TY - JOUR

T1 - Heat shock protein B1 is a key mediator of prolactin-induced beta-cell cytoprotection against oxidative stress

AU - Terra, Letícia F

AU - Wailemann, Rosangela A M

AU - Dos Santos, Ancély F

AU - Gomes, Vinicius M

AU - Silva, Railmara P

AU - Laporte, Anna

AU - Meotti, Flávia C

AU - Terra, Walter R

AU - Palmisano, Giuseppe

AU - Lortz, Stephan

AU - Labriola, Leticia

N1 - Funding Information: The authors would like to thank Martin Wirth and Maren Böger for technical assistance. This work was funded by the Research Training Group 1947 (BioX) of the Deutsche Forschungsgemeinschaft (DFG, Germany), European Foundation for the Study of Diabetes (EFSD, Germany), CNPq, Brazil [grant numbers 441878/2014-8 , 151244/2018-9] and FAPESP, Brazil [grant numbers 2013/07029-4 , 2017/03618-6 , 2013/13914-0 , 2016/02881-2 , 2014/02745-6 , 2016/04676-7 and 2014/17974-0 ]. The funding sources were not involved in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. Biomass facility at CEFAP-USP is acknowledged for providing access to the MS equipment.

PY - 2019/4

Y1 - 2019/4

N2 - Maintaining islet cell viability in vitro, although challenging, appears to be a strategy for improving the outcome of pancreatic islet transplantation. We have shown that prolactin (PRL) leads to beta-cell cytoprotection against apoptosis, an effect mediated by heat shock protein B1 (HSPB1). Since the role of HSPB1 in beta-cells is still unclear and the hormone concentration used is not compatible with clinical applications because of all the side effects displayed by the hormone in other tissues, we explored the molecular mechanisms by which HSPB1 mediates beta-cell cytoprotection. Lysates from PRL- and/or cytokine-treated MIN6 beta-cells were subjected to HSPB1 immunoprecipitation followed by identification through mass spectrometry. PRL-treated cells presented an enrichment of several proteins co-precipitating with HSPB1. Of note were oxidative stress resistance-, protein degradation- and carbohydrate metabolism-related proteins. Wild type, HSPB1 silenced or overexpressing MIN6 cells were exposed to menadione and hydrogen peroxide and analysed for several oxidative stress parameters. HSPB1 knockdown rendered cells more sensitive to oxidative stress and led to a reduced antioxidant capacity, while prolactin induced an HSPB1-mediated cytoprotection against oxidative stress. HSPB1 overexpression, however, led to opposite effects. PRL treatment, HSPB1 silencing or overexpression did not change the expression nor activities of antioxidant enzymes, it also did not lead to a modulation of total glutathione levels nor G6PD expression. However, HSPB1 levels are related to a modulation of GSH/GSSG ratio, G6PD activity and NADPH/NADP + ratio. We have shown that HSPB1 is important for pro-survival effects against oxidative stress-induced beta-cell death. These results are in accordance with PRL-induced enrichment of HSPB1-interacting proteins related to protection against oxidative stress. Finally, our results outline the need of further studies investigating the importance of HSPB1 for beta-cell viability, since this could lead to the mitigation of beta-cell death through the up-regulation of an endogenous protective pathway.

AB - Maintaining islet cell viability in vitro, although challenging, appears to be a strategy for improving the outcome of pancreatic islet transplantation. We have shown that prolactin (PRL) leads to beta-cell cytoprotection against apoptosis, an effect mediated by heat shock protein B1 (HSPB1). Since the role of HSPB1 in beta-cells is still unclear and the hormone concentration used is not compatible with clinical applications because of all the side effects displayed by the hormone in other tissues, we explored the molecular mechanisms by which HSPB1 mediates beta-cell cytoprotection. Lysates from PRL- and/or cytokine-treated MIN6 beta-cells were subjected to HSPB1 immunoprecipitation followed by identification through mass spectrometry. PRL-treated cells presented an enrichment of several proteins co-precipitating with HSPB1. Of note were oxidative stress resistance-, protein degradation- and carbohydrate metabolism-related proteins. Wild type, HSPB1 silenced or overexpressing MIN6 cells were exposed to menadione and hydrogen peroxide and analysed for several oxidative stress parameters. HSPB1 knockdown rendered cells more sensitive to oxidative stress and led to a reduced antioxidant capacity, while prolactin induced an HSPB1-mediated cytoprotection against oxidative stress. HSPB1 overexpression, however, led to opposite effects. PRL treatment, HSPB1 silencing or overexpression did not change the expression nor activities of antioxidant enzymes, it also did not lead to a modulation of total glutathione levels nor G6PD expression. However, HSPB1 levels are related to a modulation of GSH/GSSG ratio, G6PD activity and NADPH/NADP + ratio. We have shown that HSPB1 is important for pro-survival effects against oxidative stress-induced beta-cell death. These results are in accordance with PRL-induced enrichment of HSPB1-interacting proteins related to protection against oxidative stress. Finally, our results outline the need of further studies investigating the importance of HSPB1 for beta-cell viability, since this could lead to the mitigation of beta-cell death through the up-regulation of an endogenous protective pathway.

KW - Animals

KW - Apoptosis

KW - Cytoprotection

KW - Glutathione/metabolism

KW - Heat-Shock Proteins/antagonists & inhibitors

KW - Insulin-Secreting Cells/cytology

KW - Insulinoma/drug therapy

KW - Mice

KW - Molecular Chaperones/antagonists & inhibitors

KW - Oxidation-Reduction

KW - Oxidative Stress/drug effects

KW - Pancreatic Neoplasms/drug therapy

KW - Prolactin/pharmacology

KW - Protein Transport

KW - Proteolysis

KW - Tumor Cells, Cultured

KW - Oxidative stress

KW - Beta-cell

KW - HSPB1

KW - Prolactin

KW - Diabetes

UR - http://www.scopus.com/inward/record.url?scp=85060885762&partnerID=8YFLogxK

U2 - 10.1016/j.freeradbiomed.2019.01.023

DO - 10.1016/j.freeradbiomed.2019.01.023

M3 - Article

C2 - 30699366

VL - 134

SP - 394

EP - 405

JO - Free Radical Biology and Medicine

JF - Free Radical Biology and Medicine

SN - 0891-5849

ER -