Difference between revisions of "Palmeira 2019 MiP2019"

Latest revision as of 16:01, 7 October 2019

Prevention of ischemia/reperfusion injury: effects of a soluble adenylyl cyclase inhibitor LRE1.

Link: MiP2019

Rolo AP, Teodoro JS, Machado I, Steegborn C, Palmeira CM (2019)

Event: MiP2019

Ischemia/reperfusion injury (IR) is a common deleterious process that occurs after an ablation and restoration of circulation to an organ, and typically hinges on a surge of reactive oxygen species (ROS) generation, leading to cellular injury and tissue failure. Given the ubiquitous role of mitochondria as a key organelle to the survival to IR, strategies that directly target mitochondria and prime them towards stress handling might prove clinically invaluable. In fact, mitochondrial pre-conditioning has shown promising results, only marred by the need of surgical intervention. As such, a pharmacological intervention that could mimic surgical mitochondria precondition could improve mitochondrial function in a setting of rodent model of hepatic IR. Therefore, we tested LRE1 (a sAC inhibitor) administration before I/R.

Male Wistar rats were subjected to a portal vein injection with LRE1 previous to surgical IR. After 24h, liver mitochondria were isolated and several metabolic parameter assessment tests were conducted.

LRE1 was able to revert several evaluated parameters, including calcium tolerance, ROS generation and OXPHOS activity. It is clear that, by acting through different mechanisms, the inhibitor was able to acclimatize mitochondria to the insult.

It is apparent from our data that, by manipulating the generation of cAMP, we could prime mitochondria for IR injury, leading to prospective clinical investigation of the feasibility of this inhibitor for human utilization.

• Bioblast editor: Plangger M

Labels: MiParea: Pharmacology;toxicology

Stress:Ischemia-reperfusion Organism: Rat Tissue;cell: Liver Preparation: Isolated mitochondria

Affiliations and support

Anabela Pinto Rolo(1,2), João Soeiro Teodoro(1,2), Ivo Machado(1,2), Clemens Steegborn(3), and Carlos Marques Palmeira(1,2)

Dept Life Sciences
Center Neurosciences Cell Biology; Univ Coimbra, Portugal
Dept Biochemistry, Univ Bayreuth, Germany

Supported by PTDC/BIM-MEC/6911/2014 and POCI-01-0145-FEDER-007440. JST is a recipient of a FCT grant(SFRH/BPD/94036/2013).

@@ Line 1: / Line 1: @@
 {{Abstract
-|title=[[Image:KrakoN.jpg|left|90px|Nina Krako Jakovljevic]] Lower insulin sensitivity differently affects mitochondrial coupling in liver and muscle cells.
+|title=[[Image:Carlos Palmeira2.png.jpg|left|90px|Carlos Palmeira]] Prevention of ischemia/reperfusion injury: effects of a soluble adenylyl cyclase inhibitor LRE1.
 |info=[[MiP2019]]
-|authors=Krako Jakovljevic N, Pavlovic K, Zujovic T1, Isakovic AM, Jovanovic M, Markovic I, Martinovic T, Ciric D, Kravic-Stevovic T, Bumbaširevic V, Lalic NM
+|authors=Rolo AP, Teodoro JS, Machado I, Steegborn C, Palmeira CM
 |year=2019
 |event=MiP2019
 |abstract=[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MitoEAGLE]]
-Condition in which cell, tissue, organ or an organism require higher insulin concentration to obtain a quantitatively normal biological response is called insulin resistance (IR). This state of lowered insulin sensitivity leads to an inadequate response of peripheral tissues (skeletal muscle, adipose tissue and liver) to insulin and consequently to type 2 diabetes pathology. Cell viability, mitochondrial respiration, reactive oxygen species (ROS) production and mitochondrial morphology were assessed in C2C12 mouse myoblasts and Huh7 human hepatocellular carcinoma cells in order to find if there is any cell type specific difference that could potentially be ascribed to tissue specific mechanisms of IR.
+Ischemia/reperfusion injury (IR) is a common deleterious process that occurs after an ablation and restoration of circulation to an organ, and typically hinges on a surge of reactive oxygen species (ROS) generation, leading to cellular injury and tissue failure. Given the ubiquitous role of mitochondria as a key organelle to the survival to IR, strategies that directly target mitochondria and prime them towards stress handling might prove clinically invaluable. In fact, mitochondrial pre-conditioning has shown promising results, only marred by the need of surgical intervention. As such, a pharmacological intervention that could mimic surgical mitochondria precondition could improve mitochondrial function in a setting of rodent model of hepatic IR. Therefore, we tested LRE1 (a sAC inhibitor) administration before I/R.
-Cells were treated with chronic (24h) insulin or palmitate to induce IR. Insulin sensitivity was assayed by immunoblot, measuring the level of phosphorylation of Akt (Ser473). Cell viability was assessed by acid phosphatase assay. Mitochondrial respiration was measured by high resolution respirometry, ROS production by flow cytometry and mitochondrial morphology was examined by electron microscopy.
+Male Wistar rats were subjected to a portal vein injection with LRE1 previous to surgical IR. After 24h, liver mitochondria were isolated and several metabolic parameter assessment tests were conducted.
-Chronic insulin treatment in both cell lines (C2C12 and Huh7) causes a decrease in phosphorylation of Akt, which is a hallmark of insulin resistance at the cellular level. The same effect was observed in both cell lines after chronic palmitate treatment. Chronic insulin treatment does not affect viability of (C2C12 and Huh7), while palmitate treatment decreases cell viability in both cell types. Chronic insulin treatment does not affect mitochondrial respiration, at variance with chronic palmitate treatment, which decreases respiration in C2C12 and Huh7 cells. However, chronic insulin treatment causes a decrease in respiratory acceptor control ratio (RCR) in C2C12, as observed with palmitate treatment. This is not the case for Huh7 cells, where RCR is unchanged after insulin treatment, while lowered only after palmitate treatment. Total ROS production does not change significantly in either cell line. Both C2C12 and Huh7 cells showed preserved mitochondrial morphology after chronic insulin treatment, while chronic palmitate treatment causes alterations of mitochondrial morphology in both cell lines.
+LRE1 was able to revert several evaluated parameters, including calcium tolerance, ROS generation and OXPHOS activity. It is clear that, by acting through different mechanisms, the inhibitor was able to acclimatize mitochondria to the insult.
-To conclude, while chronic palmitate treatment showed to be cytotoxic in both C2C12 and Huh7 cells, chronic insulin treatment did not affect cell viability nor mitochondrial respiration. The difference found in RCR between insulin treated C2C12 and Huh7 cells is due to their OXPHOS coupling efficiency, which is an innate property of cells, it reflects differences between the metabolism of myocytes and hepatocytes and could indicate potential specificities in regulation of insulin sensitivity at the tissue level.
+It is apparent from our data that, by manipulating the generation of cAMP, we could prime mitochondria for IR injury, leading to prospective clinical investigation of the feasibility of this inhibitor for human utilization.
-|editor=[[Plangger M]], [[Tindle-Solomon L]]
+|editor=[[Plangger M]],
-|mipnetlab=RS Belgrade Lalic NM
 }}
 {{Labeling
-|area=Respiration, Comparative MiP;environmental MiP
+|area=Pharmacology;toxicology
-|diseases=Diabetes
+|injuries=Ischemia-reperfusion
-|organism=Human, Mouse
+|organism=Rat
-|tissues=Skeletal muscle, Liver
+|tissues=Liver
-|instruments=Oxygraph-2k
+|preparations=Isolated mitochondria
 }}
-== Affiliations ==
+== Affiliations and support ==
-::::Krako Jakovljević N(1), Pavlović K(1), Žujović T(1), Isaković AM(2), Jovanović M(2), Marković I(2), Martinović T(3), Ćirić D(3), Kravić-Stevović T(3), Bumbaširević V(3), Lalić NM(1)
+::::Anabela Pinto Rolo(1,2), João Soeiro Teodoro(1,2), Ivo Machado(1,2), Clemens Steegborn(3), and Carlos Marques Palmeira(1,2)
-::::#Clinic Endocrinology, Diabetes Metabolic Diseases, CCS
+::::#Dept Life Sciences
-::::#Inst Medical Clinical Biochemistry
+::::#Center Neurosciences Cell Biology; Univ Coimbra, Portugal
-::::#Inst Histology Embriology; Fac Medicine; Univ Belgrade, Serbia. - ninakrako@gmail.com
+::::#Dept Biochemistry, Univ Bayreuth, Germany
+::::Supported by PTDC/BIM-MEC/6911/2014 and POCI-01-0145-FEDER-007440. JST is a recipient of a FCT grant(SFRH/BPD/94036/2013).