Effect of radiation on hepatic fat metabolism in rat and mouse: A role of radiation-induced TNF-α in the regulation of FAT/CD36

Radiotherapy is an important method to treat tumors. The positive effects of irradiation on tumoral tissue are thought to be due to the damage of the DNA of tumor cells and the damage of tumor vessels. Radiation as single or together with anti-tumor drugs is a main therapy against cancer. Recent development in the radiotherapy such as respiratory-gated radiotherapy and the amelioration of stereotactic techniques has made radiotherapy of liver tumors possible. However, there has been little knowledge how irradiation affects healthy tissue. Accordingly, the effect of irradiation on healthy liver tissue is still poorly understood. The liver is responsible for energy metabolism and detoxification. Its lobules represent a sponge of vessels and hepatocyte cords with adjacent Kupffer cells and with a varying oxygen tension, implicating sensitivity to oxidative stress mechanisms. The liver is thus considered to be sensitive to irradiation, which together with other noxae could lead to liver damage (e.g. development of fibrosis/cirrhosis) over the course of time. Hence, irradiation might cause damage of (non-tumoral) liver tissue. Radiation-Iinduced Liver Disease (RILD) is a clinical complication characterized by the appearance of ascites and signs of decreased liver function. Histopathology shows veno-occlusive disease and atrophy of adjacent hepatocytes. Irradiation is known to induce an inflammatory response and affects fat metabolic pathways where cytokines, especially Tumor Necrosis Factor-α (TNF-α), play a key role. Although, a link between hepatic inflammation and fat accumulation has been described, the metabolic changes after irradiation have been poorly studied. The aim of the current study was to investigate changes in fat uptake and lipid profile after radiation-induced liver damage. Furthermore, the genes encoding proteins which are involved in transport of fat into the liver after selective liver irradiation were analyzed. Rat livers were selectively irradiated in vivo (25 Gy), sham-irradiated rats served as controls. In addition to the rat model, already established in our laboratory, a mouse model of selective single-dose irradiation (25 Gy) in presence or absence of TNF-α and anti-TNF-α antibody, infliximab (IFX), was established. Nile red and Sudan staining were used to observe the fat accumulation in liver tissue. Hepatic lipids were studied by colorimetric assays in liver tissue and serum. Protein level and mRNA expressions were studied by immunohistology, Western Blot analysis and RT-PCR in liver tissue, respectively. Changes in FAT/CD36 protein level were studied in vitro in a human monocyte cell line U937 after irradiation in presence or absence of anti-TNF-α antibody. In rat liver, Nile-Red staining of cryosections showed a quick (12–48 hours (h)) increase of fat droplets. Accordingly, concentrations of hepatic triglycerides (TG) and free fatty acids (FFA) were elevated. An early increase (3-6 h) in the serum level of High Density Lipoprotein-Cholesterol (HDL-C), TG and cholesterol was measured after single-dose irradiation in rat liver followed by a decrease thereafter. In addition, an increased mRNA expression of the fat transporter protein FAT/CD36 was detected, immunohistochemistry revealed basolateral and cytoplasmic localization of this transporter in hepatocytes. Moreover, apolipoprotein-B100, -C3 and key enzymes of fat metabolism (acetyl-CoA carboxylase, lipoprotein lipase, carnitine palmitoyltransferase, malonyl-CoA decarboxylase) involved in fat metabolism were induced at 12–24 h. An early activation of the NFκB pathway (IκBα) by TNF-α was seen, followed by a significant elevation of serum markers for liver damage (AST and GLDH) after single-dose irradiation in rat liver. TNF-α blockage by anti-TNF-α in cell culture (U937) prevented the increase of FAT/CD36 protein level caused by irradiation. Similar to what was observed in rat liver, an early (1-3 h) induction of TNF-α gene expression, a pro-inflammatory protein, was seen in mouse liver tissue compared to sham-irradiated controls. Increased TNF-α expression was followed by elevated hepatic TG concentration (6-12 h). In contrast, a decreased TG level was detected in the serum of irradiated animals at the same time points when liver TG were elevated. Corresponding to TG levels in mouse liver, Sudan staining of liver cryosections showed a quick (3-6 h) accumulation and increase in size of fat droplets after irradiation. In parallel, the fat transporter FAT/CD36 was increased at protein level after irradiation. In vivo, TNF-α blockage by anti-TNF-α in mice liver prevented the increase of FAT/CD36. Immunohistochemistry showed the basolateral and cytoplasmic localization in mice hepatocytes. Moreover, co-localization of FAT/CD36 was detected in CK-19+- (billary cells), SMA+- (myofibroblast) and F4/80+- (macrophages) cells in mouse liver. The results (in vivo and in vitro) suggest that IFX, by blocking soluble TNF-α, inhibits FAT/CD36 on protein level, preventing the increase of FAT/CD36 caused by TNF-α or/and irradiation in liver experiments, a prerequisite to control fat transport into tissue. The observed effect of anti-TNF-α might contribute to a reduction of inflammatory processes caused by irradiation and/or TNF-α in liver. Moreover, the presence of FAT/CD36 in different liver cell types apart from hepatocytes strongly suggests their active involvement in liver fat uptake mechanisms.