MCD Diet Research Articles

1811-P: Induction of Hepatic Steatosis by a Methionine Choline Deficient Diet Promotes FGF21-Induced Insulin Secretion Independent of Diabetes

While it is known that patients with nonalcoholic fatty liver disease exhibit enhanced insulin secretion, the factor(s) that contribute to this effect are not fully understood especially in nondiabetic subjects. Our aim was to examine crosstalk between a steatotic liver induced by a diet that is deficient in methionine choline (MCD) and pancreatic islets in the absence of diabetes/insulin resistance. Nine-week old male C57BL/6J mice were fed normal chow (CTL) or the MCD diet for 3 weeks followed by evaluation of glucose homeostasis, liver and pancreas pathology and hepatic gene expression by RNA-sequencing. Three weeks of MCD diet caused simple hepatic steatosis with hepatic triglyceride content 2-fold higher in MCD compared to CTL (n=5; p<0.05). Hepatic FGF-21 mRNA, protein and circulating FGF-21 levels were all significantly increased in MCD (129-fold, 8-fold, 5-fold respectively; n=5; p<0.05). Interestingly, while β-cell mass was not significantly altered between groups, insulin secretion as evaluated by ex-vivo GSIS in freshly isolated islets was significantly increased in MCD (5-fold; p<0.05, n=10). Examination of liver samples from patients with or without steatosis mimicked hepatic MCD data in the mouse model. Thus a significant increase in both mRNA and protein FGF-21 were evident in the steatotic group (3-fold, 5-fold respectively; n=6, p<0.05). Furthermore, 3 days treatment of nondiabetic human islets with recombinant FGF-21 protein led to a significant increase in GSIS compared to control (1.5-fold; p<0.05, n=4). FGF-21 treatment of EndoC-βH1 cells revealed significant activation of AKT and ERK1/2 signaling that likely promoted the enhanced insulin secretion. Taken together, liver steatosis significantly increases circulating FGF-21 to promote enhanced insulin secretion via improved signaling in nondiabetic beta cells. Disclosure T. Kimura: None. D.F. De Jesus: None. K. Fukuda: None. S. Kahraman: None. K. Shibue: None. K. Orime: None. R. Kulkarni: None. Funding National Institutes of Health (R01DK067536, R01DK103215)

SAT-LB124 Phosphatidylcholine Transfer Protein Interacts With PPARδ to Modulate Activity

Obesity is one of the largest public health crises in the USA. Obesity can be lethal due to the development of cardiovascular complications such as, hypertension, heart attack, and stroke. Recently, peroxisome proliferator-activated receptor δ (PPARδ), the least-characterized member of the PPAR family of nuclear receptors (NRs), has shown great promise in treating obesity and associated cardiovascular complications. Recent reports have shown that PPARδ activation is tuned, in part, through an interaction with fatty acid binding protein 5 (FABP5) in a polyunsaturated Fatty acids (PUFAs) dependent manner. This enhancement is thought to occur due to ligand transfer, however, FABP5 only binds a subset of reported PPARδ ligands. To find other candidate lipid transport proteins (LTPs), we performed a protein complementation assay (PCA) between LTPs and NRs. We uncovered a novel interaction between PPARδ and phosphatidylcholine transfer protein (PC-TP), sensitive to cellular nutrient levels. Preliminary data show that this interaction opposes canonical PPARδ signaling, leading to a decrease in PPARδ transactivation in cells and isolated mice livers. This led me to hypothesize that PC-TP senses nutrient status through membrane composition. Specific PC molecular species drive PC-TP translocation to inhibit PPARδ transactivation of genes. Utilizing our novel PCA assay I have shown that the interaction is modulated in part by cellular levels of methionine and choline, as cells cultured in media depleted of methionine and choline (MCD) show a decreased in the interaction. Using the same assay, I have shown a requirement of full length PPARδ for the interaction with PC-TP. This analysis will be complimented by mutagenesis and chemical perturbations aimed to alter PC-TP and PPARδ function. Additionally, I have assayed the in vivo relevance of PC-TP interaction with PPARδ using livers harvested from PCTP-/- and littermate control mice fed a variety of diets. Preliminary RNA-seq characterization, show interesting alterations in gene expression that suggest more complex regulation between PC-TP and PPARδ in vivo. MCD, which was shown to reduce the inhibitory interaction between PC-TP and PPARδ in vitro, seems to lead to an increased effect on PPARδ transactivation when PC-TP is depleted. Analysis comparing the effect of diet within each genotype shows a loss of differential PPARδ regulation for both CHEA and KEGG analysis when comparing WT to PCTP -/- mice supporting a role for PC-TP in differential PPARδ regulation caused by MCD diet. These studies will further the understanding of how lipid homeostasis is sensed and maintained through PPARδ by interactions with two separate LTPs.

G-Protein-Coupled Receptors 120 Agonist III Improves Hepatic Inflammation and ER Stress in Steatohepatitis.

GPR120 plays a crucial role in insulin sensitization, inflammatory responses and obesity and is considered as an attractive potential target for the treatment of metabolic dysfunctions. However, the mechanisms of GPR120 agonist III in NAFLD/NASH treatment are still unclear. We aimed to evaluate the effect and molecular mechanisms of GPR120 agonist III on NASH, and search for future treatments of human NAFLD/NASH. The effects of GPR120 agonist III on steatohepatitis were evaluated in mice fed with HFHC diet and MCD diet. The ultrastructural changes of ER were assessed by TEM. Hepatic ROS production was evaluated by DHE staining. Apoptosis and macrophage infiltration were determined by IHC staining. Inflammatory cytokines secretion were examined using mouse XL cytokine array. GPR120 agonist III significantly suppressed macrophage infiltration and ROS production and reversed hepatic inflammation, ER stress and apoptosis in dietary-induced steatohepatitis. GPR120 agonist III will be an attractive treatment method in steatohepatitis, which opens up a new sight for future treatments of human NAFLD/NASH.

RETRACTED: Role of hepatic neuropeptide Y-Y1 receptors in a methionine-cholinedeficient model of non-alcoholic steatohepatitis

NPY-Y1R plays an important role in dietary regulation. Although germline knockdown of NPY-Y1R in mice alleviates high-fat-diet-induced obesity and increases CPT1α levels in the liver, the role of the Y1 receptor in specific tissues has not been studied. MCD diet is the most widely used method to establish a model of lean NASH in a short time. We therefore evaluated the role of liver NPY-Y1R in NASH progression. In mice with liver-specific knockout of NPY-Y1R (LivKO) and wild-type control littermates fed MCD diet for 4weeks, NPY-Y1R deficiency significantly decreased body and liver weight. Moreover, NPY-Y1R deletion protected mice against hepatic steatosis and injury. LivKO decreased TG, TC, and FFA levels in the liver and alanine aminotransferase activity in plasma. To clarify the mechanism, we evaluated the key enzymes involved in triglyceride hydrolase and fatty-acid oxidase. Expression of ATGL, CPT1α, and ACO was significantly increased in LivKO mice, whereas expression of fatty-acid synthase was significantly decreased. mRNA expression analysis revealed a marked reduction of genes involved in de-novo lipogenesis and monosaturated fatty-acid synthesis, including sterol-regulatory element-binding protein 1c and fatty-acid synthase. Moreover, liver injury-related factors were significantly decreased in LivKO mice, such as TNF-α, inducible nitric oxide synthase, and MCP-1. Thus, NPY-Y1R deficiency in the liver alleviates lipid deposition and injury. However, NPY-Y1R did not affect inflammation and fibrosis. NPY-Y1R deficiency in the liver directly suppresses not only hepatic steatosis, but also liver injury, and thus provides a treatment option for NASH.

Effects of Qutan Huoxue Formula on the SOCS1/TLR4 Signaling Pathway in NASH Model Mice.

The purpose of this study was to investigate the effects of Qutan Huoxue Formula (QHF) on liver injury in mice with nonalcoholic steatohepatitis (NASH) by upregulating SOCS1 to inhibit the TLR4/NF-κB signaling pathway. Thirty male C57BL/6J mice (20–22 g) were randomly divided into the normal diet group (ND group), methionine- and choline-deficient diet group (MCD group), and Qutan Huoxue Formula group (QHF group). Mice in the ND group were fed a regular diet, while mice in other two groups were fed MCD diet. After successful molding, the QHF group was gavaged by QHF. The ND group and MCD group were gavaged by the same volume of normal saline, once a day. During the period of gavaging, all mice continue to be fed MCD fodder except for the ND group. All mice were killed at 8 w. H&E staining and Oil Red O staining were used to observe the pathological changes of liver tissues. Serum level of ALT, AST, TC, and TG was detected by enzyme-linked immunosorbent assay. The expression of liver SOCS1, TLR4, Myd88, and NF-κB was detected by real-time PCR, immunohistochemistry, and Western blot. QHF can significantly reduce the serum levels of ALT, AST, TC, and TG of NASH mice and reduce the degree of liver fat degeneration and inflammation. It also can decrease both mRNA and protein expressions of liver TLR4, Myd88, and NF-κB. The mRNA expression of SOCS1 increased, while the SOCS1 protein expression decreased. In conclusion, QHF can significantly alleviate hepatic steatosis and inflammation in NASH mice by upregulating SOCS1 to inhibit the TLR4/NF-κB signaling pathway.

1851-P: Bromocriptine (B) plus Curcumin (C) Interact to Reduce Advanced Fibrosis in a Mouse Methionine-Choline Deficient (MCD) Diet Model of Nonalcoholic Steatohepatitis (NASH)

NASH is characterized by severe hepatic fibrosis that potentiates cirrhosis and hepatocellular carcinoma. The MCD diet also potentiates severe liver fibrosis by reducing lipid secretion and antioxidant defenses, and adversely altering the gut microbiome to promote liver inflammation and damage. B, a dopamine D2 receptor agonist reduces adipose lipolysis and hepatic lipogenesis and inflammation. C improves a gut microbiome adverse to liver. C is also known to augment brain dopaminergic activity. We therefore investigated if B and C combination therapy that engages different pathological targets of NASH may provide an additive/synergistic benefit against MCD diet induced liver fibrosis. C57B6 mice were fed an MCD diet for 8 weeks to induce NASH with fibrosis then randomized to 4 treatment groups: Vehicle, B (10mg/kg), C (100mg/kg), and combination of B and C (BC) daily for 4 weeks while maintained on the diet. A group of mice on normal diet were used as normal reference control. BC therapy led to significant reductions of serum ALT (85%), serum AST (33.4%), liver steatosis (65.4%), inflammation (86.7%), ballooning degeneration (60%), and NAFLD activity score (86.4%), as well as gene expression markers each of fibrosis (αSMA, TGFβ, COL1A, TXNIP) by 88-92%, inflammation (TNFa, IL1β, MCP1, iNOS) by 91-97%, ER stress (DDIT3, ATF, sXBP1, HSPA5) by 45-93%, steatosis (ACC1, PLIN2) by 31-97%, and fatty acid oxidation (PPARα, PGC1α, CPT1, UCP1) by 65-86, and reduced the percent fibrosis area by 92.7% relative to vehicle (and resultantly similar to normal diet mice) (all above: P<0.05) and by amounts greater than B or C alone or additively. This study demonstrates the positive interactive effects of bromocriptine and curcumin on advanced liver fibrosis in an MCD diet model of NASH. Disclosure T. Tsai: Employee; Self; VeroScience LLC. A. Cincotta: Employee; Self; VeroScience LLC.

The A2B Adenosine Receptor Antagonist Alloxazine Exacerbates NASH and Disrupts Cellular Folate Metabolism in Methionine and Choline Deficient Mice

Nonalcoholic fatty liver disease is characterized by hepatic lipid accumulation of greater than 5% and is a rising healthcare problem in the United States. Approximately 25–30% of the US population may have a fatty liver and 10–15% of these patients will progress to more severe disease known as nonalcoholic steatohepatitis (NASH). There are no approved treatments for NASH and the factors that result in the transition from fatty liver to NASH are not well defined. Folate and one‐carbon metabolism are implicated in the pathogenesis of NASH and are important pathways for the biosynthesis of many important molecules like creatine, phosphatidylcholine, purines, and thymidine. Additionally, the purine adenosine modulates immune cells by signaling to cell surface receptors on macrophages in the liver. In the present study, mice were fed a methionine and choline deficient diet to induce NASH lesions and treated with a putative A2B adenosine receptor antagonist, alloxazine, to probe the role of this adenosine receptor in NASH. MCD diet feeding reduced purine and thymidine nucleotides in the liver; potentially limiting the availability of crucial molecules for regeneration of liver tissue. MCD mice showed reduced activating transcription factor 4 (ATF4) expression, which has been shown to regulate mitochondrial folate metabolism and formate production which is required for purine biosynthesis. Alloxazine treatment exacerbated hepatic lipid accumulation and caused severe lobular inflammation in MCD mice. Alloxazine also disrupted hepatic folate metabolism and reduced the concentration of formate without altering concentrations of S‐adenosylmethionine and S‐adenosylhomocysteine. These data suggest that in addition to its function as an A2B adenosine receptor antagonist, alloxazine also impairs cellular folate metabolism. These data provide a novel link between folate and lipid metabolism, and inflammation in NASH.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MiR-218-XOR-ROS Pathway Regulates the Progression of Nonalcoholic Steatohepatitis.

To investigate the role of the miR-218-xanthine oxidoreductase (XOR) pathway in the pathogenesis of nonalcoholic steatohepatitis (NASH) and to explore the potential downstream mechanisms involving oxidative stress and energy metabolism. The NASH animal model was established by feeding BALB/c mice with an MCD diet, while BRL-3A cells were cultured with a mixture of oleate and palmitate for 72 hours to mimic the steatosis and inflammation of NASH in vitro. The steatosis and inflammation levels were assessed by H-E/oil-red staining and serum/supernatant TG, ALT, and AST levels. The apoptosis degree was tested by the TUNEL/flow cytometry method both in animals and cultured cells. The XOR and miR-218 levels were detected by western blotting and qRT-PCR. Decreased miR-218 and increased XOR levels were identified in the NASH animal and cell models, while the regulation of miR-218 on XOR was also confirmed. NASH alleviation was achieved after miR-218 over-expression in vivo and in vitro, according to the declination of steatosis and inflammation-related markers. Although H2O2 and ATP levels were increased and decreased in NASH models, respectively, antagonizing miR-218 could significantly alleviate those changes. The miR-218-XOR pathway may provide a novel mechanism and treatment option for NASH.

Upregulation of non-canonical Wnt ligands and oxidative glucose metabolism in NASH induced by methionine-choline-deficient diet

Wnt ligands regulate metabolic pathways, and dysregulation of Wnt signaling contributes to chronic inflammatory disease. A knowledge gap exists concerning the role of aberrant Wnt signaling in non-alcoholic steatohepatitis (NASH), which exhibits metabolic syndrome and inflammation. Using a mouse model of methionine-choline deficient diet (MCDD)-induced NASH, we investigated the Wnt signaling pathways in relation to hepatic glucose oxidation. Mice fed the MCD diet for 6 weeks developed prominent NASH marked by macrovesicular steatosis, inflammation and lipid peroxidation. qPCR analysis reveals differential hepatic expression of canonical and non-canonical Wnt ligands. While expression of Wnt3a was decreased in NASH vs chow diet control, expression of Wnt5a and Wnt11 were increased 3 fold and 15 fold, respectively. Consistent with activation of non-canonical Wnt signaling, expression of the alternative Wnt receptor ROR2 was increased 5 fold with no change in LRP6 expression. Activities of the metabolic enzymes glucokinase, phosphoglucoisomerase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, and pyruvate dehydrogenase were all elevated by MCDD. NASH-driven glucose oxidation was accompanied by a 6-fold increase in lactate dehydrogenase (LDH)-B with no change in LDH-A. In addition, glucose-6-phosphate dehydrogenase, the regulatory and NADPH-producing enzyme of the pentose phosphate pathway, was elevated in NASH. These data support a role of accelerated glucose oxidation in the development of NASH, which may be driven by non-canonical Wnt signaling.

Administration of methyl palmitate prevents non-alcoholic steatohepatitis (NASH) by induction of PPAR-α

Background and aimsThe lack of valid therapeutic approach that can ameliorate the manifestations of NASH is a barrier to therapeutic development. Therefore, we investigate the novel role of Methyl Palmitate (MP) in preventing NASH and the possible mechanism involved. Methods50 Male C57BL/6 J mice were randomly divided into 5 groups (n = 10). The control group was fed control diet; model group was fed MCD diet; MP 1 group was fed MCD diet supplemented with MP (75 mg/kg/day); MP 2 group was fed MCD plus MP diet (150 mg/kg/day); and MP 3 group was fed MCD plus MP diet (300 mg/kg/day). Histological staining’s, and commercially available kits for serum ALT and AST and hepatic contents of TG, TC, MDA, SOD, and GSH were used to assess NASH. Furthermore, relative liver protein and gene expression levels were determined by Western Blot and qPCR, respectively. ResultsMice fed MCD diet developed NASH, which was markedly improved by MP in a dose-dependent manner. MP treatment improved hepatic content of TG, TC, MDA, SOD and GSH and serum levels of ALT and AST. In vivo studies showed that MP treatment activated PPARα expression, that in turns, promoted β-oxidation protein and gene expressions, suppressed TNFα, MCP1, TGFβ1 and Colla1 protein and gene expression levels, contributing to the prevention of NASH. ConclusionsOur results indicated that MP could successfully prevent NASH. This effect of MP was mediated through induction of PPARα pathway. This study provides a novel therapeutic target that plays pivotal role in the prevention of NASH.

Novel hepatoprotective role of Leonurine hydrochloride against experimental non-alcoholic steatohepatitis mediated via AMPK/SREBP1 signaling pathway

Background and aimsNon-alcoholic steatohepatitis (NASH) is the hepatic manifestation of metabolic syndrome and is characterized by steatosis, inflammation, and fibrosis. We aim to characterize the hepatoprotective effects of Leonurine hydrochloride (LH) and the possible pathway in a cell and rodent model of diet-induced steatohepatitis (NASH). MethodsFor in vitro studies, Palmitic acid (PA) and free fatty acid (FFA) induced HepG2 and HL7702 steatosis cell models were used. For in vivo studies, NASH was induced by feeding mice MCD diet. These mice received either placebo or LH at three different doses (50、100、200 mg/kg/day) for 6 weeks. Histological staining’s, and commercially available kits for ALT and AST and hepatic contents of TG, TC, MDA, SOD, and GSH were used to assess NASH. Furthermore, relative liver protein and gene expression levels were determined by Western Blot and qPCR, respectively. ResultsAfter establishing NASH models, LH treatment improved lipid accumulation, hepatic contents of TG, TC, and expression levels of ALT and AST in dose-dependent manner. Also, LH improved MDA, SOD, and GSH expression levels. The results of RT-PCR and Western blotting showed that LH upregulated the expression of AMPK phosphorylation and downregulated SREBP-1c and its target genes expression level. ConclusionsOur data reveal the promising role of Leonurine hydrochloride in the prevention and treatment of NASH, in vitro and in vivo. This effect may be partially mediated by the AMPK/SREBP1 pathway. These findings provide a novel therapeutic target for the clinical treatment of NASH.

Liver Histological Improvement After Administration of High-Dose Vitamin C in Guinea Pig with Nonalcoholic Steatohepatitis.

Background: Vitamin C is a strong antioxidant, and the health effects of vitamin C megadoses have not been validated despite the apparent health benefits. Therefore, the present study sought to confirm the effects of vitamin C megadoses. Materials and Methods : Four groups of six guinea pigs were used. Each group was fed one of the following diets for three weeks: normal diet, methionine choline-deficient diet, methionine choline-deficient diet + vitamin C megadose (MCD + vit C 2.5 g/kg/day), and methionine-choline deficient diet + ursodeoxycholic acid (MCD + UDCA 30 mg/kg/day). The MCD diet was given to induce nonalcoholic steatohepatitis, and UDCA was used to treat nonalcoholic steatohepatitis. Three weeks after initial diet administration, the results of biochemical tests and liver biopsy were compared between the groups. Results: The cytoplasm state was similar in the MCD + vit C and MCD + UDCA groups, exhibiting clearing of the cytoplasm and ballooning degeneration. However, macrovesicular steatosis was not observed in the MCD + vit C group. Aspartate transaminase and alanine transaminase were elevated significantly following vitamin C administration. Conclusions: The present study confirmed that alone vitamin C megadoses are potential remedies for nonalcoholic steatohepatitis, based on the liver biopsy results of guinea pigs that were unable to synthesize vitamin C.

Protective effects of Kangxian ruangan capsule against nonalcoholic fatty liver disease fibrosis in rats induced by MCD diet.

Kangxian ruangan (KXRG) capsule is a classical formula containing various herbals that play a vital role of replenishing spleen and warming Yang. Traditional Chinese medicine believes that insufficiency of the spleen, damp-heat, and phlegm and stasis are the key factors to nonalcoholic fatty liver disease (NAFLD). The objective of this study was to investigate the effects of KXRG capsule on NAFLD fibrosis rats induced by MCD diet. The liver functions (ALT, AST and GGT) and levels of blood lipids (CHOL and TG) in each treatment group rats were significantly decreased, especially those in H-KXRG group. At the same time, the KXRG capsule alleviated the inflammatory response, histopathological changes and liver fibrosis of NAFLD fibrosis rats. In addition, the apoptosis of liver cells induced by diet was obvious via TUNEL staining. However, KXRG capsule reversed that negative change. Moreover, the levels of pro-apoptotic proteins (Caspase 3, 8, 9 and Bax) were reduced by exposure to KXRG capsule, except that the anti-apoptotic proteins (Bcl-2 and Bcl-XL) were elevated. In conclusion, KXRG relieved the progression of NAFLD fibrosis via maintaining the balance of TNF-α/IL-10 further relieving the inflammatory reaction, and regulating the balance of Bcl-2/Bax or Bcl-XL/Bax in a positive direction further activating damaged hepatocytes.

Effect of Phyllanthus emblica L. fruit on methionine and choline-deficiency diet-induced nonalcoholic steatohepatitis

Phyllanthus emblica L. fruit contains abundant bioactive components and exhibits a variety of biological activities. In this study, the hepatoprotective effect of water extract of P. emblica (WEPE) on nonalcoholic steatohepatitis (NASH) was evaluated. C57BL/6 mice were fed methionine and choline-deficiency diet (MCD diet) for 4 or 8 weeks to induce NASH. Results showed that administration of WEPE could significantly reduce serum AST and ALT as compared to MCD diet-alone group. Administration of WEPE could significantly decrease lipid peroxidation and CYP2E1 mRNA expression, and elevate the antioxidant activities in mice livers. In addition, administration of WEPE after 8 weeks could significantly decrease the mRNA expressions of TNF-α and IL-1β in mice livers, but have less improving effect of hepatic steatosis and mononuclear cell infiltration. Taken together, MCD diet might cause serious hepatic steatosis and mild inflammation in mice livers, but administration of WEPE could ameliorate the rapid progression of NASH.

Decreased Hepatic Lactotransferrin Induces Hepatic Steatosis in Chronic Non-Alcoholic Fatty Liver Disease Model

Background/Aims: Non-alcoholic fatty liver disease (NAFLD) is an emerging metabolic disease. Although it leads to severe hepatic diseases including steatohepatitis, cirrhosis, and hepatic cancer, little is known about therapy to prevent and cure hepatic steatosis, the first step of NAFLD. We conducted this investigation to unveil the mechanism of hepatic steatosis. Methods: We established a novel chronic NAFLD mouse model through whole body irradiation and verified the model through histological and biochemical analysis. To find molecular mechanism for hepatic steatosis, we analyzed hepatic transcriptomic profiles in this model and selected target molecule. To induce the expression of lactotransferrin (Ltf) and regulate the NAFLD, growth hormone (GH) and coumestrol was introduced to hepatocyte and mice. The universal effect of coumestrol was confirmed by administration of coumestrol to NAFLD mouse model induced by high-fructose, high-fat, and MCD diet. Results: It was observed that decreased hepatic Ltf expression led to excessive hepatic lipid accumulation in NAFLD mouse. Furthermore, we found that GH was decreased in irradiated mice and functioned as an upstream regulator of Ltf expression. It was observed that GH could stimulate Ltf expression and prevent uptake of dietary lipids in hepatocytes, leading to rescue of NAFLD. Finally, we suggested that coumestrol, a kind of isoflavonoid, could be used as an inducer of hepatic Ltf expression through cooperation with the GH signaling pathway both in vitro and in vivo. Conclusions: Hepatic Ltf prevents hepatic steatosis through inhibition of dietary lipid uptake in radiation-induced NAFLD mouse model. We also suggest coumestrol as a drug candidate for prevention of NAFLD.

Metformin ameliorates activation of hepatic stellate cells and hepatic fibrosis by succinate and GPR91 inhibition

BackgroundChronic liver disease is becoming a major cause of morbidity and mortality worldwide. During liver injury, hepatic stellate cells (HSCs) trans-differentiate into activated myofibroblasts, which produce extracellular matrix.Succinate and succinate receptor (G-protein coupled receptor91, GPR91) signaling pathway has now emerged as a regulator of metabolic signaling. A previous study showed that succinate and its specific receptor, GPR91, are involved in the activation of HSCs and the overexpression of α-smooth muscle actin (α-SMA).Metformin, a well-known anti-diabetic drug, inhibits hepatic gluconeogenesis in the liver. Many studies have shown that metformin not only prevented, but also reversed, steatosis and inflammation in a nonalcoholic steatohepatitis (NASH) animal model. However, the role of metformin in HSC activation and succinate-GPR91 signaling has not been clarified. MethodsThe immortalized human HSCs, LX-2 cells, were used for the in vitro study. For the in vivo study, male C57BL/J6 mice were randomly divided into 3 groups and were fed with a methionine-choline-deficient diet (MCD diet group) as a nonalcoholic steatohepatitis (NASH) mouse model with or without 0.1% metformin for 12 weeks, or were fed a control methionine-choline-sufficient diet (MCS diet group). ResultsIn our study, metformin and 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR), which is an analog of adenosine monophosphate, were shown to suppress α-SMA expression via enhanced phosphorylation of AMP-activated protein kinase (AMPK) and inhibition of succinate-GPR91 signaling in activated LX-2 cells induced by palmitate- or succinate. Metformin and AICAR also reduced succinate concentration in the cell lysates when LX-2 cells were treated with palmitate. Moreover, metformin and AICAR reduced interleukin-6 and, transforming growth factor-β1 production in succinate-treated LX-2 cells. Both metformin and AICAR inhibited succinate-stimulated HSC proliferation and cell migration.Mice fed a MCD diet demonstrated increased steatohepatitis and liver fibrosis compared to that of mice fed control diet. Metformin ameliorated steatohepatitis, liver fibrosis, inflammatory cytokine production and decreased α -SMA and GPR91expression in the livers of the MCD diet-fed mice. ConclusionThis study shows that metformin can attenuate activation of HSCs by activating the AMPK pathway and inhibiting the succinate-GPR91 pathway. Metformin has therapeutic potential for treating steatohepatitis and liver fibrosis.

P2X7 Receptor-NOX2 Axis Mediates Tryptophan Oxidation in Murine and Human Nonalcoholic Fatty Liver Disease

Tryptophan oxidative metabolite kynurenine is known to be proinflammatory in neurodegeneration, autoimmune disease and sepsis. NAFLD is a proinflammatory condition where resident liver macrophages play a significant role in progression of the disease. With NAFLD being increasingly considered an epidemic in the western world and no suitable therapy available, it is important that novel approaches be studied for possible therapy. The present study shows that P2X7r causes the oxidation of tryptophan via NOX2 induced oxidative stress signaling. Further it may do so by the involvement of JAK/STAT pathway in the diseased liver macrophages. Results in both mouse (MCD Diet) and human liver biopsies showed an increase in tryptophan oxidation product n-formyl kynurenine (NFK) when compared to relevant controls. P2X7r KO mice or use of p47 phox deleted mice significantly decreased NOX2 activation and NFK immunoreactivity. Mechanistically, experiments conducted in immortalized Kupffer cells showed a significant upregulation of STAT3 phosphorylation in LPS-treated and P2X7r-Agonist-treated groups while use of P2X7r antagonist or Apocynin (NOX inhibitor) significantly decreased it. Concomitant use of apocynin and P2X7r agonist significantly decreased STAT3 phosphorylation suggesting that NOX2 acts downstream to P2X7r in these cells. Kynurenine levels in Kupffer cells decreased significantly in groups incubated with P2X7r antagonist, Apocynin or Ruxolitinib (JAK/STAT inhibitor). NASH Histology scores were significantly higher in wildtype MCD fed mice with a normal P2X7r phenotype while p47 phox KO or a P2X7r KO showed a decreased disease pathology. In conclusion, tryptophan metabolite NFK can be a strong indicator of NAFLD inflammation and can predict the stage of the disease based on the evidence in this study.

FXR controls CHOP expression in steatohepatitis

The farnesoid X receptor (FXR) and C/EBP homologous protein (CHOP) have critical functions in hepatic lipid metabolism. Here, we aimed to explore a potential relationship between FXR and CHOP. We fed wild‐type (WT) and FXR KO mice a MCD diet (model of steatohepatitis) and found that Chop mRNA expression is upregulated in WT but not FXR KO mice. The absence of FXR aggravates hepatic inflammation after MCD feeding. In HepG2 cells, we found that Chop expression is regulated in a FXR/Retinoid X receptor (RXR)‐dependent manner. We identified a FXR/RXR‐binding site in the human CHOP promoter, demonstrating a highly conserved regulatory pathway. Our study shows that FXR/RXR regulates Chop expression in a mouse model of steatohepatitis, providing novel insights into pathogenesis of this disorder.

Abstract 1909: Non-alcoholic steatohepatitis promotes EpCAM positive cancer stem cells mediated tumorigenesis in immunocompetent mouse model of hepatocellular carcinoma

Abstract Introduction: Non-alcoholic Steatohepatitis (NASH) is one of the major risk factors for cirrhosis and its progression to Hepatocellular Carcinoma (HCC). Cancer stem cells (CSCs) are sub-population of cells that bear stem-like properties, and are believed to contribute in tumor initiation and recurrence in tumor microenvironment. Understanding role of CSCs in tumorigenesis was limited to immunocompromised nude/SCID mouse models, and should be investigated in immunocompetent mouse model(s) representing clinical conditions. In this study, (1) we have investigated and confirmed CSCs properties in HCC cells lines, and (2) investigated tumor initiation capability of EpCAM positive CSCs in novel immunocompetent mouse model of HCC. Methods: In vitro CSC enrichment was achieved by treating murine (Hepa1-6) and human (HepG2, Hep3B) HCC cells in serum-free condition. To confirm CSCs, we analyzed CSC biomarkers (EpCAM,CD90,CD44,CD133) using flow-cytometry and Immunocytochemistry (ICC), and functional markers using Aldeflour assay and Hoechst-33342 efflux. Drug resistance property of CSCs was studied using Doxorubicin (anthracycline antibiotic) and Sorafenib (multikinase inhibitor) by MTT assay. To study in vivo tumorigenesis, immunocompetent mouse model was established using C57L/J mouse and copGFP expressing Hepa1-6 cells. Capability of EpCAM CSCs for tumor initiation was tested in 3 diet induced groups, i.e. control (10% dietary fat), high-fat (60% dietary fat), and NASH (60% dietary fat MCD diet with 0.1% methionine). 2 million FACS sorted EpCAM+/+ or EpCAM-/- cells were orthotopically injected into left liver lob. Tumor growth was monitored using high-frequency ultrasound and animals were euthanized after 18 days. Histology and ICC analysis was performed to confirm the tumors and findings. Results: Spheroid forming HCC-CSCs showed significant higher EpCAM expression and significant higher chemoresistance compared with control HCC cells. Our in vivo findings confirmed that EpCAM CSCs required NASH microenvironment. In NASH group mice, EpCAM+/+ CSCs have shown significant tumorigenesis compared with no tumors in EpCAM-/- (n=9, p<0.005). Histology analysis of NASH liver tissue confirmed lobular and portal inflammation, hepatocellular ballooning, and fibrosis. Control and high-fat diet group mice failed to develop tumors. Conclusions: We have developed a novel mouse model to study CSCs to overcome confounding limitations of immunocompromised mouse models for lacking functional immune system components. Our findings using immunocompetent mouse model suggest that, in HCC, (1) EpCAM+ CSCs cannot initiate tumor by itself within normal liver microenvironment in the presence of functional immune components, (2) NASH microenvironment promotes EpCAM+ CSCs mediated tumorigenesis while EpCAM-/- non-CSCs subset failed to develop tumor. Citation Format: Harshul Pandit, Yan Li, Guozhen Cui, Suping Li, Salina Li, Robert C. Martin. Non-alcoholic steatohepatitis promotes EpCAM positive cancer stem cells mediated tumorigenesis in immunocompetent mouse model of hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1909. doi:10.1158/1538-7445.AM2017-1909

Over-expressing the soluble gp130-Fc does not ameliorate methionine and choline deficient diet-induced non alcoholic steatohepatitis in mice.

Non-alcoholic steatohepatitis (NASH) is a liver disease with the potential to lead to cirrhosis and hepatocellular carcinoma. Interleukin-6 (IL-6) has been implicated in the pathogenesis of NASH, with the so-called IL-6 ‘trans-signaling’ cascade being responsible for the pro-inflammatory actions of this cytokine. We aimed to block IL-6 ‘trans-signaling’, using a transgenic mouse that overexpresses human soluble glycoprotein130 (sgp130Fc Tg mice) fed a commonly used dietary model of inducing NASH (methionine and choline deficient-diet; MCD diet) and hypothesized that markers of NASH would be ameliorated in such mice. Sgp130Fc Tg and littermate control mice were fed a MCD or control diet for 4 weeks. The MCD diet induced many hallmarks of NASH including hepatomegaly, steatosis, and liver inflammation. However, in contrast with other mouse models and, indeed, human NASH, the MCD diet model did not increase the mRNA or protein expression of IL-6. Not surprisingly, therefore, markers of MCD diet-induced NASH were unaffected by sgp130Fc transgenic expression. While the MCD diet model induces many pathophysiological markers of NASH, it does not induce increased IL-6 expression in the liver, a key hallmark of human NASH. We, therefore, caution the use of the MCD diet as a viable mouse model of NASH.