(C) 2005 BS Yandell UW Summer Research Program 1

islet insulin production in diabetes model:spatial comparison of mouse strains

• mentor: Brian S. Yandell, UW-Biostat Trainee www.stat.wisc.edu/~yandell

• scientist: Donnie Stapleton, Attie Biochem Labdsstaple@biochem.wisc.edu

• objective of project:– improve methods to distinguish "quality"– of insulin production– between two strains of mice – based on 3-color images of islets in pancreas slices

(C) 2005 BS Yandell UW Summer Research Program 2

why do this project?

• Knowledge/Skills needed– Basic statistics (t-test, ideally some nonparametrics)– R programming (a la biostat training)

• Knowledge/Skills obtained– communication skills with scientist (lab biochemist)– image analysis basics– nonparametric density estimation (mechanics at least)– experimental design considerations (nesting, balance,

etc.)

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project abstract• insulin is produced in the pancreas

– specifically the Islets of Langerhans by "beta" cells– (there are "alpha" cells as well.)

• scientists in biochemistry have imaged hundreds of islets– for two strains of obese mice

• BTBR.ob mimics type II (mature onset) diabetes mellitus• B6.ob appears healthy

• roughly a hundred islets per mouse, 3 mice per strain– can visually see differences in production of insulin in beta cells – have summary measurements on many islets to compare strains

• opportunity beyond data analysis– how to improve measurement process? – how to improve experimental design?

• goal: clearly infer differences in "quality" of insulin production– between BTBR.ob and B6.ob

• you can meet with the scientist and work with measurement system

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of mice and men

• obese strains: BTBR.ob vs. B6.ob– obese BTBR mice mimic human diabetes

• high glucose, low insulin– obese B6 mice appear non-diabetic

• congenic substrains A and B– mostly BTBR mouse genome– A congenic: insert small segment of B6– B congenic: essentially BTBR mice

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glucose insulin

(courtesy AD Attie)

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Type 2 Diabetes Mellitus

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Insulin Requirement

from Unger & Orci FASEB J. (2001) 15,312

decompensation

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islet degradation seriesA vs. B congenics

1 2 3 4 5healthy sick

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comparison of 3 type A

and 2 type B congenic

mice

healthy sick

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A and B congenic dataA congenics:Class.I Class.II Class.III Class.IV Class.V Total.IsletsA33-18 26 36 23 21 40 146A34-22 55 36 24 18 10 143A39-15 69 60 41 35 41 246

B congenics:Class.I Class.II Class.III Class.IV Class.V Total.IsletsA34-19 87 37 35 35 9 203A48-4 44 29 12 19 7 111

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B6 Female&Male OB 14wk

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Btbr Female& Male OB 14wk

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Btbr Male & Female OB 14wks

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total percent area for islets

0 50000 100000 150000

0.0

e+00

1.0

e-05

2.0

e-05

3.0

e-05

B6.1105

N = 21 Bandw idth = 6625

Den

sity

0 50000 100000 150000

0 e

+00

2 e

-05

B6.1169

N = 13 Bandw idth = 6111

Den

sity

0 50000 100000 150000

0 e

+00

2 e

-05

4 e

-05

6 e

-05

B6.1195

Den

sity

0 50000 100000 150000

0.0

e+00

1.0

e-05

2.0

e-05

BT.1419

N = 30 Bandw idth = 8943

Den

sity

0 50000 100000 150000

0.0

e+00

1.0

e-05

BT.1429

N = 34 Bandw idth = 1.246e+04

Den

sity

0 50000 100000 150000

0.0

e+00

1.0

e-05

2.0

e-05

BT.1507

Den

sity

12 13 14 15 16 17

0.00

0.10

0.20

0.30

B6.1105

N = 21 Bandw idth = 0.4719

Den

sity

12 13 14 15 16 17

0.0

0.1

0.2

0.3

0.4

B6.1169

N = 13 Bandw idth = 0.4596D

ensi

ty

12 13 14 15 16 17

0.0

0.2

0.4

0.6

B6.1195

Den

sity

12 13 14 15 16 17

0.00

0.10

0.20

0.30

BT.1419

N = 30 Bandw idth = 0.5141

Den

sity

12 13 14 15 16 17

0.00

0.05

0.10

0.15

0.20

0.25

BT.1429

N = 34 Bandw idth = 0.5427

Den

sity

12 13 14 15 16 17

0.00

0.10

0.20

BT.1507

Den

sity

log2 totalsraw totals

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percent glu/som,

dapi, insulin

by mouse

(smooth histogram +

median)

0 20 40 60 80 100

0.00

0.04

0.08

0.12

B6.1105

0 20 40 60 80 100

0.00

0.02

0.04

0.06

0.08

B6.1169

0 20 40 60 80 100

0.00

0.02

0.04

0.06

0.08

B6.1195

0 20 40 60 80 100

0.00

0.02

0.04

0.06

0.08

0.10

BT.1419

0 20 40 60 80 100

0.00

0.02

0.04

0.06

BT.1429

0 20 40 60 80 100

0.00

0.02

0.04

0.06

0.08

0.10

BT.1507

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sum of insulin, glu/som,

dapi percents

60 80 100 120 140

0.00

00.

010

0.02

00.

030

B6.1105

N = 19 Bandw idth = 5.231

Den

sity

60 80 100 120 140

0.00

00.

005

0.01

00.

015

0.02

0

B6.1169

N = 13 Bandw idth = 8.969

Den

sity

60 80 100 120 140

0.00

0.01

0.02

0.03

0.04

B6.1195

Den

sity

60 80 100 120 140

0.00

00.

010

0.02

00.

030

BT.1419

N = 30 Bandw idth = 5.164

Den

sity

60 80 100 120 140

0.00

00.

010

0.02

0

BT.1429

N = 34 Bandw idth = 6.835

Den

sity

60 80 100 120 140

0.00

00.

010

0.02

00.

030

BT.1507

Den

sity

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i

0 10 20 30 40 50 60 80 100 120 140

3050

7090

010

2030

4050

g

d

1020

3040

50

30 50 70 90

6080

100

120

140

10 20 30 40 50

tot

perc

ent

pairs

(cor

rela

tion)

plo

t

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inte

nsity

pairs

(cor

rela

tion)

plo

ti

600 620 640 660 680 700 720

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850

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g

700 750 800 850 900 1000 1200 1400 1600

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d

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calib

ratio

n st

udy

200 400 600 800 1000

1000

2000

3000

intensity

millisec

inte

nsity

200 400 600 800 1000

2000

040

000

6000

0

thresholded

millisec

thre

shol

ded

200 400 600 800 1000

2030

4050

6070

percent

millisec

perc

ent

200 400 600 800 1000

68

1012

14

signal.noise

millisec

sign

al.n

oise

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90

80

70

60

50

40

30

140x10312010080604020Area (um^2)

B6 Btbr

Dot plo:t Insulin vs

Area of islet

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50

40

30

20

10

140x10312010080604020Area (um^2)

B6 Btbr

Dot plot:

Glu/Som vs

Area of Islet

(C) 2005 BS Yandell UW Summer Research Program 22

90

80

70

60

50

40

30

5040302010Percent Glu/Som

B6 Btbr

Dot plot: Insulin vs

Glu/Som

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Female ob/ob B6

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Female ob/ob BTBR

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Male ob/ob B6

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Male ob/ob BTBR

(C) 2005 BS Yandell UW Summer Research Program 27

Summary table for ob/ob B6 and Btbr islets

All animals ob/ob and 6 wk of age.#'s show mean of guassian fit for all islets analyzed.

Islet area Islet diameterStrain # islets (m2) (m) % Gluc&Som % DAPI % Insulin Total %

B6 F 54 12864 128 6 25 71 102Btbr F 104 14242 135 8 23 61 92

B6 M 107 13545 131 10 24 61 95Btbr M 106 11520 121 17 25 58 100

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14 week Congenic Females

• Is there a difference between A & B congenics?

• Is there a difference between Btbr and the B congenic strain?

• How does the glucose and insulin values effect our comparisons?

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Btbr Female OB 14wk

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A33-18, Female OB A 14wk

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A34-19, Female OB B 14wk

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A34-22, Female OB A 14wk

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A39-15, Female OB A 14wk

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A48-4, Female OB B 14wk