Uncovering the roles of dihydropyrimidine dehydrogenase in fatty-acid induced steatosis using human cellular models

www.nature.com/scientificreports OPEN Uncovering the roles of dihydropyrimidine dehydrogenase in fatty‑acid induced steatosis using human cellular models Kelly E. Sullivan1,3, Sheetal Kumar1,4, Xin Liu1,5, Ye Zhang1,5, Emily de Koning1,6, Yanfei Li2, Jing Yuan1,7 & Fan Fan1,8* Pyrimidine catabolism is implicated in hepatic steatosis. Dihydropyrimidine dehydrogenase (DPYD) is an enzyme responsible for uracil and thymine catabolism, and DPYD human genetic variability affects clinically observed toxicity following 5-Fluorouracil administration. In an in vitro model of fatty acid-induced steatosis, the pharmacologic inhibition of DPYD resulted in protection from lipid accumulation. Additionally, a gain-of-function mutation of DPYD, created through clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR-Cas9) engineering, led to an increased lipid burden, which was associated with altered mitochondrial functionality in a hepatocarcionma cell line. The studies presented herein describe a novel role for DPYD in hepatocyte metabolic regulation as a modulator of hepatic steatosis. Abbreviations 5-FU 5-Fluorouracil CRISPR-Cas9 Clustered regularly interspaced short palindromic repeats associated protein 9 DPYD Dihydropyrimidine dehydrogenase FASN Fatty acid synthase FXR Farsenoid X receptor G0S2 G0/G1 Switch Gene 2 HMGCS1 3-Hydroxy-3-Methylglutaryl-CoA Synthase 1 HSD17B13 Hepatic lipid droplet protein hydroxysteroid 17-beta dehydrogenase 13 MID1IP1 MID1 interacting protein 1 NAFLD Nonalcoholic fatty liver disease NASH Non-alcoholic steatohepatitis PHH Primary human hepatocyte PNPLA3 Patatin Like Phospholipase Domain Containing 3 TXNIP Thioredoxin interacting protein 1 UPP1 Uridine phosphorylase-1 Hepatic steatosis results from dysregulated energy homeostasis in the liver and an accumulation of lipids in the parenchyma1. Hepatic steatosis, or fatty liver, is the first stage of the nonalcoholic fatty liver disease (NAFLD) spectrum. Some individuals with steatosis may progress to non-alcoholic steatohepatitis (NASH), the stage at which excessive levels of triglycerides become cytotoxic and trigger an inflammatory and subsequent fibrotic response2. Non-alcoholic fatty acid disease progression impairs normal hepatic function and is quickly becoming 1 Translational Systems Biology Group, Amgen Inc., Cambridge, MA 02141, USA. 2Amgen Inc., South San Francisco, CA 90408, USA. 3Present address: Vertex Pharmaceuticals, Boston, MA 02210, USA. 4Present address: Nimbus Therapeutics, Cambridge, MA 02139, USA. 5Present address: Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA. 6Present address: Amgen Inc., Thousand Oaks, CA 91320, USA. 7Present address: Pfizer Inc., Cambridge, MA 02139, USA. 8Present address: Janssen Pharmaceutical Companies of Johnson & Johnson, La Jolla, CA 92037, USA. *email: Scientific Reports | (2022) 12:14109 | https://doi.org/10.1038/s41598-022-17860-2 1 Vol.:(0123456789) www.nature.com/scientificreports/ a global epidemic with nearly 30% of the population impacted3. NASH is a precursor stage to cirrhosis, and cirrhosis from fatty liver is increasingly becoming the leading reason for liver transplantation globally4. Currently there are no approved therapies for NASH or cirrhosis. Identifying novel protein targets of hepatic lipid accumulation is key to understanding and preventing disease p athogenesis5. Pyrimidine catabolism has been implicated in liver lipid homeostasis. Transgenic mouse models identify the enzyme, uridine phosphorylase-1 (UPP1), as a key driver of pyrimidine catabolism and the salvage pathway, and a modulator of hepatic microvesicular s teatosis6. An intermediate of pyrimidine catabolism, dihydrothymine, has been identified as a metabolic signature associated with pediatric N AFLD7. Daily uridine supplementation for two weeks improved liver lipid metabolism and weight loss in mice fed a high fat diet8 and reduced drug-induced liver lipid accumulation in mice following exposure to hepatotoxic drugs such as t amoxifen9, zalcitabine10 and fenofibrate11. However, chronic uridine administration for 16 weeks induced fatty liver and glucose intolerance in mice12. In addition, plasma uridine levels are regulated by fasting and feeding cycles in mice in an adipocyte dependent manner and uridine levels can directly influence energy homeostasis and thermoregulation13. These results suggest pyrimidine catabolism regulates metabolism and sensitive pharmacologic control of uridine levels may be a promising approach to treating metabolic disorders. Uridine catabolism is accomplished through its reversible conversion to uracil via the UPP1 enzyme6. The enzyme dihydropyrimidine dehydrogenase (DPYD) is the rate limiting enzyme in uracil catabolism and therefore a major regulator of uracil and uridine levels in the l iver14. DPYD expression levels are controlled in part by the farsenoid X receptor (FXR)15, a transcription factor known to regulate hepatic energy homeostasis. A high degree of genetic variability in DPYD has been highlighted in the human population through the development of the chemotherapeutic 5-Fluorouracil (5-FU) for the treatment of colorectal cancer. The majority of 5-FU is metabolized and cleared through the liver via DPYD, thus controlling the fraction of active metabolites present in the bloodstream. Loss-of-function carriers in DPYD demonstrate significant toxicity to 5-FU due to higher levels of circulating drug, whereby gain-of-function carriers demonstrate reduced responsiveness to treatment because of higher rates of clearance16. Chemotherapy associated simple steatohepatitis is an adverse side effect associated with 5-FU treatment and patients with lower DPYD mRNA levels demonstrate significantly higher rates of steatosis. It has yet to be determined whether this toxicity is due to higher levels of circulating 5-FU (due to reduced metabolism by DPYD)17. However, it has been demonstrated that pharmacological manipulation of DPYD through the small molecule inhibitor, Gimeracil, has, clinically, demonstrated the ability to increase the efficacy of 5-FU t reatment18. Whether genetic variability and/or pharmacologic perturbation of DPYD alters uracil and uridine homeostasis, resulting in liver lipid accumulation and susceptibility to NAFLD, remains to be elucidated. Thus, these questions were explored using a variety of in vitro cell models. The results demonstrate that inhibition of DPYD results in a reduced lipid burden in the presence of free fatty acid exposure in primary human hepatocytes cultured in vitro. RNAseq and extracellular flux analysis revealed a reduction in lipogenesis and an increase in mitochondrial respiration likely due to increased beta-oxidation of fatty acids with exposure to the DPYD inhibitor, Gimeracil. A gain-of-function mutation in DPYD was engineered into a human hepatocarcinoma cell line using CRISPR-Cas9 homology-directed repair to assess (...truncated)