Short-term starvation is a strategy to unravel the cellular capacity of oxidizing specific exogenous/endogenous substrates in mitochondria

Abstract

Mitochondrial oxidation of nutrients is tightly regulated in response to the cellular environment and changes in energy demands. studies evaluating the mitochondrial capacity of oxidizing different substrates are important for understanding metabolic shifts in physiological adaptations and pathological conditions, but may be influenced by the nutrients present in the culture medium or by the utilization of endogenous stores. One such influence is exemplified by the Crabtree effect (the glucose-mediated inhibition of mitochondrial respiration) as most experiments are performed in glucose-containing media. Here, using high-resolution respirometry, we evaluated the oxidation of endogenous or exogenous substrates by cell lines harboring different metabolic profiles. We found that a 1-h deprivation of the main energetic nutrients is an appropriate strategy to abolish interference of endogenous or undesirable exogenous substrates with the cellular capacity of oxidizing specific substrates, namely glutamine, pyruvate, glucose, or palmitate, in mitochondria. This approach primed mitochondria to immediately increase their oxygen consumption after the addition of the exogenous nutrients. All starved cells could oxidize exogenous glutamine, whereas the capacity for oxidizing palmitate was limited to human hepatocarcinoma Huh7 cells and to C2C12 mouse myoblasts that differentiated into myotubes. In the presence of exogenous glucose, starvation decreased the Crabtree effect in Huh7 and C2C12 cells and abrogated it in mouse neuroblastoma N2A cells. Interestingly, the fact that the Crabtree effect was observed only for mitochondrial basal respiration but not for the maximum respiratory capacity suggests it is not caused by a direct effect on the electron transport system.

Systems biology identifies preserved integrity but impaired metabolism of mitochondria due to a glycolytic defect in Alzheimer's disease neurons

“…The reduction of mitochondrial NADH would impair mitochondrial respiratory capacity, as predicted by the model and measured by experiments. The link between glycolysis and maximal respiration has been previously reported—inhibition of glycolysis by glucose restriction or pharmacological inhibitors can impair respiration generally (Clerc & Polster, ; Pike Winer & Wu, ; Zeidler et al, ), and maximal respiration specifically (Gouarne et al, ; Tan, Xiao, Li, Zeng, & Yin, )—further validating the strong link between glucose metabolism and mitochondrial activity.…”

Anaplerotic Role of Glucose in the Oxidation of Endogenous Fatty Acids during Dengue Virus Infection

“…In order to evaluate the use of different substrates by cells, we submitted the cells to a short-term nutrient deprivation protocol, which consisted of cell incubation for 1 h in restricted medium (RM; DMEM [Dulbecco’s modified Eagle’s medium] without glucose, glutamine, and sodium pyruvate) without fetal bovine serum (FBS), before carrying out the experiments. This protocol makes the cells prone to use the chosen substrate without loss of cellular viability, overcoming the influence of the nutrients present in the culture medium ( 26 ). Huh7 cells were subjected to mock infection or DENV infection for 24 h, and the OCR was measured when the only substrate added after nutrient deprivation was palmitate, glutamine, glucose, or pyruvate ( Fig.…”

“…We used short-term cellular starvation as a strategy to prevent the possibility that the nutrients already present in the culture medium as well as the endogenous substrates would influence the observation of the metabolic alterations induced by infection. This experimental approach was recently developed by our group ( 26 ) and allowed us to evaluate the contribution of fatty acids, glutamine, glucose, and pyruvate to mitochondrial oxygen consumption during DENV infection. Interestingly, we found that DENV infection inhibited the Crabtree effect, resulting in an increase in the cellular capacity of metabolizing glucose.…”

Glucose Favors Lipid Anabolic Metabolism in the Invasive Breast Cancer Cell Line MDA-MB-231

“…In order to avoid possible interferences with other metabolites from culture media and serum, in this work, we performed the short-time experiments in very restrictive conditions, although it should be taken into account that other metabolites may also be present in an individual. Thus, this kind of methodological approximation allows for the determination of the short-time preference of a metabolic substrate in these cells when only these fuels are present at physiological concentration after a fasted period [12,13]. Combining long-and short-term experiments covers different possible scenarios, such as the short-term response of the cells to a drastic nutritional change (their immediate fuel preference) and the long-term consequence of that change (their ability to adapt to a different nutritional condition).…”