. 2025 May;301(5):108468.

doi: 10.1016/j.jbc.2025.108468. Epub 2025 Mar 28.

Activating transcription factor 3 regulates hepatic apolipoprotein A4 upon metabolic stress

Jasmine Encarnacion¹, Danielle M Smith², Joseph Choi², Joseph Scafidi³, Michael J Wolfgang⁴

Affiliations

¹ Department of Physiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
² Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
³ Department of Neurology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; The Michael V. Johnston Center for Developmental Neuroscience, Kennedy Krieger Institute, Baltimore, Maryland, USA.
⁴ Department of Physiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. Electronic address: [email protected].

PMID: 40158856
PMCID: PMC12059330
DOI: 10.1016/j.jbc.2025.108468

Activating transcription factor 3 regulates hepatic apolipoprotein A4 upon metabolic stress

Jasmine Encarnacion et al. J Biol Chem. 2025 May.

. 2025 May;301(5):108468.

doi: 10.1016/j.jbc.2025.108468. Epub 2025 Mar 28.

Authors

Jasmine Encarnacion¹, Danielle M Smith², Joseph Choi², Joseph Scafidi³, Michael J Wolfgang⁴

Affiliations

¹ Department of Physiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
² Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
³ Department of Neurology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; The Michael V. Johnston Center for Developmental Neuroscience, Kennedy Krieger Institute, Baltimore, Maryland, USA.
⁴ Department of Physiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. Electronic address: [email protected].

PMID: 40158856
PMCID: PMC12059330
DOI: 10.1016/j.jbc.2025.108468

Abstract

The liver plays essential roles in maintaining systemic glucolipid homeostasis under ever changing metabolic stressors. Metabolic dysregulation can lead to both adaptive and maladaptive changes that impact systemic physiology. Here, we examined disparate genetic and environmental metabolic stressors and identified apolipoprotein A4 (ApoA4) as a circulating protein upregulated in liver-specific KOs for carnitine palmitoyltransferase 2 and pyruvate carboxylase. We found this upregulation to be exacerbated by fasting and high-fat or ketogenic diets. Unique among these models was a concomitant increase in activating transcription factor 3 (Atf3). Liver-specific overexpression of Atf3 resulted in increased ApoA4 expression in a sex-dependent manner. To understand the requirement of Atf3 to metabolic stress, we generated liver-specific Atf3, Cpt2 double KO mice. These experiments demonstrated the requirement for Atf3 in the induction of ApoA4 mRNA, ApoA4 protein, and serum triglycerides that were also sex-dependent. These experiments reveal the roles of hepatic Atf3 and ApoA4 in response to metabolic stress in vivo.

Keywords: Atf3; apolipoprotein A4; fatty acid oxidation; gene knockout; liver metabolism; metabolic regulation; sex-dependent; transcription factor.

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Conflict of interest statement

Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.

Figures

**Figure 1**
**Serum protein screening shows ApoA4 upregulation in several models of dysregulated hepatic metabolism.** Coomassie-stained SDS-PAGE gels of serum proteins, followed by Western blots for ApoA4 with ponceau stained albumin bands for loading control. A, serum was derived from male mice, fasted 24 h, with WT, Cpt2^L−/−, PCx^L−/−, PDH^L−/− genotypes. n = 4. B, comparison of WT, TSC1^L−/−, Pparα^−/−, and InsR^L−/− genotypes; n = 4. C, serum derived from female mice, fasted 24 h, with WT, PCx^L−/−, Cpt2^L−/−, PDH^L−/−, and TSC1^L−/− genotypes; n = 3. D, serum derived from WT male mice in various dietary states: Chow-fed 4 h fast, chow-fed 24 h fast, high-fat diet–fed 4 h fast, and ketogenic diet–fed 4 h fast; n = 4. Relative values for Western blot band intensities are illustrated. Statistical significance was determined by ordinary one-way ANOVA with the Brown-Forsythe test, Bartlett's test, and Bonferroni multiple comparisons test, conducted with GraphPad prism; n = 4. Data expressed as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. ApoA4, apolipoprotein A4; Cpt2, carnitine palmitoyltransferase 2; PCx, pyruvate carboxylase; InsR, insulin receptor; PDH, pyruvate dehydrogenase; TSC1, tuberous sclerosis complex 1.

**Figure 2**
**Changes in serum ApoA4 levels vary across PCx^L−/− and Cpt2^L−/− mice fed chow, high-fat, and ketogenic diets.** Coomassie-stained SDS-PAGE gel of serum proteins, followed by Western blots for ApoA4 with ponceau-stained albumin bands for loading control. Serum was collected from male mice, fasted 4 h, in the following groups (n = 4): (A) PCx^{L f/f} (phenotypically WT) on chow or HFD, PCx^L−/− (liver knockout) on chow or HFD; (B) PCx^{L f/f} on chow or KD, PCx^L−/− on chow or KD; (C) Cpt2^{L f/f} on chow or HFD, Cpt2^L−/− on chow or HFD; (D) Cpt2^{L f/f} on chow or KD, and Cpt2^L−/− on chow or KD. Relative values for Western blot band intensities are illustrated. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test, conducted with GraphPad prism; n = 4. Data expressed as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. ApoA4, apolipoprotein A4; Cpt2, carnitine palmitoyltransferase 2; HFD, high-fat diet; KD, ketogenic diet; PCx, pyruvate carboxylase.

**Figure 3**
**Relative liver ApoA4 mRNA levels increase for PCx^L−/− and Cpt2^L−/− mice in different dietary states, as determined by reverse transcriptase-quantitative polymerase chain reaction**. Dietary states described as: chow diet, 4 h fast; chow diet, 24 h fast; high-fat diet, 4 h fast; and ketogenic diet, 4 h fast. A–F, liver mRNA (ApoA1, ApoA4, ApoA5, ApoB, ApoC3, and ApoE, respectively) of PCx^{L f/f} and PCx^L−/− mice with described dietary states, relative to chow diet 4 h fast PCx^{L f/f}. G–L, liver mRNA (ApoA1, ApoA4, ApoA5, ApoB, ApoC3, and ApoE, respectively) of Cpt2^{L f/f} and Cpt2^L−/− mice with described dietary states, relative to chow diet 4 h fast Cpt2^{L f/f}. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test, conducted using GraphPad prism. Data are expressed as mean ± SD. Single-letter difference denotes p < 0.05 between groups. ApoA4, apolipoprotein A4; Cpt2, carnitine palmitoyltransferase 2; PCx, pyruvate carboxylase.

**Figure 4**
**Hepatic ApoA4 upregulation is most prominent with a 24 h fast in the absence of fatty acid oxidation.**A, Western blots comparing serum ApoA4 levels in 24 h-fasted WT, PCx^L−/−, Cpt2^L−/−, and PCx/Cpt2^{L−/−;−/−} (double liver KO) mice. Ponceau-stained serum albumin bands were used as loading controls. B, reverse transcriptase-quantitative polymerase chain reaction comparison of ApoA4 mRNA from liver homogenate across 24 h-fasted WT (n = 5), PCx^L−/−(n = 5), Cpt2^L−/− (n = 6), and PCx/Cpt2^{L−/−;−/−} mice (n = 6). In groups with n = 5, one outlier was excluded prior to statistical analysis due to aberrant Cq values for control genes (CycloA and 18s). Data are expressed as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. ApoA4, apolipoprotein A4; Cpt2, carnitine palmitoyltransferase 2; PCx, pyruvate carboxylase.

**Figure 5**
**RT-qPCR for genes in the CREB/Atf family show consistent elevation of liver Atf3 mRNA in 24 h-fasted mouse models of hepatic stress.**A, WT and PCx^L−/− liver mRNA levels for CREB/Atf family members Atf 1 to 7, CREB, CREB3, CREB3L3, CREB5, and CREM, along with commonly associated proteins cJun and JunB were assessed using RT-qPCR. B, RT-qPCR for Atf1-7, CREB, CREB3, CREB3L3, CREB5, CREM, cJun, and JunB mRNA in WT and Cpt2^L−/− livers. C, RT-qPCR for Atf 1 to 7, CREB3, CREB3L3, CREB5, and JunB in WT and PCx/Cpt2^{L−/−;−/−} mice. Data are expressed as mean ± SD. For all graphs, Welch’s t tests with no correction for multiple comparisons were used to establish statistical significance (p < 0.05) on GraphPad prism. AFT, activating transcription factor; Cpt2, carnitine palmitoyltransferase 2; PCx, pyruvate carboxylase; RT-qPCR, reverse transcriptase-quantitative polymerase chain reaction.

**Figure 6**
**Atf3 overexpression in WT mice significantly upregulates hepatic ApoA4 in females, but not males with a 24 h fast.**A, coomassie-stained SDS-PAGE gels of serum proteins, followed by Western blots for ApoA4. Both male and female groups are shown for hepatic Atf3 or eGFP-overexpressing mice. Serum proteins are compared under a 4 h fast, followed by a 24 h fast replicate using the same mice (B) reverse transcriptase-quantitative polymerase chain reaction was used to assess hepatic mRNA abundance of apolipoproteins in 24 h-fasted female (n = 3) (C) or male (n = 4) (D) Atf3-overexpressing mice compared to GFP controls. Relevant genes in the CREB/Atf family were also assessed by reverse transcriptase-quantitative polymerase chain reaction in female (E) and male (F) groups. Data are expressed as mean ± SD and analyzed using Welch’s t test with no correction for multiple comparisons. All statistical tests were conducted using GraphPad prism. ApoA4, apolipoprotein A4; AFT, activating transcription factor; Cpt2, carnitine palmitoyltransferase 2; PCx, pyruvate carboxylase.

**Figure 7**
**Hepatic Atf3^L−/−/Cpt2 ^L−/− double KOs demonstrate a reduced induction of hepatic ApoA4 in females, with a transcriptional profile distinct from Cpt2^L−/− single knockouts.**A, validation of Atf3^L−/−/Cpt2 ^L−/− double KO model using reverse transcriptase-quantitative polymerase chain reaction to show significantly reduced levels of hepatic Atf3 and Cpt2 mRNA. B, reverse transcriptase-quantitative polymerase chain reaction of liver mRNA for ApoA4 and its documented regulators, CREB3L3, CREB3, and ERRα. C, Western blots comparing serum ApoA4 levels in Atf3^L−/−/Cpt2 ^L−/− males and females compared to Cpt2 ^L−/− mice (n = 4). Ponceau-stained serum albumin bands were used as loading controls. D, serum triglyceride assay comparing floxed, Cpt2^L−/−, and Atf3^L−/−/Cpt2^L−/− levels. Data analyzed using ordinary one-way ANOVA with the Brown-Forsythe test, Bartlett's test, and Bonferroni multiple comparisons test, conducted with GraphPad PRISM; n = 6. AFT, activating transcription factor; ApoA4, apolipoprotein A4; Cpt2, carnitine palmitoyltransferase 2; ERRα, estrogen-related receptor alpha; PCx, pyruvate carboxylase.

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Activating transcription factor 3 regulates hepatic apolipoprotein A4 upon metabolic stress

Affiliations

Activating transcription factor 3 regulates hepatic apolipoprotein A4 upon metabolic stress

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

References

MeSH terms

Substances

Grants and funding

LinkOut - more resources

Full Text Sources

Molecular Biology Databases

Research Materials

Miscellaneous