NUTRITION HAS A STRONG influence on the activity

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1 /04/$15.00/0 Endocrinology 145(7): Printed in U.S.A. Copyright 2004 by The Endocrine Society doi: /en Further Evidence for the Role of Glucose as a Metabolic Regulator of Hypothalamic Gonadotropin-Releasing Hormone Pulse Generator Activity in Goats SATOSHI OHKURA, TORU ICHIMARU, FUMIAKI ITOH, SHUICHI MATSUYAMA, AND HIROAKI OKAMURA Laboratory of Neuroendocrinology (S.O., T.I., H.O.), National Institute of Agrobiological Sciences, Tsukuba , Japan; Department of Animal Physiology and Nutrition (F.I.), National Institute of Livestock and Grassland Science, Tsukuba , Japan; and Laboratory of Animal Reproduction (S.M.), Nagoya University, Nagoya , Japan The present study examined the relative importance of blood glucose vs. free fatty acids as a metabolic signal regulating GnRH release as measured electrophysiologically by multiple-unit activity (MUA) in the arcuate nucleus/median eminence region in ovariectomized, estradiol-treated goats. MUA was recorded before, during, and after: 1) cellular glucoprivation by peripheral infusion of 2-deoxy-D-glucose (2DG; 25, 50, and 75 mg/kg h, iv); 2) peripheral hypoglycemia in response to various doses ( mu/kg h, iv) of insulin infusion; and 3) cellular lipoprivation induced by peripheral infusion of sodium mercaptoacetate (MA; 2.4 mg/kg h alone or combined with 25 mg/kg h of 2DG, iv), and effects on the interval of characteristic increases in MUA (MUA volleys) were examined. Infusion of the highest dose of 2DG increased the mean Abbreviations: CV, Coefficient of variation; 2DG, 2-deoxy-d-glucose; E 2, estradiol-17 ; EIA, enzyme immunoassay; FFA, free fatty acid; MA, mercaptoacetate; MUA, multiple-unit activity; OVX, ovariectomized. Endocrinology is published monthly by The Endocrine Society ( the foremost professional society serving the endocrine community. interval between MUA volleys, whereas the lower doses of 2DG had no effect on volley interval. The MUA volley intervals lengthened as insulin-induced hypoglycemia became profound. There was a negative correlation between MUA volley intervals and blood glucose concentrations during insulin infusion, and coinfusion of glucose with insulin returned the MUA volley interval to a normal frequency. Infusion of MA alone or MA with 2DG did not increase MUA volley intervals. These findings demonstrate that glucose availability, but not fatty acids, regulates the GnRH pulse generator activity in the ruminant. Glucose is considered a key metabolic regulator that fine-tunes pulsatile GnRH release. (Endocrinology 145: , 2004) NUTRITION HAS A STRONG influence on the activity of the hypothalamo-pituitary-gonadal axis in mammals. Reduced energy intake inhibits gonadal function through suppression of GnRH/LH secretion. Indeed, shortterm fasting suppresses pulsatile LH secretion in rats (1), sheep (2), monkeys (3), and humans (4). In immature ewes, hypogonadotropism and reduced pulsatile GnRH release in the pituitary portal circulation (5) or median eminence (6) has been revealed during growth retardation induced by dietary restriction. It is widely accepted that pulsatile GnRH discharge into the pituitary portal circulation is governed by the hypothalamic GnRH pulse generator (7), although its neural component has yet to be fully understood. Thus, information on nutritional status would be conveyed to the GnRH pulse generator and regulate its activity. Using a method for monitoring the electrical activity of the GnRH pulse generator by the hypothalamic multiple-unit activity (MUA) recording technique in the arcuate nucleus/median eminence region, which has been developed in monkeys (8), rats (9), and goats (10 12), we recently demonstrated that the activity of the GnRH pulse generator is suppressed by fasting for 4din ovariectomized (OVX), estradiol-17 (E 2 )-treated goats (13). Although the mechanism by which undernutrition inhibits the activity of the GnRH pulse generator has yet to be fully determined, a number of studies suggest a role for glucose availability in this phenomenon in both monogastric and ruminant species. Pharmacological cellular glucoprivation induced by iv administration of 2-deoxy-d-glucose (2DG), an antimetabolic glucose analog that competitively inhibits intracellular glucose oxidation (14), rapidly suppresses pulsatile LH secretion in rats (15) and sheep (16 18). When blood glucose concentrations are decreased by iv administration of insulin, LH pulses are also inhibited in rats (19, 20), sheep (21, 22), and monkeys (23). Moreover, central inhibition of the electrical activity of the GnRH pulse generator by insulininduced hypoglycemia has been demonstrated electrophysiologically in monkeys (24, 25) and rats (26). In male monkeys, the degree of restoration of pulsatile LH secretion after shortterm fasting is linearly associated with the size of the refed meal (27), suggesting that pulsatile LH secretion is regulated in a graded manner by nutrient(s) whose levels are closely related with food intake. Therefore, if glucose is important as a metabolic signal, graded changes in its availability would be expected to be accurately reflected in pulsatile GnRH/LH release. Although a number of reports have described the effects of abrupt and severe glucoprivation induced by 2DG or insulin, few studies have examined the temporal relationship between gradual changes in glucose availability and the activity of the GnRH pulse generator. In addition to glucose, other oxidizable metabolites may play some role in the nutritional control of GnRH/LH release. Recently Lado-Abeal et al. (28) reported that a paren- 3239

2 3240 Endocrinology, July 2004, 145(7): Ohkura et al. Glucose Controls GnRH Pulse Generator teral glucose supplement in fasted male monkeys only partially reversed the fasting-induced reduction in pulsatile LH secretion and suggested that other nutrients such as free fatty acids (FFAs) are required to completely restore LH pulses. In female Syrian hamsters, a combination of glucoprivation and lipoprivation is necessary for the suppression of estrous behavior to the extent observed after fasting for 48 h (29). These findings are from studies that used monogastric animals; however, the role of FFAs in the nutritional control of pulsatile GnRH/LH release in ruminants is not well understood. In the present study, to further elucidate the role of glucose in regulating pulsatile GnRH release, 2DG or insulin was continuously administered to OVX, E 2 -treated goats, and the effects of decreased glucose availability on the activity of the GnRH pulse generator were electrophysiologically assessed using a recording technique of the hypothalamic MUA that is associated with pulsatile LH secretion. Moreover, to investigate whether FFAs are involved in the nutritional control of GnRH pulse generator activity in goats, the effect of a pharmacological blockade of its use was also examined. Materials and Methods Animals and MUA recordings Five OVX adult Shiba goats weighing kg were used. They were implanted with an array of bilateral recording electrodes aimed at the arcuate nucleus/median eminence region under halothane anesthesia according to the stereotaxic procedure (30). The electrodes consisted of six Teflon-insulated platinum-iridium wires (75 m in diameter; A-M Systems, Inc., Carlsborg, WA) in each side. The method for recording MUA has been described elsewhere (10, 12, 13), and characteristic increases in the MUA (MUA volleys) were considered to be electrophysiological manifestations of the GnRH pulse generator activity (10). The MUA recording was conducted continuously throughout the experimental period. Goats were loosely tied to a stanchion and maintained in an animal room under controlled conditions: a 12-h light, 12-h dark cycle (lights on at 0800 h), 23 C, and 50% relative humidity. They were fed with a standard pelleted diet and hay wafer and had free access to water and supplemental minerals. At least 1 wk before the experiment, the goats were sc implanted with silicon tubing (inner diameter, 3.0 mm; outer diameter, 5.0 mm; length, 20 mm; Dow Corning, Midland, MI) containing crystalline E 2 (Sigma Chemical Co., St. Louis, MO). This E 2 treatment has been reported to produce plasma E 2 levels of 4 8 pg/ml (31), which are found during the luteal phase of the estrous cycle in intact goats. On the day before the experiment, each goat was fitted with bilateral jugular catheters (18 gauge; Medicut, Nippon Sherwood Medical Industries Ltd., Tokyo, Japan), one for blood sampling and the other for infusion. The catheters were maintained by daily flushing of heparinized saline (20 IU/ml) throughout the experiment. All experimental procedures were approved by the Committee on the Care and Use of Experimental Animals at the National Institute of Agrobiological Sciences. Experimental design Experiment 1: infusion of 2DG. The effects of continuous infusion of several doses of 2DG on the MUA volley intervals were examined. During a pretreatment period, the appearance of at least two MUA volleys was confirmed. Five minutes after the onset of the last MUA volley, a single dose of 2DG (1.56, 3.12, or 4.68 mg/kg ml; Wako Pure Chemical Industries, Ltd., Osaka, Japan), which was dissolved in saline, was infused iv for 2.5 h through the jugular catheter with a peristaltic pump (Atto Corp., Tokyo, Japan). The rate of infusion was 16 ml/h; thus, the doses of 2DG infused were 25, 50, and 75 mg/kg h, respectively. As an isoosmotic control, xylose (75 mg/kg h), a nonutilizable sugar, was infused by the same procedure. To apply the 2DG treatment in experiment 4 (see below), the duration of 2DG treatment (2.5 h) was made shorter than that of treatments with insulin or sodium mercaptoacetate (MA) in other experiments. The treatment was replicated four times in each goat such that it received three different doses of 2DG and the xylose infusion. Each infusion was performed on a different day with at least a 2-d interval. Blood samples (1 ml) were collected at 30-min intervals beginning at least 1 h before the onset of infusion through the indwelling jugular catheter contralateral to that used for 2DG or xylose infusion. After centrifugation, plasma was separated and stored at 30 C until assayed for insulin. In a separate experiment, 0.5-ml blood samples were collected at 5- or 10-min intervals in some goats when they received the infusion of the highest dose (75 mg/kg h) of 2DG on a different experimental day to examine the correlation of MUA volleys with plasma LH pulses. Blood was centrifuged, and plasma was stored at 30 C until the determination of LH concentrations. Experiment 2: infusion of insulin. The effects of a continuous infusion of several doses of insulin on the MUA volley intervals were analyzed. Bovine insulin (Wako) was initially dissolved in 0.1 n HCl and then further diluted with saline to make different doses ( mu/ kg ml) of solution. Final concentrations of 0.1 n HCl in the administered solutions were less than 0.2% (vol/vol). On an experimental day, the occurrence of at least two MUA volleys was observed during a pretreatment period. After a bolus injection of 1 ml of infusate, a single dose of insulin solution was infused iv with a syringe pump (Terumo Corp., Tokyo, Japan) for 5 h through the jugular catheter. The rate of infusion was 400 l/h; thus, the dose of insulin infused was mu/kg h. The effect of insulin infusion on plasma glucose concentrations was different among individuals, so that the dose of insulin varied. As a control treatment, saline was infused by the same procedure. Each goat received three to four different doses of insulin and saline infusion, and each infusion was performed on a different day with at least a 2-d interval. Blood samples (1 ml) were collected every 30 min beginning 1 h before the start of infusion. After centrifugation, plasma was separated and glucose concentrations were promptly determined with the YSI model 27 glucose analyzer (Yellow Springs Instruments Inc., Yellow Springs, OH). Experiment 3: concurrent infusion of both insulin and glucose. The effects of elevated glucose levels during insulin-induced hypoglycemia on the MUA volley intervals were examined in three goats. At first, the infusion of insulin solution ( mu/kg h) was begun as described in experiment 2. After confirmation that blood glucose concentrations had decreased less than 30 mg/dl with 1.5- to 3-h infusion of insulin as determined promptly with the glucose analyzer, a 5% glucose solution (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan) was infused concurrently for a further h with a peristaltic pump connected with the tubing for insulin infusion with a sterile three-way stopcock. The rate of glucose infusion was manually adjusted throughout the infusion period to maintain blood glucose concentrations at around 60 mg/dl. Blood samples (1 ml) were collected every 30 min beginning 1 h before the start of insulin infusion and processed as described in experiment 2. Experiment 4: infusion of MA in the absence or presence of 2DG. The effects of the infusion of MA, a depressor of the -oxidation pathway that suppresses long-chain acyl-coenzyme A dehydrogenase activity (32), on MUA volley intervals were examined in the absence or presence of 2DG. Sodium MA (Sigma) was dissolved in saline to make a single dose (2.4 mg/kg ml) of solution. On an experimental day, the occurrence of at least two MUA volleys was observed during a pretreatment period, and then the MA solution was infused iv for 5 h with a syringe pump. The rate of MA infusion was 1 ml/h; thus, the dose of MA infused were 2.4 mg/kg h. The solution of MA was replaced with a freshly prepared one halfway through the infusion period to avoid losing the efficacy of MA solution. In a separate experiment on a different day, while the MA solution was infused for 5hinthemanner described above, a low dose of 2DG (25 mg/kg h) was infused for 2.5 h in the latter half of infusion period at a rate of 16 ml/h with a peristaltic pump, which was connected with the tubing for the MA infusion via a sterile three-way stopcock. The dose of MA (2.4 mg/kg h) and the procedure for infusion was selected in a pilot study because the above treatment with MA increased plasma FFA levels (see Results) and doses higher than 2.4 mg/kg h appeared to induce side effects in some goats. Blood samples (1 ml) were collected at 30-min intervals beginning 30

3 Ohkura et al. Glucose Controls GnRH Pulse Generator Endocrinology, July 2004, 145(7): min before the start of MA infusion through the indwelling jugular catheter contralateral to that used for infusion. Plasma was separated and stored at 30 C until assayed for FFAs. Assays Plasma LH concentrations were measured in duplicate using an enzyme immunoassay (EIA) developed by Mutayoba et al. (33) with a slight modification. EIA plates of 96 wells (Corning Inc., Corning, NY) were coated with 100 l/well of coating buffer [15 mm Na 2 CO 3 10H 2 O and 35 mm NaHCO 3 (ph 9.6)] containing 5 g antirabbit IgG (H L) goat serum (Seikagaku Corp., Tokyo, Japan). Aliquots (20 l) of ovine LH standard (NIDDK-oLH-I-4) or unknown samples were mixed with 100 l antiovine LH serum (YM no. 18) (34) diluted 1:250,000 in EIA buffer [42 mm Na 2 HPO 4 2H 2 O, 8 mm KH 2 PO 4,20mm NaCl, 4.8 mm EDTA 2Na, and 0.05% BSA (ph 7.5)] in each well. Each plate was incubated at 4 C for 24 h and, after decantation and the addition of bovine LH labeled with d-biotinoyl- -aminocaproic acid-n-hydroxysuccinimide ester (1:15,000) in 100 l of EIA buffer into each well, incubated further at 4 C for 24 h. The solution was decantated and 100 l/well of EIA buffer containing 20 ng of streptavidin peroxidase (Sigma) was added into each well. After 15 min incubation at 4 C, the plate was washed four times (350 l/well) with 0.05% Tween 80 (Sigma) and further incubated at 37 C for 40 min in the dark with gentle shaking after addition of 150 l/well of substrate solution [a mixture of equal volumes of solution A (0.1% urea hydrogen peroxide, 0.1 m Na 2 HPO 4 2H 2 O, and 0.05 m citric acid H 2 O) (ph 5.0), and solution B (0.05% 3,3,5,5 -tetramethylbenzidine, 4% dimethyl sulfoxide, and 0.05 m citric acid H 2 O) (ph 2.4)]. The reaction was terminated by addition of 50 l well of 4 N H 2 SO 4, and the absorbance was measured at 450 nm with a Benchmark microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA). The lowest detectable level of LH was 0.39 ng/ml. The intra- and interassay coefficients of variation (CVs) at 7.40 ng/ml were 6.6 and 8.9%, respectively. Plasma insulin concentrations were quantified in a single determination with a commercial RIA kit (Eiken Chemical Co., Tokyo, Japan). The detectable limit for insulin was 5 U/ml, and intra- and interassay CVs for 50- l samples of a plasma pool containing U/ml of insulin were 8.0 and 10.0%, respectively. Plasma FFA concentrations were determined in duplicate by an enzymatic colorimetric assay with a commercial kit (NEFA C-Test Wako; Wako) using 96-well microtiter plates. The protocol for this assay has been described (35), although we slightly modified the incubation period (40 min for Color Reagent A and 20 min for Color Reagent B) and temperature (37 C). The detectable limit for FFAs was meq/liter, and intra- and interassay CVs for 5- l samples of a plasma pool containing meq/liter of FFAs were 7.6 and 15.9%, respectively. Data analysis For the evaluation of GnRH pulse generator activity, the intervolley interval (minutes) of the MUA was used in all experiments. In experiment 1, the mean MUA volley interval over the 2.5-h infusion period was calculated for each animal and then for each treatment group. For the purpose of statistical analysis and data presentation, values were expressed as percentages of the mean MUA volley interval observed during the control period in the nontreated condition. Significant differences between treatments were analyzed using two-way ANOVA followed by the Dunnett post hoc test for multiple comparisons. Statistical differences in mean plasma insulin concentrations over the 2.5-h infusion period between control (xylose) and 2DG treatments were analyzed using two-way ANOVA followed by the Dunnett post hoc test. In experiment 2, the correlation between mean blood glucose levels and the interval between MUA volleys was analyzed as follows. First, blood glucose levels measured every 30 min for 8 h beginning 1 h before the start of insulin/saline infusion were plotted on graph paper with clock time on the horizontal axis in a single treatment in each goat. Timings of the occurrence of the MUA volleys were marked on it, and the integrated areas under the curve of blood glucose levels between two successive volleys were calculated. Then these values were divided by the intervals (minutes) between the two volleys, and resulting values were designated as mean blood glucose levels for given MUA volley intervals. To examine the temporal correlation between mean glucose levels and MUA volley intervals in each goat, data from each treatment with three to four different doses of insulin and saline for each animal were combined and analyzed using Pearson s correlation coefficient. Due to individual variability in the frequency of MUA volleys, the number of points used to analyze the correlation between the two parameters differs across individuals. In experiment 3, the mean MUA volley interval during the infusion of insulin alone or simultaneous infusion of insulin and glucose was compared with that in the untreated condition. A statistical comparison of the interval was made as in experiment 1. In experiment 4, the mean MUA volley interval over the 2.5-h period during the infusion with MA alone or MA with 2DG was compared with that in the untreated condition. A statistical comparison of the interval was made as in experiment 1. Plasma FFA concentrations between pre- (0 h) and posttreatments (5 h after the start of MA infusion) were compared using a paired t test. Results Experiment 1: infusion of 2DG MUA volley intervals lengthened during the infusion period when a high dose (75 mg/kg h) of 2DG was administered (Fig. 1A). Infusion of a moderate dose (50 mg/kg h) of 2DG slightly prolonged the time between volleys, but a low dose (25 mg/kg h) of 2DG and xylose had no effect. Each LH pulse followed the corresponding MUA volley before, during and after 2DG infusion (Fig. 1B), indicating that pulsatile LH secretion was well correlated with GnRH pulse generator activity throughout the experimental period. The basal level of MUA remained stable during 2DG infusion. The GnRH pulse generator activity was suppressed by 2DG treatment in a dose-dependent manner (Fig. 1C). The mean volley interval was significantly (P 0.05) longer during high-dose 2DG infusion than during xylose treatment. The moderate-dose 2DG treatment tended to prolong the interval between MUA volleys, compared with xylose treatment, but the effect was not significant (P 0.05). The lowdose 2DG treatment had no effect. Mean plasma insulin concentrations during the 2.5-h infusion of high (75 mg/kg h), moderate (50 mg/kg h), and low (25 mg/kg h) doses of 2DG and xylose (75 mg/kg h) were , , , and U/ml, respectively (mean sem). There was no significant difference (P 0.05) in mean insulin concentrations between xylose and each 2DG treatments. Experiment 2: infusion of insulin Representative profiles of MUA and blood glucose levels during the infusion of insulin or saline are shown in Fig. 2. The infusion of insulin at the relatively high dose (135 mu/ kg h) for 5 h resulted in a decrease in blood glucose concentrations to less than 30 mg/dl and a marked lengthening of the interval between MUA volleys during the infusion period (Fig. 2A). After the cessation of infusion, blood glucose levels gradually increased and eventually exceeded levels before infusion, whereas the volley intervals promptly returned to the preinfusion level. The basal level of MUA remained stable during insulin infusion. The infusion of saline had no effect on the MUA volleys and blood glucose concentrations (Fig. 2B). The correlation between mean blood glucose concentra-

4 3242 Endocrinology, July 2004, 145(7): Ohkura et al. Glucose Controls GnRH Pulse Generator FIG. 1. A, Profiles of MUA and volley intervals (closed circle) in a representative OVX, E 2 -treated goat during xylose (Xyl) or 2DG infusion (0 2.5 h; shaded periods). B, A representative profile of MUA with changes in plasma LH concentrations (open circle) and volley intervals (closed circle) during infusion of 2DG at 75 mg/kg h (0 2.5 h; shaded periods). C, Mean ( SEM) volley intervals (percent of OVX E 2 value) during xylose or 2DG infusion (n 5 goats). *, P 0.05 vs. xylose infusion. tions and MUA volley intervals during treatments with insulin or saline was analyzed in individual goats as shown in Fig. 3. A significant (P 0.05) negative correlation between the two parameters was observed in all goats. FIG. 2. Representative profiles of MUA, volley intervals (closed circle), and plasma glucose concentrations (open circle) in an OVX, E 2 - treated goat during infusion of insulin at 135 mu/kg h (A) or saline (B)for5h(0 5 h;shaded periods). Experiment 3: concurrent infusion of both insulin and glucose On infusion of the high dose ( mu/kg h) of insulin, as shown in Fig. 4, blood glucose concentrations fell less than 30 mg/dl, and the interval between volleys increased. When hypoglycemia was improved by subsequent coinfusion of glucose, the frequency of MUA volleys promptly recovered to preinfusion levels in all three goats. Statistical comparison revealed that the significantly (P 0.05) prolonged MUA volley intervals during insulin infusion ( % vs. untreated condition, mean sem) were returned to normal intervals during the simultaneous infusion of insulin and glucose ( % vs. untreated condition). After the cessation of insulin and glucose infusion, blood glucose concentrations dropped for a short period, and this transient hypoglycemia was accompanied by a slight increase in the interval between MUA volleys in two goats (nos. 70 and 79).

5 Ohkura et al. Glucose Controls GnRH Pulse Generator Endocrinology, July 2004, 145(7): FIG. 3. Individual plots of the correlation between mean plasma glucose concentrations and MUA volley intervals in experiment 2. These data were collected during treatments with three to four different doses of insulin and saline for each animal. A minimum and maximum dose of insulin used for each goat is indicated in each panel. Due to individual variability in the frequency of MUA volleys, the number of points used to analyze the correlation between the two parameters differs across individuals. Significant negative correlations were found in all goats (P 0.05). Experiment 4: infusion of MA in the absence or presence of 2DG When MA (2.4 mg/kg h) was infused together with a low dose of 2DG (25 mg/kg h) for 2.5 h, no change in volley intervals was observed, although plasma FFA concentrations increased toward the end of the infusion (Fig. 5A). Infusion of MA alone (2.4 mg/kg h) for 5 h also did not cause any apparent change in the MUA (Fig. 5B). Statistical comparison revealed that there was no significant difference (P 0.05) in mean MUA volley interval over the 2.5-h period of infusion between the MA-treated, MA plus 2DG-treated and control groups (Fig. 5C). Plasma FFA concentrations (mean sem) were significantly (P 0.05, paired t test) higher in the MA-treated ( meq/liter) and MA plus 2DG-treated ( meq/liter) groups at the end (5 h after the start of MA infusion) than beginning of the treatments ( and meq/liter, respectively). Discussion By monitoring the MUA in the arcuate nucleus/median eminence region in the hypothalamus, the present study revealed that 2DG-induced inhibition of cellular glucose availability suppressed the activity of the hypothalamic GnRH pulse generator in OVX, E 2 -treated goats. Moreover, the GnRH pulse generator activity was inhibited by insulin-

6 3244 Endocrinology, July 2004, 145(7): Ohkura et al. Glucose Controls GnRH Pulse Generator FIG. 4. Individual profiles of MUA, volley intervals (closed circle), and plasma glucose concentrations (open circle) in OVX, E 2 -treated goats during insulin infusion (shaded periods) concurrent with glucose infusion (closed bar). induced hypoglycemia. These findings suggest that glucose availability is one of the metabolic regulators of the GnRH pulse generator and plays a key role in the nutritional control of reproduction in ruminant species. In ruminants, little glucose is absorbed from the digestive tract, so they have a highly efficient ability in gluconeogenesis and mobilizing glucose reserves to maintain plasma glucose concentrations about half those of most simple-stomached animals. Nevertheless, glucose turnover in ruminants does not differ substantially from that of monogastric animals (36); thus, it is reasonable to suppose that glucose is a key metabolic regulator controlling physiological functions, even in ruminants. The results in the present study are consistent with previous reports in sheep, in which the administration of 2DG (16 18) or insulin-induced hypoglycemia (21, 22) suppressed pulsatile LH secretion. It is most likely that information on the decreased availability of glucose reaches the hypothalamus and reduces GnRH pulsatility in ruminants as shown electrophysiologically in monogastric animals such as monkeys (24, 25) and rats (26) with insulin-induced hypoglycemia. The alteration of glucose availability does not seem to have a general, nonspecific effect in the brain. The fact that the basal level of MUA remained stable during 2DG or insulin infusion, whereas the volley intervals lengthened, indicates that those treatments altered the activity of the GnRH pulse generator itself but did not impair overall neuronal function. The infusion of several doses of insulin induced hypoglycemia with a variety of blood glucose concentrations. The statistical analysis of temporal changes in MUA volley intervals and mean glucose concentrations during insulin infusion revealed a significant negative correlation between the two parameters in each goat. The interval between MUA volleys lengthened as the insulin-induced hypoglycemia became more profound. This indicated that gradual changes in glucose availability would be precisely reflected in the activity of the GnRH pulse generator. Moreover, the prolonged volley intervals were promptly shortened when hypoglycemia was relieved by concurrent glucose infusion with insulin, suggesting that changes in the glucose availability were conveyed to the GnRH pulse generator with a short latency, perhaps on a minute-to-minute basis. These results provide further evidence that glucose functions as a metabolic signal to fine-tune the pulsatile release of GnRH in ruminants. The MUA volley intervals promptly returned to the preinfusion level, whereas blood glucose levels gradually increased after the cessation of insulin infusion in experiment 2 (Fig. 2A), indicating that there was a short time lag between the recovery of MUA volleys and blood glucose concentrations. This suggests that a start of increase in blood glucose levels also serves as a cue to release the suppression of the GnRH pulse generator activity after severe hypoglycemia. Clearly, further experiments will be required to investigate this notion. It is still possible that nutritional conditions are monitored as the total amount of energy available from multiple metabolites, and glucose partially contributes to the regulation of the GnRH pulse generator activity. Indeed, in monkeys, a parenteral supplement of glucose alone is not sufficient to completely reverse the fasting-induced reduction in pulsatile LH secretion (28), and the involvement of nutrients other than glucose in this phenomenon is suggested (37). Thus, it would be expected that when other metabolites are unavailable, a relatively mild blockade of glucose availability is sufficient to suppress the activity of the GnRH pulse generator. Because FFAs would also be an important energy substrate in the nutritional control of reproduction (29), its role as a metabolic signal was examined in the present study. However, neither MA alone nor the combination of MA and a low dose of 2DG changed the interval between MUA volleys. It is unlikely that the dose of MA used in this study was inadequate because a compensatory rise in plasma FFA levels was apparent, even in animals treated with MA alone. A plausible explanation for the absence of an inhibitory effect of lipoprivation on the GnRH pulse generator is that FFAs do not serve as a metabolic signal, at least not in normally fed goats. Alternatively, other metabolites such as ketone bodies and volatile (short-chain) fatty acids, the major energy-yielding substrates in ruminants, may have some role as metabolic signals. It has been shown that pulsatile LH secretion was stimulated by supplementing a maintenance diet with volatile fatty acids in mature sheep (38). Concerning the administration of insulin, it could be argued that high levels of insulin per se represent the suppression of the GnRH pulse generator. It has been shown that insulin alters LH pulse frequency in diabetic sheep (39), and insulin receptors in the brain are involved in the control of

7 Ohkura et al. Glucose Controls GnRH Pulse Generator Endocrinology, July 2004, 145(7): FIG. 5. A, Representative profiles of MUA, volley intervals (closed circle), and plasma FFA concentrations (open circle) in two OVX, E 2 -treated goats during MA (0 5 h; shaded periods) and 2DG (2.5 5 h; closed bar) infusion. B, A representative profile of MUA, volley intervals (closed circle), and plasma FFA concentrations (open circle) during the infusion of MA (0 5 h;shaded periods) alone. C, Mean ( SEM) volley intervals (percent of OVX E 2 value) during MA or MA plus 2DG infusion (n 5 goats). LH secretion in mice (40). Moreover, glucose availability might be modified during hyperinsulinemia through the insulin-dependent glucose transporter, GLUT4, in the hypothalamus (41). In the present study, however, concurrent infusion of glucose with insulin reinstated the activity of GnRH pulse generator, even though the plasma concentrations of insulin remained high. In addition, the suppressive effect of 2DG on MUA volleys was evident without any obvious fluctuations in insulin concentrations between control and 2DG treatment. These observations strongly suggest that hypoglycemia itself, not a direct action of insulin, is the cause of suppression of the GnRH pulse generator activity in goats. This notion is consistent with reports that insulininduced reduction of pulsatile LH secretion in sheep (21), and hypothalamic MUA volleys in monkeys (25) and rats (26) is prevented by supplemental glucose administration. It is likely that information on the availability of glucose eventually reaches the hypothalamus and influences the pulsatile GnRH release because direct suppression of the GnRH pulse generator in the hypothalamus, measured as a lengthening of the interval between MUA volleys, was evident in the present study. However, the location of the putative glucose-sensing mechanism, by which a change in glucose availability regulates the activity of the GnRH pulse generator, remains unknown. It has been demonstrated that the glucoprivic suppression of pulsatile GnRH/LH secretion is mediated by a glucose-sensing mechanism in the central nervous system because the intracerebroventricular infusion of 2DG into the fourth cerebral ventricle abolished LH pulses in adult sheep (18). A more precise identification of the glucose-sensing site was made in studies using rats: the infusion of 2DG targeted at the fourth cerebral ventricle suppressed the frequency of LH pulses (42), and the inhibition of pulsatile LH secretion by insulin-induced hypoglycemia was completely prevented when the area postrema, a circumventricular organ within the fourth ventricle, was removed (43). These findings in sheep and rats strongly suggest that the central glucose-sensing mechanism is located in the hindbrain, and information on glucose availability is conveyed from this neural substrate to the hypothalamic GnRH pulse generator. In conclusion, the results of the present study show that the activity of the hypothalamic GnRH pulse generator is suppressed by 2DG-induced glucoprivation or insulin-induced hypoglycemia in E 2 -treated OVX goats. The finding that there is a significant temporal correlation between blood glucose levels and the activity of the GnRH pulse generator

8 3246 Endocrinology, July 2004, 145(7): Ohkura et al. Glucose Controls GnRH Pulse Generator lends further support to the importance of glucose in the regulation of pulsatile GnRH release in ruminants. Acknowledgments We are grateful to Dr. A. F. Parlow and the National Hormone and Peptide Program (Torrance, CA) for reagents used in the LH EIA; Dr. Y. Mori (The University of Tokyo) for providing antiovine LH serum (YM no. 18); and Dr. A. Miyamoto (Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan) for his technical advice on the LH EIA and providing biotin-labeled bovine LH. We also thank Ms. Y. Sakairi and Mr. K. Sakurai for their technical assistance throughout the study. Received November 7, Accepted March 18, Address all correspondence and requests for reprints to: Dr. Satoshi Ohkura, Laboratory of Neuroendocrinology, National Institute of Agrobiological Sciences, 2 Ikenodai, Tsukuba , Japan. ohkura@affrc.go.jp. 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