Selective Blockade of the Metabotropic Glutamate Receptor mGluR5 Protects Mouse Livers in In Vitro and Ex Vivo Models of Ischemia Reperfusion Injury

Abstract

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.

The selective blockade of metabotropic glutamate receptor-5 attenuates fat accumulation in an <em>in vitro</em> model of benign steatosis

“…A glutamate accumulation may be responsible for the mGluR5 hyperactivation as already seen in the promotion of liver ischemic injury. 14 , 16 Recently, serum and liver levels of glutamate/glutamine ratio have been show to increase significantly in patients with NAFLD; the increase in glutamate/glutamine ratio significantly correlated with the severity of NAFLD and fibrosis. 53 The underlying mechanism for altered glutamate levels and the associated correlation with NAFLD severity is unclear.…”

“…Cell extracts containing the same amount of proteins were separated in SDS-PAGE on 7, 10 or 12% acrylamide gels, and transferred to PVDF membrane, as previously described. 14 Unspecific sites were blocked for 2 h with 5% BSA in tris-buffered saline (TBS, 20 mM Tris/HCl, 500 mM sodium chloride, pH 7.5, 0.1% Tween 20) at 4°C. The membranes were incubated with primary antibodies overnight at 4°C, under gentle agitation, while secondary antibodies were incubated at the same condition for 1 h. Primary antibodies against mGluR5, SREBP-1, PPAR-α, iNOS, NFkB p65 and phospho-NFkB p65, cleaved caspase- 3, tubulin and actin were used at 1:1000 dilution.…”

“… 13 More recently, it has been shown in ex vivo models of hepatic cold and warm ischemia that mGluR5 blockade stops mGluR5 hyperactivation and reduces the expression of inflammatory factors such as TNF-α and iNOS. 14 The mechanism underlying the increase in glutamate release involved in mGluR5 hyperactivation has not yet been completely elucidated; however, an increase in glutamate release has been observed under hypoxic conditions in isolated rat hepatocytes 7 and in a rat portal vein ligation model. 15 The resulting glutamate accumulation was considered the culprit for mGluR5 hyperactivation involved in the promotion of liver ischemic injury.…”

“… 15 The resulting glutamate accumulation was considered the culprit for mGluR5 hyperactivation involved in the promotion of liver ischemic injury. 14 , 16 Recently, it has been shown that an increased glutamate secretion also occurs in an alcoholinduced steatosis model, resulting in mGluR5s hyperactivation and abnormal fat accumulation in hepatocytes. 17 No study is currently available on the mGluR5 role in the development of NAFLD; then, the aim of this work was to elucidate the role of mGluR5 in an in vitro model of steatosis in HepG2 cells.…”

Molecular Mechanisms and Pathophysiology of Ischemia-Reperfusion Injury

“…An example is the mitochondrial enzyme ALDH2, which has mostly been studied in ethanol detoxification. ALDH2 activation has been associated with hepatoprotection [8], but in other cases it seems that the process of enzyme inactivation is associated with hepatoprotection; indeed, recent investigations of the blockade of PPARγ [9] or the glutamate receptor mGluR5, which can halt the excitotoxic events, have shown benefits in ischemia alone and in both warm and cold IRI [10].…”