Glutamine Breakdown as a Potential Metabolic Biomarker for Nonalcoholic Steatohepatitis.

As the obesity epidemic continues to grow, so too does the prevalence of nonalcoholic fatty liver disease (NAFLD). This condition is characterized by increased steatosis in the liver and has been associated with obesity, dyslipidemia, and type II diabetes mellitus, and generally is thought of as the hepatic manifestation of metabolic syndrome. The most predominant form of NAFLD is steatosis or simple fatty liver. Up to 40% of the US population has fatty liver, placing them at risk for the more advanced progressive stage of NAFLD, nonalcoholic steatohepatitis (NASH).

NASH is characterized by inflammation, liver cell damage, and increasing fibrosis, and, if left untreated, can progress to cirrhosis, liver failure, and increased risk for hepatocellular carcinoma. An estimated 20% of individuals with fatty liver have NASH; however, little is known regarding why some patients progress to the more advanced form of the disease while others do not. Furthermore, there are limited therapeutic options for this population outside of weight loss. Although 5% to 10% loss of one’s body weight has been associated with improvements in steatosis and inflammation, respectively, this is difficult for patients to achieve and maintain. This problem is compounded by the fact that there are no Food and Drug Administration–approved pharmacologic interventions for this constellation of diseases, highlighting the need to further our understanding of what drives the differential incidence of steatosis and NASH.

Currently, the gold standard for diagnosing steatosis vs NASH is liver biopsy. Although great efforts have been made to noninvasively diagnose, predict, and monitor disease severity, liver fibrosis, and scarring in NAFLD, repeat biopsy remains the standard for histologic assessment and disease staging. As a result, intensive investigation has focused on identifying which patients are at risk for progressive fibrosis and whether the underlying mechanisms can be targeted therapeutically. In new work from Duke University published in this issue of Cellular and Molecular Gastroenterology and Hepatology, Du et al expand on recent findings that the fibrogenic myofibroblasts in the liver show disordered glutamine metabolism. This hypothesis is particularly intriguing given the recent report of an association between plasma glutamate levels and fibrosis severity in NAFLD patients. In the current study, Du et al convincingly show an association between fibrosis severity and increased glutamine breakdown in various animal models of steatohepatitis, including treatment with Western diet rich in fat, the choline-deficient L-amino acid–defined diet, the methionine/choline-deficient diet, and carbon tetrachloride-induced hepatotoxicity. In particular, this study documents an association not only between fibrosis and changes in glutamine, glutamate, and their ratio in liver and serum, but also changes in the levels and distribution of enzymes involved in glutamine metabolism including glutaminase (Gls) and glutamine synthase. Furthermore, they found that glutamine deprivation and pharmaceutical or short hairpin RNA–mediated suppression of high affinity glutaminase (Gls1), the form predominately found in the kidney, can suppress growth in human myofibroblastic hepatic stellate cells. This suggests Gls1-dependent glutaminolysis is required for profibrogenic activity of myofibroblastic-hepatic stellate cells.

One of the most important aspects of this report is the ability of Du et al to further test the relevance of their findings in human biology. First, Du et al revisited their previously published microarray data from 70 biopsy specimens from NASH patients to discover increased Gls1 expression in patients with advanced fibrosis compared with mild fibrosis. They then expanded upon the association between the plasma changes in glutamine metabolism and fibrosis seen by other investigators by performing metabolomic analysis on a large cohort of biopsy-proven NAFLD patients. Here, they found that glutamate, the glutamate/glutamine ratio, α-ketoglutarate, and other tricarboxylic acid cycle intermediates increased with fibrosis severity even after accounting for potential cofounders such as age, sex, and severity of the metabolic syndrome. Finally, they also confirmed that plasma glutamine levels correlated with fibrosis in an independent cohort of human immunodeficiency virus/hepatitis C virus patients with differing degrees of fibrosis, highlighting the potential of this approach to monitor fibrosis activity across steatohepatitis of various causes.

As mentioned previously, the ability to prevent morbidity and mortality in NASH is limited not only by a lack of treatments, but also an inability to longitudinally monitor disease severity outside of invasive repeat biopsies. Several potential biomarkers and imaging approaches have been proposed as a potential means of monitoring NAFLD severity and progression, but none have proven more reliable for prognosis and progression than histology after biopsy., Unfortunately, serial liver biopsies are not without risk. Overall, this study by Du et al supports and expands upon previous findings that suggest altered glutamine metabolism could be used to stratify patients by disease severity, although more work is needed to validate the sensitivity of this approach as well as its ability to measure NAFLD progression in a longitudinal manner. To this end, the authors capitalized upon their findings to develop a positron emission tomography method to image changes in uptake relative to fibrosis in livers of mice, a paradigm that already is being used in oncology. Here, Du et al show that an injection of 18F-flouroglutamine was associated with increased hepatic uptake of the labeled glutamine by imaging in carbon tetrachloride-treated mice in a manner that could distinguish fibrotic mice from controls. Additional work is needed to determine the sensitivity and specificity of this positron emission tomography approach, in particular if this approach has utility in less-severe forms of NASH in rodents. If so, future studies in human beings would be essential for establishing if increased glutamine breakdown could serve as a clinical biomarker for NASH. Despite these limitations, the possibility means of reliably detecting hepatic fibrogenesis noninvasively in NASH has the potential to improve how clinicians monitor and make treatment decisions in this increasingly prevalent and morbid disease. Moreover, future studies that aim to explore the therapeutic relevance of aberrant glutamine metabolism as a metabolic driver of NAFLD pathogenesis are warranted.