How Artificial Sweeteners Reduce Diabetes-Associated Blood Vessel Injury in

the Eye

BARS conference, 27 September 2019, 11:50-12:15

Crown Plaza Hotel, Liverpool@DrHaviChichger

Dr Havovi ChichgerHavovi.Chichger@Anglia.ac.uk

Diabetic Eye Disease

• Diabetes – 4.5 million UK, ~10% global community

• Diabetic eye disease comprises a group of eye conditions including diabetic retinopathy (DR), diabetic macular edema (DME), cataract, and glaucoma.

• All forms of diabetic eye disease have the potential to cause severe vision impairment, vision loss and blindness.

• In 2014, 54.6% patients with type I diabetes had DR; and 30% patients with type II diabetes had DR

Mathur R et al, 2017, RNIB; Diabetes UK 2018; Eschenbach Optik 2019; Mayo Clinic 2019

Clinical signs of diabetic retinopathy on fundoscopic examination

Mild, non-proliferative DR

Proliferative DR

Moderative, non-proliferative DR

Diabetic macular oedema

Wong TY, 2016, Nature Reviews Disease Primers

Pathogenic events in DR

Wong TY, 2016, Nature Reviews Disease Primers

1 2

3 4

Macular oedema (fluid leak) Proliferative retinopathy (new blood vessel formation)

Diabetes

High blood glucose

Physiological stress

Release of factors(e.g. VEGF)

Breakdown of junctions between cells

Cells divideCells become stickyCells move

Cells form tubes

Clapp C, 2009, Physiological Reviews

• Afilbercept (Eylea)• Ranibizumab (Lucentis)• Bevacizumab (Avastin)• Pegaptanib (Macugen)

Methods to study DR processes in the lab

Proliferative

Moderate

Fluid leak

Tube formation

+ VEGF

Cell migration

Chichger H, 2015, FASEB, 2016, AJRCMB

Paths

Tube

formation

Joints

paths

tubes

joints

Retinal microvascular endothelial cells

Endothelial cells

Matrigel

Fluorescent dextran

Endothelial cells

+ VEGF

Cell proliferation

-400

-350

-300

-250

-200

-150

-100

-50

0

De

cre

ase

in e

nd

oth

elia

l re

sist

ance

at

10

hr

(oh

ms)

- + - +- - + +

Sucralose:LPS:

*

*#

Harrington EO & Chichger H, 2017, AJP LungIP3, inositol triphosphate; PKC, protein kinase C; LPS. Lipopolysaccharide, *p<0.05 vs LPS vehicle, #p<0.05 vs Suc vehicle

Fluid leak

Healthy barrier

Ca2+PKC

IP3

Lung endothelial cells

Lessons from the lung – the sweet taste receptor

Sweet taste receptor

• Receptor which has remained unchangedthrough evolution - important

• Oral and extra-oral localisation

• Activated by high concentration glucose or lowconcentration of acutely-sweet artificialsweeteners

Behrens, Physiol Behav, 2011; Hass, Cell Tissue Res, 2010; Dyer J, Biochem SocTrans; 2005; Henquin, Sci Signal, 2012; Masubuchi, PloS One, 2013; Rozengurt, AJP

Gastro, 2006; Ren, Front Integr Neurosci, 2009; Foster, Plos One, 2013; Elliott, J Urol, 2011; Mosinger, PNAS, 2013; Simon, PloS One, 2014; Max, Nat Genet, 2001

Sweet taste molecules

Commercialname

Sweetmolecule

Sweet taste perception relative

to table sugar

Equal Cyclamate 30-50

Equal Aspartame 160-200

Sunett Acesulfame K 200

Sweet n Low Saccharin 300-450

Splenda Sucralose 600

NHDC Neohesperidin 1500 - 1800

Aclame Alitame 2000

E961 Neotame 7000-8000

Natural sweetmolecule

Sweet taste perception relative

to table sugar

Mabinlin 400

Pentadin 500

Brazzein 500

Monelin 800-1000

Thaumatin 3000

Does the sweet taste receptor play a role in fluid leak and new vessel formation in the retina?

+ Artificial sweeteners

+ lactisole

Saccharin (0.1 µM); Sucralose (0.1 µM)

Proliferative

Moderate

Fluid leak

Tube formation

+ VEGF

Cell migration

Chichger H, 2015, FASEB, 2016, AJRCMB

Paths

Tube

formation

Joints

paths

tubes

joints

Retinal microvascular endothelial cells

Endothelial cells

Matrigel

Fluorescent dextran

Endothelial cells

+ VEGF

Cell proliferationVEGF – vascular endothelial growth factor

Sucralose reduces VEGF-induced leak

VEGF: - -+ +Vehicle Sucralose

- -+ +Vehicle Sucralose

Vehicle Lactisole

* * *

0

2

4

6

8

10

12

14

16

% p

erm

ea

bili

ty

Fluid leak

Healthy barrier

Lizunkova P, 2019, Graefe's Clinical and Experimental Ophthalmology*p<0.05 vs vehicle for VEGF; n = 6

+ Artificial sweeteners

+ lactisole

Fluorescent dextran

Endothelial cells

- dependent on sweet taste receptor

+-VEGF

0 hr

6 hr

0

10

20

30

40

50

60

70

Cel

l Mig

rati

on

(µ

m/h

r)

* * *

VEGF: - -+ +Vehicle Saccharin

- -+ +Vehicle Saccharin

Vehicle Lactisole

*p<0.05 vs vehicle for VEGF; n = 4-6. x 10 magnification, scale bar 20 µm

+ Artificial sweeteners

+ lactisole

0

2

4

6

8

10

12

14

Nu

mb

er o

f tu

bes

* * *

VEGF: - -+ +Vehicle Sucralose

- -+ +Vehicle Sucralose

Vehicle Lactisole

Artificial sweeteners reduce angiogenesis

+-

VEGF

Lizunkova P, 2019, Graefe's Clinical and Experimental Ophthalmology

Src

RhoA

VEGFR2

VEGF

Akt

AdhesionProliferation

Migration

Tube formationProliferative (tube formation)

Diabetes

Mean ± SD, *p<0.05 vs vehicle for VEGF

*

#

# #

*

V e h i c l e V e h i c l eS u c r a lo s e S u c r a lo s e

Veh

icle

VE

GF

Veh

icle

VE

GF

Veh

icle

VE

GF

Veh

icle

VE

GF

0

5

1 0

1 5

2 0

Tu

be

fo

rm

ati

on

(nu

mb

er

of

tub

es

)

S C 7 9V e h ic le

*

V e h ic le V E G F V e h ic le V E G F

0 .0

0 .5

1 .0

1 .5

Ph

os

ph

ory

late

d A

kt

(ra

tio

of

un

ph

os

ph

ory

late

d A

kt)

S u c ra lo s eV e h ic le

:Sucralose

P-Akt

Akt

:VEGF- +- +

- - + +

T1R3

SC79 – Akt activator; Akt – protein kinase B; Src – kinase; VEGFR2 – VEGF receptor Lizunkova P, 2019, Graefe's Clinical and Experimental Ophthalmology

Src

VEGFR2

PROTECTION

T1R3

VEGF

Akt

AdhesionProliferation

Migration

Tube formation

Proliferative (tube formation)

Diabetes

???

PAK VE-cadherin

PI3K p110α

Pyk2 RhoA

P

Actin remodelling

Cell contact disassembly

MLC2

Moderate (fluid leak)

Cell junction

Suez, Nature, 2014

The controversy

If people with diabetes eat more Splenda, will it stop their proliferative retinopathy getting worse?

Artificial sweeteners in the diet –negative consequences

*

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A rtif ic ia l s w e e te n e r (µ M )

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(%

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S a c c h a r in

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Mean ± SEM, *p<0.05 vs 0 µM Invasion index = CFU/Caco2 cell # Steinberg, AJP Cell, 2006

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The future: The therapeutic implications of taste sensors in the microvasculature

Acknowledgements

• Prof Elizabeth Harrington

• Alex Vang

• Julie Braza

Related publications:

Thank you for your attention