SEARCHCROSS-SECTIONALgastroenterology, hepatology

cross-sectional·gastroenterology, hepatology, endocrinology, nutrition, genetics·PMC10018489

The I148M PNPLA3 variant mitigates niacin beneficial effects: How the genetic screening in non-alcoholic fatty liver disease patients gains value

Frontiers in Nutrition · 10 authors, 6 centres

AI SUMMARY

FIDELITY 100%

POPULATIONNAFLD patients (Discovery cohort n=172, Validation cohort n=358) and hepatoma cell lines (Hep3B, HepG2, siHep3B, HepG2^I148^+^)

INTERVENTIONDietary niacin intake assessment, serum niacin measurement, niacin treatment (0.5 mM) in cell models

COMPARISONPNPLA3 I148M carriers (CG/GG) vs non-carriers (CC); PNPLA3-silenced vs wild-type cells; PNPLA3-overexpressing vs mutant cells

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TL;DR

This study found that NAFLD patients carrying the PNPLA3 I148M genetic variant have lower dietary and circulating niacin (vitamin B3) levels, especially when obese. In liver cells, the loss of functional PNPLA3 protein impaired niacin's ability to reduce fat accumulation and activate protective metabolic pathways. These findings suggest that genetic screening for PNPLA3 could identify NAFLD patients who may benefit most from niacin supplementation or niacin-fortified foods.

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**Background:** Non-alcoholic fatty liver disease (NAFLD) is a highly prevalent chronic liver disorder strongly linked to obesity and type 2 diabetes. The PNPLA3 p.I148M variant (rs738409 C>G) is the strongest genetic predictor of NAFLD, with homozygous GG carriers having a more than 2-fold greater risk of NASH and cirrhosis, up to a 12-fold increased risk for HCC, and an 18-fold increase in liver-related mortality. This variant impairs triglyceride hydrolysis, leading to hepatic fat accumulation. Niacin (vitamin B3) has been shown to reduce triglyceride synthesis in NAFLD models by inhibiting DGAT2 and activating the ERK1/2/AMPK/SIRT1 pathway, which downregulates de novo lipogenesis. Given that nutrients can modulate PNPLA3's effect, this study investigated the interplay between the I148M variant and niacin metabolism in NAFLD patients and hepatoma cells.

**Methods:** The study included a Discovery cohort of 172 NAFLD patients with non-invasive steatosis assessment (ultrasound and FibroScan) and a Validation cohort of 358 biopsy-proven NAFLD patients. Dietary niacin was collected from 3-week food diaries (Discovery cohort only), and serum niacin was quantified by ELISA in both cohorts. A Transcriptomic cohort of 183 severely obese patients (152 with NAFLD) underwent RNA-seq to assess hepatic expression of NAD metabolism genes. In vitro, Hep3B cells (wild-type PNPLA3, 148I/I) and HepG2 cells (homozygous 148M/M) were used. PNPLA3 was silenced in Hep3B (siHep3B) and overexpressed in HepG2 (HepG2^I148^+^). Cells were treated with palmitic acid (0.25 mM) to induce steatosis, with or without niacin (0.5 mM) for 24 hours. NAD content, gene expression (qRT-PCR), protein levels (Western blot), triglyceride content, and lipid droplet formation (Oil Red O) were measured.

**Key Results:** In the Discovery cohort, dietary niacin was significantly lower in patients with steatosis ≥2 (p<0.05) and in PNPLA3 I148M carriers (CG/GG vs CC, p=0.04). Serum niacin was also lower in I148M carriers (p<0.05) and negatively correlated with BMI (p<0.01). The lowest serum niacin levels were observed in obese (BMI≥30) I148M carriers (p=0.005 at ANOVA). Multivariate analysis confirmed that the PNPLA3 G allele was independently associated with lower serum niacin in obese individuals (β=-0.21, 95% CI: -0.41 to 0.004, p=0.04). These findings were validated in the larger Validation cohort (n=358), where serum niacin inversely correlated with BMI (p<0.0001) and was lowest in obese I148M carriers (p=0.0001 at ANOVA). Multivariate analysis again showed a significant association between the G allele and lower serum niacin in obese patients (β=-0.24, 95% CI: -0.43 to -0.06, p=0.009). In the Transcriptomic cohort, hepatic NAPRT1 (the key enzyme for niacin conversion to NAD) was lower in I148M carriers (p<0.01), while alternative pathway genes NADSYN1 and NMNAT1 were upregulated (p<0.05). In vitro, Hep3B cells had higher NAD content than siHep3B or HepG2 cells. Niacin increased NAD only in Hep3B and HepG2^I148^+^ cells (p<0.05), not in siHep3B or HepG2. NAPRT1 expression was induced by niacin only in Hep3B and HepG2^I148^+^ cells. Niacin reduced triglyceride content and DGAT2 expression in all models, but the effect was blunted in siHep3B and HepG2 cells. Niacin activated ERK1/2 and AMPK phosphorylation and upregulated SIRT1 only in Hep3B and HepG2^I148^+^ cells, leading to downregulation of SREBP1, ACC, FASn, and PNPLA3 (p<0.05). These effects were absent or mild in siHep3B and HepG2 cells.

**Clinical Implications:** This study demonstrates a novel gene-nutrient interaction where the PNPLA3 I148M variant impairs niacin availability and metabolism, reducing its beneficial effects on hepatic steatosis. The findings suggest that genetic screening for PNPLA3 could identify NAFLD patients who may have lower niacin levels and a diminished response to dietary niacin. For these genetically predisposed individuals, especially those with obesity, niacin supplementation or consumption of niacin-fortified foods may be a valuable personalized nutritional strategy to improve NAFLD outcomes. The study highlights the importance of considering genetic background when designing dietary interventions for NAFLD.

PICO

PPOPULATION

NAFLD patients (Discovery cohort n=172, Validation cohort n=358) and hepatoma cell lines (Hep3B, HepG2, siHep3B, HepG2^I148^+^)

IINTERVENTION

Dietary niacin intake assessment, serum niacin measurement, niacin treatment (0.5 mM) in cell models

OOUTCOME

Dietary and serum niacin levels; hepatic NAD metabolism gene expression; intracellular triglyceride content; DGAT2, SREBP1, PNPLA3 expression; ERK1/2/AMPK/SIRT1 pathway activation

STUDY TYPE

cross-sectional

SPECIALTY

gastroenterology

SUMMARISED BY

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FIDELITY CHECK

100% · A

ORIGINAL PAPER

PMC10018489

The I148M PNPLA3 variant mitigates niacin beneficial effects: How the genetic screening in non-alcoholic fatty liver disease patients gains value

Frontiers in Nutrition

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ABSTRACT

Background The PNPLA3 p.I148M impact on fat accumulation can be modulated by nutrients. Niacin (Vitamin B3) reduced triglycerides synthesis in in vitro and in vivo NAFLD models. Objectives In this study, we aimed to investigate the niacin-I148M polymorphism crosstalk in NAFLD patients and examine niacin’s beneficial effect in reducing fat by exploiting hepatoma cells with different PNPLA3 genotype. Design We enrolled 172 (Discovery cohort) and 358 (Validation cohort) patients with non-invasive and histological diagnosis of NAFLD, respectively. Dietary niacin was collected from food diary, while its serum levels were quantified by ELISA. Hepatic expression of genes related to NAD metabolism was evaluated by RNAseq in bariatric NAFLD patients ( n = 183; Transcriptomic cohort). Hep3B (148I/I) and HepG2 (148M/M) cells were silenced (siHep3B) or overexpressed (HepG2 I148 + ) for PNPLA3 , respectively. Results In the Discovery cohort, dietary niacin was significantly reduced in patients with steatosis ≥ 2 and in I148M carriers. Serum niacin was lower in subjects carrying the G at risk allele and negatively correlated with obesity. The latter result was confirmed in the Validation cohort. At multivariate analysis, the I148M polymorphism was independently associated with serum niacin, supporting that it may be directly involved in the modulation of its availability. siHep3B cells showed an impaired NAD biosynthesis comparable to HepG2 cells which led to lower niacin efficacy in clearing fat, supporting a required functional protein to guarantee its effectiveness. Conversely, the restoration of PNPLA3 Wt protein in HepG2 I148 + cells recovered the NAD pathway and improved niacin efficacy. Finally, niacin inhibited de novo lipogenesis through the ERK1/2/AMPK/SIRT1 pathway, with the consequent SREBP1 -driven PNPLA3 reduction only in Hep3B and HepG2 I148M+ cells. Conclusions We demonstrated a niacin-PNPLA3 I148M interaction in NAFLD patients which possibly pave the way to vitamin B3 supplementation in those with a predisposing genetic background.

CITATION EXPORT

Erika Paolini, Miriam Longo, Marica Meroni et al.. The I148M PNPLA3 variant mitigates niacin beneficial effects: How the genetic screening in non-alcoholic fatty liver disease patients gains value. Frontiers in Nutrition. (2023). https://doi.org/10.3389/fnut.2023.1101341

AUTHORS

10 · 6 CENTRES

Erika Paolini, Miriam Longo, Marica Meroni, Giada Tria, Annalisa Cespiati, Rosa Lombardi, Sara Badiali, Marco Maggioni, Anna Ludovica Fracanzani, Paola Dongiovanni

1General Medicine and Metabolic Diseases, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy
2Department of Pharmacological and Biomolecular Sciences, Università Degli Studi di Milano, Milan, Italy
3Department of Clinical Sciences and Community Health, Università Degli Studi di Milano, Milan, Italy
4Department of Pathophysiology and Transplantation, Università Degli Studi di Milano, Milan, Italy
5Department of Surgery, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy
6Department of Pathology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy