Angiotensin IV and the Molecular Mechanisms Involved in the Development of Insulin Resistance in 3T3-L1 Adipocytes

This study explored angiotensin IV’s (Ang IV) affects on the signaling pathways involved in the development of insulin resistance in 3T3-L1 adipocytes. Ang IV, working through the AT₄ receptor, interferes with insulin signaling through the blockade of the phosphatidylinositol 3-kinases (PI3K)/Akt pathway and through activating mitogen activated protein kinases (MAPK): extracellular signal regulated kinase (ERK) and c-Jun-N-terminal kinase (JNK) which are known to impair insulin receptor substrate-1 (IRS-1) signaling. The expression of AT₄ receptors was confirmed by reverse transcription polymerase chain reaction. Ang IV’s effects were found by treating adipocytes with combinations of Ang IV, AT₄ receptor inhibitor Norleual, and insulin. Cell lysates were resolved by SDS-PAGE electrophoresis and immunoblotted. Ang IV down-regulated the PI3K/Akt pathway. Insulin exerts its effects on adipocytes by activating this pathway, phosphorylating Akt (S473 and T308) residues. Pre-treatment with Ang IV blocked insulin’s effects, reversing Akt activation. Addition of Norleual blocked Ang IV’s inhibitory actions, leading to the phosphorylation of Akt residues. Studies show elevated MAPK levels produced by angiotensin peptides catalyze the phosphorylation of serine residues on IRS-1, impairing insulin signal transduction. Ang IV increased the activation of ERK 1/2 and JNK. This was reversed by pretreatment with Norleual. Ang IV’s effects on IRS-1 residues were found by immunoprecipitation of IRS-1 followed by SDS-PAGE immunoblotting. Ang IV increased the phosphorylation of IRS-1(S307 and S612). Pre-incubation with Norleual attenuated Ang IV’s effects. Ang IV’s stimulation of adipocytes quickly caused the phosphorylation of MAPK corresponding to serine residue phosphorylation on IRS-1, which may implicate Ang IV activation of MAPK in the development of insulin resistance. Ang IV is involved in the down-regulation of the insulin-signaling cascade by inhibiting insulin’s phosphorylation on Akt (S473 and T308). Ang IV increased phosphorylation of ERK 1/2 and JNK, corresponding with increases in serine phosphorylation of IRS-1. Pre-treatment with Norleual inhibited Ang IV’s negative effects on insulin signaling. This study elucidates a new mechanism that may lead to the development of insulin resistance in adipose tissue.