preprints.org > medicine and pharmacology > pharmacology and toxicology > doi: 10.20944/preprints202303.0012.v1

Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

Version 1 : Received: 1 March 2023 / Approved: 1 March 2023 / Online: 1 March 2023 (07:45:34 CET)

Hsp70.1 has dual functions as chaperone protein and lysosomal stabilizer. Previously, we reported that calpain-mediated cleavage of carbonylated Hsp70.1 causes ischemic neuronal death by inducing lysosomal rupture. Recently, we found that the consecutive injections of vegetable oil-peroxidation product ‘hydroxynonenal’ induces hepatocyte death via the similar cascade. As Hsp70.1 is related also to fatty acid β-oxidation in the liver, its deficiency is known to cause accumulation of fat. Genetic deletion of betaine-homocysteine S-methyltransferase (BHMT) was reported to perturb choline metabolism, inducing decrease of phosphatidylcholine with the resultant hepatic steatosis. Here, focusing on disorders of Hsp70.1 and BHMT, we studied the mechanism of hepatocyte degeneration and steatosis, using monkeys after the consecutive injections of synthetic hydroxynonenal. As these monkeys showed a significant impairment of liver functions, the liver tissues without and with hydroxynonenal injections were compared by proteomics, immunoblotting, immunohisto-chemical and electron microscopic analyses. Western blotting showed upregulation of neither Hsp70.1 nor BHMT, but an increased cleavage of both proteins. Proteomics showed downregulation of Hsp70.1, while 2-fold increments of carbonylated BHMT. Hsp70.1 carbonylation was negligible, showing a marked contrast to ischemic neurons which were associated with ~10-fold increments. The control liver histologically showed lipid droplets in Ito cells, but lipid depositions within hepatocytes were very little. In contrast, after hydroxynonenal injections, widespread fatty degeneration and focal coagulation necrosis were observed with accumulation of numerous tiny lipid droplets within and around the degenerating/dying hepatocytes. Electron microscopy showed lysosomal membrane permeabilization/rupture, remarkable dissolution of mitochondria and rough ER membrane, and proliferation of abnormal peroxisomes. It is probable that disruptions of rough ER caused impaired synthesis of Hsp70.1 and BHMT proteins, while impairments of mitochondria and peroxisomes presumably contributed to the sustained generation of reactive oxygen species. Since both Hsp70.1 and BHMT are vulnerable to the long-standing oxidative stressor, hydroxynonenal-induced disorders facilitated degeneration and steatosis of hepatocytes, respectively.

betaine-homocysteine S-methyltransferase; calpain-cathepsin hypothesis; carbonylation; cell death; lysosomal rupture; phosphatidylcholine

Medicine and Pharmacology, Pharmacology and Toxicology

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