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Abstract (english)

Chronic hepatitis C virus infection occurs in 170-200 millions of people worldwide and is the leading cause of liver disease progression, ranging from cirrhosis to hepatocarcinoma. The current standard treatment of HCV infection is based on a combination of Pegylated interferon-alpha (PEG-IFN-a) plus ribavirin. However sustained virological response (SVR) rate is achieved in 50-80% of the patients according with HCV-genotype (1). Non-response to antiviral therapy seems to be associated with several host and viral factors including HCV genotype, viral load, older age, male sex, BMI, degree of fibrosis, African ancestry.
Recent studies have shown that insulin resistance (IR) is a frequent pathological condition in CHC when compared to patients with other forms of chronic liver disease (2) and that IR prevalence is higher in patients with HCV genotype 1 infection (3). Understanding mechanisms that cause IR in the HCV patients has been made difficult due to the co-existence of several cofactors, associated with the metabolic syndrome. Shintany et al (4) suggested that HCV could be directly involved in the pathogenesis of IR through a modulation of insulin pathway induced by viral proteins (5). IR is clinically relevant as it is associated with type 2 diabetes (3, 6), and it correlates with liver fibrosis progression, cirrhosis and HCC (7, 8). Several lines of evidence indicate that IR is a negative predictor of the response to antiviral therapy in CHC patients treated with IFN plus ribavirin (9, 10). In a recent study, we have demonstrated that in HCV patients the impairment of antiviral response to PEG-IFN-a 24 h after the first injection of the drug is directly related to baseline insulin levels much more than HOMA-IR Index (11) suggesting the hypothesis that insulin itself can interferes with IFN-a signalling in infected hepatocytes probably because of common intracellular players. However the mechanisms by which insulin can affect IFN-a signalling are still unclear and are under intensive investigation.
Over-expression of intracellular factors associated with IR phenotype may play a role in the response to antiviral therapy in HCV infected patients as they are possibly involved in regulating IFN-a signalling (12). These intracellular factors include some members of SOCS family, a group of related proteins induced also by insulin treatment (13), that act as negative regulators of several intracellular pathway, including insulin signalling itself, and that have been shown to alter tyrosine kinase activity of growth factor receptors (13-17). It is known that hepatocyte-specific SOCS3 suppression by antisense-oligonucleotide treatment, in obese diabetic mice, decreased the elevated circulating insulin levels improving insulin sensitivity (18, 19). SOCS-1 and SOCS-3 proteins may inhibit insulin signalling through different mechanisms which involve both an inhibition of the insulin receptor kinase activity, ubiquitin-mediated degradation and a down-regulation of tyrosine phosphorylation of insulin receptor substrate-1 and 2 (IRS1 and IRS2) (20). Interestingly, SOCS3 over-expression in vitro was shown to be related to an impairment of IFN-a antiviral and anti-proliferative activity (21, 22) and in liver biopsies was also strongly associated with non-response to interferon therapy in HCV patients (23-28).
In response to viral infection, IFN-a is produced in epithelial cells, plasmacytoid dendritic cells and lymphocytes. The binding of IFN-a to the type I IFN receptor induces a very rapid phosphorylation and activation of some important effectors of the interferon pathway, such as JAKs and STATs, resulting in mRNA translation of IFN-stimulated genes (ISGs) which have a direct antiviral properties. ISGs include myxovirus resistance 1 (MxA), 2’-5’ oligoadenylatesynthetase 1 (OAS-1) and double-stranded RNA (dsRNA)-dependent protein kinase (PKR) (29).
On the basis of these clinical and experimental evidence we aimed to investigate in vitro the possible role of insulin in interfering with IFN-a pathway by analyzing gene and protein expression of some ISGs and to elucidate the mechanisms at the basis. Particularly we wanted to study a possible involvement of SOCS3, as a common player of both insulin and IFN pathways, which is known to be over-expressed by insulin in myocytes. For this purpose, siRNA technology was used.
Human hepatocellular carcinoma HepG2 cells were treated with IFN-a (100 IU/ml) and insulin (100nM and 1000nM) alone or in combination for different incubation-times (2, 4, 8 and 12 hours). Total RNA was isolated from cultured cells. Time course analysis of ISGs mRNA levels (PKR, MxA e OAS-1) was performed by Real Time PCR. PKR protein expression was also evaluated in the same settings by Western-blot analysis. SOCS3-siRNA experiments were set and silencing effect was evaluated through gene expression analysis of ISGs. Our results showed that IFN-a alone was able to enhance the gene expression of all the analyzed ISGs. Insulin alone induces no effect on the gene expression of all ISGs analysed, suggesting specificity of the stimuli. However, HepG2 treated with IFN-a plus insulin (100 and 1000nM) showed a dose-dependent decrease of ISGS mRNA levels compared to controls. This result is more evident when data were expressed as AUC (area under the curve), which considers the total mRNA production during the stimulation. Mean AUC were the following: IFN-a only treatment PKR = 38,97 + 8,60; MxA= 12531,89 + 4667,98; OAS-1= 24,63 + 5,58); IFN+ insulin100nM: PKR = 19,94 + 8,03 (P=0,017); MxA= 3679,34 + 1158,01 (P=0,0103); OAS-1= 7,1 + 3,76 (P=0,002); IFN+ insulin1000nM: PKR = 11,46 + 0,01 (P=0,0017).; MxA= 949,04 + 100,07 (P=0,0186); OAS-1= 1,37 + 1,36(P=0,006). Interestingly, PKR protein expression was also reduced when cells were treated with IFN-a plus insulin (100nM). In order to verify if SOCS3 is involved in the inhibition of IFN-a signalling by insulin, HepG2 were incubated with both insulin and IFN-a and then transfected with SOCS3-siRNA. Silencing of SOCS3 had no effect on ISGs mRNA levels (PKR: 3,28 + 0,43; OAS-1: 2,59 + 0,94; SOCS3: 1,43 + 1,22) if compared to controls [cells incubated with insulin plus IFN-a and cells transfected with non-targeting siRNA (negative control) in witch SOCS3 over-expression is maintained as consequence of insulin effect (PKR: 3,10 + 0,19; OAS-1: 3,1+ 0,92; SOCS3: 7,3 + 0,98)]. Therefore the modulation of IFN-a antiviral activity does not involve over-expression of SOCS3 induced by insulin.
Our results showed that in HepG2 cells insulin significantly reduces the IFN-a mediated induction of three major ISGs in a dose-dependent manner through SOCS3-independent mechanism. These data suggest that insulin levels might play a pivotal role in reduced response to IFN-a based antiviral therapy. Indeed these experimental results support the clinical evidence that control of baseline insulin levels before initiation of IFN-a therapy remains the best choice to improve the response to antiviral therapy in CHC.

Abstract (italian)

L'infezione cronica da virus dell'epatite C (CHC) colpisce 170-200 milioni di persone nel modo. Essa rappresenta una delle principali cause di malattia acuta e cronica del fegato che porta allo sviluppo di cirrosi ed epatocarcinoma (HCC) nei paesi occidentali. Attualmente il trattamento terapeutico dell'HCV è basato sull'utilizzo di interferone-alpha peghilato (PEG-IFN-a) somministrato in combinazione con l'analogo guanosidico ribavirina. Tuttavia, la risposta virologica sostenuta (SVR) si ottiene solo nel 50-80% dei pazienti in base al genotipo virale (1). Il fallimento della terapia antivirale sembra essere legato sia alle caratteristiche dell'ospite che a fattori virali che includono il genotipo virale, la carica virale, l'età, il sesso maschile, l'indice di massa corporea (BMI), il grado di fibrosi e la razza africana. Studi recenti hanno evidenziato che l'insulino-resistenza (IR) è una condizione frequente nei pazienti con CHC se paragonati a pazienti con altre forme di malattia epatica (2), inoltre la prevalenza dell'IR è maggiore nei pazienti infettati con HCV di genotipo 1 (3). Tuttavia i meccanismi che portano all'IR nei pazienti HCV sono di difficile comprensione a causa della coesistenza di numerosi cofattori coinvolti nella sindrome metabolica. Shintany et al (4) ha suggerito che l'HCV sembra essere direttamente coinvolto nella patogenesi dell'IR mediante una modulazione del segnale intracellulare dell'insulina da parte delle proteine virali (5). IR ha una certa rilevanza clinica poiché è associata al diabete di tipo 2 (3, 6) e correla con la progressione della fibrosi epatica, cirrosi e HCC (7, 8). Inoltre, parecchie evidenze cliniche dimostrano che l'IR può essere considerato un fattore predittivo negativo della risposta alla terapia antivirale in pazienti CHC trattati con PEG-IFN-a e ribavirina (9, 10). In un recente studio clinico abbiamo dimostrato che in pazienti HCV l'inefficacia della risposta alla terapia antivirale può essere dimostrata già dopo 24 ore dalla prima iniezione di PEG-IFN, esiste, infatti, una relazione lineare tra il calo viremico e i livelli di insulina al basale più che con l'indice HOMA-IR.
Questi risultati suggeriscono fortemente l'ipotesi che l'insulina, probabilmente favorendo l'over-espressione di fattori intracellulari, possa interferire con il pathway dell'IFN, bloccando l'effetto soppressivo dell'IFN sulla replica virale negli epatociti infettati (12). I meccanismi mediante cui l'insulina interferisce con il segnale dell'IFN-a non sono ancora stati chiariti e sono ancora molto studiati. Questi fattori potrebbero comprendere i membri della famiglia SOCS (Suppressor of Cytokine Signaling), un gruppo di proteine la cui espressione è indotta anche dal trattamento con l'insulina (17). Essi agiscono come regolatori negativi di numerosi pathway intracellulari, tra cui lo stesso segnale dell'insulina, poiché alterano l'attività tirosin chinasica di numerosi recettori di fattori di crescita (13-17). E' stato dimostrato che la soppressione specifica di SOCS3, mediante oligonucleotidi antisenso in topi obesi, porta ad una riduzione dei livelli di insulina in circolo migliorando la sensibilità all'insulina (18, 19). Le proteine SOCS1 e SOCS3 modulano il segnale dell'insulina tramite differenti meccanismi: inibizione dell'attività chinasica del recettore dell'insulina, attraverso la degradazione mediata dall'ubiquitina, e riduzione della fosforilazione a livello della tirosina di IRS-1 e 2 (Insulin Receptor Substrate-1 and 2) (20). Inoltre è stato dimostrato, in vitro, che l'over-espressione di SOCS3 è associata ad una riduzione dell'attività antivirale e anti-proliferativa dell'IFN-a (21, 22) e che in biopsie epatiche di pazienti HCV è strettamente associata alla ridotta risposta alla terapia antivirale (23-28). In risposta all'infezione virale L'IFN-a è prodotto dalle cellule epiteliali, dalle cellule dendritiche e dai linfociti. Il legame dell'IFN-a al recettore di classe I induce una rapida fosforilazione e attivazione di alcuni importanti effettori del segnale intracellulare dell'IFN, come le proteine JAK e STAT, questo porta alla trascrizione dei geni stimolati dall'IFN (ISGs) che hanno importanti proprietà antivirali. Gli ISGs comprendono myxovirus resistance 1 (MxA), 2’-5’ oligoadenylatesynthetase 1 (OAS-1) e double-stranded RNA (dsRNA)-dependent protein kinase (PKR) (29).
Sulla base di queste evidenze cliniche e sperimentali lo scopo di questo progetto di ricerca è stato quello di analizzare in vitro come l'espressione degli IFN-Stimulated-Genes (ISGs), effettori del segnale dell'IFN con proprietà antivirali, possa essere modulata in seguito a trattamento con insulina. Si è voluto inoltre verificare il possibile meccanismo mediante il quale l'insulina è in grado di interagire sulla risposta cellulare all'IFN- a andando a valutare il ruolo l'espressione di un membro della famiglia di Suppressor of Cytokine Signalling (SOCS), SOCS3. E' noto, infatti, che l'espressione di SOCS3 aumenta notevolmente dopo trattamento con insulina. A tal scopo la tecnologia utilizzata è stata quella degli siRNA.
La linea cellulare di epatocarcinoma HepG2 è stata trattata con IFN-a (100 IU/ml) e insulina (100nM and 1000nM) da soli o in co-stimolazione per diversi tempi di incubazione (2, 4, 8 e 12 ore). Dopo l'estrazione dell'RNA totale è stata eseguita l'analisi di espressione genica degli ISGs (PKR, MxA e OAS-1) mediante Real Time PCR. Sugli stessi campioni è stata fatta anche l'analisi dell'espressione proteica di PKR mediante Western-blot. Utilizzando lo stesso modello sperimentale sono stati condotti degli esperimenti di siRNA sui quali è stato valutato l'effetto del silenziamento di SOCS3 sull'espressione genica degli ISGs. Come da atteso il trattamento con solo IFN-a induce un aumento dei livelli di RNA di tutti i geni ISGs analizzati. Lo stimolo con sola insulina non ha alcun effetto sull'espressione genica degli ISGs; questo dimostra che la loro over-espressione è stimolo-specifica ed avvalora la specificità dell'analisi condotta. Tuttavia, il pre-trattamento della linea cellulare HepG2 con diverse concentrazioni di insulina (100 e 1000 nM) e poi lo stimolo con IFN-a (100 UI/mL) ha dimostrato chiaramente che l'insulina è in grado di diminuire l'espressione genica degli ISGs in modo dose-dipendente. Tale risultato è particolarmente evidente se espresso come AUC (area under the curve), che considera la produzione totale di trascritto durante tutto il tempo di stimolazione. AUC media: trattamento con solo IFN-a: PKR = 38,97 + 8,60; MxA= 12531,89 + 4667,98; OAS-1= 24,63 + 5,58); IFN+ insulina100nM: PKR = 19,94 + 8,03 (P=0,017); MxA= 3679,34 + 1158,01 (P=0,0103); OAS-1= 7,1 + 3,76 (P=0,002); IFN+ insulina1000nM: PKR = 11,46 + 0,01 (P=0,0017).; MxA= 949,04 + 100,07 (P=0,0186); OAS-1= 1,37 + 1,36(P=0,006). I risultati ottenuti dall’analisi quantitativa dell’mRNA di PKR hanno trovato un solido riscontro nella successiva valutazione dell’espressione di PKR proteina. Si osserva, infatti, una significativa riduzione della proteina, rispetto al trattamento con solo IFN-a, quando le cellule HepG2 vengono incubate sia con insulina (100 nM) che con IFN-a (100UI/ml). Allo scopo di verificare il ruolo di SOCS3, indotto dall'insulina, come possibile soppressore citoplasmatico dello stimolo interferonico le cellule HepG2 sono state trasfettate con SOCS3-siRNA e stimolate con insulina e IFN-a. Il silenziamento di SOCS3 non ha alcun effetto positivo sui livelli di mRNA dei geni ISGs analizzati (PKR: 3,28 + 0,43; OAS-1: 2,59 + 0,94; SOCS3: 1,43 + 1,22), se confrontati con il controllo [cellule trasfettate con non-targeting siRNA e trattate con insulina e IFN-a (controllo negativo) nelle quali l'over-espressione di SOCS3 è mantenuta come conseguenza dell'effetto dell'insulina (PKR: 3,10 + 0,19; OAS-1: 3,1+ 0,92; SOCS3: 7,3 + 0,98)]. Questi risultati dimostrano che la modulazione dell'attività antivirale dell' IFN-a, da parte dell'insulina, non è dovuta all'over-espressione di SOCS3.
I nostri risultati dimostrano che l'insulina riduce, in modo dose-dipendente, l'espressione dei tre principali geni ISGs analizzati attraverso un meccanismo SOCS3-indipendente. Questi dati suggeriscono che i livelli di insulina potrebbero svolgere un ruolo cruciale nel ridurre la risposta alla terapia antivirale basata sull'uso dell'IFN. Questi dati sperimentali sono a supporto delle evidenze cliniche in quanto sottolineano che il controllo dei livelli basali di insulina prima dell'inizio della terapia con IFN-a rimane ad oggi la scelta migliore per aumentare la risposta antivirale.

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EPrint type:Ph.D. thesis
Data di deposito della tesi:UNSPECIFIED
Anno di Pubblicazione:29 January 2010
Key Words:Virus dell'epatite C (HCV), Insulino Resistenza, SOCS3, geni correlati all'interferone (ISGs)
Settori scientifico-disciplinari MIUR:Area 06 - Scienze mediche > MED/04 Patologia generale
Struttura di riferimento:Dipartimenti > pre 2012 - Dipartimento di Scienze Chirurgiche Gastroenterologiche "Pier Giuseppe Cevese"
Codice ID:2786
Depositato il:20 Sep 2010 13:06
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