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Knockout in Embryonic Stem Cells and Adipocytes Impairs Insulin-Stimulated Glucose Transport
James A. Haley Veterans Hospital and University of South Florida College of Medicine (G.B., M.L.S., M.P.S., Y.K., A.M., R.V.F.), Tampa, Florida 33612; and Max-Planck Institute for Experimental Endocrinology (U.B., F.K., M.L.), 30625 Hannover, Germany
Address all correspondence and requests for reprints to: Robert V. Farese, M.D., 13000 Bruce B. Downs Boulevard, Tampa, Florida 33612. E-mail: rfarese{at}hsc.usf.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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alleles were knocked out by recombinant methods. Insulin activated PKC- and stimulated glucose transport in wild-type (WT) PKC-+/+, but not in knockout PKC--/-, ES cells. However, insulin-stimulated glucose transport was rescued by expression of WT PKC- in PKC--/- ES cells. Surprisingly, insulin-induced increases in both PKC- activity and glucose transport were dependent on activation of proline-rich tyrosine protein kinase 2, the ERK pathway, and phospholipase D (PLD) but were independent of phosphatidylinositol 3-kinase (PI3K) in PKC-+/+ ES cells. Interestingly, this dependency was completely reversed after differentiation of ES cells to adipocytes, i.e. insulin effects on PKC- and glucose transport were dependent on PI3K, rather than proline-rich tyrosine protein kinase 2/ERK/PLD. As in ES cells, insulin effects on glucose transport were absent in PKC--/- adipocytes but were rescued by expression of WT PKC- in these adipocytes. Our findings suggest that insulin activates aPKCs and glucose transport in ES cells by a newly recognized PI3K-independent ERK/PLD-dependent pathway and provide a compelling line of evidence suggesting that aPKCs are required for insulin-stimulated glucose transport, regardless of whether aPKCs are activated by PI3K-dependent or PI3K-independent mechanisms. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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A perhaps more rigorous approach to assess the importance of aPKCs in insulin-stimulated glucose transport would be to use gene-targeting methods to specifically knock out one or both aPKC isoforms, and/or 
. However, generalized knockout of PKC-, the major aPKC isoform in mouse tissues, is embryonic lethal, and, as of yet, there are no reports of successful knockout of PKC- in specific mouse tissues in which insulin stimulates glucose transport.
As another approach, signaling factors can be knocked out by recombinant methods in embryonic stem (ES) cells, and pluripotential ES cells can be differentiated to more terminal cell types in vitro. This approach is particularly interesting, as it not only provides an opportunity to examine the role of the deleted signaling factor in biological processes, but it also allows us to examine the development of signaling pathways as primitive ES cells proliferate and differentiate to more terminal cell types.
Recently, we generated mouse ES cells in which both PKC- alleles were knocked out by recombinant methods. Like parental wild-type (WT) PKC-+/+ ES cells, knockout PKC--/- ES cells readily differentiated to adipocytes, and, moreover, the absence of PKC-, the major aPKC in insulin-sensitive murine white adipocytes and skeletal muscles, allowed us to test the hypothesis that aPKCs are required for insulin-stimulated glucose transport in both the more primitive ES cells and ES-derived adipocytes. Using this approach, we found that insulin-stimulated glucose transport was markedly compromised in PKC--/- ES cells and ES-derived PKC--/- adipocytes. Moreover, insulin-stimulated glucose transport was fully restored by adenoviral-mediated introduction of WT PKC- into both PKC--/- ES cells and ES-derived PKC--/- adipocytes. Surprisingly, we also found that insulin uses different signaling pathways to activate aPKCs and glucose transport in parental ES cells and ES-derived adipocytes, viz., PI3K independent and PI3K dependent, respectively.
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RESULTS |
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-deficient cells are described in Materials and Methods and Fig. 1
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+/+ ES Cells.+/+ ES cells, basal glucose transport rates were relatively high, as compared with other cultured cells that we have used, including 3T3/L1 fibroblasts and adipocytes, L6 myotubes, and rat and human adipocytes (data not shown). Despite the high rates of basal transport, insulin increased [3H]2-DOG uptake by approximately 60% in PKC-+/+ ES cells (Fig. 2A). Whereas basal uptake was not significantly altered, insulin-stimulated increases in [3H]2-DOG uptake were inhibited by the PKC-/ pseudosubstrate and expression of kinase-inactive PKC- (Fig. 2A), suggesting dependence on PKC-.
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+/+ and PKC--/- ES Cells./ activity in PKC-+/+ ES cells (Fig. 2B
). In marked contrast, PKC-/ activity was markedly diminished in PKC--/- ES cells and essentially unresponsive to insulin, presumably reflecting the absence of PKC- in these PKC--/- ES cells (Fig. 2B). In the latter regard, note that the levels of PKC- are apparently much less than those of PKC- in PKC-+/+ ES cells, as indicated by the fact that total immunoreactive aPKCs (as measured with anti-C-terminal antiserum that measures both PKC- and PKC-) in PKC--/- ES cells was, at most, only about 10% of that observed in PKC-+/+ ES cells (see Fig. 2B). Also note the low level of total aPKC enzyme activity in PKC--/- ES cells (Fig. 2B). Thus, as in adipocytes and muscles of intact mice (16, 17), PKC- appears to be the major aPKC in mouse ES cells.
Impaired Activation of [3H]2-DOG Uptake in PKC--/- ES Cells and Rescue by PKC- Expression.
In contrast to findings in PKC-+/+ ES cells, and in keeping with the failure of insulin to substantially increase total aPKC activity in PKC--/- ES cells, insulin had little or no effect on [3H]2-DOG uptake in PKC--/- ES cells (Fig. 3B). However, insulin effects on [3H]2-DOG uptake were readily apparent after adenoviral-mediated introduction of WT PKC- into these knockout cells (Fig. 3B). Of further interest, the introduction of WT PKC- into WT PKC-+/+ ES cells markedly enhanced sensitivity for insulin-stimulated [3H]2-DOG uptake (see Fig. 3A for shift of insulin dose-response curve to the left).
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, adenoviral-mediated expression of WT PKC- in PKC--/- ES cells (i.e. to supplement the relatively low level of endogenous PKC- present in mouse ES cells so that total aPKC content was comparable to that of WT cells) restored insulin effects on [3H]2-DOG uptake in PKC--/-cells (data not shown). This finding supports our contention (6) that aPKCs, and , can function interchangeably in supporting insulin-stimulated glucose transport.
Signaling Requirements for Insulin-Stimulated aPKC Activation and Glucose Transport in PKC-+/+ ES Cells: Dependence on ERK and Phospholipase D (PLD).
Surprisingly, unlike what is seen in mature adipocytes (1, 3, 4) and myocytes (7), insulin effects on glucose transport and aPKC activation in PKC-+/+ ES cells were not inhibited by the PI3K inhibitor, wortmannin (Fig. 4, A and B). We therefore examined a newly recognized PI3K-independent pathway that results in increases in aPKC activity and aPKC-dependent glucose transport and involves the activation of nonreceptor proline-rich tyrosine protein kinase-2 (PYK2), which in turn activates GRB2 and thus the ERK pathway, thereby activating PLD and increasing production of phosphatidic acid, which directly activates aPKCs (12, 13, 14). In keeping with the possibility that insulin uses this PYK2/ERK/PLD signaling pathway to activate aPKCs and glucose transport in ES cells, we found that: 1) insulin effects on both [3H]2-DOG uptake and aPKC activation in PKC-+/+ ES cells were largely inhibited by the MAPK kinase 1 (MEK1) inhibitor, PD98059 (Fig. 4, A and B) and PLD inhibitor, 1% 1-butanol (Fig. 4, A and B), and partially inhibited by expression of kinase-inactive forms of ERK1 and cRAF (Fig. 5, A and B); 2) insulin activated both ERK and PLD (Fig. 6, A and B); and 3) MEK1 inhibitors, UO126 and PD98059, inhibited insulin-induced increases in both ERK and PLD activity (Fig. 6, A and B).
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Signaling Requirements for Insulin-Stimulated Glucose Transport in PKC-+/+ ES Cells: Dependence on PYK2.
Consonant with the possibility that insulin uses PYK2 to activate ERK, PLD, aPKCs, and glucose transport in PKC-+/+ ES cells, insulin provoked increases in the autophosphorylation site of PYK2, i.e. tyrosine-402, and thus activated PYK2 (Fig. 7). Moreover, adenoviral-mediated expression of a dominant-negative form of PYK2, viz., PRNK (which lacks the catalytic domain), but not WT PYK2 (which was stimulatory), inhibited insulin effects on [3H]2-DOG uptake (Fig. 8A), ERK activation (Fig. 8B), and aPKC activation (Fig. 8C) in PKC-+/+ ES cells.
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Evidence that Insulin Acts via Its Own Receptor in ES Cells.
Given our surprise that insulin used the PYK2/ERK/PLD pathway to activate aPKCs and glucose transport in PKC-+/+ ES cells, it was important to determine whether insulin was acting through its own or the IGF-I receptor. In this regard, we found substantial amounts of insulin receptor 
- and ß-subunits in plasma membranes of PKC-+/+ ES cells (Fig. 9). Moreover, IGF-I activated aPKCs by a signaling pathway different (i.e. requiring PLD, but not RAF, MEK1, and ERK) from that used by insulin in PKC-+/+ ES cells (data not shown), suggesting that insulin was not acting through the IGF-I receptor. Taken together, these findings suggested that insulin acted through its own, rather than the IGF-I, receptor in ES cells.
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, relative to microsomes, plasma membranes of ES cells contained seemingly large amounts of Glut1 and insulin did not substantially alter the levels of these transporters in either cell fraction. The apparently high basal level of Glut1 in the plasma membrane may explain the high basal level of 2-DOG uptake in PKC-+/+ ES cells (see above). We did not detect any immunoreactive insulin-sensitive Glut4 or Glut8 in ES cells.
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); as may be noted, the presence or absence of PKC- did not alter growth/differentiation processes or the microscopic appearance of undifferentiated ES cells and ES-derived adipocytes. After differentiation of PKC-+/+ ES cells to PKC-+/+ adipocytes, insulin provoked 2- to 3-fold increases in both PKC-/ activity and [3H]2-DOG uptake (Fig. 12, A and B). Interestingly, in contrast to parental ES cells, these insulin-stimulated increases in [3H]2-DOG uptake in ES-derived adipocytes were 1) wortmannin sensitive, 2) PD98059 insensitive, and 3) inhibited by the PKC-/ pseudosubstrate and adenoviral-mediated expression of kinase-inactive forms of both phosphoinositide-dependent protein kinase 1 (PDK-1) and PKC-, but not kinase-inactive ERK1 (Fig. 12B). Similarly, in contrast to ES cells, insulin-stimulated increases in PKC-/ activity in ES-derived adipocytes were 1) wortmannin sensitive, 2) PD98059 insensitive, and 3) inhibited by adenoviral-mediated expression of kinase-inactive 3-phosphoinositide-dependent protein kinase-1 (PDK-1), but not kinase-inactive ERK1 (Fig. 12A).
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+/+ and PKC--/- adipocytes contained comparable amounts of immunoreactive Glut4 (Fig. 13A), and insulin provoked decreases in microsomal Glut4 levels and increases in plasma membrane Glut4 levels in PKC-+/+ adipocytes (Fig. 13B). Thus, because insulin activates only the PI3K/PDK-1 pathway in these adipocytes, it may be surmised that insulin uses this pathway to activate aPKCs and Glut4 translocation/glucose transport in PKC-+/+ adipocytes.
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-/- adipocytes, and, moreover, this effect of insulin was readily apparent after adenovirally mediated introduction of WT PKC- into PKC--/- adipocytes (Fig. 13C). Although not directly tested, we presume that insulin activated the PKC- that had been introduced into PKC--/- adipocytes by means of the same signaling factors that are operative in PKC-+/+ adipocytes, i.e. via PI3K and PDK-1. |
DISCUSSION |
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The relatively high level of basal 2-DOG uptake and the modest increase in 2-DOG uptake in response to insulin treatment in ES cells were most likely reflections of a relatively high plasma membrane content of Glut1 in these cells. Indeed, as compared with the plasma membrane, microsomal levels of Glut 1 appeared to be relatively low, and we did not detect a significant change in either plasma membrane or microsomal content of Glut1 in response to insulin treatment. Thus, although we cannot rule out a small change in Glut1 subcellular distribution or an important change in Glut1 content in a specific microdomain of the plasma membrane in response to insulin treatment, our findings were more in keeping with the notion that insulin may have increased the activity rather than the plasma membrane content of Glut1 in ES cells. In this respect, however, we cannot rule out the possibility that our ES cells may contain glucose transporters other than Glut1, Glut4, or Glut8 that might have been activated and/or translocated in response to insulin treatment.
The finding that insulin used the PYK2/ERK/PLD in Glut-1-containing undifferentiated ES cells, and the PI3K/PDK-1 in Glut1/Glut4-containing ES-derived adipocytes, to activate aPKCs and glucose transport does not imply that the PYK2/ERL/PLD and the PI3K/PDK-1 pathways are specifically used to activate/translocate Glut1 and Glut4, respectively. To begin with, we did not observe any changes in the subcellular distribution of the Glut1 transporter in response to insulin treatment in either cell type. Second, we have found that insulin-stimulated glucose transport is dependent upon PI3K, not MEK1/ERK, in both the presently used ES-derived embryoid bodies, i.e. before terminal differentiation to adipocytes, and in mouse 3T3/L1 fibroblasts/preadipocytes, which contain Glut1, but little or no Glut4 (our unpublished observations). Third, we have reported that the PYK2/ERK/PLD, rather than the PI3K/PDK-1, pathway is used by several noninsulin agonists to stimulate Glut4 translocation and glucose transport in 3T3/L1adipocytes and L6 myocytes (12, 13, 14). Therefore it seems clear that the PYK2/ERK/PLD and PI3K/PDK-1 pathways are not specific activators/translocaters of Glut1 and Glut4, respectively.
Regardless of which signaling pathway was used by insulin to activate aPKCs and regardless of whether ES cells or ES-derived adipocytes were examined, aPKC activation was required for insulin-stimulated glucose transport. This is clear both from the findings that 1) insulin effects on glucose transport were absent in PKC--/- ES cells and PKC--/- adipocytes, and 2) insulin effects on glucose transport were restored by the introduction of PKC- into both of these knockout cells. Accordingly, these findings suggested that aPKCs serve as distal activators of glucose transport for both PYK2/ERK/PLD and PI3K/PDK1 pathways.
The fact that insulin did not use the PI3K/PDK-1 pathway to activate aPKCs in WT ES cells cannot be explained by an absence of what are generally considered to be the major upstream signaling factors required for aPKC activation in mature adipocytes and myocytes, viz., insulin receptor substrate-1, PI-kinase, and PDK-1. In this regard, it has been shown that IGF-I activates insulin receptor substrate 1 (IRS-1)-dependent PI3K and, by a PDK-1-dependent mechanism, protein kinase B in mouse ES cells (18). Moreover, we have documented that insulin activates protein kinase B, presumably via IRS-1-dependent PI3K and PDK-1, in our ES cells. It may therefore be surmised that a factor(s) in addition to IRS-1 and PDK-1 is (are) needed to couple PI3K to aPKC activation. Obviously, this factor is absent or inactive in ES cells and is acquired or activated after differentiation of ES cells to embryoid bodies, preadipocytes/fibroblasts, adipocytes, and presumably other cell types. Precisely what factors are responsible for this adaptation is presently under study.
Whereas PI3K was not effectively coupled to the activation of aPKCs in ES cells, insulin was able to activate PYK2 and thereby activate ERK, PLD, and aPKCs in these cells. In our experience, this ability of insulin to activate the PYK2/ERK/PLD/aPKC pathway is neither seen in mature adipocytes and myocytes, nor in preadipocytes or fibroblasts. It may therefore be conjectured that ES cells, but not adipocytes, myocytes, or fibroblasts, contain a factor(s) that is capable of coupling the insulin receptor to PYK2, or this coupling to PYK2 occurs as a default mechanism when coupling between PI3K and aPKC activation is absent.
As alluded to above, in our experience, whole-body knockout of PKC- is embryonic lethal, and, to date, attempts to specifically knockout PKC- in adipocytes or muscles of intact mice have not been fruitful. Moreover, knockout of PKC- in the mouse does not significantly diminish total aPKC levels in muscle and adipocytes, nor does it alter glucose metabolism appreciably (our unpublished observations), presumably reflecting that PKC-, rather than PKC-, is the major aPKC in insulin-sensitive muscles and adipocytes of mice. It is hoped that future in vivo conditional knockout studies will ultimately be feasible and allow us to determine the phenotypic/metabolic consequences of knockout of PKC- in the intact mouse. In the meantime, the present findings provide strong, if not compelling, evidence that aPKCs are essential for insulin-stimulated glucose transport in both more primitive and more terminally differentiated cells.
Although we were able to differentiate our murine ES cells to lipid-laden adipocytes, it should be realized that these ES-derived adipocytes are undoubtedly, in many respects, considerably different from other cultured adipocytes and adipocytes of intact animals. For example, relative effects of insulin on [3H]2-DOG uptake in our murine ES-derived adipocytes were substantially less than those observed in preadipocyte-derived murine 3T3/L1 adipocytes, but, on the other hand, similar to those observed in preadipocyte-derived human adipocytes (3).
Finally, although further studies are needed for more definitive mapping of signaling pathways in ES cells, we have found that a number of agonists, in addition to insulin (other growth factors and osmotically active substances such as sorbitol), use either the presently described PYK2/ERK pathway or a separate pathway to activate PLD, aPKCs, and glucose transport in undifferentiated ES cells. Accordingly, the present findings provide new information on unique signaling mechanisms that are used in primitive ES cells to ensure that glucose transport is commensurately stimulated by endocrine/paracrine/autocrine factors that control growth and subsequent differentiation of ES cells.
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MATERIALS AND METHODS |
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/-Deficient ES Cells/ locus, a 129/Ola genomic phage library (Stratagene, La Jolla, CA) was screened using full-length rat PKC-/ cDNA. Several clones were identified and purified to homogeneity and subsequently rescreened with a 5'-specific cDNA probe corresponding to nucleotides 1–300. One phage clone was shown to harbor the exon corresponding to nucleotides 110–233 of the published murine PKC-/ cDNA. A subcloned 11-kb EcoRI genomic DNA fragment obtained from this phage was used as a backbone for all cloned targeting vectors that were used subsequently. For the generation of a floxed targeting vector, the aforesaid 11-kb EcoRI fragment was modified as follows. A single loxP site was introduced into a unique HindIII site located approximately 2.5 kb 5' of the exon (nucleotides 110–233). Subsequently, a floxed neo cassette (pMC1-neo-polyA) was inserted into a BglII site located downstream of the aforesaid exon. The floxed targeting vector was functionally tested by transformation into bacteria expressing Cre constitutively.
For the generation of the LacZ targeting vector, the aforesaid 11-kb EcoRI fragment was modified as follows. A SalI site was introduced into the exon sequence by site-directed mutagenesis followed by an insertion of a ß-galactosidase/neomycin cassette (LacZ/neo) into this SalI site, resulting in a targeting vector that expressed the reporter gene (LacZ) under the control of the endogenous PKC-/ promoter after homologous recombination.
To generate PKC-/-deficient ES cells, the linearized floxed targeting vector was introduced into E14 ES cells by electroporation (9). After 10 d of selection with G418 (300 µg/ml), resistant ES cell clones were further analyzed by Southern blot analysis using the probe indicated in Fig. 1. Several independent clones were identified harboring a homologously integrated targeting vector, resulting in a floxed PKC-/ allele. A few clones were further analyzed by Southern blotting using different probes and restriction enzymes to verify that there was no additional integration of the vector into the genome and that the homologous recombination had taken place in the predicted manner (data not shown). Two ES cell clones that fulfilled these criteria were then transiently transfected with a Cre-expressing vector according to the protocol as described previously (10). Subsequently, several clones were identified that harbored the predicted deletion of the floxed region (i.e. that flanked by loxP sites) of the PKC- / gene. Two of these clones were used for the final electroporation to introduce the LacZ targeting vector to knock out the remaining unfloxed WT allele. After 10 d of selection with G418 (300 µg/ml), resistant ES cell clones were analyzed by Southern blot analysis using the probe indicated in Fig. 1. We then identified ES cell clones harboring an integrated LacZ targeting vector into the mutant allele, thus generating a heterozygote mutant LacZ PKC-/ allele, as well as ES cell clones showing an integration into the remaining WT allele, thus causing a null mutation (homozygote) of the PKC-/ gene in ES cells.
PKC-/-/- ES cell clones that were used in all later studies were again analyzed by Southern blotting using different probes and restriction enzymes to verify that there was no additional integration of the vector into the genome and that the homologous recombination had taken place in the predicted manner (data not shown).
Genotyping
Genotypic characterization of ES cells was accomplished by Southern blot analysis of either HindIII- or BamHI-digested genomic DNA. DNA was hybridized with the probe indicated in Fig. 1, thereby distinguishing WT, floxed, deleted, and LacZ alleles. The probe corresponded to a 5' 0.3-kb HindIII/EcoRI fragment hybridizing to an 8.0-kb BamHI band and a 3.0-kb HindIII band in WT PKC- DNA. The floxed allele was recognized by an additional 2.0 kb HindIII band, and, after Cre-mediated deletion, a 4.0-kb BamHI band compared with the 8.0-kb WT band identified the successfully mutated allele (del). The targeted LacZ allele was recognized by an additional 6.5-kb BamHI band.
Culture of ES Cells
Cells were cultured as monolayers on 0.1% gelatin-coated plates (Corning, Inc., Corning, NY) in DMEM (Invitrogen, San Diego, CA) containing 25 mM glucose, 2 mM glutamine (Invitrogen), 0.1 mM ß-mercaptoethanol, 0.1 mM nonessential amino acids (Invitrogen), 0.03 mM nucleoside mixture (Sigma Chemical Co., St. Louis, MO), 15% fetal bovine serum (ES-qualified, Invitrogen), 1000 U/ml leukemia-inhibitory factor (Sigma Chemical Co.) and 50 U per plate penicillin and streptomycin. There were no differences in growth characteristics of PKC-+/+ and PKC--/- cells. Heterozygote PKC-+/- cells were not studied.
Differentiation of ES Cells to Adipocytes
ES cells were differentiated to adipocytes as described by Rosen et al. (11). In brief, aliquots (20 µl) of suspended ES cells (1–2 x 103 cells in DMEM growth medium) were spotted and cultured as hanging drops on the underside of covers of plates containing PBS. After 2 d, aggregated embryoid bodies were obtained and cultured for 3 d with daily changes of leukemia-inhibitory factor-free DMEM growth medium containing 100 nM all-trans-retinoic acid. Embryoid bodies were then transferred to gelatin-coated plates and incubated for 2–3 wk (with medium changes every 2–3 d) in DMEM containing 10% fetal bovine serum, 100 nM insulin, 100 nM dexamethasone (Sigma), and 100 nM isobutylmethylxanthine (Sigma). Lipid content of differentiated adipocytes was readily apparent by staining with Oil Red O. In agreement with findings of Rosen et al. (11), 70–90% of cells were lipid-laden adipocytes after the differentiation process. There were no differences in differentiation properties of PKC-+/+ and PKC--/- ES cells.
Incubations and Assays
Before experiments, ES cells and ES-derived adipocyte cells were maintained for 48 h in DMEM containing all the aforesaid ingredients except, in the case of adipocytes, agents used for differentiation. Where indicated, cells were 1) treated for 48 h with adenoviruses encoding various signaling proteins (3, 12, 13, 14) as described in the text, or 2) incubated for 24 h with [3H]oleic acid (NEN Life Science Products, Boston, MA) to prelabel phosphatidylcholine and other phospholipids, which serve as substrates for PLD and subsequent generation of the labeled PLD product, [3H]phosphatidylbutanol, in cells incubated in the presence of 1% 1-butanol, as described previously (12, 13, 14, 15).
Immediately before experiments, cells were first incubated for 3–4 h in serum-free DMEM containing 2% BSA, and then equilibrated for 15–30 min in glucose-free Krebs Ringer phosphate medium containing 1% BSA, and finally treated with insulin or other substances as described in the text.
[3H]2-DOG uptake was measured over 5 min after a 30-min incubation with or without insulin, as described previously (1). Results of [3H]2-DOG uptake in ES cell monolayers are expressed as counts per min/well, as the protein concentration in each well was essentially constant within each experiment (
50–100 µg protein/well). Results of [3H]2-DOG uptake in differentiated adipocytes, on the other hand, are expressed as counts per min/100 µg protein, as the protein content of adipocytes (as differentiated from embryoid bodies) varied in each well.
In studies in which PLD was assayed, the incubation medium contained 1% 1-butanol to trap PLD-released phosphatidic acid as [3H]phosphatidylbutanol, as described previously (12, 13, 14, 15).
After incubation, cells were scraped and sonicated, and lysates were 1) immunoprecipitated with anti-C-terminal PKC-// or anti-ERK antibodies for determination of total aPKC (1, 4) and ERK (12, 13, 14) activity as described; or 2) extracted and examined for PLD-dependent generation of [3H]phosphatidylbutanol as described (12, 13, 14, 15).
Glut4 Translocation Studies
After incubation of ES-derived adipocytes with or without insulin as described in the text, plasma membranes and microsomes were isolated by sucrose-gradient ultracentrifugation and subjected to Western analysis for immunoreactive Glut4, as described (1).
Western Analyses
As described previously (1, 3), cell lysates were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with antibodies to 1) a common C termini amino acid sequence in PKC-// (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); 2) a specific N-terminal sequence in PKC- (kindly supplied by Dr. Todd Sactor); 3) a specific internal sequence in PKC-/ (Transduction Laboratories, Inc., Lexington, KY); 4) - and ß-subunits of the insulin receptor (Santa Cruz Biotechnology, Inc.); 5) the ß-subunit of the IGF-I receptor (Santa Cruz Biotechnology, Inc.); 6) Glut4 (Biogenesis, Kingston, NH; or Santa Cruz Biotechnology, Inc.); 7) Glut1 (kindly supplied by Dr. Ian Simpson; 8) Glut8 (kindly supplied by Dr. Kelly Moley); 9) PYK2 (kindly provided by Dr. Ivan Dikic); and 10) phospho-Y402-PYK2 (Biosource International, Camarillo, CA).
Statistical Methods
Data were expressed as mean ± SE. P values were determined by ANOVA and the least-significant multiple comparison method.
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FOOTNOTES |
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G.B., M.L., and R.F. contributed equally to this paper.
Requests for ES cells should be directed to Dr. Michael Leitges.
Abbreviations: aPKC, Atypical protein kinase C; 2-DOG, 2-deoxyglucose; ES, embryonic stem; Glut, glucose transporter; IRS, insulin receptor substrate; MEK1, MAPK kinase 1; PDK-1, 3-phosphoinositide-dependent protein kinase-1; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PLD, phospholipases D; PYK2, proline-rich tyrosine protein kinase 2; WT, wild-type.
Received for publication March 13, 2003. Accepted for publication November 4, 2003.
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