1

1

Journal club

Titikan Chukijrungroat 4836206SIPS/M

By:

Advisor:

Dr. Suwattanee Kooptiwut

On Wednesday 30 May 2006, At 9.30 am

2

Ablation of PDK1 in pancreaticβ cells induces diabetes as a result

of loss of β cell mass

Naoko Hashimoto, Yoshiaki Kido, Tohru Uchida, Shun-ichiro Asahar, Yutaka Shigeyama, TomokazuMatsuda, Akihiko Takeda, Daisuke Tsuchihashi, Akihiko Nishizawa, Wataru Ogawa, YoshitoFujimoto, Hitoshi Okamura, Karen C Arden, Pedro L Herrera, Tetsuo Noda & Masato Kasuga

Department of Clinical Molecular Medicine, Division of Diabetes and Digestive and Kidney Diseases andDepartment of Brain Sciences, Division of Molecular Brain Science, Kobe University Graduate School ofMedicine, Japan. Ludwig Institute for Cancer Research, University of California at San Diego, La Jolla,

USA. Department of Genetic Medicine and Development, University of Geneva Medical School,Switzerland. Department of Cell Biology, Cancer Institute, Japan

NATURE GENETICS 2006; 38(5): 589-593.

3

Introduction

The total mass of islets of Langerhans isreduced in individuals with type 2 diabetes( Butler et al 2003 ), possibly contributing to the

pathogenesis of this condition

4

Regulation of islet mass :

signaling pathway

insulin or insulin-like growth factor–1 receptors pathway

insulin receptor substrate pathway

phosphatidylinositol ( PI ) 3-kinase pathway

( Kulkarni et al 1999, Withers et al 1998 & Kubota et al 2000 )

5

Insulin signaling pathway

FOXO

S6K

PP

PI3-kinase

PIP2

AktP

P

PDK1

Nucleus

PIP3

Active Akt

Cell number

Cell size

Insulin

Insulin receptor

Cell membrane

IRS

P P

FOXO = Forkhead box O

P

6

FOXO

S6K

PP

PI3-kinase

PIP2

AktP

P

PDK1

Nucleus

PIP3

Active Akt

Cell number Cell size

Insulin

Insulin receptor

Cell membrane

IRS

P PP

PDK1 is a serine-threonine kinase thatmediates signaling

downstream ofPI 3-kinase

3-Phosphoinositide–dependent protein kinase 1 ( PDK1 )

2

7

FOXOS6K

PP

PI3-kinase

PIP2

AktP

P

PDK1

Nucleus

PIP3

Active Akt

Cell number Cell size

Insulin

Insulin receptor

Cell membrane

IRS

P PP

Several signaling pathway also

converges at PDK1

phosphorylates andactivates several

members of the AGCfamily of kinases

Protein kinase B ( PKB )

p70 ribosomal S6 kinase

3-Phosphoinositide–dependent protein kinase 1 ( PDK1 )8

• Furthermore, PDK1 is thought to beencoded by a single gene, and noisoforms of this protein have beendetected to date

9

Objective

• To study effect of deletion of PDK1 in pancreatic β cells

10

11

Mice

homozygous pancreatic β cell-specific PDK1 knockout mice ( βPdk1–/– )

This study was performed according to the guidelines of the Animal EthicsCommittee of Kobe University Graduate School of Medicine

male mice

plasma insulin concentrations

tail vein puncture

blood glucose level

In fed state

heterozygous pancreatic β cell-specific PDK1 knockout mice ( βPdk1+/– )

Foxo1 knockout mice

Control mice ( βPdk1+/+ )

12

Histology and immunostaining

Bouin’s solution

embedded in paraffinsectioned at a thickness of 4 to 5 μm

Sections were stained with antibodies to- insulin- glucagon- PCNA- β-catenin- Pdx1

- p27Kip1

- phospho-Akt ( Thr308 )- phospho-Akt ( Ser473 )- Foxo1- phospho-Foxo1 ( Thr24 )

3

13

Immune complexes were detected withsecondary antibodies conjugated with

Antigen

Primary antibody

Secondary antibody

- Cy3- fluorescein isothiocyanate- horseradish peroxidase

14

Immunoblot analysis

Western blot analysis measurement amount of protein

- Protein preparation: lysis of cell

ProteinPrimary antibody

Secondary antibody

- Quantitation of protein

- Separation of protein

- Transfer protein to membrane

- Hybridized with antibody ( primary & secondary )

- Expose to X-ray film

15

lysates of isolated islets

probed with antibodies to- PDK1- Akt- p70 S6 kinase- phospho–p70 S6 kinase ( Thr389 )- S6- phospho S6 ( Ser235/236 )- Foxo1- β-actin- α-tubulin

ProteinPrimary antibody

Secondary antibody

16

In situ hybridization

Complementary RNA

RNA template

UGCAUAGG

ACGUAUCC

cRNA probe

Used of a DNA or RNA to detect the presence of complementary DNA sequence in cloned

bacterial or cultured eukaryotic cell

[33P]UTP-labeled cRNA probes

UGCAUAGG

17

Statistical analysis

• Data are presented as mean ± s.e.m.• Data were analyzed by analysis of variance,

with the exception of in situ hybridization data,which were compared by the unpairedStudent’s t test

• A P value of < 0.05 was consideredstatistically significant

18

4

19

Generation of β cell-specificPDK1 knockout mice

Experimental 1

20

Generation of β cell-specific PDK1 knockout mice

Immunoblot analysis of PDK1 and β-actin in islets of control and

βPdk1–/– mice

Immunoblot analysis of PDK1, β-actin and α-tubulinin brain, hypothalamus, liver, fat, heart and skeletal

muscle of control and βPdk1–/– mice

PDK1 in islets: βPdk1-/- mice < control mice ~ 90%

PDK1 in other tissues: βPdk1-/- mice ~ control mice

21

In situ hybridization of Pdk1 mRNA in the hypothalamusof βPdk1–/– and control mice

Graphs show mean ± s.e.m. fromthree mice of each genotype

*P< 0.05

ventromedial hypothalamic nucleus ( arrow )

No difference in the amount of Pdk1 mRNA in βPdk1–/– miceand control mice

n = 3

22

• These results demonstrated that βPdk1–/–

mice indeed lack PDK1 specifically inpancreatic β cells

23

Effect of PDK1 ablation onglucose metabolism

Experimental 2

24

Blood glucose level for control, βPdk1+/– andβPdk1–/– mice at the indicated ages

control ( n = 30 )

βPdk1–/– ( n = 30 )

βPdk1+/– ( n = 10 )

*P< 0.05

No significant differences in blood glucose between βPdk1+/– and control mice

At 4 wks in fed state: blood glucose level of βPdk1–/– mice > control mice

Blood glucose increased progressively to > 500 mg dl–1 by 12 to 16 weeks

5

25

*P< 0.05

Plasma insulin concentrations for control, βPdk1+/– andβPdk1–/– mice at the indicated ages

control ( n = 30 )

βPdk1–/– ( n = 30 )

βPdk1+/– ( n = 10 )

At 12 wks in fed state: plasma insulin conc. of βPdk1–/– mice < control mice

At 24 wks in fed state: plasma insulin conc. of βPdk1+/– mice < control mice

26

*P< 0.05

Growth curve for control, βPdk1+/– and βPdk1–/– miceat the indicated ages

control ( n = 30 )

βPdk1–/– ( n = 30 )

βPdk1+/– ( n = 10 )

No significant differences in body weight between βPdk1+/– and control mice

After 8 wks of age: rate of increase in body weight βPdk1–/– mice < control mice

27

βPdk1–/– βPdk1+/–

Blood glucose level Blood glucose level

Plasma insulin conc. Plasma insulin conc.

Rate of increase in BW Rate of increase in BW

Diabetes28

Effects of PDK1 deficiency onislet characteristics

Experimental 3

29

Effects of PDK1 deficiency on islet characteristics

Immunostaining of pancreatic sections from control and βPdk1–/– mice

Scale bars, 50 μm

insulin ( red )

glucagon ( green )

At 4 wks : both the size and β cell content of islets in βPdk1–/– mice < control mice

The mass of non–β cells was similar in βPdk1–/– and control mice30

Scale bars, 10 μm

Pancreatic sections were stained with antibodies to insulin andβ-catenin to determine the size of individual β cells

insulin ( red )

β-catenin ( green )

**P< 0.01

200 β cells from each of four mice

At 4 wks: the size of individual β cells inβPdk1–/– mice < control mice ~ 20%

At 8 wks: the size of individual β cells inβPdk1–/– mice < control mice

6

31

Pancreatic sections were stained with antibodies to insulin andglucagon to determine islet density

insulin ( red )

glucagon ( green )

Scale bars, 100 μm

*P< 0.05

At 4 wks: islet density βPdk1–/– mice < control mice

At 16 wks: islet density βPdk1–/– mice < control mice ~ 60%

At least 500 islets were counted

Islet density = Total number of islets

Pancreas area

32

These results indicate that . . .

PDK1 is important in

determination of total islet mass

regulating both the number and size of β cells

regulating islet density

33

Cell proliferation in islets by immunostaining with antibodies to proliferating cell nuclear antigen ( PCNA )

Scale bars, 50 μm

at least 50 islets from a total of five mice of each genotype

*P< 0.05, **P< 0.01

At 4 wks: number of PCNA-positive β cells in islets of βPdk1–/– mice < control mice ~ 50 %

At 8 wks: number of PCNA-positive β cells in islets of βPdk1–/– mice < control mice ~ 75 %

34

DNA fragmentation of islet

At 6 wks: the extent of internucleosomal DNAfragmentation in islets of βPdk1–/– mice > control mice

Apoptosis : βPdk1–/– mice > control mice

35

Insulin content of the pancreas and of isolated isletsfrom mice at 6 weeks of age

*P< 0.05, **P< 0.01

Insulin content of pancreas Insulin content of isolated islets

from four pancreases from at least 100 islets from a total of four mice

At 6 wks: insulin content both of the pancreas and of isolated isletsin βPdk1–/– mice < control mice

36

These results suggest that . . .

PDK1

regulate proliferation of pancreatic β cells

regulate death by apoptosis of pancreatic β cells

7

37

Effects of PDK1 ablation ondownstream signaling in

pancreatic islets

Experimental 4

38

FOXO

S6K

PP

PI3-kinase

PIP2

AktP

P

PDK1

Nucleus

PIP3

Active Akt

Cell number

Cell size

Insulin

Insulin receptor

Cell membrane

IRS

P PP

PDK1 phosphorylatesAkt on Thr308 not Ser473

- arrest of the cell cycle- apoptosis- stress responses

( Accili et al 2004 )

39

Islets in pancreatic sections of control and βPdk1–/– miceat 4 weeks of age

Scale bars, 50 μm ( white ) or 10 μm ( black )

Arrows indicate nuclei

The form of Akt with phosphorylated Thr308 in islets: βPdk1–/– mice < control mice

40

Islets in pancreatic sections of control and βPdk1–/– miceat 4 weeks of age

Scale bars, 50 μm ( white ) or 10 μm ( black )

Arrows indicate nuclei

The amount of Akt phosphorylated on Ser473 in islets: βPdk1–/– mice ~ control mice

41

Islets in pancreatic sections of control and βPdk1–/– miceat 4 weeks of age

Scale bars, 50 μm ( white ) or 10 μm ( black )

Arrows indicate nuclei

The form of Foxo1 with phosphorylated Thr24in islets: βPdk1–/– mice < control mice

Resulting in accumulation of Foxo1 in the nucleus

42

These observations suggested that . . .

activation of Foxo1 might be responsiblefor cell cycle arrest in the β cells of

βPdk1–/– mice

8

43

FOXO

S6K

PP

PI3-kinase

PIP2

AktP

P

PDK1

Nucleus

PIP3

Active Akt

Cell number

Cell size

IGF-1

IGF-1 receptor

Cell membrane

IRS

P PP

The IGF-1–induced phosphorylation of both p70 S6 kinaseand ribosomal protein S6

44

Immunoblot analysis of isolated islet in the presence and absence of 1 nM IGF-1

for 1 h at 6 wks of age

pS6

protein synthesis

PDK1

β cell size

IGF-1

45

Effects of haploinsufficiency ofFoxo1 in βPdk1–/– mice

Experimental 5

46

Haploinsufficiency

• A situation in which the total level of gene product

produced by the cell is about half of the normal level

function function

that is not sufficient to permit the cell to function normally

47

IRS2 knockouts mice

β cell proliferation and expression of Pdx1

( Kitamura et al 2002 )

+ Foxo1 haploinsufficiency

48

Blood glucose and plasma insulin concentrations in control mice,βPdk1–/– mice and βPdk1–/–Foxo1+/– mice at the indicated ages

*P< 0.05, **P< 0.01

Blood glucose Plasma insulin conc.

control mice βPdk1–/– mice βPdk1–/–Foxo1+/– mice( n = 25 ) ( n = 14 ) ( n = 12 )

βPdk1–/–Foxo1+/– mice showed a significant improvement inblood glucose level compared with βPdk1–/– mice

The βPdk1–/–Foxo1+/– animals maintained a blood glucose concentration of200–300 mg dl–1 in the fed state even at an age of 20–24 weeks

9

49

Blood glucose and plasma insulin concentrations in control mice,βPdk1–/– mice and βPdk1–/–Foxo1+/– mice at the indicated ages

*P< 0.05, **P< 0.01

Blood glucose Plasma insulin conc.

control mice βPdk1–/– mice βPdk1–/–Foxo1+/– mice( n = 25 ) ( n = 14 ) ( n = 12 )

The plasma insulin level in the fed state was moderately but significantly higherin βPdk1–/–Foxo1+/– mice than in βPdk1–/– animals after 16 weeks of age

50

β cell mass in 8-week-oldcontrol, βPdk1–/– andβPdk1–/–Foxo1+/– mice

Immunostaining of islets in pancreaticsections of 8-week-old control, βPdk1–/–

and βPdk1–/–Foxo1+/– mice withantibodies to the indicated proteins

Scale bars, 50 μm

*P< 0.05**P< 0.01

p27 = negative regulator of cell cycle

51

Insulin content of the pancreas ofmice of the indicated genotypes

( n = 5 ) at 8 weeks of age

*P< 0.05**P< 0.01

Insulin content of the pancreas: βPdk1–/–Foxo1+/– mice > βPdk1–/– mice ~ 6 fold

Insulin content

52

Islet density in the pancreas ofmice at 8 weeks of age

*P< 0.05**P< 0.01

Islet density also tended to be higher in βPdk1–/–Foxo1+/– mice than in βPdk1–/–

mice, although this effect was not statistically significant

At least 12 pancreatic sections from each of four pancreases

Islet density

53

*P< 0.05**P< 0.01

Foxo1 haploinsufficiency did not affect the size of individual β cells in βPdk1–/– mice

Size of individual β cells in mice at 8 weeks of age

At least 200 β cells of each of four mice

β cell size

54

Proportion of PCNA-positive β cells among all β cells in mice at 8 weeks

*P< 0.05**P< 0.01

The number of PCNA-positive β cells was significantly increased by Foxo1haploinsufficiency in the islets of βPdk1–/– mice

At least 50 islets of each

β cell proliferation

10

55

Isolated islets from mice at 6 weeks of age were examined forinternucleosomal DNA fragmentation

The proportion of apoptotic cells, as shown by DNA fragmentation, did notseem to be affected by Foxo1 deficiency

Apoptosis

56

These results suggested that . . .

Foxo1 haploinsufficiency

increases β cell number and islet density

predominantly as a result of

increased β cell proliferation

57 58

βPdk1-/- βPdk1-/-Foxo+/-

Blood glucose level

Plasma insulin conc.

β cell mass

β cell size

Islet density

Cell proliferation

DNA frangmentation ( apoptosis )

Insulin content

59

FOXO

S6K

PP

PI3-kinase

PIP2

AktP

P

PDK1

Nucleus

PIP3

Active Akt

Cell number

Cell size

Insulin

Insulin receptor

Cell membrane

IRS

P PP

Cell number & cell size

60

FOXO

PP

PI3-kinase

PIP2PDK1

Nucleus

PIP3

Cell number

Cell size

Insulin

Insulin receptor

Cell membrane

IRS

P PP

Foxo haploinsuffficiencyCell number

Cell size

11

61

insulin receptor–IRS-Akt signaling pathway

Drosophila melanogaster

insulin receptor–IRS-Akt signaling pathway

control of cell size and number

mice

control organ size

( Stocker et al 2000 ) ( Kozma et al 2002 )

Akt1 contributes to determination of cell number in mouse brain

Akt1 deficiency in the heart

normal cell number

( Easton et al 2005 )reduced cell size

62

Mice deficient in PDK1 specifically in muscle

( decrease in cell size > cell number )

marked reduction in cardiac muscle mass

( Mora et al 2003 )

PDK1 regulates both the number and sizeof pancreatic β cells

Previous study

Present study

63

( Kulkarni et al 1999 )

Mice

lack the insulin receptor specifically in β cells

progressive decrease in both β cell mass andglucose tolerance with age

64

Mice

deficient in IRS2 specifically in β cells and the hypothalamus

reduced β cell mass

results largely from a reduced level of β cell proliferation

( Lin et al 2004 & Kubota et al 2004 )

65

Mice

lack functional receptors for both insulin andIGF-1 only in pancreatic β cells

develop pronounced hyperglycemia at 5 to 6 weeks of age

( R. Kulkarni et al & Ueki et al 2006 )

suggest that

complete pancreatic β cell–specific inactivation ofsignaling via the insulin and IGF-1 receptors

66

IRS and PDK1

sufficient to cause overt diabetes as a result of a loss ofpancreatic β cell mass

12

67

Summary

• PDK1 is important in maintenance ofpancreatic β cell mass and glucosehomeostasis

FOXO

S6K

PP

PI3-kinase

PIP2

AktP

P

PDK1

Nucleus

PIP3

Active Akt

Cell numberCell size

Insulin

Insulin receptor

Cell membrane

IRS

P P

FOXO = Forkhead box O

P

68