Regulation of astrocyte metabolism by mitochondrial translocator protein 18 kDa

There was a statistically significant impact of glucose concentration on OCR in MPAs.

“we observed a statistically significant impact of glucose concentration on OCR (Figure 4A,C; pglucose=0.044,F(1,58) = 4.232)”

There was no significant difference in NFκB phosphorylation in TSPO-/- MPAs compared to TSPO+/+ controls following either 3h (p=0.48) or 24h LPS stimulation (p=0.99).

“Post-hoc assessment confirmed no significant difference in NFκB phosphorylation in TSPO -/-MPAs compared to TSPO +/+ controls following either 3h (Figure 10A-C; p=0.48) or 24h LPS stimulation (Figure 10D-F; p=0.99).”

Mean glycolytic reserve was increased by 27.6% in TSPO -/- MPAs.

“Similarly, mean glycolytic capacity and reserve were increased by 25.8% and 27.6% respectively (Figure 3D,E; p<0.0001 (both parameters)).”

Mean basal OCR was reduced by 15.7% in TSPO -/- U373 cells compared to TSPO +/+ EV controls.

“These results were replicated in TSPO -/-U373 cells, where mean basal OCR was reduced by 15.7% (Figure 1E,F; p=0.0293)”

TNF release was enhanced in TSPO -/- MPAs at 24h LPS stimulation compared to TSPO +/+ cells.

“Compared to TSPO +/+ cells, inflammation induced by 3h lipopolysaccharide (LPS) stimulation of TSPO - /-MPAs revealed attenuated tumour necrosis factor release, which was enhanced in TSPO -/-MPAs at 24h LPS stimulation.”

TSPO is part of a protein complex containing both VDAC and CPT1a.

“Using this method, we established that TSPO is part of a protein complex containing both VDAC and CPT1a (Figure 6A, n=4).”

There was a statistically significant impact of glucose concentration on ECAR in MPAs.

“and ECAR (Figure 4A,D; pglucose=0.035,F(1,58) = 4.654)”

There were no statistically significant effects of genotype on ECAR in MPAs.

“and ECAR (Figure 4B,D; pgenotype=0.16,F(1,58) = 1.987).”

Inflammation induced by 3h LPS stimulation of TSPO -/- MPAs revealed attenuated tumour necrosis factor release compared to TSPO +/+ cells.

“Compared to TSPO +/+ cells, inflammation induced by 3h lipopolysaccharide (LPS) stimulation of TSPO - /-MPAs revealed attenuated tumour necrosis factor release, which was enhanced in TSPO -/-MPAs at 24h LPS stimulation.”

Mean glycolytic rate was increased by 18.0% in TSPO -/- MPAs.

“we found that mean glycolytic rate was increased by 18.0% in TSPO -/-MPAs (Figure 3C, p<0.0001).”

Coupling efficiency was increased by 2.3% in TSPO -/- MPAs.

“Coupling efficiency was increased by 2.3% (Figure 2F, p=0.0007)”

Mean proton leak was reduced by 19.1% in TSPO -/- U373 cells.

“and mean proton leak was reduced by 19.1% (Figure 2L, p=0.0011).”

Mitochondria-linked ATP production was reduced by 26.9% in TSPO -/- U373 cells.

“Mitochondria-linked ATP production was reduced by 26.9% (Figure 2K, p=0.0004)”

Loss of TSPO increased fatty acid oxidation (FAO).

“We found that loss of TSPO reduced basal astrocyte respiration and increased the bioenergetic response to glucose reintroduction following glucopenia, while increasing fatty acid oxidation (FAO).”

There were no statistically significant effects of genotype on OCR in MPAs.

“we observed no statistically significant effects of genotype on OCR (Figure 4A,C; pgenotype=0.64,F(1,58) = 0.2246)”

Mean basal ECAR was reduced by 38.8% in TSPO -/- MPAs compared to TSPO +/+ MPA controls.

“Similarly, mean basal extracellular acidification rate (ECAR; a proxy of glycolysis) was reduced by 38.8% (Figure 1C,D; p<0.0001).”

Loss of TSPO expression does not alter the expression of key metabolic proteins in response to inflammation.

“nor alter the expression of key metabolic proteins in response to inflammation”

TSPO interacts with CPT1a.

“For the first time, we demonstrated using coimmunoprecipitation an interaction between TSPO and the rate-limiting enzyme in FAO, CPT1a”

Loss of TSPO expression does not restrict the metabolic response of astrocytes to an inflammatory stimulus.

“We showed here that loss of TSPO expression does not restrict the metabolic response of astrocytes to an inflammatory stimulus”

Under basal conditions, mean L-lactate secretion was reduced by 20.4% in TSPO-/- U373 cells compared to TSPO+/+ controls (p=0.0024).

“Post-hoc analyses supported our results from MPAs: mean L-lactate secretion was reduced by 20.4% in TSPO -/-U373 cells compared to TSPO +/+ controls (Figure 4F, p=0.0024) under basal conditions.”

Mean basal FAO was increased by 241.0% in TSPO-/- MPAs compared with TSPO+/+ MPAs (p=0.031).

“We found that mean basal FAO was increased by 241.0% in TSPO -/-MPAs (Figure 5B, p=0.031) compared with TSPO +/+ MPAs.”

Basal OCR and ECAR of TSPO-/- MPAs were lower than TSPO+/+ MPAs.

“Although the basal OCR and ECAR of TSPO -/-MPAs were lower than TSPO +/+ MPAs”

There was no significant effect of genotype on GLUT1 expression at baseline or following 3h LPS stimulation (p=0.96).

“Post-hoc analyses found no significant effect of genotype on GLUT1 expression at baseline or following 3h LPS stimulation in these cells, which showed the same response as TSPO +/+ controls (Figure 12A,B; p=0.96).”

Mean proton leak was reduced by 46.0% in TSPO -/- MPAs.

“and mean proton leak was reduced by 46.0% (Figure 2E, p<0.0001).”

Under basal conditions U373 cells showed reduced OCR relative to MPAs.

“We found that under basal conditions U373 cells showed increased ECAR (a proxy of glycolysis) and reduced OCR (a proxy of mitochondrial respiration) relative to MPAs”

After 24h LPS stimulation, TSPO-/- MPAs released 183.9% more TNF than TSPO+/+ controls (p=0.0035).

“In contrast, after 24h LPS stimulation, we found that TSPO -/- MPAs released 183.9% more TNF than TSPO +/+ controls (Figure 9B, p=0.0035).”

The interaction between endogenous TSPO and CPT1a occurs in U373 cells not subject to transfection.

“we performed a further co-immunoprecipitation using proteins endogenous to U373 cells that were not subject to transfection and determined that this interaction also occurred between endogenous TSPO and CPT1a (Figure 6B,C) (n=3).”

In TSPO-/- U373 cells, mean maximal FAO was increased by 245.5% (p=0.13).

“however mean maximal FAO was increased by 245.5% (Figure 5F, p=0.13).”

In response to 3h LPS stimulation, TNF secretion from TSPO-/- MPAs was reduced by 52.0% compared to TSPO+/+ controls (p=0.0001).

“Post-hoc analyses confirmed that in response to 3h LPS stimulation, TNF secretion from TSPO -/-MPAs was reduced by 52.0% compared to TSPO +/+ controls (Figure 9A; p=0.0001).”

Mean glycolytic capacity was increased by 25.8% in TSPO -/- MPAs.

“Similarly, mean glycolytic capacity and reserve were increased by 25.8% and 27.6% respectively (Figure 3D,E; p<0.0001 (both parameters)).”

Following 24h LPS stimulation, TSPO genotype had no statistically significant impact on GLUT1 expression (p=0.52).

“In contrast to our expectations, following 24h LPS stimulation, TSPO genotype had no statistically significant impact on GLUT1 expression (Figure 12E,F; pgenotype=0.52,F(1,20) = 0.42).”

In U373 astrocytoma cells, TSPO genotype did not have a statistically significant effect on L-lactate secretion (p=0.20).

“In U373 astrocytoma cells, TSPO genotype did not have a statistically significant effect on L-lactate secretion (Figure 4F, pgenotype=0.20,F(1,20) = 1.748)”

In MPAs, there was a statistically significant interaction between TSPO genotype and glucose concentration on L-lactate secretion (p=0.032).

“We observed a statistically significant interaction of these variables in MPAs (pgenotype=0.032,F(1,20) = 5.298).”

There was no change in glycolytic capacity in TSPO -/- U373 cells compared to TSPO +/+ EV controls.

“we observed no change in glycolytic capacity in TSPO -/-U373 cells compared to TSPO +/+ EV controls (Figure 3I, p=0.38).”

There was no significant difference in mean coupling efficiency in TSPO -/- U373 cells compared to TSPO +/+ EV controls.

“there was no significant difference in mean coupling efficiency (Figure 2M, p=0.17) in TSPO -/-U373 cells compared to TSPO +/+ EV controls”

There was no statistically significant interaction between TSPO genotype and 24h LPS stimulation on NFκB phosphorylation (p=0.57).

“There was no statistically significant interaction between these variables (pinteraction=0.57,F(1,19) = 0.3276).”

Mean spare capacity was reduced by 27.3% in TSPO -/- U373 cells.

“mean spare capacity was reduced by 27.3% (Figure 2N, p=0.0025).”

GLUT1 expression was similarly reduced by 24h LPS stimulation in MPAs regardless of TSPO-genotype (p<0.0001; p>0.99).

“Using post-hoc analyses, we found that in line with wildtype controls, GLUT1 expression was similarly reduced by 24h LPS stimulation in MPAs (Figure 12E,F; p<0.0001) regardless of TSPO-genotype (Figure 12E,F; p>0.99).”

Mean basal OCR was reduced by 31.1% in TSPO -/- MPAs compared to TSPO +/+ MPA controls.

“mean basal oxygen consumption rate (OCR; a proxy of mitochondrial metabolism) was reduced by 31.1% in TSPO -/-MPAs compared to TSPO +/+ MPA controls (Figure 1A,B; p<0.0001).”

Mitochondrial spare capacity was reduced by 49.3% in TSPO -/- MPAs.

“however mitochondrial spare capacity was reduced by 49.3% (Figure 2G, p=0.029).”

Mean glycolytic reserve was increased by 72.9% in TSPO -/- U373 cells.

“However, mean glycolytic reserve was increased by 72.9% in TSPO -/-U373 cells (Figure 3J, p<0.0001).”

Following 3h LPS stimulation, the statistically significant impact of genotype on astrocyte metabolic parameters seen in earlier experiments was maintained (OCR p<0.0001; ECAR p=0.0019).

“Following 3h LPS stimulation, the statistically significant impact of genotype on astrocyte metabolic parameters seen in earlier experiments (Figure 1) was maintained (Figure 11A-D; OCR: pgenotype<0.0001,F(1,66) = 18.45;ECAR: pgenotype=0.0019,F(1,64) = 10.51).”

TSPO forms a complex with carnitine palmitoyltransferase 1a in U373 cells.

“Coimmunoprecipitation studies in U373 cells revealed that TSPO forms a complex with carnitine palmitoyltransferase 1a, which presents a mechanism wherein TSPO may regulate FAO in astrocytes.”

Mean glycolytic rate was reduced by 26.5% in TSPO -/- U373 cells.

“in TSPO -/-U373 cells, mean glycolytic rate was reduced by 26.5% (Figure 3H, p<0.0001)”

Loss of TSPO reduced basal astrocyte respiration.

“We found that loss of TSPO reduced basal astrocyte respiration and increased the bioenergetic response to glucose reintroduction following glucopenia, while increasing fatty acid oxidation (FAO).”

Mean maximal mitochondrial respiration was reduced by 40.2% in TSPO -/- MPAs compared to TSPO +/+ MPA controls.

“This was accompanied by a reduction in mean maximal mitochondrial respiration of 40.2% compared to TSPO +/+ MPA controls (Figure 2C, p=0.0012).”

Non-glycolytic acidification was increased by 21.1% in TSPO -/- U373 cells.

“In TSPO -/-U373 cells (Figure 3F), non-glycolytic acidification was increased by 21.1% (Figure 3G, p=0.0057).”

There was no statistically significant interaction between TSPO genotype and 3h LPS stimulation on NFκB phosphorylation (p=0.18).

“We observed no statistically significant interaction between TSPO genotype and 3h LPS stimulation (pinteraction=0.18,F(1,19) = 1.911).”

TSPO-/- astrocytes secrete less lactate than wildtype controls.

“We showed that TSPO -/-astrocytes secrete less lactate than wildtype controls”

Lactate production was significantly reduced in TSPO -/- astrocytes.

“Lactate production was significantly reduced in TSPO -/-astrocytes.”

Mean basal ECAR was reduced by 45.6% in TSPO -/- U373 cells compared to TSPO +/+ EV controls.

“and mean basal ECAR was reduced by 45.6% compared to TSPO +/+ EV controls (Figure 1G,H; p<0.0001).”

In the absence of glucose, L-lactate secretion was not altered in TSPO-/- MPAs (p=0.35).

“L-lactate secretion was not altered in TSPO -/-MPAs (Figure 4E, p=0.35).”

Mitochondria-linked ATP production was reduced by 35.8% in TSPO -/- MPAs.

“In TSPO -/-MPAs mitochondria-linked ATP production was reduced by 35.8% (Figure 2D, p=0.0009)”

In the presence of glucose, TSPO-/- MPAs secreted 5.6% less L-lactate than TSPO+/+ controls (p=0.002).

“Post-hoc analysis revealed that in the presence of glucose TSPO -/-MPAs secreted 5.6% less L-lactate than TSPO +/+ controls (Figure 4E, p=0.002).”

NFκB phosphorylation was significantly increased at both 3h and 24h LPS stimulation.

“we confirmed that MPAs were undergoing an inflammatory response to LPS stimulation by assessing phosphorylation of nuclear factor kappa-B (p65) which was significantly increased at both timepoints (NFκB; 3h Figure 7A-C”

Using the 24h LPS stimulation model, there was a statistically significant effect of genotype on CPT1a expression (p=0.017).

“In contrast, using our 24h LPS stimulation model, we observed a statistically significant effect of genotype on CPT1a expression (Figure 12 G,H; pgenotype=0.017,F(1,20) = 6.8).”

In TSPO-/- U373 cells, there was no change in basal FAO (p=0.28).

“In TSPO -/-U373 cells, we did not see any change in basal FAO (Figure 5E, p=0.28)”

Mean maximal respiration was reduced by 26.1% in TSPO -/- U373 cells compared to TSPO +/+ EV controls.

“where mean maximal respiration was reduced by 26.1% compared to TSPO +/+ EV controls (Figure 2I, p=0.0002 (basal); Figure 2J, p=0.001 (maximal)).”

Basal OCR and ECAR of TSPO-/- MPAs did not change in the absence of glucose.

“they did not change in the absence of glucose (Figure 4A-D; p=0.9823 [OCR], p>0.9999 [ECAR])”

Maximal FAO was enhanced in TSPO-/- MPAs (p=0.0004).

“We found that maximal FAO was enhanced in TSPO -/-MPAs (Figure 5C, p=0.0004).”

There was no statistically significant interaction between 3h LPS stimulation and TSPO genotype on GLUT1 expression (p=0.17).

“We did not observe a statistically significant interaction between 3h LPS stimulation and TSPO genotype on GLUT1 expression (pinteraction=0.17,F(1,19) = 2.0).”

Under basal conditions U373 cells showed increased ECAR relative to MPAs.

“We found that under basal conditions U373 cells showed increased ECAR (a proxy of glycolysis) and reduced OCR (a proxy of mitochondrial respiration) relative to MPAs”

In U373 astrocytoma cells, glucose concentration had a statistically significant effect on L-lactate secretion (p=0.0015).

“however we observed a statistically significant effect of glucose concentration on L-lactate secretion from these cells (pglucose=0.0015,F(1,20) = 7.159).”

Basal mitochondrial respiration was significantly reduced in TSPO -/- MPAs.

“basal mitochondrial respiration was significantly reduced in TSPO -/-MPAs (Figure 2B, p=0.0006).”

In the absence of glucose, TSPO+/+ MPAs reduced their L-lactate secretion by 6.7% (p=0.0003).

“In the absence of glucose, while TSPO +/+ MPAs reduced their L-lactate secretion by 6.7% (Figure 4E, p=0.0003)”

In U373 astrocytoma cells, there was a statistically significant interaction between TSPO genotype and glucose concentration on L-lactate secretion (p=0.0001).

“Moreover, we observed a statistically significant interaction between these variables (pinteraction=0.0001,F(1,20) = 21.84).”

TSPO deficiency enhances the metabolic response in astrocytes following the injection of a supraphysiological glucose concentration during the glycolysis stress test.

“Furthermore, in agreement with a recent study in microglia 9 , we found that TSPO deficiency enhances the metabolic response in astrocytes following the injection of a supraphysiological glucose concentration during the glycolysis stress test.”

• cites [9]Not matched

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In response to 24h LPS stimulation both OCR and ECAR increased in TSPO+/+ MPAs.

“However, post-hoc analyses revealed that, in response to 24h LPS stimulation both OCR and ECAR increased in TSPO +/+ (Figure 11F”

Non-glycolytic acidification was significantly increased by 24.4% in TSPO -/- MPAs.

“Non-glycolytic acidificationacidification of the media that may be attributed to other cellular or metabolic processes that produce H + , such as FAO 45 -was significantly increased by 24.4% in TSPO -/-MPAs (Figure 3B, p<0.0001).”

Loss of TSPO increased the bioenergetic response to glucose reintroduction following glucopenia.

“We found that loss of TSPO reduced basal astrocyte respiration and increased the bioenergetic response to glucose reintroduction following glucopenia, while increasing fatty acid oxidation (FAO).”

3h or 24h 2DG stimulation did not significantly affect NFκB phosphorylation.

“Neither 3h nor 24h 2DG stimulation significantly affected NFκB phosphorylation (Figure7A,D (3h), E,H (24h); p=0.39 (3h), p=0.71 (24h))”

Loss of TSPO reduced L-lactate secretion by astrocytes.

“while reducing L-lactate secretion”

Removal of glucose from the media resulted in no change in lactate secretion, OCR or ECAR in TSPO-/- astrocytes.

“Furthermore, removal of glucose from the media resulted in no change in lactate secretion, OCR or ECAR in TSPO -/-astrocytes.”

TSPO-/- astrocytes utilised more fatty acids to maintain their basal bioenergetic needs.

“Using the FAO stress test paradigm, we found that TSPO -/-astrocytes indeed utilised more fatty acids to maintain their basal bioenergetic needs”

24h LPS stimulation enhanced mitochondrial respiration in C57BL/6J MPAs.

“Figure 8: 24h LPS stimulation enhanced mitochondrial and non-mitochondrial respiration in C57BL/6J MPAs”

Inflammation-induced changes to GLUT1 and CPT1a expression were not modulated by TSPO deficiency.

“Figure 12: Inflammation-induced changes to GLUT1 and CPT1a expression were not modulated by TSPO deficiency”

TSPO-/- astrocytes used more FAO to fuel their response during the FAO paradigm when mitochondrial stress responses were assessed using FCCP.

“In our data, when mitochondrial stress responses were assessed during the FAO paradigm using the uncoupling agent FCCP -to mimic the oxidative stress that might be seen in disease states -we found that TSPO -/-astrocytes used more FAO to fuel their response (Figure 5C,F).”

TSPO-/- astrocytes produce less lactate.

“and we have reported that TSPO -/-astrocytes produce less lactate (Figure 4)”

The rate of fatty acid oxidation (FAO) is increased in TSPO -/- astrocytes.

“we have shown that the rate of FAO is increased in TSPO -/- astrocytes”

The bioenergetic phenotype of LPS stimulated TSPO-/- MPAs was not significantly different from TSPO+/+ controls.

“However, we found that the bioenergetic phenotype of LPS stimulated TSPO -/-MPAs was not significantly different from TSPO +/+ controls (Figure 11)”

TSPO deficiency modulated cytokine release from MPAs.

“Figure 9: TSPO deficiency modulated cytokine release from MPAs”

The release profile of the pro-inflammatory cytokine TNF is altered in TSPO -/- astrocytes.

“though the release profile of the pro-inflammatory cytokine TNF is altered.”

When present, TSPO forms a complex with CPT1a.

“when present, TSPO forms a complex with CPT1a”

• Also stated in: § Conclusion · plain

  “Figure 6: TSPO formed a complex with CPT1a in U373 cells”

TSPO deficiency did not modulate NFκB activation following LPS stimulation.

“Figure 10: TSPO deficiency did not modulate NFκB activation following LPS stimulation”

3h or 24h 2DG stimulation did not significantly affect TSPO expression.

“or TSPO expression (Figure7A,D (3h), E,H (24h); p=0.47 (3h), p=0.98 (24h)).”

TSPO deficiency reduced glycolytic rate in U373 astrocytoma cells.

“Figure 3: TSPO deficiency increased glycolytic rate in MPAs but reduced glycolytic rate in U373 astrocytoma cells”

24h LPS stimulation enhanced non-mitochondrial respiration in C57BL/6J MPAs.

“Figure 8: 24h LPS stimulation enhanced mitochondrial and non-mitochondrial respiration in C57BL/6J MPAs”

The basal bioenergetic rates of TSPO-/- astrocytes are reduced relative to TSPO+/+ controls.

“Regardless, our data show that the basal bioenergetic rates of TSPO -/- astrocytes are reduced relative to TSPO +/+ controls.”

The metabolic response to LPS stimulation is not impeded in TSPO -/- astrocytes.

“despite increases in basal FAO, the metabolic response to LPS stimulation is not impeded in TSPO -/-astrocytes”

• Also stated in: § Conclusion · plain

  “Figure 11: TSPO deficiency did not affect the metabolic response of MPAs to inflammatory stimulation”

• Also stated in: § Conclusion · plain

  “Despite this, there was no statistically significant difference in the metabolic responses of TSPO -/-MPAs to 24h LPS stimulation compared to TSPO +/+ controls (Figure 11F,H; p=0.87 [OCR], p=0.74 [ECAR]).”

3h LPS stimulation did not increase TSPO expression in C57BL/6J MPAs.

“While 3h LPS stimulation did not significantly increase TSPO expression in MPAs (Figure7A,D; p=0.99)”

• Also stated in: § Conclusion · plain

  “Figure 7: 24h but not 3h LPS stimulation increased TSPO expression in C57BL/6J MPAs”

24h LPS stimulation increased TSPO expression in C57BL/6J MPAs.

“Figure 7: 24h but not 3h LPS stimulation increased TSPO expression in C57BL/6J MPAs”

• Also stated in: § Conclusion · plain

  “24h LPS stimulation induced a significant increase in TSPO expression (Figure7E,H; p=0.048).”

TSPO deficiency increased glycolytic rate in MPAs.

“Figure 3: TSPO deficiency increased glycolytic rate in MPAs but reduced glycolytic rate in U373 astrocytoma cells”

We have presented data suggesting an altered temporal release profile of TNF from TSPO-/- MPAs.

“Despite this, we have presented data suggesting an altered temporal release profile of TNF from TSPO -/-MPAs (Figure 9), though we only examined one concentration of one inflammatory stimulus at two timepoints.”

OCR was increased in TSPO-/- MPAs supplemented with palmitate compared to those supplemented with palmitate and treated with etomoxir.

“OCR was increased in TSPO -/-MPAs supplemented with palmitate compared to those supplemented with palmitate and treated with etomoxir, a potent inhibitor of CPT1a”

When exogenous glucose was unavailable as a substrate, there was no significant increase in the OCR of TSPO-/- astrocytes.

“However, when exogenous glucose was unavailable as a substrate, we saw no significant increase in the OCR of TSPO - /-astrocytes (Figure 4).”

Loss of TSPO reduced the basal metabolic rates of astrocytes.

“We have shown that loss of TSPO reduced the basal metabolic rates of astrocytes”

• Also stated in: § Conclusion · plain

  “Figure 1: Loss of TSPO attenuated basal metabolism in astrocytes”

Loss of TSPO enabled astrocytes to maintain their metabolic rates in the absence of glucose.

“enabled astrocytes to maintain their metabolic rates in the absence of glucose”

During a nutrient deficit simulated by exposing the cells to a glucose-free condition for 1h, TSPO-deficient astrocytes lacked the metabolic adaptations observed in TSPO-expressing controls.

“During a nutrient deficit simulated by exposing the cells to a glucose-free condition for 1h, we found that TSPO-deficient astrocytes lacked the metabolic adaptations observed in TSPO-expressing controls.”

TSPO may regulate FAO in astrocytes.

“Coimmunoprecipitation studies in U373 cells revealed that TSPO forms a complex with carnitine palmitoyltransferase 1a, which presents a mechanism wherein TSPO may regulate FAO in astrocytes.”

TSPO may play cell-type and state-dependent roles in regulating cellular metabolism.

“The heterogeneity in these findings implies that TSPO may play cell-type and state-dependent roles in regulating cellular metabolism.”

• cites [9]Not matched

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• cites [10]Not matched

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• cites [30]Not matched

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Lipids constituted a greater proportion of the substrates used to maintain basal metabolic rates in TSPO-/- MPAs compared with TSPO+/+ MPAs.

“This suggests that lipids constituted a greater proportion of the substrates used to maintain basal metabolic rates in TSPO -/-MPAs compared with TSPO +/+ MPAs.”

Loss of TSPO may alter the temporal profile of the astrocyte inflammatory response.

“but may alter the temporal profile of the astrocyte inflammatory response.”

TSPO-/- astrocytes rely more on FAO to meet their bioenergetic requirements.

“and rely more on FAO to meet their bioenergetic requirements.”

Data from MPAs and U373 astrocytoma cells support that TSPO-/- astrocytes are less reliant on glucose than TSPO+/+ controls to maintain their metabolic requirements in the absence of glucose.

“Together, our data from MPAs and U373 astrocytoma cells supports the notion that TSPO -/-astrocytes are less reliant on glucose than TSPO +/+ controls to maintain their metabolic requirements in the absence of glucose”

Mean astrocyte purity was determined to be 98.67%.

“mean astrocyte purity was determined to be 98.67% (Supplementary Figure 2; n=10 coverslips, 2-5 images per coverslip over 3-4 individual collections).”

In other glial and non-glial cells, TSPO deficiency has been linked to reduced oxidative phosphorylation and glycolysis.

“In other glial and non-glial cells, TSPO deficiency has been linked to reduced oxidative phosphorylation and glycolysis 9,30,31,54,55 .”

• cites [9]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [30]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [31]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [54]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [55]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Like TSPO, CPT1a resides in the outer mitochondrial membrane.

“Like TSPO, CPT1a resides in the outer mitochondrial membrane 46 .”

• cites [46]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Under basal conditions TSPO -/- MPAs showed reductions in aerobic glycolysis compared to TSPO +/+ counterparts.

“This led us to infer that under basal conditions TSPO -/-MPAs showed reductions in aerobic glycolysis, and oxidative phosphorylation of its products, compared to TSPO +/+ counterparts.”

Compared to TSPO+/+ controls, TSPO-/- astrocytes may be meeting their bioenergetic requirements by preferentially metabolising substrates other than glucose.

“these data suggest that, compared to TSPO +/+ controls, TSPO -/-astrocytes may be meeting their bioenergetic requirements by preferentially metabolising substrates other than glucose.”

Loss of TSPO does not appear to modulate the metabolic response of astrocytes to inflammation, at least in response to the stimulus/time course used in this study.

“Together these data suggest that while TSPO acts as a regulator of metabolic flexibility in astrocytes, loss of TSPO does not appear to modulate the metabolic response of astrocytes to inflammation, at least in response to the stimulus/time course used in this study..”

In this study, we provide data supporting the hypothesis that TSPO regulates cellular bioenergetics in astrocytes.

“In this study, we provide data supporting the hypothesis that TSPO regulates cellular bioenergetics in astrocytes.”

TSPO acts as a regulator of metabolic flexibility in astrocytes.

“Together these data suggest that while TSPO acts as a regulator of metabolic flexibility in astrocytes, loss of TSPO does not appear to modulate the metabolic response of astrocytes to inflammation, at least in response to the stimulus/time course used in this study..”

Under basal conditions TSPO -/- MPAs showed reductions in oxidative phosphorylation of its products compared to TSPO +/+ counterparts.

“This led us to infer that under basal conditions TSPO -/-MPAs showed reductions in aerobic glycolysis, and oxidative phosphorylation of its products, compared to TSPO +/+ counterparts.”

Astrocytes undergo a bioenergetic shift to rely more heavily on mitochondrial metabolism after 24h exposure to inflammatory stimuli.

“We have shown previously that astrocytes undergo a bioenergetic shift to rely more heavily on mitochondrial metabolism after 24h exposure to inflammatory stimuli 28 .”

• cites [28]Not matched

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The different rates of FAO observed between U373 astrocytoma cells and MPAs may be due to the relatively increased reliance of MPAs on OCR to meet their bioenergetic requirements.

“The different rates of FAO we observed between U373 astrocytoma cells and MPAs may be due to the relatively increased reliance of MPAs on OCR to meet their bioenergetic requirements (Supplementary Figure 4).”

TSPO-/- MPAs may be less dependent on glucose to maintain their basal metabolic rate than TSPO+/+ controls.

“suggesting that TSPO -/-MPAs may be less dependent on glucose to maintain their basal metabolic rate than their TSPO +/+ controls.”

TSPO deficiency reduces the basal metabolic rates of astrocytes under basal conditions.

“Together, these data suggest that TSPO deficiency reduces the basal metabolic rates of astrocytes under basal conditions.”

The L-lactate data alone cannot inform whether TSPO-/- MPAs oxidise a greater amount of lactate than wildtype controls.

“As we did not directly quantify pyruvate production in these cells our L-lactate data alone cannot inform us whether TSPO -/-MPAs oxidise a greater amount of lactate than wildtype controls.”

TSPO may act as a regulator of metabolic flexibility in astrocytes.

“Taken together, these data show that TSPO may act as a regulator of metabolic flexibility in astrocytes.”

We propose that the TSPO-CPT1a interaction is a mechanism by which TSPO regulates FAO in astrocytes.

“We propose that this interaction is a mechanism by which TSPO regulates FAO in astrocytes.”

• AssumptionRests on an assumption: The existence of a protein complex containing TSPO and CPT1a has not yet been experimentally confirmed.

The decrease in L-lactate secreted by TSPO-/- astrocytes relative to TSPO+/+ controls indicates that TSPO-/- astrocytes are less dependent on glucose to meet their bioenergetic requirements.

“Thus, we are confident that the decrease in L-lactate secreted by TSPO -/- astrocytes relative to TSPO +/+ controls indicates that TSPO -/-astrocytes are less dependent on glucose to meet their bioenergetic requirements.”

TSPO may ordinarily inhibit CPT1a function to conserve intracellular fat stores.

“This suggests that TSPO may ordinarily inhibit CPT1a function to conserve intracellular fat stores.”

Our data suggest that TSPO deficiency promotes FAO.

“Overall, our data interrogating the role of TSPO in regulating astrocyte metabolism suggests that TSPO deficiency promotes FAO.”

FAO formed a larger part of the metabolic base of TSPO-/- MPAs compared to wildtype MPAs.

“implying that FAO formed a larger part of the metabolic base of TSPO -/-MPAs compared to wildtype MPAs (Figure 5B).”

The bioenergetic response of TSPO-/- MPAs to inflammatory stimulation may be modulated by enhanced FAO.

“This suggests that the bioenergetic response of TSPO -/-MPAs to inflammatory stimulation may be modulated by enhanced FAO.”

The basal metabolic response of these cells to inflammatory stimuli is not modulated by the absence of TSPO, at least using LPS as an inflammatory stimulus.

“suggesting that the basal metabolic response of these cells to inflammatory stimuli is not modulated by the absence of TSPO, at least using LPS as an inflammatory stimulus.”

Because microglia express low or no levels of CPT1a, a TSPO-CPT1a interaction is less likely to be detected/present in these cells.

“Because microglia express low or no levels of CPT1a 34,35 , a TSPO-CPT1a interaction is less likely to be detected/present in these cells.”

• cites [34]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [35]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

This may be interpreted as increased glycolytic rate in TSPO-/- MPAs.

“This may be interpreted as increased glycolytic rate in TSPO -/-MPAs.”

TSPO deficient astrocytes are less reliant on glucose to meet their energetic needs.

“Together these data suggest that TSPO deficient astrocytes are less reliant on glucose to meet their energetic needs.”

When present, TSPO may act to suppress FAO in these cells, potentially via its interaction with CPT1a.

“This implies that, when present, TSPO may act to suppress FAO in these cells, potentially via its interaction with CPT1a.”

TSPO is reported to have multiple interacting partners, including VDAC.

“TSPO is reported to have multiple interacting partners, including VDAC 2,56 , another constituent of the outer mitochondrial membrane.”

• cites [2]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [56]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.