A rapid microglial metabolic response controls metabolism and improves memory

Department of Epigenetics, Van Andel Research Institute, 333 Bostwick Ave, 49503, Grand Rapids, MI, USA
Max Planck Institute of Immunobiology and Epigenetics, Stübeweg 51, 79108 Freiburg, Germany
Department of Metabolism and Nutritional Programming, Van Andel Research Institute, 333 Bostwick Ave, 49503, Grand Rapids, MI, USA
Metabolomics and Bioenergetics Core, Van Andel Research Institute, 333 Bostwick Ave, 49503, Grand Rapids, MI, USA
Department of Neurodegenerative Sciences, Van Andel Research Institute, 333 Bostwick Ave, 49503, Grand Rapids, MI, USA
Institute of Neuropathology, Medical Faculty, University of Freiburg, Freiburg, Germany
Department of Medicine II, University Hospital Freiburg, Freiburg, Germany
Centre for NeuroModulation (NeuroModBasics), University of Freiburg, Freiburg, Germany
Signaling Research Centers BIOSS and CIBSS, University of Freiburg, Freiburg, Germany

https://doi.org/10.7554/eLife.87120.1

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Figures and data

Acute HFD-induced metabolic changes are microglia dependent
A. Schematic depicting the different treatments and diets followed by the different mice groups.
B. Graphs showing the Glucose Tolerance Test (OGTT) and the associated-insulin kinetic of C57Bl6/J male fed with control diet (Control) or fed with high fat diet for 3 days (3-day HFD) (n=8).
C. Graph showing the overnight fasted glycemia from the mice groups depicting in the A. Schematic (n=5 to 11).
D. Graph showing the 2hours-fasted glycemia from the mice groups depicting in the A. schematic (n=5 to 11).
E. Graph showing the insulin released after a glucose gavage from the mice groups depicting in the A. schematic (n=5 to 11).
F. Microglial cells staining with Iba1 (green) in the brain slices from mice fed with 3 days HFD or mice depleted from their microglial cells with 1 week control diet complexed with PLX-5662 prior the 3 days HFD (PLX-5662) (n=5).
G. sgRNAseq dataset from hypothalamic microglia cells harvested from C57bl6/J male mice fed with control diet (CT) and High Fat Diet for 3 days (HFD_3d) (n=5) merged with sgRNAseq microglia dataset from mice presenting an Experimental Autoimmune Encephalomyelitis (EAE). Data are presented as mean ±SEM. *p<0.05, **p<0.01, ***p<0.001 as determined by two-tailed Student’s test and two-way ANOVA followed by Bonferroni post hoc test.

A rapid Microglial Mitochondria Response to high fat diet
A. Facs plots depicting the ratio Mitotraker Deep Red/ Mitotraker Green from sorted microglial cells of C57Bl6/J male fed with a control diet (Control) or fed with high fat diet for 3 days (3-day HFD).
B. Graph showing the ratio Mitotraker Deep Red/ Mitotraker Green from sorted hypothalamic microglial cells of C57Bl6/J male fed with a control diet (Control) or fed with high fat diet for 12 hours,3 days or 1-4 weeks (n=5 to 12).
C. Graph showing the Mitotraker Green fluorescence from sorted hypothalamic microglial cells of C57Bl6/J male fed with a control diet (Control) or fed with high fat diet for for 12 hours,3 days or 1-4 weeks (n=5 to 12).
D. Volcano plot showing the metabolites content of cerebrospinal fluid from of C57Bl6/J male fed with a control diet (Control) or fed with high fat diet for 3 days (n=10).
E. Seahorse (+/- succinate added in the media during the experiment) on primary microglia challenged for 24hours with BSA (control) or Palmitate (experiment replicated 3times).
F. Mitochondrial Electron transport chain activity recorded with FACS after TMRM staining from sorted microglial cells of C57Bl6/J male fed with a control diet (Control) or fed with high fat diet for 3 days (n=5).
G. Mitochondrial networks from primary microglia stained with Mitotraker green after being challenged for 24hours with BSA (control), Palmitate, Oleate or LPS (n=40) and the mitochondrial length quantification graphs.
H. DRP1 colocalization with the mitochondrial network stained with TOMM20 on primary microglial cell after being challenged for 24hours with BSA (control) and Palmitate(n=40) and the colocalization quantification graphs.
Data are presented as mean ±SEM. *p<0.05, **p<0.01, ***p<0.001 as determined by two-tailed Student’s test and two-way ANOVA followed by Bonferroni post hoc test.

aMMR is required for diet induced homeostatic rewiring in vivo
A. Immunostaining of TOMM20 and DRP1 on sorted microglia from mice Drp1^MGWT or Drp1^MGKO
B. Western Blot against DRP1 on sorted microglia from mice Drp1^MGWT or Drp1^MGKO
C. Graphs showing the overnight fasted glycemia, the 2hours fasted glycemia and the insulin released from Drp1^MGWT or Drp1^MGKO fed with control diet (n=5 to 11).
D. Graphs showing the overnight fasted glycemia, the 2hours fasted glycemia and the insulin released from Drp1^MGWT or Drp1^MGKO fed with 3 day high fat diet (n=5 to 11).
Data are presented as mean ±SEM. *p<0.05, **p<0.01, ***p<0.001 as determined by two-tailed Student’s test and two-way ANOVA followed by Bonferroni post hoc test.

Palmitate induces a novel microglial lactate/succinate/itaconate release pathway
A. BODIPY staining on primary microglia challenged for 24hours with BSA or palmitate and the lipid droplets quantification graph. (n=20)
B. Schematic depicting the timeline for the tracing experiments (¹³C-palmitate or ¹³C-glucose) on primary microglial challenged for 24hours with BSA or palmitate.
C.¹³C-palmitate incorporation into palmytoilcarnitine (m+16) and acetylcarnitine (m+2) after 4 hours tracing experiment on primary microglia pretreated for 24hours with BSA or palmitate (n=3).
D. ¹³C-palmitate incorporation into acetyl serine (m+2) after 4 hours tracing experiment on primary microglia pretreated for 24hours with BSA or palmitate (n=3).
E. ¹³C-palmitate incorporation into aconitate, alpha-ketoglutarate, fumarate, malate (m+2) after 4 hours tracing experiment on primary microglia pretreated for 24hours with BSA or palmitate (n=3).
F. ¹³C-palmitate incorporation into glutamate (m+2) after 4 hours tracing experiment on primary microglia pretreated for 24hours with BSA or palmitate (n=3).
G. ¹³C-palmitate incorporation into glutamate (m+2) released during the 4 hours tracing experiment by primary microglia pretreated for 24hours with BSA or palmitate (n=3). The results are graphed in relative abundance.
H. ¹³C-palmitate incorporation into itaconate (m+1) released during the 4 hours tracing experiment by primary microglia pretreated for 24hours with BSA or palmitate (n=3).
I. ¹³C-palmitate incorporation into succinate (m+2) released during the 4 hours tracing experiment by primary microglia pretreated for 24hours with BSA or palmitate (n=3).
J. ¹³C-glucose incorporation into the intracellular glucose pool (m+6) after 6 hours tracing experiment on primary microglia pretreated for 24hours with BSA or palmitate (n=3).
K. ³C-glucose incorporation into glutamate (m+2) released during the 6 hours tracing experiment by primary microglia pretreated for 24hours with BSA or palmitate (n=3).
L. ¹³C-glucose incorporation into itaconate (m+1) released during the 6 hours tracing experiment by primary microglia pretreated for 24hours with BSA or palmitate (n=3).
M. ³C-glucose incorporation into succinate (m+2) released during the 6 hours tracing experiment by primary microglia pretreated for 24hours with BSA or palmitate (n=3).
N. ¹³C-glucose incorporation into lactate (m+3) released during the 6 hours tracing experiment by primary microglia pretreated for 24hours with BSA or palmitate (n=3).
O. Schematic depicting the metabolic pathways used by the primary microglial challenged for 24hours with BSA (black arrow) or palmitate (red arrow) during the different tracing (¹³C-palmitate or ¹³C-glucose).
Data are presented as mean ±SEM. *p<0.05, **p<0.01, ***p<0.001 as determined by two-tailed Student’s test and two-way ANOVA followed by Bonferroni post hoc test.

Acute HFD induces widespread MMR and rapid modulation of spatial and learning memory
A. Primary microglial cell media was collected after the ¹³C-glucose tracing (containing ¹³C-lactate released by microglia challenged with BSA or Palmitate) and incubated for 4 hours with primary neurons, the graph shows the ¹³C-lactate incorporation in the neurons in relative abondance, as control, primary neurons were incubated directly with ¹³C-glucose (n=6).
B. Graph showing the ratio Mitotraker Deep Red/ Mitotraker Green from sorted cortical microglial cells of C57Bl6/J male fed with a control diet (Control) or fed with high fat diet for 3 days (3-day HFD) (n=5 to 12).
C. Graph showing the ratio Mitotraker Deep Red/ Mitotraker Green from sorted hippocampic microglial cells of C57Bl6/J male fed with a control diet (Control) or fed with high fat diet for 3 days (3-day HFD) (n=5 to 12).
D. Graph showing the latency during the Barnes Test from mice fed with normal diet (Control) or 3 days HFD (3-day HFD) (n=11). The test was performed in the VAI animals facility (USA).
E. Graph showing the alternation during the T Maze Test from mice fed with normal diet (Control) or 3 days HFD (3-day HFD) (n=11). The test was performed in the VAI animals facility (USA).
F. Graph showing the distance walked during the ROTAROD test from mice fed with normal diet (Control or 3 days HFD (3-day HFD) (n=11).
G. Graph showing the number of turn before the mice fall during the ROTAROD test from mice fed with normal diet (Control) or 3 days HFD (3-day HFD) (n=11).
H. Graph showing the latency during the ROTAROD test from mice fed with normal diet (Control) or 3 days HFD (3-day HFD) (n=11).
I. Microglial staining with Iba1 (green) in the Hippocampus slices from mice fed with 3 days HFD or mice depleted from their microglial cells with 1 week control diet complexed with PLX-5662 prior the 3 days HFD (PLX-5662) (n=5).
J. Graph showing the latency during the Barnes Test from mice fed with normal diet (Control) or with depleted microglia (n=8).
K. Graph showing the latency during the Barnes Test from Drp1^MGWT or Drp1^MGKO mice fed with normal diet (Control diet) or with 3 days HFD (3-day HFD) (n=11).
Data are presented as mean ±SEM. *p<0.05, **p<0.01, ***p<0.001 as determined by two-tailed Student’s test and two-way ANOVA followed by Bonferroni post hoc test

Acute HFD-induced metabolic changes are microglia dependent
A. Graphs showing the Glucose Tolerance Test (OGTT) and the associated-insulin kinetic of C57Bl6/J male before keeping them with a control diet (Control) or before feeding them with high fat diet for 3 days (3-day HFD) (n=8).
B. Graph showing the Insulin Tolerance Test (ITT) of C57Bl6/J male before keeping them with a control diet (Control) or before feeding them with high fat diet for 3 days (3-day HFD) (n=5).
C. Graphs showing the Glucose Tolerance Test (OGTT) expressed in percentage from the basal glycemia of C57Bl6/J male fed with a control diet (Control) or fed with high fat diet for 3 days (3-day HFD) (n=8).
D. Graph showing the body weight evolution between C57Bl6/J male fed with control diet (Control) or fed with high fat diet for 3 days (3-day HFD), before and after the diet change. (n=7).
E. IBA1 immunostaining on hypothalamic slices from C57Bl6/J male fed with control diet (Control) or fed with high fat diet for 3 days (3-day HFD) and its quantification (n=10).
F. IBA1 and YFP immunostaining on hypothalamic slices from Cx3cr1creERT2-Rosa26YFP mice fed with control diet or 3 days high fat diet for 3 days (n=5).
G. sgRNAseq dataset from hypothalamic microglia cells harvested from C57bl6/J male mice fed with control diet (Control) and High Fat Diet for 3 days (HFD_3d) (n=5)
Data are presented as mean ±SEM. *p<0.05, **p<0.01, ***p<0.001 as determined by two-tailed Student’s test and two-way ANOVA followed by Bonferroni post hoc.

A rapid Microglial Mitochondria Response to high fat diet
A. IBA1 immunostaining of primary microglia cells treated with BSA (control) or Palmitate for 24hours.
B. Graph showing mRNA expression of microglial marker (TMEM119) or astrocytes marker (S100B) or macrophages marker (Arg1) in primary microglial culture challenged for 24hours with BSA or palmitate. Primary culture of astrocytes and macrophages were used as positive controls.
C. MitoSOX staining of primary microglial cells after being challenged for 24hours with BSA (control), Palmitate, Oleate or LPS (n=10) and the MitoSOX quantification graph.
D. Mitochondrial networks from primary microglia stained with Mitotraker green and stained with MitoSOX after being challenged for 2hours with BSA (control), Palmitate, Oleate or LPS (n=40) and the mitochondrial length as well as the MitoSOX quantification graphs
F. Interleukins concentrations (TNFalpha, IL-1beta, IL6) in the primary microglia cells media after being challenged for 24hours with BSA or palmitate (n=7)
Data are presented as mean ±SEM. *p<0.05, **p<0.01, ***p<0.001 as determined by two-tailed Student’s test and two-way ANOVA followed by Bonferroni post hoc.

aMMR is required for diet induced homeostatic rewiring in vivo
A Schematic depicting the timeline for the tamoxifen injection and the experiments performed on Drp1^MGWT or Drp1^MGKO
B. Graph showing the body weight evolution among all the Drp1^MGKO genotypes after the tamoxifen injection (n=6 to 11)
C. Graphs showing the fat mass and lean mass evolution among all the Drp1^MGKO genotypes after the tamoxifen injection (n=6 to 11)
D. Graphs showing the body weight before and after the 3 day HFD for the mice Drp1^MGWT and Drp1^MGKO (n=11 to 13)
Data are presented as mean ±SEM. *p<0.05, **p<0.01, ***p<0.001 as determined by two-tailed Student’s test and two-way ANOVA followed by Bonferroni post hoc.

Palmitate induces a novel microglial lactate/succinate/itaconate release pathway
A ¹³C-palmitate incorporation into palmytoilcarnitine and acetylcarnitine after 4 hours tracing experiment on primary microglia pretreated for 24hours with BSA or palmitate (n=3). The results are graphed in relative abundance.
B. ¹³C-palmitate incorporation into acetyl-serine after 4 hours tracing experiment on primary microglia pretreated for 24hours with BSA or palmitate (n=3). The results are graphed in relative abundance.
C. ¹³C-palmitate incorporation into aconitate, alpha-ketoglutarate, fumarate, malate after 4 hours tracing experiment on primary microglia pretreated for 24hours with BSA or palmitate (n=3). The results are graphed in relative abundance.
D. ¹³C-palmitate incorporation into glutamate after 4 hours tracing experiment on primary microglia pretreated for 24hours with BSA or palmitate (n=3). The results are graphed in relative abundance.
E. ¹³C-glucose incorporation into pyruvate, citrate, alpha-ketoglutarate, succinate, and malate after 6 hours tracing experiment on primary microglia pretreated for 24hours with BSA or palmitate (n=3). The results are graphed in pool size.
Data are presented as mean ±SEM. *p<0.05, **p<0.01, ***p<0.001 as determined by two-tailed Student’s test and two-way ANOVA followed by Bonferroni post hoc.

Acute HFD induces widespread MMR and rapid modulation of spatial and learning memory
A. Map2, NeuN, Iba1 and Map2, NeuN, GFAP immunostainings on primary neurons.
B. Primary microglial cell media was collected after the ¹³C-glucose tracing (containing ¹³C-metabolites released by microglia challenged with BSA or Palmitate) and incubated for 4 hours with primary neurons, the graph shows the ¹³C-citrate, ¹³C-itaconate and ¹³C-succinate incorporation in the neurons in relative abondance (n=6).
C. Graph showing the latency during the Barnes Test from mice fed with normal diet (Control), or 3 days HFD (3-day HFD) (n=11). The test was performed in the MPI animals facility (Germany).
D. Graph showing the alternation during the T Maze Test from mice fed with normal diet (Control), or 3 days HFD (3-day HFD) (n=11). The test was performed in the MPI animals facility (Germany).
E. Graph showing the distance walked during the ROTAROD test from mice fed with normal diet (Control) or depleted from their microglia (n=8).
F. Graph showing the number of turns before the mice fall during the ROTAROD test from mice fed with normal diet (Control), (Control) or depleted from their microglia (n=8).
G. Graph showing the latency during the ROTAROD test from mice fed with normal diet (Control) or depleted from their microglia (n=8).
H. Graph showing the distance walked during the ROTAROD test from Drp1^MGWT or Drp1^MGKO mice fed with normal diet (Control diet), or 3 days HFD (3-day HFD) (n=11).
I. Graph showing the number of turns before the mice fall during the ROTAROD test from Drp1^MGWT or Drp1^MGKO mice fed with normal diet (Control diet), or 3 days HFD (3-day HFD) (n=11).
I. Graph showing the latency during the ROTAROD test from Drp1^MGWT or Drp1^MGKO mice fed with normal diet (Control diet), or 3 days HFD (3-day HFD) (n=11).
Data are presented as mean ±SEM. *p<0.05, **p<0.01, ***p<0.001 as determined by two-tailed Student’s test and two-way ANOVA followed by Bonferroni post hoc.

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