Cholangiocytes express an isoform of soluble adenylyl cyclase that is N-linked glycosylated and secreted in extracellular vesicles.

Soluble adenylyl cyclase (sAC)-derived cAMP regulates various cellular processes; however, the regulatory landscape mediating sAC protein levels remains underexplored. We consistently observed a 85 kD (sAC85 ) or 75 kD (sAC75 ) sAC protein band under glucose-sufficient or glucose-deprived states, respectively, in H69 cholangiocytes by immunoblotting. Deglycosylation by PNGase-F demonstrated that both sAC75 and sAC85 are N-linked glycosylated proteins with the same polypeptide backbone. Deglycosylation with Endo-H further revealed that sAC75 and sAC85 carry distinct sugar chains. We observed release of N-linked glycosylated sAC (sACEV ) in extracellular vesicles under conditions that support intracellular sAC85 (glucose-sufficient) as opposed to sAC75 (glucose-deprived) conditions. Consistently, disrupting the vesicular machinery affects the maturation of intracellular sAC and inhibits the release of sACEV into extracellular vesicles. The intracellular turnover of sAC85 is extremely short (t1/2 ~30 min) and release of sACEV in the medium was detected within 3 h. Our observations support the maturation and trafficking in cholangiocytes of an N-linked glycosylated sAC isoform that is rapidly released into extracellular vesicles.

Role of the IgG4-related cholangitis autoantigen annexin A11 in cholangiocyte protection.

BACKGROUND & AIMS: Annexin A11 was identified as autoantigen in IgG4-related cholangitis (IRC), a B-cell driven disease. Annexin A11 modulates calcium-dependent exocytosis, a crucial mechanism for insertion of proteins into their target membranes. Human cholangiocytes form an apical 'biliary bicarbonate umbrella' regarded as defense against harmful hydrophobic bile acid influx. The bicarbonate secretory machinery comprises the chloride/bicarbonate exchanger AE2 and the chloride channel ANO1. We aimed to investigate the expression and function of annexin A11 in human cholangiocytes and a potential role of IgG1/IgG4-mediated autoreactivity against annexin A11 in the pathogenesis of IRC. METHODS: Expression of annexin A11 in human liver was studied by immunohistochemistry and immunofluorescence. In human control and ANXA11 knockdown H69 cholangiocytes, intracellular pH, AE2 and ANO1 surface expression, and bile acid influx were examined using ratio microspectrofluorometry, cell surface biotinylation, and 22,23-3H-glycochenodeoxycholic acid permeation, respectively. The localization of annexin A11-mEmerald and ANO1-mCherry was investigated by live-cell microscopy in H69 cholangiocytes after incubation with IRC patient serum containing anti-annexin A11 IgG1/IgG4-autoantibodies or disease control serum. RESULTS: Annexin A11 was strongly expressed in human cholangiocytes, but not hepatocytes. Knockdown of ANXA11 led to reduced plasma membrane expression of ANO1, but not AE2, alkalization of intracellular pH and uncontrolled bile acid influx. High intracellular calcium conditions led to annexin A11 membrane shift and colocalization with ANO1. Incubation with IRC patient serum inhibited annexin A11 membrane shift and reduced ANO1 surface expression. CONCLUSION: Cholangiocellular annexin A11 mediates apical membrane abundance of the chloride channel ANO1, thereby supporting biliary bicarbonate secretion. Insertion is inhibited by IRC patient serum containing anti-annexin A11 IgG1/IgG4-autoantibodies. Anti-annexin A11 autoantibodies may contribute to the pathogenesis of IRC by weakening the 'biliary bicarbonate umbrella'. LAY SUMMARY: We previously identified annexin A11 as a specific autoantigen in immunoglobulin G4-related cholangitis (IRC), a B-cell driven disease affecting the bile ducts. Human cholangiocytes are protected against harmful hydrophobic bile acid influx by a defense mechanism referred to as the 'biliary bicarbonate umbrella'. We found that annexin A11 is required for the formation of a robust bicarbonate umbrella. Binding of patient-derived annexin A11 autoantibodies inhibits annexin A11 function, possibly contributing to bile duct damage by weakening the biliary bicarbonate umbrella in patients with IRC.

ATP8B1 Deficiency Results in Elevated Mitochondrial Phosphatidylethanolamine Levels and Increased Mitochondrial Oxidative Phosphorylation in Human Hepatoma Cells.

ATP8B1 is a phospholipid flippase that is deficient in patients with progressive familial intrahepatic cholestasis type 1 (PFIC1). PFIC1 patients suffer from severe liver disease but also present with dyslipidemia, including low plasma cholesterol, of yet unknown etiology. Here we show that ATP8B1 knockdown in HepG2 cells leads to a strong increase in the mitochondrial oxidative phosphorylation (OXPHOS) without a change in glycolysis. The enhanced OXPHOS coincides with elevated low-density lipoprotein receptor protein and increased mitochondrial fragmentation and phosphatidylethanolamine levels. Furthermore, expression of phosphatidylethanolamine N-methyltransferase, an enzyme that catalyzes the conversion of mitochondrial-derived phosphatidylethanolamine to phosphatidylcholine, was reduced in ATP8B1 knockdown cells. We conclude that ATP8B1 deficiency results in elevated mitochondrial PE levels that stimulate mitochondrial OXPHOS. The increased OXPHOS leads to elevated LDLR levels, which provides a possible explanation for the reduced plasma cholesterol levels in PFIC1 disease.

The extracellular lactate-to-pyruvate ratio modulates the sensitivity to oxidative stress-induced apoptosis via the cytosolic NADH/NAD+ redox state.

The advantages of the Warburg effect on tumor growth and progression are well recognized. However, the relevance of the Warburg effect for the inherent resistance to apoptosis of cancer cells has received much less attention. Here, we show here that the Warburg effect modulates the extracellular lactate-to-pyruvate ratio, which profoundly regulates the sensitivity towards apoptosis induced by oxidative stress in several cell lines. To induce oxidative stress, we used the rapid apoptosis inducer Raptinal. We observed that medium conditioned by HepG2 cells has a high lactate-to-pyruvate ratio and confers resistance to Raptinal-induced apoptosis. In addition, imposing a high extracellular lactate-to-pyruvate ratio in media reduces the cytosolic NADH/NAD+ redox state and protects against Raptinal-induced apoptosis. Conversely, a low extracellular lactate-to-pyruvate ratio oxidizes the cytosolic NADH/NAD+ redox state and sensitizes HepG2 cells to oxidative stress-induced apoptosis. Mechanistically, a high extracellular lactate-to-pyruvate ratio decreases the activation of JNK and Bax under oxidative stress, thereby inhibiting the intrinsic apoptotic pathway. Our observations demonstrate that the Warburg effect of cancer cells generates an anti-apoptotic extracellular environment by elevating the extracellular lactate-to-pyruvate ratio which desensitizes cancer cells towards apoptotic insults. Consequently, our study suggests that the Warburg effect can be targeted to reverse the lactate-to-pyruvate ratios in the tumor microenvironment and thereby re-sensitize cancer cells to oxidative stress-inducing therapies.

Overexpression of the constitutive androstane receptor and shaken 3D-culturing increase biotransformation and oxidative phosphorylation and sensitivity to mitochondrial amiodarone toxicity of HepaRG cells.

The liver cell line HepaRG is one of the preferred sources of human hepatocytes for in vitro applications. However, mitochondrial energy metabolism is relatively low, which affects hepatic functionality and sensitivity to hepatotoxins. Culturing in a bioartificial liver (BAL) system with high oxygen, medium perfusion, low substrate stiffness, and 3D conformation increases HepaRG functionality and mitochondrial activity compared to conventional monolayer culturing. In addition, drug metabolism has been improved by overexpression of the constitutive androstane receptor (CAR), a regulator of drug and energy metabolism in the new HepaRG-CAR line. Here, we investigated the effect of BAL culturing on the HepaRG-CAR line by applying a simple and downscaled BAL culture procedure based on shaking 3D cultures, named Bal-in-a-dish (BALIAD). We compared monolayer and BALIAD cultures of HepaRG and HepaRG-CAR cells. CAR overexpression and BALIAD culturing synergistically or additively increased transcript levels of CAR and three of the seven tested CAR target genes in biotransformation. Additionally, Cytochrome P450 3A4 activity was 35-fold increased. The mitochondrial energy metabolism was enhanced; lactate production and glucose consumption switched into lactate elimination and glucose production. BALIAD culturing alone reduced glycogen content and increased oxygen consumption and mitochondrial content. Both CAR overexpression and BALIAD culturing decreased mitochondrial superoxide levels. HepaRG-CAR BALIADs were most sensitive to mitochondrial toxicity induced by the hepatotoxin amiodarone, as indicated by oxygen consumption and mitochondrial superoxide accumulation. These data show that BALIAD culturing of HepaRG-CAR cells induces high mitochondrial energy metabolism and xenobiotic metabolism, increasing its potential for drug toxicity studies.

Role of the bicarbonate-responsive soluble adenylyl cyclase in cholangiocyte apoptosis in primary biliary cholangitis; a new hypothesis.

Primary biliary cholangitis (PBC) is a chronic fibrosing cholangiopathy characterized by an autoimmune stereotype and defective biliary bicarbonate secretion due to down-regulation of anion exchanger 2 (AE2). Despite the autoimmune features, immunosuppressants are ineffective while two bile acid-based therapies (ursodeoxycholic acid and obeticholic acid) have been shown to improve biochemical and histological features of cholestasis and long-term prognosis. However, the etiology and pathogenesis of PBC is largely unknown. Recently, it has been shown that microRNA-506 (miR-506) on chromosome X is up-regulated in PBC cholangiocytes and suppresses AE2 expression, which sensitizes cholangiocytes to bile salt-induced apoptosis by activating soluble adenylyl cyclase (sAC), an evolutionarily conserved bicarbonate sensor. In this review, we discuss the experimental evidence for the emerging role of the miR-506-AE2-sAC axis in PBC pathogenesis. We further hypothesize that the initial disease trigger induces an X-linked epigenetic change, leading to a female-biased activation of the miR-506-AE2-sAC axis. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni and Peter Jansen.

The phospholipid flippase ATP8B1 mediates apical localization of the cystic fibrosis transmembrane regulator.

Progressive familial intrahepatic cholestasis type 1 (PFIC1) is caused by mutations in the gene encoding the phospholipid flippase ATP8B1. Apart from severe cholestatic liver disease, many PFIC1 patients develop extrahepatic symptoms characteristic of cystic fibrosis (CF), such as pulmonary infection, sweat gland dysfunction and failure to thrive. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel essential for epithelial fluid transport. Previously it was shown that CFTR transcript levels were strongly reduced in livers of PFIC1 patients. Here we have investigated the hypothesis that ATP8B1 is important for proper CFTR expression and function. We analyzed CFTR expression in ATP8B1-depleted intestinal and pulmonary epithelial cell lines and assessed CFTR function by measuring short-circuit currents across transwell-grown ATP8B1-depleted intestinal T84 cells and by a genetically-encoded fluorescent chloride sensor. In addition, we studied CFTR surface expression upon induction of CFTR transcription. We show that CFTR protein levels are strongly reduced in the apical membrane of human ATP8B1-depleted intestinal and pulmonary epithelial cell lines, a phenotype that coincided with reduced CFTR activity. Apical membrane insertion upon induction of ectopically-expressed CFTR was strongly impaired in ATP8B1-depleted cells. We conclude that ATP8B1 is essential for correct apical localization of CFTR in human intestinal and pulmonary epithelial cells, and that impaired CFTR localization underlies some of the extrahepatic phenotypes observed in ATP8B1 deficiency.

Soluble Adenylyl Cyclase Regulates Bile Salt-Induced Apoptosis in Human Cholangiocytes.

UNLABELLED: Anion exchanger 2 (AE2), the principal bicarbonate secretor in the human biliary tree, is down-regulated in primary biliary cholangitis. AE2 creates a "bicarbonate umbrella" that protects cholangiocytes from the proapoptotic effects of bile salts by maintaining them deprotonated. We observed that knockdown of AE2 sensitized immortalized H69 human cholangiocytes to not only bile salt-induced apoptosis (BSIA) but also etoposide-induced apoptosis. Because the toxicity of etoposide is pH-independent, there could be a more general mechanism for sensitization of AE2-depleted cholangiocytes to apoptotic stimuli. We found that AE2 deficiency led to intracellular bicarbonate accumulation and increased expression and activity of soluble adenylyl cyclase (sAC), an evolutionarily conserved bicarbonate sensor. Thus, we hypothesized that sAC regulates BSIA. H69 cholangiocytes and primary mouse cholangiocytes were used as models. The sAC-specific inhibitor KH7 not only reversed sensitization to BSIA in AE2-depleted H69 cholangiocytes but even completely prevented BSIA. sAC knockdown by tetracycline-inducible short hairpin RNA also prevented BSIA. In addition, sAC inhibition reversed BSIA membrane blebbing, nuclear condensation, and DNA fragmentation. Furthermore, sAC inhibition also prevented BSIA in primary mouse cholangiocytes. Mechanistically, sAC inhibition prevented Bax phosphorylation at Thr167 and mitochondrial translocation of Bax and cytochrome c release but not c-Jun N-terminal kinase activation during BSIA. Finally, BSIA in H69 cholangiocytes was inhibited by intracellular Ca(2+) chelation, aggravated by thapsigargin, and unaffected by removal of extracellular calcium. CONCLUSIONS: BSIA is regulated by sAC, depends on intracellular Ca(2+) stores, and is mediated by the intrinsic apoptotic pathway; down-regulation of AE2 in primary biliary cholangitis sensitizes cholangiocytes to apoptotic insults by activating sAC, which may play a crucial role in disease pathogenesis. (Hepatology 2016;64:522-534).

The Emerging Role of Soluble Adenylyl Cyclase in Primary Biliary Cholangitis.

BACKGROUND: Primary biliary cholangitis (PBC; previously referred to as primary biliary cirrhosis) is a chronic fibrosing cholangiopathy with the signature of an autoimmune disease and features of intrahepatic cholestasis. Immunosuppressing treatments are largely unsuccessful. Responsiveness to ursodeoxycholic acid and reduced expression of anion exchanger 2 (AE2) on canalicular membranes and small bile ducts underline the importance of bicarbonate transportation in its disease mechanism. Soluble adenylyl cyclase (sAC; ADCY10) is an evolutionarily conserved bicarbonate sensor that regulates apoptosis, barrier function and TNF signaling. Key Messages: The biliary epithelium defends against the toxic bile by bicarbonate secretion and by maintaining a tight barrier. Passive diffusion of weak acid conjugates (e.g. bile salts and other toxins) across plasma membrane is pH-dependent. Reduced AE2 expression results in both reduced bicarbonate secretion and accumulation of bicarbonate in the cells. Increased intracellular bicarbonate leads to increased sAC activity, which regulates bile salt-induced apoptosis. Reduced bicarbonate secretion causes more bile salts to enter cells, which further increase sAC activity by releasing intracellular Ca2+ store. In vitro studies demonstrate that inhibition of sAC not only corrects sensitization to bile salt-induced apoptosis as a result of AE2 down-regulation but also prevents bile salt-induced apoptosis altogether. Targeting sAC is also likely to slow down disease progression by strengthening the barrier function of biliary epithelia and by reducing oxidative stress as a result of chronic inflammation. CONCLUSIONS: sAC is a potential therapeutic target for PBC. More in vitro and in vivo studies are needed to understand how sAC regulates bile salt-induced apoptosis and to establish its therapeutic value in PBC and other cholestatic cholangiopathies.

Elevated interleukin-8 in bile of patients with primary sclerosing cholangitis.

BACKGROUND & AIMS: To better understand the pathogenesis of primary sclerosing cholangitis, anti- and pro-inflammatory factors were studied in bile. METHODS: Ductal bile of PSC patients (n = 36) and controls (n = 20) was collected by endoscopic retrograde cholangiography. Gallbladder bile was collected at liver transplantation. Bile samples were analysed for cytokines, FGF19 and biliary lipids. Hepatobiliary tissues of PSC and non-PSC patients (n = 8-11 per patient group) were collected at transplantation and were analysed for IL8 and FGF19 mRNA expression and IL8 localization. The effect of IL8 on proliferation of primary human cholangiocytes and expression of pro-fibrotic genes was studied. RESULTS: In PSC patients, median IL8 in ductal bile was 6.6 ng/ml vs. 0.24 ng/ml in controls. Median IL8 in gallbladder bile was 7.6 ng/ml in PSC vs. 2.2 and 0.3 ng/ml in two control groups. IL8 mRNA in PSC gallbladder was increased and bile ducts stained positive for IL8. In vitro, IL8 induced proliferation of primary human cholangiocytes and increased the expression of pro-fibrotic genes. CONCLUSION: Elevation of IL8 in bile of PSC patients, collected at different stages of disease, indicates an ongoing inflammatory stimulus that drives IL8 production. This challenges the idea that advanced PSC is a burned-out disease, and calls for reconsideration of anti-inflammatory therapy in PSC.

Cyclic AMP and alkaline pH downregulate carbonic anhydrase 2 in mouse fibroblasts.

BACKGROUND: The hydration of CO2 catalyzed by the ubiquitous carbonic anhydrase 2 (Ca2) is central for bicarbonate transport, bone metabolism and acid-base homeostasis in metazoans. There is evidence that in some tissues Ca2 expression can be acutely induced by cAMP, whereas in other cell types it is unresponsive to cAMP-mediated transcriptional activation. METHODS: We isolated fibroblasts from wild type and mice lacking the ubiquitous chloride/bicarbonate exchanger (Ae2a,b(-/-) mice). In these cells the regulation of carbonic anhydrase 2 by cAMP was studied. RESULTS: We show that Ca2 expression is strongly inhibited by chronic incubation with dibutyryl-cAMP, forskolin or alkaline pH in cultured mouse fibroblasts. Furthermore, fibroblasts obtained from anion exchanger 2 deficient (Ae2a,b(-/-)) mice, which display intracellular alkalosis and increased cAMP production, express less than 10% of control Ca2 mRNA and protein. Surprisingly, inhibition of the bicarbonate-sensitive soluble adenylyl cyclase (sAC) was found to reduce CA2 expression instead of increasing it. CONCLUSIONS: CA2 expression is strongly regulated by intracellular pH and by cAMP, suggesting a role for soluble adenylyl cyclase. Regulation occurs in opposite directions which may be explained by an incoherent feedforward loop consisting of activation by pCREB and repression by ICER.

Soluble adenylyl cyclase, the cell-autonomous member of the family.

Soluble adenylyl cyclase (sAC) is the evolutionarily most ancient of a set of 10 adenylyl cyclases (Adcys). While Adcy1 to Adcy9 are cAMP-producing enzymes that are activated by G-protein coupled receptors (GPCRs), Adcy10 (sAC) is an intracellular adenylyl cyclase. sAC plays a pivotal role in numerous cellular processes, ranging from basic physiological functions to complex signaling cascades. As a distinct member of the adenylyl cyclase family, sAC is not activated by GPCRs and stands apart due to its unique characteristics, regulation, and localization within cells. This minireview aims to honour Ulli Brandt, the outgoing Executive Editor of our journal, Biochimica Biophysica Acta (BBA), and longstanding Executive Editor of the BBA section Bioenergetics. We will therefore focus this review on bioenergetic aspects of sAC and, in addition, review some important recent general developments in the field of research on sAC.

Deciphering metabolic crosstalk in context: lessons from inflammatory diseases.

Metabolism plays a crucial role in regulating the function of immune cells in both health and disease, with altered metabolism contributing to the pathogenesis of cancer and many inflammatory diseases. The local microenvironment has a profound impact on the metabolism of immune cells. Therefore, immunological and metabolic heterogeneity as well as the spatial organization of cells in tissues should be taken into account when studying immunometabolism. Here, we highlight challenges of investigating metabolic communication. Additionally, we review the capabilities and limitations of current technologies for studying metabolism in inflamed microenvironments, including single-cell omics techniques, flow cytometry-based methods (Met-Flow, single-cell energetic metabolism by profiling translation inhibition (SCENITH)), cytometry by time of flight (CyTOF), cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq), and mass spectrometry imaging. Considering the importance of metabolism in regulating immune cells in diseased states, we also discuss the applications of metabolomics in clinical research, as well as some hurdles to overcome to implement these techniques in standard clinical practice. Finally, we provide a flowchart to assist scientists in designing effective strategies to unravel immunometabolism in disease-relevant contexts.

Metabolic changes underlying drug resistance in the multiple myeloma tumor microenvironment.

Multiple myeloma (MM) is characterized by the clonal expansion of malignant plasma cells in the bone marrow (BM). MM remains an incurable disease, with the majority of patients experiencing multiple relapses from different drugs. The MM tumor microenvironment (TME) and in particular bone-marrow stromal cells (BMSCs) play a crucial role in the development of drug resistance. Metabolic reprogramming is emerging as a hallmark of cancer that can potentially be exploited for cancer treatment. Recent studies show that metabolism is further adjusted in MM cells during the development of drug resistance. However, little is known about the role of BMSCs in inducing metabolic changes that are associated with drug resistance. In this Perspective, we summarize current knowledge concerning the metabolic reprogramming of MM, with a focus on those changes associated with drug resistance to the proteasome inhibitor Bortezomib (BTZ). In addition, we present proof-of-concept fluxomics (glucose isotope-tracing) and Seahorse data to show that co-culture of MM cells with BMSCs skews the metabolic phenotype of MM cells towards a drug-resistant phenotype, with increased oxidative phosphorylation (OXPHOS), serine synthesis pathway (SSP), TCA cycle and glutathione (GSH) synthesis. Given the crucial role of BMSCs in conveying drug resistance, insights into the metabolic interaction between MM and BMSCs may ultimately aid in the identification of novel metabolic targets that can be exploited for therapy.

Gastrointestinal Mast Cell Tumor in an African Dormouse (Graphiurus sp.).

Mast cell tumors (MCTs) are well-known neoplasms derived from either mucosal or connective tissue mast cells. While well studied in several domestic species, MCTs are rarely documented in rodents. A three-year-old, male African dormouse (Graphiurus sp.) presented with a history of vomiting and anorexia for 3 months. Sonography revealed thickened gastric mucosa and hyperperistalsis. The patient died after receiving symptomatic treatment for 2 months. At necropsy, locally extensive, pale, thickened mucosal foci obscuring the first half of the stomach lumen was noted. Histological examination revealed moderately polymorphic, round, oval to spindle cells with amphophilic cytoplasmic granules infiltrating the mucosa to tunica muscularis, with moderate numbers of eosinophils. The mucosa was severely ulcerated with the proliferation of granulation tissue. The granules in most tumor cells exhibited metachromasia with the toluidine blue stain. Neoplastic cells revealed positive membranous immunoreactivity to KIT. Herein, we report the first case report of MCT in dormouse but also the first gastrointestinal MCT in a rodent species.

Innate Immune Training of Human Macrophages by Cathelicidin Analogs.

Trained innate immunity can be induced in human macrophages by microbial ligands, but it is unknown if exposure to endogenous alarmins such as cathelicidins can have similar effects. Previously, we demonstrated sustained protection against infection by the chicken cathelicidin-2 analog DCATH-2. Thus, we assessed the capacity of cathelicidins to induce trained immunity. PMA-differentiated THP-1 (dTHP1) cells were trained with cathelicidin analogs for 24 hours and restimulated after a 3-day rest period. DCATH-2 training of dTHP-1 cells amplified their proinflammatory cytokine response when restimulated with TLR2/4 agonists. Trained cells displayed a biased cellular metabolism towards mTOR-dependent aerobic glycolysis and long-chain fatty acid accumulation and augmented microbicidal activity. DCATH-2-induced trained immunity was inhibited by histone acetylase inhibitors, suggesting epigenetic regulation, and depended on caveolae/lipid raft-mediated uptake, MAPK p38 and purinergic signaling. To our knowledge, this is the first report of trained immunity by host defense peptides.

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.

Improved oxygenation dramatically alters metabolism and gene expression in cultured primary mouse hepatocytes.

Primary hepatocyte culture is an important in vitro system for the study of liver functions. In vivo, hepatocytes have high oxidative metabolism. However, oxygen supply by means of diffusion in in vitro static cultures is much less than that by blood circulation in vivo. Therefore, we investigated whether hypoxia contributes to dedifferentiation and deregulated metabolism in cultured hepatocytes. To this end, murine hepatocytes were cultured under static or shaken (60 revolutions per minute) conditions in a collagen sandwich. The effect of hypoxia on hepatocyte cultures was examined by metabolites in media and cells, hypoxia-inducible factors (HIF)-1/2α western blotting, and real-time quantitative polymerase chain reaction for HIF target genes and key genes of glucose and lipid metabolism. Hepatocytes in shaken cultures showed lower glycolytic activity and triglyceride accumulation than static cultures, compatible with improved oxygen delivery and mitochondrial energy metabolism. Consistently, static cultures displayed significant HIF-2α expression, which was undetectable in freshly isolated hepatocytes and shaken cultures. Transcript levels of HIF target genes (glyceraldehyde 3-phosphate dehydrogenase [Gapdh], glucose transporter 1 [Glut1], pyruvate dehydrogenase kinase 1 [Pdk1], and lactate dehydrogenase A [Ldha]) and key genes of lipid metabolism, such as carnitine palmitoyltransferase 1 (Cpt1), apolipoprotein B (Apob), and acetyl-coenzyme A carboxylase 1 (Acc1), were significantly lower in shaken compared to static cultures. Moreover, expression of hepatocyte nuclear factor 4α (Hnf4α) and farnesoid X receptor (Fxr) were better preserved in shaken cultures as a result of improved oxygen delivery. We further revealed that HIF-2 signaling was involved in hypoxia-induced down-regulation of Fxr. Conclusion: Primary murine hepatocytes in static culture suffer from hypoxia. Improving oxygenation by simple shaking prevents major changes in expression of metabolic enzymes and aberrant triglyceride accumulation; in addition, it better maintains the differentiation state of the cells. The shaken culture is, therefore, an advisable strategy for the use of primary hepatocytes as an in vitro model. (Hepatology Communications 2018;2:299-312).

Oxygen drives hepatocyte differentiation and phenotype stability in liver cell lines.

The in vitro generation of terminally differentiated hepatocytes is an unmet need. We investigated the contribution of oxygen concentration to differentiation in human liver cell lines HepaRG and C3A. HepaRG cells were cultured under hypoxia (5%O2), normoxia (21%O2) or hyperoxia (40%O2). Cultures were analysed for hepatic functions, gene transcript levels, and protein expression of albumin, hepatic transcription factor CEBPα, hepatic progenitor marker SOX9, and hypoxia inducible factor (HIF)1α. C3A cells were analysed after exposure to normoxia or hyperoxia. In hyperoxic HepaRG cultures, urea cycle activity, bile acid synthesis, CytochromeP450 3A4 (CYP3A4) activity and ammonia elimination were 165-266% increased. These effects were reproduced in C3A cells. Whole transcriptome analysis of HepaRG cells revealed that 240 (of 23.223) probes were differentially expressed under hyperoxia, with an overrepresentation of genes involved in hepatic differentiation, metabolism and extracellular signalling. Under hypoxia, CYP3A4 activity and ammonia elimination were inhibited almost completely and 5/5 tested hepatic genes and 2/3 tested hepatic transcription factor genes were downregulated. Protein expression of SOX9 and HIF1α was strongly positive in hypoxic cultures, variable in normoxic cultures and predominantly negative in hyperoxic cultures. Conversely, albumin and CEBPα expression were highest in hyperoxic cultures. HepaRG cells that were serially passaged under hypoxia maintained their capacity to differentiate under normoxia, in contrast to cells passaged under normoxia. Hyperoxia increases hepatocyte differentiation in HepaRG and C3A cells. In contrast, hypoxia maintains stem cell characteristics and inhibits hepatic differentiation of HepaRG cells, possibly through the activity of HIF1α.

Role of the bicarbonate-responsive soluble adenylyl cyclase in pH sensing and metabolic regulation.

The evolutionarily conserved soluble adenylyl cyclase (sAC, adcy10) was recently identified as a unique source of cAMP in the cytoplasm and the nucleus. Its activity is regulated by bicarbonate and fine-tuned by calcium. As such, and in conjunction with carbonic anhydrase (CA), sAC constitutes an HCO(-) 3/CO(-) 2/pH sensor. In both alpha-intercalated cells of the collecting duct and the clear cells of the epididymis, sAC is expressed at significant level and involved in pH homeostasis via apical recruitment of vacuolar H(+)-ATPase (VHA) in a PKA-dependent manner. In addition to maintenance of pH homeostasis, sAC is also involved in metabolic regulation such as coupling of Krebs cycle to oxidative phosphorylation via bicarbonate/CO2 sensing. Additionally, sAC also regulates CFTR channel and plays an important role in regulation of barrier function and apoptosis. These observations suggest that sAC, via bicarbonate-sensing, plays an important role in maintaining homeostatic status of cells against fluctuations in their microenvironment.