* Washington State University, Pullman, Washington 991646510; and
Pacific Northwest National Laboratory, Richland, Washington 99352
Received December 31, 2001; accepted April 5, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: dichloroacetate; glycogen; insulin; insulin receptor; PKB/Akt; PI3K; hepatocyte.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Glycogen synthase is the rate-limiting enzyme of glycogen biosynthesis, and its activation is regulated by a reversible dephosphorylation mechanism in which several insulin-controlled phosphatases and kinases can be involved (Pugazenthi and Khandelwal, 1995). The principal signaling pathway by which insulin stimulates glycogen synthase is via activation of the insulin receptor (IR), leading to phosphatidylinositol-3' kinase (PI3K)-dependent activation of protein kinase B (PKB/Akt), inactivation of glycogen synthase kinase-3 (GSK-3), and increased activity of glycogen synthase (GS) (Cohen, 1999
; Cross et al., 1995
, 1997
; Lawrence and Roach, 1997
; Park et al., 1999
). PI3K-dependent activation of the p70 kDa S6 protein kinase (p70S6K) as well as activation of the Ras/Raf/MEK/Erk1/2 signaling pathway have also been linked with GSK-3 inactivation and increased glycogen synthesis (Azpiazu et al., 1996
; Dent et al., 1990
; Park et al., 1999
; Shepherd et al., 1995
; Sutherland and Cohen, 1994
; Sutherland et al., 1993
). In addition to their role in regulating metabolism, the activities of PI3K, PKB/Akt and Erk1/2 play important roles in regulating cell proliferation and apoptosis in hepatocytes (reviewed in Band et al., 1999
; Galetic et al., 1999
; Mounho and Thrall, 1999
; Roberts et al., 2000
).
DCA has been shown to markedly increase liver glycogen levels in mice within one week, and the increase is sustained with continued treatment (Kato-Weinstein et al., 1998). However, the glycogenic effect of DCA was associated with decreased glycogen synthase activity in the livers of these mice (Kato-Weinstein et al., 1998
). In more recent studies, serum insulin levels were found to be suppressed, but the effect required 2 weeks or more of DCA treatment to occur. At one week, insulin levels tended to be increased, but not significantly. Additionally, hepatic steady-state expression levels of the IR and PKB/Akt were significantly reduced during DCA treatment in vivo (Lingohr et al., 2001
). The decrease in expression of hepatic insulin signaling proteins was most dramatic after 10 weeks of DCA treatment, but readily apparent within 2 weeks (Lingohr et al., 2001
). These data suggested the accumulation of glycogen caused by DCA treatment might result in a compensatory downregulation of insulin signaling proteins and suppression of serum insulin levels. However, the lag observed in the decreased serum insulin concentrations also raised the question whether insulin and its signaling proteins were involved in the initial increase in liver glycogen caused by DCA treatment.
In the present study, the effect of DCA on glycogen levels in isolated B6C3F1 mouse hepatocytes has been examined to see if the effects of DCA could be distinguished from those of insulin. As found in livers of mice administered DCA in the drinking water, DCA increased glycogen accumulation in isolated hepatocytes. DCA-induced glycogen accumulation in hepatocytes was independent of insulin, but entirely dependent upon PI3K activity. Also, DCA downregulated IR expression in isolated hepatocytes, which could perhaps be the result of a regulatory loop involving glycogen content reminiscent of that occurring with insulin (Fleig et al., 1985; Knutson, 1991
).
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals and treatments.
All animal care, use and experiment protocols were submitted and approved by the Institutional Animal Care and Use Committee (IACUC) of Washington State University or the IACUC of Battelle, Pacific Northwest Laboratories. Hepatocytes were isolated from male B6C3F1 mice 812 weeks of age as described below.
Primary hepatocyte isolation.
Primary mouse hepatocytes were isolated by a retrograde perfusion method. Briefly, 410 week old mice were anesthetized by an ip injection of Nembutol (125 mg/kg), cannulated through the right ventricle, and livers were perfused with EGTA (0.5 mM) in Hepes-buffered Hanks Balanced Salt Solution (HBSS, pH 7.4) for 68 min (flow rate 57 ml/min). The livers were then perfused with Hepes-buffered HBSS solution containing collagenase (Worthington Type II100 units/ml, Worthington Biochemical Corp., Freehold, NJ) for another 810 min (flow rate 710 ml/min). Hepatocytes were dispersed, washed, and purified on a Percoll density gradient (Sigma, St. Louis, MO). Cells were plated at 30,000 cells/cm2 onto tissue culture dishes coated with Vitrogen (Collagen Biomaterials, Inc., Palo Alto, CA). Cell viability was >90% of cells in all experiments, as determined by trypan blue exclusion. Hepatocytes were allowed to attach 4 h in serum-free Williams media E (Sigma, St. Louis, MO) containing 1% penicillin/streptomycin and 2 mM of L-glutamine. After attachment cells were washed with HBSS and given fresh medium, with or without test chemicals. Hepatocytes were incubated in 95% air, 5% CO2 until the end of treatment. Following treatment, hepatocytes were rinsed with ice-cold HBSS and lysed on the plate with 0.125 ml of lysis buffer (10 mM Tris, 150 mM NaCl, 1% nonidet P-40, 10 mM NaF, 1 mM pepstatin, 200 mg/ml leupeptin, 10 ug/ml aprotinin, 50 mg/ml PMSF, and 200 mM sodium orthovanadate). Protein content was determined by the method of Lowry et al(1951), and lysates were frozen at 80°C until analysis.
Immunoblot analysis.
Hepatocellular protein was diluted in gel loading buffer (final concentration of 50 mM Tris-HCl, pH 6.8, 2.5% glycerol, 2% SDS, 1% mercaptoethanol, 0.001% bromophenol blue, and 0.5 mM EDTA) and resolved by SDS-polyacrylamide gel electrophoresis (SDSPAGE). Gels were transferred to nitrocellulose membrane on a semi-dry blotting apparatus (Integrated Separation Systems, Natick, MA). Membranes were blocked for 1 h in 5% nonfat milk in TBS-T (20 mM Tris, pH 7.6, 137 mM NaCl, and 0.1% Tween-20) and incubated overnight with primary antibodies directed against IR-ß (Santa Cruz, Santa Cruz, CA) or phosphorylated PKB/Akt (New England Biolabs, Beverly, MA). A separate gel was run for each individual antibody tested and equal protein loading was confirmed either by gel staining or by blotting with a primary antibody directed against a protein (i.e., epidermal growth factor receptor, Santa Cruz). Blots were washed in 5 changes of TBS-T and incubated for 1 h with goat anti-rabbit IgG horseradish peroxidase conjugated secondary antibody (Zymed Laboratories, San Francisco, CA). Detection was by horseradish peroxidase and chemiluminescence (Amersham Life Science, Arlington Heights, IL). Immunoblots were quantified using NIH Image 1.61 Software.
Glycogen analysis.
Glycogen levels in hepatocyte monolayers were measured by a spectrophotometric enzymatic assay as described by Gomez-Lechon et al(1995). Briefly, hepatocyte monolayers were washed twice with ice-cold PBS (20 mM, pH 7.4). Plates were frozen immediately in liquid nitrogen. Cells were scraped from the plates in 100 ml PBS and frozen and thawed twice. Lysates were added to 800 ml reaction buffer, containing 200 mM sodium acetate (pH 4.8). Amyloglucosidase (10 Units; Sigma, St. Louis, MO) was added to initiate enzymatic glycogen hydrolysis. Reactions were incubated for 2 h at 40°C with constant agitation. Glucose was determined by a colorimetric glucose oxidase method with reagents purchased from Sigma (St. Louis, MO). Phosphate buffer (100 mM, pH 7) containing 1 mg/ml ABTS, 0.0008U/ml peroxidase, and 0.5U/ml glucose oxidase were added to 200 ml of sample that were then incubated in the dark for 45 min. The intensity of the color reaction was measured at 405 nM. Glycogen content was normalized to protein concentration within each individual sample and expressed as glucose equivalents (nmol)/mg protein.
In experiments measuring glycogen degradation, hepatocytes were treated 16 h with DCA, followed by a 30-min treatment with 10 nM glucagon. Loss of intracellular glycogen levels was measured at the indicated time periods.
Other procedures.
Data are presented as a mean ± SEM. Statistically significant differences between samples and groups were analyzed using one-way ANOVA or Students t test as indicated in the figure legends. Statistical significance was set at p < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
As expected, insulin (100 nM) in the presence of dexamethasone (100 nM) significantly (>2-fold, p < 0.05) increased hepatocellular glycogen content after 16 h (Figs. 3A3C. Unlike DCA-stimulated glycogen synthesis, insulin-stimulated glycogen synthesis in hepatocyte monolayers was only partially inhibited (
50%) by the PI3K inhibitors (Fig. 3A
).
Alternative signaling pathways, which may lead to stimulation of glycogen synthase activity, include PI3K-dependent activation of the p70 ribosomal protein S6-kinase (p70S6K) or activation of the Ras/Raf/MEK/Erk1/2 pathway (Azpiazu et al., 1996; Dent et al., 1990
; Shepherd et al., 1995
; Sutherland and Cohen, 1994
). In primary mouse hepatocytes treated 16 h in the presence and absence of DCA and/or insulin, neither DCA- nor insulin-induced glycogen deposition was affected by rapamycin (10100 nM) (Fig. 3B
, data not shown).
Inhibition of the Erk1/2 kinase-signaling pathway via the MEK inhibitor, PD098059, also did not affect the increase in glycogen caused by DCA or insulin/dexamethasone in hepatocyte monolayers (Fig. 3C). Based on our previous work (Mounho and Thrall 1999
), the concentration of PD098059 used in these experiments was sufficient to completely block Erk1/2 activation. Immunoblot analysis demonstrated that treatment of hepatocytes with DCA does not stimulate phosphorylation of Erk1/2 (data not shown).
DCA and insulin-induced glycogen deposition is PKB/Akt-independent.
One of the most common downstream targets of PI3K reported to be involved in regulating glycogen synthesis is PKB/Akt (Cross et al., 1995, 1997
; Lawrence and Roach, 1997
). Hepatocyte monolayers were treated for 5 min with increasing concentrations of insulin in the presence of 100 nM dexamethasone, increasing concentrations of epidermal growth factor (EGF, 125 ng/ml), or 500 µM DCA. Lysates were immunoblotted with antibody that selectively recognized Ser473 phosphorylated PKB/Akt. Preliminary time-course experiments had delineated 5 min as the peak level of phosphorylation of PKB/Akt (data not shown). Insulin significantly increased PKB/Akt phosphorylation at doses of 100 nM (Fig. 4
). The relative level of phosphorylation of PKB/Akt caused by 100 nM insulin was nearly 4-fold (p < 0.05) and was completely inhibited in the presence of 25 µM LY294002 (Fig. 4
). In contrast to insulin, DCA did not cause a measurable increase in PKB/Akt phosphorylation after 5-min treatment (Fig. 4
), nor after 15, 30 or 60 min of treatment with DCA (10500 µM, data not shown). Therefore, the concentrations of DCA that stimulate hepatocellular glycogen accumulation do not cause PKB/Akt phosphorylation.
PKB/Akt is not an indicator of glycogen synthesis in isolated hepatocytes.
As with insulin, EGF (125 ng/ml) treatment of hepatocytes results in an increase in PKB/Akt phosphorylation (>6-fold, p < 0.05) that is dependent on activation of PI3K (Fig. 4). As observed in prior experiments by others (Chowdhury and Agius, 1987
; Grau et al., 1996
; Peak and Agius, 1994
), EGF significantly decreased glycogen content of hepatocyte monolayers (Fig. 6
). As before, insulin in the presence of dexamethasone increased glycogen content greatly (p < 0.5; Fig. 6
).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In vivo, the temporal relationship of DCAs effects on liver glycogen, serum insulin, and hepatic expression of the IR suggest that the accumulation of glycogen induced by DCA treatment may lead to a compensatory downregulation of the IR and suppression of insulin. In isolated hepatocytes, the DCA-induced downregulation of the IR occurred in the absence of insulin. Earlier studies (Fleig et al., 1985, 1987
) have shown that pre-incubation of hepatocyte monolayers overnight with insulin downregulates IR binding via a decrease in IR on the cell surface, which then reduces the acute glycogenic response to insulin. When this occurs, there is an inverse correlation between glycogen content and insulin-induced glycogen synthase activity. These results support the hypothesis that the increased glycogen content induced by DCA treatment may trigger a regulatory pathway that results in downregulation of the IR, but through actions subsequent to the IR, because it does not require dexamethasone.
Selective inhibitors have proven to be useful probes for characterizing target signaling pathways involved in mediating particular insulin-induced biological responses (Cohen, 1999; Ui et al., 1995
). Based upon the inhibitor studies in isolated hepatocytes, the DCA-induced increase in glycogen is dependent on activation of PI3K. The concentrations of inhibitors that completely blocked the glycogenic response to DCA also significantly inhibited the accumulation of glycogen caused by insulin (in the presence of dexamethasone) in primary hepatocytes. However, the effects of the PI3K inhibitors on insulin-induced glycogen synthesis were only partial (<40%), suggesting that PI3K-independent signaling pathways also contribute to the glycogenic response to insulin in hepatocyte monolayers. This is not surprising since insulin stimulated GS activation is also dependent on the inactivation of protein kinase A and the activation of protein phosphatase-1 (PP1) (reviewed in Saltiel, 2001
).
The serine/threonine kinase, PKB/Akt, is a downstream target of PI3K that has been shown to be involved in mediating the glycogenic response to insulin (Cheatham et al., 1994; Osawa et al., 1996
; Shepherd et al., 1995
). Upon its activation by insulin, PKB/Akt phosphorylates GSK-3, leading to its inactivation and the enhancement of GS activity (Cross et al., 1995
, 1997
; Ueki et al., 1998
). Although insulin stimulated PKB/Akt phosphorylation in hepatocyte monolayers, DCA did not affect PKB/Akt phosphorylation, even at concentrations that resulted in maximal increases in glycogen content. It is notable that the concentration of insulin required to produce a significant increase in PKB/Akt phosphorylation was 10-fold higher than that necessary to increase glycogen accumulation, suggesting that other PI3K-dependent signaling pathways (which could include PP1 activation) are more important in mediating the glycogenic response to insulin in hepatocytes.
The conflict between GSK-3 inhibition (via PKB/Akt) and increased glycogen synthesis has been described in adipocytes (Brady et al., 1998) and more recently in hepatocytes (Lavoie et al., 1999
). Activation of PI3K is required for stimulation of GS in hepatocytes, but PKB/Akt activation and subsequent GSK3 inactivation is not sufficient to induce GS activation. This was reinforced in the present study by the finding that EGF activated PKB/Akt but did not increase glycogen synthesis. While our results strongly support a role for PI3K in DCA- and insulin-stimulated glycogen synthesis, it appears that PKB/Akt phosphorylation is neither a required nor a sufficient stimulus for glycogen synthesis in primary mouse hepatocytes. Our results also do not support a role for a rapamycin-sensitive signaling pathway or one involving the Ras/Raf/MEK/Erk1/2 signaling pathway in DCA- and insulin-induced glycogen synthesis.
The most important aspect of our results is the observation that DCA stimulates glycogen accumulation in hepatic parenchymal cells in the absence of insulin. In Figure 7, a diagram outlining the pathways implicated in DCA-induced glycogen deposition is shown. In turn, we hypothesize that the marked increase in hepatocellular glycogen caused by DCA may negatively influence insulin-controlled signaling pathways by downregulating the IR. The signaling molecules involved in DCA-stimulated glycogen accumulation downstream of PI3K are unknown. However one of the most likely modifiers is PP1, whose activity is highly correlative with changes in glycogen synthase activity (Brady et al., 1998
). Insulin-stimulated PP1 activity is dependent on PI3K activation at least, in cardiomyocytes (De Luca et al., 1999
). Therefore, it is a prospective candidate for mediating DCA-stimulated glycogen deposition in isolated hepatocytes.
|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
NOTES |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Azpiazu, L., Saltiel, A. R., DePaoli-Roach, A. A., and Lawrence, J. C. (1996). Regulation of both glycogen synthase and PHAS-I by insulin in rat skeletal muscle involves mitogen-activated, protein kinase-independent, and rapamycin-sensitive pathways. J. Biol. Chem. 271, 50335039.
Band, C. J., Mounier, C., and Posner, B. I. (1999). Epidermal growth factor and insulin-induced deoxyribonucleic acid synthesis in primary rat hepatocytes is phosphatidylinositol 3-kinase-dependent and dissociated from protooncogene induction. Endocrinology 140, 56265634.
Brady, M. J., Bourbonais, F. J., and Saltiel, A. R. (1998). The activation of glycogen synthase by insulin switches from kinase inhibition to phosphatase activation during adipogenesis in 3T3L1 cells. J. Biol. Chem. 273, 1406314066.
Bull, R. J., Sanchez, I. M., Nelson, M. A., Larson, J. L. and Lansing, A. J. (1990). Liver tumor induction in B6C3F1 mice by dichloroacetate and trichloroacetate. Toxicology 63, 341359.[ISI][Medline]
Cheatham, B., Vlahos, C. J., Cheatham, L., Wang, L., Blenis, J., and Kahn, C. R. (1994). Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation. Mol. Cell. Biol. 14, 49024911.[Abstract]
Chowdhury, M. H., and Agius, L. (1987). Epidermal growth factor counteracts the glycogenic effect of insulin in parenchymal hepatocyte cultures. Biochem. J. 247, 307314.[ISI][Medline]
Cohen, P. (1999). The Croonian Lecture 1998. Identification of a protein kinase cascade of major importance in insulin signal transduction. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 354, 485495.[ISI][Medline]
Conti, J. A., and Kemeny, N. (1992). Type Ia glycogenosis associated with hepatocellular carcinoma. Cancer 69, 13201322.[ISI][Medline]
Cross, D. A. E., Alessi, D. R., Cohen, P., Andjelkovich, M., and Hemmings, B. A. (1995). Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378, 785789.[ISI][Medline]
Cross, D. A., Watt, P. W., Shaw, M., van der Kaay, J., Downes, C. P., Holder, J. C., and Cohen, P. (1997). Insulin activates protein kinase B, inhibits glycogen synthase kinase-3, and activates glycogen synthase by rapamycin-insensitive pathways in skeletal muscle and adipose tissue. FEBS Lett. 406, 211215.[ISI][Medline]
DeAngelo, A. B., Daniel, F. B., Most, B. M., and Olson, G. R. (1996). The carcinogenicity of dichloroacetic acid in the male Fischer 344 rat. Toxicology 114, 207221.[ISI][Medline]
DeAngelo, A. B., Daniel, F. B., Stober, J. A., and Olson, G. R. (1991). The carcinogenecity of dichloroacetic acid in the male B6C3F1 mouse. Fundam. Appl. Toxicol. 16, 337347.[ISI][Medline]
De Luca, J. P., Garnache, A. K., Rulfs, J., and Miller, T. B., Jr. (1999). Wortmannin inhibits insulin-stimulated activation of protein phosphatase-1 in rat cardiomyocytes. Am. J. Physiol. 276, H15201526.
Dent, P., Lavoinne, A., Nakielny, S., Caudwell, F. B., Watt, P., and Cohen, P. (1990). The molecular mechanism by which insulin stimulates glycogen synthesis in mammalian skeletal muscle. Nature 348, 302308.[ISI][Medline]
Fleig, W. E., Enderle, D., Steudter, S., Nother-Fleig, G., and Ditschuneit, H. (1987). Regulation of basal and insulin-stimulated glycogen synthesis in cultured hepatocytes: Inverse relationship to glycogen content. J. Biol. Chem. 262, 11551160.
Fleig, W. E., Nother-Fleig, G., Steudter, S., Enderle, D., and Ditschuneit, H. (1985). Regulation of insulin binding and glycogenesis by insulin and dexamethasone in cultured rat hepatocytes. Biochim. Biophys. Acta 847, 352361.[ISI][Medline]
Galetic, I., Andjelkovic, M., Meier, R., Brodbeck, D., Park, J., and Hemmings, B. A. (1999). Mechanism of protein kinase B activation by insulin/insulin-like growth factor-1 revealed by specific inhibitors of phosphoinositide 3-kinasesignificance for diabetes and cancer. Pharmacol. Ther. 82, 409425.[ISI][Medline]
Gomez-Lechon, M. J., Ponsoda, X., and Castell, J. V. (1995). A microassay for measuring glycogen in 96-well-cultured cells. Anal. Biochem. 236, 296301.[ISI]
Grau, M. Tebar, F., Ramirez, I., and Soley, M. (1996). Epidermal growth factor administration decreases liver glycogen and causes mild hyperglycaemia in mice. Biochem. J. 315, 289293.[ISI][Medline]
Kato-Weinstein, J., Lingohr, M. K., Orner, G. A., Thrall, B. D., and Bull, R. J. (1998). Effects of dichloroacetate on glycogen metabolism in B6C3F1 mice. Toxicology 130, 141154.[ISI][Medline]
Knutson, V. P. (1991). Cellular trafficking and processing of the insulin receptor. FASEB J. 5, 21302138.
Labrune, P., Trioche, P., Duvaltier, I., Chevalier, P., and Odievre, M. (1997). Hepatocellular adenomas in glycogen storage disease types I and III: A series of 43 patients and review of the literature. J. Pediatr. Gastroenterol. Nutr. 24, 276279.[ISI][Medline]
Lavoie, L., Band, C. J., Kong, M., Bergeron, J. J., and Posner, B. I. (1999). Regulation of glycogen synthase in rat hepatocytes. Evidence for multiple signaling pathways. J. Biol. Chem. 274, 2827928285.
Lawrence, J. C., Jr., and Roach, P. J. (1997). New insights into the role and mechanism of glycogen synthase activation by insulin. Diabetes 46, 541547.[Abstract]
Lingohr, M. K., Thrall, B. D., and Bull, R. J. (2001). Effects of dichloroacetate (DCA) on serum insulin levels and insulin-controlled signaling proteins in livers of male B6C3F1 mice. Toxicol. Sci. 59, 178184.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with folin phenol reagent. J. Biol. Chem. 193, 265275.
Mounho, B. J., and Thrall, B. D. (1999). The extracellular signal-regulated kinase pathway contributes to mitogenic and antiapoptotic effects of peroxisome proliferators in vitro. Toxicol. Appl. Pharmacol. 159, 125133.[ISI][Medline]
Osawa, H., Sutherland, C., Robey, R. B., Printz, R. L., and Granner, D. K. (1996). Analysis of the signaling pathway involved in the regulation of hexokinase-II gene transcription by insulin. J. Biol. Chem. 271, 1669016694.
Park, B., Kido, Y., and Accili, D. (1999). Differential signaling of insulin and IGF-1 receptors to glycogen synthesis in murine hepatocytes. Biochemistry 38, 75177523.[ISI][Medline]
Peak, M., and Agius, L. (1994). Inhibition of glycogen synthesis by epidermal growth factor. The role of cell density and pertussis toxin-sensitive GTP-binding proteins. Eur. J. Biochem. 221, 529536.[Abstract]
Pereira, M. A. (1996). Carcinogenic activity of dichloroacetic acid and trichloroacetic acid in the liver of female B6C3F1 mice. Fundam. Appl. Toxicol. 31, 192199.[ISI][Medline]
Pugazenthi, S., and Khandelwal, R. L. (1995). Regulation of glycogen synthase activation in isolated hepatocytes. Mol. Cell. Biochem. 149150, 95101.[ISI]
Roberts, R. A, James, N. H., and Cosulich, S. C. (2000). The role of protein kinase B and mitogen-activated protein kinase in epidermal growth factor and tumor necrosis factor -mediated rat hepatocyte survival and apoptosis. Hepatology 31, 420427.[ISI][Medline]
Saltiel, A. R. (2001). New perspectives into the molecular pathogenesis and treatment of type 2 diabetes. Cell. 104, 517529.[ISI][Medline]
Shepherd, P. R., Nave, B. T., and Siddle, K. (1995). Insulin stimulation of glycogen synthesis and glycogen synthase activity is blocked by wortmannin and rapamycin in 3T3L1 adipocytes: Evidence for the involvement of phosphoinositide 3-kinase and p70 ribosomal protein-S6 kinase. Biochem. J. 305, 2528.[ISI][Medline]
Stauber, A. J., and Bull, R. J. (1997). Differences in phenotype and cell replicative behavior of hepatic tumors induced by dichloracetate (DCA) and trichloroacetate (TCA). Toxicol. Appl. Pharmacol. 144, 235246.[ISI][Medline]
Sutherland, C., and Cohen, P. (1994). The aisoform of glycogen synthase kinase-3 from rabbit skeletal muscle is inactivated by p70 S6 kinase or MAP kinase-activated protein kinase-1 in vitro. FEBS Lett. 338, 3742.[ISI][Medline]
Sutherland, C., Leighton, I. A., and Cohen, P. (1993). Inactivation of glycogen synthase kinase-3 ß by phosphorylation: New kinase connections in insulin and growth-factor signalling. Biochem. J. 296, 1519.[ISI][Medline]
Ueki, K., Yamamoto-Honda, R., Kaburagi, Y., Yamauchi, T., Tobe, K., Burgering, B. M., Coffer, P. J., Komuro, I., Akanuma, Y., Yazaki, Y., and Kadowaki, T. (1998). Potential role of protein kinase B in insulin-induced glucose transport, glycogen synthesis, and protein synthesis. J. Biol. Chem. 273, 53155322.
Ui, M., Okada, T., and Hazeki, K., and Hazeki, O. (1995). Wortmannin as a unique probe for an intracellular signalling protein, phosphoinositide 3-kinase. Trends Biochem. Sci. 20, 303307.[ISI][Medline]
Vlahos, C. M., Matter, W. F., Hui, K. Y., and Brown, R. F. (1994). A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J. Biol. Chem. 269, 52415248.