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Molecular Endocrinology, doi:10.1210/me.2004-0045
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Molecular Endocrinology 18 (10): 2513-2521
Copyright © 2004 by The Endocrine Society

Tissue-Specific Differences in Activation of Atypical Protein Kinase C and Protein Kinase B in Muscle, Liver, and Adipocytes of Insulin Receptor Substrate-1 Knockout Mice

Mini P. Sajan, Mary L. Standaert, Atsushi Miura, C. Ron Kahn and Robert V. Farese

Research Service (M.P.S., M.L.S., A.M., R.V.F.), James A. Haley Veterans Hospital and the Department of Internal Medicine, University of South Florida College of Medicine, Tampa, Florida 33612; and the Research Division (C.R.K.), Joslin Diabetes Center and Harvard Medical School, Boston, Massachusetts 02215

Address all correspondence and requests for reprints to: Robert V. Farese, M.D., James A. Haley Veterans Hospital, ACOS-151, 13000 Bruce B. Downs Boulevard, Tampa, Florida 33612. E-mail: rfarese{at}hsc.usf.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Insulin receptor substrates (IRSs) 1 and 2 are postulated to control the activation of phosphatidylinositol 3-kinase (PI3K)-dependent signaling factors, namely, atypical protein kinase C (aPKC) and protein kinase B (PKB)/Akt, which mediate metabolic effects of insulin. However, it is uncertain whether aPKC and PKB are activated together or differentially in response to IRS-1 and IRS-2 activation in insulin-sensitive tissues. Presently, we examined insulin activation of aPKC and PKB in vastus lateralis muscle, adipocytes, and liver in wild-type and IRS-1 knockout mice, and observed striking tissue-specific differences. In muscle of IRS-1 knockout mice, the activation of both aPKC and PKB was markedly diminished. In marked contrast, only aPKC activation was diminished in adipocytes, and only PKB activation was diminished in liver. These results suggest that IRS-1 is required for: 1) activation of both aPKC and PKB in muscle; 2) aPKC, but not PKB, activation in adipocytes; and 3) PKB, but not aPKC, activation in liver. Presumably, IRS-2 or other PI3K activators account for the normal activation of aPKC in liver and PKB in adipocytes of IRS-1 knockout mice. These complexities in aPKC and PKB activation may be relevant to metabolic abnormalities seen in tissues in which IRS-1 or IRS-2 is specifically or predominantly down-regulated.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
DOWNSTREAM EFFECTORS OF PI3K [phosphatidylinositol (PI) 3-kinase], atypical protein kinase C (aPKC), and protein kinase B (PKB/Akt) are thought to participate in mediating important metabolic actions of insulin, including increases in transport [aPKC (1, 2) and PKB (3, 4)] and storage [PKB (5)] of glucose in muscle and/or adipocytes, and, in the liver, increases in glycogen [PKB (6)] and lipid [aPKC (7)] synthesis, and decreases in gluconeogenesis [PKB (8)] and glucose release [PKB (9)]. Mechanisms used by insulin to activate aPKCs and PKB, however, are only partly understood. Concerning general activating mechanisms, acting downstream of PI3K, and in conjunction with PI3K-dependent increases in PI-3,4,5-(PO4)3 (PIP3), 3-phosphoinositide protein kinase-1 (PDK-1) phosphorylates threonine residues in the activation loops of both PKC-{zeta}/{lambda} and PKB (10, 11, 12). Activation loop phosphorylations are then followed by secondary phosphorylations and other modifications. For aPKCs, full activation requires PI3K/PDK1/PIP3-dependent loop phosphorylation, PIP3-dependent autophosphorylation and PIP3-dependent allosteric release from autoinhibition independently of phosphorylation (11, 12, 13, 14). For PKB, activation loop phosphorylation is followed by phosphorylation of serine-473 by a still unidentified protein kinase (PDK2), but there are no allosteric effects of PIP3 that are known to be independent of phosphorylation.

Acting upstream of PI3K, there is only limited information on signaling factors that are responsible for activating aPKCs and PKB. In this regard, it is commonly assumed that insulin receptor substrate-1 (IRS-1) or IRS-2, or both, depending upon their availability and activation within specific tissues, function interchangeably to activate PI3K, which in turn activates aPKCs and PKB in a parallel coordinated manner. However, as is evident from results presented herein, such linearity and parallelism are not necessarily observed in specific insulin-sensitive tissues.

Concerning factors upstream of PI3K that activate PKB, in mice in which IRS-1 has been knocked out by gene targeting methods, PKB activation by insulin is compromised in skeletal muscle and liver, and selective correction of this defect in the liver by adenoviral transfer of the IRS-1 gene results in improvement in PKB activation and insulin-regulated hepatic glucose metabolism (15). This defect in PKB activation in the liver of IRS-1 knockout mice occurs despite compensatory increases in IRS-2 function (i.e. tyrosine phosphorylation) that seem sufficient in magnitude to offset the loss of IRS-1 function, at least with respect to acting as a substrate for the tyrosine kinase activity of the insulin receptor (16). In contrast, in muscle, wherein IRS-2 appears to be of lesser importance for insulin-stimulated glucose transport (17), compensatory increases in IRS-2 phosphorylation are not sufficient to offset decreases in IRS-1 phosphorylation, and PKB activation is deficient in IRS-1 knockout mice (16). In white adipocytes, the effects of knockout of IRS-1 and IRS-2 on PKB activation have not been reported, but, in immortalized brown adipocytes, knockout of IRS-1 (18), but not IRS-2 (19), has been reported to significantly diminish PKB activation in some (18), but not all (20), studies. Also, in immortalized hepatocytes, knockout of IRS-2 impairs PKB activation (21), and it therefore seems likely that both IRS-1 and IRS-2 are required for PKB activation in hepatocytes.

Even less information is available on factors that activate aPKCs via PI3K during insulin action. Indeed, the effects of knockout of IRS-1 or IRS-2 on aPKC activation in muscle, liver, and white adipocytes of otherwise intact mice have not been reported. On the other hand, in immortalized brown adipocytes, knockout of IRS-2 is attended by diminished activation of aPKCs, but not PKB, and this defect in aPKC activation has been proposed to explain decreases in insulin-stimulated glucose transport in these cells (19). In this regard, knockout of IRS-1 has been reported to diminish insulin-stimulated glucose transport in white adipocytes (22, 23) and in some (16) but not all (24) studies of muscle, but alterations in aPKC activation have not been reported in either cell type. In immortalized hepatocytes, knockout of IRS-2 diminishes the activation of aPKCs, as well as PKB, by insulin (21).

Interestingly, differences between aPKC and PKB activation by insulin in muscle have been seen in type 2 diabetes, i.e. partial decreases in IRS-1-dependent activation of PI3K are attended by substantial decreases in the activation of aPKCs and glucose transport in vitro or glucose disposal rates in vivo, with relatively little effect on PKB activation in adipocytes and muscles of rats (25, 26), and muscles of obese monkeys (27) and humans (28, 29, 30). In addition, we have found that PKB activation by insulin is compromised in livers of type 2 diabetic rats, whereas aPKC activation is surprisingly intact in these livers (31); this contrasts strikingly with adipocytes and muscle in which the activation of aPKCs, but not PKB, is diminished (25, 26). In view of these contrasting divergences in insulin-sensitive signaling factors in insulin-resistant diabetic tissues, there is a critical need to have a more comprehensive understanding of the roles of IRS-1 and IRS-2 during aPKC and PKB activation by insulin in muscle, liver, and adipocytes. To this end, we presently examined the activation of aPKCs and PKB in tissues of IRS-1 knockout mice.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
aPKC Activation
In control wild-type IRS-1+/+ mice, insulin provoked 2- to 3-fold increases in aPKC activity in muscle, liver, and adipocytes (Fig. 1Go). With knockout of the IRS-1 gene, basal aPKC activity was unaffected, but aPKC activation by insulin was markedly diminished in vastus lateralis muscles of these IRS-1 knockout mice. In contrast to the marked diminution in muscle, aPKC activation by insulin was only partially diminished in adipocytes, and, surprisingly, fully intact in the livers, of IRS-1 knockout mice (Fig. 1Go). As depicted, immunoreactive levels of aPKCs in muscle, liver and adipocytes were not altered by knockout of IRS-1 (Fig. 1Go).



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  		Fig. 1. Effects of Knockout (KO) of IRS-1 on Insulin-Induced Activation of aPKCs in Vastus Lateralis (VL) Muscle (A), Liver (B), and Adipocytes (C)

Mice were injected with or without insulin 15 min before they were killed, after which, muscle and liver were harvested and examined for immunoprecipitable aPKC activity. Adipocytes were prepared from epididymal and retroperitoneal fat and incubated for 15 min with or without 10 nM insulin, after which, immunoprecipitable aPKC activity was measured. Bargram values are mean ± SE of (n) determinations. Asterisks indicate P < 0.05 for comparison of insulin-stimulated values of IRS-1 knockout vs. wild-type mice. As shown in representative immunoblots, IRS-1 knockout did not alter levels of aPKCs in muscle, liver, and adipocytes. PPT, Immunoprecipitate.

 
We have previously reported that PIP3, the lipid product of PI3K, activates aPKCs in vitro nearly as effectively as insulin in vivo (13, 14, 27, 28, 32). Moreover, this activation of aPKCs by PIP3 in muscle is markedly diminished, not only in type 2 diabetes in which IRS-1-dependent PI3K activation is impaired (27, 28) but also in obesity (Farese, R. V., M. P. Sajan, A. Miura, and M. L. Standaert, unpublished observations) and after high-fat feeding in rats (32) and mice (our unpublished observations), even in the absence of significant decreases in IRS-1-dependent PI3K activation (e.g. Ref. 32). It was therefore of interest to see whether PIP3 could activate aPKCs in muscle of IRS-1 knockout mice. As seen in Fig. 2Go, and as expected from previous reports (13, 14, 27, 28, 32), PIP3 activated aPKCs nearly as effectively as insulin in immunoprecipitates prepared from muscles of control mice. More importantly, PIP3 effectively activated aPKCs in immunoprecipitates prepared from muscles of IRS-1 knockout mice, despite the inability of insulin to activate aPKCs in intact mice. These findings suggested that in IRS-1 knockout mouse, the defect in muscle aPKC activation is largely if not solely due to the loss of the PI3K activity and PIP3 generation that derives specifically from IRS-1 activation. These findings also suggested that a simple defect in IRS-1-dependent PI3K activation by insulin in intact muscle does not necessarily lead to decrease in the ability of PIP3 to directly activate aPKC activation in vitro.



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  		Fig. 2. Effects of PIP3 on Activation of aPKCs Immunoprecipitated from Lysates of Muscles of Wild-Type and IRS-1 Knockout (KO) Mice

Mice were treated with or without insulin as described in Fig. 1Go, and aPKCs were immunoprecipitated from vastus lateralis (VL) muscles and incubated with or without 10 µM PIP3, as indicated. Values are mean ± SE of (n) determinations. Asterisk indicates P < 0.05 for comparison of insulin-stimulated values of IRS-1 knockout vs. wild-type mice. PPT, Immunoprecipitate.

 
PKB Activation
Similar to aPKC activation, the activation and phosphorylation of PKB by insulin were markedly diminished in the vastus lateralis muscles of IRS-1 knockout mice (Figs. 3Go and 4Go). In the livers of IRS-1 knockout mice, however, much differently from aPKC activation, which was intact, PKB activation/phosphorylation was markedly impaired (Figs. 3Go and 4Go). Also, in adipocytes of IRS-1 knockout mice, in which aPKC activation was partially diminished, PKB activation and phosphorylation were apparently intact (Figs. 3Go and 4Go). Note that levels of immunoreactive PKB were not altered in muscle, liver, and adipocytes by knockout of IRS-1 (Fig. 4Go).



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  		Fig. 3. Effects of Knockout (KO) of IRS-1 on Insulin-Induced Activation of PKB in Vastus Lateralis (VL) Muscle (A), Liver (B), and Adipocytes (C)

Mice were injected with or without insulin 15 min before they were killed as in Fig. 1Go, after which muscle and liver were harvested and examined for immunoprecipitable PKB activity. Adipocytes were prepared from epididymal and retroperitoneal fat and incubated for 15 min with or without 10 nM insulin, after which immunoprecipitable PKB activity was measured. Values are mean ± SE of (n) determinations. Asterisks indicate P < 0.05 for comparison of insulin-stimulated values of IRS-1 knockout vs. wild-type mice. PPT, Immunoprecipitate.

 


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  		Fig. 4. Effects of Knockout (KO) of IRS-1 on Insulin-Induced Increases in Levels of Immunoreactive Phospho-Serine-473-PKB (pPKB) in Vastus Lateralis (VL) Muscles (A), Liver (B), and Adipocytes (C)

Mice were injected with or without insulin as in Figs. 1Go and 3Go, after which, muscle and liver were harvested. Adipocytes were prepared from epididymal and retroperitoneal fat and incubated for 15 min with or without 10 nM insulin. Cell lysates were subjected to SDS-PAGE and blotted with anti-p-ser-473-PKB and anti-PKB antisera. Shown here are representative blots.

 
ERK Activation
Similar to results reported previously (16), we found that ERK activation by insulin was diminished in muscle, but largely intact in liver, of IRS-1 knockout mice (Fig. 5Go). We also found that, as in liver, ERK activation was largely intact in adipocytes of IRS-1 knockout mice (Fig. 5Go).



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  		Fig. 5. Effects of Knockout (KO) of IRS-1 on Insulin-Induced Activation of ERK in Vastus Lateralis (VL) Muscle (A), Liver (B), and Adipocytes (C)

Mice were injected with or without insulin 15 min before they were killed as in Figs. 1Go and 3Go, after which muscle and liver were harvested and examined for immunoprecipitable ERK activity. Adipocytes were prepared from epididymal and retroperitoneal fat and incubated for 15 min with or without 10 nM insulin, after which immunoprecipitable ERK activity was measured. Values are mean ± SE of (n) determinations. Asterisks indicate P < 0.05 for comparison of insulin-stimulated values of IRS-1 knockout vs. wild-type. PPT, Immunoprecipitate.

 
IRS-2-Dependent PI3K Activation
To gain insight into 1) how aPKC, but not PKB, activation may be spared in the liver of IRS-1 knockout mice, and 2) how PKB, but not aPKC activation, may be spared in adipocytes of IRS-1 knockout mice, we examined parameters of IRS-2 function in these tissues/cells. As seen in Fig. 6Go, levels of IRS-2 were not significantly different in adipocytes, livers, and muscles of IRS-1 knockout and wild-type mice. On the other hand, the binding of the p85 subunit of PI3K to IRS-2 was increased 2.8-fold in livers of IRS-1 knockout mice [1.00 ± 0.26 (n = 4) in wild-type, vs. 2.83 ± 0.62 (n = 4) in IRS-1 knockout; P < 0.05;also see Fig. 6Go]. See Discussion for other data on increases in IRS-2 activity in the liver of IRS-1-deficient mice.



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  		Fig. 6. Effects of IRS-1 Knockout (KO) on (A) IRS-2 Levels in Adipocytes, Liver, and Vastus Lateralis (VL) Muscle, and (B) Binding of IRS-2 to the p85 Subunit of PI3K in Liver

Muscle, liver, and adipocytes were harvested as in Fig. 1Go. Samples in panel B were derived from immunoprecipitates of insulin-stimulated liver. Shown here are blots representative of at least four samples.

 
Analagous to findings in liver, IRS-2-dependent PI3K activity was increased in adipocytes of IRS-1 knockout mice (Fig. 7Go). In this regard, note that IRS-1-dependent PI3K activity is reported to be small relative to IRS-1-dependent PI3K in adipocytes (33) but is nevertheless required for activation of aPKC and glucose transport in brown adipocytes (19). Also see Discussion for other data on increases in IRS-2 activity in IRS-1-deficient adipocytes.



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  		Fig. 7. Effects of IRS-1 Knockout (KO) on IRS-2-Dependent PI3K Activity in Adipocytes

As in Fig. 1Go, adipocytes from wild-type and IRS-1 knockout mice were incubated for 15 min with or without 10 nM insulin, after which IRS-2 was immunoprecipitated and assayed for PI3K activity. Values are mean ± SE of (n) determinations. Asterisk indicates P < 0.05 for comparison of insulin-stimulated values found in assays of IRS-1 knockout vs. wild-type adipocytes. Shown in the inset is a representative autoradiogram of 32P-labeled PI-3-PO4. after purification by thin-layer chromatography.

 
Because the activation of all three insulin signaling factors, aPKC, PKB, and ERK, was markedly inhibited in muscles of IRS-1 knockout mice, we did not evaluate IRS-2-dependent PI3K activation because it obviously did not compensate for the loss of IRS-1 in this tissue. In keeping with the failure of IRS-2 to maintain insulin effects on these insulin-sensitive signaling factors in IRS-1 knockout mice is the fact that knockout of IRS-2 does not inhibit insulin effects on glucose transport in muscle (17), and we have found that PKB activation is intact in muscles, but not liver, of IRS-2 knockout mice (Farese, R. V., M. P. Sajan, A. Miura, and M. L. Standaert, unpublished observations).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
From the present findings, it seems clear that: 1) aPKC and PKB are not simply activated in parallel after activation of PI3K during insulin action; 2) factors that operate upstream of PI3K, i.e. IRS-1, IRS-2, and probably other factors, acting independently or in conjunction with PI3K, are important determinants of whether aPKC and/or PKB is/are activated; and 3) there are surprisingly considerable differences in patterns of aPKC and PKB activation in the insulin target tissues presently studied. Thus, in liver of IRS-1 knockout mice, PKB, but not aPKC, activation was impaired, whereas, in white adipocytes of IRS-1 knockout mice, aPKC, but not PKB, activation was impaired. In contrast to these divergences between aPKC and PKB activation in liver and white adipocytes, the activation of both aPKC and PKB was impaired in skeletal muscle of IRS-1 knockout mice.

In light of the above findings, it may be surmised that IRS-1 serves as a major determinant for activation of both aPKC and PKB in muscle of intact mice. On the other hand, IRS-1 apparently serves as an important determinant for activation of PKB but not aPKC in liver, and for activation of aPKC but not PKB in white adipocytes of intact mice. These dependencies on IRS-1 do not necessarily imply that IRS-1 is sufficient for these activations of aPKC and PKB. In this regard, it may be noted that IRS-1 and IRS-2 activate at least partly different pools of PI3K in 3T3/L1 adipocytes (34), and it is therefore reasonable to suggest that their combined actions may be required for the activation of aPKC and PKB in adipocytes. Similar differences in functions of IRS-1 and IRS-2 most likely exist in other tissues, and, suffice it to say that further studies of signaling in IRS-2 knockout mice are needed to see whether IRS-2 is corequired along with IRS-1 for aPKC and/or PKB activation in muscle, for PKB activation in liver, and for aPKC activation in white adipocytes, of intact mice.

In view of the impairment in aPKC activation in muscle and white adipocytes, the apparently normal activation of aPKC by insulin in the liver of IRS-1 knockout mice was surprising. This finding suggested that one or more factors other than IRS-1 is/are capable of maintaining the activation of one or more PI3K pools responsible for activating aPKC during insulin action in the liver. In this regard, we presently found that the binding of IRS-2 to the p85 subunit of PI3K was increased in the livers of IRS-1 knockout mice, and this complements reports of 1) compensatory increases in insulin-stimulated tyrosine phosphorylation of IRS-2 in the liver (but not muscle) of IRS-1 knockout mice (15, 16), and 2) maintenance of total phosphotyrosine- dependent PI3K activity, along with 20–30% increases in IRS-2-dependent PI3K activity, in mouse liver in which IRS-1 has been diminished by approximately 80% after in vivo administration of adenovirus expressing specific silencing RNA (35). Accordingly, it seems likely that IRS-2 contributes to maintaining the apparently normal activation of aPKC in the liver of IRS-1 knockout mice. Further studies of IRS-3 and other PI3K activators are needed to see whether they are corequired for maintaining aPKC activation in the liver of IRS-1 knockout mice.

Similarly, in view of the apparently normal activation of aPKC by insulin in the liver of IRS-1 knockout mice, it was surprising to find that the activation of PKB was impaired in this tissue. It may therefore be surmised that PKB activation is dependent on IRS-1-dependent PI3K activation in the liver, even in the presence of compensatory increases in the activation of IRS-2 (see above) and/or other factors that effectively maintain aPKC activation. In this regard, however, note that PKB activation is compromised in immortalized hepatocytes in which IRS-2 is absent (21). Accordingly, it is likely that both IRS-1 and IRS-2 are required for PKB activation in the liver.

Of further note was the observation that, unlike aPKC activation, which was diminished, the activation of PKB by insulin was intact in white adipocytes of IRS-1 knockout mice. This suggested that, whereas IRS-1 is needed for aPKC activation, IRS-2, IRS-3, and/or other factors are sufficient for PKB activation in white adipocytes of IRS-1 knockout mice. In this regard, we presently found that insulin effects on IRS-2-dependent PI3K activity are modestly increased in adipocytes of IRS-1-knockout mice. In addition, in immortalized brown adipocytes, we have found that IRS-2-dependent PI3K activation by insulin is increased approximately 2-fold in adipocytes in which IRS-1 has been knocked out (our unpublished observations). However, it has been reported that total phosphotyrosine-dependent PI3K activity is diminished by approximately 50% in adipocytes of IRS-1 knockout mice, and pp60 (IRS-3), rather than IRS-2, may be a more important PI3K activator in IRS-1 knockout adipocytes (34). Nevertheless, IRS-2 is essential for stimulation of aPKC, but not PKB, during insulin-stimulated glucose transport in brown adipocytes (19). Accordingly, the presently observed increase in IRS-2-dependent PI3K activity, perhaps along with IRS-3-dependent PI3K, may serve to fully maintain PKB activation in IRS-1 knockout adipocytes.

Although aPKC activation in white adipocytes is dependent on IRS-1, it is important to note that this dependence is only partial. Indeed, we have found that both IRS-1 and IRS-2 are corequired for full aPKC activation in immortalized brown adipocytes, and deficiency of either IRS-1 or IRS-2 results in similar partial (50–60%) decreases in insulin-induced activation of both aPKCs and glucose transport (but not PKB) in these adipocytes (Miura, A., M. P. Sajan, M. L. Standaert, G. Bandyopadhyay, C. R. Kahn, and R. V. Farese, unpublished observations).

In view of the difference between aPKC and PKB activation in liver, it may be speculated that aPKC-dependent functions, such as induction of sterol regulatory element-binding protein-1c and hepatic lipid synthesis (7), may be maintained, whereas PKB-dependent functions, such as increases in glycogen synthesis (6), glycolysis (36, 37), and glucose release (9), may be diminished in livers of IRS-1 knockout mice. Indeed, it seems clear that both IRS-1 (15) and PKB (38) have important roles in regulating glucose metabolism in the liver. Further studies are needed to test these speculations, particularly in view of the fact that we have observed a pattern of signaling in livers of type 2 diabetic rodents that is similar to that of IRS-1 knockout mice, namely, decreases in PKB, but not aPKC, activation (31). From the present results, we must consider the possibility that the activation or subsequent function of IRS-1 may be compromised more than that of IRS-2 in livers of these diabetic rodents.

In addition to aPKC and PKB, it was of interest to examine ERK activation in tissues of IRS-1 knockout mice. Similar to results reported previously (16), IRS-1 was found to be required for insulin-induced activation of ERK in muscle, but not in liver. However, note that this apparent requirement for IRS-1 in muscle may be more reflective of a defect in IRS-1-dependent activation of SH2 domain-containing factors such as Shc or GRB2, rather than PI3K because defects in IRS-1-dependent PI3K activation in obesity and type 2 diabetes in humans do not appear to be associated with defects in MAP kinase activation (39). We also presently found that ERK activation was not significantly diminished in adipocytes of IRS-1 knockout mice. Presumably, in adipocytes (40) as well as liver, IRS-2, IRS-3, Shc, or other IRSs are sufficient to satisfy requirements for ERK activation.

In summary, knockout of IRS-1 results in tissue-specific differences in activation of aPKC and PKB by insulin. In muscle, the activation of both aPKC and PKB is diminished, whereas only aPKC activation is diminished in adipocytes, and only PKB activation is diminished in liver of IRS-1 knockout mice. Further studies are needed to see whether knockout of IRS-1 results in specific alterations of cellular processes that are presumed to be dependent on activation of aPKC and/or PKB in insulin-sensitive tissues.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Experimental Mice
Male IRS-1–/– knockout mice were obtained as described (24) and compared with control IRS-1+/+ male mice that were genetically similar (Taconic, Germantown, NY), except for the presence of the IRS-1 gene. Mice were housed in a controlled environment of alternating 12-h light, 12-h dark cycles and fed the same diet for least 2 wk before experimental use. The mice were used experimentally in the unfasted (to avoid release of counterregulatory factors) state at 1000–1200 h.

In vivo Muscle and Liver Signaling Studies
Male mice, weighing approximately 30–35 g, were injected with 1 mU insulin/g body weight im 15 min before they were killed. This time was selected because it allows for optimal or near optimal activation of aPKC, PKB, and ERK by insulin in both muscle and liver. However, also note that, different from muscle and adipocytes, the optimal time for activation of IRS-1- and IRS-2-dependent PI3K by insulin in mouse liver is much earlier, i.e. at approximately 2 min, and, in our experience, such activation is not readily apparent at 15 min in normal mice. Nevertheless, despite poor ability to observe insulin effects on either IRS-1- or IRS-2-dependent PI3K at these later times, it seems most likely that these factors are operative and required for insulin action because knockout of either IRS-1 or IRS-2 markedly inhibits insulin effects on PKB activation. Also note that there appears to be an up-regulation of binding of IRS-2 to the p85 subunit of PI3K both basally and during insulin action in IRS-1 knockout mice (see Results).

As described (1, 2, 13, 25, 26, 27, 28, 31), vastus lateralis muscles and liver were rapidly removed and homogenized in ice-cold buffer, which, for aPKC assays, contained 0.25 M sucrose, 20 mM Tris/HCl (pH, 7.5), 1.2 mM EGTA, 20 mM ß-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 20 µg/ml leupeptin,10 µg/ml aprotinin, 3 mM Na4P2O7, 3 mM Na3VO4, 3 mM NaF, and 1 µM LR-microcystin. For PKB and ERK assays, the homogenizing buffer contained 50 mM Tris/HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 0.1% ß-mercaptoethanol, 50 mM NaF, 5 mM Na4P2O7, 10 mM ß-glycerophosphate, 1 mM PMSF, 10 µg/ml aprotinin, 20 µg/ml leupeptin, and 1 µM LR-microcystin. After low-speed centrifugation for 10 min at 700 x g to remove unbroken cells, debris, nuclei and floating fat, 0.15 M NaCl, 1% Triton X-100, and 0.5% Nonidet P-40 were added, and the resulting cell lysates were immunoprecipitated with antibodies that target 1) aPKCs (rabbit polyclonal antiserum from Santa Cruz Biotechnology, Inc., Santa Cruz, CA; recognizes the C termini of both PKC-{lambda} and PKC-{zeta}); 2) PKB [rabbit polyclonal antiserum obtained from Upstate Inc., Lake Placid, NY]; or 3) ERK (rabbit polyclonal antiserum obtained from Santa Cruz Biotechnology, Inc.). Immunoprecipitates were collected on Sepharose-AG beads and PI3K, PKB, PKC-{lambda}, and ERK were assayed as described below. Lysates were also used to measure levels of immunoreactive proteins, as described below.

In vitro Adipocyte Signaling Studies
As described (13), epididymal and retroperitoneal adipose tissues were digested with collagenase and isolated adipocytes were equilibrated and then incubated in glucose-free Krebs Ringer phosphate medium containing 1% BSA for 15 min with or without 10 nM insulin. After incubation, cells were sonicated, and cell lysates were prepared as described above in muscle/liver studies, and then subjected to immunoprecipitation and assay for aPKC, PKB, or ERK enzyme activity, or, as another indicator of PKB activation, used to measure immunoreactive phospho-ser-473-PKB, as in muscle/liver studies.

aPKC Activation
aPKC activity was measured as described (1, 2, 13, 25, 26, 27, 28, 31). In brief, aPKCs were immunoprecipitated from cell lysates with a rabbit polyclonal antiserum (Santa Cruz Biotechnology, Inc.) that recognizes the C-termini of both PKC-{lambda} and PKC-{zeta} (mouse adipocytes and muscle contain mainly PKC-{lambda} and little PKC-{zeta}, but mouse liver contains substantial amounts of both PKC-{lambda} and PKC-{zeta}), collected on Sepharose-AG beads (Santa Cruz Biotechnology, Inc.), and incubated for 8 min at 30 C in 100 µl buffer containing 50 mM Tris/HCl (pH,7.5), 100 µM Na3VO4, 100 µM Na4 P2O7, 1 mM NaF, 100 µM PMSF, 4 µg phosphatidylserine (Avanti Polar-Lipids, Inc., Alabaster, AL), 50 µM [{gamma}-32P]ATP (PerkinElmer, Boston, MA), 5 mM MgCl2 and, as substrate, 40 µM serine analog of the PKC-{epsilon} pseudosubstrate (BioSource, Camarillo, CA). After incubation, 32P-labeled substrate was trapped on P-81 filter papers and counted. Note that assays of knockout and wild-type samples were conducted simultaneously.

PKB Activation
PKB enzyme activity was measured using a kit obtained from UBI, as described (13, 25, 26, 27, 28, 31). In brief, PKB was immunoprecipitated with a rabbit polyclonal antiserum, collected on Sepharose-AG beads, and incubated as per directions in the PKB assay kit. PKB activation was also assessed by immunoblotting for phosphorylation of serine-473 (see Immunoblot Studies).

ERK Activation
Immunoprecipitable ERK activity was measured as described (39).

IRS-2/PI3K Activation
The activation of IRS-2 was examined either by its binding to the p85 subunit of PI3K or by the activation of the p110 catalytic subunit of PI3K and subsequent increases in the labeling of PI. With both methods, IRS-2 was immunoprecipitated with rabbit polyclonal antiserum (Upstate, Inc.) and either subjected to Western analysis or incubated with PI, MgCl2, and [{gamma}32P]ATP as described (13, 25, 26, 27, 28, 31).

Immunoblot Studies
Western analyses were conducted as described (1, 2, 13, 25, 26, 27, 28, 31), using the following: 1) rabbit polyclonal anti-PKC-{zeta}/{lambda} antiserum (obtained from Santa Cruz Biotechnology, Inc.); 2) rabbit polyclonal anti-PKB antiserum (obtained from UBI); 3) rabbit polyclonal anti-phospho-serine-473-PKB antiserum (New England BioLabs Inc., Beverly, MA); and 4) rabbit polyclonal anti-p85/PI3K antiserum (Upstate, Inc.).

Statistical Analyses
Data are presented as mean ± SEM. Means were considered as statistically different, i.e. P < 0.05, which was determined by one-way ANOVA and the least significant multiple comparison method.


   FOOTNOTES
 
This work was supported by funds from the Department of Veterans Affairs Merit Review Program (to R.V.F.) and National Institutes of Health Grants DK-38079-09A1 (to R.V.F.), DK33201 (to C.R.K.), and DK55545 (to C.R.K.).

Abbreviations: aPKC, Atypical PKC; PKB, protein kinase B; IRS, insulin receptor substrate; PDK-1, 3-phosphoinositide protein kinase-1; PI, phosphatidylinositol; PI3K, PI3-kinase; PIP3, PI-3,4,5-(PO4)3; PMSF, phenylmethylsulfonyl fluoride.

Received for publication February 3, 2004. Accepted for publication July 6, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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