Targeting protein kinase C epsilon or theta as a therapeutic strategy for insulin resistance

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Drug Discovery Today: Therapeutic Strategies Editors-in-Chief Raymond Baker – formerly University of Southampton, UK and Merck Sharp & Dohme, UK Eliot Ohlstein – GlaxoSmithKline, USA DRUG DISCOVERY

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Targeting protein kinase C epsilon or theta as a therapeutic strategy for insulin resistance Carsten Schmitz-Peiffer Cell Signalling Group, Diabetes and Obesity Program, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, Sydney, NSW 2232, Australia

Isoforms of the protein kinase C family are strong candidates for mediating the inhibitory effects of lipid oversupply on insulin action. These enzymes are lipid-

Section Editors: James Fraser – Pfizer, USA Anne Miller – Lilly Research Laboratories, IN, USA

activated, can interfere with insulin signal transduction, and several studies have highlighted an association between insulin resistance and chronic activation of specific protein kinase C isoforms, especially epsilon (e) and theta (u). The assessment of glucose homeostasis and lipid-induced insulin resistance in protein kinase C u knockout mice has now demonstrated a key role for this isoform, although it is not yet clear whether its long-term inhibition would be beneficial. Potential approaches to block the action of specific

correlated with fat oversupply [2], although high glucose and insulin levels can also cause secondary insulin resistance. Although several mechanisms have been proposed for the generation of lipid-induced insulin resistance, evidence for the involvement of specific isoforms of the protein kinase C (PKC) family of signal transduction molecules has steadily accumulated over the last decade. Initially this was through studies associating PKC activation with insulin resistance in target tissues, but more recent research has implied causative roles for particular isoforms and examined their mechanism of action.

PKC isoforms include pharmacological inhibitors, antisense oligonucleotides and bioactive peptides. Introduction Insulin resistance is a major factor in the development of type 2 diabetes and the metabolic syndrome. Because skeletal muscle accounts for a high proportion of the whole body glucose disposal after a meal [1], defects in insulin action in this tissue can give rise to hyperglycaemia and hyperinsulinaemia, with their resultant complications. Insulin resistance at the level of the liver leads to poor suppression of hepatic glucose output by the hormone, which also contributes to glucose intolerance [1]. Reduced insulin sensitivity is strongly E-mail address: C. Schmitz-Peiffer ([email protected]) 1740-6773/$ ß 2005 Elsevier Ltd. All rights reserved.

DOI: 10.1016/j.ddstr.2005.04.002

Why are PKC isoforms such good candidates? As the PKC family became defined, it was possible to classify individual isoforms into three subclasses on the basis of structural homology [3]. Now, these groupings are also useful in describing the modes of activation of the enzymes. Thus the conventional (or classical) PKC isoforms, PKCa, PKCbI, PKCbII and PKCg, share C1 domains for diacylglycerol (DAG)binding and C2 domains for Ca2+-binding, located in their N-terminal regulatory domains. Both DAG and Ca2+ are required for the full activation of conventional PKCs. In contrast, the novel PKC isoforms, PKCd, PKCe, PKCu and PKCh, possess only the DAG-binding domain and are Ca2+independent. Because DAG can be generated by de novo synthesis from fatty acids entering the cell (as opposed to www.drugdiscoverytoday.com

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Figure 1. Simplified scheme for the activation of protein kinase C (PKC) isoforms through elevation of intracellular diacylglycerol (DAG) species upon chronic exposure to lipid. PKC activity in turn might directly or indirectly cause diminished signalling through insulin receptor substrate-1 (IRS-1), by promoting its Ser phoshorylation at one or more sites. IRS-1 normally promotes glucose disposal by the activation of phosphatidylinositol 3-kinase (PI3K) and subsequently atypical PKC (aPKC) and Akt (also known as protein kinase B). See text for further details.

its acute release by phospholipase C action during activation of signal transduction pathways), both the conventional and novel PKC isoforms could be expected to exhibit chronic activation upon increased lipid availability (Fig. 1). Intracellular DAG can also become elevated during prolonged exposure to glucose or insulin, suggesting that this mechanism can also be responsible for PKC activation by these factors. Finally, the atypical PKC isoforms, PKCz and PKCi/l, are both Ca2+- and DAG-independent, and become activated in response to elevations in phosphatidylinositol 3,4,5-trisphosphate, produced by the action of phosphatidylinositol 3kinase (PI3K), for example, upon insulin stimulation (Fig. 1). This distinction between atypical and other PKC isoforms is in agreement with the roles that each plays in glucose metabolism – the atypical PKCs are required for insulin-stimulated glucose uptake, and their activation is reduced in insulinresistant states [4], whereas the conventional and especially the novel PKCs appear to act in an inhibitory fashion. Conventional and novel PKCs translocate from the cytosol to cell membranes upon activation, and therefore, translocation has been used to correlate the insulin resistance of skeletal muscle with the activation of specific PKC isoforms, notably PKCe and PKCu, observed in several animal models and human studies (reviewed in [5,6]). Importantly, when the total PKCe protein levels are maintained upon such translocation, PKCu levels in skeletal muscle were found to fall in several studies [7–9], manifested as a decrease in cytosolic protein whereas membrane-associated levels remain unchanged. This has been interpreted as chronic activation in response to elevated lipids because of the increase in the ratio of membrane-bound to cytosolic PKCu. Alternatively, however, the downregulation could indicate a reduced potential for further PKC activation, which would argue against an inhibitory role for PKCu in the long term. 106

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Although PKCu shows a restricted distribution, being expressed in only a few tissues such as skeletal muscle and haematopoietic cells, most other PKCs are more widely expressed. Translocation of PKCd [10] and, more recently, PKCe [11] has been associated with lipid-induced insulin resistance in the liver. Increased PKCe expression has also been reported in prediabetic animals of the diabetes-prone line of the sand rat (Psammomys obesus), together with translocation of the isoform in muscle from diabetic animals [12]. Because such correlative studies have implicated several PKCs other than PKCu and PKCe in insulin resistance [10,13–15], albeit to a lesser extent, new approaches have become necessary, and current efforts are focussed upon mechanism and causation.

How could increased PKC activity interfere with insulin action? Much attention has been centred on upstream insulin signal transduction because lesions at this level have been widely reported in the studies of type 2 diabetes and insulin resistance [5]. It now appears that PKC activation is one of the several pathways which lead to an increased Ser/Thr phosphorylation of insulin receptor substrate (IRS) proteins (Fig. 1), especially IRS-1 [16,17]. Such a modification of these docking proteins reduces their ability to interact with the binding partners such as the insulin receptor and downstream components of insulin signalling, especially PI3K. Ser307 is a key site on IRS-1, and is apparently phosphorylated by several different kinases, including the mammalian Target of Rapamycin (mTOR), IkB kinase b (IKKb) and c-jun N-terminal kinase (JNK), to inhibit insulin signalling. This site exhibits increased phosphorylation during the generation of insulin resistance in rats by lipid infusion although IRS-1 tyrosine phosphorylation and PI3K association is

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reduced, and PKCu redistribution is promoted [9]. Similarly, PKCe translocation is associated with a diminished IRS-1 and IRS-2 tyrosine phosphorylation in liver [11]. Although PKCs might not mediate Ser307 phosphorylation directly, both PKCe and PKCu were found to act upstream of IKKb [18,19] and JNK [20–22], and could inhibit insulin signalling through these kinases. Such a scheme has been proposed for PKCu action in fatty acid-treated adipocytes [23]. By contrast, it has recently been shown that PKCu can directly mediate the phosphorylation of IRS-1 at Ser1101 in intact cells and muscle, which also results in the inhibition of subsequent IRS-1 tyrosine phosphorylation and activation of downstream signalling [24]. In addition to the modulation of IRS-1, other mechanisms by which PKCs could inhibit insulin action have been proposed. PKCe can play a role in the regulation of insulin receptor numbers by promoting their degradation [12] although this is probably not a major cause of insulin resistance. Although the substrates for PKC isoforms are poorly defined in general, PKCu has recently been shown to specifically phosphorylate N-myc downstream regulated protein 2 (Ndrg2) in vitro and in intact muscle cells, and thus prevent its phosphorylation by Akt in response to insulin [25]. The role of Ndrg2 in insulin action, however, remains to be elucidated. As with the correlative studies referred to above, other PKC isoforms have also been shown to modulate insulin signalling, especially through the Ser-phosphorylation of IRS-1. Perhaps the most exciting advances in this area currently, however, are the demonstrations of the deletion of specific PKC isoforms causing alterations in the susceptibility of mice to glucose intolerance.

Glucose homeostasis in PKC-deficient mice Because specific pharmacological inhibitors are not currently available for most PKC isoforms, the most convincing way to demonstrate a causative role for a particular isoform in the generation of insulin resistance is to compare insulin action in wild type and PKC-gene knockout mice. The first reports of glucose homeostasis in such mice was concerned with the conventional PKCs. PKCa / mice exhibited enhanced insulin signalling and glucose uptake in muscle and fat cells [26]. The effect of these improvements on whole body glucose tolerance, however, was not described, and any protection against lipid-induced insulin resistance was not addressed. Similarly, although PKCb / mice also showed very minor improvements in glucose tolerance, it was not reported whether these mice were less susceptible to fat-diet induced insulin resistance, because, as with the study involving PKCa / mice, a major aim was to examine the role of the kinase in normal insulin action [27]. In contrast, recent studies have examined the effects of PKCu deletion or inhibition in the context of obesity and lipid-induced insulin resistance. Transgenic overexpression

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of a kinase dead mutant of PKCu in skeletal muscle, expected to compete for PKC substrates and act in a trans-dominant negative manner in this tissue, was shown to have unexpected effects on body weight and insulin sensitivity. These mice exhibited increased fasting and fed plasma insulin levels, indicative of insulin resistance, at 4 months [28]. Furthermore, the mice became obese due to the accumulation of visceral adipose tissue from 6 to 7 months, and demonstrated impaired glucose homeostasis as assessed by glucose tolerance tests. These results favour the interpretation of the association between diminished cytosolic PKCu and insulin resistance as representing a reduced capacity for PKCu function, as discussed above, which in turn reduces insulin sensitivity, albeit through an unknown mechanism. Conversely, when the effects of acute lipid-infusion were examined in a global PKCu / mouse, skeletal muscle insulin action, in terms of insulin signalling through IRS-1 and glucose disposal, was spared in comparison to that in wild type muscle, leading to protection from whole body insulin resistance induced by the lipid [29]. These findings are in agreement with a more common interpretation of the previous data that an increased membrane-associated to cytosolic PKCu ratio indicates chronic PKC activation, which interferes with insulin action, most probably through IRS-1 Ser/Thr phosphorylation. These studies [28,29] are disparate, and one major difference lies in the manner in which PKCu deficiency was generated. It could be argued that in the case of the transgenic approach, integration of the transgene into an unknown gene could have led to non-PKCu-specific effects causing insulin resistance and obesity [30]. The other major difference lies in the nature of the studies themselves. PKCu / mice undergoing acute lipid-infusion were only studied at 4 months of age [29], whereas the effects of greater age or longer term high-fat diet feeding, which might also have resulted in insulin resistance and obesity similar to that seen in the transgenic animals [28], were not addressed. More direct comparisons can now be made as a result of two further studies also using the PKCu / animals generated by Sun et al. [31], although again there are intriguing differences. In one study, these PKCu / mice were found to display enhanced insulin signalling and glucose tolerance, as well as protection against fat diet-induced insulin resistance despite a body weight gain equal to that of control mice (M. Hundt, A. Altman, pers. commun.). These data, therefore, support the findings obtained with acute lipid infusion [29], and again advocate a role for PKCu in the generation of lipidinduced insulin resistance. In a second study, however, these mice have been found to possess an increased white adipose mass, together with a decreased muscle mass and insulin sensitivity, and an increased susceptibility to hyperinsulinaemia and insulin resistance caused by fat diet-feeding (J. Ye, pers. commun.). The latter data suggest that PKCu has tissuewww.drugdiscoverytoday.com

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specific functions, promoting insulin signalling rather than insulin resistance in skeletal muscle, and is thus more in agreement with the study of transgenic mice expressing kinase dead PKCu specifically in muscle [28]. The reasons for these important discrepancies are unclear, but are unrelated to the construction of the PKCu deletion or the strain of mouse upon which it was made, as all three studies employed a common PKCu / mouse. It is possible that subtle differences in animal maintenance or diet could play a role. For example, we have shown that only certain common fatty acids are capable of activating PKCs [32] whereas others inhibit insulin action by PKC-independent means [33]. A causative role for PKCe in lipid-induced insulin resistance has not been so widely examined, although at least three independent PKCe / mice have been generated [34–36]. The extensive associative evidence concerning a role for this kinase is more easily interpreted because the enzyme is not downregulated upon chronic activation, perhaps indicating a less complex role than that apparently played by PKCu. Work in our laboratory, however, has indicated that although fatfed PKCe / mice do indeed show improved glucose tolerance in comparison to wildtype littermates, this cannot be explained solely in terms of an improvement in skeletal muscle insulin resistance (Carsten Schmitz-Peiffer et al., unpublished). Either this isoform does not play a role in the generation of insulin resistance by fat oversupply, despite the wealth of correlative data, or the other enzymes can compensate functionally for the lack of the kinase in skeletal muscle of these animals. In support of the former, we have failed to reverse the insulin resistance of lipid-pretreated myotubes by the overexpression of trans-dominant negative mutants of PKCe [32]. Finally, there have been no reports of studies addressing the involvement of PKCg or PKCd by gene-targeting methods, most probably because associative studies suggest that these isoforms are less likely to be involved. Furthermore, although generalized knockout of atypical PKCs seems to be embryonically lethal, PKCl knockout in embryonic stem cells, subsequently induced to differentiate into adipocytes, has demonstrated a requirement for this enzyme in insulin-stimulated glucose transport [37], in agreement with an earlier work indicating its role in normal insulin action rather than insulin resistance.

Conclusions Our understanding of the involvement of PKC in insulin resistance has evolved significantly from the initial findings that specific isoforms exhibited signs of chronic activation in conjunction with diminished insulin action in skeletal muscle. Numerous correlative studies have established PKCu and PKCe as the strongest candidates for mediating insulin resistance, and mechanistic details are now emerging which could explain such a role, at least in the case of PKCu. The most 108

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rigorous approach currently employed to investigate the significance of such findings has been the genetic manipulation of PKC expression. From the currently available information, it is clear that PKCu has important regulatory effects on glucose homeostasis which might not be limited to insulin action in skeletal muscle. However, the seemingly conflicting data generated using transgenic and PKCu / mice, taken together with the downregulation as well as translocation of PKCu in insulin-resistant muscle, make it difficult to draw a final conclusion concerning a protective or inhibitory role for the isoform. Full publication of the studies mentioned above (Hundt, Altman et al. and Ye et al.), together with further interventive approaches, might enable the resolution of this issue. Another point requiring clarification is the relationship between PKC activation and the inhibitory effects of other kinases such as JNK and IKKb, for which causative roles in insulin resistance have been more clearly demonstrated [38– 40] and which might lie downstream of PKC signalling in specific tissues. Understanding such relationships could simplify the targeting of these enzymes for therapeutic intervention. In the event that a beneficial effect of reduced PKCe or PKCu activity on insulin action is clearly established, then the development of specific small molecule inhibitors of these isoforms would be warranted. The major challenges are the specificity and potency of such compounds. With the exception of LY333531, which shows specificity towards PKCb [41] and can be useful in the treatment of diabetic complications due to hyperglycaemia [42], there are currently no specific inhibitors available for most PKC isoforms. This can be due in part to the fact that at present, PKC inhibitors are mostly directed at the ATP-binding site of the kinases, which might not be sufficiently different between isoforms or even between PKCs and other kinases to afford the required specificity. It might be possible to circumvent this problem by the use of siRNA or antisense oligonucleotide technology, which specifically inhibits the expression of genes at the mRNA level [43]. This approach has recently been used to block PKCa as a treatment for cancer [44]. Alternatively, the issue of specificity might be resolved by the use of isoform-specific bioactive peptides, derived for example from the pseudosubstrate regions, which help to maintain the kinases in inactive conformations [45]. Administration of a cell-permeable JNKinhibitory peptide has recently been shown to ameliorate of glucose tolerance in db/db diabetic mice [46]. Because each PKCu and PKCe deletion also results in less favourable phenotypes which appear to be unrelated to insulin action [31,35], peptides derived from IRS-1 interaction or phosphorylation sites, for example, might be also advantageous in the selective inhibition of specific effects. Therfore, in summary, although the targeting of PKCe or PKCu will affect whole body glucose disposal, and several approaches for doing this are

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Parker, P.J. and Murray-Rust, J. (2004) PKC at a glance. J. Cell Sci. 117, 131–132 Schmitz-Peiffer, C. (2002) Protein kinase C and lipid-induced insulin resistance in skeletal muscle. Ann. N. Y. Acad. Sci. 967, 146–157 Serra, C. et al. (2003) Transgenic mice with dominant negative PKCtheta in skeletal muscle: a new model of insulin resistance and obesity. J. Cell. Physiol. 196, 89–97 Kim, J.K. et al. (2004) PKC-u knockout mice are protected from fatinduced insulin resistance. J. Clin. Invest. 114, 823–827

Outstanding issues

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 Does the chronic translocation and downregulation of PKCu in insulin-resistant states indicate a diminished capacity for activation, and hence a protective rather than inhibitory role for the kinase?  Are the long-term effects of PKCu on insulin action and glucose homeostasis different to acute effects on insulin signalling?  Does PKCu act independently of or in concert with other key mediators of insulin resistance such as JNK and IKKb?

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available, further work is required to delineate the downstream signalling from these PKC isoforms in skeletal muscle and other tissues involved in glucose homeostasis.

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Acknowledgements I would like to thank Dr. Matthias Hundt and Prof. Amnon Altman, La Jolla Institute for Allergy and Immunology, and Prof. Jiangping Ye, Louisiana State University, for making their results available ahead of publication.

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