Aspartate a New Treatment Modality in the treatment of Acute Liver Failure and Acute Pancreatitis

Ahmad Farooq, M.DMedical Resident ,

University at Buffalo, Catholic Health System .

Tissue Injury Triggers Innate Immune Responses

ProCaspase-1

Pro-IL-1 Pro-IL-18

IL-1 IL-18

Caspase-1

Neighboring CellInjured Parenchymal Cell DAMP Sensing Cell

TLRs

DAMPs

Nlrp3

IL-1IL-18independent components

ASC

Signal 1

What About Amino Acids and Fatty acids ?

Activation of the N‐methyl‐d‐aspartate receptor by aspartic acid downregulates inflammasome activity and 

liver inflammation via a b‐arrestin‐2 pathway

Step 2: Cleavage of CASP1 and IL-1b release

Step 1: Gene Transcription of pro-IL-1, Nlrp3, CASP1

LPS

plasma membrane

TLR4

ASP

NMDA

?

Aspartate downregulates inflammasome components and IL‐1b in mouse macrophages

0

4000

8000

LPS LPS+ Asp 1mMLPS+ Asp 5mM LPS+ Asp15mM

LPS+ Asp25mM

LPS+ Asp50mM

Relativ

e Expression

Pro IL 1

***

0

50

100

150

200

LPS LPS+ Asp 15mM

Rel

ativ

e E

xpre

ssio

n

Nlrp3

**

0

2

4

6

8

10

12

LPS LPS+ Asp15mM

Rel

ativ

e E

xpre

ssio

n

Pro caspase 1

*

0

200

400

Untreated LPS LPS+ 15mMAsp

pg/m

L

Supernatant IL 1

***

Caspase1 P10

Beta-actin

LPS+ATP LPS+ATP+Asp Unt

A

D

CB

E

0

15

30

45

LPS LPS+Asp15mM

Rel

ativ

e E

xpre

ssio

n

Pro IL 1

*

0

75

150

Untreated LPS LPS +AA15mM

pg/m

L

Supernatant IL 1

** *

0

3

5

LPS LPS+ Asp15mM

Rel

ativ

e E

xpre

ssio

n

Nlrp3

0

1

2

3

LPS LPS+Asp15mM

Rel

ativ

e E

xpre

ssio

n

Pro caspase-1

Mouse Kupffer cells

Human Peripheral Blood Mononuclear cells

0

20

40

LPS LPS+Asp

15mM

Rel

ativ

e E

xpre

ssio

n

Pro IL 1

**

0

3

5

LPS LPS+ Asp15mM

Rel

ativ

e E

xpre

ssio

n

Nlrp3

***

0

1

2

LPS LPS+ Asp15mM

Rel

ativ

e E

xpre

ssio

n

Pro caspase 1

*

A

F

C

E

DB

G

In vivo aspartate supplementation reduces hepatic inflammasome levels and protects from acute inflammatory liver injury.

0

125

250

LPS/Gal+DPBS

LPS/Gal+Asp

Rel

ativ

e Ex

pres

sion

Pro IL 1

**

0

40

80

LPS/Gal+DPBS

LPS/Gal+Asp

Rel

ativ

e Ex

pres

sion

Nlrp3

**

0

5

10

LPS/Gal+DPBS

LPS/Gal+Asp

Rel

ativ

e Ex

pres

sion

Pro caspase 1

**

0

10

20

Untreated Asp

mM

Serum Aspartic Acid

0

1500

3000

Untreated LPS/Gal+DPBS

LPS/Gal+Asp

pg/m

L

Serum IL 1

*

LPS/Gal + DPBS LPS/Gal + Asp

10x 10x

0

5,000

10,000

15,000

LPS/Gal+DPBS

LPS/Gal+ Asp

IU/L

Serum ALT

*0

1.8

3.6

LPS/Gal+ DPBS LPS/Gal +Asp

His

tolo

gy S

core

Hemorrhage

**

In vivo aspartate supplementation reduces pancreas inflammasome levels and protects from caerulein induced 

pancreatitis

0

750

1500

Untreated LPS/Cer+ DPBS

LPS/Cer+ Asp

U/L

Am

ylas

e

Serum Amylase

*

0

25

50

LPS/Cer+DPBS

LPS/Cer+Asp

Rel

ativ

e E

xpre

ssio

n

Pro IL 1

*

0

5

10

LPS/Cer+DPBS

LPS/Cer+Asp

Rel

ativ

e E

xpre

ssio

n

Nlrp3

*

0

5

10

LPS/Cer+DPBS

LPS/Cer+Asp

Rel

ativ

e E

xpre

ssio

n

Pro caspase 1

*

LPS/CER+ DPBS LPS/CER + Asp

10x 10x

0

2

3

LPS/Cer+DPBS

LPS/Cer+Asp

Inflammation

***

0

1

2

3

LPS/Cer+DPBS

LPS/Cer+Asp

Necrosis

0

2

4

LPS/Cer+DPBS

LPS/Cer+Asp

Edema

In vivo aspartate supplementation reduces liver inflammasome levels and protects from acetaminophen induced liver failure.

0

5

10

APAP+ DPBS APAP+ Asp

Rel

ativ

e Ex

pres

sion

Pro IL 1

*

0

3

5

APAP+ DPBS APAP+ Asp

Rel

ativ

e Ex

pres

sion

Nlrp3

*

0

1

2

3

APAP+ DPBS APAP+ Asp

Rel

ativ

e Ex

pres

sion

Pro caspase-1

0

8000

16000

APAP+DPBS

APAP+Asp

IU/L

Serum ALT

**

0

1

2

3

4

APAP+ DPBS APAP+ Asp

His

tolo

gy S

core

Hemorrhage

***

10x 10x

0

1.5

3

APAP+ DPBS APAP+ Asp

Necrosis

***

APAP+ DPBS APAP + Asp

Aspartate mediated suppression of TLR4 signaling requiresthe plasma membrane receptor NMDA, and ARRB2

0

150

300

Rel

ativ

e Ex

pres

sion

Nr2a Expression

0

4

8

12

SiRNAScramble

SiRNA NR2

Rel

ativ

e E

xpre

ssio

n

Nr2a Expression

*

0

35

70

LPS LPS+Asp15mM

Untreated LPS LPS+Asp15mM

Untreated

Rel

ativ

e E

xpre

ssio

n

Pro IL 1 SiRNA Scramble SiRNA NR2a

*

NS

0

2

3

SiRNAScramble

SiRNA Arrb2

Rel

ativ

e Ex

pres

sion

Arrb2 Expression

***

‐10

40

90

LPS LPS+Asp15mM

Untreated LPS LPS+Asp15mM

Untreated

Pro IL 1SiRNA Scramble SiRNA Arrb2

*NS

P Ikk

actin

LPS LPS+Asp Untreated

A

G

FED

CB

‐arrestin‐2 induced immune regulation is providing a significant degree ofdown‐regulation of the inflammatory response,and supplementation withaspartate acid can further increase this protective effect

0

300

600

Kuffer Cells Stellate Cells HepatocytesRel

ativ

e E

xpre

ssio

n

Arrb2 Expression

05101520

SiRNA Scramble SiRNA Arrb2Rel

ativ

e E

xpre

ssio

n

Arrb2 Expression*

060120180240

Rel

ativ

e E

xpre

ssio

n

Pro IL 1SiRNA Scramble SiRNA Arrb2

0

1250

2500

Pg/

mL

Serum IL 1 SiRNA Scramble SiRNA Arrb2

*

NSNS

siR

NA

S

cram

ble

siR

NA

A

rrB

2

LPS/Gal+ DPBS LPS/Gal +Asp

0

12500

25000

IU/m

LSerum ALT

*

NS

BA

E

D

F

C

Conclusion

• Therapeutic potential of aspartate mediated NMDA signaling in limiting TLR4 driven inflammation in acute pancreatitis.  

• NMDA as a novel target and highlight selective NMDA agonists and antagonist in clinical use in the treatment of acute inflammation 

• Ethanol has been proposed to be a non‐competitive antagonist of the NMDA receptor in neuron. If the same effect is present in macrophages it will result in loss of NMDA induced down regulation of inflammasome activity and may provide another mechanism of the hepatic immune dysregulation induced by ethanol in alcoholic pancreatitis. 

• In addition use of TPN enriched with glutamate and aspartate has been shown to be associated with reduced inflammation in inflammatory bowel disease.

• Of great interest is the further possibility that the general immunosuppression seen with TPN may be due to activation of this pathway.

Thank you Dr H Woodman Dr Khalid QaziDr MehalDr GorelickDr Hoque

LPS