Anaerobic Glycolysis Research Articles

A randomized, double-blind, placebo-controlled, mechanistic trial on the safety, tolerability and pharmacodynamics of ninerafaxstat in patients with angina and chronic coronary syndrome

Abstract Background/Introduction Symptoms of angina resulting from reversible myocardial ischemia in chronic coronary syndromes have a negative impact on quality of life. Metabolic alterations are a key feature of ischemia, including reduced ATP generation by mitochondrial oxidative phosphorylation, NAD+ depletion, anaerobic glycolysis, proton & lactate accumulation leading to contractile dysfunction. Ninerafaxstat is a novel cardiac mitotrope that shifts metabolism from fatty acids towards glucose utilization, optimizing myocardial energy production during ischemia. Purpose We aimed to investigate the safety, tolerability, impact on myocardial glucose utilization and preliminary anti-ischemic efficacy of ninerafaxstat administered for 8 weeks to patients with symptomatic chronic stable angina who were on anti-anginal therapy and had evidence of myocardial stress hypoperfusion using 15O-H2O PET imaging. Methods IMPROVE-Ischemia was an international, randomized, double-blind, placebo-controlled mechanistic trial comparing ninerafaxstat vs. placebo in patients with symptomatic chronic coronary syndrome. The primary endpoint was the safety and tolerability of ninerafaxstat. Key efficacy evaluations were undertaken at baseline and end-of-treatment (Week 8) including imaging core laboratory assessments of 15O-H2O PET perfusion rest and stress imaging to quantify absolute myocardial blood flow (MBF), dobutamine stress echo with contrast for evaluation of LV contractile response during demand stress using the wall motion score index (WMSI) and, in a subgroup of subjects, 18F-FDG-PET imaging of myocardial glucose metabolism. Results The study randomized 58 subjects (mean age 68.6±7.1 years) with symptomatic stable angina and inducible hypoperfusion from 4 sites across 3 countries (Denmark, Finland, Sweden). 22% had type 2 diabetes and 50% had undergone percutaneous and/or surgical coronary revascularization. The majority (68%) were on ≥2 (up to 4) regular anti-anginals. Ninerafaxstat was well tolerated without impact on systemic hemodynamics. At Week 8, ninerafaxstat significantly increased myocardial glucose utilization and, in those with dobutamine-induced deterioration in wall motion at baseline assessment (n=25, pre-specified subgroup), resulted in a ~50% improvement in mean and peak stress WMSI, without altering rest or hyperemic stress MBF (Table 1). Conclusion(s) Treatment with ninerafaxstat was well-tolerated and showed a neutral hemodynamic profile. Ninerafaxstat significantly increased myocardial glucose utilization consistent with its mechanism of action and improved myocardial contractile response to dobutamine stress in subjects with baseline stress inducible deterioration of wall motion. These findings support a direct, metabolically-mediated, anti-ischemic effect of ninerafaxstat on top of existing anti-anginal agents, supporting its further development in cardiac pathologies characterised by ischemia.

MICU2 up-regulation enhances tumor aggressiveness and metabolic reprogramming during colorectal cancer development.

The mitochondrial Ca2+ uniporter (MCU) plays crucial role in intramitochondrial Ca2+ uptake, allowing Ca2+-dependent activation of oxidative metabolism. In recent decades, the role of MCU pore-forming proteins has been highlighted in cancer. However, the contribution of MCU-associated regulatory proteins mitochondrial calcium uptake 1 and 2 (MICU1 and MICU2) to pathophysiological conditions has been poorly investigated. Here, we describe the role of MICU2 in cell proliferation and invasion using in vitro and in vivo models of human colorectal cancer (CRC). Transcriptomic analysis demonstrated an increase in MICU2 expression and the MICU2/MICU1 ratio in advanced CRC and CRC-derived metastases. We report that expression of MICU2 is necessary for mitochondrial Ca2+ uptake and quality of the mitochondrial network. Our data reveal the interplay between MICU2 and MICU1 in the metabolic flexibility between anaerobic glycolysis and OXPHOS. Overall, our study sheds light on the potential role of the MICUs in diseases associated with metabolic reprogramming.

Investigating the effects of empagliflozin on myocardial metabolic and lipid profile in healthy and metabolic syndrome murine models following acute myocardial infarction

Abstract Background/Introduction Empagliflozin (EMPA) is a sodium/glucose co-transporter type 2 inhibitor with clinically documented cardioprotective effect. However, the mechanism of cardioprotection remains elusive. We have previously shown that Western diet induces metabolic syndrome (MS) and deteriorates cardiac function in mice, whereas EMPA treatment for 6 weeks prevents this damage. Also, EMPA administration for 6 weeks suppresses ischemia/reperfusion (I/R) injury in mice, independent of the presence of MS. Purpose The aim of the present study is to investigate whether changes in myocardial metabolic and lipid profile, induced by EMPA treatment, are responsible for its cardioprotective effect in the setting of myocardial I/R injury. Methods C57Bl6 mice received chow diet or Western diet for 14 weeks. At week 8, mice from each dietary group were randomized into 3 subgroups: 1. Control - Sham surgery (5% DMSO-SHAM), 2. Control - I/R (5% DMSO-I/R) and 3. EMPA (10mg/kg/day in 5% DMSO) - I/R (EMPA-I/R) and received the corresponding treatment by daily per os administration for 6 weeks. At week 14, mice were subjected to 30 minutes of I, followed by 2 hours of R, or sham surgery. Myocardial tissue was harvested at the end of R, and metabolomic and lipidomic analysis was performed by nuclear magnetic resonance spectroscopy and mass spectrometry, respectively. Body weight, as well as blood glucose and cholesterol levels were assessed at baseline and at the end of the administrations. Results EMPA administration reduced body weight, as well as blood glucose and cholesterol levels in animals with MS. EMPA decreased anaerobic glycolysis products (UDP-glucose, Lactic acid) and ketone bodies (β-hydroxybutyrate, acetoacetic acid) in both healthy animals and mice with MS. Additionally, an increase in ATP, accompanied by a decrease in AMP and ADP were observed in the EMPA-I/R group, compared to the DMSO-I/R group. Regarding the lipidomic analysis, gangliosides GM3 emerged as the major lipid group decreased by EMPA treatment. Lastly, despite significant changes were presented in the metabolic and lipid profile between healthy animals and those with MS, the effects of EMPA on myocardial metabolic and lipid profile were the same, regardless of the presence of MS. Conclusion(s) EMPA administration induces metabolic changes in the heart tissue and improves myocardial energetics upon myocardial I/R injury. Also, EMPA suppresses gangliosides GM3, a lipid group that has been associated with neutrophil activation and recruitment. Importantly, the effects of EMPA on myocardial metabolic and lipid profile following I/R is independent of the presence of MS.

Metabolic profile in elite badminton match play and training drills.

Aim of the study was to analyze the metabolic profile of badminton matches and training drills. Therefore, 11 male (23.2±3.8years, 182±7cm, 74.4±8.4kg) and five female (19.3±1.5years, 170±6cm, 62.6±9.2kg) elite badminton players participated in either a training match (TM; n=7) and/or three protocols of multifeeding drills (T10, T30, T50; n=13), that varied in interval and rest durations (10s/10s, 30s/30s, 50s/50s). Absolute and relative energetic costs (Wtot and Etot) and contribution to oxidative (WOxid), phosphagen (WPCr), and anaerobic glycolytic (WLa) metabolism were calculated by the three-component PCr-La-O2-method based on an indirect calorimetric approach from oxygen consumption during exercise, post exercise, and net blood lactate concentration. A novel intermittent approach was used to consider replenishment of phosphocreatine during each resting phase. Results show that during TM, Etot was 676±98J·kg-1min-1, while metabolic pathways contributed by 56.9±8.6% (WOxid), 42.7±8.7% (WPCr), and 0.4±0.6% (WLa). In the multifeeding drills Etot was comparable between T10 (1020±160J·kg-1min-1) and T30 (985±173J·kg-1min-1) but higher in T50 (1266±194J·kg-1min-1) (p<0.001). Relative contribution of WOxid was lower in T10 (47.3±7.7%) but similar in T30 (56.5±6.2%) and T50 (57.3±6.0%) (p<0.001). WPCr was highest in T10 (51.1±8.3%) followed by T30 (42.2±6.9%) and lowest in T50 (31.2±7.7%) (p<0.001). WLa was similar between T10 (1.6±1.0%) and T30 (2.1±1.0%) but higher in T50 (11.6±4.8%) (p<0.001). Concludingly, metabolic costs in badminton are predominantly covered by oxidative and phosphagen energetic pathways. Metabolic profiles of the multifeeding drills differ depending on rally/interval duration, with increasing contribution of anaerobic glycolysis and decreasing phosphagen contribution in case of longer intervals.

6885 Glucose Utilization Differs Between RAS-like and BRAF-like Thyroid Cancer

Abstract Disclosure: S. Kumari: None. E. Chuki: None. J. Klubo-Gwiezdzinska: None. Background: RAS-like thyroid cancer (TC) tends to be more differentiated and less [1][8]Fluorodeoxyglucose Positron Emission Tomography-avid as compared with BRAF-like TC. Therefore, we hypothesized that glucose metabolism may differ between RAS-like and BRAF-like tumors and performed a study aimed at metabolomic profiling of TC. Methods: Based on Institutional Review Board approved protocol, we obtained fresh frozen human TC samples with corresponding normal tissue and performed targeted metabolomics including oxidative phosphorylation (OXPHOS), glycolysis and tricarboxylic acid (TCA) markers using mass spectrometry (MS). The Trusight 500 gene panel was used to determine molecular signature of tumors. Similarly, we performed targeted metabolomics of 4 BRAF-like and 2 RAS-like human TC cell lines. The data were analyzed using Ingenuity Pathway Analysis (Qiagen). We also analyzed mRNA expression of OXPHOS and glycolytic enzymes in 496 human TC tissue samples of The Cancer Genome Atlas (TCGA). The division into BRAF-like and RAS-like TC was based on well-established BRAF-RAS score (BRS) ranging from -1 to +1 for BRAF-like and RAS-like TC, respectively. Results: The study cohort consisted of 20 patients, 13 with BRAF-like and 7 with RAS-like tumors, aged 43±12 years old, 90% (18/20) females, with an average tumor size of 3±1.9 cm, multifocal growth in 55% (11/20) patients, central neck lymph node (LN) metastases in 55% (11/20), lateral neck LN metastases in 15% (3/20) patients and no distant metastases. The tumor size tended to be larger in RAS-like cohort (BRAF-like 2.27±1.28 vs RAS-like 4.34±2.52, p=0.08), and LN metastases more prevalent in BRAF-like cohort (BRAF-like 45% vs RAS-like 10%, p=0.08). Metabolomics data revealed that compared with normal thyroid, human TC tissue samples were characterized by an increased glucose metabolism (p&amp;lt;0.001), TCA (p&amp;lt;0.001) and gluconeogenesis (p&amp;lt;0.001). RAS-like tumors were characterized by a significantly higher TCA (p&amp;lt;0.001) and lower gluconeogenesis (p&amp;lt;0.001) as compared with BRAF-like tumors. Consistently, we found that TCA (p&amp;lt;0.001) and glucose metabolism (p&amp;lt;0.001) was higher in RAS-like vs BRAF-like cell lines. Transcriptomic profiling revealed that RAS-like TC was characterized by an increased expression of majority of tested OXPHOS enzymes compared to BRAF-like tumors, as evidenced by positive correlation with BRS, ranging from r = 0.28 to 0.54, p&amp;lt;0.001. Consistently, BRAF-like TC was associated with an upregulated mRNA expression of anaerobic glycolysis markers, with lactate dehydrogenase A, characterized by the highest negative correlation (r = -0.6, p&amp;lt;0.001) with BRS. Conclusion:RAS-like TC is more likely to utilize glucose through Krebs cycle, while BRAF-like tumors are more prone towards anaerobic glycolysis. Therapeutic strategies based on diverse metabolic profiles may provide a personalized approach to TC therapy. Presentation: 6/3/2024

Smartphone-based lateral flow immunoassay for sensitive determination of lactate dehydrogenase at the point of care

Lactate dehydrogenase (LDH), a prevalent enzyme involved in anaerobic glycolysis, is released into body fluids following cell damage and has long been a general marker of tissue injury. However, due to its lack of selectivity and the advent of more accurate biomarkers, the clinical utility of LDH has been largely limited to confirming hemolysis. LDH has been recognized as a valuable prognostic biomarker for various cancers, making its monitoring crucial during cancer management. Traditional LDH methods include spectrophotometric analysis of NADH at 340 nm, native electrophoresis, or enzyme-linked immunosorbent assay. This study presents the first lateral flow immunoassay (LFIA) for the smartphone-based quantification of serum LDH levels at the point of care. Highly-affinity and specific antibodies have been produced, with 5 nM equilibrium dissociation constant and no cross-reactivity with human serum albumin and human immunoglobulin G. Utilizing carbon nanoparticles as signal transducers significantly enhanced the quantification limit 55-fold, compared to the conventional gold nanoparticles-based LFIA, achieving a quantification limit of 1.5 ng mL−1. The developed assay demonstrated a mean recovery rate of 115 ± 21 % when evaluating LDH-spiked serum samples. This method can be an interesting home-testing tool for monitoring cancer progression or therapy effectiveness.

Association of Weight-indexed Conventional Ultrafiltration Volume with Post-operative Lactate in Patients Undergoing Cardiopulmonary Bypass.

Conventional ultrafiltration (CUF) during cardiopulmonary bypass (CPB) is utilized to minimize hemodilution. However, removing high volumes leads to tissue hypoperfusion by activating the anaerobic glycolysis pathways. This study aimed to determine the association between weight-indexed CUF volumes and lactate in patients who underwent coronary artery bypass grafting (CABG). In this single-center retrospective study, 641 CABG patients, who were referred to Al-Zahra Hospital (Shiraz, Iran) and underwent CPB, during 2019-2021, were recruited. Peri-operative parameters were extracted from the patient's records. The patients with non-elective status, pre-existing liver and renal diseases, ejection fraction<35%, and repeated sternotomy were excluded from the study. An increase in post-operative lactate level≥4 mmol/L after 6 hours was defined as hyperlactatemia (HL). To predict HL, univariable and multiple logistic regression modeling, while controlling confounding factors, were employed. The patients' mean age was 58.8±11.1 years, and 39.2% were women. The incidence of HL was 14.5% (93 patients). There was a significant association between weight-indexed CUF volume and HL. The volume removed in the HL patients was almost doubled (43.37±11.32 vs. 21.41±8.15 mL/Kg, P<0.001), and the higher the weight-indexed CUF volume, the more likely to develop an HL at a rate of 1.38 (Odds ratio=1.38 [1.27-1.49], 95% CI, P<0.001). Furthermore, the multiple logistic regression model showed that HL was associated with the lowest mean arterial pressure (MAP) during CPB. A higher volume of ultrafiltration was associated with increased post-operative serum lactate levels.

Immunometabolic effects of lactate on humoral immunity in healthy individuals of different ages

Aging is characterized by chronic systemic inflammation and metabolic changes. We compare the metabolic status of B cells from young and elderly donors and found that aging is associated with higher oxygen consumption rates, and especially higher extracellular acidification rates, measures of oxidative phosphorylation and of anaerobic glycolysis, respectively. Importantly, this higher metabolic status, which reflects age-associated expansion of pro-inflammatory B cells, is found associated with higher secretion of lactate and autoimmune antibodies after in vitro stimulation. B cells from elderly individuals induce in vitro polarization of CD4+ T cells from young individuals into pro-inflammatory CD4+ T cells through metabolic pathways mediated by lactate, which can be inhibited by targeting lactate enzymes and transporters, as well as signaling pathways supporting anaerobic glycolysis. Lactate also induces immunosenescent B cells that are glycolytic, express transcripts for multiple pro-inflammatory molecules, and are characterized by a higher metabolic status. These results altogether may have relevant clinical implications and suggest alternative targets for therapeutic interventions in the elderly and patients with inflammatory conditions and diseases.

Modulating glucose metabolism and relieving hypoxia for enhanced Chemo/Photoimmunotherapy with photosensitizing enzymatic nanodrugs

Photoimmunotherapy by photodynamic therapy (PDT) induces immunogenic cell death (ICD), providing anti-tumor treatment in a safe, precise, minimally invasive way. However, PDT-demanded massive oxygen consumption exacerbates the hypoxia and creates an acidic tumor microenvironment (TME), leading to an anaerobic glycolysis and immune escape condition. In this study, a natural anti-tumor resveratrol (RSV) nanodrug was introduced via Fe3+ coordination flash nanoprecipitation (cFNP) for enhanced aqueous solubility to specifically downregulate the expression of Pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA), reduce the glucose resorption and lactate production, tipping the cellular balance from anaerobic glycolysis to oxidative phosphorylation (OXPHOS). RSV enhances the immunotherapy by suppressing tumor cell glycolysis and active the apoptotic cell death as well. Meanwhile, structural linker Fe3+ could also effectively catalyze the endogenous H2O2 in the tumor cells to generate oxygen in situ and relieve the hypoxic microenvironment, enhancing the PDT efficacy. This strategy can effectively modulate glucose metabolism and relieve hypoxia by Fe/RSV/ZnPc nanodrugs to greatly promote the synergistic chemo/photoimmunotherapy, providing a promising strategy to tackle the formulation of multi-therapeutic nanodrugs for clinical cancer therapy.

Anaerobic Glycolysis and Ischemic Stroke: From Mechanisms and Signaling Pathways to Natural Product Therapy.

Ischemic stroke is a serious condition that results in high rates of illness and death. Anaerobic glycolysis becomes the primary means of providing energy to the brain during periods of low oxygen levels, such as in the aftermath of an ischemic stroke. This process is essential for maintaining vital brain functions and has significant implications for recovery following a stroke. Energy supply by anaerobic glycolysis and acidosis caused by lactic acid accumulation are important pathological processes after ischemic stroke. Numerous natural products regulate glucose and lactate, which in turn modulate anaerobic glycolysis. This article focuses on the relationship between anaerobic glycolysis and ischemic stroke, as well as the associated signaling pathways and natural products that play a therapeutic role. These natural products, which can regulate anaerobic glycolysis, will provide new avenues and perspectives for the treatment of ischemic stroke in the future.

‘Glycolysis’- Its Role in Prosthodontics- A Narrative Review

Glycolysis, found in almost all organisms, is a metabolic pathway providing anaerobic energy. The Embden- Meyerhof pathway is also known by another name. The procedure involves the oxidation of glucose molecules, the most important organic fuel in plants, microbes, and animals. During glycolysis, 2 ATP molecules are used up, resulting in the production of 4 ATP, 2 NADH, and 2 pyruvates for every glucose molecule. Pyruvate can be utilized in the citric acid cycle or act as a precursor for additional reactions. Glycolysis involves two stages that take place within the cytosol of cells. The initial stage is known as the "investment" phase as it requires two ATP molecules, while the following stage is referred to as the "payoff" phase. All of these reactions are facilitated by their respective enzyme, with phosphofructokinase being crucial for regulating glycolysis speed. The disrupted Glycolysis pathway plays a crucial part in dental conditions. The prosthodontist should be aware of these conditions and strategically plan for patients impacted by these faulty glycolytic conditions. Key Words: Glycolysis; Aerobic Glycolysis; Anaerobic Glycolysis; Dysregulated Glycolysis.

Cellular metabolism changes in atherosclerosis and the impact of comorbidities.

Cell activation and nutrient dysregulation are common consequences of atherosclerosis and its preceding risk factors, such as hypertension, dyslipidemia, and diabetes. These diseases may also impact cellular metabolism and consequently cell function, and the other way around, altered cellular metabolism can impact disease development and progression through altered cell function. Understanding the contribution of altered cellular metabolism to atherosclerosis and how cellular metabolism may be altered by co-morbidities and atherosclerosis risk factors could support the development of novel strategies to lower the risk of CVD. Therefore, we briefly review disease pathogenesis and the principles of cell metabolic pathways, before detailing changes in cellular metabolism in the context of atherosclerosis and comorbidities. In the hypoxic, inflammatory and hyperlipidemic milieu of the atherosclerotic plaque riddled with oxidative stress, metabolism shifts to increase anaerobic glycolysis, the pentose-phosphate pathway and amino acid use. We elaborate on metabolic changes for macrophages, neutrophils, vascular endothelial cells, vascular smooth muscle cells and lymphocytes in the context of atherosclerosis and its co-morbidities hypertension, dyslipidemia, and diabetes. Since causal relationships of specific key genes in a metabolic pathway can be cell type-specific and comorbidity-dependent, the impact of cell-specific metabolic changes must be thoroughly explored in vivo, with a focus on also systemic effects. When cell-specific treatments become feasible, this information will be crucial for determining the best metabolic intervention to improve atherosclerosis and its interplay with co-morbidities.

A Pyroptosis-Inducing Arsenic(III) Nanomicelle Platform for Synergistic Cancer Immunotherapy.

Immunogenic cell death (ICD) could activate anti-tumor immune responses, which is highly attractive for improving cancer treatment effectiveness. Here, this work reports a multifunctional arsenic(III) allosteric inhibitor Mech02, which induces excessive accumulation of 1O2 through sensitized biocatalytic reactions, leading to cell pyroptosis and amplified ICD effect. After Mech02 is converted to Mech03, it could actualize stronger binding effects on the allosteric pocket of pyruvate kinase M2, further interfering with the anaerobic glycolysis pathway of tumors. The enhanced DNA damage triggered by Mech02 and the pyroptosis of cancer stem cells provide assurance for complete tumor clearance. In vivo experiments prove nanomicelle Mech02-HA NPs is able to activate immune memory effects and raise the persistence of anti-tumor immunity. In summary, this study for the first time to introduce the arsenic(III) pharmacophore as an enhanced ICD effect initiator into nitrogen mustard, providing insights for the development of efficient multimodal tumor therapy agents.

Quality and stability studies of red blood cell concentrates from umbilical cord blood compared to their adult counterparts.

Prematurity is a significant health issue due to its incidence and associated complications. Anemia is common in extremely preterm infants (EPI) and often requires transfusions. Red blood cells (RBC) from adult blood (AB) donors have been linked to oxygen-related complications in EPI, leading to the exploration of cord blood (CB) as an alternative source. However, standardization of CB-RBC manufacturing and comparison with AB-RBC characteristics are necessary before clinical studies can be conducted. This study investigated the quality and characteristics of leukoreduced, gamma-irradiated CB-RBC obtained using a commercial closed system from CB donations not meeting hematopoietic transplantation criteria. CB-RBC units were compared with AB-RBC units, both stored in saline-adenine-glucose-mannitol (SAGM). Various parameters, including hematological and biochemical characteristics, pH, 2,3-DPG levels, blood gases and potential toxicants, were evaluated during storage. CB-RBC units had acceptable initial quality parameters and a hematocrit (55±2%) comparable to AB-RBC. The main finding during storage was a faster rise in hemolysis compared to AB-RBC. Potassium (K+) significantly increased during storage in both sources. As expected, glucose levels decreased, and conversely, lactate levels increased, indicating similar patterns of anaerobic glycolysis during storage. pH decreased, affecting the oxygen dissociation curve due to reduced 2,3-DPG levels. After irradiation at 14 days of storage, CB-RBC were less stable as hemolysis and K+ significantly increased compared to AB-RBC at 24 hours. Phthalate concentrations, indicative of plasticizers, increased during storage, but significantly less in CB compared to AB-RBC. Most metals measured were within acceptable ranges. The quality of CB-RBC during storage is primarily influenced by levels of hemolysis and extracellular K+ content. Based on the analyzed parameters, we suggest that the expiration date for CB-RBC stored with SAGM should be set at 14 days, with transfusion occurring within <24 hours after irradiation.

Loss of STK11 Suppresses Lipid Metabolism and Attenuates KRAS-Induced Immunogenicity in Patients with Non-Small Cell Lung Cancer.

In patients with lung cancer, we demonstrate that KRAS mutations increase tumor immunogenicity; however, KRAS/STK11 co-mutated patients display an immune-excluded phenotype. KRAS/STK11 co-mutated patients also demonstrated significant downregulation of several key lipid metabolism genes, many of which were associated with increased immunogenicity and improved overall survival in KRAS-mutated patients. Hence, alteration to lipid metabolism warrants further study as a potential biomarker and target for therapy in patients with KRAS-mutated lung cancer.

Glycolytic PFKFB3 and Glycogenic UGP2 Axis Regulates Perfusion Recovery in Experimental Hind Limb Ischemia.

Despite being in an oxygen-rich environment, endothelial cells (ECs) use anaerobic glycolysis (Warburg effect) as the primary metabolic pathway for cellular energy needs. PFKFB (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase)-3 regulates a critical enzymatic checkpoint in glycolysis and has been shown to induce angiogenesis. This study builds on our efforts to determine the metabolic regulation of ischemic angiogenesis and perfusion recovery in the ischemic muscle. Hypoxia serum starvation (HSS) was used as an in vitro peripheral artery disease (PAD) model, and hind limb ischemia by femoral artery ligation and resection was used as a preclinical PAD model. Despite increasing PFKFB3-dependent glycolysis, HSS significantly decreased the angiogenic capacity of ischemic ECs. Interestingly, inhibiting PFKFB3 significantly induced the angiogenic capacity of HSS-ECs. Since ischemia induced a significant in PFKFB3 levels in hind limb ischemia muscle versus nonischemic, we wanted to determine whether glucose bioavailability (rather than PFKFB3 expression) in the ischemic muscle is a limiting factor behind impaired angiogenesis. However, treating the ischemic muscle with intramuscular delivery of D-glucose or L-glucose (osmolar control) showed no significant differences in the perfusion recovery, indicating that glucose bioavailability is not a limiting factor to induce ischemic angiogenesis in experimental PAD. Unexpectedly, we found that shRNA-mediated PFKFB3 inhibition in the ischemic muscle resulted in an increased perfusion recovery and higher vascular density compared with control shRNA (consistent with the increased angiogenic capacity of PFKFB3 silenced HSS-ECs). Based on these data, we hypothesized that inhibiting HSS-induced PFKFB3 expression/levels in ischemic ECs activates alternative metabolic pathways that revascularize the ischemic muscle in experimental PAD. A comprehensive glucose metabolic gene qPCR arrays in PFKFB3 silenced HSS-ECs, and PFKFB3-knock-down ischemic muscle versus respective controls identified UGP2 (uridine diphosphate-glucose pyrophosphorylase 2), a regulator of protein glycosylation and glycogen synthesis, is induced upon PFKFB3 inhibition in vitro and in vivo. Antibody-mediated inhibition of UGP2 in the ischemic muscle significantly impaired perfusion recovery versus IgG control. Mechanistically, supplementing uridine diphosphate-glucose, a metabolite of UGP2 activity, significantly induced HSS-EC angiogenic capacity in vitro and enhanced perfusion recovery in vivo by increasing protein glycosylation (but not glycogen synthesis). Our data present that inhibition of maladaptive PFKFB3-driven glycolysis in HSS-ECs is necessary to promote the UGP2-uridine diphosphate-glucose axis that enhances ischemic angiogenesis and perfusion recovery in experimental PAD.

Unraveling cysteine deficiency-associated rapid weight loss.

Forty percent of the US population and 1 in 6 individuals worldwide are obese, and the incidence of this disease is surging globally1,2. Various dietary interventions, including carbohydrate and fat restriction, and more recently amino acid restriction, have been explored to combat this epidemic3-6. We sought to investigate the impact of removing individual amino acids on the weight profiles of mice. Compared to essential amino acid restriction, induction of conditional cysteine restriction resulted in the most dramatic weight loss, amounting to 20% within 3 days and 30% within one week, which was readily reversed. This weight loss occurred despite the presence of substantial cysteine reserves stored in glutathione (GSH) across various tissues7. Further analysis demonstrated that the weight reduction primarily stemmed from an increase in the utilization of fat mass, while locomotion, circadian rhythm and histological appearance of multiple other tissues remained largely unaffected. Cysteine deficiency activated the integrated stress response (ISR) and NRF2-mediated oxidative stress response (OSR), which amplify each other, leading to the induction of GDF15 and FGF21, hormones associated with increased lipolysis, energy homeostasis and food aversion8-10. We additionally observed rapid tissue coenzyme A (CoA) depletion, resulting in energetically inefficient anaerobic glycolysis and TCA cycle, with sustained urinary excretion of pyruvate, orotate, citrate, α-ketoglutarate, nitrogen rich compounds and amino acids. In summary, our investigation highlights that cysteine restriction, by depleting GSH and CoA, exerts a maximal impact on weight loss, metabolism, and stress signaling compared to other amino acid restrictions. These findings may pave the way for innovative strategies for addressing a range of metabolic diseases and the growing obesity crisis.

AutoMitoNetwork: Software for analyzing mitochondrial networks in autofluorescence images to enable label-free cell classification.

High-resolution mitochondria imaging in combination with image analysis tools have significantly advanced our understanding of cellular function in health and disease. However, most image analysis tools for mitochondrial studies have been designed to work with fluorescently labeled images only. Additionally, efforts to integrate features describing mitochondrial networks with machine learning techniques for the differentiation of cell types have been limited. Herein, we present AutoMitoNetwork software for image-based assessment of mitochondrial networks in label-free autofluorescence images using a range of interpretable morphological, intensity, and textural features. To demonstrate its utility, we characterized unstained mitochondrial networks in healthy retinal cells and in retinal cells exposed to two types of treatments: rotenone, which directly inhibited mitochondrial respiration and ATP production, and iodoacetic acid, which had a milder impact on mitochondrial networks via the inhibition of anaerobic glycolysis. For both cases, our multi-dimensional feature analysis combined with a support vector machine classifier distinguished between healthy cells and those treated with rotenone or iodoacetic acid. Subtle changes in morphological features were measured including increased fragmentation in the treated retinal cells, pointing to an association with metabolic mechanisms. AutoMitoNetwork opens new options for image-based machine learning in label-free imaging, diagnostics, and mitochondrial disease drug development.

Modeling Glucose, Insulin, C-Peptide, and Lactate Interplay in Adolescents During an Oral Glucose Tolerance Test.

Lactate is not considered just a "waste product" of anaerobic glycolysis anymore. It has been proved to play a key role in several metabolic diseases, such as in the metabolic dysfunction-associated steatotic liver disease, obesity, and diabetes. The capability of simulating glucose-insulin-lactate interaction would be useful to design and test drugs targeting lactate metabolism in such pathological conditions. Minimal models are available, which describe and quantify glucose-lactate interaction but models to simulate postprandial glucose-insulin-C-peptide-lactate time courses are missing. The aim of this study is to fill this gap. Starting from the Padova Type 2 Diabetes Simulator (T2DS), we first added a description of glucose-lactate kinetics and then created a population of 100 in silico subjects to match glucose-insulin-C-peptide-lactate data of 44 adolescents with/without obesity who underwent a standard oral glucose tolerance test (OGTT) of 75 g. The developed model accurately predicts all molecules time courses, guaranteeing precise model parameter estimates (percent coefficient of variation [CV%] median [25th-75th percentile] = 19 [9-29]%). The generated in silico population shows good agreement with the clinical data in terms of area under the curve (AUC) (P = .6, .6, .9, .6 for glucose, insulin, C-peptide, and lactate, respectively) and parameter distributions (P > .1). We have developed a simulator to describe glucose, insulin, C-peptide, and lactate kinetics during an OGTT, which captures the behavior of a real population of adolescents with/without obesity both in terms of average and intersubject variability. Such simulator can be used to investigate the pharmacodynamics of drugs targeting lactate metabolic pathway in various pathological conditions.

Anti-fatigue activity on male mice of 70% ethanol and aqueous extract of Rusa unicolor antler from East Kalimantan

Energy supply issues, ATP+PC, anaerobic glycolysis, product accumulation, lactic acid, mechanical failure of muscles to contractions and nervous system changes contribute to fatigue. The goal of this study was to determine whether 70% ethanol and aqueous extracts of Rusa unicolor antler from East Kalimantan, Indonesia, can reduce fatigue in male mice. A total of sixty-four male mice aged 2-3 months (20-30 g) were randomly assigned to eight groups (negative control, positive control and 3 doses of treatments for each extract). After that, the swimming endurance time was assessed, and blood glucose levels were measured before and after the test. Mice's tiredness is demonstrated by their head remaining submerged for 4-7 s while their tails and legs are stretched. One-way ANOVA was used to statistically analyze the data. The results showed that there were significant differences between all treatments with the negative control group (p&lt;0.05). At a dose of 1.3 mg/kg BW of 70% ethanol and aqueous extract of Rusa unicolor antlers were able to maintain the stamina of male mice in swimming for 2 hrs 37 mins. Both extracts improve endurance training, delay tiredness, and promote recovery after exercise. Hence, Rusa unicolor antler extract can significantly improve athletic ability and facilitate recovery after exercise in male mice by increasing endurance training capacity and delaying tiredness.