NAD+ Protects Hepatocytes Against Palmitate-Induced Lipotoxicity by Preventing PARP-1 Inhibition and mTORC1-p300 Pathway Activation

Research Background

Metabolic dysfunction-associated fatty liver disease (MAFLD) encompasses a spectrum of liver diseases ranging from simple steatosis to steatohepatitis, fibrosis/cirrhosis, and even hepatocellular carcinoma. As the hepatic manifestation of metabolic syndrome, MAFLD is closely associated with obesity and insulin resistance, particularly the elevated levels of circulating free fatty acids (FFAs). Saturated fatty acids (SFAs), such as palmitate, exhibit cytotoxicity in hepatocytes, inducing cell death. Thus, palmitate is commonly used as an in vitro model for studying hepatic lipotoxicity. Despite extensive research, the mechanisms underlying palmitate-induced lipotoxicity remain incompletely understood.

In recent years, increasing evidence suggests that intracellular NAD+ (nicotinamide adenine dinucleotide) levels play a crucial role in cellular metabolism and stress responses. PARP-1 (poly(ADP-ribose) polymerase 1), a DNA damage repair enzyme, relies on NAD+ for its activity. Additionally, mTORC1 (mechanistic target of rapamycin complex 1), a central regulator of cell growth and metabolism, also plays a significant role in lipotoxicity. However, how palmitate induces hepatocyte death by modulating the PARP-1 and mTORC1 pathways remains to be further elucidated.

Research Source

This study was conducted by Rui Guo, Yanhui Li, Qing Song, Rong Huang, Xiaodong Ge, Natalia Nieto, Yuwei Jiang, and Zhenyuan Song from institutions including the University of Illinois Chicago and Purdue University. It was published in the journal American Journal of Physiology-Cell Physiology on January 28, 2025, with the DOI: 10.1152/ajpcell.00946.2024.

Research Workflow and Results

1. Palmitate Exposure Suppresses PARP-1 Expression and Activity in Hepatocytes

The study first exposed two hepatocyte lines, AML12 and HepG2, to 0.4 mM palmitate for 16 hours in vitro to examine PARP-1 expression and activity. The results showed that palmitate significantly downregulated PARP-1 at both mRNA and protein levels (Figures 1A, B, D) and inhibited its enzymatic activity, evidenced by reduced levels of poly(ADP-ribosyl)ation (PARylation) proteins (Figures 1C, E). These findings indicate that palmitate may contribute to hepatocyte lipotoxicity by suppressing PARP-1 expression and activity.

2. PARP-1 Inhibition Exacerbates Palmitate-Induced Hepatocyte Death

To further validate the role of PARP-1 in palmitate-induced lipotoxicity, the study pretreated AML12 cells with specific PARP-1 inhibitors PJ34 and Talazoparib before exposing them to palmitate. The results showed that PARP-1 inhibition significantly exacerbated palmitate-induced cell death (Figure 2A) and further reduced PARylation protein levels (Figure 2B). Conversely, pretreatment with the PARG (poly(ADP-ribose) glycohydrolase) inhibitor PDD00017273 alleviated palmitate-induced cell death (Figure 2C) and restored PARylation protein levels (Figure 2D). These results suggest that PARP-1 inhibition plays a critical role in palmitate-induced hepatocyte death.

3. Intracellular NAD+ Depletion Is Associated with PARP-1 Suppression

The study further explored the role of NAD+ in PARP-1 suppression. By pretreating hepatocytes with NAD+ precursors (such as NR and NMN) or NNMT (nicotinamide N-methyltransferase) inhibitors (such as II399 and JBSNF), the researchers successfully increased intracellular NAD+ levels and restored PARP-1 expression and activity (Figures 4A-C). These treatments also significantly mitigated palmitate-induced hepatocyte death (Figures 4D-F). This indicates that restoring NAD+ levels can protect hepatocytes from palmitate-induced lipotoxicity by activating PARP-1.

4. PARP-1 Suppression Mediates Lipotoxicity via Activation of the mTORC1-p300 Pathway

The study found that palmitate exposure activated the mTORC1 pathway, evidenced by increased levels of its downstream target p-S6 protein (Figure 5A). The PARP-1 inhibitor PJ34 further exacerbated palmitate-induced mTORC1 activation (Figure 5B), while the PARG inhibitor PDD00017273 suppressed this process (Figure 5C). Additionally, the NAD+ precursor NR and NNMT inhibitors significantly mitigated palmitate-induced mTORC1 activation (Figures 5B, F). These results suggest that PARP-1 suppression plays a key role in palmitate-induced mTORC1 activation.

Further research revealed that mTORC1 mediates lipotoxicity by activating p300, a histone acetyltransferase. Palmitate exposure significantly increased p300 activity, evidenced by elevated levels of acetylated H3-K27 protein (Figure 6A). The mTORC1 inhibitor Rapamycin suppressed this process (Figure 6A), while the p300 inhibitor C646 or gene knockdown significantly alleviated palmitate-induced hepatocyte death (Figures 6E, F). These results indicate that the mTORC1-p300 pathway plays an important role in palmitate-induced lipotoxicity.

5. TLR4-NF-κB Pathway Mediates Palmitate-Induced PARP-1 Suppression

The study also investigated how palmitate downregulates PARP-1 expression. By pretreating hepatocytes with the TLR4 (Toll-like receptor 4) antagonist CLI-095 and the NF-κB inhibitor Bay11-7082, the researchers found that activation of the TLR4-NF-κB pathway plays a critical role in palmitate-induced PARP-1 suppression (Figures 7B, D). These results suggest that palmitate downregulates PARP-1 expression by activating the TLR4-NF-κB pathway, thereby inducing hepatocyte death.

Research Conclusions and Significance

This study reveals for the first time the critical role of PARP-1 in palmitate-induced hepatocyte lipotoxicity. The findings show that palmitate downregulates PARP-1 expression and activity by activating the TLR4-NF-κB pathway, subsequently activating the mTORC1-p300 pathway, ultimately leading to hepatocyte death. Increasing intracellular NAD+ levels can restore PARP-1 activity, thereby protecting hepatocytes from lipotoxicity. These discoveries provide new molecular mechanisms for understanding the pathogenesis of MAFLD and offer potential therapeutic targets for addressing lipotoxicity.

Research Highlights

1. First Revelation of PARP-1’s Role in Palmitate-Induced Lipotoxicity: The study identifies PARP-1 inhibition as a key mechanism in palmitate-induced hepatocyte death.
2. Protective Effects of NAD+: Increasing intracellular NAD+ levels restores PARP-1 activity, alleviating lipotoxicity.
3. Regulatory Role of the mTORC1-p300 Pathway: The study uncovers a novel mechanism by which mTORC1 mediates lipotoxicity through p300 activation.
4. Involvement of the TLR4-NF-κB Pathway: The study finds that palmitate downregulates PARP-1 expression by activating the TLR4-NF-κB pathway.

Research Value

This study not only deepens our understanding of the pathogenesis of MAFLD but also provides new insights for developing therapeutic strategies targeting lipotoxicity. Increasing intracellular NAD+ levels or inhibiting the mTORC1-p300 pathway may become effective approaches for treating MAFLD. Additionally, the study highlights the critical role of the TLR4-NF-κB pathway in lipotoxicity, offering new directions for future research.