Proceedings of The Physiological Society

Physiology 2016 (Dublin, Ireland) (2016) Proc Physiol Soc 37, PCA229

Poster Communications

Heat shock protein 72 regulates mitochondrial integrity and function in the prevention of hepatic insulin resistance

A. E. Archer1, R. S. Rogers1, J. Wheatley1, H. Wilkins1, R. Swerdlow1, J. P. Thyfault1, P. Geiger1

1. KUMC, Kansas City, Missouri, United States.


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 mitochondrial dysfunction and lipid accumulation in the presence of a high fat diet (HFD). 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 (41°C for 20 min) or sham-treatments (37°C for 20 min) for the last 7 weeks. In addition, hepatocytes (n=6 per group) were isolated from 2-month old male C57BL/6 mice and treated with HSP72 siRNA (10nM) for 24 hours and analyzed for mitochondrial oxygen consumption rate using Seahorse Bioscience XF24 Analyzer (substrates used: 2 uM oligomycin, 0.5 uM FCCP, 1uM rotenone/antimycin A). Hepatocytes were also stained with MitoTracker green (100nM) and TMRE (600nM) following siRNA treatment in order to evaluate mitochondrial integrity. Values are means ± S.E.M. compared by ANOVA. Weekly in-vivo heat treatments resulted in upregulation of HSP72 protein content (p<0.01), decreased liver triglyceride storage (p<0.05), and modulation of protein content of the autophagy markers microtubule-associated protein light chain 3 (LC3-II) (p<0.01) and p62 (p<0.05). Loss of HSP72 in primary hepatocytes resulted in decreased basal, maximal, and ATP-coupled oxygen consumption (p<0.05). The siRNA treatment also reduced phosphorylation of AMP-activated protein kinase (AMPK) (p<0.05) and Akt (p<0.05), increased Parkin protein content (p<0.05), and there was a trend toward decreased LC3-II protein content (p=0.07). Decreased expression of HSP72 also reduced mitochondrial quality which was reflected through the decreased TMRE/Mitotracker ratio in siRNA-treated primary hepatocytes (p<0.05). This data suggests that HSP72 regulates mitochondrial function by maintaining mitochondrial integrity through regulation of mitophagy. Future therapies could target HSP72 and its beneficial effects on liver mitochondrial function and metabolism to prevent NAFLD and hepatic insulin resistance.

Where applicable, experiments conform with Society ethical requirements