Glycolysis is a key metabolic pathway that breaks down glucose to support cellular energy production. When oxygen supply is limited (hypoxia), mammalian cells rely predominantly on glycolysis to compensate for reduced oxidative phosphorylation. Hypoxia is a common feature of the gastrointestinal epithelium, where the hypoxia-inducible factor (HIF) enhances expression of glycolytic genes. This metabolic shift is exacerbated in cancer, such as colorectal cancer, a hallmark known as the Warburg effect. Recent work suggests hypoxia promotes the assembly of glycolytic enzyme complexes. We hypothesize that hypoxia-induced glycolytic complex formation differs between normal and cancerous colon epithelial cells, and is regulated by post-translational modifications. This study aims to define how hypoxia promotes glycolytic enzyme complex formation in colon epithelial cells and whether this response is amplified in colorectal cancer. To first determine if glycolytic complexes form in vivo, colon tissues were collected from mice kept in hypoxia (9% O 2 ) or atmospheric conditions (21% O 2 ). Immunofluorescence staining revealed an increased colocalization of two key glycolytic enzymes, Phosphofructokinase (PFKP) and Hexokinase 2 (HK2). We quantified these interactions using a proximity ligation assay (PLA), which confirmed a significant hypoxia-dependent increase in HK2-PFKP complex formation. To define molecular drivers of this phenomenon, we next studied glycolytic activity in vitro using primary and cancerous intestinal epithelial cells. Extracellular lactate levels and glycolytic enzyme expression increased after 24 hours of hypoxia (1% O 2 ) in primary and cancer colon cells, but not in primary small intestinal cells, indicating colon-specific sensitivity to hypoxia. Immunoprecipitation and PLA further demonstrated a significant increase in HK2-PFKP interactions in hypoxic primary and cancer colon cells (p< 0.05), with cancer cells exhibiting a greater response than primary cells. In contrast, small intestinal epithelial cells did not show increased enzyme interactions. We next assessed lysine lactylation, a post-translational modification derived from the glycolytic end-product, lactate. Primary colon cells displayed a hypoxia-dependent increase in lactylation, whereas cancer colon cells maintained consistently high levels over time in both normoxia and hypoxia, suggesting lactylation is already maximized in cancer. Future studies will evaluate whether lactylation promotes glycolytic complex assembly in hypoxia. Overall, our findings show that colon epithelial cells exhibit a strong hypoxic glycolytic adaptation involving enzyme complex formation, which is further amplified in colorectal cancer cells. This enhanced metabolic response in the colon may contribute to the higher incidence of colorectal cancer compared to small intestinal malignancies, and highlights a cancer-specific metabolic adaptation with potential as a therapeutic target. Ongoing work will explore how post-translational modifications regulate hypoxia-driven glycolytic organization and metabolic remodelling. Funding agencies: Natural Science and Engineering Research Council of Canada, Science Foundation of Ireland. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Chronic mucosal inflammation in inflammatory bowel disease (IBD) creates a profoundly hypoxic environment that alters epithelial cell metabolism with profound implications for epithelial cell barrier function. Hypoxia stabilizes the hypoxia-inducible factor-1α (HIF-1α) which drives an adaptive metabolic shift towards increased glycolysis, enabling cells to maintain ATP levels when mitochondrial oxidative phosphorylation is reduced due to a lack of oxygen availability. We have shown that hypoxia can also induce the formation of glycolytic complexes that may facilitate substrate channelling, improved metabolic efficiency and cell survival in hypoxia (Kierans et al. 2023). In this study, we investigated for the first time whether these glycolytic complexes formed in healthy and inflamed human mucosal tissue. Work by other groups have suggested a role for long non-coding RNA molecules (lncRNA) as scaffolds for glycolytic complexes. Therefore, we also investigated the possible role of lncRNAs in the formation of glycolytic complexes in IBD. We identified for the first time in vivo, the presence of glycolytic complexes in colonic biopsies collected from control individuals and IBD patients using both immunofluorescence (IF) and proximity ligation assay (PLA). Next, we investigated what microenvironmental feature is driving the formation of these glycolytic complexes in intestinal epithelial cells. We found that hypoxia, rather than inflammation, is the main microenvironmental driver of glycolytic complex formation and glycolytic activity, as confirmed by immunoprecipitation of PFKP followed by HK2 western blot, and via lactate assays. We next investigated whether lncRNAs might contribute to the formation of a hypoxia-induced glycolytic complex. These experiments revealed that disruption of RNA did not impair hypoxia-induced glycolytic complex formation in intestinal epithelial cells. In summary, we have demonstrated that hypoxia induces a glycolytic complex in vivo and that hypoxia induces a glycolytic complex in intestinal epithelial cells independently of RNA. Understanding the drivers and mechanisms of intestinal epithelial cell metabolism will allow us to identify new epithelium-directed therapeutic targets for IBD. 1) Kierans, S.J., et al., Hypoxia induces a glycolytic complex in intestinal epithelial cells independent of HIF-1-driven glycolytic gene expression. Proc Natl Acad Sci U S A, 2023. 120(35): p. e2208117120. Funding agencies: Research Ireland (Taighde Éireann). This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Environmental stressors in the modern world can fundamentally affect human physiology and health. Exposure to stressors like air pollution, heat, and traffic noise has been linked to a pronounced increase in non-communicable diseases. Specifically, aircraft noise has been identified as a risk factor for cardiovascular and metabolic diseases, such as arteriosclerosis, heart failure, stroke, and diabetes. Noise stress leads to neuronal activation with subsequent stress hormone release that ultimately activates the renin-angiotensin-aldosterone system, increases inflammation and oxidative stress thus substantially affecting the cardiovascular system. However, despite the epidemiological evidence of a link between noise stress and metabolic dysfunction, the consequences of exposure at the molecular, metabolic level of the cardiovascular system are largely unknown. Here, we use a murine model system of short-term aircraft noise exposure to show that noise stress profoundly alters heart metabolism. Within 4 days of noise exposure, the heart proteome and metabolome bear the hallmarks of reduced potential for generating ATP from fatty-acid beta-oxidation, the tricarboxylic acid cycle, and the electron transport chain. This is accompanied by the increased expression of glycolytic metabolites, including the end-product, lactate, suggesting a compensatory shift of energy production towards anaerobic glycolysis. Intriguingly, the metabolic shift is reminiscent of what is observed in failing and ischaemic hearts. Mechanistically, we further show that the metabolic rewiring is likely driven by reactive oxygen species (ROS), as we can rescue the phenotype by knocking out NOX-2/gp91phox, a ROS inducer, in mice. Our results suggest that within a short exposure time, the cardiovascular system undergoes a fundamental metabolic shift that bears the hallmarks of cardiovascular disease. These findings underscore the urgent need to comprehend the molecular consequences of environmental stressors, paving the way for targeted interventions to mitigate health risks associated with chronic noise exposure in modern, environments heavily disturbed by noise pollution.

The gastrointestinal epithelium relies on activation of the hypoxia-inducible factor (HIF) to promote cell survival and maintain bioenergetic homeostasis during hypoxia. While many pathogens can activate HIF, the effects of enteric protozoa on HIF activation in gastrointestinal epithelial cells remain unclear. Giardia duodenalis, a prevalent protozoan enteropathogen, causes intestinal barrier dysfunction characterized by epithelial malabsorption, mucus depletion, altered mucin glycosylation, and microbiota dysbiosis. Findings from the present study reveal an epithelial hypoxic signature upon Giardia infection. Human intestinal epithelial cells were exposed to vehicle or Giardia duodenalis isolate GS/M under normoxic (21% O2) or hypoxic (1% O2) conditions. In normoxia, infected cells displayed a time-dependent increase in HIF-1α protein expression, the oxygen-dependent subunit of HIF-1. In normoxia, Giardia infection upregulated HIF-1 target genes involved in cellular stress (i.e., VEGFA, ANKRD37, GADD45A) and glycolysis (i.e., HK2, LDHA). This was accompanied by changes in the abundance of glycolytic intermediates (i.e., glucose-6-phosphate, pyruvate, lactate). Although infection in hypoxia failed to augment the hypoxia-induced HIF-1α stabilization, HIF-1 target genes were still upregulated, albeit to a lesser degree. These findings indicate that Giardia induces a transient epithelial hypoxic response in normoxic conditions, revealing a hitherto unrecognized epithelial rescue response to this intestinal parasite.

Background/aimsACE2 is highly expressed in the gut and with known alterations in expression in IBD patients potentially linked to gut inflammation and fibrosis. In addition, little is known about the role of serum soluble ACE2 (sACE2) or its hypothetical role in SARS-CoV-2 binding. We sought to evaluate tissue and serum ACE2 profiles in IBD and healthy controls and evaluate alterations related to disease activity and medical therapy.MethodsCirculating sACE2 and intestinal tissue ACE2 was evaluated respectively in serum samples and endoscopic biopsies from patients with IBD and healthy controls in addition to murine DSS induced colitis.Results91 IBD (UC/n=41; CD n=50) and 55 controls were analyzed. Immunohistochemical ACE2 staining in controls was limited to brush border expression with markedly increased colonic ACE2 expression (and reduced ileal ACE2 expression) in IBD. This was not observed in the mouse model which demonstrated positive ileal ACE2 and negative colonic staining in healthy and DSS mice. Colonic ACE2 staining was further increased in Ulcerative Colitis in inflammation (% staining, 20(5-30) vs. 5(0-6.5), p<0.015) and in IBD patients receiving corticosteroids (% staining, 20(20-40) vs 10(0-20), p<0.052). Steroid use was associated with significantly lower sACE2 with a trend towards reduced sACE2 with biologic exposure.ConclusionWe observe significant increases in colonic ACE2 expression in IBD, especially with active colitis. Corticosteroids further modify the observed imbalance between tissue and serum ACE2 levels.

A highly-regulated and dynamic cytoskeleton is vital for functional cellular physiology and the maintenance of homeostasis. Although much is known about the mechanisms by which the cytoskeleton is regulated under physiological conditions, the effect of pathological stimuli and how this contributes to disease progression remains poorly understood. Hypoxia is a prominent microenvironmental feature of a range of pathological states including inflammation, cancer and ischaemia. In this review, we summarise what is known about the effects of hypoxia on the cytoskeleton and discuss the implications of this for physiology and disease.

Oxygen (O2)-controlled cell culture has been pivotal in studying mammalian mechanisms of O2 sensing, regulation, and utilization. We posit, however, that O2-controlled cell culture is paradoxically not controlling O2. There is overwhelming evidence that the pericellular O2 is lower than the surrounding gas phase due to cellular O2 consumption. Standard hypoxic cell culture is at high risk of inducing pericellular anoxia. We discuss the implications of poor O2 control for cellular O2 regulation mechanisms, bioenergetics, and redox signaling. We also highlight the evidence of frequent under-oxygenation in standard (i.e., normoxic) cell culture. This issue has been largely overlooked because strategies to control pericellular O2 have been lacking. Here, we propose a framework to control pericellular O2 based on our recent investigation into the nature of the gas/pericellular O2 gradient. Implementing this framework into standard practice will unlock quantitative O2 control in vitro, improving our ability to understand the role of O2 in biology.

The gastrointestinal system experiences frequent oxygenic fluctuations and must be able to maintain barrier integrity during periods of suboptimal oxygen concentration, or hypoxia. Epithelial cells rely on the Hypoxia-Inducible Factor (HIF) complex to activate genes that combat cellular stress and adapt cellular metabolism during hypoxia. Importantly, protozoan parasites can modulate host tissue oxygen tension and HIF activation, yet little is known regarding the relationship between enteric protozoa and hypoxia. This research aims to uncover the role of HIF upon Giardia duodenalis infection, a top cause of global diarrheal disease and an excellent infection model for the study of GI physiology. We hypothesize that HIF-target genes are activated upon Giardia infection, promoting alternative cellular glucose metabolism to sustain bioenergetic homeostasis. Caco-2 colonic epithelial cells were infected with Giardia isolate GS/M (MOI 10) for 1.5 or 4.5 hours under normoxic (21% O2) or hypoxic (~1%O2, STEMCELL hypoxia incubator) conditions to capture the early or peak activation of the oxygen-dependent HIF subunit (HIF-1α). RNA was extracted for assessment of transcriptional alterations of HIF-target genes via reverse transcriptase quantitative polymerase chain reaction. Investigation of HIF-mediated intracellular metabolic changes was carried out via liquid-chromatography mass-spectrometry analysis (hydrophilic-interaction chromatography method) on cocultures supplemented with a hypoxia mimetic (DMOG) or a HIF inhibitor (PX-478). Metabolomics experiments were repeated using Caco-2 cells transfected with sodium-dependent glucose cotransporter 1 (SGLT1), a key glucose transporter in the small intestine where Giardia localizes which is not reliably expressed in Caco-2 cells. Under normoxic conditions, genes that aid in cell stress responses ( VEGFA, ANKRD37, GADD45A) and glycolysis ( HK2, LDHA) are upregulated in Giardia-infected cells in a time-dependent manner (p<0.05). Fewer HIF-target genes are upregulated under hypoxic conditions (e.g., VEGFA, GADD45A, PGK1; p<0.05), indicating Giardia-infected cells exhibit a transcriptional profile similar to hypoxic uninfected cells. Interestingly, HIF1α was upregulated in Giardia-infected cells under hypoxic conditions at 1.5 hours (p<0.05). Analysis of the Caco-2 intracellular metabolome indicated HIF-dependent changes to nucleic acid (e.g., guanosine, inosine, uracil) and amino acid (e.g., glycine, threonine) metabolism. DMOG treatment increased the abundance of glycolytic intermediates (e.g., PEP, DHAP) in both cell lines and inhibited the depletion of the Kreb’s Cycle intermediate aconitate in the Caco-2-SGLT1 transfected cells, confirming the promotion of glycolytic flux by HIF. Taken together, our findings indicate Giardia-infected cells illustrate a hypoxic signature, a novel metabolic cell rescue mechanism in response to enteropathogens. Elucidating the role of HIF during enteric parasitic infections will aid in our understanding of the cellular adaptations underpinning GI pathophysiology, while also shedding light on the potential of HIF as a therapeutic target. Natural Science and Engineering Research Council of Canada. 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.

Mammalian cells utilize glucose as a primary carbon source to produce energy for most cellular functions. However, the bioenergetic homeostasis of cells can be perturbed by environmental alterations, such as changes in oxygen levels which can be associated with bacterial infection. Reduction in oxygen availability leads to a state of hypoxia, inducing numerous cellular responses that aim to combat this stress. Importantly, hypoxia strongly augments cellular glycolysis in most cell types to compensate for the loss of aerobic respiration. Understanding how this host cell metabolic adaptation to hypoxia impacts the course of bacterial infection will identify new anti-microbial targets. This review will highlight developments in our understanding of glycolytic substrate channeling and spatiotemporal enzymatic organization in response to hypoxia, shedding light on the integral role of the hypoxia-inducible factor (HIF) during host-pathogen interactions. Furthermore, the ability of intracellular and extracellular bacteria (pathogens and commensals alike) to modulate host cellular glucose metabolism will be discussed.

Glycolysis is a highly conserved metabolic pathway responsible for the anaerobic production of adenosine triphosphate (ATP) from the breakdown of glucose molecules. While serving as a primary metabolic pathway in prokaryotes, glycolysis is also utilized by respiring eukaryotic cells, providing pyruvate to fuel oxidative metabolism. Furthermore, glycolysis is the primary source of ATP production in multiple cellular states (e.g., hypoxia) and is particularly important in maintaining bioenergetic homeostasis in the most abundant cell type in the human body, the erythrocyte. Beyond its role in ATP production, glycolysis also functions as a signaling hub, producing several metabolic intermediates which serve roles in both signaling and metabolic processes. These signals emanating from the glycolytic pathway can profoundly impact cell function, phenotype, and fate and have previously been overlooked. In this review, we will discuss the role of the glycolytic pathway as a source of signaling molecules in eukaryotic cells, emphasizing the newfound recognition of glycolysis' multifaceted nature and its importance in maintaining cellular homeostasis, beyond its traditional role in ATP synthesis.

In normoxic conditions where oxygen supply exceeds demand, mammalian cells produce 38 molecules of adenosine triphosphate (ATP) per molecule of glucose through cellular respiration which is primarily oxygen-dependent. In hypoxia where oxygen demand exceeds supply, the cell enters a bioenergetic crisis due to the decrease in oxygen-dependent ATP synthesis. The cell relies on overcoming this deficiency in ATP through upregulation of glycolytic gene expression via the hypoxia inducible factor 1-a (HIF-1a). However, it is highly unlikely, if not impossible, that the bioenergetic requirements of the hypoxic cell depend solely on the increased expression of glycolytic enzymes distributed randomly in the cytoplasm. We have recently shown that in response to hypoxia, glycolytic enzymes coalesce to form a glycolytic metabolon to effciently increase ATP production in response to this metabolic challenge (Kierans SJ, et al., 2023). While the function of this metabolon has been characterised, the mechanisms underpinning formation, scaffolding, and intracellular distribution remains poorly understood. Here we investigate the possible role of the cytoskeleton as a scaffold or transport system. Microtubules are a primary method of intracellular transport and may alter the energy distribution strategies of cells in bioenergetic crisis. It is hypothesized that the microtubular network transports glycolytic enzymes and/or facilitates increased glycolytic ATP production by promoting metabolon formation to induce glycolysis in hypoxia. Through western blots, it was successfully shown that upon exposure to 1% oxygen, caco2 intestinal epithelial cells induce a robust hypoxic response by stabilising HIF-1a. To investigate the role of the microtubules, two microtubule inhibitors were used: nocodazole, a microtubule assembly and disassembly inhibitor and dynarrestin, a dynein-1 motor transport protein inhibitor. Non-toxic concentrations of nocodazole and dynarrestin were determined using cell viability assays (trypan blue and resazurin assays). Upon exposure to 24 hours of hypoxia, caco2 cells elicit a statistically significant increase in intracellular lactate (185.9% ± 7.5% SEM, n = 3), which was then supressed when treated with nocodazole or dynarrestin (20.6% ± 10.2% SEM and 74.9% ± 8.87 SEM respectively, n = 3). These results thus implicate a role for the transport and structural capabilities of the microtubules in hypoxia-induced glycolysis. Kierans, S. J., Fagundes, R. R., Malkov, M. I., Sparkes, R., Dillon, E. T., Smolenski, A.,... & Taylor, C. T. (2023). Hypoxia induces a glycolytic complex in intestinal epithelial cells independent of HIF-1-driven glycolytic gene expression. Proceedings of the National Academy of Sciences, 120(35), e2208117120. University College Dublin, School of Medicine. 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.

Investigating how cells and organisms sense and respond to O2 levels is essential to our understanding of physiology and pathology. This field has advanced considerably since the discovery of the major transcription factor family, hypoxia-inducible factor (HIF), and the enzymes that control its levels: prolyl hydroxylases (PHDs). However, with its expansion, new complexities have emerged. Herein we highlight three main areas where, in our opinion, the research community could direct some of their attention. These include non-transcriptional roles of HIFs, specificity and O2 sensitivity of 2-oxoglutarate-dependent dioxygenases (2-OGDDs), and new tools and methods to detect O2 concentrations in cells and organs. A greater understanding of these areas would answer big questions and help drive our knowledge of cellular responses to hypoxia forward.

Type 1 diabetes (T1D) is a common autoimmune disease in which dysregulated glucose metabolism is a key feature. T1D is both poorly understood and in need of improved therapeutics. Hypoxia is frequently encountered in multiple tissues in T1D patients including the pancreas and sites of diabetic complications. Hypoxia-inducible factor (HIF)-1, a ubiquitous master regulator of the adaptive response to hypoxia, promotes glucose metabolism through transcriptional and non-transcriptional mechanisms and alters disease progression in multiple preclinical T1D models. However, how HIF-1 activation in β-cells of the pancreas and immune cells (two key cell types in T1D) ultimately affects disease progression remains controversial. We discuss recent advances in our understanding of the role of hypoxia/HIF-1-induced glycolysis in T1D and explore the possible use of drugs targeting this pathway as potential new therapeutics.

Acute myeloid leukemia (AML) is an aggressive hematological cancer with limited treatment options. A study now provides compelling data and develops a therapeutic approach of targeting AML with a prolyl hydroxylase inhibitor, a strategy based on the sensitivity of myeloid cells to modulation of the transcription factor HIF.

In oxygen (O2)-controlled cell culture, an indispensable tool in biological research, it is presumed that the incubator setpoint equals the O2 tension experienced by cells (i.e., pericellular O2). However, it is discovered that physioxic (5% O2) and hypoxic (1% O2) setpoints regularly induce anoxic (0% O2) pericellular tensions in both adherent and suspension cell cultures. Electron transport chain inhibition ablates this effect, indicating that cellular O2 consumption is the driving factor. RNA-seq analysis revealed that primary human hepatocytes cultured in physioxia experience ischemia-reperfusion injury due to cellular O2 consumption. A reaction-diffusion model is developed to predict pericellular O2 tension a priori, demonstrating that the effect of cellular O2 consumption has the greatest impact in smaller volume culture vessels. By controlling pericellular O2 tension in cell culture, it is found that hypoxia vs. anoxia induce distinct breast cancer transcriptomic and translational responses, including modulation of the hypoxia-inducible factor (HIF) pathway and metabolic reprogramming. Collectively, these findings indicate that breast cancer cells respond non-monotonically to low O2, suggesting that anoxic cell culture is not suitable for modeling hypoxia. Furthermore, it is shown that controlling atmospheric O2 tension in cell culture incubators is insufficient to regulate O2 in cell culture, thus introducing the concept of pericellular O2-controlled cell culture.

Environmental stressors present in the modern world can fundamentally affect humans’ physiology and health. Exposure to stressors like air pollution, heat, and traffic noise has been linked to a pronounced increase in non-communicable diseases. Specifically, aircraft noise has been identified as a risk factor for cardiovascular and metabolic diseases, such as arteriosclerosis, heart failure, stroke, and diabetes. Noise stress leads to neuronal activation with subsequent stress hormone release that ultimately leads to activation of the renin-angiotensin-aldosterone system, increasing inflammation and oxidative stress, dramatically affecting the cardiovascular system. However, despite the epidemiological evidence of a link between noise stress and metabolic dysfunction, the consequences of exposure at the molecular, metabolic level of the cardiovascular system are largely unknown. Here we use a murine model system of aircraft noise exposure to show that noise stress profoundly alters heart metabolism. Within days of exposing animals to aircraft noise, the heart has a reduced potential for utilising fatty-acid beta-oxidation, the tricarboxylic acid cycle, and the electron transport chain for generating ATP. This is compensated by shifting energy production towards glycolysis. Intriguingly, the metabolic shift is reminiscent of what is observed in failing and ischaemic hearts. Our results demonstrate that within a relatively short exposure time, the cardiovascular system undergoes a fundamental metabolic shift that bears the hallmarks of cardiovascular disease. Overall, aircraft noise induces rapid, detrimental metabolic shifts in the heart, resembling patterns seen in cardiovascular diseases. These findings underscore the urgent need to comprehend the molecular consequences of environmental stressors, paving the way for targeted interventions aiming at mitigating health risks associated with chronic noise exposure in our modern, noisy environments. ### Competing Interest Statement The authors have declared no competing interest.

IntroductionIntestinal epithelial cells produce interleukin-18 (IL-18), a key factor in promoting epithelial barrier integrity. Here, we analyzed the potential role of gut bacteria and the hypoxia-inducible factor 1α (HIF1α) pathway in regulating mucosal IL18 expression in inflammatory bowel disease (IBD).MethodsMucosal samples from patients with IBD (n = 760) were analyzed for bacterial composition, IL18 levels and HIF1α pathway activation. Wild-type Caco-2 and CRISPR/Cas9-engineered Caco-2-HIF1A-null cells were cocultured with Faecalibacterium prausnitzii in a “Human oxygen-Bacteria anaerobic” in vitro system and analyzed by RNA sequencing.ResultsMucosal IL18 mRNA levels correlated positively with the abundance of mucosal-associated butyrate-producing bacteria, in particular F. prausnitzii, and with HIF1α pathway activation in patients with IBD. HIF1α-mediated expression of IL18, either by a pharmacological agonist (dimethyloxallyl glycine) or F. prausnitzii, was abrogated in Caco-2-HIF1A-null cells.ConclusionButyrate-producing gut bacteria like F. prausnitzii regulate mucosal IL18 expression in a HIF1α-dependent manner that may aid in mucosal healing in IBD.

The metabolic adaptation of eukaryotic cells to hypoxia involves increasing dependence upon glycolytic adenosine triphosphate (ATP) production, an event with consequences for cellular bioenergetics and cell fate. This response is regulated at the transcriptional level by the hypoxia-inducible factor-1(HIF-1)-dependent transcriptional upregulation of glycolytic enzymes (GEs) and glucose transporters. However, this transcriptional upregulation alone is unlikely to account fully for the levels of glycolytic ATP produced during hypoxia. Here, we investigated additional mechanisms regulating glycolysis in hypoxia. We observed that intestinal epithelial cells treated with inhibitors of transcription or translation and human platelets (which lack nuclei and the capacity for canonical transcriptional activity) maintained the capacity for hypoxia-induced glycolysis, a finding which suggests the involvement of a nontranscriptional component to the hypoxia-induced metabolic switch to a highly glycolytic phenotype. In our investigations into potential nontranscriptional mechanisms for glycolytic induction, we identified a hypoxia-sensitive formation of complexes comprising GEs and glucose transporters in intestinal epithelial cells. Surprisingly, the formation of such glycolytic complexes occurs independent of HIF-1-driven transcription. Finally, we provide evidence for the presence of HIF-1α in cytosolic fractions of hypoxic cells which physically interacts with the glucose transporter GLUT1 and the GEs in a hypoxia-sensitive manner. In conclusion, we provide insights into the nontranscriptional regulation of hypoxia-induced glycolysis in intestinal epithelial cells.

Background: Increased levels of CO 2 reduce macrophage function during inflammatory processes and wound healing. However, little is known how macrophages sense CO 2 and adapt upon high levels of CO 2 during differentiation and activation. We, therefore, elucidated the effects of CO 2 on gene and protein expression during basic inflammatory processes, such as monocyte differentiation and macrophage activation. Methods: Monocyte differentiation was induced by phorbol 12-myristate 13-acetate (PMA). Primary macrophages (BMDMs) were polarized using different cytokines to induce proinflammatory (M1) macrophages (lipopolysaccharides) or immunomodulatory (M2) macrophages (interleukin-4). Cells were simultaneously subjected to different levels of CO 2 . Morphological changes, mRNA and protein expression of markers for cell differentiation and macrophage polarization were determined. Cell culture medium was buffered and intracellular pH (pHi) was monitored to determine pH-dependent effects of CO 2 . Direct effects of CO 2 on PKC protein function were analyzed. Results: High levels of CO 2 attenuated PMA-induced cell differentiation of human monocytes. In BMDMs, CO 2 significantly reduced transcript levels of M1-marker. Mechanistically, CO 2 mitigated PMA-stimulated PKC activity and function. Experiments with buffered medium revealed that changes in pH were responsible for most, but not all, of the CO 2 -mediated effects on monocyte differentiation and macrophage polarization. Pharmacological and genetic inhibition of carbonic anhydrases (CA) uncoupled the CO 2 -elicited intracellular pH-response, thereby preventing CO 2 -sensitivity during monocyte differentiation, activation, and migration, which could be reversed by CRISPR-mediated upregulation of CA2. Conclusion: Carbon dioxide is highly immunomodulatory and might thus affect wound healing properties in vivo. In M1 (but not M2) macrophages, CO 2 is sensed by CA2-driven changes in pHi. MJS received funding from the German Research Foundation (DFG; STR 1570/1-1) and the Braun Foundation (Braun®; BBST-D-18-00018). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.