Human articular chondrocytes express three facilitative glucose transporter isoforms: GLUT1, GLUT3 and GLUT9

Glucose is an important metabolite and a structural precursor for articular cartilage and its transport has significant consequences for cartilage development and functional integrity. In this study the expression of facilitative glucose transporters (GLUTs) in human chondrocytes was investigated. Results showed that at least three GLUT isoforms (GLUT1, GLUT3 and GLUT9) are expressed by normal chondrocytes. Given the central role of glucose in chondrocyte physiology and metabolism, its regular provision via GLUTs will influence the metabolic activity and survival of chondrocytes in cartilage matrices.     

Glutamine fuels proliferation but not migration of endothelial cells

Endothelial metabolism is a key regulator of angiogenesis. Glutamine metabolism in endothelial cells (ECs) has been poorly studied. We used genetic modifications and ¹³C tracing approaches to define glutamine metabolism in these cells. Glutamine supplies the majority of carbons in the tricyclic acid (TCA) cycle of ECs and contributes to lipid biosynthesis via reductive carboxylation. EC‐specific deletion in mice of glutaminase, the initial enzyme in glutamine catabolism, markedly blunts angiogenesis. In cell culture, glutamine deprivation or inhibition of glutaminase prevents EC proliferation, but does not prevent cell migration, which relies instead on aerobic glycolysis. Without glutamine catabolism, there is near complete loss of TCA intermediates, with no compensation from glucose‐derived anaplerosis. Mechanistically, addition of exogenous alpha‐ketoglutarate replenishes TCA intermediates and rescues cellular growth, but simultaneously unveils a requirement for Rac1‐dependent macropinocytosis to provide non‐essential amino acids, including asparagine. Together, these data outline the dependence of ECs on glutamine for cataplerotic processes; the need for glutamine as a nitrogen source for generation of biomass; and the distinct roles of glucose and glutamine in EC biology.     

Elevated glutamate dehydrogenase flux in glucose-deprived hybridoma and myeloma cells: evidence from 1H/15N NMR

The glutamine metabolism was studied in glucose-starved and glucose-sufficient hybridoma and Sp2/0-Ag14 myeloma cells. Glucose starvation was attained by cultivating the hybridoma cells with fructose instead of glucose, and the myeloma cells with a low initial glucose concentration which was rapidly exhausted. Glutamine used in the experiments was labeled with 15N, either in the amine or in the amide position. The fate of the label was monitored by 1H/15N NMR analysis of released 15NH+4 and 15N-alanine. Thus, NH+4 formed via glutaminase (GLNase) could be distinguished from NH+4 formed via glutamate dehydrogenase (GDH). In the glucose-sufficient cells a small but measurable amount of 15NH+4 released by GDH could be detected in both cell lines (0.75 and 0.31 micromole/10(6) cells for hybridoma and myeloma cells, respectively). The uptake of glutamine and the total production of NH+4 was significantly increased in both fructose-grown hybridoma and glucose-starved myeloma cells, as compared to the glucose-sufficient cells. The increased NH+4 production was due to an increased throughput via GLNase (1.6 -1.9-fold in the hybridoma, and 2.7-fold in the myeloma cell line) and an even further increased metabolism via GDH (4.8-7.9-fold in the hybridoma cells, and 3.1-fold in the myeloma cells). The data indicate that both GLNase and GDH are down-regulated when glucose is in excess, but up-regulated in glucose-starved cells. It was calculated that the maximum potential ATP production from glutamine could increase by 35-40 % in the fructose-grown hybridoma cells, mainly due to the increased metabolism via GDH.     

Glucose, glutamine and ketone body utilisation by resting and concanavalin A activated rat splenic lymphocytes

The utilisation of glucose, glutamine, acetoacetate and D-3-hydroxybutyrate were investigated over 72 h of incubation of rat splenic lymphocytes, with and without concanavalin A. Lymphocytes consumed both ketone bodies; acetoacetate was consumed preferentially. The ketone bodies reduced glucose consumption by 30-50%, but had little effect on lactate production. Glutamine uptake was concentration dependent up to 4 mM, and consumption was increased in the presence of concanavalin. Glutamine stimulated glucose consumption and lactate production in both resting and activated cells. Complete oxidation contributed 65% of glucose-derived ATP, but less than 40% of glutamine-derived ATP. Glutamine metabolism makes only a minor contribution to lymphocyte ATP generation.     

Profile of energy metabolism in a murine hybridoma: Glucose and glutamine utilization

The antibody-secreting murine hybridoma, CC9C10, was grown in batch culture in a medium containing 20 mM glucose and 2 mM glutamine. After 2 days of exponential growth, the glutamine content of the medium was completely depleted, whereas the glucose content was reduced to 60% of the original concentration. The glucose and glutamine metabolism was analyzed at midexponential phase by use of radioactively labelled substrates. Glycolysis accounted for the metabolism of most of the glucose utilized (> 96%) with flux through the pentose phosphate pathway (3.6%) and the TCA cycle (0.6%) accounting for the remainder. Glutamine was partially oxidised via glutaminolysis to alanine (55%), aspartate (3%), glutamate (4%), lactate (9%), and CO₂ (22%). Calculation of the theoretical ATP production from these pathways indicated that glucose could provide 59% and glutamine 41% of the energy requirement of the cells. © 1994 Wiley-Liss, Inc.     

Glutamine and glucose metabolism in thymocytes from normal and spontaneously diabetic BB rats.

Metabolism of glutamine and glucose was studied in thymocytes from normal rats and BB rats with the spontaneous autoimmune diabetic syndrome to assess their potential roles as fuels. The major measured products from glucose were lactate and, to a lesser extent, CO2, and pyruvate. Glutamine had no effect on the rates of their production from glucose. Glutamine was metabolized to ammonia, aspartate, glutamate, and CO2, with aspartate being the major product of carbons from glutamine in the absence of glucose. Glucose markedly decreased the formation of ammonia, aspartate, and CO2 from glutamine, but increased that of glutamate, with an overall decrease in glutamine utilization by 55%. More glutamate than aspartate was produced from glutamine in the presence of glucose. The potential production of ATP from glucose was similar to that when glutamine was present alone. However, glucose markedly decreased production of ATP from glutamine, but not vice versa. This resulted in ATP production from glucose being 2.5 times that from glutamine when both substrates were present. The oxidation of glucose to CO2 via the Krebs cycle accounts for 75-80% of glucose-derived ATP production. Cellular ATP levels markedly decreased in the absence of exogenous substrates, but were constant throughout a 2-h incubation in the presence of glutamine, glucose, or both. There were no differences in thymocyte glucose or glutamine metabolism between normal and diabetic BB rats, in contrast to previous findings in peripheral lymphoid organs. Our results suggest that glucose is a more important fuel than glutamine for "resting" thymocytes, again in contrast to the cells of peripheral lymphoid organs in which glutamine is as important as glucose as a fuel.(ABSTRACT TRUNCATED AT 250 WORDS)     

Melatonin Cytotoxicity Is Associated to Warburg Effect Inhibition in Ewing Sarcoma Cells

Melatonin kills or inhibits the proliferation of different cancer cell types, and this is associated with an increase or a decrease in reactive oxygen species, respectively. Intracellular oxidants originate mainly from oxidative metabolism, and cancer cells frequently show alterations in this metabolic pathway, such as the Warburg effect (aerobic glycolysis). Thus, we hypothesized that melatonin could also regulate differentially oxidative metabolism in cells where it is cytotoxic (Ewing sarcoma cells) and in cells where it inhibits proliferation (chondrosarcoma cells). Ewing sarcoma cells but not chondrosarcoma cells showed a metabolic profile consistent with aerobic glycolysis, i.e. increased glucose uptake, LDH activity, lactate production and HIF-1α activation. Melatonin reversed Ewing sarcoma metabolic profile and this effect was associated with its cytotoxicity. The differential regulation of metabolism by melatonin could explain why the hormone is harmless for a wide spectrum of normal and only a few tumoral cells, while it kills specific tumor cell types.     

Pharmacological actions of 17 beta-oestradiol on articular cartilage chondrocytes and chondrosarcoma chondrocytes in the absence of oestrogen receptors

(1) Pharmacological concentrations (greater than 10(-5) M) of 17 beta-oestradiol inhibited 35S-labelled proteoglycan synthesis in bovine articular cartilage explant cultures. They also inhibited 35S-labelled proteoglycan synthesis and 3H-labelled protein synthesis in cell cultures of chondrocytes from bovine articular cartilage and Swarm rat chondrosarcoma. Maximal inhibition was about 30-50%. Physiological concentrations (10(-9)-10(-8) M) of oestradiol had no effect on the synthesis of either protein or proteoglycan. (2) The inhibitory action of high concentrations of oestradiol on these biosynthetic pathways is not common to all steroids since 10(-4) M cortisol had no effect on articular chondrocyte cell cultures. 10(-4) M testosterone had a similar action to oestradiol. (3) Neither physiological nor pharmacological concentrations of 17 beta-oestradiol had any effect on 35S-labelled proteoglycan turnover in the cartilage explant system. (4) 10(-5) M oestradiol inhibited cell division in cultures of articular chondrocytes which had entered the log growth phase. 10(-7) M oestradiol had no effect on articular chondrocyte growth. (5) In male rats implanted with silastic capsules releasing 17 beta-oestradiol, increase in body weight was retarded by about 25% over a period of 6 weeks, compared to control rats. Rat chondrosarcoma grew to the same size in oestrogen-treated rats as it did in controls. (6) Oestrogen receptors could not be detected in freshly isolated bovine articular chondrocytes or in rat chondrosarcoma. (7) In conclusion, neither the mitotic rate of articular chondrocytes nor their proteoglycan metabolism is under the direct physiological control of oestradiol. Growth and biosynthetic activity of the rat chondrosarcoma chondrocytes are independent of either direct control by the hormone or control effected by oestradiol regulation of a second hormone or growth factor.     

Gene expression profiles in chondrosarcoma cells subjected to cyclic stretching and hydrostatic pressure. A cDNA array study

Mechanical forces have a profound effect on cartilage tissue and chondrocyte metabolism. Strenuous loading inhibits the cellular metabolism, while optimal level of loading at correct frequency raises an anabolic response in chondrocytes. In this study, we used Atlas Human Cancer cDNA array to investigate mRNA expression profiles in human chondrosarcoma cells stretched 8% for 6 hours at a frequency of 0.5 Hz. In addition, cultures were exposed to continuous and cyclic (0.5 Hz) 5 MPa hydrostatic pressure. Cyclic stretch had a more profound effect on the gene expression profiles than 5 MPa hydrostatic pressure. Several genes involved with the regulation of cell cycle were increased in stretched cells, as well as mRNAs for PDGF-B, glucose-1-phosphate uridylyltransferase, Tiam1, cdc37 homolog, Gem, integrin alpha6, and matrix metalloproteinase-3. Among down-regulated genes were plakoglobin, TGF-alpha, retinoic acid receptor-alpha and Wnt8b. A smaller number of changes was detected after pressure treatments. Plakoglobin was increased under cyclic and continuous 5 MPa hydrostatic pressure, while mitogen-activated protein kinase-9, proliferating cell nuclear antigen, Rad6, CD9 antigen, integrins alphaE and beta8, and vimentin were decreased. Cyclic and continuous pressurization induces a number of specific changes. In conclusion, a different set of genes were affected by three different types of mechanical stimuli applied on chondrosarcoma cells.     

Glucose and glutamine metabolism in pre-attachment cattle embryos in relation to sex and stage of development

Individual Day-7 embryos (morulae to expanded blastocysts) were incubated with radiolabelled substrates and karyotyped to determine the sex. In Exp. 1, embryos were incubated for 3 h with D-[1-14C]glucose, as a measure of the activity of the pentose-phosphate pathway (PPP) and D-[5-3H]glucose, as a measure of total glucose metabolism. The labelled products 14CO2 and 3H2O were collected throughout the measurement period. Total glucose metabolism in male embryos was twice that in female embryos and increased between the morula and expanded-blastocyst stages. Relative to total glucose metabolism, PPP activity was four times greater in female than in male embryos. In Exp. 2, embryos were cultured with D-[1-14C]glucose, and L-[3,4-3H(N)]glutamine (a measure of Krebs cycle activity) in the presence of brilliant cresyl blue, a stimulator of the PPP. Glutamine metabolism increased from the morula to expanded-blastocyst stages. Relative to the metabolism of glutamine, the activity of the PPP was one-third greater in female than in male embryos.     

Zinc protects chondrocytes from monosodium iodoacetate-induced damage by enhancing ATP and mitophagy

Osteoarthritis (OA) is characterized with articular cartilage degradation, and monosodium iodoacetate (MIA)-treated chondrocyte is the most commonly used model for mimicking OA progression. Zinc protects chondrocytes from MIA-induced damage. Here, we explored the protective effects of 25 μM zinc on 5 μM MIA-treated SW1353 cells (human chondrosarcoma cell line) through the analysis of energy metabolism- and autophagy-related parameters. We found that the exposure of SW1353 cells to MIA decreased ATP levels, expression of glycolysis-related proteins, including glucose transporter 1, hexokinase 2, and pyruvate dehydrogenase E1 component subunit alpha, and the levels of mitochondrial complex I, II, IV, and V subunits of the oxidative phosphorylation pathway. MIA treatment also decreased the expression of autophagy-related proteins, including autophagic elongation protein 5 (ATG5), ATG7, and microtubule-associated protein 1A/1B light chain 3B (LC3-II) and mitophagy-related proteins, including phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1), ubiquitin, and p62. These results indicate that MIA interferes with energy metabolism and the autophagic clearance of dysfunctional mitochondria (so called mitophagy). Interestingly, zinc exposure could almost completely reverse the effects of MIA, suggesting its potential protective role against OA progression.     

Mice with Trp53 and Rb1 deficiency in chondrocytes spontaneously develop chondrosarcoma via overactivation of YAP signaling

Chondrosarcoma (CHS) is a rare type of soft sarcoma with increased production of cartilage matrix arising from soft bone tissues. Currently, surgical resection is the primary clinical treatment for chondrosarcoma due to the poor response to radiotherapy and chemotherapy. However, the therapeutic effect is not satisfactory due to the higher local recurrence rate. Thus, management and elucidation of the pathological mechanism of chondrosarcoma remain an ongoing challenge, and the development of effective chondrosarcoma mouse models and treatment options are urgently needed. Here, we generated a new transgenic chondrosarcoma model by double conditional deletions of Trp53 and Rb1 in chondrocyte lineage which spontaneously caused spinal chondrosarcoma and lung metastasis. Bioinformatic analysis of the human soft sarcoma database showed that Trp53 and Rb1 genes had higher mutations, reaching up to approximately 33.5% and 8.7%, respectively. Additionally, Trp53 and Rb1 signatures were decreased in the human and mouse chondrosarcoma tissues. Mechanistically, we found that YAP expression and activity were significantly increased in mouse Col2-Cre;Trp53^f/f/Rb1^f/f chondrosarcoma tissues compared to the adjacent normal cartilage. Knockdown of YAP in primary chondrosarcoma cells significantly inhibited chondrosarcoma proliferation, invasion, and tumorsphere formation. Chondrocyte lineage ablation of YAP delayed chondrosarcoma progression and lung metastasis in Col2-Cre;Trp53^f/f/Rb1^f/f mice. Moreover, we found that metformin served as a YAP inhibitor, which bound to the activity area of YAP protein, and inhibited chondrosarcoma cell proliferation, migration, invasion, and progression in vitro and significantly suppressed chondrosarcoma formation in vivo. Collectively, this study identifies the inhibition of YAP may be an effective therapeutic strategy for the treatment of chondrosarcoma.Subject terms: Bone cancer, Bone cancer     

Glycolysis and glutaminolysis in perifused Ehrlich ascites tumour cells

A perifusion system was designed in order to study glucose and glutamine metabolism by freshly harvested Ehrlich ascites tumour cells in steady state conditions. Cells were perifused in the presence of 5 mM glucose, 0.5 mM glutamine or 5 mM glucose and 0.5 mM glutamine. The results in steady state reveal that both substrates glucose and glutamine are continuously wasted by tumour cells, excreting two moles of lactate per mol of glucose and one mol of glutamate and ammonia per mol of glutamine consumed into the medium. Glutamine consumption in the presence of glucose was higher than with glutamine alone.     

Membrane-associated hyaluronate-binding activity of chondrosarcoma chondrocytes

The association of hyaluronate with the surface of chondrocytes was examined by several approaches using primary cultures of chondrocytes derived from the Swarm rat chondrosarcoma. In culture, chondrosarcoma chondrocytes produced large pericellular coats, which can be visualized by particle exclusion, and which can be removed by Streptomyces hyaluronidase. Exposure of chondrocytes, which had been metabolically labelled with 3H-acetate, to exogenous hyaluronate or to Streptomyces hyaluronidase resulted in the release of 36-38% of the endogenous, labelled chondroitin sulfate from the cell layer into the incubation solution. These results imply that at least 37% of the cell layer chondroitin sulfate proteoglycan is retained there by an interaction with hyaluronate. Thus membranes were prepared from cultured chondrocytes and examined for sites which bind 3H-hyaluronate. Binding was observed and found to be saturable, specific for hyaluronate, of high affinity (Kd = approximately 10(-10) M), and destroyed by treating the membranes with trypsin. The 3H-hyaluronate-binding activity was inhibited competitively by hyaluronate decasaccharides but not by hexasaccharides or octasaccharides, indicating that the binding sites recognize a sequence of hyaluronate composed of five disaccharide repeats. The binding activity was partially purified from a detergent extract of chondrocyte membranes by ion exchange chromatography on DEAE-cellulose, followed by affinity chromatography on wheat germ agglutinin-agarose. Analysis of the partially purified binding activity by SDS-PAGE revealed five protein bands of 48,000-66,000 daltons in silver-stained gels. SDS-PAGE followed by Western blotting and exposure to monoclonal antibodies which recognize epitopes present in link protein and in the hyaluronate-binding region of cartilage proteoglycan revealed no immunoreactive protein bands in the partially purified material. We conclude that one mechanism by which hyaluronate associates with the chondrocyte surface may be via interaction with a membrane-bound hyaluronate-binding protein which is distinct from link protein and proteoglycan.     

Dynamic compression augments interstitial transport of a glucose-like solute in articular cartilage

Solute transport through the extracellular matrix is essential for cellular activities in articular cartilage. Increased solute transport via fluid convection may be a mechanism by which dynamic compression stimulates chondrocyte metabolism. However, loading conditions that optimally augment transport likely vary for different solutes. To investigate effects of dynamic loading on transport of a bioactive solute, triangular mechanical loading waveforms were applied to cartilage explants disks while interstitial transport of a fluorescent glucose analog was monitored. Peak-to-peak compression amplitudes varied from 5-50% and frequencies varied from 0.0006-0.1 Hz to alter the spatial distribution and magnitude of oscillatory fluid flow. Solute transport was quantified by monitoring accumulation of fluorescence in a saline bath circulated around the explant. Individual explants were subjected to a series of compression protocols, so that effects of loading on solute desorption could be observed directly. Maximum increases in solute transport were obtained with 10-20% compression amplitudes at 0.1 Hz; similar loading protocols were previously found to stimulate chondrocyte metabolism in vitro. Results therefore support hypotheses relating to increased solute transport as a mediator of the cartilage biological response to dynamic compression, and may have application in mechanical conditioning of cartilage constructs for tissue engineering.     

Glutaminolysis Activates Rag-mTORC1 Signaling

Amino acids control cell growth via activation of the?highly conserved kinase TORC1. Glutamine is a particularly important amino acid in cell growth control and metabolism. However, the role of glutamine in TORC1 activation remains poorly defined. Glutamine is metabolized through glutaminolysis to?produce α-ketoglutarate. We demonstrate that glutamine in combination with leucine activates mammalian TORC1 (mTORC1) by enhancing glutaminolysis and α-ketoglutarate production. Inhibition of glutaminolysis prevented GTP loading of RagB and lysosomal translocation and subsequent activation of mTORC1. Constitutively active Rag heterodimer activated mTORC1 in the absence of glutaminolysis. Conversely, enhanced glutaminolysis or?a cell-permeable α-ketoglutarate analog stimulated lysosomal translocation and activation of mTORC1. Finally, cell growth and autophagy, two processes controlled by mTORC1, were regulated by glutaminolysis. Thus, mTORC1 senses and is activated by?glutamine and leucine via glutaminolysis and α-ketoglutarate production upstream of Rag. This may provide an explanation for glutamine addiction in cancer cells.     

Nutrient utilization by bovine articular chondrocytes: a combined experimental and theoretical approach

A combined experimental-numerical approach was adopted to characterize glucose and oxygen uptake and lactate production by bovine articular chondrocytes in a model system. For a wide range of cell concentrations, cells in agarose were supplemented with either low or high glucose medium. During an initial culture phase of 48 h, oxygen was monitored noninvasively using a biosensor system. Glucose and lactate were determined by medium sampling. In order to quantify glucose and oxygen uptake, a finite element approach was adopted to describe diffusion and uptake in the experimental model. Numerical predictions of lactate, based on simple relations for cell metabolism, were found to agree well for low glucose, but not for high glucose medium. Oxygen did not play a role in either case. Given the close association between chondrocyte energy metabolism and matrix synthesis, a quantifiable prediction of utilization can present a valuable contribution in the optimization of tissue engineering conditions.     

Biosynthesis and cell-free translation of Swarm rat chondrosarcoma and bovine cartilage link proteins

In cartilage, link protein(s) (LP) stabilize proteoglycan aggregates via their specific association with hyaluronic acid and proteoglycan monomers. Two major link glycoproteins are produced in bovine articular cartilage, designated LP1 (49.5 kDa) and LP2 (44.0 kDa), whereas rat chondrosarcoma produces a single link protein species similar in size to bovine LP2. Although multiple link proteins differ to a significant degree in carbohydrate content, it is not known whether they arise from variable glycosylation of a single common protein core or from complete glycosylation of different protein cores. Biosynthesis of these molecules has been studied under conditions where differences generated by N-linked glycosylation would not be evident. Link proteins were immunoprecipitated 1) from cell-free translation products of total cellular and size fractionated RNA and 2) from cell lysates and medium of cultured chondrocytes using short term radioactive labeling of the protein in the presence and absence of tunicamycin. A 42-kDa link protein precursor is synthesized by cell-free translation of either rat chondrosarcoma or bovine chondrocyte mRNa. An apparently single 41.5-kDa link protein is synthesized with inhibition of N-linked glycosylation by tunicamycin, whereas LP1 and LP2 are the mature products of cultured bovine chondrocytes. The size range of translatable rat chondrosarcoma LP mRNA is 4.0-5.5 kilobase pairs and bovine LP mRNA is 3.0-4.5 kilobase pairs, both much larger than required to encode the link protein molecule. These results suggest that a single link protein precursor gives rise to multiple fully glycosylated forms and that link protein is not synthesized as a significantly larger "pro" form.