A Cholecystokinin Receptor Antagonist Halts Nonalcoholic Steatohepatitis and Prevents Hepatocellular Carcinoma

Abstract

Background and Aims Nonalcoholic steatohepatitis (NASH) is a common inflammatory liver condition that may lead to cirrhosis and hepatocellular carcinoma (HCC). Risk factors for NASH include a saturated fat diet, altered lipid metabolism, and genetic and epigenetic factors, including microRNAs. Serum levels of cholecystokinin (CCK) are elevated in mice and humans that consume a high-saturated fat diet. CCK receptors (CCK-Rs) have been reported on fibroblasts which when activated can induce fibrosis; however, their role in hepatic fibrosis remains unknown. We hypothesized that elevated levels of CCK acting on the CCK-Rs play a role in the development of NASH and in NASH-associated HCC. Methods We performed a NASH Prevention study and Reversal study in mice fed a saturated fat 75% choline-deficientethionine-supplemented (CDE) diet for 12 or 18 weeks. In each study, half of the mice received untreated drinking water, while the other half received water supplemented with the CCK-R antagonist proglumide. CCK-R expression was evaluated in mouse liver and murine HCC cells. Results CCK receptor antagonist treatment not only prevented NASH but also reversed hepatic inflammation, fibrosis, and steatosis and normalized hepatic transaminases after NASH was established. Thirty-five percent of the mice on the CDE diet developed HCC compared with none in the proglumide-treated group. We found that CCK-BR expression was markedly upregulated in mouse CDE liver and HCC cells compared with normal hepatic parenchymal cells, and this expression was epigenetically regulated by microRNA-148a. Conclusion These results support the novel role of CCK receptors in the pathogenesis of NASH and HCC.

Proglumide Reverses Nonalcoholic Steatohepatitis by Interaction with the Farnesoid X Receptor and Altering the Microbiome

“…CCK receptors have been characterized on stellate cells of the pancreas [59] and on fibroblasts [60] and stimulation of these receptors results in collagen deposition and fibrosis. We have previously shown that proglumide therapy could decrease fibrosis by preventing stellate cell activation in the mouse liver [21] and in the pancreas microenvironment [61]; therefore, we assume that the anti-fibrotic effect seen in the livers of the CDE-fed mice treated with proglumide in this investigation was related to the blockade of the CCK receptor on mouse stellate cells in the liver. The reduction in hepatic inflammation observed in the CDE/Prog-treated mice of this study may be due to proglumide's action on reducing inflammatory cytokines and chemokines as previously described [27].…”

“…In rodents, proglumide increases bile flow [20] and decreases bile acid concentration. We previously showed that proglumide therapy prevented and reversed biochemical and histologic NASH in mice fed a choline deficient ethionine (CDE) diet [21]. In this model, we found that proglumide significantly reduced serum bilirubin levels, suggesting that proglumide was stimulating bile flow and reversing hepatocyte injury.…”

“…Liver tissues were fixed, paraffin embedded, and 5 µm tissue sections on slides stained with hematoxylin and eosin (H&E). Using the criteria in our previously published study [21], histologic NASH was scored for inflammation, fibrosis, and steatosis by a pathologist blinded to the treatment groups.…”

Safety and Pharmacokinetic Assessment of Oral Proglumide in Those with Hepatic Impairment

“…This prospective open-labeled clinical trial undertaken at Georgetown University was an investigator-initiated translational research study based upon a study in mice that showed reversal of hepatic fibrosis and prevention of hepatocellular carcinoma [ 19 ]. The protocol and sample size were approved by the Food and Drug Administration (FDA) and the Georgetown University Institutional Review Board (IRB), and the trial was registered on (accessed on 20 February 2022) (NCT04814602).…”

“…The cholecystokinin-B receptor (CCK-BR) is not found in normal liver tissue (mouse or human) but becomes overexpressed in NASH and HCC [ 18 ]. In fact, in [ 19 ] 35% of the mice on a NASH-inducing diet developed dysplastic nodules or HCC after 18 weeks, while none of the mice treated with a CCK-BR antagonist, proglumide, developed HCC. Further examination of the mouse NASH livers in this study showed that treatment with the CCK receptor antagonist, proglumide, rendered the liver microenvironment less carcinogenic by decreasing fibrosis, inflammatory chemokines, and cytokines and reducing M2-polarized macrophages [ 18 ].…”