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. 2019 Mar 26;26(13):3709-3725.e7.
doi: 10.1016/j.celrep.2019.02.107.

Blockade of MCU-Mediated Ca 2+ Uptake Perturbs Lipid Metabolism via PP4-Dependent AMPK Dephosphorylation

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Free PMC article

Blockade of MCU-Mediated Ca 2+ Uptake Perturbs Lipid Metabolism via PP4-Dependent AMPK Dephosphorylation

Dhanendra Tomar et al. Cell Rep. .
Free PMC article

Abstract

Mitochondrial Ca2+ uniporter (MCU)-mediated Ca2+ uptake promotes the buildup of reducing equivalents that fuel oxidative phosphorylation for cellular metabolism. Although MCU modulates mitochondrial bioenergetics, its function in energy homeostasis in vivo remains elusive. Here we demonstrate that deletion of the Mcu gene in mouse liver (MCUΔhep) and in Danio rerio by CRISPR/Cas9 inhibits mitochondrial Ca2+ (mCa2+) uptake, delays cytosolic Ca2+ (cCa2+) clearance, reduces oxidative phosphorylation, and leads to increased lipid accumulation. Elevated hepatic lipids in MCUΔhep were a direct result of extramitochondrial Ca2+-dependent protein phosphatase-4 (PP4) activity, which dephosphorylates AMPK. Loss of AMPK recapitulates hepatic lipid accumulation without changes in MCU-mediated Ca2+ uptake. Furthermore, reconstitution of active AMPK, or PP4 knockdown, enhances lipid clearance in MCUΔhep hepatocytes. Conversely, gain-of-function MCU promotes rapid mCa2+ uptake, decreases PP4 levels, and reduces hepatic lipid accumulation. Thus, our work uncovers an MCU/PP4/AMPK molecular cascade that links Ca2+ dynamics to hepatic lipid metabolism.

Keywords: AMPK; MCU; bioenergetics; calcium; diabetes; hepatocyte; lipid metabolism; metabolic diseases; mitochondrial Ca(2+) uniporter; phosphatase.

Conflict of interest statement

DECLARATION OF INTERESTS

The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Hepatocyte MCU Activity Regulates FAO-Coupled Mitochondrial Respiration
(A) Generation and confirmation of MCUΔhep mice by PCR genotyping (left) and western blotting (right). (B) Schematic representation of the patch-clamp technique for measuring MCU channel activity. (C) Representative IMCU traces derived from MCUfl/fl and MCUΔhep mitoplasts. (D) IMCU densities (picoamperes/picofarads) in mitoplasts. n = 6. (E) Representative traces of extramitochondrial Ca2+ ([Ca2+]out) clearance and DJm in permeabilized hepatocytes from MCUfl/fl and MCUΔhep. n = 3. (F) mCa2+ uptake rate calculated from (E). n = 3. (G) Representative traces of [Ca2+]out rise from MCUfl/fl and MCUΔhep. n = 3. (H) Quantification of mCa2+ after addition of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) from (G). n = 3. (I) Immunoblot for the reconstitution of MCU in MCUΔhep mice using adenovirus-mediated delivery. n = 2. (J) Reconstitution of MCU restores [Ca2+]out clearance ability. n = 4. (K) mCa2+ uptake rate calculated from (J). n = 4. (L) MCU reconstitution restores the matrix Ca2+ in MCUΔhep. Shown are representative traces of [Ca2+]out rise in response to FCCP. n = 4. (M) Quantification of mCa2+ calculated after addition of FCCP from (L). n = 4. (N) MCU reconstitution restores the OCR in MCU KO hepatocytes. n = 3. (O) Quantification of basal, ATP-coupled, and maximal OCRs in hepatocytes from (N). n = 3. (P) The FAO OCR was measured in MCUfl/fl and MCUΔhep hepatocytes using palmitate as a substrate with or without etomoxir (40 mM). n = 3. (Q) Quantification of basal, ATP-coupled, and maximal FAO-coupled OCRs from (P). n = 3. (R) Measurement of hepatocyte ATP. n = 3. (S) Measurement of AMP:ATP ratio in hepatocytes. n = 3. Statistical analysis: mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; ns, non-significant. (T) Schematic of hepatocyte deletion of MCU, showing reduction in mCa2+ and bioenergetic parameters.
Figure 2.
Figure 2.. Loss of MCU Promotes Hepatic Lipid Accumulation and Increases Total Body Fat
(A) Body mass was monitored using NMR and DEXA before and after 24 h fasting. n = 11–12 mice per group. (B) Liver triglycerides were measured using an enzymatic assay. n = 4–6 mice per group. (C) Hepatic deletion of MCU results in increased plasma TAG in the fasting state, as measured by enzymatic assay. n = 5–6 mice per group. (D) MCUΔhep mice showed decreased plasma ketones under the fasting state, as measured by enzymatic assay. n = 5 mice per group. (E) Primary hepatocytes were isolated from MCUfl/fl and MCUΔhep mice and cultured in low glucose, high glucose, and high glucose followed by starvation. Lipid droplets were visualized by confocal microscopy. n = 3. (F) Quantification of the number of lipid droplets from (E). n = 3. (G) Quantification of the size of lipid droplets from (E). n = 15–30 cells from each isolation. n = 3. (H) Representative electron micrograph showing a large number of lipid droplets in MCU KO hepatocytes. n = 3 mice per group. (I) Representative histological section image showing massive oil red O staining in MCU KO liver. n 40 images. n = 3 mice. (J) Schematic of hepatic ablation of MCU, showing increased lipid deposition in the liver, which subsequently increased the whole-body fat content. Statistical analysis: mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 3.
Figure 3.. Loss of MCU Limits mCa2+ Uptake and Elevates Total Body Fat in Zebrafish
(A) Schematic of the generation of global MCU KO zebrafish using CRISPR/Cas9. (B) Genotyping for MCU deletion. (C) Bar graph showing MCU mRNA abundance in WT, MCU+/−, and MCU−/− zebrafish. (D) Western blot for MCU expression. n = 3. (E) Measurement of mCa2+ uptake. n = 5–10. (F) Quantification of ionomycin-induced peak mCa2+ levels from (E). n = 5–10. (G) Bar graph representing cellular ATP levels in cells isolated from MCU+/+, MCU+/−, and MCU−/− zebrafish. n = 4. (H) Adult WT and MCU−/−zebrafish were homogenized and centrifuged. MCU−/−zebrafish samples show a clear yellow color lipid layer on top of the protein lysate. n = 6. (I) Adult WT and MCU−/−zebrafish were stained with Lipid Green to monitor the distribution of lipids in the whole body. n = 3. (J and K) Bar graphs represent the quantification of Lipid Green staining from the dorsal (J) or tail (K) fin. n = 3. Statistical analysis: mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 4.
Figure 4.. Ablation of mCa2+ Uptake Augments the cCa2+ Rise That Enhances AMPK Dephosphorylation
(A) Hepatocytes isolated from MCUfl/fl and MCUΔhep mice were lysed in radioimmunoprecipitation assay (RIPA) buffer and immunoblotted for the indicated antibodies. n = 6. (B) Bar graph representing the pAMPK:AMPK ratio from (A). n = 6. (C) Immunoblot analysis for phospho and total CaMKK-II. n = 6.(D) Hepatocyte lysates from AMPKα1/α2fl/fl and AMPKα1/α2fl/flCre+ mice were probed for AMPKa protein abundance. n = 3. (E) Hepatic TAG levels. n = 5. (F) Assessment of hepatic lipid accumulation by Oil-Red-O staining. n = 5. (G) Primary hepatocytes isolated from MCUfl/fl and MCUΔhep mice were infected with a CA-AMPK-expressing adenovirus. Hepatocytes were stained with oil red O and measured. n = 6–12 replicates from 3 mice. (H) Visualization of lipid droplets from CA-AMPK reconstituted MCUΔhep hepatocytes. n = 3. (I) Quantification of the number of lipid droplets from (H). n = 20–30 cells. n = 3. (J) Schematic depicting a link between MCU and AMPK phosphorylation. (K) GCaMP6-expressing hepatocytes from MCUfl/fl and MCUΔhep were stimulated with thapsigargin (Tg) or Vasopressin, and cCa2+ dynamics were monitored. n = 3. (L) Quantification of the cCa2+ clearance rate from (K). n = 15–25 cells. n = 3. (M) GCaMP6-expressing hepatocytes from MCUfl/fl and MCUΔhep were stimulated with Vasopressin and cCa2+ dynamics were monitored. n = 3. (N) Quantification of the cCa2+ clearance rate from (M). n = 15–25 cells. n = 3. (O) Hepatocytes isolated from MCUfl/fl and MCUΔhep adult mice were transduced with an adenovirus expressing the cCa2+ sensor GCaMP6, and basal cCa2+ fluorescence was quantified. n = 74–91 cells from 3 mice in each group. (P) Hepatocytes were treated with the intracellular Ca2+ chelator BAPTA-AM for various times. Cell lysates were immunoblotted for the indicated antibodies. n = 4. (Q) Hepatocytes were treated with BAPTA-AM overnight, and oil red O stain was quantified. 12 replicates from 3 mice per group. (R) Visualization of lipid droplets from control and BAPTA-AM-treated hepatocytes. n = 3 mice per group. (S) Quantification of lipid droplets from control and BAPTA-AM-treated hepatocytes. n = 20–30 cells per group. n = 3 mice per group. (T) Schematic depicting how cCa2+ determines lipid clearance, possibly through AMPK phosphorylation. Statistical analysis: mean ± SEM. **p < 0.01, ***p < 0.001.
Figure 5.
Figure 5.. AMPK Dephosphorylation in MCU KO Hepatocytes Is Likely Due to PP4
(A) Hepatocytes were treated with okadaic acid (OA) (50 nM) for 2 h. Cell lysates were immunoblotted for the indicated antibodies. n = 4. (B) MCUfl/fl and MCUΔhep hepatocytes were treated with FK506 (2 μM) for 2 h and immunoblotted for the indicated antibodies. n = 4. (C) MCUfl/fl and MCUΔhep hepatocytes were treated with cantharidin (50 μM) for 2 h and immunoblotted for the indicated antibodies. n = 4. (D) Hepatocytes isolated from MCUfl/fl and MCUΔhep mice were lysed and immunoblotted for the indicated antibodies. n = 4. (E) Densitometric analysis of PP4 protein abundance. n = 4. (F) Hepatocytes were transfected with PP4 siRNA for 72 h. Cell lysates were immunoblotted for the indicated antibodies. n = 4. (G) Hepatocytes were transfected with PP4-FLAG for 48 h. Cell lysates were immunoblotted for the indicated antibodies. n = 4. (H–J) HepG2 cells were transfected with AMPK-hemagglutinin (HA) and PP4-FLAG plasmids for 48 h. Cell lysates were immunoprecipitated with an HA antibody and probed with the indicated antibodies (H). Under a similar condition (I and J), cells were treated with BAPTA-AM in the presence or absence of ionomycin (50 nM) stimulation. The reciprocal immunoprecipitation was performed with HA or FLAG antibodies and probed with the indicated antibodies. n = 3. (K) Visualization of lipid droplets from scrambled (Scr) siRNA- and PP4 siRNA-treated hepatocytes. n = 3. (L) Quantification of lipid droplets from Scr siRNA- and PP4 siRNA-treated hepatocytes. n = 20–30 cells. n = 3. (M) Hepatocytes isolated from MCUfl/fl and MCUΔhep adult mice were transfected with PP4 siRNA for 72 h. Cells were stained with oil red O and quantified. n = 3. (N) Schematic depicting aberrant cCa2+ clearance in MCU KO hepatocytes exhibiting AMPK dephosphorylation through elevated PP4 activity. Statistical analysis: mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 6.
Figure 6.. Hepatocytes Harboring MCU-C96A Display Reduced PP4 and Augmented Hepatic Lipid Clearance during Fasting
(A) Schematic depicting the strategy used to generate MCU-C96A KI mice. Top right: genotyping of MCU-C96A KI mice. Bottom: MCU protein abundance. (B) 12-week-old MCU-C96A KI mice are viable. (C) MCUC96A-KI mice have reduced body weight at 12 weeks. n = 5. (D) MCU-mediated extramitochondrial Ca2+ clearance and mCa2+ uptake. n = 3. (E) MCU-mediated mCa2+ uptake rate was calculated from (D). n = 3. (F) Measurement of basal mCa2+ after addition of FCCP. n = 3. (G) Quantification of basal mCa2+ after addition of FCCP from (F). n = 3. (H) Western blot analysis of mitochondrial respiratory chain subunits of complex I (NDUFB8), complex II (succinate dehydrogenase complex iron sulfur subunit B [SDHB]), complex III (UQCRC2), complex IV (MTCO1), and ATP synthase subunit ATP5A. MCU and TOM20 were used as loading controls. n = 3. (I) Measurement of mitochondrial OCR. n = 3. (J) Measurement of basal, ATP coupled, and maximal OCR from (I). n = 3. (K) Hepatocytes isolated from WT and C96A-KI mice were lysed and probed with the indicated antibodies. n = 3. (L) Densitometric analysis of pAMPK/AMPK ratio from (K). n = 3. (M) Densitometric analysis of PP4 protein expression from (K). n = 3. (N) Measurement of liver and triglycerides (TAGs) under fasting conditions. Measurement of these parameters is described in Figure 2. n = 5. (O) Measurement of plasma triglycerides (TAG) under fasting conditions. n = 5. (P) Measurement of plasma ketone bodies under fasting conditions. n = 5. (Q) Visualization of lipid droplets from WT and C96A KI hepatocytes. n = 3. (R) Quantification of lipid droplets from (Q). n = 20–30 cells. n = 3. (S) Representative histological section image shows reduced lipid accumulation in C96A-KI mice. n = 3 mice per group. Statistical analysis: mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 7.
Figure 7.. Activation of AMPK by Metformin Corrects Hepatic Lipidome Remodeling in MCU KO Mice
(A) Hepatocytes isolated from MCUfl/fl and MCUΔhep treated with metformin (1 mM), AICAR (100 μM) for 16 hours. After treatment, cell lysates were probed with indicated antibodies. n = 3. (B) Bar graph showing the quantification of pAMPK in Figure 7A. n = 3. (C) Representative histological section image shows clearance of lipids in MCU KO metformin-administered mice. n = 3. (D) Visualization of lipid droplets from MCUfl/fl and MCUΔhep mice with or without metformin treatment. n = 3. (E) Quantification of lipid droplets from (D). n = 15–30 cells per mice. n = 3. (F) Lipids were analyzed as described in STAR Methods, and the identified features were subjected to a hierarchical cluster analysis. n = 4. (G) Significant top 15 lipids (false discovery rate [FDR] = 0.001) representing the relative lipid modulation among the groups. The y axis represents normalized values. *, significantly different from MCUΔhep; #, significantly different from metformin-administrated MCUΔhep. n = 4 per group. Statistical analysis: mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

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