Figure 6 - available via license: Creative Commons Attribution 4.0 International
Content may be subject to copyright.
Metabolic profiling and ATP production rate of C6-rtTA-AMPK-DN cells. The oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) were measured using the Seahorse XFe96 flux analyzer. The rate of ATP production in C6-rtTA-AMPK-DN cells was quantified by real-time ATP rate assay following 6 h of severe (0.1 mM) or total (0 mM) glucose deprivation. Data from four biological replicates are presented as means ± SEM. (A) Energetic map of not
Source publication
Energy homeostasis in the central nervous system largely depends on astrocytes, which provide metabolic support and protection to neurons. Astrocytes also ensure the clearance of extracellular glutamate through high-affinity transporters, which indirectly consume ATP. Considering the role of the AMP-activated protein kinase (AMPK) in the control of...
Contexts in source publication
Context 1
... Seahorse XF analyzer allows one to quantitatively assess the mitochondrial and glycolytic ATP production rates through the measures of changes in oxygen consumption and extracellular acidification, after sequential addition of the metabolic modulators oligomycin (inhibitor of complex V of the electron transport chain) and rotenone/antimycin A (inhibitors of complexes I and III of the electron transport chain, respectively). Under standard culture conditions, C6-rtTA-AMPK-DN cells mainly generated ATP through a glycolytic metabolism, and this was not significantly modified after partial silencing of the AMPK activity ( Figure 6A,C). Upon complete glucose depri-vation, the oxygen consumption rate increased in C6-rtTA-AMPK-DN cells, reflecting an increase in the mitochondrial ATP production rate (Figure 6B,D). ...
Context 2
... standard culture conditions, C6-rtTA-AMPK-DN cells mainly generated ATP through a glycolytic metabolism, and this was not significantly modified after partial silencing of the AMPK activity ( Figure 6A,C). Upon complete glucose depri-vation, the oxygen consumption rate increased in C6-rtTA-AMPK-DN cells, reflecting an increase in the mitochondrial ATP production rate (Figure 6B,D). The same observation was obtained 6 h after adding DOX, indicating that cells can switch to mitochondrial ATP production when glycolysis is reduced, independently of the AMPK activity ( Figure 6C,D). ...
Context 3
... complete glucose depri-vation, the oxygen consumption rate increased in C6-rtTA-AMPK-DN cells, reflecting an increase in the mitochondrial ATP production rate (Figure 6B,D). The same observation was obtained 6 h after adding DOX, indicating that cells can switch to mitochondrial ATP production when glycolysis is reduced, independently of the AMPK activity ( Figure 6C,D). This metabolic shift, however, cannot entirely compensate for the lack of glucose, as total ATP production rates are lower in both cell lines ( Figure 6D). ...
Context 4
... same observation was obtained 6 h after adding DOX, indicating that cells can switch to mitochondrial ATP production when glycolysis is reduced, independently of the AMPK activity ( Figure 6C,D). This metabolic shift, however, cannot entirely compensate for the lack of glucose, as total ATP production rates are lower in both cell lines ( Figure 6D). It is noteworthy that when treating cells with DOX, the switch to mitochondrial ATP production tends to be less efficient, and the mitochondrial ATP production rate seems slightly lower (p = 0.08) ( Figure 6D). ...
Context 5
... metabolic shift, however, cannot entirely compensate for the lack of glucose, as total ATP production rates are lower in both cell lines ( Figure 6D). It is noteworthy that when treating cells with DOX, the switch to mitochondrial ATP production tends to be less efficient, and the mitochondrial ATP production rate seems slightly lower (p = 0.08) ( Figure 6D). This suggests that the AMPK activity might contribute to the activation of mitochondria in conditions of stress. ...
Context 6
... 2022, 11, x FOR PEER REVIEW 11 of 19 transport chain, respectively). Under standard culture conditions, C6-rtTA-AMPK-DN cells mainly generated ATP through a glycolytic metabolism, and this was not significantly modified after partial silencing of the AMPK activity ( Figure 6A,C). Upon complete glucose deprivation, the oxygen consumption rate increased in C6-rtTA-AMPK-DN cells, reflecting an increase in the mitochondrial ATP production rate ( Figure 6B,D). ...
Context 7
... standard culture conditions, C6-rtTA-AMPK-DN cells mainly generated ATP through a glycolytic metabolism, and this was not significantly modified after partial silencing of the AMPK activity ( Figure 6A,C). Upon complete glucose deprivation, the oxygen consumption rate increased in C6-rtTA-AMPK-DN cells, reflecting an increase in the mitochondrial ATP production rate ( Figure 6B,D). The same observation was obtained 6 h after adding DOX, indicating that cells can switch to mitochondrial ATP production when glycolysis is reduced, independently of the AMPK activity ( Figure 6C,D). ...
Context 8
... complete glucose deprivation, the oxygen consumption rate increased in C6-rtTA-AMPK-DN cells, reflecting an increase in the mitochondrial ATP production rate ( Figure 6B,D). The same observation was obtained 6 h after adding DOX, indicating that cells can switch to mitochondrial ATP production when glycolysis is reduced, independently of the AMPK activity ( Figure 6C,D). This metabolic shift, however, cannot entirely compensate for the lack of glucose, as total ATP production rates are lower in both cell lines ( Figure 6D). ...
Context 9
... same observation was obtained 6 h after adding DOX, indicating that cells can switch to mitochondrial ATP production when glycolysis is reduced, independently of the AMPK activity ( Figure 6C,D). This metabolic shift, however, cannot entirely compensate for the lack of glucose, as total ATP production rates are lower in both cell lines ( Figure 6D). It is noteworthy that when treating cells with DOX, the switch to mitochondrial ATP production tends to be less efficient, and the mitochondrial ATP production rate seems slightly lower (p = 0.08) ( Figure 6D). ...
Context 10
... metabolic shift, however, cannot entirely compensate for the lack of glucose, as total ATP production rates are lower in both cell lines ( Figure 6D). It is noteworthy that when treating cells with DOX, the switch to mitochondrial ATP production tends to be less efficient, and the mitochondrial ATP production rate seems slightly lower (p = 0.08) ( Figure 6D). This suggests that the AMPK activity might contribute to the activation of mitochondria in conditions of stress. ...
Similar publications
Exposure to stressful stimuli promotes multi‐system biological responses to restore homeostasis. Catecholaminergic neurons in the rostral ventrolateral medulla (RVLM) facilitate sympathetic activity and promote physiological adaptations, including glycaemic mobilization and corticosterone release. While it is unclear how brain regions involved in t...
Citations
... Based on previous studies highlighting an altered control of CNS metabolism in ALS, we herein characterized the expression of AMPKα in primary cultures of astrocytes derived from non-transgenic (Ntg) and transgenic rats carrying an ALS-associated mutated form of the superoxide dismutase 1 gene (hSOD1 G93A ). Our previous work suggested that AMPK regulates the metabolic adaptation to glucose deprivation, as an experimental model of metabolic stress, in an astrocyte-like cell line [37]. Therefore, we hypothesized that a deficiency in AMPK expression or function in astrocytes derived from the transgenic rat model of ALS might alter the astrocyte response to stress and impact on their metabolic plasticity, contributing to the loss of physiological support of nearby neurons. ...
... The reverse transcription was then performed using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer's instructions. Real-time PCR amplifications were carried out as previously described [37]. The following primers were used in the RT-qPCR reactions: Prkaa1: 5 -TTAAACCCACAGAAATCCAAACAC-3 (forward), 5 -CTTCGCACACGCAAATAATAGG-3 (reverse); Prkaa2: 5 -GTGGTGTTATCCTGTATGCCCTTCT-3 (forward), 5 -CTGTTTAAACCATTCATGCTCTCGT-3 (reverse); Gapdh: ...
... Cells were seeded into 60 mm cultures dishes at a density of 500,000 cells/dish and Western blot experiments were performed as previously described [37]. Guided by the molecular weight of the protein of interest, the membranes were cut horizontally into several pieces. ...
Alterations in the activity of the regulator of cell metabolism AMP-activated protein kinase (AMPK) have been reported in motor neurons from patients and animal models of amyotrophic lateral sclerosis (ALS). Considering the key role played by astrocytes in modulating energy metabolism in the nervous system and their compromised support towards neurons in ALS, we examined whether a putative alteration in AMPK expression/activity impacted astrocytic functions such as their metabolic plasticity and glutamate handling capacity. We found a reduced expression of AMPK mRNA in primary cultures of astrocytes derived from transgenic rats carrying an ALS-associated mutated superoxide dismutase (hSOD1G93A). The activation of AMPK after glucose deprivation was reduced in hSOD1G93A astrocytes compared to non-transgenic. This was accompanied by a lower increase in ATP levels and increased vulnerability to this insult, although the ATP production rate did not differ between the two cell types. Furthermore, soliciting the activity of glutamate transporters was found to induce similar AMPK activity in these cells. However, manipulation of AMPK activity did not influence glutamate transport. Together, these results suggest that the altered AMPK responsiveness in ALS might be context dependent and may compromise the metabolic adaptation of astrocytes in response to specific cellular stress.
... While previous understanding of mechanisms focused on the effects of ES on neuronal depolarization, a new methodology, based on transcriptomic and proteomic analyses, supports the effects of electrical signals beyond neurons involving glial cells and neuroinflammation [5][6][7][8]. We previously used C6 glioma cells in culture subjected to ES as a model of astrocytes [9][10][11] and showed modulation of gene expression that was dependent on the anodic content of the stimulating biphasic pulses [9]. This work indicated that the effects on gene expression of a selection of gliarelated genes were dependent on the ES waveform composition. ...
This research focused on the development of an astrocyte cell model system (C6 glioma) for the assessment of molecular changes in response to cathodic passively balanced pulsed electrical stimulation at a rate of 50 Hz (60 µs duration, 0.15 mA intensity). Cells treated with selected neurotransmitters (glutamate, adenosine, D-serine, and γ-aminobutyric acid) were monitored (using specific fluorescent probes) for changes in levels of intracellular nitric oxide, calcium ions, and/or chloride. ES exerted an inhibitory effect on NO, increased calcium and had no effect on chloride. Using this model, cells can be assessed qualitatively and quantitatively for changes and these changes can be correlated with the putative molecular effects that electrical stimulation has on astrocytes and their role in glia-mediated diseases. This model system allows for faster and cheaper experiments than those involving animal models due to the potential to easily vary the conditions, reduce the number of variables (especially problematic in animal models), and closely monitor the cellular effects.
The occurrence and progression of tumors are inseparable from glucose metabolism. With the development of tumors, the volume increases gradually and the nutritional supply of tumors cannot be fully guaranteed. The tumor microenvironment changes and glucose deficiency becomes the common stress environment of tumors. Here, we discuss the mutual influences between glucose deprivation and other features of the tumor microenvironment, such as hypoxia, immune escape, low pH, and oxidative stress. In the face of a series of stress responses brought by glucose deficiency, different types of tumors have different coping mechanisms. We summarize the tumor studies on glucose deficiency in the last decade and review the genes and pathways that determine the fate of tumors under harsh conditions. It turns out that most of these genes help tumor cells survive in glucose-deprivation conditions. The development of related inhibitors may bring new opportunities for the treatment of tumors.
