Connecting the dots in the pathogenesis of Connecting the dots in the pathogenesis of type 2 diabetes : the need for systems biologytype 2 diabetes : the need for systems biology

Pierre De Meyts, MD, PhDPierre De Meyts, MD, PhDReceptor Systems Biology LaboratoryReceptor Systems Biology Laboratory

Hagedorn Research InstituteHagedorn Research Institute

Novo Nordisk A/SNovo Nordisk A/S

TNO WorkshopTNO Workshop

Zeist, December 4, 2008Zeist, December 4, 2008

?

Islet of Islet of LangerhansLangerhans

Beta cellBeta cell

GlucoseGlucose

INSULININSULIN

INSULININSULIN

INSULIN RECEPTORINSULIN RECEPTOR

Target cell (muscle, Target cell (muscle, liver, fat)liver, fat)

GLUCOSE GLUCOSE TRANSPORTERTRANSPORTER

GLUCOSEGLUCOSE

Theme of the talk:

Reductionist approaches have failed to provide an understanding of the pathogenesis of diabetes mellitus, methods to prevent it or cure it, and an adequate pipeline of drugs to treat it optimally.

A systems biology approach is warranted.

AgendaAgenda

Towards a systems understanding of T2DTowards a systems understanding of T2D

• What is systems biology?What is systems biology?

• Insulin resistance versus beta cell defect in T2DInsulin resistance versus beta cell defect in T2D

• Is there an insulin signal transduction defect in T2D?Is there an insulin signal transduction defect in T2D?

• The beta cell defect in type 2 diabetesThe beta cell defect in type 2 diabetes

• Insights from genome-wide scans for association (GWSA)Insights from genome-wide scans for association (GWSA)

• Epigenetic aspects of T2DEpigenetic aspects of T2D

• Tissue interactions, metabonomics and the microbiomeTissue interactions, metabonomics and the microbiome

Conclusion: need for an integrative approach in Conclusion: need for an integrative approach in order to understand the multi-layered complexity of order to understand the multi-layered complexity of

diabetes mellitusdiabetes mellitus

What is systems biology?What is systems biology?

A scientific discipline that endeavours to A scientific discipline that endeavours to quantify all the molecular elements of a quantify all the molecular elements of a

biological system to assess their biological system to assess their interactions and to integrate that interactions and to integrate that

information into models that explain and information into models that explain and predict emergent behaviours that cannot be predict emergent behaviours that cannot be understood by looking at the properties of understood by looking at the properties of the individual components of the system. the individual components of the system.

Disease like diabetes mellitusDisease like diabetes mellitus

Candidate genes

Traditional thinking about diabetes mellitusTraditional thinking about diabetes mellitus

• Find ”the” causeFind ”the” cause

• Find ”the” cureFind ”the” cure

Current statusCurrent status

• Pathogenesis unknownPathogenesis unknown

• Few drugsFew drugs

• No cureNo cure

• No predictionNo prediction

• No preventionNo prevention

• Type 2 epidemicsType 2 epidemics

What causes type 2 diabetes?What causes type 2 diabetes?

The beta cells do not produce enough insulin

The target cells (muscle, fat, liver) do not

respond well to insulin

Genes

Nutrition

Lifestyle

Environment

Type 2Type 2DiabetesDiabetes

It’s the fat cell

It’sGLUT4!

It’sthe liver!

It’s the insulin

receptor!

It’sglucokinase!

It’s themuscle!

It’s the betacell!

De Meyts, P. The diabetogenes concept of NIDDM. Adv. Exp. Med. Biol. 334:89 (1993)De Meyts, P. The diabetogenes concept of NIDDM. Adv. Exp. Med. Biol. 334:89 (1993)

the brain!

Etiology of T2DEtiology of T2D

• Multiple genes (”diabetogenes”)Multiple genes (”diabetogenes”)

• Unfavorable combination of common Unfavorable combination of common allelesalleles

De Meyts, P. The diabetogenes concept of NIDDM. Adv. Exp. Med. De Meyts, P. The diabetogenes concept of NIDDM. Adv. Exp. Med. Biol. 334:89 (1993)Biol. 334:89 (1993)

Conventional wisdom regarding the respective roles of Conventional wisdom regarding the respective roles of insulin resistance and beta cell function in insulin resistance and beta cell function in type 2 diabetestype 2 diabetes

INSULIN RESISTANCE -CELL FUNCTION

STARLING LAW OF THE BETA CELLSTARLING LAW OF THE BETA CELL

INS

ULI

NIN

SU

LIN

FASTING GLYCEMIAFASTING GLYCEMIANORMAL ELEVATED

NORMOGLYCEMIA NORMOGLYCEMIA IGTIGT TYPE 2 DIABETESTYPE 2 DIABETES

DIABETOGENES DIABETOGENES

Lifestyle/nutrition/exerciseObesityEnvironment

EnvironmentGlucose toxicityLipotoxicityLipotoxicity

Biddinger SB and Kahn CR. Ann Rev Physiol 68:123-158 (2006)

The ”insulin resistance-centric” view of T2D

The ptolemaic geocentric view of the Universe

Frayling TM. Nat Rev Gen 8:657-662 (2007)

The distributions of obesity and T2D overlap but do not coincide

Development of obesity and diabetes in NZO mice

CFD: 68% fat, 20% prot.

HFD: 15% fat, 47% carb., 17% prot.

StD: 4% fat, 51% carb., 19% prot.

Jürgens HS et al, Diabetologia 50:1481-1489 (2007)

Yudkin JS Diabetologia 50:1576-1586 (2007)

The ”inflammation-centric” view of T2D

This review alludes to several problems inherent in the epidemiological method in understanding disease mechanisms. (…) Integrated systems biology needs more

complex approaches to investigate disease mechanisms, involving cell, organ, whole

organism and population studies.

Nature doi:10.1038/nature06098, 2007

Biddinger SB and Kahn CR. Ann Rev Physiol 68:123-158 (2006)

The ”brain-centric” view of T2D

?

x = constant

Bergman’s disposition index

Review: Bergman RN, Horm Res 64 (suppl. 3) 8-15, 2005

The hyperbolic law of glucose tolerance

Bergman’s disposition index

Maps to chromosome 11

Conventional wisdom regarding the respective roles of Conventional wisdom regarding the respective roles of insulin resistance and beta cell function in insulin resistance and beta cell function in type 2 diabetestype 2 diabetes

INSULIN RESISTANCE -CELL FUNCTION

STARLING LAW OF THE BETA CELLSTARLING LAW OF THE BETA CELL

INS

ULI

NIN

SU

LIN

FASTING GLYCEMIAFASTING GLYCEMIANORMAL ELEVATED

NORMOGLYCEMIA NORMOGLYCEMIA IGTIGT TYPE 2 DIABETESTYPE 2 DIABETES

DIABETOGENES DIABETOGENES

Lifestyle/nutrition/exerciseObesityEnvironment

EnvironmentGlucose toxicityLipotoxicityLipotoxicityType 2 diabetes is a Type 2 diabetes is a

signal transduction signal transduction diseasedisease

Type 2 diabetes is a Type 2 diabetes is a gene transcription gene transcription

diseasedisease

The signal transduction defect(s) in type 2 diabetes: understanding the combinatorial

nature of signalling

www.hprd.org

• Binding kinetics

• BRET

• Mathematical modelling

• Phosphoproteomics

• Microarray gene profiling

• siRNAs

Network inference by reverse engineering

0

500

1000

1500

20000.01

0.1

1

10

100

1,000

10,000

0.25

0.5

0.75

1

0

500

1000

1500

2000

Time

Concentration

Fraction Bound

Modelling of insulin receptor dissociation kinetics and negative cooperativity

Kizelyov VV and De Meyts P, 2008

Screening for candidate type 2 diabetogenes Screening for candidate type 2 diabetogenes affecting insulin signal transductionaffecting insulin signal transduction

Picture from Jan Nygaard Jensen and Oluf B. Pedersen, Picture from Jan Nygaard Jensen and Oluf B. Pedersen, Steno Diabetes Center, Gentofte, DenmarkSteno Diabetes Center, Gentofte, Denmark

PPARG

The beta cell defect in type 2 diabetes: The beta cell defect in type 2 diabetes: transcriptional, functional and systemic transcriptional, functional and systemic

aspectsaspects

Organellar relationships in the Golgi region of a HIT-T15 beta cell by high-Organellar relationships in the Golgi region of a HIT-T15 beta cell by high-resolution electron tomographyresolution electron tomography

Marsh BJ et al, PNAS 98:2399-2406, 2001

The Visible CellTM Project at University of Queensland,

Australia

Noske AB et al, J Struct Biol 161:298-313, 2008.

Transcriptional networks controlling beta cell development and functionTranscriptional networks controlling beta cell development and function

Servitja JM and Ferrer J , Diabetologia 47:597-613 (2004)Servitja JM and Ferrer J , Diabetologia 47:597-613 (2004)

MODY 1

MODY 2MODY 3

MODY 4

MODY 6

MODY 5

Insulin

Positive feedback of insulin secretion on beta cell functionPositive feedback of insulin secretion on beta cell function

Leibiger B et al, Biochem Soc Trans 30:312-317, 2002

Beta cell differentiation Beta cell differentiation and regenerationand regeneration

Insulin

Beta cell Beta cell secretion and secretion and functionfunction

Peripheral insulin actionPeripheral insulin action

Multi-layered complexity in pathogenesis of type 2 diabetesMulti-layered complexity in pathogenesis of type 2 diabetes

Time for BetaSys?

Need for in silico beta cell

(”virtual beta cell”)

Insights from genome-wide scans for Insights from genome-wide scans for association in T2Dassociation in T2D

Sydney Brenner

Translational medicine:

from bench to bedsideXFrom bedside to bench!

Forget rodents!

There are 6.7 x 109 human

genomes/phenomes on this planet!

The Tokyo Declaration"Recent advances in Systems Biology indicate that the time is now ripe to initiate a grand challenge project to create over the next thirty years a comprehensive, molecules-based, multi-scale, computational model of the human (‘the virtual human’), capable of simulating and predicting, with a reasonable degree of accuracy, the consequences of most of the perturbations that are relevant to healthcare.The JST and BBSRC are encouraged to join forces in order to create a collaborative program between Japan and the UK to drive this project forward.”

Workshop on Future Challenges in Systems Biology

Tokyo, February 4-6, 2008

First Individual Diploid Human Genome Published By Researchers at J. Craig Venter Institute

Sequence Reveals that Human to Human Variation is Substantially Greater than Earlier Estimates

Independent sequence and assembly of the six billion base pairs from the genome of one person ushers in

the era of individualized genome-based medicine

ROCKVILLE, MD—September 3, 2007—ROCKVILLE, MD—September 3, 2007—Researchers at the J. Craig Venter Institute (JCVI), along with collaborators at The

Hospital for Sick Children (Sick Kids) in Toronto and the University of California, San Diego (UCSD), have

published a genome sequence of an individual, J. Craig Venter, Ph.D., that covers both of his chromosome pairs (or diploid genome), one set being inherited from each of

his parents. 

J. Craig Venter

500.000 dollars reward!

Apr 23, 2007UK-MVA Bioscience Alliance, Malmo

PPARGPPARG TCF7L2TCF7L2

HHEXHHEXSLC30A8SLC30A8CDKAL1CDKAL1IGF2BP2IGF2BP2CDKN2ACDKN2AFTOFTOWFS1WFS1TCF2TCF2

KCNJ11KCNJ11

19981998

20072007

2006200620032003

At least 18 validated At least 18 validated type 2 diabetes genes/loci on June 2008type 2 diabetes genes/loci on June 2008

Several of the diabetes-Several of the diabetes-associated variants are associated variants are

intronic or intergenic and intronic or intergenic and the actual causal the actual causal variant(s)/gene(s) variant(s)/gene(s)

remains to be identifiedremains to be identified

JAZF1JAZF1CDC123CDC123TSPAN8TSPAN8THADATHADAADAMTS9ADAMTS9NOTCH2NOTCH2KCNQ1KCNQ1

20082008

Apr 23, 2007UK-MVA Bioscience Alliance, Malmo

PPARGPPARG TCF7L2TCF7L2

HHEXHHEXSLC30A8SLC30A8CDKAL1CDKAL1IGF2BP2IGF2BP2CDKN2ACDKN2AFTOFTOWFS1WFS1TCF2TCF2

KCNJ11KCNJ11

19981998

20072007

2006200620032003

At least 18 validated At least 18 validated type 2 diabetes genes/loci on June 2008type 2 diabetes genes/loci on June 2008

What Do theDiabetes-Associated

Variants Do?

JAZF1JAZF1CDC123CDC123TSPAN8TSPAN8THADATHADAADAMTS9ADAMTS9NOTCH2NOTCH2KCNQ1KCNQ1

20082008

Apr 23, 2007UK-MVA Bioscience Alliance, Malmo

PPARGPPARG TCF7L2TCF7L2

HHEXHHEXSLC30A8SLC30A8CDKAL1CDKAL1IGF2BP2IGF2BP2CDKN2ACDKN2AFTOFTOWFS1WFS1TCF2TCF2

KCNJ11KCNJ11

19981998

20072007

2006200620032003

At least 1 T2D variantAt least 1 T2D variantassociates with insulin resistance associates with insulin resistance

JAZF1JAZF1CDC123CDC123TSPAN8TSPAN8THADATHADAADAMTS9ADAMTS9NOTCH2NOTCH2KCNQ1KCNQ1

20082008

Apr 23, 2007UK-MVA Bioscience Alliance, Malmo

PPARGPPARG TCF7L2TCF7L2

HHEXHHEXSLC30A8SLC30A8CDKAL1CDKAL1IGF2BP2IGF2BP2CDKN2ACDKN2A

FTO FTO WFS1WFS1TCF2TCF2

KCNJ11KCNJ11

19981998

20072007

2006200620032003

At least 1 T2D variantAt least 1 T2D variantassociates with increased fat massassociates with increased fat mass

JAZF1JAZF1CDC123CDC123TSPAN8TSPAN8THADATHADAADAMTS9ADAMTS9NOTCH2NOTCH2KCNQ1KCNQ1

20082008

Apr 23, 2007UK-MVA Bioscience Alliance, Malmo

PPARGPPARG TCF7L2TCF7L2

HHEXHHEXSLC30A8SLC30A8CDKAL1CDKAL1IGF2BP2IGF2BP2CDKN2ACDKN2AFTOFTO

WFS1WFS1TCF2TCF2

KCNJ11KCNJ11

19981998

20072007

2006200620032003

At least 13 T2D variantsAt least 13 T2D variantsassociates with decreased beta-cell functionassociates with decreased beta-cell function

JAZF1JAZF1CDC123CDC123TSPAN8TSPAN8THADATHADAADAMTS9ADAMTS9NOTCH2NOTCH2

KCNQ1KCNQ1

20082008

Impairedinsulin biosynthesis/

releaseObesity

PPARG2

KCNJ11 KCNJ11 TCF7L2 TCF7L2 HHEX HHEX SLC30A8 SLC30A8 CDKAL1 CDKAL1 IGF2BP2 IGF2BP2 CDKN2A CDKN2A WFSWFSTCF2TCF2JAZF1 CDC123/CAMK1D LGR5THADA ?ADAMTS9 ?NOTCH2 ?KCNQ1

FTO

Impairedinsulin action

Validated type 2 diabetes genesrelative risk 1.10-1.50

Eighteen confirmed type 2 diabetes associatedEighteen confirmed type 2 diabetes associatedSNPs examined in Danish cases (n=4093)SNPs examined in Danish cases (n=4093)and glucose tolerant controls (n=5302)and glucose tolerant controls (n=5302)

0.8 1.0 1.2 1.4 1.6 1.8

NOTCH2 (rs10923931)

WFS1 (rs10010131)

FTO (rs8050136)

TCF2 (rs7501939)

PPARG (rs1801282)

IGF2BP2 (rs4402960)

SLC30A8 (rs13266634)

ADAMTS9 (rs4607103)

HHEX (rs1111875)

CDC123 (rs12779790)

TSPAN8 (rs7961581)

KCNJ11 (rs5215)

JAZF1 (rs864745)

Chr 11p15 (-)

CDKAL1 (rs10946398)

THADA (rs7578597)

CDKN2A/2B (rs10811661)

TCF7L2 (rs7903146)

0.8 1.0 1.2 1.4 1.6 1.8

OR (CI 95%)

PPARG Nuclear receptor for thiazolidinediones

KCNJ11 Kir 6.2, ATP-sensitive potassium channel

TCFL2 Transcription factor

HHEX/IDE Homeobox, hematopoietically expressed: Wnt signalling (pancreatic development)

SLC30A8 Zinc transporter ZnT8

CDKAL1 Cyclic dependent kinase5 regulatory subunit associated protein 1-like 1 (regulation of beta cell function)

IGF2BP2 Regulates IGF-2 translation

CDKN2A/B Tumor suppressor (p16INK4a and b)(regulation of beta cell regeneration)

FTO Adiposity gene

WFS1 Wolfram syndrome

T2D associated genes identified in genome-wide association scans

PNAS 104, 15040-15044, 2007

A24D

G32S

G32R

C43G

G47V

F48C

R89C

G90C

C96Y

Y108C

(proinsulin nomenclature)

(insulin nomenclature)

B8

B8

B19

B23

B24

A1

A7

A19

Zinc

Zn T8

Zinc

A zinc- and insulin centric vision of the beta cell (and universe)…

Type 2Type 2DiabetesDiabetes

It’s the fat cell!

It’sGLUT4!

It’sthe liver!

It’s the insulin

receptor!

It’sglucokinase!

It’s themuscle!

It’s the betacell!

De Meyts, P. The diabetogenes concept of NIDDM. Adv. Exp. Med. Biol. 334:89 (1993)De Meyts, P. The diabetogenes concept of NIDDM. Adv. Exp. Med. Biol. 334:89 (1993)

the brain!

From bedside to bench…

HUMANC. elegans BLAST or ensembl

score

IMP2 M88.5 (UBRH) E-36, 28% ID

SLC30A8 T13D3.3 (UBRH), Y39E4A.2B/ttm-1

E-71, 48% IDE-59, 40% ID

TCF7L2 W10C8.2/pop-1 E-35, 40% ID (C’)

PKN2 F46F6.2 E-166, 47% ID

KCNJ11 M02A10.2/irk-2R03E9.4/irk-1

E-76, 44% IDE-69, 42% ID

CDKAL1 Y92H12BL.1 Ensembl 1:1

HHEX M6.3/pha-2 Ensembl 1:1

CDKN2A D2021.8Y17G7B.15/cnt-1B0350.2/unc-44

E-7, 35% ID (ankyrin)E-5, 33% ID (ankyrin)E-5, 32% ID (ankyrin)

CDKN2B D2021.8K12C11.4/dapk-1

E-10, 31% ID (ankyrin)E-7, 30% ID (ankyrin)

PPARG nhr-49 and others

GCKR No hits

FLJ39370 No hits

Data from Kaveh Ashrafi, UCSF

Diabetes susceptibility genes alter C. elegans fat content

Control RNAi ttm-1 RNAi(SLC30A8?)

Zinc transporter

M88.5 RNAi (IMP2)IGF-II mRNA binding

protein

Data from Kaveh Ashrafi, UCSF

112 genes inactivation increases fat storage, many of which have human homologues.

Nature 421:268-272, 2003

Epigenetic aspects of T2DEpigenetic aspects of T2D

Genetic versus epigenetic mechanisms in Type 2 diabetesGenetic versus epigenetic mechanisms in Type 2 diabetes

Diabetes/Metabolism Research and Reviews, 21:416-433, 2005.

Tissue interactions, metabonomics and the microbiomeTissue interactions, metabonomics and the microbiome

Diabetes, 53 suppl.3: S6-S15, 2004

The gut!G

LP-1

Metabonomics and the microbiome

Highly complex animals such as humans can be considered ”superorganisms” with an internal ecosystem of diverse symbiotic microbiota and parasites that have interactive metabolic processes.

We now need novel approaches to measure and model metabolic compartments in interactive cell types and genomes that are

connected by cometabolic processes in symbiotic mammalian systems.

Nicholson JK, Holmes E, Lindon JC and Wilson D

The challenges of modeling mammalian biocomplexity

Nat Biotechnol 22:1268-1274, 2004

Our body contains 10 times more microbial cells than human cells!

Turnbaugh PJ, Ley RE, Hamadi M, Fraser-Liggett CM, Knight R and Gordon JI.

Nature 449:18 Oct. 2007 online.

The gut bug-centric view of the universe…

Sequencing the intestinal flora:

MetaHit

”Intestinal metagenome”

20 million Euros/4 years

European – China project

Take home messageTake home message

• Avoid unidimensional thinkingAvoid unidimensional thinking

• GWSA will sooner than later establish a complete GWSA will sooner than later establish a complete nomenclature of the common gene alleles that in nomenclature of the common gene alleles that in unfavorable combination predispose to disturbed unfavorable combination predispose to disturbed metabolismmetabolism

• These will reveal new critical nodes/pathways in These will reveal new critical nodes/pathways in metabolic regulationmetabolic regulation

• Connecting the dots = Connecting the dots = systems biologysystems biology

• New drug targets

• Pharmacogenomics

• Biomarkers

• Personalized medicine/prevention

Biddinger SB and Kahn CR. Ann Rev Physiol 68:123-158 (2006)

The ”insulin resistance-centric” view of T2D

Goh KI et al, PNAS 104:8685-8690, 2007

Systems biology of diabetes: what is needed?

• Transdisciplinarity

• Computing power

• Sharing/compatibility of data/databases/representations/models

• Resources

• International cooperation

• Rethinking teaching of biology and medicine

• Training new generation of system biologists

• Some success stories

What is simpleWhat is simple

is wrong, andis wrong, and

what iswhat is

complicatedcomplicated

cannot becannot be

understood.understood.

Paul Valery (1871 – 1945)Paul Valery (1871 – 1945)

Thank you!

pdm@novonordisk.com

0.0 2.5 5.0 7.5 10.0 12.50.0

0.2

0.4

0.6

0.8

1.0

L1

CR

L1

L2

Fn1

Fn2

Fn3

Fn1

Fn2

Fn3

Site 2Site 2

Site 1

A

Site 2

Site 2

Site 1

Site 1

B C

Figure 1

Energy (kJ/mol)

dP

/dE

(1

/kT

)

Activation energy 5 % fraction of receptor molecules in the active state

D

S1

S2 S1

S2

r1

S1

S2 S1

S2

r2

S1

S2 S1

S2

r3

S1

S2 S1

S2

r4

S1

S2 S1

S2

r13

S1

S2 S1

S2

r14

S1

S2 S1

S2

r23

S1

S2 S1

S2

r24

Figure 2

S1

S2 S1

S2

r0

S1

S2S1

S2

S1

S2 S1

S2

r1x2 r3x4

S1

S2S1

S2 S1

S2S1

S2 S1

S2S1

S2

S1

S2 S1

S2 S1

S2 S1

S2

S1

S2S1

S2

S1

S2 S1

S2S1

S2 S1

S2

r1x23 r1x24 r1x24d r13x4 r23x4 r2d3x4 r1x234 r123x4

Figure 3

S1

S2 S1

S2 S1

S2 S1

S2a1

d1S1

S2S1

S2

kcr

d2S1

S2S1

S2

a1

d1

S1

S2 S1

S2

a2d2

d1

S1

S2 S1

S2

S1

S2S1

S2

a1

a2d2

kcr

d2

a1

d1

d2a2

S1

S2S1

S2

a2d2

S1

S2 S1

S2 S1

S2 S1

S2

S1

S2 S1

S2 S1

S2 S1

S2

kcrd1kcrd1

a1

d1

a2d2

a1

d1

a2d2

Simplified scheme of the insulin receptor binding

10-12 10-11 10-10 10-9 10-8 10-7 10-6

0.00

0.25

0.50

0.75

1.00

Hot

insu

lin b

ound

Cold insulin (M)

10-11 10-10 10-9 10-8 10-7 10-6 10-50

20

40

60

80

100

Hot

insu

lin r

emai

ning

afte

r di

ssoc

iatio

n (%

)

Cold insulin (M)

Dissociation time: 20 min 60 min

0 1 2 3

0

1x109

2x109

3x109

4x109

Bou

nd/fr

ee (

1/M

)

Bound

A B C

D E

Figure 4

10-13 10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5

0.0

0.2

0.4

0.6

0.8

1.0

Figure 5

Kd= 0.19 nM, kcr = 0.33 s-1

Kd= 1.8 nM, kcr = 0.033 s-1

Kd= 0.019 nM, kcr = 3.3 s-1

Cold insulin (M)

Ho

t in

sulin

bo

un

d

Parameter Average SD SD %

Apparent KD 0.19 nM 0.005 nM 2.6

Kcr 0.33 s-1 0.099 s-1 29.7

Site 1 KD 6.41 nM 0.78 nM 12.1

Site 2 KD 399 nM 76 nM 19.0

a1 490 637 s-1 M-1 25 945 s-1 M-1 5.3

d1 0.0031 s-1 0.00031 s-1 10.2

a2 27 890 s-1 M-1 12 125 s-1 M-1 43.5

d2 0.010 s-1 0.0024 s-1 23.3

Endocytosis rate 12.1 % per hour 3.2 % per hour 26.5

Exocytosis rate 8.3 % per hour 12.1 % per hour 145

Table 1

d 2

r0 r1r3

r2

r4

a1

d1a1

d1

a2 d2

a2d2

1.

r1 r1x2r14

r0

r13

kcr

d2a2

d2

d1 a1

a1d1

2.

r2 r1x2r24

r0

r23

kcr

d1a2

d2

d2 a2

a1d1

3.

r3 r3x4r23

r0

r13

kcr

d2a2

d2

d1 a1

a1d1

4.

r4 r3x4r24

r0

r14

kcr

d1a2

d2

d2 a2

a1d1

5.

r1x2

r1r1x23

r2

r1x24d

d2

k cra1

d1

d1 kcr

a 2

d 2

6.

a 2

d 2

r1x24

r14 r1r13x4

r4

d2

a2kcr

d1

d1 a1

kcrd2

7.

r1x24

r13 r1r13x4

r3

d1

a1kcr

d2

d1a1

kcrd2

8.

r1x23

r23 r2r23x4

r3

d1

a1kcr

d2

d2 a2

kcrd1

9.

r1x23

r24 r2r23x4

r4

d2

a2kcr

d1

d2 a2

kcrd1

10.

r1x24

r3x4

r3r23x4

r4

r13x4

d2

k cra

2

d2

d1 kcr

a 1

d 1

11.

a 2

r2d3x4

r1x23 r1x2r23

r13

d1

a1d1

kcr

d2 kcr

a2d2

12.

r1x234

r1x24

r1x2r24

r14

r1x234

d2

a2d1

kcr

d2 kcr

a 1

d 1

13.

k 4

k 8

r1x24d

r13x4 r3x4r13

r14

d1

a1d2

kcr

d1 kcr

a2d2

14.

r123x4

r23x4

r3x4r23

r24

r123x4

d2

a2d

2

kcr

d1 kcr

a 1

d 1

15.

k 4

k 8

r2d3x4

r1x24d r1x2r1x24

d2

a2k8

k416.

r2d3x4 r3x4r23x4

d2

a2k8

k417.

r1x234 r1x23r1x24

d2

a2d1

a118.

r123x4 r13x4r23x4

d2

a2d1

a119.

Figure S1

Figure S2

1. r0' -a1r0im+d1r1-a2r0im+d2r4-a1r0im+d1r3-a2r0im+d2r2+kexr0cyt, 2. r1' -kcrr1+d2r1x2-a1r1im+d1r13-a2r1im+d2r14-d1r1+a1r0im, 3. r2' -kcrr2+d1r1x2-a1r2im+d1r23-a2r2im+d2r24-d2r2+a2r0im, 4. r3' -kcrr3+d2r3x4-a1r3im+d1r13-a2r3im+d2r23-d1r3+a1r0im, 5. r4' -kcrr4+d1r3x4-a1r4im+d1r14-a2r4im+d2r24-d2r4+a2r0im, 6. r1x2' -d2r1x2+kcrr1-a2r1x2id+d2r1x24d-a2r1x2im+d2r1x24-a1r1x2im+d1r1x23-d1r1x2+kcrr2-kendr1x2, 7. r14' -d2r14+a2r1im-kcrr14+d2r1x24-kcrr14+d1r13x4-d1r14+a1r4im, 8. r13' -d1r13+a1r1im-kcrr13+d2r1x23-kcrr13+d2r13x4-d1r13+a1r3im, 9. r23' -d1r23+a1r2im-kcrr23+d1r1x23-kcrr23+d2r23x4-d2r23+a2r3im, 10. r24' -d2r24+a2r2im-kcrr24+d1r1x24-kcrr24+d1r23x4-d2r24+a2r4im, 11. r3x4' -d2r3x4+kcrr3-a1r3x4im+d1r13x4-a2r3x4id+d2r2d3x4-a2r3x4im+d2r23x4-d1r3x4+kcrr4-kendr3x4, 12. r1x23' -d1r1x23+a1r1x2im-a2r1x23im+d2r1x234-d1r1x23+kcrr23-d2r1x23+kcrr13-kendr1x23, 13. r1x24' -d2r1x24+a2r1x2im-a1r1x24im+d1r1x234-k4r1x24im+k8r1x24d-d1r1x24+kcrr24-d2r1x24+kcrr14-kendr1x24, 14. r13x4' -d1r13x4+a1r3x4im-a2r13x4im+d2r123x4-d2r13x4+kcrr13-d1r13x4+kcrr14-kendr13x4, 15. r23x4' -d2r23x4+a2r3x4im-a1r23x4im+d1r123x4-k4r23x4im+k8r2d3x4-d2r23x4+kcrr23-d1r23x4+kcrr24-kendr23x4, 16. r1x24d' -d2r1x24d+a2r1x2id-k8r1x24d+k4r1x24im-kendr1x24d, 17. r2d3x4' -d2r2d3x4+a2r3x4id-k8r2d3x4+k4r23x4im-kendr2d3x4, 18. r1x234' -d2r1x234+a2r1x23im-d1r1x234+a1r1x24im-kendr1x234, 19. r123x4' -d2r123x4+a2r13x4im-d1r123x4+a1r23x4im-kendr123x4, 20. r0cyt' -kexr0cyt+kendr1x2+kendr3x4+kendr1x23+kendr1x24+kendr13x4+kendr23x4+kendr1x24d+kendr2d3x4+kendr1x234+kendr123x4, 21. iend' -kexiend+kendr1x2+kendr3x4+2kendr1x23+2kendr1x24+2kendr13x4+2kendr23x4+3kendr1x24d+3kendr2d3x4+3kendr1x234+3kendr123x4

Figure S3Hot and cold insulin

S1

S2S1

S2

r1x2h

S1

S2S1

S2 S1

S2S1

S2 S1

S2S1

S2

S1

S2 S1

S2 S1

S2 S1

S2

r1x2h4 r1x2h3 r1x2h4d r1h r2h

S1

S2S1

S2

S1

S2 S1

S2 S1

S2 S1

S2

S1

S2 S1

S2 S1

S2 S1

S2

r1x2h34 r1h3 r1h4

r2h3 r2h4

S1

S2 S1

S2 S1

S2 S1

S2

r1h3x4 r2h3x4

S1

S2 S1

S2 S1

S2 S1

S2 S1

S2 S1

S2

r1h23x4 r12h3x4 r2hd3x4

r1x2h

r1x2h3r1x2h4d

r1x2h4

r2h

a1

d1a2

d2

a2 d2

d 1

k cr

20.d 2

k cr

r1h

r1hr0r1h3

r1h4

d1

a1

d1

kcr d2

a2d2

21.r1x2h

r2h r0r2h3

r2h4

d2

a1

d1

kcr d1

a2d2

22.r1x2h

r1x2h3 r1x2h34r1h3

r2h3

a2

d2

kcr

d1 a1

d1kcr

23.r1x2h

d2

r1x2h4

r1x2h34r1h4

r1x2h

r1x2h4d

a1

d1d2

kcr

d2 a2

k 4

k 8

24.

d 1

k cr

r2h4

r1x2h4d r1x2hr1x2h4

d2

a2k8

k425.

r1x2h34 r1x2h3r1x2h4

d2

a2d1

a126.

r1h4 r4r1h

r1h3x4

d1

d2

a2

kcr d2

kcrd1

27.r1x2h4

r2h4 r4r2h

r2h3x4

d2

d2

a2

kcr d1

kcrd1

28.r1x2h4

r2h3 r3r2h

r2h3x4

d2

d1

a1

kcr d1

kcrd2

29.r1x2h3

r1h3 r3r1h

r1h3x4

d1

d1

a1

kcr d2

kcrd2

30.r1x2h3

r1h3x4 r3x4r1h23x4

r1h4

d1

a2

d2

d2 kcr

d1kcr

31.r1h3

r2h3x4

r3x4r12h3x4

r2h4

r2h3

d2

a1

d1

d1 kcr

d 2

k cr

32.

k 4

k 8

r2hd3x4

r1h23x4 r23x4r1h3x4 d2

a233.

d1 r12h3x4 r13x4r2h3x4 d1

a134.

d2 r2hd3x4 r3x4r2h3x4

k8

k435.

d2

Figure S4

Figure S51. r0' -a1r0im+d1r1-a2r0im+d2r4-a1r0im+d1r3-a2r0im+d2r2+d1r1h+d2r2h+kexr0cyt, 2. r1' -kcrr1+d2r1x2-a1r1im+d1r13-a2r1im+d2r14-d1r1+a1r0im, 3. r2' -kcrr2+d1r1x2-a1r2im+d1r23-a2r2im+d2r24-d2r2+a2r0im, 4. r3' -kcrr3+d2r3x4-a1r3im+d1r13-a2r3im+d2r23-d1r3+a1r0im+d2r2h3+d1r1h3, 5. r4' -kcrr4+d1r3x4-a1r4im+d1r14-a2r4im+d2r24-d2r4+a2r0im+d1r1h4+d2r2h4, 6. r1x2' -kendr1x2-d2r1x2+kcrr1-a2r1x2id+d2r1x24d-a2r1x2im+d2r1x24-a1r1x2im+d1r1x23-d1r1x2+kcrr2, 7. r14' -d2r14+a2r1im-kcrr14+d2r1x24-kcrr14+d1r13x4-d1r14+a1r4im, 8. r13' -d1r13+a1r1im-kcrr13+d2r1x23-kcrr13+d2r13x4-d1r13+a1r3im, 9. r23' -d1r23+a1r2im-kcrr23+d1r1x23-kcrr23+d2r23x4-d2r23+a2r3im, 10. r24' -d2r24+a2r2im-kcrr24+d1r1x24-kcrr24+d1r23x4-d2r24+a2r4im, 11. r3x4' -kendr3x4-d2r3x4+kcrr3-a1r3x4im+d1r13x4-a2r3x4id+d2r2d3x4-a2r3x4im+d2r23x4-d1r3x4+kcrr4+d1r1h3x4+d2r2h3x4+ d2r2hd3x4, 12. r1x23' -kendr1x23-d1r1x23+a1r1x2im-a2r1x23im+d2r1x234-d1r1x23+kcrr23-d2r1x23+kcrr13, 13. r1x24' -kendr1x24-d2r1x24+a2r1x2im-a1r1x24im+d1r1x234-k4r1x24im+k8r1x24d-d1r1x24+kcrr24-d2r1x24+kcrr14, 14. r13x4' -kendr13x4-d1r13x4+a1r3x4im-a2r13x4im+d2r123x4-d2r13x4+kcrr13-d1r13x4+kcrr14+d2r12h3x4, 15. r23x4' -kendr23x4-d2r23x4+a2r3x4im-a1r23x4im+d1r123x4-k4r23x4im+k8r2d3x4-d2r23x4+kcrr23-d1r23x4+kcrr24+d1r1h23x4, 16. r1x24d' -kendr1x24d-d2r1x24d+a2r1x2id-k8r1x24d+k4r1x24im, 17. r2d3x4' -kendr2d3x4-d2r2d3x4+a2r3x4id-k8r2d3x4+k4r23x4im, 18. r1x234' -kendr1x234-d2r1x234+a2r1x23im-d1r1x234+a1r1x24im, 19. r123x4' -kendr123x4-d2r123x4+a2r13x4im-d1r123x4+a1r23x4im, 20. r1x2h' -kendr1x2h-a1r1x2him+d1r1x2h3-d1r1x2h+kcrr2h-d2r1x2h+kcrr1h-a2r1x2hid+d2r1x2h4d-a2r1x2him+d2r1x2h4, 21. r1h' -d1r1h-a2r1him+d2r1h4-a1r1him+d1r1h3-kcrr1h+d2r1x2h, 22. r2h' -d2r2h-a2r2him+d2r2h4-a1r2him+d1r2h3-kcrr2h+d1r1x2h, 23. r1x2h3' -kendr1x2h3-a2r1x2h3im+d2r1x2h34-d1r1x2h3+kcrr2h3-d2r1x2h3+kcrr1h3-d1r1x2h3+a1r1x2him, 24. r1x2h4' -kendr1x2h4-a1r1x2h4im+d1r1x2h34-k4r1x2h4im+k8r1x2h4d-d1r1x2h4+kcrr2h4-d2r1x2h4+kcrr1h4-d2r1x2h4+a2r1x2him, 25. r1x2h4d' -kendr1x2h4d-d2r1x2h4d+a2r1x2hid-k8r1x2h4d+k4r1x2h4im, 26. r1x2h34' -kendr1x2h34-d2r1x2h34+a2r1x2h3im-d1r1x2h34+a1r1x2h4im, 27. r1h4' -d1r1h4-kcrr1h4+d1r1h3x4-d2r1h4+a2r1him-kcrr1h4+d2r1x2h4, 28. r2h4' -d2r2h4-kcrr2h4+d1r2h3x4-d2r2h4+a2r2him-kcrr2h4+d1r1x2h4, 29. r2h3' -d2r2h3-kcrr2h3+d2r2h3x4-d1r2h3+a1r2him-kcrr2h3+d1r1x2h3, 30. r1h3' -d1r1h3-kcrr1h3+d2r1h3x4-d1r1h3+a1r1him-kcrr1h3+d2r1x2h3, 31. r1h3x4' -kendr1h3x4-d1r1h3x4-d1r1h3x4+kcrr1h4-a2r1h3x4im+d2r1h23x4-d2r1h3x4+kcrr1h3, 32. r2h3x4' -kendr2h3x4-d2r2h3x4-d2r2h3x4+kcrr2h3-k4r2h3x4im+k8r2hd3x4-a1r2h3x4im+d1r12h3x4-d1r2h3x4+kcrr2h4, 33. r1h23x4' -kendr1h23x4-d1r1h23x4-d2r1h23x4+a2r1h3x4im, 34. r12h3x4' -kendr12h3x4-d2r12h3x4-d1r12h3x4+a1r2h3x4im, 35. r2hd3x4' -kendr2hd3x4-d2r2hd3x4-k8r2hd3x4+k4r2h3x4im, 36. r0cyt' -kexr0cyt+kendr1x2+kendr3x4+kendr1x23+kendr1x24+kendr13x4+kendr23x4+kendr1x24d+kendr2d3x4+kendr1x234+kendr123x4+kendr1x2h+ kendr1x2h3+kendr1x2h4+kendr1x2h4d+ kendr1x2h34+ kendr1h3x4+kendr2h3x4+kendr1h23x4+kendr12h3x4+kendr2hd3x4, 37. iend' -kexiend+kendr1x2h+kendr1x2h3+kendr1x2h4+kendr1x2h4d+kendr1x2h34+kendr1h3x4+kendr2h3x4+ kend r1h23x4+kendr12h3x4+ kendr2hd3x4

10-11 10-10 10-9 10-8 10-7 10-6 10-5

0

20

40

60

80

100

Hot

insu

lin r

emai

ning

afte

r di

ssoc

iatio

n (%

)

Cold insulin (M)

Dissociation time: 10 min

10-11 10-10 10-9 10-8 10-7 10-6 10-5

0

20

40

60

80

100

Hot

insu

lin r

emai

ning

afte

r di

ssoc

iatio

n (%

)

Cold insulin (M)

Dissociation time: 20 min

10-11 10-10 10-9 10-8 10-7 10-6 10-5

0

20

40

60

80

100

Hot

insu

lin r

emai

ning

afte

r di

ssoc

iatio

n (%

)

Cold insulin (M)

Dissociation time: 30 min

10-11 10-10 10-9 10-8 10-7 10-6 10-5

0

20

40

60

80

100

Hot

insu

lin r

emai

ning

afte

r di

ssoc

iatio

n (%

)

Cold insulin (M)

Dissociation time: 60 min

Figure S6