Adenosine A2a receptor stimulation prevents hepatocyte lipotoxicity and non-alcoholic steatohepatitis (NASH) in rats

Abstract

NEFA (non-esterified 'free' fatty acid)-mediated lipotoxicity plays a critical role in the pathogenesis of NASH (non-alcoholic steatohepatitis). In the light of the growing need for new therapeutic options for NASH, we investigated the action of A2aR (adenosine A(2a) receptor) stimulation against lipotoxicity. The effects of the A(2a)R agonist CGS21680 [2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxyamidoadenosine] were evaluated 'in vitro' in liver cells exposed to SA (stearic acid) and 'in vivo' in rats with NASH induced by 8 weeks of feeding with an MCD diet (methionine/choline-deficient diet). In cultured hepatocytes, SA promoted apoptosis by inducing MKK4 (mitogen-activated protein kinase kinase 4)/SEK1 (stress-activated protein kinase/extracellular-signal-regulated kinase kinase-1) and JNK-1/2 (c-Jun N-terminal kinase-1/2) activation. CGS21680 addition prevented JNK-1/2 activation and reduced apoptosis without interfering with lipid accumulation. CGS21680 action required PI3K (phosphoinositide 3-kinase)/Akt-mediated block of MKK4/SEK1. Consistently, PI3K inhibition with wortmannin abolished the cytoprotective action of CGS21680 and reverted MKK4 inhibition. SA lipotoxicity was also prevented by transfecting HTC cells with a specific MKK4/SEK1 siRNA (small interfering RNA). In rats receiving the MCD diet, the development of NASH was associated with MKK4/SEK1 and JNK-1/2 activation. CGS21680 (0.5 mg/kg of body weight, intraperitoneal) administration to MCD-fed rats prevented JNK-1/2 activation by acting on MKK4/SEK1. CGS21680 also effectively reduced NASH-associated ALT (alanine aminotransferase) release, hepatocyte apoptosis, liver inflammation and fibrosis without affecting hepatic steatosis. Taken together, these results demonstrate that, by inhibiting JNK-1/2, A(2a)R stimulation reduces lipotoxicity and ameliorates NASH, giving a rationale to investigate A(2a)R agonists as possible new therapeutic agents in preventing fatty liver progression to NASH.

The balance between IL-17 and IL-22 produced by liver-infiltrating T-helper cells critically controls NASH development in mice

“…This feature allowed us to characterize the kinetic and the hepatic levels of different CD4 + T-cell subsets during the early phase of steatohepatitis development (first week), established steatohepatitis (second to fourth weeks) and evolution steatohepatitis/fibrosis (fourth to eighth week) as measured by ALT transaminase release, hepatic TG content, histological changes and pro-inflammatory/profibrotic cytokine content. In accordance with previous observations [26], the MCD diet induced a progressive increase in hepatic TG content ( Figure 1A) that paralleled the extension of steatosis ( Figure 1B). There was a significant increase in ALT release after just 1 week on the MCD diet, which peaked at the second week and remained significantly higher than that of mice receiving the ND (normal diet) at the fourth and eighth weeks ( Figure 1A).…”

“…Hepatocytes were obtained by differential centrifugation at 50 g for 5 min at 4 • C. Hepatocyte suspensions (purity >95 %) were plated on collagen-coated culture dishes and cultured for 48 h in DMEM (Dulbecco's modified Eagle's medium)/Ham's F12 containing 10 % (v/v) FBS, 1 % penicillin/streptomycin and 1 % glutamine. For lipotoxicity assays [26], hepatocytes were incubated with fresh medium supplemented with 50 μmol/l PA (palmitic acid) (Sigma) in the presence or absence of 10 nmol/l recombinant murine IL-17 and or IL-22 (eBioscience).…”

Ursodeoxycholyl Lysophosphatidylethanolamide Inhibits Lipoapoptosis by Shifting Fatty Acid Pools toward Monosaturated and Polyunsaturated Fatty Acids in Mouse Hepatocytes

“…GPCR activation leads to an elevation of cAMP (Hutchinson et al, 2008), which mediates apoptosis inhibition (Webster and Anwer, 1998;Kwon et al, 2004). The observed UDCA-LPE lipoprotection via cAMP/PKA signaling supports the possibility that UDCA-LPE may interact with specific GPCRs, among which are lipid-sensing GPCRs [e.g., sphingosine-1-phosphate receptor 2 (Studer et al, 2012) and adenosine A2a receptor (Imarisio et al, 2012)]. UDCA-LPE (or cAMP generated by UDCA-LPE) may interact with these GPCRs to induce PKA signaling leading to activation of cytoprotective signals Akt and ERK (Dent et al, 2005).…”

Mouse hepatocytes and LSEC proteome reveal novel mechanisms of ischemia/reperfusion damage and protection by A2aR stimulation

“…This suggests that the genomic changes induced by A2aR stimulation accomplish a full-protected phenotype only in presence of cell stress. Indeed, recent results showed that A2aR stimulation might also effectively prevent pathological conditions different from IR through the activation of noxious-specific mechanisms of protection [36].…”