Insulin dose dependent responses

β-actin (45 kDa) Phospho-Akt

(60 kDa)

0 3 1 60 5 10 20 30 90 120 (min)

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.

Anti-phosphorylated Akt antibody detected a time dependent response of Anti-phosphorylated

Akt in insulin-treated cells.

31

Ʌ

Ʌƨ

1i=&#&IgG&#&F0VTQU [1,2-³H]-2-Deoxy-D-glucose 4

^(1B

Vehicle)'ȇ!HSBS-Ǡá,<?gTɣɝɁɠ1-[³H]-2-DOG-ô>ʮ7ˊ.˼ǴÑȂ-Ɂ ɠ,?ô>ʮ7ˊ˺100%˻)34úȶ˺105.46.6%˻(>˼vehicleǠá,<?ô>ʮ7ˊđá .+$!G†X†10^-7 M-ìǻǠá,<?Ɂɠ1- [³H]-2-DOG-ô>ʮ7ˊ.˼vehicleǠá ,<?ô>ʮ7ˊ,Dž2'ɶđá!˺p<0.01, Fig. 2-6A˻$5˼ȵ1ÓȊ<0ȵ2 ÓȊ-ìǻǠá,<?Ɂɠ1- [³H]-2-DOG-ô>ʮ7ˊ.˼Ʊʍ!ǯŇ˺10^-12 M, 10^-11 M<010^-10 M˻

,'vehicleǠá,<?ô>ʮ7ˊ)úȶ(˼Ȼô>ʮ7ˊBđá+$!˺Fig. 2-6A˻G†

X†Ǡá,<?Ɂɠ1-[³H]-2-DOG-ô>ʮ7ˊ.˼ȵ1ÓȊB):,Ǡá?)(ɶđá

!˺p<0.01˻˼đáˊ.ȇˊ±ğś(.+$!˺Fig. 2-6B˻˼G†X†Ǡá,<? Ɂɠ

1-[³H]-2-DOG-ô>ʮ7ˊ.˼ȵ2ÓȊB):,Ǡá':˼Ʊʍ!2'-ǯŇ(˼G†X†

ìǻǠá,<?ô>ʮ7ˊ)úȶ($!˺Fig. 2-6B˻

32

Low-affinity IgG High-affinity IgG 0

100 200 300

N V I

[3H]- 2-DOG uptake percentage of non-treatment control

A) Insulin alone and anti-insulin IgG fractions alone

Non-treatment

control

Vehicle

Anti-insulin IgG 1st fraction

Anti-insulin IgG 2nd fraction Insulin

10^-7 M 10^-12 10^-11 10^-10 M 10^-12 10^-11 10^-10 M

Low-affinity IgG High-affinity IgG 0

100 200 300 400

Insulin [3H]- 2-DOG uptake percentage of non-treatment control

B) Insulin with or without anti-insulin IgG fractions

Insulin 10^-7 M Insulin 10^-7 M Insulin

10^-7 M

+ +

10^-10 M 10^-11

10^-12 10^-12 10^-11 10^-10 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.

Anti-insulin IgG 1st fraction

Anti-insulin IgG 2nd fraction p<0.01

††

**

*

*

††

††

33

2. =&#&IgG&#&F0VTQU RTK$D;3(1B

Vehicle)'ȇ!HSBS-gTɣɝɁɠ1-Ǡá,<?†ˇæIRS-1-ƱÒˊ.˼Ǡá=60

ÓˏĔæ+$!˺Fig. 2-7 A˻ɣɝɁɠ1-G†X†-ìǻǠá,<?†ˇæIRS-1-ƱÒˊ .˼˾Ó=3Ó,'vehicleǠá,<?ƱÒˊ<>:ƞš,Ė$!˺p<0.05˻˼5Ó¢˗, 'úƵ,Ĕæ!˺Fig. 2-7 B˻ůG†X†IgG-ȵ1ÓȊ<0ȵ2ÓȊ-ɣɝɁɠ1-ìǻǠ á,<?†ˇæ IRS-1 -ƱÒˊ.˼60 ÓˏBʳ'˼vehicle Ǡá,<?ƱÒˊ)úƵ,Ĕæ!

˺Fig.2-7C<0D˻$5˼ɣɝɁɠ1-G†X†Ǡá,<?†ˇæIRS-1-ƱÒˊ.˼ȵ1 ÓȊB):,Ǡá?)˼5Óő,Ɯė,ʻ'˼@¢˗˼Œ,ǣİ!˼Ǡá=30Ó6(G

†X†ìǻǠá,<?ƱÒˊ<>ƞš,Ė$!˺Fig. 2-8B˻6!˼ɣɝɁɠ,G†X†)ȵ2Ó ȊB):,Ǡá?)˼†ˇæIRS-1-ƱÒˊ.˼60ÓˏBʳ'˼G†X†ìǻǠá,<?ƱÒ ˊ)úƵ,Ĕæ!(Fig. 2-8C)

Vehicle)'ȇ!HSBS-gTɣɝɁɠ1-Ǡá,<?†ˇæAkt-ƱÒˊ.˼Ǡá=60

ÓˏĔæ+$!˺Fig. 2-9A˻ɣɝɁɠ1-G†X†-ìǻǠá,<?†ˇæAkt-ƱÒˊ.˼

5Ó(Ɯė,ʻ!ő˼Œ,¨!˼Ǡá= 60ÓˏBʳ'˼vehicleǠá,<?ƱÒˊ<>

ƞš,Ė$!˺Fig. 2-9B˻ůG†X†IgG-ȵ1ÓȊ-ɣɝɁɠ1-ìǻǠá,<?†ˇæAkt -ƱÒˊ.˼15Ó˺p<0.05˻)30Ó˺p<0.01˻,'˼vehicleǠá,<?ƱÒˊ<>@ @Ė

$! (Fig. 2-9C)6!˼ȵ2ÓȊ-ɣɝɁɠ1-ìǻǠá,<?†ˇæAkt -ƱÒˊ.˼5Ó˺p<0.05˻

)15Ó˺p<0.01˻,'˼vehicleǠá,<?ƱÒˊ<>@ @Ė$!˺Fig. 2-9D˻$5˼

ɣɝɁɠ1-G†X†Ǡá,<?†ˇæAkt-ƱÒˊ.˼ȵ1ÓȊB):,Ǡá?)˼1 Óő-ƱÒˊ.G†X†ìǻǠá<>:ƞš,İ+$!˺p<0.01˻˼-őđá˼5 Óő,Ɯė,ʻ

!˺Fig. 2-10B˻˼ɣɝɁɠ1-G†X†ìǻǠá,<?ƱÒˊǣİĝ9!15Ó¢˗˼

G†X†)ȵ1ÓȊ-Ǡá,<?†ˇæAkt-ƱÒˊ.Ǡá=30Óő6(G†X†ìǻǠá

<>ƞš,Ė$!˺p<0.05˻6!˼ɣɝɁɠ,G†X†)ȵ2ÓȊB):,Ǡá?)˼†ˇæ Akt-ƱÒˊ.60ÓˏBʳ'˼G†X†ìǻǠá)úƵ,Ĕæ!˺Fig. 2-10C˻

34 0

100 200 300 400

0 15 30 45 60 Phosphorylated IRS-1 percentage of non-treatment cells

Time (min)

0 100 200 300 400

0 15 30 45 60 Phosphorylated IRS-1 percentage of non-treatment cells

Time (min)

0 100 200 300 400

0 15 30 45 60 Phosphorylated IRS-1 percentage of non-treatment cells

Time (min ) B) Insulin alone (●) and vehicle alone (○)

C) 1st fraction alone () and vehicle alone (○)

D) 2nd fraction alone () and vehicle alone (○) A) Vehicle (○)

0 1 3 5 15 30 60 Time (min)

(●) (○)

0 1 3 5 15 30 60 Time (min)

§ §

()

0 1 3 5 15 30 60 Time (min)

()

0 1 3 5 15 30 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 (○).

35 0

500 1,000 1,500

0 15 30 45 60 Phosphorylated IRS-1 percentage of non-treatment cells

Time (min)

0 500 1,000 1,500

0 15 30 45 60 Phosphorylated IRS-1 percentage of non-treatment cells

Time (min ) B) 1st fraction + insulin (▲) and insulin (●)

C) 2nd fraction + insulin () and insulin(●)

*

*

**

** *

0 1 3 5 15 30 60 Time (min)

0 1 3 5 15 30 60 Time (min)

0 1 3 5 15 30 60 Time (min)

A) Insulin (●)

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 (●).

(▲) (●)

()

36 0

500 1000 1500 2000

0 15 30 45 60

Phosphorylated Akt percentage of non-treatment cells

Time (min)

0 500 1000 1500 2000

0 15 30 45 60

Phosphorylated Akt percentage of non-treatment cells

Time (min) 0

500 1000 1500 2000

0 15 30 45 60

Phosphorylated Akt percentage of non-treatment cells

Time (min) B) Insulin alone (●) and vehicle alone (○)

C) 1st fraction alone () and vehicle alone (○)

D) 2nd fraction alone () and vehicle alone (○) A) Vehicle (○)

0 1 3 5 15 30 60 Time (min)

(●) (○)

0 1 3 5 15 30 60 Time (min)

()

()

0 1 3 5 15 30 60 Time (min)

0 1 3 5 15 30 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 (○).

37 0

500 1000 1500 2000

0 15 30 45 60

Phosphorylated Akt percentage of non-treatment cells

Time (min)

0 500 1000 1500 2000

0 15 30 45 60

Phosphorylated Akt percentage of non-treatment cells

Time (min)

0 1 3 5 15 30 60 Time (min)

B) 1st fraction group + insulin (▲) and insulin alone (●) A)  Insulin alone (●)

0 1 3 5 15 30 60 Time (min)

C) 2nd fraction + insulin () and insulin alone (●)

0 1 3 5 15 30 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 (●).

(●)

(▲)

()

38

ɗ

ɗ Ī

ȵ2Ȳ(., ¼ň+gT-ɹǡ=EoFe`FP„wbQ€oFŠ)HPLC,<>G†X†,Ĭ

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ȂȈ.˼G†X†-õħ«Ʌùő,?RTK

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?ÓȊ)˼đŌ+ÓȊ?)Ɣ=)+$!

gT-ůG†X†ɯǵɯĻů«)G†X†-Ŏŧ?ůîů«ʁù«*-<+žɃ7(G†

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!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!3T3ɍɊɳɁɠBȇ!ȥȯ[46],'˼EGF,ʈĀśBȨů EGFů«EGF)ʁù«BŎŧ?)(˼EGF-ãƨBđŌ?)Ďý@'?ȵ2Ȳ,

?gT-ůG†X†IgG-ȵ1ÓȊ,<?G†X†-¬ȇđŌãƨ,.˼IgG)G†X†,

<?ĖÓĞʁù«Ŏŧː˼G†X†õħ«Ð˝-ʖĮ+*Bʳ'˼RTKVQd‚-ǗśæB

39 Ō9˼ɁɠË1-Ȼô>ʮ7ɢBđŌ!öɢś?

Fig. 2-11. The presumed multivalent complex formed of insulin and 1st fraction of anti-insulin IgG. Anti-anti-insulin IgGs are bound with anti-insulin as polyvalent antigen by binding to multiple antigenic sites at a time.

gT-ůG†X†IgG-ȵ1ÓȊ)ȵ2ÓȊ,?G†X†-¬ȇ,?ãƨ-ʼ.˼

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-#˼gT-ɹǡ=Ó˟!ůG†X†IgG-ȵ1ÓȊ)ȵ2ÓȊ,'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†-KD¹.5.4410.310^-5˺łĊ¹ƶǩºĺ˻M(>˼¨ʈĀś,Ó˫@![26]

gT-ůG†X†IgG-ȵ1ÓȊ)ȵ2ÓȊ)G†X†-KD¹.˼@ @1.6110^-4 M˼2.00 10^-5 M(>˼):,¨ʈĀś,Ó˫(!˼G†X†-¬ȇđŌãƨ-ʕ9=@!ȵ1 ÓȊ-ʈĀś.˼ȵ2 ÓȊ<>¨$!G†X†,Ĭ?ɅùʈĀś¢ĕ-ůîů«ˏ-ÓĞˏȝ–¬ȇ

Insulin

Anti-insulin IgG 1st fraction

40

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,Ʊʍ?Řʃ?

ȏȭäǸȈƥ-G†X†Ů,òŚ'Ȇȅ@!ůG†X†ů«,%'.˼ُG†X†

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G†X†¬ȇ-ǣŋ)đŌ-Ǝ-ãƨĎý@'?˼gT-ůG†X†ɯǵɯĻů

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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(?

41

RTKVQd‚\†kPʥ-ƱÒ,'˼ȵ1ÓȊ<0ȵ2ÓȊ-ìǻǠá,<>†ˇæAkt -ƱÒˊA,đá!˼†ˇæIRS-1-ƱÒˊ.ÈĔæ˼ɁɠË1-Ȼ-ô>ʮ7:

đá+$!Ʊʍ@!ǯŇ-ůG†X†IgGÓȊ-ìǻǠá.˼Ɂɠ,?AktBȜſǗś æ(?öɢś.?˼AktǗśæ-ȬŇ.ŋ˼GLUT4-translocationB'ɁɠË1-Ȼô

>ʮ7B´)(+˼ȅȂĠȘ,.šþBŷ!+†ˇæAkt-ȖȀˊđá)ɗ=@!

42

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į Ŷ

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@?ĖÓĞʁù«-ƴʵȘǹŖ-ʼː³'?öɢśɗ=@!

43

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ůG†X†IgGG†X†-¬ȇ,?ãƨBƱʍ!ÓȊ!2ȭ˫-ůG†X†IgG

-#˼G†X†,Ĭ?ʈĀś-¨ȵ1ÓȊ.˼G†X†-¬ȇBđŌ?ãƨBȨ!gT -ůG†X†IgG-ȵ1ÓȊ˼*-<,'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 ,%'.˼ůEGFů«[46];ůT†Ldj†Aů«[48]˼6!ȏȭäǸȈƥG†X†Ů,òŚ 'Ȇȅ@?ůG†X†ů«[18, 46],'Ďý@'?ůG†X†IgG-ȵ1ÓȊ, ':˼G†X†-¬ȇđŌãƨ,˼IgG )G†X†,<>Ŏŧ@?ĖÓĞʁù«ː³'?

öɢś?

ȵ3Ȳ(.˼ȵ2Ȳ,'ʕ9=@!ůG†X†IgG-ȵ1ÓȊ,<?G†X†-¬ȇđŌã ƨ,˼IgG )G†X†,<?ĖÓĞʁù«Ŏŧː³?*BƱʍ!IgG .˼kkG†Ñ Ȃ,<>l†W˃Nơȳ½Ôƌ@˼1ÓĞō!>2%-Fabo€Qz†b)1%-Fco€Qz†

b,Ó˟?Fabo€Qz†bæ!IgG.˼ůîBƩƹ!ĖÓĞʁù«BŎŧ?)(+

+?gTŧǷɣɝɁɠ)Fabo€Qz†bæ!ůG†X†IgG-ȵ1ÓȊBȇ'˼G†X

†¬ȇđŌ?*BƱʍ!

44

ƣ

ƣƋ<0Ǝǐ

1. =&#&IgG O1.M Fab" &3

ȵ2Ȳ,'˼¼ň+gT-ɹǡ=EoFe`FP„wbQ€oFŠ)HPLC,<$'ÓȊ

!ůG†X†IgG-ȵ1ÓȊB®ȇ!ģ˴,.ǴÑɒ-ȵ1ÓȊ)kkG†ÑȂ,<>Fabo€

Qz†bæ!ȵ 1 ÓȊBȇ!kkG†ÑȂ.˼Pierce Fab Preparation Kit˺Thermo Fisher Scientific, Rochester, NY, USA˻Bȇ'žƵƛ,œ$'ģƏ˼ 1ÓĞō!>2%-Fabo€Qz†

b)1%-Fco€Qz†b,Ó˟!˺Fig. 3-1˻Ó˟!Fab<0Fco€Qz†bÓĞ-ğĈB Ȧʕ?!9,˼ǴÑɒ-ȵ1ÓȊ)kkG†ÑȂő-ȵ1ÓȊ-Fabo€Qz†bÓȊ<0Fco

€Qz†bÓȊB15%SDSEP‚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,˼4Ɩˏ˼

ɹǡ)G†X†Bû6+ɊŷČĉ(˭˱Č˰!

45 3. 2-Deoxy-D-glucose uptake assay

gTŧǷɣɝɁɠ1-Ȼô>ʮ7ɢ-ʓ².˼ȵ2Ȳ)úƵ-Ǝǐ(ģƏ!˭˱Č˰!ɣɝɁ ɠ,˼HSBS(ɋˊ100 μl,ʛƉ!¢-7ȭ˫-ǪǜB¢-ǯŇ(Ǡá˼=,30ÓˏČ˰

!; 1) ǴÑȂ˼2) ÛČ˰,®ȇ!HSBS˺vehicle˻˼3) G†X†10^-7 M-ìǻ˼4) ǴÑɒ-ȵ 1ÓȊ10^-12 M, 10^-11 M, 10^-10 Mìǻ˼5) G†X†10^-7 M)ǴÑɒ-ȵ1ÓȊ10^-12 M, 10^-11 M, 10^-10 M -ǟù˼6) Fabo€Qz†bæ!ȵ1ÓȊ10^-12 M, 10^-11 M, 10^-10 Mìǻ˼7) G†X†10^-7 M)Fab o€Qz†bæ!ȵ1ÓȊ10^-12 M, 10^-11 M, 10^-10 M-ǟù

4. VTQU#&_3%&&]fAktg C-

Fabo€Qz†bæ!ůG†X†IgG-ȵ1ÓȊ-ğĈ˼gTŧǷɣɝɁɠ1-G†X†Ǡ á,<?RTK VQd‚Ǘśæ,?ãƨBʓ²?!9,˼ȵ2Ȳ)úƵ-Ǝǐ(Ɂɠ-†ˇæ Akt-ɄƖȘ+ƱÒˊ-ĔæBʛ2!˭˱Č˰!ɣɝɁɠ,, ˭˱Č˰,ȇ!ɹǡ)G†X†B û6+ɊŷČĉ(ɋˊ100 μl,ʛƉ!¢-6ȭ˫-ǪǜBǠá˼=,0Ó, 1Ó, 3Ó, 5Ó, 15 Ó, 30Ó, 60ÓˏČ˰!; 1) ˭˱Č˰,®ȇ!ɊŷČĉ˺vehicle˻˼2) G†X†10^-7 M ìǻ˼3) Ǵ Ñɒ-ȵ1ÓȊ10^-10 Mìǻ˼4) G†X†10^-7 M)ǴÑɒ-ȵ1ÓȊ10^-10 M-ǟù˼5) Fabo€Q z†bæ!ȵ1ÓȊ10^-10 Mìǻ˼6) G†X†10^-7 M)Fabo€Qz†bæ!ȵ1ÓȊ10^-10 M -ǟù

5. RZ.A

gTɣɝɁɠ1-G†X†˼ǴÑɒ-ȵ1ÓȊ<0Fabo€Qz†bæ!ȵ1ÓȊ-ìǻǠá ,<?Ɂɠ1-[³H]-2-DOGô>ʮ7ˊ.˼øģ˴ɔ-aŠ\-ȶÓƇśBBartlettƱĢ,<>Ȧʕ!

ő,˼vehicleǠá,<?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\-ȶÓƇśBBartlettƱĢ,<>Ȧʕ!ő,˼G†X

46

†ìǻǠá,<?ô>ʮ7ˊ)-ĺB‹Ã˄ɒÓƇÓƧ)Dunnett ǐ,<>Džʪ!=,˼Ʊʍ

!ǴÑɒ-ȵ1ÓȊ<0Fabo€Qz†bæ!ȵ1ÓȊ,'ǠáǯŇ˺10^-12 M, 10^-11 M, 10^-10 M˻ˏ-[³H]-2-DOG ô>ʮ7ˊ-ĺ.˼‹Ã˄ɒÓƇÓƧ)Tukey-Kramerǐ,<>@ @Džʪ

!Ɂɠ1-ǴÑɒ-ȵ1ÓȊ<0Fabo€Qz†bæ!ȵ1ÓȊ-gTɣɝɁɠ1-ìǻǠá,

<?†ˇæAkt-ƱÒˊ.˼Ǡáő-ɄʺƖˏ˺Ó˻DŽ,aŠ\BFƱĢ,<>ȶÓƇśBȦʕ!

ő,˼ vehicleìǻǠá,<?ƱÒˊ)-ĺBStudent-t-ƱĢ,<>Džʪ!6!˼ǴÑɒ-ȵ1 ÓȊ6!. Fab o€Qz†bæ!ȵ 1 ÓȊ-ğĈ,<?G†X†Ǡáő-Ɂɠ,?†ˇæ Akt-ƱÒˊ.˼Ǡáő-ɄʺƖˏDŽ,FƱĢ,<>aŠ\-ȶÓƇśBȦʕ!ő,˼G†X†ì ǻǠá,<?ƱÒˊ)-ĺBStudent-t-ƱĢ,<>Džʪ!p<0.05BɇʌȘ,ƞš)!

47

Ʌ

Ʌ ƨ

1. =&#&IgG O1.M Fab" &3

Figure 3-2.˼ůG†X†IgG-ǴÑɒ-ȵ1ÓȊ<0kkG†ÑȂ,<>Fabo€Qz†b) Fco€Qz†b,Ó˟!ȵ1ÓȊ-SDS-PAGE<0CBBƪɲ-ɅƨBȨ˺Fig. 3-2˻ǴÑɒ -ȵ1ÓȊ-Ʌƨ.˼IgG-HˌBȨ50 kDa ʯ)Lˌ-25 kDa ʯ,•ȝś-j†cBʕ9!

kkG†ÑȂ!ůG†X†IgG-ȵ1ÓȊ,'˼Fabo€Qz†bÓȊ-Ʌƨ.Fabo€Qz

†b)LˌBȨ25 kDa ʯ-‹ȝś-j†cBʕ9!6!˼ Fco€Qz†bÓȊ-Ʌƨ.Fco

€Qz†bBȨ28-30 kDa ʯ-‹ȝś-j†cBʕ9!kkG†ÑȂ,<>˼ůG†X†IgG -ȵ1ÓȊBFabo€Qz†b)Fco€Qz†b,Ó˟!)Ȧʕ(!

2. =&#&IgG O1.M Fab" &&#&F0VTQU [1,2-³H]-2-Deoxy-D-glucose 4^(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.˼vehicleǠ á,<?ô>ʮ7ˊ,Dž2'ɶđá!˺p<0.01, Fig. 3-3A˻$5˼ůG†X†IgG-ǴÑ ɒ-ȵ1ÓȊ<0Fabo€Qz†bæ!ȵ1ÓȊ-ìǻǠá,<?Ɂɠ1-[³H]-2-DOG-ô>ʮ 7ˊ.˼Ʊʍ!ǯŇ˺10^-12 M, 10^-11 M, 10^-10 M˻,'vehicleǠá,<?ô>ʮ7ˊ)úȶ(˼Ȼ ô>ʮ7ˊBđá+$!˺Fig. 3-3A˻G†X†Ǡá,<?Ɂɠ1-[³H]-2-DOG-ô>ʮ7ˊ .˼ǴÑɒ-ȵ1ÓȊB):,Ǡá?)(ɶđá!˺p<0.01˻˼đáˊ.ȇˊ±ğś(.

+$!˺Fig. 3-3B˻˼G†X†Ǡá,<?Ɂɠ1-[³H]-2-DOG-ô>ʮ7ˊ.˼Fabo€

Qz†bæ!ȵ1ÓȊB):,Ǡá':˼Ʊʍ!2'-ǯŇ(˼G†X†ìǻǠá,<?ô

>ʮ7ˊ)úȶ($!˺Fig. 3-3B˻

48

75

50 37

25 20

(kDa) 1 2 3 4

Fab fragment

+ L-chain (near 25 kDa) Fc fragment (near 28-30 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.

ドキュメント内 ネコの抗インスリン自然自己抗体（IgG）に関する研究 (ページ 33-51)