PPARγ regulates PNPLA3 in adipocytes in vitro and in vivo in healthy obese patients and mice with metabolic dysfunction-associated steatohepatitis

The mechanisms regulating the patatin-like phospholipase domain-containing protein 3 (PNPLA3) are currently unclear. We uncovered that peroxisome proliferator-activated receptor gamma (PPARγ) transcriptionally regulates PNPLA3 in vitro and in vivo in humans and diabetic and hyperlipidaemic mice. We observed increased PNPLA3 mRNA transcripts in the subcutaneous femoral but not in the abdominal fat of healthy women with obesity who received 30 mg/day of pioglitazone over 16 weeks in “The Apple & Pear trial (“Cellular Dynamics of Subcutaneous Fat Distribution in Obese Women”; ClinicalTrials.gov ID- NCT01748994)”. Likewise, pioglitazone treatment upregulated Pnpla3 in adipose tissue but not the liver of the STAM mice with metabolic dysfunction-associated steatohepatitis. Since pioglitazone has a moderate effect on human PPARγ, we used the most potent PPARγ agonist, rosiglitazone, to show that PPARγ regulation of PNPLA3 involves a direct transcriptional mechanism. Mechanistically, we further interrogated the interaction between PNPLA3 and PPARγ using in silico analysis, luciferase reporter assay, gene expression analysis, and gel shift assays. Rosiglitazone increased luciferase activity in PNPLA3 and PPARγ co-transfected cells, demonstrating PPARγ-mediated induction of PNPLA3 promoter activity. Unexpectedly, we identified two binding sites (PPREs), bp -713 to -707 and bp -790 to -780, in the PNPLA3 promoter recognized by PPARγ. Mutation in these newly identified sites abolishes the PPARγ-mediated induction and binding activities. Collectively, these results establish a novel role of PPARγ in PNPLA3 gene regulation.