Statin-associated regulation of hepatic PNPLA3 in patients without known liver disease

Abstract

Background and objectives: Statins are used for metabolic dysfunction-associated steatotic liver disease (MASLD) (NAFLD) treatment, but their role in this context is unclear. Genetic variants of patatin-like phospholipase domain containing 3 (PNPLA3) are associated with MASLD susceptibility and statin treatment efficacy. Access to liver biopsies before established MASLD is limited, and statins and PNPLA3 in early liver steatosis are thus difficult to study.

Methods: Liver biopsies were collected from 261 patients without known liver disease at surgery and stratified based on statin use and criteria for the metabolic syndrome (MS). Genotypes and transcript levels were measured using Illumina and Affymetrix arrays, and metabolic and lipoprotein profiles by clinical assays. Statin effects on PNPLA3, de novo lipogenesis (DNL), and lipid accumulation were further studied in vitro.

Results: The PNPLA3^I148M genetic variant was associated with significantly lower hepatic levels of cholesterol synthesis-associated transcripts. Patients with MS had significantly higher hepatic levels of MASLD and lipogenesis-associated transcripts than non-MS patients. Patients with MS on statin therapy had significantly higher hepatic levels of PNPLA3, acetyl-CoA carboxylase alpha, and ATP citrate lyase, and statin use was associated with higher plasma fasting glucose, insulin, and HbA1c. Exposure of hepatocyte-like HepG2 cells to atorvastatin promoted intracellular accumulation of triglycerides and lipogenesis-associated transcripts. Atorvastatin-exposure of HepG2, sterol O-acyltransferase (SOAT) 2-only-HepG2, primary human hepatic stellate, and hepatic stellate cell-like LX2 cells significantly increased levels of PNPLA3 and SREBF2-target genes, whereas knockdown of SREBF2 attenuated this effect.

Conclusions: Collectively, these observations suggest statin-associated regulation of PNPLA3 and DNL in liver. The potential interaction between PNPLA3 genotype and metabolic status should be considered in future studies in the context of statin therapy for MASLD.

Keywords: MASLD; NAFLD; lipogenesis; metabolic syndrome.

Fig. 1

Inclusion and stratification of patients.

Fig. 2

PNPLA3^I148M was associated with altered hepatic levels of metabolic dysfunction–associated steatotic liver disease (MASLD)‐associated and cholesterol synthesis–associated transcripts. Liver biopsy homogenates from individuals without known liver disease were analyzed using human transcriptome array and grouped by genotype for the PNPLA3^I148M. (a and b) Transcript levels of MASLD associated genes patatin‐like phospholipase domain containing 3(PNPLA3), transmembrane 6 superfamily member 2 (TM6SF2), glucokinase regulator (GCKR), and membrane bound O‐acyltransferase domain containing 7 (MBOAT7) (c) Transcript levels of cholesterol synthesis associated genes, in patients without statin therapy, 3‐hydroxy‐3‐methylglutaryl‐CoA synthase (HMGCS), 3‐hydroxy‐3‐methylglutaryl‐CoA reductase (HMGCR) and (d) SREBF2. 148II—homozygote for the enzymatically active allele, 148I/M—heterozygote, and 148MM—homozygote for the enzymatically inactive alleles. Data are expressed as min to max box plots with middle line at median, each dot represents one patient. One‐way ANOVA, Fisher's least significant difference (LSD) test. n.s., not significant. *p < 0.05 and **p < 0.01 (n = 15–83/group).

Fig. 3

Hepatic levels of metabolic dysfunction–associated steatotic liver disease (MASLD)‐ and de novo lipogenesis (DNL)‐associated transcripts were higher in human metabolic syndrome patients. Liver biopsy homogenates from individuals without known liver disease and without statin therapy were analyzed using human transcriptome array and grouped by meeting criteria for the metabolic syndrome (MS) or not (non‐MS). (a and b) Transcript levels of MASLD associated genes patatin‐like phospholipase domain containing 3 (PNPLA3), transmembrane 6 superfamily member 2 (TM6SF2), glucokinase regulator (GCKR), and membrane bound O‐acyltransferase domain containing 7 (MBOAT7). (c) Transcript levels of key enzymes involved in DNL ATP citrate lyase (ACLY), acetyl‐CoA carboxylase alpha (ACACA), ACACB, stearoyl‐CoA desaturase (SCD), and FASN. (d) Transcript levels of SREBF1 transcriptional regulator of DNL associated genes. Data are expressed as min to max box plots with middle line at median, each dot represents one patient. Mann Whitney U test. n.s., not significant. *p < 0.05, **p < 0.01, and ***p < 0.001 (n = 26–115/group).

Fig. 4

Hepatic expression of metabolic dysfunction‐associated steatotic liver disease (MASLD)‐ and de novo lipogenesis (DNL)‐associated genes in patients with metabolic syndrome was elevated in the statin‐treated group. Liver biopsy homogenates from individuals without known liver disease were analyzed using human transcriptome array. (a and b) Transcript levels of MASLD associated genes patatin‐like phospholipase domain containing 3 (PNPLA3), transmembrane 6 superfamily member 2 (TM6SF2), glucokinase regulator (GCKR), and membrane bound O‐acyltransferase domain containing 7 (MBOAT7). (c) Transcript levels of key regulators of hepatic cholesterol metabolism (SREBF2, 3‐hydroxy‐3‐methylglutaryl‐CoA synthase (HMGCS1), 3‐hydroxy‐3‐methylglutaryl‐coa reductase (HMGCR), low‐density lipoprotein receptor (LDLR), and PCSK9) in metabolic syndrome (MS) patients with or without statin treatment. (d and e) Transcript levels of transcriptionally regulated key enzymes involved in DNL ATP citrate lyase (ACLY), acetyl‐CoA carboxylase alpha (ACACA), ACACB, stearoyl‐CoA desaturase (SCD), FASN, and SREBF1. Data are expressed as min to max box plots with the middle line at the median, each dot representing one patient. Mann Whitney U test. n.s., not significant. *p < 0.05, **p < 0.01, ****p < 0.0001 (n = 24–26/group).

Fig. 5

Statin treatment associated with hyperglycemic signs in patients with metabolic syndrome. (a) Plasma levels of fasting blood glucose (n = 57–115 and 24–26/treatment group, insulin (n = 39–70 and 13–17/treatment group) and HbA1c (n = 51–95 and 22–24/treatment group) in metabolic and non‐metabolic syndrome patients treated with (blue dots) or without (gray dots) statins. Data are expressed as min to max box plots, and each dot represents one patient. One‐way ANOVA with Šídák's multiple comparisons test. (b) Plasma levels of fasting blood glucose (n = 15–26/treatment group), insulin (n = 9–17/treatment group) and HbA1c (n = 14–24/treatment group) in metabolic syndrome (MS) patients, after exclusion of individuals diagnosed with diabetes, treated with or without statins. Data are expressed as min to max box plots, and each dot represents one patient. Student's t test. n.s., not significant. *p < 0.05.

Fig. 6

Statin regulation of patatin‐like phospholipase domain containing 3 (PNPLA3) in liver cells required SREBF2. (a and b) HepG2 was exposed to atorvastatin (Atrov.) or vehicle dimethylsulfoxide (DMSO), with and without SREBF2 knockdown, and the mRNA levels of SREBF2, 3‐hydroxy‐3‐methylglutaryl‐CoA reductase (HMGCR), PCSK9, low‐density lipoprotein receptor (LDLR), and PNPLA3 were quantified using qPCR. Data are expressed as mean ± SEM. One‐way ANOVA, Fisher's least significant difference (LSD) post hoc test. n.s., not significant; siC, scrambled siRNA; siSR, SREBF2 siRNA. **p < 0.01, ***p < 0.001, and ****p < 0.0001 (n = 10).

Fig. 7

Statin regulation of patatin‐like phospholipase domain containing 3 (PNPLA3) in primary human HSC required SREBF2. (a and b) Primary human hepatic stellate cells (HSC) were exposed to atorvastatin (Atrov.) or vehicle dimethylsulfoxide (DMSO), with and without SREBF2 knockdown, and the mRNA levels of SREBF2, 3‐hydroxy‐3‐methylglutaryl‐CoA reductase (HMGCR), PCSK9, low‐density lipoprotein receptor (LDLR), and PNPLA3 were quantified using qPCR. Data are expressed as mean ± SEM. One‐way ANOVA, Fisher's least significant difference (LSD) post hoc test. n.s., not significant; siC, scrambled siRNA; siSR, SREBF2 siRNA. **p < 0.01, ***p < 0.001, and ****p < 0.0001 (n = 3–4).

Fig. 8

Statin regulation of patatin‐like phospholipase domain containing 3 (PNPLA3) in sterol O‐acyltransferase (SOAT) 2‐only‐HepG2 cells involved SREBF2. (a and b) SOAT2‐only‐HepG2 cells were exposed to atorvastatin (Atrov.) or vehicle (DMSO), with and without SREBF2 knockdown, and the mRNA levels of SREBF2, 3‐hydroxy‐3‐methylglutaryl‐CoA reductase (HMGCR), PCSK9, low‐density lipoprotein receptor (LDLR), and PNPLA3 were quantified using qPCR. Data are expressed as mean ± SEM. One‐way ANOVA, Fisher's least significant difference (LSD) post hoc test. n.s., not significant; siC, scrambled siRNA; siSR, SREBF2 siRNA. **p < 0.01, ***p < 0.001, and ****p < 0.0001 (n = 3–4).