1

The ROLE of GLUCAGON in DIABETES :An Update

Pierre Lefèbvre

Cairo, Egypt, September 2007

2

3

4

1923

Murlin et al The discovery

5

1942Czyste Hospital Warsaw

GhettoStudies on « Hunger’s disease » by Dr

Emil Apfelbaum et al

6

7

8

1949

Foà* et alCross-circulation experiments

*Only Author I know to have published a paper when in utero …

9

10

11

Insulin-inducedhypoglycemia inthe « donor » dog

Paradoxical hyperglycemia in the dog receiving PDV blood

12

1959-1960

Unger et al The first polypeptide radio-immunoassay

13

Roger UNGER

14

1973

15

Topics

Importance of glucagon for the maintenance of glucose Importance of glucagon for the maintenance of glucose homeostasishomeostasis

The physiology of glucagon production and how glucagonexerts its effects

Abnormalities of glucagon secretion in patients with T2DM

16

Pancreatic Islet Hormones Are Critical for Normal Glucose Tolerance

HGO = hepatic glucose outputAdapted from Unger RH. Metabolism. 1974;23:581–593

Glucose

-Cells -Cells

Insulin Glucagon–+

Glucose

uptake

HGO

+

17

+Glucagon*

(plasma concentration)

–

–Insulin*

(plasma concentration)

+Glucose

(plasma concentration)

The Physiological Balance Between Insulin and Glucagon is Essential for Normal Metabolic Control

*Insulin and glucagon secretion are also influenced by other nutrients, hormones, and neural input Adapted from Berne RM, Levy MN, eds. Physiology. St. Louis, Mo: Mosby, Inc; 1998:822–847

18

Glucagon is Important to Maintain Adequate FPG Levels Between Meals

Adapted from Unger RH. Diabetes. 1983;32:575–583

α-Cell

Blood glucose4.5 mmol/L (81 mg/dL)

4 g/h

6 g/h

Glucagon

10 g/h

+

Glucose production = Glucose

utilization

19

Normal Reciprocal Response of Insulin and Glucagon in Persons Without Diabetes

CHO = carbohydrateAdapted from Unger RH, et al. N Engl J Med. 1971;285:443–449

Glucose

mg%

12010080

–60 0 60 120 180 240Time (min)

CHO Meal

Insulin

μU/m

L

12080400

Glucagon

90

pg/m

L 120110100

Postprandial Insulin and Glucagon Responses in Persons Without Diabetes

20

Topics

Importance of glucagon for the maintenance of glucose homeostasis

The physiology of glucagon production and how glucagonThe physiology of glucagon production and how glucagonexerts its effectsexerts its effects

Abnormalities of glucagon secretion in patients with T2DM

21

α-Cell Function is Regulated by Nutrient, Neural and Hormonal Influences

Adapted from Dunning BE, et al. Diabetologia. 2005;48:1700–1713

Stimulatory Factors (↑ Glucagon)Stimulatory Factors (↑ Glucagon) Inhibitory Factors (↓ Glucagon)Inhibitory Factors (↓ Glucagon)

Hypoglycaemia

Protein meal

Amino acids

Stress; adrenaline (epinephrine)

Sympathetic/parasympathetic nerves

GIP

Glucose

Carbohydrate meal

Ketones

Insulin

Somatostatin

GLP-1

22

Glucagon Stimulation of Hepatocytes Leads to Increased Hepatic Glucose Production

TCA = trichloroacetic acidAdapted from McMurry J, et al. Fundamentals of General, Organic, and Biological Chemistry. 4th ed. Upper Saddle River, NJ:Prentice Hall; 2006Jiang G, Zhang BB. Am J Physiol Endocrinol Metab. 2003;284:E671-E678

Glycolysis

Glycogenolysis

HGP

Glucose

Glucagon

-+

Glycogenesis

Gluconeogenesis

-+

23

Glucagon From α-Cells is Responsible for 75%of HGP

HGP = hepatic glucose production*Somatostatin and insulin were delivered to induce selective glucagon deficiency †Healthy, non-obese men age 18–30Adapted from Liljenquist JE, et al. J Clin Invest. 1977;39:369–374

150

100

50Net

Spl

anch

nic

Glu

cose

Pro

duct

ion

(mg/

min

)

Time (min)–30 0 30 60 90 120

START INFUSION*

<.01 <.01 <.01 <.01<.01

<.01 <.01 <.01

75% in HGP

vs baseline

N = 8†

24

Changes in Glucagon/Insulin Ratio Regulate HGO and Glycogen Synthesis in Normal Physiology

HGO = hepatic glucose output*All measurements made after a standard mixed meal in healthy volunteers ages 18–40 years†HGO completely suppressed after 30 minutes of meal intakeAdapted from Taylor R, et al. J Clin Invest. 1996;97:126–132

Liver Glycogen (mmol/L)*HGO (mg/kg.min)*†Glucagon/Insulin ratio*

| | | | | |0 120 240 360 480 600

40 –

30 –

20 –

10 –

0 –

Minutes

(n= 6) (n= 6) (n= 8)

2.5 –

2.0 –

1.5 –

1.0 –

0.5 –

0 –

Minutes

| | | | | |0 120 240 360 480 600

350 –

300 –

250 –

200 –

150 –

Minutes

| | | | | |0 120 240 360 480 600

25

Topics

Importance of glucagon for the maintenance of glucose homeostasis

The physiology of glucagon production and how glucagonexerts its effects

Abnormalities of glucagon secretion in patients with T2DMAbnormalities of glucagon secretion in patients with T2DM

26

T2DM = type 2 diabetes mellitusAdapted from Rhodes CJ. Science. 2005; 307:380–384

α-Cells (glucagon)

Normal T2DM

β-Cells(insulin)

• Disorganised and misshapen • Marked reduction in β-cell number • Amyloid plaques

Amyloid plaque

Pancreatic Islet Morphology: Structural Defects are Evident in T2DM

27

Islet Dysfunction Leads to Hyperglycaemiain T2DM

HGO = hepatic glucose outputAdapted from Unger RH. Metabolism. 1974;23:581–593

T2DM pancreatic islet

Glucose

Fewer -cells -cell hypertrophy

Insufficient insulin Excess glucagon–+

Less effective glucose uptake

HGO↑

↑

28

In T2DM, Insufficient Insulin and Elevated Glucagon Secretion Result in Hyperglycaemia

Adapted from Müller WA, et al. N Engl J Med. 1970;283:109–115

CHO meal

Glucagon

Time (min)

75

100

125

150

–60 0 60 120 180 240

pg/m

L

Insulin0

50

100

150

μU/m

L

0Glucose100

200

300

400

mg/

dL NGTT2DM

NGTT2DM

NGTT2DM

29

Decreased Glucose Disposal and Increased HGP Contribute to Increased FPG in T2DM

HGP = hepatic glucose production; FPG = fasting plasma glucose; T2DM = type 2 diabetes mellitusAdapted from DeFronzo RA. Diabetes. 1988;37:667–687

1.01.52.02.53.03.54.04.5

50 100 150 200 250 300FPG (mg/dL)

Glycogenolysis andGluconeogenesis

(mg/kg • min)

Excessive glucagon-mediatedglucose output

0.81.21.62.02.42.8

50 100 150 200 250 300

Glucose Clearance

(mL/kg • min)Impairedinsulin-mediated glucose disposal

Diagnosis

30

Suppression of Endogenous Glucose Production is Impaired in T2DM

HGO = hepatic glucose outputAdapted from Kelley D, et al. Metabolism. 1994;43:1549–1557

Time (minutes)-30 -15 0 30 60 90 120 150 180 210 240 270 300

Meal

2

6

10

14

18

Endo

geno

us G

luco

se(µ

mol

/min

/kg)

NGT (n= 12)

T2DM (n= 18)

31

α-Cell Sensitivity to Glucose is Reduced in T2DM

AGRarg= mean acute glucagon response to arginine from 2 to 5 min; PG50 = plasma glucose level required for half-maximal suppression of AGRargT2DM = type 2 diabetes mellitus; *Healthy men ages 18–29Adapted from Ward WK, et al. J Clin Invest. 1984;74:1318–1328. Dunning B, et al. Diabetologia. 2005;48:1700–1713

NGT* (n = 8)T2DM (n = 8)

180 -

150 -

120 -

90 -

60 -

30 -0 100 200 300 400 500 600 700

AGR a

rg (p

g/m

L)

Plasma Glucose Level (mg/dL)

PG50

32

In Patients with T2DM, Suppression of Glucagon Reduces Glycogenolysis and Plasma Glucose Levels

†P <0.0001 between groups; ‡P <0.001between groups§As assessed by [14C] glucose appearanceProtocol: somatostatin and insulin delivered to mimic glucagon deficiency, then glucagon infused to simulate a non-suppressed day,or delayed by 2 hours to create transient glucagon deficiency (suppressed day)Adapted from Shah P, et al. J Clin Endocrinol Metab. 2000;85:4053–4059

Glucagon GlucoseGlycogenolysis§

Glu

cose

(m

mol

/L)

-60 0 60 120 180 240 300 3604

6

8

10

12

Time (min)

↓50-g glucose‡

Glu

cago

n (n

g/L)

-60 0 60 120 180 240 3003600

100

200

Time (min)

50

150

↓

†† † †

50-g glucose

dpm

/kg/

min

0 60 120 180 240 300 3600

4,500

3,000

1,500

Time (min)

‡

‡‡

‡

↓50-g glucose

(n= 9)

Non-suppressed glucagon Suppressed glucagon

33

Effect of a glucagon monoclonal antibody on glucose excursion during an OGTTIn ob/ob mice. Data from Sorensen et al , Diabetes 2006, 55: 2843-2848 m

34

35

Hyperglucagonemia is a feature of all forms of diabetes

« Dual A- and B-cell abnormality » ?A-cell insulin resistance ?Hyperglycemia-induced desensitization of the A-cell (Glucotoxicity ? )

36

An alternative hypothesis*

-Insulin and glucagon are secreted in a pulsatile manner-Normal intra-islet insulin pulsatility helps avoiding excessive glucagon release-Normal insulin pulsatility is lost in diabetesThus, loss of normal intra-islet insulin pulsatility may be involved in the hyperglucagonemia of diabetes *Lefèbvre, Paolisso and Scheen 1991

37

Lelio ORCI

38

39

40

Pulsatile insulin and glucagon-induced glucagon release*

Nine Type 1 diabetic patients Very small insulin infusion given either continuously (circulating insulin levels ~15 µU/ml) or same total amount of insulin given in 13 min pulses and both compared to saline infusion No significant effect on blood glucose After 52 minutes, 5gm iv arginine pulse and evaluation of integrated glucagon response

*Paolisso et al , JCEM1988,66: 1220-26

41

42

43

New support for the intra-islet insulin hypothesis

MEIER JJ, KJEMS LL, VELDHUIS JD , LEFEBVRE PJ and BUTLER PC : Postprandial Suppression of Glucagon Secretion Depends on Intact Pulsatile Insulin Secretion : Further Evidence for the Intraislet Insulin Hypothesis*

*Diabetes , 55:1051-1056,2006

44

-40 -35 -30 -25 -20 -15 -10 -5 00.0

2.5

5.0

7.5

10.0

12.5

15.0

Post alloxanPre alloxan

p = 0.067

Glu

cose

[mm

ol/l]

0 10 20 30 40 50 60 70 80 900.0

2.5

5.0

7.5

10.0

12.5

15.0

Post alloxanPre alloxan

Testmeal

p = 0.021

Glu

cose

[mm

ol/l]

-40 -35 -30 -25 -20 -15 -10 -5 00

200

400

600

800

1000

1200

1400

p = 0.032

Insu

lin [p

mol

/l]

0 10 20 30 40 50 60 70 80 900

200

400

600

800

1000

1200

1400p = 0.079

Insu

lin [p

mol

/l]

-40 -35 -30 -25 -20 -15 -10 -5 00

20406080

100120140160

Time [min]

Glu

cago

n [p

g/m

l]

p = 0.0045

0 10 20 30 40 50 60 70 80 900

20406080

100120140160

Time [min]

Glu

cago

n [p

g/m

l]

p = 0.0037

A B

C D

E F

45

-40 -30 -20 -10 080

90

100

110

120

130

Glu

cago

n [p

g/m

l]

-40 -30 -20 -10 00

10

20

30

40

50

60

70

Time [min]

Glu

cago

n se

cret

ion

[pg/

ml/m

in]

A

B

Glucagon pulsesin the basal stateprior to alloxan in a representative pig

46

GLUCAGONPulse mass, heightand interval in the fasting state and after a meal studiedboth before and after alloxan in 7 pigs*

*Meier et al , 2005submitted

47

-20 -10 0 10 20-0.8-0.6-0.4-0.2-0.00.20.40.60.8

Lag time [min]

Cor

rela

tion

coef

ficie

nt

-20 -10 0 10 20-0.8-0.6-0.4-0.2-0.00.20.40.60.8

Lag time [min]

Cor

rela

tion

coef

ficie

nt

A

B

Pre alloxan

Post alloxan

Mean cross-correlogrammsbetween insulin and glucagon concentrationstime series in 7 pigsstudied over 90 min aftera test meal, each prior andafter alloxan treatment.

The relationship between insulin and glucagon levelsis significant before but disappears after alloxan

48

1.0

1.5

2.0

2.5p = 0.0035

Forward Reverse

Cros

s-Ap

En

1.0

1.5

2.0

2.5p = 0.80

Forward Reverse

Cros

s-Ap

En

Pre alloxan Post alloxan

« Forward » and « reverse » cross-AsPen analyses imply that, in the post-prandial state,pulsatile insulin secretion directly suppresses glucagon secretion, but that this associationis lost after a ~60% reduction in beta cell mass

49

In conclusion

It is confirmed that glucagon is secreted in distinct pulses both in the fasting state and after a meal In healthy minipigs, suppression of glucagon secretion after a meal is likely accomplished, at least in part, by increased insulin secretion This action of insulin is lost in the minipig alloxan model of Type 2 diabetes mellitus in which post-prandial insulin secretion is impaired as a consequence of an ~60% decrease in beta-cell mass

We conclude that post-prandial hyperglucagonemia in diabetes is likely due ,at least in part, to impaired insulin secretion leading to a loss of intra-islet driven suppression of glucagon secretion

50

Potential Clinical Consequence:The best way to normalize the hyperglucagonemia of diabetesmay be to restore normal insulinsecretion , including pulsatility …Increase in amplitude of insulin pulses, without changes infrequency,reported by a 6wk GLP1 infusion in 5 old patients with T2DM by Meneilly et alJCEM, 2005, 90: 6251-56

51

Glucagon: Summary

Glucagon plays an essential role in glycaemic control α-Cell sensitivity to glucose is impaired in T2DM, resulting in

excessive glucagon secretion Excessive glucagon secretion contributes to hyperglycaemia

in both IGT and T2DM Lowering glucagon levels will be a valuable therapeutic target

in the treatment of T2DM

IGT = impaired glucose tolerance; T2DM = type 2 diabetes mellitus