- The insulin pathway
- Negative regulators and termination of insulin signaling
Insulin is a hormone released by pancreatic beta cells in response to elevated levels of nutrients in the blood. Insulin triggers the uptake of glucose, fatty acids and amino acids into liver, adipose tissue and muscle and promotes the storage of these nutrients in the form of glycogen, lipids and protein respectively. Failure to uptake and store nutrients results in diabetes. Type-1 diabetes is characterised by the inability to synthesise insulin, whereas in type-2 diabetes the body becomes resistant to the effects of insulin presumably because of defects in the insulin signaling pathway.
The insulin receptor is composed of two extracellular a subunits and two transmembrane b subunits linked together by disulphide bonds (Figure 1). Binding of insulin to the a subunit induces a conformational change resulting in the autophosphorylation of a number of tyrosine residues present in the b subunit (Van Obberghen et al., 2001). These residues are recognised by phosphotyrosine-binding (PTB) domains of adaptor proteins such as members of the insulin receptor substrate family (IRS) (Saltiel and Kahn 2001; Lizcano and Alessi 2002). Receptor activation leads to the phosphorylation of key tyrosine residues on IRS proteins, some of which are recognised by the Src homology 2 (SH2) domain of the p85 regulatory subunit of PI 3-kinase (a lipid kinase). The catalytic subunit of PI 3-kinase, p110, then phosphorylates phosphatidylinositol (4,5) bisphosphate (PtdIns(4,5)P2) leading to the formation of Ptd(3,4,5)P3. A key downstream effector of Ptd(3,4,5)P3 is AKT (otherwise known as PKB), which is recruited to the plasma membrane. Activation of AKT also requires the protein kinase 3-phosphoinositide-dependent protein kinase-1 (PDK1), which in combination with an as yet unidentified kinase leads to the phosphorylation of AKT (Figure 2). Once active, AKT enters the cytoplasm where it leads to the phosphorylation and inactivation of glycogen synthase kinase 3 (GSK3) (Figure 3). A major substrate of GSK3 is glycogen synthase, an enzyme that catalyses the final step in glycogen synthesis. Phosphorylation of glycogen synthase by GSK3 inhibits glycogen synthesis; therefore the inactivation of GSK3 by AKT promotes glucose storage as glycogen.
Figure 1: Insulin signaling pathway
Activation of the insulin receptor evokes increased transcription of SREBP and the phosphorylation of members of the IRS family, SHC and Cbl. Upon tyrosine phosphorylation, these proteins interact with signaling molecules through their SH2 domains, which results in the activation of a variety of signaling pathways, including PI 3-kinase signaling, MAPK activation and the activation of the Cbl/CAP complex. These pathways act in a coordinated manner to regulate glucose, lipid and protein metabolism.
In addition to promoting glucose storage, insulin inhibits the production and release of glucose by the liver by blocking gluconeogenesis and glycogenolysis (Saltiel and Kahn 2001). Insulin directly controls the activities of a set of metabolic enzymes by phosphorylation and dephosphorylation events and also regulates the expression of genes encoding hepatic enzymes involved in gluconeogenesis. Recent evidence suggests that forkhead transcription factors, which are excluded from the nucleus following phosphorylation by AKT, play a role in hepatic enzyme regulation by insulin (Schmoll et al., 2000; Barthel et al., 2001).
A key action of insulin is to stimulate glucose uptake into cells by inducing translocation of the glucose transporter, GLUT4, from intracellular storage to the plasma membrane. PI 3-kinase and AKT are known to play a role in GLUT4 translocation (Lizcano and Alessi 2002). In addition, a PI 3-kinase independent pathway provides a second cue for GLUT4 recruitment to the plasma membrane (Saltiel and Kahn 2001). In this pathway, insulin receptor activation leads to the phosphorylation of Cbl, which is associated with the adaptor protein CAP. Following phosphorylation the Cbl-CAP complex translocates to lipid rafts in the plasma membrane. Cbl then interacts with the adaptor protein Crk, which is constitutively associated with the Rho-family guanine nucleotide exchange factor, C3G. C3G in turn activates members of the GTP-binding protein family, TC10, which promote GLUT4 translocation to the plasma membrane through the activation of as yet unknown adaptor molecules.
Primary cortical neurones were treated with insulin (50 nM) and lysates were prepared at the indicated time points. Western blotting was subsequently performed using anti-pAKT and anti-AKT antibodies, the latter to demonstrate equal loading. p-AKT antibody recognises AKT phosphorylated at Ser473.
Primary cortical neurones were treated with insulin (50 nM) and lysates were prepared at the indicated time points. Western blotting was subsequently performed using anti-pGSK3 and anti-GSK3 antibodies, the latter to demonstrate equal loading. GSK3a is phosphorylated at serine 21, whereas GSK3b is phosphorylated at serine 9 in response to insulin treatment.
Insulin stimulates amino acid uptake into cells, inhibits protein degradation (through an unknown mechanism) and promotes protein synthesis (Saltiel and Kahn 2001). Under basal conditions the constitutive activity of GSK3 leads to the phosphorylation and inhibition of a guanine nucleotide exchange factor eIF2B, which regulates the initiation of protein translation. Therefore, upon receipt of an insulin signal, inactivation of GSK3 by AKT leads to the dephosphorylation of eIF2B thereby promoting protein synthesis and the storage of amino acids (Lizcano and Alessi 2002). AKT also activates mammalian target of rapamycin (mTOR), which promotes protein synthesis through p70 ribosomal S6 kinase (p70s6k) and inhibition of eIF-4E binding protein (4E-BP1) (Asnaghi et al., 2004).
Insulin promotes the uptake of fatty acids and the synthesis of lipids, whilst inhibiting lipolysis. Recent studies indicate that lipid synthesis requires an increase in the transcription factor steroid regulatory element-binding protein (SREBP)-1c (Shimomura et al., 1999). However, the pathway leading to changes in SREBP expression are unknown. Insulin inhibits lipid metabolism through decreasing cellular concentrations of cAMP by activating a cAMP specific phosphodiesterase in adipocytes (Kitamura et al., 1999).
Other signal transduction proteins interact with IRS including GRB2, an adaptor protein that contains SH3 domains, which in turn associates with the guanine nucleotide exchange factor son-of sevenless (sos) and elicits activation of the MAPK cascade leading to mitogenic responses (Ogawa et al., 1998). SHC is another substrate for the insulin receptor. Upon phosphorylation SHC associates with GRB2 and can therefore activate the MAPK pathway independently of IRS.
Enzymes that are important in the attenuation of PtdIns(3,4,5)P3 signaling are phosphatase and tensin homologue on chromosome 10 (PTEN, a 3’ phosphatase) and the family of SRC homology 2 containing inositol 5’-phosphatase (SHIP, a 5’ phosphatase) proteins, which include two gene products SHIP1 and SHIP2 (Lazar and Saltiel 2006). These phosphatases degrade PtdIns(3,4,5)P3 to PtdIns(4,5)P2 or PtdIns(3,4)P2 respectively. Termination of insulin signaling is also achieved by internalization of the insulin-insulin receptor complex into endosomes and the degradation of insulin by insulin degrading enzyme (IDE) (Bevan 2001).
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