Endoplasmic Reticulum Stress Is Involved In Hepatic SREBP-1c Activation And Lipid Accumulation In Fructose-fed Mice: Critical Role Of Kupffer Cells

Objective To investigate the molecular mechanism of fructose-evoked NAFLD and to explore the effects of Kupffer cell.Methods The present study included three separate experiments. In experiment 1, To investigate the effects of fructose drinking on hepatic TG synthesis and lipid accumulation, twenty-four mice were divided into two groups. Mice had free access to tap water containing 30% fructose or plain tap water for eight weeks. Body weight was assessed weekly over the 8-week feeding period. In experiment 2, To investigate the role of ndoplasmic reticulum (ER) stress on fructose-induced hepatic SREBP-1c activation, TG synthesis and lipid accumulation, forty-eight mice were divided into four groups. In fructose alone group, all mice had free access to tap water containing 30% fructose for eight weeks. In control group, mice had free access to plain tap water for eight weeks. In fructose+ PBA group, mice were free access to tap water containing 30% fructose and administered with PBA (100 mg/kg) for another two week after mice were administered with 30% fructose for six weeks. In PBA alone group, mice were free access to plain tap water and administered with PBA (100 mg/kg) for another two week after mice were administered with plain tap water for six weeks. In experiment 3, To investigate the role of Kupffer cells on hepatic ER stress, SREBP-1c activation, TG synthesis and lipid accumulation, forty-eight mice were divided into four groups. In fructose alone group, mice had free access to tap water containing 30% fructose for eight weeks. In control group, mice had free access to plain tap water for eight weeks. In GdCl₃ alone group, mice were free access to plain tap water for eight weeks and administered with GdCl₃ (10 mg/kg) twice a week. In GdCl₃+fructose group, mice were free access to tap water containing 30% fructose for eight weeks and administered with GdCl₃ (10 mg/kg) twice a week. Body weight was assessed weekly over the 8-week feeding period. All mice were sacrificed after feeding for eight weeks. Blood serum was collected for serum TG measurement. Liver was collected and either frozen immediately in liquid nitrogen for RT-PCR, Western blot and hepatic TG measurement, or fixed in neutral-buffered formalin for immunohistochemistry and histological examination, or frozen-fixed in OCT mounting media for Oil red O staining.Results The level of serum and hepatic TG was significantly increased in mice fed with fructose solution as compared with those drinking plain tap water. An obvious hepatic lipid accumulation, as determined by Oil Red O staining, was observed in fructose-fed mice. Chronic fructose drinking upregulated the expression of enzymes for TG synthesis , such as fas,acc and scd-1. There was no significant difference on the level of hepatic cd36, cpt1α, cyp4a10 and cyp4a14 mRNA between fructose-fed mice and controls. For further study chronic fructose drinking induced hepatic SREBP-1c activation.But fructose did not affect the level of nuclear ChREBP and LXR-αin liver. There was no significant difference on the level of hepatic insig-1 and insig-2 mRNA between fructose-fed mice and controls. Surprisingly, chronic fructose drinking significantly reduced the level of hepatic Insig-1 protein. The expression of hepatic grp78 was significantly increased in fructose-fed mice. The level of hepatic phosphorylated PERK (pPERK) and phosphorylated eIF2α(peIF2α) were significantly increased in fructose-fed mice. The level of nuclear XBP-1 was significantly increased in liver of mice fed with fructose solution, indicating that fructose could induce hepatic ER Stress. PBA reduced ER Stress and inhibited hepatic TG synthesis. PBA inhibited hepatic SREBP-1c activation and hepatic fatty acid and TG synthetic enzymes. Fructose-induced hepatic Insig-1 depletion was significantly alleviated in mice administered with PBA. The level of hepatic TNF-α, iNOS and P50 were significantly increased in mice fed with fructose solution for eight weeks, indicating that Kupffer Cells were activated. Fructose-evoked ER Stress and UPR were almost completely inhibited by GdCl₃, a selective Kupffer cell toxicant. Importantly, GdCl₃ pretreatment significantly attenuated SREBP-1c activation and the expression of SREBP-1c target genes. In addition, GdCl₃ pretreatment prevented hepatic TG synthesis and lipid accumulation.Conclusion In summary, the present study demonstrated that the elevation of de novo fatty acid syntheses contributes to hepatic lipid accumulation in fructose-induced NAFLD. The increased hepatic TG synthesis is mainly attributed to hepatic SREBP-1c activation. Hepatic ER stress and the UPR induces SREBP-1c activation. Kupffer cell activation, play an important role on fructose-evoked SREBP-1c activation.