Extracellular flux analyses reveal differences in mitochondrial PBMC metabolism between high-fit and low-fit females

Analyzing metabolism of peripheral blood mononuclear cells (PBMCs) can possibly serve as a cellular metabolic read-out for lifestyle factors and lifestyle interventions. However, the impact of PBMC composition on PBMC metabolism is not yet clear, neither is the differential impact of a longer-term lifestyle factor versus a short-term lifestyle intervention. We investigated the effect of aerobic fitness level and a recent exercise bout on PBMC metabolism in females. PBMCs from 31 young female adults divided into a high-fit (V̇O2peak ≥ 47 mL/kg/min, N = 15) and low-fit (V̇O2peak ≤ 37 mL/kg/min, N = 16) group were isolated at baseline and overnight after a single bout of exercise (60 minutes, 70% V̇O2peak). Oxygen consumption rate (OCR) and glycolytic rate (GR) were measured using extracellular flux (XF) assays and PBMC subsets were characterized using fluorescence-activated cell sorting (FACS). Basal OCR, FCCP-induced OCR, spare respiratory capacity, ATP-linked OCR, and proton leak were significantly higher in high-fit compared to low-fit females (all P < 0.01), while no significant differences in glycolytic rate (GR) were found (all P > 0.05). A recent exercise bout did not significantly affect GR or OCR parameters (all P > 0.05). The overall PBMC composition was similar between high-fit and low-fit females. Mitochondrial PBMC function was significantly higher in PBMCs from high-fit compared to low-fit females, which was unrelated to PBMC composition and not impacted by a recent bout of exercise. Our study reveals a link between PBMC metabolism and levels of aerobic fitness, increasing the relevance of PBMC metabolism as a marker to study the impact of lifestyle factors on human health.

Glycolysis Rate-Limiting Enzymes: Novel Potential Regulators of Rheumatoid Arthritis Pathogenesis

Rheumatoid arthritis (RA) is a classic autoimmune disease characterized by uncontrolled synovial proliferation, pannus formation, cartilage injury, and bone destruction. The specific pathogenesis of RA, a chronic inflammatory disease, remains unclear. However, both key glycolysis rate-limiting enzymes, hexokinase-II (HK-II), phosphofructokinase-1 (PFK-1), and pyruvate kinase M2 (PKM2), as well as indirect rate-limiting enzymes, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), are thought to participate in the pathogenesis of RA. In here, we review the latest literature on the pathogenesis of RA, introduce the pathophysiological characteristics of HK-II, PFK-1/PFKFB3, and PKM2 and their expression characteristics in this autoimmune disease, and systematically assess the association between the glycolytic rate-limiting enzymes and RA from a molecular level. Moreover, we highlight HK-II, PFK-1/PFKFB3, and PKM2 as potential targets for the clinical treatment of RA. There is great potential to develop new anti-rheumatic therapies through safe inhibition or overexpression of glycolysis rate-limiting enzymes.

Altered Metabolic Phenotype of Immune Cells in a Spontaneous Autoimmune Uveitis Model

As one of the leading causes of blindness worldwide, uveitis is an important disease. The exact pathogenesis of autoimmune uveitis is not entirely elucidated to date. Equine recurrent uveitis (ERU) represents the only spontaneous animal model for autoimmune uveitis in humans. As the metabolism of immune cells is an emerging field in research and gains more and more significance to take part in the pathogenesis of various diseases, we conducted experiments to investigate the metabolism of immune cells of ERU cases and healthy controls. To our knowledge, the link between a deviant immunometabolism and the pathogenesis of autoimmune uveitis was not investigated so far. We showed that PBMC of ERU cases had a more active metabolic phenotype in basal state by upregulating both the oxidative phosphorylation and the glycolytic pathway. We further revealed an increased compensatory glycolytic rate of PBMC and CD4+ T cells of ERU cases under mitochondrial stress conditions. These findings are in line with metabolic alterations of immune cells in other autoimmune diseases and basic research, where it was shown that activated immune cells have an increased need of energy and molecule demand for their effector function. We demonstrated a clear difference in the metabolic phenotypes of PBMC and, more specifically, CD4+ T cells of ERU cases and controls. These findings are another important step in understanding the pathogenesis of ERU and figuratively, human autoimmune uveitis.

Investigation of energy metabolic dynamism in hyperthermia-resistant ovarian and uterine cancer cells under heat stress

Despite progress in the use of hyperthermia in clinical practice, the thermosensitivity of cancer cells is poorly understood. In a previous study, we found that sensitivity to hyperthermia varied between ovarian and uterine cancer cell lines. Upon hyperthermia, glycolytic enzymes decreased in hyperthermia-resistant SKOV3 cells. However, the mechanisms of glycolysis inhibition and their relationship with thermoresistance remain to be explored. In this study, metabolomic analysis indicated the downregulation of glycolytic metabolites in SKOV3 cells after hyperthermia. Proteomic and pathway analyses predicted that the ubiquitin pathway was explicitly activated in resistant SKOV3 cells, compared with hyperthermia-sensitive A2780 cells, and STUB1, a ubiquitin ligase, potentially targeted PKM, a glycolytic rate-limiting enzyme. PKM is degraded via ubiquitination upon hyperthermia. Although glycolysis is inactivated by hyperthermia, ATP production is maintained. We observed that oxygen consumption and mitochondrial membrane potential were activated in SKOV3 cells but suppressed in A2780 cells. The activation of mitochondria could compensate for the loss of ATP production due to the suppression of glycolysis by hyperthermia. Although the physiological significance has not yet been elucidated, our results demonstrated that metabolomic adaptation from the Warburg effect to mitochondrial oxidative phosphorylation could contribute to thermoresistance in ovarian and uterine cancer cells.

Rigor development and meat quality of Murrah buffalo from different production systems

The purpose of this study was to evaluate the rigor mortis development and its effects on striploins quality of Murrah buffalos from meat (males) and dairy (females) productions. The rigor was monitored during carcass chilling (up to 22 h postmortem) and the meat physicochemical traits was accessed after 72 h postmortem. Glycolytic rate, measured by decreasing pH and glycogen content, was higher (p < 0.05) in female than in male buffalo, leading to a different onset rigor time (5-6 h and 9-10 h, respectively). Male meat had (p < 0.05) lower fat (1.41 vs 3.58%) and insoluble collagen (1.18 vs 1.58 mg g-1) contents, but higher soluble collagen content (5.16 vs 20.38%), sarcomere length (1.65 vs 1.84 µm), myofibrillar fragmentation (334 vs 295) and shear force (39.38 vs 25.92 N) than females. These results suggest that dairy buffalo meat is more tender and can be used on the market as high-quality meat such as from buffalo raised to meat production.

Lipopolysaccharide promotes metastasis via acceleration of glycolysis by the nuclear factor-κB/snail/hexokinase3 signaling axis in colorectal cancer

Background Cancer cell is generally characterized by enhanced glycolysis. Inflammasome activation is interaction with glycolysis. The concentration of lipopolysaccharide (LPS), a classic inflammasome activator, is significantly higher in colorectal cancer tissue than in normal intestinal mucosa. However, the mechanism of LPS on glycolysis and metastasis has not been fully elucidated. This study aimed to investigate the roles of LPS on inflammasome activation, glycolysis, and metastasis, and unravel metformin’s potential in treatment of CRC. Methods We detected inflammasome activation and cell motility following LPS exposure in CRC cell lines. Glycolysis analysis was performed, and the key glycolytic rate-limiting enzymes were detected. Dual-luciferase reporter gene assay, co-immunoprecipitation, chromatin immunoprecipitation (ChIP) analysis, and ChIP-reChIP assay were performed to identify the specific mechanisms of LPS on glycolysis. Mouse metastasis models were used to determine the effects of LPS and metformin on metastasis. Correlation analysis of the expression of various molecules was performed in 635 CRC samples from The Cancer Genome Atlas and 83 CRC samples from our lab. Results LPS activates caspase-1 through NF-κB and upregulates the expression of Snail and HK3 depending on caspase-1 activation. LPS potentiates migration and invasion depending on accelerated glycolysis, which could be reversed by knockdown of glycolytic rate-limiting enzyme HK3. Nuclear Snail is upregulated by NF-κB under LPS treatment and then forms a complex with NF-κB, then directly binds to the HK3 promoter region to upregulate the expression of HK3. Metformin suppresses the NF-κB/Snail/HK3 signaling axis that is activated by LPS and then inhibits LPS-induced metastasis. In vivo, LPS-treated cells form more metastasis in the lungs of mice, and metformin completely reverses this effect of LPS. Conclusion LPS activates inflammasomes in cancer cells through NF-κB and promotes metastasis through glycolysis enhanced by the NF-κB/Snail/HK3 signaling pathway in CRC. Metformin could prevent this effect of LPS.

ARRB1 Regulates Metabolic Reprogramming to Promote Glycolysis in Stem Cell-Like Bladder Cancer Cells

β-arrestin 1 (ARRB1) is a scaffold protein that regulates signaling downstream of G protein-coupled receptors (GPCRs). In the current work, we investigated the role of ARRB1 in regulating the metabolic preference of cancer stem cell (CSC)-like cells in bladder cancer (BC). We show that ARRB1 is crucial for spheroid formation and tumorigenic potential. Furthermore, we measured mitochondrial respiration, glucose uptake, glycolytic rate, mitochondrial/glycolytic ATP production and fuel oxidation in previously established ARRB1 knock out (KO) cells and corresponding controls. Our results demonstrate that depletion of ARRB1 decreased glycolytic rate and induced metabolic reprogramming towards oxidative phosphorylation. Mechanistically, the depletion of ARRB1 dramatically increased the mitochondrial pyruvate carrier MPC1 protein levels and reduced the glucose transporter GLUT1 protein levels along with glucose uptake. Overexpression of ARRB1 in ARRB1 KO cells reversed the phenotype and resulted in the upregulation of glycolysis. In conclusion, we show that ARRB1 regulates the metabolic preference of BC CSC-like cells and functions as a molecular switch that promotes reprogramming towards glycolysis by negatively regulating MPC1 and positively regulating GLUT1/ glucose uptake. These observations open new therapeutic avenues for targeting the metabolic preferences of cancer stem cell (CSC)-like BC cells.

The Glycolytic Pathway as a Target for Novel Onco-Immunology Therapies in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal forms of human cancer, characterized by unrestrained progression, invasiveness and treatment resistance. To date, there are limited curative options, with surgical resection as the only effective strategy, hence the urgent need to discover novel therapies. A platform of onco-immunology targets is represented by molecules that play a role in the reprogrammed cellular metabolism as one hallmark of cancer. Due to the hypoxic tumor microenvironment (TME), PDA cells display an altered glucose metabolism—resulting in its increased uptake—and a higher glycolytic rate, which leads to lactate accumulation and them acting as fuel for cancer cells. The consequent acidification of the TME results in immunosuppression, which impairs the antitumor immunity. This review analyzes the genetic background and the emerging glycolytic enzymes that are involved in tumor progression, development and metastasis, and how this represents feasible therapeutic targets to counteract PDA. In particular, as the overexpressed or mutated glycolytic enzymes stimulate both humoral and cellular immune responses, we will discuss their possible exploitation as immunological targets in anti-PDA therapeutic strategies.

Inhibition of mitochondrial function by metformin increases glucose uptake, glycolysis and GDF-15 release from intestinal cells

Even though metformin is widely used to treat type2 diabetes, reducing glycaemia and body weight, the mechanisms of action are still elusive. Recent studies have identified the gastrointestinal tract as an important site of action. Here we used intestinal organoids to explore the effects of metformin on intestinal cell physiology. Bulk RNA-sequencing analysis identified changes in hexose metabolism pathways, particularly glycolytic genes. Metformin increased expression of Slc2a1 (GLUT1), decreased expression of Slc2a2 (GLUT2) and Slc5a1 (SGLT1) whilst increasing GLUT-dependent glucose uptake and glycolytic rate as observed by live cell imaging of genetically encoded metabolite sensors and measurement of oxygen consumption and extracellular acidification rates. Metformin caused mitochondrial dysfunction and metformin’s effects on 2D-cultures were phenocopied by treatment with rotenone and antimycin-A, including upregulation of GDF15 expression, previously linked to metformin dependent weight loss. Gene expression changes elicited by metformin were replicated in 3D apical-out organoids and distal small intestines of metformin treated mice. We conclude that metformin affects glucose uptake, glycolysis and GDF-15 secretion, likely downstream of the observed mitochondrial dysfunction. This may explain the effects of metformin on intestinal glucose utilisation and food balance.

Novel standardized method for extracellular flux analysis of oxidative and glycolytic metabolism in peripheral blood mononuclear cells

Analyzing metabolism of peripheral blood mononuclear cells (PBMCs) provides key opportunities to study the pathophysiology of several diseases, such as type 2 diabetes, obesity and cancer. Extracellular flux (XF) assays provide dynamic metabolic analysis of living cells that can capture ex vivo cellular metabolic responses to biological stressors. To obtain reliable data from PBMCs from individuals, novel methods are needed that allow for standardization and take into account the non-adherent and highly dynamic nature of PBMCs. We developed a novel method for extracellular flux analysis of PBMCs, where we combined brightfield imaging with metabolic flux analysis and data integration in R. Multiple buffy coat donors were used to demonstrate assay linearity with low levels of variation. Our method allowed for accurate and precise estimation of XF assay parameters by reducing the standard score and standard score interquartile range of PBMC basal oxygen consumption rate and glycolytic rate. We applied our method to freshly isolated PBMCs from sixteen healthy subjects and demonstrated that our method reduced the coefficient of variation in group mean basal oxygen consumption rate and basal glycolytic rate, thereby decreasing the variation between PBMC donors. Our novel brightfield image procedure is a robust, sensitive and practical normalization method to reliably measure, compare and extrapolate XF assay data using PBMCs, thereby increasing the relevance for PBMCs as marker tissue in future clinical and biological studies, and enabling the use of primary blood cells instead of immortalized cell lines for immunometabolic experiments.