Glioblastoma (GBM) is the most aggressive primary brain tumor, with a median survival at diagnosis of 16–20 months. Metabolism represents a new attractive therapeutic target; however, due to high intratumoral heterogeneity, the application of metabolic drugs in GBM is challenging. We characterized the basal bioenergetic metabolism and antiproliferative potential of metformin (MF), dichloroacetate (DCA), sodium oxamate (SOD) and diazo-5-oxo-L-norleucine (DON) in three distinct glioma stem cells (GSCs) (GBM18, GBM27, GBM38), as well as U87MG. GBM27, a highly oxidative cell line, was the most resistant to all treatments, except DON. GBM18 and GBM38, Warburg-like GSCs, were sensitive to MF and DCA, respectively. Resistance to DON was not correlated with basal metabolic phenotypes. In combinatory experiments, radiomimetic bleomycin exhibited therapeutically relevant synergistic effects with MF, DCA and DON in GBM27 and DON in all other cell lines. MF and DCA shifted the metabolism of treated cells towards glycolysis or oxidation, respectively. DON consistently decreased total ATP production. Our study highlights the need for a better characterization of GBM from a metabolic perspective. Metabolic therapy should focus on both glycolytic and oxidative subpopulations of GSCs.

Glioblastoma (GBM) is the most aggressive primary brain tumor in adults, with a universally lethal prognosis despite maximal standard therapies. Here, we present a consensus treatment protocol based on the metabolic requirements of GBM cells for the two major fermentable fuels: glucose and glutamine. Glucose is a source of carbon and ATP synthesis for tumor growth through glycolysis, while glutamine provides nitrogen, carbon, and ATP synthesis through glutaminolysis. As no tumor can grow without anabolic substrates or energy, the simultaneous targeting of glycolysis and glutaminolysis is expected to reduce the proliferation of most if not all GBM cells. Ketogenic metabolic therapy (KMT) leverages diet-drug combinations that inhibit glycolysis, glutaminolysis, and growth signaling while shifting energy metabolism to therapeutic ketosis. The glucose-ketone index (GKI) is a standardized biomarker for assessing biological compliance, ideally via real-time monitoring. KMT aims to increase substrate competition and normalize the tumor microenvironment through GKI-adjusted ketogenic diets, calorie restriction, and fasting, while also targeting glycolytic and glutaminolytic flux using specific metabolic inhibitors. Non-fermentable fuels, such as ketone bodies, fatty acids, or lactate, are comparatively less efficient in supporting the long-term bioenergetic and biosynthetic demands of cancer cell proliferation. The proposed strategy may be implemented as a synergistic metabolic priming baseline in GBM as well as other tumors driven by glycolysis and glutaminolysis, regardless of their residual mitochondrial function. Suggested best practices are provided to guide future KMT research in metabolic oncology, offering a shared, evidence-driven framework for observational and interventional studies.

Aberrant metabolism is recognized as a hallmark of cancer, a pillar necessary for cellular proliferation. Regarding bioenergetics (ATP generation), most cancers display a preference not only toward aerobic glycolysis (“Warburg effect”) and glutaminolysis (mitochondrial substrate level-phosphorylation) but also toward other metabolites such as lactate, pyruvate, and fat-derived sources. These secondary metabolites can assist in proliferation but cannot fully cover ATP demands. The concept of a static metabolic profile is challenged by instances of heterogeneity and flexibility to meet fuel/anaplerotic demands. Although metabolic therapies are a promising tool to improve therapeutic outcomes, either via pharmacological targets or press-pulse interventions, metabolic plasticity is rarely considered. Lack of bioenergetic analysis in vitro and patient-derived models is hindering translational potential. Here, we review the bioenergetics of cancer and propose a simple analysis of major metabolic pathways, encompassing both affordable and advanced techniques. A comprehensive compendium of Seahorse XF bioenergetic measurements is presented for the first time. Standardization of principal readouts might help researchers to collect a complete metabolic picture of cancer using the most appropriate methods depending on the sample of interest.

Most studies on ketosis have focused on short-term effects, male athletes, or weight loss. Hereby, we studied the effects of short-term ketosis suppression in healthy women on long-standing ketosis. Ten lean (BMI 20.5 ± 1.4), metabolically healthy, pre-menopausal women (age 32.3 ± 8.9) maintaining nutritional ketosis (NK) for > 1 year (3.9 years ± 2.3) underwent three 21-day phases: nutritional ketosis (NK; P1), suppressed ketosis (SuK; P2), and returned to NK (P3). Adherence to each phase was confirmed with daily capillary D-beta-hydroxybutyrate (BHB) tests (P1 = 1.9 ± 0.7; P2 = 0.1 ± 0.1; and P3 = 1.9 ± 0.6 pmol/L). Ageing biomarkers and anthropometrics were evaluated at the end of each phase. Ketosis suppression significantly increased: insulin, 1.78-fold from 33.60 (± 8.63) to 59.80 (± 14.69) pmol/L (p = 0.0002); IGF1, 1.83-fold from 149.30 (± 32.96) to 273.40 (± 85.66) µg/L (p = 0.0045); glucose, 1.17-fold from 78.6 (± 9.5) to 92.2 (± 10.6) mg/dL (p = 0.0088); respiratory quotient (RQ), 1.09-fold 0.66 (± 0.05) to 0.72 (± 0.06; p = 0.0427); and PAI-1, 13.34 (± 6.85) to 16.69 (± 6.26) ng/mL (p = 0.0428). VEGF, EGF, and monocyte chemotactic protein also significantly increased, indicating a pro-inflammatory shift. Sustained ketosis showed no adverse health effects, and may mitigate hyperinsulinemia without impairing metabolic flexibility in metabolically healthy women.

Invasion of high-grade glioma (HGG) cells through the brain and spinal cord is a leading cause of cancer death in children. Despite advances in treatment, survivors often suffer from lifelong adverse effects of the current toxic therapies used for management. This study investigated the influence of nutritional ketosis on the therapeutic action of mebendazole (MBZ) and devimistat (CPI-613) against the highly invasive VM-M3 and non-invasive CT-2A glioblastoma cells grown orthotopically in juvenile syngeneic mice. Additionally, both drugs were tested in the human pediatric GBM cell line SF-188. DON (6-Diazo-5-oxo-L-norleucine) was used as a positive drug control for glutamine targeting. Cerebral implantation of the VM-M3 cells, which are mesenchymal origin, invaded throughout the brain and the spinal column similar to that seen in children with HGG. Neither the CT-2A nor the VM-NM1 glioblastoma stem cell tumors showed distal invasion in syngeneic juvenile mouse brains. The maximum therapeutic benefit of MBZ and CPI-613 on tumor invasion, growth, and mouse survival occurred only when the drugs were administered together with a ketogenic diet (KD). MBZ treatment inhibited both the glutaminolysis and the glycolysis pathways in VM-M3 cells grown either in vivo or in vitro . Both MBZ and CPI-613 significantly reduced the in vitro growth and viability of the SF-188 cells. Moreover, drug administration together with the KD allowed for lower dosing thus minimizing toxicity while improving overall survival of the mice. This preclinical study in two different HGGs, grown in syngeneic juvenile mice, highlights the potential importance of diet/drug therapeutic strategies for managing childhood brain cancer.

Background: As the growing global population continues to age, the risk of chronic metabolic diseases, including cardiovascular disease, neurodegenerative disorders, type 2 diabetes mellitus, and fatty liver disease, increases considerably. Driven largely by lifestyle factors and metabolic dysfunction, this escalating health crisis is placing mounting pressure on healthcare systems and contributing to significant economic costs. Insulin resistance and hyperinsulinaemia are major drivers of these disorders, emphasising the need for early detection and intervention. Changes in liver enzymes, such as alanine aminotransferase (ALT) and gamma-glutamyl transferase (GGT), commonly assessed in routine laboratory testing, can serve as biomarkers of early-stage insulin resistance, offering a potentially underutilised window for intervention and disease prevention. Correspondingly, low-carbohydrate ketogenic diets have shown to be effective in reversing insulin resistance, metabolic disease, and liver disease. Objectives: We chose to explore the relationship between suppressing ketosis and changes in liver enzymes in the Ketosis Suppression and Ageing cohort. Methods: Ten lean (BMI 20.5 kg/m² ± 1.4), healthy young women (age 32.3 ± 8.9 years) who habitually followed a ketogenic diet maintaining nutritional ketosis (NK) for an average of 3.9 years (±2.3) were exposed to a higher carbohydrate diet, in line with standard healthy eating guidelines for a 21-day phase and then transitioned back to a ketogenic diet. Results: Carbohydrate challenge and suppression of ketosis increased insulin resistance score HOMA-IR by 2.13-fold (p = 0.0008), GKI by 22.28-fold (p = 0.0024), and liver markers ALT by 1.85-fold (p = 0.0010), GGT, 1.29-fold (p = 0.0087) and the ALT/AST, 1.30-fold (p = 0.0266), reflecting an adverse pattern suggestive of hepatic insulin resistance. Conclusions: These results support the clinical utility of liver markers as early and directional signs of hyperinsulinaemia.