Effects of glycated insulin on adipogenic differentiation. A. Piccoli1, T. Bisogno1,2, M. Maccarrone1,3, P. Pozzilli1,4, N. Napoli1,5, R. Strollo1. 1Universita Campus Bio-Medico di Roma, Rome, Italy; 2National Research Council, Pozzuoli, Italy; 3European Center for Brain Research (CERC)/Santa Lucia Foundation IRCCS, Rome, Italy; 4Queen Mary, University of London, London, UK; 5Washington University in St Louis, St Louis, MO, USA Hyperglycaemia and chronic low-grade inflammation associated with obesity and diabetes are a main source of reactive oxidants (ROS). High levels of ROS may generate oxidative post-translational modification (oxPTMs) that may impair proteins biological functions. We have shown that insulin could also be a target of these modifications that include glycation, oxidation and chlorination. Adipose tissue is a primary target of this hormone and a key player in insulin resistance (IR): evidence from animal studies show that the glycation of insulin or other proteins result in a significant impairment of biological function of adipose tissue, affecting adipokines expression and insulin sensitivity. However, how oxPTMs such as glycation interfere with insulin function and their involvement in IR and type 2 diabetes (T2D) is still unknown. We hypothesize that insulin glycation may be involved in insulin resistance and in the pathogenesis of T2D. Therefore, the aim of this study was to assess the possible effects of glycated vs. native insulin on adipocyte differentiation. Human recombinant insulin was glycated in vitro following our established protocol. Insulin glycation was monitored through PAGE. Human preadipocytes (HPAd), a primary cell line from subcutaneous adipose tissue, and adipose-derived stem cells (ASCs), isolated from tissues of lean and obese subjects (BMI<25 and BMI>30), were differentiated with standard adipogenic medium in presence of native or glycated insulin. Cellular differentiation was assessed by gene and protein expression analyses of adipogenic markers (adiponectin, peroxisome proliferator-activated receptor gamma -Ppar?-, fatty acid binding protein 4 -Fabp4-, glucose transporter 4 -Glut4-) and of receptors for adiponectin (AdipoR1) and for insulin (InsR), through real time RT-PCR and immunoblot respectively. Our preliminary results showed that preadipocytes (both HPAd and ASCs) exposed to glycated insulin had impaired differentiation capacities: Oil Red O-staining showed that these cells had lower number of lipid droplets compared to those exposed to native insulin, suggesting an impaired adipocyte differentiation. Considering gene expression, we observed an overall downregulation of adipogenic markers, with a significant decrease of adiponectin both in leans- and in obese-derived cells (p=0.007 and p=0.017 respectively), lower levels of Glut4 and a significant lower expression of Fabp4 (p=0.002) in cells from leans differentiated with glycated insulin. Immunoblotting showed a similar pattern to that observed with gene expression analysis but, regarding insulin receptor, protein levels of the receptor presented a significant down regulation in cells treated with glycated insulin, with a p value of 0.0266 in leans and p=0.011 in obese subjects. Conversely, gene expression of insulin receptor did not present any difference between the native and glycated insulin. In conclusion, our data suggest that glycated insulin could affect adipocytes differentiation and impair insulin receptor expression, acting at translational/post-translational level or increasing its degradation. Its role and mechanisms that lead to these effects should be investigated as a possible mediator in the pathogenesis of T2D.
Effects of glycated insulin on adipogenic differentiation
Bisogno T;
2018
Abstract
Effects of glycated insulin on adipogenic differentiation. A. Piccoli1, T. Bisogno1,2, M. Maccarrone1,3, P. Pozzilli1,4, N. Napoli1,5, R. Strollo1. 1Universita Campus Bio-Medico di Roma, Rome, Italy; 2National Research Council, Pozzuoli, Italy; 3European Center for Brain Research (CERC)/Santa Lucia Foundation IRCCS, Rome, Italy; 4Queen Mary, University of London, London, UK; 5Washington University in St Louis, St Louis, MO, USA Hyperglycaemia and chronic low-grade inflammation associated with obesity and diabetes are a main source of reactive oxidants (ROS). High levels of ROS may generate oxidative post-translational modification (oxPTMs) that may impair proteins biological functions. We have shown that insulin could also be a target of these modifications that include glycation, oxidation and chlorination. Adipose tissue is a primary target of this hormone and a key player in insulin resistance (IR): evidence from animal studies show that the glycation of insulin or other proteins result in a significant impairment of biological function of adipose tissue, affecting adipokines expression and insulin sensitivity. However, how oxPTMs such as glycation interfere with insulin function and their involvement in IR and type 2 diabetes (T2D) is still unknown. We hypothesize that insulin glycation may be involved in insulin resistance and in the pathogenesis of T2D. Therefore, the aim of this study was to assess the possible effects of glycated vs. native insulin on adipocyte differentiation. Human recombinant insulin was glycated in vitro following our established protocol. Insulin glycation was monitored through PAGE. Human preadipocytes (HPAd), a primary cell line from subcutaneous adipose tissue, and adipose-derived stem cells (ASCs), isolated from tissues of lean and obese subjects (BMI<25 and BMI>30), were differentiated with standard adipogenic medium in presence of native or glycated insulin. Cellular differentiation was assessed by gene and protein expression analyses of adipogenic markers (adiponectin, peroxisome proliferator-activated receptor gamma -Ppar?-, fatty acid binding protein 4 -Fabp4-, glucose transporter 4 -Glut4-) and of receptors for adiponectin (AdipoR1) and for insulin (InsR), through real time RT-PCR and immunoblot respectively. Our preliminary results showed that preadipocytes (both HPAd and ASCs) exposed to glycated insulin had impaired differentiation capacities: Oil Red O-staining showed that these cells had lower number of lipid droplets compared to those exposed to native insulin, suggesting an impaired adipocyte differentiation. Considering gene expression, we observed an overall downregulation of adipogenic markers, with a significant decrease of adiponectin both in leans- and in obese-derived cells (p=0.007 and p=0.017 respectively), lower levels of Glut4 and a significant lower expression of Fabp4 (p=0.002) in cells from leans differentiated with glycated insulin. Immunoblotting showed a similar pattern to that observed with gene expression analysis but, regarding insulin receptor, protein levels of the receptor presented a significant down regulation in cells treated with glycated insulin, with a p value of 0.0266 in leans and p=0.011 in obese subjects. Conversely, gene expression of insulin receptor did not present any difference between the native and glycated insulin. In conclusion, our data suggest that glycated insulin could affect adipocytes differentiation and impair insulin receptor expression, acting at translational/post-translational level or increasing its degradation. Its role and mechanisms that lead to these effects should be investigated as a possible mediator in the pathogenesis of T2D.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
