Effects of Dichloroacetate (DCA) on Serum Insulin Levels and Insulin-Controlled Signaling Proteins in Livers of Male B6C3F1 Mice

Melissa K. Lingohr*,{dagger}, Brian D. Thrall*,{dagger} and Richard J. Bull*,{dagger},1

* Washington State University, Pullman, Washington 99164-6510; and {dagger} Pacific Northwest National Laboratory, Richland, Washington 99352

Received July 20, 2000; accepted October 5, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DCA is hepatocarcinogenic in rodents. At carcinogenic doses, DCA causes a large accumulation of liver glycogen. Thus, we studied the effects of DCA treatment on insulin levels and expression of insulin-controlled signaling proteins in the liver. DCA treatment (0.2–2.0 g/l in drinking water for 2 weeks) reduced serum insulin levels. The decrease persisted for at least 8 weeks. In livers of mice treated with DCA for 2-, 10-, and 52-week periods, insulin receptor (IR) protein levels were significantly depressed. Additionally, protein kinase B (PKB{alpha}) expression decreased significantly with DCA treatment. In normal liver, glycogen levels were increased as early as at 1 week, and this effect preceded changes in insulin and IR and PKB{alpha}. In contrast to normal liver, IR protein was elevated in DCA-induced liver tumors relative to that in liver tissue of untreated animals and to an even greater extent when compared to adjacent normal liver in the treated animal. Mitogen-activated protein kinase (MAP kinase) phosphorylation was also increased in tumor tissue relative to normal liver tissue and tissue from untreated controls. These data suggest that normal hepatocytes down-regulate insulin-signaling proteins in response to the accumulation of liver glycogen caused by DCA. Futhermore, these results suggest that the initiated cell population, which does not accumulate glycogen and is promoted by DCA treatment, responds differently from normal hepatocytes to the insulin-like effects of this chemical. The differential sensitivity of the 2 cell populations may contribute to the tumorigenic effects of DCA in the liver.

Key Words: dichloroacetate; glycogen; insulin; signaling; insulin receptor PKB; hepatocarcinogen.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dichloroacetate (DCA) is a contaminant of chlorinated drinking water (Singer and Chang, 1989Go) and is carcinogenic in the liver of male and female B6C3F1 mice (Bull et al., 1990Go; Daniel et al., 1992Go; DeAngelo et al., 1991Go; Pereira et al., 1997Go; Pereira and Phelps, 1996Go) and male F344 rats (DeAngelo et al., 1996Go). The weight of evidence suggests DCA is not genotoxic in in vitro tests at concentrations <=1 mM or in in vivo tests at doses <3.5 g/l of drinking water (DeMarini et al., 1994Go; Fox et al., 1996Go; Fuscoe et al., 1996Go; Giller et al., 1997Go; Harrington-Brock et al., 1998Go; Leavitt et al., 1997Go; Rapson et al., 1980Go; Waskell et al., 1978). DCA was shown to promote N-methyl-N-nitrosurea-initiated liver tumors in mice (Pereira et al., 1997Go; Pereira and Phelps, 1996Go). The development of liver tumors in mice administered DCA, without prior treatment of an initiator, occurs by increasing clonal expansion of spontaneously initiated liver cell populations (Miller et al., 2000Go; Stauber et al., 1998Go). Lending support to the hypothesis that DCA-induced liver tumorigenesis results form DCA-induced increases in the rates of clonal expansion (Miller et al., 2000Go), Stauber and Bull (1997) found that DCA-treatment increases the rates of replication of cells within altered hepatic foci, while suppressing replication in normal portions of the liver. Snyder et al. (1995) demonstrated that DCA treatment suppresses the apoptotic rate of normal hepatocytes in mice. Subsequent studies have shown that DCA at concentrations as low as 20 µM promotes the growth of anchorage-independent colonies from hepatocyte suspensions (Stauber et al., 1998Go). The present study investigates factors that might account for differential effects of DCA treatment on the replication of normal hepatocytes and initiated hepatocytes and/or tumor cells.

In livers of DCA-treated mice, tumor cells are differentiated from the normal cell population by glycogen content (Stauber and Bull, 1997Go). Normal cells accumulate large amounts of glycogen, whereas altered hepatic foci and tumors induced by DCA are glycogen-poor (Kato-Weinstein et al., 1998Go). The doses of DCA required to produce liver tumorigenesis in mice coincide with those that produce glycogenosis in normal hepatocytes (Kato-Weinstein et al., 1998Go). 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, 1995Go; Koontz and Iwahashi, 1981Go; Massague et al., 1982Go) and has been shown to suppress apoptosis (Wu et al., 1995Go). Insulin induces biological responses via activation of its tyrosine kinase receptor and downstream serine/threonine kinase-signaling pathways (Kahn and White, 1995Go).

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{alpha}). 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
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and materials.
Analytical grade dichloroacetic acid (Fluka Chemical Corp., Ronkonkoma, NY) was dissolved in double distilled water to concentrations of 0.1–2.0 g/l. This solution was neutralized with 1 N NaOH to a pH 6.8–7.2, and supplied to mice as their drinking water. In all experiments, a concurrent control group was included in which mice received double distilled water, pH 6.8–7.2, as their drinking water.

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 (0.1–2.0 g/l) in their drinking water for 2–10 weeks. Water consumption was monitored and animals weighed weekly for the duration of the studies. Consistent with previous studies (Bull et al., 1990Go), 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., 1998Go).

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., 1998Go). 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{alpha}; 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{alpha} 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
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Administration of DCA in the Drinking Water Decreases Serum Insulin Levels
In the first experiment (conducted at WSU), mice were 7-weeks old at the start of treatment and were sacrificed at ages 9, 11, and 15 weeks, corresponding to 2-, 4-, and 8-week treatment periods. Blood glucose concentrations tended to decrease with age, independent of DCA treatment, which is characteristic of the developing animal (Fig. 1cGo). Note that the age-related increase in serum insulin levels (Fig. 1aGo) in control mice corresponds with the decrease in blood glucose (Fig. 1cGo). In animals administered 2.0 g/l DCA for 2, 4, and 8 weeks serum insulin levels were decreased significantly below that of age-matched controls (Fig. 1aGo). The effect of DCA-treatment on serum insulin levels was greater than 50% at 4 and 8 weeks.



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FIG. 1. Serum insulin levels were significantly decreased by DCA treatment. Serum was collected from mice administered DCA, and insulin and glucose levels were measured as described in the experimental section. In a and c, mice were treated with 2.0 g/l DCA for 1, 2, 4, or 8 weeks and sacrificed at 9:00 A.M. In b and d, mice were treated with 0.1–2.0 g/l DCA for 2 weeks and sacrificed at 3:00 A.M. The number of animals used in all treatment groups was &GE; 5. Values are mean ± SEM. The data within a and c were analyzed using 2-way analysis of variance and a Tukey test. The data within b were analyzed using 1-way analysis of variance and Students t-test; *indicates p < 0.05.

 
All subsequent experiments were conducted at Battelle, Pacific Northwest Laboratories with mice purchased from Charles River Laboratories. Additionally, short-term experiments were designed such that all mice were 16 weeks old at the time of sacrifice. This avoided the age-dependent changes in these parameters that were observed in the first experiment. Sacrifice in this experiment occurred at 3:00 A.M. Serum insulin levels were markedly decreased in animals treated 2-weeks with 2.0 g/l DCA in these experiments as well (Fig. 1bGo). However, the effect was even greater at night, and at the 2.0-g/l dose, serum insulin levels were only 20% of control values. Moreover, the dose-response curve appeared more complex, displaying a biphasic character. As the concentration in drinking water increased to 1 g/l, the depression of serum insulin was more marked than would be predicted from the dose response at lower doses. The most likely explanation of this is the rapid increase in blood concentrations of DCA that occurs when animals drink >=1 g DCA/l of drinking water as a result of suicide inhibition of the glutathione S-transferase zeta that is responsible for DCA metabolism (Kato-Weinstein, 1998; Tong et al., 1998Go). Glucose levels were not significantly affected by DCA-treatment at 3:00 A.M. (Fig. 1dGo).

In no instance were IGF-I levels significantly affected by DCA-treatment (Table 1Go). 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.


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TABLE 1 Circulating Blood Levels of IGF-I following a 2-week Administration of DCA
 
Administration of DCA in the Drinking Water Decreases Insulin Receptor Protein Levels
In a sub-group of randomly selected mice (n = 3), in which serum insulin levels were measured at 2 weeks, IR expression levels in livers were also measured. After 2 weeks of treatment, a trend towards decreased expression of IR protein was observed in livers of mice treated with DCA (Figs. 2a and 2cGo). The decrease in IR was significant at 0.5 and 2.0 g/l DCA. IR levels were also measured after 10 weeks of treatment. In the 10-week experiment, the high dose of DCA was decreased to 1.0 g/l because doses >= 2.0 g/l led to blood levels of DCA that approached the apparent Ki of pyruvate dehydrogenase kinase (Kato-Weinstein et al., 1998Go; Pratt et al., 1979). After 10 weeks of treatment, a substantial decrease in IR was observed at both 0.5 and 1 g/l DCA (Figs. 2b and 2cGo).



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FIG. 2. DCA decreases liver IR expression. Mice were administered (a) 0, 0.2, 0.5, and 2.0 g/l DCA for 2 weeks, and (b) 0, 0.5, and 1.0 g/l DCA for 10 weeks. Animals were sacrificed at 3:00 A.M. Liver homogenates were prepared and protein samples were resolved by SDS–PAGE and immunoblotted with anti-IR antibody (a and b). In c, relative levels of IR were quantified from Western blot analyses via NIH Image software. Data were analyzed using analysis of variance and Students t-test; *indicates p < 0.05. In all analyses, n >= 3.

 
Additionally, PKB{alpha} expression was measured in livers of animals treated for 10 weeks with DCA. Similar to the effect on IR expression, PKB{alpha} expression was significantly decreased in the livers of DCA-treated mice (Figs. 3a and 3bGo).



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FIG. 3. DCA decreases liver PKB{alpha} expression. Mice were administered 0, 0.5, or 1.0 g/l DCA for 10 weeks. Animals were sacrificed at 3:00 A.M. Liver homogenates were prepared and protein samples were resolved by SDS–PAGE and immunoblotted with anti-PKB{alpha} antibody (a). In b, relative levels of PKB{alpha} were quantified from Western blot analyses via NIH Image software. Data were analyzed using analysis of variance and Students t-test; *indicates p < 0.05. In all analyses, n = 3.

 
Insulin Receptor Expression in DCA-Induced Liver Tumors
IR levels were also examined in livers of mice from a previously conducted carcinogenesis study (Orner et al., 1998Go). In this experiment, DCA was administered at 0.5 or 2.0 g/l for 87 and 52 weeks, respectively. Animals on chronic DCA-treatment were from a prior study (conducted at WSU). In this case animals had been sacrificed between 9:00 and 11:00 A.M. Analysis of the livers from mice treated at the high dose of DCA revealed a decrease in IR protein expression in normal portions of the liver (approximately 30% relative to the concurrent control; p < 0.05 at 2.0 g/l; Fig. 4aGo). At 0.5 g/l, IR expression was also decreased, but the decrease was not significantly below that observed in livers from untreated animals (data not shown). The lack of significance of this change could be partially due to the fact that the sacrifice time occurred at a time when DCA concentrations in blood were reduced (Kato-Weinstein et al., 1998Go).



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FIG. 4. IR protein levels were increased in liver tumors. Mice were administered 2.0 g/l DCA for 52 weeks and animals were sacrificed at 9:00 A.M. Liver homogenates were prepared and protein samples were resolved by SDS–PAGE and immunoblotted with anti-IR antibody. In a, a representative Western blot analysis of IR in non-tumor bearing tissue (N), tumor tissue (T) from the same treated animals (DCA = 2.0 g/l) as non-tumor-bearing liver tissue, and from livers of concurrent controls (Control). In b, relative levels of IR protein were quantified from Western blot analyses via NIH Image software. Data were analyzed using analysis of variance and Students t-test; y indicates normal tissue from treated mice was significantly different from control tissue; p < 0.05; z indicates that IR expression in tumors was significantly different from adjacent normal tissue in the same animals, p < 0.05. In all analyses, n = 6.

 
In contrast to findings in normal liver, liver tumors induced by 2.0 g/l DCA in the drinking water expressed higher amounts of IR than normal liver tissue taken from the same animal (Figs. 4a and 4bGo). The same was observed for tumors induced by 0.5 g/l DCA treatment (data not shown). The amount of IR protein present in tumors was nearly 2-fold greater than that of non-tumor-bearing portions of the same liver (p < 0.05; Figs. 4a and 4bGo). The amount of IR protein in tumors also tended to be higher (35%) than that in livers of mice that hand no DCA treatment (p < 0.06; Fig. 4aGo).

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 5bGo).



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FIG. 5. p42/44MAPK phosphorylation was increased in liver tumors. Mice were administered 2.0 g/l DCA for 52 weeks and sacrificed at 9:00 A.M. Liver homogenates were prepared and protein samples were resolved by SDS–PAGE and immunoblotted with anti-phosphorylated p42/44MAPK antibody. In a, a representative Western blot analysis of phosphorylated p42/44MAPK in non-tumor bearing tissue (N), tumor tissue (T) from the same treated animals (DCA = 2.0 g/l) as non-tumor-bearing liver tissue, and from livers of concurrent controls (Control). In b, relative levels of phosphorylated p42/44MAPK were quantified from Western blot analyses via NIH Image software. Data were analyzed using analysis of variance and Students t-test; *indicates p < 0.05. In all analyses, n >= 5.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, DCA consistently suppressed levels of circulating insulin. The decrease in serum insulin levels required a minimum of a 2-week treatment with DCA. No significant decrease occurred after 1 week of DCA administration. However, hepatic glycogen levels are increased within 1 week (Kato-Weinstein et al., 1998Go). Therefore the accumulation in liver glycogen precedes the decrease of serum insulin concentrations. Previous work has shown that DCA treatment does not inhibit the release of insulin in response to glucose (Kato-Weinstein et al., 1998Go). Thus, serum insulin changes due to DCA-treatment are consistent with a feedback inhibition of insulin secretion that develops over time. Such feedback could be resulting from the accumulation of glycogen in the liver. However, no specific mechanism for such negative feedback could be identified in the literature.

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, 1995Go). 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{alpha}, a downstream signaling component of the insulin-stimulated kinase cascade involved in mediating the glycogenic response to insulin (Cohen et al., 1997Go; Cross et al., 1995Go; Lawrence and Roach, 1997Go). Most recent work has suggested that insulin-stimulated glycogen synthesis occurs via phosphatidylinositol-3 kinase (PI3-K)-dependent activation of PKB{alpha}, which inactivates glycogen synthase kinase-3 (GSK-3), thereby reducing the level of phosphorylation of glycogen synthase and increasing glycogen synthesis (Cross et al., 1995Go; Lawrence and Roach, 1997Go; Park et al., 1999Go).

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., 1998Go). 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., 1998Go). 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., 1997Go; Kato-Weinstein et al., 1998Go).

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., 1990Go). 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., 1984Go, 1997Go). 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, 1995Go) and even more so in malignant liver cells (Koontz and Iwahashi, 1981Go; Massague et al., 1982Go). 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, 1997Go) 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., 1995Go). Also, increased IR expression is characteristic of hepatoma cells in culture (Taouis et al., 1994Go; Khamzina and Borgeat, 1998Go) 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., 1998Go).

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{alpha} 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
 
We are grateful for the support of the Environmental Management Science Program, project 26748, under D.O.E. contract DE-ACO6-76RLO 1830.


    NOTES
 
1 To whom correspondence should be addressed at MoBull Consulting, 8382 Gage Blvd., Suite O, Box 511, Kennewick, WA 99336. E-mail: rjrdbull2{at}bossig.com. Back


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