福島県立医科大学 学術機関リポジトリ
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Title Liver-specific G0S2 expression exacerbates hepatic insulin resistance in high fat induced insulin resistance rats( 本文 )
Author(s) 菅谷, 芳幸
Citation
Issue Date 2014-03-25
URL http://ir.fmu.ac.jp/dspace/handle/123456789/602
Rights
This is the pre-peer reviewed version of the following article: [J Diabetes. 2017 Aug;9(8):754-763], which has been published in final form at [https://doi.org/10.1111/1753-0407.12482].
This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self- Archived Versions.
DOI
Text Version ETD
学 位 論 文
Liver-specific G0S2 expression exacerbates hepatic insulin resistance in high fat induced insulin-resistant rats.
(肝臓における G0S2
の 剰発現は、 脂肪 誘発性インスリン抵抗性モデルラットにおいて肝臓でのインスリン抵抗性を増悪させる
)
福島県立医科大学医学研究科
腎臓 血圧・糖尿病内分泌代 内科 座 菅 芳幸
ABSTRUCT
OBJECTIVE: Hepatic steatosis is strongl associated with insulin resistance.
Recentl it has been reported that G0S2 inhibited the lipol sis activit of adipose trigl ceride lipase (ATGL), which is a major hepatic lipase as well as in adipoc tes.
Moreover, we confirmed that G0S2 protein contents were increased in the livers of high fat diet (HFD)-fed rats. However, the precise ph siological role of hepatic G0S2 is still unknown.
In the current studies, we investigated the effect of hepatic G0S2 on insulin sensitivit in normal chow diet (NCD) or HFD-fed male Wistar rats b overexpressing G0S2 protein using an adenovirus (Ad) encoding mouse G0S2.
RESEARCH DESIGN AND METHODS: Male Wistar rats were fed with NCD or
60% HFD for a total of 4 weeks. After 3 weeks feeding, the were injected with control Ad-GFP or Ad-G0S2. On da 7 post injection, intravenous glucose tolerance test (ivGTT) and eugl cemic-h perinsulinemic clamp studies (at 25 mU/kg/min insulin infusion rate) were performed after a 8-hour fast.
RESULTS: The bod weight and fasting glucose levels were not significantl
different between the Ad-GFP and the Ad-G0S2 groups. In the HFD-fed rats, during the clamp studies, the glucose infusion rate (GIR) required to eugl cemia was significantl decreased b 16% (from 34.9 2.3 to 29.4 0.8 mg/kg/min, P <
0.05) in the Ad-G0S2 group compared with the Ad-GFP group. Insulin-suppressed hepatic glucose output (HGO) was significantl increased from 5.1 0.6 to 6.7 0.5 mg/kg/min (p < 0.05) in the Ad-G0S2 group, but insulin-stimulated glucose disposal rate (IS-GDR) was not significantl different between two groups. On the other hand, under the NCD conditions, there were no significant changes in the GIR,
IS-GDR, and HGO between two groups. Consistent with the clamp data, the insulin-stimulated phosphor lation of Akt (Ser473) was significantl decreased in the livers of the HFD-fed rats injected with Ad-G0S2. Furthermore, the Oil-Red O stain indicated that overexpression of G0S2 protein in liver promoted hepatic steatosis b 2.5-fold in HFD-fed rats.
CONCLUSION: These results indicate that hepatic G0S2 protein might promote hepatic insulin resistance b the exacerbation of hepatic steatosis.
INTRODUCTION
T pe 2 diabe e i a p og e i e di ea e, cha ac e i ed b in lin e i ance and impai ed in lin ec e ion, hich lead o ch onic h pe gl cemia and inc ea ed i k of mic o- and mac o- a c la complica ion [1]. In lin e i ance, hich i cha ac e i ed b an impai ed abili of in lin o inhibi gl co e o p f om he li e and o p omo e gl co e p ake in m cle [1], i f e en l ell e abli hed in indi id al i h impai ed gl co e in ole ance (IGT). In addi ion, In lin e i ance i a cen al componen defining he me abolic nd ome, a con ella ion of abno mali ie ha incl de obe i , h pe en ion, gl co e in ole ance, and d lipidemia [2].
Hepa ic ea o i , defined a ec opic acc m la ion of igl ce ide (TG ) in hepa oc e , i he fi ep in a pec m of non-alcoholic fa li e di ea e (NAFLD).
In he o-hi model of NAFLD pa hogene i , hepa ic fa acc m la ion i he fi hi and hepa ic ea o i lead o econd hi ha e l in hepa oc e inj [3]. Hepa ic ea o i i mo e f e en among people i h diabe e and obe i [4, 5], and p e alence of hepa ic ea o i i inc ea ing in Japan [6]. Acc m la ion of TG in he li e lead o hepa ic in lin e i ance [7], and mo eo e , hepa ic ea o i i ho gh o be an independen de e minan of ca dio a c la di ea e [8, 9]. Hepa ic ea o i de elop hen f ee fa acid (FFA) p ake and n he i in he li e e ceed i elimina ion a e [10]. A la ge n mbe of molec le can be in ol ed in he eg la ion of lipid me aboli m in he li e .
G0/G1 i ch gene 2 (G0S2) i one of he G0/G1 i ch (G0S) gene ha e e fi iden ified in l mphoc e d ing hei lec in-ac i a ed i ch f om he G0 o he G1 pha e of he cell c cle [11]. Se e al epo ha e gge ed ha G0S2 i a m l iface ed p o ein i h di pa a e f nc ion ela ed o p olife a ion, me aboli m, inflamma ion, and
ca cinogene i . The G0S2 gene a i epigene icall ilenced in head and neck cance [12], amo l ng cance , and ci pla in- e i an cance cell [13]. On he o he hand, he G0S2 e p e ion a ele a ed in endome io i [14], b onchial epi helial cell ea ed i h e inoic acid [15], ene cen de mal fib obla [16], bone ma o cell f om pa ien i h he ma oid a h i i [17], and pe iphe al monon clea cell f om pa ien
i h a c li i [18] and p o ia i [19].
G0S2 a la e iden ified a a no el p a i e a ge gene of pe o i ome p olife a o-ac i a ed ecep o (PPAR ) b compa ing li e mRNA of ild- pe and PPAR -n ll mice ing mic oa a , and i a epo ed ha he G0S2 mRNA le el a highe in adipo e i e and a p- eg la ed d ing adipogenic diffe en ia ion of mo e 3T3-L1 cell and ha G0S2 i a di ec PPAR , e peciall PPAR , a ge gene [20].
Recen l , i a e ealed ha G0S2 inhibi ed adipo e igl ce ide lipa e (ATGL) [21], hich a he a e-limi ing en me fo TG h d ol i in adipoc e [22] and a ho n o be a majo hepa ic lipa e ha eg la e TG no e [23-25]. Hepa ic e p e ion of G0S2 a ma kedl inc ea ed nde high c o e die (HSD) in Nago a-Shiba a-Ya da (NSY) mice [26]. Wang e al. epo ed ha Adeno i -media ed e p e ion of G0S2 (Ad-G0S2) po en l ind ced hepa ic ea o i in mice [27]. The e die gge ha G0S2 ha an impo an ole in eg la ion of hepa ic TG me aboli m. Ho e e , he ole of G0S2 in in lin ignaling i nclea . In hi d , e in e iga ed he effec of hepa ic G0S2 on in lin en i i i in no mal cho die (NCD) o high fa die (HFD)-fed male Wi a a b o e e p e ing G0S2 ing Ad-G0S2.
MATERIALS AND METHODS Materials
AdEasy Adenoviral Vector System was purchased from Stratagene Inc. (La Jolla, CA, USA). Adeno-X Virus Purification and Rapid Titer Kits were purchased from TAKARA BIO INC. (Shiga, JAPAN). Male Wistar rats were procured from Charles River Laboratory Japan, Inc. (Kanagawa, Japan). Insulin (Novolin R) was purchased from Novo Nordisk (Copenhagen, Denmark). High fat diet (HFD) (60%
w/w, #D12492) was purchased from Research Diet Inc. (New Brunswick, NJ, USA). The catheter (Micro-Renathane MRE-033, 0.033 cm in OD and 0.014 cm in ID) was purchased from Braintree Scientific (Braintree, MA, USA). D-[3-3H] glucose was purchased from PerkinElmer Inc. (Waltham, MA, USA). Pentobarbital was purchased from Kyoritsu Pharmaceutical Co. (Tokyo, Japan). The 50% dextrose was purchased from Otsuka Pharmaceutical Co. (Tokushima, Japan). BCA protein assay reagent was purchased from Thermo Fisher Scientific Inc. (Rockford, IL, USA).
Complete Mini (Phosphatase and protease inhibitors) was purchased from Roche Applied Science (Mannheim, Germany). Polyvinylidine difluoride (PVDF) transfer membranes were purchased from Millipore Corp (Bedford, MA, USA). ImmunoBlock was purchased from DS Pharma Biomedical Co. Ltd (Osaka, Japan). Anti-phospho-specific Akt (Ser473), Akt, and -actin antibodies were purchased from Cell Signaling Technology (Boston, MA, USA). G0S2 (#sc-133424) antibody and horseradish peroxidase-conjugated secondary antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). TRIzol reagent and pCR2.1-TOPO vector were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). The RNeasy kit was
purchased from QIAGEN Inc. (Valencia, CA, USA). All other reagents were purchased from Sigma (St. Louis, MO, USA).
Subcloning of the mouse adiponectin cDNA by RT-PCR.
The mouse full-length G0S2 cDNA (312 bp) was amplified from mouse liver using the 5'-CAGATGGAAAGTGTGCAGGAGCTG-3' sense and 5'-CCGGCCTTAAG AGGCGTGCTGCCG-3' antisense primers, subcloned into the pCR2.1-TOPO (Invitrogen) and sequenced, confirming that the clones corresponded to the mouse adiponectin (GenBank(TM) accession number BC117038).
Construction of recombinant adenoviruses.
Adeno i e p e ing mo e G0S2 (Ad-G0S2) a gene a ed f om he f ll-leng h cDNA, bcloned i h an AdEa Adeno i al Vec o S em (S a agene Inc.), p opaga ed in HEK293 cell , and p ified i h Adeno-X Vi P ifica ion and Rapid Ti e Ki (TAKARA BIO INC). Con ol Ad-GFP a i ola ed ing he ame p oced e. Bo h ecombinan i e e e dial ed in PBS, pH 7.4, and o ed in 10%
gl ce ol/PBS a 80 C n il e.
Animal studies.
Si - eek old male Wi a a (Cha le Ri e Labo a o Japan, Inc.) e e ho ed indi id all nde con olled ligh /da k (12/12 h) and empe a e condi ion (25 C), and had f ee acce o a e and no mal cho die (NCD) o 60% HFD (Re ea ch Die Inc.). Ra ecei ed a f e h die e e 3 da , and food con mp ion a e and bod eigh gain e e moni o ed e e 3da . Af e feeding he indica ed cho fo 3 eek ,
he a e e implan ed i h 3 ca he e (Mic o-Rena hane MRE-033, 0.033 cm in OD and 0.014 cm in ID; B ain ee Scien ific, B ain ee, MA), a p e io l de c ibed [28].
In b ief, 2 ca he e e e placed in o he igh j g la ein, and ano he one a placed in o he lef ca o id a e nde ingle-do e ane he ia (pen oba bi al 50 mg/kg; K o i Pha mace ical Co.) gi en in ape i oneall . Ca he e e e nneled bc aneo l , e e io i ed a he back of he neck, and filled i h hepa ini ed aline. The j g la and ca o id ca he e e e ed fo inf ion and blood ampling, e pec i el . And b e en l , he e e injec ed in a-a e iall f om he ca he e of he lef ca o id a e i h 1 109 p.f. . of G0S2 o GFP i e pe a . The animal e e gi en 7 da o f ll eco e f om he ge . All p oced e e e pe fo med in acco dance i h he G ide fo Ca e and U e of Labo a o Animal of he NIH and e e app o ed b he Animal S bjec Commi ee of he F k hima Medical Uni e i , Japan.
Glucose and insulin tolerance tests and eugl cemic-h perinsulinemic clamp procedures.
On da 7 af e he adeno i injec ion, gl co e ole ance and in lin en i i i a a e ed ing an in a eno gl co e ole ance e (i GTT) and e gl cemic-h pe in linemic clamp. The a e e fa ed fo 8 ho befo e he a of all e pe imen .
Fo he i GTT, he a e e injec ed i h 0.5 g/kg bod eigh of de o e (50% de o e; O ka Pha mace ical Co.) in he j g la ein. Blood ample e e collec ed a ime 0, 15, 30, 60, 90, and 120 min f om he ca o id a e .
The e gl cemic-h pe in linemic clamp e pe imen began i h a con an inf ion (0.04 Ci/min) of D-[3-3H] gl co e (Ne England N clea ). Af e 120 min of
ace e ilib a ion and ba al ampling a ime -10 min, and 0 min, gl co e (50%
de o e, a iableinf ion; O ka Pha mace ical Co.) and ace (0.12 Ci/min) pl in lin (25 mU/kg/min, No olin R; No o No di k) e e inf ed in o he j g la ein a p e io l de c ibed [29, 30].
Small blood ample (60 l) e e d a n a 10-min in e al and immedia el anal ed fo gl co e (Compac Elec ode Blood S ga Anal e An en e: HORIBA L d, K o o, Japan) o main ain he in eg i of he gl co e clamp h o gho he d a ion of he e pe imen . Blood ample e e aken a -120 min ( a of e pe imen ), -10 min and 0 min (ba al), and 100 min, 110 min and 120 min (end of e pe imen ), fo de e mina ion of gl co e pecific ac i i , in lin and FFA, con en . To en e acc ac , ba al and e minal ampling a pe fo med ice, a a 10-min e in e al. We confi med ha ead - a e condi ion e e achie ed a he end of he clamp befo e ob aining he e minal blood pecimen b mea ing blood gl co e e e en min e and a ing ha ead a e fo gl co e inf ion and pla ma gl co e le el a main ained fo a minim m of 20 min e befo e final ampling. We define a ead - a e blood gl co e concen a ion a one he e he gl co e concen a ion and inf ion a e fl c a e b 3 mg/dl o le and b 5% o le , e pec i el , o e 10 min e . All blood ample e e immedia el cen if ged, and pla ma a o ed a -80 C fo b e en anal i . Af e e minal blood ampling a 120 min, animal e e p omp l e hani ed i h pen oba bi al (180 mg/kg). Ti e e e aken and immedia el f o en in li id ni ogen and o ed a -80 C fo b e en me abolic anal i .
Analytical procedures.
Ra in lin, h man in lin, adiponec in, f ee fa acid (FFA), a pa a e
amino an fe a e (AST), alanine amino an fe a e (ALT), -gl am l an pep ida e ( -GT) e e anal ed b a p i a e labo a o (SRL Labo a o , Tok o, Japan). Pla ma gl co e pecific ac i i a mea ed in d plica e af e inc lfa e and ba i m h d o ide dep o eini a ion.
Immunoblotting anal sis.
Li e , ed ad icep m cle, and hi e adipo e i e ed fo me abolic anal i e e aken f om animal immedia el af e e hana ia, in ed e e al ime in cold aline o emo e blood, f o en in li id ni ogen and o ed a -80 C. Ca e a aken o a oid aking ec ion of he li e lobe con aining la ge blood e el . Ti e e e homogeni ed in li id ni ogen and l ed in b ffe con aining pho pha a e and p o ea e inhibi o (Comple e Mini; Roche Applied Science) acco ding o he p o ocol of he man fac e . The p o ein concen a ion a de e mined b BCA p o ein a a eagen (The mo Fi he Scien ific Inc.). E al amo n of p o ein e e epa a ed b 10%
odi m dodec l lfa e-pol ac lamide gel elec opho e i (SDS-PAGE) and an fe ed o pol inidene difl o ide memb ane (Immobilon; Millipo e), and e e hen blocked i h Imm noBlock (DS Pha ma Biomedical Co. L d.) o e nigh. The memb ane e e p obed i h p ima Ak and pho pho-Ak (Se 473) an ibodie (Cell Signaling Technolog ) follo ed b a Ho e adi h pe o ida e-conj ga ed econda an ibod (San a C Bio echnolog ). The imm nocomple e e e i ali ed i h ECL e e n blo ing de ec ion eagen (Ame ham; GH Heal hca e, UK). -ac in e ed a an in e nal con ol p o ein. Band in en i ie e e an ified b den i ome ing he Image-J of a e (NIH, Be he da, MD, USA).
Histological examination.
A po ion of li e a fi ed i h 10% fo malin and embedded in pa affin. 3 m ec ion e e c and ained i h hema o lin and eo in fo e amina ion of li e hi olog (BX-50, Ol mp Co po a ion, Tok o, Japan). The ec ion e e al o inc ba ed i h a abbi monoclonal G0S2 an ibod (San a C , CA, USA) fo 12 o 16 ho a 4 and ained b Simple S ain MAX-PO (Nichi ei Bio cience Inc., Tok o, Japan). To e amine lipid acc m la ion, 6 m f o en ec ion e e ained i h Oil Red O. Ten field in e e indi id al ec ion e e andoml elec ed, and he Oil Red O- ained a ea and he o al i e a ea e e mea ed ing he Image J of a e. The
a io of he Oil Red O- ained a ea o he o al i e a ea a calc la ed (%).
Statistical anal sis.
The glucose infusion rate (GIR) required to maintain euglycemia reflects the systemic insulin sensitivity. Hepatic glucose output (HGO), which reflects the hepatic insulin sensitivity, and glucose disposal rate (GDR) were calculated for the basal period and steady-state portion of the glucose clamp using the Steele equation for steady-state conditions (HGO = GDR GIR) [31]. The insulin-stimulated GDR (IS-GDR) reflects the ability of insulin to increase GDR above the basal value, which means that IS-GDR reflects the muscle insulin sensitivity. IS-GDR is calcula ed b b ac ing each animal basal HGO value from the final GDR achieved at the end of the clamp period (IS-GDR = total GDR basal HGO value).
Data are presented as mean SEM. Statistical differences between two groups were calculated by the unpaired Student's test. All data are distributed to ensure that the use of means and t-test is appropriate. Statistical significance was defined as P<0.05.
RESULTS
G0S2 e e e e , c e, a d h e ad e e he ba a a e.
We fi in e iga ed he e p e ion of G0S2 p o ein in he li e , kele al m cle and hi e adipo e i e of con ol a b e e n blo anal i . In li e and hi e adipo e i e , G0S2 p o ein le el of HFD-fed a e e ignifican l highe han ho e of NCD-fed a (Fig e 1A and 1C). Ho e e , he G0S2 p o ein le el in kele al m cle e e no ignifican change in NCD- and HFD-fed a (Fig e 1B).
To confi m he G0S2 p o ein e p e ion b Ad-G0S2, e pe fo med e e n blo ing of cell l a e ob ained f om ei he Ad-G0S2- o Ad-GFP- infec ed HEK 293 cell . The e p e ion of G0S2 p o ein a de ec ed in cell infec ed i h Ad-G0S2, b did no de ec ed in cell infec ed i h Ad-GFP (Fig e 1C).
Rep e en a i e e l of imm nohi ochemi of G0S2 p o ein in li e of a infec ed i h Ad-G0S2 o Ad-GFP a e ho n in Fig e 4E. S aining fo G0S2 p o ein a locali ed o he c opla m of hepa oc e . G0S2 p o ein le el e e highe in he li e i e infec ed i h Ad-G0S2 han i h Ad-GFP. G0S2 p o ein le el of Ad-G0S2-infec ed li e i e e e de e mined b e e n blo anal i (Fig e 1F).
O e a a a cha ac e c .
Table 1 ho ome of he gene al cha ac e i ic of he a in he ba al a e on da 7 af e injec ion of Ad-G0S2 o con ol Ad-GFP nde he NCD o HFD condi ion . The bod eigh , li e eigh , and food in ake (da a no ho n) e e no diffe en be een he Ad-G0S2 and con ol Ad-GFP g o p , hile he bod eigh and li e eigh of he HFD-fed a e e ignifican l g ea e han ho e of he NCD-fed a . The e e e no ignifican diffe ence in gl co e, in lin, and HOMA-IR le el be een
he Ad-G0S2-infec ed a and he Ad-GFP-infec ed a , al ho gh ho e le el of he HFD-fed a ended o be highe han ha of NCD-fed a . Adiponec in, FFA, AST, ALT, and -GTT le el did no diffe ignifican l be een o g o p .
NCD-feed g d e .
In a eno gl co e ole ance e .
On 7 da af e injec ion of adeno i e , i GTT e e pe fo med on 8-h-fa ed a . 0.5 g/kg gl co e a injec ed in o he j g la ein. The e e e no ob io diffe ence in gl co e le el be een he Ad-G0S2-infec ed a and he Ad-GFP-infec ed a nde he NCD condi ion (Fig e 2A). I a he ame e l fo in lin le el (Fig e 2B).
E gl cemic-h pe in linemic clamp .
To di ec l e amine he me abolic impac of G0S2 p o ein on in lin en i i i , e ne bjec ed bo h g o p of a o e gl cemic-h pe in linemic clamp a ma imal (25 mU/kg/min) in lin inf ion a e. S ead - a e gl co e and in lin le el d ing he clamp die e e imila in he o g o p (Table 1). D ing he e die , e mea ed in lin im la ion of hole-bod GDR and pp e ion of HGO. The GIR e i ed o main ain e gl cemia didn diffe be een he o g o p (Fig e 3A). To a e he in lin- im la ed componen of gl co e di po al, he IS-GDR a calc la ed.
The IS-GDR didn diffe be een he o g o p , ei he (Fig e 3B).
Ba al HGO al e e e no diffe en be een he Ad-G0S2-infec ed a and he Ad-GFP-infec ed a . D ing he clamp die , HGO al e didn diffe be een he
o g o p , ei he (Fig e 3C).
HFD-feed g d e .
In a eno gl co e ole ance e .
Fa ing gl co e and in lin le el ho ed no ignifican diffe ence be een he Ad-G0S2-infec ed a and he Ad-GFP-infec ed a nde he HFD condi ion (Fig e 4A, 4B). Ho e e , af e he 0.5 g/kg gl co e load, he gl co e le el e e ignifican l highe in he Ad-G0S2-infec ed a a 15 and 30 min (283.3 11.4 . 244.9 10.7 mg/dl and 226.2 9.4 . 195.9 8.7 mg/dl e pec i el , p < 0.05) (Fig e 4A). Con a o o e pec a ion, he e e e no ignifican diffe ence in in lin le el d ing i GTT die be een he o g o p (Fig e 4B).
E gl cemic-h pe in linemic clamp .
S ead - a e gl co e and in lin le el d ing he clamp die e e imila in he o g o p (Table 1). The GIR e i ed o main ain e gl cemia a ignifican l dec ea ed b 16% (f om 34.9 2.3 o 29.4 0.8 mg/kg/min, P < 0.05) in he Ad-G0S2-infec ed a , ho ing impai ed o e all in lin en i i i (Fig e 5A). The IS-GDR didn diffe be een he o g o p (Fig e 5B).
Ba al HGO al e e e no diffe en be een he Ad-G0S2-infec ed a and he Ad-GFP-infec ed a (9.9 0.6 and 9.5 0.6 mg/kg/min) (Fig e 5C). D ing he clamp die , HGO al e e e ignifican l highe in he Ad-G0S2-infec ed a han in he Ad-AGF-infec ed a (6.7 0.5 . 5.1 0.6 mg/kg/min, P < 0.05) (Fig e 3C),
gge ing ha he G0S2 p o ein p omo e hepa ic in lin e i ance.
I g a g d e .
To a e he po en ial cell la mechani m of G0S2-ind ced impai men in in lin en i i i , e ob ained li e , kele al m cle and hi e adipo e i e ample
f om he a a he end of he e gl cemic-h pe in linemic clamp. The e ample , hich ep e en he f ll in lini ed a e a he e mina ion of he gl co e clamp d , e e homogeni ed and he p o ein l a e e e bjec ed o imm noblo ing.
We mea ed Ak pho pho la ion (Se 473), hich i he mo impo an molec le of he in lin ignaling. In lin led o a ma ked im la ion of Ak pho pho la ion in li e , kele al m cle, and hi e adipo e i e f om con ol ba al a e a nde he NCD condi ion (Fig e 6A, 6B, and 6C). The e e e no ignifican diffe ence in Ak pho pho la ion be een he Ad-G0S2-infec ed a and he Ad-GFP-infec ed a in li e , m cle, and hi e adipo e i e . On he o he hand, hi effec of Ak pho pho la ion a Se 437 a ignifican l dec ea ed b 65% (P < 0.05) in li e nde he HFD condi ion (Fig e 7A), b no in m cle and hi e adipo e i e of he Ad-G0S2-infec ed a (Fig e 7B, 7C).
H g ca e a a .
Mac o copicall , he li e e ha e ed f om he Ad-G0S2-infec ed a and he Ad-GFP-infec ed a looked he ame, al ho gh li e of he HFD-fed a looked fa ie han ho e of he NCD-fed a (Fig e 8A). To e al a e lipid acc m la ion, he li e e e b e en l ec ioned and ained i h Oil Red O, hich ain ne al lipid (Fig e 8B). Oil Red O aining a ea of he HFD-fed a e e ignifican l la ge han ho e of NCD-fed a . Thi inc ea e of lipid acc m la ion a a gmen ed b 2.5-fold in he G0S2-infec ed a nde he HFD condi ion , b no nde he NCD condi ion . Thi e l gge hepa ic in lin e i ance i o en in he Ad-G0S2-infec ed a nde he HFD condi ion , and ppo he e l of i GTT, e gl cemic h pe in linemic clamp, and e e n blo ing die .
DISCUSSION
Re l f om he p e en d demon a ed ha nde HDF condi ion, no nde NCD condi ion, G0S2 ind ced he lipid acc m la ion in he a li e and de eloped hepa ic ea o i . G0S2 o e e p e ion impai ed he o al in lin en i i i b p omo ing he hepa ic in lin e i ance. The e finding gge ha G0S2 i an impo an eg la o of hepa ic gl colipid me aboli m.
The li e i one of he p ima o gan e pon ible fo con olling ene g homeo a i in mammal . Hepa ic ea o i a i e f om an imbalance be een TG ac i i ion and emo al. The fa acid ed fo hepa ic TG fo ma ion a e de i ed f om die , de no o n he i , and adipo e i e. Die a fa aken p in he in e ine a e packaged in o TG- ich ch lomic on and deli e ed o he emic ci c la ion, and 20%
of he TG in ch lomic on i deli e ed o he li e [32]. Ca boh d a e feeding p omo e de no o n he i of FFA f om ace l-coen me A (CoA) b inc ea ing he le el of in lin and he a ailabili of b a e. In lin im la e he an c ip ion fac o e ol eg la o elemen-binding p o ein-1c (SREBP-1c) ia a ignaling ca cade in ol ing Ak 2, LXR, and mTOR [33]. SREBP-1c p- eg la e he en me ha ca al e lipogene i [34]. Gl co e al o p omo e lipogene i b ac i a ing he an c ip ion fac o ca boh d a e e pon i e elemen-binding p o ein (ChREBP) [35]. D ing fa ing, pla ma le el of in lin fall, he ea le el of gl cagon and epineph ine inc ea e, im la ing TG h d ol i in adipoc e . The fi ep in TG h d ol i i ca al ed b ATGL ha i
he a e-limi ing en me [22].
ATGL i e p e ed a lo e le el in non-adipo e i e ch a hea , m cle, and li e [36]. In mo i e of ATGL n ll mice, he ec opic lipid acc m la ion a inc ea ed (21-fold in ca diac m cle, 3-fold in kele al m cle, and 2.3-fold in li e ) [37].
Adeno i -media ed knockdo n of ATGL epo edl de eloped hepa ic ea o i in mice [23]. On he o he hand, i a epo ed ha adeno i -media ed o e e p e ion of ATGL dec ea ed hepa ic ea o i in HFD-fed mice [24]. Li e - pecific ATGL knocko mice e e ho n o ha e e e e p og e i e pe ipo al mac o e ic la and pe icen al mac o e ic la hepa ic ea o i [25]. The e die gge ha ATGL i a majo hepa ic lipa e ha eg la e TG no e .
ATGL ac i i i ongl infl enced b eg la o p o ein . La e al. iden ified compa a i e gene iden ifica ion-58 (CGI-58) a coac i a o of ATGL, hich i e i ed fo efficien lipol i [38]. CGI-58 n ll mice inc ea ed fa ma and die a fe ho af e bi h, mo likel d e o a e e e kin defec . The e die gge ha CGI-58 i e en ial fo ATGL ac i a ion [39]. On he o he hand, mo e ecen l , Yang e al.
iden ified G0S2, hich had been fi iden ified a a po en ial cell c cle eg la o [11], a an inhibi o p o ein of ATGL [21].
When o e e p e ed in HeLa cell , G0S2 locali ed o lipid d ople and p e en ed ATGL-media ed TG h d ol i b binding o ATGL [21]. Al ho gh G0S2 i mainl e p e ed in b o n and hi e adipo e i e, G0S2 mRNA i ea onabl ell e p e ed in o he i e , ch a l ng, li e , and hea . [20]. Hepa ic e p e ion of G0S2 a ma kedl inc ea ed in onl HSD-fed NSY mice, no in HSD-fed C3H mice, hile HSD de eloped hepa ic ea o i and p omo ed in lin e i ance [26]. We epo ed he e ha Ad-G0S2-infec ed a , onl nde HFD condi ion , de eloped hepa ic ea o i and p omo ed in lin e i ance. Hepa ic e p e ion of G0S2 a p- eg la ed b fa ing and b PPAR agoni hile G0S2 e p e ion a p-eg la ed d ing he adipogene i of mo e 3T3-L1 cell [20]. Recen l , Wang e al. epo ed ha Adeno i -media ed e p e ion of G0S2 po en l ind ced hepa ic ea o i in mice, and G0S2 a fo nd o be
co-locali ed i h ATGL a he face of lipid d ople [27]. In hi d he e, con i en i h ob e a ion of Wang e al., he Ad-G0S2-infec ed a de eloped hepa ic ea o i nde HFD condi ion , al ho gh hi effec a n ob e ed nde NCD condi ion . Thi effec of lipid acc m la ion in li e migh be d e o G0S2 inhibi ing AGTL ac i i .
A n mbe of die ha e epo ed a ong ela ion hip be een hepa ic ea o i and in lin e i ance [5, 7, 40-43]. Fa acc m la ion in he li e i a ocia ed i h defec in in lin pp e ion of HGO independen of obe i [44, 45]. Hepa ic in lin e i ance can occ independen l of change in ci c la ing adipoc okine ch a mo nec o i fac o - (TNF- ), in e le kin-6 (IL-6), e i in, adiponec in, and e inol binding p o ein-4 (RBP-4) [43]. Hepa ic e i ance a a ocia ed i h an inc ea e in hepa ic digl ce ide (DG) con en [41]. The link be een hepa ic DAG acc m la ion and hepa ic in lin e i ance co ld be a ib ed o ac i a ion of p o ein kina e C (PKC ) [46], hich a p edominan PKC i ofo m ac i a ed in li e follo ing fa feeding [7].
The mechani m fo lipid-ind ced in lin e i ance i imila o ha i ob e ed in kele al m cle he e PKC ha been ho n o be he p edominan no el PKC i ofo m ac i a ed d ing lipid-ind ced m cle in lin e i ance [47, 48]. Ac i a ed b DG, PKC inhibi he in lin ecep o kina e. Thi hen lead o dec ea ed in lin- im la ed o ine pho pho la ion of in lin ecep o b a e 1 and 2 (IRS1, IRS2), e l ing in ed ced in lin ac i a ion of 1-pho phoino i ol 3-kina e (PI 3-kina e), and Ak 2.
Red ced Ak 2 ac i a ion e l in dec ea ed gl cogen n ha e (GS)-media ed gl cogen n he i and dec ea ed pp e ion of gl coneogene i , hich in n lead o gl co e elea e h o gh gl co e an po e 2 (GLUT2).
Con ide ing ha G0S2 inhibi ATGL ac i i , inac i a ion of ATGL migh be in ol ed in modif ing in lin e i ance in he Ad-G0S2-infec ed a . Ho e e , he
a ocia ion be een ATGL and in lin e i ance i con o e ial. Haemme le e al.
epo ed ha gl co e and in lin ole ance e e imp o ed in ATGL-KO mice nde NCD condi ion [37]. W e al. epo ed ha he e e e no diffe ence in li e - pecific ATGL-KO mice nde bo h NCD and HFD condi ion d ing GTT and ITT [25]. On he o he hand, Ong e al. epo ed ha adeno i -media ed o e e p e ion of ATGL imp o ed in lin ole ance nde NCD condi ion [24]. Wi h ega d o G0S2, Wang e al.
epo ed ha , d ing o al GTT, G0S2 o e e p e ion in he li e ignifican l imp o ed gl co e ole ance in mice nde anda d oden cho condi ion , he ea he e e e no ignifican diffe ence in gl co e le el d ing in lin ole ance e [27]. In he e die , DG con en in he li e a n a e ed, and mo die e e in e iga ed nde onl NCD condi ion . The a e ed in lin e i ance b GTT o ITT. The e me hod a e indi ec fo an if ing in lin en i i i . In he p e en d , e ed e gl cemic-h pe in linemic clamp, hich i he gold anda d fo an if ing hepa ic in lin en i i i . We fo nd ha he Ad-G0S2-infec ed a p omo ed hepa ic in lin e i ance nde HFD condi ion , hich a con i en i h he e l of IV-GTT and We e n blo ing anal i in hi d . Fed i h HFD, he amo n of TG , hich a e de i ed f om ch lomic on, i inc ea ed in li e i h he e l of g o ing lipid d ople . In he Ad-G0S2 infec ed a , nde HDF condi ion , G0S2 inhibi ATGL-media ed TG h d ol i in d ople and TG con en i inc ea ed. A he ame ime, in he endopla mic e ic l m (ER), TG a e al o n he i ed f om DG , hich i a pa of de no o lipogene i [49]. The n he i of TG f om DG i ca al ed b ac l CoA:
diac lgl ce ol ac l an fe a e (DGAT) en me . W e al. epo ed ha DGAT2 mRNA a ma kedl dec ea ed in li e - pecific ATGL-KO mice [25], hich indica e ha TG n he i migh be do n- eg la ed b ome kind of nega i e feedback mechani m. Then,
DG con en migh be inc ea ed in li e and p omo e hepa ic in lin e i ance. Ho e e , DG con en in li e i e o be a e ed, and he de ail mechani m of p omo ing hepa ic in lin e i ance b G0S2 ill emain o be el cida ed.
In concl ion, G0S2 migh p omo e hepa ic in lin e i ance b he e ace ba ion of hepa ic ea o i nde HFD condi ion . The f he in e iga ion a e needed o e eal
he mechani m of G0S2 fo in lin en i i i .
ACKNOWLEDHMENTS
I hanked T o hi Wa anabe MD, PhD and Hi oaki Sa oh MD, PhD fo e pe a i ance, Y ko Oha hi and A ko Ha himo o fo e cellen echnical a i ance.
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FIGURE LEGENDS
Figure 1. Expression of G0S2 in each tissues of male Wistar rats, and overexpression of G0S2 in HEK293 cells and livers of male Wister rats.
Si eek-old male Wi a a e e fed i h NCD (open ba ) o HFD (clo ed ba ) fo 4 eek . G0S2 p o ein le el in he li e (A), kele al m cle (B), and WAT (C) e e de e mined i h e e n blo ing. (D) G0S2 o GFP p o ein le el in HEK293 cell , hich e e infec ed i h Ad-G0S2 o Ad-GFP fo 48 ho e pec i el , e e al o de e mined. (E) Imm nological de ec ion of G0S2 p o ein in li e of male Wi a a . (F) G0S2 p o ein le el in li e of Ad-GFP o Ad-G0S2-infec ed a e e de e mined
i h e e n blo ing. Val e a e mean SE (n = 4). *P < 0.05 . NCD-fed a .
Figure 2. IV-GTT in NCD-fed rats on postinjection da 7.
Gl co e (A) and in lin (B) c e f om he GTT in Ad-GFP (open ci cle, n = 7) and Ad-G0S2 (clo ed ci cle, n = 7)-infec ed a injec ed i h 0.5 g/kg bod eigh af e 8-h fa . Val e a e mean SE.
Figure 3. Eugl cemic h perinsulinemic clamp in NCD-fed rats on postinjection da 7.
GIR (A), IS-GDR (B), and in Ad-GFP (open ba , n = 10) and Ad-G0S2 (clo ed ba , n = 10)-infec ed a af e 8-h fa . HGO (C) a calc la ed a he ba al a e (open ba ) and d ing clamp (clo ed ba ). In lin inf ion a e e e 25 mU/kg/min. Val e a e mean SE. P < 0.05, P < 0.01 . ba al a e.
Figure 4. IV-GTT in HFD-fed rats on postinjection da 7.
Gl co e (A) and in lin (B) c e f om he GTT in Ad-GFP (open ci cle, n = 7) and Ad-G0S2 (clo ed ci cle, n = 9)-infec ed a injec ed i h 0.5 g/kg bod eigh af e 8-h fa . Val e a e mean SE. *P < 0.05 . Ad-GFP-infec ed a .
Figure 5. Eugl cemic h perinsulinemic clamp in HFD-fed rats on postinjection da 7.
GIR (A), IS-GDR (B), and in Ad-GFP (open ba , n = 8) and Ad-G0S2 (clo ed ba , n = 10)-infec ed a af e 8-h fa . HGO (C) a calc la ed a he ba al a e (open ba ) and d ing clamp (clo ed ba ). In lin inf ion a e e e 25 mU/kg/min. Val e a e mean SE. *P < 0.05 . Ad-GFP-infec ed a ; P < 0.05, P < 0.01 . ba al a e.
Figure 6. Effect of G0S2 protein on Akt phosphor lation (Ser473) in each tissues of NCD-fed rats on postinjection da 7.
Ak and pAk (Se 473) p o ein le el in he li e (A), kele al m cle (B), and WAT (C) of Ad-GFP (open ba ) and Ad-G0S2 (clo ed ba )-infec ed a e e de e mined i h
e e n blo ing. Val e a e mean SE (n = 4). P < 0.05, P < 0.01 . ba al a e.
Figure 7. Effect of G0S2 protein on Akt phosphor lation (Ser473) in each tissues of HFD-fed rats on postinjection da 7.
Ak and pAk (Se 473) p o ein le el in he li e (A), kele al m cle (B), and WAT (C) of Ad-GFP (open ba ) and Ad-G0S2 (clo ed ba )-infec ed a e e de e mined i h e e n blo ing. Val e a e mean SE (n = 4). *P < 0.05 . Ad-GFP-infec ed a ; P <
0.05 . ba al a e.
Figure 8. Macroscopic and microscopic aspect of livers postinjection da 7.
(A) Pho og aph of li e a mac o copic cale. (B) The li e ec ion e e ained i h Oil Red O ( 400). (C) The a io of he Oil Red O- ained a ea o he o al i e a ea a calc la ed (%) (open ba fo Ad-GFP-infec ed a , clo ed ba fo Ad-G0S2 infec ed a ).
Val e a e mean SE (n = 6). **P < 0.01 . Ad-GFP-infec ed a ; P < 0.05 . NCD-fed a .
Ta 1
P -
A -GFP ( ) A -G0 2 ( )
B 408.4 8.5(21) 407.8 6.1 (24)
L 14.3 0.5 (6) 14.7 0.7 (6)
B
G / L 178.1 7.8 (13) 180.7 6.8 (16)
I / L 4.48 0.32 (13) 4.75 0.76 (16)
HOMA-I 2.01 0.23 (13) 2.09 0.48 (16)
G / L 57.3 5.2 (6) 73.5 4.7 (6) *
A / L 1.42 0.10 (6) 1.35 0.07 (6)
FFA EQ/L 262.7 42.2 (6) 270.8 42.0 (6)
G / L 56.0 10.3 (6) 59.7 10.2 (6)
C / L 63.2 5.8 (6) 57.1 5.9 (6)
HDL-C / L 18.7 1.0 (6) 17.3 1.4 (6)
L
A I /L 81.7 10.9(6) 60.8 4.6(6)
AL I /L 26.5 1.7(6) 28.3 1.6(6)
-G I /L 3.0 0.0(6) 3.0 0.0(6)
C
G / 146.9 1.7 (8) 149.8 1.1 (10)
H I / L 1039.7 87.5 (6) 1260.0 63.9 (6)
FFA EQ/L 63.8 13.4 (6) 55.7 17.9 (6)
% 75.7 79.4
E. *P < 0.01 . A -GFP-
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