ABSTRACT
BACKGROUND: Precision-cut liver slices present different cell types of liver in a physiological context, and they have been explored as effective in vitro model systems to study liver fibrosis. Inducing fibrosis in the liver slices using toxicants like carbon tetrachloride is of less relevance to human disease conditions. Our aim for this study was to establish physiologically relevant conditions in vitro to induce fibrotic phenotypes in the liver slices. RESULTS: Precision-cut liver slices of 150 µm thickness were obtained from female C57BL/6 J mice. The slices were cultured for 24 hours in media containing a cocktail of 10 nM each of TGF-ß, PDGF, 5 µM each of lysophosphatidic acid and sphingosine 1 phosphate and 0.2 µg/ml of lipopolysaccharide along with 500 µM of palmitate and were analyzed for triglyceride accumulation, stress and inflammation, myofibroblast activation and extracellular matrix (ECM) accumulation. Incubation with the cocktail resulted in increased triglyceride accumulation, a hallmark of steatosis. The levels of Acta2, a hallmark of myofibroblast activation and the levels of inflammatory genes (IL-6, TNF-α and C-reactive protein) were significantly elevated. In addition, this treatment resulted in increased levels of ECM markers - collagen, lumican and fibronectin. CONCLUSIONS: This study reports the experimental conditions required to induce fibrosis associated with steatohepatitis using physiologically relevant inducers. The system presented here captures various aspects of the fibrosis process like steatosis, inflammation, stellate cell activation and ECM accumulation and serves as a platform to study the liver fibrosis in vitro and to screen small molecules for their antifibrotic activity.
ABSTRACT
BACKGROUND: AMP activated protein kinase (AMPK) regulates key metabolic reactions and plays a major role in glucose homeostasis. Activating the AMPK is considered as one of the potential therapeutic strategies in treating type-2 diabetes. However, targeting AMPK by small molecule mediated approach can be challenging owing to diverse isoforms of the enzyme and their varied combination in different tissues. In the current study we employ a novel strategy of achieving AMPK activation through increasing the levels of cellular AMP (an allosteric activator of AMPK) levels by activating the enzyme involved in AMP biosynthesis namely Adenylosuccinate lyase (ADSL). METHODS: Rat primary hepatocytes were cultured under metabolic overload conditions (500 µM palmitate) to induce insulin resistance. ADSL was overexpressed in these hepatocytes and its effect on hepatic glucose output, and triglyceride accumulation was checked. In addition to this, ADSL was overexpressed in high fat diet induced obese mice by hydrodynamic tail vein injection and its effect on fasting glucose, glucose tolerance and pyruvate tolerance were checked. RESULTS: Rat primary hepatocytes when cultured under metabolic overload conditions developed insulin resistance as measured in terms of failure of insulin to suppress the glucose output. Overexpressing the ADSL in these hepatocytes resulted in increased AMPK phosporylation and improved the insulin sensitivity and also resulted in reduced triglyceride accumulation and inflammatory cytokine levels. In addition to this, when ADSL was overexpressed in high fat diet induced obese mice, it resulted in reduced the fasting hyperglycemia (20% reduction), and increased glucose and pyruvate tolerance. CONCLUSIONS: This study indicates that activating ADSL can be a potential mechanism to achieve the activation of AMPK in the cells. This leads to a novel idea of exploring the purine nucleotide metabolic pathway as a promising therapeutic target for diabetes and metabolic syndrome.
