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Peer-reviewed journal article

APOE4 drives widespread changes to the hepatic proteome and alters metabolic function

Colton R LysakeriD, Chelsea N. JohnsoniD, Vivien CsikósiD, Edziu FranczakiD, Ashley L. TetlowiD, Maggie N. Benson, John P. ThyfaultiD, Paige C. GeigeriD, Jill K. MorrisiD, Heather WilkinsiD

Abstract

Apolipoprotein E (APOE) is essential for lipid homeostasis and has been extensively studied in the central nervous system, particularly in the context of Alzheimers disease (AD). Individuals carrying an APOE4 allele have an increased risk of AD and exhibit deficits in energy metabolism, including glucose utilization and mitochondrial dysfunction. While the role of APOE in the liver is well characterized, the impact of APOE genetic variation on hepatic health and metabolism remains poorly understood. We sought to investigate this using young female and male APOE3 and APOE4 targeted replacement mice. We also used APOE isogenic induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (iHLCs) to examine specific effects in a human-relevant cell model. Proteomic and functional assays show that APOE4 causes extensive changes to liver mitochondrial function in a sex-specific manner in mice and alters glucose and lipid metabolism. APOE4 also impairs mitochondrial function in iHLCs and shifts metabolism towards glycolysis while modifying expression of extracellular matrix proteins. Additionally, APOE4 iHLCs display a greater reliance on fatty acids as an energy source and show increased lipid accumulation. Taken together, our findings show that APOE genetic variation causes mitochondrial dysfunction and rewires hepatic metabolism.

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Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (19 Jun 2025) · CC BY 4.0

In 4-month-old APOE targeted-replacement mice, APOE4 remodels the liver proteome sex-dependently; in isogenic iPSC-derived hepatocytes it impairs respiration and raises glycolysis and lipid droplets.

APOE4 increases Alzheimer's disease risk and is tied to deficits in energy metabolism, yet its impact on the liver (hepatocytes are the main peripheral APOE source) is poorly understood, and prior hepatoma and mouse studies gave mixed results. The work pairs sex-stratified mouse liver proteomics and respirometry with what the authors call the first use of iPSC-derived hepatocyte-like cells to study APOE genotype and hepatic metabolism.

Key results

  1. ShownAPOE4 changed the young mouse liver proteome far more in females than in males without altering hepatic APOE protein levels, and oxidative phosphorylation pathways shifted in opposite directions by sex.

    Whole-liver proteomics (n=3 per group): 402 DE proteins in female APOE4 vs APOE3 vs 58 in males; 15 shared up and 6 shared down. IPA: electron transport/oxidative phosphorylation and mTOR signaling up in APOE4 females; oxidative phosphorylation and NRF2 response down in APOE4 males. ELISA APOE unchanged (n=8 per group). · APOE genotype and sex alter the hepatic proteome…; Discussion; Figure legends

  2. ShownIn isolated liver mitochondria, APOE4 effects on respiration and mitochondrial pathways depended on sex: some respiratory states rose in females, while fatty acid-driven maximal flux fell in males.

    Seahorse, n=8 per genotype and sex: palmitoyl-CoA uncoupled rate reduced in APOE4 males (sex x genotype interaction); pyruvate/malate state 3S and uncoupled rates higher in APOE4 vs APOE3 females. Mitochondrial proteomics (n=3): TCA cycle and complex III assembly down in APOE4 females; complex I biogenesis and oxidative phosphorylation down in APOE4 males. · Mitochondrial proteins and pathways are modified…; APOE genotype and sex interact to alter…; Figure legends

  3. ShownIn isogenic iPSC-derived hepatocyte-like cells (iHLCs), APOE4 altered thousands of proteins. Glycolysis, inflammation and extracellular matrix (ECM) proteins rose consistently, while ribosomal and mitochondrial translation/respiration pathways fell.

    Pair A, two differentiation batches (n=5 per group): Batch 1 had 1258 up/902 down and Batch 2 had 1097 up/1115 down proteins vs APOE3. Shared IPA pathways: neddylation, autophagy, cGAS-STING and glycolysis up; mitochondrial translation, oxidative phosphorylation and complex I biogenesis down; GO showed ECM component enrichment; APP reduced. · APOE genotype drives significant changes to the…; Discussion; Figure legends

  4. ShownAPOE4 iHLCs from both isogenic pairs had lower mitochondrial respiration, more hydrogen peroxide and higher glycolysis, with impaired mitochondrial pyruvate oxidation.

    Seahorse in Pairs A and B: basal, maximal, proton-leak and ATP-linked respiration and complex IV flux fell; basal glycolysis and glycolytic capacity rose; acute response to UK5099 was smaller. Amplex Red H2O2 rose in both pairs; membrane potential and superoxide rose only in Pair A, and mitochondrial Ca2+ only in Pair B. · APOE4 isogenic iHLCs display impaired…; Figure legends

  5. ShownAPOE4 iHLCs showed impaired long-chain fatty acid oxidation alongside a greater apparent reliance on these fatty acids, accumulated more lipid droplets per cell, and upregulated proteins in the SREBP2 cholesterol-synthesis network.

    Etomoxir (CPT1 inhibitor) gave a smaller acute response in APOE4 cells and lowered their maximal respiration, which did not happen in APOE3 cells, in both pairs. LipidTOX imaging across Pairs A and B showed more droplets per cell with unchanged diameter. SREBF2 network proteins and FDFT1 were up in APOE4 iHLCs (Fdft1 was also up in female APOE4 mice). · Cholesterol and lipid metabolism are altered by…; Discussion; Figure legends

Methods, data and reproducibility

Whole liver and isolated liver mitochondria from 4-month-old female and male APOE3/APOE4 targeted-replacement mice underwent data-independent acquisition LC-MS proteomics and Seahorse respirometry. Two isogenic iPSC pairs were differentiated into hepatocyte-like cells (iHLCs) for substrate stress tests, mitochondrial probes and lipid droplet imaging, with proteomics on Pair A only.

  • Homozygous APOE3 and APOE4 targeted-replacement mice (B6.129P2 background), females and males aged 4 months on chow; whole liver and isolated liver mitochondria · mouse (endogenous Apoe replaced by human APOE) · n = n=8 per genotype and sex; n=3 for proteomic outcomes
  • Isogenic CRISPR-edited APOE3/APOE4 iPSC Pair A (JIPSC001162, JIPSC001150; KOLF2.1J male parental line) differentiated to iHLCs; proteomics (two batches) and functional assays · human (iPSC-derived) · n = n=5 per group for proteomics
  • Isogenic APOE3/APOE4 iPSC Pair B (JIPSC001268, JIPSC001142; KOLF2.1J background) differentiated to iHLCs for respirometry, mitochondrial probes and lipid droplet imaging · human (iPSC-derived)
Methods & tools
Spectronaut 18.3 (Biognosys), proteiNorm, MitoCarta 3.0, QIAGEN Ingenuity Pathway Analysis (IPA), STRING, KEGG, UniProt (mouse and human), Orbitrap Exploris 480 LC-MS with data-independent acquisition, Agilent Seahorse XFe96 / XF Pro with Wave software, HCS LipidTOX Red with BioTek Cytation 1 / Gen5, TMRE, MitoSOX Red, Amplex Red and Rhod-2 AM probes, GraphPad Prism 10
Reagents & environment
Spectronaut 18.3 with UniProt mouse/human databases; proteiNorm QC/normalization; MitoCarta 3.0; QIAGEN IPA; GraphPad Prism 10; Agilent Seahorse Wave; BioTek Gen5.
Reproducibility gaps
The text gives no proteomics repository accession and no statement on data or code availability. Mass spectrometry was done by an external core (UAMS).
Limitations (6)
  • Only male-derived iHLCs were used (both isogenic pairs come from the male KOLF2.1J line), so sex differences at the hepatocyte level could not be examined. · stated by the authors
  • Only APOE3 and APOE4 homozygotes were studied. APOE2 and the other allele combinations found in the population were not examined. · stated by the authors
  • The authors note that iHLCs are isolated cells lacking the many cell types of whole liver, which may explain why glycolysis rose in APOE4 iHLCs while glucose metabolism pathways fell in APOE4 mouse livers. · stated by the authors
  • In APOE4 females, mitochondrial protein import pathways moved in opposite directions in whole liver versus isolated mitochondria, and the TCA cycle-respiration link needs follow-up on succinate dehydrogenase activity. Both mechanisms remain unresolved. · stated by the authors
  • Mouse proteomics used n=3 per group, nominal p < 0.05 for DE calls and p < 0.2 as pathway input, with no stated multiple-testing correction. The DE counts and pathway calls may therefore include false positives. · noted in this summary
  • iHLC proteomics covered only isogenic Pair A, and both pairs share one male parental background. Several mitochondrial readouts (membrane potential, superoxide, Ca2+) differed between the pairs, so the results may not generalize. · noted in this summary

Who should read:Read this if you study peripheral effects of APOE4 or sex differences in liver mitochondrial metabolism, or need an isogenic iPSC-hepatocyte model for APOE-dependent bioenergetics and lipid handling.

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