Endogenous Carbon Monoxide Signaling Modulates Mitochondrial Function and Intracellular Glucose Utilization: Impact of the Heme Oxygenase Substrate Hemin.

David Stucki,Peter Brenneisen,Heide Krahl,Andreas S Reichert,Wilhelm Stahl,Annika Massenberg,Andreas P M Weber,Julia Steinhausen,Dominik Brilhaus,Philipp Westhoff

doi:10.3390/antiox9080652

Abstract

Stress-inducible heme oxygenase-1 (HO-1) catalyzes the oxidative cleavage of heme yielding biliverdin, ferrous iron, and carbon monoxide (CO). Heme oxygenase activity has been attributed to antioxidant defense via the redox cycling system of biliverdin and bilirubin. There is increasing evidence that CO is a gaseous signaling molecule and plays a role in the regulation of energy metabolism. Inhibitory effects of CO on the respiratory chain are well established, but the implication of such a process on the cellular stress response is not well understood. By means of extracellular flux analyses and isotopic tracing, we studied the effects of CO, either released from the CO donor CORM-401 or endogenously produced by heme oxygenases, on the respiratory chain and glucose metabolism. CORM-401 was thereby used as a tool to mimic endogenous CO production by heme oxygenases. In the long term (>60 min), CORM-401-derived CO exposure inhibited mitochondrial respiration, which was compensated by increased glycolysis accompanied by a loss of the ATP production rate and an increase in proton leakage. This effect pattern was likewise observed after endogenous CO production by heme oxygenases. However, in the present setting, these effects were only observed when sufficient substrate for heme oxygenases (hemin) was provided. Modulation of the HO-1 protein level was less important. The long-term influence of CO on glucose metabolism via glycolysis was preceded by a short-term response (<30 min) of the cells to CO. Stable isotope-labeling experiments and metabolic flux analysis revealed a short-term shift of glucose consumption from glycolysis to the pentose phosphate pathway (PPP) along with an increase in reactive oxygen species (ROS) generation. Overall, we suggest that signaling by endogenous CO stimulates the rapid formation of reduction equivalents (NADPH) via the PPP, and plays an additional role in antioxidant defense, e.g., via feed-forward stimulation of the bilirubin/biliverdin redox cycling system.

Highlights

Heme oxygenases (HOs) catalyze the oxidative cleavage of heme to yield biliverdin, ferrous iron, and carbon monoxide (CO)
We first examined in Murine embryonic fibroblasts (MEFs) the effects of CO-releasing molecules (CORMs)-401 and inactive
Under the conditions applied here, the modulation of mitochondrial respiration was dependent on the amount of the substrate hemin but not on the expression level of we suggest that availability of the substrate plays a role in the regulation of heme oxygenase-mediated CO signaling

Summary

Introduction

Heme oxygenases (HOs) catalyze the oxidative cleavage of heme to yield biliverdin, ferrous iron, and carbon monoxide (CO). Among them are several reactive oxygen species (ROS), such as superoxide, hydrogenperoxide, and lipidperoxides, and transition metals, ultraviolet light, secondary plant constituents, and the natural substrate of HOs, namely heme molecules [3,4,5]. In this context, an induction of HO-1 expression has been associated with the cellular stress response, in particular as an adaptive mechanism of antioxidant defense [6]. Scavenging of ROS has been attributed to the redoxcycling system of biliverdin and bilirubin, which comprises oxidation of bilirubin by ROS to yield biliverdin and enzymatically catalyzed reduction of biliverdin to bilirubin with nicotinamide adenine dinucleotide phosphate (NADPH) as an electron donor [7]

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Results

Conclusion