Insulin like growth factor-1 increases fatty liver preservation in IGL-1 solution

AIM: To investigate the benefits of insulin like growth factor-1 (IGF-1) supplementation to serum-free institut georges lopez-1 (IGL-1) solution to protect fatty liver against cold ischemia reperfusion injury. METHODS: Steatotic livers were preserved for 24 h in IGL-1  solution supplemented with or without IGF-1 and then perfused "ex vivo " for 2 h at 37℃. We examined the effects of IGF-1 on hepatic damage and function (transaminases, percentage of sulfobromophthalein clearance in bile and vascular resistance). We also studied other factors associated with the poor tolerance of fatty livers to cold ischemia reperfusion injury such as mitochondrial damage, oxidative stress, nitric oxide, tumor necrosis factor-α (TNF-α) and mitogen-activated protein kinases.RESULTS: Steatotic livers preserved in IGL-1 solutionsupplemented with IGF-1 showed lower transaminase levels, increased bile clearance and a reduction in vascular resistance when compared to those preserved in IGL-1solution alone. These benefits are mediated by activation of AKT and constitutive endothelial nitric oxide synthase (eNOS), as well as the inhibition of inflammatory cytokines such as TNF-α. Mitochondrial damage and oxidative stress were also prevented.CONCLUSION: IGL-1  enrichment with IGF-1 increasedfatty liver graft preservation through AKT and eNOS activation, and prevented TNF-α release during normothermic reperfusion.

Hypoxia inducible factor-1αaccumulation in steatotic liver preservation:Role of nitric oxide

AIM:To examine the relevance of hypoxia inducible factor(HIF-1)and nitric oxide(NO)on the preservation of fatty liver against cold ischemia-reperfusion injury(IRI). METHODS:We used an isolated perfused rat liver model and we evaluated HIF-1αin steatotic and non-steatotic livers preserved for 24 h at 4℃in University of Wisconsin and IGL-1 solutions,and then subjected to 2 h of normothermic reperfusion.After normoxic reperfusion,liver enzymes,bile production,bromosulfophthalein clearance,as well as HIF-1αand NO[endothelial NO synthase(eNOS)activity and nitrites/nitrates]were also measured.Other factors associated with the higher susceptibility of steatotic livers to IRI,such as mitochondrial damage and vascular resistance were evaluated. RESULTS:A significant increase in HIF-1αwas found in steatotic and non-steatotic livers preserved in IGL-1 after cold storage.Livers preserved in IGL-1 showed a significant attenuation of liver injury and improvement in liver function parameters.These benefits were enhanced by the addition of trimetazidine(an antiischemic drug),which induces NO and eNOS activation, to IGL-1 solution.In normoxic reperfusion,the presence of NO favors HIF-1αaccumulation,promoting also the activation of other cytoprotective genes,such as hemeoxygenase-1. CONCLUSION:We found evidence for the role of the HIF-1α/NO system in fatty liver preservation,especially when IGL-1 solution is used.

Role of sirtuins in ischemia-reperfusion injury

Ischemia-reperfusion injury(IRI)remains an unresolved and complicated situation in clinical practice,especially in the case of organ transplantation.Several factors contribute to its complexity;the depletion of energy during ischemia and the induction of oxidative stress during reperfusion initiate a cascade of pathways that lead to cell death and finally to severe organ injury.Recently,the sirtuin family of nicotinamide adenine dinucleotide-dependent deacetylases has gained increasing attention from researchers,due to their involvement in the modulation of a wide variety of cellular functions.There are seven mammalian sirtuins and,among them,the nuclear/cytoplasmic sirtuin 1(SIRT1)and the mitochondrial sirtuin 3(SIRT3)are ubiquitously expressed in many tissue types.Sirtuins are known to play major roles in protecting against cellular stress and in controlling metabolic pathways,which are key processes during IRI.In this review,we mainly focus on SIRT1 and SIRT3 and examine their role in modulating pathways against energy depletion during ischemia and their involvement in oxidative stress,apoptosis,microcirculatory stress and inflammation during reperfusion.We present evidence of the beneficial effects of sirtuins against IRI and emphasize the importance of developing new strategies by enhancing their action.

Polyethylene glycol rinse solution:An effective way to prevent ischemia-reperfusion injury

AIM: To test whether a new rinse solution containing polyethylene glycol 35 (PEG-35) could prevent ischemia-reperfusion injury (IRI) in liver grafts.

Sirtuin 1 in rat orthotopic liver transplantation:An I GL-1 preservation solution approach

AIM:To investigate the possible involvement of Sirtuin1(SIRT1)in rat orthotopic liver transplantation(OLT),when Institute Georges Lopez 1(IGL-1)preservation solution is enriched with trimetazidine(TMZ).METHODS:Male Sprague-Dawley rats were used as donors and recipients.Livers were stored in IGL-1 preservation solution for 8h at 4℃,and then underwent OLT according to Kamada’s cuff technique without arterialization.In another group,livers were stored in IGL-1 preservation solution supplemented with TMZ,at10-6 mol/L,for 8 h at 4℃and then underwent OLT.Rats were sacrificed 24 h after reperfusion,and liver and plasma samples were collected.Liver injury(transaminase levels),mitochondrial damage(glutamate dehydrogenase activity)oxidative stress(malondialdehyde levels),and nicotinamide adenine dinucleotide(NAD+),the cofactor necessary for SIRT1 activity,were determined by biochemical methods.SIRT1 and its substrates(acFox O1,ac-p53),the precursor of NAD+,nicotinamide phosphoribosyltransferase(NAMPT),as well as the phosphorylation of adenosine monophosphate activated protein kinase(AMPK),p-m TOR,p-p70S6K(direct substrate of m TOR),autophagy parameters(beclin-1,LC3B)and MAP kinases(p-p38 and p-ERK)were determined by Western blot.RESULTS:Liver grafts preserved in IGL-1 solution enriched with TMZ presented reduced liver injury and mitochondrial damage compared with those preservedin IGL-1 solution alone.In addition,livers preserved in IGL-1+TMZ presented reduced levels of oxidative stress.This was consistent with enhanced SIRT1 protein expression and elevated SIRT1 activity,as indicated by decreased acetylation of p53 and Fox O1.The elevated SIRT1 activity in presence of TMZ can be attributed to the enhanced NAMPT protein and NAD+/NADH levels.Up-regulation of SIRT1 was consistent with activation of AMPK and inhibition of phosphorylation of m TOR and its direct substrate(p-p70S6K).As a consequence,autophagy mediators(beclin-1 and LC3B)were overexpressed.Furthermore,MAP kinases were regulated in livers preserved with IGL-1+TMZ,as they were characterized by enhanced p-ERK and decreased p-p38protein expression.CONCLUSION:Our study shows that IGL-1 preservation solution enriched with TMZ protects liver grafts from the IRI associated with OLT,through SIRT1 up-regulation.

Relevance of proteolysis and proteasome activation in fatty liver graft preservation: An Institut Georges Lopez-1 vs University of Wisconsin appraisal

To compare liver proteolysis and proteasome activation in steatotic liver grafts conserved in University of Wisconsin (UW) and Institut Georges Lopez-1 (IGL-1) solutions.METHODSFatty liver grafts from male obese Zücker rats were conserved in UW and IGL-1 solutions for 24 h at 4 °Cand subjected to “ex vivo” normo-thermic perfusion (2 h; 37 °C). Liver proteolysis in tissue specimens and perfusate was measured by reverse-phase high performance liquid chromatography. Total free amino acid release was correlated with the activation of the ubiquitin proteasome system (UPS: measured as chymotryptic-like activity and 20S and 19S proteasome), the prevention of liver injury (transaminases), mitochondrial injury (confocal microscopy) and inflammation markers (TNF 1 alpha, high mobility group box-1 (HGMB-1) and PPAR gamma), and liver apoptosis (TUNEL assay, cytochrome c and caspase 3).RESULTSProfiles of free AA (alanine, proline, leucine, isoleucine, methionine, lysine, ornithine, and threonine, among others) were similar for tissue and reperfusion effluent. In all cases, the IGL-1 solution showed a significantly higher prevention of proteolysis than UW (P < 0.05) after cold ischemia reperfusion. Livers conserved in IGL-1 presented more effective prevention of ATP-breakdown and more inhibition of UPS activity (measured as chymotryptic-like activity). In addition, the prevention of liver proteolysis and UPS activation correlated with the prevention of liver injury (AST/ALT) and mitochondrial damage (revealed by confocal microscopy findings) as well as with the prevention of inflammatory markers (TNF1alpha and HMGB) after reperfusion. In addition, the liver grafts preserved in IGL-1 showed a significant decrease in liver apoptosis, as shown by TUNEL assay and the reduction of cytochrome c, caspase 3 and P62 levels.CONCLUSIONOur comparison of these two preservation solutions suggests that IGL-1 helps to prevent ATP breakdown more effectively than UW and subsequently achieves a higher UPS inhibition and reduced liver proteolysis.

Losartan activates sirtuin 1 in rat reduced-size orthotopic liver transplantation

AIM: To investigate a possible association between losartan and sirtuin 1(SIRT1) in reduced-size orthotopic liver transplantation(ROLT) in rats.METHODS: Livers of male Sprague-Dawley rats(200-250 g) were preserved in University of Wisconsin preservation solution for 1 h at 4 ℃ prior to ROLT.In an additional group,an antagonist of angiotensin Ⅱ type 1 receptor(AT1R),losartan,was orally administered(5 mg/kg) 24 h and 1 h before the surgical procedure to both the donors and the recipients.Transaminase(as an indicator of liver injury),SIRT1 activity,and nicotinamide adenine dinucleotide(NAD+,a co-factor necessary for SIRT1 activity) levels were determined by biochemical methods.Protein expression of SIRT1,acetylated Fox O1(ac-Fox O1),NAMPT(the precursor of NAD+),heat shock proteins(HSP70,HO-1) expression,endoplasmic reticulum stress(GRP78,IRE1 a,p-e IF2) and apoptosis(caspase 12 and caspase 3) parameters were determined by Western blot.Possible alterations in protein expression of mitogen activated protein kinases(MAPK),such as p-p38 and p-ERK,were also evaluated.Furthermore,the SIRT3 protein expression and m RNA levels were examined.RESULTS: The present study demonstrated that losartan administration led to diminished liver injury when compared to ROLT group,as evidenced by the significant decreases in alanine aminotransferase(358.3 ± 133.44 vs 206 ± 33.61,P < 0.05) and aspartate aminotransferase levels(893.57 ± 397.69 vs 500.85 ± 118.07,P < 0.05).The lessened hepatic injury in case of losartan was associated with enhanced SIRT1 protein expression and activity(5.27 ± 0.32 vs 6.08 ± 0.30,P < 0.05).This was concomitant with increased levels of NAD+(0.87 ± 0.22 vs 1.195 ± 0.144,P < 0.05) the co-factor necessary for SIRT1 activity,as well as with decreases in ac-Fox O1 expression.Losartan treatment also provoked significant attenuation of endoplasmic reticulum stress parameters(GRP78,IRE1 a,p-e IF2) which was consistent with reduced levels of both caspase 12 and caspase 3.Furthermore,losartan administration stimulated HSP70 protein expression and attenuated HO-1 expression.However,no changes were observed in protein or m RNA expression of SIRT3.Finally,the protein expression pattern of p-ERK and p-p38 were not altered upon losartan administration.CONCLUSION: The present study reports that losartan induces SIRT1 expression and activity,and that it reduces hepatic injury in a ROLT model.