- Email: [email protected]
P0935 : Non-alcoholic steatohepatitis (NASH): 2 complementary dietary mouse models
POSTERS Results: NGM282 significantly reduced hepatic expression of proinflammatory cytokines (TNFa, IL-1b; Figure 1A) and chemokines (Ccl2, Cxcl2) as well as key pro-fibrotic genes including TGFb1 and Galectin-3 (Figure 1B). mRNA levels of collagen (Col1a1, Col1a2, Col3a1, Col14a1, Figure 1C), matrix metallopeptidases (Mmp2, Mmp12) and tissue inhibitors of matrix metallopeptidases (Timp1, Timp2) were normalized by NGM282 treatment. Histological analysis demonstrated that NGM282 reduced hepatic steatosis, immune cell infiltration, and bile duct proliferation in FXR-null mice. Conclusions: NGM282 protects aged FXR-null mice from developing steatosis, hepatic inflammation and fibrosis. These activities, in addition to the metabolic and BA synthetic actions, are supportive of the clinical development of NGM282 in NASH.
Figure 1.
P0934 LYSOPHOSPHATIDYLCHOLINE (LPC) AS CENTRAL PLAYER FOR HEPATIC FAT ACCUMULATION AND INFLAMMATION: IMPLICATION FOR PATHOGENESIS OF NASH W. Stremmel1 , S. Staffer1 , A. Wannhoff1 , A. Pathil-Warth1 , W. Chamulitrat1 . 1 Internal Medicine, University Clinics of Heidelberg Prof. Dr. W. Stremmel, Heidelberg, Germany E-mail: [email protected] Background and Aims: In NASH the intracellular ratio of phosphatidylcholine (PC): lysophosphatidylcholine (LPC) is decreased due to activation of the membrane localized phospholipase A2 (iPLA2b). Here we aim the evaluation of LPC levels in development and reversal of fat accumulation and inflammation. Methods: In HepG2 cells the bile acid-phospholipid conjugate ursodeoxycholate-lysophosphatidylethanolamide (UDCA-LPE) as iPLA2 b inhibitor was used to modify intracellular LPC levels. We examined the impact of LPC on JNK1-p and transcription of the heterotetrameric fatty acid transport complex constituted of CD36, FABPPM , caveolin1 and iPLA2 b. A NASH phenotype was generated by exposure of HepG2 cells with high concentrations of oleate bound to albumin (4:1) for 3 h. Results: Addition of 1–10 mM LPC to delipidated cytosolic extracts revealed a dose dependent increase of JNK1-p a central promoter of fatty acid metabolism and lipoapoptosis. In vitro transcription with native HepG2 nuclear extracts exposed to these LPC conditioned cytosolic samples resulted in synthesis stimulation of all members of the fatty acid uptake complex. In contrast, the NASH phenotype was reversed or prevented by incubation with the iPLA2 b inhibitor UDCA-LPE (1 h, 100 mM) suppressing cytosolic LPC levels. Accordingly JNK1-p and transcription of fatty acid transporter biosynthesis were reduced, concomitantly with disappearance of
lipid droplets, triglyceride accumulation and LDH release in the medium. Conclusions: iPLA2 b mediated generation of LPC represents a central regulator for hepatic steatosis and inflammation via JNK1-p. Inhibition of iPLA2b by nontoxic UDCA-LPE could be an ideal therapeutic strategy against NASH. P0935 NON-ALCOHOLIC STEATOHEPATITIS (NASH): 2 COMPLEMENTARY DIETARY MOUSE MODELS M.V. Machado1,2 , G.A. Michelotti2 , G. Xie2 , J. Boursier2,3 , T. Pereira de Almeida2 , L. Kruger2 , G. Karaca2 , C. Guy4 , A.M. Diehl2 . 1 Gastrenterologia, Hospital de Santa Maria, Lisboa, Portugal; 2 Gastroenterology, Duke University, Durham, United States; 3 HIFIH Laboratory, LUNAM University, Angers, France; 4 Pathology, Duke University, Durham, United States E-mail: [email protected] Background and Aims: Non-alcoholic steatohepatitis (NASH), the potentially progressive form of nonalcoholic fatty liver disease (NAFLD), is the pandemic liver disease of our times. However, its ideal treatment has yet to be found. To better understand the physiology of NASH and develop therapeutic strategies, a good animal model is needed that is reproducible, easy to implement, and that causes not only hepatic steatosis, but also the characteristics of NASH (cellular injury/death, inflammation), as well as fibrosis (the pathology that confers bad prognosis in humans). We compared features of NASH in 2 mouse models most widely used to study the disease: methionine-choline deficient (MCD) diet and Western diet. Methods: 36 C57Bl6 mice were fed either chow diet, MCD diet for 8 weeks or Western diet (45% energy from fat, predominantly saturated fat, with 0.2% cholesterol, plus drinking water supplemented with fructose and glucose) for 16 weeks. All animals were sacrificed at 20 weeks of age. Liver pathology and metabolic profile were compared. Results: The metabolic profile associated with human NASH was best mimicked by Western diet, which induced weight gain, hyperleptinemia, insulin resistance and dyslipidemia. Hepatic steatosis (i.e., triglyceride accumulation) was also more severe in Western diet than MCD diet. However, liver non-esterified fatty acids content was higher in the MCD diet group. Hepatic steatosis associated with increased de novo lipogenesis in Western diet and decreased VLDL secretion in MCD diet. NASH was much more severe and reproducible in the MCD model, as evidenced by greater liver cell death/apoptosis and inflammation. Compared to mice fed Western diets, MCD diet-fed mice developed an enhanced ductular response, and significantly more fibrosis. Various mechanisms implicated in human NASH pathogenesis/progression were also significantly more robust in the MCD diet model, including oxidative stress, ER stress, autophagy deregulation, and hedgehog pathway activation. Conclusions: MCD and Western diet are two complementary models to study NASH. Only Western diet reproduces the metabolic disturbances seen in human NASH. Western diet induces severe hepatic steatosis but after 4 months of diet exposure, liver injury is limited and minimal fibrosis is evident. MCD diet more efficiently reproduces the pathophysiologic mechanisms that drive human NASH pathogenesis/progression and causes substantial liver injury and fibrosis in 2 months.
Journal of Hepatology 2015 vol. 62 | S263–S864
S695
Figure 1.
P0934 LYSOPHOSPHATIDYLCHOLINE (LPC) AS CENTRAL PLAYER FOR HEPATIC FAT ACCUMULATION AND INFLAMMATION: IMPLICATION FOR PATHOGENESIS OF NASH W. Stremmel1 , S. Staffer1 , A. Wannhoff1 , A. Pathil-Warth1 , W. Chamulitrat1 . 1 Internal Medicine, University Clinics of Heidelberg Prof. Dr. W. Stremmel, Heidelberg, Germany E-mail: [email protected] Background and Aims: In NASH the intracellular ratio of phosphatidylcholine (PC): lysophosphatidylcholine (LPC) is decreased due to activation of the membrane localized phospholipase A2 (iPLA2b). Here we aim the evaluation of LPC levels in development and reversal of fat accumulation and inflammation. Methods: In HepG2 cells the bile acid-phospholipid conjugate ursodeoxycholate-lysophosphatidylethanolamide (UDCA-LPE) as iPLA2 b inhibitor was used to modify intracellular LPC levels. We examined the impact of LPC on JNK1-p and transcription of the heterotetrameric fatty acid transport complex constituted of CD36, FABPPM , caveolin1 and iPLA2 b. A NASH phenotype was generated by exposure of HepG2 cells with high concentrations of oleate bound to albumin (4:1) for 3 h. Results: Addition of 1–10 mM LPC to delipidated cytosolic extracts revealed a dose dependent increase of JNK1-p a central promoter of fatty acid metabolism and lipoapoptosis. In vitro transcription with native HepG2 nuclear extracts exposed to these LPC conditioned cytosolic samples resulted in synthesis stimulation of all members of the fatty acid uptake complex. In contrast, the NASH phenotype was reversed or prevented by incubation with the iPLA2 b inhibitor UDCA-LPE (1 h, 100 mM) suppressing cytosolic LPC levels. Accordingly JNK1-p and transcription of fatty acid transporter biosynthesis were reduced, concomitantly with disappearance of
lipid droplets, triglyceride accumulation and LDH release in the medium. Conclusions: iPLA2 b mediated generation of LPC represents a central regulator for hepatic steatosis and inflammation via JNK1-p. Inhibition of iPLA2b by nontoxic UDCA-LPE could be an ideal therapeutic strategy against NASH. P0935 NON-ALCOHOLIC STEATOHEPATITIS (NASH): 2 COMPLEMENTARY DIETARY MOUSE MODELS M.V. Machado1,2 , G.A. Michelotti2 , G. Xie2 , J. Boursier2,3 , T. Pereira de Almeida2 , L. Kruger2 , G. Karaca2 , C. Guy4 , A.M. Diehl2 . 1 Gastrenterologia, Hospital de Santa Maria, Lisboa, Portugal; 2 Gastroenterology, Duke University, Durham, United States; 3 HIFIH Laboratory, LUNAM University, Angers, France; 4 Pathology, Duke University, Durham, United States E-mail: [email protected] Background and Aims: Non-alcoholic steatohepatitis (NASH), the potentially progressive form of nonalcoholic fatty liver disease (NAFLD), is the pandemic liver disease of our times. However, its ideal treatment has yet to be found. To better understand the physiology of NASH and develop therapeutic strategies, a good animal model is needed that is reproducible, easy to implement, and that causes not only hepatic steatosis, but also the characteristics of NASH (cellular injury/death, inflammation), as well as fibrosis (the pathology that confers bad prognosis in humans). We compared features of NASH in 2 mouse models most widely used to study the disease: methionine-choline deficient (MCD) diet and Western diet. Methods: 36 C57Bl6 mice were fed either chow diet, MCD diet for 8 weeks or Western diet (45% energy from fat, predominantly saturated fat, with 0.2% cholesterol, plus drinking water supplemented with fructose and glucose) for 16 weeks. All animals were sacrificed at 20 weeks of age. Liver pathology and metabolic profile were compared. Results: The metabolic profile associated with human NASH was best mimicked by Western diet, which induced weight gain, hyperleptinemia, insulin resistance and dyslipidemia. Hepatic steatosis (i.e., triglyceride accumulation) was also more severe in Western diet than MCD diet. However, liver non-esterified fatty acids content was higher in the MCD diet group. Hepatic steatosis associated with increased de novo lipogenesis in Western diet and decreased VLDL secretion in MCD diet. NASH was much more severe and reproducible in the MCD model, as evidenced by greater liver cell death/apoptosis and inflammation. Compared to mice fed Western diets, MCD diet-fed mice developed an enhanced ductular response, and significantly more fibrosis. Various mechanisms implicated in human NASH pathogenesis/progression were also significantly more robust in the MCD diet model, including oxidative stress, ER stress, autophagy deregulation, and hedgehog pathway activation. Conclusions: MCD and Western diet are two complementary models to study NASH. Only Western diet reproduces the metabolic disturbances seen in human NASH. Western diet induces severe hepatic steatosis but after 4 months of diet exposure, liver injury is limited and minimal fibrosis is evident. MCD diet more efficiently reproduces the pathophysiologic mechanisms that drive human NASH pathogenesis/progression and causes substantial liver injury and fibrosis in 2 months.
Journal of Hepatology 2015 vol. 62 | S263–S864
S695