* Washington State University, Pullman, Washington 99164-6510; and
Pacific Northwest National Laboratory, Richland, Washington 99352
Received July 20, 2000; accepted October 5, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: dichloroacetate; glycogen; insulin; signaling; insulin receptor PKB; hepatocarcinogen.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In livers of DCA-treated mice, tumor cells are differentiated from the normal cell population by glycogen content (Stauber and Bull, 1997). Normal cells accumulate large amounts of glycogen, whereas altered hepatic foci and tumors induced by DCA are glycogen-poor (Kato-Weinstein et al., 1998
). The doses of DCA required to produce liver tumorigenesis in mice coincide with those that produce glycogenosis in normal hepatocytes (Kato-Weinstein et al., 1998
). Insulin is the primary hormone involved in shifting intermediary liver metabolism from glycogen depletion to glycogen storage. It is also a mitogen for normal and malignant liver cells (Kahn and White, 1995
; Koontz and Iwahashi, 1981
; Massague et al., 1982
) and has been shown to suppress apoptosis (Wu et al., 1995
). Insulin induces biological responses via activation of its tyrosine kinase receptor and downstream serine/threonine kinase-signaling pathways (Kahn and White, 1995
).
In the present study, the effect of DCA treatment on the level of circulating insulin in male B6C3F1 mice and on insulin-controlled signaling proteins in normal liver tissue and DCA-induced liver tumor tissue were examined. Our results demonstrate that DCA treatment results in decreases in serum insulin levels and hepatocyte expression of the insulin receptor (IR) and protein kinase B (PKB). We also report that liver tumor cells are refractory to the DCA-induced changes that occur in normal hepatocytes. The differential sensitivity of the 2 cell populations to DCA's insulin-like activity may contribute to the tumorigenic effects of DCA in the liver.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals and treatments.
Male B6C3F1 mice were purchased from Simonsen Laboratories (Gilroy, CA) or Charles River (Raleigh, NC), allowed to acclimate for 1 week to a 12-h light/dark cycle, and placed on treatment. All animal care, use, and experiment protocols were submitted to and approved by the Institutional Animal and Use Committee (IACUC) of Washington State University or the IACUC of Battelle, Pacific Northwest Laboratories. Mice were treated with DCA (0.12.0 g/l) in their drinking water for 210 weeks. Water consumption was monitored and animals weighed weekly for the duration of the studies. Consistent with previous studies (Bull et al., 1990), DCA treatment does not significantly alter body weight or drinking water/food consumption for the duration of the studies at concentrations of DCA up to and including 2 g/l. At termination of treatment animals were sacrificed by CO2 asphyxiation. Blood was collected by cardiac puncture. Livers were excised and immediately frozen in liquid nitrogen. Most of these animals were sacrificed at 3:00 A.M. after discovering that blood concentrations of DCA at low doses rapidly decrease when lights are turned on and animals are no longer drinking water (Kato-Weinstein et al., 1998
).
Livers and tumors were available from prior experiments where mice were treated with 0.5 and 2.0 g/l DCA for 87 and 52 weeks, respectively, and age-matched controls (Orner et al., 1998). These animals were sacrificed during normal daylight hours.
Immunoblot analysis.
Livers, previously frozen at 80°C, were weighed, minced, and homogenized in 1.0 ml ice-cold homogenization buffer (50 mM HEPES, pH 7.6, 150 mM NaCl, 50 units/ml bacitracin, 1 µM pepstatin, 200 µg/ml leupeptin, 10 µg/ml aprotinin, 50 µg/ml PMSF, and 200 µM sodium orthovanadate) per 100 mg tissue (10% homogenate). Protein content was determined by the method of Lowry et al. (1951). Homogenate protein was diluted in 0.1 mM Tris-HCl (pH 7.4, 4°C) and in 2x SDS loading-gel buffer (100 mM Tris-HCl, pH 6.8, 5% glycerol, 3% SDS, 2% mercaptoethanol, 0.002% bromophenol blue, 1 mM EDTA) and 30 µg was resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Gels were transferred to either nitrocellulose or PVDF 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, 0.1% Tween-20) and incubated overnight with primary antibody directed against insulin receptor-ß (IR; Santa Cruz, Santa Cruz, CA), or protein kinase B (PKB; Santa Cruz, Santa Cruz, CA), or phosphorylated p42/44mitogen-activated protein kinase (MAP kinase; Promega, Madison, WI). 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, Santa Cruz, CA), which did not change between samples. Blots were washed in 5 changes of TBS-T and incubated for 1 h with goat anti-(rabbit IgG) alkaline phosphatase conjugated secondary antibody (Zymed Laboratories, San Francisco, CA), or goat anti-(rabbit IgG) horseradish peroxidase conjugated secondary antibody (Biorad, Hercules, CA). Detection was done by either alkaline phosphatase methodology and analyzed with a fluorescent imager or by horseradish peroxidase and chemiluminescence (Amersham Life Science, Arlington Heights, IL). Relative IR, PKB
and phosphorylated MAP kinase protein levels were quantified via NIH Image 1.61 software.
Measurement of hormones and glucose in serum.
Serum hormones were measured by radioimmunoassay with commercially available assays from Linco Research, Inc., (St. Charles, MO) for insulin or from Diagnostic Systems Laboratories (Webster, TX) for IGF-I. Serum glucose was measured by glucose oxidase determination (Sigma, St. Louis, MO).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
In no instance were IGF-I levels significantly affected by DCA-treatment (Table 1). Although, there was an increase in circulating levels of IGF-1 during the night in comparison to daytime levels, this effect was not treatment-related since it occurred in both control and DCA-exposed animals.
|
|
|
|
Phosphorylation of MAP Kinase Is Elevated in DCA-Induced Tumors
Since increased expression of the insulin receptor can indicate increased activation of its signaling pathway, the relative phosphorylation level of MAP kinase, a downstream target protein, was measured. Phosphorylation of MAP kinase was increased nearly 4-fold within liver tumors when compared to adjacent normal liver tissue of the same animal (p < 0.05) and also to that of normal liver from untreated animals (p < 0.05; Figs. 5a and 5b).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In addition to decreasing serum insulin levels, DCA-treatment resulted in decreased hepatic expression of the IR. The decrease in IR expression was significant at 2 weeks of DCA-treatment but progressed to an even lower level at 10 weeks of DCA-treatment. IR protein levels would normally be expected to increase with a fall in serum insulin levels (Kahn and White, 1995). However, it may be that the accumulation of glycogen in normal hepatocytes caused by DCA down-regulates the receptor responsible for initiating the activation of the signaling pathway leading to glycogen synthesis.
In support of this hypothesis, DCA decreased hepatic expression of PKB, a downstream signaling component of the insulin-stimulated kinase cascade involved in mediating the glycogenic response to insulin (Cohen et al., 1997
; Cross et al., 1995
; Lawrence and Roach, 1997
). Most recent work has suggested that insulin-stimulated glycogen synthesis occurs via phosphatidylinositol-3 kinase (PI3-K)-dependent activation of PKB
, which inactivates glycogen synthase kinase-3 (GSK-3), thereby reducing the level of phosphorylation of glycogen synthase and increasing glycogen synthesis (Cross et al., 1995
; Lawrence and Roach, 1997
; Park et al., 1999
).
The decrease in IR expression was still apparent at the high dose of DCA (2.0 g/l) after 52 weeks of DCA-treatment. The decrease in IR expression was not significant after 87 weeks of treatment with 0.5 g/l DCA. However, this is most likely explained by the fact that the concentration of DCA in the blood is highest at night (Kato-Weinstein et al., 1998). At the 9:00 A.M. time of sacrifice, DCA had essentially disappeared from the blood of animals treated with 0.5 g/l DCA (Kato-Weinstein et al., 1998
). The changes in IR protein are still evident at the higher dose of DCA, where the concentrations of DCA in blood are sharply increased and remain significantly elevated into the daylight hours as a result of auto-inhibition of metabolism (Gonzalez-Leon et al., 1997
; Kato-Weinstein et al., 1998
).
In contrast to normal hepatocytes of treated mice, insulin receptor protein was somewhat increased in liver tumors relative to livers from untreated animals and even increased significantly when compared to adjacent normal liver of the treated mice. As mentioned, DCA-induced tumors are uniformly glycogen-poor (Bull et al., 1990). In other studies, lesions expressing the glycogen-poor phenotype have been shown to have a significant reduction in activity of glycogen synthase and several other enzymes involved in glycogen metabolism (Bannasch et al., 1984
, 1997
). If the down-regulation of IR in normal hepatocytes is the result of increased glycogen content, then the elevated expression in DCA-induced tumors could be related to their glycogen-poor character.
Insulin is mitogenic in normal liver (Kahn and White, 1995) and even more so in malignant liver cells (Koontz and Iwahashi, 1981
; Massague et al., 1982
). The suppression of cell division in normal liver associated with extended DCA-treatment described by Stauber and Bull (1997) might be attributable to the decreases in serum insulin and IR expression in hepatocytes. In contrast, the increase in cell division in tumors induced by DCA (Stauber and Bull, 1997
) may be explained by the greater sensitivity of these tumors to the mitogenic effects of insulin as a result of the higher expression of IR relative to the surrounding tissue.
Increased binding of insulin to its receptor has been observed in hepatocarcinomas in prior studies (Kurtaran et al., 1995). Also, increased IR expression is characteristic of hepatoma cells in culture (Taouis et al., 1994
; Khamzina and Borgeat, 1998
) and insulin-induced DNA synthesis in these cells is blocked by inhibitors of insulin-stimulated signaling pathways. Finally, the data shows that MAP kinase phosphorylation is significantly higher in DCA-induced tumors than in adjacent normal liver and also higher than that from liver of control animals. MAP kinase is a downstream target of the IR and its level of activity has been positively correlated to that of insulin-induced increases in cell proliferation (Porras et al., 1998
).
In summary, the results of this study indicate that glycogen accumulation induced by DCA precedes decreases in serum insulin levels and expression of insulin-signaling proteins in normal liver. These data strongly suggest DCA-induced alterations in insulin, IR, and perhaps PKB are a result of a compensatory down-regulation of the insulin pathway triggered by high glycogen levels in the liver. The mechanisms for compensatory down-regulation of the insulin-signaling pathway as a result of DCA-induced glycogen accumulation are not clear. Such mechanisms may be important, since cells promoted by DCA to form tumors are refractory to both glycogen accumulation and decreased IR expression.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
NOTES |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bannasch, P., Hacker, H. J., Klimek, F., and Mayer, D. (1984). Hepatocellular glycogenosis and related pattern of enzymatic changes during hepatocarcinogenesis. Adv. Enzyme Regul. 22, 97121.[ISI][Medline]
Bannasch, P., Klimek, F., and Mayer, D. (1997). Early bioenergetic changes in hepatocarcinogenesis: Preneoplastic phenotypes mimic responses to insulin and thyroid hormone. J. Bioenerg. Biomembr. 29, 303313.[ISI][Medline]
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]
Cohen, P., Alessi, D. R., and Cross, D. A. (1997). PDK1, one of the missing links in insulin signal transduction? FEBS Lett. 410, 310.[ISI][Medline]
Cross, D. A., 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]
Daniel, F. B., DeAngelo, A. B., Stober, J. A., Olson, G. R., and Page, N. P. (1992). Hepatocarcinogenicity of chloral hydrate, 2-chloroacetaldehyde, and dichloroacetic acid in the male B6C3F1 mouse. Fundam. Appl. Toxicol. 19, 159168.[ISI][Medline]
DeAngelo, A. B., Daniel, F. B., Most, B. M., and Olson, G. R. (1996). The carcinogenicity of dichloroacetic acid in the male Fisher 344 rat. Toxicology 114, 207221.[ISI][Medline]
DeAngelo, A. B., Daniel, F. B., Stober, J. A., and Olson, G. R. (1991). The carcinogenicity of dichloroacetic acid in the male B6C3F1 mouse. Fundam. Appl. Toxicol. 16, 337347.[ISI][Medline]
DeMarini, D. M., Perry, E., and Shelton, M. L. (1994). Dichloroacetic acid and related compounds: Induction of prophage in E.coli and mutagenicity and mutation spectra in Salmonella TA 100. Mutagenicity 9, 429437.
Farber, E., and Sarma, D. S. R. (1987). Hepatocarcinogenesis: A dynamic cellular perspective. Lab. Invest. 56, 422.[ISI][Medline]
Fox, A. W., Yang, X., Murli, H., Lawlor, T. E., Cifone, M. A., and Reno, F. E. (1996). Absence of mutagenic effects of dichloroacetate. Fundam. Appl. Toxicol. 32, 8795.[ISI][Medline]
Fuscoe, J. C., Afshari, A. J., George, M. H., DeAngelo, A. B., Tice, R. R., Salma, T., and Allen, J. W. (1996). In vivo genotoxicity of dichloroacetic acid: Evaluation with the mouse peripheral blood micronucleus assay and the single cell gel assay. Environ. Mol. Mutagen. 27, 19.[ISI]
Giller, S., LeCurieux, F., Erb, F., and Marzin, D. (1997). Comparative genotoxicity of halogenated acetic acids found in drinking water. Mutagenesis 12, 321328.[Abstract]
Gonzalez-Leon A., Schultz, I. R., Xu, G., and Bull, R. J. (1997). Pharmacokinetics and metabolism of dichloroacetate in the F344 rat after prior administration in drinking water. Toxicol. Appl. Pharmacol. 146, 189195.[ISI][Medline]
Harrington-Brock, K., Doerr, C. L., and Moore, M. M. (1998). Mutagenicity of three disinfection by-products: Di- and trichloroacetic acid and chloral hydrate in L5178Y/TK+/-(-)3. 7.2C mouse lymphoma cells. Mutat. Res. 413, 265276.[ISI][Medline]
Kahn, C. R., and White, M. F. (1995). Molecular mechanism of insulin action. In Endocrinology (L. J. DeGroot, Ed.), pp. 25902623. Saunders, Philadelphia.
Kato-Weinstein, J., Lingohr, M. K., Thrall, B. D., and Bull, R. J. (1998). Effects of dichloroacetate on carbohydrate metabolism in B6C3F1 mice. Toxicology 130, 141154.[ISI][Medline]
Khamzina, L., and Borgeat, P. (1998). Correlation of alpha-fetoprotein expression in normal hepatocytes during development with tyrosine phosphorylation and insulin-receptor expression. Mol. Biol. Cell. 9, 10931105.
Koontz, J. W., and Iwahashi, M. (1981). Insulin as a potent, specific growth factor in a rat hepatoma cell line. Science 211, 947949.[ISI][Medline]
Kurtaran, A., Li, S. R., Raderer, M., Leimer, M., Muller, C., Pidlich, J., Neuhold, N., Hubsch, P., Angelberger, P., Scheirhauer, W., and Virgolini, I. (1995). Technetium-99m-galactosyl-neoglycoalbumin combined with iodine-123-Tyr-(A14)-insulin visualizes human hepatocellular carcinomas. J. Nucl. Med. 36, 18751881.[Abstract]
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]
Leavitt, S. A., DeAngelo, A. B., George, M. H., and Ross, J. A. (1997). Assessment of the mutagenicity of dichloroacetic acid in lacI transgenic B6C3F1 mouse liver. Cacinogenesis 18, 21012106.
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.
Massague, J., Blinderman, L. A., and Czech, M. P. (1982). The high-affinity insulin receptor mediates growth stimulation in rat hepatoma cells. J. Biol. Chem. 257, 1395813963.
Miller, J. H., Minard, K., Wind, R. A., Orner, G. A., Sasser, L. B., and Bull, R. J. (2000). In vivo MRI measurements of tumor growth induced by dichloroacetate: Implications for mode of action. Toxicology 145, 115125.[ISI][Medline]
Orner, G. A., Stillwell, L. C, Cheng, R. S., Sasser, L. B., Bull, R. J., and Thrall, B. D. (1998). Effects of trichloroacetate (TCA) and dichloroacetate (DCA) on H-ras in male B6C3F1 mice. Toxicol. Sci. 42, 12.
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]
Pereira, M.A., Li, K., and Kramer, P.M. (1997). Promotion by mixtures of dichloroacetic acid and trichloroacetic acid of N-methyl-N-nitrosourea-initiated cancer in the liver of female B6C3F1 mice. Cancer Lett. 115, 1523.[ISI][Medline]
Pereira, M.A., and Phelps, J.B. (1996). Promotion by dichloroacetic acid and trichloroacetic acid of methylnitrosourea-initiated cancer in the liver of female B6C3F1 mice. Cancer Lett. 102, 133141.[ISI][Medline]
Porras, A., Alvarez, A. M., Valladares, A., and Benito, M. (1998). p42/p44Mitogen-activated protein kinases activation is required for the insulin-like growth factor-1/insulin induced proliferation, but inhibits differentiation in rat fetal brown adipocytes. Mol. Endocrinol. 12, 825834.
Pratt, M. L., and Roche, T. E. (1979). Mechanism of pyruvate inhibition of kidney pyruvate dehydrogenase kinase and synergistic inhibition by pyruvate and ADP. J. Biol. Chem. 254, 71917196.[ISI][Medline]
Ragolia, L., and Begum, N. (1998). Protein phosphatase-1 and insulin action. Mol. Cell. Biochem. 182, 4958.[ISI][Medline]
Rapson, W. H., Nazar, M. A., and Butsky, V. V. (1980). Mutagenicity produced by aqueous chlorination of organic compounds. Bull. Environ. Contam. Toxicol. 24, 590596.[ISI][Medline]
Singer, P. C., and Chang, S. D. (1989). Correlations between trihalomethanes and total organic halides formed during water treatment. J. Am. Water Works Assoc. 81, 6165.
Snyder, R. D., Pullman, J., Carter, J. H., Carter, H. W., and DeAngelo, A. B. (1995). In vivo administration of dichloroacetic acid suppresses spontaneous apoptosis in murine hepatocytes. Cancer Res. 55, 37023705.[Abstract]
Stauber, A. J., and Bull, R. J. (1997). Differences in phenotype and cell replicative behavior of hepatic tumors induced by dichloroacetate and trichloroacetate. Toxicol. Appl. Pharmacol. 144, 235246.[ISI][Medline]
Stauber, A. J., Bull, R. J., and Thrall B. D. (1998). Dichloroacetate and trichloroacetate promote clonal expansion of anchorage-independent hepatocytes in vivo and in vitro. Toxicol. Appl. Pharmacol. 150, 287294.[ISI][Medline]
Taouis, M., Derouet, M., Caffin, J. P., and Simon, J. (1994). Increased insulin receptor number and insulin responsiveness in a chicken hepatoma cell line. J. Endocrinol. 140, 119124.[Abstract]
Tong, Z., Board, P. G., and Anders, M. W. (1998). Glutathione S-transerase zeta-catalyzed biotransformation of dichloroacetic acid and other -haloacids. Chem. Res. Toxicol. 11, 13321338.[ISI][Medline]
Waskell, L. (1978). A study of the mutagenicity of anesthetics and their metabolites. Mutat. Res. 57, 141153.[ISI][Medline]
Wilden, P. A., Backer, J. M., Kahn, C. R., Cahill, D. A., Schroeder, G. J., and White, M. F. (1990). The insulin receptor with phenylalanine replacing tyrosine-1146 provides evidence for separate signals regulating cellular metabolism and growth. Proc. Natl. Acad. Sci. U.S.A. 87, 33583362.[Abstract]
Wu, X., Fan, Z., Masui, H., Rosen, N., and Mendelsohn, J. (1995). Apoptosis induced by an anti-epidermal growth-factor receptor monoclonal antibody in a human colorectal carcinoma cell line and its delay by insulin. J. Clin. Invest. 95, 18971905.[ISI][Medline]
Yenush, L., Fernandez, R., Myers, M.G., Jr., Grammer, T. C., Sun, X. J., Blenis, J., Pierce, J. H., Schlessinger, J., and White, M. F. (1996). The Drosophila insulin receptor activates multiple signaling pathways but requires insulin-receptor substrate proteins for DNA synthesis. Mol. Cell. Biol. 16, 25092517.[Abstract]