Acetate alters adenosine metabolism in BV-2 microglia cells

Grace E. Postma, Dhaval P. Bhatt, and Thad A. Rosenberger

Department of Pharmacology, Physiology, and Therapeutics, chool of !edicine and "ealth ciences, Grand #or$s, %D, &'()* .

Abstract
Acetate supplementation increases rat plasma acetate levels, brain acetyl-CoA, and reduces activation of neuroglia in a rat model of neuroinflammation. Since adenosine is a known endogenous anti-inflammatory agent we postulated that the anti-inflammatory effect of acetate may be due to an increase in adenosine (AD ! metabolism. "o begin to test this hypothesis we investigated the effect of acetate in presence and absence of inflammation on AD metabolism in #$-% microglia cultures. &e treated cultures with sodium chloride ('aCl, ()m*!, sodium acetate ('aAc, ()m*!, 'aCl plus a bacterial lipopolysaccharide (+,S, (-g.m+! which induces inflammation, and 'aAc plus +,S for (%, %/, and /0 hours. 1ollowing the treatment cell lysates were collected and en2ymes involved in the formation 3ecto-45-nucleotidase (CD67!, ecto-apyrase (CD78!9 and removal 3AD deaminase (ADA!, AD kinase (A:!9 of AD as well as the adenosine receptors A( and A%A were analy2ed using western blot analysis. &e found that after /0 hours, acetate prevents the increase in e;pression of CD67 and A: en2ymes, and A%A receptor that are increased by +,S to %, %, and (.84 fold over control levels, respectively. 1urther, e;pression of ADA was increased (.7 fold with +,S and was reduced to ).4 fold below control levels with acetate and +,S. "hese results suggest that acetate modulates adenosine metabolism in #$-% microglia cells and can potentially contribute towards the antiinflammatory effect of acetate supplementation. 1uture studies will measure absolute adenosine levels in #$-% cultures using high performance li<uid chromatography.

Results
Acetate pre ents !"#-induced e$pression o% en&ymes in ol ed in adenosine remo al Acetate modulates e$pression o% en&ymes in ol ed in adenosine %ormation in presence o% !"# Acetate alters adenosine receptors e$pression in presence o% !"#

b b ab

Background
'euroinflammation is a common feature found in Al2heimer=s, ,arkinson, >untington, amyotrophic lateral sclerosis, and multiple sclerosis which afflicts appro;imately 6 million Americans.(,%,7 Acetate supplementation (glyceryl triacetate (?"A!, @g.kg by oral gavage! for %0 days reduces the markers of inflammation, which are activated microglia and astrocyte cells./ ?"A increases rat brain acetate and acetyl-CoA levels./,4 A single dose of ?"A after / hr of treatment increased brain phosphocreatine levels and reduced A*, levels
(unpublished results!.

a b

ab

Adenosine is a known endogenous anti-inflammatory agent. and a metabolic product of adenine nucleotides.

Hypothesis:

Acetate modulates adenosine metabolism in BV-2 microglia cells.

'igure (: B;pression of en2ymes involved in adenosine removal in #$-% microglia after /0 hr treatment. AbbreviationsF A:, adenosine kinaseI ADA, adenosine deaminaseI 'aCl, sodium chlorideI 'aAc, sodium acetateI +,S, lipopolysaccharide

'igure 2: B;pression of en2ymes involved in adenosine formation in #$-% microglia after /0 hr treatment. AbbreviationsF CD67, ecto-45nucleotidaseI CD78, ecto-apyraseI 'aCl, sodium chlorideI 'aAc, sodium acetateI +,S, lipopolysaccharide.

'igure ): B;pression of adenosine receptors in #$-% microglia after /0 hr treatment. AbbreviationsF A(A, adenosine A( receptorI A%AA, adenosine A%A receptorI 'aCl, sodium chlorideI 'aAc, sodium acetateI +,S, lipopolysaccharide.

Statistical analysis was performed using one way A' $A followed by "ukey5s post hoc test (nL 4!. a, statistically significant difference from control group ('aCl!I and b, statistical significance from inflammation group ('aClM+,S!3p N ).)4 9.

Conclusions
Abbre iations: A:, adenosine kinaseI ADA, adenosine deaminaseI CD67, ecto-45-nucleotidaseI CD78, ectoapyrase, A(A, adenosine A( receptorI A%AA, adenosine A%A receptor

Re%erences
+. %. ,orld report on violence and health- summary. ?eneva, &orld >ealth rgani2ation, %))%. &ilhelmsen :C, 1orman *S, Aosen >J, et al. (6<-+inked 1rontotemporal DementiaKAmyotrophic +ateral Sclerosis &ithout "au *utations &ith "au and G-Synuclein Dnclusions. Arch %eurol. %))/I@((7!F780-/)@. >ui-*ing ?ao, Jau-Shyong >ong, &hy neurodegenerative diseases are progressiveF uncontrolled inflammation drives disease progression. Trends in .mmunology. %))0I%8(0!F746-7@4. Aeisenauer CJ, #hatt, D,, *itteness DJ, et al. Acetate supplementation attenuates lipopolysaccharideinduced neuroinflammation. / %eurochem. %)((I((6F%@/-%6/ *athew A, Arun ,, *adhavarao C', et al. ,rogress toward Acetate Supplementation "herapy for Canavan DiseaseF ?lyceryl "riacetate Administration Dncreases Acetate, but 'ot %-Acetylaspartate, +evels in #rain. /PET. %))4I7(4((!F%86-7)7

Methods
G

+,S increases en2ymes involved in the removal and formation of adenosine, A:, ADA, and CD67, while acetate reduces those levels to control (A:, CD67! or below control (ADA!. +,S increases adenosine A%A receptor e;pression but acetate reduces it back to control levels.

Cell culture and acetate treatment: #$-% microglia (immortali2ed murine cell line! were cultured and treated with sodium chloride ('aCl, () m*!, sodium acetate ('aAc, ()m*!, 'aCl and lipopolysaccharide (+,S, (-g.m+! which induces inflammation, and 'aAc plus +,S for (%, %/ and /0 hrs. #$-% microglia were collected in AD,A buffer containing protease and phosphatase inhibitors. Soluble protein supernatant was collected after sonication and centrifugation. #radford assay was performed to determine protein concentration and loading samples were prepared in loading buffer (84E +aemmli and 4E beta-mercaptoethanol!. Western blotting analysis: 4) -g.well protein was loaded and separated using SDS-,A?B (4E. ?els were then transferred to nitrocellulose membranes and blocked with 4E milk in ""#S ("ween tris-buffered saline!. #lots were incubated overnight with primary antibodiesF goat anti human anti-CD67, A(A, AD:, and ADA (goat anti-human, (F()))!, CD78 (goat anti-mouse,(F()))!, A%A-A (mouse anti-human, (F()))!. +oading control was G-tubulin (primaryF mouse anti-porcine, (F7))) and secondaryF goat anti-mouse, (F4)))!. "he secondary antibodies for CD67, CD78, A(A, AD:, and ADA (donkey anti-goat!, A%AA (goat anti-mouse!, (F4))). ,rotein bands were visuali2ed with a SuperSignalH &est ,ico and.or 1emto Chemiluminescent Substrate (,ierce, Aockford, D+! using a C$, #ioimaging System (Cpland, CA!. Dmage capturing and analysis was performed with +ab&orks"* imaging software (version /.4, Cpland, CA!. ptical density obtained for all proteins were normali2ed to G-tubulin optical densities (loading control! and data is e;pressed as fold change over control levels.

7. Acetate reduces adenosine A( receptors below control levels in presence of +,S. /. "hese data support our hypothesis that acetate can modulate adenosine metabolism but we need to measure the activity of these en2ymes and measure absolute adenosine levels to completely test the hypothesis. 4.

'uture +irections
"o determine en2yme activity of adenosine metaboli2ing en2ymes in #$-% microglia. "o determine e;pression and activity of adenosine metaboli2ing en2ymes, and e;pression of adenosine receptors in primary astrocyte cultures. "o determine e;tracellular adenosine levels in #$-% microglia and primary astrocyte cultures.

Ackno*ledgments
"his study was supported by 'S1 ABC Site ?rant )04(0@8 and 'D> ?rant ,%)?*()7//%.,%)AA)(@/6(.