Vegetable Oil-Peroxidation Product 'Hydroxynonenal' Causes Hepatocyte Injury and Steatosis via Hsp70.1 and BHMT Disorders in the Monkey Liver.

Hsp70.1 has a dual function as a chaperone protein and lysosomal stabilizer. In 2009, we reported that calpain-mediated cleavage of carbonylated Hsp70.1 causes neuronal death by inducing lysosomal rupture in the hippocampal CA1 neurons of monkeys after transient brain ischemia. Recently, we also reported that consecutive injections of the vegetable oil-peroxidation product 'hydroxynonenal' induce hepatocyte death via a similar cascade in monkeys. As Hsp70.1 is also related to fatty acid ß-oxidation in the liver, its deficiency causes fat accumulation. The genetic deletion of betaine-homocysteine S-methyltransferase (BHMT) was reported to perturb choline metabolism, inducing a decrease in phosphatidylcholine and resulting in hepatic steatosis. Here, focusing on Hsp70.1 and BHMT disorders, we studied the mechanisms of hepatocyte degeneration and steatosis. Monkey liver tissues with and without hydroxynonenal injections were compared using proteomics, immunoblotting, immunohistochemical, and electron microscopy-based analyses. Western blotting showed that neither Hsp70.1 nor BHMT were upregulated, but an increased cleavage was observed in both. Proteomics showed a marked downregulation of Hsp70.1, albeit a two-fold increase in the carbonylated BHMT. Hsp70.1 carbonylation was negligible, in contrast to the ischemic hippocampus, which was associated with ~10-fold increments. Although histologically, the control liver showed very little lipid deposition, numerous tiny lipid droplets were seen within and around the degenerating/dying hepatocytes in monkeys after the hydroxynonenal injections. Electron microscopy showed permeabilization/rupture of lysosomal membranes, dissolution of the mitochondria and rough ER membranes, and proliferation of abnormal peroxisomes. It is probable that the disruption of the rough ER caused impaired synthesis of the Hsp70.1 and BHMT proteins, while impairment of the mitochondria and peroxisomes contributed to the sustained generation of reactive oxygen species. In addition, hydroxynonenal-induced disorders facilitated degeneration and steatosis in the hepatocytes.

Hydroxynonenal Causes Hepatocyte Death by Disrupting Lysosomal Integrity in Nonalcoholic Steatohepatitis.

BACKGROUND & AIMS: The lipid oxidation is a key factor for damaging hepatocytes and causing cell death. However, the mechanisms underlying hepatocyte death and the role of the most popular lipid peroxidation product 4-hydroxy-2-nonenal (HNE) in nonalcoholic steatohepatitis (NASH) remains unclear. METHODS: We demonstrated using hepatoma cell lines, a NASH mouse model, HNE-treated monkeys, and biopsy specimens from patients with NASH that HNE induced hepatocyte death by disintegrating the lysosomal limiting membrane. RESULTS: The degree of HNE deposition in human NASH hepatocytes was more severe in cases with high lobular inflammation, ballooning, and fibrosis scores, and was associated with enlargement of the staining of lysosomes in hepatocytes. In in vitro experiments, HNE activated µ-calpain via G-protein coupled receptor (GPR) 120. The resultant rupture/permeabilization of the lysosomal limiting membrane induced the leakage of cathepsins from lysosomes and hepatocyte death. The blockade of G-protein coupled receptor 120 (GPR120) or µ-calpain expression suppressed lysosomal membrane damage and hepatocyte death by HNE. Alda-1, which activates aldehyde dehydrogenase 2 to degrade HNE, prevented HNE-induced hepatocyte death. Intravenous administration of HNE to monkeys for 6 months resulted in hepatocyte death by a mechanism similar to that of cultured cells. In addition, intraperitoneal administration of Alda-1 to choline-deficient, amino-acid defined treated mice for 8 weeks inhibited HNE deposition, decreased liver inflammation, and disrupted lysosomal membranes in hepatocytes, resulting in improvement of liver fibrosis. CONCLUSIONS: These results provide novel insights into the mechanism of hepatocyte death in NASH and will contribute to the development of new therapeutic strategies for NASH.

Hsp70.1 carbonylation induces lysosomal cell death for lifestyle-related diseases.

Alzheimer's disease, type 2 diabetes, and non-alcoholic steatohepatitis (NASH) constitute increasingly prevalent disorders. Individuals with type 2 diabetes are well-known to be susceptible to Alzheimer's disease. Although the pathogenesis of each disorder is multifactorial and the causal relation remains poorly understood, reactive oxygen species (ROS)-induced lipid and protein oxidation conceivably plays a common role. Lipid peroxidation product was recently reported to be a key factor also for non-alcoholic steatohepatitis, because of inducing hepatocyte degeneration/death. Here, we focus on implication of the representative lipid-peroxidation product 'hydroxynonenal' for the cell degeneration/death of brain, pancreas, and liver. Since Hsp70.1 has dual roles as a chaperone and lysosomal membrane stabilizer, hydroxynonenal-mediated oxidative injury (carbonylation) of Hsp70.1 was highlighted. After intake of high-fat diets, oxidation of free fatty acids in mitochondria generates ROS which enhance oxidation of ω-6 polyunsaturated fatty acids (PUFA) involved within biomembranes and generate hydroxynonenal. In addition, hydroxynonenal is generated during cooking deep-fried foods with vegetable oils especially containing linoleic acids. These intrinsic and exogenous hydroxynonenal synergically causes an increase in its serum and organ levels to induce Hsp70.1 oxidation. As it is amphiphilic; being water-soluble but displays strong lipophilic characteristics, hydroxynonenal can diffuse within the cells and react with targets like senile and/or atheromatous plaques outside the cells. Hydroxynonenal can deepen and expand lysosomal injuries by facilitating 'calpain-mediated cleavage of the carbonylated Hsp70.1'. Despite the unique anatomical, physiological, and biochemical characteristics of each organ for its specific disease, there should be a common cascade of the cell degeneration/death which is caused by hydroxynonenal. This review aims to implicate hydroxynonenal-mediated Hsp70.1 carbonylation for lysosomal membrane permeabilization/rupture and the resultant cathepsin leakage for inducing cell degeneration/death. Given the tremendous number of worldwide people suffering various lifestyle-related diseases, it is valuable to consider how ω-6 PUFA-rich vegetable oils is implicated for the organ disorder.

Hydroxynonenal causes Langerhans cell degeneration in the pancreas of Japanese macaque monkeys.

BACKGROUND: For their functions of insulin biosynthesis and glucose- and fatty acid- mediated insulin secretion, Langerhans ß-cells require an intracellular milieu rich in oxygen. This requirement makes ß-cells, with their constitutively low antioxidative defense, susceptible to the oxidative stress. Although much progress has been made in identifying its molecular basis in experimental systems, whether the oxidative stress due to excessive fatty acids plays a crucial role in the Langerhans cell degeneration in primates is still debated. METHODS: Focusing on Hsp70.1, which has dual functions as molecular chaperone and lysosomal stabilizer, the mechanism of lipotoxicity to Langerhans cells was studied using macaque monkeys after the consecutive injections of the lipid peroxidation product 'hydroxynonenal'. Based on the 'calpain-cathepsin hypothesis' formulated in 1998, calpain activation, Hsp70.1 cleavage, and lysosomal integrity were studied by immunofluorescence histochemistry, electron microscopy, and Western blotting. RESULTS: Light microscopy showed more abundant vacuole formation in the hydroxynonenal-treated islet cells than the control cells. Electron microscopy showed that vacuolar changes, which were identified as enlarged rough ER, occurred mainly in ß-cells followed by δ-cells. Intriguingly, both cell types showed a marked decrease in insulin and somatostatin granules. Furthermore, they exhibited marked increases in peroxisomes, autophagosomes/autolysosomes, lysosomal and peroxisomal membrane rupture/permeabilization, and mitochondrial degeneration. Disrupted peroxisomes were often localized in the close vicinity of degenerating mitochondria or autolysosomes. Immunofluorescence histochemical analysis showed an increased co-localization of activated µ-calpain and Hsp70.1 with the extralysosomal release of cathepsin B. Western blotting showed increases in µ-calpain activation, Hsp70.1 cleavage, and expression of the hydroxynonenal receptor GPR109A. CONCLUSIONS: Taken together, these data implicate hydroxynonenal in both oxidation of Hsp70.1 and activation of µ-calpain. The calpain-mediated cleavage of the carbonylated Hsp70.1, may cause lysosomal membrane rupture/permeabilization. The low defense of primate Langerhans cells against hydroxynonenal and peroxisomally-generated hydrogen peroxide, was presumably overwhelmed to facilitate cell degeneration.