Activities Of Glycolytic Enzymes Research Articles

Integrated multiple-omics reveals the regulatory mechanism underlying the effects of homologous Bacillus tequilensis (GCB-3) on growth performance of grass carp (Ctenopharyngodon idellus)

The purpose of this study was to investigate the regulatory mechanism of Bacillus tequilensis (GCB-3) in improving the growth performance of grass carp by microbiome and targeted metabolomics analysis. Fish were fed with different amounts of GCB-3 including B0 (0 CFU/g), B6 (1×106 CFU/g), B7 (1×107 CFU/g), B8 (1×108 CFU/g), and B9 (1×109 CFU/g) for 8 weeks. The results indicate that with the increase of dietary GCB-3 concentration, grass carp showed a significant linear trend in growth performance, and appetite factors showed a significant linear and quadratic trend, the growth performance and appetite-promoting factors in the B8 group reach their maximum value in the B8 group. Microbiome analysis found that Proteobacteria and Actinobacteria were the most abundant phylum in the gut microbiota of grass carp. The functional potential predicted that GCB-3 affected the synthesis and degradation of carbohydrates, amino acids, fatty acids and lipids. It found that GCB-3 also affected the tricarboxylic acid (TCA) cycle VII (acetate-producers) pathway. The results of targeted metabolomics showed a significant linear and quadratic increase in intestinal acetate with GCB-3 concentration and a maximum in the B8 group. The results of the correlation analysis showed a significant positive correlation between Actinobacteria abundance and acetic acid content, and also between TCA cycle VII (acetate-producers) pathway abundance and Actinobacteria abundance and acetic acid content. In B8 group, grass carp showed a faster regulation speed of blood glucose levels after glucose injection, and the GLP-1 content remained stable. The activities of glycolytic enzymes, lipolytic enzymes, protein metabolism enzymes, and their related mRNA expression levels in grass carp showed a linear or quadratic increasing trend. However, the expression levels of gluconeogenic enzymes and fatty acid synthesis enzyme related mRNA showed an opposite trend. In summary, the addition of 1×108 CFU/g GCB-3 in grass carp feed can improve the abundance of dominant bacterial phyla in the intestine, especially Actinobacteria, increase the concentration of intestinal acetate, provide substrate for the TCA cycle VII (acetate-producers) pathway, thereby regulating the nutrient metabolism of grass carp and ultimately promoting growth.

Vibration Emissions Reduce Boar Sperm Quality via Disrupting Its Metabolism

Artificial insemination (AI) with liquid-preserved semen has recently become common in pig breeding. The semen doses are produced in a centralized manner at the boar stud and then subsequently distributed and transported to sow farms. However, vibration emissions during transportation by logistic vehicles may adversely affect the quality of boar sperm. Therefore, this study aimed to explore the impact of vibration-induced emissions on sperm quality and function under simulated transportation conditions. Each time, ejaculates from all 15 boars were collected and then pooled together to minimize individual variations, and the sample was split using an extender for dilution. Different rotational speeds (0 rpm, 80 rpm, 140 rpm, 200 rpm) were utilized to simulate varying intensities of vibration exposure using an orbital shaker, considering different transportation times (0 h, 3 h, and 6 h). Subsequently, evaluations were conducted regarding sperm motility, plasma membrane integrity, acrosome integrity, mitochondrial function, adenosine triphosphate (ATP) levels, mitochondrial reactive oxygen species (ROS) levels, pH, glycolytic pathway enzyme activities, and capacitation following exposure to vibration emissions. Both vibration time and intensity impact sperm motility, plasma membrane integrity, and acrosomal integrity. Vibration exposure significantly reduced sperm ATP levels, mitochondrial membrane potential, and the levels of mitochondria-encoded proteins (MT-ND1, MT-ND6) (p < 0.05). After vibration emission treatment, the pH value and mitochondrial ROS levels significantly increased (p < 0.05). Inhibition of sperm glycolysis was observed, with reduced activities of hexokinase (HK), pyruvate kinase (PK), and lactate dehydrogenase (LDH), along with decreased lactate levels (p < 0.05). Additionally, sperm tyrosine phosphorylation levels were significantly reduced by vibration emissions compared to the control group (p < 0.05). After the vibration emission treatment, the number of sperm bound to each square millimeter of oviduct explants decreased significantly compared to the control group (p < 0.05). Similarly, compared to the control group, using semen subjected to vibration stress for AI results in significantly reduced pregnancy rates, total born litter size, live-born litter size, and healthy born litter size (p < 0.05).

Regulation of carbohydrate metabolism and insulin secretion in diabetic rats following treatment with Hypericum perforatum L. hairy root aqueous methanol extract

Hairy root (HR) cultures of Hypericum perforatum L. have shown promise in controlling hyperglycemia, regulating blood lipid and enzyme profiles, and improving metabolic function in vivo. These benefits are mainly attributed to the phenolic acids, flavonoids, and xanthones present in HR extracts. However, the specific mechanism underlying these effects remains unclear. This study was performed to elucidate the biochemical and molecular mechanisms driving HR the antihyperglycemic and antidiabetic effects of HR extracts. HR extract (200 mg/kg body weight) was administered daily for 14 days to healthy rats and rats with streptozotocin-induced diabetes, with glibenclamide serving as a positive control. The phenolic composition of the HR extracts was confirmed through high-performance liquid chromatography/diode array detection/electrospray ionization mass spectrometry (HPLC/DAD/ESI-MSn ) analysis. The results showed that HR extract treatment increased the plasma insulin level and pancreatic poly (adenosine diphosphate-ribose) polymerase (PARP) activity in diabetic rats, thus normalizing blood glucose levels. Additionally, it reduced the activity of gluconeogenic enzymes, increased the activity of glycolytic enzymes, and normalized the glycogen content in the liver. HR extract-treated rats also exhibited increased hepatic adenosine monophosphate-activated protein kinase (AMPK) mRNA expression and a decreased protein kinase Cε (PKCε) concentration. In conclusion, HR extract demonstrated insulinotropic effects and effectively regulated hepatic carbohydrate metabolism in diabetic rats by modulating AMPK expression and the PKCε concentration. These findings suggest the potential use of HR extract as an herbal medicine for diabetes treatment and a source of antidiabetic drug development.

Better maintenance of enzymatic capacity and higher levels of substrate transporter proteins in skeletal muscle of aging female mice.

This study investigated sex-specific differences in high-energy phosphate, glycolytic, and mitochondrial enzyme activities and also metabolite transporter protein levels in the skeletal muscles of adult (5 months old), middle-aged (12 months old), and advanced-aged (24 months old) mice. While gastrocnemius glycogen content increased with age regardless of sex, gastrocnemius triglyceride levels increased only in advanced-aged female mice. Aging decreased creatine kinase and adenylate kinase activities in the plantaris muscle of both sexes and in the soleus muscle of male mice but not in female mice. Irrespective of sex, phosphofructokinase and lactate dehydrogenase (LDH) activities decreased in the plantaris and soleus muscles. Additionally, hexokinase activity in the plantaris muscle and LDH activity in the soleus muscle decreased to a greater extent in aged female mice compared with those in aged male mice. Mitochondrial enzyme activities increased in the plantaris muscle of aged female mice but did not change in male mice. The protein content of the glucose transporter 4 in the aged plantaris muscle and fatty acid translocase/cluster of differentiation 36 increased in the aged plantaris and soleus muscles of both sexes, with a significantly higher content in female mice. These findings suggest that females possess a better ability to maintain metabolic enzyme activity and higher levels of metabolite transport proteins in skeletal muscle during aging, despite alterations in lipid metabolism. Our data provide a basis for studying muscle metabolism in the context of age-dependent metabolic perturbations and diseases that affect females and males differently.

An integrated analysis of transcriptome and metabolome to reveal the effects of temperature stress on energy metabolism and physiological responses in Schizothorax wangchiachii muscles

The unique hydrological situation of Jinsha River and the construction and operation of large hydropower stations may cause resident Schizothorax wangchiachii to experience acute changes in water temperature. To reveal the response mechanisms of S. wangchiachii to high and low temperature stress, we conducted a stress experiment. Compared with the control group, the activity of antioxidant enzymes (SOD\\CAT) increased first and then decreased at high temperature, while MDA activity in the low temperature group continued to increase slowly. The activities of glycolytic enzymes were increased initially and then decreased including HK and PFK, but PK activity continued to increase throughout the stress period. Transcriptome sequencing showed that a large number of genes in muscle tissues were differentially expressed after high and low temperature stress, and the main enriched pathways included glycosaminoglycan biosynthesis, lysine degradation, glycerolipid metabolism, the PPAR signaling pathway, and the adipocytokine signaling pathway. Significant metabolic pathways were identified as enriched according to the metabolic differences,such as glycerophospholipid metabolism, qRT-PCR was used to verify gene expression. The results suggested that both high temperature and low temperature stress induced physiological responses in S. wangchiachii that maintained homeostasis by regulating glucolipid metabolism and amino acid degradation. This study revealed the response mechanisms of S. wangchiachii to high and low temperature stress, information that may serve as a reference for the study of responses in other fish to environmental changes in the upper reaches of the Yangtze River or where they are displaced by hydropower station construction.

Alpha-ketoglutarate supplementation in long-lived Drosophila melanogaster: Impact on lifespan and metabolic responses.

Studies on antiaging remedies in insect models sometimes show discrepancies in results. These discrepancies could be explained by different responses of short- and long-lived strains on the antiaging remedies. The purpose of the study was to test whether life-prolonging effects of alpha-ketoglutarate (AKG), observed in nematodes and fruit flies, would be reproduced in long-lived Drosophila melanogaster flies. Lifespan was assayed in flies kept in demographic cages. Fecundity, proportion of flies capable of negative geotaxis, starvation resistance, time of heat coma onset, levels of triacyglycerols, body glucose, glycogen, activities of glutamate dehydrogenase, catalase, glutathione-S-transferase, hexokinase, phosphofructokinase, pyruvate kinase, lactate, and glutamate dehydrogenases were assessed. Dietary AKG did not affect fly lifespan on the diet with 5% yeast and 5% sucrose (5Y:5S) and on the diet with 9% yeast and 1% sucrose (9Y:1S), but increased lifespan on the low-protein diet (1Y:9S). Twenty-five-day-old female flies fed a 5Y:5S diet with 10 mM AKG for 3 weeks, did not differ from the control group (without AKG) in climbing activity, resistance to heat stress, and starvation. The levels of glucose and glycogen were unaffected but the levels of triacylglycerols were lower in AKG-fed female flies. No differences in activities of glycolytic enzymes, NADPH-producing enzymes, glutamate dehydrogenase, oxygen consumption, and levels of oxidative stress markers were observed between the control and AKG-fed flies. However, AKG-fed flies had lower activities of catalase and glutathione-S-transferase. These results suggest that potential antiaging remedies, such as AKG, may not extend lifespan in long-living organisms despite influencing several metabolic parameters.

Lack of Sestrins Is Associated with Increased Glycolytic Flux

Introduction: Sestrin1, 2, and 3 are a family of stress-response proteins implicated in many diseases. How Sestrins affect cell survival through AMP-activated kinase (AMPK) and protein synthesis through mechanistic target of rapamycin in complex 1 (mTORC1) is most studied; however, how Sestrins affect metabolism is poorly understood. Hypothesis: Independent of AMPK/mTORC1 activation, Sestrins negatively regulate glycolytic flux by inhibiting glycolytic enzyme activity through binding inhibition. Results: Wildtype (WT) HEK293T cells were transfected with plasmids expressing individual FLAG-tagged Sestrins and then deprived of glucose for 180 min or deprived of glucose for 150 min followed by glucose addback for 30 min. FLAG-tagged Sestrins were immunoprecipitated and found to directly interact with Hexokinase2 and Phosphofructokinase such that binding inversely correlates with glucose concentration. Additionally, WT and Sestrin-knockout (TKO) HEK293T cells were subjected to the glycolytic and mitochondrial stress tests and analyzed for metabolic flux via Seahorse analysis. TKO cells demonstrated higher glycolytic flux, but lower mitochondrial flux, compared to WT cells. Cells were then treated with inhibitors against AMPK and mTORC1 and subjected to the glycolytic stress test. Surprisingly, glycolytic flux in TKO cells was unaffected by the inhibitors while WT cells reduced glycolytic flux suggesting Sestrins can regulate glycolytic flux independent of AMPK/mTORC1 activation. Thereafter, WT and TKO cells were glucose-deprived for 120 min or glucose-deprived for 110 min followed by glucose addback for 10 min and then characterized by steady-state metabolomics. As expected, TKO cells possessed elevations in glycolytic metabolites with glucose addback compared to WT cells. Similarly, WT and TKO cells were deprived of glucose for 110 min followed by stable isotope tracing with [13C6]glucose for 10- and 30-minutes and metabolites were profiled by mass spectrometry. TKO cells possessed higher abundances of glycolytic metabolites as well as elevated labeled extracellular lactate. Conclusions: These data support a model wherein Sestrins directly act as negative regulators for glycolytic enzyme activity, thus regulating glycolytic flux. This work is funded by F32DK126312 (P.A.R.), P30DK048520 Pilot and Feasibility Award (P.A.R.), T32DK120521 (A.E.L.), P30CA046934 Cancer Center Support Grant, R01AR080051 (A.B.), R01DK015658 (S.R.K.), P30DK048520 (P.S.M.). This is the full abstract presented at the American Physiology Summit 2024 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.

Insect flight energetics and the evolution of size, form and function.

Flying insects vary greatly in body size and wing proportions, significantly impacting their flight energetics. Generally, the larger the insect, the slower its flight wingbeat frequency. However, variation in frequency is also explained by differences in wing proportions, where larger-winged insects tend to have lower frequencies. These associations affect the energy required for flight. The correlated evolution of flight form and function can be further defined using a lineage of closely related bee species varying in body mass. The decline in flight wingbeat frequency with increasing size is paralleled by flight mass-specific metabolic rate. The specific scaling exponents observed can be predicted from the wing area allometry, where a greater increase (hyperallometry) leads to a more pronounced effect on flight energetics, and hypoallometry can lead to no change in frequency and metabolic rate across species. The metabolic properties of the flight muscles also vary with body mass and wing proportions, as observed from the activity of glycolytic enzymes and the phospholipid compositions of muscle tissue, connecting morphological differences with muscle metabolic properties. The evolutionary scaling observed across species is recapitulated within species. The static allometry observed within the bumblebee Bombus impatiens, where the wing area is proportional and isometric, affects wingbeat frequency and metabolic rate, which is predicted to decrease with an increase in size. Intraspecific variation in flight muscle tissue properties is also related to flight metabolic rate. The role of developmental processes and phenotypic plasticity in explaining intraspecific differences is central to our understanding of flight energetics. These studies provide a framework where static allometry observed within species gives rise to evolutionary allometry, connecting the evolution of size, form, and function associated with insect flight.

Coculture with Colon-26 cancer cells decreases the protein synthesis rate and shifts energy metabolism toward glycolysis dominance in C2C12 myotubes.

Cancer cachexia is the result of complex interorgan interactions initiated by cancer cells and changes in patient behavior such as decreased physical activity and energy intake. Therefore, it is crucial to distinguish between the direct and indirect effects of cancer cells on muscle mass regulation and bioenergetics to identify novel therapeutic targets. In this study, we investigated the direct effects of Colon-26 cancer cells on the molecular regulating machinery of muscle mass and its bioenergetics using a coculture system with C2C12 myotubes. Our results demonstrated that coculture with Colon-26 cells induced myotube atrophy and reduced skeletal muscle protein synthesis and its regulating mechanistic target of rapamycin complex 1 signal transduction. However, we did not observe any activating effects on protein degradation pathways including ubiquitin-proteasome and autophagy-lysosome systems. From a bioenergetic perspective, coculture with Colon-26 cells decreased the complex I-driven, but not complex II-driven, mitochondrial ATP production capacity, while increasing glycolytic enzyme activity and glycolytic metabolites, suggesting a shift in energy metabolism toward glycolysis dominance. Gene expression profiling by RNA sequencing showed that the increased activity of glycolytic enzymes was consistent with changes in gene expression. However, the decreased ATP production capacity of mitochondria was not in line with the gene expression. The potential direct interaction between cancer cells and skeletal muscle cells revealed in this study may contribute to a better fundamental understanding of the complex pathophysiology of cancer cachexia.NEW & NOTEWORTHY We explored the potential direct interplay between colon cancer cells (Colon-26) and skeletal muscle cells (C2C12 myotubes) employing a noncontact coculture experimental model. Our findings reveal that coculturing with Colon-26 cells substantially impairs the protein synthesis rate, concurrently instigating a metabolic shift toward glycolytic dominance in C2C12 myotubes. This research unveils critical insights into the intricate cellular cross talk underpinning the complex pathophysiology of cancer cachexia.

Abstract 4743: Targeting a novel TWIST1-Hexokinase II pathway to overcome MET TKI resistance in NSCLC

Abstract Non-small cell lung cancer (NSCLC) is the most common primary tumor to metastasize to the brain, with up to 40% of NSCLC patients developing brain metastases (BM). These BM portend a poor prognosis, even when extracranial disease is well-controlled. While targeted therapy and immunotherapy have improved lung cancer survival, there are no therapies specifically targeting NSCLC BM. Our lab identified a significant enrichment of MET amplification in lung adenocarcinoma (LUAD) BM (16%) compared to primary NSCLC (3%) and liver metastases (5%). MET, a receptor tyrosine kinase, and its ligand, hepatocyte growth factor (HGF), promote proliferation, epithelial-mesenchymal transition (EMT), angiogenesis, and metastasis. Although MET tyrosine kinase inhibitors (TKIs) are approved for use in METaltered NSCLC, half of patients with MET alterations fail to respond or inevitably acquire TKI resistance. We have found that MET pathway activation by MET alterations leads to increased expression of TWIST1, an EMT transcription factor required for tumorigenesis in MET-dependent NSCLC. Together, the HGF/MET/TWIST1 axis promotes cancer survival and metastasis. We found that MET amplified BM had distinct transcriptional signatures reflecting increased glycolysis. A similar shift toward glycolysis was observed in the MET amplified metastatic LUAD cell line (H1993) compared to the MET wild type primary LUAD line (H2073) derived from the same patient. Furthermore, LUAD cell lines with high MET expression demonstrated increased expression and activity of glycolytic enzymes, as well as increased susceptibility to glucose deprivation and glycolytic inhibitors (2DG, PFK158, AT-101, V-9302). Conversely, treatment of MET amplified cells with MET TKI, capmatinib, reduced glycolysis and oxidation phosphorylation to the levels observed in MET wild type LUAD lines. Activation of the MET pathway with HGF or TWIST1 overexpression specifically increased the mRNA and protein expression of the key glycolytic enzyme, Hexokinase II (HK2), compared to other glycolytic pathway proteins. Conversely, inhibition of the MET pathway with capmatinib or the TWIST1 inhibitor, harmine, decreased HK2 protein expression through a MYC-independent pathway. However, TWIST1 was required for MET-dependent transcriptional upregulation of HK2. Remarkably, in two novel MET altered models (cell line and patient derived xenograft) of acquired MET TKI resistance, TWIST1 and HK2 expression were increased at the time of resistance. Both models remained sensitive to pharmacologic inhibition of either TWIST1 or glycolysis at the time of acquired resistance to MET TKIs. These findings suggest that continued upregulation of the TWIST1-HK2 axis is required for acquired MET TKI resistance. In summary, these studies suggest a targetable, metabolic reprogramming in MET altered LUAD BM mediated through a novel TWIST1-HK2 pathway. Citation Format: Purva H. Rumde, Kasey R. Cargill, Vinod Kumar, Princey Devadassan, Jason Chen, Leviticus McGraw-Sapp, Xiaxuan Wu, Sanja Dacic, Riyue Bao, Bharathri Sivakama, Eric S. Goetzman, Sameer Agnihotri, Steven J. Mullett, Stacey G. Wendell, Laura P. Stabile, Timothy F. Burns. Targeting a novel TWIST1-Hexokinase II pathway to overcome MET TKI resistance in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4743.

Abstract 432: The noncanonical mechanism of metabolic enzyme LDHA in cancer progression

Abstract Metabolic reprogramming is a hallmark of cancer cells and a key contributor to cancer progression, which is a promising target for cancer therapies. The enhanced glycolysis, also known as the Warburg effect, represents the most widely recognized metabolic alteration in cancer, wherein the majority of cancer cells undergoes reprogramming to heavily rely on glycolysis for their accelerated growth and proliferation. The glycolytic enzyme lactate dehydrogenase A (LDHA) catalyzes the conversion of pyruvate to lactate in the final stage of glycolysis. LDHA is frequently overexpressed in cancers, which plays a critical role in promoting glycolysis and cancer progression. To deepen our understanding of the oncogenic mechanism of LDHA, we screened for LDHA-interacting proteins in human breast cancer cells by liquid chromatography mass spectrometry (LC/MS) analysis. Through this strategy, small GTPase Rac1 was identified as an LDHA-interacting protein. LDHA interacted with the active form of Rac1, Rac1-GTP, and impaired the interaction of Rac1-GTP with its negative regulators, GTPase-activating proteins, leading to Rac1 activation independently of the glycolytic enzyme activity of LDHA. In addition, LDHA overexpression was significantly associated with increased Rac1 activity in clinical breast cancer specimens. Inhibiting Rac1 significantly attenuated the oncogenic effect of LDHA. Combining small-molecule inhibitors targeting LDHA enzyme activity and Rac1 activity displayed a synergistic inhibitory effect on breast cancers with LDHA overexpression in mouse models. Thus, our results revealed a novel and critical noncanonical mechanism of LDHA in promoting cancer progression through its direct interaction and activation of Rac1. Given that LDHA is frequently overexpressed in cancer, LDHA overexpression constitutes an important mechanism contributing to aberrant Rac1 activation in cancer. Importantly, our findings also suggest that pharmacologically targeting LDHA enzyme activity and Rac1 activity simultaneously is a promising strategy to treat breast cancers with LDHA overexpression. Citation Format: Juan Liu, Wenwei Hu, Zhaohui Feng. The noncanonical mechanism of metabolic enzyme LDHA in cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 432.

Monocarboxylate transporter 4 deficiency enhances high-intensity interval training-induced metabolic adaptations in skeletal muscle.

High-intensity exercise stimulates glycolysis, subsequently leading to elevated lactate production within skeletal muscle. While lactate produced within the muscle is predominantly released into the circulation via the monocarboxylate transporter 4 (MCT4), recent research underscores lactate's function as an intercellular and intertissue signalling molecule. However, its specific intracellular roles within muscle cells remains less defined. In this study, our objective was to elucidate the effects of increased intramuscular lactate accumulation on skeletal muscle adaptation to training. To achieve this, we developed MCT4 knockout mice and confirmed that a lack of MCT4 indeed results in pronounced lactate accumulation in skeletal muscle during high-intensity exercise. A key finding was the significant enhancement in endurance exercise capacity at high intensities when MCT4 deficiency was paired with high-intensity interval training (HIIT). Furthermore, metabolic adaptations supportive of this enhanced exercise capacity were evident with the combination of MCT4 deficiency and HIIT. Specifically, we observed a substantial uptick in the activity of glycolytic enzymes, notably hexokinase, glycogen phosphorylase and pyruvate kinase. The mitochondria also exhibited heightened pyruvate oxidation capabilities, as evidenced by an increase in oxygen consumption when pyruvate served as the substrate. This mitochondrial adaptation was further substantiated by elevated pyruvate dehydrogenase activity, increased activity of isocitrate dehydrogenase - the rate-limiting enzyme in the TCA cycle - and enhanced function of cytochrome c oxidase, pivotal to the electron transport chain. Our findings provide new insights into the physiological consequences of lactate accumulation in skeletal muscle during high-intensity exercises, deepening our grasp of the molecular intricacies underpinning exercise adaptation. KEY POINTS: We pioneered a unique line of monocarboxylate transporter 4 (MCT4) knockout mice specifically tailored to the ICR strain, an optimal background for high-intensity exercise studies. A deficiency in MCT4 exacerbates the accumulation of lactate in skeletal muscle during high-intensity exercise. Pairing MCT4 deficiency with high-intensity interval training (HIIT) results in a synergistic boost in high-intensity exercise capacity, observable both at the organismal level (via a treadmill running test) and at the muscle tissue level (through an ex vivo muscle contractile function test). Coordinating MCT4 deficiency with HIIT enhances both the glycolytic enzyme activities and mitochondrial capacity to oxidize pyruvate.

Uncoordinated 51-like kinase 1a/b and 2 in fish Megalobrama amblycephala: Molecular cloning, functional characterization, and their potential roles in glucose metabolism

Uncoordinated (Unc) 51-like kinase (ulk1) and ulk2 are closely involved in autophagy activation, but little is known about their roles in regulating glucose homeostasis. In this study, the genes of ulk1a, ulk1b and ulk2 were cloned and characterized in fish Megalobrama amblycephala. All the three genes shared the approximate N-terminal kinase domain and the C-terminal Atg1-like_tMIT domain structure, while the amino acid sequence identity of them are different between M. amblycephala and other vertebrates. Their transcripts were widely observed in various tissues (brain, muscle, gill, heart, spleen, eye, liver, intestine, abdominal adipose and kidney), but differed in tissue expression patterns. During the glucose tolerance test and the insulin tolerance test, the up-regulated transcriptions of ulk1a, ulk1b and ulk2 were all found despite some differences in the temporal patterns. At the same time, the activities of glycolytic enzymes like hexokinase and phosphofructokinase both showed parallel increases. Furthermore, the feeding of a high-carbohydrate diet decreased the transcriptions of ulk1a, ulk1b and ulk2. Collectively, this study demonstrated that ulk1a, ulk1b and ulk2 in M. amblycephala had similar molecular characterizations, but with different conservation and tissue expression patterns. In addition, ulk1/2 might play important roles in maintaining the glucose homeostasis in fish through regulating the glycolytic pathway.

Potential Effects of NO-Induced Hypoxia-Inducible Factor-1α on Yak Meat Tenderness during Post-Mortem Aging.

The objective of this study was to investigate the mechanism underlying nitric oxide (NO)-induced hypoxia-inducible factor-1α (HIF-1α) and its impact on yak muscle tenderness during post-mortem aging. The Longissimus thoracis et lumborum (LTL) muscle of yak were incubated at 4 °C for 0, 3, 6, 9, 12, 24, and 72 h after treatment with 0.9% saline, NO activator, or a combination of the NO activator and an HIF-1α inhibitor. Results indicated that elevated NO levels could increase HIF-1α transcription to achieve stable expression of HIF-1α protein (P < 0.05). Additionally, elevated NO triggered HIF-1α S-nitrosylation, which further upregulated the activity of key glycolytic enzymes, increased glycogen consumption, accelerated lactic acid accumulation, and decreased pH (P < 0.05). These processes eventually improved the tenderness of yak muscle during post-mortem aging (P < 0.05). The results demonstrated that NO-induced activation of HIF-1α S-nitrosylation enhanced glycolysis during post-mortem aging and provided a possible pathway for improving meat tenderness.

SARS-CoV-2 Infection and Anemia-A Focus on RBC Deformability and Membrane Proteomics-Integrated Observational Prospective Study.

The multifaceted impact of COVID-19 extends beyond the respiratory system, encompassing intricate interactions with various physiological systems. This study elucidates the potential association between SARS-CoV-2 infection and anemia, with a particular emphasis on the deformability of red blood cells (RBCs), stability of hemoglobin, enzymatic activities, and proteomic profiles. The study encompasses a cohort of 74 individuals, including individuals positive for COVID-19, a control group, and patients with other viral infections to discern the specific effects attributable to COVID-19. The analysis of red blood cells was focused on deformability measured by osmotic gradient ektacytometry, hemoglobin stability, and glycolytic enzyme activity. Furthermore, membrane proteins were examined using advanced proteomics techniques to capture molecular-level changes. Findings from the study suggest a correlation between anemia and exacerbated outcomes in COVID-19 patients, marked by significant elevations in d-dimer, serum procalcitonin, creatinine, and blood urea nitrogen (BUN) levels. These observations suggest that chronic kidney disease (CKD) may play a role in the development of anemia in COVID-19 patients, particularly those of advanced age with comorbidities. Furthermore, the proteomic analyses have highlighted a complex relationship between omics data and RBC parameters, enriching our understanding of the mechanisms underlying the disease. This research substantiates the complex interrelationship between COVID-19 and anemia, with a specific emphasis on the potential repercussions of SARS-CoV-2 infection on RBCs. The findings contribute to the growing body of evidence supporting the extensive impact of COVID-19 on RBCs.

Icariin ameliorates glycolytic dysfunction in Alzheimer's disease models by activating the Wnt/β-catenin signaling pathway.

It was reported that the Wnt/β-catenin pathway is involved in the regulation of aerobic glycolysis and that brain glycolytic dysfunction results in the development of Alzheimer's disease (AD). Icariin (ICA), an active component extracted from Epimedii Folium, has been reported to produce neuroprotective effects in multiple models of AD, but its underlying mechanism remains to be fully described. We aimed to investigate the protective effects of ICA on animal and cell models of AD and confirm whether the Wnt/β-catenin pathway has functions in the neuroprotective function of ICA. The 3 × Tg-AD mice were treated with ICA. HT22 cells, the Aβ25-35 peptide and Dickkopf-1 (DKK1) agent (a specific inhibitor of the Wnt/β-catenin pathway) were used to further explore the underlying mechanism of ICA that produces anti-AD effects. Behavioral examination, western blotting assay, staining analysis, biochemical test, and lactate dehydrogenase (LDH) assays were applied. We first demonstrated that ICA significantly improved cognitive function and autonomous behavior, reduced neuronal damage, and reversed the protein levels and activities of glycolytic key enzymes, and expression of protein molecules of the canonical Wnt signaling pathway, in 3 × Tg-AD mice back to wild-type levels. Next, we further found that ICA increased cell viability and effectively improved the dysfunctional glycolysis in HT22 cells injured by Aβ25-35 . However, when canonical Wnt signaling was inhibited by DKK1, the above effects of ICA on glycolysis were abolished. In summary, ICA exerts neuroprotective effects in 3 × Tg-AD animals and AD cellular models by enhancing the function of glycolysis through activation of the Wnt/β-catenin pathway.

Hexokinase-linked glycolytic overload and unscheduled glycolysis in hyperglycemia-induced pathogenesis of insulin resistance, beta-cell glucotoxicity, and diabetic vascular complications.

Hyperglycemia is a risk factor for the development of insulin resistance, beta-cell glucotoxicity, and vascular complications of diabetes. We propose the hypothesis, hexokinase-linked glycolytic overload and unscheduled glycolysis, in explanation. Hexokinases (HKs) catalyze the first step of glucose metabolism. Increased flux of glucose metabolism through glycolysis gated by HKs, when occurring without concomitant increased activity of glycolytic enzymes-unscheduled glycolysis-produces increased levels of glycolytic intermediates with overspill into effector pathways of cell dysfunction and pathogenesis. HK1 is saturated with glucose in euglycemia and, where it is the major HK, provides for basal glycolytic flux without glycolytic overload. HK2 has similar saturation characteristics, except that, in persistent hyperglycemia, it is stabilized to proteolysis by high intracellular glucose concentration, increasing HK activity and initiating glycolytic overload and unscheduled glycolysis. This drives the development of vascular complications of diabetes. Similar HK2-linked unscheduled glycolysis in skeletal muscle and adipose tissue in impaired fasting glucose drives the development of peripheral insulin resistance. Glucokinase (GCK or HK4)-linked glycolytic overload and unscheduled glycolysis occurs in persistent hyperglycemia in hepatocytes and beta-cells, contributing to hepatic insulin resistance and beta-cell glucotoxicity, leading to the development of type 2 diabetes. Downstream effector pathways of HK-linked unscheduled glycolysis are mitochondrial dysfunction and increased reactive oxygen species (ROS) formation; activation of hexosamine, protein kinase c, and dicarbonyl stress pathways; and increased Mlx/Mondo A signaling. Mitochondrial dysfunction and increased ROS was proposed as the initiator of metabolic dysfunction in hyperglycemia, but it is rather one of the multiple downstream effector pathways. Correction of HK2 dysregulation is proposed as a novel therapeutic target. Pharmacotherapy addressing it corrected insulin resistance in overweight and obese subjects in clinical trial. Overall, the damaging effects of hyperglycemia are a consequence of HK-gated increased flux of glucose metabolism without increased glycolytic enzyme activities to accommodate it.

Effect of glycolysis on water holding capacity during postmortem aging of Jersey cattle-yak meat.

Postmortem muscle moisture loss leads to a decrease in carcass weight and can adversely impact overall meat quality. Therefore, it is critical to investigate water holding capacity (WHC) to enhance meat quality. Current research has primarily focused on examining the correlation between signaling molecules and meat quality in relation to the glycolysis effect on muscle WHC. But there exists a significant knowledge gap regarding the mechanism of WHC in Jersey cattle-yak meat. Jersey cattle-yak meat pH decreased and then increased during postmortem aging. Lactate content, cooking loss, pressing loss, drip loss and centrifuging loss of Jersey cattle-yak meat increased and then decreased during postmortem aging. The glycogen content of Jersey cattle-yak meat was significantly higher than that of yak meat at 6-120 h, being 8.40% higher than that of yak meat at 120 h. The activity of key glycolytic enzymes hexokinase (HK), pyruvate kinase (PK), phosphofructokinase (PFK) and lactate dehydrogenase (LDH) in Jersey cattle-yak meat was lower than that in yak meat. Correlation analysis showed that Jersey cattle-yak meat WHC was positively correlated with the activity of HK, PK, PFK and LDH. The WHC of Jersey cattle-yak meat was higher than that of Gannan yak meat, and it was significantly positively correlated with the activity of key enzymes of the glycolytic signaling pathway. Therefore, the glycolysis rate can be reduced by inhibiting enzyme activity to improve Jersey cattle-yak meat WHC and meat quality. © 2023 Society of Chemical Industry.

Glycolytic enzymes in non-glycolytic web: functional analysis of the key players.

To survive in the tumour microenvironment, cancer cells undergo rapid metabolic reprograming and adaptability. One of the key characteristics of cancer is increased glycolytic selectivity and decreased oxidative phosphorylation (OXPHOS). Apart from ATP synthesis, glycolysis is also responsible for NADH regeneration and macromolecular biosynthesis, such as amino acid biosynthesis and nucleotide biosynthesis. This allows cancer cells to survive and proliferate even in low-nutrient and oxygen conditions, making glycolytic enzymes a promising target for various anti-cancer agents. Oncogenic activation is also caused by the uncontrolled production and activity of glycolytic enzymes. Nevertheless, in addition to conventional glycolytic processes, some glycolytic enzymes are involved in non-canonical functions such as transcriptional regulation, autophagy, epigenetic changes, inflammation, various signaling cascades, redox regulation, oxidative stress, obesity and fatty acid metabolism, diabetes and neurodegenerative disorders, and hypoxia. The mechanisms underlying the non-canonical glycolytic enzyme activities are still not comprehensive. This review summarizes the current findings on the mechanisms fundamental to the non-glycolytic actions of glycolytic enzymes and their intermediates in maintaining the tumor microenvironment.

Comparative effects of phosphorylation and acetylation on glycolysis and myofibrillar proteins degradation in postmortem muscle

The study investigated the different effects between protein phosphorylation and acetylation on glycolytic enzyme activity and myofibrillar protein degradation. Lamb longissimus thoracis lumborum muscles were homogenized and then inhibitors were added for incubation at 4 °C. Phosphatase inhibitor was added to produce a high phosphorylation level (PI group) and lysine deacetylase inhibitor was added to produce a high acetylation level (DI group). The lactate and ATP content in the PI group was inhibited compared with that in the DI group (P < 0.05). Phosphofructokinase (PFK) activity was negatively related with the phosphorylation level and was positively related with the acetylation level in the DI group (P < 0.05). The degradation of troponin T and desmin of the DI group were restrained when compared to that in the PI group (P < 0.05). Compared with initial PFK and desmin, the simulation of phosphorylation and acetylation of PFK and desmin showed different electrostatic potential at the surface and a more unstable structure. The phosphorylation level of the DI group was increased, suggesting that the changes of protein acetylation altered protein phosphorylation. In conclusion, compared with protein phosphorylation, protein acetylation had a greater effect on promoting glycolysis and inhibiting protein degradation.