P t ti f Gl li f H h t i i I d d I j i A id S hi liProtection of Glomeruli from Hyperhomocysteinemia Induced Injury in Acid SphingomyelinaseProtection of Glomeruli from Hyperhomocysteinemia-Induced Injury in Acid SphingomyelinaseProtection of Glomeruli from Hyperhomocysteinemia Induced Injury in Acid Sphingomyelinase yp y j y p g yG K k t MiGene Knockout MiceGene Knockout MiceGene Knockout Mice

K i h M B i i Ch Zh Mi Xi d Pi L LiKrishna M Boini Chun Zhang Min Xia and Pin Lan LiKrishna M. Boini, Chun Zhang, Min Xia and Pin-Lan LiKrishna M. Boini, Chun Zhang, Min Xia and Pin Lan Li , g,f i i i C i i i C i A 23298Department of Pharmacology Virginia Commonwealth University Medical Center Richmond VA 23298Department of Pharmacology, Virginia Commonwealth University Medical Center, Richmond, VA 23298Department of Pharmacology, Virginia Commonwealth University Medical Center, Richmond, VA 23298p gy, g y , ,

ABSTRACT BACKGROUND 3 2AA BB Desmin PodocinABSTRACT BACKGROUND 3.2 **AA BB Desmin PodocinABSTRACT BACKGROUND

DI)

N l Di Normal Diet FF diet ND FF dietGDNormal Diet FF Diet Normal Diet FF diet ND FF diet

x (G

2.4ex

C id h b d i i i l l id i i j i l li d diff H h t i i i k iti l th i f t i th i f d ndCeramide has been reported to initiate local oxidative injury in glomeruli under different Hyperhomocysteinemia is known as a critical pathogenic factor in the progression of end e InAsm+/+Ceramide has been reported to initiate local oxidative injury in glomeruli under different

th l i l diti Th t t d f d t l th l f idyp y p g p gstage renal disease (ESRD) and in the development of cardiovascular complications ge ##pathological conditions. The present study was performed to explore the role of acid stage renal disease (ESRD) and in the development of cardiovascular complications Asm+/+ Asm+/+1.6m

ag ##p g p y p phi li (ASM) i th d l t f h h t i i (hH ) i d d

g ( ) p prelated to ESRD

1.6

amsphingomyelinase (ASM) in the development of hyperhomocysteinemia (hHcys)-induced related to ESRD. Dap g y ( ) p yp y ( y )

glomerular injury Uninephrectomized ASM knockout ( -/-) and wild type ( +/+) ar

glomerular injury. Uninephrectomized ASM knockout (asm-/-) and wild type (asm+/+) 0 8ula

Asm /g j y p ( ) yp ( )mice were fed a folate free (FF) diet or normal chow for 8 weeks to produce hHcys Recently we demonstrated that ceramide importantly contributes to the development of

0.8

eruAsm-/-

mice were fed a folate free (FF) diet or normal chow for 8 weeks to produce hHcys. Recently we demonstrated that ceramide importantly contributes to the development of me

HPLC analysis revealed that plasma homocysteine (Hcy) was significantly elevated in chronic glomerular injury associated with hyperhomocysteinemia and thereby ceramide loHPLC analysis revealed that plasma homocysteine (Hcy) was significantly elevated in chronic glomerular injury associated with hyperhomocysteinemia and thereby ceramidei h i f d l di hibi f d Asm-/- Asm-/-0G

both asm-/- and asm+/+ mice However the plasma Hcy levels were significantly may serve as an important mechanism of end-stage renal disease. Inhibitors of de novo Asm / Asm-/-0N l Di t FF Di tboth asm and asm mice. However, the plasma Hcy levels were significantly may serve as an important mechanism of end stage renal disease. Inhibitors of de novo

h i f id d L h i i d d id f i iNormal Diet FF Diet

attenuated in asm-/- than in asm+/+ mice FF diet significantly increased the renal ASMase synthesis of ceramide prevented L-homocysteine-induced ceramide formation inattenuated in asm than in asm mice. FF diet significantly increased the renal ASMase/ /

y p yi l ll ll i i i th kid d tt t d l l i j d Asm+/+

mRNA expression and ASMase activity in asm+/+ mice than in asm-/- mice. mesangial cells as well as in vivo in the kidney and attenuated glomerular injury and Asm-/-Asm

mRNA expression and ASMase activity in asm mice than in asm mice.M h l i l i i h d h FF di i d d f d i j i l li f

g y g j yt i i Th h d id d ti h NADPH id ti it d

Asm /

Morphological examinations showed that FF diet induced profound injury in glomeruli of proteinuria. The enhanced ceramide production enhances NADPH oxidase activity andFi 6 I hi t h i l l i d t t th t d t i jo p o og ca e a at o s s owed t at d et duced p o ou d ju y g o e u o

+/+ i hi h k dl tt t d i / i (th l l d i dp p ygenerates superoxide production and ultimately causes glomerular injury (1) Fig. 6: Immunohistochemical analysis demonstrate that podocyte injury wasasm+/+ mice which was markedly attenuated in asm-/- mice (the glomerular damage index generates superoxide production and ultimately causes glomerular injury (1). g y p y j y

tt t d i / i T i l i f d i (l ft l) d i ( i hty ( g g(GDI) i +/+ 2 95± 0 09 / 1 51 ± 0 11) Th d d l l i j Fig 3: Attenuation of glomerular injury in asm-/- mice Photomicrographs (panel A) show attenuated in asm-/- mice. Typical images of desmin (left panel) or podocin (right(GDI) in asm+/+: 2.95± 0.09 vs. asm-/-: 1.51 ± 0.11). The decreased glomerular injury Fig. 3: Attenuation of glomerular injury in asm mice. Photomicrographs (panel A) show yp g ( p ) p ( g

panel) staining in glomeruli from asm-/- and asm+/+ mice fed a normal (ND) or( ) ) g j yin asm-/- mice was accompanied by attenuated proteinuria associated with hHcy Ceramide production is mediated by the hydrolysis of membrane sphingomyelin by acid typical glomerular structure in normal (ND) or folate free diet (FF) fed asm-/- and asm+/+ mice panel) staining in glomeruli from asm / and asm+/+ mice fed a normal (ND) orin asm / mice was accompanied by attenuated proteinuria associated with hHcy. Ceramide production is mediated by the hydrolysis of membrane sphingomyelin by acid typical glomerular structure in normal (ND) or folate free diet (FF) fed asm and asm mice.

folate free diet (FF Diet) treatment FF diet treatment increased the desminImmunohistochemical analysis showed that FF diet decreased expression of podocin but sphingomyelinase (ASMase) or neutral sphingomyelinase (NSMase) or by de novo Panel B depicts semi- quantitative score of glomerular damage index (n=6). Morphological folate free diet (FF Diet) treatment. FF diet treatment increased the desminImmunohistochemical analysis showed that FF diet decreased expression of podocin, but sphingomyelinase (ASMase) or neutral sphingomyelinase (NSMase) or by de novo Panel B depicts semi quantitative score of glomerular damage index (n 6). Morphologicall i h d h / i h d l l l i j d h +/+ i f d staining and decreases the podocin staining in asm+/+ than in asm-/- mice Theseincreased desmin and ceramide levels in glomeruli of asm+/+ mice than in asm-/- mice synthesis via serine palmitoyltransferase (SPT) and ceramide synthase. Ceramide is analysis showed that asm-/- mice had less glomerular injury compared to the asm+/+ mice fed a staining and decreases the podocin staining in asm than in asm mice. Theseincreased desmin and ceramide levels in glomeruli of asm mice than in asm mice. synthesis via serine palmitoyltransferase (SPT) and ceramide synthase. Ceramide is

b l b li d i hi i b id d hi i ba a ys s s owed t at asm ce ad ess g o e u a ju y co pa ed to t e asm ce ed aFF di t Th l l d i d i il i b th / d +/+ i l di t f d data reveal that FF diet induced podocyte injury was attenuated in asm-/- miceElectron spin resonance analysis demonstrated that FF diet significantly increased the subsequently metabolized into sphingosine by ceramidases, and sphingosine can be FF diet. The glomerular damage index was similar in both asm-/- and asm+/+ in normal diet fed data reveal that FF diet induced podocyte injury was attenuated in asm mice

/Electron spin resonance analysis demonstrated that FF diet significantly increased the/

subsequently metabolized into sphingosine by ceramidases, and sphingosine can bef th t d t S1P i hi i ki C id i t d i t

g gi A / i i ifi tl tt t d th hH i d d GDI d t th +/+ i than in asm+/+ miceNADPH oxidase-dependent superoxide (O2.-) production in the kidneys of asm+/+ than in further converted to S1P via sphingosine kinase. Ceramide is generated in response to mice. Asm-/- mice significantly attenuated the hHcy-induced GDI compared to the asm+/+ mice. than in asm mice.NADPH oxidase dependent superoxide (O2 ) production in the kidneys of asm than in

/ i l i h b i l i l l f AS i h hp g g p

i t f di t i l di i fl t t ki id ti t d i dg y y p

* significant difference (P<0 05) from normal diet # significant difference (P<0 05) fromasm-/- mice. In conclusion, the observations reveal a pivotal role of ASM in the hHcy- variety of mediators including proinflammatory cytokines, oxidative stress, and increased * significant difference (P<0.05) from normal diet, # significant difference (P<0.05) from3 **asm mice. In conclusion, the observations reveal a pivotal role of ASM in the hHcy

i d d l l i j d ASM b th ti t t f t t ty g p y y , ,

levels of free fatty acids asm+/+ 3 **induced glomerular injury and ASMase may be a therapeutic target for treatment or levels of free fatty acids. asm / .g j y y p g

ti f l l l i i t d ith hH ( t d b NIH t HLy

2 5t)prevention of glomerulosclerosis associated with hHcys (supported by NIH grants HL- 2.5ietp g y ( pp y g

091464 HL 75316 and DK54927) However nothing is known about the hyperhomocysteinemia induced glomerular injury l d091464, HL-75316 and DK54927). However, nothing is known about the hyperhomocysteinemia-induced glomerular injury 2on

mal)

on mice lacking the acid sphingomyelinase gene Hence the present study was tio rmon mice lacking the acid sphingomyelinase gene. Hence, the present study was1 5uc

tN

or

##performed to explore the role of acid sphingomyelinase (ASM) in the development of 1.5

odu

T N ##

A +/+performed to explore the role of acid sphingomyelinase (ASM) in the development ofh h i i (h ) i d d l l i j

400 ro WT Asm+/+

METHODS hyperhomocysteinemia (hHcys)-induced glomerular injury.400

n) * 1-p s W Asm-/-METHODS hyperhomocysteinemia (hHcys) induced glomerular injury.

350ein 1

O2. - vsMETHODS 350ot

e O ld

pro

0.5Fol

RESULTS300g

p (F

RESULTS mg # 0RESULTS 250g/ 0RESULTS 250

(n Normal Diet FF Diet

A i l Ei ht k ld i h t i d l ASM i d i th t Asm+/+200c.

Animals: Eight weeks old uninephrectomized male ASM mice were used in the presentA /Asm+/+200

oncg p p

st d (Jackson Laboratories Bar Harbor Maine) Mice ere fed either a normal dietAsm-/-costudy (Jackson Laboratories, Bar Harbor, Maine). Mice were fed either a normal diet Fig 7: Effects of normal and FF Diet on the glomerular O .- production in150de

y ( )(Normal Diet Dyets Inc Bethlehem PA) or a folate free diet (FF diet Dyets Inc

Fig. 7: Effects of normal and FF Diet on the glomerular O2 production in150

mid50(Normal Diet, Dyets Inc, Bethlehem, PA) or a folate free diet (FF diet, Dyets Inc, asm-/- and asm+/+ mice NADPH dependent O2.- production was measured by100am**

Bethlehem PA) for 8 weeksasm and asm mice. NADPH dependent O2 production was measured by

/100

era

Bethlehem, PA) for 8 weeks. ESR spectrometry. Summarized data demonstrated that asm-/- miceCe

40M) ESR spectrometry. Summarized data demonstrated that asm mice

i ifi l d h O d i h i +/+ i f d i h FF50

40

µM significantly attenuated the O2.- production than in asm+/+ mice fed with a FFNormal Diet FF Diet (µ

Morphological examinations. The fixed kidneys were paraffin-embedded sections weresignificantly attenuated the O2 production than in asm mice fed with a FFdi t ti th t / i l l id ti t i th kid

Normal Diet FF Diet30ve

lMorphological examinations. The fixed kidneys were paraffin embedded, sections were diet suggesting that asm-/- mice suppresses local oxidative stress in the kidney.30ev

prepared and stained with periodic acid–Schiff stain. Glomerular injury index wasgg g pp y

* i ifi t diff (P<0 05) f l di t # i ifi t diffy l

## Asm+/+prepared and stained with periodic acid Schiff stain. Glomerular injury index wasl l d f 0 4 h b i f h d f l l l i d i l

* significant difference (P<0.05) from normal diet, # significant difference20H

cy ##/

Asm+/+

calculated from 0 to 4 on the basis of the degree of glomerulosclerosis and mesangialg ( ) , g

(P<0 05) from asm+/+20

a H Asm-/-ca cu ated o 0 to o t e bas s o t e deg ee o g o e u osc e os s a d esa g a

t i i d ib d i l I l t d t t l f 80 100(P<0.05) from asm+/+ .

Fi 4 Eff t f l di t d FF Di t id d ti i th l li i +/+ma

matrix expansion as described previously. In general, we counted a total of 80-100 Fig. 4: Effects of normal diet and FF Diet on ceramide production in the glomeruli in asm+/+asmp p y g ,

l li i h kid li d i h h l l d d l lg p g

and asm-/- mice To explore whether the asm-/- mice attenuates the FF induced ceramide10Pla

glomeruli in each kidney slice under microscope, when each glomerulus was graded level and asm / mice. To explore whether the asm / mice attenuates the FF induced ceramidePg y p , g g0 4 damages 0 represents no lesion 1+ represents sclerosis of <25% of the glomerulus production Using LC/MS spectroscopy we determined the total ceramide concentrations in0-4 damages. 0 represents no lesion, 1+ represents sclerosis of <25% of the glomerulus,

CONCLUSIONproduction. Using LC/MS spectroscopy we determined the total ceramide concentrations in0

while 2+ 3+ and 4+ represent sclerosis of 25% to 50% >50% to 75% and >75% of the CONCLUSIONrenal tissues The total ceramide concentration was similar in both asm+/+ and asm-/- mice fed a0

Normal Diet FF Dietwhile 2+, 3+, and 4+ represent sclerosis of 25% to 50%, >50% to 75%, and >75% of the CONCLUSIONrenal tissues. The total ceramide concentration was similar in both asm and asm mice fed aNormal Diet FF Diet

glomerulus A whole kidney average sclerosis index was obtained by averaging scoresCONC US ON

normal diet However FF diet treatment significantly enhanced the renal ceramide productionglomerulus. A whole kidney average sclerosis index was obtained by averaging scoresFi 1 Pl H i d i +/+ d / i f d i h

normal diet. However, FF diet treatment significantly enhanced the renal ceramide production/ /from counted glomeruli The immunohistochemical analysis of renal tissue was Fig. 1: Plasma Hcy concentrations were measured in asm+/+ and asm-/- mice fed with or in asm+/+ than in asm-/- mice * significant difference (P<0 05) from normal diet,from counted glomeruli. The immunohistochemical analysis of renal tissue was Fig. 1: Plasma Hcy concentrations were measured in asm and asm mice fed with or

ith t f l t f di t i HPLC Pl H t ti i il i b thin asm than in asm mice. significant difference (P<0.05) from normal diet,# i ifi diff (P 0 05) f +/+performed to detect podocin and desmin staining using anti-podocin (Sigma 1: 50 The observations reveal a pivotal role of ASM in the hHcy-inducedwithout folate free diet using HPLC. Plasma Hcy concentration was similar in both # significant difference (P<0.05) from asm+/+.performed to detect podocin and desmin staining using anti podocin (Sigma, 1: 50

dil i ) d i d i (Ab 1 0 dil i ) ib diThe observations reveal a pivotal role of ASM in the hHcy-inducedg y

+/+ d / i f d l di t FF di t t t t i ifi tl i d thsignificant difference (P 0.05) from asm .

dilution) and anti-desmin (Abcam, 1: 50 dilution) antibodies. glomerular injury and ASMase may be a therapeutic target for treatmentasm+/+ and asm-/- mice fed a normal diet. FF diet treatment significantly increased thedilution) and anti desmin (Abcam, 1: 50 dilution) antibodies. glomerular injury and ASMase may be a therapeutic target for treatmentg yplasma Hcy levels in both asm+/+ and asm-/- mice compared to the normal diet fed mice AA BB or prevention of glomerulosclerosis associated with hHcys.plasma Hcy levels in both asm+/+ and asm / mice compared to the normal diet fed mice. AA BB

ESR D t ti f S id (O ) P d ti H t f l ti lor prevention of glomerulosclerosis associated with hHcys.

However the plasma Hcy levels were significantly attenuated in asm-/- than in asm+/+ESR Detection of Superoxide (O2.−) Production. Homogenates from renal cortical However the plasma Hcy levels were significantly attenuated in asm / than in asm /

2.4* 0 08e **p ( 2 ) g

tiss es ere prepared b sing s crose b ffer and res spended ith defero imine (25 mice * significant difference (P<0 05) from normal diet # significant difference on * 0.08

ate

n)tissues were prepared by using sucrose buffer and resuspended with deferoximine (25 mice. significant difference (P<0.05) from normal diet, significant difference 2sio ra einp p y g p (

µmol/L metal chelator) and resuspended with modified Kreb's Hepes buffer containing (P<0 05) from asm+/+ ess

on

ote

µmol/L, metal chelator) and resuspended with modified Kreb s–Hepes buffer containing (P<0.05) from asm .1 6pr

eol

)

0 06tio pro REFERENCESdeferoximine (100 μM) and diethyldithiocarbamate (5 μM) The NADPH oxidase

1.6

xp tro 0.06

ma

n p REFERENCES**deferoximine (100 μM) and diethyldithiocarbamate (5 μM). The NADPH oxidase-

1 2Aex on

t

orm

min ##0 49

REFERENCES25 **

dependent O .− production was examined by addition of 1 mM NADPH as a substrate in1.2

NA co

#

fo g/m ##0.49

*25

dependent O2 production was examined by addition of 1 mM NADPH as a substrate in0 8R

Nvs

* #0 04ne

m

g **0 42*

50 μg protein and incubated for 15 min at 37°C in the presence or absence of SOD (200 0.8mR (v 0.04lin e/m0.42

2050 μg protein and incubated for 15 min at 37 C in the presence or absence of SOD (2000 4M

m

ho ole20) Asm+/+

U/ml), and then supplied with 1 mM O2.− specific spin trap 1-hydroxy-3- 0.4SM ch mo

0.35h) 1 Yi F Zhang AY Li N Muh RW Fillet M Renert AF Li PL Inhibition ofW)

Asm-/-U/ml), and then supplied with 1 mM O2 specific spin trap 1 hydroxy 3h b l 2 2 5 5 h l lidi (CMH Si S L i MO USA)

A 4C

(nm0.35

4h 1. Yi F, Zhang AY, Li N, Muh RW, Fillet M, Renert AF, Li PL. Inhibition of1

BW Asm

methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH, Sigma, St. Louis, MO, USA). 0 0.0214(

0 28g/2 ceramide-redox signaling pathway blocks glomerular injury in15/g et o yca bo y , ,5,5 tet a et y py o d e (C , S g a, St. ou s, O, US ).

Th i t l d d i l ill i d i di t l l d f O d ti Normal Diet FF Diet Normal Diet FF Diet0.28

mg ceramide redox signaling pathway blocks glomerular injury in

4h/

#The mixture was loaded in glass capillaries and immediately analyzed for O2.− productionNormal Diet FF Diet Normal Diet FF Diet

0 21

(m hyperhomocysteinemic rats. Kidney Int. 70: 88-96, 2006./24 #g p y y 2 p

ki ti ll f 10 i0.21

ein # hyperhomocysteinemic rats. Kidney Int. 70: 88 96, 2006.

2 Yi F Zh AY J h JL Li PL d Z AP H i i10µg/

kinetically for 10 min. Asm+/+rote 2. Yi F, Zhang AY, Janscha JL, Li PL, and Zou AP. Homocysteine activates10(µy

A /Asm+/+

0.14Up 2. Yi F, Zhang AY, Janscha JL, Li PL, and Zou AP. Homocysteine activates

NADH/NADPH id th h id ti l t d R GTP ti itmin

Asm-/-U NADH/NADPH oxidase through ceramide-stimulated Rac GTPase activity5bum

Biochemical Analysis Plasma concentrations of hcy were determined using HPLC 0 07g y

i t i l ll Kid I t 66 1977 1987 20045

UA

lb

Biochemical Analysis. Plasma concentrations of hcy were determined using HPLCFi 5 ASM k k t ( / ) i tt t d th FF i d d ASM RNA d ASM

0.07 in rat mesangial cells. Kidney Int 66: 1977–1987, 2004U

method Renal ceramide concentrations were determined using LC/MS spectroscopy as Fig. 5: ASM knockout (asm-/- ) mice attenuated the FF induced ASMase mRNA and ASMase0g y ,

3 Zhang AY Yi F Jin S Xia M Chen QZ Gulbins E Li PL Acid0method. Renal ceramide concentrations were determined using LC/MS spectroscopy as g ( )ti it Th ASM RNA d ASM ti it i il i b th +/+ d / i

0 3. Zhang AY, Yi F, Jin S, Xia M, Chen QZ, Gulbins E, Li PL. Acid0we described previously (2) activity. The ASMase mRNA and ASMase activity was similar in both asm+/+ and asm-/- miceNormal Diet FF Diet sphingomyelinase and its redox amplification in formation of lipid raftNormal Diet FF Dietwe described previously (2). y y

fed a normal diet FF Diet treatment significantly increased the ASMase mRNA (A) andNormal Diet FF Diet sphingomyelinase and its redox amplification in formation of lipid raftNormal Diet FF Diet

fed a normal diet. FF Diet treatment significantly increased the ASMase mRNA (A) and redox signaling platforms in endothelial cells Antioxid Redox Signal 9: 817A i i i i i h i i f hi li d i d ASMase activity (B) in asm+/+ mice compared to the asm+/+ mice fed a normal diet The asm-/-Fig 2: ASM knockout attenuated the FF diet induced urinary total protein and albumin

redox signaling platforms in endothelial cells, Antioxid Redox Signal 9: 817-Acid sphingomyelinase activity: The activity of sphingomyelinase was determined as ASMase activity (B) in asm / mice compared to the asm / mice fed a normal diet. The asm /Fig. 2: ASM knockout attenuated the FF diet induced urinary total protein and albumin 828 2007Acid sphingomyelinase activity: The activity of sphingomyelinase was determined as

d ib d i l Th 14C h li h h f i l l d mice significantly attenuated the FF diet induced ASMase mRNA and ASM activity Valuesexcretion In parallel to the elevation of plasma Hcys level the 24-hour urinary total828, 2007.

we described previously. The 14C-choline phosphate formation rate was calculated to mice significantly attenuated the FF diet induced ASMase mRNA and ASM activity. Valuesexcretion. In parallel to the elevation of plasma Hcys level, the 24-hour urinary total 4 Zhang C Hu JJ Xia M Boini KM Brimson C and Li PL Redox signalingp y p pt th ti it (3) are arithmetic means ± SEM (n=3-4 each group) of ASMase mRNA and ASMase activityprotein and albumin excretions were significantly higher in both asm+/+ and asm-/- mice

4. Zhang C, Hu JJ, Xia M, Boini KM, Brimson C, and Li PL. Redox signalingi li id f l i i h i i d d i j f drepresent the enzyme activity (3). are arithmetic means ± SEM (n 3-4 each group) of ASMase mRNA and ASMase activity.

#protein and albumin excretions were significantly higher in both asm and asm mice via lipid raft clustering in homocysteine-induced injury of podocytes.p y y ( )

* significant difference (P<0 05) from normal diet # significant difference (P<0 05) fromfed a FF diet However the urinary total protein and albumin excretion wasvia lipid raft clustering in homocysteine induced injury of podocytes.Bi hi Bi h A 1803 482 491 2010significant difference (P<0.05) from normal diet, significant difference (P<0.05) from

+/+fed a FF diet. However, the urinary total protein and albumin excretion wasi ifi l d i / i h i +/+ i * i ifi diff

Biochim Biophys Acta 1803: 482-491, 2010a.Real time reverse transcription polymerase chain reaction (RT PCR): RT PCR asm+/+ .significantly attenuated in asm-/- mice than in asm+/+ mice. * significant difference

p y ,Real-time reverse transcription polymerase chain reaction (RT-PCR): RT-PCR asm .significantly attenuated in asm mice than in asm mice. significant difference

(P 0 05) f l di t # i ifi t diff (P 0 05) f +/+experiments were performed as described previously (4) (P<0.05) from normal diet, # significant difference (P<0.05) from asm+/+ .experiments were performed as described previously (4). ( ) , g ( )