Rate Of Lactate Production Research Articles

Central metabolism of functionally heterogeneous mesenchymal stromal cells

Metabolism and mitochondrial biology have gained a prominent role as determinants of stem cell fate and function. In the context of regenerative medicine, innovative parameters predictive of therapeutic efficacy could be drawn from the association of metabolic or mitochondrial parameters to different degrees of stemness and differentiation potentials. Herein, this possibility was addressed in human mesenchymal stromal/stem cells (hMSC) previously shown to differ in lifespan and telomere length. First, these hMSC were shown to possess significantly distinct proliferation rate, senescence status and differentiation capacity. More potential hMSC were associated to higher mitochondrial (mt) DNA copy number and lower mtDNA methylation. In addition, they showed higher expression levels of oxidative phosphorylation subunits. Consistently, they exhibited higher coupled oxygen consumption rate and lower transcription of glycolysis-related genes, glucose consumption and lactate production. All these data pointed at oxidative phosphorylation-based central metabolism as a feature of higher stemness-associated hMSC phenotypes. Consistently, reduction of mitochondrial activity by complex I and III inhibitors in higher stemness-associated hMSC triggered senescence. Finally, functionally higher stemness-associated hMSC showed metabolic plasticity when challenged by glucose or glutamine shortage, which mimic bioenergetics switches that hMSC must undergo after transplantation or during self-renewal and differentiation. Altogether, these results hint at metabolic and mitochondrial parameters that could be implemented to identify stem cells endowed with superior growth and differentiation potential.

P2.04-42 PD-L1-Mediated Enhancement of Hexokinase 2 Expression Is Inversely Related to T-Cell Effector Gene Expression in Non-Small-Cell Lung Cancer

The PD-1/PD-L1 pathway contributes to the metabolic reprogramming of immune cells and tumor cells. However, the mechanism by which PD-L1 regulates glycolysis in human cancer and the relationship between PD-L1 expression, glycose metabolism and anti-tumor immune response remain unclear. We addressed these issues in human non-small-cell lung cancer (NSCLC). Changes in glycolysis-related molecules and glycolytic activity were evaluated in PD-L1low and PD-L1high NSCLC cells after transfection or knockdown of PD-L1, respectively. RNA seq and gene set enrichment analyses (GSEA) was also performed. The association between PD-L1 and immune response-related molecules or glycolysis were analyzed in patients with NSCLC and The Cancer Genome Atlas (TCGA). Transfecting PD-L1 in PD-L1low cells enhanced hexokinase-2 (HK2) expression, lactate production, and extracellular acidification rates, but minimally altered GLUT1 and PKM2 expression and oxygen consumption rates. Consistently, GSEA revealed that the glycolytic pathway was enhanced in PD-L1low cells after PD-L1 transfection. By contrast, knocking-down PD-L1 in PD-L1high cells decreased HK2 expression and glycolysis by suppressing PI3K/Akt and Erk pathways. Immunohistochemistry revealed that PD-L1 expression was positively correlated with HK2 expression in NSCLC tissues from patients (p<0.001). In TCGA analyses, HK2 exhibited a positive linear association with CD274 (PD-L1) expression (p<0.001) but an inverse correlation with the expression of CD4, CD8A, and T-cell effector function-related genes in the CD274high rather than CD274low group. Consistent with this, there were fewer CD8+ T-cells in PD-L1positive/HK2high tumors compared to PD-L1positive/HK2low tumors in squamous cell carcinoma. PD-L1 enhances glycolysis in NSCLC by upregulating HK2, which might dampen anti-tumor immunity. PD-L1 may contribute to NSCLC oncogenesis by inducing metabolic reprogramming and immune checkpoint pathway.

The use of the SLC16A1 gene as a potential marker to predict race performance in Arabian horses

BackgroundArabian horses are commonly believed to be one of the oldest and the most popular horse breeds in the world, characterized by favourable stamina traits and exercise phenotypes. During intensive training, the rates of lactate production and utilization are critical to avoid muscle fatigue and a decrease in exercise performance. The key factor determining transmembrane lactate transport is the monocarboxylate transporter 1 protein coded for by the SLC16A1 gene. The aim of the present research was to identify polymorphisms in the coding sequence and UTRs in the equine SLC16A1 gene and to evaluate their potential association with race performance traits in Arabian horses. Based on RNA-seq data, SNPs were identified and genotyped using PCR-RFLP or PCR-HRM methods in 254 Arabian horses that competed in flat races. An association analysis between polymorphisms and racing results was performed.ResultsNovel polymorphisms in the equine SLC16A1 locus have been identified (missense and 5’UTR variants: g.55601543C > T and g.55589063 T > G). Analysis showed a significant association between the 5’UTR polymorphism and several racing results as follows: the possibility of winning first or second place, the number of races in which horses started and total financial benefits. The analysis also showed differences in genotype distribution depending on race distance. In the studied population, the shorter distance races were only won by TT horses. The GG and TG horses took first and second places in middle- and long-distance races, and the percentage of winning heterozygotes increased from 19.5 to 27% at the middle and long distances, respectively. The p.Val432Ile (g.55601543C > T) polymorphism was not significantly related to the analysed racing results.ConclusionOur results showed that g.55589063 T > G polymorphism affected the possibility of winning first or second place and of competing in more races. The different distribution of genotypes depending on race distance indicated the possibility of using a SNP in the SLC16A1 gene as a marker to predict the best race distance for a horse. The presented results provide a basis for further research to validate the use of the SLC16A1 gene as a potential marker associated with racing performance.

Targeted inhibition of MCT4 disrupts intracellular pH homeostasis and confers self-regulated apoptosis on hepatocellular carcinoma

The high lactate production rate in hepatocellular carcinoma cells (HCC) have a profound impact on their malignant properties. In adaptation to the enhanced lactate stress, lactate-effusing monocarboxylate transporter 4(MCT4) is usually overexpressed in a broad range of HCC subtypes. In this study, the MCT4-mediated lactate efflux in HCC was blocked using microRNA-145(miR-145), which would force the endogenously generated lactate to accumulate within tumor cells in a self-regulated manner, resulting in the acidification of the cytoplasmic compartment as well as partial neutralization for pH in the tumor extracellular environment. Evaluations on multiple representative HCC subtypes (HepG2, Hep3B and HuH7) suggested that the disrupted pH homeostasis would amplify the lactate stress to initiate HCC apoptosis, while at the same time also suppressing their migration and invasion abilities. Moreover, safety tests on 7702 cells and living animals revealed that MCT4-blockade treatment has no cytotoxicity against healthy cells/tissues. The results indicate the MCT4-inhibition-induced disruption of tumor intracellular pH holds promise as a therapy against not only HCC, but a broader spectrum of MCT4-overexpressing hyperglycolytic tumors.

PS1528 INCREASED T CELLS GLYCOLYSIS IN PATIENTS WITH ACUTE GRAFT VERSUS HOST DISEASE AFTER ALLOGENEIC HEMATOPOIETIC STEM CELL

Background:Acute graft‐versus‐host disease (aGVHD) is a major complication limiting successful allogeneic hematopoietic stem cell transplantation (allo‐HSCT), the pathogenesis of aGVHD is commonly considered to be T cell‐mediated. Cell metabolism determines T cell fate and function, the metabolic profile of T cells varies in different immunological disorders such as lupus, rheumatoid arthritis. Previous murine transplantation study found that alloreactive donor T cells increased both oxidative phosphorylation (OXPHOS) and aerobic glycolysis and appeared bioenergetically similar to the lymphocytes that mediate lupus. Recently, another murine study suggested that alloreactive donor T cells use glycolysis as the predominant metabolic process. However, the metabolic profile of T cells in aGVHD patients still remains unclear.Aims:To determine the metabolic adaptations that occur in activated T cells in patients with aGVHD and approach for modulating their metabolism.Methods:In this prospective case‐control study, a total of 20 patients with aGVHD after allo‐HSCT were enrolled, for each case, 1 matched transplant recipients without aGVHD (non‐aGVHD; N = 20) were selected at random. CD3+ T cells were isolated by microbeads, the metabolic profile of sorted T cells was determined using a Seahorse XF96 Analyze. RNA sequencing (RNA‐Seq) was performed to analyze the gene expression profiles of the T cells. Glucose consumption and lactate production rates were detected by glucose assay kit and lactate assay kit. The levels of intracellular cytokines, such as IFN‐r, IL‐4 and IL‐17, were detected by flow cytometry. Protein expressions for CTP1a and PFKFB3 were measured by flow cytometry. 3‐PO, as a glycolysis inhibitor at PFKFB3, was administrated to T cells from aGVHD patients.Results:Firstly, we detected the intracellular levels of proinflammatory cytokines (IFN‐γ, and IL‐17) in the peripheral T cells. There were significantly elevated proportions of Tc1(CD8+IFN‐γ+), Th1 (CD4+IFN‐γ+) and Th17(CD4+IL17+) in patients with aGVHD. Switching from OXPHOS to glycolysis is the hallmark of in vitro–activated T cell metabolism. Hence, we determined the rates of glycolysis and OXPHOS in T cells by measuring extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). T cells isolated from aGVHD patients exhibited comparable OCR values and higher ECAR values. In addition, glucose consumption rate and lactate production rate were both higher in aGVHD group, implying higher glycolytic activity of T cells in aGVHD patients. Moreover, RNA‐seq were performed to further understand the metabolic profile of T cells in aGVHD patients. Consistent with the elevated glycolytic activity observed in vitro, the levels of the mRNAs encoding metabolic enzymes involved in the glycolytic pathway were substantially elevated in aGVHD group. Protein expression of PFKFB3, which is a glycolytic activator, was significantly higher in T cells, especially in naïve T cells of aGVHD patients. Furthermore, 3‐PO significantly diminished PFKFB3 expression, glycolytic activity of T cells, and reduced the expression of proinflammatory cytokines in T cells from aGVHD patients.Summary/Conclusion:In the current study, we found that T cells in patients with aGVHD preferentially depend on glycolysis to meet bioenergetic demands. Furthermore, inhibition of glycolysis through targeting PFKFB3 ameliorates the activity of T cells from aGVHD patients. Therefore, the current work indicated that glycolysis promises to be a novel therapeutic target for aGVHD patients after allo‐HSCT.

Induction of Pyruvate Dehydrogenase Kinase 1 by Hypoxia Alters Cellular Metabolism and Inhibits Apoptosis in Endometriotic Stromal Cells.

Endometriosis is a common gynecological disease, which is defined as the growth of endometrial tissues outside the uterine cavity. It often causes dysmenorrhea, dyspareunia, chronic pelvic pain, and infertility in reproductive-age women. However, the pathogenesis of endometriosis remains largely unclear. Since our previous study revealed that ectopic endometriotic stromal cells experience greater hypoxic stress than their eutopic counterparts, we aim to investigate whether the metabolic properties are changed in the ectopic endometriotic stromal cell when compared to its eutopic counterpart. Here, we found the expression of pyruvate dehydrogenase kinase 1 (PDK1), a critical enzyme in regulating glucose metabolism, was increased in ectopic stromal cells. Molecular characterization reveals that overexpression of PDK1 is induced by hypoxia through transcriptional regulation. Upregulation of PDK1 in ectopic endometriotic stromal cells was accompanied by increases in lactate production and oxygen consumption rate when compared to eutopic endometrial stromal cells. Furthermore, our data showed that inhibition of PDK1 activity by treatment with dichloroacetate inhibits the lactate production and oxygen consumption rate of ectopic stromal cells. In addition, hypoxia-induced PDK1 expression prevented cells from H2O2- and low nutrient-induced cell death. These data indicate that ectopic endometriotic cells may adapt to hypoxic microenvironment via upregulating PDK1 and reprogramming metabolism, which provides a survival advantage in the hostile peritoneal microenvironment.

Metformin enhances antitumor action ofsodiumdichloroacetate against glioma C6.

To investigate the anti-glioma activity of sodium dichloroacetate (DCA) and metformin (MTF) used in combination in vitro and in vivo. Cell survival, cell cycle, apoptosis, mitochondrial membrane potential (Δψm), ATP level, the glucose consumption rate, and lactate production rate were determined in vitro in cultured glioma C6cells. The antitumor action of agents in vivo was evaluated routinely by the prolongation of the life span of rats with transplanted intracerebral glioma C6and was confirmed by histological examination of tumor tissue. The half maximal inhibitory concentration (IC50) for DCA and MTF used separately was 79.2±2.1mM and 78.4±4.0mM, respectively, whereas IC50for DCA used in combination with 7.8mM MTF was 3.3fold lower (24.0±1.2mM, p < 0.05). The 1-day incubation of cells with DCA at a concentration close to IC50 (25mM), in combination with MTF at a concentration by order lower than IC50 (7.8mM), in contrast to their separate use, resulted in a decrease in the number of viable cells by 40% (p < 0.05); redistribution of the cells by the cell cycle phases toward decreased proportion of cells in the S-phase by 46% (p < 0.05) and an increased percentage of cells in the G0/G1phase by 24% (p < 0.05) compared to similar indices in the control. High proapoptotic activity of DCA in combination with MTF was supported by a significantly higher percentage of apoptotic cells in vitro than in the control (18.9±4.4% vs 5.7±1.3%, p < 0.05) and a high number of tumor cells with signs of apoptosis revealed during the histological examination of tumor pathomorphosis. The combined effect of DCA and MTF resulted in almost 4-fold decrease of the glucose consumption rate by glioma C6cells (0.23±0.05μmol/106cells/h vs 0.91 ± 0.12μmol/106cells/h, p < 0.05) compared to the corresponding parameters in the control, and 2-fold increased rate of lactate production (1.06 ± 0.03μmol/106cells/h vs 0.53 ± 0.03μmol/106cells/h, p < 0.05). At the same time, both Δψm and the level of intracellular ATP in the glioma C6cells treated with DCA and MTF, both separately and in combination, did not differ significantly from those indices in the control. In in vivo studies, the average life span of rats with intracranial transplanted glioma C6, treated with DCA in combination with MTF in a total dose of 1.1and 2.6g/kg body weight, respectively, was 50% higher (p < 0.001) than in the control group. In contrast, in the case of single-use (at a dose of 2.6g/kg), MTF increased the life span of tumor-bearing animals just by 19% (p < 0.01), whereas DCA alone (at a dose of 1.1g/kg) did not significantly change the survival time of rats. The obtained data indicate synergism of anti-glioma action of DCA and MTF in a case of their combined use both in vitro and in vivo and may be considered a starting point for the development of effective treatment regimens for malignant brain tumors based on the combined use of DCA and MTF.

Effect of Sustained Joint Loading on TMJ Disc Nutrient Environment

The temporomandibular joint (TMJ) disc nutrient environment profoundly affects cell energy metabolism, proliferation, and biosynthesis. Due to technical challenges of in vivo measurements, the human TMJ disc extracellular nutrient environment under load, which depends on metabolic rates, solute diffusion, and disc morphometry, remains unknown. Therefore, the study objective was to predict the TMJ disc nutrient environment under loading conditions using combined experimental and computational modeling approaches. Specifically, glucose consumption and lactate production rates of porcine TMJ discs were measured under varying tissue culture conditions (n = 40 discs), and mechanical strain-dependent glucose and lactate diffusivities were measured using a custom diffusion chamber (n = 6 discs). TMJ anatomy and loading area were obtained from magnetic resonance imaging of healthy human volunteers (n = 11, male, 30 ± 9 y). Using experimentally determined nutrient metabolic rates, solute diffusivities, TMJ anatomy, and loading areas, subject-specific finite element (FE) models were developed to predict the 3-dimensional nutrient profiles in unloaded and loaded TMJ discs (unloaded, 0% strain, 20% strain). From the FE models, glucose, lactate, and oxygen concentration ranges for unloaded healthy human TMJ discs were 0.6 to 4.0 mM, 0.9 to 5.0 mM, and 0% to 6%, respectively, with steep gradients in the anterior and posterior bands. Sustained mechanical loading significantly reduced nutrient levels (P < 0.001), with a critical zone in which cells may die representing approximately 13.5% of the total disc volume. In conclusion, this study experimentally determined TMJ disc metabolic rates, solute diffusivities, and disc morphometry, and through subject-specific FE modeling, revealed critical interactions between mechanical loading and nutrient supply and metabolism for the in vivo human TMJ disc. The results suggest that TMJ disc homeostasis may be vulnerable to pathological loading (e.g., clenching, bruxism), which impedes nutrient supply. Given difficulties associated with direct in vivo measurements, this study provides a new approach to systematically investigate homeostatic and degenerative mechanisms associated with the TMJ disc.

MiR-150-5p regulates melanoma proliferation, invasion and metastasis via SIX1-mediated Warburg Effect

Aerobic glycolysis is a hallmark of cancer. Sine oculis homeobox 1 (SIX1), a key transcription factor in terms of regulating aerobic glycolysis (the Warburg Effect), plays a critical role in tumorigenesis of various cancer types, including breast cancer, liver cancer, and lung cancer. However, the upstream regulating mechanisms of SIX1 in melanoma remain to be determined. MicroRNAs (miRNAs) have emerged as key regulators in tumorigenesis and progression. Here, we initially showed that microRNA-150-5p (miR-150-5p) inhibits SIX1 expression by directly targeting its 3′-UTR in melanoma cells. miR-150-5p suppressed melanoma cell proliferation, migration, and invasion through inhibition of SIX1. Mechanistically, miR-150-5p dampens glycolysis by decreasing the glucose uptake, lactate production, ATP generation, and extracellular acidification rate (ECAR), and increasing oxygen consumption rate (OCR) by targeting SIX1. Importantly, glycolysis regulated by miR-150-5p/SIX1 axis is critical for its regulation of melanoma growth and metastasis both in vitro and in vivo. Collectively, our study demonstrates the importance of miR-150-5p/SIX1 axis in melanoma, which could be a promising therapeutic target in melanoma.

B7-H3 promotes aerobic glycolysis and chemoresistance in colorectal cancer cells by regulating HK2

Accumulating evidence suggests that aerobic glycolysis is important for colorectal cancer (CRC) development. However, the underlying mechanisms have yet to be elucidated. B7-H3, an immunoregulatory protein, is broadly overexpressed by multiple tumor types and plays a vital role in tumor progression. In this study, we found that overexpression of B7-H3 effectively increased the rate of glucose consumption and lactate production, whereas knockdown of B7-H3 had the opposite effect. Furthermore, we showed that B7-H3 increased glucose consumption and lactate production by promoting hexokinase 2 (HK2) expression in CRC cells, and we also found that HK2 was a key mediator of B7-H3-induced CRC chemoresistance. Depletion of HK2 expression or treating cells with HK2 inhibitors could reverse the B7-H3-induced increase in aerobic glycolysis and B7-H3-endowed chemoresistance of cancer cells. Moreover, we verified a positive correlation between the expression of B7-H3 and HK2 in tumor tissues of CRC patients. Collectively, our findings suggest that B7-H3 may be a novel regulator of glucose metabolism and chemoresistance via controlling HK2 expression in CRC cells, a result that could help develop B7-H3 as a promising therapeutic target for CRC treatment.

Integrated Computational Model of Lung Tissue Bioenergetics.

Altered lung tissue bioenergetics plays a key role in the pathogenesis of lung diseases. A wealth of information exists regarding the bioenergetic processes in mitochondria isolated from rat lungs, cultured pulmonary endothelial cells, and intact rat lungs under physiological and pathophysiological conditions. However, the interdependence of those processes makes it difficult to quantify the impact of a change in a single or multiple process(es) on overall lung tissue bioenergetics. Integrated computational modeling provides a mechanistic and quantitative framework for the bioenergetic data at different levels of biological organization. The objective of this study was to develop and validate an integrated computational model of lung bioenergetics using existing experimental data from isolated perfused rat lungs. The model expands our recently developed computational model of the bioenergetics of mitochondria isolated from rat lungs by accounting for glucose uptake and phosphorylation, glycolysis, and the pentose phosphate pathway. For the mitochondrial region of the model, values of kinetic parameters were fixed at those estimated in our recent model of the bioenergetics of mitochondria isolated from rat lungs. For the cytosolic region of the model, intrinsic parameters such as apparent Michaelis constants were determined based on previously published enzyme kinetics data, whereas extrinsic parameters such as maximal reaction and transport velocities were estimated by fitting the model solution to published data from isolated rat lungs. The model was then validated by assessing its ability to predict existing experimental data not used for parameter estimation, including relationships between lung nucleotides content, lung lactate production rate, and lung energy charge under different experimental conditions. In addition, the model was used to gain novel insights on how lung tissue glycolytic rate is regulated by exogenous substrates such as glucose and lactate, and assess differences in the bioenergetics of mitochondria isolated from lung tissue and those of mitochondria in intact lungs. To the best of our knowledge, this is the first model of lung tissue bioenergetics. The model provides a mechanistic and quantitative framework for integrating available lung tissue bioenergetics data, and for testing novel hypotheses regarding the role of different cytosolic and mitochondrial processes in lung tissue bioenergetics.

Energy Demands during Maximal Sprint Cycling in Youth Cyclists

PURPOSE The purpose of this study was to determine all different energy systems contribution during the maximal sprint cycling in youth cyclists. METHODS Fifteen male and female youth cyclists participated in this study. A simulated maximal 10 sec maximal sprint cycling was carried out. During maximal sprint cycling, energy contribution (phosphagen, WPCR; glycolytic, WLa -; oxidative system WOXI in kJ and %), maximal lactate production rate (VLamax), difference of lactate (ΔLa-), highest lactate (Peak La-), peak power (Wpeak) and anaerobicalactic time (tAlac) were analyzed using off VO2 kinetics, trapezoidal method, O2-lactate equivalent, and caloric quotient. RESULTS During the maximal sprint cycling, WPCR was significantly higher than WLa - and WOXI (p=.008; d: 0.98, p<.001; d: 2.79, respectively). Also, WLa - was significantly higher than WOXI (p<.001; d: 2.56). Additionally, low negative and moderate positive correlations were observed between tAlac, Wpeak and WPCR in kJ (r=.43, r=.63, respectively). Furthermore, high and moderate positive correlations were calculated for ΔLa-, Peak La-, VLamax and WLa - (r=.91, r=.86, r=.68, respectively). CONCLUSIONS Phosphagen and glycolytic energy systems were predominantly used during maximal sprint cycling. Therefore, physical training program for cycling athletes should be directed to the development of anaerobic metabolism because anaerobic performance is crucial during the short term cycling sprint phase. 색인어: 초당 최대 ì –ì‚° ìƒì„±ë¥ , 포스파ì ì‹œìŠ¤í œ, 포스파ì 무산소성 운동시간, í•´ë‹¹ê³¼ì • 에너지 Keywords: Maximal lactate production rate, Phosphagen system, Anaerobic-alactic time, Glycolytic energy

Deficiency in IL-33/ST2 Axis Reshapes Mitochondrial Metabolism in Lipopolysaccharide-Stimulated Macrophages

The polarization and function of macrophages play essential roles in controlling immune responses. Interleukin (IL)-33 is a member of the IL-1 family that has been shown to influence macrophage activation and polarization, but the underlying mechanisms are not fully understood. Mitochondrial metabolism has been reported to be a central player in shaping macrophage polarization; previous studies have shown that both aerobic glycolysis and oxidative phosphorylation uniquely regulate the functions of M1 and M2 macrophages. Whether IL-33 polarizes macrophages by reshaping mitochondrial metabolism requires further investigation. In this work, we examined the mitochondrial metabolism of bone marrow-derived macrophages (BMDMs) from either wild type (WT), Il33-overexpressing, or IL-33 receptor knockout (St2−/−) mice challenged with lipopolysaccharide (LPS). We found that after LPS stimulation, compared with WT BMDMs, St2−/− BMDMs had reduced cytokine production and increased numbers and activity of mitochondria via the metabolism regulator peroxisome proliferator-activated receptor-C coactivator-1 α (PGC1α). This was demonstrated by increased mitochondrial DNA copy number, mitochondria counts, mitochondria fission- and fusion-related gene expression, oxygen consumption rates, and ATP production, and decreased glucose uptake, lactate production, and extracellular acidification rates. For Il33-overexpressing BMDMs, the metabolic reprogramming upon LPS stimulation was similar to WT BMDMs, and was accompanied by increased M1 macrophage activity. Our findings suggested that the pleiotropic IL-33/ST2 pathway may influence the polarization and function of macrophages by regulating mitochondrial metabolism.

Integrated Computational Model of Lung Tissue Bioenergetics

Altered lung tissue bioenergetics plays a key role in the pathogenesis of lung diseases. A wealth of information exists regarding the bioenergetic processes in mitochondria isolated from rat lungs, cultured pulmonary endothelial cells, and intact rat lungs under physiological and pathophysiological conditions. However, the interdependence of those processes makes it difficult to quantify the impact of a change in a single or multiple process(es) on overall lung tissue bioenergetics. Integrated computational modeling provides a mechanistic and quantitative framework for the bioenergetic data at different levels of biological organization. The objective of this study was to develop and validate an integrated computational model of lung bioenergetics using existing experimental data from isolated perfused rat lungs. The model expands our recently developed computational model of the bioenergetics of mitochondria isolated from rat lungs by accounting for glucose uptake and phosphorylation, glycolysis, and the pentose phosphate pathway. For the mitochondrial region of the model, values of kinetic parameters were fixed at those estimated in our recent model of the bioenergetics of mitochondria isolated from rat lungs. For the cytosolic region of the model, intrinsic parameters such as apparent Michaelis constants were determined based on previously published enzyme kinetics data, whereas extrinsic parameters such as maximal reaction and transport velocities were estimated by fitting the model solution to published data from isolated rat lungs. The model was then validated by assessing its ability to predict existing experimental data not used for parameter estimation, including relationships between lung nucleotides content, lung lactate production rate, and lung energy charge under different experimental conditions. In addition, the model was used to gain novel insights on how lung tissue glycolytic rate is regulated by exogenous substrates such as glucose and lactate, and assess differences in the bioenergetics of mitochondria isolated from lung tissue and those of mitochondria in intact lungs. To the best of our knowledge, this is the first model of lung tissue bioenergetics. The model provides a mechanistic and quantitative framework for integrating available lung tissue bioenergetics data, and for testing novel hypotheses regarding the role of different cytosolic and mitochondrial processes in lung tissue bioenergetics.

Betulinic acid suppresses breast cancer aerobic glycolysis via caveolin-1/NF-κB/c-Myc pathway

Emerging evidence has suggested that targeting glycolysis may be a promising strategy for cancer treatment. Betulinic acid (BA) is a natural pentacyclic terpene that has been reported to be active in inhibiting various malignancies. Here, we showed that BA could inhibit aerobic glycolysis activity in breast cancer cell lines MCF-7 and MDA-MB-231 by hampering lactate production, glucose uptake and extracellular acidification rate (ECAR), as well as suppressing aerobic glycolysis-related proteins including c-Myc, lactate dehydrogenase A (LDH-A) and p-PDK1/PDK1 (pyruvate dehydrogenase kinase 1). Mechanistic studies validated Caveolin-1 (Cav-1) as one of key targets of BA in suppressing aerobic glycolysis, as BA administration resulted in Cav-1 upregulation, whereas silencing Cav-1 abrogated the inhibitory effect of BA on aerobic glycolysis. Further investigations demonstrated that BA suppressed aerobic glycolysis in breast cancer cells by regulating the Cav-1/NF-κB/c-Myc pathway. More meaningfully, BA significantly inhibited breast cancer growth and glycolytic activity in both the transgenic MMTV-PyVT+/− breast cancer spontaneous model and the zebrafish breast cancer xenotransplantation model without any detectable side effects in vivo. Taken together, our study sheds novel insights into BA as a promising candidate drug for suppressing aerobic glycolysis, highlighting Cav-1 as a potential molecular target of BA and aerobic glycolysis regulation.

Lactate clearance in infants undergoing surgery for congenital heart disease.

Measurement of lactate concentrations during cardiac surgery with cardiopulmonary bypass (CPB) is a reliable monitoring tool for the assessment of the adequacy of perfusion, and a predictor of poor outcome. However, increased lactate production, which is multifactorial (anaerobic metabolism, hyperglycemia), increased lactate load by packed red blood cell (PRBC) transfusions, and decreased lactate clearance may all result in hyperlactatemia. The aim of this study was to estimate the clearance of lactate in infants undergoing surgery with CPB, using the lactate load from the PRBCs transfusions received during CPB. Retrospective cohort of infants <1year of age with repeated lactate measurements during CPB, and a known lactate concentration in the PRBCs used during CPB were evaluated. All patients received PRBCs in the prime and during CPB to maintain hematocrit >35% and venous saturation >70%. Lactate kinetics were estimated across several time intervals between two lactate measurements, using a single compartment model. The lactate load was calculated as the product: PRBC-lactate concentration * volume. The rate of endogenous lactate production was assumed to be unchanged (maintenance of high oxygen deliveries and normoglycemia throughout CPB). Overall, 87 calculations were performed in 27 patients, then averaged per patient. The mean lactate half-life was 12.36min [10.67-14.06], the mean clearance was 0.09 L/min [0.06-0.11], the indexed lactate clearance was 0.36 L/min/m2 [0.28-0.44]. Lactate clearance increased significantly with age. The half-life of lactate in infants is comparable with that reported in adults with CPB, and lactate clearance is higher. Knowing the high lactate content of PRBCs, lactate clearance rather than absolute concentration is potentially a better indicator of the adequacy of perfusion during CPB in infants.

Effects of Ganoderma lucidum and ‘Essence of Chicken’ on Physical Fatigue Recovery and Exercise Performance Improvement.

A fast-paced lifestyle, pressure from the environment and a heavy work load often cause extreme tiredness in modern life. Different kinds of nutritional supplements in the form of functional foods or traditional Chinese medicine, such as ‘essence of chicken’ and Ganoderma lucidum have been claimed to benefit physical performance and promote health. Previous studies have revealed that ‘essence of chicken’ or G. lucidum have a wide spectrum of biological activities. In this study, we combined these two ingredients together (designated as CEG) to evaluate their synergistic effects on physiological adaption on exercise fatigue and physical activities. The ICR strain mice were allocated as 0, 833, 1666, and 4165 mg/kg dose groups and administrated by oral gavage consecutively for 4 weeks. Physical activities including grip strength and aerobic endurance were evaluated. Various fatigue-associated biochemical variables such as lactate, BUN or CK were also evaluated. The levels of liver and muscle glycogen were measured as an indicator of energy storage at the end of the experiment. Safety assessments for supplementation were also evaluated. CEG supplementation significantly increased the endurance and grip strength and demonstrated beneficial effects on lactate production and clearance rate after an acute exercise challenge. The CEG supplementation significantly mitigated the BUN and CK indexes after extended exercise and elevated the glycogen content in the liver and muscle tissues. According to body composition, biochemical and histopathological data, daily administration of CEG for over 28 days (subacute toxicity) also demonstrated reasonable safety results for supplementation. Combined G. lucidum and ‘essence of chicken’ can significantly increase the exercise performance and improve fatigue recovery. It may also provide a viable alternative nutritional supplement for health promotion.

Real-time ALT and LDH activities determined in viable precision-cut mouse liver slices using hyperpolarized [1-13 C]pyruvate-Implications for studies on biopsied liver tissues.

Precision-cut liver slices (PCLS) are widely used in liver research as they provide a liver model with all liver cell types in their natural architecture. The purpose of this study was to demonstrate the use of PCLS for hyperpolarized metabolic investigation in a mouse model, for potential future application in liver biopsy cores. Fresh normal liver was harvested from six mice. 500μm PCLS were prepared and placed in a 10mm NMR tube in an NMR spectrometer and perfused continuously. 31 P spectra were acquired to evaluate the presence of adenosine triphosphate (ATP) and validate viability in all samples. Hyperpolarized [1-13 C]pyruvate was flushed into the NMR tube in the spectrometer. Consecutive 13 C NMR spectra were acquired immediately after the injection using both non-selective (five injections, two livers) and selective RF excitation (six injections, three livers). The 31 P spectra showed the characteristic signals of ATP, confirming the viability of the PCLS for more than 2.5h in the spectrometer. After each of the [1-13 C]pyruvate injections, both [1-13 C]lactate and [1-13 C]alanine signals were detected. Selective RF excitation aimed at both [1-13 C]lactate and [1-13 C]alanine enabled better visualization and quantification of the metabolic activity. Using this acquisition approach only the newly formed metabolites are observed upon excitation, and their intensities relative to those of hyperpolarized pyruvate enable quantification of metabolite production rates. This rate of lactate and alanine production appeared to be constant throughout the measurement time, with alanine production about 2.3 times higher than lactate. In summary, the viability of PCLS in an NMR spectrometer was demonstrated and hyperpolarized [1-13 C]pyruvate metabolism was recorded. This study opens up the possibility of evaluating alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) activities in human liver biopsies, while preserving the tissue architecture and viability. In healthy, well-perfused liver slices the ratio of ALT to LDH activity is about 2.3.

An Optimized Bistable Metabolic Switch To Decouple Phenotypic States during Anaerobic Fermentation.

Metabolic engineers aim to genetically modify microorganisms to improve their ability to produce valuable compounds. Despite the prevalence of growth-coupled production processes, these strategies can significantly limit production rates. Instead, rates can be improved by decoupling and optimizing growth and production independently, and operating with a growth stage followed by a production stage. Here, we implement a bistable transcriptional controller to decouple and switch between these two states. We optimize the controller in anaerobic conditions, typical of industrial fermentations, to ensure stability and tight expression control, while improving switching dynamics. The stability of this controller can be maintained through a simulated seed train scale-up from 5 mL to 500 000 L, indicating industrial feasibility. Finally, we demonstrate a two-stage production process using our optimal construct to improve the instantaneous rate of lactate production by over 50%, motivating the use of these systems in broad metabolic engineering applications.

Persistent Organic Pollutant Burden, Experimental POP Exposure, and Tissue Properties Affect Metabolic Profiles of Blubber from Gray Seal Pups.

Persistent organic pollutants (POPs) are toxic, ubiquitous, resist breakdown, bioaccumulate in living tissue, and biomagnify in food webs. POPs can also alter energy balance in humans and wildlife. Marine mammals experience high POP concentrations, but consequences for their tissue metabolic characteristics are unknown. We used blubber explants from wild, gray seal ( Halichoerus grypus) pups to examine impacts of intrinsic tissue POP burden and acute experimental POP exposure on adipose metabolic characteristics. Glucose use, lactate production, and lipolytic rate differed between matched inner and outer blubber explants from the same individuals and between feeding and natural fasting. Glucose use decreased with blubber dioxin-like PCBs (DL-PCB) and increased with acute experimental POP exposure. Lactate production increased with DL-PCBs during feeding, but decreased with DL-PCBs during fasting. Lipolytic rate increased with blubber dichlorodiphenyltrichloroethane and its metabolites (DDX) in fasting animals, but declined with DDX when animals were feeding. Our data show that POP burdens are high enough in seal pups to alter adipose function early in life, when fat deposition and mobilization are vital. Such POP-induced alterations to adipose metabolic properties may significantly alter energy balance regulation in marine top predators, with the potential for long-term impacts on fitness and survival.