Ammonia, a nitrogen-containing compound produced during amino acid catabolism, plays a key role in nitrogen homeostasis. The primary source of ammonia in humans is the gut, where it is produced from dietary sources, luminal bacterial urease activity and glutamine deamination in enterocytes.1 This gut-derived ammonia is transported to the liver via the portal vein, and because ammonia is potentially neurotoxic, rapid and efficient removal is essential. Among others organs, the liver is the main organ responsible for its detoxification via the urea cycle. Ureagenesis occurs specifically in periportal hepatocytes by the action of urea cycle enzymes (UCEs), which involves two mitochondrial enzymes (carbamoyl phosphate synthetase 1 (CPS1) and ornithine transcarbamylase (OTC)) and three cytosolic enzymes (arginosuccinate synthetase (ASS), arginosuccinate lyase (ASL) and arginase 1 (ARG1)). In health, ammonia is converted into urea, which is excreted by the kidneys maintaining the systemic nitrogen balance.2
Glutamine functions as a major non-toxic interorgan nitrogen carrier. Glutamine synthetase (GS) converts glutamate and ammonia into glutamine, detoxifying ammonia. By contrast, glutaminase (GLS), the main regulatory enzyme of hepatic glutamine catabolism, converts glutamine into glutamate and ammonia.3
Liver dysfunction can compromise the efficiency of this detoxification process. When an imbalance between ammonia production and detoxification occurs, a condition known as hyperammonemia is generated, which might lead to complications such as hepatic encephalopathy. In recent years, ammonia has emerged as a potential contributor to metabolic dysfunction-associated steatotic liver disease (MASLD), a condition associated with metabolic syndrome.4
Ammonia, apart from being viewed as a hepatic product, needs to be considered as a systemic metabolite, with implications in multiple organs in physiological and pathological contexts (please refer to Fig. 1). Skeletal muscle, kidney and brain play key roles in maintaining ammonia homeostasis. In physiological conditions, skeletal muscle, through the action of GS, converts ammonia into glutamine. The kidneys contribute to both ammonia production and excretion: renal GLS generates ammonia to maintain the acid–base equilibrium. In the brain, astrocytes detoxify ammonia converting glutamate into glutamine. However, due to its properties, plasma ammonia can cross the blood-brain barrier, leading to hepatic encephalopathy under hyperammonemic conditions.5
In this review, we specifically focus on (i) the integration of hepatic and extrahepatic routes of ammonia in MASLD, (ii) the mechanistic links between ammonia, hepatic stellate cell activation and fibrogenesis, and (iii) the rationale for targeting ammonia as a therapeutic strategy in MASLD.
Role of ammonia in the development and progression of MASLDMASLD is the most prevalent chronic liver disease, with a prevalence of 30% of the world population.6 It is a spectrum of liver diseases that ranges from steatosis to metabolic dysfunction-associated steatohepatitis (MASH), which can evolve into cirrhosis and lastly, hepatocellular carcinoma.7
Ammonia and glutamine metabolism are closely linked to the progression and pathogenesis of MASLD. In this condition, the equilibrium between ammonia production and detoxification is compromised due to reduced urea cycle activity and increased ammonia production. Both of them are associated with clinical manifestations of the disease together with the progression of liver injury and fibrosis. In liver biopsies from patients with MASLD hepatic ammonia levels showed a fivefold increase compared with controls and an additional twofold rise in patients with MASH when compared to patients with steatosis. Moreover, plasma ammonia levels were higher in patients with MASH than in those with steatosis.8 In a mouse model of MASH, animals fed with a high-fat, high-cholesterol (HFHC) diet showed a threefold increase in plasma ammonia levels compared with animals receiving a normal chow diet.9
In the liver, GLS converts glutamine into glutamate, generating ammonia as a byproduct. This enzyme exists in two isoforms: GLS1, the kidney-type glutaminase, and GLS2, the liver-type glutaminase localized in healthy livers.10 In MASLD, GLS1 expression is upregulated and GLS2 is downregulated, associated with an increase in hepatic glutaminase activity.11 Moreover, in MASLD there is an impairment of the hepatic urea cycle characterized by a decrease in the expression of UCEs, particularly OTC and CPS1, limiting effective ammonia detoxification.12,13 Hypermethylation of the promoter regions of UCE genes is one of the regulatory mechanisms responsible for the observed changes in their expression.8 The combination of a deficit in hepatic detoxification (UCEs downregulation) and an increase in ammonia production (GLS1 overexpression) results in hyperammonemia.
Hepatic stellate cells (HSCs) coordinate important functions in the liver and their activation and consequent dysfunction is associated with liver fibrosis, portal hypertension and liver cancer. In vitro studies have shown that increased ammonia levels can activate HSCs and transform them into a myofibroblastic phenotype, increasing proliferation and contractility and stimulating extracellular matrix production and fibrotic progression. Progressive fibrosis disrupts the hepatic architecture, compromising ammonia detoxification and promoting a vicious cycle of metabolic imbalance.14
Beyond its role in promoting fibrogenesis, hyperammonemia in MASLD also exerts extrahepatic effects in distant tissues. Pathological ammonia levels lead to sarcopenia due to a depletion of tricarboxylic acid (TCA) cycle intermediates, impaired ATP generation and increased muscle protein breakdown. Systemic hyperammonemia further contributes to cognitive impairment,15 compromises immune function with a deleterious effect on neutrophil function and is associated with an increased risk of cancer in general16,17 and particularly of hepatocellular carcinoma.18
Overall, ammonia appears to act as a mediator of MASLD progression. Its capacity to alter hepatocellular function and stimulate fibrosis suggests that restoring hepatic ammonia homeostasis could be an attractive therapeutic goal in MASLD management.
Ammonia as a target for MASLD treatmentThe management of MASLD involves a multifaceted approach, including lifestyle modifications, control of cardiometabolic risk factors and the prevention of hepatic and extrahepatic complications.7 Given the emerging role of ammonia in MASLD progression, therapeutic strategies to lower ammonia levels have become an important area of research. These approaches include the removal of the injurious stimulus to restore hepatic ammonia-detoxifying capacity; the use of pharmacological ammonia-lowering agents; and the targeting of glutaminolysis via GLS1 inhibition.
In a preclinical model of MASLD induced by a choline deficient high-fat diet (CDA-HFD), CPS1 expression was downregulated, leading to an impaired capacity for ureagenesis and ammonia clearance.19 Hyperammonemia promotes MASLD progression, especially liver fibrosis, by activating HSCs.9 The removal of the main trigger of liver damage when replacing the CDA-HFD diet with a control diet showed a normalization of CPS1 expression and a regression of liver fibrosis 4 weeks after the administration of the control diet.19
In parallel, pharmacological ammonia scavenging has shown strong therapeutic potential. In preclinical models of MASLD, lowering hepatic ammonia through agents such as ornithine phenylacetate (OP) restores urea-cycle enzyme activity, reduces ammonia accumulation in liver tissue and attenuates fibrosis progression in MASLD. OP modulates inter-organ ammonia metabolism, combining the action of the amino acid ornithine, which participates in glutamine synthesis, and phenylacetate which, in conjugation with glutamine, forms phenylacetylglutamine, allowing nitrogen excretion through the kidneys.20 Therefore, the efficacy of OP mainly relies on the ability of ornithine to increase the glutamine synthesis. An in vivo model of MASLD induced by a HFHC diet showed a reduction in plasma ammonia levels after OP administration, a restoration of OTC enzyme activity, a significant reduction in fibrosis progression and reduced expression of HSC activation markers. In vitro model of MASLD using precision-cut liver slices (PCLS) showed a reduction in ammonia concentrations in the medium as well as reduced collagen deposition after the addition of OP.9 Moreover, a study in vitro using primary human HSCs shows that ammonia lowering drug OP causes a downregulation of HSC activation markers.14 A different therapeutic option is to target hepatic GLS activity to reduce hepatic ammonia content. Specific silencing or inhibition of the high-activity GLS1 has been shown to reduce hepatic ammonia content in a preclinical mouse model of steatohepatitis.21 These findings identify GLS1 as a promising therapeutic target, although clinical data are still lacking.
Concluding remarksEmerging evidence suggests ammonia is an important contributor to the pathogenesis of MASLD, with an active role in hepatic injury and fibrogenesis, whereby increased ammonia production and impaired detoxification might create a self-perpetuating vicious circle. Hepatic ammonia accumulation in MASLD is associated with impaired urea cycle function, evidenced by downregulation of UCEs expression and increased hepatic GLS activity. In hyperammonemia, mitochondrial respiration is compromised and oxidative stress is increased, leading to reduced hepatocyte viability and function.22 Moreover, high ammonia levels have been implicated in the activation of HSCs, promoting extracellular matrix deposition and fibrosis progression.
Cumulative evidence suggests that targeting the urea cycle and restoring ammonia homeostasis might be a promising therapeutic strategy. Experimental studies demonstrate the therapeutic potential of ammonia-lowering therapies for preventing the progression of MASLD. OP, an ammonia scavenger, reduces ammonia levels towards normal, prevents the progression of fibrosis and improves mitochondrial function, reducing inflammatory or oxidative signalling pathways in animal and in vitro preclinical models.
Taken together, these findings suggest that ammonia could represent a promising therapeutic target for preventing liver fibrosis progression, and clinical trials will be warranted to confirm whether ammonia scavenging could improve outcomes in patients with MASLD.
Future studies should identify MASLD phenotypes with clinically relevant hyperammonemia, validate reliable biomarkers of hepatic ammonia, and evaluate ammonia-lowering therapies on top of standard lifestyle and pharmacological interventions. In addition to liver histology, endpoints should include the evaluation of muscle mass and function, cognitive performance, and infection risk, reflecting the systemic nature of ammonia dysregulation.
The research leading to these results has received funding from Instituto de Salud Carlos III under grant agreements PI24/02008, PI22/01342, from Agencia Estatal de Investigación under grant agreement PID2024-162275OB-I00 and European Partnership for Personalized Medicine under grant agreement EPPERMED2024-496. RGD and DMM are both recipients of EMERGIA fellowships from Consejería de Universidad, Investigación e Innovación from Junta de Andalucía under grant agreements DGP_EMEC_2023_00394 and DGP_EMEC_2023_00472.
