A Molecular Basis for Insulin Resistance

Abstract

Tumor necrosis factor (TNF) or chronic hyperinsu-linemia that induce insulin resistance trigger increased Ser/Thr phosphorylation of the insulin receptor (IR) and of its major insulin receptor substrates, IRS-1 and IRS-2. To unravel the molecular basis for this uncoupling in insulin signaling, we undertook to study the interaction of Ser/Thr-phosphorylated IRS-1 and IRS-2 with the insulin receptor. We could demonstrate that, similar to IRS-1, IRS-2 also interacts with the juxtamembrane (JM) domain (amino acids 943-984) but not with the carboxyl-terminal region (amino acids 1245-1331) of IR expressed in bacteria as His 6 fusion peptides. Moreover, incubation of rat hepatoma Fao cells with TNF, bacterial sphingomyelinase, or other Ser(P)/Thr(P)-elevating agents reduced insulin-induced Tyr phosphorylation of IRS-1 and IRS-2, markedly elevated their Ser(P)/Thr(P) levels, and significantly reduced their ability to interact with the JM region of IR. Withdrawal of TNF for periods as short as 30 min reversed its inhibitory effects on IR-IRS interactions. Similar inhibitory effects were obtained when Fao cells were subjected to prolonged (20-60 min) pretreatment with insulin. Incubation of the cell extracts with alkaline phosphatase reversed the inhibitory effects of insulin. These findings suggest that insulin resistance is associated with enhanced Ser/Thr phosphorylation of IRS-1 and IRS-2, which impairs their interaction with the JM region of IR. Such impaired interactions abolish the ability of IRS-1 and IRS-2 to undergo insulin-induced Tyr phosphorylation and further propagate the insulin receptor signal. Moreover, the reversibility of the TNF effects and the ability to mimic its action by exogenously added sphingomyeli-nase argue against the involvement of a proteolytic cascade in mediating the acute inhibitory effects of TNF on insulin action. The insulin receptor (IR) 1 is an heterotetrameric transmem-brane glycoprotein composed of two extracellular subunits and two transmembrane subunits linked by disulfide bonds. The subunits contain the insulin-binding domain while the transmembrane subunits function as a tyrosine-specific protein kinase (IRK) that undergoes autophosphorylation following insulin binding (reviewed in Ref. 1). Autophosphorylation activates the IRK (2) and enables it to phosphorylate endoge-nous protein substrates, including Shc (3) and the insulin receptor substrates IRS-1 (4) and IRS-2 (5), to further propagate the insulin signal. IRS-1 and IRS-2, two related protein sub-strates of IRK, have a highly conserved amino terminus, which contains a pleckstrin homology domain and a phosphotyrosine-binding (PTB) domain, and a poorly conserved carboxyl terminus with several tyrosine phosphorylation motifs. IRS-1 and IRS-2 also contain over 30 Ser/Thr residues in consensus phos-phorylation sites (4, 5). The relative roles of IRS-1 and IRS-2 in mediating insulin action is presently unknown, although IRS-2 functions as an alternative substrate of IR in IRS-1 null mice (6), which manifest a mild form of insulin resistance. Insulin resistance is a state in which target cells fail to respond to ordinary levels of circulating insulin (7). At the molecular level, impaired insulin signaling results from mutations or post-translation modifications of the insulin receptor itself or any of its downstream effector molecules. A major negative regulatory role to insulin action is attributed to agents that enhance Ser/Thr phosphorylation of either the receptor itself or of its downstream effectors, which reduce IRK activity or its ability to phosphorylate substrate proteins (see Refs. 8 and 9 for reviews). For example, insulin's counter-regulatory hormones such as epinephrine or glucagon increase cAMP levels , activate the cAMP-dependent protein kinase, and increase the Ser(P)/Thr(P) content of the insulin receptor, which results in an insulin-resistant state. Similarly, okadaic acid, an inhib-itor of protein phosphatases, inhibits insulin-induced Tyr phos-phorylation of IRS-1 while increasing the Ser/Thr phosphoryl-ation level of this protein (10, 11). Tumor necrosis factor-(TNF), a mediator of insulin resistance in infection, tumor cachexia, and obesity, also acts in a similar manner. TNF diminishes insulin-induced Tyr phosphorylation of IRS-1 while it induces Ser/Thr phosphorylation of IRS-1, which decreases