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J. Anim Sci. 2008. 86:1124-1131. doi:10.2527/jas.2007-0638
© 2008 American Society of Animal Science
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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION

Dietary restriction reduces the rate of estradiol clearance in sheep (Ovis aries)

B. J. Renquist, T. E. Adams, B. M. Adams and C. C. Calvert1

Department of Animal Science, University of California, One Shields Avenue, Davis 95616


   Abstract
Top
 Abstract
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Three experiments were designed to test the effect of dietary restriction on clearance of 17β-estradiol (E2) in sheep. A preliminary experiment examined the effect of a 4-d fast on the rate of E2 clearance in wethers. The second experiment tested the hypothesis that either long-term restriction (7 wk) or a 5-d fast would increase steroid-binding capacity of serum by increasing the concentration of sex hormone-binding globulin (SHBG) in the blood of ovariectomized ewes. In Exp. 3, we hypothesized that nutrition-dependent regulation of E2 clearance by the liver would result in divergence in biliary extraction of E2 in fed and fasted wethers receiving comparable levels of exogenous E2. A marked difference in E2 clearance between fed and fasted wethers was noted in the preliminary study. Relative to ad libitumfed wethers, a 4-d fast decreased E2 clearance by 52%. Serum concentrations of SHBG were increased in long-term energy-restricted and fasted ewes, relative to the concentration in maintenancefed ewes (P = 0.015). Furthermore, a 5-d fast nearly doubled serum steroid-binding capacity in wethers. The E2 concentration in bile was 2 times greater in fasted than in fed wethers. This fasting-dependent increase in biliary E2 may be reflective of the increased serum E2 in fasted animals, because each 1 pg/mL increase in serum E2 increased bile E2 by 0.86 ± 0.12 pg/mL, independent of nutrition (P = 0.002). Our results demonstrate that the rate of clearance of E2 is decreased during nutritional restriction. Additionally, these data indicate that altered SHBG expression, enterohepatic recirculation, or both are involved in the decreased E2 clearance during dietary restriction.

Key Words: clearance • enterohepatic recirculation • estradiol • restriction • sex hormone-binding globulin • sheep


   INTRODUCTION
Top
 Abstract
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
High levels of dietary energy intake inhibit reproduction. Elevated levels of feed intake have been implicated in the development of short estrous cycles and increased difficulty of estrus detection in cattle, decreased embryo survival in swine, and decreased pregnancy rates in ewes (den Hartog and van Kempen, 1980Go; Dyck and Strain, 1983Go). These observations have led to the suggestion that excessive energy intake decreases reproductive performance by increasing the rate of progester-one and 17β-estradiol (E2) clearance.

Studies have suggested that a high rate of progesterone clearance may result in subthreshold serum progesterone concentrations required to maintain pregnancy (Parr et al., 1987Go). Similarly, high E2 clearance may eliminate embryonic signals essential for maternal recognition of pregnancy (Geisert et al., 1982Go). It is important to note that the liver extracts 96% of the progesterone and 85% of the estrogen that it encounters (Pardridge, 1981Go; Parr et al., 1993). Therefore, increased hepatic blood flow, associated with improved nutrition, is directly related to the steroid hormone clearance rate (Sangsritavong et al., 2002Go). Despite the critical role of hepatic tissue in steroid clearance, limited work has focused on the role of steroid-binding proteins, biliary excretion, and enterohepatic recirculation of E2 in domestic species.

Three experiments were designed to examine the effect of dietary restriction on clearance of steroid hormones in sheep given exogenous E2. First, we enumerated the effect of a 4-d fast on the E2 clearance rate. Second, we hypothesized that dietary restriction would increase steroid-binding capacity of the serum by increasing serum concentrations of sex hormone-binding globulin (SHBG). Finally, we examined the effect of fasting on bile E2 concentration.


   MATERIALS AND METHODS
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
All experimental procedures involving the use of animals were conducted in accordance with the Universities Federation for Animal Welfare Handbook on the Care and Management of Laboratory Animals and were approved by the Animal Use and Care Committee for the University of California.

Experimental Design

Three experiments were conducted to examine the effect of nutrition on the rate of serum E2 clearance and to elucidate the mechanisms by which nutrition affects E2 clearance in the gonadectomized sheep (Ovis aries).

Experiment 1. Twelve orchidectomized, crossbred, castrate male lambs (wethers) were either individually fed or fasted (n = 6/dietary treatment) in an experiment designed to enumerate the effect of a 4-d fast on the rate of E2 clearance. Wethers had been castrated for at least 4 mo before the beginning of the experiment. For 3 wk before the onset of the experiment, all wethers were acclimated to an alfalfa-based pelleted ration designed to meet the requirements for growth (NRC, 1985Go). Cannulae were inserted into the right and left jugular veins (Intramedic PE 190, Clay Adams, Parsippany, NJ) 1 d before beginning the fast. One cannula was used for infusion of E2 and the other for blood collection. Four days after cannulation, E2 (Sigma Chemical Co., St. Louis, MO) administration was begun, with 1 lamb in each dietary treatment receiving a constant infusion of 0, 0.156, 0.312, 0.625, 1.25, or 2.646 µg of E2•50 kg of BW–1•h–1 in 1 mL of 10% ethanol-saline (vol/vol). Administration of the E2-containing solutions during the 24-h study period was effected by using a Harvard infusion pump (Model 2265, Harvard Bioscience, South Natick, MA). Blood was collected at 0, 1, 3, 6, 12, 18, and 24 h after the onset of infusion. At the conclusion of the E2 infusion period, the delivery pump was stopped and blood (3 mL) was collected at 10-min intervals for 1 h. Serum for E2 analysis was isolated after allowing the blood to clot at 4°C for 24 h.

Experiment 2. Mature (n = 35; 2 to 5 yr of age) Targhee ewes (initial BW = 58.7 ± 4.9 kg) were bilaterally ovariectomized (ovx) by laparoscopy in early August, 2 to 3 wk before the onset of dietary treatments (n = 5 ewes/treatment, Restricted E; Restricted S; Fast S; and Fed S; n = 6 ewes/treatment; Fast E and Fed E). During the 2 to 3 wk after ovariectomy, all ewes were individually penned in an open-sided barn and fed a diet that satisfied their maintenance energy requirements (NRC, 1985Go). The maintenance diet was formulated to maintain BW, providing 2 MCal/kg of energy and 140 g of protein/kg (as fed). At the conclusion of the acclimation period, the ewes were randomized by BW into a 3 x 2 factorial experimental design, with 3 nutritional treatments and 2 hormone treatments. The 3 nutritional treatments were imposed on individually housed ewes for 7 wk. Dietary treatments were dietary restriction designed to induce a 15% decrease in BW, maintenance feeding for 44 d with a fasting period during the last 5 d of feeding, or maintenance feeding for the entire 49-d experimental period (treatment groups designated as Restriction, Fast, and Maintenance, respectively). Dietary restriction was imposed by feeding 1.2 kg of a low-energy-density sheep pellet (described previously by Renquist et al., 2008Go) that was designed to provide 1.225 MCal/kg of energy and 140 g of protein/kg as fed. These dietary formulations ensured that dietary energy varied among the groups while holding the protein content and quantity consumed constant for restricted- and maintenance-fed ewes.

After 7 wk of controlled feeding, each ewe received 2 jugular cannulae that were used for blood collection and E2 administration. Fasting began at 0600 h on the day after cannulation, whereas maintenance- and restricted-fed ewes were maintained on their respective diets. Four days after cannulation, one-half of the ewes in each nutritional treatment group received exogenous E2 by continuous infusion [0.31 µg of E2•50 kg of BW–1•h–1 in a 10% ethanol-saline (vol/vol) vehicle]. This dosage of E2 was chosen because it had been shown previously to induce diestrous concentrations of E2 in fed animals (Beckett et al., 1997Go). Estradiol levels resulting from this infusion in fed, fasted, and restricted ewes have been reported previously (Renquist et al., 2008Go). Control ewes from each dietary treatment received infusion of the vehicle alone. Estradiol and vehicle were infused at a rate of 1 mL/h using the Harvard infusion pumps. Blood samples (3 mL) for analysis of serum steroid-binding capacity were collected 54 h after the onset of continuous E2 infusion. Serum was isolated as described for Exp. 1.

Experiment 3. Wether lambs (n = 42) were used in Exp. 3, which was designed to examine the effect of nutrition on biliary clearance of E2 and circulating serum-binding proteins. Wethers were castrated for at least 4 mo before the beginning of the experiment. For 3 wk before the beginning of the experiment, all wethers were acclimated to an alfalfa-based pelleted ration designed to meet the requirements for growth (NRC, 1985Go). Immediately before the experiment, the wethers were blocked by BW into 7 treatment groups (n = 6 wethers/treatment). The 5-d period of controlled feeding was initiated 1 d after insertion of the jugular cannulae and the blank or E2-containing implants. During the period of controlled feeding, wethers in 4 treatments were fasted, whereas wethers in the other 3 treatments were individually allowed ad libitum access to feed. Data from Exp. 1 showed that a 4-d fast decreased E2 clearance by 53.5%; therefore, the implant lengths were shorter in fasted than in fed wethers. Fasted wethers received either a blank implant or an implant packed with crystalline E2 0.2, 0.7, or 2.5 cm in length, as described below. Fully fed wethers received either a sham implant or implants packed with E2 1.5 or 6 cm in length (n = 6/implant length). The implants in the fed and fasted wethers were designed to result in a similar range (0 to 26 pg/mL) of serum E2 concentrations. Blood (3 mL) for serum analysis of E2 and steroid-binding capacity was collected 126 h after the fast was imposed.

At the end of blood collection, wethers in both dietary treatments with blank implants, fasted wethers with 0.7-cm implants, and fully fed wethers with 1.5-cm implants were stunned by electrical shock and killed by exsanguination. During processing, the gall bladder was removed and bile was aspirated by syringe with an 18-gauge needle. Bile was stored at –20°C for analysis of E2.

Construction of E2 Implants

Estradiol-containing implants were produced according to the procedure described by Karsch et al. (1973)Go. Briefly, Silastic tubing (i.d. 0.33 cm, o.d. 0.46 mL, Dow-Corning Corp., Midland, MI), sealed at one end with 0.5 cm of Type A adhesive (Dow-Corning Corp.) was packed with crystalline E2 to the desired length and the second end was sealed with the Silastic adhesive. The implants were washed for 24 h in sterile saline before being inserted s.c. into the axillary region.

Cannulation

The animals were sedated with Xylazine (0.1 mg/50 kg of BW), and a polyethylene cannula (Intramedic PE 190, Clay Adams) was inserted into the jugular vein to a depth of 20 cm. The cannula was passed through a protective Tygon tubing sheath along a 1-m lead to the outside of the animal pens. The animals were freely mobile at the end of the 1-m lead. Heparinized sterile saline (1 IU/mL; Baxter Healthcare Corp., Deerfield, IL) was used to maintain the patency of the cannulae.

E2 and Serum-Binding Analysis

Serum and bile E2 concentrations were assessed by using previously validated procedures (Sakurai et al., 1992Go; Renquist et al., 2008Go). Briefly, serum samples (250 uL) were extracted once with anhydrous diethyl ether (2 mL mixed by vortexing twice for 60 s). The ether phase was decanted after freezing the aqueous phase in a dry ice-acetone bath. The ether phase was evaporated to dryness at 37°C, and the sample was reconstituted by using 100 µL of 0.1% gelatin-PBS. Subsequently, E2 antiserum (1:2,500,000 dilution) was added in 100 µL of 0.1% gelatin-PBS and the samples were incubated at 4°C. Twenty-four hours after the addition of antiserum, 125I- E2 (approximately 5,000 cpm, GE Healthcare, Buckinghamshire, UK) in 100 µL of 0.1% gelatin-PBS was added to each sample for overnight incubation at 4°C. Antibody-bound radioactivity was separated from free radioactivity by using dextran-coated charcoal, and the radioactivity associated with the free fraction was determined. Bound radioactivity was calculated by subtracting the free counts from the total counts. Figure 1Go shows the validation of this assay for measurement of bile E2 concentrations. The relationship between ln(E2 concentration + 1) and the relative proportion of binding was similar among all 3 diluents [bile: relative binding = –0.16x + 0.99; serum: relative binding = –0.17x + 0.99; gelatin-PBS: relative binding = –0.17x + 0.99, where x = ln(E2 concentration + 1)]. The E2 assay had an intraassay CV of 19.9%, an interassay CV of 22.3%, and a minimum sensitivity of 0.5 pg/mL.


Figure 1
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  		Figure 1. Validation of the estradiol (E2) assay for measurement of a range of E2 concentrations in 0.1% gelatin in PBS, serum, or bile. Data are plotted as the relationship between ln(standard estradiol concentration + 1) and relative 125I-E2 binding for standards made in 0.1% gelatin-PBS, serum, or bile.

 
Steroid-binding capacity of the serum was assayed by allowing 100 µL of serum to incubate with 100 µL of 3H-E2 (Amersham Biosciences, Piscataway, NJ) at 4°C for 2 h (Murayama et al., 1979Go). Subsequently, dextran-coated charcoal was added to separate free from bound E2 and the bound fraction was counted on a Beckman LS3801 β-counter (Beckman Coulter Inc., Fullerton, CA). The binding of albumin was determined by using the same method, with serum samples heated at 60°C for 30 min to deactivate SHBG (Sodergard et al., 1982Go). The specific binding to SHBG was calculated by subtracting the albumin-bound fraction from the total bound fraction. All steroid-binding data are presented as the percentage of the 3H- E2 bound by serum.

Statistical Analysis

Experiment 1. A mixed ANOVA model (PROC MIXED, SAS Inst. Inc., Cary, NC) was used to evaluate the relationship between serum and infused E2 in fasted and fed wethers. The time at which the plateau concentrations of circulating E2 were reached was identified by using a model with serum E2 as the dependent variable and time, dose, and nutrition as independent fixed variables. The time to plateau was determined when the least squares means for serum E2 were statistically similar for 2 consecutive time points.

Further analyses of the dose relationship between E2 infusion rate and serum E2 were performed only on samples collected after the plateau concentration (6 to 24 h after the onset of infusion) was established. To specifically identify differences in E2 clearance between fed and fasted wethers, serum E2 was regressed on E2 infusion rate independently for fed and fasted wethers by using PROC REG of SAS. To determine whether the relationship between dose and serum E2 differed for fed and fasted wethers, we evaluated the interaction between nutrition and dose in a mixed ANOVA model including dose, nutrition, and dose x nutrition interaction as fixed effects, and serum E2 as the dependent variable. The P-value for the interaction between nutrition and dose indicated the probability that the relationship between infused E2 and serum E2 was similar between nutritional treatments.

Experiment 2. A mixed ANOVA model was used to analyze the effects of nutrition (categorical variable), serum E2 (continuous variable), and their interaction on total steroid-binding capacity of the serum- and albumin-binding capacity. The random effect of animal nested with nutrition was included, because binding was assayed in triplicate. When P > 0.20, interactions were removed from the model. The model for SHBG-binding capacity did not include the random effect of animal, because the SHBG-binding value was obtained by subtracting the mean of the total binding from the mean of the albumin binding. The probability of a difference between treatments was estimated by using the PDIFF option of SAS adjusted by using the Bonferroni method.

Experiment 3. Steroid-binding capacity of the serum was analyzed as described for Exp. 2 by using a mixed ANOVA model with fixed effects of nutrition (categorical variable) and implant length (continuous variable), as well as their interaction, with the random effect of animal nested within nutrition x implant length. The analysis of serum E2 effects on bile E2 was performed as described above, replacing the main effect of implant length with serum E2. The magnitude of the effects was estimated by using either the LSMEANS or SOLUTION function of SAS. The PROC REG of SAS was used to regress bile E2 on serum E2; because there was no interaction between nutrition and serum E2 (P = 0.15) in the mixed model described above, data from both fed and fasted animals were pooled for this analysis.


   RESULTS
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Experiment 1

Serum concentrations of E2 were increased to a stable plateau within 6 h of beginning infusion, and E2 concentrations were maintained at that concentration for the remainder of the infusion period (P = 0.29). Therefore, analysis of the influence of infused E2 on serum E2 concentration was performed only on samples collected between 6 and 24 h. Regressing serum E2 (pg/mL) on 50 kg of BW–1•h–1) showed that the infused E2 (µg of E2• increase in serum E2 across infusion rates was 46.5% less in fed than in fasted wethers (P < 0.001; Figure 2Go). In fact, serum E2 increased by 3.1 and 5.8 pg/mL for every 1 µg of E2• 50 kg of BW–1•h–1 infused in fed and fasted wethers, respectively.


Figure 2
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  		Figure 2. Relationship between estradiol (E2) infusion rate (µg of E2•50 kg of BW–1•hr–1) and total serum estradiol (pg/mL) in 4-d fasted ({diamond}) and maintenance-fed ({blacksquare}) wethers (Exp. 1; error bars indicate SD; n = 6/dietary treatment).

 
Experiment 2

Serum Steroid-Binding Capacity. Dietary treatment (P < 0.001), but not serum E2 (P = 0.89), significantly affected total steroid-binding capacity of the serum (Table 1Go). Although the steroid-binding capacity of the albumin fraction was not affected by either nutrition or serum E2 concentration (P = 0.08 and P = 0.88, respectively), both fasting and caloric restriction increased the SHBG-binding capacity relative to maintenance feeding (P = 0.015; Table 1Go). In contrast, serum E2 did not affect SHBG-binding (P = 0.99) or total steroid-binding capacity of the serum.


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  		Table 1. Total serum- and sex steroid-binding globulin (SHBG)-associated estradiol-binding capacity in the serum1 of fasted (5 d), fed (maintenance energy and protein), and restricted (7 wk) ewes (Exp. 2) and wethers (Exp. 3)
 
Experiment 3

Serum Steroid-Binding Capacity. Total steroid-binding capacity of the serum was greater in fasted wethers than in fed wethers (P < 0.001; Table 1Go), and here was no effect of serum E2 or an interaction between serum E2 and nutrition on total SHBG (P = 0.53 and P = 0.24, respectively). Albumin binding was not affected by either nutrition or E2 (P = 0.93 and P = 0.11, respectively). Similarly, E2 did not affect SHBG binding (P = 0.85). Yet SHBG binding was nearly 2-fold greater in fasted animals than in fed animals (P = 0.001).

Bile E2. Biliary concentration of E2 increased 31.5 ± 5.3 pg/mL for every 1-cm increase in E2 implant length in fasted animals, but was unaffected by implant length in fed animals (P < 0.001; Figure 3Go). If examined in relation to serum E2, every 1 pg/mL increase in serum E2 increased biliary E2 by 0.86 ± 0.12 pg/mL (P = 0.002, R2 = 0.69; Figure 4Go), independent of nutrition (P > 0.15).


Figure 3
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  		Figure 3. Bile estradiol concentration (pg/mL) of fed and fasted wethers (n = 6) implanted with a blank implant (0 cm) or implants of 0.7 or 1.5 cm in length for fed and fasted wethers, respectively (Exp. 3). a,bMeans ± SE differ (P < 0.0001).

 

Figure 4
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  		Figure 4. Regression of bile estradiol (pg/mL) on serum estradiol (pg/mL; Exp. 3). Data for both fasted and fed wethers with all implant lengths are included (n = 24). Bile estradiol = 0.86 x serum estradiol (R2 = 0.69).

 

   DISCUSSION
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Nutrition has a dramatic impact on the clearance of steroid hormones. Several studies have examined the clearance of exogenous E2, testosterone, progesterone, or their combination in nutritionally restricted animals. Additional work has focused on the mechanism by which poor nutrition decreases steroid hormone clearance. Because the liver clears 85% of the E2 that enters, a large body of work has identified changes in liver mass and blood flow, and alterations in the serum steroid-binding proteins as possible modulators of E2 clearance (Pardridge, 1981Go). The present experiment focused on SHBG and possible alterations in biliary excretion of E2.

In E2-infused lactating cows, feeding induced a decrease in circulating E2 within 1 h and elevated the mean clearance rate of E2 within 2 h (Sangsritavong et al., 2002Go). Additionally, restricted feeding in E2-implanted ovx ewes and ovx rats increased circulating E2 relative to ad libitum feeding (Bronson, 1988Go; Adams et al., 1997Go). In fact, in E2-implanted ovx ewes, mean E2 concentration was negatively associated with ME intake (Adams et al., 1997Go). Surprisingly, studies using bolus administration of 3H-E2 have been unable to show differences in steroid clearance between nutrient-restricted and well-fed ewes or rats (Bronson, 1988Go; Adams et al., 1994Go). This may be due to transfer of the 3H from E2 to other molecules, because Adams et al. (1994)Go showed that the free steroid disappeared very quickly and the remaining circulating 3H was not ether extractable and appeared to be bound to protein.

Nutritional restriction also inhibits clearance of both progesterone and testosterone. Indeed, 5-d-fasted rats require a testosterone implant one-half the size of fed rats to achieve similar circulating testosterone concentrations (Pirke and Spyra, 1981Go). These observations, using a rat model, are similar to the nearly 50% decrease in E2 clearance noted in the preliminary sheep-based study presented here. Moreover, serum concentrations of progesterone during the luteal phase are increased in ewes and heifers during fasting (McCann and Hansel, 1986Go; Kiyma et al., 2004Go). A similar effect of fasting on serum progesterone has been noted in cows during pregnancy (Vasconcelos et al., 2003Go). In fact, providing pregnant cows with either 100 or 50% of their maintenance requirement decreased progesterone concentrations within 1 h of feeding, whereas feeding 25% of the maintenance energy requirement did not affect circulating progesterone (Vasconcelos et al., 2003Go). In ewes given PGF2{alpha} to induce luteal regression and eliminate progesterone production, circulating progesterone concentrations decreased below 0.75 ng/mL 60% more quickly in fed than in fasted ewes (Kiyma et al., 2004Go). Furthermore, mean clearance rate of exogenously administered progesterone is directly related to the level of nutrition. Parr et al. (1993a)Go noted that serum concentrations of progesterone were inversely related to nutritive status in ewes fed 50, 100, or 200% of maintenance and receiving similar amounts of exogenous pro-gesterone. The effect of dietary restriction on progesterone clearance seems to apply to swine, because similar results have been reported for gilts, in which a 66% reduction in feed intake resulted in a 32% reduction in progesterone clearance (Prime and Symonds, 1993Go).

As noted previously, 85% of the E2 and 96% of the progesterone that enters the liver is cleared (Pardridge, 1981Go; Parr et al., 1993bGo). In the lactating cow, liver blood flow is highly correlated (r = 0.92) with the mean clearance rate for progesterone (Sangsritavong et al., 2002Go). Because the liver is the main organ responsible for steroid hormone clearance from the blood, a great deal of research has examined the relationship between hormone clearance and nutrition-dependent changes in liver mass, hepatic blood flow, and overall hepatic metabolism.

Dietary energy restriction decreases wet liver mass in wethers (Wester et al., 1995Go), steers (Johnson et al., 1985Go), rams (Ferrell et al., 1986Go), barrows (Koong and Nienaber, 1985Go), and rats (Ferrell and Koong, 1986Go). The percentage change in liver mass is greater than the percentage change in total BW because liver weight loss is 30 and 48% greater than the empty BW change in steers and wethers fed a maintenance or ad libitum diet for 45 and 21 d, respectively (Johnson et al., 1985Go; Burrin et al., 1990Go). In fact, the loss in liver weight occurs quite rapidly. In wethers and rats, more than 50% of the loss in liver mass noted after a 21-d restriction occurs during the first week (Coward et al., 1977Go; Burrin et al., 1990Go). The capacity of the liver to alter tissue mass to conform to change in nutritive status is clearly evident in the energy-restricted and refed wether. Wester et al. (1995)Go reported that liver mass was markedly reduced by 2 wk of limited energy intake. Interestingly, hepatic mass returned to normal after 2 d of ad libitum feeding.

The robust changes in liver mass reported above affect the total metabolic rate of the liver. A 6-d fast in dairy cows reduced the hepatic metabolic rate by 70%. Three days of refeeding increased liver metabolism by more than 2-fold, yet this remained 32% less than before the fast (Lomax and Baird, 1983Go). In ewes, rams, wethers, barrows, and steers, dietary restriction has also been shown to decrease liver metabolism by 23 to 70%. The magnitude of the nutrition-dependent decrease in hepatic metabolism is dependent on the duration and level of energy restriction (Koong et al., 1982Go; Reynolds et al., 1992Go; Freetly et al., 1995Go). Results from a study in restricted-fed wethers suggest that the decrease in metabolic rate is a result of decreased blood flow to the liver (Freetly et al., 1995Go). Depending on the duration and degree of restriction, limited energy provision decreases liver blood flow by 30 to 60% in ewes (Freetly and Ferrell, 1994Go), rams (Burrin et al., 1989Go), wethers (Freetly et al., 1995Go), heifers (Reynolds and Tyrrell, 1987Go), and gilts (Prime and Symonds, 1993Go). Additionally, fasting for as little as 2 d decreases liver blood flow in cows (Lomax and Baird, 1983Go). The ability of the liver to alter blood flow, metabolic rate, and mass, as a reflection of changes in nutritive status, suggests that hepatic tissue may play a critical role in the nutrient-dependent change in the rate of clearance of E2 noted in this experiment.

Alterations in liver function may also affect the metabolic clearance rate of steroid hormones by decreasing either production or clearance of the circulating steroid-binding proteins. In the circulation, only 1 to 3% of the E2 is free, not bound to albumin or SHBG (Pardridge, 1981Go). Steroid-binding proteins influence the rate of steroid clearance. For example, the metabolic clearance rate for E2 is greater in men than in women, whereas the relative binding of E2 to SHBG is less in men than women (Hembree et al., 1969Go; Sodergard et al., 1982Go). Our results show that total serum binding to 3H-E2 is greater in ovx ewes than in wethers.

Albumin-bound E2 is available to many tissues and should be considered an effective part of the free E2 pool, because the KD (Equilibrium dissociation constant) for E2 is high (2.9 mM; Pardridge, 1986Go). Fasting decreases the rate of synthesis of albumin by the liver (Rothschild et al., 1968Go; Yap et al., 1978Go). Furthermore, both fasting and long-term dietary restriction decrease circulating albumin concentrations in humans and rats (Ballantyne et al., 1973Go; Hubert et al., 2000Go). However, long-term fasting also decreases clearance of albumin (Kirsch et al., 1968Go). Therefore, our finding that neither fasting nor long-term energy restriction resulted in a significant change in serum albumin is likely the combined result of decreased albumin production and concurrent reduction in rate of clearance.

Similarly, SHBG is produced by the liver and has a circulating half-life of 3.95 d (Namkung, et al., 1989Go). The E2-binding affinity of SHBG is 10,000-fold greater than that of albumin (Pardridge, 1981Go; Pardridge and Landaw, 1984Go). As a consequence, SHBG more selectively transports E2 to tissues than does albumin. Specifically, SHBG-bound E2 is not readily available to the brain and is preferentially cleared by the liver (Pardridge, 1986Go). The data presented here demonstrate that a 5-d fast increased SHBG-specific binding of 3H-E2 in ewes and wethers by 199 and 93%, respectively. Furthermore, we showed that 7-wk dietary restriction increased SHBG-specific binding by 78%. These results are consistent with a previous study noting that long-term restriction of nonobese human subjects increased SHBG, whereas a return to a nonrestricted diet had the opposite effect (Walford et al., 2002Go).

The high concentration of E2 in the bile of fasted animals was surprising. In fact, we expected that fasting-induced decreases in liver clearance of E2 would result in lower concentrations of E2 in the bile. However, despite evidence that the liver is important for clearance of hormones, a great deal of evidence suggests that extraction by the liver may contribute only modestly to permanent E2 clearance from the body. In fact, biliary E2 is readily reabsorbed in the large intestine. Up to 65 to 70% of 14C-E2 given intraduodenally to female rats is found in the gall bladder 1.5 h after administration (Brewster et al., 1977Go). Additionally, only 4 to 26% of the E2 found in the bile is recovered in the feces (Adlercreutz et al., 1979Go). In rats and rabbits, an i.v. injection of 3H-ethinyl estradiol, which does not bind SHBG, resulted in a biphasic pattern of radioactive steroid in the serum, namely, a rapid decrease followed by an increase 6 to 9 h after administration (Back et al., 1982Go). Although much of the biliary steroid is conjugated to glucuronide, the large microbial population in the large intestine ensures relatively efficient deconjugation and rapid reabsorption in the large intestine (Brewster et al., 1977Go; Adlercreutz et al., 1979Go). Our results suggest much greater concentrations of E2 in the bile of fasted animals than in fed animals receiving comparable concentrations of exogenous E2. This may be a result of increased transport of E2 to the liver associated with the elevated SHBG concentrations, or slowed passage time through the intestines and therefore greater reabsorption of E2. Alternatively, dietary restriction may depress the enzymes that affect glucuronide- E2 conjugation.

This experiment confirms that nutritional restriction decreases E2 clearance in sheep. Additionally, the results presented here suggest that dietary-induced alterations in SHBG expression, enterohepatic recirculation, or both may be involved in the decreased E2 clearance during restriction. Restriction-induced decreases in liver mass, blood flow, and metabolic rate are well documented, yet increased SHBG expression in both fasted and restricted animals and increased bile E2 concentrations emphasize that liver blood flow may explain only a portion of the steroid hormone clearance differences. Future work is focused on more precisely defining the interplay between nutritive status, hepatic function, SHBG, and enterohepatic recirculation of steroid hormones.

1 Corresponding author: cccalvert{at}ucdavis.edu

Received for publication October 6, 2007. Accepted for publication February 4, 2008.


   LITERATURE CITED
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 


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