MASTER’S THESIS University of Copenhagen · 2025
LIPID SIGNALING & LIVER INFLAMMATION
N-acyl taurines in liver inflammation: regulation and cellular effects
N-acyl Taurines in LPS-Induced Hepatic Inflammation:
Alterations and Potential Effects
My master’s thesis examined N-acyl taurines (NATs), a class of endogenous bioactive lipids in which a fatty acyl group is linked to taurine through an amide bond. I studied how NAT abundance and hepatic NAT synthesis capacity respond to LPS-induced inflammation, and whether selected NAT species alter inflammatory gene expression in hepatocyte models.
01 / BACKGROUND
Why study NATs in liver inflammation?
Lipids can act as biological signals as well as energy stores and structural components. NATs are one such family of bioactive lipids: their fatty acyl chains differ in length and saturation, and individual NAT species can have different biological activities.
What was already known: published studies had linked selected NAT species to glucose regulation, intestinal lipid handling, and tissue repair. For example, C18:1 NAT was reported to improve glucose tolerance and stimulate GPR119-dependent hormone secretion in mice, while other NAT species promoted wound repair in mouse and cell models. These findings provided evidence of biological activity; they did not establish an anti-inflammatory effect of NATs in the liver.
Why the liver? The liver is a site of NAT synthesis and a central organ in metabolism and inflammatory responses. BAAT (bile acid-CoA:amino acid N-acyltransferase) had been identified as a hepatic enzyme supporting polyunsaturated NAT synthesis, while FAAH (fatty acid amide hydrolase) participates in NAT degradation. This made it possible to investigate NAT regulation by altering synthesis and degradation separately.
The research gap: taurine and selected fatty acids or their derived mediators have reported effects on inflammation in specific experimental settings. Their conjugation into a NAT does not mean those effects are retained. Whether NATs themselves modulate inflammatory responses in hepatocytes, and how their abundance changes when hepatic synthesis is impaired, therefore remained questions to test.
Background from thesis Introduction, pp. 6–14. Selected primary studies: Grevengoed et al., 2019 (glucose regulation); Sasso et al., 2016 (wound repair); Trammell et al., 2023 (hepatic synthesis).
02 / RESEARCH QUESTIONS
How are NATs regulated, and what do they do in hepatocytes?
The project addressed two complementary questions. The first concerned endogenous NAT regulation during inflammatory stress; the second tested the effects of adding defined NAT species to liver cells.
- Regulation: how does LPS exposure affect plasma and biliary NAT abundance and hepatic NAT synthase activity, and how do these responses differ when BAAT-dependent synthesis or FAAH-mediated degradation is impaired?
- Cellular effects: does pretreatment with C18:1 NAT or C20:4 NAT change inflammatory gene expression after LPS exposure in Huh7 cells and primary mouse hepatocytes?
The experimental stimulus: lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria, was used to induce an inflammatory response. NAT abundance, enzyme activity, and gene expression were treated as distinct measurements.
03 / EXPERIMENTAL APPROACH
Connecting each question to an experimental readout
NAT abundance in the mouse study
LPS- and saline-treated mice were compared across wild-type, BAAT-knockout, FAAH-S268D knock-in, and double-mutant groups. Plasma and bile NAT measurements assessed whether inflammatory responses differed between compartments and genetic backgrounds.
Hepatic synthesis capacity
I optimized protein input and reaction time for liver-lysate NAT synthase activity assays, then compared activity across the study groups. These ex vivo assays measured synthesis capacity under the assay conditions, rather than NAT production rates in living animals.
Responses to selected NAT species
I tested C18:1 NAT and C20:4 NAT pretreatment before LPS exposure in human Huh7 cells and primary mouse hepatocytes. RNA extraction, cDNA synthesis, and qPCR were used to assess cytokine and inflammasome-related transcripts, with relative expression compared in GraphPad Prism.
Methods, interpretation & collaboration
Why these genetic models? BAAT loss tested the contribution of hepatic NAT synthesis. The FAAH-S268D mutation selectively impairs NAT hydrolysis, helping distinguish effects associated with altered NAT degradation from those caused by conventional FAAH knockout.
Assay interpretation: activity assays used supplied substrates and conditions selected within a linear range. NAT accumulation can reflect synthesis, degradation, transport, or clearance; a concentration change alone does not identify its source.
Cell readout: qPCR expression was normalized to GAPDH/Gapdh using the ΔΔCt approach. Transcript changes were used to assess the response to NAT pretreatment; protein signaling, cytokine release, and inflammasome activation were not established by these measurements alone.
My contribution: I carried out liver activity assay optimization and measurements, cell experiments, gene-expression analysis, and interpretation of the combined findings. Anna Skab Hassing performed animal handling, plasma and bile collection, and primary hepatocyte isolation. Samuel Addison Jack Trammell performed mass-spectrometric NAT measurements.
Source: thesis Methods, pp. 16–21, and assay optimization, pp. 25–26.
04 / RESULTS
What the experiments showed
- 01
NAT responses differed between blood and bile
Plasma NATs rose after LPS across the mouse genotypes, including those without BAAT. Biliary NAT increases depended on functional BAAT. The plasma response therefore persisted despite impaired BAAT-dependent hepatic synthesis. These measurements did not establish the tissue source of circulating NATs.
View dataHide data ＋Plasma and bile NAT profilesPanels A–D show total NATs; E–H show individual NAT species. The plasma and bile responses differed across the genetic backgrounds.Thesis Fig. 2, p. 24. Mean ± SEM; n = 1–7 in the BAATKO experiment and n = 2–6 in the KIKO experiment. No statistical analysis was performed because some saline groups had fewer than three samples. - 02
BAAT supported hepatic PUFA-NAT synthesis
BAAT-deficient liver samples showed reduced capacity to produce polyunsaturated NATs. Baat mRNA increased after LPS, providing a transcriptional readout alongside the enzyme activity measurements; mRNA abundance alone does not establish enzyme activity or in vivo flux. Other tissue sources remain a hypothesis, rather than a directly measured result.
View dataHide data ＋Hepatic activity and Baat expressionPanels A–B show liver-lysate NAT synthase activity; C–D show Baat mRNA. These are separate readouts of synthesis capacity and transcription.Thesis Fig. 4, p. 29. Mean ± SEM; n = 1–7 in the BAATKO experiment and n = 2–6 in the KIKO experiment. No statistical analysis was performed because some saline groups had fewer than three samples. WT Baat expression data were provided by Anna Skab Hassing. The original caption proposes a non-hepatic source; this study did not directly measure tissue sources of circulating NATs. - 03
Selected NATs lowered specific inflammatory transcripts
C18:1 NAT and C20:4 NAT reduced TNF-α expression in Huh7 cells and Tnf-α expression in primary hepatocytes. Primary cells also showed lower Nlrp3 and Casp1 expression. Neither Huh7 IL-6 comparison was statistically significant. In primary hepatocytes, Il-6 decreased significantly with C20:4 NAT but not C18:1 NAT. These findings concern mRNA responses under the tested conditions.
View dataHide data ＋Huh7 cellsBoth NAT species reduced TNF-α mRNA. Neither IL-6 comparison reached statistical significance.Thesis Fig. 6, p. 34. Three independent experiments with technical replicates. Primary hepatocytesTnf-α, Nlrp3 and Casp1 mRNA decreased with both NAT species. Il-6 decreased significantly with C20:4 NAT, but not C18:1 NAT.Thesis Fig. 7, p. 35. Triplicate measurements from two independent experiments. Original thesis figures. Error bars show SEM. The figures report unpaired two-tailed t-tests against the LPS-only group. Technical replicates do not represent additional independent experiments.
05 / INTERPRETATION
A promising starting point, with questions still open
The results suggest that NATs may help regulate the response of liver cells to inflammatory stress. They do not yet establish a treatment effect in animals or people. Changes in Nlrp3 and Casp1 mRNA also do not, by themselves, demonstrate reduced inflammasome activation.
Limitations & next questions
- Small saline-control groups limited statistical power in the mouse study.
- Background NATs in liver lysates complicate the separation of existing lipids from newly synthesized products.
- Extrahepatic synthesis, including a possible contribution from the kidney, was not measured directly.
- Further experiments could test protein signaling, additional NAT species, and NAT administration in animal models.
METHODS I WORKED WITH
Based on my master’s thesis (10 August 2025) and defence presentation (22 August 2025). Figure captions and detailed results take precedence where short summaries are less specific.