Nicotinamide mononucleotide attenuates HIF-1α activation and fibrosis in hypoxic adipose tissue via NAD+/SIRT1 axis

Abstract

Background: Fibrosis is increasingly considered as a major contributor in adipose tissue dysfunction. Hypoxic activation of hypoxia-inducible factor 1α (HIF-1α) induces a profibrotic transcription, leading to adipose fibrosis. Nicotinamide mononucleotide (NMN), a member of the vitamin B₃ family, has been shown to relieve hepatic and cardiac fibrosis, but its effects on hypoxic adipose fibrosis and the underlying mechanism remain unclear. We aimed to elucidate the roles of NMN in regulating HIF-1α and fibrosis in hypoxic adipose tissue.

Methods: Mice were placed in a hypobaric chamber for four weeks to induce adipose fibrosis. NMN (500 mg/kg, every three days) was administered by intraperitoneal injection. In vitro, Stromal vascular fractions (SVF) cells were treated by hypoxia with or without NMN (200μM), sirtinol (25μM, a SIRT1 inhibitor) and CoCl₂ (100μM, a HIF1α enhancer). The effects of NMN on hypoxia-associated adipose fibrosis, inflammation, NAD⁺/SIRT1 axis alteration, and HIF-1α activation were evaluated by real-time polymerase chain reaction (PCR), western blots, immunohistochemistry staining, immunoprecipitation, and assay kits.

Results: Mice placed in a hypoxic chamber for four weeks showed obvious adipose fibrosis and inflammation, which were attenuated by NMN. NMN also restore the compromised NAD⁺/SIRT1 axis and inhibited the activation of HIF-1α induced by hypoxia. In hypoxia-induced SVFs, the SIRT1 inhibitor sirtinol blocked the anti-fibrotic and anti-inflammatory effects of NMN, upregulated the HIF-1α and its acetylation level. The HIF1α stabilizer CoCl₂ showed similar effects as sirtinol.

Conclusion: NMN effectively attenuated HIF-1α activation-induced adipose fibrosis and inflammation by restoring the compromised NAD⁺/SIRT1 axis.

Keywords: HIF-1α activation; NAD +/SIRT1 axis; adipose tissue fibrosis; inflammation; nicotinamide mononucleotide.

Figure 1

NMN alleviates the abnormal degradation and synthesis of ECM components in the eWAT of hypoxia-induced mice. (A, B) Representative images of eWAT adipocytes morphology and fibrosis as reflected by the H&E staining and Masson staining (400×magnification, scale bar=50 μm). (C) Statistical result for the interstitial fibrosis of eWAT (n = 6). (D) The relative mRNA levels of Col1a1, Col3a1, MMP-2, MMP-9, TIMP-1, LOX, and FN normalized to β-actin in eWAT (n = 3). (E, F) The relative protein levels of MMP9 and TIMP1 normalized to β-actin in eWAT (n = 5). *P < 0.05 versus control group, ^# P < 0.05 versus hypoxia group.

Figure 2

NMN alleviates dysregulated adipokines secretion and macrophage infiltration in the eWAT of hypoxia induced mice. (A, B) The relative protein levels of APN, TGFβ, and IL-6 normalized to β-actin in eWAT (n = 5). (C) The relative mRNA levels of APN, leptin, ang, resistin, IL-6, and TGF-β normalized to β-actin in eWAT (n = 6). (D) Representative images of macrophage marker F4/80 in eWAT as reflected by the IHC staining (400 × magnification, scale bar = 50μm). (E) The relative mRNA levels of F4/80 in eWAT (n = 6). (F, G) The relative mRNA levels of genes encoding TNFα, iNOS, Arg1 and Ym1 in eWAT (n = 6). *P < 0.05 versus control group, ^# P < 0.05 versus hypoxia group.

Figure 3

NMN restores NAMPT/NAD+/SIRT1 axis and inhibited HIF-1α acetylation in eWAT of hypoxia induced mice. (A) The relative protein levels of NAMPT normalized to β-actin in eWAT (n = 5). (B, C) Statistical results for NAD⁺/NADH contents and NAD⁺/NADH ratio in eWAT (n = 6). (D) Representative images of immunohistochemical staining for the SIRT1 protein in eWAT sections (400× magnification, scale bar=50μm). (E–G) The relative protein levels of SIRT1 and HIF1α in eWAT (n = 5). *P < 0.05 versus control group, ^# P < 0.05 versus hypoxia group. NS indicates no significant difference compared with the matched group.

Figure 4

NMN restores NAMPT/NAD+/SIRT1 axis and inhibited HIF-1α acetylation in SVF cells. (A) The relative protein levels of NAMPT normalized to β-actin in SVF cells (n = 3). (B, C) Statistical results for NAD⁺/NADH contents and NAD⁺/NADH ratio in SVF cells (n = 3). (D–F) The relative protein levels of SIRT1 and HIF1α in SVF cells (n = 3). *P < 0.05 versus control group, ^# P < 0.05 versus hypoxia group. NS indicates no significant difference compared with the matched group.

Figure 5

NMN suppresses HIF-1α signalling-associated upregulation of fibrogenic and inflammatory gene in a SIRT1-dependent manner. (A–F) The relative mRNA levels of Col1α, Fibronectin, IL-6, TGF-β, MIF and TNF-α normalized to β-actin in SVF cells (n = 3). *P < 0.05 versus control group, ^# P < 0.05 versus hypoxia group.

Figure 6

The proposed mechanisms for the protective role of NMN in hypoxia-induced adipose tissue remodeling. Hypoxia led to compromised NAMPT/NAD⁺/SIRT1 axis, which further promoted HIF1α activation. As a result, the fibrotic and inflammatory response are significantly increased, accompanied by adipocytokine dysregulation, which ultimately lead to adipose tissue remodeling. NMN restored the NAMPT/NAD⁺/SIRT1 axis and inhibited HIF1α activation, which further attenuated adipose tissue remodeling.