The ongoing COVID-19 pandemic, caused by SARS-CoV-2, has resulted in millions of infections and significant morbidity and mortality worldwide. While primarily known as a respiratory illness, SARS-CoV-2 infection often leads to multi-organ dysfunction, with liver damage being a significant concern. Studies have shown that approximately 50% of COVID-19 patients exhibit elevated levels of liver enzymes (transaminases), which is an indicator of liver injury and correlates with the severity of the disease. The mechanisms underlying this liver damage remain unclear, with several contributing factors implicated, including the direct cytopathic effect of the virus on hepatocytes, exacerbated immune responses, and drug toxicity from antiviral treatments or other medications. Previous studies with other coronaviruses, such as SARS-CoV and MERS-CoV, have demonstrated their ability to infect liver cells. While some evidence suggests SARS-CoV-2 can infect the liver, the lack of sufficient samples and the use of less-than-ideal cell models have hampered definitive conclusions. Angiotensin-converting enzyme 2 (ACE2) serves as the primary receptor for SARS-CoV-2 entry into host cells, through interaction with the receptor-binding domain (RBD) of the viral spike protein. Other factors such as transmembrane protease serine 2 (TMPRSS2) and neuropilin-1 (NRP1) also play a role in viral entry. There's evidence suggesting that patients with pre-existing chronic liver disease, such as metabolic-associated fatty liver disease (MAFLD), alongside type 2 diabetes (T2D), are at increased risk of severe COVID-19. Metformin, a common medication for managing hyperglycemia in T2D patients, has also shown potential benefits in reducing the mortality and severity of COVID-19, although the mechanisms involved are not fully understood. This study uses robust primary cell models to investigate the direct effect of SARS-CoV-2 infection on liver cells and to explore the potential of metformin as a therapeutic intervention.

Existing literature reveals a range of evidence regarding SARS-CoV-2's impact on the liver. Studies showing elevated liver enzyme levels in a substantial portion of COVID-19 patients indicate hepatic involvement. However, the direct causation of this liver damage by SARS-CoV-2 remains debated. While some studies have shown the presence of viral particles in liver biopsies, the small sample sizes limit the strength of these findings. Other coronaviruses have demonstrated liver tropism, strengthening the hypothesis that SARS-CoV-2 may directly infect hepatocytes. The role of pre-existing liver conditions, especially MAFLD, in increasing the severity of COVID-19 is increasingly recognized. Research has also explored potential repurposing of existing drugs, notably metformin, for COVID-19 treatment, although the underlying mechanisms remain uncertain. This study aims to address these knowledge gaps and provide clarity on the virus's direct impact on the liver and on the possible protective role of metformin.

This research employed multiple experimental models to assess SARS-CoV-2 infectivity in hepatocytes. The study used THLE-2 cells (a human hepatocyte cell line expressing ACE2, TMPRSS2, and NRP1), primary hepatocytes derived from humanized ACE2 (hACE2) mice, and upcyte second-generation human hepatocytes. Pseudotyped lentiviral particles expressing the full-length spike protein of SARS-CoV-2, and control particles lacking the spike protein, were used to infect hepatocytes. Flow cytometry was used to measure the infection rate, and binding assays (using biotinylated spike protein subunits S1 and RBD) were conducted to assess the interaction between the viral spike protein and ACE2 on the hepatocyte surface. Western blotting was used to analyze the expression levels of ACE2, NRP1, TMPRSS2 and phospho-AMPKa. Proteomic analysis via LC-MS/MS was performed on infected and control hACE2 primary hepatocytes to identify differentially expressed proteins and associated pathways. Metabolic flux analysis using labeled glucose ([U-13C]-glucose) and extracellular flux analysis (measuring oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)) were used to examine alterations in energy production and metabolic pathways in infected hepatocytes. To induce steatotic conditions, primary hepatocytes were incubated in methionine-choline deficient (MCD) media. The effect of metformin on infection and related metabolic changes was evaluated by treating hepatocytes with metformin before or during infection. Extracellular angiotensin 1-7 (ANG(1-7)) levels were measured by ELISA to evaluate the impact of infection on the renin-angiotensin system. Finally, the effect of the ACE2/ANG(1-7)/Mas axis inhibitor A779 on infection was also assessed.

The study's key findings demonstrate that hepatocytes are indeed susceptible to SARS-CoV-2 infection. Both primary human hepatocytes and hepatocytes from hACE2 mice were successfully infected by pseudotyped viral particles expressing the full-length spike protein. The RBD of the spike protein directly interacted with ACE2 on the surface of the hepatocytes, confirming the role of ACE2 in viral entry. Proteomic analysis revealed that infection with SARS-CoV-2 resulted in significant changes in the proteome of hACE2 hepatocytes, with dysregulation predominantly impacting mitochondrial activity, antiviral immunity, inflammatory response, and iron metabolism. Metabolic flux analysis showed a clear shift towards a glycolytic phenotype in infected hepatocytes, characterized by increased glucose uptake, lactate production, and enhanced TCA cycle activity. This metabolic reprogramming suggests a significant alteration in energy production in infected liver cells. Furthermore, the study found that hepatocytes under steatotic conditions (induced by MCD media), exhibiting increased ACE2 and NRP1 expression, were more susceptible to infection compared to healthy hepatocytes. Importantly, metformin treatment significantly reduced both ACE2 and NRP1 levels, consequently reducing the infection rate in both steatotic and healthy hepatocytes. The findings also showed that inhibition of the renin-angiotensin system using the ACE2/ANG(1-7)/Mas axis inhibitor A779 increased hepatocyte susceptibility to infection, suggesting a protective role of the RAS system.

The findings directly address the research question by providing definitive evidence of SARS-CoV-2 infectivity in primary human hepatocytes. The observed metabolic reprogramming towards glycolysis, along with impaired mitochondrial function, sheds light on the potential mechanisms of liver damage in COVID-19. The increased susceptibility of steatotic hepatocytes highlights the increased risk in patients with MAFLD. The demonstration of metformin's ability to reduce infection in these vulnerable hepatocytes suggests a promising avenue for therapeutic intervention. This effect could be partly explained by metformin's ability to reduce ACE2 and NRP1 levels and the potential influence on the renin-angiotensin system. These findings have significant implications for understanding the pathophysiology of COVID-19-associated liver injury and for potential therapeutic strategies. The study's findings contribute significantly to the field by confirming the direct role of SARS-CoV-2 infection in liver damage and suggesting metformin as a potential preventative and treatment option.

This study provides compelling evidence that hepatocytes are susceptible to SARS-CoV-2 infection, leading to metabolic reprogramming and mitochondrial dysfunction. Steatotic hepatocytes are more vulnerable, and metformin shows promise in reducing infection rates. These findings highlight the direct link between SARS-CoV-2 and liver damage in COVID-19 patients, particularly those with MAFLD. Further investigation into the precise mechanisms underlying metformin's protective effects and the clinical translation of these findings are warranted.

While this study provides strong evidence for SARS-CoV-2 infection of hepatocytes, the use of pseudotyped viruses might not perfectly replicate the complexities of authentic SARS-CoV-2 infection. The in vitro nature of the study might not entirely recapitulate the in vivo environment. Furthermore, the study focuses on the direct impact of SARS-CoV-2 on hepatocytes; the contribution of indirect effects (e.g., immune response) is not fully explored. The sample size of some experiments could be considered relatively small, despite the use of robust statistical analysis.