Apolipoprotein E Codetermines Tissue Tropism of Hepatitis C Virus and Is Crucial for Viral Cell-to-Cell Transmission by Contributing to a Postenvelopment Step of Assembly

Hepatitis C virus (HCV) predominantly infects human hepatocytes, although extrahepatic virus reservoirs are being discussed. Infection of cells is initiated via cell-free and direct cell-to-cell transmission routes. Cell type-specific determinants of HCV entry and RNA replication have been reported. Moreover, several host factors required for synthesis and secretion of lipoproteins from liver cells, in part expressed in tissue-specific fashion, have been implicated in HCV assembly. However, the minimal cell type-specific requirements for HCV assembly have remained elusive. Here we report that production of HCV trans-complemented particles (HCV_TCP) from nonliver cells depends on ectopic expression of apolipoprotein E (ApoE). For efficient virus production by full-length HCV genomes, microRNA 122 (miR-122)-mediated enhancement of RNA replication is additionally required. Typical properties of cell culture-grown HCV (HCVcc) particles from ApoE-expressing nonliver cells are comparable to those of virions derived from human hepatoma cells, although specific infectivity of virions is modestly reduced. Thus, apolipoprotein B (ApoB), microsomal triglyceride transfer protein (MTTP), and apolipoprotein C1 (ApoC1), previously implicated in HCV assembly, are dispensable for production of infectious HCV. In the absence of ApoE, release of core protein from infected cells is reduced, and production of extracellular as well as intracellular infectivity is ablated. Since envelopment of capsids was not impaired, we conclude that ApoE acts after capsid envelopment but prior to secretion of infectious HCV. Remarkably, the lack of ApoE also abrogated direct HCV cell-to-cell transmission. These findings highlight ApoE as a host factor codetermining HCV tissue tropism due to its involvement in a late assembly step and viral cell-to-cell transmission.     

Different Requirements for Scavenger Receptor Class B Type I in Hepatitis C Virus Cell-Free versus Cell-to-Cell Transmission

Hepatitis C virus (HCV) is believed to initially infect the liver through the basolateral side of hepatocytes, where it engages attachment factors and the coreceptors CD81 and scavenger receptor class B type I (SR-BI). Active transport toward the apical side brings the virus in close proximity of additional entry factors, the tight junction molecules claudin-1 and occludin. HCV is also thought to propagate via cell-to-cell spread, which allows highly efficient virion delivery to neighboring cells. In this study, we compared an adapted HCV genome, clone 2, characterized by superior cell-to cell spread, to its parental genome, J6/JFH-1, with the goal of elucidating the molecular mechanisms of HCV cell-to-cell transmission. We show that CD81 levels on the donor cells influence the efficiency of cell-to-cell spread and CD81 transfer between neighboring cells correlates with the capacity of target cells to become infected. Spread of J6/JFH-1 was blocked by anti-SR-BI antibody or in cells knocked down for SR-BI, suggesting a direct role for this receptor in HCV cell-to-cell transmission. In contrast, clone 2 displayed a significantly reduced dependence on SR-BI for lateral spread. Mutations in E1 and E2 responsible for the enhanced cell-to-cell spread phenotype of clone 2 rendered cell-free virus more susceptible to antibody-mediated neutralization. Our results indicate that although HCV can lose SR-BI dependence for cell-to-cell spread, vulnerability to neutralizing antibodies may limit this evolutionary option in vivo. Combination therapies targeting both the HCV glycoproteins and SR-BI may therefore hold promise for effective control of HCV dissemination.     

The mitochondria-targeted imidazole substituted oleic acid ‘TPP-IOA’ affects mitochondrial bioenergetics and its protective efficacy in cells is influenced by cellular dependence on aerobic metabolism

A variety of mitochondria-targeted small molecules have been invented to manipulate mitochondrial redox activities and improve function in certain disease states. 3-Hydroxypropyl-triphenylphosphonium-conjugated imidazole-substituted oleic acid (TPP-IOA) was developed as a specific inhibitor of cytochrome c peroxidase activity that inhibits apoptosis by preventing cardiolipin oxidation and cytochrome c release to the cytosol. Here we evaluate the effects of TPP-IOA on oxidative phosphorylation in isolated mitochondria and on mitochondrial function in live cells. We demonstrate that, at concentrations similar to those required to achieve inhibition of cytochrome c peroxidase activity, TPP-IOA perturbs oxidative phosphorylation in isolated mitochondria. In live SH-SY5Y cells, TPP-IOA partially collapsed mitochondrial membrane potential, caused extensive fragmentation of the mitochondrial network, and decreased apparent mitochondrial abundance within 3 h of exposure. Many cultured cell lines rely primarily on aerobic glycolysis, potentially making them less sensitive to small molecules disrupting oxidative phosphorylation. We therefore determined the anti-apoptotic efficacy of TPP-IOA in SH-SY5Y cells growing in glucose or in galactose, the latter of which increases reliance on oxidative phosphorylation for ATP supply. The anti-apoptotic activity of TPP-IOA that was observed in glucose media was not seen in galactose media. It therefore appears that, at concentrations required to inhibit cytochrome c peroxidase activity, TPP-IOA perturbs oxidative phosphorylation. In light of these data it is predicted that potential future therapeutic applications of TPP-IOA will be restricted to highly glycolytic cell types with limited reliance on oxidative phosphorylation.     

Claudin-6 and Occludin Natural Variants Found in a Patient Highly Exposed but Not Infected with Hepatitis C Virus (HCV) Do Not Confer HCV Resistance In Vitro

The clinical course of Hepatitis C Virus (HCV) infection is highly variable between infected individual hosts: up to 80% of acutely HCV infected patients develop a chronic infection while 20% clear infection spontaneously. Spontaneous clearance of HCV infection can be predicted by several factors, including symptomatic acute infection, favorable IFNL3 polymorphisms and gender. In our study, we explored the possibility that variants in HCV cell entry factors might be involved in resistance to HCV infection. In a same case patient highly exposed but not infected by HCV, we previously identified one mutation in claudin-6 (CLDN6) and a rare variant in occludin (OCLN), two tight junction proteins involved in HCV entry into hepatocytes. Here, we conducted an extensive functional study to characterize the ability of these two natural variants to prevent HCV entry. We used lentiviral vectors to express Wildtype or mutated CLDN6 and OCLN in different cell lines and primary human hepatocytes. HCV infection was then investigated using cell culture produced HCV particles (HCVcc) as well as HCV pseudoparticles (HCVpp) expressing envelope proteins from different genotypes. Our results show that variants of CLDN6 and OCLN expressed separately or in combination did not affect HCV infection nor cell-to-cell transmission. Hence, our study highlights the complexity of HCV resistance mechanisms supporting the fact that this process probably not primarily involves HCV entry factors and that other unknown host factors may be implicated.     

Adenylate energy charge of rat and human cultured hepatocytes

Summary A simple and rapid method for the assay of adenine nucleotides (ATP, ADP, and AMP) was established to evaluate the adenylate energy charge (ATP+ADP/2)/(ATP+ADP+AMP) of cultured hepatocytes. The effects of inhibitors of glycolysis, fatty acid oxidation, or oxidative phosphorylation on the energy charge were examined. The energy charges of cultured hepatocytes in rats and human were almost identical and were maintained at a high level between 6 and 24 h after changing the media (rat: 0.908±0.008n=9, human: 0.918±0.014n=6, mean ± SD). Inhibition of glycolysis with sodium fluoride or oxidative phosphorylation with antimycin A irreversibly reduced both the adenine nucleotide contents and the energy charge. However, the inhibition of fatty acid oxidation with 2-tetradecylglycidic acid did not affect the nucleotide contents, and the energy charge only decreased transiently to recover within 8 h. When the inhibitor of oxidative phosphorylation was removed, the recovery in the energy charge preceded the recovery in the adenine nucleotide contents. These findings suggest that the adenylate energy charge is a more sensitive measure of the changes in energy metabolism than the adenine nucleotide contents. Furthermore, energy charge regulates adenine nucleotide contents in cultured hepatocytes. It is important to confirm that the high energy charge of the cultured hepatocytes is maintained when these cells are used for metabolic studies.     

Survival of Primary Human Hepatocytes and Death of Induced Pluripotent Stem Cells in Media Lacking Glucose and Arginine

Background

Tumorigenicity is an associated risk for transplantation of hepatocytes differentiated from human induced pluripotent stem (hiPS) cells. Hepatocytes express the enzymes galactokinase and ornithine transcarbamylase (OTC) to aid in their own survival. However, hiPS cells do not express these enzymes, and therefore, are not be expected to survive in a medium containing galactose and ornithine and lacking glucose and arginine.

Materials and Methods

Real-time quantitative polymerase chain reaction (PCR) was performed to analyze the expression of galactokinase 1 (GALK1)1 and GALK2, ornithine carbamyltransferase, and phenylalanine hydroxylase (PAH). The hiPS cell line 201B7 was cultured in hepatocyte selection medium (HSM), which lacks glucose and arginine but contains galactose and ornithine. Furthermore, microscopic analysis of the cultured cells was performed after hematoxylin and eosin (H&E) staining, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL). The hiPS cells were immunostained to assess their pluripotency in HSM. In addition, the primary human hepatocytes were cultured with or without hiPS cells in HSM.

Results

The expression levels of GALK1, GALK2, OTC, and PAH in 201B7 were 22.2±5.0 (average ± standard deviation), 14.2% ±1.1%, 1.2% ±0.2%, and 8.4% ±0.7% respectively, compared with those in the adult liver. The hiPS cell population diminished when cultured in HSM and completely disappeared after 3 days. The cultured cells showed condensation or fragmentation of their nuclei, thereby suggesting apoptosis. TUNEL staining confirmed that the cells had undergone apoptosis. The 201B7 cells were positive for Nanog, SSEA-4, and TRA-1-60. The primary human hepatocytes survived when cultured alone in HSM and when co-cultured with hiPS cells.

Conclusion

Therefore, HSM is and ideal medium for eliminating hiPS cells and purifying hepatocytes without inducing any damage.     

Long chain acyl-CoA synthetase 3-mediated phosphatidylcholine synthesis is required for assembly of very low density lipoproteins in human hepatoma Huh7 cells

Hepatocytes play a crucial role in regulating lipid metabolism by exporting cholesterol and triglyceride into plasma through secretion of very low density lipoproteins (VLDL). VLDL production is also required for release of hepatitis C virus (HCV) from infected hepatocytes. Here, we show that long chain acyl-CoA synthetase 3 (ACSL3) plays a crucial role in secretion of VLDL and HCV from hepatocytes. In cultured human hepatoma Huh7 cells, ACSL3 is specifically required for incorporation of fatty acids into phosphatidylcholine. In cells receiving small interfering RNA targeting ACSL3, secretion of apolipoprotein B, the major protein component of VLDL, was inhibited and the lipoprotein was rapidly degraded. This inhibition in secretion was completely eliminated when these cells were treated with phosphatidylcholine. Treatment of cells with small interfering RNA targeting ACSL3 also inhibited secretion of HCV from Huh7-derived cells. These results identify ACSL3 as a new enzymatic target to limit VLDL secretion and HCV infection.     

Receptor-mediated endocytosis of galactose and mannose exposing ligands: an electron microscopic study on adult and neonatal cultured rat hepatocytes.

We compared the receptor-mediated endocytosis for galactose and mannose exposing ligands in primary cultures of hepatocytes from newborn and adult rats. The endocytic pathway was revealed ultrastructurally using colloidal gold particles coupled to lactosylated bovine serum albumin and invertase. The binding activity on the cell surfaces is observed by keeping the cells at 4 degrees C. For both ligands used, the binding capacity for hepatocytes from adult rats was greater than for neonatal cultured cells. Increasing the temperature to 37 degrees C, we observed that the protein-gold complexes entered the intracellular endocytic organelles. Within 5-15 min, the marker was confined in vesicles close to the cell surface and in the endosome, while after 60 min, the marker is found in lysosome-like compartments. We found that the process of endocytosis is similar for galactose and mannose exposing ligands. The organelles involved in the process of endocytosis in newborn cultured hepatocytes are not different in shape from those of cultured cells of adult rats, but the process of internalization is slower.     

Specific proto-oncogenic tyrosine kinases of src family are enriched in cell-to-cell adherens junctions where the level of tyrosine phosphorylation is elevated

To approach the transmembrane signaling pathway in the cell-to-cell adherens junctions (AJ), AJ-specific tyrosine phosphorylation was analyzed. When various types of rat adult tissues were pretreated with sodium orthovanadate, a potent inhibitor of tyrosine phosphatase, immunofluorescence microscopy showed that anti-phosphotyrosine polyclonal antibody specifically stained the undercoat of the cell-to-cell AJ. This indicates that the tyrosine kinase activity is elevated at the undercoat of the cell-to-cell AJ of adult tissues. To identify tyrosine kinases responsible for the high level of tyrosine phosphorylation at AJ, we have performed in vitro phosphorylation experiments with cell-to-cell AJ isolated from rat liver (Tsukita, Sh. and Sa. Tsukita. 1989. J. Cell Biol. 108:31-41) and immunoblotting analyses with specific antibodies for tyrosine kinases. As a result, three proto-oncogenic tyrosine kinases of src family, c-yes, c-src, and lyn kinases, were identified as major tyrosine kinases in the cell-to-cell AJ of hepatocytes. Furthermore, it was immunofluorescently shown that at least two of these kinases, c-yes and c-src kinases, were enriched at the cell-to-cell AJ of various types of cells including hepatocytes. Based on these findings, it is concluded that, in various types of cells, specific proto-oncogenic tyrosine kinases of src-family (c-yes and c-src) are enriched to work as signal mediators in the cell-to-cell AJ where the level of tyrosine phosphorylation is elevated.     

Circumventing the Crabtree Effect: A method to induce lactate consumption and increase oxidative phosphorylation in cell culture

Most cells grown in glucose-containing medium generate almost all their ATP via glycolysis despite abundant oxygen supply and functional mitochondria, a phenomenon known as the Crabtree effect. By contrast, most cells within the body rely on mitochondrial oxidative phosphorylation (OXPHOS) to generate the bulk of their energy supply. Thus, when utilising the accessibility of cell culture to elucidate fundamental elements of mitochondria in health and disease, it is advantageous to adopt culture conditions under which the cells have greater reliance upon OXPHOS for the supply of their energy needs. Substituting galactose for glucose in the culture medium can provide these conditions, but additional benefit can be gained from alternate in vitro models. Herein we describe culture conditions in which complete autonomous depletion of medium glucose induces a lactate-consuming phase marked by increased MitoTracker Deep Red staining intensity, increased expression of Kreb’s cycle proteins, increased expression of electron transport chain subunits, and increased sensitivity to the OXPHOS inhibitor rotenone. We propose these culture conditions represent an alternate accessible model for the in vitro study of cellular processes and diseases involving the mitochondrion without limitations incurred via the Crabtree effect.     

MicroRNA-7 Promotes Glycolysis to Protect against 1-Methyl-4-phenylpyridinium-induced Cell Death

Parkinson disease is associated with decreased activity of the mitochondrial electron transport chain. This defect can be recapitulated in vitro by challenging dopaminergic cells with 1-methyl-4-phenylpyridinium (MPP⁺), a neurotoxin that inhibits complex I of electron transport chain. Consequently, oxidative phosphorylation is blocked, and cells become dependent on glycolysis for ATP production. Therefore, increasing the rate of glycolysis might help cells to produce more ATP to meet their energy demands. In the present study, we show that microRNA-7, a non-coding RNA that protects dopaminergic neuronal cells against MPP⁺-induced cell death, promotes glycolysis in dopaminergic SH-SY5Y and differentiated human neural progenitor ReNcell VM cells, as evidenced by increased ATP production, glucose consumption, and lactic acid production. Through a series of experiments, we demonstrate that targeted repression of RelA by microRNA-7, as well as subsequent increase in the neuronal glucose transporter 3 (Glut3), underlies this glycolysis-promoting effect. Consistently, silencing Glut3 expression diminishes the protective effect of microRNA-7 against MPP⁺. Further, microRNA-7 fails to prevent MPP⁺-induced cell death when SH-SY5Y cells are cultured in a low glucose medium, as well as when differentiated ReNcell VM cells or primary mouse neurons are treated with the hexokinase inhibitor, 2-deoxy-d-glucose, indicating that a functional glycolytic pathway is required for this protective effect. In conclusion, microRNA-7, by down-regulating RelA, augments Glut3 expression, promotes glycolysis, and subsequently prevents MPP⁺-induced cell death. This protective effect of microRNA-7 could be exploited to correct the defects in oxidative phosphorylation in Parkinson disease.     

Bystander CD4 T-cell death is inhibited by broadly neutralizing anti-HIV antibodies only at levels blocking cell-to-cell viral transmission

The progressive loss of CD4+ T cells during HIV infection of lymphoid tissues involves both the apoptotic death of activated and productively infected CD4 T cells and the pyroptotic death of large numbers of resting and abortively infected bystander CD4 T cells. HIV spreads both through cellular release of virions and cell-to-cell transmission involving the formation of virological synapses. Cell-to-cell transmission results in high-level transfer of large quantities of virions to the target cell exceeding that achieved with cell-free virions. Broadly neutralizing anti-HIV antibodies (bNAbs) binding to HIV envelope protein capably block cell-free virus spread, and when added at higher concentrations can also interdict cell-to-cell transmission. Exploiting these distinct dose–response differences, we now show that four different bNAbs block the pyroptotic death of bystander cells, but only when added at concentrations sufficient to block cell-to-cell transmission. These findings further support the conclusion that HIV killing of abortively infected bystander CD4 T cells requires cell-to-cell transfer of virions. As bNAbs attract more interest as potential therapeutics, it will be important to consider the higher concentrations of these antibodies required to block the inflammatory death of bystander CD4 T cells.     

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.     

Loss of Infectivity of HIV-1 Particles Produced by Mobile Lymphocytes

HIV-1 spreads by cell-free particles and through direct cell contacts. To discriminate between these two modes of dissemination, an assay in which the cells are cultured under shaking conditions impairing cell-to-cell transmission has been described. We addressed the impact of shaking on HIV-1 particle infectivity. Kinetics of HIV-1 infection in static or shaking conditions confirmed that HIV-1 replication is reduced in mobile lymphocyte T cells. Strikingly, the infectivity of viruses produced by mobile lymphocytes was dramatically reduced. In parallel, the amount of envelope protein present on these particles showed a continuous decrease over time. We conclude that inefficient HIV-1 replication in mobile lymphocytes in this experimental system is not only due to avoidance of viral cell-to-cell transfer but also to the loss of infectivity of the viral particles due to the alteration of the composition and functionality of the particles produced by these lymphocytes. It is important to take these observations into account when studying viral transmission under shaking conditions.     

Human alveolar macrophages do not rely on glucose metabolism upon activation by lipopolysaccharide

Most macrophages generate energy to mount an inflammatory cytokine response by increased glucose metabolism through intracellular glycolysis. Previous studies have suggested that alveolar macrophages (AMs), which reside in a glucose-poor natural environment, are less capable to utilize glycolysis and instead rely on other substrates to fuel oxidative phosphorylation (OXPHOS) for energy supply. At present, it is not known whether AMs are capable to use glucose metabolism to produce cytokines when other metabolic options are blocked. Here, we studied human AMs retrieved by bronchoalveolar lavage from healthy subjects, and examined their glucose metabolism in response to activation by the gram-negative bacterial component lipopolysaccharide (LPS) ex vivo. The immunological and metabolic responses of AMs were compared to those of cultured blood monocyte-derived macrophages (MDMs) from the same subjects. LPS stimulation enhanced cytokine release by both AMs and MDMs, which was associated with increased lactate release by MDMs (reflecting glycolysis), but not by AMs. In agreement, LPS induced higher mRNA expression of multiple glycolytic regulators in MDMs, but not in AMs. Flux analyses of [¹³C]-glucose revealed no differences in [¹³C]-incorporation in glucose metabolism intermediates in AMs. Inhibition of OXPHOS by oligomycin strongly reduced LPS-induced cytokine production by AMs, but not by MDMs. Collectively, these results indicate that human AMs, in contrast to MDMs, do not use glucose metabolism during LPS-induced activation and fully rely on OXPHOS for cytokine production.     

Exosomes from Hepatitis C Infected Patients Transmit HCV Infection and Contain Replication Competent Viral RNA in Complex with Ago2-miR122-HSP90

Antibodies targeting receptor-mediated entry of HCV into hepatocytes confer limited therapeutic benefits. Evidence suggests that exosomes can transfer genetic materials between cells; however, their role in HCV infection remains obscure. Here, we show that exosomes isolated from sera of chronic HCV infected patients or supernatants of J6/JFH1-HCV-infected Huh7.5 cells contained HCV RNA. These exosomes could mediate viral receptor-independent transmission of HCV to hepatocytes. Negative sense HCV RNA, indicative of replication competent viral RNA, was present in exosomes of all HCV infected treatment non-responders and some treatment-naïve individuals. Remarkably, HCV RNA was associated with Ago2, HSP90 and miR-122 in exosomes isolated from HCV-infected individuals or HCV-infected Huh7.5 cell supernatants. Exosome-loading with a miR-122 inhibitor, or inhibition of HSP90, vacuolar H⁺-ATPases, and proton pumps, significantly suppressed exosome-mediated HCV transmission to naïve cells. Our findings provide mechanistic evidence for HCV transmission by blood-derived exosomes and highlight potential therapeutic strategies.     

Bivascular liver perfusion in the anterograde and retrograde modes: Zonation of the response to inhibitors of oxidative phosphorylation

The action of cyanide (500 μM ), 2,4-dinitrophenol (50 μM ) and atractyloside (100 μM ) on glycogen catabolism and oxygen uptake was investigated in the bivascularly perfused liver of fed rats. Cyanide, 2,4-dinitrophenol and atractyloside were infused at identical rates into the hepatic artery in either the anterograde or retrograde perfusion. The accessible aqueous cell spaces were determined by means of the multiple-indicator dilution technique. Glucose release, oxygen uptake and glycolysis were measured as metabolic parameters. Oxygen uptake changes per unit cell space caused by atractyloside (inhibition) and 2,4-dinitrophenol (stimulation) were equal in the retrograde perfusion (periportal cells) and the anterograde perfusion (space enriched in perivenous cells); the decreases caused by cyanide were higher in the retrograde perfusion. Glucose release from periportal cells was not increased upon inhibition of oxidative phosphorylation, a phenomenon which was independent of the mechanism of action of the inhibitor. There were nearly identical changes in glycolysis in the periportal and perivenous cells. It was concluded that: (1) oxygen concentration in the perfused rat liver, if maintained above 100 μM , had little influence on the zonation of the respiratory activity; (2) in spite of the lower activities of the key enzymes of glycolysis in the periportal hepatocytes, as assayed under standard conditions, these cells were as effective as the perivenous ones in generating ATP in the cytosol when oxidative phosphorylation was impaired; (3) the key enzymes of glycogenolysis and glycolysis in periportal and perivenous cells responded differently to changes in the energy charge.     

Decreasing glycolysis increases sensitivity to mitochondrial inhibition in primary cultures of renal proximal tubule cells

Summary We have previously shown that shaking the culture plates (SHAKE) of rabbit renal proximal tubule cells (RPTC) to maintain adequate aeration increased aerobic metabolism and decreased the induction of glycolysis compared to RPTC cultured under standard conditions (STILL). However, glycolysis in SHAKE RPTC remained elevated compared to glycolysis in proximal tubules in vivo. In the present study the contribution of culture medium sugar composition and concentration to glycolytic metabolism was assessed in RPTC. SHAKE and STILL RPTC cultured in 5 mM glucose contained lactate levels equivalent to the respective SHAKE and STILL RPTC cultured in standard culture medium which contains 17.5 mM glucose. Similarly, the activity of lactate dehydrogenase was unchanged by lowering the medium glucose concentration. Substituting 5 mM galactose for 5 mM glucose in the culture medium significantly reduced the lactate content of both SHAKE and STILL RPTC but had no effect on lactate dehydrogenase activity. Cell growth was equivalent under all culture conditions. Sensitivity to mitochondrial inhibition was determined for each culture condition by measuring cell death after exposure to the respiratory inhibitor antimycin A. The results showed a hierarchy of sensitivity to antimycin A (5 mM galactose SHAKE >5 mM glucose SHAKE >17.5 mM glucose SHAKE = 17.5 mM glucose STILL), which was generally inversely correlated with the level of glycolysis as measured by lactate content (17.5 mM glucose STILL >17.5 mM glucose SHAKE = 5 mM glucose SHAKE >5 mM galactose SHAKE).