Biomedical subjects
A J Carrillo
Publications and source records attributed to A J Carrillo.
Serotonergic involvement in the regulation of prolactin and vasoactive intestinal peptide mRNA expression in the rat anterior pituitary.
These studies examined the contribution of serotonin (5-HT) to the control of prolactin (PRL) and vasoactive intestinal peptide (VIP) messenger RNA expression in rat anterior pituitary. Daily injection of rats with the biosynthetic precursor to serotonin, 5-hydroxytryptophan (5-HTP; 25 mg/kg, q.i.d.), resulted on day 5 in a 50% increase in the expression of PRL mRNA in the pituitary while at the same time reducing the levels of both the 1.0 and 1.7 kb VIP mRNA transcripts. Co-treatment of rats with 5-HTP plus the catecholamine biosynthesis inhibitor, alpha-methyl-tyrosine (alpha-MT; 150 mg/kg, q.d. x 2 days), or the dopamine receptor antagonist haloperidol (1.25 mg/kg, b.i.d. x 5 days), resulted in increases in pituitary PRL message levels that were greater than those observed with either anti-dopaminergic agent alone. In contrast, 5-HTP was unable to reverse the inhibition of PRL mRNA expression caused by treatment with the dopamine receptor agonist bromocriptine (2.5 mg/kg, b.i.d. x 5 days). Neither alpha-MT, haloperidol nor bromocriptine had a significant effect on pituitary VIP mRNA expression. Administration of the direct-acting 5-HT receptor agonist quipazine (5 mg/kg, b.i.d.) for 14 consecutive days caused a significant increase in pituitary PRL mRNA levels on day 1 and reached a plateau of 90% above control levels on days 7 and 14. VIP mRNA levels rose significantly on day 1 of quipazine treatment but thereafter fell to a minimum of 22% (1.0 kb) and 52% (1.7 kb) of control by day 14.(ABSTRACT TRUNCATED AT 250 WORDS)
Ablation of the hypothalamic arcuate-median eminence region reduces the concentration of vasoactive intestinal peptide in the anterior pituitary gland of male rats.
This study was designed to determine the influence of the hypothalamus on the content of vasoactive intestinal peptide (VIP) in the anterior pituitary. Disruption of the hypothalamic-pituitary connection was performed by ablating the arcuate-median eminence (ARC-ME) region in adult male rats. Fifteen days later, there was a significant reduction in pituitary mass, adrenal and testicular weight and an increase in water consumption and serum prolactin levels indicating the elimination of hypothalamic influence on the pituitary gland in the ARC-ME group when compared to controls. Anterior pituitary VIP content was also significantly reduced in the lesion group. These data suggest that the hypothalamus is involved in the regulation of pituitary VIP.
Para-chlorophenylalanine treatment inhibits the expression of vasoactive intestinal peptide messenger RNA in rat anterior pituitary.
Adult male Sprague-Dawley rats were treated with para-chlorophenylalanine (pCPA) or alpha-methyl tyrosine (alpha-MT) to study the effect of serotonin or catecholamine depletion on the expression of vasoactive intestinal peptide (VIP) messenger RNA in the anterior pituitary. Single injections of pCPA (300 mg/kg) for two consecutive days resulted on the third day in a dramatic depletion of serotonin in the medial basal hypothalamus, and a significant reduction in the pituitary content of VIP mRNA (1.0 and 1.7 kb). The effect of pCPA on VIP mRNA appeared to be relatively specific for the anterior pituitary since VIP message levels in the cerebral cortex did not decrease. alpha-MT treatment, (150 mg/kg) for 2 consecutive days, reduced dopamine concentrations in the MBH but had no significant effect on pituitary VIP levels. In a time-course study, hypothalamic serotonin and pituitary VIP mRNA levels were significantly depressed 1-3 days after initiation of pCPA treatment; however, 12 days after pCPA treatment, serotonin concentrations in the hypothalamus approached control values and pituitary VIP mRNA content increased an average of 2-fold over control levels in an apparent rebound effect. pCPA-treated rats injected i.p. twice a day with 5-hydroxytryptophan (5-HTP; 50 mg/kg) experienced a partial reversal in the decline in the 1.7 kb VIP mRNA seen 24 h after the first pCPA injection. However, at 72 h, supplementation with 5-HTP did not prevent the pCPA-induced decrease of pituitary VIP mRNA. These data indicate that serotonergic pathways have a major role in the control of VIP mRNA expression in the rat anterior pituitary.
Quantification of vasoactive intestinal peptide immunoreactivity in the anterior pituitary glands of intact male and female, ovariectomized, and estradiol benzoate-treated rats.
There are considerable data suggesting that vasoactive intestinal peptide (VIP) is involved in the regulation of PRL secretion; however, the role and cell of origin of anterior pituitary VIP remain to be determined. Immunocytochemical (ICC) studies have generally failed to detect VIP-immunoreactive (IR) cells in the pituitary of the untreated rat, although VIP-IR cells have been observed in the pituitaries of hypothyroid or estrogen-treated rats. This study was designed to examine the cellular distribution and tissue content of VIP in the anterior pituitary gland of rats under selected endocrine conditions known to alter the rates of PRL and VIP synthesis and secretion. To this end, anterior pituitary VIP and PRL content (ICC and RIA) and serum PRL levels were determined in ovariectomized (OVX) and OVX rats 3 days after treatment with 7 or 70 micrograms estradiol benzoate (EB). For comparison, pituitary VIP and PRL content (ICC and RIA) and serum PRL levels in untreated male and diestrous female rats were determined. Immunostaining for VIP was accomplished using a newly developed primary antiserum. Significant numbers of VIP-IR cells per 5-microns section were found in the anterior pituitary glands of all animals examined (275 +/- 33 in diestrous to 481 +/- 103 cells in male rats). VIP was not colocalized with PRL in any of the pituitaries regardless of steroid treatment or sex. Furthermore, the number of VIP-IR cells per pituitary gland was not significantly correlated with sex or EB treatment. Treatment with 70 micrograms, but not 7 micrograms, EB significantly increased the pituitary content of VIP and serum PRL levels compared to those after ovariectomy. However, both EB treatments resulted in a significant increase in pituitary PRL content compared to that in untreated OVX rats. Pituitaries from male rats had several-fold more VIP and less PRL content than pituitaries from diestrous rats. These data show that 1) in contrast to previous ICC studies, VIP-IR cells are readily detected in the anterior pituitary of intact male and female and OVX as well as EB-treated rats; 2) VIP is localized to cells other than lactotrophs, regardless of the steroid background; and 3) marked changes in anterior pituitary VIP content are not accompanied by changes in VIP-IR cell number.
Preferential increase in pituitary prolactin versus vasoactive intestinal peptide as a function of estradiol benzoate dose in the ovariectomized rat.
Vasoactive intestinal peptide (VIP) is synthesized in various tissues, including the anterior pituitary gland, where it may stimulate the release of PRL. Because estrogen plays a central role in the regulation of PRL, it becomes important to determine the effects of this steroid on both pituitary VIP and PRL. To study this, pituitary VIP and PRL and plasma PRL were assayed in ovariectomized rats after treatment with estradiol benzoate (EB; 0.007, 0.07, 0.7, 7 or 70 microgram/rat). Pituitary and plasma TSH were also determined as well as VIP content in the medial basal hypothalamus, suprachiasmatic region, cerebral cortex, and jejunum. Oil-treated rats served as controls. Injection of 0.7 or 7 microgram EB resulted in a significant increase in pituitary PRL without changing plasma PRL levels or pituitary VIP content compared to values in the control group. Only treatment with 70 microgram EB produced a significant increase in both pituitary VIP and PRL as well as in plasma PRL compared to control values. EB treatment at any of the doses used had no significant effect on pituitary and plasma TSH or VIP content in any of the other tissues examined. These data show that pituitary PRL and VIP are differentially regulated in response to estrogen. The increases in pituitary VIP and basal plasma PRL after treatment with the highest dose of EB suggest that pituitary VIP may be involved in the development of estrogen-induced hyperprolactinemia. These data also show that the regulations of pituitary VIP and TSH are independent of each other in the estrogen-treated rat.
Developmental changes in levels of growth hormone mRNA in Zucker rats.
Levels of pituitary growth hormone (GH) messenger RNA (mRNA) were compared in groups of genetically obese (fa/fa) and lean (Fa/-) littermate male Zucker rats at four different ages, 3, 5, 9, and 11 weeks, in order to determine the earliest age at which a difference between the two groups could be detected. No difference was seen in three-week-old animals. Five weeks of age was the earliest time at which the level of GH mRNA was significantly decreased in the obese rats; this decrease was present at all subsequent ages. Mean serum growth hormone levels were lower in obese animals at all ages, but the differences were not statistically significant because of the large individual variation associated with the pulsatile nature of GH release. The earliest occurrence of differences in GH mRNA level is later than some of the obesity associated abnormalities present in adipose tissue. The earliest time of the GH mRNA differences can be associated with the time when decreased protein deposition is initially seen in the obese rats. Because of this association, decreased GH mRNA may enhance the development of obesity.
Obesity- and sex-related alterations in growth hormone messenger RNA levels.
The secretion of growth hormone (GH) is abnormal in genetically obese Zucker rats. Measurements of pulsatile GH release and circulating GH levels in lean (Fa/?) and obese (fa/fa) rats have shown that both are reduced in the latter. We have studied pituitary GH gene expression in order to understand the role of GH synthesis in this abnormality. Obese animals have lower pituitary GH mRNA levels than lean controls. Within each genotype a sex difference was observed with the female animals having lower GH mRNA levels than the males. It is unlikely that the GH abnormality is due to a generalized pituitary defect because prolactin mRNA levels were the same in all four groups of rats.
Dual functions of a cis-acting element within the rat prolactin gene promoter.
Within the promoter region of the rat prolactin gene lies a TA-rich imperfect palindrome. The possible functions of the 18-base-pair symmetrical sequence were investigated by using an in vitro transcription system. Prolactin templates with and without the palindrome were transcriptionally assayed in both pituitary and nonpituitary extracts. Our results indicated that the palindromic sequence has at least two functions in the regulation of prolactin transcription.
In vivo and in vitro examination of an autoregulatory mechanism for luteinizing hormone-releasing hormone.
Recent evidence has been presented that demonstrates the existence of ultrashort feedback circuits for a number of neuropeptides in the central nervous system. The present investigation was undertaken to examine the possible existence of an autoregulatory mechanism for LHRH. In the first experiment, long term ovariectomized rats bearing lateral ventricular and atrial cannulae received intracerebroventricular (icv) injections of saline or LHRH every hour from 1200-1500 h. Blood was collected at 10-min intervals from 1200-1500 h to assess the effects of icv LHRH on pulsatile LH and FSH release. After the 1500 h collection, LHRH was injected iv and blood was collected to determine the effects of icv LHRH on pituitary responsiveness to LHRH. Central injections of 0.1 pg and 1 ng LHRH, but not 10 pg LHRH, significantly suppressed mean LH levels, trough LH levels, and LH pulse frequency compared to those in saline-treated control rats. The amplitude of LH pulses was not significantly affected by any dose of LHRH. In contrast to LH, multiple icv injections of LHRH failed to alter pulsatile FSH release. Pituitary LH and FSH responses to iv LHRH injection were not suppressed by icv LHRH. In a second experiment, hourly icv injections of 1 ng LHRH into proestrous rats markedly suppressed preovulatory LH release only during the middle to latter phases of the surge. In a final study using an in vitro superfusion system, addition of a LHRH agonist to the superfusion medium at a concentration that does not cross-react in the LHRH RIA suppressed basal and K+-stimulated LHRH release from medial basal hypothalamic fragments, but not from median eminence explants. These results support the existence and operation of an autoregulatory mechanism for LHRH in the central nervous system which may participate in the control of episodic LHRH release in ovariectomized rats and preovulatory LHRH release in proestrous rats. Seemingly, ultrashort-loop negative feedback regulation of LHRH requires the presence of structures other than nerve terminals in the MBH (i.e. cell bodies).
Correlation of rat pituitary prolactin messenger ribonucleic acid and hormone content with serum levels during the estrogen-induced surge.
The model of the serum PRL surge generated in the ovariectomized rat after estradiol benzoate (EB) treatment was used to study the relationship between serum and pituitary PRL levels and pituitary PRL mRNA levels. Adult ovariectomized rats were injected sc with 7 micrograms EB or vehicle at noon on day 0. Three days later (day 3), the rats were decapitated every 4 h over a 24-h period (0800 h on day 3 to 0400 h on day 4) for determination of serum and pituitary PRL and GH levels by RIA. In addition, PRL and GH mRNA content was determined using dot blot hybridization with cDNAs. Administration of EB resulted in a significant rise in serum PRL levels at 1200, 1600, and 2000 h on day 3 compared to control values. At other times, serum PRL levels in the EB group were the same as control values. EB treatment also elicited a marked increase in pituitary PRL content at all time periods examined except during (1600 and 2000 h) and after the PRL surge (2400 h on day 3) when there was a significant reduction in stored pituitary PRL. The pituitary PRL mRNA content in the EB-treated group was significantly elevated (4- to 6-fold) over control levels throughout the study. Furthermore, PRL mRNA levels in EB-treated rats were significantly higher at 2000 and 2400 h on day 3 than at other time periods. In contrast to its effects on PRL, EB treatment had a slight inhibitory effect on pituitary GH content at 2000 and 2400 h on day 3 compared to control values; otherwise, this steroid had no effect on serum GH levels and pituitary GH mRNA content. Interestingly, serum GH levels and pituitary GH mRNA content in both treatment and control groups fluctuated in a pattern consistent with circadian rhythms, with peak values occurring during the lights-on hours. These data show that estrogen has a stimulatory effect on pituitary content of PRL and its corresponding mRNA in the rat 3 days after injection. These elevated PRL mRNa levels may be necessary for the occurrence of PRL surges. Furthermore, the facts that serum PRL levels were elevated only at certain times (1200-2000 h on day 3) while PRL mRNA content was increased at all times in the EB-treated rats suggest a differential regulation between PRL release and biosynthesis.
Hyperprolactinemia attenuates ovarian steroid stimulation of region-specific hypothalamic serotonin synthesis and luteinizing hormone release in ovariectomized rats.
Hyperprolactinemia adversely affects reproductive functions, presumably through an effect at the hypothalamic level. Given the numerous published reports linking hypothalamic serotonin (5-hydroxytryptamine, 5HT) mechanisms to the regulation of gonadotrophin secretion, we sought to determine the effects of experimentally induced hyperprolactinemia on ovarian steroid-induced increases in serum LH levels and region-specific hypothalamic 5HT synthesis in ovariectomized rats. In the first study, bilaterally ovariectomized Sprague-Dawley rats either received two pituitary homografts implanted beneath the left kidney capsule or were sham-grafted. Both groups of rats were injected subcutaneously with 5 micrograms/100 g of estradiol benzoate (E2) in corn oil vehicle at 08.00 h, 1 and 2 days before serum collection and 5 mg/100 g of progesterone (P) in corn oil vehicle at 07.00 h on the day of serum collection. Blood samples were collected via chronic indwelling jugular cannulae from each rat at 10.00, 12.00, 13.00, 14.00, 16.00 and 18.00 h. A statistically significant elevation in serum LH levels was detected at 13.00, 14.00 and 16.00 h. This increase in serum LH levels was significantly attenuated in rats bearing pituitary homografts, an effect attributed to the high serum PRL levels measured in these animals. In the second study, bilaterally ovariectomized Sprague-Dawley rats were divided into three experimental groups: (1) rats bearing two pituitary homografts and injected with E2 and P on the schedule and at the dosages previously described, (2) sham-grafted rats injected with E2 and P on the schedule and at dosages previously described and (3) sham-grafted rats injected with corn oil vehicle only.(ABSTRACT TRUNCATED AT 250 WORDS)
Vasoactive intestinal peptide increases prolactin messenger ribonucleic acid content in GH3 cells.
The influence of vasoactive intestinal peptide (VIP), TRH, and dexamethasone (Dex) on PRL mRNA was investigated in PRL-producing GH3 cells using cytoplasmic dot hybridization and RNA blot analysis. Total cytoplasmic RNA was transferred to nitrocellulose filter paper and quantitated by hybridization to PRL recombinant DNA probes labeled with 32P. Incubation of GH3 cells with VIP for 25 h increased the content of cytoplasmic PRL mRNA. This increase was dose dependent, being significant at 2 X 10(-8) M and reaching a maximum at 2 X 10(-7) M. VIP at 2 X 10(-9) M had no effect on cytoplasmic PRL mRNA content. TRH (2 X 10(-7) M) also increased whereas Dex (2 X 10(-7) M) decreased the content of PRL mRNA. The inhibitory effect of Dex (2 X 10(-7) M) on cytoplasmic PRL mRNA was reversed by VIP (2 X 10(-7) M). Changes in medium PRL levels after these various hormone treatments paralleled those changes observed in PRL mRNA content. Examination of total poly(A)+ RNA demonstrated that incubation with VIP (2 X 10(-7) M) for 6 h increased the content of the mature PRL mRNA and its processing intermediates. Dex (2 X 10(-7) M) decreased the content of all species of PRL mRNA. These data suggest that VIP-stimulated PRL release is the result of an increase in the content of PRL mRNA and its precursors.
Estrogen induced prolactin mRNA accumulation in adult male rat pituitary as revealed by in situ hybridization.
Adult male rats were injected with different doses (1, 10, and 100 micrograms) of 17 beta-estradiol daily for 5 days, and the changes in prolactin (PRL) mRNA levels were examined by in situ hybridization and cytoplasmic dot blot hybridization using cloned cDNA for rat prolactin mRNA. An increase in cytoplasmic PRL mRNA content was evident in all the animals treated with estrogen as revealed with cytoplasmic dot blot analysis. There were however, no significant differences in PRL mRNA content among the three estradiol treated groups. Cytoplasmic PRL mRNA was also demonstrated by in situ hybridization on the frozen pituitary sections using a 3H-labeled PRL cDNA probe. The number of grains per cell was increased after estrogen treatment. 3H-thymidine uptake into pituitary cells was also examined in vivo using combined techniques of immunocytochemistry and autoradiography. Although the percentage of immunoreactive PRL cells which took up thymidine in their nuclei increased to more than double after estrogen treatment, the increase in the total number of immunoreactive PRL cells was small. These results suggest that the major effect of estrogen on PRL cells is an increase in the accumulation of PRL mRNA in the individual PRL cells. The number of grains per cell was found to vary from cell to cell, both in control and estrogen treated animals. This variability is discussed in relation to the functional heterogeneity within the PRL cell population.
Hypothalamic deafferentation inhibits the stimulatory influence of prolactin on follicle-stimulating hormone release in the golden hamster.
We have examined the effects of PRL-producing ectopic pituitary grafts on plasma FSH and LH levels in male golden hamsters with anterior (AHD) and anteolateral hypothalamic deafferentation (ALHD). After hypothalamic deafferentation or sham deafferentation, hamsters received pituitary transplants under the kidney capsule. In the first experiment, the animals were unilaterally nephrectomized 5-6 weeks after transplantation to remove the ectopic pituitaries. In a second experiment the ectopic pituitaries remained under the kidney capsule for the duration of the experiment. Serial blood samples were collected before and after pituitary transplantation and after transplant removal. In the first experiment, ALHD resulted in a significant (P less than 0.01) suppression of plasma LH levels that lasted through the entire study, but had no significant effect on plasma FSH levels or on testicular and seminal vesicle weight. The presence of pituitary grafts resulted in a significant (P less than 0.01) elevation in plasma FSH levels in the sham-deafferented hamsters but had no effect on plasma FSH levels in those with ALHD. After transplant removal there was a significant (P less than 0.01) drop in plasma FSH levels in the hamsters that had been sham deafferented but not in those with a deafferented hypothalamus. Plasma LH levels were not affected by the pituitary transplants or their removal in either group. In the second experiment, ALHD but not AHD significantly (P less than 0.05) reduced testicular and seminal vesicle weights. Transplantation of an anterior pituitary elevated plasma FSH levels in the control group but not in the AHD and ALHD groups. Both types of deafferentations suppressed plasma LH levels below the limit of sensitivity of the assay for the duration of the experiment. Plasma PRL levels were markedly elevated only in the groups with an ectopic pituitary transplant. These results suggest that the anterior connections of the medial basal hypothalamus are necessary in order for PRL-producing pituitary transplants to influence FSH secretion from the in situ pituitary.
Dopaminergic stimulation of pituitary but not hypothalamic estrogen receptors in ovariectomized rats.
This study was designed to examine the role of catecholamines and serotonin in the regulation of estrogen receptors (ER) in the medial basal hypothalamus (MBH) and anterior pituitary gland (AP) of adult ovariectomized rats. Plasma PRL and LH were also determined. Injection of alpha-methyl tyrosine (alpha-MT) resulted in the significant reduction of norepinephrine and dopamine (DA) content in the MBH, and of plasma LH levels as well as in the significant elevation of plasma PRL levels. This treatment also resulted in the significant reduction of ER concentration in the AP. The elevation in plasma PRL by alpha-MT was reversed by the simultaneous injection of bromocriptine (BC), a DA agonist, which also partially reversed the alpha-MT reduction of ER concentrations in the AP. BC had no effect on plasma LH levels. The ER concentration in the MBH was not significantly changed by any of these treatments. The reduction of serotonin content in the MBH by the injection of p-chlorophenylalanine had no effect on the ER concentration in either the MBH or AP, nor did it have any effect on plasma PRL levels. However, p-chlorophenylalanine treatment did decrease plasma LH levels. Neonatal treatment of female rats with monosodium glutamate which has been reported to destroy part of the MBH, resulted in a significant reduction in body and pituitary weight and in a significant elevation of plasma PRL levels in adults (2 months old). This treatment also resulted in a significant reduction in the AP and MBH ER concentration. Injection of BC to adults reversed the effects of neonatal monosodium glutamate treatment on plasma PRL levels and on the pituitary ER concentration, but had no effect on the ER concentration in the MBH. BC had no effect on the AP or MBH ER concentration in control rats, although it did as expected reduce the plasma PRL levels in these animals. Plasma LH levels were not significantly changed by any of these treatments. Injection of the DA antagonist, haloperidol, to adult rats resulted in a significant elevation of plasma PRL and in a significant reduction of ER concentration in the AP. Haloperidol treatment did not affect the binding affinity of these receptors. Overall, these data suggest that DA is involved with the regulation of ER in the AP.
Plasma LH levels in the long term ovariectomized rat after anterolateral deafferentation of the medial basal hypothalamus and lesions in the medial preoptic area.
This study was designed to investigate the effect of anterolateral hypothalamic deafferentiation (ALHD) and medial preoptic area (MPOA) lesions on plasma LH levels in the long term ovariectomized rat. The deafferentations were carried out with a Halasz-Pupp knife (radius of 1.5 mm and height of 2.0 mm) and the MPOA lesions with a platinum electrode. Sham treated and an intact group served as controls. Blood samples were obtained from the jugular vein under light ether anesthesia before and at 1, 2, 4 and 6 weeks after brain surgery. After the sixth week sample all rats were treated with 50 micrograms of estradiol benzoate (EB) and two days later blood samples were collected during the morning and afternoon. Hypothalamic deafferentation resulted in a more significant (p less than 0.01) drop in plasma LH levels in one half of the group (ALHD-1) than in the other half (p less than 0.05) (ALHD-2) when compared to the controls. Treatment of the controls with EB resulted in a significant (p less than 0.01) depression of LH levels in the morning and an LH surge during the afternoon. EB also resulted in a suppression (p less than 0.01) of LH levels during the morning in all of the ALDH rats; however, only the ALHD-1 group had an LH surge during the afternoon following EB. Plasma LH levels in the ALHD-2 remained suppressed during the afternoon after EB treatment. Lesions in the MPOA had no effect on plasma LH levels at 1 to 6 weeks when compared to controls. Treatment of the MPOA lesion group with EB resulted in a significant (p less than 0.01) drop in plasma LH levels during the morning as well as the afternoon. These data suggest that the fibers that are critical for the control of tonic and phasic LH secretion enter the medial basal hypothalamus laterally and that the deafferentations carried out here were selective in interrupting fibers involved with tonic LH secretion in some rats and those involved with the phasic secretion in others. These data also suggest that the MOPA components involved with tonic LH secretion are separate from those controlling phasic LH secretion.
The hypophyseal-adrenal axis in the protein-calorie malnourished rat.
Male rats were fed an 8% low protein diet for 30 consecutive days beginning at 20 days of age. On day 50, the effect of the diet was evaluated on the hypophyseal-adrenal axis and the observations compared with those noted in age-matched controls fed a standard laboratory diet. The malnourished animals had significantly larger adrenal gland weights, relative to body size, and higher levels of serum corticosterone. Additionally, the number of ACTH-secreting cells in the pituitary glands of these rats was 63.8% greater than that present in the controls. These results suggest that ACTH secretion and adrenocortical physiology are stimulated under conditions of experimentally induced protein-calorie malnutrition.