0013-7227/90/1264-1934$02.00/0 Endocrinology Copyright© 1990 by The Endocrine Society
Vol. 126, No. 4 Printed in U.S.A.
Effects of Free Fatty Acids on Luteinizing Hormone and Growth Hormone Secretion in Ovariectomized Lambs* M. J. ESTIENNE, K. K. SCHILLO, S. M. HILEMAN, M. A. GREEN, S. H. HAYES, AND J. A. BOLING Department of Animal Sciences, University of Kentucky, Lexington, Kentucky 40546-0215
ABSTRACT. The effects of FFA on circulating LH and GH concentrations in ovariectomized ewe lambs were investigated. Lambs (n = 14) were weaned at 2.5 months, ovariectomized at 6.5 months, and used at 8.5 months of age. From weaning until day 0 of the experiment, lambs were fed to maintain body weights (23 kg). On day 0, serum FFA concentrations and mean serum LH concentrations and number and amplitude of LH pulses, as assessed in blood samples collected every 12 min for 4 h, were 6.4 ± 0.6 mg/100 ml, 0.57 ± 0.08 ng/ml, 0.45 ± 0.09 pulses/h, and 0.73 ± 0.11 ng/ml, respectively. Double the maintenance feeding, beginning day 1, increased (P < 0.01) body weights by 16% and LH pulse frequency by 82%, but had no effect (P > 0.1) on FFA concentrations, mean LH concentrations, or LH pulse amplitude by day 14. On day 14, lambs were infused with
I
NADEQUATE nutrition is associated with aberrant reproductive function in several species (1-4). Foster and Olster (5) demonstrated that the nutritionally growth-restricted female lamb failed to reach puberty and proposed that the inhibition of sexual maturation was due to inadequate secretion of LH. They hypothesized that the frequency of LH pulses was not sufficient to stimulate development of ovarian follicles to the preovulatory stage, and as a consequence, circulating concentrations of estradiol failed to reach levels necessary for induction of the preovulatory surge of LH (6). The mechanisms by which poor nutrition influences LH secretion in the developing lamb are only partially understood. Restricted food intake prolongs hypersensitivity of the hypothalamic-pituitary axis to the estradiol negative feedback characteristic of prepubertal lambs (5). Nutritionally growth-restricted lambs, however, have infrequent LH pulses after ovariectomy (5, 6), a finding consistent with a steroid-independent suppressive effect Received November 11,1989. Address requests for reprints to: Dr. Keith K. Schillo, Department of Animal Sciences, 807 Agricultural Science Building South, University of Kentucky, Lexington, Kentucky 40546-0215. * Presented in part at the Neuroendocrine Control of Reproduction Symposium, November 1989, Napa, CA. This work was supported by a grant from the USDA (87-CRCR-1-2567), and this paper (no. 89-5208) is published with the approval of the director of the Kentucky Agricultural Experiment Station.
lipid (n = 9; 95.8 mg/min) or 0.9% saline solution (n = 5) for 8 h. Blood samples were collected at 12-min intervals for 12 h,
beginning 4 h before infusions. FFA levels increased (P < 0.01) in lipid-infused animals to 27.6 ± 2.9 mg/100 ml by 4 h of infusion. Mean LH concentrations and LH pulse frequency and amplitude were unaffected (P > 0.1) by treatment. In contrast, mean GH concentrations and GH pulse frequency, which were similar (P > 0.1) between groups before infusion (14.0 ± 0.8 ng/ ml and 0.36 ± 0.07 pulses/h, respectively) were decreased by FFA treatment by 51% (P < 0.01) and 81% (P < 0.006), respectively. GH pulse amplitude was highly variable and unaffected (P > 0.1) by treatment. In summary, elevated FFA levels appear to inhibit the release of GH, but not LH, in the ovariectomized ewe lamb. (Endocrinology 126: 1934-1940, 1990)
of poor nutrition on LH secretion. It is likely that the effects of nutrition on pulsatile LH release involve central mechanisms controlling hypothalamic discharge of LHRH, rather than impaired pituitary function, since pituitary responsiveness to LHRH was not compromised in the nutritionally growth-restricted lamb (6). Cameron et al. (7) proposed that nutritional influences on LH secretion may be attributed to changes in circulating concentrations of metabolites. Previous studies (8-10) have demonstrated that an inverse relationship exists between the level of feeding and circulating concentrations of FFA. Thus, we hypothesized that the reduced frequency of LH pulses in the nutritionally growth-restricted lamb may be due to inhibition of the hypothalamic-pituitary axis by FFA. Therefore, the first objective of the following experiment was to assess, in nutritionally growth-restricted lambs, changes that occur in steroid-independent LH secretion after an increased level of feeding and relate these changes to circulating concentrations of FFA. Secondly, we sought to determine the effects of lipid infusion on LH secretion after an increased level of feeding. We have previously demonstrated that elevated FFA concentrations caused by lipid infusion are associated with decreased levels of GH (11). In that study, mature ewes (mean age and weight, 3.3 yr and 53.8 kg, respec-
1934
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FFA EFFECTS ON LH AND GH IN LAMBS
tively) were used (11). The effect of exogenous lipid on GH release may differ in animals of differing age and/or metabolic status. Thus, as a third objective of this experiment we characterized the effects of lipid infusion on GH concentrations in nutritionally growth-restricted lambs after an increased level of feeding.
Materials and Methods General Fourteen cross-bred ewe lambs, predominantly of the Hampshire breed, were used. Lambs were born on approximately February 1, 1988, and were weaned at 8-10 weeks of age. Ewe lambs in our flock typically reach puberty by 28 weeks of age. After weaning, ewes were housed in a barn with access to a drylot and exposed to natural photoperiod and ambient temperatures of Lexington, KY (30° 2' N latitude). On August 5, 1988, lambs were transported to an environmentally controlled building where they were housed for the remainder of the experiment. In this building ewes were exposed to a constant ambient temperature (20 C) and artificial photoperiod that simulated the natural photoperiod for Lexington, KY during August, September, and October. Ewes were ovariectomized at approximately 6.5 months of age. Diet From weaning until the initiation of the experiment, feed intake of lambs was restricted to 0.5 kg daily in order to maintain constant body weights. The experimental diet used is described in Table 1. Beginning on day 1 of the experiment (October 18, 1988), the daily amount of feed each lamb was allowed was gradually increased. By day 8 of the experiment, each lamb was receiving 1 kg experimental diet (i.e. twice maintenance) daily. This level of feed intake was continued until the termination of the study. As per accepted animal husbandry practices, lambs received injections of vitamins A, D, and E as well as selenium at appropriate ages. Animals were allowed water ad libitum before and during the experiment. Catheterization On the days before sequential blood samples were collected, ewes received indwelling catheters (16-gauge Angiocath, Becton Dickinson Co., Sandy, UT) in either one (for day 0) or both (for day 14) jugular veins. Catheters were used for collecting TABLE 1. Composition of diet fed to lambs
0 6
Ingredient
% Composition
Cottonseed hulls Cracked corn Soybean meal" Trace mineralized salt Ground limestone Crude protein*
15.0 78.0 5.6 0.5 0.9 10.4
Crude protein = 45%. KjeldahlNx6.25.
1935
blood (5.1-cm length) or infusing lipid or vehicle (8.3-cm length). Experimental
design
To monitor the pulsatile pattern of LH release in chronically feed-restricted lambs (n = 14), blood samples were collected at 12-min intervals for 4 h on day 0. Blood was sampled between 0800-1200 h. After the twice maintenance feeding period, sequential blood samples were again collected. Blood was sampled every 12 min from 0800-2000 h on day 14. Additionally, 8-h iv infusions of lipid (n = 9) or 0.9% saline solution (n = 5) were given between 1200-2000 h. Lipid or 0.9% saline solution was infused continuously at a rate of 0.48 ml/min (95.8 mg fatty acids/min) using a peristaltic pump (model 1203, Harvard Apparatus, South Natick, MA). This lipid infusion regimen has been demonstrated to increase circulating FFA concentrations to levels characteristic of fasted sheep (11). The lipid emulsion (20% Intralipid, Kabi Vitrum, Inc., Alameda, CA) consisted of the following fatty acids: linoleic (50%), oleic (26%), palmitic (10%), linolenic (9%), stearic (3.5%), and less than 1% each of myristic, arachidic, and behenic. The fatty acid content of Intralipid is generally comparable to that present in the circulation of fasted sheep (12), with the exception of stearic and linoleic acid which comprised 35% and 4%, respectively, of the total FFA in plasma. Blood-handling procedures and assays Blood samples were allowed to clot at 4 C for 24 h; serum was harvested and then stored at -20 C until assayed. All samples were quantified for LH using a previously reported RIA procedure (11). The intra- and interassay coefficients of variation were 5.2% and 21.1%, respectively. The assay sensitivity was 0.02 ng/tube. Samples collected on day 14 were analyzed for GH concentrations using a RIA described previously (11). The assay sensitivity and intra- and interassay coefficients of variation were 0.34 ng/tube, 9.2%, and 17.0%, respectively. Long chain FFA concentrations were determined for samples collected at 1048 h on day 1 and at 1200, 1400,1600, 1800, and 2000 h on day 14 using a radiochemical assay (11). Intra- and interassay coefficients of variation were 3.5% and 11.6%, respectively. Statistical analyses For the day 14 analysis, the 12-h experiment was divided into three 4-h periods corresponding to the 4-h period before initiating infusions (period 1) and the two consecutive 4-h periods following the initiation of infusions (periods 2 and 3, respectively). For each ewe, mean hormone concentrations and the number and amplitude of hormone pulses, as determined by PULSAR (11, 13), were calculated for each period. Data were then subjected to analysis of variance for a repeated measures design (14). The model included treatment (FFA vs. saline), period, and treatment X period interaction as possible sources of variation. The effect of treatment was tested against animal within treatment. The effects of period and treatment
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1936
FFA EFFECTS ON LH AND GH IN LAMBS 30
(kg)
X period interaction were tested using animal within treatment X period as the error term. If significant treatment X period interactions were detected, then additional analyses were conducted. Separate one-way analyses of variance were conducted to determine the main effect of treatment on mean hormone concentrations, number of hormone pulses, and hormone pulse amplitude in each individual period. One-way analyses of variance were also used to determine the main effect of period on the various indices of hormone secretion within each of the two treatments. Finally, period effects were analyzed for significant linear and quadratic components to assess changes in hormone secretion over time. Serum FFA concentrations on day 14 were analyzed using a split plot in time analysis of variance (14). The statistical model included treatment, sample (time), and the treatment X sample interaction. The effect of treatment was tested using animal within treatment as the error term. Sample and treatment X sample were tested against animal within treatment X sample. If significant treatment X sample interactions were detected, then one-way analyses of variance were conducted to determine the main effect of treatment within each sample and the main effect of sample within each treatment. Sample effects within treatments were analyzed for significant linear and quadratic components. Mean LH concentrations, pulse frequency and amplitude, FFA concentrations, and body weights on day 0 and during the 4-h period before infusions began on day 14 were compared using paired t tests.
Endo • 1990 Voll26«No4
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On day 0, mean body weight, mean LH concentrations, LH pulse frequency, and LH pulse amplitude were 22.4 ± 0.5 kg, 0.57 ± 0.08 ng/ml, 0.45 ± 0.09 pulses/h, and 0.73 ±0.11 ng/ml, respectively (Fig. 1). The mean serum FFA concentration was 6.4 ± 0.6 mg/100 ml. An increased level of feeding, beginning on day 1, increased (P < 0.01) mean body weight by 16%, and this growth was associated with an 82% increase in LH pulse frequency by day 14 (Fig. 1). Mean LH concentrations, LH pulse amplitude, and serum FFA concentrations (6.4 ± 0.9 mg/100 ml) on day 14 were similar (P > 0.1) to those on day 0. Depicted in Fig. 2 are profiles of serum LH concentrations for two ewes on days 0 and 14. Analysis of variance revealed an interaction (P < 0.0001) between treatment and sample for FFA concentrations on day 14. Serum concentrations of FFA increased linearly (P < 0.01) after initiation of lipid treatment and reached a peak level of 27.6 ± 2.9 mg/100 ml by the fourth hour of infusion (Fig. 3). Saline infusion had no effect (P > 0.1) on FFA concentrations. Mean serum LH concentrations, LH pulse frequency, and LH pulse amplitude on day 14 were similar (P > 0.1) for lipid- and saline-infused ewes (Fig. 4). Serum LH concentrations in individual ewes receiving each treatment are shown in Fig. 2. Mean serum GH concentrations, GH pulse frequency,
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Day FIG. 1. Mean body weight, mean serum LH concentrations, LH pulse frequency, and LH pulse amplitude in nutritionally growth-restricted lambs (n = 14) before (day 0) and after (day 14) twice maintenance feeding. Blood samples were collected at 12-min intervals for 4 h on day 0 and 12 h on day 14. Values are the mean ± SE. Only the data for the first 4 h on day 14 (i.e. before initiation of lipid or 0.9% saline infusion) are shown. Paired t tests revealed an increase (P < 0.01, denoted by horizontal bars and asterisks) in body weight and LH pulse frequency between days 0 and 14.
and GH pulse amplitude for lipid- and saline-infused ewes on day 14 are shown in Fig. 5. Depicted in Fig. 6 are profiles of serum GH concentrations in ewes from each treatment group. Analyses of variance revealed treatment x period interactions for mean GH concentrations (P < 0.0002) and GH pulse frequency (P < 0.02). Mean serum GH concentrations were similar (P > 0.1) between groups before infusion and averaged 14.0 ± 0.8 ng/ml. Lipid infusion decreased (P < 0.01) mean GH
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FFA EFFECTS ON LH AND GH IN LAMBS Day O
1937
lipid-infused compared to saline-infused animals during period 2 (P < 0.01), but not during period 3 (P > 0.1). The amplitude of GH pulses was highly variable and was not affected (P > 0.1) by treatment.
Day 14
Discussion
4
0
10
12
Hour FIG. 2. Serum LH concentrations in samples collected at 12-min intervals from representative ovariectomized ewe lambs during restricted feed intake (day 0) and after 14 days of twice maintenance feeding (day 14). The top and bottom panels represent animals infused iv, beginning at 4 h on day 14, with lipid and saline, respectively. Arrows indicate pulses, as determined by PULSAR.
concentrations by 51%, with saline-treated ewes having higher levels (P < 0.001) than lipid-treated ewes during periods 2 and 3. Saline infusion had no effect (P > 0.1) on mean GH concentrations. GH pulse frequency was similar between treatment groups before infusions began (0.36 ± 0.07 pulses/h). The frequency of GH pulses decreased 81% (P < 0.006) during lipid infusion and was lowest during period 2. This resulted in a quadratic effect of period (P < 0.01). Saline infusion had no effect (P > 0.1) on GH pulse frequency. The frequency of GH pulses was lower in ^
Previous studies have demonstrated that undernutrition inhibits pulsatile LH secretion in ovariectomized ewe lambs (5, 6). Only when nutritionally growth-restricted animals are given access to ad libitum feed does LH pulse frequency increase (6). Similarly, in the present study LH pulse frequency increased with an increased level of feeding. This increase in LH secretion was almost certainly due to enhanced nutrition and was not an agerelated steroid-independent increase in gonadotropin release (15). Even before they were allowed ad libitum feed, the lambs in this study were at an age at which well fed flockmates were neuroendocrinologically postpubertal. Thus, nutritional constraints aside, the ovariectomized animals in the current investigation would be expected to display a high frequency of LH pulses. Furthermore, in an experiment conducted at a similar time as the investigation described here, ovariectomized ewe lambs not given increased amounts of feed displayed infrequent LH pulses ( i_
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FIG. 4. Mean serum LH concentrations, LH pulse frequency, and LH pulse amplitude determined in blood samples collected at 12-min intervals from ovariectomized ewe lambs infused with lipid (n = 9) or 0.9% saline solution (n = 5) iv. Values are the mean ± SE for the 4-h period before infusion (•; period 1) and the two consecutive 4-h periods after initiation of infusion (M, period 2; • , period 3). All indices of LH secretion were similar (P > 0.1) for lipid- and saline-treated animals, as determined by analysis of variance.
ing, FFA levels would decrease, and LH release would increase. In the current investigation, however, FFA concentrations did not change with increased feeding, although LH pulse frequency increased dramatically. These results provide evidence that FFA are not responsible for the low frequency of LH pulses in the nutritionally growth-restricted lamb. The lack of an inhibitory effect of FFA on LH release is further supported by the fact that lipid infusion increased FFA concentrations in twice maintenance-fed lambs to levels characteristic of fasted sheep (11), yet the high frequency mode of pulsatile LH secretion was
FFA
Saline
FIG. 5. Serum GH concentrations, GH pulse frequency, and GH pulse amplitude determined in blood samples collected at 12-min intervals from ovariectomized ewe lambs infused with lipid (n = 9) or 0.9% saline solution (n = 5) iv. Values are the mean ± SE for the 4-h period before infusion (D; period 1) and the two consecutive 4-h periods (ffl, period 2; • , period 3) after initiation of infusion. Analyses of variance revealed a decrease in mean GH concentrations (P < 0.01) and frequency of GH pulses (P < 0.006) in lipid-infused animals. Horizontal bars denote periods in which there were significant effects of treatment (**, P< 0.01;***, P< 0.001).
unaffected. Similarly, we previously reported that LH secretion was unaffected by FFA in mature ovariectomized ewes (11). Thus, acute increases in FFA concentrations caused by lipid administration do not appear to influence gonadotropin release in sheep. These results, however, do not negate the prospect that long-term treatment with lipid might have an inhibitory effect on LH secretion. Nutrition may influence LH secretion via blood-borne signals that reflect metabolic status (7). While the results
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FFA EFFECTS ON LH AND GH IN LAMBS 301
FFA
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Vol. 126, No. 4 Printed in U.S.A.
Effects of Free Fatty Acids on Luteinizing Hormone and Growth Hormone Secretion in Ovariectomized Lambs* M. J. ESTIENNE, K. K. SCHILLO, S. M. HILEMAN, M. A. GREEN, S. H. HAYES, AND J. A. BOLING Department of Animal Sciences, University of Kentucky, Lexington, Kentucky 40546-0215
ABSTRACT. The effects of FFA on circulating LH and GH concentrations in ovariectomized ewe lambs were investigated. Lambs (n = 14) were weaned at 2.5 months, ovariectomized at 6.5 months, and used at 8.5 months of age. From weaning until day 0 of the experiment, lambs were fed to maintain body weights (23 kg). On day 0, serum FFA concentrations and mean serum LH concentrations and number and amplitude of LH pulses, as assessed in blood samples collected every 12 min for 4 h, were 6.4 ± 0.6 mg/100 ml, 0.57 ± 0.08 ng/ml, 0.45 ± 0.09 pulses/h, and 0.73 ± 0.11 ng/ml, respectively. Double the maintenance feeding, beginning day 1, increased (P < 0.01) body weights by 16% and LH pulse frequency by 82%, but had no effect (P > 0.1) on FFA concentrations, mean LH concentrations, or LH pulse amplitude by day 14. On day 14, lambs were infused with
I
NADEQUATE nutrition is associated with aberrant reproductive function in several species (1-4). Foster and Olster (5) demonstrated that the nutritionally growth-restricted female lamb failed to reach puberty and proposed that the inhibition of sexual maturation was due to inadequate secretion of LH. They hypothesized that the frequency of LH pulses was not sufficient to stimulate development of ovarian follicles to the preovulatory stage, and as a consequence, circulating concentrations of estradiol failed to reach levels necessary for induction of the preovulatory surge of LH (6). The mechanisms by which poor nutrition influences LH secretion in the developing lamb are only partially understood. Restricted food intake prolongs hypersensitivity of the hypothalamic-pituitary axis to the estradiol negative feedback characteristic of prepubertal lambs (5). Nutritionally growth-restricted lambs, however, have infrequent LH pulses after ovariectomy (5, 6), a finding consistent with a steroid-independent suppressive effect Received November 11,1989. Address requests for reprints to: Dr. Keith K. Schillo, Department of Animal Sciences, 807 Agricultural Science Building South, University of Kentucky, Lexington, Kentucky 40546-0215. * Presented in part at the Neuroendocrine Control of Reproduction Symposium, November 1989, Napa, CA. This work was supported by a grant from the USDA (87-CRCR-1-2567), and this paper (no. 89-5208) is published with the approval of the director of the Kentucky Agricultural Experiment Station.
lipid (n = 9; 95.8 mg/min) or 0.9% saline solution (n = 5) for 8 h. Blood samples were collected at 12-min intervals for 12 h,
beginning 4 h before infusions. FFA levels increased (P < 0.01) in lipid-infused animals to 27.6 ± 2.9 mg/100 ml by 4 h of infusion. Mean LH concentrations and LH pulse frequency and amplitude were unaffected (P > 0.1) by treatment. In contrast, mean GH concentrations and GH pulse frequency, which were similar (P > 0.1) between groups before infusion (14.0 ± 0.8 ng/ ml and 0.36 ± 0.07 pulses/h, respectively) were decreased by FFA treatment by 51% (P < 0.01) and 81% (P < 0.006), respectively. GH pulse amplitude was highly variable and unaffected (P > 0.1) by treatment. In summary, elevated FFA levels appear to inhibit the release of GH, but not LH, in the ovariectomized ewe lamb. (Endocrinology 126: 1934-1940, 1990)
of poor nutrition on LH secretion. It is likely that the effects of nutrition on pulsatile LH release involve central mechanisms controlling hypothalamic discharge of LHRH, rather than impaired pituitary function, since pituitary responsiveness to LHRH was not compromised in the nutritionally growth-restricted lamb (6). Cameron et al. (7) proposed that nutritional influences on LH secretion may be attributed to changes in circulating concentrations of metabolites. Previous studies (8-10) have demonstrated that an inverse relationship exists between the level of feeding and circulating concentrations of FFA. Thus, we hypothesized that the reduced frequency of LH pulses in the nutritionally growth-restricted lamb may be due to inhibition of the hypothalamic-pituitary axis by FFA. Therefore, the first objective of the following experiment was to assess, in nutritionally growth-restricted lambs, changes that occur in steroid-independent LH secretion after an increased level of feeding and relate these changes to circulating concentrations of FFA. Secondly, we sought to determine the effects of lipid infusion on LH secretion after an increased level of feeding. We have previously demonstrated that elevated FFA concentrations caused by lipid infusion are associated with decreased levels of GH (11). In that study, mature ewes (mean age and weight, 3.3 yr and 53.8 kg, respec-
1934
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FFA EFFECTS ON LH AND GH IN LAMBS
tively) were used (11). The effect of exogenous lipid on GH release may differ in animals of differing age and/or metabolic status. Thus, as a third objective of this experiment we characterized the effects of lipid infusion on GH concentrations in nutritionally growth-restricted lambs after an increased level of feeding.
Materials and Methods General Fourteen cross-bred ewe lambs, predominantly of the Hampshire breed, were used. Lambs were born on approximately February 1, 1988, and were weaned at 8-10 weeks of age. Ewe lambs in our flock typically reach puberty by 28 weeks of age. After weaning, ewes were housed in a barn with access to a drylot and exposed to natural photoperiod and ambient temperatures of Lexington, KY (30° 2' N latitude). On August 5, 1988, lambs were transported to an environmentally controlled building where they were housed for the remainder of the experiment. In this building ewes were exposed to a constant ambient temperature (20 C) and artificial photoperiod that simulated the natural photoperiod for Lexington, KY during August, September, and October. Ewes were ovariectomized at approximately 6.5 months of age. Diet From weaning until the initiation of the experiment, feed intake of lambs was restricted to 0.5 kg daily in order to maintain constant body weights. The experimental diet used is described in Table 1. Beginning on day 1 of the experiment (October 18, 1988), the daily amount of feed each lamb was allowed was gradually increased. By day 8 of the experiment, each lamb was receiving 1 kg experimental diet (i.e. twice maintenance) daily. This level of feed intake was continued until the termination of the study. As per accepted animal husbandry practices, lambs received injections of vitamins A, D, and E as well as selenium at appropriate ages. Animals were allowed water ad libitum before and during the experiment. Catheterization On the days before sequential blood samples were collected, ewes received indwelling catheters (16-gauge Angiocath, Becton Dickinson Co., Sandy, UT) in either one (for day 0) or both (for day 14) jugular veins. Catheters were used for collecting TABLE 1. Composition of diet fed to lambs
0 6
Ingredient
% Composition
Cottonseed hulls Cracked corn Soybean meal" Trace mineralized salt Ground limestone Crude protein*
15.0 78.0 5.6 0.5 0.9 10.4
Crude protein = 45%. KjeldahlNx6.25.
1935
blood (5.1-cm length) or infusing lipid or vehicle (8.3-cm length). Experimental
design
To monitor the pulsatile pattern of LH release in chronically feed-restricted lambs (n = 14), blood samples were collected at 12-min intervals for 4 h on day 0. Blood was sampled between 0800-1200 h. After the twice maintenance feeding period, sequential blood samples were again collected. Blood was sampled every 12 min from 0800-2000 h on day 14. Additionally, 8-h iv infusions of lipid (n = 9) or 0.9% saline solution (n = 5) were given between 1200-2000 h. Lipid or 0.9% saline solution was infused continuously at a rate of 0.48 ml/min (95.8 mg fatty acids/min) using a peristaltic pump (model 1203, Harvard Apparatus, South Natick, MA). This lipid infusion regimen has been demonstrated to increase circulating FFA concentrations to levels characteristic of fasted sheep (11). The lipid emulsion (20% Intralipid, Kabi Vitrum, Inc., Alameda, CA) consisted of the following fatty acids: linoleic (50%), oleic (26%), palmitic (10%), linolenic (9%), stearic (3.5%), and less than 1% each of myristic, arachidic, and behenic. The fatty acid content of Intralipid is generally comparable to that present in the circulation of fasted sheep (12), with the exception of stearic and linoleic acid which comprised 35% and 4%, respectively, of the total FFA in plasma. Blood-handling procedures and assays Blood samples were allowed to clot at 4 C for 24 h; serum was harvested and then stored at -20 C until assayed. All samples were quantified for LH using a previously reported RIA procedure (11). The intra- and interassay coefficients of variation were 5.2% and 21.1%, respectively. The assay sensitivity was 0.02 ng/tube. Samples collected on day 14 were analyzed for GH concentrations using a RIA described previously (11). The assay sensitivity and intra- and interassay coefficients of variation were 0.34 ng/tube, 9.2%, and 17.0%, respectively. Long chain FFA concentrations were determined for samples collected at 1048 h on day 1 and at 1200, 1400,1600, 1800, and 2000 h on day 14 using a radiochemical assay (11). Intra- and interassay coefficients of variation were 3.5% and 11.6%, respectively. Statistical analyses For the day 14 analysis, the 12-h experiment was divided into three 4-h periods corresponding to the 4-h period before initiating infusions (period 1) and the two consecutive 4-h periods following the initiation of infusions (periods 2 and 3, respectively). For each ewe, mean hormone concentrations and the number and amplitude of hormone pulses, as determined by PULSAR (11, 13), were calculated for each period. Data were then subjected to analysis of variance for a repeated measures design (14). The model included treatment (FFA vs. saline), period, and treatment X period interaction as possible sources of variation. The effect of treatment was tested against animal within treatment. The effects of period and treatment
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 18:44 For personal use only. No other uses without permission. . All rights reserved.
1936
FFA EFFECTS ON LH AND GH IN LAMBS 30
(kg)
X period interaction were tested using animal within treatment X period as the error term. If significant treatment X period interactions were detected, then additional analyses were conducted. Separate one-way analyses of variance were conducted to determine the main effect of treatment on mean hormone concentrations, number of hormone pulses, and hormone pulse amplitude in each individual period. One-way analyses of variance were also used to determine the main effect of period on the various indices of hormone secretion within each of the two treatments. Finally, period effects were analyzed for significant linear and quadratic components to assess changes in hormone secretion over time. Serum FFA concentrations on day 14 were analyzed using a split plot in time analysis of variance (14). The statistical model included treatment, sample (time), and the treatment X sample interaction. The effect of treatment was tested using animal within treatment as the error term. Sample and treatment X sample were tested against animal within treatment X sample. If significant treatment X sample interactions were detected, then one-way analyses of variance were conducted to determine the main effect of treatment within each sample and the main effect of sample within each treatment. Sample effects within treatments were analyzed for significant linear and quadratic components. Mean LH concentrations, pulse frequency and amplitude, FFA concentrations, and body weights on day 0 and during the 4-h period before infusions began on day 14 were compared using paired t tests.
Endo • 1990 Voll26«No4
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Results
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On day 0, mean body weight, mean LH concentrations, LH pulse frequency, and LH pulse amplitude were 22.4 ± 0.5 kg, 0.57 ± 0.08 ng/ml, 0.45 ± 0.09 pulses/h, and 0.73 ±0.11 ng/ml, respectively (Fig. 1). The mean serum FFA concentration was 6.4 ± 0.6 mg/100 ml. An increased level of feeding, beginning on day 1, increased (P < 0.01) mean body weight by 16%, and this growth was associated with an 82% increase in LH pulse frequency by day 14 (Fig. 1). Mean LH concentrations, LH pulse amplitude, and serum FFA concentrations (6.4 ± 0.9 mg/100 ml) on day 14 were similar (P > 0.1) to those on day 0. Depicted in Fig. 2 are profiles of serum LH concentrations for two ewes on days 0 and 14. Analysis of variance revealed an interaction (P < 0.0001) between treatment and sample for FFA concentrations on day 14. Serum concentrations of FFA increased linearly (P < 0.01) after initiation of lipid treatment and reached a peak level of 27.6 ± 2.9 mg/100 ml by the fourth hour of infusion (Fig. 3). Saline infusion had no effect (P > 0.1) on FFA concentrations. Mean serum LH concentrations, LH pulse frequency, and LH pulse amplitude on day 14 were similar (P > 0.1) for lipid- and saline-infused ewes (Fig. 4). Serum LH concentrations in individual ewes receiving each treatment are shown in Fig. 2. Mean serum GH concentrations, GH pulse frequency,
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Day FIG. 1. Mean body weight, mean serum LH concentrations, LH pulse frequency, and LH pulse amplitude in nutritionally growth-restricted lambs (n = 14) before (day 0) and after (day 14) twice maintenance feeding. Blood samples were collected at 12-min intervals for 4 h on day 0 and 12 h on day 14. Values are the mean ± SE. Only the data for the first 4 h on day 14 (i.e. before initiation of lipid or 0.9% saline infusion) are shown. Paired t tests revealed an increase (P < 0.01, denoted by horizontal bars and asterisks) in body weight and LH pulse frequency between days 0 and 14.
and GH pulse amplitude for lipid- and saline-infused ewes on day 14 are shown in Fig. 5. Depicted in Fig. 6 are profiles of serum GH concentrations in ewes from each treatment group. Analyses of variance revealed treatment x period interactions for mean GH concentrations (P < 0.0002) and GH pulse frequency (P < 0.02). Mean serum GH concentrations were similar (P > 0.1) between groups before infusion and averaged 14.0 ± 0.8 ng/ml. Lipid infusion decreased (P < 0.01) mean GH
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FFA EFFECTS ON LH AND GH IN LAMBS Day O
1937
lipid-infused compared to saline-infused animals during period 2 (P < 0.01), but not during period 3 (P > 0.1). The amplitude of GH pulses was highly variable and was not affected (P > 0.1) by treatment.
Day 14
Discussion
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Hour FIG. 2. Serum LH concentrations in samples collected at 12-min intervals from representative ovariectomized ewe lambs during restricted feed intake (day 0) and after 14 days of twice maintenance feeding (day 14). The top and bottom panels represent animals infused iv, beginning at 4 h on day 14, with lipid and saline, respectively. Arrows indicate pulses, as determined by PULSAR.
concentrations by 51%, with saline-treated ewes having higher levels (P < 0.001) than lipid-treated ewes during periods 2 and 3. Saline infusion had no effect (P > 0.1) on mean GH concentrations. GH pulse frequency was similar between treatment groups before infusions began (0.36 ± 0.07 pulses/h). The frequency of GH pulses decreased 81% (P < 0.006) during lipid infusion and was lowest during period 2. This resulted in a quadratic effect of period (P < 0.01). Saline infusion had no effect (P > 0.1) on GH pulse frequency. The frequency of GH pulses was lower in ^
Previous studies have demonstrated that undernutrition inhibits pulsatile LH secretion in ovariectomized ewe lambs (5, 6). Only when nutritionally growth-restricted animals are given access to ad libitum feed does LH pulse frequency increase (6). Similarly, in the present study LH pulse frequency increased with an increased level of feeding. This increase in LH secretion was almost certainly due to enhanced nutrition and was not an agerelated steroid-independent increase in gonadotropin release (15). Even before they were allowed ad libitum feed, the lambs in this study were at an age at which well fed flockmates were neuroendocrinologically postpubertal. Thus, nutritional constraints aside, the ovariectomized animals in the current investigation would be expected to display a high frequency of LH pulses. Furthermore, in an experiment conducted at a similar time as the investigation described here, ovariectomized ewe lambs not given increased amounts of feed displayed infrequent LH pulses ( i_
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FIG. 4. Mean serum LH concentrations, LH pulse frequency, and LH pulse amplitude determined in blood samples collected at 12-min intervals from ovariectomized ewe lambs infused with lipid (n = 9) or 0.9% saline solution (n = 5) iv. Values are the mean ± SE for the 4-h period before infusion (•; period 1) and the two consecutive 4-h periods after initiation of infusion (M, period 2; • , period 3). All indices of LH secretion were similar (P > 0.1) for lipid- and saline-treated animals, as determined by analysis of variance.
ing, FFA levels would decrease, and LH release would increase. In the current investigation, however, FFA concentrations did not change with increased feeding, although LH pulse frequency increased dramatically. These results provide evidence that FFA are not responsible for the low frequency of LH pulses in the nutritionally growth-restricted lamb. The lack of an inhibitory effect of FFA on LH release is further supported by the fact that lipid infusion increased FFA concentrations in twice maintenance-fed lambs to levels characteristic of fasted sheep (11), yet the high frequency mode of pulsatile LH secretion was
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Saline
FIG. 5. Serum GH concentrations, GH pulse frequency, and GH pulse amplitude determined in blood samples collected at 12-min intervals from ovariectomized ewe lambs infused with lipid (n = 9) or 0.9% saline solution (n = 5) iv. Values are the mean ± SE for the 4-h period before infusion (D; period 1) and the two consecutive 4-h periods (ffl, period 2; • , period 3) after initiation of infusion. Analyses of variance revealed a decrease in mean GH concentrations (P < 0.01) and frequency of GH pulses (P < 0.006) in lipid-infused animals. Horizontal bars denote periods in which there were significant effects of treatment (**, P< 0.01;***, P< 0.001).
unaffected. Similarly, we previously reported that LH secretion was unaffected by FFA in mature ovariectomized ewes (11). Thus, acute increases in FFA concentrations caused by lipid administration do not appear to influence gonadotropin release in sheep. These results, however, do not negate the prospect that long-term treatment with lipid might have an inhibitory effect on LH secretion. Nutrition may influence LH secretion via blood-borne signals that reflect metabolic status (7). While the results
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FFA EFFECTS ON LH AND GH IN LAMBS 301
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