Role of Cellular Mg2+ in Modulating Collagen Deposition and Enzyme Efficiency in Hepatocytes and Kupffer Cells

Steven Ewart1, Andrea Romani2 1Dept. Physics, 2Dept. Physiology and Biophysics, Case Western Reserve University,

Cleveland, OH 44106Abstract

Acute and chronic ethanol administration reduces total Mg2+ content in liver tissue. A decrease in cellular Mg2+ content has been associated with increased pro-inflammatory cytokines release and altered collagen deposition. Increased collagen deposition can lead to liver fibrosis, while increased inflammation caused by overproduction of interleukin-2 (Il-2) and interleukin-6(Il-6) may cause hepatitis. The two conditions combined may trigger cirrhosis and liver function failure, which can be fatal.

The long term goal of this study is to test the hypothesis that reduced cellular Mg2+ content within Kupffer cells and hepatocytes is sufficient to promote an increased production of interleukin-2 (Il-2), interleukin-6 (Il-6), and NFκ-β, and an increased deposition of collagen, respectively, mimicking the effect of prolonged alcohol exposure. Alternatively, it can be expected that a low cellular Mg2+ concentration will enhance the effect of ethanol in eliciting the changes.

Materials and Methods-Hepatocytes

-Overexpressing various enzymes involved in EtOH metabolism- Effect of enzyme-specific inhibitors: 4-MP and CMZ

-Kupffer- Chronic (3 weeks) ethanol exposure and control (P = pair-fed)- Varying extracellular Mg2+ Concentration

-Mg2+ concentration measured by Atomic Absorbance Spectroscopy-Protein concentration measured by Biorad and Insoluble Lowry Assays

Fig. 1: EtOH metabolism by Alcohol dehydrogenase (ADH) and CYP2E1 within Microsomal Ethanol Oxidating System (MEOS): Consequence for Mg2+ homeostasis in the hepatocyte.

Fig. 2: MEOS-mediated generation of LOOH, MDA, and HNE vis lipid peroxidation.[1]

Fig. 4: Net Mg2+ extrusion in Kupffer cells from pair-fed (P, Control) and EtOH-fed (E) animals. Different extracellular Mg2+ concentrations and EtOH doses were tested (n=3).

Future Directions Higher doses of inhibitors, especially CMZ, need to be

tested to determine whether a more effective inhibition of EtOH-induced Mg2+ extrusion can be attained. Glucose metabolism and ATP levels will also be assessed before and after exposure to EtOH. Because cyP4502E1 generates ROS as byproducts of EtOH oxidation, ROS formation will be assessed using fluorometric indicators.

Acknowledgments: I would like to thank the Department of Physiology and Biophysics for

their cooperation throughout the project, especially everyone in Romani Lab, without their help this project would not have been possible.

[1] Konishi Masahiro, Ishii Hiromasa. Journal of Gastroenterology and Heptology 22 (2007) Suppl. 1; S7-S10[2] Friedman S. Gastroenterology 2008; 134: 1655-16669

Fig. 5: Net Mg2+ extrusion in VI-7 (Overexpression of ADH) cells (Top) VL-17a (Overexpression of ADH and CYP2E1) cells (Bottom) and incubated in the presence of various concentrations of EtOH and various inhibitors.

Results and Conclusions (Kupffer)Mg2+ concentration had a significant impact in alcohol metabolism

of Kupffer cells. At low Mg2+ concentration EtOH metabolism results in a smaller extrusion from the cells, whereas a larger extrusion is observed in the presence of higher Mg2+ concentration. Increasing EtOH concentration increased Mg2+ extrusion under similar experimental conditions.

Fig. 3: Activated stellate cells (HSC) response following cellular injury like the damage caused by EtOH via MEOS. [2]

The Story Thus Far

Table 1: Various liver cell lines utilized in the study.

Table 2: Cell types, Time points, EtOH concentrations, and Inhibitors used.

Results and Conclusions (Hepatocyte)Exposure to increasing concentrations of EtOH results

in HepG2 cells overexpressing ADH, or CyP4502E1 and ADH. The presence of CMZ as a specific inhibitor of CyP4502E1 attenuates the amplitude of Mg2+ extrusion in VI-7 but not in VL-17a cells. Inhibition of ADH by 4-MP appears to be more effective in both cell lines.