Iñigo San-Millán

Background: Physical inactivity is a major contributor to cardiometabolic disease and mortality. Although mitochondrial dysfunction characterizes overt pathology, whether sedentarism constitutes a distinct and measurable bioenergetic disease state, rather than simply reduced fitness, has not been established. Methods: Nine sedentary (SED) and ten physically active (AC) healthy males (42 ± 14 yr) were studied. Skeletal muscle bioenergetics were assessed using high-resolution respirometry, fluxomics, metabolomics, and protein expression analyses. Whole-body physiology was evaluated using cardiopulmonary exercise and lactate testing (CPELT). Results: At rest, SED exhibited marked reductions in mitochondrial capacity, including Complex I (−36%), Complex II (−28%), electron transport system capacity (−34%), and ATP-synthase-coupled respiration (−30%, all p < 0.01). The most pronounced alteration was a 49% reduction in mitochondrial pyruvate carrier (MPC1) expression, which closely correlated with reduced pyruvate oxidation (−37%, p = 0.006) and lower TCA intermediates. SED also showed reduced MCT1 abundance, impaired fatty-acid oxidation capacity (−32% to −35%), decreased CPT1 activity (−51%), altered cardiolipin composition, and elevated ROS/O₂ flux ratios. During exercise, SED demonstrated lower VO₂max (−38%), reduced fat oxidation (−35%), and higher blood lactate accumulation (>60%, p < 0.001). Mitochondrial function was strongly associated with exercise performance (r = 0.57–0.78, p < 0.01). Conclusions: Healthy sedentary adults displayed a coordinated reduction in tissue-level mitochondrial oxidative capacity, substrate-handling markers, cardiolipin abundance, and metabolic flexibility. These findings should be interpreted as an integrated per-mg skeletal-muscle bioenergetic phenotype in which lower mitochondrial density may account for much of the observed reduction. Within this phenotype, the 49% reduction in MPC1 alongside preserved GLUT4, LDHA, and LDHB abundance represents an outstanding differential observation that future studies with direct mitochondrial-content normalization should test. CPELT-derived fat oxidation and blood lactate responses reflected this tissue-level bioenergetic phenotype, providing candidate noninvasive physiological markers for future longitudinal and interventional studies.

Cancer cachexia affects 50–80% of patients with advanced malignancy and contributes to 20–30% of cancer-related deaths, yet therapies targeting the predominant cytokine-centric framework have not yielded clinically meaningful, sustained reversal. This persistent therapeutic gap suggests that an upstream driver may have been incompletely characterized. We propose a substrate-level mechanistic hierarchy: (1) the tumor Warburg phenotype, through accelerated glycolysis, with constitutive GLUT1/GLUT3-mediated insulin-independent glucose uptake, imposes a continuous and progressively escalating glucose drain on the host that scales with tumor burden and metastatic spread, as quantified by FDG-PET total lesion glycolysis; (2) host glycogen reserves (~400–500 g) are progressively depleted under this continuous demand; (3) once depleted, hepatic gluconeogenesis becomes mandatory, because even-chain fatty acids cannot generate net glucose in humans; (4) skeletal muscle proteolysis supplies the alanine and glutamine required for gluconeogenesis and TCA-cycle anaplerosis, mobilizing muscle as the only scalable gluconeogenic substrate; (5) inflammatory cytokines (TNF-α, IL-6, IL-1β) function as downstream amplifiers of an already-engaged proteolytic program rather than as its primary drivers, consistent with the recent demonstration that cachexia proceeds in the colon carcinoma 26 model despite genetic ablation of IL-6, LIF, and IL-11.The novelty proposed here is not the recognition of tumor glucose consumption or of gluconeogenesis in cachexia, both well established, but the identification of the limited glycogen reserve, combined with the biochemical inability to derive net glucose from fat, as the substrate-level constraint that converts tumor glucose demand into mandatory proteolysis. A conceptual flux-constraint model is offered to organize the relevant variables, together with six falsifiable predictions and immediate research implications as well as clinical applications spanning glycogen monitoring as a candidate clinical biomarker and nutritional strategies oriented toward glycogen repletion.

Tumor-derived lactate is increasingly recognized as an immunosuppressive metabolite within the tumor microenvironment (TME), with emerging evidence highlighting its role beyond metabolism to include epigenetic and immune regulatory functions. While prior studies have primarily focused on individual immune checkpoints, most prominently PD-L1, it remains unclear whether lactate broadly coordinates the expression of multiple immune regulatory pathways across distinct tumor types, particularly in the context of chronic exposure mimicking glycolytic tumors. Here, we investigated the relationship between lactate-producing metabolism and immune checkpoint gene expression in four human cancer cell lines representing breast and lung cancer: MCF7 (estrogen receptor-positive breast), MDA-MB-231 (triple-negative breast), A549 (non-small cell lung), and H82 (small cell lung). By manipulating glucose availability and exposure duration to model acute (6 h) versus chronic (48 h) lactate production, and by pharmacologically inhibiting lactate dehydrogenase (LDH) with oxamate, we quantified extracellular lactate accumulation and assessed transcriptional responses of a panel of immune checkpoints (PD-L1, CD80, CD73, LGALS9, VISTA, PVR, CD47, FGL1, STING) and lactate-associated genes (MCT1, MCT4, LDHA, HCAR1) via qPCR. Chronic high-glucose conditions produced robust, LDH-dependent lactate accumulation and were associated with coordinated, lineage-specific remodeling of multiple checkpoint transcripts, whereas acute exposure induced minimal changes. MDA-MB-231 and A549 cells displayed striking but distinct checkpoint patterns under chronic lactate-producing conditions: MDA-MB-231 cells showed strong co-induction of PD-L1 and CD80, while A549 cells exhibited dominant CD80 induction with modest PD-L1 upregulation. H82 cells upregulated PD-L1 alongside CD73, LGALS9, CD47, and CD80, whereas MCF7 cells demonstrated more modest yet coordinated increases across several checkpoints. Chronic glucose exposure resulted in sustained, LDH-dependent lactate accumulation and coordinated induction of multiple immune checkpoint genes, with distinct lineage-specific patterns, e.g., robust PD-L1/CD80 upregulation in MDA-MB-231 versus CD80 dominance in A549. Unsupervised clustering and principal component analysis revealed that duration of glucose exposure, rather than acute glucose availability, was the primary axis of variation and that MCT4 and HCAR1 clustered with strongly induced checkpoints, consistent with a transcriptional program linking lactate export and sensing to immune regulation. These findings support a model in which lactate acts as an upstream regulator of a broader immune escape program, potentially via mechanisms like lactylation and HCAR1 signaling. This work highlights the limitations of single-checkpoint blockade strategies in solid tumors and underscores the potential of targeting lactate metabolism to enhance immunotherapy efficacy in breast and lung cancers.

Lack of physical activity has been associated with multiple diseases including cardiovascular disease (CVD), cancer, Alzheimer’s disease (AD), type 2 diabetes (T2D), Parkinson’s disease, depression, dementia and even cancer. Mitochondrial impairment or dysfunction is associated with lack of physical activity and considered to be involved in the pathogenesis of the most prevalent non-communicable diseases (NCDs) aflicting our societies such as T2D, CVD, metabolic syndrome, and even AD. To our knowledge, there is a scarcity of studies on the metabolic, mitochondrial and cellular characteristics of “healthy sedentary” individuals living without clinical symptoms. Hence, the main aim of our study herein was to characterize multiple metabolic, mitochondrial and cellular bioenergetic signatures in “healthy sedentary” individuals which could already be downregulated compared to moderately active individuals. Nineteen subjects, 9 sedentary (SED) and 10 moderately active (AC) volunteered for multiple assessments including muscle biopsies, in order to assess muscle metabolism, mitochondrial respiration and bioenergetics both at rest and during exercise. For our exercise studies, we performed graded exercise testing (GXT) to assess carbohydrate and fat oxidation capacity as well as lactate clearance capacity according to our previously developed methodology. Resting studies showed decreased mitochondrial respiration including decreases in complex I (-36%) and II (-28%) as well as total electron system capacity (-34%) and electron system capacity coupled to ATP production via ATP synthase (-30%). Regarding muscle carbohydrate metabolism, SED individuals showed a decrease in mitochondrial pyruvate oxidation (-37%) as well as reduced expression (-49%) of mitochondrial pyruvate carrier (MPC). Regarding fatty acid metabolism, SED showed decreased activity of carnitine palmitoyltransferase I (CPT1)(-51%) and CPT2 (-44%) as well as decreased mitochondrial fatty acid oxidation (-35%). Metabolomics analysis also confirmed downregulation of carbohydrate and fat metabolism. Partial Least-Squares Discriminant Analysis (PLS-DA) identified distinct metabolic phenotypes through intermediates of glycolysis and fatty acid oxidation. Further, we found significant differences in cardiolipin (CL) species expression between SED and AC groups, which, due to the important role of CL in mitochondrial structure, function, biogenesis and bioenergetics, deserves further attention. Exercise studies also showed significant differences in substrate utilization between groups where SED possessed a significantly lower fat oxidation capacity as well as lactate clearance capacity. The correlation of different bioenergetic parameters between resting and exercise conditions were robust, suggesting the possibility of performing cardiopulmonary exercise testing (CPET) as a non-invasive methodology to indirectly assess metabolic function in multiple populations. In summary, in our study herein, we show that “healthy sedentary” individuals already possess a significant decrease in cellular metabolism, mitochondrial respiration and bioenergetics compared to moderately active individuals both during resting and exercising conditions. Since large numbers of sedentary individuals evolve to develop cardiometabolic disease, a better understanding of decreased cellular bioenergetics and mitochondrial function is needed in order to improve both diagnosis and treatment of multiple metabolic diseases. CONCLUSIONS - Sedentary individuals possess significant decreases in mitochondrial respiration as well as muscle bioenergetics during resting conditions compared to moderately active individuals. - Despite similar GLUT-4 concentrations, sedentary individuals show significant decreased pyruvate oxidation as well as expression of mitochondrial pyruvate carrier (MPC). - During exercise studies, sedentary individuals show significantly decreased levels of fat oxidation and lactate clearance capacity which correlate with mitochondrial and bioenergetics parameters from resting muscle biopsies. Hence, cardiopulmonary exercise testing (CPET) accompanied by measuring blood lactate levels could be a practical manner to assess muscle mitochondrial function and bioenergetics in a non-invasive and ambulatory manner. - Identifying early signatures of decreased mitochondrial function and bioenergetics capacity could be an important approach to prevent or improve different metabolic diseases through lifestyle changes mainly from exercise and nutrition.

There is a growing interest in studies involving carbohydrate (CHO) manipulation and subsequent adaptations to endurance training. This study aimed to analyze whether a periodized carbohydrate feeding strategy based on a daily training session has any advantages compared to a high-carbohydrate diet in well-trained cyclists. Seventeen trained cyclists (VO_2peak = 70.8 ± 6.5 mL·kg⁻¹·min⁻¹) were divided into two groups, a periodized (PCHO) group and a high-carbohydrate (HCHO) group. Both groups performed the same training sessions for five weeks. In the PCHO group, 13 training sessions were performed with low carbohydrate availability. In the HCHO group, all sessions were completed following previous carbohydrate intake to ensure high pre-exercise glycogen levels. In both groups, there was an increase in the maximal lactate steady state (MLSS) (PCHO: 244.1 ± 29.9 W to 253.2 ± 28.4 W; p = 0.008; HCHO: 235.8 ± 21.4 W to 246.9 ± 16.7 W; p = 0.012) but not in the time to exhaustion at MLSS intensity. Both groups increased the percentage of muscle mass (PCHO: p = 0.021; HCHO: p = 0.042) and decreased the percent body fat (PCHO: p = 0.021; HCHO: p = 0.012). We found no differences in carbohydrate or lipid oxidation, heart rate, and post-exercise lactate concentration. Periodizing the CHO intake in well-trained cyclists during a 5-week intervention did not elicit superior results to an energy intake-matched high-carbohydrate diet in any of the measured outcomes.

Background and Objective Metabolomics studies of recreational and elite athletes have been so far limited to venipuncture-dependent blood sample collection in the setting of controlled training and medical facilities. However, limited to no information is currently available to determine if findings in laboratory settings are translatable to a real-world scenario in elite competitions. The goal of this study was to define molecular signatures of exertion under controlled exercise conditions and use these signatures as a framework for assessing cycling performance in a World Tour competition. Methods To characterize molecular profiles of exertion in elite athletes during cycling, we performed metabolomics analyses on blood isolated from 28 international-level, elite, World Tour professional male athletes from a Union Cycliste Internationale World Team taken before and after a graded exercise test to volitional exhaustion and before and after a long aerobic training session. Moreover, established signatures were then used to characterize the metabolic physiology of five of these cyclists who were selected to represent the same Union Cycliste Internationale World Team during a seven-stage elite World Tour race. Results Using dried blood spot collection to circumvent logistical hurdles associated with field sampling, these studies defined metabolite signatures and fold change ranges of anaerobic or aerobic exertion in elite cyclists, respectively. Blood profiles of lactate, carboxylic acids, fatty acids, and acylcarnitines differed between exercise modes. The graded exercise test elicited significant two- to three-fold accumulations in lactate and succinate, in addition to significant elevations in free fatty acids and acylcarnitines. Conversely, the long aerobic training session elicited a larger magnitude of increase in fatty acids and acylcarnitines without appreciable increases in lactate or succinate. Comparable signatures were revealed after sprinting and climbing stages, respectively, in a World Tour race. In addition, signatures of elevated fatty acid oxidation capacity correlated with competitive performance. Conclusions Collectively, these studies provide a unique view of alterations in the blood metabolome of elite athletes during competition and at the peak of their performance capabilities. Furthermore, they demonstrate the utility of dried blood sampling for omics analysis, thereby enabling molecular monitoring of athletic performance in the field during training and competition.

The Warburg Effect is characterized by accelerated glycolytic metabolism and lactate production and is a hallmark of cancer cells. Recently, we have demonstrated the role of endogenous, glucose-derived lactate as an oncometabolite which regulates gene expression in the estrogen receptor positive (ER+) MCF7 cell line cultivated in glucose media (San-Millan, Julian et al. 2019). Presently, with the addition of a triple negative breast cancer (TNBC) cell line, MDA-MB-231, we further confirm the effect of lactate on gene expression patterns, but extend results to include lactate effects on protein expression. As well, we report effects of lactate on the expression of E-cadherin and vimentin, proteins associated with epithelial-to-mesenchymal transition (EMT). Endogenous lactate regulates the expression of multiple genes involved in carcinogenesis. In MCF7 cells, lactate increased the expression of EGFR, VEGF HIF-1a, KRAS, MIF, mTOR, PIK3CA, TP53, and CDK4 (The Ital is used for genes) as well as decreased the expression of ATM, BRCA1, BRCA2, E2F1, MET, MYC, and RAF mainly at 48h of exposure. On the other hand, in the MDA-MB-231 cell line, lactate increased the expression of PIK3CA, VEGF, EGFR, mTOR, HIF-1α, ATM, E2F1, TP53 and decreased the expression of BRCA1, BRCA2, CDK4, CDK6, MET, MIF, MYC, and RAF after 48h of exposure. Protein expression of representative genes corroborated mRNA expressions. Finally, lactate decreased E-cadherin protein expression in MCF7 cells and increased vimentin expression in MDA-MB-231 cells. In this study, we demonstrate that endogenous lactate produced under aerobic conditions (Warburg Effect) is capable of eliciting important regulation of gene and protein expression in both ER+ and TNBC cell lines. The regulation of multiple genes by lactate is widespread and involves those involved in carcinogenesis including DNA repair, cell growth, proliferation, angiogenesis, and metastasis. Furthermore, both cell lines demonstrated changes in the expression of EMT biomarkers representing a shift to a more mesenchymal phenotype with exposure to endogenous lactate. Simple Summary This study shows how endogenous lactate is a major regulator of key genes involved in two main breast cancer cell types: estrogen receptor positive (ER⁺) and triple negative breast cancer cells (TPBC). Lactate regulates both gene and protein expression in these cells. Furthermore, lactate is also an important player in the regulation of epithelial-to-mesenchymal transition (EMT), a process involved in metastasis. Targeting lactate production and exchange within and among cancer cells should offer novel therapeutics opportunities.

The role of mitochondrial function in health and disease has become increasingly recognized, particularly in the last two decades. Mitochondrial dysfunction as well as disruptions of cellular bioenergetics have been shown to be ubiquitous in some of the most prevalent diseases in our society, such as type 2 diabetes, cardiovascular disease, metabolic syndrome, cancer, and Alzheimer’s disease. However, the etiology and pathogenesis of mitochondrial dysfunction in multiple diseases have yet to be elucidated, making it one of the most significant medical challenges in our history. However, the rapid advances in our knowledge of cellular metabolism coupled with the novel understanding at the molecular and genetic levels show tremendous promise to one day elucidate the mysteries of this ancient organelle in order to treat it therapeutically when needed. Mitochondrial DNA mutations, infections, aging, and a lack of physical activity have been identified to be major players in mitochondrial dysfunction in multiple diseases. This review examines the complexities of mitochondrial function, whose ancient incorporation into eukaryotic cells for energy purposes was key for the survival and creation of new species. Among these complexities, the tightly intertwined bioenergetics derived from the combustion of alimentary substrates and oxygen are necessary for cellular homeostasis, including the production of reactive oxygen species. This review discusses different etiological mechanisms by which mitochondria could become dysregulated, determining the fate of multiple tissues and organs and being a protagonist in the pathogenesis of many non–communicable diseases. Finally, physical activity is a canonical evolutionary characteristic of humans that remains embedded in our genes. The normalization of a lack of physical activity in our modern society has led to the perception that exercise is an “intervention”. However, physical activity remains the modus vivendi engrained in our genes and being sedentary has been the real intervention and collateral effect of modern societies. It is well known that a lack of physical activity leads to mitochondrial dysfunction and, hence, it probably becomes a major etiological factor of many non–communicable diseases affecting modern societies. Since physical activity remains the only stimulus we know that can improve and maintain mitochondrial function, a significant emphasis on exercise promotion should be imperative in order to prevent multiple diseases. Finally, in populations with chronic diseases where mitochondrial dysfunction is involved, an individualized exercise prescription should be crucial for the “metabolic rehabilitation” of many patients. From lessons learned from elite athletes (the perfect human machines), it is possible to translate and apply multiple concepts to the betterment of populations with chronic diseases.

Exercise intolerance is a major manifestation of post-acute sequelae of severe acute respiratory syndrome coronavirus infection (PASC, or “long-COVID”). Exercise intolerance in PASC is associated with higher arterial blood lactate accumulation and lower fatty acid oxidation rates during graded exercise tests to volitional exertion, suggesting altered metabolism and mitochondrial dysfunction. It remains unclear whether the profound disturbances in metabolism that have been identified in plasma from patients suffering from acute coronavirus disease 2019 (COVID-19) are also present in PASC. To bridge this gap, individuals with a history of previous acute COVID-19 infection that did not require hospitalization were enrolled at National Jewish Health (Denver, CO, USA) and were grouped into those that developed PASC (n = 29) and those that fully recovered (n = 16). Plasma samples from the two groups were analyzed via mass spectrometry-based untargeted metabolomics and compared against plasma metabolic profiles of healthy control individuals (n = 30). Observational demographic and clinical data were retrospectively abstracted from the medical record. Compared to plasma of healthy controls or individuals who recovered from COVID-19, PASC plasma exhibited significantly higher free- and carnitine-conjugated mono-, poly-, and highly unsaturated fatty acids, accompanied by markedly lower levels of mono-, di- and tricarboxylates (pyruvate, lactate, citrate, succinate, and malate), polyamines (spermine) and taurine. Plasma from individuals who fully recovered from COVID-19 exhibited an intermediary metabolic phenotype, with milder disturbances in fatty acid metabolism and higher levels of spermine and taurine. Of note, depletion of tryptophan—a hallmark of disease severity in COVID-19—is not normalized in PASC patients, despite normalization of kynurenine levels—a tryptophan metabolite that predicts mortality in hospitalized COVID-19 patients. In conclusion, PASC plasma metabolites are indicative of altered fatty acid metabolism and dysfunctional mitochondria-dependent lipid catabolism. These metabolic profiles obtained at rest are consistent with previously reported mitochondrial dysfunction during exercise, and may pave the way for therapeutic intervention focused on restoring mitochondrial fat-burning capacity.

Introduction Metabolomics studies of recreational and elite athletes have been so far limited to venipuncture-dependent blood sample collection in the setting of controlled training and medical facilities. However, limited to no information is currently available if findings in laboratory settings are translatable to real world scenario in elite competitions. Methods To characterize molecular profiles of exertion in elite athletes during cycling, we performed metabolomics analyses on blood isolated from twenty-eight international-level elite World Tour professional male athletes from a Union Cycliste Internationale (UCI) World Team taken before and after a graded exercise test (GXT) to volitional exhaustion and before and after a long aerobic training session. Moreover, established signatures were then used to characterize the metabolic physiology of five of these cyclists that were selected to represent the same UCI World Team during a 7-stage elite World Tour race. Results Using dried blood spot collection to circumvent logistical hurdles associated with field sampling, these studies defined metabolite signatures and fold change ranges of anaerobic or aerobic exertion in elite cyclists, respectively. Blood signatures derived in controlled settings enabled comparison with blood sampled during competition, thus providing insight into fatigue status of the cyclists during the course of the race. Collectively, these studies provide a unique view of alterations in the blood metabolome of elite athletes during competition and at the peak of their performance capabilities. Summary Nemkov et al. leveraged field sampling and blood metabolomics and lipidomics approaches to follow a professional Team of elite cyclists upon graded exercise test to volitional exhaustion, field aerobic training (180 km) and a during multi-stage World Tour race. They identify markers of cycling performance beyond lactate thresholds (ranging from 3.75 to 6.5 watts per kilogram in this group), including carboxylic acids, fatty acids and acylcarnitines. Key points We profiled metabolism of 28 international-level elite World Tour professional male athletes from a Union Cycliste Internationale UCI World Team during training and World Tour multi-stage race; Dried blood spot sampling affords metabolomics analyses to monitor exercise performance; Determination of lactate thresholds during graded exercise test (GXT) to volitional exhaustion shows a range of from 3.75 to 6.5 watts per kilogram in this group; Blood profiles of lactate, carboxylic acids, fatty acids and acylcarnitines differed between different exercise modes (GXT and 180 km aerobic training session); Metabolic profiles were affected by stage-specific challenges (sprint vs climbing) during a World Tour multi-stage race.