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Endocrinology Vol. 148, No. 1 300-309
Copyright © 2007 by The Endocrine Society

Severity of the Catabolic Condition Differentially Modulates Hypothalamic Expression of Growth Hormone-Releasing Hormone in the Fasted Mouse: Potential Role of Neuropeptide Y and Corticotropin-Releasing Hormone

Raul M. Luque, Seungjoon Park and Rhonda D. Kineman

Section of Endocrinology and Metabolism Department of Medicine (R.M.L., R.D.K.), University of Illinois at Chicago, Chicago, Illinois; Research and Development Division (R.M.L., R.D.K.), Jesse Brown Veterans Administration Medical Center, Chicago, Illinois 60612; and Department of Pharmacology and Institute for Basic Medical Science (S.P.), Kyunghee University School of Medicine, Seoul 130-701, Korea

Address all correspondence and requests for reprints to: Rhonda D. Kineman, Ph.D., Jesse Brown Veterans Administration Medical Center, Research and Development Division, mail posted 151, West Side, Suite 6215, 820 South Damen Avenue, Chicago, Illinois 60612. E-mail: Kineman{at}uic.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To determine whether the severity of the catabolic condition differentially regulates the GH axis, male mice were either fed ad libitum or fasted for 12, 24, and 48 h. Hypothalami, pituitaries, and stomachs were collected for assessment of mRNA levels by quantitative real-time RT-PCR, and blood collected for measurement of plasma hormone and metabolite levels by commercial assay kits. Overnight (12 h) fasting resulted in a significant suppression of circulating glucose, insulin, IGF-I, and leptin levels and an increase in corticosterone, free fatty acids, and n-octanoyl ghrelin levels, and these directional changes were maintained at the 24- and 48-h time points. Fasting (24 h) also increased circulating GH levels, which was associated with an increase in pituitary mRNA levels for GHRH receptor and ghrelin receptor and a decrease in mRNA levels for somatostatin (SST) receptor (SSTR) subtypes, SSTR2, SSTR3, and SSTR5, where the changes in ghrelin receptor and SSTR expression persisted after 48 h fasting. Hypothalamic SST mRNA levels were not altered by fasting, whereas there was a transient rise in stomach SST mRNA levels 24 h after food withdrawal. In contrast, there was a biphasic effect of fasting on GHRH expression. GHRH mRNA levels were significantly elevated at 12 and 24 h but fell to ~50% of fed controls 48 h after food withdrawal. A sequential rise in hypothalamic neuropeptide Y (NPY) and CRH mRNA levels preceded the fall in GHRH expression, where fasting-induced changes in CRH and GHRH mRNA levels were not observed in 48-h-fasted NPY knockout mice. These observations, in light of previous reports showing both NPY and CRH can inhibit GHRH expression and GH release, suggest that these neuronal systems may work in concert to control the ultimate impact of fasting on GH axis function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE EFFECT OF fasting on circulating GH levels is species dependent. In male rats, fasting results in progressive suppression of GH pulse amplitude, associated with a decrease in expression of hypothalamic GHRH, no change or reduced expression of hypothalamic somatostatin (SST), and an increase in systemic SST tone (1, 2, 3, 4, 5, 6, 7). We have previously observed that fasting (48 h) suppressed hypothalamic GHRH mRNA levels in mice similar to that observed in rats (8). However, measurement of GH levels from single trunk blood samples revealed GH was not suppressed but in fact tended to be elevated in the fasted mouse, similar to that observed in the majority of mammalian species studied to date (9, 10, 11, 12, 13, 14, 15). Given the mouse has become an animal model that is commonly used to investigate the interrelationship between metabolism and the endocrine system, we sought to clarify these divergent results by determining whether the duration of fasting differentially regulates the GH axis of the mouse.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
All experimental procedures were approved by the animal care and use committees of the University of Illinois at Chicago and the Jesse Brown Veterans Administration Medical Center. Male C57BL/6J mice were purchased from Jackson Laboratories (Bar Harbor, ME) at 7 wk of age and allowed to acclimate to the facility, personnel, and daily handling. During this acclimation period, mice were housed under standard conditions of light (12-h light, 12-h dark cycle; lights on at 0700 h) and temperature (22–24 C), with free access to standard rodent chow (LabDiet, St. Louis, MO; catalog no. 5008; fat, 17 kcal%, carbohydrate, 56 kcal%; protein, 27 kcal%) and tap water. At 10 wk of age, mice were assigned to one of four groups: 12-h fast (food removed at 1900 h), 24- or 48-h fast (food removed at 0700 h), or ad libitum fed. Mice were weighed at the time of food withdrawal and just before death. All mice were killed by decapitation without anesthesia between 0700 and 0900 h on the same day. Trunk blood was collected for hormone and metabolite determinations, whereas hypothalami, pituitaries, and stomachs were collected for analysis of mRNA by quantitative real-time RT-PCR (qrtRT-PCR; see below for details).

RNA isolation and RT
Tissues were processed for recovery of total RNA using the Absolutely RNA RT-PCR Miniprep Kit (Stratagene, La Jolla, CA), with Deoxyribonuclease treatment as previously described (16, 17). The amount of RNA recovered was determined using the Ribogreen RNA quantification kit (Molecular Probes, Eugene, OR). Total RNA (1 µg) was reversed transcribed in a 20-µl volume using random hexamer primers, with enzyme and buffers supplied in the cDNA First Strand Synthesis kit (MRI Fermentas, Hanover, MD). cDNA was treated with ribonuclease H, and duplicate aliquots (1 µl) were amplified by qrtRT-PCR, where samples were run against synthetic standards to estimate mRNA copy number (see below). To study the role of neuropeptide Y (NPY) on fasting-induced changes in GHRH and CRH mRNA levels, RNA samples previously extracted from hypothalami of fed and fasted (48 h only) wild-type and NPY knockout (KO) of the 129/sv background strain (8) were purified using the absolutely RNA RT-PCR Miniprep Kit and processed as described above.

qrtRT-PCR
Details regarding the development, validation, and application of a qrtRT-PCR to measure expression levels of mouse GHRH, SST, NPY, CRH, proopiomelanocorticotropin (POMC), GHRH receptor (GHRH-R), ghrelin receptor (GHS-R), ghrelin, and cyclophilin A have been reported previously (16, 17). Primer sets for mouse SST receptor (SSTR) 1–5 used in this study have not been previously reported and therefore are provided in Table 1Go. For real-time PCR, Brilliant SYBR Green QPCR Master Mix (Stratagene) was used, where thermocycling and fluorescence detection was performed using a Stratagene Mx3000p real-time PCR machine. The final volume of the PCR was 25 µl: 1 µl RT sample, 12.5 µl QPCR Master Mix, 0.375 µl each primer (10 µM stock solution), 0.375 µl reference dye, and 10.375 µl dH2O. Thermal cycling profile consisted of a preincubation step at 95 C for 10 min, followed by 40 cycles of denaturation (95 C, 30 sec), annealing (61 C, 1 min), and extension (72 C, 30 sec). Final PCR products were subjected to graded temperature-dependent dissociation to verify that only one product was amplified. To determine the starting copy number of cDNA, RT samples were PCR amplified, and the signal was compared with that of the standard curve run on the same plate. Standard curves consisted of 101, 102, 103, 104, 105, and 106 copies of synthetic cDNA template for each of the transcripts of interest. Standard curves were generated by the Stratagene Mx3000p Software, and the slopes were approximately 1, indicating the efficiency of amplification was 100%, meaning within the detectable range, all templates in each cycle were copied. In addition, total RNA samples that were not reversed transcribed and a no DNA control were run on each plate to control for genomic DNA contamination and to monitor potential exogenous contamination, respectively. Also, to control for variations in the amount of RNA used in the RT reaction and the efficiency of the RT reaction, mRNA copy number of the transcript of interest was adjusted by the mRNA copy number of cyclophilin A (a peptidyl isomerase), where cyclophilin A mRNA levels did not significantly vary between experimental groups, within tissue type (data not shown).


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  		TABLE 1. Mouse-specific primers for somatostatin receptor subtypes (SSTR1–5) used for quantitative real-time RT-PCR

 
Assessment of hormones and metabolites
Glucose levels were determined in fresh whole-blood samples, using the SureStep glucometer (Johnson & Johnson, Milpitas, CA). The remaining trunk blood (200–500 µl) was immediately mixed with 15 µl MiniProtease inhibitor (Roche, Nutley, NJ) and placed on ice until centrifugation. Plasma was collected and stored at –80 C until analysis of insulin (rat/mouse ELISA; LINCO, St. Charles, MO), corticosterone (Octeia rat/mouse ELISA; IDS Fountain Hills, AZ), free fatty acids (FFAs) (WAKO, Richmond, VA), leptin (mouse ELISA; LINCO), total and n-octanoyl ghrelin (rat/mouse ELISA; LINCO; catalog nos. EZGDAC-87K and EZGAC-86K, respectively), GH (mouse/rat ELISA, DSL, Webster, TX), and IGF-I (Octeia rat/mouse ELISA; IDS).

Data analysis
The effect of time of fasting on mRNA and circulating hormone and metabolite levels in C57BL6/SJ mice was assessed by one-way ANOVA, whereas the effects of fasting on GHRH and CRH mRNA levels in NPY-intact vs. NPY KO mice were assessed by two-way ANOVA, followed by Fisher’s least significant difference test for multiple comparisons. In the case of circulating GH levels, which were pulsatile in nature, the values in fed and 24-h-fasted mice did not fit a normal distribution and therefore were normalized by log transformation before statistical analysis by Student’s t test. P < 0.05 was considered significant. All statistical analyses were performed using the GB-STAT software package (Dynamic Microsystems, Inc., Silver Spring, MD).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of fasting on metabolic endpoints
Mice fasted for 12 and 24 h exhibited a comparable loss in body weight [9.5 ± 0.8% and 10.9 ± 0.5% of starting weight (27.1 ± 0.4 g), respectively], whereas mice fasted for 48 h lost 17.3 ± 0.8% of prefast weight. Consistent with the catabolic condition, circulating glucose, insulin, and leptin levels were significantly suppressed, whereas circulating FFAs were significantly increased after an overnight (12 h) fast, where all endpoints were maximally affected by 24 h (Fig. 1Go).


Figure 1
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  		FIG. 1. Circulating glucose (A), insulin (B), leptin (C), and FFAs (D) of fed and fasted (12, 24, and 48 h) 10-wk-old male C57BL/6J mice (n = 5–8 mice/group). Values are presented as mean ± SEM, and group means that do not share a common letter (a, b) significantly differ (P < 0.05).

 
Effect of fasting on the primary components of the GH axis
As illustrated in Fig. 2AGo, short-term fasting (12 and 24 h) resulted in a significant increase in hypothalamic GHRH mRNA levels. However, by 48 h fasting, GHRH mRNA levels tended to fall below those of fed controls, consistent with the inhibitory effect of 48 h fasting on GHRH mRNA levels in the NPY-intact (wild type) mice previously reported by our laboratory (8) and confirmed in the present study (Fig. 7BGo). In contrast, fasting did not alter hypothalamic expression of SST or SSTR subtypes (SSTR1–5). However, fasting did result in a transient rise in stomach SST mRNA levels after 24 h fasting (Fig. 2AGo, inset). Because our laboratory has previously reported that endogenous NPY is required for the suppression of GHRH mRNA levels observed after a 48-h fast (8), we also examined whether the duration of fasting differentially regulated hypothalamic NPY mRNA levels. A significant increase in NPY expression was observed as early as 12 h after food withdrawal, and NPY mRNA levels achieved 200% of fed controls by 48 h.


Figure 2
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  		FIG. 2. GHRH, SST, NPY, and SST receptor (SSTR1–5) mRNA levels in the hypothalamus (A), SST mRNA levels in the stomach (A, inset), and GH, GHRH-R, and SST receptor (SSTR1–5) mRNA levels in the pituitary (B) of fed and fasted (12, 24, and 48 h) 10-wk-old male C57BL/6J mice (n = 5–8 mice/group). mRNA copy number was determined by qrtRT-PCR, and the values were adjusted by cyclophilin A mRNA copy number as an internal control. Values represent the mean ± SEM and are expressed as percentage of fed controls (set at 100%). Group means that do not share a common letter (a) significantly differ (P < 0.05).

 

Figure 7
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  		FIG. 7. Hypothalamic NPY (A), GHRH (B), and CRH (C) mRNA levels of fed and fasted (48 h) NPY+/+ and NPY–/– male 129/sv mice (n = 5–6 mice/group). mRNA copy number was determined by qrtRT-PCR, and the values were adjusted by cyclophilin A mRNA copy number as an internal control. Values represent the mean ± SEM and are expressed as percentage of fed mice controls (set at 100%). Group means that do not share a common letter (a) significantly differ (P < 0.05).

 
At the level of the pituitary, fasting did not alter GH mRNA levels (Fig. 2BGo). However, fasting did transiently augment the expression of the GHRH-R (24 h), whereas having an overall inhibitory effect on SST receptor expression (Fig. 2BGo). The fasting-induced suppression of SST receptors was isoform specific, where a significant suppression of SSTR2, SSTR3, and SSTR5 mRNA levels was observed as early as 12 h fasting, whereas SSTR1 and SSTR4 mRNA levels were resistant and only tended to be suppressed at the 48-h time point.

Consistent with the elevations in the expression of hypothalamic GHRH and the reciprocal shift in expression of pituitary GHRH-R and SST receptors at 24 h fasting, circulating GH levels were elevated (P < 0.002; Fig. 3AGo). Despite the overall stimulatory effect of short-term fasting on hypothalamic and pituitary components of the GH axis, circulating levels of total IGF-I were reduced after 12 h fasting and remained suppressed throughout the duration of the fast (Fig. 3BGo), whereas fasting failed to alter hypothalamic IGF-I and IGF-IR expression (Fig. 3CGo). The dissociation between circulating GH and IGF-I in the fasted state has been attributed to a decrease in hepatic sensitivity to GH stimulation that leads to a decrease in hepatic expression of IGF-I (18).


Figure 3
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  		FIG. 3. Circulating GH (n = 16–17 mice/group; A) and IGF-I (n = 5–8 mice/group; B) levels and hypothalamic IGF-I and IGF-I receptor (IGF-IR) mRNA levels (n = 5–8 mice/group; C) of fed and fasted 10-wk-old male C57BL/6J mice. GH values were normalized by log transformation, and data are presented as mean ± SEM, as well as individual data points (A). Absolute values for circulating IGF-I levels are shown as the mean ± SEM (B). Hypothalamic IGF-I and IGF-IR mRNA copy numbers were determined by qrtRT-PCR, and the values were adjusted by cyclophilin A mRNA copy number as an internal control and are expressed as percentage of fed controls (set at 100%; C). Group means that do not share a common letter (a) significantly differ (P < 0.05). **, Significant effect of 24 h fasting from fed controls, P < 0.002.

 
Effects of fasting on ghrelin/GHS-R
The n-octanoyl form of ghrelin stimulates the release of GH by acting at both the hypothalamic and pituitary levels via the GH secretagogue receptor, GHS-R1a (for review, see Ref. 19). In the mammalian species studied to date, fasting increases circulating ghrelin levels, which may in turn contribute to fasting-induced changes in the GH axis. The bulk of ghrelin found in the circulation is produced in the gastrointestinal tract. However, ghrelin produced in regions of the hypothalamus (20) and within the anterior pituitary gland (21) may also contribute to regulation of GH secretion. Therefore, we assessed the impact of fasting on circulating n-octanoyl ghrelin and total ghrelin levels and stomach ghrelin mRNA levels (Fig. 4Go), as well as hypothalamic and pituitary ghrelin and GHS-R1a mRNA levels (Fig. 5Go). Fasting did not alter circulating levels of total ghrelin or stomach ghrelin mRNA levels (Fig. 4Go, B and C, respectively) but did have an overall stimulatory impact on circulating levels of n-octanoyl ghrelin, which reached significance at 12 h fasting (Fig. 4AGo). Fasting also enhanced pituitary expression of ghrelin but had no significant effect on hypothalamic ghrelin mRNA levels (Fig. 5Go). The fasting-induced rise in circulating n-octanoyl ghrelin and pituitary ghrelin mRNA was accompanied by a rise in GHS-R1a mRNA at both the pituitary and hypothalamic levels.


Figure 4
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  		FIG. 4. Circulating n-octanoyl ghrelin (A), circulating total ghrelin (B), and stomach ghrelin mRNA (C) levels of fed and fasted (12, 24, and 48 h) 10-wk-old male C57BL/6J mice (n = 5–8 mice/group). Values are shown as mean ± SEM. Stomach ghrelin mRNA copy number was determined by qrtRT-PCR, and the values were adjusted by cyclophilin A mRNA copy number as an internal control and are expressed as percentage fed mice controls (set at 100%). Group means that do not share a common letter (a, b) significantly differ (P < 0.05).

 

Figure 5
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  		FIG. 5. Hypothalamic and pituitary ghrelin and GHS-R mRNA levels of fed and fasted (12, 24, and 48 h) 10-wk-old male C57BL/6J mice (n = 5–8 mice/group). mRNA copy numbers were determined by qrtRT-PCR, and the values were adjusted by cyclophilin A mRNA copy number as an internal control. Values represent the mean ± SEM and are expressed as percentage of fed mice controls (set at 100%). Group means that do not share a common letter (a–c) significantly differ (P < 0.05).

 
Effects of fasting on the adrenal axis
As illustrated in Fig. 6AGo, fasting dramatically increased circulating corticosterone levels, with maximal effects observed at 12 h. Changes in circulating corticosterone levels were not accompanied by significant changes in pituitary expression of POMC (Fig. 6BGo). Also, the rise in corticosterone levels observed at 12 h was not accompanied by an increase in hypothalamic CRH mRNA levels (Fig. 6CGo). However, CRH expression was increased after 24 h fasting.


Figure 6
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  		FIG. 6. Circulating corticosterone levels (A), pituitary POMC (B), and hypothalamic CRH (C) mRNA levels of fed and fasted (12, 24, and 48 h) 10-wk-old male C57BL/6J mice (n = 5–8 mice/group). CRH and POMC mRNA copy numbers were determined by qrtRT-PCR, and the values were adjusted by cyclophilin A mRNA copy number as an internal control. mRNA levels are expressed as percentage of fed controls (set at 100%). Group means that do not share a common letter (a–d) significantly differ (P < 0.05).

 
Effects of 48 h fasting on hypothalamic GHRH and CRH mRNA levels in NPY KO mice
This is the first report demonstrating a biphasic effect of fasting on hypothalamic GHRH expression, where short-term (12 and 24 h) fasting augments and more prolonged (48 h) fasting suppresses GHRH mRNA levels (Fig. 2AGo). The question arises, what drives this reciprocal shift in GHRH expression? CRH represents a potential candidate because 1) a CRH antagonist enhanced GHRH mRNA levels (22); 2) NPY, which is required for fasting-induced suppression of GHRH mRNA level (8), also stimulates CRH neuronal activity (23, 24); and 3) there was a delayed rise in CRH mRNA levels in response to fasting that preceded the fall in GHRH expression (Fig. 6Go). To further explore the interrelationship among fasting, GHRH, CRH, and NPY, we used qrtRT-PCR to measure NPY, GHRH, and CRH mRNA levels in hypothalamic RNA samples previously generated from fed and 48-h-fasted wild-type and NPY KO mice of the 129/sv background strain (8). Fasting increased NPY mRNA levels and suppressed GHRH mRNA levels in wild-type mice but failed to alter GHRH expression in NPY KO mice (Fig. 7Go, A and B). These results are consistent with our previous findings using ribonuclease protection assays to measure hypothalamic NPY and GHRH mRNA levels in these same samples (8) and therefore confirm the quality of the samples and the accuracy of the qrtRT-PCR assay system. As shown in Fig. 7CGo, a 48-h fast enhanced CRH mRNA levels in wild-type 129/sv mice, a response comparable with that observed in fasted C57BL/6J mice (Fig. 6Go). However, in the absence of NPY, fasting did not alter CRH expression.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Fasting enhances GH release in the majority of mammalian species studied to date, including humans, pigs, guinea pigs, sheep, cows, rabbits, and dogs (9, 10, 11, 12, 13, 14, 15). The current results support our previous findings (8) and those of others (25), indicating circulating GH levels also rise in the mouse in response to food deprivation. In addition, GH levels have been reported to increase in response to nutrient deprivation at the cellular level, as observed in streptozotocin-induced diabetic mice and nonobese diabetic mice (26, 27), a response similar to that observed in humans with uncontrolled type 1 diabetes (28). Exactly what potentiates GH release in response to nutritional stress remains a subject of debate.

In humans, acute fasting results in an increase in GH peak frequency and peak height, in addition to an elevation in interpulse GH release (9, 29). A similar pattern of GH release is observed in anorexia (30) and uncontrolled type 1 diabetes (28). The fasting-induced changes in interpulse GH release are thought to be mediated by a reduction in SST tone, whereas the increase in GH peak frequency and height has been attributed to a rise in pulsatile GHRH release. Experimental data supports a major role of SST in mediating changes in GH output in response to nutritional stress. In the food-restricted sheep, GH levels rise in association with a reduction in SST mRNA levels specifically within the rostral periventricular nucleus and ventromedial nucleus of the hypothalamus (31), which is reflected by a fall in hypophyseal portal concentrations of SST (32). In the current study, fasting did not significantly alter hypothalamic SST mRNA levels in the mouse. However, we cannot exclude the possibility that our measurement of whole hypothalamic extracts may have masked nuclei specific changes in SST expression. In fact, in a previous study we observed a small (15%) but significant decrease in hypothalamic SST mRNA levels in 48-h-fasted mice of the 129/sv background (8). In the rat, fasting also has been reported to decrease or have no effect on hypothalamic expression of SST (6, 7). However, in this species, circulating SST levels are elevated due to an increase SST production from the gastrointestinal tract (33, 34), and this rise in systemic SST is thought to significantly contribute to the reduction in GH pulsatile release observed in this species (1). It is possible that systemic levels of SST may also rise in the fasted mouse, in that we observed a transient increase in SST mRNA levels in the stomach, at the 24-h time point.

Experimental data also support a role for GHRH in mediating changes in GH output in response to nutritional stress. Specifically, in food-restricted sheep, the fasting-induced suppression of hypothalamic SST synthesis and release is accompanied by an increase in GHRH mRNA levels within neurons of the arcuate and ventromedial nuclei of the hypothalamus (31). These findings are consistent with the results of the current study demonstrating that short-term fasting (12 and 24 h) can augment hypothalamic GHRH expression in mice. However, it should be noted that in food-restricted sheep, the rise in GHRH mRNA levels was not accompanied by a detectable rise in hypophyseal portal vascular concentrations of GHRH (32). This discrepancy may be more apparent than real, in that interpulse GHRH peptide levels in the hypophyseal portal vasculature of both ad libitum-fed and food-restricted sheep were below the sensitivity of the assay used; therefore, a rise in baseline levels or increases in the number of low amplitude pulses would not have been detected (32). Interestingly, a fasting-induced rise in GHRH mRNA levels is also observed in the spontaneous dwarf rat, an animal model characterized by low basal IGF-I levels due to an inactivating mutation in the GH gene (7). This is in contrast to the inhibitory effects of fasting on GHRH expression in normal rats (2, 5, 6, 7), suggesting there are both positive and negative regulators of GHRH expression in the fasted state, where under certain physiologic conditions, the balance between these regulators can be skewed to favor GHRH synthesis.

In the current study, the increase in hypothalamic GHRH mRNA levels observed in the mouse after 24 h fasting was associated with a reciprocal shift in the pituitary receptor expression profile (increased GHRH-R and GHS-R and decreased SSTR2, SSTR3, and SSTR5 mRNA levels) that would be predicted to favor GH synthesis and release. The current results are consistent with the results of a previous study using a semiquantitative RT-PCR method (8). It is interesting to note that our laboratory (7), as well as others (35, 36, 37), have also reported similar changes in pituitary receptor expression in the fasted male rat, suggesting that fasting-mediated changes in the pituitary responsiveness are common across species, despite the species-dependent impact of fasting on GHRH expression and GH release. These changes in pituitary receptor expression are likely functionally relevant because nutrient deprivation has been shown to increase the GH response to exogenous administration of GHRH and GHS-R agonist and decrease GH response to SST (15, 33, 35, 38).

The current findings, coupled with the experimental and clinical observations of others, support the hypothesis that changes in hypothalamic GHRH and SST input, as well as changes in pituitary sensitivity to that input, collectively contribute to elevations in circulating GH concentrations in response to nutritional stress. Review of the literature suggests multiple factors may work in concert to mediate these changes. Although IGF-I is clearly important in negative feedback regulation of the GH axis under fed conditions, the fall in circulating IGF-I is unlikely to mediate fasting-induced effects on GHRH expression because GHRH and SST mRNA levels are unaltered in liver-specific, IGF-I KO mice, where circulating IGF-I levels are less than 10% of wild-type controls (39). However, it has long been recognized that IGF-I can directly inhibit somatotrope function by suppressing both GH release and synthesis in rat pituitary cell lines, primary rat pituitary cell cultures, and human somatotropinomas (40). IGF-I has also been shown to decrease GHRH-R (41) and GHS-R (42) mRNA levels in primary rat pituitary cell cultures; more recently, our laboratory has reported that IGF-I decreases GH synthesis and release, as well as GHRH-R and GHS-R mRNA levels, in primary pituitary cell cultures of mice and baboons (43, 44). The inhibitory actions of IGF-I on somatotrope function can be mimicked by insulin at doses not predicted to bind and activate the IGF-I receptor (43, 44). These results, taken together with the fact that pituitaries express receptors for both insulin and IGF-I (43, 44), support the hypothesis that the fasting-mediated increase in GH release may be explained, in part, by a decrease in IGF-I and insulin inhibitory tone. Finally, the rise in circulating glucocorticoids, in the face of falling IGF-I and insulin levels, may also directly enhance somatotrope function in response to fasting. In vitro, glucocorticoids have been shown to increase GHRH-R and GHS-R mRNA levels in primary pituitary cell cultures from rats and baboons, whereas they have a predominately inhibitory effect on SST receptor expression in rat pituitary cell cultures (45, 46, 47, 48).

Another factor that may be involved in the fasting-induced changes in the GH axis is ghrelin. In many species, both total and n-octanoylated ghrelin levels rise in response to fasting (for review, see Ref. 19). However, as observed in the current study, fasting resulted in a rise in n-octanoyl ghrelin in mice without significant changes in total circulating ghrelin levels, consistent with our previous observations and those of others showing no effect of fasting on total circulating ghrelin or stomach ghrelin mRNA levels in mice (17, 49, 50). Nonetheless, it is the n-octanoyl form of ghrelin, acting via GHS-R1a, that enhances GH release by both central and pituitary actions (19). Centrally, ghrelin enhances pulsatile GH release, and this stimulatory effect is thought to be due to an increase in GHRH neuronal activity (51). A positive effect of ghrelin on GHRH expression is further supported by the recent findings that transgenic rats with attenuated expression of hypothalamic GHS-R1a levels have reduced GHRH expression, compared with wild-type controls (52). Also, ghrelin can rapidly stimulate the expression of the GHS-R in the arcuate nucleus (53) and thus may account for fasting-induced rise in hypothalamic GHS-R mRNA observed in mice (current report) and in rats (53). At the level of the pituitary, ghrelin directly stimulates basal GH release and augments GHRH-stimulated cAMP production (19). Although the primary source of circulating ghrelin is the gastrointestinal tract, ghrelin is also produced within the pituitary (21) and in neurons within the hypothalamus (20); therefore, local changes in ghrelin production may mediate both central and pituitary regulation of GH release. In the mouse, we observed an early and sustained rise in circulating n-octanoylated ghrelin, in addition to an increase in pituitary ghrelin expression. Therefore, an increase in pituitary production of ghrelin and an increase in pituitary and hypothalamic sensitivity to ghrelin’s actions may work in concert with enhanced GHRH input to promote GH release. However, it should be noted that central and systemic infusion of ghrelin or its synthetic analogs can also increase both NPY and CRH neuronal activity (54, 55), where both NPY and CRH have been shown to have inhibitory effects on GHRH expression and GH release (22, 52, 56, 57, 58, 59, 60, 61). Therefore, although the elevation in ghrelin levels observed after an acute fast may directly serve to increase GHRH neuronal activity and enhance GHRH-stimulated pituitary GH release, chronic elevations in ghrelin may indirectly serve to suppress GHRH neuronal function, leading to a suppression of GH output in states of chronic nutrient deprivation (see below).

Changes in the hypothalamic-pituitary GH axis of the mouse after short-term fasting (12 and 24 h) would be predicted to favor GH release and synthesis. However, a more prolonged fast (48 h) resulted in a fall in GHRH mRNA levels, reminiscent of the male rat, in which fasting decreases hypothalamic GHRH expression, as well as GH pulse release (1, 2, 3, 4, 5, 6, 7). The biphasic effects of fasting on hypothalamic GHRH mRNA levels in mice suggest that the severity of the catabolic stress dictates the ultimate impact of fasting on GH axis function. A differential sensitivity of the GH axis to the catabolic condition may also occur in humans. The majority of studies report enhanced GH release in patients with anorexia nervosa (62). However, Argente et al. (63) observed a subset of anorexic patients with hyposecretion of GH. A biphasic effect of the catabolic condition on GH output is also observed in critical illness (64). In the acute phase of critical illness, circulating GH levels are elevated (64, 65), similar to that that observed in fasting. Of interest is the observation that the number of immunodetectable GHRH neurons in postmortem samples was shown to be positively correlated with the duration of critical illness in a sample set in which mean duration of illness was 19 ± 19 d (66). However, after prolonged critical illness, characterized by muscle wasting (despite adequate nutritional support and maintenance of visceral fat stores) circulating GH levels are suppressed (64). A similar shift in GH release with advance critical illness is also observed in a rabbit model (67). These results, taken together with the current findings showing that the duration of fasting has a biphasic effect on GHRH expression, suggest that central changes in GHRH may in part explain differences in GH output observed in clinical conditions of catabolic stress.

What factors could account for the shift in GHRH expression with prolonged fasting? In stark contrast to the direct stimulatory effects of glucocorticoids on somatotrope function discussed above, chronic hypercortisolemia due to Cushing’s syndrome or immunosuppressive glucocorticoid therapy is associated with reduced GH secretion in humans (68). Also, in normal fasted men, circulating cortisol levels are negatively correlated with circulating GH (69). In rats, chronic treatment with dexamethasone decreases hypothalamic GHRH and SST mRNA levels (70, 71). Therefore, we speculate that the fasting-induced rise in glucocorticoids may directly or indirectly lead to suppressed GHRH expression, where relative sensitivity to this inhibitory effect may in part explain the time- and species-dependent effects of fasting on GHRH expression.

Of the endpoints examined in this study, only hypothalamic CRH mRNA exhibited a delayed response to fasting, rising after 24 h food deprivation. Huang et al. (72) failed to see a significant effect of 24 h fasting on CRH mRNA levels in lean mice; however, fasting did enhance CRH expression in leptin-deficient ob/ob mice, and this response could be blocked by leptin infusion. A similar situation was observed in the leptin receptor defective, obese Zucker (fa/fa) rat, where fasting increased c-fos immunoreactivity and CRH mRNA levels in CRH neurons but failed to alter c-fos expression and led to a decrease in CRH mRNA levels in lean (fa/?) controls (73, 74). These findings indicate that fasting-mediated changes in CRH neuronal activity are regulated by leptin input. Nutrient deprivation has also been reported to enhance CRH mRNA levels within the paraventricular nucleus of hamsters and guinea pigs (31, 75, 76). In hamsters, the stimulatory effect of fasting on CRH is negatively correlated with prefast body weight and is dependent on photoperiod (75, 76). Taken together, these results suggest that body composition and metabolic rate before fasting dictate the ultimate response of the adrenal (stress) axis to nutrient deprivation. The rise in CRH expression observed after prolonged fasting may represent an early signal that leads to suppression of GH release. Central administration of CRH decreases pulsatile GH release (56, 57), and intraventricular infusion of a CRH antagonist was shown to increase GHRH mRNA levels and GH pulse release in rats (22). Although these findings indicate that endogenous CRH is inhibitory to GHRH neuronal activity in the rat, it should be noted that CRH mRNA levels do not rise or are reported to be reduced in the normal fasted rat, where the decrease in CRH expression is attributed to negative feedback regulation of glucocorticoids (77).

The current observation that 48 h fasting fails to alter CRH and GHRH mRNA levels in the NPY KO mouse, coupled with the experimental data showing a CRH antagonist can increase GHRH mRNA levels (22), suggests that there is a functional connection between these neuronal systems. Central administration of NPY inhibits pulsatile GH release in rats and decreases GHRH mRNA levels in both rats and mice (59, 60, 61). Also, NPY decreases GHRH expression in primary rat hypothalamic cell cultures (52). Although GHRH and NPY neurons are both located within the arcuate nucleus, to date, there is no evidence that GHRH neurons express NPY receptors or form close contacts with NPY neurons. However, NPY fibers arising from the arcuate nucleus form synaptic contacts with paraventricular CRH neurons that express the NPY receptor subtype, Y5 (23, 24, 78). Also, intracerebroventricular administration of NPY stimulates CRH gene expression and immunoreactivity in the paraventricular nucleus (79). From these anatomical and functional studies, we might speculate that the fasting-induced rise in NPY mediates its inhibitory effects on GHRH expression by activation of CRH neurons. CRH receptors (CRHR1 and CRHR2) are expressed in the hypothalamus of the mouse and rat, including the arcuate nucleus, where the majority of GHRH neurons are located (80, 81). However, further studies are required to determine whether fasting-induced activation of paraventricular CRH neurons are directly responsible for suppression of GHRH because the arcuate nucleus does not appear to be innervated by CRH fibers arising from the paraventricular nucleus (81).

In summary, the results of the current study show for the first time that the duration of fasting can differentially regulate GHRH expression in the mouse. Short-term fasting (12 and 24 h) enhanced GHRH mRNA levels, which was associated with an increase in circulating GH levels and a reciprocal shift in pituitary expression of GH-stimulatory and -inhibitory receptors to favor GH output. However, more prolonged fasting (48 h) suppressed GHRH mRNA levels, similar to that reported in the rat (2, 5, 6, 7), where this fall was preceded by a sequential rise in NPY and CRH mRNA levels. Because both CRH and NPY have been previously shown to negatively impact GHRH expression and GH release (22, 56, 57, 58, 59, 60, 61), coupled with our novel observations that NPY is required for the fasting-induced rise in CRH, as well as the fall in GHRH expression, we hypothesize that these neuronal systems work in concert to modulate GH release and synthesis in response to nutrient deprivation.


   Footnotes
 
This work was supported by the Secretaria de Universidades, Investigación y Tecnología de la Junta de Andalucia (to R.M.L.), and by the National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases (Grant 30677 to R.D.K.).

All authors confirm that there are no potential conflicts of interest to disclose.

First Published Online October 12, 2006

Abbreviations: FFA, Free fatty acid; GHRH-R, GHRH receptor; GHS-R, ghrelin receptor; KO, knockout; NPY, neuropeptide Y; POMC, proopiomelanocorticotropin; qrtRT-PCR, quantitative real-time RT-PCR; SST, somatostatin.

Received May 4, 2006.

Accepted for publication October 2, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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