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The extracellular redox state modulates mitochondrial function, gluconeogenesis, and glycogen synthesis in murine hepatocytes.

Nocito L, Kleckner AS, Yoo EJ, Jones Iv AR, Liesa M, Corkey BE - PLoS ONE (2015)

Bottom Line: Thus, primary hepatocytes were treated with different ratios of the following physiological extracellular redox couples: β-hydroxybutyrate (βOHB)/acetoacetate (Acoc), reduced glutathione (GSH)/oxidized glutathione (GSSG), and cysteine/cystine.On the other hand, addition of more reduced ratios of extracellular βOHB/Acoc led to increased NAD(P)H and maximal mitochondrial respiratory capacity in hepatocytes.Greater βOHB/Acoc ratios were also associated with decreased β-oxidation, as expected with enhanced lipogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Boston University, Boston, Massachusetts, United States of America.

ABSTRACT
Circulating redox state changes, determined by the ratio of reduced/oxidized pairs of different metabolites, have been associated with metabolic diseases. However, the pathogenic contribution of these changes and whether they modulate normal tissue function is unclear. As alterations in hepatic gluconeogenesis and glycogen metabolism are hallmarks that characterize insulin resistance and type 2 diabetes, we tested whether imposed changes in the extracellular redox state could modulate these processes. Thus, primary hepatocytes were treated with different ratios of the following physiological extracellular redox couples: β-hydroxybutyrate (βOHB)/acetoacetate (Acoc), reduced glutathione (GSH)/oxidized glutathione (GSSG), and cysteine/cystine. Exposure to a more oxidized ratio via extracellular βOHB/Acoc, GSH/GSSG, and cysteine/cystine in hepatocytes from fed mice increased intracellular hydrogen peroxide without causing oxidative damage. On the other hand, addition of more reduced ratios of extracellular βOHB/Acoc led to increased NAD(P)H and maximal mitochondrial respiratory capacity in hepatocytes. Greater βOHB/Acoc ratios were also associated with decreased β-oxidation, as expected with enhanced lipogenesis. In hepatocytes from fasted mice, a more extracellular reduced state of βOHB/Acoc led to increased alanine-stimulated gluconeogenesis and enhanced glycogen synthesis capacity from added glucose. Thus, we demonstrated for the first time that the extracellular redox state regulates the major metabolic functions of the liver and involves changes in intracellular NADH, hydrogen peroxide, and mitochondrial respiration. Because redox state in the blood can be communicated to all metabolically sensitive tissues, this work confirms the hypothesis that circulating redox state may be an important regulator of whole body metabolism and contribute to alterations associated with metabolic diseases.

No MeSH data available.


Related in: MedlinePlus

Extracellular incubation with increasingly oxidized ratios of redox couples increased ROS production.Intracellular ROS measurements in primary cultured hepatocytes treated with A) 20 mM total active ketone bodies (-355 mV = 2:1 d-βOHB:Acoc; -346 mV = 1:1 d-βOHB:Acoc; -337 mV = 1:2 d-βOHB:Acoc), B) 200 μM cysteine (cysteine + cystine), or C) 110 μM glutathione (reduced + oxidized glutathione). Cells were plated in 24 well plates. Five hours later, the cells were loaded with H2-DCF-DA for 30 min, rinsed, and then treated with the compounds of interest. Data represent the fluorescence from DCF after 40 min of treatment with the compounds. Data represent avg ± SE of three independent experiments. Different letters indicate statistical significance, ANOVA, Tukey’s posthoc analysis.
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pone.0122818.g002: Extracellular incubation with increasingly oxidized ratios of redox couples increased ROS production.Intracellular ROS measurements in primary cultured hepatocytes treated with A) 20 mM total active ketone bodies (-355 mV = 2:1 d-βOHB:Acoc; -346 mV = 1:1 d-βOHB:Acoc; -337 mV = 1:2 d-βOHB:Acoc), B) 200 μM cysteine (cysteine + cystine), or C) 110 μM glutathione (reduced + oxidized glutathione). Cells were plated in 24 well plates. Five hours later, the cells were loaded with H2-DCF-DA for 30 min, rinsed, and then treated with the compounds of interest. Data represent the fluorescence from DCF after 40 min of treatment with the compounds. Data represent avg ± SE of three independent experiments. Different letters indicate statistical significance, ANOVA, Tukey’s posthoc analysis.

Mentions: Given the intimate connection between changes in the intracellular redox state and ROS, we next investigated whether the external redox state can directly affect internal ROS production in primary hepatocytes. DCF is a ROS-sensitive dye that fluoresces according to hydrogen peroxide (H2O2) levels. In addition to βOHB/Acoc, we tested cysteine/cystine, and GSH/GSSG and their ratios are expressed as the steady-state redox potential calculated from the Nernst equation with the standard cell potential (E0) of -346 mV for βOHB/Acoc, -264 mV for GSH/GSSG, and -250 mV for cysteine/cystine. Addition of redox pairs with increasing oxidative potential led to increases in DCF fluorescence for ketone bodies (Fig. 2A), cysteine/cystine (Fig. 2B), and reduced/oxidized glutathione (Fig. 2C). These data demonstrate that the external redox potential conveyed by ketone bodies or thiol redox couples cyst(e)ine and glutathione can all impact ROS production.


The extracellular redox state modulates mitochondrial function, gluconeogenesis, and glycogen synthesis in murine hepatocytes.

Nocito L, Kleckner AS, Yoo EJ, Jones Iv AR, Liesa M, Corkey BE - PLoS ONE (2015)

Extracellular incubation with increasingly oxidized ratios of redox couples increased ROS production.Intracellular ROS measurements in primary cultured hepatocytes treated with A) 20 mM total active ketone bodies (-355 mV = 2:1 d-βOHB:Acoc; -346 mV = 1:1 d-βOHB:Acoc; -337 mV = 1:2 d-βOHB:Acoc), B) 200 μM cysteine (cysteine + cystine), or C) 110 μM glutathione (reduced + oxidized glutathione). Cells were plated in 24 well plates. Five hours later, the cells were loaded with H2-DCF-DA for 30 min, rinsed, and then treated with the compounds of interest. Data represent the fluorescence from DCF after 40 min of treatment with the compounds. Data represent avg ± SE of three independent experiments. Different letters indicate statistical significance, ANOVA, Tukey’s posthoc analysis.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4376787&req=5

pone.0122818.g002: Extracellular incubation with increasingly oxidized ratios of redox couples increased ROS production.Intracellular ROS measurements in primary cultured hepatocytes treated with A) 20 mM total active ketone bodies (-355 mV = 2:1 d-βOHB:Acoc; -346 mV = 1:1 d-βOHB:Acoc; -337 mV = 1:2 d-βOHB:Acoc), B) 200 μM cysteine (cysteine + cystine), or C) 110 μM glutathione (reduced + oxidized glutathione). Cells were plated in 24 well plates. Five hours later, the cells were loaded with H2-DCF-DA for 30 min, rinsed, and then treated with the compounds of interest. Data represent the fluorescence from DCF after 40 min of treatment with the compounds. Data represent avg ± SE of three independent experiments. Different letters indicate statistical significance, ANOVA, Tukey’s posthoc analysis.
Mentions: Given the intimate connection between changes in the intracellular redox state and ROS, we next investigated whether the external redox state can directly affect internal ROS production in primary hepatocytes. DCF is a ROS-sensitive dye that fluoresces according to hydrogen peroxide (H2O2) levels. In addition to βOHB/Acoc, we tested cysteine/cystine, and GSH/GSSG and their ratios are expressed as the steady-state redox potential calculated from the Nernst equation with the standard cell potential (E0) of -346 mV for βOHB/Acoc, -264 mV for GSH/GSSG, and -250 mV for cysteine/cystine. Addition of redox pairs with increasing oxidative potential led to increases in DCF fluorescence for ketone bodies (Fig. 2A), cysteine/cystine (Fig. 2B), and reduced/oxidized glutathione (Fig. 2C). These data demonstrate that the external redox potential conveyed by ketone bodies or thiol redox couples cyst(e)ine and glutathione can all impact ROS production.

Bottom Line: Thus, primary hepatocytes were treated with different ratios of the following physiological extracellular redox couples: β-hydroxybutyrate (βOHB)/acetoacetate (Acoc), reduced glutathione (GSH)/oxidized glutathione (GSSG), and cysteine/cystine.On the other hand, addition of more reduced ratios of extracellular βOHB/Acoc led to increased NAD(P)H and maximal mitochondrial respiratory capacity in hepatocytes.Greater βOHB/Acoc ratios were also associated with decreased β-oxidation, as expected with enhanced lipogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Boston University, Boston, Massachusetts, United States of America.

ABSTRACT
Circulating redox state changes, determined by the ratio of reduced/oxidized pairs of different metabolites, have been associated with metabolic diseases. However, the pathogenic contribution of these changes and whether they modulate normal tissue function is unclear. As alterations in hepatic gluconeogenesis and glycogen metabolism are hallmarks that characterize insulin resistance and type 2 diabetes, we tested whether imposed changes in the extracellular redox state could modulate these processes. Thus, primary hepatocytes were treated with different ratios of the following physiological extracellular redox couples: β-hydroxybutyrate (βOHB)/acetoacetate (Acoc), reduced glutathione (GSH)/oxidized glutathione (GSSG), and cysteine/cystine. Exposure to a more oxidized ratio via extracellular βOHB/Acoc, GSH/GSSG, and cysteine/cystine in hepatocytes from fed mice increased intracellular hydrogen peroxide without causing oxidative damage. On the other hand, addition of more reduced ratios of extracellular βOHB/Acoc led to increased NAD(P)H and maximal mitochondrial respiratory capacity in hepatocytes. Greater βOHB/Acoc ratios were also associated with decreased β-oxidation, as expected with enhanced lipogenesis. In hepatocytes from fasted mice, a more extracellular reduced state of βOHB/Acoc led to increased alanine-stimulated gluconeogenesis and enhanced glycogen synthesis capacity from added glucose. Thus, we demonstrated for the first time that the extracellular redox state regulates the major metabolic functions of the liver and involves changes in intracellular NADH, hydrogen peroxide, and mitochondrial respiration. Because redox state in the blood can be communicated to all metabolically sensitive tissues, this work confirms the hypothesis that circulating redox state may be an important regulator of whole body metabolism and contribute to alterations associated with metabolic diseases.

No MeSH data available.


Related in: MedlinePlus