Vitamin E Stimulates Luteinizing Hormone-Releasing Hormone and Ascorbic Acid Release from Medial Basal Hypothalami of Adult Male Rats 1

Abstract

Vitamin E, a dietary factor, is essential for reproduction in animals. It is an antioxidant present in all mammalian cells. Previously, we showed that ascorbic acid (AA) acted as an inhibitory neurotransmitter in the hypothalamus by scavenging nitric oxide (NO). Earlier studies have shown the antioxidant synergism between vitamin E and ascorbic acid (AA). Therefore, it was of interest to evaluate the effect of vitamin E on luteinizing hormone-releasing hormone (LHRH) and AA release. Medial basal hypothalami from adult male rats of the Sprague Dawley strain were incubated with Krebs-Ringer bicarbonate buffer or graded concentrations of a water soluble form of vitamin E, tocopheryl succinate polyethylene glycol 1000 (TPGS, 22–176 μM) for 1 hr. Subsequently, the tissues were incubated with vitamin E or combinations of vitamin. E + N-methyl-D-aspartic acid (NMDA), an excitatory amino acid for 30 min to study the effect of prior and continued exposure to vitamin E on NMDA-induced LHRH release. AA and LHRH released into the incubation media were determined by high-performance liquid chromatography and radioimmunoassay, respectively. Vitamin E stimulated both LHRH and AA release. The minimal effective concentrations were 22 and 88 μM, respectively. NMDA stimulated LHRH release as previously shown and this effect was not altered in the combined presence of vitamin E plus NMDA. However, AA release was significantly reduced in the combined presence of vitamin E plus NMDA. To evaluate the role of NO in vitamin E-induced LHRH and AA release, the tissues were incubated with vitamin E or combinations of vitamin E + N^G-monomethyl-L-arginine (NMMA), a competitive inhibitor of NO synthase. NMMA significantly suppressed vitamin E-induced LHRH and AA release indicating a role of NO in the release of both LHRH and AA. The data suggest that vitamin E plays a role in the hypothalamic control of LHRH and AA release and that the release is mediated by NO.

The term Vitamin E was introduced to describe a factor in the diet that is important for reproduction in animals and was given the name tocopherol, meaning child birth in Greek (1). Vitamin E deficiency produces degeneration of the seminiferous epithelium in male and fetal resorption in female rats (1, 2). Different forms of tocopherols and tocotrienols have been identified and of these, alpha-tocopherol is the most biologically active member of the vitamin E family (3–7). It is found in polyunsaturated vegetable oils, major mammalian cell types, and cell membranes (8–10). It passively reaches the blood stream and liver after emulsifying together with the fat soluble components of the food and shows tissue-specific distribution (11, 12). A selective transfer of α-tocopherol into lipoproteins was mediated by the specific α-tocopherol transfer protein (α-TTP) in the hepatocyte (13). In addition, a protein capable of specifically binding tocopherol, a tocopherol-associated protein, was found in a large number of tissues, such as liver, prostate, and neural tissues (14).

The mechanism of the physiological action of vitamin E was not very clear but some of the biological activities are attributed to its antioxidant activity (15, 16). It plays a major role in the prevention of lipid peroxidation in biological membranes and is an important intramembrane antioxidant, membrane stabilizer, and lipid-soluble antioxidant (17, 18). Numerous in vitro experiments have demonstrated antioxidant synergism between alpha-tocopherol and ascorbate, reduced glutathione, NADPH, and cellular electron transport proteins (19, 20). Studies of vitamin E regeneration in a protein-denaturing system revealed that ascorbate regenerates vitamin E by a nonenzymatic mechanism (21). These studies suggest that significant interaction occurs between water- and lipid-soluble vitamins at the membrane cytosol interface and that ascorbic acid (AA) may function in vivo to repair membrane-bound oxidized vitamin E (4, 22).

A water-soluble form of vitamin E, tocopheryl succinate polyethylene glycol 1000 (TPGS), is used as a vitamin supplement in children with cholestatic liver disease who are incapable of absorbing alpha-tocopherol or alpha-tocopheryl acetate (23–25). TPGS does not depend on fat absorption for uptake into intestinal cells because TPGS forms micellar solutions at low concentrations, thereby making the need for bile acids for vitamin E absorption is eliminated (26). Supplementation with TPGS improves neurologic function in vitamin E-deficient children with cholestatic liver disease (27).

The role of vitamin E in the release of hypothalamic releasing or inhibiting hormones is not known. Previously, we reported the role of AA as an inhibitory transmitter in the hypothalamus to inhibit NMDA-stimulated luteinizing hormone-releasing hormone (LHRH) release (28). Because there is antioxidant synergism between vitamin E and AA and vitamin E is important for reproduction, it was of interest to study the effect of vitamin E on AA and LHRH release. In the present investigation, medial basal hypothalami (MBH) were incubated with varying concentrations of TPGS, a water-soluble form of vitamin E. AA and LHRH released into the incubation medium were measured by radioimmunoassay and high-performance liquid chromatography, respectively.

Materials and Methods

Adult male rats of the Sprague-Dawley strain (Holtzman, Madison, WI, 200–250 g) were housed two per cage under controlled conditions of temperature (23–25°C) and lighting (on 05:00–17:00 h). The animals had free access to a pellet diet consisting of Purina Lab rat chow 5001 and tap water.

Chemicals.

N-methyl-D-aspartic acid (NMDA), AA, N^G-monomethyl-L-arginine (NMMA), and bacitracin were obtained from Sigma (St. Louis, MO). A water miscible form of vitamin E (TPGS) was obtained from Acros Organics (Fisher Scientific).

In Vitro Studies

Incubation of MBH.

Animals were killed by decapitation and the brain was exposed by a dorsal incision. MBH were dissected by vertical cuts along the lateral hypothalamic sulci, posterior edge of the optic chiasm and the anterior edge of the mammillary bodies. A horizontal cut 1 mm from the base separated the island. The explants (8–12 mg) were incubated in vitro as previously reported (28). In brief, one MBH/tube was placed in 0.5 ml of Krebs-Ringer bicarbonate (KRB) buffer (pH 7.4) supplemented with 20 μM bacitracin in an atmosphere of 95% O₂/5% CO₂ and incubated in a Dubnoff shaker (50 cycles/minute) for a period of 60 min. Following this preincubation, the tissues were incubated with 0.5 ml of KRB or KRB containing various concentrations of vitamin E (22–176 μM) for 60 min. Subsequently, the tissues were incubated with KRB or vitamin E or vitamin E plus NMDA (10 mM) for 30 min to evaluate the influence of vitamin E in the presence of NMDA. The dose of NMDA was chosen as per our previous work (28). A simultaneous incubation of MBH with vitamin E + NMMA (300 μM) for 60 min was also performed to evaluate the role of NO in vitamin E-induced AA and LHRH release. NMMA (300 μM) was previously shown to be effective at this concentration (28). At the end of this medium was aspirated and medium and tissues were stored at −80°C. The number of tissues for each group was 8 in all the experiments. Ascorbic acid and LHRH were analyzed by high-performance liquid chromatography and radioimmunoassay, respectively.

MBH Homogenates.

MBH were weighed and placed in 600 μl of sample buffer containing 1 mg/ml of potassium metabisulfite and homogenized in a glass homogenizer. The sample buffer consisted of 5 mM each of metaphosphoric acid and sodium salt of ethylene diamine tetra acetic acid (Na₂ EDTA). The homogenate was thoroughly mixed and centrifuged at 1000 g for 10 min at 0°C. The supernatant was decanted and passed through syringe filters before placing in the vials for determining AA by HPLC.

Chromatography.

Isocratic analyzes were conducted with Beckman system gold HPLC equipped with 126 module and diode array detector 168 operating at 254 nm at a sensitivity of 0.016 a.u.f.s (Beckman Instruments Incorporation, Fullerton, CA). The separation was performed on a μBoundopack Beckman ultrasphere C18 column (average particle size 5 μm, 25 cm × 4.6 mm). The mobile phase was a buffer consisting of 0.1M sodium dihydrogen phosphate (NaH₂PO₄) and 0.2 mM Na₂EDTA adjusted to pH 3.1 with orthophosphoric acid. The buffer was filtered through 0.45 μm membrane filter (Gelman Sciences, MI) and degassed prior to use. The column was maintained at room temperature and the mobile phase was used at a constant flow rate of 1.0 ml/min.

Preparation of Standard.

A sample buffer consisting of 5 mM each of metaphosphoric acid and Na₂EDTA was prepared in HPLC grade water (V.W.R Scientific products, TX) and was used for preparing AA standards and MBH homogenates. This buffer was previously shown to stabilize AA for 3–4 hr and all the estimations were completed within this time (29). A standard curve for AA was prepared from a stock solution of 1 mg/ml and was found to be linear from 487.5 to 7800 ng. AA in standards, incubation medium and homogenates was measured using 507 ASE auto sampler (Beckman) and the samples were used at a volume of 30 μl. Each sample (unknown) was passed through syringe filters (Gelman Sciences) before placing in the vial for measurement. A standard calibration plot was obtained for AA concentrations (μg/ml) versus peak area (numerical units on 126 module). Multiple plots for standard curve were constructed using freshly prepared samples on different days.

LHRH Assay.

A highly specific antibody to LHRH was kindly provided by Dr. A. Barnea (U.T. Southwestern Medical Center, Dallas, TX). The minimal detectable LHRH was 0.2 pg/tube and the curve was linear to 100 ng/tube. The inter and intra assay coefficient of variations were 5% and 4%, respectively.

Statistics.

Results were analyzed by one way analysis of variance or paired t test wherever, applicable and P < 0.05 was considered significant.

Results

Effect of Different Concentrations of Vitamin E on LHRH Release and the Response to NMDA.

Incubation of MBH with graded concentrations of vitamin E (22, 88, and 176 μM) for 1 hr significantly stimulated LHRH release (Fig. 1A). The lowest tested effective dose was 22 μM and LHRH release induced by the highest concentration (176 μM) was significantly greater than that of the group incubated with 88 μM illustrating a dose-dependent effect. Subsequent to this incubation, the tissues were incubated with NMDA (10 mM) or combinations of NMDA + Vitamin E (22, 88, and 176 μM) for 30 min to ascertain the role of vitamin E in NMDA-induced LHRH release. A significant increase in LHRH release was observed in the group incubated with NMDA alone (Fig. 1B ). LHRH release by the groups incubated with NMDA or the same concentration of vitamin E + NMDA was similar to the group treated with NMDA alone illustrating that prior or continuous exposure to Vitamin E resulted in no additional release of LHRH upon addition of NMDA to the incubation medium.

Effect of Combined Incubation with Vitamin E + NMDA on LHRH Release.

MBH were incubated with either vitamin E or a combination of vitamin E + NMDA for 1h. This experimental protocol was different from that mentioned earlier (Fig. 1 ) because the tissues were not incubated with vitamin E for 1 hr followed by incubation with vitamin E + NMDA for 30 min. The results showed that NMDA and vitamin E stimulated LHRH release as before (Fig. 2 ) and that the groups incubated with vitamin E + NMDA showed a similar increase in LHRH release as compared to the group incubated with vitamin E or NMDA. Therefore it is apparent that the LHRH response remained the same irrespective of whether the tissues were initially exposed to vitamin E for 1 hr followed by incubation with vitamin E + NMDA for 30 min or simultaneously exposed to vitamin E + NMDA for 1 hr.

Effect of NMMA on Vitamin E-Induced LHRH Release.

To assess the role of NO in vitamin E-induced LHRH release, MBH were incubated with NMMA (300 μM), a competitive inhibitor of NO synthase (NOS), or a combination of NMMA + vitamin E for 1 hr. Vitamin E stimulated LHRH release as reported above (Figs. 1 and 2 , respectively). LHRH release was not altered in the group incubated with NMMA (Fig. 3 ); however, a combination of vitamin E + NMMA completely blocked LHRH release suggesting a role of NO in vitamin E-induced LHRH release.

Effect of Vitamin E on AA Content and Release.

Addition of varying concentrations of vitamin E to MBH failed to alter AA content in the MBH (Fig. 4A ). However, incubation of MBH with vitamin E stimulated AA release and the minimal effective dose was 88 μM (Fig. 4B ). Increasing the concentration of vitamin E to 176 μM did not increase the release of AA above that obtained with the 88 μM concentration. Comparison of AA content in the tissue and medium showed that the tissue AA content was significantly higher (70- to 80-fold) than that of the medium. Therefore, only a very small percentage of the AA stored in the tissue was released into the incubation medium.

Effect of Combined Incubation with Vitamin E + NMDA on AA Release.

Incubation of MBH with NMDA (10 mM) failed to alter AA release (Fig. 5 ). Vitamin E (88 μM) stimulated AA release as before. A combination of vitamin E + NMDA completely blocked vitamin E-induced AA release.

Effect of NMMA or Combination of Vitamin E + NMMA on AA Release.

To ascertain the role of NO in vitamin E-induced AA release, MBH were incubated with NMMA (300 μM) or a combination of vitamin E (88 μM) + NMMA. Vitamin E stimulated AA release as before (Fig. 6 ). NMMA failed to alter basal AA release, but a combination of vitamin E + NMMA significantly lowered AA release to basal levels.

Discussion

Our results showed that vitamin E stimulated both LHRH and AA release from MBH. This to our knowledge, is the first report showing a stimulatory action of vitamin E on LHRH and AA release demonstrating its acute action on the hypothalamus. Vitamin E was discovered as a lipid-soluble dietary factor essential for reproduction (1). Our results support this view and it is most likely that by stimulating LHRH release from the hypothalamus it may stimulate LH and FSH release from the pituitary. Additional work is necessary to assess its action at the pituitary level.

Vitamin E-induced LHRH release was in contrast to the suppressive effect of AA on stimulated LHRH release. Recently, we reported that AA was ineffective in altering basal LHRH release but suppressed NMDA or sodium nitroprusside (NP)-induced LHRH release (28). Because NMDA and NP stimulated LHRH release via NO, the suppression of LHRH release in the presence of AA, a reducing agent indicated that AA was able to reduce chemically NO released by NMDA and NP.

Incubation of MBH with NMDA (10 mM) in the present study stimulated LHRH release and this is in agreement with our earlier reports and those of others (28, 30–32). NMDA also stimulated LHRH release from immortalized hypothalamic neuronal cells (GTI-1) that can be activated to release LHRH (33). In addition, the stimulatory action of NO on LHRH release was shown earlier (34, 35). Because both NMDA and vitamin E stimulated LHRH release, it was possible that the releasable pool of LHRH was exhausted by either stimulus alone, thereby preventing further stimulation of LHRH but since a further release of LHRH occurred with a higher dose of vitamin E this is ruled out. However, we earlier showed that AA suppressed NMDA-induced LHRH release, presumably by reducing NO chemically. Therefore, the AA released by vitamin E in the present experiments may have blocked the action of NMDA by chemically reducing the NO that mediated the LHRH release induced by NMDA. Our results also showed that the response to NMDA was similar irrespective of whether the tissues were exposed to vitamin E + NMDA initially or subjected to NMDA addition after prior and continued exposure to vitamin E. However, a decreased release of AA was observed when MBH were incubated with a combination of vitamin E + NMDA. Since NMDA stimulated LHRH release via NO, the increased NO released may oxidize AA to dehydroascorbic acid that is not measurable by our method thus accounting for a decreased concentration of AA in the medium.

We previously showed that NO induces LHRH release since it was induced by a releaser of NO, sodium nitroprusside and blocked by hemoglobin, an inactivator of NO or NMMA, a competitive inhibitor of NOS (35). In addition to inducing LHRH release NO also induces AA release as was confirmed in the present study. Here, we showed that NO also mediates the action of vitamin E to induce AA release since this release was blocked by NMMA.

The capability of vitamin E to stimulate AA release showed a close association between the two antioxidants. Previous studies showed that a high dietary AA elevated plasma and tissue vitamin E levels in guinea pigs (36, 37). The sparing effect of AA on vitamin E was reported in a mutant strain of Wistar rat defective in AA synthesis (38). In addition, AA was shown to play an important role in the regeneration of vitamin E from the tocopherol-free radical, suggesting a synergistic interaction between the two vitamins’ antioxidant activities (39, 40). The influence of graded dietary vitamin E intake on AA concentration in specific regions of the brain was also assessed previously (41). Rats treated with different doses of vitamin E for 2 months showed a significant increase in vitamin E concentration in the brain and peripheral tissue (41). Blood and liver showed a dose-dependent increase that paralleled the different concentrations of vitamin E in the diet. The central nervous system also followed the same pattern when the dietary supplements were 5, 30, and 60 mg/kg of diet except that the higher dose had no further effect on vitamin E concentration. On the contrary, the higher doses of vitamin E, 250, or 500 mg/kg diet lowered AA concentration in brain cortex, cerebellum, plasma, liver and heart (41).

Deficiency of vitamin E markedly impaired both humoral and cellular immunity thus emphasizing the importance of this micronutrient in the diet (42–45). Nerve cells in the central nervous system were the primary site of pathology in animals having vitamin E deficiency as well as in humans with very low vitamin E levels (46). The central nervous system requirement of vitamin E during aging was shown to increase. Aging was associated with a decrease in vitamin E levels (47–51) and a higher intake was needed to maintain and improve the decreased cellular immunity functions (47–51). Considerable evidence suggested that oxidative stress played a significant role in the pathogenesis of Alzheimer’s disease, a neurodegenerative disorder associated with aging (52–54). A few investigations reported that vitamin E concentrations in plasma, brain and cerebrospinal fluid were significantly lowered in Alzheimer’s patients (55, 56). Treatment of Alzheimer’s patients with vitamin E slowed the progression of the disease (57). These studies showed the critical role played by this vitamin in maintaining the structure and metabolic integrity of nerve cells (46, 57, 58).

The results are summarized in Figure 7. Earlier we hypothesized the mechanism of action of AA to inhibit the release of LHRH produced by high [K⁺] medium and NMDA (28). According to this concept the axons of the glutamatergic neurons synapse on the axons of the noradrenergic neurons that in turn synapse on the NOergic neurons. Therefore, NMDA would cause the release of norepinephrine by acting on α₁-noradrenergic receptors would increase intracellular calcium [Ca²⁺] in the NOergic neuron. This increase would activate NOS, causing generation of NO that would diffuse to the LHRH neuron and activate release by stimulating GC that causes conversion of GTP to cGMP. cGMP activates protein kinase G that releases LHRH and AA colocalized in LHRH secretory granules. However, the effects of AA and vitamin E on LHRH release were differential. AA had no effect on basal LHRH release but inhibited NMDA and NP-stimulated LHRH release by scavenging NO (28). In contrast, vitamin E stimulated LHRH and AA release from MBH by the stimulation of NOS followed by NO release. Our data suggest that vitamin E plays a role in the release of AA and LHRH and acts as a neurotransmitter in the hypothalamus.

Figure 1.

Effect of graded concentrations of vitamin E (22–176 μM) on LHRH release from MBH after 1 hr of incubation (A). The response to NMDA is depicted in B. In this and subsequent figures, the results are the mean ± SEM and the number of tissues for each group was 8. *P < 0.05 or **P < 0.01 and *** P < 0.001 versus KRB. + +P < 0.01 versus the group incubated with vitamin E (88 μM).

Figure 2.

Effect of vitamin E or a combination of vitamin E + NMDA on LHRH release after 1 hr of incubation. *P < 0.05 or ***P < 0.001 versus KRB.

Figure 3.

Influence of vitamin E or a combination of vitamin E + NMMA (300 μM) on LHRH release after 1 hr of incubation. **P < 0.01 or +P < 0.05 versus the group incubated with vitamin E.

Figure 4.

Effect of vitamin E on AA content in the tissue and medium after 1 hr of incubation (A and B, respectively). *P < 0.05 versus KRB.

Figure 5.

Effect of vitamin E (22 μM) or a combination of vitamin E + NMDA (10 mM) on AA release after 1 hr of incubation. ** P < 0.01 versus KRB. +++P < 0.001 versus the group incubated with vitamin E.

Figure 6.

Effect of vitamin E or a combination of vitamin E + NMMA on AA release after 1 hr of incubation. ***P < 0.001 versus KRB. ++P < 0.01 versus the group incubated with vitamin E.

Figure 7.

A schematic diagram of the hypothesized mechanism of action of vitamin E induced LHRH and AA release. Glu n, glutamergic neuron; NMDA r, NMDA receptor; SV, synaptic vesicle; NE, norepinephrine; α₁ r, α ₁- adrenergic receptor; AA, ascorbic acid; NO, nitric oxide; NO n, NOergic neuron; Arg, arginine; NOS, nitric oxide synthase; cit, citrulline; gc, guanylyl cyclase; cGMP, cyclic guanosine monophosphate; PKG, protein kinase G; pv, portal vesicle.

Footnotes

This work was supported by the National Institutes of Health Grant MH51853.

1

A preliminary report of this research was given by S. Karanth, W.H. Yu, C.A. Mastronardi, and S.M. McCann, “Vitamin E stimulates the release of luteinizing hormone-releasing hormone and ascorbic acid from medial basal hypothalamic explants.” 30th Annual Meeting Society for Neuroscience, New Orleans, LA, November 2–9, 2000. Abstract #544.5, p1454.

2

To whom requests for reprints should be addressed at Pennington Biomedical Research Center (Louisiana State University), 6400 Perkins Road, Baton Rouge, LA 70808–4124. E-mail:

Acknowledgements

We would like to thank Judy Scott and Nicole Mestayer for their excellent secretarial assistance.

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