Increased hepatic putrescine levels as a new potential factor related to the progression of metabolic dysfunction-associated steatotic liver disease

Abstract

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a chronic liver condition that often progresses to more advanced stages, such as metabolic dysfunction-associated steatohepatitis (MASH). MASH is characterized by inflammation and hepatocellular ballooning, in addition to hepatic steatosis. Despite the relatively high incidence of MASH in the population and its potential detrimental effects on human health, this liver disease is still not fully understood from a pathophysiological perspective. Deregulation of polyamine levels has been detected in various pathological conditions, including neurodegenerative diseases, inflammation, and cancer. However, the role of the polyamine pathway in chronic liver disorders such as MASLD has not been explored. In this study, we measured the expression of liver ornithine decarboxylase (ODC1), the rate-limiting enzyme responsible for the production of putrescine, and the hepatic levels of putrescine, in a preclinical model of MASH as well as in liver biopsies of patients with obesity undergoing bariatric surgery. Our findings reveal that expression of ODC1 and the levels of putrescine, but not spermidine nor spermine, are elevated in hepatic tissue of both diet-induced MASH mice and patients with biopsy-proven MASH compared with control mice and patients without MASH, respectively. Furthermore, we found that the levels of putrescine were positively associated with higher aspartate aminotransferase concentrations in serum and an increased SAF score (steatosis, activity, fibrosis). Additionally, in in vitro assays using human HepG2 cells, we demonstrate that elevated levels of putrescine exacerbate the cellular response to palmitic acid, leading to decreased cell viability and increased release of CK-18. Our results support an association between the expression of ODC1 and the progression of MASLD, which could have translational relevance in understanding the onset of this disease. © 2024 The Pathological Society of Great Britain and Ireland.

Keywords: liver metabolism; metabolic dysfunction‐associated steatohepatitis; metabolic dysfunction‐associated steatotic liver disease; obesity; ornithine decarboxylase; polyamine.

Figure 1.. Levels of polyamines and Odc1 in mouse liver samples.

Levels of (A) putrescine, (B) spermidine, and (C) spermine determined in mouse liver samples under LFCF diet (N=4) versus HFCF diet (N=5) performed by high-performance liquid chromatography and high-resolution mass spectrometry and tandem mass spectrometry represented as % relative to LFCF. Odc1 mRNA relative expression in (D) mouse liver samples and (E) mouse primary hepatocytes (MPH, N=3). Statistical differences were calculated using the Mann–Whitney U-test test considering p < 0.05 significant.

Figure 2.. Levels of polyamines human liver tissue samples.

Levels of (A) putrescine, spermidine, and spermine determined in human liver tissue samples between no MASLD (N=10), MASL (N=9) and MASH (n=28) patients by ultra-performance liquid chromatography high-resolution time-of-flight mass spectrometry represented as ng/mg protein. Correlation between levels of hepatic putrescine and (B) aspartate aminotransferase (AST) and (C) SAF score. Statistical differences were calculated using the one-way ANOVA test for multiple comparison flowed by a Tukey’s post hoc analysis fand considering p < 0.05 significant. Correlations were performed using the Spearman correlation coefficient. MASH, metabolic dysfunction-associated steatohepatitis; MASL, metabolic dysfunction-associated steatotic liver; MASLD, metabolic dysfunction-associated steatotic liver disease; SAF, steatosis, activity, and fibrosis.

Figure 3.. Ornithine decarboxylase mRNA and protein levels in human liver tissue samples.

Differences in relative ODC1 mRNA expression between patients with (A) no MASLD versus MASLD (N=25 versus N=94); (B) No MASLD, MASL and MASH (N=25, N=42 and N=52, respectively) and (C) mild versus severe MASH (N=29 versus. N=23). Values are represented as ODC1 mRNA expression relative to 18S. (D) Values of ODC1 expression represented as percentage of positive surface. (E) Representative images of ODC1 expression (brown signals, scale bar, 100 μm). Statistical differences were calculated using an unpaired t-test or one-way ANOVA test for multiple comparison followed by a Tukey’s post hoc analysis considering p < 0.05 significant. MASH, metabolic dysfunction-associated steatohepatitis; MASL, metabolic dysfunction-associated steatotic liver; MASLD, metabolic dysfunction-associated steatotic liver disease; ODC1, ornithine decarboxylase.

Figure 4.. Effect of putrescine on an in vitro model of palmitic acid-induced steatosis.

Correlation between palmitic acid and (A) putrescine (N=39) and (B) ODC1 mRNA levels in human liver tissue samples (N=90). (C) Effect of palmitic acid on ODC1 mRNA levels in HepG2 cells. Data were normalized to the vehicle control group (5% albumin), which was set to 1. (D) Effect of 500 μM palmitic acid, 10 μM putrescine, or the combination of both after 24 h of exposure on cell viability determined using an MTT assay. Data were normalized to the control group (untreated HepG2 cells), which was set to 1. (E) Effect of 500 μM palmitic acid, 10 μM putrescine, or the combination of both after 24 h of exposure on secreted cytokeratin-18 (CK-18) levels. All the in vitro experiments using HepG2 cells were performed in triplicate in three independent experiments. Statistical differences were calculated using unpaired t-tests or one-way ANOVA test for multiple comparison followed by a Tukey’s post hoc analysis considering p < 0.05 significant. ODC1, ornithine decarboxylase; PA, palmitic acid (C16:0); Put, putrescine.