Cholesterol-responsive NFE2L1-INSIG1 interaction controls VLDL secretion and metabolic dysfunction–associated steatohepatitis pathogenesis in mice

Q: What is the clinical evidence from this study?

Population: Metabolic dysfunction-associated steatohepatitis (MASH) and hepatic cholesterol overload. Intervention: Hepatic NFE2L1 overexpression (via adenoviral vector AV-NFE2L1) and NFE2L1-WT/ΔNHB2 rescue (via AAV) vs. AV-LacZ control vector, AAV-GFP control, or NFE2L1-ΔNHB2 mutant. Primary outcome: Hepatic SREBP1 activation, VLDL secretion, and MASH histopathology (CD68+ macrophage infiltration and alpha-SMA+ stellate cell activation) (p=<0.0001).

Shijun Deng; Jessica E. Freed; Grace Y. Lee; Gizel Askin; Zhe Cao; Özgür Cakici; Bo Yuan; Sheng Tony Hui; Karen E. Inouye; Isabel Graupera; Gökhan S. Hotamışlıgil

doi:10.1172/jci197094

July 14, 2026Journal of Clinical InvestigationOpen Access

Cholesterol-responsive NFE2L1-INSIG1 interaction controls VLDL secretion and metabolic dysfunction–associated steatohepatitis pathogenesis in mice

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Key result

Hepatic NFE2L1 overexpression promoted INSIG1 degradation to sustain SREBP1-mediated VLDL secretion, reducing liver macrophage infiltration by 45% and activated stellate cells by 24% in MASH mice.

Why the study?

Does hepatic NFE2L1 activation preserve VLDL secretion and attenuate hepatic cholesterol accumulation, liver injury, inflammation, and fibrosis in murine models of MASH?

Key Points

To resolve how hepatic VLDL secretion persists during cholesterol overload and determine how the interaction between NFE2L1 and INSIG1 governs lipid homeostasis and MASH progression.
Generated hepatocyte-specific NFE2L1-knockout (LKO) and wild-type (WT) mice evaluated under overnight fasting and 6-hour refeeding conditions, alongside Hepa1-6 hepatoma cell models.
Assessed de novo lipogenesis via deuterated water labeling, measured VLDL export using tyloxapol-induced lipid accumulation assays, and traced INSIG1 degradation rates with cycloheximide chase assays.
Evaluated rescue with wild-type NFE2L1 or an NHB2-deleted mutant (ΔNHB2), and tested therapeutic NFE2L1 overexpression in a murine MASH model.
Refed LKO mice exhibited more than a 50% reduction in nuclear SREBP1(N) (P < 0.0001 vs. refed WT) and a 2-fold elevation of endogenous INSIG1 protein, leading to defective VLDL secretion and elevated hepatic cholesterol.
INSIG1 showed a half-life of approximately 30 minutes in WT hepatocytes but remained stable and 2-fold higher than WT after 60 minutes in NFE2L1-deficient cells.
In a murine MASH model, NFE2L1 overexpression activated SREBP1/2, reduced hepatic cholesterol, and attenuated liver injury, inflammation, and fibrosis without elevating atherogenic lipoproteins.

Structured PICO

Does hepatic NFE2L1 activation preserve VLDL secretion and attenuate hepatic cholesterol accumulation, liver injury, inflammation, and fibrosis in murine models of MASH?

P

Population

8- to 10-week-old male liver-specific NFE2L1-knockout mice and 8-week-old male db/db mice on a methionine/choline-deficient diet for 2 weeks, alongside cultured murine Hepa1-6 hepatocytes.

I

Intervention

Hepatic NFE2L1 genetic modulation: adenoviral NFE2L1 overexpression (AV-NFE2L1, 1 x 10^9 particles/mouse i.v. via retro-orbital plexus for 14 days in db/db MCD mice) and adeno-associated viral rescue (AAV-NFE2L1-WT or AAV-NFE2L1-ΔNHB2, 1 x 10^11 viral genomes i.v. for 3 weeks in LKO mice).

C

Comparator

Control viral vectors (AV-LacZ in db/db MCD mice; AAV-GFP or INSIG1-binding-deficient mutant AAV-NFE2L1-ΔNHB2 in LKO mice) and wild-type (WT) littermate controls.

O

Outcome

Hepatic SREBP1 proteolytic activation, VLDL secretion rate, hepatic cholesterol accumulation, and MASH severity (serum ALT/AST, inflammatory/fibrotic gene expression, and blinded histological assessment of CD68+ macrophage infiltration and alpha-SMA+ hepatic stellate cell activation).surrogate

Main Result

p-value: p=<0.0001

Limitations

In vivo therapeutic experiments were performed in leptin receptor-deficient db/db mice on an MCD diet, which does not fully recapitulate human MASH pathophysiology.
All animal studies were conducted exclusively in male mice; relevance to female animals and humans remains untested.
The detailed biochemical mechanism of NFE2L1-mediated INSIG1 degradation, including the specific E3 ubiquitin ligase, remains unknown.
Cardiovascular effects were not tested in atherosclerosis-prone animal models.
Therapeutic in vivo studies relied on leptin receptor-deficient db/db mice fed an MCD diet, which does not fully recapitulate human MASH pathophysiology.
All animal experiments used only male mice, leaving sex-specific or hormonal effects unexamined.
The specific E3 ligase and regulatory cofactors mediating NFE2L1-driven INSIG1 degradation were not identified.
Vascular safety was not evaluated in dedicated atherosclerosis-prone models.

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Implication

Identifies the ER-resident NFE2L1-INSIG1 interaction as a cholesterol-responsive mechanism that maintains VLDL export and mitigates MASH liver injury and fibrosis in mice without increasing atherogenic apoB/non-HDL lipoproteins.

Abstract

Cholesterol overload contributes to metabolic dysfunction–associated steatohepatitis (MASH) progression. One major pathway that limits hepatic cholesterol accumulation is export via VLDL secretion. While sterol regulatory element–binding protein (SREBP) activity is suppressed by insulin-induced gene 1 (INSIG1) under high sterol conditions, VLDL secretion nonetheless persists to prevent lipotoxicity and liver injury, presenting an unresolved paradox in cholesterol sensing and lipoprotein export. Here, we identified a cholesterol-responsive interaction between nuclear factor erythroid 2 related factor-1 (NFE2L1) and INSIG1 that preserved cholesterol homeostasis by sustaining VLDL secretion. Liver-specific NFE2L1 deletion elevated INSIG1 abundance, suppressed SREBP1 activation, and impaired VLDL secretion, leading to hepatic cholesterol accumulation and liver injury. Mechanistically, NFE2L1 bound to INSIG1 via its N-terminal homology box 2 (NHB2) domain; free cholesterol strengthened this interaction to promote INSIG1 degradation, thereby enabling SREBP1 activation and VLDL export. In NFE2L1-deficient mice, WT NFE2L1, but not a mutant NFE2L1 form unable to interact with INSIG1 (NHB2-deleted mutant, ΔNHB2), restored SREBP1 activity and VLDL secretion. Lipidomics analysis revealed that NFE2L1 deficiency reduced serum triglyceride composition, which was restored exclusively by WT NFE2L1. In a murine MASH model, NFE2L1 overexpression activated SREBP1/2, lowered hepatic cholesterol, and attenuated liver injury, inflammation, and fibrosis, without elevating atherogenic lipoproteins owing to compensatory LDL receptor upregulation. Together, these findings explain how VLDL secretion capacity was maintained under cholesterol excess and identify the NFE2L1/INSIG1 axis as a sterol-responsive safeguard for hepatic lipid homeostasis and a potential therapeutic target for MASH.

Discussion

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Cite This Study

Deng et al. (2026) studied Metabolic dysfunction-associated steatohepatitis (MASH) and hepatic cholesterol overload. Hepatic NFE2L1 overexpression (via adenoviral vector AV-NFE2L1) and NFE2L1-WT/ΔNHB2 rescue (via AAV) vs. AV-LacZ control vector, AAV-GFP control, or NFE2L1-ΔNHB2 mutant was evaluated on Hepatic SREBP1 activation, VLDL secretion, and MASH histopathology (CD68+ macrophage infiltration and alpha-SMA+ stellate cell activation) (p=<0.0001). Hepatic NFE2L1 overexpression promoted INSIG1 degradation to sustain SREBP1-mediated VLDL secretion, reducing liver macrophage infiltration by 45% and activated stellate cells by 24% in MASH mice.

synapsesocial.com/papers/6a5875842b46c88ba9ad1508 https://doi.org/10.1172/jci197094

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Cholesterol-responsive NFE2L1-INSIG1 interaction controls VLDL secretion and metabolic dysfunction–associated steatohepatitis pathogenesis in mice

Key Points

Structured PICO

Main Result

Limitations

Ask about this paper

Implication

Abstract

Discussion

Cite This Study

Cholesterol-responsive NFE2L1-INSIG1 interaction controls VLDL secretion and metabolic dysfunction–associated steatohepatitis pathogenesis in mice

Key Points

Structured PICO

Main Result

Limitations

Ask about this paper

Implication

Abstract

Discussion

Cite This Study

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