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The events which follow insulin binding and transmembrane signalling and culminate in the metabolic actions o f insulin are less well understood. Many actions involve the phosphorylation and dephosphorylation of enzymes and this has led to the theory o f a p h o sp h o ry latio n cascade in itiated by the recep to r tyrosine kinase. T hese phosphorylation/dephosphorylation processes are mediated by serine/threonine kinases (Czech et al. 1988), with insulin-induced receptor autophosphorylation as the initiating event in a cascade o f phosphorylation of cellular substrates. This results in amplification o f the signal and alteration of the phosphorylation state, and hence the activity, o f target enzymes (Lane et al. 1990) (Figure 1.4). Many proteins have been proposed as substrates in this phosphorylation cascade (Kasuga et al. 1990).

Figure 1.4. Post-receptor phosphorylation cascade i n s u l i n

I

G3 GD

The first endogenous cellular substrate of the insulin receptor, insulin re c e p to r

s u b s tra te 1 (IRS-1), has recently been identified (Sun et al. 1991). This cytosolic protein o f m olecular weight 131 000, contains over 30 potential serine/threonine p h o sp h o ry latio n sites. In sulin stim ulates the asso ciatio n o f IRS-1 w ith phosphatidylinositol (Ptdlns) 3 ’-kinase, an enzyme which phosphorylates the Ptdlns of the myoinositol ring by an alternative route to the classical phospholipase C pathway discussed below. Ptdlns 3 ’-kinase is composed of 2 subunits, a 110 000 m olecular weight catalytic subunit and a regulatory protein of molecular weight 85 000 (p 8 5 a ). The latter contains two SH (src homology) 2 domains which effect protein-protein interactions by binding to Tyr-phosphorylated protein motifs, and in particular, those with the sequence YMXM (Tyr-Met-Xaa-Met).

Figure 1.5. Insulin signalling initiated by insulin receptor substrate 1. insulin insulin ^ receptor Ptdlns ► Ptdlns-3P SH2 p85 SH2 IRS-1 pllO SIGNAL IRS-I SH2 p85 SH2 pi 10 Ptdlns 3' kinase Ptdlns-3P = phosphatidylinositol 3’-phosphate

W hen the insulin receptor is activated, the receptor kinase phosphorylates specific tyrosine residues with the sequence YMXM in IRS-1 and these phosphorylated sites associate with the SH2 domains o f p 8 5 a , thus activating Ptdlns 3 '-kinase. IR S-I contains six YM XM motifs, so it may potentially bind m ultiple Ptdlns 3 ’-kinase m olecules, or simultaneously bind several different proteins containing an SH2 domain. Subunit p85 exists as two isoforms, a and 13. Only isoform p 8 5 a associates with p i 10 subunit to form the Ptdlns 3 ’-kinase enzyme. The p856 does not, but may act as an adaptor molecule linking IRS-1 to a different signalling enzyme. An increasing num ber o f different proteins containing SH2 domains are currently being identified which can interact with IRS-1 after insulin stimulation (Myers and W hite 1993). Although IRS-I has been studied in relation to insulin stimulation of cell proliferation, it may also be involved in insulin-mediated metabolic events, with the particular pattern o f IRS-1 phosphorylation determining the specific metabolic actions in individual tissues.

Alternatively, insulin’s metabolic actions may be the result of the production o f a second messenger which conveys the signal to the target enzyme. Autophosphorylation of the insulin receptor results in a conformational change and this may allow its non-covalent interaction with cellular proteins that can act as signal transducers (Perlman et al. 1989). The addition o f insulin to m yocytes stim ulates the hydrolysis o f an inositol glycophospholipid, designated glycosylphosphatidylinositol (GPI), thereby generating diacylglycerol and the polar headgroup of the molecule. Glycosylphosphatidylinositol is present in many other tissues and its structure is similar to that o f a membrane bound glycosyl phosphatidylinositol. This membrane-linked phosphoinositol is hydrolysed by a specific phospholipase C releasing diacylglycerol, inositol phosphate glycan and over 40 membrane proteins with diverse functions (Saltiel 1990). The link between insulin receptor activity and stim ulation o f this phospholipase C is still speculative. Phospholipase C m ay be activated as p art o f the tyrosine kinase-induced phosphorylation cascade or it may result from a receptor conform ational change, possibly following GTP binding (Goren et al. 1985; Kom et al. 1987).

Amongst the numerous proteins yielded by phosphoinositol hydrolysis is lipoprotein lipase (Saltiel 1990). The observation that both insulin and phospholipase C release lipoprotein lipase with identical kinetics in vitro is o f significance as insulin causes lipoprotein lipase release from adipocytes in vivo (Chan et al. 1988). Phospholipase C hydrolysis also yields diacylglycerol which may itself act as a second messenger by promoting protein kinase C activity. Activation of protein kinase C by other means, such as by phorbol esters, has the effect in some cases of mimicking insulin action and in others, o f opposing insulin’s action (Van de W erve et al. 1985). This apparent paradox may be resolved by the existence of distinct chemical forms of diacylglycerol possibly deriving from different sources; each chemical form of diacylglycerol may selectively activate different isoforms o f protein kinase C (Pelosin et al. 1987) with different substrate affinities and tissue distributions.

Finally it may be that the diversity of insulin’s actions lends support to the hypothesis that no single mechanism accounts for the transmission o f insulin’s signal to target enzymes but rather, a combination o f phosphorylation cascade, conformational change

and second messengers act synergistically.