Inhibition of hepatic lipogenesis enhances liver tumorigenesis by increasing antioxidant defence and promoting cell survival.
ABSTRACT: The metabolic pathway of de novo lipogenesis is frequently upregulated in human liver tumours, and its upregulation is associated with poor prognosis. Blocking lipogenesis in cultured liver cancer cells is sufficient to decrease cell viability; however, it is not known whether blocking lipogenesis in vivo can prevent liver tumorigenesis. Herein, we inhibit hepatic lipogenesis in mice by liver-specific knockout of acetyl-CoA carboxylase (ACC) genes and treat the mice with the hepatocellular carcinogen diethylnitrosamine (DEN). Unexpectedly, mice lacking hepatic lipogenesis have a twofold increase in tumour incidence and multiplicity compared to controls. Metabolomics analysis of ACC-deficient liver identifies a marked increase in antioxidants including NADPH and reduced glutathione. Importantly, supplementing primary wild-type hepatocytes with glutathione precursors improves cell survival following DEN treatment to a level indistinguishable from ACC-deficient primary hepatocytes. This study shows that lipogenesis is dispensable for liver tumorigenesis in mice treated with DEN, and identifies an important role for ACC enzymes in redox regulation and cell survival.
Project description:BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH) is a chronic liver disease characterized by hepatic lipid accumulation, inflammation, and progressive fibrosis. Acetyl-CoA carboxylase (ACC) catalyzes the rate-limiting step of de novo lipogenesis and regulates fatty-acid -oxidation in hepatocytes. ACC inhibition reduces hepatic fat content and markers of liver injury in NASH patients; however, the effect of ACC inhibition on liver fibrosis has not been reported. METHODS: A direct role for ACC in fibrosis was evaluated by measuring de novo lipogenesis, procollagen production, gene expression, glycolysis, and mitochondrial respiration in hepatic stellate cells (HSCs) in the absence or presence of small-molecule inhibitors of ACC. ACC inhibitors were evaluated in rodent models of liver fibrosis induced by diet or the hepatotoxin, DEN. Fibrosis and hepatic steatosis were evaluated by histological and biochemical assessments. RESULTS: In TGF--stimulated HSCs, ACC inhibition reduced activation and collagen production independent of mitochondrial -oxidation by blocking de novo lipogenesis. ACC inhibition prevented a metabolic switch necessary for induction of glycolysis and oxidative phosphorylation during HSC activation. Consistent with this direct anti-fibrotic mechanism in HSCs, ACC inhibition reduced liver fibrosis in a rat CDHFD model and in response to chronic DEN-induced liver injury that lacked hepatic lipid accumulation. CONCLUSIONS: In addition to reducing lipid accumulation in hepatocytes, ACC inhibition also directly impairs the pro-fibrogenic activity of HSCs. Small molecule inhibitors of ACC may reduce liver fibrosis by both reducing lipotoxicity in hepatocytes and directly reducing HSC activation, providing a mechanistic rationale for the treatment of patients with advanced liver fibrosis due to NASH. Overall design: Liver samples from mice fed a normal diet or fed an FFD and treated with vehicle or an ACC inhibitor. Three day time course of primary hepatic stellate cells treated with an ACC inhibitor, TGF beta, or a TGF inhibitor.
Project description:BACKGROUND & AIMS:Although there are associations among oxidative stress, reduced nicotinamide adenine dinucleotide phosphate oxidase (NOX) activation, and hepatocellular carcinoma (HCC) development, it is not clear how NOX contributes to hepatocarcinogenesis. We studied the functions of different NOX proteins in mice after administration of a liver carcinogen. METHODS:Fourteen-day-old Nox1-/- mice, Nox4-/- mice, Nox1-/-Nox4-/- (double-knockout) mice, and wild-type (WT) C57BL/6 mice were given a single intraperitoneal injection of diethylnitrosamine (DEN) and liver tumors were examined at 9 months. We also studied the effects of DEN in mice with disruption of Nox1 specifically in hepatocytes (Nox1?Hep), hepatic stellate cells (Nox1?Hep), or macrophages (Nox1?Mac). Some mice were also given injections of the NOX1-specific inhibitor ML171. To study the acute effects of DEN, 8-12-week-old mice were given a single intraperitoneal injection, and liver and serum were collected at 72 hours. Liver tissues were analyzed by histologic examination, quantitative polymerase chain reaction, and immunoblots. Hepatocytes and macrophages were isolated from WT and knockout mice and analyzed by immunoblots. RESULTS:Nox4-/- mice and WT mice developed liver tumors within 9 months after administration of DEN, whereas Nox1-/- mice developed 80% fewer tumors, which were 50% smaller than those of WT mice. Nox1?Hep and Nox1?HSC mice developed liver tumors of the same number and size as WT mice, whereas Nox1?Mac developed fewer and smaller tumors, similar to Nox1-/- mice. After DEN injection, levels of tumor necrosis factor, interleukin 6 (IL6), and phosphorylated signal transducer and activator of transcription 3 were increased in livers from WT, but not Nox1-/- or Nox1?Mac, mice. Conditioned medium from necrotic hepatocytes induced expression of NOX1 in cultured macrophages, followed by expression of tumor necrosis factor, IL6, and other inflammatory cytokines; this medium did not induce expression of IL6 or cytokines in Nox1?Mac macrophages. WT mice given DEN followed by ML171 developed fewer and smaller liver tumors than mice given DEN followed by vehicle. CONCLUSIONS:In mice given injections of a liver carcinogen (DEN), expression of NOX1 by macrophages promotes hepatic tumorigenesis by inducing the production of inflammatory cytokines. We propose that upon liver injury, damage-associated molecular patterns released from dying hepatocytes activate liver macrophages to produce cytokines that promote tumor development. Strategies to block NOX1 or these cytokines might be developed to slow hepatocellular carcinoma progression.
Project description:Inhibiting lipogenesis prevents hepatic steatosis in rodents with insulin resistance. To determine if reducing lipogenesis functions similarly in humans, we developed MK-4074, a liver-specific inhibitor of acetyl-CoA carboxylase (ACC1) and (ACC2), enzymes that produce malonyl-CoA for fatty acid synthesis. MK-4074 administered to subjects with hepatic steatosis for 1 month lowered lipogenesis, increased ketones, and reduced liver triglycerides by 36%. Unexpectedly, MK-4074 increased plasma triglycerides by 200%. To further investigate, mice that lack ACC1 and ACC2 in hepatocytes (ACC dLKO) were generated. Deletion of ACCs decreased polyunsaturated fatty acid (PUFA) concentrations in liver due to reduced malonyl-CoA, which is required for elongation of essential fatty acids. PUFA deficiency induced SREBP-1c, which increased GPAT1 expression and VLDL secretion. PUFA supplementation or siRNA-mediated knockdown of GPAT1 normalized plasma triglycerides. Thus, inhibiting lipogenesis in humans reduced hepatic steatosis, but inhibiting ACC resulted in hypertriglyceridemia due to activation of SREBP-1c and increased VLDL secretion.
Project description:Prominin 1 (PROM1) is one of a few clinically relevant progenitor markers in human alcoholic hepatitis (AH) and hepatocellular carcinoma (HCC), and mouse liver tumor initiating stem cell-like cells (TICs). However, the origin, fate and functions of PROM1+?cells in AH and HCC are unknown. Here we show by genetic lineage tracing that PROM1+?cells are derived in part from hepatocytes in AH and become tumor cells in mice with diethyl nitrosamine (DEN)-initiated, Western alcohol diet-promoted liver tumorigenesis. Our RNA sequencing analysis of mouse PROM1+?cells, reveals transcriptomic landscapes indicative of their identities as ductular reaction progenitors (DRPs) and TICs. Indeed, single-cell RNA sequencing reveals two subpopulations of Prom1+?Afp- DRPs and Prom1+?Afp+?TICs in the DEN-WAD model. Integrated bioinformatic analysis identifies Discodin Domain Receptor 1 (DDR1) as a uniquely upregulated and patient-relevant gene in PROM1+?cells in AH and HCC. Translational relevance of DDR1 is supported by its marked elevation in HCC which is inversely associated with patient survival. Further, knockdown of Ddr1 suppresses the growth of TICs and TIC-derived tumor growth in mice. These results suggest the importance of PROM1+?cells in the evolution of liver cancer and DDR1 as a potential driver of this process.
Project description:Transforming growth factor beta (TGF-?) signaling activates Smad- and TGF-?-activated kinase 1 (TAK1)-dependent signaling to regulate cell survival, proliferation, fibrosis, and tumorigenesis. The effects of TGF-? signaling on metabolic syndrome, including nonalcoholic fatty liver disease, remain elusive. Wild-type (WT) and hepatocyte-specific TGF-? receptor type II-deficient (Tgfbr2?HEP) mice were fed a choline-deficient amino acid (CDAA)-defined diet for 22 weeks to induce NASH. WT mice fed a CDAA diet displayed increased activation of Smad2/3 and had marked lipid accumulation, inflammatory cell infiltration, hepatocyte death, and fibrosis; in comparison, Tgfbr2?HEP mice fed a CDAA diet had suppressed liver steatosis, inflammation, and fibrosis. Both palmitate-induced steatotic hepatocytes and hepatocytes isolated from WT mice fed a CDAA diet had increased susceptibility to TGF-?-mediated death. TGF-?-mediated death in steatotic hepatocytes was inhibited by silencing Smad2 or blocking reactive oxygen species (ROS) production and was enhanced by inhibiting TAK1 or nuclear factor kappa B. Increased hepatic steatosis in WT mice fed a CDAA diet was associated with the increased expression of lipogenesis genes (Dgat1 and Srebp1c), whereas the decreased steatosis in Tgfbr2?HEP mice was accompanied by the increased expression of genes involved in ?-oxidation (Cpt1 and Acox1). In combination with palmitate treatment, TGF-? signaling promoted lipid accumulation with induction of lipogenesis-related genes and suppression of ?-oxidation-related genes in hepatocytes. Silencing Smad2 decreased TGF-?-mediated lipid accumulation and corrected altered gene expression related to lipid metabolism in hepatocytes. Finally, we confirmed that livers from patients with nonalcoholic steatohepatitis (NASH) displayed phosphorylation and nuclear translocation of Smad2/3.TGF-? signaling in hepatocytes contributes to hepatocyte death and lipid accumulation through Smad signaling and ROS production that promote the development of NASH.
Project description:Chronic liver inflammation is a crucial event in the development and growth of hepatocellular carcinoma (HCC). Compelling evidence has shown that interleukin-6 (IL-6)/gp130-dependent signaling has a fundamental role in liver carcinogenesis. Thus, in the present study we aimed to investigate the role of gp130 in hepatocytes for the initiation and progression of HCC. Hepatocyte-specific gp130 knockout mice (gp130(?hepa)) and control animals (gp130(f/f)) were treated with diethylnitrosamine (DEN). The role of gp130 for acute injury (0-144?h post treatment), tumor initiation (24 weeks) and progression (40 weeks) was analyzed. After acute DEN-induced liver injury we observed a reduction in the inflammatory response in gp130(?hepa) animals as reflected by decreased levels of IL-6 and oncostatin M. The loss of gp130 slightly attenuated the initiation of HCC 24 weeks after DEN treatment. In contrast, 40 weeks after DEN treatment, male and female gp130(?hepa) mice showed smaller tumors and reduced tumor burden, indicating a role for hepatocyte-specific gp130 expression during HCC progression. Oxidative stress and DNA damage were substantially and similarly increased by DEN in both gp130(f/f) and gp130(?hepa) animals. However, gp130(?hepa) livers revealed aberrant STAT5 activation and decreased levels of transforming growth factor-? (TGF?), pSMAD2/3 and SMAD2, whereas phosphorylation of STAT3 at Tyr705 and Ser727 was absent. Our results indicate that gp130 deletion in hepatocytes reduces progression, but not HCC initiation in the DEN model. Gp130 deletion resulted in STAT3 inhibition but increased STAT5 activation and diminished TGF-dependent signaling. Hence, blocking gp130 in hepatocytes might be an interesting therapeutic target to inhibit the growth of HCC.
Project description:Liver cancer is a major health-care concern and its oncogenic mechanisms are still largely unclear. Persistent hepatocyte cell death is a common feature among various chronic liver diseases, the blocking of which presents as logical treatment. Therefore, we aimed at investigating tumor development in mice with hepatocyte-specific Bid depletion--a BH3-only Bcl-2 family member that amplifies apoptotic death signals. Hepatocyte-specific conditional Bid-knockout mice (Bid(?hep)) were injected with 25 mg/kg diethylnitrosamine (DEN) at 14 days of age, and liver tumorigenesis was investigated 9 months later. Additionally, different models of acute liver injury were used including: acute high-dose DEN challenge, 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet and carbon tetrachloride (CCL4) injection. Bid(?hep) mice developed significantly fewer tumors, showed smaller maximal and average tumor size and reduced tumor incidence. In the acute DEN model, 48 h post injection we observed a significant reduction in liver injury in Bid(?hep) animals, assessed via serum transaminases and liver histopathology. Furthermore, TNF-?, IL-1ß, cJUN and IL-6 mRNA expression was reduced. These findings were accompanied by reduced compensatory hepatocyte proliferation in Bid(?hep) mice when compared with controls by immunohistochemistry for Ki67 and proliferating cell nuclear antigen 48 h after DEN injection. In the acute CCL4 model, Bid(?hep) mice displayed reductions in liver injury and inflammation when compared with controls. No differences in liver injury and serum bilirubin levels were detected in Bid(?hep) and Bid(flo/flo) mice fed with DDC, which induces bile duct injury and a ductular reaction. Our study demonstrates that in DEN-induced hepatocellular carcinoma, the inhibition of hepatocyte death pathways through Bid deletion protects animals from tumorigenesis. These results suggest that reducing hepatocyte cell death, liver inflammation and compensatory proliferation has a stronger beneficial effect than the potential side effect of enhancing tumor cell survival.
Project description:<h4>Background and aims</h4>G protein-coupled receptor (GPR) 55 is a putative cannabinoid receptor, and l-?-lysophosphatidylinositol (LPI) is its only known endogenous ligand. Although GPR55 has been linked to energy homeostasis in different organs, its specific role in lipid metabolism in the liver and its contribution to the pathophysiology of nonalcoholic fatty liver disease (NAFLD) remains unknown.<h4>Approach and results</h4>We measured (1) GPR55 expression in the liver of patients with NAFLD compared with individuals without obesity and without liver disease, as well as animal models with steatosis and nonalcoholic steatohepatitis (NASH), and (2) the effects of LPI and genetic disruption of GPR55 in mice, human hepatocytes, and human hepatic stellate cells. Notably, we found that circulating LPI and liver expression of GPR55 were up-regulated in patients with NASH. LPI induced adenosine monophosphate-activated protein kinase activation of acetyl-coenzyme A carboxylase (ACC) and increased lipid content in human hepatocytes and in the liver of treated mice by inducing de novo lipogenesis and decreasing ?-oxidation. The inhibition of GPR55 and ACC? blocked the effects of LPI, and the in vivo knockdown of GPR55 was sufficient to improve liver damage in mice fed a high-fat diet and in mice fed a methionine-choline-deficient diet. Finally, LPI promoted the initiation of hepatic stellate cell activation by stimulating GPR55 and activation of ACC.<h4>Conclusions</h4>The LPI/GPR55 system plays a role in the development of NAFLD and NASH by activating ACC.
Project description:Metformin is the mainstay therapy for type 2 diabetes (T2D) and many patients also take salicylate-based drugs [i.e., aspirin (ASA)] for cardioprotection. Metformin and salicylate both increase AMP-activated protein kinase (AMPK) activity but by distinct mechanisms, with metformin altering cellular adenylate charge (increasing AMP) and salicylate interacting directly at the AMPK ?1 drug-binding site. AMPK activation by both drugs results in phosphorylation of ACC (acetyl-CoA carboxylase; P-ACC) and inhibition of acetyl-CoA carboxylase (ACC), the rate limiting enzyme controlling fatty acid synthesis (lipogenesis). We find doses of metformin and salicylate used clinically synergistically activate AMPK in vitro and in vivo, resulting in reduced liver lipogenesis, lower liver lipid levels and improved insulin sensitivity in mice. Synergism occurs in cell-free assays and is specific for the AMPK ?1 subunit. These effects are also observed in primary human hepatocytes and patients with dysglycaemia exhibit additional improvements in a marker of insulin resistance (proinsulin) when treated with ASA and metformin compared with either drug alone. These data indicate that metformin-salicylate combination therapy may be efficacious for the treatment of non-alcoholic fatty liver disease (NAFLD) and T2D.
Project description:PTEN is a tumor suppressor gene mutated in many human cancers, and its expression is reduced or absent in almost half of hepatoma patients. We used the Cre-loxP system to generate a hepatocyte-specific null mutation of Pten in mice (AlbCrePten(flox/flox) mice). AlbCrePten(flox/flox) mice showed massive hepatomegaly and steatohepatitis with triglyceride accumulation, a phenotype similar to human nonalcoholic steatohepatitis. Adipocyte-specific genes were induced in mutant hepatocytes, implying adipogenic-like transformation of these cells. Genes involved in lipogenesis and beta-oxidation were also induced, possibly as a result of elevated levels of the transactivating factors PPARgamma and SREBP1c. Importantly, the loss of Pten function in the liver led to tumorigenesis, with 47% of AlbCrePten(flox/flox) livers developing liver cell adenomas by 44 weeks of age. By 74-78 weeks of age, 100% of AlbCrePten(flox/flox) livers showed adenomas and 66% had hepatocellular carcinomas. AlbCrePten(flox/flox) mice also showed insulin hypersensitivity. In vitro, AlbCrePten(flox/flox) hepatocytes were hyperproliferative and showed increased hyperoxidation with abnormal activation of protein kinase B and MAPK. Pten is thus an important regulator of lipogenesis, glucose metabolism, hepatocyte homeostasis, and tumorigenesis in the liver.