Seminario all’Università di Pavia

Insulino-resistenza e deficit

beta-cellulare: chi è più importante

nella patogenesi del diabete tipo 2?

Pavia, 4 Novembre 2013

Enzo Bonora

Endocrinologia, Diabetologia e Metabolismo

Università e Azienda Ospedaliera Universitaria

Integrata di Verona

Hyperglycemia

LiverIncreased

endogenous glucose

production

Skeletal muscleImpaired glucose utilization

(transport, storage, oxidation)

Endocrine pancreasImpaired insulin secretion

Exaggerated glucagon secretion

GutImpaired incretin effect

KidneyIncreased glucose reabsorption

BrainAbnormal metabolic control

Adipose tissueRelease of

diabetogenic molecules

(FFA, adipocytokines)

T2DM: a Complex and Systemic Pathogenesis

Insulin

resistance

Impaired

insulin

secretion

Key Pathogenetic Defects of T2DM

Quesito: Chi è più importante fra insulino-resistenza e deficit beta-cellulare per losviluppo di diabete tipo 2?

Risposta: Il deficit beta-cellulare ma….

Evidenze: studi genetici, studi epidemiologici,studi di fisiopatologia clinica, studi su modellianimali e umani

Studi genetici

Beta-cell

Dysfunction

Insulin

Resistance

Genes Environment

Diabetes Milieu:

Hyperglycemia, Dyslipidemia, Inflammation, Oxidant stress

Environment Genes

Diabete tipo 2: malattia poligenica

Geni della disfunzione beta cellulare

• KCNJ11

• TCF7L2

• IGF2BP2

• WSF1

• SLC30A8

• CDC123

• TSPAN8

• KCNQ1

• MTNR1B

• HHEX

• CDKAL1

• CDKN2A/B

• HNF1B

• JAZF1

• GCKR

• ecc.

Geni dell’ insulino-resistenza

• ENPP1 (PC-1)

• PPARG

• ADAMTS9

Sensibilità insulinica (M-clamp) nei soggetti diabetici del

VNDS in funzione della presenza o meno dell’allele di

rischio di PPARG2 e dell’allele di rischio di ADAMTS9VNDS – Trombetta et al - Acta Diabetologica 50: 401, 2013

PPARG2

ADAMTS9

Association of T2DM

with Common

Genetic VariantsAhlqvist E – Clin Chem 2011; 57: 241-254

TCF7L2 is expressed in human islets and risk

genotypes are associated to deterioration of

-cell function over timeLyssenko et al – J Clin Invest 2007; 117: 2155-2163

Human

islet

Beta-cell

TCF7L2 merge

P = 0.003

-.5

0.5

1

log d

isposi

tion in

dex

0 5 10 15

Years from screening

rs7903146=CC

rs7903146=CT or TT

Association of rs7901695 and rs7903146 Risk

Alleles of TCF7L2 with Glucose Stimulated

Insulin Secretion in Newly-Diagnosed T2DM

p=0.022 p=0.019

N=464

VNDS - Bonetti et al – JCE&M 2011; 96: E389

Haplotype analysis of TCF7L2 polymorphisms and glucose

stimulated insulin secretion in newly-diagnosed T2DM

rs7901695 rs7903146 rs11196205 rs12255372 Haplotype

freqency

haplo.4 C T C G 0,0383

haplo.36 C T C T 0,3479

haplo.9 T C C G 0,0864

haplo.45 T C G G 0,4559

PROPORTIONAL CONTROL

0

100

200

300

400

500

600

5,5 8 11 15 20

Glycaemia (mmol/l)

Insu

lin S

ecre

tion

Rate

ISR

(pm

ol/m

in/m

2)

HAPLO.45

HAPLO.36

HAPLO.9

HAPLO.4

VNDS - Bonetti et al – JCE&M 2011; 96: E389

0

200

400

600

800

1000

5.5 8 11 15 20

GG

GA+AA

INS

UL

IN S

EC

RE

TIO

N R

AT

E (

ISR

)

[pm

ol. m

in-1

. m-2

BS

A]

0

200

400

600

800

1000

5.5 8 11 15 20

AA

AC+CC

rs6717980 rs2384628

p<0.02 p<0.04

Association of rs6717980 and rs2384628 risk allels

of GCKR with glucose stimulated insulin secretion

in newly diagnosed T2DMVNDS - Bonetti et al – Diabetes Care 2011

Plasma glucose (mmol/l)Plasma glucose (mmol/l)

0

200

400

600

800

1000

1200

1400

5.5 8 11 15 20

0

1

2

3

4

INS

UL

IN S

EC

RE

TIO

N R

AT

E (

ISR

)

[pm

ol. m

in-1

. m-2

BS

A]

p=0.012

Association of GCKR risk allele score with glucose

stimulated insulin secretion in newly diagnosed T2DM VNDS - Bonetti et al – Diabetes Care 2011

Plasma glucose (mmol/l)

No. of

Risk

Alleles

Association of CACNA1E risk allele score with glucose

stimulated insulin secretion in newly diagnosed T2DM VNDS – Trombetta et al – PLoSOne 7:e32755

No. of

Risk

Alleles

CACNA1E = major subunit of the voltage-dependent Ca2+ channel CaV2.3

SNP Nearest geneNot-risk/risk

allelesMAF

rs679931 CACNA1E A/T 0.37

rs1801282 PPARG T/C 0.07

rs1044498 ENPP1 A/C 0.16

rs10946398 CDKAKL1 A/C 0.30

rs1111875 HHEX G/A 0.38

rs10010131 WFS1 A/G 0.35

rs4430796 TCF2 A/G 0.43

rs4402960 IGF2BP2 G/T 0.35

We gave different values to the alleles for each SNP: 0 to the not-risk allele and 1 to the risk allele. In this

way we were able to calculate a sort of “genetic score”, summing up all the risk alleles values for each

patients. Since we considered 8 SNPs, the genetic load score potentially ranges between 0 (patients with

no risk alleles) and 16 (patients homozygous for risk alleles in all SNPs). We then divided the studied

population into three groups on the basis of the number of risk alleles: Low (LGL: ≤6, n=76),

Intermediate (IGL: 7-9, n=226) and High (HGL: ≥10, n=69) “Genetic Load” score.

Genetic load assessment: 8 SNPs - 0 to 16 risk alleles

PROPORTIONAL CONTROL

0

200

400

600

800

1000

0 5 10 15 20 25

GLYCAEMIA (mmol/l)

Insu

lin

Secre

tio

n R

ate

IS

R

(pm

ol/

min

/m2)

LGL

IGL

HGL

Effect of “genetic load” on proportional control of

insulin secretion in newly-diagnosed T2DM

LGL: ≤6, n=76

IGL: 7-9, n=226

HGL: ≥10, n=69

VNDS - Bonetti et al – Diabetes Care 2011

Studi epidemiologici

0

5

10

15

20

25

30

35

Low M clamp (<median) Low AIR (<median)

Risk of T2DM Associated to Insulin Resistance and Impaired Insulin Secretion in Pima Indians

Lillioja et al - NEJM 1993; 329: 1988

N=200; follow-up 6 years; data are adjusted for sex, % body fat and WTR

Rela

tive H

azard

p<0.05

2.8

p<0.01

30.8

Risk of Type 2 Diabetes According to Insulin Sensitivity and Insulin Secretion in Caucasians

Bruneck Study - Bonora et al - Diabetes 2004

N=888; follow-up 10 years; data are adjusted for sex and age

Odds r

atios

0

5

10

15

Q1 Q2 Q3 Q4

HOMA-IR

Q1 Q2 Q3 Q4

CIR-OGTT

1.0

3.0

10.3

2.8

p<0.001

4.8

1.72.2

1.0

p=0.003

Low sensitivity

Low secretion

0

1

2

3

4

5

6

7

8

Normal Low Normal Low

HOMA-IR, CIR Index and 10-yr Incidence of T2DM in the General Population Aged 40-79 yr

Bruneck Study; Bonora et al, Diabetes 2004

sex and age-adjusted

OR

p=0.007

Insulin secretion

Normal Normal Low LowInsulin sensitivity

Haffner et al, Circulation 101:975, 2000

Insulin resistanceLow insulin secretion

0

10

20

30

40

50

60P

erc

en

tag

e o

f c

on

vert

ers

Incidence of type 2 diabetes according to insulin resistance and insulin secretion at baseline

16

28.5

54

1.5

no

yes

yes

no

yes

yes

no

no

Insulin resistantInsulin sensitive

Insulin resistance = HOMA-IR; insulin secretion = ΔIns/Δglucose 0-30 min OGTT

Studi di fisiopatologia clinica

Loss of First-Phase Insulin Secretion after

i.v. Glucose in T2DMWard WK et al - Diabetes Care 1984; 7:491

Type 2 diabetes

–30 0 30 60 90 120

Normal

120

100

80

60

40

20

0

Time (minutes)

Pla

sm

a in

su

lin

(µ

U/m

l) 20 g

glucose

Impaired Insulin Response during

Hyperglycemic Clamp in T2DMBonora et al; unpublished data

-20 -10 0 2 4 6 8 10 20 30 40 50 60 70 80 90 100 110 1200

100

200

300

400

500

Time (min)

Insulin

(pm

ol/l)

T2DM

Controls

Impaired Insulin Respose to Mixed Meal in T2DM

Vilsbøll T et al - Diabetes 2001; 50: 609–613

Insu

lin

(pm

ol/L

)

Mixed

mealNormal subjects

Type 2 diabetics

Time (min)

500

400

300

200

100

0

0 60 120 180

Dose-Response Curves for Glucose-Stimulated Insulin

Secretion during OGTT in Different ConditionsFerrannini et al - JCEM 2005; 90: 493

Ferrannini E et al. Med Clin North Am 2011;95:327-39

0

25

50

75

100

125

150

175

200

225

250

NGT IFG IGT

Glu

cose

sensitiv

ity

(pm

ol. m

in-1

. m-2

. mM

-1)

T2D

Beta-cell glucose sensitivity in T2DM

according to glucose tolerance

Increased Ratio of Fasting

Proinsulin to Insulin Ratio in T2DM

NGT

(n=77)

IGT

(n=46)

Type 2 diabetes

(n=46)

Saad et al, J Clin Endocrinol Metab 70: 1247–1253, 1990

p<0.001

p<0.365

p<0.001

0

5

10

15

20

25

Tru

e P

I/IR

I (%

)*

Pulsatility of Insulin and Glucagon Secretion is Distrupted

in the Fasting State and After Meal in T2DMMenge BA et al – Diabetes 2011; 60: 2160-2168

Fasting Post-

prandial

Non diabetic Diabetic Non diabetic Diabetic

Minimal Model of -Cell Function During OGTT

PLASMA

GLUCOSE

[G(t)]

X(t)

PLASMA

C-PEPTIDE

s [G(t) - q]

t -1

REMOTE

C-PEPTIDE

POOL

CA

TA

BO

LIS

M

-CELL

OGTT -INDEX = ln {[s (11 - q)]/(2.72 + t)}

Cretti et al - Eur J Clin Invest 2001; 31:405

K21

K12

K01

What does OGTT -Index Represent?

• Secretory force of the beta-cell

• Beta-cell competence for glucose

• Efficiency of beta-cell

0

1

2

3

4

5

6

7

50 g OGTT 100 g OGTT 150 g OGTT

OGTT Insulin Secretion Rate (ISR)OGTT -index (efficiency)

OGTT-ISR and -Index Values during

Oral Glucose Loads of 50, 100 and 150 g

Cretti et al - Eur J Clin Invest 2001; 31:405

0

1.5

3.0

4.5

6.0

Controls

(n=125)

T2DM

(n=292)

Impaired Beta-cell Efficiencyin Newly-Diagnosed T2DM

-in

de

x (

log

pm

ol/m

in)

Bonora and Bonadonna - unpublished data

0

5

10

15

20

-0.8 -1.3 -1.8 -2.3 -2.8 -3.3 -3.8 -4.3 -4.8 >4.8

Frequency Distribution of -Index in Subjects with Newly-Diagnosed T2DM

-index (log units)

%

No -cell defect

Threshold of β-cell defect = upper limit of bottom quintile of β-index in 125 non-obese healthy subjects

Bonora and Bonadonna - unpublished data

0

4

8

12

16

-200 -300 -400 -500 -600 -700 -800 -900 -1000 -1100 >1100

Frequency Distribution of M-clamp in Subjects with Newly-diagnosed T2DM

M-clamp (µmol/min·m2 BSA)

%

No insulin resistance

Threshold of insulin resistance = upper limit of bottom quintile of M-clamp in 515 non-obese healthy subjects

Bonora and Bonadonna - unpublished data

Proportion of Pathogenic Phenotypes

in Newly-Diagnosed T2DM (n=577)

00

20

40

60

80

100

(%)

Isolated insulin

resistance

Isolatedbeta-cell

dysfunction

Bothdefects

10.6 8.2

81.2

Insulin resistance = 89.4 %

Beta-cell dysfunction = 91.8 %

insulin sensitivity assessed by euglycemic clamp (M)

insulin secretion assessed by OGTT and minimal model (β-index)

Bonora and Bonadonna - unpublished data

Relationship between M-clamp and

-Index in Newly-Diagnosed T2DM

r=-0.003

p=NS

Beta

in

dex

M clamp (pmol/min.m2BSA)

0

1

2

3

4

5

6

7

0 500 1000 1500 2000 2500

Bonora et al, unpublished data

(log p

mol/m

in.m

2B

SA

)

Years from diagnosis

Beta-Cell Function Declines While Insulin Sensitivity Remains Stable in Type 2 Diabetes

10-year follow-up of the Belfast Diet Study: Data from newly diagnosed subjects with type 2 diabetes mellitus (N=67) who required

oral antihyperglycemic therapy or insulin due to secondary failure of diet therapy at 5–7 years.

0

40

60

HO

MA

-S (

%)

20

0

0

40

60

80

HO

MA

-B (

%)

20

2 4 60 2 4 6

Years from diagnosis

Beta-Cell FunctionInsulin Sensitivity

Levy et al - Diabet Med 1988; 15: 290

IEq= Islet Equivalents; PW= Pancreas Weight

7,000

6,000

5,000

4,000

3,000

2,000

1,000

0

0 1 2 3 4 5 6 7 8 9

R2= 0.4079

Duration of Diabetes (yrs)

IEq

/gra

m o

f P

WIslets Mass Declines with Progression f T2DM

Deng et al - Diabetes 2004; 53: 624

*

*

*

*

**

*

*

**

*

*

**

United Kingdom Prospective Diabetes Study (UKPDS)

Progressive Loss of glucose control

06

7

8

9

0 3 6 9 12 15

HbA

1c (

%)

Years from randomisation

Conventional

Intensive

6.2% upper limit of normal range

Insults to the Beta-Cell

Glucotoxicity Lipotoxicity

Inflammation Oxidant stress

Insulin Resistance

Islet Amyloid Polypeptide

10 µm~ 10,000

granules

Micrograph: Lelio Orci, Geneva

The Normal Beta-Cell

Relationship of Fasting Plasma Glucose to Beta-Cell

Volume in Pancreas from Untreated Obese HumansRitzel et al - Diabetes Care 2006; 29: 717

Monoesponential fit

R=0.50, p<0.001

Beta-Cell Volume in Pancreas from Obese and

Lean Subjects with IFG and T2DMButler et al - Diabetes 2003; 52:102

0

0.5

1.0

1.5

2.0

2.5

3.0

Non-diabetic

(n = 35)

IFG

(n = 15)

T2DM

(n = 41)

β-C

ell

are

a/e

xocrine a

rea (

%)

OBESE

Non-diabetic

(n = 17)

T2DM

(n = 16)

LEAN

Apoptosis and Neogenesis of Beta-Cell in Pancreas

from Nondiabetic and Diabetic subjectsButler et al - Diabetes 2003; 52:102

Deng et al; Diabetes 53: 624, 2004

52% -cells

48% β-cells

P <0.01

T2DM

Beta-Cells are Reduced and Alpha-Cells are

Increased in Human Pancreatic Islets from T2DM

35% -cells

65% β-cells

Nondiabetic

Islets of

Baboons with Type 2

Diabetes Have

Increased Proliferation

of Alpha-Cells and

Increased Apoptosis of

Beta-Cells

Guardado-Mendoza R et al

PNAS 2009; 106: 13992-13997

Marker of proliferation Ki67 only

in alpha cells

Marker of apoptosis M30 only in

beta-cells

Insulin Secretory Granules Are Diminished

in Human Diabetic Beta-CellsMarchetti et al; NMCD 2006

Control T2DM

The Progression of Beta-Cell Dysfunction in T2DMUKPDS 16 - Diabetes 1995; 44: 1249

0

20

40

60

80

100

Years from diagnosis

Beta

-cell f

un

cti

on

(H

OM

A-B

%)

–10 –8 –6 –4 –2 0 2 4 6–12

Insulin Response to Intravenous Glucose According

to Glucose Tolerance in 434 Pima IndiansWeyer et al - Diabetes 1999; 48: 2197

p<0.05

0

100

200

300

400

Controls Isolated IFG Isolated IGT IFG + IGT

p<0.001

p<0.01

First- and Second-Phase Beta-Cell Secretion during

Hyperglycemic Clamp and Beta-Cell Performance after

OGTT in Normoglycemic Offspring of T2DMBonadonna et al - Diabetes 2003; 52: 470

Controls OfT2DM p

FPG (mmol/l) 4.5 4.6 NS

2h-PG (mmol/l) 4.5 4.8 NS

1st Phase Secretion (pmol/m2 BSA) 4459 3174 0.01

68334 50286 0.02

OGTT -index (log pmol/min.m2BSA) 5.6 5.1 0.01

2nd Phase Secretion (pmol/m2BSA)

N=38, Age 46, BMI 26.5

Relationship of First-Phase Insulin Secretion after i.v.

Glucose and Insulin Sensitivity in Healthy SubjectsKahn et al - Diabetes 1993; 42: 1663

100

200

300

400

500

00 1 2 3 4 5

M-low (mg/kg EMBS per min)

AIR

(

U/m

L)

Weyer C et al - J Clin Invest 1999; 104:787

Changes in Insulin Secretion and Sensitivity are

Associated to the Development of T2DM

NGTNGT

IGT

DM

NGT - Non converters to

T2DM

NGT - Converters to T2DM

Indipendent Predictors of HbA1c in Newly

Diagnosed Type 2 Diabetic Subjects

Independent

variables

Standardized

-Coefficientp

Age - 0.234 0.001

β-index - 0.386 0.001

M-clamp -0.177 0.001

Explained variance: 24.6%

Bonora and Bonadonna - unpublished data

Studi in modelli animali e umani

Fasting p

lasm

a insulin

(m

U/L

)

Age (years)

0

250

500

750

1000

1250

1500

1750

0 6 12 18 24

A Proof-of-Concept from Mother Nature:

A Girl with LeprechaunismF

astin

g p

lasm

a g

lucose (m

g/d

l)

50

100

150

200

250

300

350

0

Insulin

Glucose

Insulin Resistance

- not sufficient even when extremely severe

- perhaps not necessary, if beta-cell dysfunction is

very severe

Beta-Cell Dysfunction

- absolutely necessary

- probably sufficient, if severe enough

The main underlying disorders

in T2DM

IRS-1 genetic variant reduces mature insulin

granules in human islets

Gly/Gly Gly972Arg

Marchetti P et al - Diabetes 2002; 51:1419

Control MIRKO

Transgenic mice and glucose regulation

0

100

200

300

2 h

Glu

co

se

(m

g/d

l)

βIRKO

Mauvais-Jarvis et al - Diabetes 2000; 49: 2126

With a dynamic, rather than static, assessment of parameters, beta-cell dysfunction clearly appears to contribute to T2DM developmentmore strongly than insulin resistance.

T2DM cannot develop without beta-cell dysfunction.

Virtually all subjects with T2DM have beta-cell dysfunction.

In T2DM beta-cell dysfunction contributes to hyperglycemia morethan insulin resistance.

Beta-cell dysfunction and reduced beta-cell mass can be foundmany years before diabetes is diagnosed and they progressivelyworsen over the years dictating the loss of metabolic control.

However, insulin resistance and beta-cell dysfunction are intimallyassociated and insulin resistance is an importat promoting effect.

Conclusions

Fine

Grazie per l’attenzione