Elevated levels of extracellular heat-shock protein 72 (eHSP72) are positively correlated with insulin resistance in vivo and cause pancreatic β-cell dysfunction and death in vitro

Abstract

eHSP72 (extracellular heat-shock protein 72) is increased in the plasma of both types of diabetes and is positively correlated with inflammatory markers. Since aging is associated with a low-grade inflammation and IR (insulin resistance), we aimed to: (i) analyse the concentration of eHSP72 in elderly people and determine correlation with insulin resistance, and (ii) determine the effects of eHSP72 on β-cell function and viability in human and rodent pancreatic β-cells. Fasting blood samples were collected from 50 older people [27 females and 23 males; 63.4±4.4 years of age; BMI (body mass index)=25.5±2.7 kg/m2]. Plasma samples were analysed for eHSP72, insulin, TNF (tumour necrosis factor)-α, leptin, adiponectin and cortisol, and glycaemic and lipid profile. In vitro studies were conducted using rodent islets and clonal rat and human pancreatic β-cell lines (BRIN-BD11 and 1.1B4 respectively). Cells/islets were incubated for 24 h with eHSP72 (0, 0.2, 4, 8 and 40 ng/ml). Cell viability was measured using three different methods. The impact of HSP72 on β-cell metabolic status was determined using Seahorse Bioscience XFe96 technology. To assess whether the effects of eHSP72 were mediated by Toll-like receptors (TLR2/TLR4), we co-incubated rodent islets with eHSP72 and the TLR2/TLR4 inhibitor OxPAPC (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; 30 μg/ml). We found a positive correlation between plasma eHSP72 and HOMA-IR (homoeostasis model assessment of IR) (r=0.528, P<0.001), TNF-α (r=0.389, P<0.014), cortisol (r=0.348, P<0.03) and leptin/adiponectin (r=0.334, P<0.03). In the in vitro studies, insulin secretion was decreased in an eHSP72 dose-dependent manner in BRIN-BD11 cells (from 257.7±33 to 84.1±10.2 μg/mg of protein per 24 h with 40 ng/ml eHSP72), and in islets in the presence of 40 ng/ml eHSP72 (from 0.48±0.07 to 0.33±0.009 μg/20 islets per 24 h). Similarly, eHSP72 reduced β-cell viability (at least 30% for BRIN-BD11 and 10% for 1.1B4 cells). Bioenergetic studies revealed that eHSP72 altered pancreatic β-cell metabolism. OxPAPC restored insulin secretion in islets incubated with 40 ng/ml eHSP72. In conclusion, we have demonstrated a positive correlation between eHSP72 and IR. In addition, we suggest that chronic eHSP72 exposure may mediate β-cell failure.