Energy crisis: The role of oxidative phosphorylation in acute inflammation and sepsis

Mitochondrial dysfunction is increasingly recognized as an accomplice in most of the common human diseases including cancer, neurodegeneration, diabetes, ischemia/reperfusion injury as seen in myocardial infarction and stroke, and sepsis. Inflammatory conditions, both acute and chronic, have recently been shown to affect mitochondrial function. We here discuss the role of oxidative phosphorylation (OxPhos), focusing on acute inflammatory conditions, in particular sepsis and experimental sepsis models. We discuss mitochondrial alterations, specifically the suppression of oxidative metabolism and the role of mitochondrial reactive oxygen species in disease pathology. Several signaling pathways including metabolic, proliferative, and cytokine signaling affect mitochondrial function and appear to be important in inflammatory disease conditions. Cytochrome c oxidase (COX) and cytochrome c, the latter of which plays a central role in apoptosis in addition to mitochondrial respiration, serve as examples for the entire OxPhos system since they have been studied in more detail with respect to cell signaling. We propose a model in which inflammatory signaling leads to changes in the phosphorylation state of mitochondrial proteins, including Tyr304 phosphorylation of COX catalytic subunit I. This results in an inhibition of OxPhos, a reduction of the mitochondrial membrane potential, and consequently a lack of energy, which can cause organ failure and death as seen in septic patients.     

Mammalian liver cytochrome c is tyrosine-48 phosphorylated in vivo, inhibiting mitochondrial respiration

Cytochrome c (Cyt c) is part of the mitochondrial electron transport chain (ETC), accepting electrons from bc(1) complex and transferring them to cytochrome c oxidase (CcO). The ETC generates the mitochondrial membrane potential, which is used by ATP synthase to produce ATP. In addition, the release of Cyt c from the mitochondria often commits a cell to undergo apoptosis. Considering its central role in life (respiration) and death (apoptosis) decisions one would expect tight regulation of Cyt c function. Reversible phosphorylation is a main cellular regulatory mechanism, but the effect of cell signaling targeting the mitochondrial oxidative phosphorylation system is not well understood, and only a small number of proteins that can be phosphorylated have been identified to date. We have recently shown that Cyt c isolated from cow heart tissue is phosphorylated on tyrosine 97 in vivo, which leads to inhibition of respiration in the reaction with CcO. In this study we isolated Cyt c from a different organ, cow liver, under conditions preserving the physiological phosphorylation state. Western analysis with a phosphotyrosine specific antibody suggested that liver Cyt c is phosphorylated. Surprisingly, the phosphorylation site was unambiguously assigned to Tyr-48 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry (IMAC/nano-LC/ESI-MS), and not to the previously identified phospho-Tyr-97 in cow heart. As is true of Tyr-97, Tyr-48 is conserved in eukaryotes. As one possible consequence of Tyr-48 phosphorylation we analyzed the in vitro reaction kinetics with isolated cow liver CcO revealing striking differences. Maximal turnover of Tyr-48 phosphorylated Cyt c was 3.7 s(-1) whereas dephosphorylation resulted in a 2.2 fold increase in activity to 8.2 s(-1). Effects of Tyr-48 phosphorylation based on the Cyt c crystal structure are discussed.     

Regulation of mitochondrial respiration and apoptosis through cell signaling: cytochrome c oxidase and cytochrome c in ischemia/reperfusion injury and inflammation

Cytochrome c (Cytc) and cytochrome c oxidase (COX) catalyze the terminal reaction of the mitochondrial electron transport chain (ETC), the reduction of oxygen to water. This irreversible step is highly regulated, as indicated by the presence of tissue-specific and developmentally expressed isoforms, allosteric regulation, and reversible phosphorylations, which are found in both Cytc and COX. The crucial role of the ETC in health and disease is obvious since it, together with ATP synthase, provides the vast majority of cellular energy, which drives all cellular processes. However, under conditions of stress, the ETC generates reactive oxygen species (ROS), which cause cell damage and trigger death processes. We here discuss current knowledge of the regulation of Cytc and COX with a focus on cell signaling pathways, including cAMP/protein kinase A and tyrosine kinase signaling. Based on the crystal structures we highlight all identified phosphorylation sites on Cytc and COX, and we present a new phosphorylation site, Ser126 on COX subunit II. We conclude with a model that links cell signaling with the phosphorylation state of Cytc and COX. This in turn regulates their enzymatic activities, the mitochondrial membrane potential, and the production of ATP and ROS. Our model is discussed through two distinct human pathologies, acute inflammation as seen in sepsis, where phosphorylation leads to strong COX inhibition followed by energy depletion, and ischemia/reperfusion injury, where hyperactive ETC complexes generate pathologically high mitochondrial membrane potentials, leading to excessive ROS production. Although operating at opposite poles of the ETC activity spectrum, both conditions can lead to cell death through energy deprivation or ROS-triggered apoptosis.     

Mitochondrial AKAP121 links cAMP and src signaling to oxidative metabolism

AKAP121 focuses distinct signaling events from membrane to mitochondria by binding and targeting cAMP-dependent protein kinase (PKA), protein tyrosine phosphatase (PTPD1), and mRNA. We find that AKAP121 also targets src tyrosine kinase to mitochondria via PTPD1. AKAP121 increased src-dependent phosphorylation of mitochondrial substrates and enhanced the activity of cytochrome c oxidase, a component of the mitochondrial respiratory chain. Mitochondrial membrane potential and ATP oxidative synthesis were enhanced by AKAP121 in an src- and PKA-dependent manner. Finally, siRNA-mediated silencing of endogenous AKAP121 drastically impaired synthesis and accumulation of mitochondrial ATP. These findings indicate that AKAP121, through its role in enhancing cAMP and tyrosine kinase signaling to distal organelles, is an important regulator in mitochondrial metabolism.     

The role of mitochondrial membrane potential in ischemic heart failure

The molecular events occurring during myocardial infarction and cardioprotection are described with an emphasis on the changes of the mitochondrial membrane potential (ΔΨ_m). The low ΔΨ_m values of the normal beating heart (100–140 mV) are explained by the allosteric ATP-inhibition of cytochrome c oxidase (CcO) through feedback inhibition by ATP at high [ATP]/[ADP] ratios. During ischemia the mechanism is reversibly switched off by signaling through reactive oxygen species (ROS). At reperfusion high ΔΨ_m values cause a burst of ROS production leading to apoptosis and/or necrosis. Ischemic preconditioning is suggested to cause additional phosphorylation of CcO, protecting the enzyme from immediate dephosphorylation via ROS signaling.     

Protein phosphorylation and prevention of cytochrome oxidase inhibition by ATP: coupled mechanisms of energy metabolism regulation

Rapid regulation of oxidative phosphorylation is crucial for mitochondrial adaptation to swift changes in fuels availability and energy demands. An intramitochondrial signaling pathway regulates cytochrome oxidase (COX), the terminal enzyme of the respiratory chain, through reversible phosphorylation. We find that PKA-mediated phosphorylation of a COX subunit dictates mammalian mitochondrial energy fluxes and identify the specific residue (S58) of COX subunit IV-1 (COXIV-1) that is involved in this mechanism of metabolic regulation. Using protein mutagenesis, molecular dynamics simulations, and induced fit docking, we show that mitochondrial energy metabolism regulation by phosphorylation of COXIV-1 is coupled with prevention of COX allosteric inhibition by ATP. This regulatory mechanism is essential for efficient oxidative metabolism and cell survival. We propose that S58 COXIV-1 phosphorylation has evolved as a metabolic switch that allows mammalian mitochondria to rapidly toggle between energy utilization and energy storage.     

A suggested role for mitochondria in Noonan syndrome

Noonan syndrome (NS) is an autosomal dominant disorder, and a main feature is congenital heart malformation. About 50% of cases are caused by gain-of-function mutations in the tyrosine phosphatase SHP2/PTPN11, a downstream regulator of ERK/MAPK. Recently it was reported that SHP2 also localizes to the mitochondrial intercristae/intermembrane space (IMS), but the role of SHP2 in mitochondria is unclear. The mitochondrial oxidative phosphorylation (OxPhos) system provides the vast majority of cellular energy and produces reactive oxygen species (ROS). Changes in ROS may interfere with organ development such as that observed in NS patients. Several phosphorylation sites have been found in OxPhos components including cytochrome c oxidase (CcO) and cytochrome c (Cytc), and we hypothesized that OxPhos complexes may be direct or indirect targets of SHP2. We analyzed mitochondrial function using mouse fibroblasts from wild-types, SHP2 knockdowns, and D61G SHP2 mutants leading to constitutively active SHP2, as found in NS patients. Levels of OxPhos complexes were similar except for CcO and Cytc, which were 37% and 28% reduced in the D61G cells. However, CcO activity was significantly increased, as we also found for two lymphoblast cell lines from NS patients with two independent mutations in PTPN11. D61G cells showed lower mitochondrial membrane potential and 30% lower ATP content compared to controls. ROS were significantly increased; aconitase activity, a marker for ROS-induced damage, was decreased; and catalase activity was increased in D61G cells. We propose that decreased energy levels and/or increased ROS may explain, at least in part, some of the clinical features in NS that overlap with children with mitochondrial disorders.     

Signal transduction to mitochondrial ATP synthase: evidence that PDGF-dependent phosphorylation of the delta-subunit occurs in several cell lines, involves tyrosine, and is modulated by lysophosphatidic acid

Although signal transduction mechanisms originating from receptors on the plasma membrane and targeted to metabolic and other enzymes/proteins localized in the cytoplasm or the nucleus have been extensively studied in animal cells, few such studies have focused on the mitochondrial energy producing machinery, i.e. the electron transport chain and ATP synthase complex (F0F1). Significantly, it was shown in an earlier collaborative study that platelet-derived growth factor (PDGF), which is linked in signal transduction pathways to tyrosine kinase-dependent phosphorylations, regulates the phosphorylation of the mitochondrial ATP synthase delta subunit in cortical neurons (Zhang et. al., 1995. J. Neurochem. 65, 2812-2815). This is a particularly intriguing finding in light of more recent reports demonstrating that ATP synthases are nanomotors with a central rotor, one component of which is the delta subunit. In this report, evidence is provided that the PDGF-dependent phosphorylation of the ATP synthase delta subunit is not confined to neuronal cells but can be demonstrated also in studies with PDGF-treated NIH3T3 and kidney cells. Evidence is provided also that phosphorylation of the ATP synthase delta subunit may involve its single tyrosine residue, and that this phosphorylation is modulated when the cell based assay includes lysophosphatidic acid (LPA), a phospholipid signaling molecules. Finally, results are presented of an analysis which revealed a number of potential tyrosine phosphorylation sites on three other subunits (alpha, beta, and gamma) of the F1 (catalytic) moiety of the mitochondrial ATP synthase, thus making this important complex a most attractive target for future signal transduction studies.     

Metabolic network control of oxidative phosphorylation: multiple roles of inorganic phosphate

Phosphate (Pi) is a putative cytosolic signaling molecule in the regulation of oxidative phosphorylation. Here, by using a multiparameter monitoring system, we show that Pi controls oxidative phosphorylation in a balanced fashion, modulating both the generation of useful potential energy and the formation of ATP by F1F0-ATPase in heart and skeletal muscle mitochondria. In these studies the effect of Pi was determined on the mitochondria [NADH], NADH generating capacity, matrix pH, membrane potential, oxygen consumption, and cytochrome reduction level. Pi enhanced NADH generation and was obligatory for electron flow under uncoupled conditions. Pi oxidized cytochrome b (cyto-b) and reduced cytochrome c (cyto-c), potentially improving the coupling between the NADH free energy and the proton motive force. The apparent limitation in reducing equivalent flow between cyto-b and cyto-c in the absence of Pi was confirmed in the intact heart by using optical spectroscopic techniques under conditions with low cytosolic [Pi]. These results demonstrate that Pi signaling results in the balanced modulation of oxidative phosphorylation, by influencing both deltaGH+ generation and ATP production, which may contribute to the energy metabolism homeostasis observed in intact systems.     

Regulation of oxidative phosphorylation,the mitochondrial membrane potential,and their role in human disease

Thirty years after Peter Mitchell was awarded the Nobel Prize for the chemiosmotic hypothesis, which links the mitochondrial membrane potential generated by the proton pumps of the electron transport chain to ATP production by ATP synthase, the molecular players involved once again attract attention. This is so because medical research increasingly recognizes mitochondrial dysfunction as a major factor in the pathology of numerous human diseases, including diabetes, cancer, neurodegenerative diseases, and ischemia reperfusion injury. We propose a model linking mitochondrial oxidative phosphorylation (OxPhos) to human disease, through a lack of energy, excessive free radical production, or a combination of both. We discuss the regulation of OxPhos by cell signaling pathways as a main regulatory mechanism in higher organisms, which in turn determines the magnitude of the mitochondrial membrane potential: if too low, ATP production cannot meet demand, and if too high, free radicals are produced. This model is presented in light of the recently emerging understanding of mechanisms that regulate mammalian cytochrome c oxidase and its substrate cytochrome c as representative enzymes for the entire OxPhos system.     

Role of mitochondrial Ca2+ in the regulation of cellular energetics

Calcium is an important signaling molecule involved in the regulation of many cellular functions. The large free energy in the Ca(2+) ion membrane gradients makes Ca(2+) signaling inherently sensitive to the available cellular free energy, primarily in the form of ATP. In addition, Ca(2+) regulates many cellular ATP-consuming reactions such as muscle contraction, exocytosis, biosynthesis, and neuronal signaling. Thus, Ca(2+) becomes a logical candidate as a signaling molecule for modulating ATP hydrolysis and synthesis during changes in numerous forms of cellular work. Mitochondria are the primary source of aerobic energy production in mammalian cells and also maintain a large Ca(2+) gradient across their inner membrane, providing a signaling potential for this molecule. The demonstrated link between cytosolic and mitochondrial Ca(2+) concentrations, identification of transport mechanisms, and the proximity of mitochondria to Ca(2+) release sites further supports the notion that Ca(2+) can be an important signaling molecule in the energy metabolism interplay of the cytosol with the mitochondria. Here we review sites within the mitochondria where Ca(2+) plays a role in the regulation of ATP generation and potentially contributes to the orchestration of cellular metabolic homeostasis. Early work on isolated enzymes pointed to several matrix dehydrogenases that are stimulated by Ca(2+), which were confirmed in the intact mitochondrion as well as cellular and in vivo systems. However, studies in these intact systems suggested a more expansive influence of Ca(2+) on mitochondrial energy conversion. Numerous noninvasive approaches monitoring NADH, mitochondrial membrane potential, oxygen consumption, and workloads suggest significant effects of Ca(2+) on other elements of NADH generation as well as downstream elements of oxidative phosphorylation, including the F(1)F(O)-ATPase and the cytochrome chain. These other potential elements of Ca(2+) modification of mitochondrial energy conversion will be the focus of this review. Though most specific molecular mechanisms have yet to be elucidated, it is clear that Ca(2+) provides a balanced activation of mitochondrial energy metabolism that exceeds the alteration of dehydrogenases alone.     

Down-regulated energy metabolism genes associated with mitochondria oxidative phosphorylation and fatty acid metabolism in viral cardiomyopathy mouse heart

The majority of experimental and clinical studies indicates that the hypertrophied and failing myocardium are characterized by changes in energy and substrate metabolism that attributed to failing heart changes at the genomic level, in fact, heart failure is caused by various diseases, their energy metabolism and substrate are in different genetic variations, then the potential significance of the molecular mechanisms for the aetiology of heart failure is necessary to be evaluated. Persistent viral infection (especially coxsackievirus group B3) of the myocardium in viral myocarditis and viral dilated cardiomyopathy has never been neglected by experts. This study aimed to explore the role and regulatory mechanism of the altered gene expression for energy metabolism involved in mitochondrial oxidative phosphorylation, fatty acid metabolism in viral dilated cardiomyopathy. cDNA Microarray technology was used to evaluate the expression of >35,852 genes in a mice model of viral dilated cardiomyopathy. In total 1385 highly different genes expression, we analyzed 33 altered genes expression for energy metabolism involved in mitochondrial oxidative phosphorylation, fatty acid metabolism and further selected real-time-PCR for quantity one of regulatory mechanisms for energy including fatty acid metabolism—the UCP2 and assayed cytochrome C oxidase activity by Spectrophotometer to explore mitochondrial oxidative phosphorylation function. We found obviously different expression of 33 energy metabolism genes associated with mitochondria oxidative phosphorylation, fatty acid metabolism in cardiomyopathy mouse heart, the regulatory gene for energy metabolism: UCP2 was down-regulated and cytochrome C oxidase activity was decreased. Genes involved in both fatty acid metabolism and mitochondrial oxidative phosphorylation were down-regulated, mitochondrial uncoupling proteins (UCP2) expression did not increase but decrease which might be a kind of adaptive protection response to regulate energy metabolism for ATP produce.     

Control of mitochondrial membrane potential and ROS formation by reversible phosphorylation of cytochrome c oxidase

Phosphorylation of isolated cytochrome c oxidase from bovine kidney and heart, and of the reconstituted heart enzyme, with protein kinase A, cAMP and ATP turns on the allosteric ATP-inhibition at high ATP/ADP ratios. Also incubation of isolated bovine liver mitochondria only with cAMP and ATP turns on, and subsequent incubation with Ca²⁺ turns off the allosteric ATP-inhibition of cytochrome c oxidase. In the bovine heart enzyme occur only three consensus sequences for cAMP-dependent phosphorylation (in subunits I, III and Vb). The evolutionary conservation of RRYS⁴⁴¹ at the cytosolic side of subunit I, together with the above results, suggest that phosphorylation of Ser⁴⁴¹ turns on the allosteric ATP-inhibition of cytochrome c oxidase. The results support the 'molecular-physiological hypothesis' [29], which proposes a low mitochondrial membrane potential through the allosteric ATP-inhibition. A hormone- or agonist-stimulated increase of cellular [Ca²⁺]] is suggested to activate a mitochondrial protein phosphatase which dephosphorylates cytochrome c oxidase, turns off the allosteric ATP-inhibition and results in increase of mitochondrial membrane potential and ROS formation.     

Regulation of mitochondrial oxidative phosphorylation through cell signaling

The mitochondrial oxidative phosphorylation (OxPhos) system plays a key role in energy production, the generation of free radicals, and apoptosis. A lack of cellular energy, excessive radical production, and dysregulated apoptosis are found alone or in combination in most human diseases, including neurodegenerative diseases, stroke, cardiovascular disorders, ischemia/reperfusion, and cancer. In the context of its relevance to human disease, this article reviews current knowledge about the regulation of OxPhos with a focus on cell signaling and discusses identified phosphorylation sites with the aid of crystal structures of OxPhos complexes. Several recent studies have shown that all OxPhos components can be phosphorylated; even the small electron carrier cytochrome c is tyrosine phosphorylated in vivo. We propose that in higher organisms, in contrast to bacteria, cell signaling pathways are the main regulator of energy production, triggered for example by hormones. Pathways that have been identified to act on OxPhos include protein kinases A and C and growth factor activated receptor tyrosine kinase signaling. Present knowledge about kinases and phosphatases that execute signals at the level of the mitochondrial OxPhos system, and newly emerging concepts, such as the translocation of kinases to the mitochondria upon stimulation of a signaling pathway, are discussed.     

Phosphocreatine protects against LPS-induced human umbilical vein endothelial cell apoptosis by regulating mitochondrial oxidative phosphorylation

Phosphocreatine (PCr) is an exogenous energy substance, which provides phosphate groups for adenosine triphosphate (ATP) cycle and promotes energy metabolism in cells. However, it is still unclear whether PCr has influenced on mitochondrial energy metabolism as well as oxidative phosphorylation (OXPHO) in previous studies. Therefore, the aim of the present study was to investigate the regulation of PCr on lipopolsaccharide (LPS)-induced human umbilical vein endothelial cells (HUVECs) and mitochondrial OXPHO pathway. PCr protected HUVECs against LPS-induced apoptosis by suppressing the mitochondrial permeability transition, cytosolic release of cytochrome c (Cyt C), Ca²⁺, reactive oxygen species and subsequent activation of caspases, and increasing Bcl₂ expression, while suppressing Bax expression. More importantly, PCr significantly improved mitochondrial swelling and membrane potential, enhanced the activities of ATP synthase and mitochondrial creatine kinase (CK_mt) in creatine shuttle, influenced on respiratory chain enzymes, respiratory control ratio, phosphorus/oxygen ratio and ATP production of OXPHO. Above PCr-mediated mitochondrial events were effectively more favorable to reduced form of flavin adenine dinucleotide (FADH₂) pathway than reduced form of nicotinamide-adenine dinucleotid pathway in the mitochondrial respiratory chain. Our results revealed that PCr protects against LPS-induced HUVECs apoptosis, which probably related to stabilization of intracellular energy metabolism, especially for FADH₂ pathway in mitochondrial respiratory chain, ATP synthase and CK_mt. Our findings suggest that PCr may play a certain role in the treatment of atherosclerosis via protecting endothelial cell function.     

Tyrosine phosphorylation of mitochondrial pyruvate dehydrogenase kinase 1 is important for cancer metabolism

Many tumor cells rely on aerobic glycolysis instead of oxidative phosphorylation for their continued proliferation and survival. Myc and HIF-1 are believed to promote such a metabolic switch by, in part, upregulating gene expression of pyruvate dehydrogenase (PDH) kinase 1 (PDHK1), which phosphorylates and inactivates mitochondrial PDH and consequently pyruvate dehydrogenase complex (PDC). Here we report that tyrosine phosphorylation enhances PDHK1 kinase activity by promoting ATP and PDC binding. Functional PDC can form in mitochondria outside of the matrix in some cancer cells and PDHK1 is commonly tyrosine phosphorylated in human cancers by diverse oncogenic tyrosine kinases localized to different mitochondrial compartments. Expression of phosphorylation-deficient, catalytic hypomorph PDHK1 mutants in cancer cells leads to decreased cell proliferation under hypoxia and increased oxidative phosphorylation with enhanced mitochondrial utilization of pyruvate and reduced tumor growth in xenograft nude mice. Together, tyrosine phosphorylation activates PDHK1 to promote the Warburg effect and tumor growth.     

Preservation of NADH ubiquinone-oxidoreductase activity by Src kinase-mediated phosphorylation of NDUFB10

The tyrosine kinase Src is upregulated in several cancer cells. In such cells, there is a metabolic reprogramming elevating aerobic glycolysis that seems partly dependent on Src activation. Src kinase was recently shown to be targeted to mitochondria where it modulates mitochondrial bioenergetics in non-proliferative tissues and cells. The main goal of our study was to determine if increased Src kinase activity could also influence mitochondrial metabolism in cancer cells (143B and DU145 cells). We have shown that 143B and DU145 cells produce most of the ATP through glycolysis but also that the inhibition of OXPHOS led to a significant decrease in proliferation which was not due to a decrease in the total ATP levels. These results indicate that a more important role for mitochondria in cancer cells could be ensuring mitochondrial functions other than ATP production. This study is the first to show a putative influence of intramitochondrial Src kinase on oxidative phosphorylation in cancer cells. Indeed, we have shown that Src kinase inhibition led to a decrease in mitochondrial respiration via a specific decrease in complex I activities (NADH-ubiquinone oxidoreductase). This decrease is associated with a lower phosphorylation of the complex I subunit NDUFB10. These results suggest that the preservation of complex I function by mitochondrial Src kinase could be important in the development of the overall phenotype of cancer.     

Sphingosine kinase 1 regulates pro-inflammatory responses triggered by TNFalpha in primary human monocytes

Monocytes play an important role in inflammation, angiogenesis, and atherosclerosis. During these processes monocytes release pre-formed proinflammatory mediators from granules, and synthesize de novo cytokines and chemokines important in the amplification of the inflammatory response. One of the most prominent triggers of inflammatory responses is the cytokine TNFalpha. However, the intracellular signaling cascades triggered by TNFalpha are not fully understood. In this study we investigated the roles of SPHK on the TNFalpha-triggered responses on human primary monocytes. We show that TNFalpha rapidly triggers S1P generation and activation of SPHK. Moreover, our data shows that SPHK1 is the isoform activated by TNFalpha, and plays an essential role on the TNFalpha-triggered intracellular Ca2+ signals, degranulation, cytokine production, and activation of NFkappaB, thus suggesting a pivotal role for SPHK1 on the proinflammatory responses triggered by TNFalpha.     

Soluble adenylyl cyclase regulates the cytosolic NADH/NAD+ redox state and the bioenergetic switch between glycolysis and oxidative phosphorylation

The evolutionarily conserved soluble adenylyl cyclase (sAC, ADCY10) mediates cAMP signaling exclusively in intracellular compartments. Because sAC activity is sensitive to local concentrations of ATP, bicarbonate, and free Ca²⁺, sAC is potentially an important metabolic sensor. Nonetheless, little is known about how sAC regulates energy metabolism in intact cells. In this study, we demonstrated that both pharmacological and genetic suppression of sAC resulted in increased lactate secretion and decreased pyruvate secretion in multiple cell lines and primary cultures of mouse hepatocytes and cholangiocytes. The increased extracellular lactate-to-pyruvate ratio upon sAC suppression reflected an increased cytosolic free [NADH]/[NAD⁺] ratio, which was corroborated by using the NADH/NAD⁺ redox biosensor Peredox-mCherry. Mechanistic studies in permeabilized HepG2 cells showed that sAC inhibition specifically suppressed complex I of the mitochondrial respiratory chain. A survey of cAMP effectors revealed that only selective inhibition of exchange protein activated by cAMP 1 (Epac1), but not protein kinase A (PKA) or Epac2, suppressed complex I-dependent respiration and significantly increased the cytosolic NADH/NAD⁺ redox state. Analysis of the ATP production rate and the adenylate energy charge showed that inhibiting sAC reciprocally affects ATP production by glycolysis and oxidative phosphorylation while maintaining cellular energy homeostasis. In conclusion, our study shows that, via the regulation of complex I-dependent mitochondrial respiration, sAC-Epac1 signaling regulates the cytosolic NADH/NAD⁺ redox state, and coordinates oxidative phosphorylation and glycolysis to maintain cellular energy homeostasis. As such, sAC is effectively a bioenergetic switch between aerobic glycolysis and oxidative phosphorylation at the post-translational level.