Increased Lactate Accumulation Research Articles

Short-Term Flow Velocity Stress on the Behavioral, Physiological, and Biochemical Responses of the Large Yellow Croaker (Larimichthys crocea)

Flow velocity is a key environmental factor that affects the behavioral strategies and physiological homeostasis of fish. To study the effects of flow velocity on the behavioral changes and blood physiology of the yellow croaker, the behavioral patterns of yellow croakers in response to flow velocity stress were obtained by analyzing changes in tail wagging frequency and amplitude. Differences in blood glucose, lactate, and cortisol were compared to determine their appropriate flow rate ranges. The juvenile stage of the large yellow croaker is crucial, as environmental changes can affect the physiology of fish. Therefore, juvenile yellow croakers were selected as the experimental subjects for this study. Twenty-four healthy and uniformly sized large yellow croakers with body weights of (90.26 ± 9.91) g and body lengths of (19.91 ± 0.69) cm were randomly assigned to one control group and three experimental groups, with five fish in each group. The experimental group was set with three flow rates, namely 1 bl/s (20 cm/s), 2 bl/s (40 cm/s), and 3 bl/s (60 cm/s), with a flow rate stress duration of 1 h. The results showed that: (1) Under different flow velocities, the fish exhibited different tail wagging patterns. At low flow velocities, their tail fins exhibited a “C”-shaped swing, while at high flow velocities, their bodies exhibited an “S”-shaped swing. (2) Oscillation frequency and amplitude both increased with increasing flow velocity. At a flow velocity of 2 bl/s, the oscillation frequency significantly increased. When the flow velocity reached 3 bl/s, the oscillation amplitude significantly increased (p < 0.05). (3) Blood physiology showed significant changes with increased flow rate, and blood glucose content continuously decreased with increased flow rate, significantly decreasing at a flow rate of 2 bl/s (p < 0.05). Lactic acid and cortisol both increased with increasing flow rate, and significantly increased at a flow rate of 3 bl/s (p < 0.05). In summary, under high-flow velocity stress, significant changes occurred in the behavior and physiology of large yellow croakers, which were consistent with physiological changes in the blood. A flow rate higher than 2 bl/s can lead to intense swimming behavior, decreased blood sugar concentration, and increased lactate accumulation and stress levels. Therefore, the short-term tolerance of yellow croakers is 2 bl/s, and a flow rate of 1 bl/s is more suitable.

Overcoming the inconsistent cell culture performance caused by single-use bioreactors during scaling-up the manufacturing process for a therapeutic bispecific antibody

Single use technology has been widely used in modern biopharmaceutical process development and manufacturing. However, there are concerns about the adverse effects of disposable materials on bioprocess, product quality and patient safety. Recently, we observed slow cell growth, increased lactate accumulation, and decreased production titer during scaling up a bispecific antibody (BsAb) upstream process from 3 L to 500 L bioreactor. To investigate the phenomenon, we conducted medium incubation and leachable spike-in experiments. We found that cell culture medium incubated in CX5–14 bag caused poor cell culture performance, which was correlated with the concentration of bis(2,4-di-tert-butylphenyl)-phosphate (bDtBPP), a breakdown product of the antioxidant Irgafos®168 in polyethylene film. In addition, when the media were spiked with 0.15 μg/ml or a greater concentration of bDtBPP, slow cell growth and reduced BsAb expression level were observed. In contrast, Aegis5–14 film produced bDtBPP at a level of below detection during medium incubation. Thus, the bispecific-expressing CHO cell line restores normal cell culture performance and production titer when Aegis5–14 single-use bioreactor was employed in large-scale manufacturing. Our results reveal the importance of evaluating leachable profiles from disposable materials during process development of bispecifics and other novel therapeutic modalities.

Mimicking CHO large-scale effects in the single multicompartment bioreactor: A new approach to access scale-up behavior.

During the scale-up of biopharmaceutical production processes, insufficiently predictable performance losses may occur alongside gradients and heterogeneities. To overcome such performance losses, tools are required to explain, predict, and ultimately prohibit inconsistencies between laboratory and commercial scale. In this work, we performed CHO fed-batch cultivations in the single multicompartment bioreactor (SMCB), a new scale-down reactor system that offers new access to study large-scale heterogeneities in mammalian cell cultures. At volumetric power inputs of 20.4-1.5 W m-3, large-scale characteristics like long mixing times and dissolved oxygen (DO) heterogeneities were mimicked in the SMCB. Compared to a reference bioreactor (REFB) set-up, the conditions in the SMCB provoked an increase in lactate accumulation of up to 87%, an increased glucose uptake, and reduced viable cell concentrations in the stationary phase. All are characteristic for large-scale performance. The unique possibility to distinguish between the effects of changing power inputs and observed heterogeneities provided new insights into the potential reasons for altered product quality attributes. Apparently, the degree of galactosylation in the evaluated glycan patterns changed primarily due to the different power inputs rather than the provoked heterogeneities. The SMCB system could serve as a potent tool to provide new insights into scale-up behavior and to predict cell line-specific drawbacks at an early stage of process development.

MCT4 Deficiency Enhances High-intensity Interval Training-induced Metabolic Adaptations in Skeletal Muscle

The purpose of this study was to understand the effect of increased lactate accumulation in skeletal muscle by MCT4 deficiency on the adaptation of skeletal muscle to high-intensity interval training. To address this challenge, we first developed MCT4-deficient mice with a genetic background of the Jcl:ICR strain which is capable of performing high-intensity exercise. We found that MCT4 deficiency enhances high-intensity interval training-induced improvement of endurance exercise capacity at high intensity. Furthermore, it was also shown that the combination of MCT4 deficiency and high-intensity interval training, coupled with the physiological context of such adaptation, increases enzyme activity of the glycolysis and the mitochondrial oxidative capacity of pyruvate. The findings of this study would help us understand the physiological significance of lactate accumulation in skeletal muscle with high-intensity exercise. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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.

Interleukin 6-independent metabolic reprogramming as a driver of cancer-related fatigue

Fatigue is a common and debilitating symptom of cancer with few effective interventions. Cancer-related fatigue (CRF) is often associated with increases in inflammatory cytokines, however inflammation may not be requisite for this symptom, suggesting other biological mediators also play a role. Because tumors are highly metabolically active and can amplify their energetic toll via effects on distant organs, we sought to determine whether CRF could be explained by metabolic competition exacted by the tumor. We used a highly metabolically active murine E6/E7/hRas model of head and neck cancer for this purpose. Mice with or without tumors were submitted to metabolic constraints in the form of voluntary wheel running or acute overnight fasting and their adaptive behavioral (home cage activity and fasting-induced wheel running) and metabolic responses (blood glucose, ketones, and liver metabolic gene expression) were monitored. We found that the addition of running wheel was necessary to measure activity loss, used as a surrogate for fatigue in this study. Tumor-bearing mice engaged in wheel running showed a decrease in blood glucose levels and an increase in lactate accumulation in the skeletal muscle, consistent with inhibition of the Cori cycle. These changes were associated with gene expression changes in the livers consistent with increased glycolysis and suppressed gluconeogenesis. Fasting also decreased blood glucose in tumor-bearing mice, without impairing glucose or insulin tolerance. Fasting-induced increases in wheel running and ketogenesis were suppressed by tumors, which was again associated with a shift from gluconeogenic to glycolytic metabolism in the liver. Blockade of IL-6 signaling with a neutralizing antibody failed to recover any of the behavioral or metabolic outcomes. Taken together, these data indicate that metabolic competition between the tumor and the rest of the organism is an important component of fatigue and support the hypothesis of a central role for IL-6-independent hepatic metabolic reprogramming in the pathophysiology of CRF.

Fluctuations in dissolved oxygen concentration during a CHO cell culture process affects monoclonal antibody productivity and the sulfhydryl-drug conjugation process.

During a CHO cell culture production process, important parameters are generally well controlled by a feedback mechanism (PID loop) in order to ensure consistency in both productivity and product quality. These parameters typically include pH, dissolved oxygen (DO), and temperature. While most of these parameters are very well controlled within their specific deadband, stable DO control can be challenging. Oscillations in DO concentration are not uncommon and these fluctuations can be exacerbated with an efficient mass transfer aeration strategy. In this study, where an IgG2 producing cell line was used, we observed increased lactate accumulation accompanied by decreased titer production in lots with fluctuations in DO concentration (DOF ) when compared with lots with stable DO control (DOS ). We demonstrate that DOF had a greater impact on performance with respect to titer production and lactate accumulation than DO setpoint. Furthermore, we report that estimated specific oxygen uptake rates (qOURs) were lower in DOF lots when compared with DOS lots. We also report that purified mAb sourced from DOF lots yielded lower drug-to-antibody ratio (DAR) after the sulfhydryl-targeted maleimide conjugation process when equivalent reducing agent was used. All mAb lots were within the analytical specifications for release, though a slight increase in measureable trisulfides were observed in DOF mAb lots. DO control aimed to minimize fluctuations around DO setpoint was essential for us to produce consistent DAR without adjusting the conjugation process. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1427-1437, 2018.

Lactate in cardiogenic shock - current understanding and clinical implications.

Adenosine triphosphate (ATP) is an essential substrate metabolite in human beings. Mitochondrial oxidative phosphorylation provides > 95% of ATP with the remainder derived from glycolysis or tricarboxylic acid cycle (TCA). In normal hearts, acetyl-CoA is synthesized from the β-oxidation of free fatty acids (FFA) and the oxidation of pyruvate. Pyruvate is synthesized from glycolysis and can be submitted either for decarboxylation to acetyl-CoA or for dehydrogenation to lactate. Moreover, pyruvate, as well as lactate, plays a key role in aerobic glucose metabolism which is highly dependent on ubiquitous regulatory mechanisms. Many recent advances in molecular biology, genetics, and physiology have revealed new insights into the metabolic flux of lactate. The initial perception characterized by increased lactate production and accumulation in peripheral tissues in anaerobic conditions has been recently contested. The paradigm of increased lactate concentration in the anaerobic setting is discussed according to contemporary reports. Nevertheless, the clinical role of lactate as a prognostic factor in cardiovascular diseases is undisturbed, especially in the field of innovative technology of left/bi ventricular-assist devices and biochips where it reassured its diagnostic and prognostic impact on the cardiovascular system.

Hepatic glucose-6-phosphatase-α deficiency leads to metabolic reprogramming in glycogen storage disease type Ia

Glycogen storage disease type Ia (GSD-Ia) is caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α or G6PC), a key enzyme in endogenous glucose production. This autosomal recessive disorder is characterized by impaired glucose homeostasis and long-term complications of hepatocellular adenoma/carcinoma (HCA/HCC). We have shown that hepatic G6Pase-α deficiency-mediated steatosis leads to defective autophagy that is frequently associated with carcinogenesis. We now show that hepatic G6Pase-α deficiency also leads to enhancement of hepatic glycolysis and hexose monophosphate shunt (HMS) that can contribute to hepatocarcinogenesis. The enhanced hepatic glycolysis is reflected by increased lactate accumulation, increased expression of many glycolytic enzymes, and elevated expression of c-Myc that stimulates glycolysis. The increased HMS is reflected by increased glucose-6-phosphate dehydrogenase activity and elevated production of NADPH and the reduced glutathione. We have previously shown that restoration of hepatic G6Pase-α expression in G6Pase-α-deficient liver corrects metabolic abnormalities, normalizes autophagy, and prevents HCA/HCC development in GSD-Ia. We now show that restoration of hepatic G6Pase-α expression normalizes both glycolysis and HMS in GSD-Ia. Moreover, the HCA/HCC lesions in L-G6pc−/− mice exhibit elevated levels of hexokinase 2 (HK2) and the M2 isoform of pyruvate kinase (PKM2) which play an important role in aerobic glycolysis and cancer cell proliferation. Taken together, hepatic G6Pase-α deficiency causes metabolic reprogramming, leading to enhanced glycolysis and elevated HMS that along with impaired autophagy can contribute to HCA/HCC development in GSD-Ia.

Ginsenoside Rg5 increases cardiomyocyte resistance to ischemic injury through regulation of mitochondrial hexokinase-II and dynamin-related protein 1

Hexokinase-II (HK-II) and dynamin-related protein 1 (Drp1) regulate mitochondrial function differently. This study was designed to investigate the cardioprotective effect of ginsenoside Rg5 (Rg5) with emphasis on the regulation of mitochondrial HK-II and Drp1. Saturated acid palmitate (PA) stimulation increased lactate accumulation and induced cellular acidification by impairing the activity of pyruvate dehydrogenase (PDH) in cardiomyocytes, leading to HK-II dissociation from mitochondria. Rg5 improved PDH activity and prevented cellular acidification by combating fatty-acid oxidation, contributing to protecting mitochondrial HK-II. HK-II binding to mitochondria prevented mitochondrial Drp1 recruitment, whereas Drp1 activation decreased the content of mitochondrial HK-II, demonstrating the reciprocal control for binding to mitochondria. Rg5 promoted Akt translocation to mitochondria and increased HK-II binding to mitochondria while coordinately suppressing Drp1 recruitment and mitochondrial fission. Akt inhibitor triciribine or knockdown of Akt with small interfering RNA diminished the effects of Rg5, indicating that Rg5 inhibited Drp1 activation and promoted HK-II mitochondrial binding through Akt activation. Rg5 prevented the opening of mitochondrial permeability transition pore and increased ATP production, resultantly increasing cardiomyocyte resistance to hypoxia/reoxygenation injury. Meanwhile, Rg5 prevented cell apoptosis with increased HK-II binding and reduced Drp1 recruitment to mitochondria in isoproterenol-induced ischemic heart of mice. Taken together, these findings not only established a previously unrecognized role of ginsenosides in cardioprotection but also suggest that mitochondrial HK-II binding and Drp1 recruitment could be targeted therapeutically to prevent ischemic injury in the heart.

Abstract 325: Evaluating the consequences of MCT1 inhibition in Burkitt lymphoma following treatment with AZD3965

Abstract Many tumors display an altered metabolic phenotype with an increased reliance on glycolysis resulting in a greater production of the waste product, lactate [1]. Use of glycolysis, as opposed to oxidative phosphorylation, represents a less efficient means of ATP generation but provides a selective advantage to cancer cells in that it rapidly supplies intermediates to support anabolic pathways necessary for increased cellular proliferation. Lactate efflux, is facilitated by monocarboxylate transporters 1-4 and is essential to prevent feedback inhibition of major energy generating pathways. A sub-group of cancers express only MCT1 and are therefore exclusively reliant on this transporter to export lactate. Elevated MCT1 expression has previously been identified in Burkitt lymphoma (BL), a highly aggressive form of non-Hodgkin's lymphoma characterised by MYC translocations [2]. We have studied the effects of a potent, selective and orally available inhibitor of MCT1, AZD3965, in models of BL that predominantly express MCT1. MCT1 inhibition was associated with intracellular lactate accumulation in vitro and a significant growth inhibitory effect (AZD3965 72h GI50 <10 nM). MCT1 inhibition was also associated with a number of changes in intracellular metabolites, including increases in TCA cycle and early glycolytic intermediates, indicating potential feedback mechanisms following lactate accumulation. A measurable increase in lactate accumulation was also detectable in vivo following acute treatment with AZD3965 (100 mg/kg p.o.) using NOD/LtSz-scid IL-2Rγ -/- (NSG) mice bearing subcutaneous CA46 human xenografts. The efficacy of AZD3965 was subsequently investigated in vivo using a more clinically representative form of this BL xenograft model. Here, CA46 cells were engineered to express firefly luciferase (SLIEW lentivector) and then inoculated intravenously into NSG mice. Tumor engraftment was confirmed 6 days after inoculation by the detection of luciferase-based bioluminescence using the IVIS® Spectrum. Oral treatment with either AZD3965 (100mg/kg) or vehicle (both administered twice-daily Monday to Friday and once-daily at weekends) was then initiated for a period of 24 days. Bioluminescence was measured 3 days after the start of treatment and at weekly intervals to determine total tumor burden. Tumor growth relative to control xenografts was inhibited significantly by AZD3965 treatment (>99% P<0.005 Student's two-tailed t-test). In support of this efficacy, post-mortem macroscopic measurements revealed greatly reduced tumor cell engraftment of bone marrow and spleen in MCT1 inhibitor treated animals. AZD3965 has potent growth inhibitory activity in both in vitro and in vivo models of Burkitt lymphoma that are reliant upon MCT1 for lactate transport. [1] Science (1956) 123, 309-314 [2] Cancer Res (2014) 74, 908-920 Citation Format: Richard A. Noble, Natalie Bell, Helen Blair, Huw D. Thomas, Simon Bomken, Susan E. Critchlow, Stephen R. Wedge. Evaluating the consequences of MCT1 inhibition in Burkitt lymphoma following treatment with AZD3965. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 325.

Mechanisms of hepatic encephalopathy and thiamine deficiency.

Hepatic encephalopathy (HE) and thiamine deficiency (TD) represent two disorders in which metabolic derangements play a major role in their pathophysiology. They also define the areas of primary focus of Dr. Roger F. Butterworth, Ph.D., D.Sc. who retired from the University of Montreal in the summer of 2013. This special issue of Metabolic Brain Disease commemorates his valuable contribution to the study of these maladies. Although HE is considered a metabolic disorder, new evidence is emerging to show that it has a molecular basis as well. Vemuganti et al reports altered cerebral profiles of microRNAs after acute liver failure (ALF) that likely contribute in a significant way to gene expression changes associated with the ensuing HE. Disrupted ammonia detoxification is considered a major precipitator of neurological dysfunction in HE, and current understanding of the contribution of ammonia in liver diseases is discussed in an article by Ott and Vilstrup, while the effects of hyperammonemia on cerebral metabolic function are focussed on in an article by Schousboe and colleagues. In addition, impaired brain energy metabolism is a serious complicating feature of liver failure, with lactate production being a major consequence of this problem. In relation to this, the role of increased lactate accumulation and its pathogenetic basis in HE is discussed by Bosoi and Rose. Also, a major and often lethal complication of ALF is brain edema, and the significance of this edema and its underlying basis in ALF-induced HE is examined by Rama Rao et al, while the usefulness of magnetic resonance imaging as a tool in both acute and chronic liver failure is considered by Chavarria and Cordoba. Minimal hepatic encephalopathy (MHE) represents the mildest of the spectrum of HE forms in which patients do not display clinical symptoms but have mild cognitive and psychomotor deficits. It is also associated with poor survival rates. Two papers examine aspects of MHE, in which cognitive impairment and the issue of cognitive variability as a contributing factor in these patients is a focus of attention in Bisiacchi et al, and in an experimental model of chronic liver failure in which focal increases in the binding of translocator (18kD) protein in brain is reported by Agusti et al. Increasingly, the phenomenon of acute-on-chronic liver failure, in which acute deterioration of liver function in patients with cirrhosis is a consequence, is being considered a distinct clinical entity. The subject is discussed in Wright et al. In addition, considerable evidence has shown that astrocyte dysfunction plays a major role in HE. Karabara and colleagues explore the potential role of GlcNAcylation in these cells, while the protective effect of inhibition of glutamine synthesis is examined in neurological diseases, including HE, by Jeitner and Cooper. Development of well-defined biomarkers and improved therapeutic targets represents an important subject regarding HE. In this regard, the paper by Cooper and Kuhara shows that α-ketoglutaramate is a good marker of HE, and the paper by Mondal and Trigun demonstrates that pannexin-1 can be a marker as well as a target for HE. Although the mechanism(s) underlying the beneficial effects of rifaximin in HE remain largely unknown, the topic is tackled using multi-modal MRI in Ahluwalia et al. In addition, the subject of microglial proliferation and its significance in alcoholics with HE is explored experimentally in Dennis et al. Dienel and Cruz address the subject of decreased proteolysis as a result of hyperammonemia due to liver failure, and the effects of thioacetamide-induced ALF on citrulline uptake is examined in a study by Zielinska et al. Finally, Roger’s interest in the pathophysiology of TD has been long and enduring. A number of papers in this special issue deal with different aspects of TD. Afadlal et al shows that astrocytes play a significant role in TD-induced encephalopathy, while Bettendorff explores the role of thiamine triphosphate in cells. In addition, since mitochondrial dysfunction is an important aspect of both TD and Alzheimer’s disease in which thiamine-dependent enzyme activity is reduced, Huang et al explores how cellular calcium stores are affected in a fibroblast cell line, an important first step towards the eventual determination of how this process comes about in the AD patient and development of protective/treatment strategies targeting this mechanism, which may also have relevance for the future treatment of TD.

High expression of the aspartate–glutamate carrier Aralar1 favors lactate consumption in CHO cell culture

Process performance of mammalian cell cultures can be strongly impacted by high lactate accumulation, which can be a clone or media-dependent characteristic. In this study, the expression of specific genes was measured in several Chinese hamster ovary cell lines under culture conditions leading to different lactate profiles. A reduced expression of two genes was observed under conditions of high lactate accumulation: AGC1/Aralar1, a member of the malate–aspartate shuttle (MAS) and Timm8a1. Overexpression of either of these two genes in the lactate-producing cell line diminished lactate accumulation. This was achieved by promoting a metabolic switch to lactate consumption after day 6, while maintaining a glycolytic rate similar to the parental cells. On the other hand, the biochemical inhibition of MAS activity increased lactate accumulation. All together, these results indicate MAS as a key factor to promote a shift to lactate consumption in cultivated Chinese hamster ovary cells.

Abstract 2436: Acute Metformin Treatment Worsens Short-term Stroke Outcome: Comparison Of Underlying Mechanisms In Control And Type 2 Diabetic Rats

Background: Admission hyperglycemia aggravates neurovascular injury and worsens outcome in acute ischemic stroke (AIS). Current stroke guidelines recommend glycemic control as an important therapeutic strategy to prevent cerebrovascular complications. We have previously shown that chronic glucose control with metformin prior to AIS prevents cerebrovascular remodeling, reduces hemorrhagic transformation (HT), and improves neurological outcome. The goal of this study was to test the hypothesis that acute glucose control in diabetes prior to AIS will also provide neurovascular protection and improve functional outcome. Methods: Ten-week-old male control and type 2 diabetic Goto-Kakizaki (GK) rats (n=6-9 each group) were treated with or without metformin (300mg/kg/day in drinking water) for 3 days prior to stroke. Infarct size, edema ratio (% edema/infarct), and HT frequency and severity were evaluated in all groups after 3 hr middle cerebral artery occlusion (MCAO) and 21 hr reperfusion. AMPK activity and lactate levels--biomarkers of ischemic brain metabolism--were also measured. Results: Compared to controls, metformin treatment decreased blood glucose levels in diabetic animals (160.4±36.5 vs. 120.1±31.1 mg/dL, p<0.0001). Metformin treatment decreased edema ratio in GKs (0.90±0.71 vs. 1.55±0.70, p<0.05), but increased infarct size (23.3±17.5 vs. 12.5±9.5%, p<0.05). Untreated diabetic rats had lower composite scores for neurological tests (3.8±2.4 vs. 5.9±1.2, p<0.05), indicating worse outcome. Metformin treatment worsened this deficit in both diabetic and control animals (2.4±1.6 vs. 1.9±1.0, p=0.0008). AMPK activity was increased only in metformin-treated GK rats while lactate levels were increased in both groups (p<0.05). Results are expressed as mean ± SD. Conclusion: Worsening of stroke outcome by acute metformin treatment is associated with increased lactate accumulation in both control and diabetic rats. Increased infarct size in diabetic but not control rats may be due to greater AMPK activation in diabetes. Whether this effect is specific to metformin or general to acute glycemic control needs to be confirmed with other glucose control measures.

Second window of preconditioning normalizes palmitate use for oxidation and improves function during low-flow ischaemia

Although a major mechanism for cardioprotection is altered metabolism, little is known regarding metabolic changes in ischaemic preconditioning and subsequent ischaemia. Our objective was to examine the effects of the second window of preconditioning (SWOP), the delayed phase of preconditioning against infarction and stunning, on long-chain free fatty acid (LCFA) oxidation during ischaemia in chronically instrumented, conscious pigs. We studied three groups: (i) normal baseline perfusion (n = 5); (ii) coronary artery stenosis (CAS; n = 5); (iii) CAS 24 h following 2 × 10 min coronary occlusions and 10 min reperfusion (n = 7). Ischaemia was induced by a left anterior descending (LAD) stenosis (40% flow reduction) for 90 min, dropping systolic wall thickening by 72%. LCFA oxidation was assessed following LAD infusion of (13)C palmitate, i.e. during control or stenosis, by in vitro nuclear magnetic resonance of the sampled myocardium. Stenosis reduced subendocardial blood flow subendocardially, but not subepicardial, yet induced transmural reductions in LCFA oxidation and increased non-oxidative glycolysis. During stenosis, preconditioned hearts showed normalized contributions of LCFA to oxidative ATP synthesis, despite increased lactate accumulation. SWOP induced a shift towards LCFA oxidation during stenosis, despite increased malonyl-CoA, and marked protection of contractile function with a significant improvement in systolic wall thickening. Thus, the second window of preconditioning normalized oxidative metabolism of LCFA during subsequent ischaemia despite elevated non-oxidative glycolysis and malonyl-CoA and was linked to protection of regional contractile function resulting in improved mechanical performance. Interestingly, the metabolic responses occurred transmurally while ischaemia was restricted solely to the subendocardium.

Functional characteristics of apheresis‐derived platelets treated with ultraviolet light combined with either amotosalen‐HCl (S‐59) or riboflavin (vitamin B2) for pathogen‐reduction

To examine if different pathogen-reduction technologies (PRTs) induce different degrees of platelet (PLT) storage lesion. Twenty-seven split triple-dose apheresis PLTs were PRT treated using ultraviolet light with either riboflavin (M) or psoralen (I) or remained untreated (C). Samples taken on days (d) 0 to 8 were analysed for PLT count, blood gas (pH, pO(2) and pCO(2)), metabolism (lactate, glucose, ATP content), in vitro function [swirling, hypotonic shock response (HSR) and aggregation], activation (p-selectin expression) and cellular integrity (JC-1 signal, annexin A5 release). Platelet counts of all study groups remained unchanged during storage indicating that PRT treatment did not induce relevant cell lysis. Although M units demonstrated the highest values for HSR until d5, PRT treatment lowered all parameters examined with significant differences to untreated controls by d7 of storage. During final storage, M was significantly superior over I for HSR, aggregation with TRAP-6 as agonist (collagen was similar), annexin A5 release and JC-1 signal. Regarding blood gas and metabolic analysis, the most evident effect of PRT was an elevated glycolytic flux combined with higher acidity due to increased lactate accumulation. Most likely due to impaired O(2) consumption, pH and ATP decreased more rapidly in I relative to C and M. Pathogen reduction technology-treated PLTs remained comparable to untreated units throughout 7 days of storage. Mitochondria-based oxidative respiration appeared up-regulated after the riboflavin-based PRT. Compared to the psoralen-based PRT, this resulted in significantly better ATP maintenance and in vitro function during the last storage period (d7, d8).

Effect of various prostaglandins on the metabolism of fructose by epididymal and ejaculated ram spermatozoa in vitro.

The effects of a number of prostaglandins on the metabolism of fructose by epididymal and ejaculated ram spermatozoa were investigated using conventional Warburg techniques. Spermatozoa from the two sources were collected concurrently during electrical stimulation; the epididymal spermatozoa were harvested via a cannula inserted chronically into one vas deferens and thus were free from exposure to the seminal prostaglandins. In general, the metabolism of fructose by the spermatozoa was little affected by a wide range of prostaglandins at a concentration of 20 micrograms/ml. Of the PGs present in semen, PGE2, PGE3 and PGF2alpha had no significant effects whereas PGE1 and PGF1alpha significantly increased lactate accumulation and the latter increased oxygen uptake, in both cases without significantly changing fructose utilization, fructose oxidized or CO2 production. Both 15-methyl-substituted PGE2 and PGF2alpha and their corresponding methyl esters failed to change fructose metabolism and it is unlikely therefore that the lack of response to PGE2 and PGF2alpha was due to their being metabolized by the spermatozoa during the incubation. Of a number of breakdown products tested, PGA1 and to a lesser extent PGA2 appeared to inhibit the Krebs tricarbocyclic acid cycle and 15-keto 13,14-dihydro-PGF2alpha appeared to stimulate it. In general, epididymal spermatozoa responded to the PGs in the same way as ejaculated spermatozoa. Thus we did not confirm a suggestion in the literature that spermatozoa have lowered sensitivity to PGs in vitro once they have been in contact with the PGs in seminal plasma.

Functional Characterization of Muscle Fibres from Patients with Chronic Fatigue Syndrome: Case-Control Study

Chronic fatigue syndrome (CFS) is a disabling condition characterized by unexplained chronic fatigue that impairs normal activities. Although immunological and psychological aspects are present, symptoms related to skeletal muscles, such as muscle soreness, fatigability and increased lactate accumulation, are prominent in CFS patients. In this case-control study, the phenotype of the same biopsy samples was analyzed by determining i) fibre-type proportion using myosin isoforms as fibre type molecular marker and gel electrophoresis as a tool to separate and quantify myosin isoforms, and ii) contractile properties of manually dissected, chemically made permeable and calcium-activated single muscle fibres. The results showed that fibre-type proportion was significantly altered in CSF samples, which showed a shift from the slow- to the fast-twitch phenotype. Cross sectional area, force, maximum shortening velocity and calcium sensitivity were not significantly changed in single muscle fibres from CSF samples. Thus, the contractile properties of muscle fibres were preserved but their proportion was changed, with an increase in the more fatigue-prone, energetically expensive fast fibre type. Taken together, these results support the view that muscle tissue is directly involved in the pathogenesis of CSF and it might contribute to the early onset of fatigue typical of the skeletal muscles of CFS patients.

Modelling Dysregulated Na+ Absorption in Airway Epithelial Cells with Mucosal Nystatin Treatment

In cystic fibrosis (CF), the absence of functional CFTR leads to dysregulated Na(+) absorption across airway epithelia. We established an in vitro model of dysregulated Na(+) absorption by treating polarized normal human bronchial epithelial cells (HBEs) with nystatin (Nys), a polyene antibiotic that enables monovalent cations to permeate biological membranes. Acute mucosal Nys produced a rapid increase in short circuit current (I(sc)) that reflected increased transepithelial Na(+) absorption and required Na(+)/K(+)ATPase activity. The acute increase in I(sc) was associated with increased mucosal liquid absorption. Prolonged mucosal Nys treatment resulted in sustained Na(+) hyperabsorption, associated with increased mucosal liquid absorption in comparison with naïve (nontreated, kept under air-liquid interface conditions) or vehicle-treated cultures. Nys treatment was not toxic. Increased lactate accumulation in Nys-treated culture media suggested a higher metabolic rate associated with the higher energy demand for Na(+) transport. After chronic Nys treatment, the increased I(sc) was rapidly lost when the cultures were mounted in Ussing chambers, indicating that Nys could be rapidly removed from the apical membrane. Importantly, chronic Nys treatment promoted sustained mucosal liquid depletion and caused mucus dehydration, compaction, and adhesion to the apical surface of Nys-treated cultures. We conclude that mucosal Nys treatment of HBEs provides a simple in vitro model to recapitulate the Na(+) and volume hyperabsorptive features of CF airway epithelia.

Ammonia effects on pyruvate/lactate production in astrocytes—Interaction with glutamate

Ammonia exerts a multitude of metabolic and non-metabolic effects on brain tissue. In the present communication we have investigated its effect on lactate production rates, pyruvate production rates and pyruvate/lactate ratios in mouse cerebrocortical astrocytes and neurons in primary cultures. No effects were found in neurons. All three parameters were affected by ammonia in astrocytes, but less potently and to a smaller degree in cells that had been treated with dibutyryl cyclic AMP (morphologically differentiated cells) than in untreated cells (morphologically undifferentiated cells). In the differentiated cells ammonia had virtually no effect up to a concentration of 1.0 mM, but at 3.0 mM it increased lactate production and decreased pyruvate/lactate ratio significantly. In the undifferentiated cells ammonia greatly increased lactate accumulation (by 80% at 3.0 mM) and it inhibited pyruvate accumulation (by 40% at 3.0 mM). It thereby reduced the pyruvate/lactate ratio progressively within the entire range 0.1–3.0 mM ammonia. In support of the hypothesis that the ammonia-induced reduction of pyruvate/lactate ratio is secondary to depletion of cellular glutamate by formation of glutamine (and glutathione) and a resulting interruption of the malate–aspartate shuttle (MAS), the addition of glutamate to the incubation medium significantly diminished the ammonia-induced reduction of pyruvate/lactate ratio, whereas it had no effect on the increased lactate production. It is discussed that MAS interruption may have additional consequences in astrocytes.