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Heat Shock Protein 72 Regulates Lipid Handling in Hepatocytes
Author(s) -
Archer Ashley E.,
Rogers Robert S.,
Wheatley Joshua L.,
Wilkins Heather M.,
Swerdlow Russell H.,
Thyfault John P.,
Geiger Paige C.
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.lb719
Subject(s) - nonalcoholic fatty liver disease , heat shock protein , medicine , insulin resistance , endocrinology , lipid metabolism , fatty liver , mitochondrion , liver function , skeletal muscle , type 2 diabetes , lipid droplet , biology , inflammation , chemistry , insulin , diabetes mellitus , biochemistry , disease , gene
Nonalcoholic Fatty Liver Disease (NAFLD) is characterized by an excessive accumulation of lipids in the liver which can lead to inflammation, hepatic insulin resistance, and type 2 diabetes. Mitochondria are critical to liver function and decreased mitochondrial function can contribute to NAFLD and metabolic disease. Induction of the chaperone Heat Shock Protein 72 (HSP72) through heat treatment, exercise, or transgenic overexpression improves glucose tolerance, insulin sensitivity and mitochondrial function in skeletal muscle. The role of HSP72 in liver metabolism is largely unknown. The purpose of this study was to determine the ability of HSP72 to protect against hepatic lipid accumulation and mitochondrial dysfunction in the presence of a high fat diet (HFD) or lipid exposure in culture. Male Wistar rats (~160g, n=9 per group) were fed a HFD for 15 weeks and were anesthetized with pentobarbitol (5mg/100g bw) and given weekly heat treatments (HT, 41°C for 20 min) or sham‐treatments (ST, 37°C for 20 min) for the last 7 weeks. We also evaluated the role of HSP72 in primary hepatocytes. Hepatocytes (n=6 per group) were first treated with 30 min of HT, AdCMV‐HSP72, siRNA for HSP72, or relevant controls and 24 h later exposed to Palmitate (250 μM) or Vehicle for an additional 24 h. Cells were them stained with Bodipy (3.5 μM) and imaged at 40×. In addition, hepatocytes were treated with HSP72 siRNA for 24 hours and then analyzed for mitochondrial oxygen consumption rate using Seahorse Bioscience XF24 Analyzer (substrates used: 2 uM oligomycin, 0.5 uM FCCP, 1uM rotenone/antimycin A). Cells were also harvested and frozen for western blot analysis. Following siRNA, we also stained cells with MitoTracker green (100nM) and TMRE (600nM) in order to evaluate mitochondrial integrity. Values are means ± S.E.M. compared by unpaired t‐tests or ANOVA. Weekly in‐vivo HT in rats resulted in upregulation of HSP72 protein content, decreased liver triglyceride storage, and decreased JNK phosphorylation. 24 h of palmitate exposure increased lipid accumulation in control primary hepatocytes, while lipid accumulation was significantly decreased in palmitate‐treated cells exposed to HT or HSP72 o/e. In siHSP72 treated cells, lipid accumulation was increased above hepatocytes treated with control siRNA. Loss of HSP72 in primary hepatocytes also resulted in decreased basal, maximal, and ATP‐coupled oxygen consumption while protein content levels of mitochondrial ETC Complex proteins, Cytochrome C, or PGC1α were unchanged. HSP72 siRNA also reduced phosphorylation of AMP‐activated protein kinase (AMPK) and Akt. Decreased expression of HSP72 reduced mitochondrial quality as well which was reflected through the decreased TMRE/Mitotracker ratio in siRNA‐treated primary hepatocytes. Lastly, knockdown of HSP72 increased protein expression of pJNK and JNK. This data suggests that HSP72 activation can improve lipid handling and mitochondrial function in hepatocytes. Future therapies could target HSP72 and its beneficial effects on liver mitochondrial function and metabolism to prevent NAFLD and hepatic insulin resistance. Support or Funding Information This research was supported in part by AG‐031575 and the Self Graduate Fellowship.