molecular mechanisms of adipogenesis in obesity and the metabolic syndrome

8/13/2019 molecular mechanisms of adipogenesis in obesity and the metabolic syndrome

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1. H, D.W. & J, W.P. O. 3, 11971209 (2005).

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66. M, S. & P, R.G. L : . , 373378 (2006).

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. , 12651270 (1981).

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74. M L, W.D., . C . 3, 15001508 (2009).

75. V, M.C., . A . , 12171222 (2004).

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77. K, M., B, P., S, L. & S, U. I . I . , 11501162(1983).


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29

78. S, S.R., . E :

. 4, 2432 (2005).79. W, S.P., .O .

, 17961808 (2003).

80. R, E.D. & MD, O.A. A . , 885896 (2006).

81. K, Y., K, S., N, I., T, K. & N, F. O .

3, 3844 (2000).82. K, T., .M : VI.

, 15751591 (2009).

83. S, A., S, J. & B, C. M :

. , 103111 (2005).

84. A, R.S. A . ( )4 , 242S249S

(2006).85. R, E.D. & S, B.M. A

. 444, 847853 (2006).

86. L, R.L., B, M., M, H. & M, M.G., J. L . ( )4 , 208S212S (2006).

87. M, Y., .L AMP

. 4, 339343 (2002).

88. H, K., .C 2 . , 11261133 (2001).

89. F, A., .V: .3, 426430 (2005).

90. Y, R.Z., .I

: . , E12531261 (2006).

91. Y, Q., .S 4 2 . 43, 356362 (2005).

92. J, J., . H / H: D A T G.

, 1000324 (2009).93. S, K.L., .D . 43, 783787 (2008).

94. T, P., H, E.D. & S, B.M. S A F P

G2, LA T F. , 11471156 (1994).

95. T, T., Y, N., K, T. & A, S. D C/EBP / C/EBP . , 74327443 (1997).

96. L, K., . I

1 (P1). , 453461 (2003).

97. S, C.M. & S, H.S. M

1. , 281298 (1997).98. T, Q., . F GATA ., 134138 (2000).

99. Y, E.J., C, J.J., C, S.S., K, K.H. & K, J.B. D

. , 66086615 (2006).

100. A, T., .PPAR

. 3, 846855 (2004).101. J, M.J., .A

R2 . , 2327023277

(2003).

102. L, K.A., . W10 . , 3550335509 (2004).


30/144

C 1

30

103. SJ, S., . S

. 4, 20852094 (2001).

104. C, L., S, J. & D, R. R TGF S

. 4, 667682 (2000).105. K, Q., .A BMP

. , 545559 (2009).106. K, N., . D

. , 10621066 (1998).107. S, H., .R 10

. , 908912 (2002).

108. L, J., S, D. & W, Z. M : .

, 15511554 (2002).109. C, V. & L, H.R. R

. , 254259 (2006).

110. S, S., L, L.R., T, T., C, C.S. & W, Z. A . 3, 267275 (2001).

111. C, T.H., . A 3

. , 577591 (2006).112. A K. L., R.M.C. T C D P C B C 19

"N I MMP A". (2008).

113. T, W., .W F P C R A V. (2008).

114. W, A., .D .

, 6475 (2007).115. T, K., . C +

. , 330338 (2008).

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. , 32203228 (2006).118. R, M.S., B, K. & F, J.M. I

. 3, 240249 (2008).

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. 4, E863873 (2003).

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(2008).

125. P, S. & M, W. F . 4, 429

434 (2008).

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127. C, A., . A FFA

FFA . , E10551066 (1999).

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31

129. Y, T., . H

J 2 . , 12741278 (2003).130. F, J.N., M, A.K., H, M.L., C, P. & B, S.W. C

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136. WF, L., .M . 4, 12811289 (2004).

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. 4, 13921399 (2005).138. K, T.R., . M

. , 4556 (2008).

139. H, C., .A

. , R14 (2008).140. C, C., .M

. , 1188811896 (2003).

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., 10931101 (2002).

142. K, A.D., . T . , 41454150 (2005).

143. S, N., .I . , 16091616 (2008).

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? ,10201025 (2001).

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, 1399& (1971).146. S, L.B., K, J.L. & H, J. T

. 4, 153165 (1968).

147. J, M., .S :

. , 1540+ (2006).

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. , 314320 (2006).

150. F, N., . I GLUT4

. ,

17161722 (2007).151. O'C, J., .T

. , 9997 (2010).

152. D, R., . H . ()3, 283291 (2008).

153. V H, V., R, K. & H, H. C

. 3, 632637 (2004).


32/144

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154. T, T., .F

. , 25712578 (2006).155. K, J.L., T, T., P, T., H, J.R. & K, I. A :

MAD? 3, 757767 (2002).

156. D, M.N., P, R., L, M.C. & G, Y. O :

1 2. 4, 649656 (2000).


33/144

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RRE1 AGC CAC TAC CAG ATC CTT GC RRE1 GGA AAA ACG GGA GTG AAA GG,

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R34

.

RNA DNA MMACS DNA

(M). RNA PCR SG

(I) . RNA

. T (= 60C): ATG

AAA TCA CCG CAG ACG ACA G, GCC TTT CAT AAC ACA TTC CAC CAC, /

GGT CGA GCG CAA CAA CAT C, / CTC GGG CAG CTG CTT GAA CAA,

CAT CGG CAG AAG GGG CAA AGT A, GTC AGC TCC CTC CTC CGA TTC C,

ACC GCC GCG CCA AGA ACT CG, AAC TGG GTG GGG GGT CTG TGT AG,

GCT GTG GGG TAG ATG GGA TA, CCC ATG GGG AAA TAC AAT GA, 3

ACA TGG AGA CTT TGT CCC TTT TG, CTG GCC GTG GGG ATG TCT CG,

AGG CTG CTG CTG GTG GTC TCG, GAT CGA TCG AAT TGC ACA GAA CAT,

GCC TCC CTG GAC TTG CTT TGA TAC 3 TTG GCT GAG TGG TAG AGT CCC,

CTG ACG TGC CGC CTG GAG AAA, CCG GCA TCG AAG GTG GAA GAG T

F R M3 TM G E A

.

T RNA T (I)

. G DNA DNA (A) RNA

A ( ) OC

RNA SI K (A) . L RNA

U133 (A) 44 (A).


40/144

C 2

40

G 32,35

.

B, 20 M ( ) 50 M (3T3

L1 ) 10 . C 1 1M

D[114C KR 1% BSA

. S 3T3L1 , ,

14C BCA A (S)

. P ,

. O 36

C C C. G

(S)

.

S

37

. B, ,

II 1 37C. P

40 , 106

100

M [BD. C . A

46 M

. F 3

C P S.

A 5%

10 . H&E

38

. M

C P S. F

CTS (LT). A 2000

.


41/144

C 2

41

B (C). I

R/M I ELISA (C C). T

T/GB K C.... (R/H) NEAFAHR (W). F

(L) .

R +/ . S

S . L G P

P.


42/144

C 2

42

R

I ,

SVF . T

R

(S. T. I). I R

RNA

(F. 1.1). A

(SVF), ROR

. T ,

3T3L1. D RNA R

3T3L1 . U , R

ROR

(F 1.1). W ROR 3T3L1

. ROR 3T3L1

(AFABP) (F. 1.2,, S. F.1,). T

, ,

, ROR PPAR (F. 1.2,). C, RNA

ROR 3T3L1

(F. 1.2, S. F. 1). N

ROR ROR, ROR

(S. F. 1).


43/144

C 2

43

1.1 . )R

(BMI35) ( = 9). )E

R, M3 AF 3T3L1 .

a

-48 -24 0 24 48 72 96 120 144 1680.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Mmp3Ror A-Fabp

hours

normalized

mRNAexpression

induction ofdifferentiation

BMI 350.0

2.5

5.0

7.5

10.0

12.5

15.017.5

20.0

22.5***

normalzied

Ror

mRNA

expression

b


44/144

C 2

44

1.2 . )F (: , :

, : , : 100 ) 3T3L1 ROR

7 . )W

3T3L1

(AFABP) T

. , ) F AFABP 3T3L1

RNA RNA ROR

. V +/ SD, * 0.05, ** 0.01, *** 0.001.

c

d

e

f

A-FABP

-Tubulin

pcDNA3 ROR

- rosiglitazone

A-FABP

-Tubulin

pcDNA3 ROR

+ rosiglitazone

A-FABP

-Tubulin

siRNA ctrl siRNA1 siRNA2

scramble siRNA 1 siRNA20

10

20

30

4050

60

70

%

differentiation

****

pcDNA3 ROR pcDNA3 ROR0

5

10

15

20

25

30

35

40

45

%

differentiation

- rosiglitazone + rosiglitazone

***

**

pcDNA3 ROR

- rosiglitazone

pcDNA3 ROR

+ rosiglitazone

siRNA scramble

siRNA1 RORsiRNA2 ROR


45/144

C 2

45

T ROR ,

. E

J, F, C/ , PPAR AF ROR

ROR (F. 1.3). T ROR PPAR

PPAR ROR

. PPAR ROR

(F. 1.3). O , ROR

PPAR PPAR

ROR (S. F. 2). A R

PPAR30, ROR PPAR

. ROR PPAR ,

IL17 ROR

(F. 1.3). T , ROR

,

PPAR .


46/144

C 2

46

1.3 . ) PCR J,

F, C/, C/, P AF G

3T3L1 ROR . )

PPAR (PPRE) ,

3T3L1 ROR

. ) IL17 ( ),

PPAR PPRE ( ) 3T3L1

ROR (=4). V +/ SD,

* 0.05, ** 0.01, *** 0.001.

c-Jun

ay 0 ay 1 ay 2 ay 3 ay 60

1

2

3

4

5

6control

ROR

*

*

normalized

mRNAexpression

C/ebp

ay 0 ay 1 ay 2 ay 3 ay 60.0

0.1

0.2

0.3

0.4control*

*

*

normali

zedmRNAexpression

ROR

c-Fos

ay 0 ay 1 ay 2 ay 3 ay 60.0

0.5

1.0

1.5

control*

normalized

mRNAexpression

ROR

ROR

A-Fabp

ay 0 ay 1 ay 2 ay 3 ay 60.0

0.5

1.0

1.5

2.0 control

**

*

*

*norm

alizedmRNAexpression

g

ay 0 ay 1 ay 2

5

15

25control

ROR

PPREluciferaseactivity

**

***

***

C/ebp

ay 0 ay 1 ay 2 ay 3 ay 60

1

2

3

4 controlROR

**

*

normalizedmRNAexpression

Ppar

ay 0 ay 1 ay 2 ay 3 ay 60.0

0.5

1.0

1.5

2.0

2.5

ROR

**

**

*

**norm

alizedmRNAexpression

control

IL-17 PPAR prr PPRE0

1

2

3

4

5

control

ROR

***

normalizedluciferaseactivity

h

i j

k l

m n


47/144

C 2

47

T ROR

3T3L1

ROR (S. T. II). G ROR SVF RNA

(F. 2.1). S

3 (MMP3) 3T3L1

. W M3 RNA

ROR (F. 2.1), ROR

. I, M3 3T3L1

R (F. 1.1),

M3 ROR .

T MMP3 ROR MMP3

ROR (RRE). T

RRE; RRE PGGTCA

AT , RRE

PGGTAA. U ROR MMP3

RRE ROR IL17 (F. 2.2).

T CIP ROR

MMP3 (F. 2.2). A,

M3 ROR

(F. 2.2).


48/144

C 2

48

2.1 3 . ) RNA

ROR . 3T3L1 RNA ,

. ) E M3 3T3L1

ROR ROR ,

. V +/ SD, * 0.05, ** 0.01, *** 0.001.

a

control control 0

1

2

3

4

5

transient lentivirus

**

***

normalizedMmp

3expression

ROR ROR

scrambl

eRo

rAb

at

Accn1

Arhg

ap30

Arhg

ap6

Cxcl9Eg

r3FapGc

h1

Gcnt1Gp

r4Ifn

g

Mfsd4

Mmp3

Mst1r

Nxph

3Pb

p2Rtp3

Slc28a2

Slc4a1

Stmn3

0

10

20

30

40

5060

70

80

90*** ***

%

differentia

tion

b


49/144

C 2

49

2.2 3 . ) MMP3

ROR (RRE)

. L 3T3L1 ROR

48 . (=4) )

PCR RRE1 3T3L1 DNA

6 ROR . (=3) )N

3T3L1 ROR

. V +/ SD, *** 0.001.

I

III

IV

luciferase

-2460 bp -1697 bp

luciferase

luciferase

II luciferase

RRE1 RRE2

RRE2

RRE2

I

III

IV

luciferase

-2460 bp -1697 bp

luciferase

luciferase

II luciferase

RRE1 RRE2

RRE2RRE2

RRE2RRE2

MMP3prI MMP3prII MMP3prIII MMP3prIV IL170

5

10

15

20

25 control

luciferaseactivity

*** ***

ROR

c

d

e control

Ror

Mmp3

Gapdh

C/ebp

Pref1

C/ebp

Ppar

Lrh1

ROR

input control ROR ROR

anti-HA Ab IgG ctrl. Ab

control ROR0

5

10

15

20

25

30

35

ROR

DNAenrichment

***


50/144

C 2

50

T ROR MMP3,

MMP3 . I MMP3

3T3L1

(S. F. 2). M, ROR

MMP3 (F. 2.3),

ROR MMP3. T ,

MMP3 ROR

ROR MMP3.

2.3 3 . ) F

3T3L1 ROR

20 M MMP3 2

. (=4) S: 100 . V +/ SD,

* 0.05, ** 0.01, *** 0.001.

f

control ROR ROR0

25

50

75

100

125

+ MMP3 inhib

%

nor

malizeddifferentiation

*** **

*

control ROR ROR + MMP3 inhib


51/144

C 2

51

T ROR ,

SVF

. SVF GFP

46 . C GFP

(F. 3.1). L

(

). O ,

, GFP AFABP

(F. 3.1). T ,

. T ROR

GFP SVF C57B/6J

. A 4 /

,

ROR

(F. 3.1, ). Q

2.5

ROR (F. 3.1).


52/144

C 2

52

3.1

SVF

C57B/6J . F

. ) Q

V +/

( )

6 4 HFD

SVF

SD, *** 0.001.

b

w ildtype ROR -/-0

5

10

15

***

%a

dipocytes/nuclei

***

. )I

. (=5)


53/144

C 2

53

A ROR ,

/

. T ,

(DIO,

60% ) /

. M /

(F. 3.2). T

CT

(F. 3.2, S. F. 3). W

,

/

. T ROR

.

T

,

3T3L1 ROR.

N ROR (S. F. 3,

). D , 12

/

,

( ).

A MMP3 ROR

, RNA MMP3 SVF

12 /

. MMP3

2.4 (F. 3.2), MMP3

ROR . T , ROR

,

.


54/144

C 2

54

3.2 . ) M

/

/

,

/

DIO ( = 47). ) F

/

8 ( = 510). ) M3 SVF

/. (=5) V +/ SD, * 0.05, ** 0.01, *** 0.001.

wildtype ROR -/-0.0

0.1

0.2

0.3

0.4

0.5

relativeMmp3expression

*

c

d e

male

DIO

subc

ut

maleDIO

viscer

al

fem

aleDIO

subc

ut

fem

aleD

IOviscer

al

maleob/ob

subc

ut

maleob/

obviscer

al0

10000

20000 +/+-/-

meanadipocytesize[m2]

***

*** *****

*** ***

maleDIO

subcut

maleDIO

visceral

femaleDIO

subcut

femaleDIO

visceral

maleob/ob

subcut

maleob/ob

visceral

0

1

2

3

4

5

6

+/+

-/-

fatpadweight[g] **

**

** **

200

+/+

-/-

subcutaneous visceral

200


55/144

C 2

55

T

, /

.

M (F. 4). I

/

(F. 4)

(F. 4, S. F. 4). I ,

/

8

(F. 4). B ,

/

. A ,

/

(F. 4). I ,

/

(F. 4, S. F. 4). I ,

(S. F. 4).

A ROR ,

, TNF, INF, IL1,

IL2, IL12 IL6. S ,

E1 ,

(S. F. 4, ).

T , ROR

, . A ,

, ,

.


56/144

C 2

56

4

. )B /

(12 )

/ (DIO) 8 ( = 510). , )

F / /

( = 510). ) I /

8

.. 0.75U/ ( = 510). )

/ /

100% ( = 4). ) F

/ /

( = 510). V +/ SD, * 0.05, ** 0.01, *** 0.001.

male

DIO

+/+

male

DIO

-/-

female

DIO

+/+

fem

ale

DIO

-/-

male

ob/ob

+/+

male

ob/ob

-/-0

5

10

15

***

fastingbloodglucose[mM]

*

male

DIO

+/+

male

DIO

-/-

female

DIO

+/+

female

DIO

-/-

male

ob/ob

+/+

male

ob/ob

-/-0

1

2

3

4

56

7

8

9

***

insulin[ng/ml]

***

*

a b

c d

male

DIO

+/+

male

DIO

-/-

fem

aleDIO

+/+

fem

aleDIO

-/-

male

ob/ob

+/+

male

ob/ob

-/-0

10

20

30

40

50

*

weight[g]

**

male

DIO

+/+

male

DIO

-/-

fem

aleDIO

+/+

female

DIO

-/-

m

aleob/ob

+/+

male

ob/ob

-/-0.0

0.5

1.0

1.5

2.0

***

freefattyacids[mM]

*****

+/+ -/-0

20

40

60

80

100

120

basal***

%

basallipolysis/cellnumber

20nM insuline f

0 30 60 90 120 1500

25

50

75

100

125 +/+-/-

**

*

time [min]

%

initalblood

glucose


57/144

C 2

57

S ROR

,

R . T , 21 (11 , 10 )

(BMI) 3256 /2. A ,

HOMA10

(F. 5). T

SVF

,

. A

SVF,

(F. 5). S , R

(F. 5,). MMP3 , M3

SVF R

(F. 5,). W , R SVF

HOMA < 3.5

HOMA > 3.5. I, R BMI,

R (F. 5).


58/144

C 2

58

5

. ) S

HOMA . (=19) ) S

SVF. (=15)

) R RNA G SVF

. (=20) ) R RNA

SVF

SVF. (=16) ) R RNA

M3 SVF . (=21) )

M3 RNA SVF

. (=20) ) R SVF BMI

HOMA 3.5. (=19) V +/ SD,

* 0.05.

0 10 20 3017000

19000

21000

23000

25000R2= 0.47p=0.001

HOMA

meanad

ipocytesize[m

2]

0 1 2 3 4 5 6 717500

20000

22500

25000

R2=0.49p=0.004

% differentiation

meanad

ipoyctesize[m

2]

a b

c d

e f

g

17000 19000 21000 23000 250000

1

2

3

4

5

6

7

8

9 R2= 0.47

p=0.0008

adipocyte size [m2]

normalizedRorexpression

0 1 2 3 4 5 6 70

1

2

3

4

5

6

7

8

9

R2=0.38

p=0.011

% differentiation

normalizedRorexpression

0.15 0.25 0.35 0.45 0.55 0.650

1

2

3

4

5

6

7

89 R2= 0.34

p=0.005

normalized Mmp3 expression

normalizedRorexpressio

n

17000 19000 21000 23000 250000.0

0.1

0.2

0.3

0.4

0.5

0.6 R2= 0.35p=0.006

adipocyte cell size [m2]

normalizedMmp3expressi

on

HOMA 3.50

1

2

3

4

5

6

7

*

0

10

20

30

40

50

RorexpressionBMI

normalizedRorexpressio

n

BMI[kg/m2]

*


59/144

C 2

59

D

O ROR

. T MMP3, ROR

. T

(F. 6). O

,

. I

3940

. O

ROR SVF

. T, ROR

,

.

/

. A ROR

, . A ,

.

ROR/

/


60/144

C 2

60

I , ROR

. I ROR

, PPAR

4142. G

12,4344

, ROR

. R

ROR , .. 7, 24S

T0901317, ROR 2022

.

T

ROR ,

.

I, 12123 ROR

2 45

. O ROR

.

W R ,

R 3T3L1

(F 2B). I R

28

. H,

, .. 3T3L1

. T R

146

ROR . T

ROR PPAR,

. I R,

PPAR 30

, PPAR

ROR. T ROR

PPAR . T,


61/144

C 2

61

ROR PPAR

,

PPAR. T ROR , ROR

C/EBP, PPAR C/EBP.

A ROR

,

ROR . E ROR

, ROR

. A MMP3

, ROR

MMP3. I

47

. T, MMP3

,

ROR .

T MMP3 ROR ,

MMP .

MMP ,

15

. W MMP 48

, MMP3/

35%

18

MMP3

. W MMP3

3T3L1 RNA MMP3.

G MMP3 ROR,

.

A ROR

. S

4950,


62/144

C 2

62

ROR

51

. T

.

T , ROR

. E ROR

,

,

.


63/144

C 2

63

R

1. D, M.Y., .A , , .

97, 835838 (2006).

2. M, J.B., . B , , 2

. 91, 29062912 (2006).

3. S, J.K. & VP, A.J. T : A A

.. .4, 12531262 (2007).

4. J, J., . H / H: D A T G.

5, 1000324 (2009).

5. S, K.L., .D . 453, 783787 (2008).

6. G, S., T, Y. & K, C.R. D : .

131, 242256 (2007).

7. G, G.H. T

. & 4, 206218 (2008).

8. T, H. & M, A.R. A: ,

. , 772783 (2006).

9. P, G., . A

NIDDM. 3, 12131217 (1995).10. R, R., . M :

. 52, 882890 (2009).

11. U, R.H. M: :

. 144, 51595165 (2003).

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. 11, 32203228 (2006).

13. J, J.A., T, S.E., F, A.W., J. & V, J.R. P

. ( , 15,

26532663 (2007).

14. K, Y., K, S., N, I., T, K. & N, F. O

. 3, 3844 (2000).

15. L, J., S, D. & W, Z. M : .

10, 15511554 (2002).

16. B, A., S, C., P, G., G, J. & L, M. A

2 9: . 50, 20802086

(2001).

17. L, H.R., . M

. 22, 374379 (2002).

18. A, C.M., S, S., M, J. & W, Z. S1

. 152, 693703 (2001).

19. J, A.M. R (ROR): , ,

, . , 003 (2009).20. K, N., . T T0901317 ROR/ I

A. (2009).

21. W, Y., K, N., C, C., G, P.R. & B, T.P. A

: R ROR ROR 24S

(). (2010).

22. W, Y., . M ROR ROR 7

. (2009).

23. Z, F., M, G. & S, W. I R1,

ROR F3 17 T .

, 12971306 (2008).

24. Z, L., . TGF F3 T(H)17

ROR . 453, 236240 (2008).


64/144

C 2

64

25. M, B. & W, C. T .

110, 191205 (2008).

26. R, S., L, P., S, B. & M, G.E.O. R

:

. 3, 2944 (2007).

27. B, W., B, R.J., K, J.W. & T, M.B. T

C . 15, 843

853 (2007).

28. A, S., . I ROR

D1 3T3L1 . & , 267276 (1998).

29. S, Z.M., . R ROR

. 2, 23692373 (2000).

30. W, J. & L, M.A. B R .

2, 22132220 (2008).

31. K, J.B., W, H.M., W, M. & S, B.M. ADD1/SREBP1 PPAR

. 5, 43334337 (1998).

32. W, C., . R F2 .

112, 345356 (2003).

33. N, L., .I

. 22, 263267 (1996).

34. R, R.H. & R, R.G. R RNA . , 433441

(1972).

35. U, B., . D

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24, 3086430873 (1999).

36. M, H.J. & M, M.D. V E A S N

P C. 2, 980991 (1986).

37. H, L.H., M, B., T, B., N, J.H. & B, N. C

.

12, 11401149 (1998).

38. C, H.C. & F, R.V. D . . .43, 986989 (2002).

39. L, M., M, K., B, C. & L, L. A 2

. 24, 326331 (2010).

40. O'C, J., .T

. 5, 9997 (2010).

41. CM, C., . C

2 : .

, , , 191 (2004).

42. Z, A., D, A.G., P, C., M, J.P. & B, M. T

.

23, E9195 (2007).

43. G, A. S . 1, 129137(2008).

44. W, Y., C, L.W. & E, R.M. PPAR .

13, 14961503 (2007).

45. W, H., . M

(RORC): 2 .

, 176182 (2003).

46. S, C.M. & S, H.S. P1, EGF ,

. 3, 725734 (1993).

47. T, F., . B

C80/DRO1/URB. 24, 81368147 (2009).


65/144

C 2

65

48. C, C., .M

. 2, 11888

11896 (2003).

49. R, E. & S, S.R. I , ,

, , 2 .

, 363378 (2002).

50. H, L., S, S.R. & R, E. F , , II .

2 4, S1221 (2004).

51. G, M., HM, J.J., L, J., T, F.J. & Z, S. R

/ . ()30, 899905 (2006).

52. A: AFABP N. H. IL17

T K. T ERCA

(CW), SNF3100A0117979 (CW), FP7LN (CW, DG), EASD (GR, CW), BMBF (PN)

R R F (BM).


66/144

C 3

66

T 1 (TIMP1)

B M, L S, E CS, C

W

S F I T, ETH Z, I M S

B, WP S. 16, 8093 Z, S

C 3


67/144

C 3

67

A

E

MMP/TIMP . T

TIMP1 , TIMP1

,

. W

TIMP1

. TIMP1

3T3L1 . C,

TIMP1 . M,

TIMP1

. A , TIMP1

,

. I , TIMP1

. I TIMP1

,

.


68/144

C 3

68

I

O 2

. E

. S

12

. O

. T

, . A

, ,

36

. N

,

(SVF) 78

. F

, , (ECM)

. T, ECM

910

. I ,

11

. M,

I III IV VI

1214

. T

(MMP) ,

(TIMP)10

.

A MMP

MMP

15

. T, MMP

,

10

. N, MMP3, MMMP11 MMP19

1618

. I ,

, MMP

. L MMP3, ,

19

. M, , ,


69/144

C 3

69

MMP, 20

. F,

MMP

21

. T MMP BB94 3T3L1

15.

MMP 1:1 TIMP

(TIMP14) . TIMP1,

SVF, MMP

MMP14, MMP16 MMP1922

. E

TIMP1 23

.

C TIMP1

. W TIMP1

, TIMP1

2425

. O ,

TIMP1

26

. N TIMP1

3T3F442A , , TIMP1 3T3L1

19,26. A

TIMP1 . T,

MMP .

A TIMP1 ,

. T,

TIMP1 ,

TIMP1

.


70/144

C 3

70

E P

RNA DNA MMACS DNA (M).

RNA PCR SG (I)

. RNA G.

T (= 60C): CTG ACG TGC CGC CTG

GAG AAA, CCG GCA TCG AAG GTG GAA GAG T, 1 GAT ATG CCC ACA

AGT CCC AGA ACC, 1 GCA CAC CCC ACA GCC AGC ACT AT, AGG CGA

GGG CGA TCT TGA CAG, AAT TCG GAT GGC CAC CTC TTT G, ATG

AAA TCA CCG CAG ACG ACA G, GCC TTT CAT AAC ACA TTC CAC CAC.

S 27

2 /

II 1 , . C

D

E 10%

/. T , OMEM

(I) 24. TIMP1

Q M TIMP1 ELISA (R&D S)

.

MMP9 MMP9 A. I MMP9 T1

[R&D S SL 570 G MMP A K

F (A) .

M T1

DNA : T1 AAG CTT TTG TAG CTG TGC CCC ACC

CCA T1 CTC GAG TCG GGC CCC AAG GGA TCT CC. U HIII XI

T1 STA (I). F

TIMP1 HEK293T


71/144

C 3

71

(I) . OMEM

. TIMP1 N

NTA (GE H) FPLC . TIMP1

SAL D C (T S) PBS W B (C S) C

12% SDS . I MMP9 TIMP1

SL G MMP A K C (A).

3T3L1

96 D E

10% /.

A 48 3T3L1 80%

(1 / , 1 M

, 115 / ) 2 , 2

(1 /) 2 . TIMP1

, TIMP1 (R&D ) IG1

(MBL) 0.5 / 2 . D 5 %

4 M H (), 5 M S60 () 2 M

B ( ) O R O. A 2 1.5% BSA, 9

(CW). P C P S.

M C57BL/6J 12/12 /

. M (P K AG) 60%

3 . D TIMP1

(200 /) . F

, TIMP1 3

VO2, VCO2,

O (C I).


72/144

C 3

72

B (C). F ITT, 0.75 U/

3 TIMP1 .

I R/M I ELISA (C C).

T T/GB K C....

(R/H) NEAFAHR (W).

TIMP1 Q M TIMP1 ELISA (R&D

S).

L 150 1 : (3:2) TL. S

0.5 :. 0.5 15% N2SO4

. L 2

TX100:: (1:1:3) T/GB K

(R/H)28

.

A 5%

10 . H&E

29

. M

C P S. F CT

S (LT).

R +/ . S

S .


73/144

C 3

73

R

I

3031

,

. T, T1 RNA

. W T1 RNA

(SVF),

, TIMP1

(F. 1.1). W

T1 RNA . A ,

T1 RNA .

F, T1 RNA

/ .

T1 RNA SVF, ,

(F. 1.1).

S SVF, T1 RNA 3T3L1

. I,

, T1 RNA

3T3L1 .

H, T1 RNA

(P)

(AF),

(F. 1.1).


74/144

C 3

74

. )RNA

T1

C57BL/6J . E G. (=4) ) RNA

T1, P AF G 3T3L1

. (=5)

a

b

wildty

pe

ob/ob

DIO

wildty

pe

ob/ob

DIO

0.0

0.5

1.0

1.5

2.0

2.5 subcutaneous

visceral

SVF adipocytes

normalizedTimp1e

xpression

******

** **

**

** *

-48 -24 0 24 48 72 96 120 144 168 1920.0

0.5

1.0

1.5

2.0

2.5

3.0 Ppar A-FabpTimp1

time [h]

normalizedmRNAexpression

induction ofdifferentiation


75/144

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75

T SVF TIMP1

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. I RNA , TIMP1 SVF

SVF. H, TIMP1 SVF

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TIMP1 .

I / TIMP1

(F. 1.2).

. )TIMP1

. C

OMEM 24 . TIMP1 ELISA. (=3) )

TIMP1 ELISA.

(=5) V SD *


76/144

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76

I TIMP1

. H,

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. G

TIMP1 72 % (F. 2),

TIMP1 MMP

. T , TIMP1 (MMP9)

MMP9 (MMP9) MMP . A F. 2, TIMP1

MMP9

MMP9. T

MMP/TIMP1

MMP .

I TIMP1 ,

TIMP1 (TIMP1) 34D

(F. 2,). TIMP1

MMP9 ( ).


77/144

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77

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MMP FRET . (=3)) C TIMP1. ) W

TIMP1. TIMP1 T . V

SD *


78/144

C 3

78

0 1 10 50 0 1 10 500

5

10

15

20

25

30

35

g/ml mTIMP1

%

differentiatedcells

**

subcutaneous visceral

0 3 6 13 25 50 1000

5

10

15

20

25

30

35

***

** ***

g/ml mTIMP1

%

differentia

tedcells

a

b

c

c)

control 100 g/ml TIMP150 g/ml TIMP125 g/ml TIMP13T3-L1 cells

control 50 g/ml TIMP125 g/ml TIMP112.5 g/ml TIMP1

subcutaneous stromal-vascular fraction


79/144

C 3

79

) 3T3L1

TIMP1. L

. E

, (=4). ) SVF

C57BL/6J

TIMP1 (=4). )O O SVF

TIMP1. ) SVF

C57BL/6J TIMP1

(=4). E , .A . V

SD. S: 100 *


80/144

C 3

80

(F. 4.1). T

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T TIMP1 ,

.

)

TIMP1 ELISA (=5) , )

CT C57BL/6J TIMP1 PBS

3 (=5).

a

0 10 20 30 40 50 60 70

0

1

2

3

4

5

6

7

8

time [h]

injections

TIMP1serum

conc.

[ng/ml]

subcutanous visceral0.0

2.5

5.0

7.5

10.0

12.5 PBS

TIMP1

fatpa

dweight[g]

b

c

PBS TIMP1

0

10

20

30 3 weeks HFD

start weight

weight[g]


81/144

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81

)

,

C57BL/6J TIMP1 PBS 3 )

3 . (=5) V

SD. *


82/144

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82

A , TIMP1

,

TIMP1 (F. 5.1). I ,

TIMP1 ,

TIMP1 (F. 5.1,).

S (F. 5.1).

, )

TIMP1 PBS ) ITT

TIMP1 PBS 3 (0.75U /

) (=5). ) ,

PBS TIMP1 4 (=5).

a

PBS TIMP10.0

0.5

1.0

1.5

2.0

2.5

insulin[ng/ml]

*

PBS TIMP10

1

2

3

FFA[mM]

**

PBS TIMP10

1

2

3

4

56

7

8

9

bloodglucose

[mM]

*

PBS TIMP10

25

50

75

100

triglycerides[mg/dl]

0 30 60 90 1200

50

100

150PBSTIMP1

time [min]

glucose(%

ofinitial)

** ***

PBS TIMP10

10

20

3040

50

60

70

mgTG

/gw

etliver

**

b

c d

e f


83/144

C 3

83

I TIMP1 MMP ,

MMP2 MMP9,

32

. T MMP

, MMP MMP9 MMP2

(F. 5.2).

) W B

MMP2, MMP9 ,

, . T

. V

SD. *


84/144

C 3

84

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,

TIMP1 (3 ) 3 TIMP1

. N ( ) (F. 6) TIMP1

, ,

TIMP1

.

. ) )

) C57BL/6J TIMP1 PBS

3 . (=5)

a

day night day night0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500 PBS

TIMP1

VO2 VCO2

VO2

/VCO2

[ml/kg/h]

day night

0

5000

10000

15000

20000

25000

30000

35000PBS

TIMP1

xtot

[counts]

day night

0.0

0.5

1.0

1.5

2.0

2.5PBS

TIMP1

foodintake[g]

b

c


85/144

C 3

85

I , TIMP1, ,

. I TIMP1

,

. T , .


86/144

C 3

86

D

T MMP

TIMP1 . T TIMP1

TIMP1

. W

TIMP1 SVF

15,23,3335

. S,

TIMP1 ( ,23

), TIMP1

RNA SVF TIMP1 SVF

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,

TIMP1 RNA . . T

M .35

, TIMP1 SVF

. T

. A,

TIMP1 , TIMP1 .

A , TIMP1

, TIMP1

15

. W TIMP1

3T3L1 SVF,

TIMP1 .

A TIMP1 MMP,

MMP 15,32

. I

, D .26

TIMP1

3T3F332A , A .19

MMP GM6001 TIMP1. O

TIMP1

. M TIMP1 72%


87/144

C 3

87

TIMP1 MMP9

, MMP9. MMP9 MMP2

32,36

, MMP

TIMP1 . I SVF, TIMP1 TIMP1

SVF. I

8,37

. A SVF

( ,38

), TIMP1

.

, TIMP1 ,

,

TIMP1

2425

. O TIMP1

, ,

. O , TIMP1

, TIMP1 . S TIMP1

26

, A .19

,

TIMP1. A,

. H, TIMP1

3

, ,

TIMP1. I,

. A

TIMP1 ,

.


88/144

C 3

88

T, TIMP1, ,

. I

56,3940. I ,

,

. A , TIMP1

.

A TIMP1 MMP , TIMP1

22

. L (MMP2/9),

32,36,

MMP, TIMP1

MMP . F

MMP

.

I

4142

. I 3T3L1 TIMP1 TNF,

IL6 IL14344

. I TIMP1

,

. I , SVF

,

45

.

I , TIMP1 ,

. T

,

.


89/144

C 3

89

R

1. G, G.H. T

. , 206218 (2008).

2. S, J.K. & VP, A.J. T : . A

. , 12531262 (2007).

3. J, J., . H / H: D A T G.

, 1000324 (2009).

4. S, K.L., .D . , 783787 (2008).

5. S, L.B. & D.J. E I U G M A C

D S I C L P C, A, N S.

, 1399& (1971).

6. L, M., . F

''. , 625633 (2007).

7. G, F.M. A : .

(), 9971002 (2001).

8. G, S., T, Y. & K, C.R. D : .

, 242256 (2007).9. C, T.H., . A 3

. , 577591 (2006).

10. L, J., S, D. & W, Z. M : .

, 15511554 (2002).

11. S, B.M. & G, C.A. F

3T3. , 657666 (1983).

12. A, Y. & K, Y. E IV

3T3L1 .

, 1616316169 (1988).

13. K, Y., K, S., N, I., T, K. & N, F. O

.

, 3844 (2000).14. W, F.R., S, A., S, P.J., R, C.S. & Z, M.A. R

3T3L1 . E .

, 40944099 (1989).

15. C, C., .M

. , 1188811896 (2003).

16. L, H.R., V, H.B., F, L., R, M.C. & C, D. A

3 . , 530535 (2002).

17. M, E., D, D., V, G., C, D. & L, H.R. E

1 .

, 696704 (2003).

18. P, A.M., . D

19 . , 53045313 (2004).19. A, C.M., S, S., M, J. & W, Z. S1

. , 693703 (2001).

20. S, S., L, L.R., T, T., C, C.S. & W, Z. A

. , 267275 (2001).

21. L, H.R., . M

. , 374379 (2002).

22. B, K. & N, H. T (TIMP):

. , 5571 (2010).

23. K, S., . T 1 .

, 257261 (2007).

24. L, H.R., D, D., V H, B., C, D. & M, E. D TIMP1

. , 249255 (2003).


90/144

C 3

90

25. G, I., . H

1. (2008).

26. D, D., . O 1

(TIMP1) .

, 10191024 (2006).

27. H, L.H., M, B., T, B., N, J.H. & B, N. C

.

, 11401149 (1998).

28. H, A. & R, N.S. L . ,

420426 (1978).

29. C, H.C. & F, R.V., J. D .

, 986989 (2002).

30. P, P.A., . S

.

, E958E962 (2004).

31. W, C., L, E. & S, M. R 2

. , A5A5 (2004).

32. B, A., S, C., P, G., G, J. & L, M. A

2 9: . , 20802086

(2001).

33. K, E., .P

PPAR . , 897

902 (2007).

34. M, E., M, C., C, A., C, D. & L, H.R. M

.

, 10931101 (2002).

35. M, E., . A .

, E656665 (2007).

36. B, V., . P

9 . , 12721279

(2005).37. W, B.L. S :

. , 697738 (2000).

38. J, A.W., Y, L., E, Y., V, A.W. & R, F.M. D

. , 25632570

(2009).

39. B, J., L, S., R, F. & E, J.W. I

2 :

? , 239245 (2003).

40. W, C., F, J.E., B, C., T, P.A. & P, R.E. E

, , II

. , 14981506 (2000).

41. S, R., D, C., M, M. & K, S. PPAR, O, I. , 95974 (2007).

42. Z, M. & S, T.M. O, , .

, 379386 (2009).

43. K, S., . P TIMP1 3T3L1

. , 64176422 (2005).

44. W, S., .T 1 RNA

1 3T3L1 . , 169

174 (2008).

45. W, C., .R F2 . ,

345356 (2003).


91/144

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91

B M, H M, A P, M G,

C W

S F I T, ETH Z, I M S

B, WP S. 16, 8093 Z, S

C 4

R


92/144

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92

A

V

. T,

SVF, . T

LS1

+CD29

+CD34

+P1

+

,

SVF. U

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SVF. I

, SVF

113 42%

SVF. C RNA SVF

16

SVF. A

7 , .. (B), (D),

1 (B1),

2 (E2), 1 (E1),

(M) S 1 (S1). F ,

S1 D

. F

.

F, , SVF

.


93/144

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,

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+ ( ) T119

( ), CD29 ( 1),

CD34 1 (S1/L6)

APC1618

. A, 1 (P1/D),

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,

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94/144

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94

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, ,

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. T ,

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, SVF. U

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.


95/144

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95

M

C57B/6J 1214 J L

12/12 / .

SVF 48

. B,

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2

DMEM 10% FBS /.

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F , SVF 6

. A , , 48 SVF. A ,

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(2 M) , H (4 M) S60 (5 M)

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(CW). P C P

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10 . H&E


96/144

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96

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. M

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FJ .

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20 L HPLC 0.1% .

S LTQ (T

E, S J, CA) (T E)

A 1100 HPLC . P

A 2 C18 (M C18 AQ 5

; M B, A, CA, USA). A 80 5%

40% H2O 0.1% 10

C18

0.5 /. T MS

. T MS

MS/MS . T MS

4001600 /. D ,

2 30. F MS


97/144

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97

: 1.95 V, 230 C,

35%, 0.25 30 .

T XML RAW

IPI 3.26 S

. T

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TPP PP PP

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5%.

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. F PANTHER C .

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DNA MMACS DNA (M).

RNA PCR SG PCR

(I). E G . F

S. T I.

F RNA , RNA

RNA (I) RNA ENTRU6

ERI A I (S. T II). ENTRU6

ABLOCKTDEST LRII (I).

C HEK293A 1 PI

. C

HEK293A

A MINI P VK (V).


98/144

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. C , .

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I /

. DNA ENTRCMVMCSTPA (I)

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HEK293A FGENE (R)

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R +/ . S

S .


99/144

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99

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100/144

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101/144

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101

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102/144

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102

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(),

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) %

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103/144

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104/144

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105/144

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41

24

subcutaneous

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c

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106/144

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N G . RNA

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152

353

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SVF 3T3L1

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,

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2 RNA SVF 3T3L1 (2 ) (S. T VI). F

7 (B), (D), 1 (B1), 2 (E2),

1 (E1), (M) SPARC 1

(S1), RNA PCR 3T3L1 (F. 4). >50%

RNA , B1.


107/144

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50

subcutaneous

visceral

ctin

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d

Ace

Adam

19Bg

n Bmp1

Cc18 Cdh

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1Dcn Ecm

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p1 Efem

p2 Emilin1 Enp

pFn

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p3 Igfbp

4 Igf

bp7 Il15ra Il1r

l1Lb

p Lcn

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p Ptp

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a3er

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20

30

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***

*

*

*

*

*

*

*

*

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C 4

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108/144

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V SD, *


109/144


110/144

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110

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111/144

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112/144

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113/144

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molecular mechanisms of adipogenesis in obesity and the metabolic syndrome