Nutr Res Pract.  2019 Feb;13(1):3-10. 10.4162/nrp.2019.13.1.3.

Nicotinamide riboside regulates inflammation and mitochondrial markers in AML12 hepatocytes

• Lee HJ
• Yang SJ

• KMID: 2434070
• DOI: http://doi.org/10.4162/nrp.2019.13.1.3

Abstract

BACKGROUND/OBJECTIVES
The NAD+ precursor nicotinamide riboside (NR) is a type of vitamin B3 found in cow's milk and yeast-containing food products such as beer. Recent studies suggested that NR prevents hearing loss, high-fat diet-induced obesity, Alzheimer's disease, and mitochondrial myopathy. The objective of this study was to investigate the effects of NR on inflammation and mitochondrial biogenesis in AML12 mouse hepatocytes.
MATERIALS/METHODS
A subset of hepatocytes was treated with palmitic acid (PA; 250 µM) for 48 h to induce hepatocyte steatosis. The hepatocytes were treated with NR (10 µM and 10 mM) for 24 h with and without PA. The cell viability and the levels of sirtuins, inflammatory markers, and mitochondrial markers were analyzed.
RESULTS
Cytotoxicity of NR was examined by PrestoBlue assay. Exposure to NR had no effect on cell viability or morphology. Gene expression of sirtuin 1 (Sirt1) and Sirt3 was significantly upregulated by NR in PA-treated hepatocytes. However, Sirt1 activities were increased in hepatocytes treated with low-dose NR. Hepatic pro-inflammatory markers including tumor necrosis factor-alpha and interleukin-6 were decreased in NR-treated cells. NR upregulated anti-inflammatory molecule adiponectin, and, tended to down-regulate hepatokine fetuin-A in PA-treated hepatocytes, suggesting its inverse regulation on these cytokines. NR increased levels of mitochondrial markers including peroxisome proliferator-activated receptor γ coactivator-1α, carnitine palmitoyltransferase 1, uncoupling protein 2, transcription factor A, mitochondrial and mitochondrial DNA in PA-treated hepatocytes.
CONCLUSIONS
These data demonstrated that NR attenuated hepatic inflammation and increased levels of mitochondrial markers in hepatocytes.

Keyword

Fatty liver; inflammation; mitochondria; niacin

MeSH Terms

Adiponectin
alpha-2-HS-Glycoprotein
Alzheimer Disease
Animals
Beer
Carnitine O-Palmitoyltransferase
Cell Survival
Cytokines
DNA, Mitochondrial
Fatty Liver
Gene Expression
Hearing Loss
Hepatocytes*
Inflammation*
Interleukin-6
Mice
Milk
Mitochondria
Mitochondrial Myopathies
Niacin
Niacinamide*
Obesity
Organelle Biogenesis
Palmitic Acid
Peroxisomes
Sirtuin 1
Sirtuins
Transcription Factors
Tumor Necrosis Factor-alpha
Adiponectin
Carnitine O-Palmitoyltransferase
Cytokines
DNA, Mitochondrial
Interleukin-6
Niacin
Niacinamide
Palmitic Acid
Sirtuin 1
Sirtuins
Transcription Factors
Tumor Necrosis Factor-alpha
alpha-2-HS-Glycoprotein

Figure

• 

  Fig. 1 Effects of nicotinamide riboside (NR) treatment on (A) cell viability, (B) cell morphology, and (C) Oil Red O staining in AML12 mouse hepatocytes. Cell viability was analyzed by PrestoBlue assay. The cells were incubated with the PrestoBlue reagent, and the absorbance was measured using a microplate reader at 570 nm. Data are expressed as Mean±SE. Morphological changes in the cells were captured using a microscope attached with camera (original magnification 200 x). (C) Representative images of Oil Red O staining in AML12 mouse hepatocytes (original magnification 40 x). NR_L, low-dose (10 µM) NR; NR_H, high-dose (10 mM) NR; PA, palmitic acid.

• 

  Fig. 2 Effects of nicotinamide riboside (NR) treatment on (A) sirtuin1 (Sirt1) activity, and the gene expression of Sirt1 (B), Sirt3 (C) and Sirt6 (D) in AML12 mouse hepatocytes. Gene expression of each target was normalized to that of 18S. Representative Western blots (E) and densitometric analysis for Sirt1 (F) and Sirt6 (G). Relative densitometry data of each target were normalized to those of beta-actin. Data are expressed as Mean±SE (n = 5–6 per group). Different letters within a variable are significantly different at P < 0.05. NR_L, low-dose (10 µM) NR; NR_H, high-dose (10 mM) NR; PA, palmitic acid.

• 

  Fig. 3 Effects of NR treatment on inflammation in AML12 mouse hepatocytes. The gene expression of (A) tumor necrosis factor-alpha (TNF-α), (B) interleukin-6 (IL-6), (C) caspase1 (CASP1), (D) peroxisome proliferator-activated receptor-alpha (PPAR-α), (E) adiponectin, (F) fetuin-A, and (G) nuclear factor kappa B (NF-κB) (n = 5–6 per group). Gene expression of each target was normalized to that of 18S. (H) Representative Western blots and (I) densitometric analysis for nucleotide binding and oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome. Relative densitometry data of each target were normalized to those of beta-actin. (J) Protein concentrations of TNF-α in PA-untreated and -treated AML12 mouse hepatocytes analyzed by enzyme-linked immunosorbent assay. Data are expressed as Mean±SE. Different letters within a variable are significantly different at P < 0.05. NR_L, low-dose (10 µM) NR; NR_H, high-dose (10 mM) NR; PA, palmitic acid.

• 

  Fig. 4 Effects of NR treatment on the gene expression levels of (A) peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), (B) carnitine palmitoyltransferase 1 (CPT-1), (C) uncoupling protein 2 (UCP2), (D) transcription factor A, mitochondrial (TFAM), (E) nuclear respiratory factor 1 (Nrf1), and (F) mitochondrial DNA (mtDNA). Gene expression of each target was normalized to that of 18S. Data are expressed as Mean±SE (n = 5–6 per group). Different letters within a variable are significantly different at P < 0.05. NR_L, low-dose (10 µM) NR; NR_H, high-dose (10 mM) NR; PA, palmitic acid.

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