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Regulation of the Amino Acid Exchanger xCT (Slc7a11) in Acute Pancreatitis


Kuster, Evelyne. Regulation of the Amino Acid Exchanger xCT (Slc7a11) in Acute Pancreatitis. 2018, University of Zurich, Faculty of Science.

Abstract

Acute pancreatitis is a potentially lethal, inflammatory disease of the exocrine pancreas. A major hallmark during the pathogenesis of acute pancreatitis is the development of oxidative stress. Thereby, increased levels of reactive oxygen species (ROS) have deleterious effects by damaging lipids, proteins and nucleic acids. ROS can be neutralised by glutathione (GSH), the most abundant intracellular antioxidant, but GSH concentrations themselves are depleted rapidly in acute pancreatitis. To restore the intracellular GSH pool, GSH is synthesised from the amino acids cysteine, glutamate and glycine. The intracellular availability of cysteine is considered as the rate limiting factor of GSH synthesis.
In the healthy state, the pancreas is the organ with the highest protein synthesis rate. The majority of these proteins are digestive enzymes that are released within the pancreatic juice into the duodenum to facilitate meal digestion. To enable such a high protein translation rate, the pancreas is required to import extensive amounts of amino acids from the plasma, which is mediated by amino acid transporters. Importantly, pancreatic acinar cells dedifferentiate during acute pancreatitis, which is reflected by a reduced protein synthesis rate.
We postulated that acinar cells have altered metabolic needs during acute pancreatitis due to the reduced protein synthesis rate and simultaneously increased GSH synthesis. These alterations may require adaptations in amino acid transporter expression to satisfy the particular demands of amino acids in the inflammatory state. It was therefore the aim of this thesis project to characterise amino acid transporter expression during acute pancreatitis, with a particular focus on the transport of cysteine and its oxidised form cystine.
To address this aim, acute pancreatitis was induced in wild type mice by repetitive administrations of supraphysiological doses of cerulein, an analogue of cholecystokinin. Among the cysteine and cystine transporters screened at 6 hours of acute pancreatitis, a prominent up regulation of the cystine transporter xCT (Slc7a11) mRNA was observed. Moreover, this mRNA increase led to enhanced xCT protein levels, which was quantified by Western blot analysis. During acute pancreatitis, the up regulated transporter xCT was localised to the acinar cell membrane via immunofluorescence technique. In the control mice, the protein could not be visualised. The functional consequences of the increased xCT protein expression was investigated with ex vivo uptake experiments using radiolabelled cystine and freshly isolated acinar cells. Indeed, the enhanced xCT protein levels resulted in higher cystine uptake rates after the induction of acute pancreatitis. Moreover, using isolated acinar cells derived from xCT-/- mice, we could demonstrate that xCT is the only cystine transporter expressed in the pancreas.
We further aimed to determine whether up regulation of xCT is required to efficiently restore the GSH pool during acute pancreatitis and we therefore induced the disease in xCT-/- and xCT+/+ littermates. Pancreatic GSH concentrations did not differ in the two genotypes at any of the investigated time points of pancreatitis, hence xCT expression is not a prerequisite to overcome GSH depletion in this model. Interestingly, amino acid analysis revealed a decrease of the cysteine precursors (cystathionine, methionine and serine) in the pancreata of the cerulein treated xCT-/- compared to xCT+/+ mice. This data suggests a compensatory mechanism via an increased utilisation of the transsulfuration pathway in the xCT-/- animals.
Furthermore, we aimed to uncover the transcriptional activator of xCT during acute pancreatitis. Nrf2 is a transcription factor belonging to the cap'n'collar basic-region leucine zipper (CNC-bZIP) family and it is considered as one of the master regulators during oxidative stress situations. After activation, Nrf2 induces the transcription of a wide range of different antioxidative target genes, amongst which xCT had also been identified in several models. In our study using wild type mice, we could demonstrate the induction of Nrf2 mRNA expression early during acute pancreatitis and we further took advantage of Nrf2 knockout mice to reveal targets of Nrf2. We found significantly blunted mRNA levels of xCT and other target genes in Nrf2-/- mice at 6h of acute pancreatitis. However, in the healthy state as well as during the disease, xCT protein levels in Nrf2-/- pancreata were unchanged compared to the samples of Nrf2+/+ littermates, as was demonstrated by Western blot analysis. We considered a possible compensatory mechanism in Nrf2-/- mice via other members of CNC-bZIP family. Nrf1 and Nrf3 mRNA expression remained at basal levels after inducing pancreatitis, in Nrf2-/- as well as Nrf2+/+ pancreata, speaking against a compensation via these members after loss of Nrf2.
Taken together, our data showed that the expression and function of the cystine transporter xCT is increased during acute pancreatitis, resulting in enhanced substrate availability for GSH synthesis. This activation is at least partially mediated via the upstream regulator Nrf2. After loss of xCT or Nrf2, acinar cells may activate alternative mechanisms to replenish the intracellular cysteine pool for the de novo synthesis of GSH during acute pancreatitis.

Abstract

Acute pancreatitis is a potentially lethal, inflammatory disease of the exocrine pancreas. A major hallmark during the pathogenesis of acute pancreatitis is the development of oxidative stress. Thereby, increased levels of reactive oxygen species (ROS) have deleterious effects by damaging lipids, proteins and nucleic acids. ROS can be neutralised by glutathione (GSH), the most abundant intracellular antioxidant, but GSH concentrations themselves are depleted rapidly in acute pancreatitis. To restore the intracellular GSH pool, GSH is synthesised from the amino acids cysteine, glutamate and glycine. The intracellular availability of cysteine is considered as the rate limiting factor of GSH synthesis.
In the healthy state, the pancreas is the organ with the highest protein synthesis rate. The majority of these proteins are digestive enzymes that are released within the pancreatic juice into the duodenum to facilitate meal digestion. To enable such a high protein translation rate, the pancreas is required to import extensive amounts of amino acids from the plasma, which is mediated by amino acid transporters. Importantly, pancreatic acinar cells dedifferentiate during acute pancreatitis, which is reflected by a reduced protein synthesis rate.
We postulated that acinar cells have altered metabolic needs during acute pancreatitis due to the reduced protein synthesis rate and simultaneously increased GSH synthesis. These alterations may require adaptations in amino acid transporter expression to satisfy the particular demands of amino acids in the inflammatory state. It was therefore the aim of this thesis project to characterise amino acid transporter expression during acute pancreatitis, with a particular focus on the transport of cysteine and its oxidised form cystine.
To address this aim, acute pancreatitis was induced in wild type mice by repetitive administrations of supraphysiological doses of cerulein, an analogue of cholecystokinin. Among the cysteine and cystine transporters screened at 6 hours of acute pancreatitis, a prominent up regulation of the cystine transporter xCT (Slc7a11) mRNA was observed. Moreover, this mRNA increase led to enhanced xCT protein levels, which was quantified by Western blot analysis. During acute pancreatitis, the up regulated transporter xCT was localised to the acinar cell membrane via immunofluorescence technique. In the control mice, the protein could not be visualised. The functional consequences of the increased xCT protein expression was investigated with ex vivo uptake experiments using radiolabelled cystine and freshly isolated acinar cells. Indeed, the enhanced xCT protein levels resulted in higher cystine uptake rates after the induction of acute pancreatitis. Moreover, using isolated acinar cells derived from xCT-/- mice, we could demonstrate that xCT is the only cystine transporter expressed in the pancreas.
We further aimed to determine whether up regulation of xCT is required to efficiently restore the GSH pool during acute pancreatitis and we therefore induced the disease in xCT-/- and xCT+/+ littermates. Pancreatic GSH concentrations did not differ in the two genotypes at any of the investigated time points of pancreatitis, hence xCT expression is not a prerequisite to overcome GSH depletion in this model. Interestingly, amino acid analysis revealed a decrease of the cysteine precursors (cystathionine, methionine and serine) in the pancreata of the cerulein treated xCT-/- compared to xCT+/+ mice. This data suggests a compensatory mechanism via an increased utilisation of the transsulfuration pathway in the xCT-/- animals.
Furthermore, we aimed to uncover the transcriptional activator of xCT during acute pancreatitis. Nrf2 is a transcription factor belonging to the cap'n'collar basic-region leucine zipper (CNC-bZIP) family and it is considered as one of the master regulators during oxidative stress situations. After activation, Nrf2 induces the transcription of a wide range of different antioxidative target genes, amongst which xCT had also been identified in several models. In our study using wild type mice, we could demonstrate the induction of Nrf2 mRNA expression early during acute pancreatitis and we further took advantage of Nrf2 knockout mice to reveal targets of Nrf2. We found significantly blunted mRNA levels of xCT and other target genes in Nrf2-/- mice at 6h of acute pancreatitis. However, in the healthy state as well as during the disease, xCT protein levels in Nrf2-/- pancreata were unchanged compared to the samples of Nrf2+/+ littermates, as was demonstrated by Western blot analysis. We considered a possible compensatory mechanism in Nrf2-/- mice via other members of CNC-bZIP family. Nrf1 and Nrf3 mRNA expression remained at basal levels after inducing pancreatitis, in Nrf2-/- as well as Nrf2+/+ pancreata, speaking against a compensation via these members after loss of Nrf2.
Taken together, our data showed that the expression and function of the cystine transporter xCT is increased during acute pancreatitis, resulting in enhanced substrate availability for GSH synthesis. This activation is at least partially mediated via the upstream regulator Nrf2. After loss of xCT or Nrf2, acinar cells may activate alternative mechanisms to replenish the intracellular cysteine pool for the de novo synthesis of GSH during acute pancreatitis.

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Additional indexing

Item Type:Dissertation (monographical)
Referees:Camargo Simone M R, Verrey François, Graf Rolf, Herrera Pedro L, Stoeckli Esther
Communities & Collections:04 Faculty of Medicine > Institute of Physiology
07 Faculty of Science > Institute of Physiology

UZH Dissertations
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:2018
Deposited On:22 Feb 2019 13:23
Last Modified:25 Aug 2020 14:36
Number of Pages:131
Additional Information:Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich
OA Status:Closed

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