Insulin Regulation in AhR-null Mice: Embryonic Cardiac Enlargement, Neonatal Macrosomia, and Altered Insulin Regulation and Response in Pregnant and Aging AhR-null Females

E. A. Thackaberry*, E. J. Bedrick{dagger}, M. B. Goens{ddagger}, L. Danielson§, A. K. Lund, D. Gabaldon, S. M. Smith* and M. K. Walker,1

* Molecular and Environmental Toxicology Center, Department of Nutritional Sciences, University of Wisconsin, 1415 Linden Drive, Madison, Wisconsin 53706; {dagger} Department of Mathematics and Statistics, MSC03 2150, 1 University of New Mexico, Albuquerque, New Mexico 87131; {ddagger} Department of Pediatrics, University of New Mexico, 2211 Lomas Blvd NE, Albuquerque, New Mexico 87131; § Medical Lab Sciences, MSC09 5250, 1 University of New Mexico, Albuquerque, New Mexico 87131; and College of Pharmacy, MSC09 5360, 1 University of New Mexico, Albuquerque, New Mexico 87131

Received July 1, 2003; accepted August 19, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The aryl hydrocarbon receptor (AhR) was originally characterized because of its high affinity binding of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin. However, studies using AhR-null mice have demonstrated the importance of this protein in normal physiology and development. Here we demonstrate that AhR-null embryos develop cardiac enlargement, and that this phenotype is dependent, at least in part, on the maternal genotype. Neonates born to AhR-null females had increased heart weights regardless of the neonatal genotype, an outcome also observed in gestational diabetes. The cardiac hypertrophy markers, beta-myosin heavy chain and atrial natriuretic factor, and the cardiac proliferative index were increased in AhR-null embryos, indicating that the cardiac enlargement is associated with myocyte hypertrophy and hyperplasia, which begin prior to birth. Importantly, two- to three-month-old pregnant and seven-month-old nonpregnant females, but not nonpregnant three-month-old AhR-null females had significantly decreased fasting plasma insulin levels and a reduced ability to respond to exogenous insulin compared to controls. Despite these alterations in insulin regulation and responsiveness, pregnant AhR females did not have abnormal glucose tolerance tests and did not develop hyperglycemia, classic characteristics of gestational diabetes. However, twenty-three percent of seven-month-old AhR-null females did have altered glucose tolerance tests, but did not show hyperglycemia or increased hemoglobin A1C concentration under normal feeding conditions. While the ultimate cause of the neonatal phenotype remains unclear, these studies establish that the AhR is required for normal insulin regulation in pregnant and older mice and for cardiac development in embryonic mice.

Key Words: insulin regulation; embryonic cardiac enlargement; neonatal macrosomia; AhR.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The aryl hydrocarbon receptor (AhR) is a cytoplasmic protein, originally characterized because of its high affinity binding for 2, 3, 7, 8, -tetrachlorodibenzo-p-dioxin (TCDD), and similar polyhalogenated aromatic hydrocarbons. Upon binding TCDD, the AhR translocates into the nucleus, dimerizes with the AhR nuclear translocator protein (ARNT), binds dioxin-responsive elements, and upregulates the expression of genes like cytochrome P4501A1 (Denison et al., 1988Go; Schmidt and Bradfield, 1996Go). AhR is required for most, if not all, of the toxic effects of TCDD (Fernandez-Salguero et al., 1996Go; Peters et al., 1999Go; Shimizu et al., 2000Go), although the mechanisms underlying TCDD-induced toxicity have not been fully elucidated.

The AhR is a member of the basic-helix-loop-helix PAS (Per-ARNT-SIM) transcription family, which also includes its dimerization partner ARNT, hypoxia-inducible factor 1-alpha (HIF1{alpha}), PER1-3, and others. Many members of this protein family, including AhR, act as environmental sensors, regulating downstream responses to environmental cues (Gu et al., 2000Go). For example, HIF1{alpha} is involved in the sensing and mediating the response to hypoxia (Wang et al., 1995Go), while the PER proteins as well as CLOCK and NPAS are involved in the control of circadian rhythmicity in response to day/night cycles (King et al., 1997Go; Reick et al., 2001Go; Zheng et al., 1999Go). Studies using AhR-null mice have identified new roles for the AhR in normal physiology and development, distinct from its role as an environmental sensor of toxicants (Abbott et al., 1999Go; Fernandez-Salguero et al., 1997Go; Lahvis et al., 2000Go).

The AhR and its dimerization partner ARNT have been shown to be expressed in the developing mouse and avian heart (Abbott and Probst, 1995Go; Abbott et al., 1995Go; Walker et al., 2000Go). Treatment of chick embryos with TCDD has been demonstrated to result in cardiac malformations, and the expression of AhR and ARNT in the chick cardiovascular system is consistent with a potential role in this toxicity (Walker and Catron, 2000Go; Walker et al., 1997Go). Indeed, TCDD has been demonstrated to be a cardiovascular teratogen in all species tested (Harris et al., 1973Go; Hassoun et al., 1984Go; Hornung et al., 1999Go; Walker and Catron, 2000Go). Interestingly, mice lacking the AhR have been demonstrated to develop cardiac hypertrophy and hypertension, suggesting that the AhR is required for cardiovascular homeostasis in the adult mouse (Lund et al., in press; Thackaberry et al., 2002Go). Given these data, we investigated the role of the AhR in cardiac development using AhR-null mice.

Here we show that AhR-null embryos develop increased heart weight, and that pregnant mice lacking the AhR develop altered insulin regulation and responsiveness as seen by decreased fasting plasma insulin levels and insulin resistance. However, hyperglycemia and altered glucose tolerance, characteristics of gestational diabetes, were not observed. These results suggest that, although the embryonic cardiac enlargement in mice born to AhR-null females was not associated with an overt diabetic condition, alterations in insulin regulation and tissue responsiveness cannot be eliminated as potential causative factors. In addition, we also report that 23% of nonpregnant seven-month-old female AhR-/- mice develop glucose intolerance, and that all AhR-null mice of this age show significantly reduced fasting plasma insulin and tend to exhibit insulin resistance. Despite these alterations in insulin regulation and responsiveness, nonpregnant seven-month-old AhR-/- mice secrete normal amounts of insulin in response to a bolus dose of glucose and do not exhibit hyperglycemia. These data demonstrate that AhR-null females develop altered insulin regulation during pregnancy or as they age, but that these effects are subtle and do not result in overt diabetes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
AhR-/- mice were obtained from Dr. Frank Gonzalez (National Cancer Institute), and maintained at the University of Wisconsin-Madison and University of New Mexico (Fernandez-Salguero et al., 1995Go). Experiments were approved by the appropriate animal care committees. Mice were maintained on a 12-h light cycle and given food and water ad libitum except where noted. Two- to three-month-old nulliparous AhR-/-, AhR+/-, and AhR+/+ (C57Bl/6N controls) females were mated with males of the appropriate genotype; the morning that a plug was observed was defined as d0.5. Embryos were collected at d14.5 or d17.5, and pups at postnatal day 1. Hearts were dissected from d14.5 and d17.5 embryos and frozen for mRNA analysis or fixed in 10% neutral buffered formalin for histological analysis (n = 4 litters per genotype) or proliferating cell nuclear antigen (PCNA) immunohistochemical staining. Neonatal body weights were measured, hearts were dissected, and heart weights were recorded. All reported weights are litter averages; n-values are listed in Table 1Go. Hearts were then frozen for mRNA analysis or fixed for histology. Histological samples were embedded in paraplast extra (Fisher Scientific, Pittsburgh, PA), sectioned at 8 µm, and stained with hematoxylin and eosin (Fisher Scientific). All experiments were carried out using AhR-null mice backcrossed to the C57Bl6N background at least seven, and as many as eleven generations.


View this table:
[in this window]
[in a new window]
 
TABLE 1 Crosses Used for Mating Experiments. Number of Litters Examined Per Cross Are Indicated
 
PCNA staining.
Embedded d14.5 hearts were sectioned at 7 um and stained using an anti-PCNA antibody (BD PharMingen, Cat # 555567). Nuclei were counter-stained with Hoescht dye, and PCNA and total nuclei were counted. Results were expressed as PCNA positive nuclei/total nuclei. Hearts from five litters per genotype were used.

mRNA analysis.
mRNA was isolated from pooled (4–6 hearts) d14.5, d17.5, or neonatal hearts using Trizol (Life Technologies, Rockville, MD). Atrial natriuretic factor (ANF), beta-myosin heavy chain (ß-MHC), myosin light chain 2V (MLC-2V), and 28S ribosomal RNA (28S), were measured by ribonuclease protection assay (RPA), using an RPA II kit from Ambion (Austin, TX). The probe for MLC-2V was supplied by Dr. Gary Lyons (University of Wisconsin-Madison). Probes for murine ANF, ß-MHC, and 28S were generated by polymerase chain reaction (PCR), as previously described (Thackaberry et al., 2002Go). Four litters per genotype were used for each transcript. Blots were quantified using a Cyclone Storage Phosphor System phosphoimager (Packard) with OptiQuant software. Values were normalized against 28S RNA.

Glucose measurements.
All mice were maintained on a 12-h dark/light cycle, with lights off at 1800 h. Mice were deprived of food for 15 h prior to blood collection (fasted), or fed ad libitum. All samples collected from timed-pregnant mice were taken at d14.5 of gestation, and correct timing of the pregnancies was confirmed at parturition. For ad libitum fed glucose measurements, blood samples were taken at 2400 h (n = 4 for both genotypes), 0400 h (n = 4 for both genotypes), 1000 h (n = 19 for AhR+/+, 11 for AhR-/-), 1500 h (n = 19 for AhR+/+, 11 for AhR-/-), and 2000 h (n = 4 for both genotypes). For nonpregnant mice, plasma glucose was measured from seven-month-old female mice following 15 h of fasting (n = 5 for AhR+/+, 8 for AhR+/-, 9 for AhR-/-) or when fed ad libitum at or around 1200 (n = 4 for all genotypes). Plasma was collected by centrifugation at 2, 750 x g for 10 min at 4°C, and frozen at -20°C until analyzed. Glucose was measured using a Vitros Systems GLU DT enzymatic assay and analyzer (Endocrine Sciences Products, Calabasas Hills, CA). The upper detection limit was 450 mg/dl, and all values that exceeded this limit were reported as 450 mg/dl.

For glucose tolerance tests, two- to three-month-old pregnant mice (n = 4 per genotype), and three- (n = 6 for all genotypes) and seven-month-old (n = 14 for AhR+/+, 13 for AhR+/-, 17 for AhR-/-) nonpregnant mice were fasted for 15 h. An initial blood sample was collected, and mice were then given 2 g/kg glucose by oral gavage. Blood samples were taken at 15, 30, 60, 90, 120, and 150 min after gavage and analyzed for glucose. For insulin tolerance tests, randomly fed two- to three-month-old pregnant females or seven-month-old nonpregnant females were given 0.75 units of porcine insulin (Sigma, St. Louis, MO) per kg of body weight in phosphate buffered saline via ip injection, and blood samples were taken at 15, 30, and 60 min, analyzed for glucose, and compared to preinjection values.

C-Peptide measurements.
Two- to three-month-old pregnant (d14.5 of gestation, four mice per genotype), nonpregnant three- month-old (n = 11 for AhR+/+, 6 for AhR+/-, 14 for AhR-/-) and nonpregnant seven-month-old (n = 12 for AhR+/+, 8 for AhR+/-, 15 for AhR-/-) female mice were fasted for 15 h, anesthetized, and blood was collected via cardiac puncture using a heparinized syringe. Correct timing of the pregnancies was confirmed by morphological analysis of the embryos at the time of sacrifice. Plasma was collected by centrifugation at 2, 750 x g for 10 min at 4°C, and blood was then frozen at -20°C until analyzed. C-Peptide was measured using a radioimmunoassay (RIA, Linco Research, St. Charles, MO). For secretory insulin response to glucose, mice were fasted for 15 h, given 2 g/kg glucose via oral gavage, and blood was collected and C-peptide measured 15 min after glucose administration.

Hemoglobin A1c quantification.
Whole blood from nonpregnant female AhR-/- and wild-type mice at 3 (n = 5 for AhR+/+, 6 for AhR-/-), 4.5 (n = 3 for both genotypes) and 6.5 (n = 6 for AhR+/+, 4 for AhR-/-) months of age was collected via tail clip. Hemoglobin A1c was isolated using a boronate affinity column (Endocrine Sciences) and quantified using a spectrophotometer at 414 nm. Results are expressed as a percent of total hemoglobin concentration.

Statistics.
For analysis of the nonpregnant glucose tolerance tests, a Bonferroni adjustment of a mean-shift outlier test, accounting for censoring at the upper detection limit of 450 mg/dl, was used to segregate the outlying "glucose intolerant" AhR-/- mice from the "normal" AhR-/- mice (Weisberg, 1985Go). All other data sets and comparisons of individual time points from the glucose tolerance tests were compared using Students t test. A Kolmogorov-Smirnov (KS-distance) test was used to confirm normal distribution of the data for all other data sets. Statistical significance was set at p < 0.05 for all comparisons.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neonatal Macrosomia and Increased Cardiac Weight Are Partially Dependent on Maternal Genotype
Initial data showed that AhR-/- neonates born to AhR-/- females exhibited significantly increased heart weight, body weight, and heart-to-body weight ratios, compared to AhR+/+ neonates born to AhR+/+ females. However, when AhR+/- mice were crossed to each other, AhR-/- neonates failed to show a difference in body, heart weights, or heart-to-body weight ratio compared to AhR+/+ or AhR+/- littermates. We then performed the following crosses: AhR+/- female x AhR-/- male, AhR-/- female x AhR+/- male and the pure and heterozygous crosses described above. Neonate heart, body, and heart/body weights were grouped by the genotype of the mother and neonate (Fig. 1Go). Neonates born to AhR-/- females, regardless of the neonatal genotype, showed an increase in heart weight (Fig. 1AGo). AhR+/- neonates born to AhR-/- females showed an increase in body weight (macrosomia, Fig. 1BGo), while AhR-/- neonates born to AhR-/- females did not have increased body weights (p = 0.09), but did have an increase in heart-to-body weight ratio (Fig. 1CGo). In contrast, neonates born to AhR+/- females, regardless of the neonatal genotype, failed to exhibit any differences in heart, body, or heart-to-body weight ratio (Fig. 1Go).



View larger version (19K):
[in this window]
[in a new window]
 
FIG. 1. Heart weights, body weights, and heart-to-body weight ratios of neonates born to AhR+/+, AhR+/-, and AhR-/- females, grouped by maternal and neonatal genotype. Genotypes indicated on the x-axis are the genotypes of the dams, while genotypes indicated by bar shading are neonatal. (A) AhR+/- and AhR-/- neonates born to AhR-/- females have increased heart weights. (B) AhR+/- neonates born to AhR-/- females have increased body weights; however, AhR-/- neonates born to AhR-/- females did not have increased body weights. (C) AhR-/- neonates, but not AhR+/- neonates born to AhR-/- females, have increased heart/body weight ratio. All statistical comparisons are made to wild-type mice born to wild-type females. Bars and associated vertical lines represent mean ± standard error. *p < 0.05; n = 12 litters for AhR+/+ born to AhR+/+, 7 for AhR+/+ born to AhR+/-, 10 for AhR+/- born to AhR+/-, 8 for AhR-/- born to AhR+/-, 8 for AhR+/- born to AhR-/-, and 21 for AhR-/- born to AhR-/-.

 
Altered Morphology of AhR-/- Embryonic and Neonatal Hearts
To further investigate the cardiac phenotype of AhR-/- mice born to AhR-/- females, we examined the morphology of embryonic and neonatal hearts. Hearts from AhR-/- neonates exhibited an increase in ventricle wall thickness and an increase in overall size of the heart compared to AhR+/+ controls (Fig. 2Go). This thickening of ventricle walls was evident as early as d14.5 in some embryos, but was not associated with overt cardiac malformations (data not shown).



View larger version (77K):
[in this window]
[in a new window]
 
FIG. 2. Representative morphology of neonatal AhR+/+ and AhR-/- hearts. (A) Neonatal heart of an AhR+/+ mouse born to an AhR+/+ female, compared to (B) heart of an AhR-/- neonate born to an AhR-/- female. Note the increased thickness of the ventricular compact layer (arrows) and ventricular septa (double-ended arrows). RV = right ventricle, LV = left ventricle, VS = ventricular septa.

 
Increased Proliferation in AhR-Null Embryonic Hearts
We next investigated the possibility that the increased ventricular wall thickness seen in embryonic AhR-null hearts may be to due to increased proliferation in these hearts. To do this, we used immunohistochemical staining of day 14.5 hearts for PCNA, a marker of S phase. AhR-null embryonic hearts had significantly higher proliferative index compared to control hearts: AhR+/+, 10.3 ± 2.5 proliferating cells per total cell number x100; AhR-/- 39.6 ± 5.0 proliferating cells per total cell number x100; n = 5 litters per genotype. The increased proliferation was most evident in the developing ventricular septa, which was the most actively proliferating area of the heart at this stage of development.

Increased Expression of Cardiac Hypertrophy Genes in the Developing AhR-/- Heart
To further characterize the cardiac phenotype of embryonic and neonatal AhR-/- mice, we measured the expression of ß-MHC, ANF, and MLC-2V transcripts in the hearts of d14.5 and d17.5 embryos, as well as in neonates (Fig. 3Go). ß-MHC was upregulated 10–30-fold at all developmental time points studied (Fig. 3AGo), while ANF was upregulated significantly on d14.5 and in neonates (Fig. 3BGo). MLC-2V transcript levels were not significantly increased in null mutant hearts at any time point examined (Fig. 3CGo).



View larger version (18K):
[in this window]
[in a new window]
 
FIG. 3. Expression of cardiac hypertrophy markers in the embryonic and neonatal AhR+/+ and AhR-/- mice. All hearts for this study were obtained from AhR+/+ females mated to AhR+/+ males and AhR-/- females mated to AhR-/- males. (A) Expression of ß-MHC, significant upregulation was seen at all developmental time points. (B) ANF expression was upregulated at d14.5 and at birth. (C) MLC-2V was not upregulated at any time point. Expression levels for each individual sample were normalized to 28S controls, and then the means were expressed as a percentage of AhR+/+ control expression. Bars and associated vertical lines represent mean ± standard error; open bars, AhR+/+; solid bars, AhR-/- *p < 0.05; n = 4 litters for all transcripts.

 
Pregnant AhR-/- Females Have Normal Glucose Tolerance Tests
We then investigated whether pregnant AhR-/- females exhibited changes consistent with gestational diabetes. To determine whether pregnant AhR-/- females could clear plasma glucose normally, we performed glucose tolerance tests on time-pregnant (d14.5) females of all three AhR genotypes. We used d14.5 of gestation for these and all future studies of pregnant female insulin regulation because this is the earliest time point that we observed altered morphology in AhR-null embryos. No differences in glucose clearance were seen among any of the three genotypes (Fig. 4Go).



View larger version (19K):
[in this window]
[in a new window]
 
FIG. 4. Glucose tolerance tests performed on pregnant AhR+/+ and AhR-/- females. Pregnant (d14.5) mice were fasted for 15 h, then given 2 g/kg glucose orally. Blood glucose was measured prior to glucose administration and at 15, 30, 60, 90, 120, and 150 min afterwards. No differences were seen among the three AhR genotypes. Symbols and associated vertical lines represent mean ± standard error. {circ} = AhR+/+; {blacktriangleup} = AhR+/-; and • = AhR-/- mice. n = 4 mice per genotype.

 
Pregnant AhR-/- Females Exhibit Insulin Resistance
Insulin tolerance tests were performed to determine if AhR-/- females developed insulin resistance. Pregnant (d14.5) AhR-/- females showed significantly reduced glucose clearance 30 min following insulin injection compared to wild-type females, indicating that these mice exhibited a reduced ability to respond to insulin (Fig. 5Go). By 60 min, wild-type females mice had largely recovered to their original plasma glucose levels, while AhR-null females appeared to only begin to significantly clear glucose, indicating that these mice also exhibit a delay in their response to insulin.



View larger version (15K):
[in this window]
[in a new window]
 
FIG. 5. Insulin response tests performed on pregnant (d14.5) AhR+/+ and AhR-/- females. Randomly fed pregnant female mice were injected with 0.75 units of insulin ip. Plasma glucose levels were measured prior to injection and at 15, 30, and 60 min afterwards. Results are expressed as percentage of preinjection plasma glucose levels. Pregnant AhR-/- females showed a significant delay in glucose clearance in response to insulin. Symbols and associated vertical lines represent mean ± standard error. {circ} = AhR+/+; • = AhR-/-. *p < 0.05. n = 4 mice per genotype.

 
Pregnant AhR-/- Females Show Decreased Fasting C-Peptide Levels
To investigate whether altered insulin regulation may be associated with the macrosomia and cardiac hypertrophy in neonates born to AhR-/- female mice, we measured C-peptide levels in pregnant AhR-/- females. C-Peptide is an indicator of plasma insulin, since it is produced in equimolar concentrations to insulin, but is significantly more stable in plasma. Pregnant (d14.5) AhR-/- females tended to exhibit a reduction in fasting plasma C-peptide (Fig. 6Go) compared to pregnant AhR+/+ controls (p < 0.08). To test the insulin secretory response to glucose in AhR-null females, fasted mice were given 2 g/kg glucose by oral gavage, and C-peptide was measured 15 min afterwards. No difference was seen between the three genotypes in their ability to secrete insulin in response to glucose administration (Fig. 6Go).



View larger version (16K):
[in this window]
[in a new window]
 
FIG. 6. Plasma C-peptide levels in pregnant (d14.5) AhR+/+ and AhR-/- females. Pregnant mice were fasted for 15 h, blood was collected and plasma C-peptide was measured by RIA. Pregnant AhR-/- mice tended to show a decrease in fasting plasma C-peptide levels (open bars). Following the 15 h fast, mice were also given 2g/kg glucose by oral gavage, and C-peptide levels were measured 15 min later (shaded bars). No difference was seen between AhR+/+ and AhR-/- mice. Bars and associated vertical lines represent mean ± standard error. {dagger}p = 0.081. n = 4 mice per genotype for fasting insulin and insulin response experiments.

 
Pregnant AhR-/- Mice Do Not Experience Hyperglycemia under Normal Feeding Conditions
To determine whether reduced fasting insulin and insulin resistance in pregnant (d14.5) AhR-/- females was associated with hyperglycemia, we measured plasma glucose levels of ad libitum fed mice at set hours during a 24-h period. Pregnant AhR-/- mice failed to show hyperglycemia (defined as plasma glucose greater than 225 mg/dl) at any time. The average daily plasma glucose levels (average of all time points throughout the 24-h period) were 163.8 ± 8.1 mg/dl for pregnant AhR+/+ females, and 159.4 ± 8.2 mg/dl for AhR-/-.

AhR-/- Females Develop Glucose Intolerance with Age
To determine whether a diabetic phenotype developed with age, we performed glucose tolerance tests on three- and seven-month-old females of all three AhR genotypes. Although the peak levels of glucose were higher on average in AhR-null females at 15 and 30 min following an oral glucose load, no statistical difference was seen in glucose clearance among the three genotypes at three months of age (Fig. 7AGo). At seven months of age, however, AhR-null mice exhibited elevated but highly variable glucose levels at 15, 30, and 60 min after glucose administration (Fig. 7BGo). Using a Bonferroni outlier test, we determined that this variability resulted from 23% (4/17) of the seven- month-old AhR-/- females exhibiting an impairment of glucose clearance. When these four AhR-null females were analyzed separately, their ability to clear glucose was significantly impaired compared to wild types, AhR+/-, and the remaining AhR-/- female mice (Fig. 7CGo).



View larger version (16K):
[in this window]
[in a new window]
 
FIG. 7. Age-related glucose tolerance in AhR+/+, AhR+/-, and AhR-/- mice. Mice were fasted for 15 h and given 2 g/kg of glucose orally. Blood samples were taken at 15, 30, 60, and 90 min post dosing. (A) No difference in glucose tolerance was seen among three-month-old AhR+/+, AhR+/-, and AhR-/- mice. n = 6 for all genotypes. (B) Glucose tolerance of all seven-month-old mice tested, showing no significant difference among the three genotypes. n = 14 for AhR+/+, 13 for AhR+/-, 17 for AhR-/-. (C) Glucose tolerances for the four glucose-intolerant AhR-null mice compared to "normal" AhR-null, heterozygous, and control mice. Symbols and vertical lines represent mean ± standard error. *p < 0.05. {circ} = AhR+/+; {blacktriangledown} = AhR+/-; • = All AhR-/- (in A & B); • = "normal" AhR-/- (in C); {triangledown} = "glucose intolerant" AhR-/-.

 
AhR+/- and AhR-/- Females Exhibit Reduced Circulating C-Peptide with Age
To determine if insulin regulation was altered with age, we next investigated C-peptide levels in three- and seven-month-old females of all three AhR genotypes. No differences were seen in plasma C-peptide levels among three-month-old AhR+/+, AhR+/-, and AhR-/- mice (Fig. 8AGo); however, seven-month-old AhR+/- and AhR-/- females showed significantly lower fasting plasma C-peptide levels compared to AhR wild-type females (Fig. 8BGo). The results for both AhR+/- and AhR-/- females were evenly distributed, with no significant outliers representing responders versus nonresponders. In addition, all AhR-null mice were able to secrete insulin normally 15 min following a bolus dose of exogenous glucose (data not shown).



View larger version (15K):
[in this window]
[in a new window]
 
FIG. 8. Fasting plasma C-peptide levels in three- and seven-month-old AhR+/+, AhR+/-, and AhR-/- mice. Mice were fasted for 15 h, blood was collected, and plasma C-peptide was measured by RIA. (A) AhR-null and heterozygous mice had normal fasting C-peptide levels at three months of age. n = 11 for AhR+/+, 6 for AhR+/-, and 14 for AhR-/-. (B) Seven-month-old AhR-/- females had significantly decreased plasma C-peptide levels. Bars and vertical lines represent mean ± standard error. * p < 0.05. n = 12 for AhR+/+, 8 for AhR+/-, 15 for AhR-/-.

 
AhR-/- Females Exhibit a Delayed Insulin Response
Insulin tolerance tests were performed to determine if seven-month-old AhR-/- females developed insulin resistance in association with glucose intolerance. Mice were injected with 0.75 U/kg porcine insulin, and plasma glucose clearance was measured. AhR-/- females showed significantly reduced glucose clearance in response to insulin compared to controls at 30 min and no apparent increase in clearance by 60 min (Fig. 9Go). Glucose clearance in AhR+/- mice was similar to that of wild-type controls, and the values for all genotpyes were normally distributed.



View larger version (16K):
[in this window]
[in a new window]
 
FIG. 9. Insulin tolerance in seven-month-old AhR+/+, AhR+/-, and AhR-/- female mice. Fed female mice were injected with 0.75 units of insulin ip, and plasma glucose levels were measured prior to injection, and 15 and 30 min post injection. Results are expressed as percentage of preinjection plasma glucose levels. AhR-/- females did not clear plasma glucose as quickly as AhR+/+ or AhR+/- mice. Symbols and vertical lines represent mean ± standard error. *p < 0.05. {circ} = AhR+/+; {blacktriangleup} = AhR+/-; • = AhR-/-. n = 4 for all groups.

 
AhR-/- Females Do Not Experience Hyperglycemia
The reduced fasting insulin, slower insulin response, and glucose intolerance seen in some AhR-null females could lead to hyperglycemia. We therefore measured fasting and ad libitum fed plasma glucose concentrations in wild-type and AhR-null mice. Under fasting conditions, seven-month-old AhR-/- females did not develop hyperglycemia (AhR+/+, 163.0 ±1 1.7 mg/dl, n = 5; AhR+/-, 153.0 ± 8.5 mg/dl, n = 8; AhR-/-, 158.6 ± 9.4 mg/dl, n = 9), despite reduced fasting plasma insulin. In addition, ad libitum fed AhR-/- mice did not develop hyperglycemia at midday (AhR+/+, 181.8 ± 10.3 mg/dl; AhR+/- 181.3 ± 8.7 mg/dl; AhR-/-, 167.3 ± 6.5 mg/dl; n = 4 for all genotypes).

AhR-/- Females Do Not Develop Increased Glycosylated Hemoglobin Concentrations
We next measured glycosylated hemoglobin A1c concentrations as a measurement of chronic hyperglycemia. Hemoglobin is glycosylated at higher rates in animals experiencing chronic hyperglycemia, and can be quantified as an indicator of hyperglycemia over time (Dan et al., 1997Go). Our results showed that AhR-/- female mice did not experience increased hemoglobin A1c levels at three, four-and-a-half, or six-and-a-half months of age (Fig. 10Go). Interestingly, AhR-null females actually have significantly lower hemoglobin A1c levels at six-and-a-half months of age compared to wild types.



View larger version (16K):
[in this window]
[in a new window]
 
FIG. 10. Age-related hemoglobin A1c concentrations in AhR+/+, AhR+/-, and AhR-/- female mice. Hemoglobin A1c levels were measured at 3, 4.5, and 6.5 months of age. No difference is seen between AhR-/- and AhR+/+ at 3 and 4.5 months of age. AhR-null females showed significantly decreased hemoglobin A1c levels at 6.5 months of age. Symbols and vertical lines represent mean ± standard error. *p < 0.05. AhR+/+: n = 5 at 3 months, 3 at 4.5 months, 6 at 6.5 months. AhR-/-: n = 6 at 3 months, 5 at 4.5 months, 4 at 6.5 months.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
While the AhR was originally identified because of its high affinity for TCDD and its role in TCDD-induced toxicity, AhR-/- knockout mice have demonstrated the importance of this protein in normal development and physiology, including vascular development, reproductive capacity, and adult cardiovascular homeostasis (Abbott et al., 1999Go; Fernandez-Salguero et al., 1997Go; Lahvis et al., 2000Go; Thackaberry et al., 2002Go). We report here for the first time that the AhR also has a role in embryonic cardiac development, and this phenotype is dependent, in part, on the maternal AhR genotype. Furthermore, pregnant AhR-/- mice exhibit altered insulin regulation and responsiveness, which may contribute to the embryonic phenotype.

AhR-/- mice born to AhR-/- females exhibited significantly enlarged hearts. This cardiac enlargement was associated with thicker ventricular walls and upregulation of the cardiac hypertrophy markers ß-MHC and ANF. ANF and ß-MHC are highly expressed during normal fetal cardiac development and are subsequently downregulated early postnatally (Mercadier et al., 1989Go). Both ß-MHC and ANF are markers of cardiac hypertrophy in adult animals (Sugden and Clerk, 1998Go), and increased expression of ANF has been reported in neonatal hearts undergoing hypertrophy (Bruneau et al., 2001Go; Walker and Catron, 2000Go). To our knowledge, these findings are the first to report upregulation of ß-MHC and ANF associated with both embryonic and neonatal cardiac hypertrophy. In contrast, in normal animals MLC-2V is not highly expressed in the fetal heart, but is upregulated in the adult hypertrophied heart (Lee et al., 1988Go). Thus, it is not inconsistent that MLC-2V was not increased in embryonic and neonatal AhR-/- hypertrophic hearts. The increased expression of ß-MHC and ANF, taken together with the thickening of the ventricular walls on d14.5 and d17.5 embryos indicate that AhR-/- mice experience cardiac hypertrophy prior to birth.

The increase in heart size in AhR-null neonates can also be explained by an increased in proliferation in the hearts of AhR-null embryos. At d14.5, AhR-null embryos had a significantly increased proliferation index, as measured by PCNA staining. This suggests that the cardiac enlargement seen in AhR-null mice may be a result of hyperplasia. While the induction of the cardiac hypertrophy markers ß-MHC and ANF suggests that this is a hypertrophic phenotype, it seems likely that both hypertrophy and hyperplasia are involved, since embryonic cardiomyocytes, unlike adult cardiomyocytes, are capable of cell division.

A progressive cardiac hypertrophy occurs in adult male AhR-null mice (Fernandez-Salguero et al., 1997Go; Thackaberry et al., 2002Go). This adult cardiac hypertrophy appears to be mechanistically distinct from the neonatal hypertrophy, since the adult hypertrophy is manifested beginning at five months of age, while three-month-old AhR-null males have normal heart weights. Thus, embryonic and neonatal cardiac hypertrophy in AhR-/- mice may resolve following birth, when the fetus is removed from the maternal environment, and results in normal heart weights by three months of age.

A series of mating experiments revealed that the neonatal cardiac enlargement was dependent, at least in part, on the maternal AhR genotype. Mice born to AhR-/- females, including both AhR-/- and AhR+/- neonates, exhibited significantly increased heart weights compared to mice born to AhR+/+ females. In contrast, mice born to AhR+/- females, including AhR+/+, AhR+/-, and AhR-/- neonates, fail to exhibit cardiac enlargement, compared to mice born to AhR+/+ females. The effects of maternal AhR genotype on neonatal body weight and heart-to-body weight ratio were more complex. Heterozygous neonates born to AhR-/- females had significantly increased body weights, while their AhR-/- littermates did not. However, only AhR-/- neonates born to AhR-/- females showed an increase in heart/body weight ratio. The increase in heart-to-body weight ratio in these mice suggests that the cardiac enlargement seen in AhR-/- mice is dependent on a combination of both maternal and neonatal loss of AhR. The AhR+/- mice born to AhR-/- females show cardiac hypertrophy that is increased in proportion to body weight. In contrast, AhR-/- neonates show cardiac hypertrophy that is increased to a larger degree than their body weights. Neonates born to AhR+/- dams show no increases in heart weight, body weight, or heart-to-body weight ratio, further suggesting that the cause of the neonatal macrosomia is dependent on both maternal and embryonic AhR status.

Since diabetic pregnancy has been shown to increase neonatal heart and body weight in a manner dependent on maternal genotype alone (Rizzo et al., 1992Go; Spellacy et al., 1985Go), we investigated glucose metabolism in pregnant AhR-/- females. Two hallmarks of gestational diabetes are glucose intolerance, or the inability to effectively clear a bolus dose of glucose, and insulin resistance, a reduced ability of peripheral tissues to uptake glucose following insulin release. To determine whether pregnant AhR-null females experienced gestational diabetes, we performed tests for glucose tolerance and insulin resistance. Pregnant AhR-/- mice are able to clear a bolus dose of plasma glucose normally; however, they exhibited a substantial delay in their ability to clear glucose in response to insulin. This suggests that the peripheral tissues in pregnant AhR-/- mice do not uptake plasma glucose in response to insulin as efficiently as controls, but that this insulin resistance was not sufficient to retard glucose clearance following a bolus dose of glucose. Thus, it appears that pregnant AhR-null mice exhibit a partial insulin resistance.

Impaired glucose uptake in response to insulin could be overcome by significantly increased insulin production, or hyperinsulinemia. Indeed, hyperinsulinemia is often seen in insulin resistant gestational diabetes (Buchanan et al., 1990Go). However, fasting C-peptide levels were significantly reduced, rather than elevated, in pregnant AhR-null mice. This result is contradictory to what would be expected if AhR-null mice were overcoming insulin resistance with increased insulin production. Furthermore, when challenged with a bolus dose of glucose, pregnant AhR-/- mice secreted normal levels of insulin. Thus, while the pregnant AhR-null mice had an apparent insulin resistance, this was not compensated for by increased insulin production.

While the decreased fasting insulin levels and insulin resistance seen in pregnant AhR-/- mice were not associated with abnormal glucose tolerance, we hypothesized that these abnormalities may be correlated with hyperglycemia under ad libitum feeding conditions. To investigate this possibility, we measured plasma glucose concentrations throughout a 24-h period. Our results demonstrate that pregnant AhR-null mice do not experience hyperglycemia at any time, and thus, the altered insulin regulation does not lead to hyperglycemia under any conditions studied.

One form of gestational diabetes, mature onset diabetes of the young-2 (MODY-2) shares many similarities with the phenotype seen in pregnant AhR-null mice. MODY-2 results when a single copy of the glucokinase gene is disrupted, leading to altered glucose sensing and reduced insulin secretion (Froguel et al., 1993Go). Thus, like pregnant AhR-/- mice, MODY-2 mice have altered basal insulin levels and normal ad libitum fed glucose levels. However, unlike AhR-null mice, MODY-2 mice develop mild hyperglycemia under fasting conditions (Bali et al., 1995Go), exhibit abnormal glucose tolerance, and do not develop insulin resistance. While the similarities between the MODY-2 mouse model and pregnant AhR-null mice are intriguing, further studies are needed to characterize the defect leading to reduced insulin production in AhR-null animals.

We next investigated whether the AhR was also required for insulin regulation and responsiveness in nonpregnant mice. Our results demonstrate that nonpregnant female AhR-null mice exhibited decreased fasting plasma insulin levels and decreased sensitivity to insulin-mediated glucose uptake by seven months of age, and that 23% of seven-month-old AhR-/- female mice develop overt glucose intolerance. However, none of these effects were observed at three months of age.

While all three-month-old AhR-null females tested had normal glucose tolerances, 23% (4/17) of seven-month-old AhR-/- females showed significant glucose intolerance, suggesting that age increases the dysregulation of glucose in AhR-null females. The incomplete penetrance of this phenotype suggests that other factors may be involved, and that the percentage of animals exhibiting this phenotype could continue to increase with age. Glucose intolerance was not seen in pregnant 3- to 4-month-old AhR-null females, indicating that age, but not pregnancy, is one important risk factor for development of this abnormality. Unfortunately, due to the reduced reproductive capacity of AhR-null mice (Abbott et al., 1999Go), studies cannot be preformed on pregnant seven-month-old AhR-null females.

In addition to glucose intolerance, seven-month-old AhR heterozygous and null females also have decreased fasting plasma insulin levels that were not apparent at three months. This suggests that, like the development of glucose intolerance, the development of reduced fasting insulin production occurs with age. Interestingly, while three-month-old AhR-null females show no alteration in fasting insulin levels, pregnant AhR-null mice of similar age do exhibit decreased fasting insulin. This suggests that both pregnancy and increased age are contributing risk factors in the development of this phenotype. The reduction in fasting plasma insulin seen in AhR+/- mice is also significant. This suggests that loss of a single AhR allele is sufficient to alter insulin regulation in mice. The only other reported abnormality seen in AhR+/- mice is cardiac hypertrophy (Thackaberry et al., 2002Go), which, interestingly, is also seen at seven months of age.

Despite the reduction in fasting plasma insulin, impaired insulin secretion in response to glucose was not observed. These results indicate that AhR-/- mice are able to secrete normal amounts of insulin in response to a bolus dose of glucose. Therefore, it seems unlikely that the decreased fasting insulin plays a role in the impaired glucose tolerance in seven-month-old AhR-null females.

Seven-month-old AhR-null female mice also demonstrate a significantly reduced sensitivity to exogenous insulin. A similar insulin insensitivity is seen in pregnant AhR-/- mice, implicating age and pregnancy as contributing factors involved in this phenotype. Thus, it seems likely that reduced responsiveness to insulin plays a causative role in the impaired glucose tolerance.

The phenotype seen in AhR-null females resembles some characteristics of type II diabetes, including a reduced ability of peripheral tissues to absorb glucose in response to insulin (decreased insulin response) and impaired glucose tolerance. Furthermore, the reduced insulin sensitivity and glucose intolerant phenotypes in AhR-null females develop with age, while reduced insulin sensitivity also develops with pregnancy. Notably, age and pregnancy have been demonstrated to increase the risk of developing type II diabetes (Bertoni et al., 2002Go). This suggests that the AhR-null phenotype and type II diabetes share certain risk factors, and therefore may have some commonalities. Despite the similarities between type II diabetes and the female AhR-null phenotype, type II diabetes typically results in hyperglycemia, increased hemoglobin A1C, and hyperinsulinemia. Seven-month-old AhR-null females fail to exhibit any of these characteristics. In fact, they exhibit normal glycemic control, reduced hemoglobin A1C, and hypoinsulinemia.

It has been demonstrated that TCDD exposure and AhR activation are linked to alterations in insulin and glucose regulation, but these data fail to provide clear evidence of a mechanism by which the AhR regulates carbohydrate metabolism. TCDD causes hypoinsulinemia and hypoglycemia in animal models (Ebner et al., 1988Go; Gorski and Rozman, 1987Go; Viluksela et al., 1999Go), but is associated with hyperinsulinemia, hyperglycemia, insulin resistance, and increased risk for type II diabetes in humans (Bertazzi et al., 2001Go; Cranmer et al., 2000Go; Henriksen et al., 1997Go; Michalek et al., 1999Go). Thus, the role of the AhR in insulin regulation and tissue responsiveness is unclear.

These data demonstrate that pregnant AhR-null mice have altered insulin regulation, but do not exhibit overt gestational diabetes, and that nonpregnant AhR-null females develop altered insulin regulation by seven months of age, and 23% of these mice develop glucose intolerance. The partial dependence of the neonatal phenotype on the maternal genotype suggests a metabolic abnormality in the mother that promotes the development of macrosomia and cardiac hypertrophy; however, the ultimate cause of the neonatal cardiac hypertrophy and macrosomia is uncertain. In addition, nonpregnant seven-month-old AhR-null female mice do not develop hyperglycemia or increased hemoglobin A1C, indicating that they do not exhibit overt diabetes. These data demonstrate that the AhR is required for normal cardiovascular development, as well as insulin regulation in pregnant and nonpregnant female mice.


    ACKNOWLEDGMENTS
 
We would like to thank Dr. Irena Ivnitski-Steele for conducting the PCNA analysis and Kim Doctorman, Katherine Debelak, and Joel Earnest-DeYoung for their invaluable assistance. Contribution # 348, Molecular and Environmental Toxicology Center, University of Wisconsin, Madison, WI 53726–4087. Supported, in part, by NIH training grant # ES07015, ES10433 to MKW, and New Mexico NIEHS Center (ES12072).


    NOTES
 
1 To whom correspondence should be addressed. Fax: (505) 272-6749. E-mail: mkwalker{at}unm.edu. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Abbott, B. D., Birnbaum, L. S., and Perdew, G. H. (1995). Developmental expression of two members of a new class of transcription factors: I. Expression of aryl hydrocarbon receptor in the C57BL/6N mouse embryo. Develop. Dyn. 204, 133–143.[ISI][Medline]

Abbott, B. D., and Probst, M. R. (1995). Developmental expression of two members of a new class of transcription factors: II. Expression of aryl hydrocarbon receptor nuclear translocator in the C57BL/6N mouse embryo. Develop. Dyn. 204, 144–155.[ISI][Medline]

Abbott, B. D., Schmid, J. E., Pitt, J. A., Buckalew, A. R., Wood, C. R., Held, G. A., and Diliberto, J. J. (1999). Adverse reproductive outcomes in the transgenic Ah receptor-deficient mouse. Toxicol. Appl. Pharmacol. 155, 62–70.[CrossRef][ISI][Medline]

Bali, D., Svetlanov, A., Lee, H. W., Fusco-DeMane, D., Leiser, M., Li, B., Barzilai, N., Surana, M., Hou, H., and Fleischer, N. (1995). Animal model for maturity-onset diabetes of the young generated by disruption of the mouse glucokinase gene. J. Biol. Chem. 270, 21464–21467.[Abstract/Free Full Text]

Bertazzi, P. A., Consonni, D., Bachetti, S., Rubagotti, M., Baccarelli, A., Zocchetti, C., and Pesatori, A. C. (2001). Health effects of dioxin exposure: A 20-year mortality study. Am. J. Epidemiol. 153, 1031–1044.[Abstract/Free Full Text]

Bertoni, A. G., Krop, J. S., Anderson, G. F., and Brancati, F. L. (2002). Diabetes-related morbidity and mortality in a national sample of U. S. elders. Diabetes Care. 25, 471–475.[Abstract/Free Full Text]

Bruneau, B. G., Bao, Z. Z., Fatkin, D., Xavier-Neto, J., Georgakopoulos, D., Maguire, C. T., Berul, C. I., Kass, D. A., Kuroski-de Bold, M. L., de Bold, A. J., et al. (2001). Cardiomyopathy in Irx4-deficient mice is preceded by abnormal ventricular gene expression. Mol. Cell. Biol. 21, 1730–1736.[Abstract/Free Full Text]

Buchanan, T. A., Metzger, B. E., Freinkel, N., and Bergman, R. N. (1990). Insulin sensitivity and B-cell responsiveness to glucose during late pregnancy in lean and moderately obese women with normal glucose tolerance or mild gestational diabetes. Am. J. Obstet. Gynecol. 162, 1008–1014.[ISI][Medline]

Cranmer, M., Louie, S., Kennedy, R. H., Kern, P. A., and Fonseca, V. A. (2000). Exposure to 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) is associated with hyperinsulinemia and insulin resistance. Toxicol. Sci. 56, 431–436.[Abstract/Free Full Text]

Dan, K., Fujita, H., Seto, Y., and Kato, R. (1997). Relation between stable glycated hemoglobin A1C and plasma glucose levels in diabetes-model mice. Exp. Anim. 46, 135–140.[ISI][Medline]

Denison, M. S., Fisher, J. M., and Whitlock, J. P., Jr. (1988). Inducible, receptor-dependent protein–DNA interactions at a dioxin-responsive transcriptional enhancer. Proc. Natl. Acad. Sci. U.S.A. 85, 2528–2532.[Abstract]

Ebner, K., Brewster, D. W., and Matsumura, F. (1988). Effects of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin on serum insulin and glucose levels in the rabbit. J. Environ. Sci. Health Part B 23, 427–438.[ISI]

Fernandez-Salguero, P. Pineau, T. Hilbert, D. M., McPhail, T., Lee, S. S., Kimura, S., Nebert, D. W., Rudikoff, S., Ward, J. M., and Gonzalez, F. J. (1995). Immune system impairment and hepatic fibrosis in mice lacking the dioxin-binding Ah receptor. Science 268, 722–726.[ISI][Medline]

Fernandez-Salguero, P. M., Ward, J. M., Sundberg, J. P., and Gonzalez, F. J. (1997). Lesions of aryl-hydrocarbon receptor-deficient mice. Vet. Pathol. 34, 605–614.[Abstract]

Fernandez-Salguero, P. M., Hilbert, D. M., Rudikoff, S., Ward, J. M., and Gonzalez, F. J. (1996). Aryl-hydrocarbon receptor-deficient mice are resistant to 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin-induced toxicity. Toxicol. Appl. Pharmacol. 140, 173–179.[CrossRef][ISI][Medline]

Froguel, P., Zouali, H., Vionnet, N., Velho, G., Vaxillaire, M., Sun, F., Lesage, S., Stoffel, M., Takeda, J., and Passa, P. (1993). Familial hyperglycemia due to mutations in glucokinase. Definition of a subtype of diabetes mellitus. N. Engl. J. Med. 328, 697–702.[Abstract/Free Full Text]

Gorski, J. R., and Rozman, K. (1987). Dose-response and time course of hypothyroxinemia and hypoinsulinemia and characterization of insulin hypersensitivity in 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD)-treated rats. Toxicology 44, 297–307.[CrossRef][ISI][Medline]

Gu, Y. Z., Hogenesch, J. B., and Bradfield, C. A. (2000). The PAS superfamily: sensors of environmental and developmental signals. Annu. Rev. Pharmacol. Toxicol. 40, 519–561.[CrossRef][ISI][Medline]

Harris, M. W., Moore, J. A., Vos, J. G., and Gupta, B. N. (1973). General biological effects of TCDD in laboratory animals. Environ. Health Perspect. 5, 101–109.[Medline]

Hassoun, E., d’Argy, R., and Dencker, L. (1984). Teratogenicity of 2, 3, 7, 8-tetrachlorodibenzofuran in the mouse. J. Toxicol. Environ. Health Part A 14, 337–351.

Henriksen, G. L., Ketchum, N. S., Michalek, J. E., and Swaby, J. A. (1997). Serum dioxin and diabetes mellitus in veterans of Operation Ranch Hand. Epidemiology 8, 252–258.[ISI][Medline]

Hornung, M. W., Spitsbergen, J. M., and Peterson, R. E. (1999). 2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin alters cardiovascular and craniofacial development and function in sac fry of rainbow trout (Oncorhynchus mykiss). Toxicol. Sci. 47, 40–51.[Abstract]

King, D. P., Zhao, Y., Sangoram, A. M., Wilsbacher, L. D., Tanaka, M., Antoch, M. P., Steeves, T. D., Vitaterna, M. H., Kornhauser, J. M., Lowrey, P. L., et al. (1997). Positional cloning of the mouse circadian clock gene. Cell 89, 641–653.[ISI][Medline]

Lahvis, G. P., Lindell, S. L., Thomas, R. S., McCuskey, R. S., Murphy, C., Glover, E., Bentz, M., Southard, J., and Bradfield, C. A. (2000). Portosystemic shunting and persistent fetal vascular structures in aryl hydrocarbon receptor-deficient mice. Proc. Natl. Acad. Sci. U.S.A. 97, 10442–10447.[Abstract/Free Full Text]

Lee, H. R., Henderson, S. A., Reynolds, R., Dunnmon, P., Yuan, D., and Chien, K. R. (1988). Alpha 1-adrenergic stimulation of cardiac gene transcription in neonatal rat myocardial cells. Effects on myosin light chain-2 gene expression. J. Biol. Chem. 263, 7352–7358.[Abstract/Free Full Text]

Lund, A. K., Goens, M. B., Kanagy, N. L., and Walker, M. K. (2003). Cardiac hypertrophy in aryl hydrocarbon receptor (AhR) null mice is correlated with elevated angiotensin II, endothelin-1 and mean arterial blood pressure. Toxicol. Appl. Pharmacol. (in press).

Mercadier, J. J., Samuel, J. L., Michel, J. B., Zongazo, M. A., de la, Bastie, D., Lompre, A. M., Wisnewsky, C., Rappaport, L., Levy, B., and Schwartz, K. (1989). Atrial natriuretic factor gene expression in rat ventricle during experimental hypertension. Am. J. Physiol. 257, H979–987.[ISI][Medline]

Michalek, J. E., Akhtar, F. Z., and Kiel, J. L. (1999). Serum dioxin, insulin, fasting glucose, and sex hormone-binding globulin in veterans of Operation Ranch Hand. J. Clin. Endocrinol. Metab. 84, 1540–1543.[Abstract/Free Full Text]

Peters, J. M., Narotsky, M. G., Elizondo, G., Fernandez-Salguero, P. M., Gonzalez, F. J., and Abbott, B. D. (1999). Amelioration of TCDD-induced teratogenesis in aryl hydrocarbon receptor (AhR)-null mice. Toxicol. Sci. 47, 86–92.[Abstract]

Reick, M., Garcia, J. A., Dudley, C., and McKnight, S. L. (2001). NPAS2: An analog of clock operative in the mammalian forebrain. Science 293, 506–509.[Abstract/Free Full Text]

Rizzo, G., Arduini, D., and Romanini, C. (1992). Accelerated cardiac growth and abnormal cardiac flow in fetuses of type I diabetic mothers. Obstet. Gynecol. 80, t-76.

Schmidt, J. V., and Bradfield, C. A. (1996). Ah receptor signaling pathways. Annu. Rev. Cell Dev. Biol. 12, 55–89.[CrossRef][ISI][Medline]

Shimizu, Y., Nakatsuru, Y., Ichinose, M., Takahashi, Y., Kume, H., Mimura, J., Fujii-Kuriyama, Y., and Ishikawa, T. (2000). Benzo[a]pyrene carcinogenicity is lost in mice lacking the aryl hydrocarbon receptor. Proc. Natl. Acad. Sci. U.S.A. 97, 779–782.[Abstract/Free Full Text]

Spellacy, W. N., Miller, S., Winegar, A., and Peterson, P. Q. (1985). Macrosomia–maternal characteristics and infant complications. Obstet. Gynecol. 66, 158–161.[Abstract]

Sugden, P. H., and Clerk, A. (1998). Cellular mechanisms of cardiac hypertrophy. J. Mol. Med. 76, 725–746.[CrossRef][ISI][Medline]

Thackaberry, E. A., Galbaldon, D. M., Walker, M. K., and Smith, S. M. (2002). Aryl hydrocarbon receptor null mice develop cardiac hypertrophy and increased hypoxia-inducible factor 1-alpha in the absence of cardiac hypoxia. Cardiovasc. Toxicol. 2, 263–273.[Medline]

Viluksela, M., Unkila, M., Pohjanvirta, R., Tuomisto, J. T., Stahl, B. U., Rozman, K. K., and Tuomisto, J. (1999). Effects of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) on liver phosphoenolpyruvate carboxykinase (PEPCK) activity, glucose homeostasis and plasma amino acid concentrations in the most TCDD-susceptible and the most TCDD-resistant rat strains. Arch. Toxicol. 73, 323–336.[CrossRef][ISI][Medline]

Walker, M. K. and Catron, T. F. (2000). Characterization of cardiotoxicity induced by 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin and related chemicals during early chick embryo development. Toxicol. Appl. Pharmacol. 167, 210–221.[CrossRef][ISI][Medline]

Walker, M. K., Heid, S. E., Smith, S. M., and Swanson, H. I. (2000). Molecular characterization and developmental expression of the aryl hydrocarbon receptor from the chick embryo. Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 126, 305–319.[ISI][Medline]

Walker, M. K., Pollenz, R. S., and Smith, S. M. (1997). Expression of the aryl hydrocarbon receptor (AhR) and AhR nuclear translocator during chick cardiogenesis is consistent with 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin-induced heart defects. Toxicol. Appl. Pharmacol. 143, 407–419.[CrossRef][ISI][Medline]

Wang, G. L., Jiang, B. H., Rue, E. A., and Semenza, G. L. (1995). Hypoxia-inducible factor 1 is a basic-helix–loop–helix-PAS heterodimer regulated by cellular O2 tension. Proc. Natl. Acad. Sci. U.S.A. 92, 5510–5514.[Abstract]

Weisberg, S. (1985). Applied Linear Regression, 2nd ed. John Wiley, New York.

Zheng, B., Larkin, D. W., Albrecht, U., Sun, Z. S., Sage, M., Eichele, G., Lee, C. C., and Bradley, A. (1999). The mPer2 gene encodes a functional component of the mammalian circadian clock. Nature 400, 169–173.[CrossRef][ISI][Medline]