Lactate Release Research Articles

PLX4032 resistance of patient-derived melanoma cells: crucial role of oxidative metabolism.

Malignant melanoma is the most lethal form of skin cancer which shows BRAF mutation in 50% of patients. In this context, the identification of BRAFV600E mutation led to the development of specific inhibitors like PLX4032. Nevertheless, although its initial success, its clinical efficacy is reduced after six-months of therapy leading to cancer relapse due to the onset of drug resistance. Therefore, investigating the mechanisms underlying PLX4032 resistance is fundamental to improve therapy efficacy. In this context, several models of PLX4032 resistance have been developed, but the discrepancy between in vitro and in vivo results often limits their clinical translation. The herein reported model has been realized by treating with PLX4032, for six months, patient-derived BRAF-mutated melanoma cells in order to obtain a reliable model of acquired PLX4032 resistance that could be predictive of patient's treatment responses. Metabolic analyses were performed by evaluating glucose consumption, ATP synthesis, oxygen consumption rate, P/O ratio, ATP/AMP ratio, lactate release, lactate dehydrogenase activity, NAD+/NADH ratio and pyruvate dehydrogenase activity in parental and drug resistant melanoma cells. The intracellular oxidative state was analyzed in terms of reactive oxygen species production, glutathione levels and NADPH/NADP+ ratio. In addition, a principal component analysis was conducted in order to identify the variables responsible for the acquisition of targeted therapy resistance. Collectively, our results demonstrate, for the first time in patient-derived melanoma cells, that the rewiring of oxidative phosphorylation and the maintenance of pyruvate dehydrogenase activity and of high glutathione levels contribute to trigger the onset of PLX4032 resistance. Therefore, it is possible to hypothesize that inhibitors of glutathione biosynthesis and/or pyruvate dehydrogenase activity could be used in combination with PLX4032 to overcome drug resistance of BRAF-mutated melanoma patients. However, the identification of new adjuvant targets related to drug-induced metabolic reprogramming could be crucial to counteract the failure of targeted therapy in metastatic melanoma.

Lactate as Key Metabolite in Prostate Cancer Progression: What Are the Clinical Implications?

Advanced prostate cancer represents the fifth leading cause of cancer death in men worldwide. Although androgen-receptor signaling is the major driver of the disease, evidence is accumulating that disease progression is supported by substantial metabolic changes. Alterations in de novo lipogenesis and fatty acid catabolism are consistently reported during prostate cancer development and progression in association with androgen-receptor signaling. Therefore, the term "lipogenic phenotype" is frequently used to describe the complex metabolic rewiring that occurs in prostate cancer. However, a new scenario has emerged in which lactate may play a major role. Alterations in oncogenes/tumor suppressors, androgen signaling, hypoxic conditions, and cells in the tumor microenvironment can promote aerobic glycolysis in prostate cancer cells and the release of lactate in the tumor microenvironment, favoring immune evasion and metastasis. As prostate cancer is composed of metabolically heterogenous cells, glycolytic prostate cancer cells or cancer-associated fibroblasts can also secrete lactate and create "symbiotic" interactions with oxidative prostate cancer cells via lactate shuttling to sustain disease progression. Here, we discuss the multifaceted role of lactate in prostate cancer progression, taking into account the influence of the systemic metabolic and gut microbiota. We call special attention to the clinical opportunities of imaging lactate accumulation for patient stratification and targeting lactate metabolism.

Metabolic Responses of Normal Rat Kidneys to a High Salt Intake.

In this study, novel methods were developed, which allowed continuous (24/7) measurement of arterial blood pressure and renal blood flow in freely moving rats and the intermittent collection of arterial and renal venous blood to estimate kidney metabolic fluxes of O2 and metabolites. Specifically, the study determined the effects of a high salt (HS; 4.0% NaCl) diet upon whole kidney O2 consumption and arterial and renal venous plasma metabolomic profiles of normal Sprague-Dawley rats. A separate group of rats was studied to determine changes in the cortex and outer medulla tissue metabolomic and mRNAseq profiles before and following the switch from a 0.4% to 4.0% NaCl diet. In addition, targeted mRNA expression analysis of cortical segments was performed. Significant changes in the metabolomic and transcriptomic profiles occurred with feeding of the HS diet. A progressive increase of kidney O2 consumption was found despite a reduction in expression of most of the mRNA encoding enzymes of TCA cycle. A novel finding was the increased expression of glycolysis-related genes in Cx and isolated proximal tubular segments in response to an HS diet, consistent with increased release of pyruvate and lactate from the kidney to the renal venous blood. Data suggests that aerobic glycolysis (eg, Warburg effect) may contribute to energy production under these circumstances. The study provides evidence that kidney metabolism responds to an HS diet enabling enhanced energy production while protecting from oxidative stress and injury. Metabolomic and transcriptomic analysis of kidneys of Sprague-Dawley rats fed a high salt diet.

Sf9 Cell Metabolism Throughout the Recombinant Baculovirus and Rabies Virus-Like Particles Production in Two Culture Systems.

This work aimed to assess the Sf9 cell metabolism during growth, and infection steps with recombinant baculovirus bearing rabies virus proteins, to finally obtain rabies VLP in two culture systems: Schott flask (SF) and stirred tank reactor (STR). Eight assays were performed in SF and STR (four assays in each system) using serum-free SF900 III culture medium. Two non-infection growth kinetics assays and six recombinant baculovirus infection assays. The infection runs were carried out at 0.1 pfu/cell multiplicity of infection (MOI) for single baculovirus bearing rabies glycoprotein (BVG) and matrix protein (BVM) and a coinfection with both baculoviruses at MOI of 3 and 2 pfu/cell for BVG and BVM, respectively. The SF assays were done in triplicate. The glucose, glutamine, glutamate, lactate, and ammonium uptake or release specific rates were quantified over the exponential growth phase and infection stage. The highest uptake specific rate was observed for glucose (42.5 × 10-12mmol cell/h) in SF and for glutamine (30.8 × 10-12mmol/cell/h) in STR, in the exponential growth phases. A wave pattern was observed for assessed analytes throughout the infection phase and the glucose had the highest wave amplitude within the 10-10mmol cell/h order. This alternative uptake and release behavior is in harmony with the lytic cycle of baculovirus in insect cells. The virus propagation and VLP generation were not limited by glucose, glutamine, and glutamate, neither by the toxicity of lactate nor ammonium under the conditions appraised in this work. The findings from this work can be useful to set baculovirus infection processes at high cell density to improve rabies VLP yield, purity, and productivity.

KATP channels are necessary for glucose-dependent increases in amyloid-β and Alzheimer's disease-related pathology.

Elevated blood glucose levels, or hyperglycemia, can increase brain excitability and amyloid-β (Aβ) release, offering a mechanistic link between type 2 diabetes and Alzheimer's disease (AD). Since the cellular mechanisms governing this relationship are poorly understood, we explored whether ATP-sensitive potassium (KATP) channels, which couple changes in energy availability with cellular excitability, play a role in AD pathogenesis. First, we demonstrate that KATP channel subunits Kir6.2/KCNJ11 and SUR1/ABCC8 were expressed on excitatory and inhibitory neurons in the human brain, and cortical expression of KCNJ11 and ABCC8 changed with AD pathology in humans and mice. Next, we explored whether eliminating neuronal KATP channel activity uncoupled the relationship between metabolism, excitability, and Aβ pathology in a potentially novel mouse model of cerebral amyloidosis and neuronal KATP channel ablation (i.e., amyloid precursor protein [APP]/PS1 Kir6.2-/- mouse). Using both acute and chronic paradigms, we demonstrate that Kir6.2-KATP channels are metabolic sensors that regulate hyperglycemia-dependent increases in interstitial fluid levels of Aβ, amyloidogenic processing of APP, and amyloid plaque formation, which may be dependent on lactate release. These studies identify a potentially new role for Kir6.2-KATP channels in AD and suggest that pharmacological manipulation of Kir6.2-KATP channels holds therapeutic promise in reducing Aβ pathology in patients with diabetes or prediabetes.

The effects of excess salt intake on the kidney metabolism in normal Sprague Dawley rats.

Introduction: This study aimed to determine the effects of a high salt diet upon the metabolism of O 2 and substances in the kidney in normal Sprague Dawley (SD) rats. Methods were developed enabling the intermittent collection of renal arteriovenous (A-Vr) blood and urine samples and to measure renal blood flow (RBF) and arterial pressure (BP) in conscious freely moving rats with GFR determined in a separate group of conscious rats. Other groups were studied to obtain cortical (Cx) and outer medullary (OM) tissue samples for associated transcriptomic and metabolomic profiles. Methods: SD rats were instrumented with a renal arterial ultrasonic flow probe, a femoral arterial and renal venous catheters, and RBF and BP measured continuously (24/7). Arterial (A) and renal venous (Vr) blood and urine were collected repeatedly before and 7, 14, and 21 days after increasing the salt diet from 0.4% (LS) to 4% (HS) NaCl. GFR was measured in age-matched rats and used as control shams for normalization. Blood O 2 content was determined by radiometry. A global metabolomic analysis was performed on plasma and Cx and OM tissue samples at JAX laboratories (Thermo Q-Exactive Orbitrap coupled to Vanquich UPLC system) and together with mRNAseq analysis for Cx and OM by Novogene Inc. Results: BP increased less than 5 mmHg by day 3 of the HS diet while RBF rose nearly 20% (P<0.05) and both were sustained at this level throughout the study. O 2 delivery, O 2 consumption (VO 2 ) and O 2 extraction rose over the 21 days of the HS diet (P<0.05). Total GFR increased from 0.64 to 0.83 mL/min/100 g bw (P<0.05) at HS 7 and remained elevated at HS 21. Whole kidney Na + reabsorption and kidney VO 2 were highly correlated positively (r=0.99). A sparse partial least squares discriminant analysis (sPLS-DA) of metabolomics for each of the 4 modes (C18 +/- and HILIC +/-) revealed clear distinctions between LS, HS14, and HS21 within Cx and OM. The mRNAseq tissue analysis found that the HS diet resulted in reduced expression of genes related to the TCA cycle and metabolism of amino acids and fatty acids. This was consistent with the metabolomic data showing a reduction of tissue citrate and succinate levels. In contrast, glycolysis-related genes including hexokinase, pyruvate kinase and lactate dehydrogenase were upregulated in the Cx tissue by HS. Minimal changes in metabolic pathways were observed in the OM tissue. A-Vr x RBF enabled metabolomic flux determination which showed the release of glucose on LS fed rats which was not evident with the HS diet. Uptake of citrate which was seen on LS was less evident on HS but there was a clear tendency observed for increased release of lactate, pyruvate, and α-ketoglutarate. Conclusions: Normal SD rats fed a HS underwent changes of RBF, O 2 extraction and utilization of metabolic substrates, most notably transitioning ATP production predominantly driven from TCA cycle to non-TCA cycle pathways which we are currently exploring more deeply. HL-116264, HL-151587 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.

Immediate-sustained lactate release using alginate hydrogel assembled to proteinase K/polymer electrospun fibers

This work proposes a microfibers-hydrogel assembled composite as delivery vehicle able to combine into a single system both burst and prolonged release of lactate. The prolonged release of lactate has been achieved by electrospinning a mixture of polylactic acid and proteinase K (26.0 mg of proteinase K and 0.99 g of PLA dissolved in 6 mL of 2:1 chloroform:acetone in the optimal case), which is a protease that catalyzes the degradation of polylactic acid into lactate. The degradation of microfibers into lactate reflects that proteinase K preserves its enzymatic activity even after the electrospinning process because of the mild operational conditions used. Besides, burst release is obtained from the lactate-loaded alginate hydrogel. The successful assembly between the lactate-loaded hydrogel and the polylactic acid/proteinase K fibers has been favored by applying a low-pressure (0.3 mbar at 300 W) oxygen plasma treatment, which transforms hydrophobic fibers into hydrophilic while the enzymatic activity is still maintained. The composite displays both fast (< 24 h) and sustained (> 10 days) lactate release, and allows the modulation of the release by adjusting either the amount of loaded lactate or the amount of active enzyme.

Cross-talk between energy and redox metabolism in astrocyte-neuron functional cooperation

Astrocytes show unique anatomical, morphological, and metabolic features to take up substrates from the blood and metabolize them for local delivery to active synapses to sustain neuron function. In the present review, we specifically focus on key molecular aspects of energy and redox metabolism that facilitate this astrocyte-neuronal coupling in a controlled manner. Basal glycolysis is co-ordinated by the anaphase-promoting complex/cyclosome (APC/C)-Cdh1, a ubiquitin ligase that targets the proglycolytic enzyme 6-phosphofructokinase-2,6-bisphosphastate-3 (PFKFB3) for degradation. APC/C-Cdh1 activity is more robust in neurons than in astrocytes, which determine that PFKFB3 abundance and glycolytic rate are weaker in neurons. The low PFKFB3 activity in neurons facilitates glucose-6-phosphate oxidation via the pentose-phosphate pathway, which promotes antioxidant protection. Conversely, the high PFKFB3 activity in astrocytes allows the production and release of glycolytic lactate, which is taken up by neurons that use it as an oxidizable substrate. Importantly, the mitochondrial respiratory chain is tighter assembled in neurons than in astrocytes, thus the bioenergetic efficiency of mitochondria is higher in neurons. Because of this, the production of reactive oxygen species (mROS) by mitochondrial complex I is very low in neurons and very high in astrocytes. Such a naturally occurring high abundance of mROS in astrocytes physiologically determines a specific transcriptional fingerprint that contributes to sustaining cognitive performance. We conclude that the energy and redox metabolism of astrocytes must complementarily match that of neurons to regulate brain function and animal welfare.

Melatonin modulates the Warburg effect and alters the morphology of hepatocellular carcinoma cell line resulting in reduced viability and migratory potential

AimsHepatocellular Carcinoma (HCC) is a primary neoplasm derived from hepatocytes with low responsiveness and recurrent chemoresistance. Melatonin is an alternative agent that may be helpful in treating HCC. We aimed to study in HuH 7.5 cells whether melatonin treatment exerts antitumor effects and, if so, what cellular responses are induced and involved. Main methodsWe evaluated the effects of melatonin on cell cytotoxicity and proliferation, colony formation, morphological and immunohistochemical aspects, and on glucose consumption and lactate release. Key findingsMelatonin reduced cell motility and caused lamellar breakdown, membrane damage, and reduction in microvillus. Immunofluorescence analysis revealed that melatonin reduced TGF and N-cadherin expression, which was further associated with inhibition of epithelial-mesenchymal transition process. In relation to the Warburg-type metabolism, melatonin reduced glucose uptake and lactate production by modulating intracellular lactate dehydrogenase activity. SignificanceOur results indicate that melatonin can act upon pyruvate/lactate metabolism, preventing the Warburg effect, which may reflect in the cell architecture. We demonstrated the direct cytotoxic and antiproliferative effect of melatonin on the HuH 7.5 cell line, and suggest that melatonin is a promising candidate to be further tested as an adjuvant to antitumor drugs for HCC treatment.

Hypoxia enhances IPF mesenchymal progenitor cell fibrogenicity via the lactate/GPR81/HIF1α pathway.

Hypoxia is a sentinel feature of idiopathic pulmonary fibrosis (IPF). The IPF microenvironment contains high lactate levels, and hypoxia enhances cellular lactate production. Lactate, acting through the GPR81 lactate receptor, serves as a signal molecule regulating cellular processes. We previously identified intrinsically fibrogenic mesenchymal progenitor cells (MPCs) that drive fibrosis in the lungs of patients with IPF. However, whether hypoxia enhances IPF MPC fibrogenicity is unclear. We hypothesized that hypoxia increases IPF MPC fibrogenicity via lactate and its cognate receptor GPR81. Here we show that hypoxia promotes IPF MPC self-renewal. The mechanism involves hypoxia-mediated enhancement of LDHA function and lactate production and release. Hypoxia also increases HIF1α levels, and this increase in turn augments the expression of GPR81. Exogenous lactate operating through GPR81 promotes IPF MPC self-renewal. IHC analysis of IPF lung tissue demonstrates IPF MPCs expressing GPR81 and hypoxic markers on the periphery of the fibroblastic focus. We show that hypoxia enhances IPF MPC fibrogenicity in vivo. We demonstrate that knockdown of GPR81 inhibits hypoxia-induced IPF MPC self-renewal in vitro and attenuates hypoxia-induced IPF MPC fibrogenicity in vivo. Our data demonstrate that hypoxia creates a feed-forward loop that augments IPF MPC fibrogenicity via the lactate/GPR81/HIF1α pathway.

Irisin modulates glucose metabolism and inhibits steroidogenesis in bovine granulosa cells.

Fertility in the dairy cow is low during the post-partum period of negative energy balance and high plasma irisin concentrations. This study shows irisin modulates granulosa cell glucose metabolism and impairs steroidogenesis. Fibronectin type III domain-containing 5 (FNDC5) is a transmembrane protein discovered in 2012 that is cleaved to release the adipokine-myokine, irisin. Originally described as an exercise hormone that browns white adipose tissue and increases glucose metabolism, irisin secretion also increases during periods of rapid adipose mobilization, such as the post-partum period in dairy cattle when ovarian activity is suppressed. The effect of irisin on follicle function is unclear and may be species-dependent. In this study, we hypothesized that irisin may compromise granulosa cell function in cattle using a well-established in vitro cell culture model. We detected FNDC5 mRNA and both FNDC5 and cleaved irisin proteins in follicle tissue and in follicular fluid. The abundance of FNDC5 mRNA was increased by the treatment of cells with the adipokine visfatin but not by other adipokines tested. The addition of recombinant irisin to granulosa cells decreased basal and insulin-like growth factor 1- and follicle-stimulating hormone-dependent estradiol and progesterone secretion and increased cell proliferation but had no effect on viability. Irisin decreased GLUT1, GLUT3, and GLUT4 mRNA levels in granulosa cells and increased lactate release in the culture medium. The mechanism of action is in part through MAPK3/1 but not Akt, MAPK14, or PRKAA. We conclude that irisin may regulate bovine folliculogenesis by modulating granulosa cell steroidogenesis and glucose metabolism.

Beneficial Effects of Rosmarinic Acid In Vitro and In Vivo Models of Epileptiform Activity Induced by Pilocarpine

Epilepsy is characterized by a predisposition to generate recurrent and spontaneous seizures; it affects millions of people worldwide. Status epilepticus (SE) is a severe type of seizure. In this context, screening potential treatments is very important. In the present study, we evaluated the beneficial effects of rosmarinic acid (RA) in pilocarpine-induced in vitro and in vivo models of epileptiform activity. Using an in vitro model in combined entorhinal cortex-hippocampal from Wistar rats we evaluated the effects of RA (10 µg/mL) on the lactate release and a glucose fluorescent analogue, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NDBG), after incubation in high potassium aCSF supplemented or not with pilocarpine. In the in vivo model, SE was induced in male C57BL/6 mice by pilocarpine. At 1, 24, and 48 h after the end of SE mice were treated with RA (30 mg/kg/v.o.). We evaluated the neuromotor impairment by neuroscore tests and protein carbonyl levels in the cerebral cortex. In both in vitro models, RA was able to decrease the stimulated lactate release, while no effect on 2-NBDG uptake was found. RA has beneficial effects in models of epileptiform activity in vivo and in vitro. We found that RA treatment attenuated SE-induced neuromotor impairment at the 48 h timepoint. Moreover, post-SE treatment with RA decreased levels of protein carbonyls in the cerebral cortex of mice when compared to their vehicle-treated counterparts. Importantly, RA was effective in a model of SE which is relevant for the human condition. The present data add to the literature on the biological effects of RA, which could be a good candidate for add-on therapy in epilepsy.

Pancreatic stellate cell-induced gemcitabine resistance in pancreatic cancer is associated with LDHA- and MCT4-mediated enhanced glycolysis

BackgroundProfound resistance to chemotherapy remains a major challenge in achieving better clinical outcomes for patients with pancreatic ductal adenocarcinoma (PDAC). Recent studies indicate that gemcitabine (GEM) resistance is promoted both by pancreatic stellate cells (PSCs) and through increased glycolysis. However, it remains unknown whether PSCs affect GEM sensitivity via glycolytic regulation.MethodsHuman pancreatic cancer cell (PCC) lines (BxPC-3, Capan-2, HPAF-II, Mia PaCa-2, Panc-1, SW-1990) were exposed to three different PSC-conditioned media (PSC-CM; PSC-1, PSC-2, HPaSteC), following either pre-treatment with glycolysis inhibitor NV-5440 or transfection for transient silencing of key glycolytic regulators (LDHA and MCT4). Proliferation, glucose transport, extracellular lactate, and GEM sensitivity were assessed. Protein expression was determined by Western blot and immunostaining. Moreover, secreted proteins in PSC-CMs were profiled by mass spectrometry (MS).ResultsWhile exposure to PSC-CMs did not affect glucose transport in PCCs, it increased their lactate release and proliferation, and reduced the sensitivity for GEM. Both NV-5440 treatment and transient silencing of LDHA and MCT4 inhibited these PSC-induced changes in PCCs. MS analysis identified 688 unique proteins with differential expression, of which only 87 were common to the three PSC-CMs. Most PSC-secreted proteins were extracellular matrix-related, including SPARC, fibronectin, and collagens. Moreover, exposure to PSC-CMs increased the phosphorylation of ERK in PCCs, but the treatment of PCCs with the MEK/ERK inhibitor PD98059 resulted in a reduction of PSC-CM-induced glycolysis and improved GEM sensitivity.ConclusionsThe study findings suggest that PSC-secreted factors promote both glycolysis and GEM resistance in PCCs, and that glycolysis inhibition by NV-5440 and blocking of ERK phosphorylation by PD98059 protect PCCs from PSC-CM-induced loss of GEM sensitivity. Taken together, PSCs appear to promote GEM resistance in PDAC via glycolysis. Thus, targeting glycolysis may improve the effect of chemotherapy in PDAC.

ACE2 negatively regulates the Warburg effect and suppresses hepatocellular carcinoma progression via reducing ROS-HIF1α activity.

Aerobic glycolysis has pleiotropic roles in the pathogenesis of hepatocellular carcinoma (HCC). Emerging studies revealed key promoters of aerobic glycolysis, however, little is known about its negative regulators in HCC. In this study, an integrative analysis identifies a repertoire of differentially expressed genes (DNASE1L3, SLC22A1, ACE2, CES3, CCL14, GYS2, ADH4, and CFHR3) that are inversely associated with the glycolytic phenotype in HCC. ACE2, a member of the rennin-angiotensin system, is revealed to be downregulated in HCC and predicts a poor prognosis. ACE2 overexpression significantly inhibits the glycolytic flux as evidenced by reduced glucose uptake, lactate release, extracellular acidification rate, and the expression of glycolytic genes. Opposite results are noticed in loss-of-function studies. Mechanistically, ACE2 metabolizes Ang II to Ang-(1-7), which activates Mas receptor and leads to the phosphorylation of Src homology 2-containing inositol phosphatase 2 (SHP-2). SHP2 activation further blocks reactive oxygen species (ROS)-HIF1α signaling. Addition of Ang-(1-7) or the antioxidant N-acetylcysteine compromises in vivo additive tumor growth and aerobic glycolysis induced by ACE2 knockdown. Moreover, growth advantages afforded by ACE2 knockdown are largely glycolysis-dependent. In clinical settings, a close link between ACE2 expression and HIF1α or the phosphorated level of SHP2 is found. Overexpression of ACE2 significantly retards tumor growth in patient-derived xenograft model. Collectively, our findings suggest that ACE2 is a negative glycolytic regulator, and targeting the ACE2/Ang-(1-7)/Mas receptor/ROS/HIF1α axis may be a promising therapeutic strategy for HCC treatment.

Measuring the Release of Lactate from Wild-Type and rd1 Mouse Retina.

The retina has the highest energy consumption of any tissue in the human body. Remarkably, to satisfy its energy demand, the retina appears to rely mostly on aerobic glycolysis, which results in the production and release of large amounts of lactate. In the present study, we compared two different methods to assess lactate release from in vitro organotypic retinal explants cultured under entirely controlled, serum-free conditions. We used a standard lactate assay kit and 1H-nuclear magnetic resonance(NMR) spectroscopy-based analysis. We found that during the culturing of retinal explants derived from wild-type mice, lactate was released in large amounts and that the two different methods agreed well with each other. When comparing wild-type retina with degenerating rd1 mouse retina, we found the latter to release significantly higher amounts of lactate. Hence, degenerating retina may have an even higher energy demand and metabolic rate compared to healthy retina. We conclude that the use of lactate measurement can be a reliable and simple readout to evaluate ongoing retinal metabolism.

Serotonergic neurons control cortical neuronal intracellular energy dynamics by modulating astrocyte-neuron lactate shuttle

The central serotonergic system has multiple roles in animal physiology and behavior, including sleep-wake control. However, its function in controlling brain energy metabolism according to the state of animals remains undetermined. Through invivo monitoring of energy metabolites and signaling, we demonstrated that optogenetic activation of raphe serotonergic neurons increased cortical neuronal intracellular concentration of ATP, an indispensable cellular energy molecule, which was suppressed by inhibiting neuronal uptake of lactate derived from astrocytes. Raphe serotonergic neuronal activation induced cortical astrocytic Ca2+ and cAMP surges and increased extracellular lactate concentrations, suggesting the facilitation of lactate release from astrocytes. Furthermore, chemogenetic inhibition of raphe serotonergic neurons partly attenuated the increase in cortical neuronal intracellular ATP levels as arousal increased in mice. Serotonergic neuronal activation promoted an increase in cortical neuronal intracellular ATP levels, partly mediated by the facilitation of the astrocyte-neuron lactate shuttle, contributing to state-dependent optimization of neuronal intracellular energy levels.

Ubiquitin-like protein MNSFβ regulates glycolysis and promotes cell proliferation with HSC70 assistance

Monoclonal non-specific suppressor factor β (MNSFβ) is a universally expressed ubiquitin-like protein that has multiple biological functions. MNSFβ modifies its target molecules through covalent conjugation. Most recently, we identified a molecular chaperone, HSC70, that facilitates the stabilization of aggregable MNSFβ. In the current study, we determined the role of HSC70 in stabilizing unstable MNSFβ. HSC70 promoted the correct folding of MNSFβ both in vitro and in vivo. We also examined the regulatory function of MNSFβ in cell proliferation and glycolysis. MNSFβ siRNA and HSC70 siRNA treatment attenuated lactate release from Raw264.7 macrophage-like cells. MNSFβ siRNA inhibited glucose uptake in Raw264.7 cells. We found that glucose transporter 1 (GLUT1) is an important membrane protein involved in the regulatory function of MNSFβ during glycolysis. MNSFβ siRNA inhibited the increased GLUT1 expression in LPS-stimulated cells, suggesting that MNSFβ controls the inflammatory response through GLUT1 regulation. We identified several important molecules, including lactate dehydrogenase A, which are regulated by MNSFβ and involved in glucose metabolism. Here we firstly report that MNSFβ regulates glycolysis and promotes cell proliferation.

EPO has multiple positive effects on astrocytes in an experimental model of ischemia

Erythropoietin (EPO) has neuroprotective effects in central nervous system injury models. In clinical trials EPO has shown beneficial effects in traumatic brain injury (TBI) as well as in ischemic stroke. We have previously shown that EPO has short-term effects on astrocyte glutamatergic signaling in vitro and that administration of EPO after experimental TBI decreases early cytotoxic brain edema and preserves structural and functional properties of the blood-brain barrier. These effects have been attributed to preserved or restored astrocyte function. Here we explored the effects of EPO on astrocytes undergoing oxygen-glucose-deprivation, an in vitro model of ischemia. Measurements of glutamate uptake, intracellular pH, intrinsic NADH fluorescence, Na,K-ATPase activity, and lactate release were performed. We found that EPO within minutes caused a Na,K-ATPase-dependent increase in glutamate uptake, restored intracellular acidification caused by glutamate and increased lactate release. The effects on intracellular pH were dependent on the sodium/hydrogen exchanger NHE. In neuron-astrocyte co-cultures, EPO increased NADH production both in astrocytes and neurons, however the increase was greater in astrocytes. We suggest that EPO preserves astrocyte function under ischemic conditions and thus may contribute to neuroprotection in ischemic stroke and brain ischemia secondary to TBI.

Role of Hypoxia-inducible factor 1α in host defense during pneumococcal pneumonia.

Hypoxia-inducible factor (HIF)1α is a transcription factor involved in cellular metabolism and regulation of immune cell effector functions. Here, we studied the role of HIF1α in myeloid cells during pneumonia caused by the major causative pathogen, Streptococcus pneumoniae (Spneu). Mice deficient for HIF1α in myeloid cells (LysMcreHif1αfl/fl) were generated to study the in vitro responsiveness of bone marrow-derived macrophages (BMDMs) and alveolar macrophages (AMs) to the Gram-positive bacterial wall component lipoteichoic acid (LTA) and heat-killed Spneu, and the in vivo host response after infection with Spneu via the airways. Both BMDMs and AMs released more lactate upon stimulation with LTA or Spneu, indicative of enhanced glycolysis; HIF1α-deficiency in these cells was associated with diminished lactate release. In BMDMs, HIF1α-deficiency resulted in reduced secretion of tumor necrosis factor (TNF)α and interleukin (IL)-6 upon activation with Spneu but not LTA, while HIF1α-deficient AMs secreted less TNFα and IL-6 in response to LTA, and TNFα after Spneu stimulation. However, no difference was found in the host response of LysMcreHif1αfl/fl mice after Spneu infection as compared to controls. Similar in vivo findings were obtained in neutrophil (Mrp8creHif1αfl/fl) HIF1α-deficient mice. These data suggest that myeloid HIF1α is dispensable for the host defense during pneumococcal pneumonia.

Lactate: A pearl dropped in the ocean: An overlooked signal molecule in physiology and pathology.

Lactate, once recognized as a wasty product from anaerobic glycolysis, is proved to be a pivotal signal molecule. Lactate accumulation occurs in diverse physiological and pathological settings due to the imbalance between lactate production and clearance. Under the condition with drastic changes in local microenvironment, such as tumorigenesis, inflammation, and microbial infection, the glycolysis turns to be active in surrounding cells leading to increased lactate release. Meanwhile, lactate can be utilized by these cells as an energy substrate and acts as a signal molecule to regulate cell functions through receptor-dependent or independent pathways. In this review, we tended to tease out the contribution of lactate in tumor progression and immunomodulation. And we also discussed the accessory role of lactate, beyond as the energy source only, in the growth of invading pathogens.