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2-Methyl-6-(phenylethynyl)pyridine (MPEP), a negative allosteric modulator of the metabotropic glutamate receptor (mGluR) 5, protects hepatocytes from ischemic injury. In astrocytes and microglia, MPEP depletes ATP. These findings seem to be self-contradictory, since ATP depletion is a fundamental stressor in ischemia. This study attempted to reconstruct the mechanism of MPEP-mediated ATP depletion and the consequences of ATP depletion on protection against ischemic injury. We compared the effects of MPEP and other mGluR5 negative modulators on ATP concentration when measured in rat hepatocytes and acellular solutions. We also evaluated the effects of mGluR5 blockade on viability in rat hepatocytes exposed to hypoxia. Furthermore, we studied the effects of MPEP treatment on mouse livers subjected to cold ischemia and warm ischemia reperfusion. We found that MPEP and 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) deplete ATP in hepatocytes and acellular solutions, unlike fenobam. This finding suggests that mGluR5s may not be involved, contrary to previous reports. MPEP, as well as MTEP and fenobam, improved hypoxic hepatocyte viability, suggesting that protection against ischemic injury is independent of ATP depletion. Significantly, MPEP protected mouse livers in two different ex vivo models of ischemia reperfusion injury, suggesting its possible protective deployment in the treatment of hepatic inflammatory conditions. [ABSTRACT FROM AUTHOR]

Background: We previously showed that increased asymmetric dimethylarginine (ADMA) biliary excretion occurs during hepatic ischemia/reperfusion (I/R), prompting us to study the effects of the farnesoid X receptor (FXR) agonist obeticholic acid (OCA) on bile, serum and tissue levels of ADMA after I/R. Material and methods: Male Wistar rats were orally administered 10mg/kg/day of OCA or vehicle for 5 days and were subjected to 60 min partial hepatic ischemia or sham-operated. After a 60 min reperfusion, serum, tissue and bile ADMA levels, liver mRNA and protein expression of ADMA transporters (CAT-1, CAT-2A, CAT-2B, OCT-1, MATE-1), and enzymes involved in ADMA synthesis (protein-arginine-N-methyltransferase-1, PRMT-1) and metabolism (dimethylarginine-dimethylaminohydrolase-1, DDAH-1) were measured. Results: OCA administration induced a further increase in biliary ADMA levels both in sham and I/R groups, with no significant changes in hepatic ADMA content. A reduction in CAT-1, CAT-2A or CAT-2B transcripts was found in OCA-treated sham-operated rats compared with vehicle. Conversely, OCA administration did not change CAT-1, CAT-2A or CAT-2B expression, already reduced by I/R. However, a marked decrease in OCT-1 and increase in MATE-1 expression was observed. A similar trend occurred with protein expression. Conclusion: The reduced mRNA expression of hepatic CAT transporters suggests that the increase in serum ADMA levels is probably due to decreased liver uptake of ADMA from the systemic circulation. Conversely, the mechanism involved in further increasing biliary ADMA levels in sham and I/R groups treated with OCA appears to be MATE-1-dependent. [ABSTRACT FROM AUTHOR]

We compared static preservation (cold storage, CS, 4 °C) with dynamic preservation (machine perfusion, MP, 20 °C) followed by reperfusion using marginal livers: a model of donation after cardiac death (DCD) livers and two models of fatty livers, the methionine-choline deficient (MCD) diet model, and obese Zucker (fa/fa) rats. CS injury in DCD livers was reversed by an oxygenated washout (OW): hepatic damage, bile flow, and the ATP/ADP ratio in the OW + CS group was comparable with the ratio obtained with MP. Using fatty livers, CS preservation induced a marked release in hepatic and biliary enzymes in obese Zucker rats when compared with the MCD group. The same trend occurred for bile flow. No difference was found when comparing MP in MCD and obese Zucker rats. Fatty acid analysis demonstrated that the total saturated (SFA)/polyunsaturated fatty acid (PUFA) ratio was, respectively, 1.5 and 0.71 in obese Zucker and MCD rats. While preservation damage in DCD livers is associated with the ATP/ADP recovered with OW, injury in fatty livers is linked to fatty acid constituents: livers from obese. Zucker rats, with greater content in saturated FA, might be more prone to CS injury. On the contrary, MCD livers with elevated PUFA content might be less susceptible to hypothermia. [ABSTRACT FROM AUTHOR]

We investigated changes in fatty acid desaturases, D5D, D6D, D9-16D and D9-18D, and their relationship with oxidative stress, matrix metalloproteinases (MMPs) and serum TNF-alpha in two rat models of non-alcoholic fatty liver disease NAFLD. Eight-week-old male Wistar rats fed for 3 weeks with methionine-choline–deficient (MCD) diet and eleven-week-old Obese male Zucker rats were used. Serum levels of hepatic enzymes and TNF-alpha were quantified. Hepatic oxidative stress (ROS, TBARS and GSH content) and MMP-2 and MMP-9 (protein expression and activity) were evaluated. Liver fatty acid profiling, performed by GC-MS, was used for the quantification of desaturase activities. Higher D5D and D9-16D were found in Obese Zucker rats as well as an increase in D9-18D in MCD rats. D6D was found only in MCD rats. A negative correlation between D5D and D9-16D versus TBARS, ROS and TNF-alpha and a positive correlation with GSH were shown in fatty livers besides a positive correlation between D9-18D versus TBARS, ROS and TNF-alpha and a negative correlation with GSH. A positive correlation between D5D or D9-16D or D9-18D versus protein expression and the activity of MMP-2 were found. NAFLD animal models showed comparable serum enzymes. These results reinforce and extend findings on the identification of therapeutic targets able to counteract NAFLD disorder. [ABSTRACT FROM AUTHOR]

While it is well established that various factors can impair the production and flow of bile and lead to cholestatic disease in hepatic and extrahepatic sites, an enhanced assessment of the biomarkers of the underlying pathophysiological mechanisms is still needed to improve early diagnosis and therapeutic strategies. Hence, we investigated fluorescing endogenous biomolecules as possible intrinsic biomarkers of molecular and cellular changes in cholestasis. Spectroscopic autofluorescence (AF) analysis was performed using a fiber optic probe (366 nm excitation), under living conditions and in serum, on the livers of male Wistar rats submitted to bile duct ligation (BDL, 24, 48, and 72 h). Biomarkers of liver injury were assayed biochemically. In the serum, AF analysis distinctly detected increased bilirubin at 24 h BDL. A continuous, significant increase in red-fluorescing porphyrin derivatives indicated the subversion of heme metabolism, consistent with an almost twofold increase in the serum iron at 72 h BDL. In the liver, changes in the AF of NAD(P)H and flavins, as well as lipopigments, indicated the impairment of mitochondrial functionality, oxidative stress, and the accumulation of oxidative products. A serum/hepatic AF profile can be thus proposed as a supportive diagnostic tool for the in situ, real-time study of bio-metabolic alterations in bile duct ligation (BDL) in experimental hepatology, with the potential to eventually translate to clinical diagnosis. [ABSTRACT FROM AUTHOR]

It has been previously found that the blockade of metabotropic glutamate receptor type 5 (mGluR5) protects against hepatic ischemia/reperfusion injury and acetaminophen toxicity. The role of mGluR5 in NAFLD has not yet been elucidated. Here, we evaluated the effects of mGluR5 blockade in an in vitro model of steatosis. HepG2 cells were pre-incubated for 12 h with an mGluR5 agonist, a negative allosteric modulator (DHPG and MPEP, respectively) or vehicle, then treated with 1.5 mM oleate/palmitate (O/P) for another 12 h. Cell viability was evaluated with the MTT assay; fat accumulation was measured using the fluorescent dye nile red; SREBP-1, PPAR-α, iNOS and Caspase-3 protein expression were evaluated by Western blot; NFkB activity was evaluated as pNFkB/NFkB ratio. mGluR5 modulation did not alter cell viability in O/P-incubated cells; MPEP prevented intracellular lipid accumulation in O/P treated cells; MPEP administration was also associated with a reversion of O/P-induced changes in SREBP-1 and PPAR-α expression, involved in free fatty acid (FFA) metabolism and uptake. No changes were observed in iNOS and Caspase-3 expression, or in NFkB activity. In conclusion, mGluR5 pharmacological blockade reduced fat accumulation in HepG2 cells incubated with O/P, probably by modulating the expression of SREBP-1 and PPAR-α.

We compared static preservation (cold storage, CS, 4 °C) with dynamic preservation (machine perfusion, MP, 20 °C) followed by reperfusion using marginal livers: a model of donation after cardiac death (DCD) livers and two models of fatty livers, the methionine-choline deficient (MCD) diet model, and obese Zucker (fa/fa) rats. CS injury in DCD livers was reversed by an oxygenated washout (OW): hepatic damage, bile flow, and the ATP/ADP ratio in the OW + CS group was comparable with the ratio obtained with MP. Using fatty livers, CS preservation induced a marked release in hepatic and biliary enzymes in obese Zucker rats when compared with the MCD group. The same trend occurred for bile flow. No difference was found when comparing MP in MCD and obese Zucker rats. Fatty acid analysis demonstrated that the total saturated (SFA)/polyunsaturated fatty acid (PUFA) ratio was, respectively, 1.5 and 0.71 in obese Zucker and MCD rats. While preservation damage in DCD livers is associated with the ATP/ADP recovered with OW, injury in fatty livers is linked to fatty acid constituents: livers from obese. Zucker rats, with greater content in saturated FA, might be more prone to CS injury. On the contrary, MCD livers with elevated PUFA content might be less susceptible to hypothermia., Competing Interests: The authors declare no conflict of interest.

Metabotropic glutamate receptor subtype 5 (mGluR5) is a Group I mGlu subfamily of receptors coupled to the inositol trisphosphate/diacylglycerol pathway. Like other mGluR subtypes, mGluR5s contain a phylogenetically conserved, extracellular orthosteric binding site and a more variable allosteric binding site, located on the heptahelical transmembrane domain. The mGluR5 receptor has proved to be a key pharmacological target in conditions affecting the central nervous system (CNS) but its presence outside the CNS underscores its potential role in pathologies affecting peripheral organs such as the gastrointestinal (GI) tract and accessory digestive organs such as the tongue, liver and pancreas. Following identification of mGluR5s in the mouth, various studies have subsequently demonstrated its involvement in mechanical allodynia, inflammation, pain and oral cancer. mGluR5 expression has also been identified in gastroesophageal vagal pathways. Indeed, experimental and human studies have demonstrated that mGluR5 blockade reduces transient lower sphincter relaxation and reflux episodes. In the intestine, mGluR5s have been shown to be involved in the control of intestinal inflammation, visceral pain and the epithelial barrier function. In the liver, mGluR5s have a permissive role in the onset of ischemic injury in rat and mice hepatocytes. Conversely, livers from mice treated with selective negative allosteric modulators and mGluR5 knockout mice are protected against ischemic injury. Similar results have been observed in experimental models of free-radical injury and in vivo mouse models of acetaminophen intoxication. Finally, mGluR5s in the pancreas are associated with insulin secretion control. The picture is, however, far from complete as the review attempts to establish in particular as regards identifying specific targets and innovative therapeutic approaches for the treatment of GI disorders., Competing Interests: Conflict-of-interest statement: Ferrigno A, Berardo C, Di Pasqua LG, Siciliano V, Richelmi P and Vairetti M declare no conflict of interest related to this publication.