Changes In Lactate Metabolism Research Articles

Elevated endoplasmic reticulum pH is associated with high growth and bisAb aggregation in CHO cells.

Chinese hamster ovary (CHO) bioprocesses, the dominant platform for therapeutic protein production, are increasingly used to produce complex multispecific proteins. Product quantity and quality are affected by intracellular conditions, but these are challenging to measure and often overlooked during process optimization studies. pH is known to impact quality attributes like protein aggregation across upstream and downstream processes, yet the effects of intracellular pH on cell culture performance are largely unknown. Recently, advances in protein biosensors have enabled investigations of intracellular environments with high spatiotemporal resolution. In this study, we integrated a fluorescent pH-sensitive biosensor into a bispecifc (bisAb)-producing cell line to investigate changes in endoplasmic reticulum pH (pHER). We then investigated how changes in lactate metabolism impacted pHER, cellular redox, and product quality in fed-batch and perfusion bioreactors. Our data show pHER rapidly increased during exponential growth to a maximum of pH 7.7, followed by a sharp drop in the stationary phase in all perfusion and fed-batch conditions. pHER decline in the stationary phase was driven by an apparent loss of cellular pH regulation that occurred despite differences in redox profiles. Finally, we found protein aggregate levels correlated most closely with pHER which provides new insights into product aggregate formation in CHO processes. An improved understanding of the intracellular changes impacting bioprocesses can ultimately help guide media optimizations, improve bioprocess control strategies, or provide new targets for cell engineering.

Early Monitoring of Redox Status Based on Reduced Molecules Using In Vivo DNP-MRI for Cancer Treatment

<h3>Purpose/Objective(s)</h3> Radiation treatment effectiveness is assessed through the observation of morphological changes with CT or MRI images after treatment. The treatment efficacy of radiotherapy depends on redox modulation in tumor tissue, such as hydroxyl radicals and of reactive oxygen species generated by irradiation. Therefore, redox status may be a prognostic factor response to radiotherapy. However, spatiotemporal response of the redox metabolism in tumor tissue post-irradiation remains unknown. The purpose of this study was to detect early changes in the redox status in tumor tissue after irradiation using <i>in vivo</i> dynamic nuclear polarization (DNP)-MRI, to clarify the key molecules that cause these changes, and to compare these changes with early changes in lactate metabolism using hyperpolarized ¹³C magnetic resonance spectroscopy (MRS). <h3>Materials/Methods</h3> To monitor the response of tumor redox status after irradiation, redox imaging using in vivo DNP MRI/ Carbamoyl PROXYL (CmP) as redox sensitive DNP probe was performed after 5 Gy irradiation. To clarify the relationship between redox status alteration and treatment efficacy, in vivo DNP MRI was performed before and after irradiation by 2 and 5 Gy. In addition, tissue GSH and AsA levels on day 1 post irradiation were determined by HPLC. To observe the Warburg effect after radiation treatment, dissolution DNP study using hyperpolarized ¹³C pyruvate was performed before and after irradiation. <h3>Results</h3> The DNP image intensity in the tumor demonstrated a maximum value approximately four minutes after CmP administration, thereafter the image intensity was decreased by the redox reaction. The tumor GSH and AsA level on day 1 post irradiation were decreased compared to the control group. Production of hyperpolarized ¹³C lactate in tumor tissue did not change before and after irradiation. <h3>Conclusion</h3> Our study highlights that redox status in tumor tissue is already significantly decreased on day 1 before the detection of any morphological changes, even after irradiation with a dose as low as 2 Gy. Early changes in this redox status may be related decreases in glutathione and ascorbic acid levels in the tumor tissue after irradiation, which may alter earlier than ¹³C pyruvate hyperpolarized MRS, based on lactate metabolism. Therefore, early assessment of change in decay rate using in vivo DNP-MRI/CmP probing may predict the subsequent radiotherapy effect.

Prevention of Cell Death by Activation of Hydroxycarboxylic Acid Receptor 1 (GPR81) in Retinal Explants.

Background: Progressive retinal ganglion cell (RGC) dysfunction and death are common characteristics of retinal neurodegenerative diseases. Recently, hydroxycarboxylic acid receptor 1 (HCA1R, GPR81) was identified as a key modulator of mitochondrial function and cell survival. Thus, we aimed to test whether activation of HCA1R with 3,5-Dihydroxybenzoic acid (DHBA) also promotes RGC survival and improves energy metabolism in mouse retinas. Methods: Retinal explants were treated with 5 mM of the HCA1R agonist, 3,5-DHBA, for 2, 4, 24, and 72 h. Additionally, explants were also treated with 15 mM of L-glutamate to induce toxicity. Tissue survival was assessed through lactate dehydrogenase (LDH) viability assays. RGC survival was measured through immunohistochemical (IHC) staining. Total ATP levels were quantified through bioluminescence assays. Energy metabolism was investigated through stable isotope labeling and gas chromatography-mass spectrometry (GC-MS). Lactate and nitric oxide levels were measured through colorimetric assays. Results: HCA1R activation with 3,5-DHBAincreased retinal explant survival. During glutamate-induced death, 3,5-DHBA treatment also increased survival. IHC analysis revealed that 3,5-DHBA treatment promoted RGC survival in retinal wholemounts. 3,5-DHBA treatment also enhanced ATP levels in retinal explants, whereas lactate levels decreased. No effects on glucose metabolism were observed, but small changes in lactate metabolism were found. Nitric oxide levels remained unaltered in response to 3,5-DHBA treatment. Conclusion: The present study reveals that activation of HCA1R with 3,5-DHBA treatment has a neuroprotective effect specifically on RGCs and on glutamate-induced retinal degeneration. Hence, HCA1R agonist administration may be a potential new strategy for rescuing RGCs, ultimately preventing visual disability.

Abstract 2484: Hyperpolarized magnetic resonance spectroscopy assessment of the metabolic effects of glutaminase inhibitor therapy in an ovarian cancer model

Abstract Introduction: Ovarian cancer accounts for more deaths than any other cancer of the female reproductive system despite advances in targeted drugs such as anti-VEGF antibody (AVA). Additionally, resistance to AVA therapy is nearly ubiquitous given the occurrence of metabolic adaptations such as a dependence on glutamine metabolism via the enzyme glutaminase. Objectives: To examine the metabolic vulnerability of AVA-resistant cancer with glutaminase inhibitor (GLSi) therapy and to test the utility of hyperpolarized magnetic resonance spectroscopy (HP-MRS) for assessing changes in lactate metabolism associated with GLSi treatment following injection of hyperpolarized [1-13C] pyruvate. Methods: We used a well-characterized SKOV3ip1 orthotopic mouse model of high-grade serous ovarian cancer with adaptive resistance to AVA (bevacizumab) treatment. Following three weeks of GLSi and bevacizumab treatment, we performed HP-MRS imaging to directly and non-invasively compare metabolic changes between the treatment groups of bevacizumab monotherapy versus a combined treatment of bevacizumab plus GLSi therapy. Upon injection of hyperpolarized [1-13C] pyruvate, we quantitatively assessed a normalized lactate ratio (nLac), defined as the 13C resonance signal of lactate divided by the sum of the pyruvate and lactate signals, sixty seconds after injection. GLSi treatment effect was subsequently compared between GLSi monotherapy, bevacizumab monotherapy and control mice receiving no therapy. This was performed via gross necropsy to assess disease burden and quantification of tumor weight and nodule number. Results: Significance analysis microarrays (SAM) of bevacizumab treated tumors from the mouse models revealed greater glutamine and glutamate abundance in all tissues suggestive of a glutamine dependence in AVA resistant tumors that could be susceptible to glutaminase inhibition. The in vivo HP-MRS provided a direct and non-invasive investigation of pyruvate metabolism of tumors in situ. Our analysis indicated that the pyruvate-to-lactate conversion was significantly reduced in vivo by GLSi treatment (0.337 vs 0.178, p=0.0002), suggestive of therapeutic efficacy of GLSi in the setting of AVA-resistant tumors. Consistent with this, we observed a statistically significant reduction in tumor weight (0.05g vs 0.62g and 0.64g, p&amp;lt;0.01) and tumor nodule number (n=3.3 vs n=12.8 and n=13.9, p&amp;lt;0.05) in mice treated with a combination of bevacizumab and GLSi compared to the control group, or GLSi monotherapy, respectively. Conclusions: Using HP-MRS in vivo, we directly monitored metabolic changes occurring after GLSi treatment. These findings are consistent with the notion of glutamine dependence being an important resistance mechanism to AVA therapy and imply potential future use of HP-MRS in monitoring therapeutic efficacy in a clinical realm. Citation Format: Deanna Glassman, Mark S. Kim, Prasanta Dutta, Charles V. Kingsley, Emine Bayraktar, Yutuan Wu, Elaine Stur, Lingegowda S. Mangala, Katherine Foster, Sanghoon Lee, Timothy A. Yap, Shannon N. Westin, Pratip Bhattacharya, Anil K. Sood. Hyperpolarized magnetic resonance spectroscopy assessment of the metabolic effects of glutaminase inhibitor therapy in an ovarian cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2484.

Development of a miniature bioreactor model to study the impact of pH and DOT fluctuations on CHO cell culture performance as a tool to understanding heterogeneity effects at large-scale.

Understanding the impact of spatial heterogeneities that are known to occur in large‐scale cell culture bioreactors remains a significant challenge. This work presents a novel methodology for mimicking the effects of pH and dissolved oxygen heterogeneities on Chinese hamster ovary (CHO) cell culture performance and antibody quality characteristics, using an automated miniature bioreactor system. Cultures of 4 different cell lines, expressing 3 IgG molecules and one fusion protein, were exposed to repeated pH and dissolved oxygen tension (DOT) fluctuations between pH 7.0–7.5 and DOT 10%–30%, respectively, for durations of 15, 30, and 60 min. Fluctuations in pH had a minimal impact on growth, productivity, and product quality although some changes in lactate metabolism were observed. DOT fluctuations were found to have a more significant impact; a 35% decrease in cell growth and product titre was observed in the fastest growing cell line tested, while all cell lines exhibited a significant increase in lactate accumulation. Product quality analysis yielded varied results; two cell lines showed an increase in the G0F glycan and decrease in G1F, G2F, and Man5; however, another line showed the opposite trend. The study suggests that the response of CHO cells to the effects of fluctuating culture conditions is cell line specific and that higher growing cell lines are most impacted. The miniature bioreactor system described in this work therefore provides a platform for use during early stage cell culture process development to identify cell lines that may be adversely impacted by the pH and DOT heterogeneities encountered on scale‐up. This experimental data can be combined with computational modeling approaches to predict overall cell culture performance in large‐scale bioreactors.

Carnitine chloride reduces the severity of experimental hyperhomocysteinemia and promotes lactate utilization by mitochondrial fraction of the rat epididymis

Hyperhomocysteinemia is a risk factor for many diseases, including reproductive disorders in men. L-carnitine is used in medical practice to correct impaired bioenergetic conditions; in patients with idiopathic forms of infertility its effects are associated with improvement of the sperm parameters. However, the effect of exogenous L-carnitine on the level of homocysteine in the gonadal tissues, as a risk factor for impaired fertility, has not been investigated yet. The aim of this study was to investigate activity of bioenergetic enzymes in the epididymal mitochondrial fraction, the dynamics of changes in the cytoplasmic and mitochondrial lactate levels and LDH activity, the total carnitine content, as well as the oxidative status of these cells under conditions of oxidative stress caused by hyperhomocysteinemia, and to assess the effect of carnitine chloride on these parameters under conditions of methionine administration to male Wistar rats. Methionine administration to animals for three weeks at a dose of 3 g/kg, resulted in development of the severe forms of hyperhomocysteinemia with serum homocysteine concentrations exceeding 100 μmol/L. This was accompanied by a decrease in the activity of enzymes involved in the bioenergetic processes of the cell: tissue respiration (succinate dehydrogenase) and oxidative phosphorylation (H+-ATPase) in the epididymal head and tail. The change in lactate metabolism included an increase in its level in both the mitochondrial and cytoplasmic fractions of the epididymal head and mitochondria of the epididymal tail, and also simultaneous statistically significant decrease in LDH activity in the mitochondria and cytoplasm of the epididymal head. In male rats with severe hyperhomocysteinemia, an increase in the activity of mitochondrial SOD accompanied by an increase in the carbonylation of mitochondrial proteins in the head and tail of the epididymis was noted. Modeling of hyperhomocysteinemia under conditions of carnitine chloride of administration led to different reactions of the cells of the studied tissues assayed in the epididymal head and tail homogenate. In the epididymal head, carnitine chloride promoted an increase in the mitochondrial lactate concentration and a decrease in the cytoplasmic lactate concentration, as well as an increase in the LDH activity associated with the mitochondrial fraction. These changes were accompanied by an increase in the activity of H+-ATPase in the epididymal, thus suggesting that carnitine chloride stimulated lactate transport of into the mitochondria and its use as an energy substrate under conditions of oxidative stress caused by hyperhomocysteinemia. In the tail tissues, the changes were protective in nature and were associated with a decrease in the formation of oxidatively modified proteins.

Response of lactate metabolism in brain glucosensing areas of rainbow trout (Oncorhynchus mykiss) to changes in glucose levels.

There is no evidence in fish brain demonstrating the existence of changes in lactate metabolism in response to alterations in glucose levels. We induced in rainbow trout through intraperitoneal (IP) treatments, hypoglycaemic or hyperglycaemic changes to assess the response of parameters involved in lactate metabolism in glucosensing areas like hypothalamus and hindbrain. To distinguish those effects from those induced by peripheral changes in the levels of metabolites or hormones, we also carried out intracerebroventricular (ICV) treatments with 2-deoxy-D-glucose (2-DG, a non-metabolizable glucose analogue thus inducing local glucopenia) or glucose. Finally, we also incubated hypothalamus and hindbrain in vitro in the presence of increased glucose concentrations. The changes in glucose availability were in general correlated to changes in the amount of lactate in both areas. However, when we assessed in these areas the response of parameters related to lactate metabolism, the results obtained were contradictory. The increase in glucose levels did not produce in general the expected changes in those pathways with only a minor increase in their capacity of lactate production. The decrease in glucose levels was, however, more clearly related to a decreased capacity of the pathways involved in the production and use of lactate, and this was especially evident after ICV treatment with 2-DG in both areas. In conclusion, the present results while addressing the existence of changes in lactate metabolism after inducing changes in glucose levels in brain glucosensing areas only partially support the possible existence of an astrocyte-neuron lactate shuttle in hypothalamus and hindbrain of rainbow trout relating glucose availability to lactate production and use.

Physiological regulation linked with physical activity and health

Physiological regulation linked with physical activity and health

Effects of a Supplementation during Exercise and Recovery

The present study was designed to investigate whether a protein hydrolysate enriched in branched chain amino acids and antioxidants, trace and mineral elements, and vitamins would affect performance and fatigue. Eighteen sportsmen underwent testing before and after 28 days supplementation with either treatment in protein hydrolysate or placebo. Testing included exhaustive aerobic and anaerobic exercises with determination of blood lactate concentration through exercise and recovery and antioxidant status, but also measurements of maximal oxidative capacity (V. (max)) and citrate synthase activity (CS) from a resting muscle biopsy. Protein hydrolysate resulted in a significant decrease in fatigue indices, without affecting performances. A significant increase in enzymatic antioxidant and a decrease in oxidative damage were observed at rest after treatment but not with a placebo. Decrease in maximal blood lactate and improvement of blood lactate removal were only observed after protein hydrolysate treatment. Furthermore, CS increased significantly, whereas no change was observed in V. (max). In conclusion, this protein hydrolysate treatment induced adaptations that may promote a decrease in fatigue during exercises, potentially explained by changes in parameters used to represent oxidative damage and antioxidant status at rest and changes in lactate metabolism.

Proteome analysis of antibody‐producing CHO cell lines with different metabolic profiles

Two-dimensional gel electrophoresis and tandem mass spectrometry were used to identify proteins associated with a metabolic shift during fed-batch cultures of two recombinant antibody-producing CHO cell lines. The first cell line underwent a marked change in lactate metabolism during culture, initially producing lactate and then consuming it, while the second cell line produced lactate for a similar duration but did not later consume it. The first cell line displayed a declining specific antibody productivity during culture, correlating to the 2-D gel results and the intracellular antibody concentration determined by HPLC. Several statistical analysis methods were compared during this work, including a fixed fold-change criterion and t-tests using standard deviations determined in several ways from the raw data and mathematically transformed data. Application of a variance-stabilizing transformation enabled the use of a global empirical standard deviation in the t-tests. Most of the protein spots changing in each cell line did not change significantly in the other cell line. A substantial fraction of the changing proteins were glycolytic enzymes; others included proteins related to antibody production, protein processing, and cell structure. Enolase, pyruvate kinase, BiP/GRP78, and protein disulfide isomerase were found in spots that changed over time in both cell lines, and some protein changes differed from previous reports. These data provide a foundation for future investigation of metabolism in industrially relevant mammalian cell culture processes, and suggest that along with differences between cell types, the proteins expressed in cultures with low lactate concentrations may depend on how those conditions were generated.

Sequential Alterations in Myocardial Lactate Metabolism, S‐T Segments, and Left Ventricular Function During Angina Induced by Atrial Pacing

SUMMARYMyocardial lactate metabolism was monitored before, during, and after a period of atrial pacing with a continuous automated sampling technic. The electrocardiogram was recorded, and left ventricular function was evaluated by relating left ventricular end‐diastolic pressure to left ventricular stroke work. Seventeen of 21 patients with coronary artery disease experienced angina during pacing, and 13 of these showed lactate production by the myocardium during angina. The abnormalities in lactate metabolism developed early in the pacing period, along with S‐T segment depression and impairment of left ventricular function, all of which usually occurred before the onset of pain. In the patients with angina, the mean lactate extraction was 14.1% during the control period and —15.1% during the pacing period. Abnormalities in myocardial lactate metabolism have been demonstrated with greater regularity during pacing‐induced angina than during angina precipitated by exercise or isoproterenol. This investigation has demonstrated the sequences of changes in lactate metabolism during and after a period of angina and their relation to electrocardiographic and hemodynamic events.

Pathophysiology of metabolic acidosis: effect of low pH on the hepatic uptake of lactate, pyruvate and alanine.

The uptake of lactate, pyruvate and alanine in perfused rat liver was investigated under normal perfusion conditions (pH 7.4, PCO2 40 mmHg) and under conditions mimicking partially compensated metabolic acidosis (pH 6.9, PCO2 20 mmHg). At 1 mM lactate as well as 10 mM lactate in the medium a lowering of pH from 7.4 to 6.9 did not affect the lactate plus pyruvate uptake. A significant effect of the low pH was seen on pyruvate uptake which at 1 mM lactate was increased from 0.027 +/- 0.008(4) mumol/min per g liver at normal pH to 0.084 +/- 0.013(4) at low pH. At 10 mM lactate the liver produced pyruvate, but the production was significantly reduced by a lowering of the pH, being 0.45 +/- 0.13(8) mumol/min per g liver at pH 7.4 and 0.22 +/- 0.06(4) at pH 6.9. THe counterbalancing changes in lactate metabolism were too small to attain statistical significance. The stimulation of gluconeogenesis by an increase in FFA from 0 to 1 mM in the medium was unaffected by pH. Alanine uptake was decreased from 0.48 +/- 0.05(6) to 0.39 +/- 0.07(3) by lowering the pH from 7.4 to 6.9. We conclude that metabolic acidosis does not in itself inhibit the capacity of the perfused rat liver to remove lactate and pyruvate from the blood. If the same is true in man, no beneficial effect of bicarbonate treatment on lactate clearance in patients with lactic acidosis should be expected.

Diagnostics of ischemic heart disease: Influence of myocardial ischemia on AMP catabolite release and hemodynamics

AbstractThe purpose of this study was to answer the questions: What is the correlation between adenosine monophosphate (AMP) breakdown and hemodynamic changes caused by myocardial ischemia? Can AMP catabolites released by the heart be used to diagnose ischemic heart disease?We studied changes in metabolism and hemodynamics in the intact ischemic pig heart and the isolated, perfused rat heart made hypoxic or ischemic. We measured the AMP catabolites adenosine, inosine, and hypoxanthine, which were released by the oxygen‐deprived heart.Inosine is a sensitive (and early) indicator of wall‐thickness changes in the ischemic pig heart. In addition to a change in lactate metabolism, myocardial inosine production is a good marker for myocardial ischemia because it correlates well with carbohydrate extraction and ventricular function, and because these occlusion‐induced changes are relatively large. We tried unsuccessfully to develop a model to study interventions during myocardial ischemia in which the pig served as its own control.

The effects of adenosine-5'-ethylcarboxamide on liver blood flow and hepatic glucose, lactate and pyruvate balances in dogs.

The actions of adenosine-5'-ethylcarboxamide (744-96), a long-acting adenosine analogue, on liver, portal and intestinal balances of glucose, lactate and pyruvate and on hepatic and portal blood flow were investigated in 6 chloralose-anaesthetized mongrel dogs. 744-96 led to an increase in portal and hepatic blood flow. Glucose release by the liver and glucose uptake by the non-hepatic splanchnic area (portal balance) were markedly increased by 744-96. Hepatic lactate and pyruvate balances were reversed from uptake to release by the adenosine analogue. The changes in glucose balances compare closely to the actions of glucagon, which is known to be released by 744-96. Apart from these possible glucagon-mediated actions, a direct action of the adenosine analogue must be assume from the changes in lactate metabolism. The results of this study are indicative of a substrate-mobilising action of adenosine in addition to its well-known vasodilatory action.

Myocardial nucleoside and carbohydrate metabolism and hemodynamics during partial occlusion and reperfusion of pig coronary artery

The effect of local ischemia on myocardial metabolism and hemodynamics was studied in open-chested pigs. Coronary artery flow was reduced by 74% with a screw clamp for one hour, after which period the clamp was released. Arterial and local coronary venous samples were taken repeatedly during control, occlusion and reperfusion. During control, the myocardium extracted lactate (20%) and inosine (24%). No or minor arteriovenous differences were measured for glucose, potassium, inorganic phosphate and hypoxanthine. During ischemia, the heart consumed glucose (about 25%); lactate and inosine extraction decreased to minimal values of −126% and −642%, respectively. Hypoxanthine, potassium and inorganic phosphate showed relatively small changes in arteriovenous difference. Extraction patterns approached control values during release. Myocardial oxygen uptake decreased by 68% after occlusion. Immediately following release, uptake returned to control value, but subsequently it fell again by 20 to 30%. Extensive and long-lasting reactive hyperemia was observed during reperfusion. However, flow debt was only partially repaid. Throughout the experiment heart rate increased, and cardiac output and mean aortic pressure decreased. During occlusion, the peak derivative of left ventricular pressure decreased about 24%; peripheral resistance and left ventricular end-diastolic pressure increased 20 to 30%. These changes were only partially reversed during reperfusion. It is concluded that, in addition to changes in lactatemetabolism, myocardial inosine production is a good marker for myocardial ischemia in the pig, because it correlates well with carbohydrate extraction and ventricular function, and the occlusion-induced changes are relatively large.

Sequential alterations in myocardial lactate metaboli, S-T segments, and left ventricular function during angina induced by atrial pacing.

Myocardial lactate metabolism was monitored before, during, and after a period of atrial pacing with a continuous automated sampling technic. The electrocardiogram was recorded, and left ventricular function was evaluated by relating left ventricular end-diastolic pressure to left ventricular stroke work. Seventeen of 21 patients with coronary artery disease experienced angina during pacing, and 13 of these showed lactate production by the myocardium during angina. The abnormalities in lactate metabolism developed early in the pacing period, along with S-T segment depression and impairment of left ventricular function, all of which usually occurred before the onset of pain. In the patients with angina, the mean lactate extraction was 14.1% during the control period and —15.1% during the pacing period. Abnormalities in myocardial lactate metabolism have been demonstrated with greater regularity during pacing-induced angina than during angina precipitated by exercise or isoproterenol. This investigation has demonstrated the sequences of changes in lactate metabolism during and after a period of angina and their relation to electrocardiographic and hemodynamic events.