2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD) and 1,2,3,4,7,8-Hexachlorodibenzo-p-Dioxin (HxCDD) Alter Body Weight by Decreasing Insulin-Like Growth Factor I (IGF-I) Signaling

Claire R. Croutch*, Margitta Lebofsky*, Karl-Werner Schramm{dagger}, Paul F. Terranova{ddagger} and Karl K. Rozman*,§,1

* Department of Pharmacology, Toxicology and Experimental Therapeutics; {dagger} GSF- Institute of Ecological Chemistry, Neuherberg, Germany; {ddagger} Center for Reproductive Sciences and Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS 66160 USA; § Section of Environmental Toxicology, GSF-Institut für Toxikologie, Neuherberg, Germany

1 To whom correspondence should be addressed at Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160. Fax: (913) 588-7501. E-mail: krozman{at}kumc.edu.

Received November 18, 2004; accepted January 27, 2005


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) affects glycemia due to reduced gluconeogenesis; when combined with a reduction in feed intake, this culminates in decreased body weight. We investigated the effects of steady-state levels of TCDD (loading dose rates of 0.0125, 0.05, 0.2, 0.8, and 3.2 µg/kg) or approximately isoeffective dose rates of 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (HxCDD) (loading dose rates of 0.3125, 1.25, 5, 20, and 80 µg/kg) on body weight, phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression and activity, and circulating concentrations of insulin, glucose, and insulin-like growth factor-I (IGF-I), and expression of hepatic phosphorylated AMP kinase-{alpha} (p-AMPK) protein in female Sprague-Dawley rats (~250 gm) at 2, 4, 8, 16, 32, 64, and 128 days after commencement of treatment. At the 0.05 and 1.25 µg/kg loading dose rates of TCDD and HxCDD, respectively, there was a slight increase in body weight as compared to controls, whereas at the 3.2 and 80 µg/kg loading dose rates of TCDD and HxCDD, respectively, body weight of the rats was significantly decreased. TCDD and HxCDD also inhibited PEPCK activity in a dose-dependent fashion, as demonstrated by reductions in PEPCK mRNA and protein. Serum IGF-I levels of rats treated initially with 3.2 µg/kg TCDD or 80 µg/kg HxCDD started to decline at day 4 and decreased to about 40% of levels seen in controls after day 16, remaining low for the duration of the study. Eight days after initial dosing, hepatic p-AMPK protein was increased in a dose-dependent manner with higher doses of TCDD and HxCDD. There was no effect with any dose of TCDD or HxCDD on circulating insulin or glucose levels. In conclusion, doses of TCDD or HxCDD that began to inhibit body weight in female rats also started to inhibit PEPCK, inhibited IGF-I, while at the same time inducing p-AMPK.

Key Words: TCDD; HxCDD; insulin-like growth factor I; IGF-I.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) belongs to the aromatic hydrocarbon family which includes polycyclic dibenzo-p-dioxins (PCDDs), polychloro-dibenzofurans (PCDFs), and polychlorinated biphenyls (PCBs). The PCDD class contains 75 congeners (Ryan et al., 1991Go). The most toxic of the PCDD congeners is TCDD. Congeners with chlorines in addition to those at the 2, 3, 7, and 8 positions are less toxic than TCDD, unless they are administered at higher doses, in which case they display the same spectrum of effects (Couture et al., 1990Go; McConnell et al., 1978Go). PCDDs with three or fewer chlorine substituents are rapidly metabolized and therefore are biologically much less potent (Goldstein, 1980Go).

PCDDs are ubiquitously found in the environment (Podoll et al., 1986Go) at low background levels (ppt or ppq) in air, water, and soil (Pohl, 2000Go). TCDD in the environment usually occurs together with other congeners of PCDDs and PCDFs. The mixtures are highly persistent in animals as well as in soil. The half-life of TCDD in animals ranges from weeks to years; its half-life on the soil surface is estimated to be 9–15 years, while the half-life in subsurface soil may range from 25 to 100 years (Paustenbach et al., 1992Go).

Insulin-like growth factors (IGFs) are members of the family of insulin-related peptides that include relaxin and several peptides isolated from lower invertebrates (Blundell and Humbel, 1980Go). IGF-I is a premier progression and survival growth factor found in serum and all major tissues in mammals (Bolander, 2004Go; Iatropoulos, 1994Go; Iatropoulos and Williams 1996Go, 2004Go). IGF-I mRNA has been detected in liver, kidney, spleen, thymus, heart, brain, skeletal muscle, testes, and epididymal (white) adipose tissue (WAT) from male rats (Gosteli-Peter et al., 1994Go). IGF-I production is induced by growth hormone (GH) in the liver; GH also regulates the paracrine production of IGF-I in many other tissues. However, IGF-I gene expression is also modified by other hormonal, tissue-specific, and developmental factors (Daughaday et al., 1989).

There are no reports of TCDD or related congeners affecting circulating IGF-I, and reports are mixed regarding effects on circulating GH. Circulating GH levels are difficult to evaluate, particularly in male rats because of its pulsatile release. Probably as a result of this variability, there has been little focus on GH in TCDD-treated rats. Gorski et al. (1988a) reported that 4 days after the administration of a nonlethal dose of TCDD to male Sprague Dawley rats there was a steep, but insignificant, increase in serum GH. GH levels decreased to levels below those of both pair-fed and ad libitum-fed controls by day 8 and remained there throughout the remainder of the 32-day study. Moore et al. (1989)Go also evaluated plasma GH in male rats 7 days after treatment with TCDD and found GH levels to be higher than in pair-fed control rats after the lowest dose; at higher doses GH concentrations were lower as compared to pair-fed controls (Moore et al., 1989Go). This group also found that these differences in GH were not statistically significant due to variability in the results. However, both studies show the same trend, indicating that TCDD administration probably has an impact on circulating GH levels. Thus, if GH was reduced following TCDD administration, then this would lead to decreased circulating IGF-I serum levels.

AMP-activated protein kinase (AMPK) has been called the "metabolic master switch" of the cell monitoring cellular energy charge (Hardie and Carling, 1997Go; Winder and Hardie, 1999)Go. AMPK is activated by phosporylation when the AMP:ATP ratio is elevated (Hardie, 1999Go). Phosphorylated AMP kinase {alpha} (p-AMPK) is believed to be the "fuel gauge" of the cell. AMPK is activated not only by treatments which deplete cellular ATP levels such as heat shock or arsenite in hepatocytes (Corton et al., 1994Go), exercise in skeletal muscle (Winder and Hardie, 1996Go), ischemia in heart (Kudo et al., 1995Go), and glucose deprivation in pancreatic ß-cell lines (Salt et al., 1998Go), but also by treatment with the nucleoside 5-aminoimidazole-4-carboxamide riboside (AICAR) (Corton et al., 1995Go; Henin et al., 1995Go). In tissues such as liver, adipose, and muscle, AMPK phosphorylates key enzymes such as acetyl-CoA carboxylase, 3-hydroxy-3-methylglutaryl-CoA reductase, glycogen synthase, and creatine kinase (Hardie and Carling, 1997Go; Kemp et al., 1999Go; Ponticos et al., 1998Go), which control the synthesis of fatty acids, cholesterol, glycogen, and phosphocreatine, respectively. AMPK also phosphorylates adipose tissue hormone-sensitive lipase (Garton et al., 1989Go) and phosphorylates and activates the endothelial isoform of nitric oxide synthase (Chen et al., 1999Go).

Hawley et al. (2002)Go reported that the antidiabetic drug metformin activated the AMPK cascade via an adenine nucleotide-independent mechanism. Similarities between metformin (Fulgencio et al., 2001Go; Minassian et al., 1998Go) and TCDD (Viluksela et al., 1999bGo; Weber et al., 1995Go) include inhibition of phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase), resulting in a reduced gluconeogenesis leading to a lowering of blood glucose, reduction in plasma insulin and free IGF-I levels, in addition to activation of AMPK. The evaluation of p-AMPK expression was also included in this study, because PEPCK gene expression has been shown to be repressed following activation of AMPK by AICAR in a similar manner to repression of PEPCK by insulin (Hubert et al., 2000Go; Lochhead et al., 2000Go). Lochhead et al. (2000)Go found that AMPK did not link the insulin receptor to the PEPCK and glucose-6-phosphatase (G6Pase) gene promoters; instead, they proposed that AMPK and insulin more likely lie on distinct pathways that converge at a point upstream of these two aforementioned gene promoters (Lochhead et al., 2000Go). Thus activation of AMPK could inhibit hepatic gluconeogenesis in an insulin-independent manner and help to reverse the hyperglycemia associated with type 2 diabetes (Lochhead et al., 2000Go).

At higher doses, TCDD induces a wasting syndrome in rats with associated changes in energy metabolism including effects on gluconeogenesis, de novo fatty acid synthesis, serum free fatty acids and triglycerides, as well as glycogen and fat storage in liver and adipose tissue, respectively (Gorski et al., 1988bGo, 1990Go; Muzi et al., 1989Go; Tuomisto et al., 1999Go; Weber et al., 1991aGo,bGo). It was of interest to determine if metabolic effects also occurred at low doses of TCDD or iso-equivalent doses of 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (HxCDD) under conditions of kinetic steady state. Therefore, the effect of TCDD and HxCDD was investigated on body weight, serum insulin, serum IGF-I, serum glucose, and hepatic PEPCK and p-AMPK.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Test chemicals.
TCDD (CAS 1746–01–6; MW 321.9; purity >99%) and 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (HxCDD; CAS 39227–28–6; MW 390.9; purity >98.5%) were obtained from Cambridge Isotope Laboratories Inc. (Woburn, MA). TCDD or HxCDD were dissolved in corn oil (Sigma, St. Louis, MO).

Animals.
A total of 375 female Sprague-Dawley rats (~250 gm) were obtained from Harlan (Indianapolis, IN) and adapted to stainless steel wire bottom cages and a light/dark cycle (6:00 to 18:00 h lights on) for at least 2 weeks prior to treatment. The animal room was maintained at an ambient temperature of 21–22°C and a relative humidity of 40–60%. Rats had free access to tap water and 8604 Rodent Diet (Harlan Teklad, Madison, WI).

Dosing regimen.
The half-life of TCDD in female Sprague-Dawley rats is approximately 20 days (Geyer et al., 2002Go; Li et al., 1995bGo), whereas that of HxCDD is about 60 days (Viluksela et al., 1998Go). Loading dose rate and maintenance dose rates were calculated according to Gibaldi and Perrier (1982), and similar dosing regimens have been used previously in our laboratory (Rozman et al., in press; Viluksela et al., 1997Go, 1998Go):

where is the loading dose rate, x0 is the maintenance dose rate, k is the elimination rate constant, and {tau} is the time interval between maintenance dose rates.

Experimental design.
Rats were randomly allocated into experimental groups of five. Body weights were recorded every 3 days in the morning. Five iso-effective loading dose rates of TCDD/HxCDD in corn oil via oral gavage (0.0125/0.3125, 0.05/1.25, 0.2/5, 0.8/20, or 3.2/80 µg/kg, respectively) were given to the animals; doses (loading dose rate plus the sum of maintenance dose rates) were selected based on their toxic equivalence. Controls were dosed with vehicle alone (4 ml/kg). Maintenance dose rates were one tenth of the loading dose rate. Animals were administered maintenance dose rates of TCDD or HxCDD every third or ninth day, respectively, to maintain pharmacokinetic steady state throughout the entire study (Viluksela et al., 1997Go). HxCDD animals were dosed with corn oil alone on every third and sixth day to provide them with an equal number of vehicle dose rates as those administered maintenance dose rates of TCDD every third day.

After 2, 4, 8, 16, 32, 64, or 128 days of initial dosing, animals were sacrificed via decapitation, trunk blood was collected, and livers were removed, weighed, and snap frozen in liquid nitrogen. Aliquots of liver tissue were stored at –80°C until subsequent biochemical analysis. Blood was centrifuged, and serum was aliquoted and frozen at –80°C for future analyses. Day 2 serum samples from HxCDD-treated rats were not collected, and therefore IGF-I, insulin, and glucose values are not reported for this time point.

Analysis of TCDD in liver.
About 0.2 gm of liver from rats treated with 0.05 µg/kg TCDD loading dose rate were lyophilized and analyzed for TCDD by the GSF-Institute of Ecological Chemistry as previously described (Rozman et al., 1995Go).

Glucose assay.
Serum samples were assayed for glucose content using a glucose hexokinase assay from Sigma (St. Louis, MO).

IGF-I and insulin radioimmunoassays (RIA).
Rat serum samples were assayed for IGF-I with kits purchased from Diagnostic Systems Laboratories (Webster, TX). Serum samples were extracted with an ethanolic hydrochloric acid solution prior to assaying by IGF-I RIA in order to remove insulin-like growth factor-binding proteins (IGFBPs). Serum samples were also assayed for insulin using a commercially available rat RIA kit purchased from LINCO Research Inc (St. Louis, MO).

Development of rat PEPCK oligonucleotide probe sets for branched DNA (bDNA) analysis.
The rat PEPCK gene sequences were accessed from GenBank (GenBank No.NM198780, Target 1828–2172). The target sequences were then analyzed by Probe-Designer Software Version 1.0 (Bayer Diagnostics, East Walpole, MA). The oligonucleotide probes designed were specific to a single mRNA transcript (Table 1). All oligonucleotide probes were designed with a Tm of approximately 63°C, enabling hybridization conditions to be held constant (i.e., 53°C) during each hybridization step and for the oligonucleotide probe set. The probe developed in ProbeDesigner was submitted to the National Center for Biotechnological Information for nucleotide comparison by the basic logarithmic alignment search tool (BLASTn; http://www.ncbi.nlm.nih.gov/BLAST/), to ensure minimal cross-reactivity with other known rat sequences and expressed sequence tags.


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TABLE 1 Oligonucleotide Probes Generated for Analysis of PEPCK Expression by bDNA Signal Amplification

 
PEPCK mRNA analysis using branched DNA (bDNA) assay.
Total RNA was isolated from liver samples using TRIzol® Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The specific oligonucleotide probe set for PEPCK was diluted in 10 mM Tris–1 mM EDTA (TE) buffer according to manufacturer's instructions provided by QuantigeneTM High Volume Kit (Bayer Diagnostics, East Walpole, MA). Total RNA (1 µg/ml; 10 µl) was added to each well of a 96-well plate which contained 50 µl of capture hybridization buffer and 50 µl of each diluted probe set. Hybridization of the PEPCK probe set to the liver RNA took place overnight at 53°C. The following day the plate was processed according to protocol supplied by the manufacturer. Luminescence was measured with a QuantiplexTM 320 bDNA luminometer interfaced with a QuantiplexTM Data Management Software Version 5.02 for analysis of results.

Western blot.
Protein was isolated from liver samples, fractionated on SDS–polyacrylamide gels, and transferred to a PVDF membrane. After blocking with 5% nonfat dry milk in Tris-buffered saline Tween-20 (TBST), blots were incubated with a polyclonal goat anti-rat PEPCK antibody (kindly provided by Dr. D. Granner, Vanderbilt University School of Medicine) or rabbit polyclonal antibody against p-AMPK (Cell Signaling Technology, Beverly, MA) at 1:1000 in 1% nonfat dry milk in TBST. Following washing, PEPCK blots were incubated with anti-goat-HRP conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:1000 in 1% nonfat dry milk in TBST; p-AMPK blots were incubated with goat anti-rabbit-HRP conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:1000 in 1% nonfat dry milk in TBST. Bands were visualized by enhanced chemiluminescence (Santa Cruz Biotechnology, Santa Cruz, CA). Western blots were then stripped and reprobed with a primary antibody against ß-actin (Santa Cruz Biotechnology, Santa Cruz, CA). Blots were quantitated using Gel Pro Software Image Analysis.

PEPCK (E.C 4.1.1.32) activity.
PEPCK activity was measured according to Wimmer, using a bioluminescent method previously described by our laboratory (Viluksela et al., 1995Go; Wimmer, 1988Go).

Statistical analysis.
Data on Figures 1–4GoGoGo are presented as mean ± SEM. Error bars on graphs represent the standard error of the mean. Results for bDNA were analyzed by two-tailed Student's t-test. For body weight, PEPCK activity, glucose, insulin, and IGF-I assays, comparisons between all groups were performed by two-way ANOVA. Subsequent to analysis of variance, the significant differences between experimental groups were determined by Student-Newman-Keuls multiple comparisons. A p value of ≤0.05 was considered statistically significant.



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FIG. 1. Effect of TCDD and HxCDD on body weight. (A) Effects of 0, 0.0125, 0.05, 0.2, 0.8, and 3.2 µg/kg loading dose rates of TCDD on body weight (n = 5). Body weight was measured every 3 days for ~20 weeks. Data points on this figure are an average of body weights for the week; a indicates statistically significant difference versus corn oil treated rats at p ≤ 0.05. (B) Effects of 0, 0.05, and 3.2 µg/kg loading dose rates of TCDD on body weight (n = 5). Body weight was measured every 3 days for ~20 weeks. Data points on this figure are an average of body weights for the week. (C) Effects of 0, 0.3125, 1.25, 5, 20, and 80 µg/kg loading dose rates of HxCDD on body weight (n = 5). Body weight was measured every 3 days for ~20 weeks. Data points on this figure are an average of body weights for the week; b indicates statistically significant difference of 0.3125 versus 80 µg/kg loading dose rates at p ≤ 0.05; c indicates statistically significant difference of 5 versus 80 µg/kg loading dose rates at p ≤ 0.05. (D) Effects of 0, 1.25, and 80 µg/kg loading dose rates of HxCDD on body weight (n = 5). Body weight was measured every 3 days for ~20 weeks. Data points on this figure are an average of body weights for the week.

 


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FIG. 2. Hepatic PEPCK activity and mRNA in TCDD- and HxCDD-treated rats. (A) Effects of 0, 0.0125, 0.05, 0.2, 0.8, and 3.2 µg/kg loading dose rates of TCDD (n = 5) on PEPCK activity on days 2–128 after initiation of dosing; a indicates statistically significant difference as compared to 0.2 µg/kg loading dose rate (p ≤ 0.05); b indicates statistically significant difference as compared to corn oil (p ≤ 0.05). (B) Effects of 0, 0.0125, 0.05, 0.2, 0.8, and 3.2 µg/kg loading dose rates of TCDD (n = 5) on PEPCK mRNA on days 2–128 after initiation of dosing. (C) Effects of 0, 0.3125, 1.25, 5, 20, and 80 µg/kg loading dose rates of HxCDD (n = 5) on PEPCK activity on days 2–128 after initiation of dosing. (D) Effects of 0, 0.3125, 1.25, 5, 20, and 80 µg/kg loading dose rates of HxCDD (n = 5) on PEPCK mRNA on days 2–128 after initiation of dosing; * indicates statistically significant difference versus rats administered a loading dose rate of 5 µg/kg HxCDD at p ≤ 0.05.

 


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FIG. 3. Serum IGF-I concentrations in TCDD- and HxCDD-treated rats. (A) Effects of various doses of TCDD on levels of circulating IGF-I on days 2–128 after treatment (n = 5); a indicates statistically significant difference as compared to corn oil, 0.0125, 0.05, 0.2, or 0.8 µg/kg TCDD loading dose rate at p ≤ 0.01; b indicates statistically significant difference as compared to 0.0125, 0.05, 0.2, or 0.8 µg/kg TCDD loading dose rate at p ≤ 0.01; c indicates statistically significant difference as compared to corn oil, 0.0125, or 0.05 µg/kg TCDD loading dose rate at p ≤ 0.01. (B) Effects of various doses of TCDD on levels of circulating IGF-I on days 2–128 after treatment (n = 5). (C) Effects of various doses of HxCDD on levels of circulating IGF-I on days 2–128 after treatment (n = 5); a indicates statistically significant difference as compared to corn oil alone at p ≤ 0.05; b indicates statistically significant difference as compared to 0.3125, 1.25, or 5 µg/kg HxCDD loading dose rate at p ≤ 0.01; c indicates statistically significant difference as compared to corn oil, 1.25, or 20 µg/kg HxCDD loading dose rate at p ≤ 0.01; d indicates statistically significant difference as compared to 0.3125, 1.25, 5, or 20 µg/kg HxCDD loading dose rate at p ≤ 0.01; e indicates statistically significant difference as compared to corn oil, 0.3125, 1.25, 5, or 20 µg/kg HxCDD loading dose rate at p ≤ 0.01. (D) Effects of various doses of HxCDD on levels of circulating IGF-I on days 2–128 after treatment (n = 5).

 


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FIG. 4. Serum insulin and glucose concentrations in TCDD- and HxCDD-treated rats. (A) Effects of 0, 0.0125, 0.05, 0.2, 0.8, and 3.2 µg/kg loading dose rates of TCDD (n = 5) on circulating insulin on days 2–128 after initiation of dosing. (B) Effects of 0, 0.3125, 1.25, 5, 20, and 80 µg/kg loading dose rates of HxCDD (n = 5) on circulating insulin on days 2–128 after initiation of dosing. (C) Effects of 0, 0.0125, 0.05, 0.2, 0.8, and 3.2 µg/kg loading dose rates of TCDD (n = 5) on circulating glucose on days 2–128 after initiation of dosing. (D) Effects of 0, 0.3125, 1.25, 5, 20, and 80 µg/kg loading dose rates of HxCDD (n = 5) on circulating glucose on days 2–128 after initiation of dosing.

 

    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Analysis of TCDD in Liver
Livers from rats treated with 0.05 µg/kg TCDD loading dose rate were analyzed on days 2, 8, and 32 for TCDD concentration by GC-MS. Table 2 shows the concentration (pg/gm dry weight) of TCDD in each group of five samples. The concentration of TCDD in liver samples from day 8 were slightly lower than those of day 2 and day 32. However, these animals received their last maintenance dose rate two days earlier, in contrast to the day 2 and 32 animals that had received the loading dose rate or the last maintenance dose rate only one day prior to collecting the liver samples. Finally, it is important to note that, in agreement with expectations, the hepatic concentration of TCDD remained quite constant between day 8 and 32, even though 8 maintenance dose rates had been administered during this time period (Table 2).


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TABLE 2 Analytical Results of Liver Concentrations in Rats Treated with TCDD

 
Effects of TCDD or HxCDD on Body Weight under Conditions of Kinetic Steady State
All animals were weighed every 3 days throughout the 128-day study. The effect of TCDD or HxCDD administration on body weight of rats is shown in Figures 1A/1B and 1C/1D, respectively. Body weight was decreased after the second week of the 3.2 µg/kg TCDD loading dose rate; two-way ANOVA indicated an overall effect of TCDD on body weight, and Student-Newman-Keuls showed a significant difference beginning at week 9 after the administration of the 3.2 µg/kg TCDD loading dose rate as compared to controls. There was also a statistically significant difference (p ≤ 0.05) in body weight at weeks 10, 11, 13, 18, and 19 in the 3.2 µg/kg TCDD loading dose rate group.

The pattern was similar for the 80 µg/kg HxCDD dosage group, although the decrease in body weight was statistically not significant at any of the time points as compared to controls. However, body weight was decreased after the second week of the 80 µg/kg HxCDD loading dose rate as compared to the 0.3125 µg/kg HxCDD loading dose rate at weeks 7, 8, and 9; Student-Newman-Keuls also indicated a significant difference between the 5 and 80 µg/kg HxCDD loading dose rates at weeks 14, 15, and 18. There was an apparent trend in the low-dose animals, especially in the 0.05 µg/kg TCDD dosage group, showing a slight but consistent increase in body weight as compared to controls. There was a less pronounced but also persistent trend toward higher body weights than controls in other dosage groups.

Effects of TCDD or HxCDD on PEPCK Activity, PEPCK mRNA, and Protein in Liver
Hepatic PEPCK activity appeared to be inhibited dose-dependently after TCDD or HxCDD administration beginning at approximately day 16 (Figs. 2A and 2C, respectively). There was a statistically significant (p ≤ 0.05) inhibition of PEPCK activity on day 4 in rats administered a loading dose rate of 0.8 or 3.2 µg/kg TCDD as compared to those given a loading dose rate of 0.2 µg/kg TCDD. On day 16, there was a statistically significant (p ≤ 0.05) inhibition of PEPCK activity in rats administered a loading dose rate of 3.2 µg/kg TCDD as compared to those given a loading dose rate of 0.2 µg/kg TCDD or corn oil. However, inhibition of PEPCK activity at other doses or time points for either TCDD or HxCDD was not statistically significant (Fig. 2).

In TCDD-treated rats, there was a dose-dependent decrease in PEPCK mRNA expression (Fig. 2B). Similarly, HxCDD-treated rats also showed a dose-dependent decrease in PEPCK mRNA expression, especially at the 32-day time point and at 64 and 128 days of treatment (Fig. 2D). At day 64, the decrease in PEPCK mRNA expressed in rats treated with loading and maintenance dose rates of HxCDD at 20 µg/kg and at 80 µg/kg was statistically significant as compared to the level in rats treated with 5 µg/kg HxCDD.

PEPCK protein (determined by Western blot) was dose-dependently inhibited by both TCDD and HxCDD administration (data not shown). At the highest loading dose rate of TCDD and HxCDD administered (3.2 or 80 µg/kg, respectively) there was a decrease in the amount of hepatic PEPCK protein which paralleled that seen for its mRNA and its activity.

Effects of TCDD or HxCDD on Circulating Levels of IGF-I
Figures 3A and 3B show that, in rats initially treated with 3.2 µg/kg TCDD, there was a sharp decline in their circulating IGF-I levels by day 8 as compared to corn oil- and lower-dose TCDD-treated groups. In 3.2 µg/kg TCDD-treated rats, this decrease in IGF-I continued to decline to 42% of controls by day 16 of the study. The decrease in IGF-I remained at this level (about 350 ng/ml) through day 128, a 66% decrease, on average, as compared to IGF-I levels in controls. Beginning on day 8 and at every time point throughout the remainder of the 128-day study, the decrease in IGF-I was statistically significant when compared to controls and also when compared to 0.0125 or 0.05 µg/kg TCDD-treated groups. As shown in Figures 3A and 3B, serum IGF-I concentrations of the 0.05 µg/kg TCDD-treated rats remained either slightly higher than or approximately the same as levels in controls.

In the 80 µg/kg HxCDD-treated rats, serum IGF-I levels were also significantly lower than in controls by day 16 (Figs. 3C and 3D). IGF-I levels in the 80 µg/kg HxCDD-treated rats remained at a concentration of about 470 ng/ml throughout the remainder of the 128-day study. The concentration of IGF-I in the 80 µg/kg HxCDD loading dose rate group initially decreased by 37% on day 16 and further declined to about 45% below control levels thereafter. On day 16, there was a statistically significant difference in IGF-1 levels in the 80 µg/kg HxCDD loading dose rate group as compared to the 0.3125, 1.25, and 5 µg/kg HxCDD loading dose rate groups. The 80 µg/kg HxCDD loading dose rate group was statistically different from the controls and from the 1.25 and 20 µg/kg HxCDD loading dose rate groups also on day 32. On days 64 and 128, the decrease in IGF-I was statistically significant when compared to controls and also when compared to all HxCDD- treated groups. Finally, although only statistically significant at day 4, HxCDD loading dose rates of ≤5 µg/kg consistently had IGF-I serum concentrations slightly higher than the concentration found in controls.

Effects of TCDD or HxCDD on Insulin and Glucose Concentrations
Circulating concentrations of insulin and glucose were unaffected by either TCDD or HxCDD at any of the doses administered (Fig. 4).

Active AMPK Protein Increases Following the Administration of TCDD or HxCDD
Western blot analysis clearly demonstrates (Fig. 5) that the active or phosphorylated form of AMPK-{alpha} protein increased in a dose-dependent manner with both TCDD and HxCDD administration. Blots were stripped and reprobed with antibody against ß-actin to show equal loading of protein in each lane.



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FIG. 5. Hepatic phospho-AMPK{alpha} after TCDD or HxCDD administration to rats; effects of TCDD and HxCDD on hepatic p-AMPK protein levels evaluated by Western blot at the 8-day time point. Bars on graphs are a single representation of each sample. Blots were stripped and reprobed with antibody against ß-actin to assess accuracy of loading of protein in each lane of the gels.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
As indicated by Table 2, the dosing regimen of TCDD with a loading dose rate of 0.05 µg/kg followed every third day by maintenance dose rates of 0.005 µg/kg resulted in a reasonably good steady-state concentration for 32 days, which represents about 2 half-lives of this compound. This implies that most likely all other dosing regimens yielded similarly good steady-state concentrations for the entire duration of each study.

The results of this study demonstrate that administration of low doses of TCDD or HxCDD to female rats resulted in metabolic changes which affected body weight. By day 8 after initial treatment with TCDD, IGF-I serum levels were significantly reduced (Figs. 3A and 3B), with a trend to reduction in HxCDD-treated rats as well (Figs. 3C and 3D). By day 21 into TCDD treatment and day 28 into HxCDD treatment, body weight started to decrease; however prior to that, on day 8 into treatment, hepatic p-AMPK protein was dose-dependently increased. Throughout the entire study, there was no evidence of changes in circulating insulin or glucose levels after any of the treatments, although both TCDD and HxCDD have been shown to cause hypoinsulinemia as well as a paradoxical hypoglycemia at higher doses (Gorski et al., 1988aGo; Viluksela et al., 1995Go). Thus the herein-described body-weight effect concomitant with effects on PEPCK, IGF-I, and p-AMPK represent a group of dose responses which occur at lower doses than the actual body weight loss which occurs at higher doses and is due to toxicity (Stahl et al., 1992). The less pronounced body weight effect in HxCDD-treated rats is also reflected in a less pronounced effect on PEPCK, IGF-I, and p-AMPK. This is the result of a slight (by a factor of 2) overestimation of the relative potency of HxCDD in comparison to TCDD.

Inhibition of gluconeogenesis by TCDD (Gorski et al., 1988a) as a result of a dose-dependent decrease in the activity of PEPCK has been demonstrated previously (Cimbala et al., 1981Go; Lamers et al., 1982Go; Loose et al., 1985Go; Stahl et al., 1993Go; Viluksela et al., 1998Go, 1999aGo). Initial dose-response studies suggested that this was a high-dose effect (Stahl et al., 1993), although clearly nonlethal doses had already a significant inhibitory effect (Viluksela et al., 1995Go). Figures 2B and 2D show by a more sensitive method that PEPCK mRNA was decreased at every time point after the 3.2 and 80 µg/kg loading dose rates of TCDD and HxCDD, respectively, albeit only at one time point significantly. This suggests that inhibition of PEPCK begins well below doses causing acute toxicity.

Prior to this study, circulating IGF-I levels in TCDD- or HxCDD-treated rats (Fig. 3) had not been evaluated. However there was some in vitro evidence that TCDD could affect IGF-I signaling pathways. Firstly, although variable, the trend in serum GH was toward a decrease following the administration of TCDD, which would lead to a decrease in circulating IGF-I (Gorski et al., 1988aGo; Moore et al., 1989Go). Secondly, there were some prior in vitro reports showing that TCDD affected IGF-I. In the human mammary epithelial cell line, MCF-10A, under insulin-deficient conditions for six days, both benzo[a]pyrene and TCDD activated IGF-I signaling pathways (Tannheimer et al., 1998Go). In MCF-7 human breast cancer cells, TCDD and IGF-I coadministration for 72 h resulted in a significant decrease in mitogen-induced cell proliferation and 3H-thymidine uptake as compared to treatment of the cells with IGF-I alone (Liu et al., 1992Go). Although TCDD did not change the mRNA levels of the IGF-I receptor or the Kd values for binding of 125I-IGF-I to the IGF-I receptor, there was a significant decrease in the number of IGF-I-induced IGF-I receptor binding sites after TCDD treatment (Liu et al., 1992Go).

There are many IGF-I actions which have been demonstrated both in vitro and in vivo. IGF-I stimulates a mitogenic response in fibroblasts, chrondrocytes, osteoblasts, keratinocytes, thyroid follicular cells, smooth muscle cells, skeletal muscle cells, neuronal cells, mammary epithelial cells, mesangial cells, erythroid progenitor cells, thymic epithelium, oocytes, granulosa cells, spermatogonia, Sertoli cells, and several cancer cell lines (Cohick et al., 1993Go; Giudice, 1992Go; Lowe, 1991Go; McCauley, 1992Go; Rechler, 1993Go). Additionally, IGF-I is also able to inhibit cell death via apoptosis; this action has been best characterized in hematopoietic cells (Williams et al., 1990Go). Both IGF-I and IGF-II can promote differentiation in myoblasts (Florini and Magri, 1989Go), osteoclasts (Mochizuki et al., 1992Go), chondrocytes (Geduspan and Solursh, 1993Go), neural cells (Pahlman et al., 1991Go), adipocytes, and osteoblasts (Sara and Hall, 1990Go). Infusion of IGF-I in humans results in effects comparable to those seen in rats (Jones and Clemmons, 1995Go). Some of the effects of IGF-I administration to humans include an increase in glucose uptake, a decrease in hepatic glucose production, an increase in protein synthesis, an increase in body and organ weight, improved wound healing, a decrease in circulating GH, and an increase in catecholamines (Jones and Clemmons, 1995Go).

IGFs also regulate hormone secretion from many cell types. In ovarian granulosa and theca cells, IGF-I and IGF-II stimulate hormone synthesis and secretion; these effects are synergized when the IGFs are combined with follicle stimulating hormone (FSH) and estrogen (Giudice, 1992Go). Hormone secretion from Leydig and thyroid follicular cells is also stimulated by IGF-I (Lowe, 1991Go). Conversely, IGF-I directly inhibits growth hormone (GH) secretion in cultured pituitary somatotrophes (Yamasaki et al., 1991Go).

The regulation of the biologically active free fraction of IGF-I occurs by different IGF binding proteins (IGFBPs) in serum and tissues. Currently, there are seven known IGFBPs, some of which are characterized well, others not. IGFBPs act as a reservoir for IGF-I, especially when bound to the extracellular matrix, and can control IGF-I activity locally, which is critically involved in the switching of energy signals (Bolander, 2004Go). The energy supply process is stimulated in mammals mainly by hypothermia, but also by hypoxia and in primates by hypoglycemia (Himms-Hagen, 1995Go; Iatropoulos and Williams, 2004Go). Normothermia, plasma catecholamines, and erythroid component behavior (as previously mentioned) are all modulated by IGF-I (Bolander, 2004Go). TCDD administration to rats causes hypothermia (Potter et al., 1983Go) and in some distinct brain regions there are changes in catecholamines (Rozman et al., 1991Go; Unkila et al., 1995Go). While the doses of TCDD used for the aforementioned studies were higher than those used for this study, these changes, in addition to hypoglycemia induced by TCDD administration, may be due to IGF-I signaling already having been turned off.

Activation of hepatic p-AMPK by TCDD and HxCDD has not been reported previously (Fig. 5). In 2002, Hawley et al. showed that the antidiabetic drug metformin activated the AMPK cascade via an adenine nucleotide-independent mechanism (Hawley et al., 2002Go). Similarities between metformin and TCDD (as previously mentioned) include reduced gluconeogenesis via inhibition of PEPCK and activation of AMPK. It has been known for some time that AMPK controls the regulation of glucose-responsive genes in mammals as well as in yeast (Leclerc et al., 1998Go). Adiponectin (Acrp30), an adipose-secreted hormone, has been shown to activate AMPK in the liver, leading to an inhibition of PEPCK and G6Pase activity, resulting in the inhibition of gluconeogenesis (Kamon et al., 2003Go; Lochhead et al., 2000Go; Shklyaev et al., 2003Go). Thus, it is quite possible that TCDD and HxCDD also activate the AMPK cascade via an adenine-nucleotide-independent mechanism, since TCDD did not seem to affect hepatic ATP levels (Neal et al., 1979Go).

It is important to note that equivalent doses of TCDD and HxCDD, which decreased serum IGF-I and body weight in these female rats, have been reported to reduce reproduction in female rats. The ED50 for the inhibition of ovulation was determined to be between 3.0 and 10.0 µg/kg TCDD by toxic equivalents of other TCDD congeners (Gao et al., 1999Go, 2000Go; Li et al., 1995aGo). It is well known that IGF-I has a major role in regulating ovarian function by enhancing responses to gonadotropins (Lowe, 1991Go). Recently, Rozman et al. (in press) reported that an equivalent dose of 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (HpCDD; 1.0 mg/kg = 8.0 µg/kg TCDD equivalent; Viluksela et al., 1997Go) to female rats had a hormetic, life-prolonging effect which was accompanied by a sustained decrease in body weight very similar to that seen in Figure 1, suggesting that the effects on reproduction and longevity could be due to decreased circulating IGF-I levels in TCDD-, HxCDD-, HpCDD- and possibly in all planar TCDD congener-treated rats.

Arking proposed that caloric restriction–induced changes resulted in animals "shifting their energy resources from growth and reproduction to somatic repair and maintenance" (Arking, 2003Go). Arking hypothesized that the IGF-I signaling system functions as the switch for two sets of genes that direct the animal to either undergo growth and reproduction, or repair (fighting stress, such as reactive oxygen species or oxidative stress) and ultimately increased longevity when IGF-I is "off" and reproduction is reduced (Arking, 2003Go). According to Arking's theory, conditions favorable to growth and successful reproduction require the IGF-I signaling system to be "on," which at the same time represses stress-resistance and other maintenance genes, leading to successful reproduction but premature aging. Conditions unfavorable to reproduction are related to the IGF-I signaling systems being turned "off," leading to the derepression of stress-resistance genes and the repression of genes related to growth and reproduction, resulting in increased longevity at the cost of reduced reproduction. This switch system for increased growth and reproduction versus increased maintenance and longevity describes very well the effects of low doses of TCDD and congeners which tended to increase serum IGF-I levels, possibly thereby enhancing reproduction, but shortening life span, whereas higher (but still nontoxic) doses did reduce IGF-I signaling, leading to decreased reproduction (Gao et al., 1999Go, 2000Go) and prolongation of life (Rozman et al., in press). Higher doses of TCDD and congeners approaching the toxicity threshold would lead to a maximum and, hence, constant response in terms of IGF. In that higher dose range, both reduced life span and decreased reproduction would be due to overt toxicity by mechanisms other than IGF-I signaling. It is very likely that mixtures of TCDD congeners would exert their effects on IGF-I signaling in accordance with their toxic equivalents, as has been demonstrated for their effect on reproduction (Gao et al., 1999Go, 2000Go) and energy metabolism (Stahl et al., 1993Go; Viluksela et al., 1995Go, 1998Go, 1999).


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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