Glucose competition between endothelial cells in the blood-spinal cord barrier and infiltrating regulatory T cells is linked to sleep restriction-induced hyperalgesia.

BACKGROUND: Sleep loss is a common public health problem that causes hyperalgesia, especially that after surgery, which reduces the quality of life seriously. METHODS: The 48-h sleep restriction (SR) mouse model was created using restriction chambers. In vivo imaging, transmission electron microscopy (TEM), immunofluorescence staining and Western blot were performed to detect the status of the blood-spinal cord barrier (BSCB). Paw withdrawal mechanical threshold (PWMT) was measured to track mouse pain behavior. The role of infiltrating regulatory T cells (Tregs) and endothelial cells (ECs) in mouse glycolysis and BSCB damage were analyzed using flow cytometry, Western blot, CCK-8 assay, colorimetric method and lactate administration. RESULTS: The 48-h SR made mice in sleep disruption status and caused an acute damage to the BSCB, resulting in hyperalgesia and neuroinflammation in the spinal cord. In SR mice, the levels of glycolysis and glycolysis enzymes of ECs in the BSCB were found significantly decreased [CON group vs. SR group: CD31+Glut1+ cells: p < 0.001], which could cause dysfunction of ECs and this was confirmed in vitro. Increased numbers of infiltrating T cells [p < 0.0001] and Treg population [p < 0.05] were detected in the mouse spinal cord after 48-h SR. In the co-cultured system of ECs and Tregs in vitro, the competition of Tregs for glucose resulted in the glycolysis disorder of ECs [Glut1: p < 0.01, ENO1: p < 0.05, LDHα: p < 0.05; complete tubular structures formed: p < 0.0001; CCK8 assay: p < 0.001 on 24h, p < 0.0001 on 48h; glycolysis level: p < 0.0001]. An administration of sodium lactate partially rescued the function of ECs and relieved SR-induced hyperalgesia. Furthermore, the mTOR signaling pathway was excessively activated in ECs after SR in vivo and those under the inhibition of glycolysis or co-cultured with Tregs in vitro. CONCLUSIONS: Affected by glycolysis disorders of ECs due to glucose competition with infiltrating Tregs through regulating the mTOR signaling pathway, hyperalgesia induced by 48-h SR is attributed to neuroinflammation and damages to the barriers, which can be relieved by lactate supplementation.

From Glycolysis to Viral Defense: The Multifaceted Impact of Glycolytic Enzymes on Human Immunodeficiency Virus Type 1 Replication.

Viruses require host cells to replicate and proliferate, which indicates that viruses hijack the cellular machinery. Human immunodeficiency virus type 1 (HIV-1) primarily infects CD4-positive T cells, and efficiently uses cellular proteins to replicate. Cells already have proteins that inhibit the replication of the foreign HIV-1, but their function is suppressed by viral proteins. Intriguingly, HIV-1 infection also changes the cellular metabolism to aerobic glycolysis. This phenomenon has been interpreted as a cellular response to maintain homeostasis during viral infection, yet HIV-1 efficiently replicates even in this environment. In this review, we discuss the regulatory role of glycolytic enzymes in viral replication and the impact of aerobic glycolysis on viral infection by introducing various host proteins involved in viral replication. Furthermore, we would like to propose a "glyceraldehyde-3-phosphate dehydrogenase-induced shock (G-shock) and kill strategy" that maximizes the antiviral effect of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to eliminate latently HIV-1-infected cells.

Glucose Regulation of ß-Cell KATP Channels: It Is Time for a New Model!

An agreed-upon consensus model of glucose-stimulated insulin secretion from healthy ß-cells is essential for understanding diabetes pathophysiology. Since the discovery of the KATP channel in 1984, an oxidative phosphorylation (OxPhos)-driven rise in ATP has been assumed to close KATP channels to initiate insulin secretion. This model lacks any evidence, genetic or otherwise, that mitochondria possess the bioenergetics to raise the ATP/ADP ratio to the triggering threshold, and conflicts with genetic evidence demonstrating that OxPhos is dispensable for insulin secretion. It also conflates the stoichiometric yield of OxPhos with thermodynamics, and overestimates OxPhos by failing to account for established features of ß-cell metabolism, such as leak, anaplerosis, cataplerosis, and NADPH production that subtract from the efficiency of mitochondrial ATP production. We have proposed an alternative model, based on the spatial and bioenergetic specializations of ß-cell metabolism, in which glycolysis initiates insulin secretion. The evidence for this model includes that 1) glycolysis has high control strength over insulin secretion; 2) glycolysis is active at the correct time to explain KATP channel closure; 3) plasma membrane-associated glycolytic enzymes control KATP channels; 4) pyruvate kinase has favorable bioenergetics, relative to OxPhos, for raising ATP/ADP; and 5) OxPhos stalls before membrane depolarization and increases after. Although several key experiments remain to evaluate this model, the 1984 model is based purely on circumstantial evidence and must be rescued by causal, mechanistic experiments if it is to endure.

A negative feedback loop underlies the Warburg effect.

Aerobic glycolysis, or the Warburg effect, is used by cancer cells for proliferation while producing lactate. Although lactate production has wide implications for cancer progression, it is not known how this effect increases cell proliferation and relates to oxidative phosphorylation. Here, we elucidate that a negative feedback loop (NFL) is responsible for the Warburg effect. Further, we show that aerobic glycolysis works as an amplifier of oxidative phosphorylation. On the other hand, quiescence is an important property of cancer stem cells. Based on the NFL, we show that both aerobic glycolysis and oxidative phosphorylation, playing a synergistic role, are required to achieve cell quiescence. Further, our results suggest that the cells in their hypoxic niche are highly proliferative yet close to attaining quiescence by increasing their NADH/NAD+ ratio through the severity of hypoxia. The findings of this study can help in a better understanding of the link among metabolism, cell cycle, carcinogenesis, and stemness.

Calcium saccharate/DUSP6 suppresses renal cell carcinoma glycolytic metabolism and boosts sunitinib efficacy via the ERK-AKT pathway.

Current therapeutic options for renal cell carcinoma (RCC) are very limited, which is largely due to inadequate comprehension of molecular pathological mechanisms as well as RCC's resistance to chemotherapy. Dual-specificity phosphatase 6 (DUSP6) has been associated with numerous human diseases. However, its role in RCC is not well understood. Here, we show that diminished DUSP6 expression is linked to RCC progression and unfavorable prognosis. Mechanistically, DUSP6 serves as a tumor suppressor in RCC by intervening the TAF10 and BSCL2 via the ERK-AKT pathway. Further, DUSP6 is also transcriptionally regulated by HNF-4a. Moreover, docking experiments have indicated that DUSP6 expression is enhanced when bound by Calcium saccharate, which also inhibits RCC cell proliferation, metabolic rewiring, and sunitinib resistance. In conclusion, our study identifies Calcium saccharate as a prospective pharmacological therapeutic approach for RCC.

Transport activity regulates mitochondrial bioenergetics and biogenesis in renal tubules.

Renal tubules are featured with copious mitochondria and robust transport activity. Mutations in mitochondrial genes cause congenital renal tubulopathies, and changes in transport activity affect mitochondrial morphology, suggesting mitochondrial function and transport activity are tightly coupled. Current methods of using bulk kidney tissues or cultured cells to study mitochondrial bioenergetics are limited. Here, we optimized an extracellular flux analysis (EFA) to study mitochondrial respiration and energy metabolism using microdissected mouse renal tubule segments. EFA detects mitochondrial respiration and glycolysis by measuring oxygen consumption and extracellular acidification rates, respectively. We show that both measurements positively correlate with sample sizes of a few centimeter-length renal tubules. The thick ascending limbs (TALs) and distal convoluted tubules (DCTs) critically utilize glucose/pyruvate as energy substrates, whereas proximal tubules (PTs) are significantly much less so. Acute inhibition of TALs' transport activity by ouabain treatment reduces basal and ATP-linked mitochondrial respiration. Chronic inhibition of transport activity by 2-week furosemide treatment or deletion of with-no-lysine kinase 4 (Wnk4) decreases maximal mitochondrial capacity. In addition, chronic inhibition downregulates mitochondrial DNA mass and mitochondrial length/density in TALs and DCTs. Conversely, gain-of-function Wnk4 mutation increases maximal mitochondrial capacity and mitochondrial length/density without increasing mitochondrial DNA mass. In conclusion, EFA is a sensitive and reliable method to investigate mitochondrial functions in isolated renal tubules. Transport activity tightly regulates mitochondrial bioenergetics and biogenesis to meet the energy demand in renal tubules. The system allows future investigation into whether and how mitochondria contribute to tubular remodeling adapted to changes in transport activity.

Imbalance in Glucose Metabolism Regulates the Transition of Microglia from Homeostasis to Disease-Associated Microglia Stage 1.

Microglia undergo two-stage activation in neurodegenerative diseases, known as disease-associated microglia (DAM). TREM2 mediates the DAM2 stage transition, but what regulates the first DAM1 stage transition is unknown. We report that glucose dyshomeostasis inhibits DAM1 activation and PKM2 plays a role. As in tumors, PKM2 was aberrantly elevated in both male and female human AD brains, but unlike in tumors, it is expressed as active tetramers, as well as among TREM2+ microglia surrounding plaques in 5XFAD male and female mice. snRNAseq analyses of microglia without Pkm2 in 5XFAD mice revealed significant increases in DAM1 markers in a distinct metabolic cluster, which is enriched in genes for glucose metabolism, DAM1, and AD risk. 5XFAD mice incidentally exhibited a significant reduction in amyloid pathology without microglial Pkm2 Surprisingly, microglia in 5XFAD without Pkm2 exhibited increases in glycolysis and spare respiratory capacity, which correlated with restoration of mitochondrial cristae alterations. In addition, in situ spatial metabolomics of plaque-bearing microglia revealed an increase in respiratory activity. These results together suggest that it is not only glycolytic but also respiratory inputs that are critical to the development of DAM signatures in 5XFAD mice.

Phosphate sensing in health and disease.

PURPOSE OF REVIEW: Disruptions of phosphate homeostasis are associated with a multitude of diseases with insufficient treatments. Our knowledge regarding the mechanisms underlying metazoan phosphate homeostasis and sensing is limited. Here, we highlight four major advancements in this field during the last 12-18 months. RECENT FINDINGS: First, kidney glycolysis senses filtered phosphate, which results in the release of glycerol 3-phosphate (G-3-P). Circulating G-3-P then stimulates synthesis of the phosphaturic hormone fibroblast growth factor 23 in bone. Second, the liver serves as a postprandial phosphate reservoir to limit serum phosphate excursions. It senses phosphate ingestion and triggers renal excretion of excess phosphate through a nerve-dependent mechanism. Third, phosphate-starvation in cells massively induces the phosphate transporters SLC20A1/PiT1 and SLC20A2/PiT2, implying direct involvement of cellular phosphate sensing. Under basal phosphate-replete conditions, PiT1 is produced but immediately destroyed, which suggests a novel mechanism for the regulation of PiT1 abundance. Fourth, Drosophila melanogaster intestinal cells contain novel organelles called PXo bodies that limit intracellular phosphate excursions. Phosphate starvation leads to PXo body dissolution, which triggers midgut proliferation. SUMMARY: These studies have opened novel avenues to dissect the mechanisms that govern metazoan phosphate sensing and homeostasis with the potential to identify urgently needed therapeutic targets.

The Molecular Mechanisms behind Advanced Breast Cancer Metabolism: Warburg Effect, OXPHOS, and Calcium.

Altered metabolism represents a fundamental difference between cancer cells and normal cells. Cancer cells have a unique ability to reprogram their metabolism by deviating their reliance from primarily oxidative phosphorylation (OXPHOS) to glycolysis, in order to support their survival. This metabolic phenotype is referred to as the "Warburg effect" and is associated with an increase in glucose uptake, and a diversion of glycolytic intermediates to alternative pathways that support anabolic processes. These processes include synthesis of nucleic acids, lipids, and proteins, necessary for the rapidly dividing cancer cells, sustaining their growth, proliferation, and capacity for successful metastasis. Triple-negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer, with the poorest patient outcome due to its high rate of metastasis. TNBC is characterized by elevated glycolysis and in certain instances, low OXPHOS. This metabolic dysregulation is linked to chemotherapeutic resistance in TNBC research models and patient samples. There is more than a single mechanism by which this metabolic switch occurs and here, we review the current knowledge of relevant molecular mechanisms involved in advanced breast cancer metabolism, focusing on TNBC. These mechanisms include the Warburg effect, glycolytic adaptations, microRNA regulation, mitochondrial involvement, mitochondrial calcium signaling, and a more recent player in metabolic regulation, JAK/STAT signaling. In addition, we explore some of the drugs and compounds targeting cancer metabolic reprogramming. Research on these mechanisms is highly promising and could ultimately offer new opportunities for the development of innovative therapies to treat advanced breast cancer characterized by dysregulated metabolism.

The impact of ultrasound treatment on glycolytic enzymes when applied to crude extracts from early post-mortem bovine muscle.

The rate of pH decline post - mortem and its interaction with temperature influences the final tenderness of meat, and therefore, the manipulation of the rate of pH decline is a strategy of interest in order to obtain consistent high quality meat. Ultrasound is a potential early post - mortem carcass intervention, which may alter the rate of glycolysis based on its ability to alter enzyme activity. In this study, homogenates (prepared from early post-mortem Longissimus thoracis et lumborum muscle) were subjected to different ultrasound intensities (0 %/60 %/100 % amp) and treatment durations (15/ 30 min). The effect of these treatments on the inherent activity of the glycolytic enzymes was investigated using an in vitro glycolytic buffer model system. It was found that ultrasound treatment intensity and duration had a significant interactive effect on the rate of pH decline, and on reducing sugars and lactic acid concentrations, specifically following the 100 % amp ultrasound for 30 min treatment and between 30 and 240 min incubation. No significant differences in pH or metabolites content were observed between treatments after 1440 min of incubation. No effect of ultrasound intensity or treatment duration was observed on the degradation of glycogen. Under the reported conditions of this trial, it can be concluded that the application of ultrasound has limited potential to have an impact on the glycolytic pathways in bovine muscle.

GLUT and HK: Two primary and essential key players in tumor glycolysis.

Cancer cells reprogram their metabolism to become "glycolysis-dominant," which enables them to meet their energy and macromolecule needs and enhancing their rate of survival. This glycolytic-dominancy is known as the "Warburg effect", a significant factor in the growth and invasion of malignant tumors. Many studies confirmed that members of the GLUT family, specifically HK-II from the HK family play a pivotal role in the Warburg effect, and are closely associated with glucose transportation followed by glucose metabolism in cancer cells. Overexpression of GLUTs and HK-II correlates with aggressive tumor behaviour and tumor microenvironment making them attractive therapeutic targets. Several studies have proven that the regulation of GLUTs and HK-II expression improves the treatment outcome for various tumors. Therefore, small molecule inhibitors targeting GLUT and HK-II show promise in sensitizing cancer cells to treatment, either alone or in combination with existing therapies including chemotherapy, radiotherapy, immunotherapy, and photodynamic therapy. Despite existing therapies, viable methods to target the glycolysis of cancer cells are currently lacking to increase the effectiveness of cancer treatment. This review explores the current understanding of GLUT and HK-II in cancer metabolism, recent inhibitor developments, and strategies for future drug development, offering insights into improving cancer treatment efficacy.

Interleukin-6 classic and trans-signaling utilize glucose metabolism reprogramming to achieve anti- or pro-inflammatory effects.

Interleukin (IL)-6 has anti- and pro-inflammatory functions, controlled by IL-6 classic and trans-signaling, respectively. Differences in the downstream signaling mechanism between IL-6 classic and trans-signaling have not been identified. Here, we report that IL-6 activates glycolysis to regulate the inflammatory response. IL-6 regulates glucose metabolism by forming a complex containing signal-transducing activators of transcription 3 (STAT3), hexokinase 2 (HK2), and voltage-dependent anion channel 1 (VDAC1). The IL-6 classic signaling directs glucose flux to oxidative phosphorylation (OxPhos), while IL-6 trans-signaling directs glucose flux to anaerobic glycolysis. Classic IL-6 signaling promotes STAT3 translocation into mitochondria to interact with pyruvate dehydrogenase kinase-1 (PDK1), leading to pyruvate dehydrogenase α (PDHA) dissociation from PDK1. As a result, PDHA is dephosphorylated, and STAT3 is phosphorylated at Ser727. By contrast, IL-6 trans-signaling promotes the interaction of sirtuin 2 (SIRT2) and lactate dehydrogenase A (LDHA), leading to the dissociation of STAT3 from SIRT2. As a result, LDHA is deacetylated, and STAT3 is acetylated and phosphorylated at Tyr705. IL-6 classic signaling promotes the differentiation of regulatory T cells via the PDK1/STAT3/PDHA axis, whereas IL-6 trans-signaling promotes the differentiation of Th17 cells via the SIRT2/STAT3/LDHA axis. Conclusion: IL-6 classic signaling generates anti-inflammatory functions by shifting energy metabolism to OxPhos, while IL-6 trans-signaling generates pro-inflammatory functions by shifting energy metabolism to anaerobic glycolysis.

The SOX2/PDIA6 axis mediates aerobic glycolysis to promote stemness in non-small cell lung cancer cells.

Non-small cell lung cancer (NSCLC) is an aggressive and rapidly expanding lung cancer. Abnormal upregulation or knockdown of PDIA6 expression can predict poor prognosis in various cancers. This study aimed to investigate the biological function of PDIA6 in NSCLC. SOX2 and PDIA6 expression in NSCLC tissues and regulatory relationship between them were analyzed using bioinformatics. GSEA was performed on the enrichment pathway of PDIA6. qRT-PCR was utilized to examine expression of SOX2 and PDIA6 in NSCLC tissues and cells, and dual-luciferase reporter assay and ChIP experiments were performed to validate their regulatory relationship. CCK-8 experiment was conducted to assess cell viability, western blot was to examine levels of stem cell markers and proteins related to aerobic glycolysis pathway in cells. Cell sphere formation assay was used to evaluate efficiency of cell sphere formation. Reagent kits were used to measure glycolysis levels and glycolysis products. High expression of PDIA6 in NSCLC was linked to aerobic glycolysis. Knockdown of PDIA6 reduced cell viability, expression of stem cell surface markers, and cell sphere formation efficiency in NSCLC. Overexpression of PDIA6 could enhance cell viability and promote aerobic glycolysis, but the addition of 2-DG could reverse this result. Bioinformatics predicted the existence of upstream transcription factor SOX2 for PDIA6, and SOX2 was significantly upregulated in NSCLC, and they had a binding relationship. Further experiments revealed that PDIA6 overexpression restored repressive effect of knocking down SOX2 on aerobic glycolysis and cell stemness. This work revealed that the SOX2/PDIA6 axis mediated aerobic glycolysis to promote NSCLC cell stemness, providing new therapeutic strategies for NSCLC.

Diabetes mellitus and Alzheimer's disease: Vacuolar adenosine triphosphatase as a potential link.

Globally, the incidence of diabetes mellitus (DM) and Alzheimer's disease (AD) is increasing year by year, causing a huge economic and social burden, and their pathogenesis and aetiology have been proven to have a certain correlation. In recent years, more and more studies have shown that vacuolar adenosine triphosphatases (v-ATPases) in eukaryotes, which are biomolecules regulating lysosomal acidification and glycolipid metabolism, play a key role in DM and AD. This article describes the role of v-ATPase in DM and AD, including its role in glycolysis, insulin secretion and insulin resistance (IR), as well as its relationship with lysosomal acidification, autophagy and ß-amyloid (Aß). In DM, v-ATPase is involved in the regulation of glucose metabolism and IR. v-ATPase is closely related to glycolysis. On the one hand, v-ATPase affects the rate of glycolysis by affecting the secretion of insulin and changing the activities of key glycolytic enzymes hexokinase (HK) and phosphofructokinase 1 (PFK-1). On the other hand, glucose is the main regulator of this enzyme, and the assembly and activity of v-ATPase depend on glucose, and glucose depletion will lead to its decomposition and inactivation. In addition, v-ATPase can also regulate free fatty acids, thereby improving IR. In AD, v-ATPase can not only improve the abnormal brain energy metabolism by affecting lysosomal acidification and autophagy but also change the deposition of Aß by affecting the production and degradation of Aß. Therefore, v-ATPase may be the bridge between DM and AD.

[Immunometabolism in Sepsis]. / Immunmetabolismus in der Sepsis.

Immunometabolism is a fascinating field of research that investigates the interactions between metabolic processes and the immune response. This intricate connection plays a pivotal role in regulating inflammatory reactions and consequently exerts a significant impact on the course of sepsis. The proinflammatory response during an immune reaction is closely tied to a high energy demand in immune cells. As a result, proinflammatory immune cells rapidly require substantial amounts of energy in the form of ATP, necessitating a fundamental and swift shift in their metabolism, i.e., their means of generating energy. This entails a marked increase in glycolysis within the proinflammatory response, thereby promptly meeting the energy requirements and providing essential metabolic building blocks for the biosynthesis of macromolecules. Alongside glycolysis, there is heightened activity in the pentose phosphate pathway (PPP). The PPP significantly contributes to NADPH production within the cell, thus maintaining redox equilibrium. Elevated PPP activity consequently leads to an increased NADPH level, resulting in enhanced production of reactive oxygen species (ROS) and nitric oxide (NO). While these molecules are crucial for pathogen elimination, an excess can also induce tissue damage. Simultaneously, there are dual interruptions in the citric acid cycle. In the cellular resting state, the citric acid cycle acts as a sort of "universal processor", where metabolic byproducts of glycolysis, fatty acid breakdown, and amino acid degradation are initially transformed into NADH and FADH2, subsequently yielding ATP. While the citric acid cycle and its connected oxidative phosphorylation predominantly generate energy at rest, it becomes downregulated in the proinflammatory phase of sepsis. The two interruptions lead to an accumulation of citrate and succinate within cells, reflecting mitochondrial dysfunction. Additionally, the significantly heightened glycolysis through fermentation yields lactate, a pivotal metabolite for sepsis diagnosis and prognosis. Conversely, cells in an anti-inflammatory state revert to a metabolic profile akin to the resting state: Glycolysis is attenuated, PPP is suppressed, and the citric acid cycle is reactivated. Of particular interest is that not only does the immune reaction influence metabolic pathways, but this connection also operates in reverse. Thus, modulation of metabolic pathways also modulates the immunity of the corresponding cell and thereby the state of the immune system itself. This could potentially serve as an intriguing avenue in sepsis therapy.

Cell via Cell Viability Assay Changes Cellular Metabolic Characteristics by Intervening with Glycolysis and Pentose Phosphate Pathway.

The Cell Counting Kit-8 (CCK-8) cell viability assay, also known as WST-8, is widely recognized for its nontoxic nature, making it suitable for further studies on treated cells. This practice is commonly observed in the field of tissue engineering. While live/dead imaging may not readily reveal macroscopic differences, our investigation has uncovered significant intracellular metabolic changes. Notably, we observed substantial down-regulation of metabolites within the glycolysis and pentose phosphate pathways. These metabolic alterations predominantly affect energy metabolism and may potentially impact the cellular redox environment. In light of these findings, we strongly recommend that researchers exercise caution when using cells treated with CCK-8 in subsequent experiments.

USP43 stabilizes c-Myc to promote glycolysis and metastasis in bladder cancer.

A hallmark of tumor cells, including bladder cancer (BLCA) cells, is metabolic reprogramming toward aerobic glycolysis (Warburg effect). The classical oncogene MYC, which is crucial in regulating glycolysis, is amplified and activated in BLCA. However, direct targeting of the c-Myc oncoprotein, which regulates glycolytic metabolism, presents great challenges and necessitates the discovery of a more clarified regulatory mechanism to develop selective targeted therapy. In this study, a siRNA library targeting deubiquitinases identified a candidate enzyme named USP43, which may regulate glycolytic metabolism and c-Myc transcriptional activity. Further investigation using functional assays and molecular studies revealed a USP43/c-Myc positive feedback loop that contributes to the progression of BLCA. Moreover, USP43 stabilizes c-Myc by deubiquitinating c-Myc at K148 and K289 primarily through deubiquitinase activity. Additionally, upregulation of USP43 protein in BLCA increased the chance of interaction with c-Myc and interfered with FBXW7 access and degradation of c-Myc. These findings suggest that USP43 is a potential therapeutic target for indirectly targeting glycolytic metabolism and the c-Myc oncoprotein consequently enhancing the efficacy of bladder cancer treatment.

Kinetic modelling of glycolytic oscillations.

Glycolytic oscillations have been studied for well over 60 years, but aspects of their function, and mechanisms of regulation and synchronisation remain unclear. Glycolysis is amenable to mechanistic mathematical modelling, as its components have been well characterised, and the system can be studied at many organisational levels: in vitro reconstituted enzymes, cell free extracts, individual cells, and cell populations. In recent years, the emergence of individual cell analysis has opened new ways of studying this intriguing system.

Role of miRNAs in glucose metabolism of mouse cumulus cells†.

It is known that the oocyte has a limited capacity to acquire and metabolize glucose, and it must rely on cumulus cells (CCs) to take up glucose and produce pyruvate for use to produce ATP through oxidative phosphorylation. We therefore propose that miRNAs might regulate glucose metabolism (GM) in CCs and might be used as markers for oocyte quality assessment. Here, mouse CC models with impaired glycolysis or pentose phosphate pathway (PPP) were established, and miRNAs targeting the key enzymes in glycolysis/PPP were predicted using the miRNA target prediction databases. Expression of the predicted miRNAs was compared between CCs with normal and impaired glycolysis/PPP to identify candidate miRNAs. Function of the candidate miRNAs was validated by transfecting CCs or cumulus-oocyte-complexes (COCs) with miRNA inhibitors and observing effects on glucose metabolites of CCs and on competence of oocytes. The results validated that miR-23b-3p, let-7b-5p, 34b-5p and 145a-5p inhibited glycolysis, and miR-24-3p, 3078-3p,183-5p and 7001-5p inhibited PPP of CCs. Our observation using a more physiologically relevant model (intact cultured COCs) further validated the four glycolysis-targeting miRNAs we identified. Furthermore, miR-let-7b-5p, 34b-5p and 145a-5p may also inhibit PPP, as they decreased the production of glucose-6-phosphate. In conclusion, miRNAs play critical roles in GM of CCs and may be used as markers for oocyte quality assessment. Summary sentence:  We identified and validated eight new miRNAs that inhibit glycolysis and/or pentose phosphate pathways in cumulus cells (CCs) suggesting that miRNAs play critical roles in glucose metabolism of CCs and may be used for oocyte quality markers.

Protein lysine acetylation does not contribute to the high rates of fatty acid oxidation seen in the post-ischemic heart.

High rates of cardiac fatty acid oxidation during reperfusion of ischemic hearts contribute to contractile dysfunction. This study aimed to investigate whether lysine acetylation affects fatty acid oxidation rates and recovery in post-ischemic hearts. Isolated working hearts from Sprague Dawley rats were perfused with 1.2 mM palmitate and 5 mM glucose and subjected to 30 min of ischemia and 40 min of reperfusion. Cardiac function, fatty acid oxidation, glucose oxidation, and glycolysis rates were compared between pre- and post-ischemic hearts. The acetylation status of enzymes involved in cardiac energy metabolism was assessed in both groups. Reperfusion after ischemia resulted in only a 41% recovery of cardiac work. Fatty acid oxidation and glycolysis rates increased while glucose oxidation rates decreased. The contribution of fatty acid oxidation to ATP production and TCA cycle activity increased from 90 to 93% and from 94.9 to 98.3%, respectively, in post-ischemic hearts. However, the overall acetylation status and acetylation levels of metabolic enzymes did not change in response to ischemia and reperfusion. These findings suggest that acetylation may not contribute to the high rates of fatty acid oxidation and reduced glucose oxidation observed in post-ischemic hearts perfused with high levels of palmitate substrate.