Proliferating Astrocytes in Primary Culture Do Not Depend upon Mitochondrial Respiratory Complex I Activity or Oxidative Phosphorylation

1 μg/mL oligomycin causes a minor nonspecific effect on mitochondrial and cellular function.

“the use of this oligomycin concentration is common in the literature and causes a minor nonspecific effect on mitochondrial and cellular function [15,25,26,27].”

• cites [15] Hao, 2010, Oligomycin-Induced Bioenergetic Adaptation in Cancer Cells with Heterogeneous Bioenergetic Organization, J. Biol. Chem. sourceNot checked — no full text of the source
• cites [25] Matsui, 1996, Mechanism Responsible for Oligomycin-Induced Occlusion of Na+ within Na/K-ATPase, J. Biol. Chem. sourceCould not verify

  The Results and Discussion report concentration-dependent effects of oligomycin on Na/K-ATPase and ion exchange, not on mitochondrial or overall cellular function. Searches for 1 μg/mL and mitochondrial or nonspecific effects found no passage assessing whether that dose causes a minor nonspecific effect. (LLM, conf=0.75)

  Experimental — accuracy not yet measured
• cites [26] Ruas, J.S., Siqueira-Santos, E.S., Amigo, I., Rodrigues-Silva, E., Kowaltowski, A.J., and Castilho, R.F. (2016). Underestimation of the Maximal Capacity of the Mitochondrial Electron Transport System in Oligomycin-Treated Cells. PLoS ONE, 11. sourceDisputed

  The Results report that oligomycin markedly inhibited CCCP-stimulated oxygen consumption in intact T98G cells, with spare respiratory capacity underestimated by 43.7 ± 2.3%; this is contrary to describing its effect as minor. The Discussion says effects on nonspecific targets are possible, not that a minor nonspecific effect was established. The paper reserves its finding of a minor inhibitory effect for permeabilized cells or isolated mitochondria. (LLM, conf=0.87)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured
• cites [27] Castilho, 1998, Mitochondrial Control of Acute Glutamate Excitotoxicity in Cultured Cerebellar Granule Cells, J. Neurosci. sourcePartly supported

  The Figure 1 caption reports that oligomycin at 2.5 μg/mL did not affect one glutamate-induced calcium transient in cultured cerebellar granule cells. The paper also identifies oligomycin as an ATP-synthase inhibitor. It does not establish that 1 μg/mL is commonly used or that its effects on mitochondrial and cellular function are minor and nonspecific. (LLM, conf=0.79)

  Experimental — accuracy not yet measured

The inhibition of complexes III and IV affects the dihydroorotate dehydrogenase pathway involved in ubiquinone-mediated de novo pyrimidine synthesis.

“The inhibition of complexes III and IV affects the dihydroorotate dehydrogenase pathway involved in ubiquinone-mediated de novo pyrimidine synthesis [49,50,51].”

• cites [49] Kennedy, 1973, Distribution, Subcellular Localization, and Product Inhibition of Dihydroorotate Oxidation in the Rat, Arch. Biochem. Biophys. sourceNot checked — no full text of the source
• cites [50] Miyake, 2022, Inhibition of Mitochondrial Complex III or Dihydroorotate Dehydrogenase (DHODH) Triggers Formation of Poly (A) RNA Foci Adjacent to Nuclear Speckles Following Activation of ATM (Ataxia Telangiectasia Mutated), RNA Biol. sourcePartly supported

  The Introduction (page unavailable) links DHODH-mediated de novo pyrimidine synthesis to coenzyme Q and respiratory-chain complex III; the Results (page unavailable) state that complex III inhibition decreased DHODH activity. The paper does not establish the same effect for complex IV inhibition: its complex IV inhibitor did not produce the nuclear RNA accumulation seen with complex III inhibitors. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [51] Khutornenko, 2010, Pyrimidine Biosynthesis Links Mitochondrial Respiration to the P53 Pathway, Proc. Natl. Acad. Sci. USA sourceDisputed

  The Results show that complex III inhibition impairs DHODH-linked pyrimidine biosynthesis, but explicitly report that the complex IV inhibitor KCN did not affect pyrimidine levels. The Discussion likewise says KCN did not affect pyrimidine nucleotide levels, contrary to the claim that inhibition of both complexes affects this pathway. (LLM, conf=0.88)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured

The availability of NAD+ as an electron acceptor enables aspartate synthesis through aspartate aminotransferase and rescues the proliferation of cultured cells with dysfunctions or inhibition of the electron transport system.

“The availability of NAD+ as an electron acceptor enables aspartate synthesis through aspartate aminotransferase and rescues the proliferation of cultured cells with dysfunctions or inhibition of the electron transport system [16,17].”

• cites [16] Birsoy, 2015, An Essential Role of the Mitochondrial Electron Transport Chain in Cell Proliferation Is to Enable Aspartate Synthesis, Cell sourcePartly supported

  The Results and Discussion sections report that pyruvate restores aspartate synthesis through GOT1, an aspartate aminotransferase, and rescues proliferation in cultured cells with inhibited or defective ETC activity. The proposed role of regenerated NAD+ in driving this pathway through MDH1 is described as a model, however; the claim presents NAD+ availability as an established cause. Page numbers are unavailable in our copy. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [17] Sullivan, 2015, Supporting Aspartate Biosynthesis Is an Essential Function of Respiration in Proliferating Cells, Cell sourceSupported

Mature astrocytes are naturally glycolytic cells that can survive by aerobic glycolysis for an extended period without functional impairments.

“Previous studies have shown that mature astrocytes are naturally glycolytic cells that can survive by aerobic glycolysis for an extended period without functional impairments [18].”

• cites [18] Supplie, 2017, Respiration-Deficient Astrocytes Survive as Glycolytic Cells in Vivo, J. Neurosci. sourcePartly supported

  The Abstract reports that respiration-deficient cerebellar Bergmann glia survived for a year by aerobic glycolysis without glial pathology or nonspecific signs of neurodegeneration; it extends this to other astrocytes only as a presumption. The Discussion describes adult steady-state Bergmann glia without signs of cerebellar dysfunction and calls natural glycolysis a supported model. The claim presents survival without functional impairments as established for mature astrocytes generally, beyond what the study directly tested. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

When the electron transport system is inhibited, reversed carbon flux through the mitochondrial dehydrogenases of Krebs cycle can provide oxidized redox equivalents to support glutamate dehydrogenase activity and cell viability.

“Additionally, when the electron transport system is inhibited, reversed carbon flux through the mitochondrial dehydrogenases of Krebs cycle can provide oxidized redox equivalents to support glutamate dehydrogenase activity and cell viability [42].”

• cites [42] Vandekeere, 2022, Reversal of Mitochondrial Malate Dehydrogenase 2 Enables Anaplerosis via Redox Rescue in Respiration-Deficient Cells, Mol. Cell sourceSupported

No difference among treatments is observed when ATP content is used to estimate cell proliferation.

“No difference among treatments was observed when ATP content was used to estimate cell proliferation (Figure 4B).”

In astrocytes chronically treated with oligomycin, acute injection of oligomycin does not further inhibit OCR.

“the injection of 1 µg/mL oligomycin did not cause further inhibition of OCR in cells chronically treated with oligomycin.”

• Also stated in: § 3.2. The Effect of the Mitochondrial Inhibitors Oligomycin and Piericidin A on the Rate of Astrocytes’ Oxygen Consumption (OCR) · plain

  “adding oligomycin in astrocytes chronically treated with this compound did not further inhibit OCR.”

Cell growth evaluated by trypan blue significantly decreases by 20% after 7 days in the presence of mitochondrial inhibitors compared with DMSO controls.

“When oligomycin and piericidin A treatments were compared with DMSO controls, cell growth evaluated by trypan blue significantly decreased by 20% after 7 days in the presence of mitochondrial inhibitors (Figure 4A).”

1 μM antimycin causes further inhibition of OCR due to arrest of the electron transport system by respiratory complex III inhibition.

“The presence of 1 µM antimycin caused a further inhibition of OCR due to an arrest of the electron transport system caused by respiratory complex III inhibition.”

The presence of mitochondrial inhibitors oligomycin or piericidin A for up to 6 days in culture medium elicits only minor effects on astrocyte growth.

“The presence of these mitochondrial inhibitors for up to 6 days in a culture medium elicited only minor effects on astrocyte growth.”

• Also stated in: § 3.3. Astrocytes Can Grow and Survive despite the Inhibition of ATP Synthase and Respiratory Complex I · plain

  “These data show that astrocytes can proliferate regardless of the presence of mitochondrial inhibitors.”

Astrocytes in primary culture have large spare respiratory capacity.

“Metabolic characterization of the astrocytes showed a relevant glycolytic metabolism under basal conditions, despite functional oxidative phosphorylation and large spare respiratory capacity.”

Piericidin A or oligomycin treatment does not affect astrocyte morphology.

“neither the morphology nor the proportion of glial fibrillary acidic protein-positive astrocytes in culture was affected by piericidin A or oligomycin.”

• Also stated in: § 3.3. Astrocytes Can Grow and Survive despite the Inhibition of ATP Synthase and Respiratory Complex I · plain

  “The GFAP labeling of astrocytes cultured in the presence of oligomycin and piericidin A (Figure 4D) showed that these treatments did not affect astrocyte morphology.”

4 μM piericidin A does not result in additional inhibition of OCR beyond 2 μM.

“In this set of experiments, the presence of 4 µM piericidin A did not result in additional inhibition of OCR.”

Piericidin A or oligomycin treatment does not affect the proportion of GFAP-positive astrocytes in culture.

“neither the morphology nor the proportion of glial fibrillary acidic protein-positive astrocytes in culture was affected by piericidin A or oligomycin.”

• Also stated in: § 3.3. Astrocytes Can Grow and Survive despite the Inhibition of ATP Synthase and Respiratory Complex I · plain

  “More than 95% of the cells were positive for GFAP both in control conditions and in the two treatments (Figure 4E).”

In astrocytes chronically treated with piericidin A, acute injection of piericidin A does not elicit additional inhibition of OCR.

“the acute injection of 2 µM piericidin A did not elicit any additional inhibition of OCR.”

Astrocytes in primary culture have functional oxidative phosphorylation under basal conditions.

“Metabolic characterization of the astrocytes showed a relevant glycolytic metabolism under basal conditions, despite functional oxidative phosphorylation and large spare respiratory capacity.”

Chronic treatment with oligomycin causes significantly lower inhibition of OCR compared with acute treatment.

“The chronic treatment with oligomycin caused a significantly lower inhibition of OCR when compared with the acute treatment.”

Cell proliferation measured by MTT reduction is 37% and 53% higher in astrocytes cultured for 7 days with oligomycin and piericidin A, respectively, compared with controls.

“cell proliferation measured by MTT reduction was 37% and 53% higher in astrocytes cultured for 7 days in the presence of oligomycin and piericidin A, respectively (Figure 4C).”

Astrocytes in primary culture show a relevant glycolytic metabolism under basal conditions.

“Metabolic characterization of the astrocytes showed a relevant glycolytic metabolism under basal conditions, despite functional oxidative phosphorylation and large spare respiratory capacity.”

MTT reduction increases by 3.4, 4.7, and 5.2 from D1 to D7 in DMSO, oligomycin, and piericidin A treatments, respectively.

“In the MTT assay, cell proliferation increased by 3.4, 4.7, and 5.2 from D1 to D7 in the DMSO, oligomycin, and piericidin A treatment, respectively.”

ATP content increases approximately 2.5 times from D1 to D7 independent of treatment.

“When measuring ATP content, an increase of approximately 2.5 times was observed from D1 to D7 independent of treatment.”

Titration of piericidin A in astrocyte OCR shows progressive inhibition from 0.02 μM and maximum inhibition at 2.0 μM.

“A progressive inhibition of OCR was induced by adding 0.02 µM piericidin A, and a maximum inhibition was achieved a few minutes after the addition of 2.0 µM piericidin A.”

The oligomycin-insensitive fraction of OCR increases in chronic exposure to oligomycin.

“However, the oligomycin-insensitive fraction increased in chronic exposure to this inhibitor.”

In astrocytes treated with piericidin A, no significant difference in OCR is observed between chronic and acute treatment.

“In astrocytes treated with piericidin A, no significant difference in OCR was observed due to chronic or acute treatment.”

More than 95% of cells are positive for GFAP in control conditions and in oligomycin and piericidin A treatments.

“More than 95% of the cells were positive for GFAP both in control conditions and in the two treatments (Figure 4E).”

As little as 0.1 μg/mL oligomycin would significantly inhibit mitochondrial respiration.

“Based on this titration, as little as 0.1 µg/mL oligomycin would significantly inhibit mitochondrial respiration.”

The presence of oligomycin or piericidin A did not elicit noticeable morphological changes in cultured astrocytes.

“Moreover, the presence of oligomycin or piericidin A did not elicit noticeable morphological changes or increased cell death in cultured astrocytes (Figure 5).”

In the presence of piericidin A, the increased non-phosphorylating respiration effect was not observed.

“In the presence of piericidin A, this effect was not observed, and possibly other mechanisms have been established to circumvent the increased superoxide production due to complex I inhibition.”

Glycolysis sustains approximately 45% of the cellular ATP yielding in proliferating astrocytes.

“In this sense, glycolysis sustains approximately 45% of the cellular ATP yielding in proliferating astrocytes.”

The growth and survival of astrocytes did not differ among Media 1, 2, and 3 as evaluated by trypan blue, ATP content, and MTT assay.

“The growth and survival of astrocytes did not differ among Media 1, 2, and 3 when viability underwent evaluation by trypan blue (Figure 1B), total ATP content in cells (Figure 1C), and MTT assay (Figure 1D).”

When piericidin A was in the medium, glucose consumption increased by 130%.

“When piericidin A was in the medium, glucose consumption increased by 130%, and the lactate release increased by 160%.”

In the presence of oligomycin, lactate release increased by 145%.

“In the presence of oligomycin, glucose consumption increased by 129%, and the lactate release increased by 145%.”

Figure 6 shows a significant increase in glucose consumption in the presence of respiratory chain inhibitors.

“Figure 6 shows a significant increase in glucose consumption and lactate release in the presence of respiratory chain inhibitors.”

Oxidative phosphorylation produces 220.2 ± 49.0 pmol ATP/sec/106 cells (N = 4) in proliferating astrocytes cultivated in control conditions.

“According to these measurements, 179.2 ± 57.4 pmol ATP/sec/106 cells (N = 7) are generated by glycolysis in proliferating astrocytes cultivated in control conditions, and 220.2 ± 49.0 pmol ATP/sec/106 cells (N = 4) are produced during oxidative phosphorylation at the same conditions.”

The piericidin A concentration used was the minimum required to reach a maximal inhibitory effect.

“On the other hand, the piericidin A concentration used was the minimum required to reach a maximal inhibitory effect.”

Figure 5 shows no statistical difference in LDH activity in the medium when comparing oligomycin and piericidin A conditions to the control condition.

“Figure 5 shows no statistical difference in LDH activity in the medium when comparing oligomycin and piericidin A conditions to the control condition.”

In the presence of oligomycin, glucose consumption increased by 129%.

“In the presence of oligomycin, glucose consumption increased by 129%, and the lactate release increased by 145%.”

Proliferating astrocytes consume glucose and release lactate to similar extents even in the control condition.

“Notably, data in Figure 6 also indicates that proliferating astrocytes consume glucose and release lactate to similar extents even in the control condition.”

When piericidin A was in the medium, lactate release increased by 160%.

“When piericidin A was in the medium, glucose consumption increased by 130%, and the lactate release increased by 160%.”

The culture in different media did not elicit any noticeable morphological change in astrocytes.

“The culture in these different media did not elicit any noticeable morphological change in astrocytes.”

Glycolysis generates 179.2 ± 57.4 pmol ATP/sec/106 cells (N = 7) in proliferating astrocytes cultivated in control conditions.

“According to these measurements, 179.2 ± 57.4 pmol ATP/sec/106 cells (N = 7) are generated by glycolysis in proliferating astrocytes cultivated in control conditions, and 220.2 ± 49.0 pmol ATP/sec/106 cells (N = 4) are produced during oxidative phosphorylation at the same conditions.”

Glucose concentration in the culture medium did not interfere with the growth and survival of astrocytes.

“Here, glucose concentration in the culture medium did not interfere with the growth and survival of astrocytes.”

Figure 6 shows a significant increase in lactate release in the presence of respiratory chain inhibitors.

“Figure 6 shows a significant increase in glucose consumption and lactate release in the presence of respiratory chain inhibitors.”

Astrocytes chronically treated with oligomycin showed larger non-phosphorylating respiration (H+ leak).

“In our experiments, astrocytes chronically treated with oligomycin showed larger non-phosphorylating respiration (H+ leak).”

Despite inhibition of oxidative phosphorylation in astrocytes cultured with oligomycin or piericidin A, changes in ATP content were not observed.

“Despite the inhibition of oxidative phosphorylation in astrocytes cultured with oligomycin or piericidin A, changes in ATP content were not observed under these conditions (Figure 4B).”

The mass of lactate release was similar to that of glucose consumed, regardless of the condition.

“When astrocytes cultured in the absence or presence of mitochondrial inhibitors were evaluated concerning glucose consumption and lactate production (Figure 6), the mass of lactate release was similar that of glucose consumed, regardless of the condition.”

The growth and survival of primary astrocytes in culture do not require electron flux through respiratory complex I or oxidative phosphorylation.

“Our data suggest that astrocytes in primary culture can sustainably proliferate when their energy metabolism relies only on aerobic glycolysis since their growth and survival do not require electron flux through respiratory complex I or oxidative phosphorylation.”

• Also stated in: § 1. Introduction · boosted

  “Here, we demonstrated that proliferative mouse astrocytes in culture can sustainably proliferate when their energy metabolism relies only on aerobic glycolysis since their growth and survival do not require electron flux through respiratory complex I or oxidative phosphorylation.”

• Also stated in: § 3.3. Astrocytes Can Grow and Survive despite the Inhibition of ATP Synthase and Respiratory Complex I · plain

  “These data show that astrocytes can proliferate regardless of the presence of mitochondrial inhibitors.”

Astrocytes in primary culture can sustainably proliferate when their energy metabolism relies only on aerobic glycolysis.

“Our data suggest that astrocytes in primary culture can sustainably proliferate when their energy metabolism relies only on aerobic glycolysis since their growth and survival do not require electron flux through respiratory complex I or oxidative phosphorylation.”

• Also stated in: § 1. Introduction · boosted

  “Here, we demonstrated that proliferative mouse astrocytes in culture can sustainably proliferate when their energy metabolism relies only on aerobic glycolysis since their growth and survival do not require electron flux through respiratory complex I or oxidative phosphorylation.”

Compounds inhibiting mitochondrial respiration at sites downstream to complex I would impair cell viability and proliferation by a mechanism other than bioenergetics.

“Thus, compounds inhibiting mitochondrial respiration at sites downstream to complex I would impair cell viability and proliferation by a mechanism other than bioenergetics.”

In the presence of piericidin A, NAD can become highly reduced, causing a stronger reduction of MTT to formazan by dehydrogenases, thereby producing an increase in this measurement that does not reflect an increase in cell proliferation.

“In the presence of piericidin A, for example, the NAD can become highly reduced, causing a stronger reduction of MTT to formazan by dehydrogenases, thereby producing an increase in this measurement that does not reflect an increase in cell proliferation.”

GFAP labeling rules out the possibility that measured cell growth was due to proliferation of non-astrocytic cells.

“this labeling ruled out the possibility that the measured cell growth was due to the proliferation of non-astrocytic cells.”

This model is not representative of neuron-glial cultures, organotypic slices, or the whole brain.

“However, this model is not representative of neuron-glial cultures, organotypic slices, or the whole brain.”

The presence of mitochondrial inhibitors in the culture medium did not induce cell death other than the cell death resulting from the plating process and normal cell aging.

“Thus, the presence of mitochondrial inhibitors in the culture medium did not induce cell death other than the cell death resulting from the plating process and normal cell aging.”

Concentrations of piericidin A above 2 μM can inhibit the ubiquinol-citochrome c reductase activity of complex III.

“Even if a slightly better inhibitory effect could be reached with 4 µM piericidin A, concentrations of this compound above 2 µM can inhibit the ubiquinol-citochrome c reductase activity of complex III [28].”

• cites [28] Esposti, 1993, Complex I and Complex III of Mitochondria Have Common Inhibitorns Acting as Ubiquinone Antagonists, Biochem. Biophys. Res. Commun. sourceNot checked — no full text of the source

Our results are more valid in a context of reactive astrocytes.

“It is worth highlighting that our results are more valid in a context of reactive astrocytes since pure astrocyte cultures present typically reactive phenotype.”

• AssumptionRests on an assumption: Pure astrocyte cultures present typically reactive phenotype.

The glycolytic profile of these astrocytes could sustain the cell proliferation in the absence of mitochondrial respiration.

“The glycolytic profile of these astrocytes could sustain the cell proliferation in the absence of mitochondrial respiration.”

Astrocytes sustainably proliferate when their energy metabolism relies only on aerobic glycolysis.

“Here, we demonstrated that astrocytes sustainably proliferate when their energy metabolism relies only on aerobic glycolysis since their growth and survival do not require electron flux through respiratory complex I or oxidative phosphorylation.”

The presence of oligomycin and piericidin A had minor effects on astrocyte growth.

“Taken together, the results indicate that the presence of oligomycin and piericidin A did not affect cell survival and had minor effects on astrocyte growth.”

Primary astrocytes can be cultured with lower concentrations of glucose, closer to the physiological ones.

“Moreover, our results support that primary astrocytes can be cultured with lower concentrations of glucose, closer to the physiological ones.”

The inhibition of mitochondrial ADP phosphorylation or respiratory chain strongly stimulated glucose consumption and lactate release in cultured astrocytes.

“The inhibition of mitochondrial ADP phosphorylation or respiratory chain strongly stimulated glucose consumption and lactate release in cultured astrocytes.”