-ɹǡ=Ó˟!ůG†X† IgG -ȵ 1 ÓȊ)ȵ 2 ÓȊ, 'şĎ ?-.G †X†,Ĭ?ɅùʈĀś-7( $!G†X†)ůG†X†ů«-Ʌùʊ˟?),< ?¨ɹȻȖ¬BɄ˴!ȻıȒŞɘ, '˼ɽˤq€Y{†É˸Ŭɻ,<>ǧĢ!ůG†X†ů «)G†X†- KD ¹. 5.4410.310-5˺łĊ¹ƶǩºĺ˻M ( >˼¨ʈĀś,Ó˫@![26] gT-ůG†X† IgG -ȵ 1 ÓȊ)ȵ 2 ÓȊ)G†X†- KD ¹.˼@ @ 1.6110-4 M˼2.00 10-5 M ( >˼):,¨ʈĀś,Ó˫(!˼G†X†-¬ȇđŌãƨ-ʕ9=@!ȵ 1 ÓȊʈĀś.˼ȵ 2 ÓȊ<>¨$!G†X†,Ĭ?ɅùʈĀś¢ĕ-ůîů«ˏ-ÓĞˏȝ–¬ȇ. 39.

(63) -ʼ ˼G†X†)Ŏŧ?ůîů«ʁù«-ƴʵNjĢ,ː' ?öɢś: ?6!˼ȵ 2 Ȳ(.˼ ůG†X†IgG -ȵ1 ÓȊ)ȵ2 ÓȊG†X†-¬ȇ, ?ãƨB×,ʓ²!˼ ȵ 1 ÓȊ)ȵ 2 ÓȊǟĈ?ďù,.˼ǟĈ+ ďù)G†X†)Ŏŧ?ʁù«-ƴʵȏ+ >˼=,.G†X†-¬ȇ, ?ãƨȏ+?öɢśɗ =@?-şĎ.˼gT-ɹ‘, ?ůG†X†ɯǵɯĻů«G†X†-¬ȇ, ?ãƨBɗ ?Œ(Řʃ)+?!9˼= ,Ʊʍ?Řʃ ?  ȏȭäǸȈƥ-G†X†Ů,òŚ'Ȇȅ@!ůG†X†ů«,% '.˼ُG†X† -õħ«Ʌù-˕Ħ[6, 7, 13]˼ ? .õħ«Ʌù) RTK VQd‚Ǘśæ-đŌ[18, 21, 46]) $!˼ G†X†¬ȇ-ǣŋ)đŌ-Ǝ-ãƨĎý@' ?˼gT-ůG†X†ɯǵɯĻů «.˼ȏȭäǸȈƥG†X†Ů,òŚ'Ȇȅ@!ů«).˼ŧ>ȱ#ƢʥȘ,ȏ+?!9˼ G†X†-¬ȇ, ?ãƨ:ȏ+?öɢś ?ɯǵů«.˼äǸ-ƽŁ+ȅÿǗä-ɊŷBų

(64) )ɗ =@' ?[30]!9˼gT-ůG†X†ɯǵɯĻů«gT-ȅÿɊŷ,ƞȇ+ãƨBŷ%ö ɢś ?ȵ 2 Ȳ, 'ʕ9=@!gT-ůG†X† IgG -ȵ 1 ÓȊ,<?G†X†-¬ȇđ Ōãƨ.˼ůG†X†ɯǵɯĻů«G†X†-¬ȇ, ?ãƨ-‹%( ?˼ȅ«Ë, '-ÓȊ*-<

(65) ,ƞȇ,ƺɢ' ?Ʊʍ?Řʃ ?gTȟśɚˬäǸ( ?)B ɗť?)˼ůG†X†ɯǵɯĻů«Exhˇ)ɣʥB‘ŗ)!gTȪäǸǹƞ-Ƭ˰¡ʞ[5], ːA?ŐßBŷ%öɢśƀĢ@?  gTŧǷɣɝɁɠ,Ǡá!G†X†ǯŇ.˼“¾ʔ˴, 'ǴÑȂ-Ɂɠ-Ƚ 2.5 ·ˊ[3H]-2-DOG ô>ʮ7BÚǭ! 10-7 M Bžȇ!G†X†Úǭ,<?Ɂɠ1-Ȼô>ʮ7ɢBƔ ȡ,ʓ²(?ǯŇ)' 10-7 M Bžȇ!˼ȵ 1 Ȳ, 'ɹǫ‘-ůG†X† IgG -{‚ǯŇ G†X†<>:ɶ˵ $!)Bɗť?)˼ ůG†X† IgG ÓȊ-ƜėǯŇ 10-10 M .˼ G†X†)-ǟùßù)'ȅȂȘ+Ƥ¤=ʊ˟' !öɢś ?6!˼ůîů«òŚ, '˼ůî-ßùů«,Dž'ɶ˵ )ʁù«-Ŏŧ˕Ħ@?) ?[1]ƻ-Ʊʍʚ˪ )'˼G†X†) IgG -ȅ«Ë,?ğĈDžBòƕ!ǟùƤ¤,<?ģ˴Řʃ( ?. 40.

(66)  RTK VQd‚\†kPʥ-ƱÒ, '˼ȵ 1 ÓȊ<0ȵ 2 ÓȊ-ìǻǠá,<>†ˇæ Akt -ƱÒˊA,đá!˼†ˇæ IRS-1 -ƱÒˊ.ÈĔæ˼ɁɠË1-Ȼ-ô>ʮ7: đá+$!Ʊʍ@!ǯŇ-ůG†X† IgG ÓȊ-ìǻǠá.˼Ɂɠ,? Akt BȜſǗś æ(?öɢś. ?˼Akt Ǘśæ-ȬŇ.ŋ˼GLUT4 - translocation B'ɁɠË1-Ȼô >ʮ7B´)(+ ˼ȅȂĠȘ,.šþBŷ!+ †ˇæ Akt -ȖȀˊđá)ɗ =@!. 41.

(67) į Ŷ  ȵ 2 Ȳ(.˼¼ň+gT-ɹǡ=EoFe`FP„wbQ€oFŠ) HPLC ,<>Ŕ=@!G†X †,Ĭ?ʈĀś-ȏ+? 2 ȭ˫-ůG†X† IgG ÓȊ-ğĈ˼G†X†-¬ȇ, ?ãƨ ,% 'gTŧǷɣɝɁɠBȇ 'Ʊʍ!G†X†,Ĭ?ʈĀś-¨ ůG†X† IgG -ȵ 1 ÓȊ.˼Ɂɠ1-G†X†Ǡá,ʖĮ@? RTK VQd‚-ǗśæBđŌ?),<>Ȼô>ʮ 7ɢBđŌ!˼ʈĀś-˵ ȵ 2 ÓȊ.úƵ-ãƨBȨ+$!¼ň+gTɹǡ‘,µƞ ?ůG†X†ɯǵɯĻů«,.˼G†X†-¬ȇBđŌ?ãƨ- ?ÓȊ)˼đŌ?ãƨ-+ ÓȊ ?)Ɣ=)+$!gT-ůG†X† IgG .ĖP„Š†ś<0ĖòŚś( ? )=˼ ůG†X†IgG)ʁƈ-Ʌù˃§Bƞ?G†X†.ÓĞŒ-ůî˃§,ůG†X†IgG BúƖ,Ʌù'˼ĖÓĞʁù«BŎŧ?)ƀĢ@?gT-ůG†X† IgG -ȵ 1 ÓȊ)ȵ 2 ÓȊ, '˼G†X†-¬ȇ, ?ãƨȏ+$!ȂȈ)'˼IgG )G†X†,<>Ŏŧ @?ĖÓĞʁù«-ƴʵȘǹŖ-ʼ ː³' ?öɢśɗ =@!. 42.

(68) ȵ3Ȳ gT-ůG†X† IgG )G†X†,<?ʁù«ŎŧG†X†-¬ȇđŌãƨ,ð4 ŏ˥. Ɍ ʋ  ȵ˿Ȳ(.˼gT-ůG†X†ɯǵɯĻů«-ȅȂȘŐßBʊƔ?!9,˼¼ň+gT-ɹǡ =EoFe`FP„wbQ€oFŠ) HPLC ,<>ÓȊ!G†X†,ʈĀśBȨ IgG Bȇ '˼ ůG†X† IgG G†X†-¬ȇ, ?ãƨBƱʍ!ÓȊ! 2 ȭ˫-ůG†X† IgG

(69) #˼G†X†,Ĭ?ʈĀś-¨ ȵ 1 ÓȊ.˼G†X†-¬ȇBđŌ?ãƨBȨ!gT -ůG†X† IgG -ȵ 1 ÓȊ˼*-<

(70) ,'G†X†-õħ«Ʌù¢˗-VQd‚¥ʻBǗś æ!-˼-ƺņ,% '.ŽƔ( ?  gT-ůG†X† IgG .ĖP„Š†ś<0ĖòŚś( ?[36])=˼G†X†)ĖÓĞʁ ù«BŎŧ?)ɗ =@?˺Fig. 2-11˻ ů«ůî)-ʁù«Ŏŧ,<>ůî-¬ȇBđŌ?ãƨ ,% '.˼ů EGF ů«[46];ůT†Ldj† A ů«[48]˼6!ȏȭäǸȈƥG†X†Ů,òŚ 'Ȇȅ@?ůG†X†ů«[18, 46], 'Ďý@' ?ůG†X† IgG -ȵ 1 ÓȊ, ':˼G†X†-¬ȇđŌãƨ,˼IgG )G†X†,<>Ŏŧ@?ĖÓĞʁù«ː³' ? öɢś ?  ȵ 3 Ȳ(.˼ȵ 2 Ȳ, 'ʕ9=@!ůG†X† IgG -ȵ 1 ÓȊ,<?G†X†-¬ȇđŌã ƨ,˼IgG )G†X†,<?ĖÓĞʁù«Ŏŧː³?*

(71) BƱʍ!IgG .˼kkG†Ñ Ȃ,<>l†W˃ N ơȳ½Ôƌ@˼1 ÓĞō!> 2 %- Fab o€Qz†b) 1 %- Fc o€Qz† b,Ó˟?Fab o€Qz†bæ! IgG .˼ůîBƩƹ!ĖÓĞʁù«BŎŧ?)(+ +?gTŧǷɣɝɁɠ) Fab o€Qz†bæ!ůG†X† IgG -ȵ 1 ÓȊBȇ '˼G†X †¬ȇđŌ?*

(72) BƱʍ!. 43.

(73) ƣƋ<0Ǝǐ 1. =&#& IgG O 1 .M Fab " &3  ȵ 2 Ȳ, '˼¼ň+gT-ɹǡ=EoFe`FP„wbQ€oFŠ) HPLC ,<$'ÓȊ !ůG†X† IgG -ȵ 1 ÓȊB®ȇ!ģ˴,.ǴÑɒ-ȵ 1 ÓȊ)kkG†ÑȂ,<> Fab o€ Qz†bæ!ȵ 1 ÓȊBȇ !kkG†ÑȂ.˼Pierce Fab Preparation Kit˺Thermo Fisher Scientific, Rochester, NY, USA˻Bȇ 'žƵƛ,œ$'ģƏ˼ 1 ÓĞō!> 2 %- Fab o€Qz† b) 1 %- Fc o€Qz†b,Ó˟!˺Fig. 3-1˻ Ó˟! Fab <0 Fc o€Qz†bÓĞ-ğĈB Ȧʕ?!9,˼ǴÑɒ-ȵ 1 ÓȊ)kkG†ÑȂő-ȵ 1 ÓȊ- Fab o€Qz†bÓȊ<0 Fc o €Qz†bÓȊB 15%SDS EP‚ExcS‚˺SuperSep TM Ace; ĀÄȾɷ˻,<?ˡdžǔä) CBB ƪɲ,°!. Fig. 3-1. Schematic for preparation and purifying Fab using papain and protein A. The Fab generation of anti-insulin IgG by Pierce Fab Preparation Kit (Thermo Fisher Scientific). Digestion by papain produces 50 kDa Fab and Fc fragments. . 2. VTQU 6c.3  gTȈƥ-ɣɝÛ˳Ɂɠ˺Primary Normal Feline Preadipocytes, Zen-Bio, Research Triangle, NC, USA˻.˼ļʢ-Ȗɞ˼Óæ˼ɊŷČĉ˺Zen-Bio˻B®$'ŧǷČ˰!2'-Č˰ĹȬ.˼5% CO2˼ 37°C -Ƥ¤(ɺ$!ŧǷɣɝɁɠ.˼ģ˴Û,ɹǡ)G†X†-ŏ˥B˚ï?!9,˼4 Ɩˏ˼ ɹǡ)G†X†Bû6+ ɊŷČĉ(˭˱Č˰!. 44.

(74) 3. 2-Deoxy-D-glucose uptake assay  gTŧǷɣɝɁɠ1-Ȼô>ʮ7ɢ-ʓ².˼ȵ 2 Ȳ)úƵ-Ǝǐ(ģƏ!˭˱Č˰!ɣɝɁ ɠ,˼HSBS (ɋˊ 100 μl ,ʛƉ!¢- 7 ȭ˫-ǪǜB¢-ǯŇ(Ǡá˼=, 30 ÓˏČ˰ !; 1) ǴÑȂ˼2) ÛČ˰,®ȇ! HSBS˺vehicle˻ ˼3) G†X† 10-7 M -ìǻ˼4) ǴÑɒ-ȵ 1 ÓȊ 10-12 M, 10-11 M, 10-10 M ìǻ˼ 5) G†X† 10-7 M )ǴÑɒ-ȵ 1 ÓȊ 10-12 M, 10-11 M, 10-10 M -ǟù˼6) Fab o€Qz†bæ!ȵ 1 ÓȊ 10-12 M, 10-11 M, 10-10 M ìǻ˼7) G†X† 10-7 M ) Fab o€Qz†bæ!ȵ 1 ÓȊ 10-12 M, 10-11 M, 10-10 M -ǟù. 4. VTQU#&_3%&&]fAktg C Fab o€Qz†bæ!ůG†X† IgG -ȵ 1 ÓȊ-ğĈ˼gTŧǷɣɝɁɠ1-G†X†Ǡ á,<? RTK VQd‚Ǘśæ, ?ãƨBʓ²?!9,˼ȵ 2 Ȳ)úƵ-Ǝǐ(Ɂɠ-†ˇæ Akt -ɄƖȘ+ƱÒˊ-ĔæBʛ2!˭˱Č˰!ɣɝɁɠ,, ˭˱Č˰,ȇ !ɹǡ)G†X†B û6+ ɊŷČĉ(ɋˊ 100 μl ,ʛƉ!¢- 6 ȭ˫-ǪǜBǠá˼ =, 0 Ó, 1 Ó, 3 Ó, 5 Ó, 15 Ó, 30 Ó, 60 ÓˏČ˰!; 1) ˭˱Č˰,®ȇ!ɊŷČĉ˺vehicle˻ ˼2) G†X† 10-7 M ìǻ˼3) Ǵ Ñɒ-ȵ 1 ÓȊ 10-10 M ìǻ˼4) G†X† 10-7 M )ǴÑɒ-ȵ 1 ÓȊ 10-10 M -ǟù˼5) Fab o€Q z†bæ!ȵ 1 ÓȊ 10-10 M ìǻ˼6) G†X† 10-7 M ) Fab o€Qz†bæ!ȵ 1 ÓȊ 10-10 M -ǟù. 5. RZ.A  gTɣɝɁɠ1-G†X†˼ǴÑɒ-ȵ 1 ÓȊ<0 Fab o€Qz†bæ!ȵ 1 ÓȊ-ìǻǠá øģ˴ɔ-aŠ\-ȶÓƇśB Bartlett ƱĢ,<>Ȧʕ! ,<?Ɂɠ1-[3H]-2-DOG ô>ʮ7ˊ.˼ ő,˼vehicle Ǡá,<?ô>ʮ7ˊ)-ĺB‹Ã˄ɒÓƇÓƧ) Dunnett ǐ,<>Džʪ!6!˼ ǴÑɒ-ȵ 1 ÓȊ6!. Fab o€Qz†bæ!ȵ 1 ÓȊ-ğĈ,<?G†X†Ǡáő-Ɂɠ[3H]-2-DOG ô>ʮ7ˊ.˼øģ˴ɔ-aŠ\-ȶÓƇśB Bartlett ƱĢ,<>Ȧʕ!ő,˼G†X. 45.

(75) †ìǻǠá,<?ô>ʮ7ˊ)-ĺB‹Ã˄ɒÓƇÓƧ) Dunnett ǐ,<>Džʪ!=,˼Ʊʍ !ǴÑɒ-ȵ 1 ÓȊ<0 Fab o€Qz†bæ!ȵ 1 ÓȊ, 'ǠáǯŇ ˺10-12 M, 10-11 M, 10-10 M˻ˏ-[3H]-2-DOG ô>ʮ7ˊ-ĺ.˼‹Ã˄ɒÓƇÓƧ) Tukey-Kramer ǐ,<>@ @Džʪ !Ɂɠ1-ǴÑɒ-ȵ 1 ÓȊ<0 Fab o€Qz†bæ!ȵ 1 ÓȊ-gTɣɝɁɠ1-ìǻǠá, <?†ˇæ Akt -ƱÒˊ.˼Ǡáő-ɄʺƖˏ˺Ó˻DŽ,aŠ\B F ƱĢ,<>ȶÓƇśBȦʕ! ő,˼ vehicle ìǻǠá,<?ƱÒˊ)-ĺB Student - t-ƱĢ,<>Džʪ!6!˼ǴÑɒ-ȵ 1 ÓȊ6!. Fab o€Qz†bæ!ȵ 1 ÓȊ-ğĈ,<?G†X†Ǡáő-Ɂɠ,?†ˇæ Akt -ƱÒˊ.˼Ǡáő-ɄʺƖˏDŽ, F ƱĢ,<>aŠ\-ȶÓƇśBȦʕ!ő,˼G†X†ì ǻǠá,<?ƱÒˊ)-ĺB Student - t-ƱĢ,<>Džʪ!p<0.05 BɇʌȘ,ƞš)!. 46.

(76) Ʌ ƨ 1. =&#& IgG O 1 .M Fab " &3  Figure 3-2 .˼ůG†X† IgG -ǴÑɒ-ȵ 1 ÓȊ<0kkG†ÑȂ,<> Fab o€Qz†b) Fc o€Qz†b,Ó˟!ȵ 1 ÓȊ- SDS-PAGE <0 CBB ƪɲ-ɅƨBȨ˺Fig. 3-2˻ ǴÑɒ -ȵ 1 ÓȊ-Ʌƨ.˼ IgG - H ˌBȨ 50 kDa  ʯ) L ˌ- 25 kDa  ʯ,•ȝś-j†cBʕ9! kkG†ÑȂ!ůG†X† IgG -ȵ 1 ÓȊ, '˼Fab o€Qz†bÓȊ-Ʌƨ. Fab o€Qz †b) L ˌBȨ 25 kDa  ʯ-‹ȝś-j†cBʕ9!6!˼ Fc o€Qz†bÓȊ-Ʌƨ. Fc o €Qz†bBȨ 28-30 kDa  ʯ-‹ȝś-j†cBʕ9!kkG†ÑȂ,<>˼ůG†X† IgG -ȵ 1 ÓȊB Fab o€Qz†b) Fc o€Qz†b,Ó˟!)Ȧʕ(!. 2. =&#& IgG O 1 .M Fab " &&#&F0VTQU [1,2-3H]-2-Deoxy-D-glucose 4^

(77) (1B  Vehicle )'ȇ ! HSBS -Ǡá,<?gTɣɝɁɠ,?[3H]-2-DOG -ô>ʮ7ˊ.˼ǴÑɒ -Ɂɠ,?ô>ʮ7ˊ˺100%˻)34úȶ˺96.43.4%˻( >˼vehicle Ǡá,<?ô>ʮ7ˊ đá.+$!G†X† 10-7 M -ìǻǠá,<?Ɂɠ1-[3H]-2-DOG -ô>ʮ7ˊ.˼vehicle Ǡ á,<?ô>ʮ7ˊ,Dž2'ɶđá!˺p<0.01, Fig. 3-3A˻  $5

(78) ˼ůG†X† IgG -ǴÑ ɒ-ȵ 1 ÓȊ<0 Fab o€Qz†bæ!ȵ 1 ÓȊ-ìǻǠá,<?Ɂɠ1-[3H]-2-DOG -ô>ʮ 7ˊ.˼Ʊʍ!ǯŇ˺10-12 M, 10-11 M, 10-10 M˻, ' vehicle Ǡá,<?ô>ʮ7ˊ)úȶ(˼Ȼ ô>ʮ7ˊBđá+$!˺Fig. 3-3A˻ G†X†Ǡá,<?Ɂɠ1-[3H]-2-DOG -ô>ʮ7ˊ .˼ǴÑɒ-ȵ 1 ÓȊB):,Ǡá?)(ɶđá!˺p<0.01˻˼đáˊ.ȇˊ±ğś(. +$!˺Fig. 3-3B˻ ˼G†X†Ǡá,<?Ɂɠ1-[3H]-2-DOG -ô>ʮ7ˊ.˼Fab o€ Qz†bæ!ȵ 1 ÓȊB):,Ǡá':˼Ʊʍ!2'-ǯŇ(˼G†X†ìǻǠá,<?ô >ʮ7ˊ)úȶ( $!˺Fig. 3-3B˻ . 47.

(79) 1. (kDa). 2. 3. 4. 75 50 37 Fc fragment (near 28-30 kDa) 25 20 Fab fragment + L-chain (near 25 kDa) Fig. 3-2. SDS-PAGE results of reduced intact and papain-treated 1st fraction of anti-insulin IgG. Fab fragments migrated near 25 kDa and Fc fragments migrate at 28-30 kDa. Lane 1: Molecular weight marker. Lane 2: Intact 1st fraction. Lane 3: Fab fragment + L-chain (papain-treated 1st fraction). Lane 4: Fc fragment (papain-treated 1st fraction).. 48.

(80) [3H]-2-DOG uptake percentage of non-treatment control. A) Insulin alone and intact or Fab fragmentary 1st fraction of anti-insulin IgG alone 300. p<0.01. 200. 100. 0. NonVehicle treatment control. Insulin 10-7 M. 10-12. 10-12. 10-11 10-10 M. Anti-insulin IgG intact 1st fraction. 10-11. 10-10 M. Anti-insulin IgG Fab fragment of 1st fraction. B) Insulin with or without intact or Fab fragmentary 1st fraction of anti-insulin IgG. [3H]-2-DOG uptake percentage of non-treatment control. * **. *. 400 ††. †† ††. 300. 200. 100. 0. Insulin 10-7 M. 10-12. 10-11 10-10 M. Intact 1st fraction + Insulin 10-7 M. 10-12. 10-11 10-10 M. Fab fragment of 1st fraction + Insulin 10-7 M. Fig. 3-3. Mean (+SD) percentages of [1,2-3H]-2-Deoxy-D-glucose ([3H]-2-DOG) uptake in feline adipocytes. Results (n=3) describe non-treatment control as 100%. †† denote significant differences from insulin alone at p<0.01. * and ** denote significant differences from other concentration at p<0.05 and p<0.01 respectively. A) Insulin alone and intact or Fab fragmentary 1st fraction of anti-insulin IgG alone. B) Insulin with or without intact or Fab fragmentary 1st fraction of anti-insulin IgG. . 49.

(81) 3. =&#&IgG O1 .M Fab " &&#&F0VTQU RTK $D;3(1B  Vehicle )'ȇ ! HSBS -Ǡá,<?gTɣɝɁɠ-†ˇæ Akt -ƱÒˊ.˼60 ÓˏĔæ+ $!˺Fig. 3-4A˻ G†X†-ìǻǠá,<?†ˇæ Akt -ƱÒˊ.˼˾Ó(đá˼3 Ó(Ɯė ,ʻ!ő˼Œ,¨!˼60 ÓˏBʳ' vehicle Ǡá,<?ƱÒˊ<>:ƞš,Ė$!˺Fig. 3-4B˻ ǴÑɒ-ȵ 1 ÓȊ-ìǻǠá,<?†ˇæ Akt -ƱÒˊ.˼15 Ó˺p<0.05˻) 30 Ó˺p<0.01˻ , '˼vehicle Ǡá,<? 15 Ó) 30 Ó-ƱÒˊ<>@ @Ė$!(Fig. 3-4C)Fab o€Qz† bæ!ȵ 1 ÓȊ-ìǻǠá,<?†ˇæ Akt -ƱÒˊ.˼vehicle Ǡá,<?ƱÒˊ)úȶ( $! ˺Fig. 3-4D˻  $5

(82) ˼G†X†Ǡá,<?†ˇæ Akt -ƱÒˊ.˼ǴÑɒ-ȵ 1 ÓȊB):, Ǡá?)˼G†X†ìǻǠá,<?ƱÒˊ<>:˼1 Ó˺p<0.01˻) 3 Ó˺p<0.01˻(ƞš,İ+ $!˼5 Ó(úȶ-ƒr‚,đá!˺Fig. 3-5B˻ ˼G†X†ìǻǠá,<?ƱÒˊǣ İĝ9! 15 Ó¢˗:˼G†X†)ǴÑɒ-ȵ 1 ÓȊ-Ǡá,<?†ˇæ Akt -ƱÒˊ.˼đá !ǺţBµ#˺p<0.05˻ ˼30 Ó(úȶ-ƒr‚)+$!Fab o€Qz†bæ!ȵ 1 ÓȊBG†X †)):,Ǡá?)˼†ˇæ Akt -ƱÒˊ.˼60 ÓˏBʳ'˼G†X†ìǻǠá,<?ƱÒ ˊ)úƵ-ĔæBȨ!˺Fig. 3-5C˻ . 50.

(83) A) Vehicle (○) Time (min) (○). 0. 1. 3. 5. 15. 30. 60. 1. 3. 5. 15. 30. 60. Phosphorylated Akt percentage of non-treatment cells . B) Insulin alone (●) and vehicle alone (○) Time (min). 2000. §§. (●). 1500 §§. 1000. 0. §§ §§. §§. 15. 30. §§. 500 0 0. 45. 60. Time (min). Phosphorylated Akt percentage of non-treatment cells . C) Intact 1st fraction alone () and vehicle alone (○) 2000. Time (min). 1500. 0. 1. 3. 5. 15. 30. 60. (). 1000 500. §§. §. 15. 30. 0 0. 45. 60. Time (min). Phosphorylated Akt percentage of non-treatment cells . D) Fab fragmentary 1st fraction alone (□) and vehicle alone (○) 2000. Time (min) 1500. 0. 1. 3. 5. 15. 30. 60. (□). 1000 500 0 0. 15. 30. 45. 60. Time (min). Fig. 3-4. Mean (±SD) percent changes and representative western blots of phosphorylated Akt in adipocytes derived from cats. Results (n=3) describe time 0 as 100%. § and §§ denote significant differences from vehicle at p<0,05, p<0.01respectively. A) Vehicle alone (○). B) Insulin 10-7 M alone (●) and vehicle alone (○). C) Intact 1st fraction 10-10 M alone () and vehicle alone (○). D) Fab fragmentary 1st fraction 10-10 M alone (□) and vehicle alone (○). 51.

(84) A) Insulin alone (●) Time (min). 0. 1. 3. 5. 15. 30. 60. 5. 15. 30. 60. (●) B) Intact 1st fraction + insulin (▲)and insulin alone (●) Time (min) (▲). Phosphorylated Akt percentage of non-treatment cells . 2000. 0. 1. 3. *. 1500. 1000. **. 500. ** 0 0. 15. 30. 45. 60. Time (min). C) Fab fragment of 1st fraction + insulin (■) and insulin alone (●) Phosphorylated Akt percentage of non-treatment cells . 2000. Time (min) (■). 0. 1. 3. 5. 15. 30. 60. 1500. 1000. 500. 0 0. 15. 30. 45. 60. Time (min). Fig. 3-5. Mean (±SD) percent changes and representative western blots of phosphorylated Akt in adipocytes derived from cats. Results (n=3) describe time 0 as 100%. * and ** denote significant differences from vehicle at p<0,05, p<0.01respectively.   A) Insulin 10-7 M alone (●). B) Intact 1st fraction 10-10 M + insulin (▲)and insulin 10-7 M alone (●). C) Fab fragmentary 1st fraction 10-10 M + insulin (■) and insulin 10-7 M alone (●). 52.

(85) ɗ Ī  ȵ 3 Ȳ(., ȵ 2 Ȳ, 'ʕ9=@!ůG†X† IgG -ȵ 1 ÓȊ,<?G†X†-¬ȇđŌã ƨ,˼IgG )G†X†,<?ĖÓĞʁù«Ŏŧː³' ?*

(86) BƱʍ!Fab o€Qz† bæ!ůG†X† IgG -ȵ 1 ÓȊ.˼G†X†BƩƹ!ĖÓĞʁù«BŎŧ(+ ůG† X† IgG -ȵ 1 ÓȊ,<?G†X†-¬ȇđŌãƨ,˼IgG )G†X†,<?ĖÓĞʁù«Ŏŧ ː' ?-( @/˼Fab o€Qz†bæ!ȵ 1 ÓȊ,.G†X†-¬ȇđŌãƨ+  ),+?ȵ 3 Ȳ, '˼ Fab o€Qz†bæ!ůG†X† IgG -ȵ 1 ÓȊ.G†X†-¬ ȇBđŌ+$!˺Fig. 3-6˻ gT-ůG†X† IgG -ȵ 1 ÓȊ,<?G†X†-¬ȇđŌãƨ ,.˼ȵ 1 ÓȊBƴŧ? IgG )G†X†,<?ĖÓĞʁù«Ŏŧː?öɢśȨ@!  ůG†X†ɯǵɯĻů«(.+ ˼ȏȭäǸȈƥ-G†X†Ů,òŚ'Ȇȅ@!ůîǹ ȏś-˵ ůG†X†ů«.˼G†X†)-ʁù«Ŏŧ,<>G†X†-õħ«Ʌù)¬ȇBđ Ō?)Ďý@' ?[18, 46]@=-Ďý(.˼“Š@?ůG†X†ů«-G†X†¬ ȇđŌ-îĄ)'˼ů«)G†X†-Ŏŧ?ʁù«,<?ɁɠɪŒ,?G†X†õħ«Ð ˝-ʖĮBŹ' ?-£ʙ.˼ɁɠɪŒ-sq^ct‚{†õħ«ȅȂȘ+Ǧҍ, 'ö äś( >˼t‚{†Ʌùő,P€X\ŠǺ<0k_^Ǻ-ŎǺB)$'Ð˝?)BȨ!Ďý [22]Bƭŵ)' ?G†X†õħ«-Ð˝Ř RTK VQd‚-ǗśæBđŌ?*

(87) .Ȧ 9=@' + ˼gT-ůG†X† IgG -ȵ 1 ÓȊG†X†)Ŏŧ?ĖÓĞʁù« ˼ɁɠɪŒ-G†X†õħ«-Ð˝BʖĮ?)Ȧʕ@@/˼RTK VQd‚ǗśæđŌ-ƞ à+îĄ)'Ź?)(?gT-ůG†X† IgG )G†X†,<>Ŏŧ@!ĖÓĞʁ ù«-ğĈ, '˼gT-G†X†ƶȘɁɠɪŒ-G†X†õħ«Ð˝ʖĮ-ƞǴBöʆȘ, ʉĪ?Řʃ ?. 53.

(88) Insulin. Insulin. +. +. Anti-insulin IgG intact 1st fraction. Anti-insulin IgG Fab fragment of 1st fraction. Monovalent complex. Multivalent complex. Enhanced insulin action. Insulin alone. >. Normal insulin action. =. Normal insulin action. Fig. 3-6. Diagrammatic representation of insulin-induced reaction with or without intact 1st fraction or Fab fragment of 1st fraction of anti-insulin IgG in feline adipocytes. Intact 1st fraction enhanced insulin-induced reaction in feline adipocytes, but Fab fragment of 1st fraction did not enhance insulin-induced action. . 54.

図

Fig. 1-2. The dilution curves of plasma anti-insulin IgG concentrations.
Table 1-1. Plasma anti-insulin IgG concentrations in 84 clinically healthy cats categorized by gender.
Fig. 1-4. Correlation between plasma anti-insulin IgG and total IgG concentrations.
Fig. 1-5. The results of intravenous glucose tolerance test (1 g/kg) in three normal domestic cats
+7

参照

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Outline

Insulin dose dependent responses Fc fragment (papain-treated 1st fraction)

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