Alemu Fite1, Abdul Badi Abou-Samra2, Berhane Seyoum3. 1. Wayne State University School of Medicine, Detroit, Michigan, USA. Electronic address: afite@med.wayne.edu. 2. Wayne State University School of Medicine, Detroit, Michigan, USA; Hamad Medical Corporation, Doha, Qatar. 3. Hamad Medical Corporation, Doha, Qatar.
Abstract
OBJECTIVES: The interaction of immune cells with adipocytes within the adipose tissues in obese persons with diabetes mellitus may play a role in insulin resistance. We examined in vitro whether nitric oxide (NO) and inducible nitric oxide synthase (iNOS) play a role in impaired insulin signalling in adipocytes exposed to activated macrophages. METHODS: We used a co-culture system in which Raw264.7 macrophages were plated over differentiated, low passage 3T3-L1 cells (dif3T3) at a cell density ratio of 1:2. Inflammation was induced by a challenge with bacterial lipopolysaccharide. RESULTS: Significantly (p<0.001) enhanced iNOS expression and NO synthesis was observed in activated co-cultures. In the co-cultures as compared with Raw264.7 cells alone, iNOS protein was induced up to 11-fold above background, and NO release was significantly (p<0.001) increased up to 2.8-fold. Co-culturing dif3T3 and Raw264.7 cells as compared to dif3T3 alone reduced insulin-induced Akt phosphorylation by 50% and AS160 phosphorylation by 42%. This was correlated with reduced glucose consumption when dif3T3 was exposed to 1,3-morpholinosydnonimine. Adiponectin, GLUT4 and AS160 mRNA were reduced by 4-fold, 5-fold and 2-fold, respectively, in co-cultures as compared to dif3T3 alone. On the contrary, GLUT1 mRNA levels were increased by 2-fold in co-cultures as compared to dif3T3. NG-monomethyl-L-arginine abolished NO production with modest reversal of Akt/AS160 phosphorylation. CONCLUSIONS: This study demonstrated a potential association between iNOS/NO-mediated inflammation and insulin resistance.
OBJECTIVES: The interaction of immune cells with adipocytes within the adipose tissues in obesepersons with diabetes mellitus may play a role in insulin resistance. We examined in vitro whether nitric oxide (NO) and inducible nitric oxide synthase (iNOS) play a role in impaired insulin signalling in adipocytes exposed to activated macrophages. METHODS: We used a co-culture system in which Raw264.7 macrophages were plated over differentiated, low passage 3T3-L1 cells (dif3T3) at a cell density ratio of 1:2. Inflammation was induced by a challenge with bacterial lipopolysaccharide. RESULTS: Significantly (p<0.001) enhanced iNOS expression and NO synthesis was observed in activated co-cultures. In the co-cultures as compared with Raw264.7 cells alone, iNOS protein was induced up to 11-fold above background, and NO release was significantly (p<0.001) increased up to 2.8-fold. Co-culturing dif3T3 and Raw264.7 cells as compared to dif3T3 alone reduced insulin-induced Akt phosphorylation by 50% and AS160 phosphorylation by 42%. This was correlated with reduced glucose consumption when dif3T3 was exposed to 1,3-morpholinosydnonimine. Adiponectin, GLUT4 and AS160 mRNA were reduced by 4-fold, 5-fold and 2-fold, respectively, in co-cultures as compared to dif3T3 alone. On the contrary, GLUT1 mRNA levels were increased by 2-fold in co-cultures as compared to dif3T3. NG-monomethyl-L-arginine abolished NO production with modest reversal of Akt/AS160 phosphorylation. CONCLUSIONS: This study demonstrated a potential association between iNOS/NO-mediated inflammation and insulin resistance.
Authors: Tamires M Zanotto; Paula G F Quaresma; Dioze Guadagnini; Lais Weissmann; Andressa C Santos; Juliana F Vecina; Kelly Calisto; Andrey Santos; Patrícia O Prada; Mario J A Saad Journal: Mol Metab Date: 2016-12-19 Impact factor: 7.422