Increased dietary leucine changes the rhythm of prolactin secretion

Increased

Dietary Leucine Changes the Rhythm of Prolactin Nathan

Miller, P. Ann Smith,

and Ameae

Secretion

M. Walker

As we have recently shown that prolactin secretion in vitro is regulated by extracellular leucine, we wanted to determine whether leucine had a similar regulatory action on prolactin secretion in the intact animal. Leucine was administered to cycling female rats by way of their drinking water (0.5%) for a period of 24 days. At daily intervals during this time, six control and six leucine-treated rats were killed and their trunk blood subsequently assayed for prolactin content. On days 7, 14, and 21 during this period, pituitaries from the sacrificed rats were fixed and processed for electron microscopy. Leucine treatment resulted in an alteration in the normal four-day cyclicity in serum prolactin levels and a stimulation of prolactin synthesis as evidenced by ultrastructural changes in the pituitary mammotrophs. In a parallel set of experiments, daily vaginal smears were taken from nonsynchronized animals for 24 days before and 24 days during leucine administration. Leucine treatment resulted in variable alterations in the estrus cycle depending upon the stage of the cycle when the leucine was first administered. The changes in serum prolactin and the disturbances of the estrus cycle in the leucine-treated animals persisted for approximately 20 of the 24 days. In a shorter control experiment (seven days), alanine treatment (0.5%) was found to have no effect on prolactin levels. It is concluded that a mild elevation in dietary leucine can affect prolactin synthesis and release and the normal progression of the estrus cycle. (D 1986 by Grune & Stratton,

Inc.

T

HE CONCENTRATION of leucine in the plasma of laboratory rodents has been shown to exhibit a diurnal fluctuation generated largely by the consumption of food.’ The composition of each meal can affect plasma amino acids by directly contributing amino acids to the circulation and/ or by causing a stimulation of insulin secretion, which will in turn affect the mobilization of amino acids from peripheral tissues.’ A high protein meal’ or glucose infusion4 can, for example, elevate plasma leucine levels by as much as 600%. As we have previously shown that leucine regulates the secretion of prolactin in vitro,’ we wanted to investigate the possibility that dietary leucine could regulate prolactin secretion in the intact animal. In this study, we present evidence that modest increases in dietary leucine disrupt both the normal four-day rhythm of prolactin secretion and the normal estrus cycle in otherwise healthy adult cycling female rats. MATERIALS AND METHODS

Animals Female Sprague-Dawley-derived rats (Simonson-Gilroy, Gilroy, Calif) with an initial body weight of approximately 225 g were used. The animals were housed in divided cages such that the control and treated rats to be killed on any given day came from the same cage. The animals were exposed to constant light-dark cycles (light 7 AM to 7 PM) and were allowed free access to Purina rat lab chow (Ralston Purina Co, Richmond, Ind). The animals were housed together and handled daily for a period of three weeks prior to the onset of leucine treatment and became accustomed to placing their heads in the guillotine without ill effect.

From the Division of Biomedical Sciences, University of California, Riverside, Calif. Supported by NIH Grants AM 28534, RR 05816, and RR 07010 and by a grant from the University of California, Riverside, Academic Senate. Address reprint requests to A.M. Walker, PhD, Division of Biomedical Sciences, University of California, Riverside, Ca 925210121. o I986 by Grune & Stratton, Inc. 0026-0495/86/3507-0009%03.00/0

622

Leucine Administration and Sample Collection On day 0, six rats were killed by decapitation, and their trunk blood was collected into precooled glass centrifuge tubes. The rats were killed at five-minute intervals (between 10 AM and 11 AM) with all signs of the previous death removed before the next rat was brought into the laboratory. The blood was allowed to clot for 15 minutes at 4 “C before separation of the serum. The serum was stored at - 20 OC until assayed for prolactin content. After the initial group was killed, the water bottles supplying drinking water to half the animals were replaced with bottles containing 0.5% leucine (Sigma Chemical Co, St Louis) in tap water. The leucine solution was replaced every two days. From day 0 on, all animals (six control and six treated with leucine per time point) were killed at the same time of day and under the same conditions as the initial group. Radioimmunoassay Prolactin was measured in a homologous radioimmunoassay using rat prolactin (RP-I-5) as the standard and an antiserum raised against highly purified rat prolactin. Both were kindly provided by Dr A.F. Parlow through the rat Pituitary Hormone Distribution Program of the NIADDK. The assay has an intra-assay variation of 5.2% and an interassay variation of 8.1%. Antibody bound antigen was separated from free antigen using heat-killed and fixed Staphylococcus aureus as described previously.6 Electron Microscopy At 7, 14, and 21 days the pituitaries from two control and two leucine-treated rats were removed, cut into approximately l-mm squares, and fixed for two hours in 50:50 Dulbecco’s modified Eagle’s medium (Grand Island Biological Co, Grand Island, NJ) and Karnovsky’s fixative.’ Following this first fixation, the pieces of tissue were postfixed in Palade’s osmium’ (one hour at 4 “C), stained en bloc with Kellenberger’s uranyl acetate9 (one hour at 4 “C), dehydrated in an alcohol series, and then embedded in polyembed 812 (Polysciences, Warrington, Pa). Silver-gold sections were cut on an LKB Ultratome V (LKB Instruments, Inc, Rockville, Md) and stained with uranyl acetate and lead citrate” before being viewed in a Siemens 101 electron microscope. Cells were photographed at a magnification x 4,000 and then printed at a final magnification x 12,000. Blocks for sectioning were chosen at random from those derived from the same pituitary. For the quantitative cytometry, the photographs were generated from at least three blocks from each of the control and leucine-treated pituitaries.

Metabolism, Vol 35, No 7 (July). 1986: pp 622-626

LEUCINE CHANGES PROLACTIN RHYTHM

Cytometry Because the borders of the mammotrophs were so irregular and frequently difficult to discern, a pseudo cell boundary was produced by drawing a circle around the center of the mammotroph nucleus. Within the enscribed circle having a 4-cm radius, the area occupied by endoplasmic reticulum, nucleus and extracellular space was determined using a graphics tablet attached to an Apple II microcomputer (Apple Computer Inc, Cupertino, Calif). Fifty cells from the control animals and fifty cells from the leucine-treated animals were analyzed. Vaginal Smears Groups of six rats were individually marked and then smeared every day for 24 days. The same animals were then given leucine water and were smeared daily for an equivalent period of time. Smears were obtained by the insertion of 3.1 mm diameter silicone rubber tubing. The smears were fixed in methanol and subsequently stained with toluidine blue. Statistical Analysis All numerical results were analyzed for statistical significance by the r-test. RESULTS

Serum Prolactin Levels

In the untreated animals, serum prolactin levels ranged from about 10 ng/mL to 40 ng/mL according to a regular four-day cycle (Fig 1, dashed line). In the leucine-treated rats, the levels of prolactin remained within the same range, but the cycle length was reduced to two days (Fig 1, solid line). In other words, prolactin levels peaked twice as often in the leucine-treated animals. This alteration in prolactin secretion persisted for approximately 20 of the 24 days. In a shorter control experiment (seven days), alanine administration (0.5% in tap water) was found to have no effect on prolactin secretion (data not shown).

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8 TIME

12

16

20

24

(days)

Fig 1. Serum prolactin levels in control and leucine-treated rats. Each point represents the mean value from six animals. The error bars have been omitted for clarity. The standard errors of these means were, however, always less than 9% of the value on the ordinate. Note the four-day rhythm in the control animals W---O) and the two-day rhythm in the leucine-treated animals (Cl--0). This graph is a representative example of two similar studies.

Prolactin Cell Ultrastructure

Consumption of Food and Water

The pituitaries from the leucine-treated animals contained mammotrophs with signs of very active protein synthesis. The cisternal spaces of the rough endoplasmic reticulum (RER) were greatly enlarged and contained flocculent material (Fig 2). This was true for all three time points, but was most evident at the 1Cday sampling. When assessed by quantitative cytometry at the 1Cday time point, the area of the cell occupied by RER in the control cells was 3.6% compared to 10.8% in the treated cells (Table 1). Even though the RER was distended, the overall cell size and nuclear size in the treated cells was unaffected (Table 1).

Although we did not specifically monitor the consumption of food and water, we observed no significant differences between the control and leucine-treated rats. The body weights of the control and leucine-treated groups increased at the same rate throughout the experimental period (data not shown).

Vaginal Smears

Analysis of the vaginal smears showed the rats to be cycling according to a four-day pattern. Leucine treatment uniformly resulted in a disruption of the cycle, but the exact pattern of the disruption was dependent on the stage of the cycle the animal was in when the Ieucine was first administered. If around diestrus, the cycle was prolonged. If in early proestrus, the cycle was held up in proestrus (see Fig 3 for example).

DISCUSSION

In this study we have demonstrated that leucine does indeed regulate the secretion of prolactin in vivo as well as in vitro. The potential mechanisms by which this in vivo regulation could occur, however, are manyfold. 1. As we have previously shown that leucine directly regulates the release of prolactin from pituitary mammotrophss it is possible that this is also the mechanism in vivo. If this were the case, however, one might simply expect prolactin levels to be elevated rather than that there be an alteration in the rhythm of prolactin secretion. Although some secretory rhythms are generated by the pituitary itself,“.” it seems more likely that the two- and four-day rhythms are of neuronal origin.

624

MILLER ET AL

Fig 2.

Mammotrophs from control and leucine-treated rats.

Note the distention of the RER in the cell from the leucine-treated rat (B) (magnification x 9,200). (A) control (magnification x 8,800). N, nucleus.

2. As dopamine is the predominant hypothalamic factor controlling prolactin release, the simplest explanation for a doubling in the pulsatile secretion of prolactin would be a reduction in the release of dopamine from the hypothalamus. How then would blood leucine levels alter the release of dopamine? The rate of dopamine synthesis is governed by the availability of its precursor, tyrosine,“.14 and the large neutral amino acids, among them leucine, compete with tyrosine for the transport system, which allows passage across the blood-brain barrier.14,15Thus, increased blood leucine levels would decrease the availability of tyrosine for dopamine synthesis. Evidence in support of this mechanism includes the lack of effect of alanine (a small neutral amino acid) and

work showing that leucine administration lowers brain dopa levels (see reference 16 for review). 3. Alternatively or additionally, leucine could directly stimulate prolactin secretion from the pituitary and this could in turn regulate the synthesis of dopamine in the tuberoinfundibular neurons.17-‘9 4. Also, as leucine can be a direct precursor of cholester01,~’it is possible that leucine alters steroid metabolism and that this in turn affects the estrus cycle and then the prolactin “surge center.“““’ 5. Lastly, as leucine is also a potent and physiologic stimulus for insulin release,23*24it is also possible that the effects of leucine on prolactin secretion occur via effects on

625

LEUCINE CHANGES PROLACTIN RHYTHM

Table 1. Effect of Leucine Treatment Mammotroph

LeucirwTreated

Control

Total cell area

20,173

RER

it 93

19.689

730 + 56*

Nucleus

on

Ultrastructure

3,931

f

176

c 221

2,139

+ 142*

3,583

* 169

Values are given as arbitrary units + SEM. *Difference of P -C 0.001.

insulin. The relevant effects of insulin include changes in the blood levels of glucose (see reference 25 for relationship to prolactin secretion) and other amino acids,26 changes in lipid metabolism,27 as well as an effect on protein synthesis in the pituitary.2* Further experimentation is required to determine which of these various possibilities comes closest to describing the actual situation. There is an apparent anomaly in our results: while the mammotrophs showed signs of a massive increase in the synthesis of prolactin (with no signs of increased storage or degradation), the total amount of prolactin measured during the 24-day period was the same for the control and leucinetreated animals (631 + 45.6 ng/mL control, 607 + 50.4 ng/mL leucine treated). This may be because we were only looking once a day (a protocol based on the findings of :.

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Neil129) and may have missed other changes such as an increased number of smaller peaks per 24-hour period. However, when Stewart et al.” looked at the effects of branched chain amino acids on growth hormone secretion during a nine-hour period, they did not observe an increase in the total amount of growth hormone released even though there was an increase in peak frequency. Whether prolactin is secreted in larger quantities (as is implied by the ultrastructural results), as well as with an altered rhythm, will have to be determined by further experimentation. The effects of the leucine treatment lasted for 20 of the 24 days, after which time the animals seemed able to adapt to the increased intake. Such an adaptation has been previously demonstrated by Tannous et al,3’ who fed rats a 5% leucine diet and found them to adapt to the increased intake after about 12 days. These authors also reported growth depression due to a lowered food intake in the leucine-treated rats. Administration of one-tenth the amount of leucine in our studies had no effect on the growth rate of the animals. The finding that the time of initial leucine administration caused different changes in the estrus cycle is difficult to definitively interpret at present. Even though prolactin is considered to be the main leuteotropic factor in the rat,32-‘4 our results do not allow us to conclude that the changes in prolactin secretion are causative of the changes in the estrus cycle. In fact, they may be secondary to an effect on the estrus cycle mediated via an e&t on steroid biosynthesis2’ and gonadotropin secretion. 22If altered prolactin levels are the cause of the changes in the estrus cycle, however, then one can explain the extended time in diestrus as a result of prolonged progesterone secretion and the extended time in proestrus as the result of an inhibition of the LH surge.3’*36 With regard to the effects on the estrus cycle, our studies also correlate nicely with an observation by Cooper and Linoila,37 who found that dietary supplements of leucine cause changes in vaginal smears in old rats. In conclusion, we have demonstrated that a mild elevation in dietary leucine can have a profound effect on prolactin secretion and the control of the estrus cycle. Continued investigation will focus on the mechanism of this effect.

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24

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32

36

40

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48

52

Fig 3. The effect of leucine-treatment on the estrus cycle. The cycle is graphed to emphasize periodicity. Each stage was assigned a number: early proestrus, 1: late proestrus 2: estrus. 3: metestrus. 4; diestrus, 5. As smears were taken daily at the same time of day, two consecutive points of equal value indicate that more than 24 hours were spent at that stage. The errow indites the time of administration of the leucine solution.

ACKNOWLEDGMENT

The authors would like to thank Nancy Price for typing the manuscript and Alison Tait and Neitchy Mora for contributions to the early stages of the project.

REFERENCES

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MILLER

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ET AL

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