Dataset Information

Blockade of MCU-Mediated Ca2+ Uptake Perturbs Lipid Metabolism via PP4-Dependent AMPK Dephosphorylation.

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 (MCU?hep) 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 MCU?hep 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 MCU?hep 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.

SUBMITTER: Tomar D

PROVIDER: S-EPMC6512325 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Publications

Blockade of MCU-Mediated Ca2+ Uptake Perturbs Lipid Metabolism via PP4-Dependent AMPK Dephosphorylation.

Tomar Dhanendra D Jaña Fabián F Dong Zhiwei Z Quinn William J WJ Jadiya Pooja P Breves Sarah L SL Daw Cassidy C CC Srikantan Subramanya S Shanmughapriya Santhanam S Nemani Neeharika N Carvalho Edmund E Tripathi Aparna A Worth Alison M AM Zhang Xueqian X Razmpour Roshanak R Seelam Ajay A Rhode Stephen S Mehta Anuj V AV Murray Michael M Slade Daniel D Ramirez Servio H SH Mishra Prashant P Gerhard Glenn S GS Caplan Jeffrey J Norton Luke L Sharma Kumar K Rajan Sudarsan S Balciunas Darius D Wijesinghe Dayanjan S DS Ahima Rexford S RS Baur Joseph A JA Madesh Muniswamy M

Cell reports 20190301 13

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 ...[more]

PMID: 30917323

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Project description:Methods: We performed mRNA sequence analysis by deep sequencing, in triplicate, using Illumina HiSeq to assess the impact of PHLPP1 knockdown in regulating gene expression in RAW 264.7 cells treated with/without OxLDL. The transcriptome libraries were constructed using the NEB adapters and were sequenced on at 150 nucleotide read length using the paired-end chemistry. The raw reads were subjected to Adapter and low-quality reads removal by Trimmomatic -0.36v. The raw reads were subjected to contamination [structural RNA / low complexity sequences] removal by mapping with bowtie 2-2.2.1. The decontaminated data set was mapped to the mouse genome. Reads mapping to gene list were counted using feature count module of sub reads package. The read counts were normalized in DESeq2-3.5, and subject to differential expression analysis. Differentially expressed genes were selected based on log2-ratio change with p-value <0.05 (Student’s t-test, unpaired). Hierarchical clustering was performed with the programs Cluster (uncentered correlation; average linkage clustering) and Treeview. Results: To gain a deeper understanding of the role of PHLPP1 in lipid metabolism in macrophages, we performed mRNA Seq analysis to assess the gene expression changes in a mouse macrophage cell line. The experiment has been carried out in triplicates. Alignment of the reads showed 97% alignment to the reference mouse genome. Identification of the differentially expressed genes were then carried out by DESeq2 version 1.2.10 with stringent criteria (FDR <0.05% with 2 fold up and down regulation) which reveals in control treated vs. control (121-down, 396-Up), Test vs. Control - (326-down, 569 Up) and Test treated vs. control treated (1314-down, 1328 up) of DEG to be altered in expression respectively. Inference: RNA-seq analysis revealed that PHLPP1 knockdown parallel with OxLDL treatment resulted in diminished expression profile of cholesterol biosynthesis and lipid metabolism genes.

Dataset Information

Blockade of MCU-Mediated Ca2+ Uptake Perturbs Lipid Metabolism via PP4-Dependent AMPK Dephosphorylation.

Publications

Blockade of MCU-Mediated Ca<sup>2+</sup> Uptake Perturbs Lipid Metabolism via PP4-Dependent AMPK Dephosphorylation.

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