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Endocrinology, doi:10.1210/en.2008-0016
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Endocrinology Vol. 149, No. 6 3016-3024
Copyright © 2008 by The Endocrine Society

Central Lipoprivation-Induced Suppression of Luteinizing Hormone Pulses Is Mediated by Paraventricular Catecholaminergic Inputs in Female Rats

Somchai Sajapitak1, Kinuyo Iwata1, Mohammad Shahab, Yoshihisa Uenoyama, Shunji Yamada, Mika Kinoshita, Farida Y. Bari, Helen I'Anson, Hiroko Tsukamura and Kei-ichiro Maeda

Graduate School of Bioagricultural Sciences (S.S., K.I., M.S., Y.U., S.Y., M.K., F.Y.B., H.T., K.-i.M.), Nagoya University, Nagoya 464-8601, Japan; and Department of Biology (H.I.), Washington and Lee University, Lexington, Virginia 24450

Address all correspondence and requests for reprints to: Kei-ichiro Maeda, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan. E-mail: keimaeda{at}agr.nagoya-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The present study aims to clarify the role of fatty acids in regulating pulsatile LH secretion in rats. To produce an acute central lipoprivic condition, mercaptoacetate (MA), an inhibitor of fatty acids oxidation, was administered into the fourth cerebroventricle (4V) in ad libitum fed ovariectomized (OVX) rats (0.4, 2, and 10 µmol/rat) with or without an estradiol (E2) implant producing diestrus plasma E2 levels. Pulsatile LH secretion was suppressed by 4V MA administration in a dose-dependent manner in both OVX and OVX plus E2 rats. Mean LH levels and LH pulse frequency and amplitude were significantly reduced by the highest dose of MA in OVX rats, and by the middle and highest dose of MA in E2-treated rats, suggesting that estrogen enhanced LH suppression. Blood glucose levels increased immediately after the highest dose of MA in both groups. Fourth ventricular injection of trimetazidine (2 and 3 µmol/rat), another inhibitor of fatty acids oxidation, also inhibited pulsatile LH release, resulting in significant and dose-dependent suppression of LH pulse frequency and an increase in blood glucose levels in OVX plus E2 rats. In contrast, peripheral injection of the highest 4V dose of MA (10 µmol/rat) did not alter LH release or blood glucose levels. Microdialysis of the hypothalamic paraventricular nucleus (PVN) revealed that norepinephrine release in the region was increased by 4V MA administration. Preinjection of {alpha}-methyl-p-tyrosine, a catecholamine synthesis inhibitor, into the PVN completely blocked the lipoprivic inhibition of LH and the counter-regulatory increase in blood glucose levels in OVX plus E2 rats. Together, these studies indicate that fatty acid availability may be sensed by a central detector, located in the lower brainstem to maintain reproduction, and that noradrenergic inputs to the PVN mediate this lipoprivic-induced suppression of LH release.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT IS WELL accepted that energy availability is one of the critical factors regulating gonadal function in peripubertal and adult animals in a variety of physiological states (1, 2, 3, 4, 5, 6, 7). Experimentally, food restriction (1, 8) or acute food deprivation (9) inhibits pulsatile LH secretion in rats through noradrenergic (NE) neurons projecting to the hypothalamic paraventricular nucleus (PVN) (10). Pulsatile LH release is sensitive to the availability of oxidizable fuels, such as glucose (7), because pharmacological glucoprivation and insulin-induced hypoglycemia are well-established inhibitors of LH release and gonadal activity (11, 12, 13). Glucose availability may be monitored by a specific detector(s) located in the brain (14, 15), including the hindbrain, to control feeding (16), estrous cyclicity (17), and LH release (18). The information on glucose availability may be conveyed by a specific neural pathway(s), such as neuropeptide Y (NPY)/NE ascending inputs to the PVN (6). Norepinephrine or epinephrine released in the PVN has mediated glucoprivic suppression of LH release (19), whereas NPY plays an important role in relaying glucoprivic information to the hypothalamus to control food intake in mice (20).

Previous studies have demonstrated that pharmacological blockade of the oxidation of fatty acids, another metabolic fuel, increases food intake through a vagally mediated pathway in rats, suggesting that a fatty acid-sensing mechanism is located in peripheral organs, such as digestive organs (16). Estrous cyclicity and sexual behavior are abolished by peripheral lipoprivation in rats and hamsters (4, 7). In addition, we have previously demonstrated that peripheral administration of mercaptoacetate (MA), an inhibitor of fatty acid oxidation, suppresses pulsatile LH release in ad libitum fed female rats (21). Thus, fatty acids may provide an additional metabolic signal to control reproductive functions. However, little is known about the fatty acid-sensing system and central pathways responsible for the lipoprivic regulation of reproduction.

The present study aims to clarify specific detectors and pathways that may mediate lipoprivic suppression of LH secretion. We first examined whether central lipoprivation with MA or trimetazidine (TMZ) blocks pulsatile LH secretion in the female rat to explore the possibility of a central detector to monitor fatty acid availability. Because administration of MA or TMZ, inhibitors of fatty acid oxidation, into the fourth cerebroventricle (4V) inhibited pulsatile LH secretion in the first experiment, we tested whether neurons projecting to the PVN mediate the lipoprivic inhibition of LH release. Specifically, we aimed to determine if PVN NE release from these ascending neurons suppressed LH release during MA-induced lipoprivation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult female Wistar-Imamichi strain rats (Imamichi Institute for Animal Reproduction, Ibaraki, Japan) weighing 230–270 g were housed under constant conditions of photoperiod and temperature (14-h light, 10-h dark; lights on 0500 h; temperature 24 ± 2 C), and fed ad libitum a pelleted food containing 25.4% crude protein, 4.4% fat, and 50.3% soluble nonnitrogenous matter (CE-2; CLEA Japan Inc., Tokyo, Japan). Water, but not the food, was available throughout the blood sampling. All surgical procedures were performed under ether or isoflurane anesthesia and aseptic condition.

Animals having shown at least two consecutive 4-d estrous cycles were bilaterally ovariectomized (OVX) 2 wk before blood sampling to serve as the OVX group. Some OVX animals immediately received a sc SILASTIC brand implant (inner diameter, 1.5 mm; outer diameter, 3.0 mm; length 25.0 mm; Dow Corning, Midland, MI) containing estradiol-17β (E2) (Sigma-Aldrich, St. Louis, MO) dissolved in peanut oil at 20 µg/ml for 1 wk to serve as the OVX plus E2 group. The E2 implant has produced plasma E2 levels found at diestrus (22). The estrogen treatment was chosen because it caused a significant suppression of LH pulses when animals were subjected to 48-h fasting (22), and enhanced the glucoprivic suppression of LH pulses (12).

The present studies were approved by the Committee on Animal Experiments of the Graduate School of Bioagricultural Sciences, Nagoya University.

Effect of 4V MA or TMZ injection on LH pulses
One week before blood sampling, a stainless-steel guide cannula (23 gauge; Plastics One Inc., Roanoke, VA) was stereotaxically implanted into the 4V in OVX and OVX plus E2 rats (n = 5–6 per group), as previously described (18). The tip of the guide cannula was placed at 12.5-mm posterior and 8.0- mm ventral to bregma at midline. An internal cannula (26 gauge; Plastics One) was inserted into the guide cannula to allow administration of sodium 2-MA (Sigma-Aldrich), a blocker of fatty acid β-oxidation by mitochondrial acyl-coenzyme A dehydrogenase inhibition (23, 24). The MA was freshly prepared in ultrapure water at doses of 0.4, 2, or 10 µmol/2 µl and infused with a microinfusion pump at a flow rate of 1 µl/min for 2 min into the 4V of the unstrained rats immediately after the first blood sample was taken. The 1-(2, 3, 4-trimethoxybenzyl) piperazine dihydrochloride (TMZ; Wako Chemical, Tokyo, Japan) was neutralized with sodium hydrate, diluted with ultrapure water at doses of 2 or 3 µmol/2 µl, and injected into the 4V. Control animals were infused with isotonic saline in the same manner. Blood samples (100 µl) were collected for determination of plasma LH levels every 6 min for 3 h from 1300 h through an indwelling atrial cannula (silicon tubing: inner diameter 0.5 mm; outer diameter, 1.0 mm; Shin-Etsu Polymer Co., Tokyo, Japan) that had been inserted the day before blood sampling through the right jugular vein. Blood glucose levels were measured in an additional volume (50 µl) of blood obtained at 12- and 30-min intervals during the first hour and last 2 h of the sampling period, respectively. Each blood sample was replaced with an equivalent volume of washed red blood cells obtained from other rats to keep the hematocrit constant. Plasma samples were obtained by immediate centrifugation and stored at –20 C until assayed for LH in all experiments. Plasma glucose levels were assayed immediately after blood sampling.

At the end of the blood sampling period, to ascertain if MA was infused into the 4V, the rats were anesthetized and infused with 2 µl 3% brilliant blue dye solution at the same rate and same volume as drug and saline infusions. Immediately after brilliant blue infusion, the rats were euthanized, and the brain was removed and sectioned to allow visual inspection of cannula placement and infusion site. Only the data from animals with appropriate cannula placement and dye infusion in the 4V were used.

Effect of iv injection of MA on LH pulses and blood levels of glucose and ketone body
To determine whether MA injected into the 4V leaked into the general circulation to exert its effect on LH secretion, 10 µmol MA (n = 6) or saline (n = 5) was iv injected through an atrial cannula at 1300 h in OVX plus E2 animals. Blood sampling was conducted in the same manner as in the previous 4V MA or TMZ studies. Plasma levels of 3-β-hydroxybutylate (3HB), a ketone body, were measured in addition to plasma glucose and LH levels.

Determination of NE release in the PVN after 4V MA injection with microdialysis
To determine whether NE release in the PVN increases after 4V MA injection, microdialysis of the PVN was conducted in OVX plus E2 rats as described previously (25). Briefly, a guide cannula (AG-12; Eicom Corp., Kyoto, Japan) was stereotaxically implanted unilaterally into the PVN. After a 7-d recovery period, and 2 h before the microdialysis period, a microdialysis probe (2 mm in length, A-I-12-02; Eicom) was inserted into the PVN through the guide cannula. The PVN was then perfused continuously through the probe with Ringer’s solution, consisting of 147 mM NaCl, 4 mM KCl, and 2.3 mM CaCl2, using a microinfusion pump at 1 µl/min. Dialysate was collected into tubes containing 5 µl 0.02 N HCl every 20 min for 3 h starting at 1300 h and immediately after the 4V MA injection. The dialysate was centrifuged, and the supernatant was taken for NE assay.

Effect of blockade of catecholamine synthesis in the PVN on lipoprivic LH suppression
One week before blood sampling, two guide cannulas for microinjection into the PVN and 4V were stereotaxically implanted in OVX plus E2 animals. A guide cannula (26 gauge; Plastics One) was unilaterally implanted into the PVN with its tip placed at 2.3-mm posterior and 7.5-mm ventral to bregma and 0.5-mm lateral to midline. A guide cannula was implanted into the 4V as described previously. Three hours before the onset of blood sampling and 4V drug infusion, 50 µg {alpha}-methyl-p-tyrosine (AMPT) (Sigma-Aldrich) in 0.5 µl saline or an equal volume of the vehicle was infused for 2 min at a rate of 0.25 µl/min into the PVN through an inner cannula (31 gauge, Plastics One). The dose of AMPT was chosen based on our previous study, in which a single infusion of the same dose of AMPT reversed LH pulse suppression during acute fasting (26). MA (2 µmol/2 µl) or saline was injected into the 4V immediately after the first blood sample was withdrawn. This treatment regimen resulted in four groups of rats: PVN saline plus 4V saline (n = 5); PVN saline plus 4V MA (n = 4); PVN AMPT plus 4V saline (n = 5); and PVN AMPT plus 4V MA (n = 5). Blood sampling was performed as described previously.

At the end of the experiment, animals were infused with 2% brilliant blue dye into the PVN and 4V cannulas, and perfused with saline, followed by 10% formalin under deep anesthesia. Coronal sections of the brain were made at 50 µm and stained with thionin, and the placement of the PVN cannula was verified under a microscope. The location of the 4V cannula was verified with visual inspection in the aforementioned manner. Only the data from animals with appropriate cannula placement in the PVN and 4V were used.

Assays
Plasma LH concentrations were determined with a RIA kit provided by the National Hormone and Peptide Program, Harbor-University of California Los Angeles Medical Center, Los Angeles, CA. LH concentrations were expressed in terms of NIDDK-rLH-RP-3, and the least detectable concentration of LH in a 50-µl plasma sample was 0.16 ng/ml. The intraassay and interassay coefficients of variation were 6.2 and 12.3% (n = 5 assays), respectively. Plasma glucose level was determined with the glucose oxidase method using a commercial kit (Glucose B-Test; Wako, Osaka, Japan). Plasma 3HB concentrations were determined by a commercial kit (Sanwa, Tokyo, Japan).

A HPLC-electrochemical detector system was used to quantify NE levels. The system consisted of a reverse-phase column (Eicompack CA-5ODS, outer diameter 2.1 mm and 150 mm in length; Eicom) and an electrochemical detector (ECD300; Eicom) with an oxidation potential of +450 mV. The mobile phase consisted of 0.1 M phosphate buffer (pH 6.0) containing EDTA-2Na (50 mg/liter), sodium 1-octanesulfonate (400 mg/liter), and 5% methanol. The least detectable NE level was 12.5 pg, and the intraassay and interassay coefficients of variation were 5.6 and 14.4% (n = 10 assays), respectively.

Data analysis
LH pulses were identified with the PULSAR computer program (27) as previously described (28). Mean LH concentrations, frequency and amplitude of LH pulses, plasma glucose and 3HB concentrations, and NE in dialysate were compared between treatments by one-way ANOVA, followed by the Bonferroni multiple comparison test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of 4V administration of MA or TMZ on pulsatile LH release
Frequent LH pulses were observed in all animals with saline infusion into the 4V in both OVX and OVX plus E2 groups (Fig. 1AGo). In contrast, 4V MA infusion suppressed LH pulses in a dose-dependent manner. The highest dose of MA (10 µmol) immediately suppressed LH pulses in OVX rats regardless of estrogen replacement (Fig. 1AGo), and significantly (P < 0.05) suppressed all pulse parameters in both OVX and OVX plus E2 animals except for amplitude in the OVX group (Fig. 1BGo). The middle dose of MA (2 µmol) suppressed LH pulses only in OVX plus E2 animals and significantly (P < 0.05) lowered all LH pulse parameters (Fig. 1Go, A and B), whereas the same treatment did not significantly affect any pulse parameters in OVX rats without estrogen treatment. The lowest dose of MA (0.4 µmol/rat) did not affect pulsatile LH release or LH pulse parameters in either group (Fig. 1Go, A and B). Plasma glucose levels were increased by 4V MA injection in a dose-dependent manner (Fig. 1CGo). The highest dose of MA (10 µmol) significantly (P < 0.05) increased plasma glucose levels at 12–48 min and 1.5 h after MA in OVX rats, and at 12–36 min and 1.5–2 h after MA in OVX plus E2 rats compared with the vehicle-injected controls. The middle dose of MA caused a slight and transient increase in blood glucose levels in both groups but only resulted in a significant increase at 12 min after MA infusion in OVX rats. Plasma glucose levels did not fluctuate after 4V infusion of either saline or the lowest dose of MA.


Figure 1
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  		FIG. 1. A, Plasma LH profiles in representative OVX and E2-treated OVX (OVX plus E2) rats injected with saline (Sal) or MA at doses of 0.4, 2, and 10 µmol into the 4V. Drugs were injected immediately after the onset of blood sampling (arrows). Blood samples were collected for 3 h at 6-min intervals. Arrowheads indicate the peaks of LH pulses identified by Pulsar. B, Mean plasma LH concentrations for 3 h and the frequency and amplitude of LH pulses in OVX and OVX plus E2 rats receiving 4V injection of MA or saline. Values are means ± SEM. The numbers in each column represent the number of animals used. Values with different letters are significantly (P < 0.05) different from each other. C, Changes in mean plasma glucose levels after MA injection into the 4V in OVX and OVX plus E2 rats. Values are means ± SEM. *, P < 0.05, vs. saline-treated controls.

 
Injection of TMZ, another β-oxidation blocker (29), into the 4V caused a suppression of LH pulses in OVX plus E2 rats (Fig. 2AGo). Pulse analysis showed that LH pulse frequency was significantly decreased by the highest dose (3 µmol) of TMZ when injected into the 4V (Fig. 2BGo). Mean LH concentrations and LH pulse amplitude showed a slight suppression in TMZ-treated groups, but the effect was not significant. Both doses of TMZ caused a significant increase in plasma glucose concentrations compared with vehicle-injected controls (Fig. 2CGo).


Figure 2
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  		FIG. 2. A, Plasma LH profiles in representative OVX plus E2 rats injected with saline or TMZ at doses of 2 and 3 µmol into the 4V. Drugs were injected immediately after the onset of blood sampling (arrows). Blood samples were collected for 3 h at 6-min intervals. Arrowheads indicate the peaks of LH pulses identified by the Pulsar computer program. B, Mean plasma LH concentrations for 3 h and the frequency and amplitude of LH pulses in OVX plus E2 rats receiving 4V injection of TMZ or saline. Values are means ± SEM. The numbers in each column represent the number of animals used. Values with different letters are significantly (P < 0.05) different from each other. C, Changes in mean plasma glucose levels after TMZ injection into the 4V in OVX plus E2 rats. Values are means ± SEM. *, P < 0.05, vs. saline-treated controls.

 
Effect of iv injection of MA on pulsatile LH release
Peripheral injection of MA at the highest dose injected into the 4V did not cause any change in LH pulse patterns (Fig. 3AGo). Mean LH concentrations and LH pulse frequency and amplitude all remained unchanged after peripheral MA administration (Fig. 3BGo). In addition, plasma glucose and 3HB levels were not significantly different in MA-treated compared with control rats (Fig. 3Go, C and D).


Figure 3
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  		FIG. 3. A, Plasma LH profiles in representative OVX plus E2 rats receiving iv injection of saline (Sal) or MA at a dose of 10 µmol. Drugs were injected immediately after the onset of blood sampling (arrows). Blood samples were collected for 3 h at 6-min intervals. Arrowheads indicate the peaks of LH pulses identified by Pulsar. B, Mean plasma LH concentrations for 3 h and the frequency and amplitude of LH pulses in OVX plus E2 rats receiving iv injection of MA or saline. Values are means ± SEM. The numbers in each column represent the number of animals used. No significant differences are found between groups. C, Changes in mean plasma glucose levels after iv MA injection in OVX plus E2 rats. Values are means ± SEM. No significant differences are found between groups. D, Changes in mean plasma 3HB levels after iv MA injection in OVX plus E2 rats. Values are means ± SEM. No significant differences are found between groups.

 
Microdialysis determination of NE release in the PVN after 4V MA injection
Norepinephrine release in the PVN began to increase within 20 min after 4V MA injection and reached its peak 40 min after injection (Fig. 4Go). The peak value was significantly (P < 0.05) higher compared with the preinjection value (one-way ANOVA followed by the Bonferroni test).


Figure 4
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  		FIG. 4. Norepinephrine (NE) release in the PVN after 4V injection of MA at 10 µmol in OVX plus E2 rats using microdialysis. The arrow indicates the 4V MA injection at 1300 h immediately after the first sampling. The dialysate was collected every 20 min, and the NE content was expressed as a percent change to preinjection value. Values are means ± SEM. *, P < 0.05, vs. preinjection values (one-way ANOVA followed by the Bonferroni test).

 
Vehicle-treated control animals showed a significant (P < 0.05, Bonferroni test) increase in PVN NE release 2 h after injection at 1500 h compared with the preinjection value, which is probably related to the beginning of feeding.

Effect of PVN blockade of catecholamine synthesis on lipoprivic LH suppression
Figure 5AGo shows the placement of the cannula tip for AMPT injection into the PVN of a representative rat brain.


Figure 5
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  		FIG. 5. A, Photomicrograph of a frontal section of the brain in a representative animal implanted with a cannula into the PVN. The asterisk indicates the site of AMPT injection (50 µg/0.5 µl) in the PVN. 3V, third ventricle. Scale bar, 200 µm. B, Representative plasma LH profiles from OVX plus E2 rats injected with MA (2 µmol) or saline (Sal) into the 4V and receiving a preinjection of either AMPT or saline into the PVN. AMPT or saline was administered 3 h before the start of blood sampling. MA and saline were injected into the 4V at 1300 h immediately after the onset of blood sampling (arrows). Arrowheads indicate the peaks of LH pulses identified by Pulsar. C, Mean plasma LH concentrations for 3 h and the frequency and amplitude of LH pulses in OVX plus E2 rats treated with a 4V MA or saline injection and an AMPT or saline preinjection into the PVN. Values are means ± SEM. The numbers in each column represent the number of animals used. Values with different letters are significantly (P < 0.05) different from each other. D, Changes in mean plasma glucose levels after MA or saline injection into the 4V in OVX and OVX plus E2 rats receiving a preinjection with AMPT or saline into the PVN. Values are means ± SEM. *, P < 0.05, vs. saline-treated controls.

 
Fourth ventricular MA injection (2 µmol) alone caused an immediate suppression of LH release (Fig. 5BGo), with significant reduction of all pulse parameters (Fig. 5CGo). When the animals were pretreated with AMPT in the PVN, the 4V MA injection failed to inhibit LH release (Fig. 5BGo), and all LH pulse parameters were comparable with 4V saline-treated controls with or without PVN AMPT pretreatment (Fig. 5CGo). The increase in plasma glucose levels induced by 4V MA injection was also blocked by the PVN AMPT injection (Fig. 5DGo). Control animals with a 4V saline injection showed regular LH pulses and normal plasma glucose levels regardless of PVN AMPT treatment.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our first study showed that 4V administration of MA suppresses pulsatile LH secretion in a dose-dependent manner and, therefore, suggests that fatty acid oxidation in the area of the 4V is needed to maintain pulsatile LH secretion in the female rats. In addition, peripheral injection of MA did not affect LH secretion profiles, plasma glucose, or 3HB levels at the highest dose that was injected centrally, suggesting that MA injected into the 4V acts locally in the brainstem region. These data further indicate that any leakage into the periphery after 4V MA administration is unlikely to affect fatty acid metabolism in peripheral tissues to alter LH secretion patterns. In addition, 4V injection of TMZ, another β-oxidation blocker (29), suppressed LH release, suggesting that MA and TMZ exerted specific actions on local fatty acid metabolism in the brain, probably in the area around the 4V, thereby turning on inhibitory neural inputs to the GnRH/LH neurosecretory system.

Fatty acid availability has been well established to regulate food intake via peripheral sensory cells because systemic injection of MA increases food intake in rats, and the increase is abolished by sensory vagotomy using capsaicin (16). On the other hand, inhibition of fatty acid oxidation inhibits estrous cyclicity in hamsters only when combined with glucoprivation or fasting (13). Unlike food intake regulation by fatty acid oxidation, vagotomy does not eliminate lipoprivic-induced anestrus in fasted, fat, or lean hamsters, or lipoprivic-induced deficits in estrous behavior (30, 31). In addition, our previous study showed that peripheral MA injection acutely suppresses pulsatile LH secretion in female rats on a normal diet, and this suppression is not reversed by the vagotomy (21). Together with our current data, these studies suggest that lipoprivation induces feeding through a peripheral metabolic detector but suppresses the reproductive axis through a central detector.

The present study showed that MA or TMZ infused into the 4V suppressed pulsatile LH secretion. Thus, it is likely that lipoprivic information is sensed by the brain, and most likely by the lower brainstem. Importantly, the dye infused into the 4V after each experiment was always found in the 4V, indicating that MA was delivered only to the 4V and not back toward the third ventricle, further supporting the role of the lower brainstem as a site of metabolic sensing of fatty acid oxidation in the regulation of reproductive function (17, 18). The exact location of the metabolic detector sensing fatty acid availability is still unknown. Ritter et al. (32) first demonstrated that glucoreceptors are located in the hindbrain and control food intake and blood glucose levels. Specifically, the area postrema and solitary tract nucleus (NTS) regions have previously been proposed to be areas where the metabolic detector resides to regulate reproductive functions. Lesion of the area postrema/NTS area prevents suppression of the gonadal axis induced by nutritional deficiency (6). Our previous study also showed that central glucoprivation by 4V injection of 2-deoxyglucose, a glucose antagonist, suppresses pulsatile LH release (18). Pancreatic glucokinase, an enzyme considered to play a central role in sensing blood glucose level in B cells, was immunocytochemically localized in ependymocytes of the lower brainstem (33), and ependymocytes taken from the wall of the 4V showed changes in intracellular calcium levels in response to a change in extracellular glucose levels (15). Thus, we have hypothesized that the lower brainstem, and in particular the ependymocytes, sense glucose availability (34) and transmit this information via ascending neuronal pathways to regulate reproductive function. It is possible that these cells may also sense free fatty acid levels in cerebrospinal fluid and, thus, total energy availability to regulate reproductive functions. Acute central lipoprivation increased plasma glucose levels in the present study. It still remains unknown how those two energy signals, such as glucose and free fatty acid, are integrated in a sensing mechanism.

The ascending NE/NPY pathway has mediated glucoregulatory feeding responses (35, 36, 37, 38). Thus, our second study determined if this same pathway mediates the suppressive effects of lipoprivation on reproductive function. In particular, we determined if PVN NE release increased after 4V MA injection and if NE mediates suppression of LH release during MA-induced lipoprivation. We demonstrated that NE release was increased by 4V MA injection, and an injection of AMPT, a tyrosine hydroxylase inhibitor, into the PVN blocks the inhibitory effect of MA on pulsatile LH secretion. This suggests that NE inputs to the PVN mediate the suppressive effect of central lipoprivation on LH pulse patterns. This notion is further supported by a previous report, in which MA induces c-fos expression in the NTS, a major brainstem nucleus located on the dorsal surface of the medulla close to the 4V that sends NE projections to the PVN (39). Interestingly, our previous studies indicate that AMPT treatment into the PVN reverses LH suppression induced by fasting or peripheral 2-deoxyglucose-induced glucoprivation (19, 26). In addition, central injection of NE antagonists blocks fasting-induced suppression of LH pulses (40). Neurotoxic lesion of the NE/adrenergic (E) pathway, which originates in the brainstem and projects to the PVN (41, 42), suggests that the pathway is activated to inhibit reproductive activity during energy deficiency. These data, together with the results of our current study, suggest that central lipoprivation signals may be transmitted along the same inhibitory NE/E pathway to the PVN as glucoprivic and fasting signals. It should be noted that NPY is colocalized in the NE/E neuronal inputs to the PVN and may play a role in modulating the inhibitory effect of NE/E on GnRH/LH release during energy deficiency. NPY release before feeding during a refeeding period is abolished by neurochemical lesion of NE/E inputs to the PVN (43). In addition, NPY has potentiated the PVN NE release in rats (44). NPY may play a neuromodulatory role in stimulating NE/E release in the PVN and thereby stimulating feeding and suppress GnRH/LH release during energy deficiency. Thus, evidence is accumulating to suggest that energy availability, in a variety of forms, is sensed by the lower brainstem, and activates NE/E neurons projecting to the PVN to stimulate NE/E and/or NPY release and suppress LH secretion and, thus, reproduction. The present study should not negate the possibility for the involvement of dopaminergic inputs to the PVN in mediating lipoprivation-induced suppression of LH release because dopaminergic fibers are closely associated with PVN CRH neurons (45), and dopamine agonists affect the activity of PVN CRH neurons via a central action (46).

The present results also suggest that estrogen augments MA-induced lipoprivic suppression of LH secretion because the middle dose (2 µmol) of MA was able to suppress LH secretion in the presence, but not in the absence, of estrogen. The mechanism by which estrogen enhances the suppressive effect of lipoprivation on LH pulses is unknown. Our previous studies reveal that estrogen enhances LH suppression induced by 48-h fasting or glucoprivation (12, 22, 47). Estrogen feedback on the PVN and A2 regions enhances fasting- or glucoprivation-induced LH suppression by increasing the sensitivity to catecholaminergic inputs to the PVN (48, 49). In addition, the enhancement of LH suppression may be due to an increase in estrogen receptor-{alpha} expression in the PVN and A2 region with these treatments (41, 50, 51). Given that lipoprivic suppression of LH secretion was enhanced by the presence of estrogen, it is possible that lipoprivation also induces central estrogen receptor-{alpha} expression in these same regions. Another possibility is that estrogen alters lipid metabolism in energy sensing cells and, thereby, alters the response of the LH-releasing mechanism to MA because estrogen has altered the lipid oxidative pathways (52). These possibilities remain to be investigated.

The present study showed an increase in plasma glucose levels after central MA- or TMZ-induced lipoprivation. The MA-induced increase was blocked by catecholamine synthesis inhibition due to AMPT in the PVN. These data suggest that counter-regulatory responses to energy deficiency are induced by central lipoprivation and that these responses may also be mediated by NE/E inputs to the PVN. It is possible that the lipoprivic increase in plasma glucose levels could be at least partly mediated by stimulation of the hypothalamo-pituitary-adrenal axis because activation of CRH neurons by NE/E inputs to the PVN has been reported in fasted and glucoprivic animals (19, 26, 49). This is supported by a finding that the ascending NE/E pathway, but not the descending one, is involved in glucoprivic-induced feeding (37). On the other hand, adrenomedullary activation to increase peripheral glucose levels occurs due to descending NE/E fiber activation during glucoprivation and is not due to ascending NE/E activation to the PVN. The present results support the notion that the increase in plasma glucose levels during lipoprivation is partly dependent on activation of the hypothalamo-pituitary-adrenal axis. The NE projection to the PVN may also cause adrenomedullary activation to increase blood glucose level through sympathetic nerve outflow from the PVN (53). The present study should not negate the possibility that the plasma glucose increase after MA or TMZ administration in the hindbrain is partially mediated by a nonspecific stress response. Further studies will be required to clarify the mechanism mediating the glucose counter-regulatory responses to lipoprivation.

In conclusion, the present results suggest that fatty acids are sensed by a central detector that may be located in the lower brainstem, and this information is transmitted to the PVN in the hypothalamus by catecholaminergic pathways to maintain reproduction. Fatty acids may be another metabolic signal regulating the activity of the reproductive axis.


   Acknowledgments
 
We thank the National Hormone and Peptide Program for the LH assay kit, and Drs. G. R. Merriam and K. W. Watcher for the Pulsar computer program. The RIA and pulse analyses were performed at the Nagoya University Radioisotope Center and Computation Center, respectively.


   Footnotes
 
This work is supported in part by Grants-in-Aid for Scientific Research 18208025 (to K.-i.M.), and 19380157 and 18658106 (to H.T.) from the Japanese Ministry of Education, Culture, Sports, Science and Technology. M.S. was supported by the postdoctoral fellowship from the Japan Society for the Promotion of Science.

Disclosure Statement: The authors have nothing to declare.

First Published Online February 28, 2008

1 S.S. and K.I. contributed equally to this project and should be considered co-first authors. Back

Abbreviations: AMPT, {alpha}-Methyl-p-tyrosine; E, adrenergic; E2, estradiol; 4V, fourth cerebroventricle; 3HB, 3-β-hydroxybutylate; MA, mercaptoacetate; NE, noradrenergic; NPY, neuropeptide Y; NTS, solitary tract nucleus; OVX, ovariectomized; PVN, paraventricular nucleus; TMZ, trimetazidine.

Received January 4, 2008.

Accepted for publication February 21, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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