Role of Mammalian Target of Rapamycin Signaling Pathway in Regulation of Fatty Acid Oxidation in a Preeclampsia-Like Mouse Model Treated with Pravastatin

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Role of Mammalian Target of Rapamycin Signaling Pathway in Regulation of Fatty Acid Oxidation in a Preeclampsia-Like Mouse Model Treated with Pravastatin

Preeclampsia (PE) is a severe pregnancy complication characterized by hypertension and systemic organ dysfunction, contributing to significant maternal and fetal morbidity and mortality. Despite its clinical importance, the molecular mechanisms underlying PE remain poorly understood. Recent evidence suggests that dysregulation of fatty acid oxidation (FAO) plays a critical role in PE pathogenesis. In this context, the mammalian target of rapamycin (mTOR) signaling pathway has emerged as a key regulator of cellular metabolism, including lipid homeostasis. This study investigates the interplay between mTOR signaling and FAO in a PE-like mouse model induced by Nv-nitro-L-arginine methyl ester (L-NAME) and explores the therapeutic potential of pravastatin (Pra), a statin with pleiotropic effects beyond lipid lowering.

Background and Rationale

PE is associated with metabolic disturbances, including elevated serum free fatty acids (FFA) and lipid deposition in maternal organs such as the liver and placenta. Long-chain 3-hydroxyacyl-coenzyme A dehydrogenase (LCHAD), a rate-limiting enzyme in mitochondrial FAO, is downregulated in severe early-onset PE, correlating with elevated FFA levels. mTOR, a serine/threonine kinase, integrates nutrient and energy signals to regulate cell growth, proliferation, and metabolism. Dysregulated mTOR activity has been implicated in gestational diabetes, fetal growth restriction, and PE. Previous studies demonstrated that mTOR inhibition with rapamycin reduces FFA levels and lipid accumulation in PE models, suggesting mTOR’s role in lipid metabolism.

Pravastatin, an HMG-CoA reductase inhibitor, ameliorates endothelial dysfunction, oxidative stress, and angiogenic imbalances in PE. In L-NAME-induced PE-like mice, Pra alleviates hypertension, proteinuria, and FAO defects by upregulating LCHAD and reducing FFA. However, the molecular mechanisms linking Pra’s effects to mTOR signaling remained unexplored. This study fills this gap by examining mTOR pathway activation in PE-like mice treated with Pra.

Experimental Design and Methods

Animal Model and Treatment

Pregnant C57BL/6J mice were divided into four groups (n=8/group):

  1. Control+NS: Normal saline injections from gestational day (GD) 7–18.
  2. Control+Pra: Pra (5 mg/kg/day) via intragastric gavage from GD8–18.
  3. L-NAME+NS: L-NAME (50 mg/kg/day, GD7–18) to induce PE-like symptoms.
  4. L-NAME+Pra: L-NAME + Pra (GD8–18).

Mice were sacrificed on GD18. Maternal liver and placental tissues were collected for molecular analyses. Serum FFA levels, LCHAD expression, and mTOR pathway activity were evaluated.

Molecular Analyses

  • Western Blot: Quantified protein levels of mTOR, phosphorylated (p)-mTOR (Ser2448), and downstream substrates (S6K1, p-S6K1 Thr389/Ser371, 4EBP-1, p-4EBP1).
  • qPCR: Assessed mTOR mRNA expression using β-actin as a reference.
  • Immunohistochemistry (IHC): Localized mTOR and p-mTOR in liver and placenta.
  • Statistical Analysis: Data expressed as mean ± SD. Differences analyzed via t-test or ANOVA. Pearson correlation tested relationships between mTOR activation, LCHAD, and FFA.

Key Findings

mTOR Signaling Activation in PE-Like Mice

  • Western Blot:

    • Liver: p-mTOR/mTOR ratio increased in L-NAME+NS (0.85 ± 0.06) vs. Control+NS (0.53 ± 0.09; P<0.05). Pra reduced this ratio in L-NAME+Pra (0.74 ± 0.08 vs. L-NAME+NS; P<0.05).
    • Placenta: Similar activation patterns: p-mTOR/mTOR ratio rose in L-NAME+NS (0.77 ± 0.06 vs. Control+NS 0.57 ± 0.07; P<0.05) and declined with Pra (0.63 ± 0.06; P<0.05).

  • IHC:

    • p-mTOR localized to hepatocyte membranes and placental trophoblasts. Optical density ratios (p-mTOR/mTOR) confirmed increased activation in L-NAME+NS (liver: 1.10 ± 0.13; placenta: 1.79 ± 0.09) vs. controls. Pra normalized these ratios (liver: 0.93 ± 0.09; placenta: 1.64 ± 0.13).

  • mTOR mRNA: No significant differences across groups, indicating post-translational regulation.

Downstream Substrate Activation

  • S6K1 Phosphorylation:

    • Thr389 (Liver): Elevated in L-NAME+NS (0.69 ± 0.11 vs. Control+NS 0.42 ± 0.07; P<0.05), reduced by Pra (0.59 ± 0.08).
    • Ser371 (Liver): Increased in L-NAME+NS (1.16 ± 0.10 vs. 0.94 ± 0.15; P<0.05), normalized by Pra (0.90 ± 0.13).
    • Placental S6K1 followed similar trends but with weaker significance.

  • 4EBP-1 Phosphorylation:

    • Reduced in L-NAME+NS (liver: 0.62 ± 0.11; placenta: 0.98 ± 0.22 vs. Control+NS 0.89 ± 0.09 and 1.36 ± 0.07; P<0.05). Pra partially restored phosphorylation (liver: 0.79 ± 0.11; placenta: 1.20 ± 0.13).

Correlation Between mTOR Activation and FAO Markers

  • L-NAME+NS Group:

    • mTOR activation (p-mTOR/mTOR) inversely correlated with LCHAD in liver (r=−0.745, P<0.05) and placenta (r=−0.833, P<0.05).
    • No significant correlation with serum FFA.

  • L-NAME+Pra Group:

    • Liver mTOR activation negatively correlated with LCHAD (r=−0.733, P<0.05) and positively with FFA (r=0.841, P<0.05).
    • Placental correlations were non-significant.

Mechanistic Insights and Implications

mTOR Overactivation in PE Pathogenesis

The study demonstrates hyperactivation of mTOR signaling in L-NAME-induced PE-like mice, consistent with clinical observations in severe PE. Elevated p-mTOR and downstream S6K1 phosphorylation suggest enhanced anabolic signaling, potentially driving lipid synthesis over oxidation. Reduced LCHAD and elevated FFA in PE models align with impaired FAO, creating a lipid-rich milieu that exacerbates oxidative stress and endothelial dysfunction.

Pravastatin’s Dual Role: Lipid Lowering and mTOR Modulation

Pra’s beneficial effects extend beyond cholesterol reduction. By inhibiting HMG-CoA reductase, Pra depletes isoprenoid intermediates required for Rheb GTPase activation, a critical mTORC1 regulator. This mechanism explains the observed suppression of mTOR activity in L-NAME+Pra mice. Restored LCHAD expression and reduced FFA levels correlate with normalized mTOR signaling, highlighting Pra’s ability to rebalance lipid metabolism.

Tissue-Specific Responses

Notably, Pra’s effects were more pronounced in maternal liver than placenta. The liver’s central role in systemic lipid metabolism may render it more responsive to mTOR modulation. Placental mTOR, crucial for nutrient transport, exhibited partial normalization, suggesting tissue-specific regulatory mechanisms.

Clinical Relevance and Future Directions

This study provides preclinical evidence supporting mTOR inhibition as a therapeutic strategy for PE. Pravastatin’s ability to improve FAO and mitigate PE-like symptoms positions it as a candidate for repurposing in PE prevention. Future studies should validate these findings in human placental models and explore combinatorial therapies targeting mTOR and FAO pathways.

Conclusion

In L-NAME-induced PE-like mice, aberrant mTOR signaling contributes to FAO dysfunction, evidenced by reduced LCHAD and elevated FFA. Pravastatin ameliorates these defects by suppressing mTOR hyperactivation, restoring lipid homeostasis, and alleviating disease manifestations. These findings deepen our understanding of PE’s metabolic underpinnings and highlight mTOR as a therapeutic target.

doi.org/10.1097/CM9.0000000000000129

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