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International Textbook of Diabetes Mellitus, 4th Ed., Excerpt #60: Mechanisms of Insulin Signal Transduction Part 4 of 8

Jan 31, 2017

PI-3 kinase pathways: regulation of metabolism and gene expression

Phosphatidylinositide-3 kinase   


Signal transduction for insulin’s metabolic effects also diverges from insulin receptor substrate proteins and proceeds via the PI-3 kinase pathway (Figure 12.2). The first committed step involves type 1A PI-3 kinase, a heterodimer consisting of a p85 regulatory subunit and a p110 catalytic subunit [64]. In quiescent cells, the regulatory subunit maintains a state of low activity for the catalytic subunit. Upon insulin-mediated tyrosine phosphorylation, IRS-1 and IRS-2 (and other docking proteins) bind PI-3 kinase via interaction with the SH-2 domain on the p85 regulatory subunit [65]. Insulin stimulation increases the amount of PI-3 kinase associated with IRS, and the binding process increases the specific activity of the p110 catalytic subunit. Activation of PI-3 kinase is critical for transducing the metabolic actions of insulin, including stimulation of GLUT4 translocation to the plasma membrane with subsequent stimulation of glucose transport activity. This has been demonstrated in several lines of investigation. Treatment of adipocytes with (i) inhibitors of PI-3 kinase including wortmannin or LY-294002, (ii) hyperexpression of dominant negative mutants of PI-3 kinase that interfere with endogenous wild-type enzyme function, and (iii) microinjection of neutralizing antibodies to the p110 catalytic subunits all result in abrogation of insulin’s biologic effect [9]. Conversely, hyperexpression of wild-type PI-3 kinase or a constitutively active mutant leads to stimulation of both glucose transport activity and GLUT4 translocation from 50 – 100% of insulin’s maximal effect depending on the study [66].

Several proteins can serve as the regulatory subunit of PI-3 kinase [28]. Multiple isoforms arise through alternative splicing of the p85α gene (p85α, p55α/AS53, and p50α each occurring with and without a spliced insert), and two other isoforms represent products of different genes (p85β, p55PIK). All regulatory subunit isoforms associate with IRS phospho tyrosines and mediate activation of the associated p110 catalytic subunit. While p85α is ubiquitously expressed and is the dominant form in many tissues, the other isoforms exhibit differential tissue-specific expression. The various isoforms of the regulatory subunit could serve to modulate or direct signal transduction via different affinities for IRS proteins or differences in subcellular compartmentalization. In fact, studies have further illuminated novel mechanisms of regulation that involve different regulatory subunits of PI 3-kinase, and it has also become clear that the relative abundance of catalytic and regulatory subunits can influence insulin action [67 – 73]. For example, investigators have explored the roles of p85α (Pik3r1) and p85β (Pik3r2) regulatory subunits by using brown adipose cell lines with disruption of these genes [72]. These studies revealed key differences in the way the p85 regulatory subunit isoforms influence downstream signaling effects. Other studies have demonstrated that the balance between p85α regulatory and p110 catalytic subunit abundance regulates both insulin and IGF-1 signaling [71]. These data suggest that lowering the abundance of the p85α regulatory subunit can enhance insulin sensitivity and could present a new therapeutic target for insulin resistance.

The function of the activated PI-3 kinase is to phosphorylate the inositol ring in plasma membrane glycolipids at the D-3 position, thereby converting phosphatidylinositol 4,5 bisphosphate to phosphatidylinositol 3,4,5 trisphosphate (PI(3,4,5)P3 ), and to a lesser extent phosphatidylinositol 4-phosphate to phosphatidylinositol 3,4 bisphosphate (PI(3,4)P2) [74,75]. In skeletal muscle, the PI-3 kinase inhibitor, wortmannin, has been shown to prevent both the formation of 3′ phospholipids and block stimulation of glucose uptake in response to insulin. While the proximal physiologic targets for the D-3 phosphoinositides have not been fully elucidated, phosphorylation of the inositol ring at the 3′ position in membrane glycolipids recruits certain signaling proteins with pleckstrin homology domains to the plasma membrane. Binding to membrane-associated phosphoinositides both activates these proteins and positions them for downstream signal transduction. In addition, phospholipase C may participate in the release of the 3′ inositol phosphate moieties and may be activated by insulin through an IRS-independent mechanism [76,50], although siRNA-based protein knockdown of phospholipase Cγ did not affect insulin signaling to the glucose transport system in 3T3-L1 adipocytes [77].

While the role of PI-3 kinase in stimulation of glucose transport has been widely recognized, it has also become clear that PI-3 kinase activation is necessary for mediating multiple other metabolic effects of insulin including regulation of gene transcription. Thus, PI-3 kinase is the second major step, downstream of insulin receptor substrates, providing for broad divergence of insulin signal transduction, as illustrated in Figure 12.3. This is well demonstrated by experiments wherein suppression of PI-3 kinase has been shown to impact a host of insulin bioeffects including stimulation of glucose transport, glycogen synthesis, and glycolysis; promotion of lipogenesis via effects on fatty acid synthase and acetyl CoA carboxylase; suppression of gluconeogenesis; stimulation of protein synthesis; stimulation of DNA synthesis; attenuation (e.g. phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase) and induction (e.g. glucose-6-phosphate dehydrogenase and hexokinase II) of gene transcription; cell survival; and cell cycle progression. In order to propagate many of these effects, PI-3 kinase activates three serine-threonine kinases and their downstream pathways, namely, Akt or protein kinase B (Akt/PKB), atypical PKC isoforms (ζ,λ), and phosphoinositide-dependent kinases (PDK1 and PDK2).

ITDM Fig. 12.3

3-Phosphoinositide-dependent protein kinase-1 (PDK1)

Insulin-mediated activation of PI-3 kinase and generation of PI(3,4,5)P3 in membrane glycolipids results in the recruitment of AGC superfamily members (protein kinase A/protein kinase G/protein kinase C family) which contain pleckstrin homology domains. This activates these serine/threonine protein kinases and positions them for downstream signal transduction. With regard to insulin signaling, the most critical of the AGC kinases activated by interaction with PI(3,4,5)P3 is the PDK1, which participates in the downstream activation of Akt/PKB and aPKCs.

The AGC superfamily includes Akt/PKB and atypical PKCs.

Akt/protein kinase B   

There are three isoforms of Akt/PKB; Akt1 is expressed in a variety of tissues, while Akt2 is most highly expressed in adipocytes and Akt3 in certain cultured cell models [78 – 81]. Phosphorylation and activation of Akt/PKB isoforms involve both 3′ phosphoinositides and the action of PDK. 3′ Phosphoinositides in membrane glycolipids bind directly to the PH domain of Akt/PKB, and this process results in the uncovering of phosphorylation sites. At the same time, 3′ phosphoinositides activate PDK1, which in turn phosphorylates the activation loops of Akt/PKB on Thr308, enhancing the activity of the kinase [82]. However, Akt/PKB requires a second phosphorylation on Ser473 for full activation, and this could be accomplished in either of three ways. This could arise through the action of another putative phosphoinositide-dependent protein kinase, PDK2, or could be accomplished by PDK1 following a change in substrate recognition that results from threonine 308 phosphorylation, or may be the consequence of autophosphorylation. Recently, it has been suggested that rapamycin insensitive companion of mTOR (rictor) is necessary for the Ser473 phosphorylation step [83].

Regardless of the mechanism of phosphorylation, there are several lines of evidence that implicate a role for Akt/PKB activation in the stimulation of glucose transport. Expression of constitutively active membrane-bound forms of Akt results in persistent translocation of GLUT4 to the plasma membrane in muscle, fat, and cultured cell systems [84]. Interestingly, insulin stimulation is accompanied by association of Akt2 with GLUT4-containing vesicles in rat adipocytes resulting in phosphorylation of constituent proteins [85]. Conversely, cellular introduction of neutralizing antibodies, substrate peptides, and dominant negative mutant blocked insulin-stimulated GLUT4 translocation in adipocytes, albeit by only ∼50%. These experiments constitute strong evidence for a role of Akt/PKB in insulin stimulation of glucose transport. However, these results are somewhat controversial because other investigators demonstrated that a dominant-interfering mutant, while blocking insulin-stimulated protein synthesis, did not inhibit GLUT4 translocation [86]. Furthermore, in muscle from insulin-resistant diabetic humans, insulin-stimulated Akt phosphorylation was intact despite gross impairment in glucose uptake [87]. These discrepant results regarding a role for Akt/PKB do not exclude a role in the stimulation of glucose transport, perhaps working in concert with parallel activating pathways (e.g. PKC λ/ζ, CAP/Cbl/TC10). Mice deficient in Akt2 exhibit mild insulin resistance in muscle, glucose intolerance, and an inability to suppress hepatic glucose production, whereas Akt1 gene disruption produces growth reduction but has little effect on glucose tolerance [88,89]. These results are indicative of an important role for Akt/PKB in insulin regulation of glucose homeostasis and also suggest that Akt1 and Akt2 may compensate for each other’s absence to varying degrees. Nevertheless, the downstream targets of Akt/PKB connecting this signaling pathway to stimulation of the glucose transport effector system have not been identified with certainty.

Akt/PKB activation is involved in multiple other insulin responses [89]. One of the first substrates identified for Akt/ PKB was a serine 9 residue in glycogen synthase kinase 3β (GSK3β) [90,91]. Thus, one pathway that could contribute to insulin-stimulated glycogen synthesis involves Akt/ PKB-mediated phosphorylation and inactivation of glycogen synthase kinase 3β resulted in activation of glycogen synthase activity (see Chapters 13 and 14 concerning regulation of carbohydrate metabolism). Akt/PKB is also capable of phosphorylating and activating mammalian target of rapamycin (mTOR), which in turn phosphorylates 4E-BP1/PHAS-1, leading to release of translation initiation factor eIF-4E, and recruitment of mRNA to the 40S ribosomal subunit [92]. The ribosomal S6 protein (p70S6K ) is phosphorylated and activated either through PDK1, Akt/PKB, or mTOR, and similarly enhances translation of select populations of mRNA by increasing their interaction with the 40S ribosome [93]. In this way, Akt/PKB helps mediate insulin’s effect to stimulate protein synthesis (see Chapter 16: Insulin regulation of protein metabolism). Another apparent action is the phosphorylation and activation of endothelial NO synthase leading to increased NO generation and vasodilatation [94]. Additional substrates for Akt/PKB include BAD and caspase-9, which are involved in anti-apoptotic effects and alteration of cell survival pathways [95]. Finally, Akt/PKB phosphorylates forkhead transcription factors [96,97] altering expression patterns of genes involved in carbohydrate and lipid metabolism. In fact, the gene regulatory effects of MAP kinase and Akt/PKB-activated forkhead transcription factors generally oppose each other. In this way, Akt/PKB represents an important mechanism for “cross-talk” between the PI-3 kinase pathway, and other pathways regulating gene transcription and mitogenic effects, in the web of insulin signal transduction (Figure 12.3).

Protein kinase C

The IRS/PI-3 kinase pathway also activates PKCζ and PKCλ, two serine-threonine protein kinase C isoforms in the “atypical” class that is not activated by calcium binding, diacylglycerol, or, phorbol esters [98,99]. PI-3 kinase-dependent activation of PKC ζ/λ has been observed in multiple target tissues and cultured cell models in response to insulin [100]. The activation of PKC ζ/λ occurs proximal to Akt/PKB as a result of direct interaction with 3′ phosphoinositides and/or through phosphorylation and activation by PDK1, which phosphorylates PKCζ on Thr 410 enhancing the activity of the kinase [82]. This is relevant to the stimulation of glucose transport since overexpression of constitutively active forms of PKCζ or PKCλ increase glucose transport activity and GLUT4 translocation by 50–100% of the extent observed in response to maximal insulin [100]. Also, expression of dominant-interfering PKC mutants, lacking a critical lysine in the kinase domain or at the site phosphorylated by PDK1, inhibits insulin’s ability to promote glucose transport and GLUT4 translocation by ∼50%. As is common in the insulin action field, discrepant results have also been reported where overexpression of wild-type or dominant negative mutant forms in adipocytes did not affect basal or insulin-stimulated glucose transport [101]. This type of recurring controversy indicates that hyperexpression and gene knockout experiments should be interpreted cautiously especially when expression levels are altered well above or below the physiologic range, and also reflects overlapping and complementary pathways that comprise the web of insulin signal transduction. Nevertheless, on balance, available data indicate that PKC ζ/λ does participate in insulin’s metabolic signaling pathways [100]. As is the case for Akt/PKB, the downstream targets of PKC ζ/λ have not been fully identified, and this will be necessary for understanding of the relative participation of these two signaling proteins in the stimulation of glucose transport and other metabolic effects. Atypical PKCs can also mediate insulin simulation of general protein synthesis in a rapamycin-insensitive manner [102].

Degradation of the PI-3 kinase signal

At least two different types of phosphatases can degrade phosphoinositides leading to deactivation of the PI-3 kinase signal [75]. Overactivity of these enzymes has the potential for inhibiting insulin signal transduction distal to the generation of 3′ phosphoinositides and causing insulin resistance. Src-homology 2-containing inositol phosphatases (SHIP1 and SHIP2) dephosphorylate the 5′ position of PI(3,4,5)P3 to form PI(3,4)P2 . Genetic disruption and loss of SHIP2 results in a marked increase in insulin sensitivity, implying that PI-3 kinase-mediated formation of PI(3,4,5)P3 , rather than PI(3,4)P2, is the critical 3′ phosphoinositide in insulin signaling. Another phosphatase, phosphatase and tensin homologue (PTEN), dephosphorylates the 3′ position converting PI(3,4,5)P3 to PI(4,5)P2. Some human cancers are associated with loss of PTEN expression suggesting that unrestrained production of 3′ phosphoinositides could result in oncogenesis.

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