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Endocrinology Vol. 147, No. 6 2754-2763
Copyright © 2006 by The Endocrine Society

Impact of Obesity on the Growth Hormone Axis: Evidence for a Direct Inhibitory Effect of Hyperinsulinemia on Pituitary Function

Raul M. Luque and Rhonda D. Kineman

Section of Endocrinology and Metabolism, Department of Medicine, University of Illinois at Chicago, and Research and Development Division, Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois 60612

Address all correspondence and requests for reprints to: Dr. Rhonda D. Kineman, Jesse Brown Veterans Affairs Medical Center, Research and Development Division, M.P. 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
 
There is a negative relationship between obesity and GH. However, it is not known how metabolic changes, associated with obesity, lead to a reduction in GH output. This study examined the GH axis of two mouse models of obesity, the leptin-deficient (ob/ob) mouse and the diet-induced obese (DIO; high-fat fed) mouse. Both models displayed hyperglycemia and hyperinsulinemia with reduced expression of GH as well as reduced expression of pituitary receptors important for GH synthesis and release [GHRH receptor (DIO only) and the ghrelin receptor (ob/ob and DIO)]. These pituitary changes were not accompanied by changes in hypothalamic expression of GHRH or somatostatin; suggesting that alterations in pituitary function may be precipitated in part by direct effects of systemic signals. Of the metabolic and hormonal parameters examined (insulin, glucose, corticosterone, free fatty acids, ghrelin, and IGF-I), only insulin/glucose showed a significant, and negative, correlation with pituitary expression. Pituitaries of DIO mice remained responsive to the acute in vivo actions of insulin, as assessed by phosphorylation of Akt, despite systemic (skeletal muscle and fat) insulin resistance. In addition, treating primary pituitary cell cultures from lean mice with insulin reduced GH release as well as GH, GHRH receptor, and ghrelin receptor mRNA levels compared with vehicle-treated controls, where the magnitude of suppression of pituitary mRNA levels was similar to that observed in the DIO mouse. These results coupled with the fact that the pituitary expresses the insulin receptor at levels comparable to tissues classically considered insulin sensitive, indicates high circulating insulin levels can directly contribute to the suppression of GH synthesis and release in the obese state.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THERE IS A NEGATIVE relationship between adiposity and GH in individuals with environmentally induced, or "simple" obesity (i.e. increased energy intake and decreased energy expenditure) (1, 2, 3, 4, 5, 6, 7). Given the lipolytic and protein anabolic actions of GH (8, 9), it has been suggested that suppressed GH may serve to promote additional weight gain, thereby helping to promote a vicious cycle (10). Obese patients not only have low basal GH output, but also display severely blunted or absent responses to all known GH stimuli, including fasting, acute exercise, somatostatin (SRIF) infusion withdrawal, insulin tolerance tests, GHRH and ghrelin or its synthetic analogs [GH secretagogues (GHS)] (1, 2, 3, 4, 5, 6, 7, 11, 12, 13, 14, 15, 16). Significant weight loss results in the recovery of basal and stimulated GH release (1, 2, 3, 4, 5), indicating that metabolic alterations associated with weight gain are the precipitating events leading to low GH output, and these changes are completely reversible.

Clinical data support a negative role of insulin in obesity-associated GH suppression (5, 13, 17). Several hypotheses have been put forth regarding how obesity-associated hyperinsulinemia may suppress GH production; these include direct pituitary actions on GH synthesis and release and/or indirect actions via 1) modulation of hypothalamic function, 2) alterations in the availability of IGF-I, and/or 3) suppression of circulating ghrelin levels (18, 19, 20). To determine which, if any, of these mechanisms could be involved in obesity-associated GH suppression, we compared the metabolic profile and GH axis (hypothalamic, pituitary, and systemic components) in two mouse models of obesity: the leptin-deficient ob/ob mouse and a mouse made obese by feeding a high-fat diet.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
All experimental procedures were approved by the animal care and use committees of University of Illinois at Chicago and the Jesse Brown Veterans Affairs Medical Center. All 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 tap water and food provided as described below.

Monogenic model of obesity
Male ob/ob mice were obtained from The Jackson Laboratory (Bar Harbor, ME) at 8 wk of age, group-housed in shoebox cages, and provided a standard pellet diet [catalog no. 5008, LabDiet (Purina Mills, Inc., Richmond, IN); fat, 17 kcal%; carbohydrate, 56 kcal%; protein, 27 kcal%]. Lean littermates, representing mice either heterozygote for the leptin mutation or homozygote for the wild-type leptin allele were used as controls and therefore are designated +/?. Mice were handled daily to acclimate them to laboratory personnel. At 10 wk of age, the animals were killed by decapitation between 0800 and 1100 h under fed conditions. Trunk blood was collected for hormone and metabolite analyses, and pituitaries and hypothalami were collected for mRNA analysis by quantitative real-time RT-PCR (qrtRT-PCR).

Diet-induced obesity (DIO)
C57BL/6J male mice were obtained at weaning (d 21) from The Jackson Laboratory and housed individually in shoebox cages. At 4 wk of age, mice were placed on either a high-fat (HF; fat, 60 kcal%; carbohydrate, 20 kcal%; protein, 20 kcal%) or a low-fat (LF; fat, 10 kcal%; carbohydrate, 70 kcal%; protein, 20 kcal%) diet supplied as palatable pellets (LF diet, catalog no. D12450B; HF diet, catalog no. D12492; Research Diets, Brunswick, NJ). The sources of fat were soybean (LF diet, 2.3%/wt; HF diet, 3.2%/wt) and lard (LF diet, 1.9%/wt; HF diet, 31.6%/wt). Mice and food were weighed weekly. To acclimate the mice to the experimental procedures, they were handled daily, 1 wk before blood sampling or death (by decapitation). Three separate experiments were performed. In experiment 1 (Expt 1), mice were fed either an LF (n = 4) or HF (n = 4) diet for 10 wk and were killed between 0800 and 1100 h in the fed state. Trunk blood was collected for evaluation of circulating hormones and metabolites, and pituitaries and hypothalami were collected for evaluation of mRNA levels by qrtRT-PCR. In Expt 2, mice were fed either an LF (n = 7) or HF (n = 6) diet for 14 wk, food was withdrawn overnight (16 h). The following morning topical anesthesia (Emla Cream, AstraZeneca, Wilmington, DE) was applied to the tail. Five minutes later the tip of the tail was nicked with a scalpel blade, and a single blood sample was collected from the tail vein, into EDTA-treated capillary tubes, for evaluation of fasting glucose, insulin, GH, and corticosterone. All mice were returned to their respective diets. Two weeks later (a total of 16 wk of diet), mice were killed between 0800 and 1100 h in the fed state. Trunk blood was collected for evaluation of circulating hormones and metabolites, and pituitaries and hypothalami were collected for evaluation of mRNA levels by qrtRT-PCR. In Expt 3, mice were fed either an LF (n = 13) or HF (n = 12) diet for 14 wk, and food was withdrawn overnight (16 h). The following morning blood samples were taken from the tail vein of all mice for evaluation of fasting glucose and insulin (time zero). A subset of mice (n = 4 mice/diet) was also administered a bolus dose of glucose (1 g/kg, ip), and tail vein blood samples were taken 15, 30, 60, 90, and 120 min after injection. All mice were returned to their respective diets. Three weeks later (a total of 17 wk of diet), mice were again fasted overnight and the following morning were injected with vehicle or insulin (10 U/kg, ip; 1 U = 36 µg; Novolin R, Novo Nordisk, Princeton, NJ); 8 min later, mice were killed, and blood was collected for hormone and metabolite determinations. Pituitary, skeletal muscle (tibialis anterior), intraabdominal fat pad, and liver were collected for protein analysis by Western blot.

Primary pituitary cell cultures
To determine whether insulin can directly regulate pituitary expression of genes important in GH synthesis and release, pituitaries of 8-wk-old, C57BL/6J male mice (n = 7–10 pooled/experiment, four separate experiments) were dispersed into single cells and plated at 2 x 105/well in {alpha}-MEM (Invitrogen Life Technologies, Inc., Grand Island, NY) containing 2.5% FBS (Sigma-Aldrich Corp., St. Louis, MO), 0.1% BSA (Sigma-Aldrich Corp.), transferrin (125 nM; Sigma-Aldrich Corp.), T3 (0.6 nM; Sigma-Aldrich Corp.), hydrocortisone (275 nM; Sigma-Aldrich Corp.), and penicillin-streptomycin antibiotic (Invitrogen Life Technologies, Inc.). After a 24-h incubation, cultures were preincubated in serum-free medium for 2 h, and subsequently, the medium was replaced with serum-free medium containing 0 (control group), 0.5, 1, 5, 10, or 50 nM insulin (three or four wells per treatment group). Cultures were incubated for an additional 24 h, medium was collected for GH determination on select samples, and cells were extracted to determine total RNA recovery.

Assessment of tissue-specific expression of insulin receptors (INSR)
To determine whether the pituitary expresses INSR at levels comparable to those in tissues classically considered to be insulin sensitive, the pituitary, hypothalamus, cortex, liver, fat, and skeletal muscle (n = 5–8) from 8-wk-old male C57BL/6J mice, under fed conditions, were collected and processed for analysis of INSR mRNA copy number by qrtRT-PCR.

RNA isolation and RT
Tissues and pituitary cell cultures were processed for recovery of total RNA using the Absolutely RNA RT-PCR Miniprep Kit (Stratagene, La Jolla, CA) with deoxyribonuclease treatment. The amount of RNA recovered was determined using the Ribogreen RNA Quantification Kit (Molecular Probes, Eugene, OR). Total RNA (1 µg for whole tissues and 0.25 µg for pituitary cell cultures) 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).

Development and validation of qrtRT-PCR
Primers sets were selected using Primer 3 software (Rosen, S., and H. J. Skaletsky, 2000; Whitehead Institute for Biomedical Research) with mouse genomic sequences as templates. BLAST (National Center for Biotechnology Information) searches were used to verify that the selected primers were specific for the designated target. Specific primer sequences, GenBank accession numbers, and product sizes were as follows: GHRH: forward, 5'-TGCCATCTTCACCACCAAC-3'; reverse, 5'-TCATCTGCTTGTCCTCTGTCC-3' (M31654; 158 bp); SRIF: forward, 5'-TCTGCATCGTCCTGGCTTT-3'; reverse, 5'-CTTGGCCAGTTCCTGTTTCC-3' (NM_009215; 113 bp); neuropeptide Y (NPY): forward, 5'-CTCGTGTGTTTGGGCATTCT-3'; reverse, 5'-CTTGCCATATCTCTGTCTGGTG-3' (NM_023456; 107 bp); GH: forward, 5'-CCTCAGCAGGATTTTCACCA-3'; reverse, 5'-CTTGAGGATCTGCCCAACAC-3' (NM_008117; 142 bp); GHRH receptor (GHRH-R): forward, 5'-ACCCGTATCCTCTGCTTGCT-3'; reverse, 5'-AGGTGTTGTTGGTCCCCTCT-3' (XM_132546; 133 bp): GHS-R: forward, 5'-TCAGGGACCAGAACCACAAA-3'; reverse, 5'-CCAGCAGAGGATGAAAGCAA-3' (NM_177330; 71 bp); INSR: forward, 5'-TCATGGATGGAGGCTATCTGG-3'; reverse, 5'-CCTTGAGCAGGTTGACGATTT-3' (NM_010568; 129 bp); and cyclophilin A: forward, 5'-TGGTCTTTGGGAAGGTGAAAG-3'; reverse, 5'-TGTCCACAGTCGGAAATGGT-3' (NM_008907; 109 bp). Primers were used in a standard PCR with the 2x Master Mix PCR reagent (MRI Fermentas) and cDNA from the tissue of interest as template. The thermocycling profile consisted of one cycle of 95 C for 10 min, followed by 35 cycles of 95 C for 1 min, 61 C for 1 min, and 72 C for 1 min and a final cycle of 72 C for 10 min. Products were run on agarose gels and stained with ethidium bromide to confirm that only one band was amplified and no primer dimers formed. PCR products were then column-purified (QIAGEN, Valencia, CA) and sequenced to confirm target specificity. For real-time PCRs, SYBR PCR Master Mix (Bio-Rad Laboratories, Hercules, CA) was used, and thermocycling and fluorescence detection was performed using a Stratagene Mx3000p real-time PCR machine. Initial screening of primer efficiency using real-time detection was performed by amplifying 2-fold dilutions of RT products, where optimal efficiency was demonstrated by a difference of one cycle threshold between dilutions. The thermocycling profile consisted of one cycle of 95 C for 10 min; 40 cycles of 95 C for 30 sec, 61 C for 1 min, and 72 C for 30 sec; followed by a graded temperature-dependent dissociation to verify that only one product was amplified. After confirmation of primer efficiency and specificity, the concentration of purified products generated by standard PCR was determined using Molecular Probe’s Picogreen DNA quantification kit, and PCR products were serial diluted to obtain standards containing 101, 102, 103, 104, 105, and 106 copies of synthetic template. Standards were then amplified by real-time PCR, and standard curves were generated using Stratagene Mx3000p software. The slope of a standard curve for each template examined was approximately 1, indicating that the efficiency of amplification was 100%, meaning that all templates in each cycle were copied. To determine the starting copy number of cDNA, RT samples were PCR amplified, and the signal was compared with that of a standard curve run on the same plate. Standard curves were constructed for all transcripts examined, including cyclophilin A, which was used as a housekeeping gene (see Data analysis below). 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.

Western blot
Tissues were homogenized in T-Per Tissue Extraction Reagent (Pierce Chemical Co., Rockford, IL) containing Halt protease inhibitor mixture (Pierce Chemical Co.), 0.05 M EDTA, 2 mmol/liter Na3VO4, 10 mmol/liter sodium fluoride, and 10 mmol/liter Na4P2O7 (Sigma-Aldrich Corp.). Protein concentrations were assessed using the Pierce bicinchoninic acid protein assay kit. Samples (10 µg) were mixed with 2x Laemmli sample buffer, boiled, size separated on 10% Tris-HCl SDS Ready Gels (Bio-Rad Laboratories), and electrophoretically transferred to Hybond enhanced chemiluminescence nitrocellulose membranes (ECL, Amersham Biosciences, Piscataway, NJ). Blots were blocked in 5% nonfat dry milk (wt/vol) dissolved in Tris-buffered saline containing 0.1% Tween 20 (TBS-T). Blots were subsequently washed (three times, 5 min each time) in TBS-T and incubated overnight (4 C) with primary antibodies against phospho-Ser473 Akt or total Akt (Cell Signaling Technology, Beverly, MA; 1:1000) in TBS-T and 5% BSA. Blots were then washed and incubated with horseradish peroxidase-conjugated, goat antirabbit IgG [Cell Signaling Technology; 1:2000 dilution in 5% (wt/vol) dry milk and TBS-T] for 1 h, then washed and exposed (1 min) to ECL Western Blotting Detection Reagent (Amersham Biosciences). Blots were exposed to Hyperfilm ECL (Amersham Biosciences) and developed. The films were scanned using the Bio-Rad Gel Doc EQ system, and the image was analyzed using Quantity One 1-D analysis software (Bio-Rad).

Assessments of hormones and metabolites
Glucose levels were determined in fresh whole blood samples (tail vein or trunk blood) 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, and plasma was stored at –80 C until analysis of insulin (rat/mouse ELISA; Linco Research, Inc., St. Charles, MO), corticosterone (rat/mouse ELISA; IDS Octeia, Fountain Hills, AZ), free fatty acids (FFA; WAKO, Richmond, VA), ghrelin (rat/mouse ELISA; Linco Research, Inc.), IGF-I (rat/mouse ELISA; IDS Octeia), and GH (mouse/rat ELISA, Diagnostic Systems Laboratories, Webster, TX) levels. The GH ELISA was also used to determine the impact of 10 nM insulin (representing the minimum dose that had the maximal effect on GH, GHS-R, and GHRH-R mRNA levels in vitro) on GH release from primary mouse pituitary cell cultures.

Data analysis
Experiments examining the effects of treatment on mRNA copy number of the transcript of interest within whole pituitary and hypothalamic extracts or from primary pituitary cell cultures were adjusted by the mRNA copy number of cyclophilin A (as a housekeeping gene) to control for the amount of RNA used in the RT reaction and the efficiency of the RT reaction, where cyclophilin A copy number was not altered between groups. When comparing the expression levels of INSR across tissue types, values are reported as absolute mRNA copy numbers, not adjusted by a housekeeping gene, in that we found the expression of cyclophilin A as well as other commonly used housekeeping genes (glyceraldehyde-3-phosphate dehydrogenase, ß-actin, and hypoxanthine phosphoribosyltransferase), varied across tissue type, consistent with other reports.

Differences between lean (+/?) and ob/ob mice or LF- and HF-fed mice, with respect to hypothalamic and pituitary mRNA levels and circulating hormone and metabolite levels (in the fed state), were assessed by Student’s t test. The effects of diet (LF vs. HF) and fasting on hormone and metabolite levels and the effects of diet and insulin treatment (vehicle vs. insulin) on Akt phosphorylation were assessed by two-way ANOVA, whereas the in vitro insulin dose response was assessed by one-way ANOVA, with replication. The Scheffé comparison was used as the post hoc test. Multiple linear regression analyses were performed with pituitary GH, GHRH-R, or GHS-R mRNA level as the dependent variable and hormone (insulin, IGF-I, ghrelin, and corticosterone) or metabolite (glucose and FFA) level as the independent variable. 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
 
Metabolic profile and GH axis of the ob/ob mouse
As shown in Table 1Go, ob/ob mice displayed elevated levels of circulating insulin, glucose, and corticosterone in the fed state, whereas FFA did not differ from lean control values (+/?). Evaluation of the expression of key components of the GH axis revealed that ob/ob mice had lower levels of circulating GH without alterations in total IGF-I (Table 1Go). Circulating ghrelin levels also tended to be suppressed; however, these differences did not reach statistical significance (P < 0.15; Table 1Go). Consistent with a decrease in circulating GH, pituitary GH and GHS-R mRNA levels were significantly reduced in ob/ob mice compared with lean controls (Fig. 1Go). However, pituitary GHRH-R mRNA levels and hypothalamic GHRH or SRIF mRNA levels did not differ between genotypes, whereas hypothalamic NPY mRNA levels were increased in ob/ob mice compared with lean (+/?) controls (Fig. 1Go).


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  		TABLE 1. Circulating hormone and metabolite levels in lean (+/?) and ob/ob male mice under fed conditions

 

Figure 1
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  		FIG. 1. Hypothalamic GHRH, SRIF, and NPY mRNA levels (left panel) and pituitary GH, GHRH-R, and GHS-R mRNA levels (right panel) in 10-wk-old lean (+/?) and obese (ob/ob) male mice. mRNA copy numbers were determined by qrtRT-PCR and were adjusted by cyclophilin A mRNA copy number as a housekeeping gene. Data are shown as the mean ± SEM of five or six mice per group. Asterisks indicate obese values that significantly differ from lean control values: *, P < 0.05; **, P < 0.01.

 
Effect of HF diet on body weight
Growth curves (for Expt 3) are shown in Fig. 2AGo, demonstrating that mice fed an HF diet were significantly heavier than LF-fed controls. Similar growth curves were observed in Expt 1 and 2 (data not shown). The increase in weight was due to a dramatic increase in sc and visceral fat mass, where the intraabdominal fat pad weight of HF-fed mice was twice that in LF-fed controls (Fig. 2BGo). Over the course of the experimental diet (Expt 3, 17 wk), HF-fed mice gained 50% more weight than LF-fed mice (HF, 32.0 ± 1.2 g; LF, 20.8 ± 0.6 g; P < 0.01). The dramatic weight gain in HF-fed mice was associated with a 34% increase in total calories consumed (HF, 1807 ± 37 kcal; LF, 1343 ± 19 kcal; P < 0.01).


Figure 2
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  		FIG. 2. Growth curves (A; n = 12–13 mice/group), intraabdominal, fat pad weight (B; n = 12–13 mice/group), and the results of glucose tolerance tests (GTT) (C; n = 4/group) in C57BL/6J male mice fed a LF or HF diet are shown. Asterisks indicate HF values that significantly differ from LF control values: *, P < 0.05; **, P < 0.01.

 
Effect of HF diet on metabolic parameters
After 10 wk of the diet (Expt 1), HF-fed mice were hyperinsulinemic, but not hyperglycemic (data not shown). After 16 wk of the HF diet, both insulin and glucose levels were elevated in the fed state compared with those in mice fed the LF diet, whereas FFA and corticosterone levels were not altered (Table 2Go). After an overnight fast, HF-fed mice also displayed elevated insulin and glucose levels (Table 2Go) and delayed glucose clearance (Fig. 2CGo) compared with LF-fed controls.


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  		TABLE 2. Circulating hormone and metabolite levels in C57BL/6J male mice fed an LF or HF diet, under fed and fasted conditions

 
Effect of HF diet on the GH axis
After 16 wk of the diet (Expt 2), HF-fed mice showed a significant reduction in GH, GHRH-R, and GHS-R mRNA levels compared with LF-fed controls, without significant alterations in hypothalamic expression of GHRH, SRIF, or NPY (Fig. 3Go). The suppression of GH and GHS-R mRNA was also observed after 10 wk of HF feeding (Expt 1; data not shown). As in ob/ob mice, total IGF-I levels in HF-fed mice did not differ from control values, whereas circulating ghrelin levels were reduced to 50%; however, these differences did not reach statistical significance (P < 0.13; Table 2Go). GH levels in HF-fed mice did not significantly differ from those in LF-fed controls (Table 2Go). However, given that GH is released in a pulsatile fashion, it is difficult to observe differences in mean values from a small sample set. Therefore, we compared frequency distributions of the GH values obtained from single random samples between each treatment group using the {chi}2 test of independence. The minimum and maximum GH values observed were 2.8 and 92 ng/ml, respectively. Therefore, we calculated the frequency distribution (percentage) of values that fell within four equal ranges (≤25, 26–50, 51–75, and ≥76 ng/ml). As shown in Table 3Go, there tended to be more GH values that were 25 ng/ml or less and fewer values that were 76 ng/ml or more in the HF group compared with the LF group under fed (P = 0.2) and fasted (P = 0.1) conditions.


Figure 3
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  		FIG. 3. Hypothalamic GHRH, SRIF, and NPY mRNA levels (left panel) and pituitary GH, GHRH-R, and GHS-R mRNA levels (right panel) in C57BL/6J male mice fed a LF or HF diet for 16 wk (starting at 4 wk of age) are shown. mRNA copy numbers were determined by qrtRT-PCR and were adjusted by cyclophilin A mRNA copy number as a housekeeping gene. Data are shown as the mean ± SEM of six or seven mice per group. Asterisks indicate HF values that significantly differ from LF control values: *, P < 0.05; **, P < 0.01.

 

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  		TABLE 3. Frequency distributions of plasma GH levels in mice fed a LF or HF diet

 
Effect of HF diet on tissue-specific insulin sensitivity
Pituitaries of DIO mice remained responsive to the acute actions of insulin despite peripheral insulin resistance (Fig. 4Go). Specifically, pituitaries of HF-fed mice displayed a robust response to a bolus insulin injection, as assessed by phosphorylation of Akt, and this response did not differ from that in LF-fed controls. However, consistent with the hyperglycemia/hyperinsulinemia and delayed glucose clearance of HF-fed mice, the acute effects of insulin on Akt activation was significantly blunted in the skeletal muscle and visceral fat of HF-fed mice, whereas the hepatic response to insulin was not significantly altered. Measurement of circulating insulin levels after bolus insulin injection confirmed that similar levels were achieved in LF (618 ± 113) and HF (540 ± 68) groups, indicating that the differences in muscle and fat response were not due to differences in the amount of insulin delivered.


Figure 4
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  		FIG. 4. Acute insulin sensitivity of pituitaries (n = 6–7/group), skeletal muscle (tibialis anterior; n = 3–4/group), liver (n = 3–4/group), and intraabdominal fat (n = 3–4/group) of LF- and HF-fed mice, as determined by Western blot analysis of phosphorylated Akt. Tissues were removed 8 min after an injection of vehicle (V) or insulin (I; 10 U/kg, ip) from mice that had been fasted overnight. Representative immunoblots are shown for each tissue, with graphs indicating the response expressed as phospho-Akt/total Akt signal in arbitrary units. Asterisks indicate values that differ significantly from their respective vehicle-treated control values (**, P < 0.01); significant differences in tissue response to insulin between LF- and HF-fed mice are indicated by horizontal brackets.

 
Regression analysis of pituitary mRNA levels vs. circulating hormones and metabolites, in lean (+/?, LF-fed) and obese (ob/ob, HF-fed) mice
Collective evaluation of the relationship between pituitary expression and circulating hormones (insulin, corticosterone, ghrelin, and IGF-I) and metabolites (glucose and FFA) in lean and obese mice in the fed state revealed that pituitary expression of GH, GHRH-R, and GHS-R was not associated with circulating FFA, corticosterone, ghrelin, or IGF-I levels (data not shown), but GH and GHS-R mRNA levels were negatively correlated with glucose (r = –0.41; P < 0.02 and r = –0.34; P < 0.05, respectively; data not shown) and insulin (Fig. 5Go).


Figure 5
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  		FIG. 5. Correlation between pituitary mRNA levels (GH, GHRH-R, and GHS-R) and circulating insulin levels in lean (+/?, LF fed) and obese (ob/ob, HF fed) mice under fed conditions. Correlation coefficients were calculated by linear regression analysis.

 
Direct effects of insulin on pituitary mRNA levels
As shown in Fig. 6AGo, GH, GHRH-R, and GHS-R mRNA levels were inhibited by insulin treatment in a dose-dependent manner. Low doses of insulin (0.5 and 1 nM), representing values that would be achieved in vivo in fasted and fed mice, respectively, resulted in a small (~15%), but significant, suppression of GH mRNA levels. Higher doses of insulin (5 and 10 nM), representative of circulating levels observed in DIO and ob/ob mice, respectively, further suppressed GH mRNA to less than 60% of vehicle-treated control values. A similar dose relationship was observed for insulin-mediated GHRH-R expression. However, the magnitude of this response exceeded that for GH mRNA. In contrast to the dose-dependent regulation of GH and GHRH-R expression by insulin, GHS-R mRNA levels were maximally suppressed to approximately 50% of vehicle-treated control values at the lowest dose of insulin tested (0.5 nM). The reduction in GH mRNA levels achieved after 24-h exposure to 10 nM insulin (which represents the minimum dose that had a maximal effect on all mRNA end points examined) was reflected by a significant suppression of GH released into the medium (controls, 100 ± 1.7%; 10 nM insulin, 79 ± 4.5%; P < 0.001; representing the mean of three separate experiments, where values are expressed as 100% of the control value, within experiment). These effects may, in fact, be mediated via the INSR, in that pituitaries of lean mice express the INSR at levels comparable to those in tissues commonly thought of as insulin sensitive (i.e. fat, skeletal muscle, and liver; Fig. 6BGo).


Figure 6
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  		FIG. 6. A, Effects of insulin treatment (24 h) on GH, GHRH-R, and GHS-R mRNA levels in primary mouse pituitary cell cultures. mRNA copy numbers were determined by qrtRT-PCR and were adjusted by cyclophilin A mRNA copy number as a housekeeping gene. Values are expressed as a percentage of the vehicle-treated control value (set at 100%) and represent the mean ± SEM of four independent experiments (three or four wells per treatment per experiment). The insulin dose response was assessed by one-way ANOVA with replication. Values that differ significantly (P < 0.05) are designated by different letters (a and b). B, Absolute INSR mRNA copy number per 0.05 µg of total RNA in the pituitary (PIT), hypothalamus (HPT), cortex (CTX), liver (LIV), fat (FAT), and skeletal muscle (MUS; tibialis anterior) taken from 8- to 9-wk-old male mice under fed conditions, as assessed by qrtRT-PCR (n = 5–8 samples/tissue tested).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The leptin-deficient ob/ob mouse represents a research model commonly used to study the impact of obesity on the metabolic and endocrine systems. In the 1970s, the ob/ob mouse was shown to have reduced circulating GH levels accompanied by a decrease in pituitary GH content (21), consistent with the GH deficiency observed in obese humans (1, 2, 3, 4, 5, 6, 7). Our current results expand and extend these early findings, showing low circulating GH levels in ob/ob mice are also accompanied by a decrease in pituitary GH mRNA levels as well as a decline in pituitary expression of the GHS-R. Despite these pituitary changes, hypothalamic expression of GHRH and SRIF in ob/ob mice does not differ from that in lean controls, whereas central expression of NPY is increased, consistent with previous reports (22). Together, these observations suggest that a reduction in pituitary GH gene expression and in pituitary sensitivity to ghrelin input may contribute to GH deficiency in this monogenic model of obesity. It should be noted that the GH axis of the ob/ob mouse differs from that reported in the leptin receptor mutant, obese, Zucker rat. In the Zucker rat, a reduction in pituitary GH mRNA levels and in vivo responsiveness to GHRH and ghrelin analogs (23, 24, 25, 26) is associated with a reduction in hypothalamic GHRH expression, whereas central SRIF expression is not altered (26, 27, 28, 29). The differences in the GH axis of the ob/ob mouse and the Zucker rat may be related to species-dependent effects of obesity or the severity of the metabolic condition at the time of death. Nonetheless, it could be argued that obesity brought about by the total lack of leptin (as in the ob/ob mouse) or insensitivity to leptin (as in the Zucker rat) may not be representative of DIO as commonly occurs in humans.

To more closely model the major form of obesity observed in humans, we characterized the metabolic phenotype and GH axis of a DIO (HF-fed) mouse model. In our feeding paradigm, HF-fed mice consumed more total calories and gained 15% more weight per calorie consumed compared with LF-fed controls. These results indicate that weight gain in the HF-fed mouse is not solely due to an increase in caloric intake, consistent with a previous report showing that HF-fed mice have a lower metabolic rate and physical activity compared with LF-fed controls (30). Excess weight gain in HF-fed mice led to insulin resistance, as demonstrated by an increase in circulating insulin and glucose as well as a decrease in glucose clearance after a bolus glucose challenge. Under these metabolic conditions, pituitary GH, GHRH-R, and GHS-R mRNA levels were suppressed without significant alterations in hypothalamic GHRH and SRIF expression. Despite these pituitary changes, mean GH levels did not significantly differ in HF-fed mice compared with LF-fed controls. However, caution should be exercised in interpreting these results, because in the mouse (31, 32), as in other species, GH release is pulsatile, making it difficult to detect changes in single random blood samples. The ideal way to examine GH output would be to obtain serial samples sufficient to assess GH changes over time (a method commonly employed in larger animals, i.e. rats and humans). However, serial blood sampling in the mouse is very stressful, and therefore, it is difficult to collect the volume required for accurate measurement without depleting blood volume. As an alternative, we compared frequency distributions of GH values obtained from single random samples, a method previously used to assess the impact of liver-specific, IGF-I knockout on circulating GH levels in mice (33). These comparisons suggest that the peak GH release is reduced in response to an HF diet, consistent with a reduction in pituitary GH, GHRH-R, and GHS-R mRNA levels. However, it is recognized that the magnitude of suppression of GH output in the HF-fed mouse was modest compared with that in the ob/ob mouse and therefore represents a limitation of this study. Several factors that may be responsible for this differential response, includes differences in animal model (monogenic obesity vs. DIO) and/or severity and age of onset of the metabolic phenotype. First, GH levels are known to decline with age (34), consistent with our current observation that mean GH levels in 10-wk-old, lean (+/?) mice were twice those in 20-wk-old, LF-fed controls of the same background strain (C57BL/6J). Given that GH levels have already begun to decline in LF-fed mice, the negative impact of HF feeding may be masked. Second, the severity of the insulin-resistant state is greater in the ob/ob mouse than in the HF-fed mouse, as indicated by 4-fold greater increase in circulating insulin levels. In fact, elevated insulin levels are observed in the ob/ob mouse as early as postnatal d 6, and glucose levels begin to rise after d 28 (22). Therefore, metabolic disturbances before full maturation may alter the development of the GH axis in the ob/ob mouse. Finally, we cannot ignore the fact that the lack of leptin, which has been shown to have a positive impact on GH at both central and pituitary levels (35, 36, 37), in conjunction with elevated glucocorticoids, which act centrally to suppress GHRH and increase SRIF (38), may contribute to the more dramatic reduction in circulating GH in the ob/ob mouse compared with the HF-fed mouse. However, by comparing the metabolic profile and GH axis of the ob/ob and HF-fed mouse, we can conclude that the absolute lack of leptin or elevations in glucocorticoids are not required for suppression of GH, GHRH-R, and GHS-R mRNA levels in DIO mice, and these changes may represent early alterations in somatotrope function as excess weight accumulates.

The mechanism by which obesity leads to a decline in GH output is poorly understood. Multiple theories based on clinical and animal studies provide evidence implicating defects in hypothalamic input (suppressed GHRH and enhanced SRIF) and/or defects in somatotrope function, where both central and pituitary changes may be mediated by changes in circulating FFA, glucocorticoids, ghrelin, IGF-I, or insulin, as previously reviewed (18, 19). All of these factors may ultimately contribute to obesity-associated GH deficiency depending on the experimental model or severity of the condition. However, examination of the GH axis of ob/ob and DIO mice revealed that obesity can be associated with a defect in somatotrope function (i.e. decreased expression of GH, GHRH-R, and GHS-R) independent of changes in hypothalamic GHRH and SRIF expression. Although we cannot rule out the possibility that GHRH and SRIF release may be modified independent of changes in their gene expressions, our current results support the contribution of a primary pituitary defect as a key component in GH deficiency observed in the obese state. These findings are consistent with a previous observation that normal male rats made obese by feeding a cafeteria-style diet had normal hypothalamic GHRH and SRIF expressions, but were insensitive to exogenous GHRH treatment (39). Also, 8-d treatment with GHRH failed to restore the GH response of obese patients to subsequent challenge with GHRH alone or in combination with an agent (arginine) that suppresses endogenous SRIF release (40). Together, these observations favor the theory that early obesity-associated defects in somatotrope function may be directly mediated by systemic signals.

Of the systemic hormones and metabolites examined in this study, only glucose and insulin levels were significantly correlated (negatively) with GH and GHS-R expression; these in vivo associations suggest, but do not prove, a cause and effect relationship. Glucose has been shown to suppress GHRH-stimulated GH release, whereas glucose concentrations do not alter basal GH release in pituitary cell cultures of rats and pigs (41, 42), suggesting that glucose does not directly impact GH synthesis. However, early work by Melmed and colleagues (43, 44, 45) demonstrated that insulin can suppress GH mRNA levels as well as GH release in rat pituitary cell lines and primary rat pituitary cell cultures and immunoneutralization of the INSR blocked the inhibitory effects of insulin. Our current results confirm and significantly extend these early findings. We observed that INSR are expressed in the mouse pituitary at levels comparable to those in tissues classically considered insulin sensitive. Also, insulin can suppress GH mRNA levels and GH release in primary mouse pituitary cell cultures as well as decrease the expression of receptors important in GH release and synthesis (i.e. GHRH-R and GHS-R) at doses well within the physiological range that are not predicted to bind to the IGF-I receptor (46, 47). The fact that the in vitro effects of insulin mimicked the pituitary changes observed in DIO/hyperinsulinemic mice, strengthens the theory that elevated circulating insulin levels significantly contribute to reduced somatotrope function in the obese state.

The hypothesis that hyperinsulinemia drives the suppression of somatotrope function in obesity is predicated on the assumption that the pituitary remains responsive to the inhibitory actions of insulin in the face of systemic insulin resistance. In our hands, HF-fed mice were indeed insulin resistant compared with LF-fed controls, as indicated by elevated fed and fasted insulin concentrations and delayed glucose clearance. Measurement of insulin-induced Akt activity, as a direct assessment of tissue sensitivity, revealed a blunted response in skeletal muscle and fat, whereas hepatic sensitivity to acute insulin challenge was not significantly reduced. Maintenance of hepatic insulin sensitivity in HF-fed mice despite muscle resistance has been observed by some (48), whereas others have reported significant suppression of Akt phosphorylation in muscle, fat, and liver (49) after HF feeding. These differential responses could be attributed to differences in age at diet introduction, duration of diet, diet composition, genetic background, insulin dose, and time of sampling. Nonetheless, the pituitary of the HF-fed mouse remained fully responsive to the acute actions of insulin in the face of systemic insulin resistance, thus providing additional evidence that hyperinsulinemia can directly suppress somatotrope function in the obese state.

The results of the current study strongly suggest that the rise in circulating insulin observed in the insulin-resistant obese state plays a major role in directly suppressing GH synthesis and release. However, we cannot exclude the possibility that other systemic factors may also contribute to this response. For example, we observed that obese mice tended to have reduced ghrelin levels, consistent with a previous reports showing low ghrelin levels in ob/ob mice (50) and variable, but low, levels of ghrelin in DIO mice (51) as well as in obese humans (52). Exogenous administration of ghrelin or its synthetic analogs (GHS) enhance GH release by both central and pituitary actions (for review, see Ref. 53). At the hypothalamic level, ghrelin is thought to regulate GH release by stimulating GHRH and/or inhibiting SRIF neuronal activity. At the pituitary level, ghrelin directly stimulates GH secretory vesicle release via a phospholipase C-mediated increase in intracellular Ca2+. In addition, ghrelin augments GHRH-stimulated cAMP production. Therefore, a fall in circulating ghrelin in conjunction with a reduction in pituitary expression of its receptor may directly contribute to the reduction in basal as well as ghrelin- and GHRH-stimulated GH release in obesity (1, 6, 7, 12, 14, 16). However, the role of endogenous ghrelin in regulating the GH axis, at least in the mouse, is lessened by the observation that mice with constitutive inactivation of the ghrelin gene show no alteration in growth or circulating IGF-I levels (54), and GHS-R-null mice display only modest reductions in growth and IGF-I (55).

It is well recognized that IGF-I plays a critical role in negative feedback regulation of GH synthesis and release; IGF-I acts directly at the pituitary to inhibit GH, GHRH-R, and GHS-R synthesis (56, 57, 58, 59). In the obese human, total circulating IGF-I levels are reported to be in the normal range (for review, see Ref. 18), consistent with the results of the current study. However, the majority of IGF-I in the circulation is bound to IGF-I binding proteins (IGFBPs); the remaining free IGF-I is believed to be critical for its biological actions, including negative feedback regulation of somatotrope function. Levels of free IGF-I have been reported by some (60, 61) to be elevated in obesity. This increase has been attributed to a direct inhibitory effect of insulin on IGFBP1 production by the liver (62) consistent with the observation that IGFBP1 levels are low in hyperinsulinemic obese subjects (63). Therefore, it has been hypothesized that more free IGF-I may be available to negatively mediate somatotrope function in the obese state. In the current study, limitations in sample size did not allow determination of free IGF-I (requiring pooling of two or three mice) and IGFBP1; therefore, how these factors may participate in obesity-associated suppression of somatotrope function in ob/ob and DIO mice remains to be determined.

In summary, simple (diet-induced) obesity is characterized by a reduction in basal and stimulated GH release in humans. The results of the current study, using obese mouse models, provide the first direct evidence that these changes can be due to a reduction in the expression of GH and pituitary receptors important for GH synthesis and release. The fact that these pituitary changes occurred independently of changes in the expression of hypothalamic regulators of GH secretion (i.e. GHRH and SRIF) suggests that obesity-associated GH suppression is at least in part due to a primary pituitary defect that may be initiated by systemic signals. Of the systemic factors examined, we found that 1) insulin negatively correlated with pituitary GH, GHRH-R, and GHS-R gene expression in obese mice; 2) mouse pituitaries express the INSR at levels comparable to insulin-sensitive tissues; 3) pituitaries of obese mice remain responsive to the acute actions of insulin despite systemic insulin resistance; and 4) the changes in pituitary expression observed in hyperinsulinemic, obese mice can be replicated by insulin administration in vitro. Together, these results strongly support the hypothesis that high circulating insulin levels may be a major determinant in the suppression of GH output in the obese state by direct down-regulation of somatotrope function, which includes a reduction in the expression (mRNA levels) of GH, GHRH-R, and GHS-R.


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

Both authors have nothing to declare.

First Published Online March 2, 2006

Abbreviations: DIO, Diet-induced obese; Expt, experiment; FFA, free fatty acid; GHRH-R, GHRH receptor; GHS, GH secretagogue; GHS-R, ghrelin receptor; HF, high fat; IGFBP, IGF-I-binding protein; INSR, insulin receptor; LF, low fat; NPY, neuropeptide Y; qrtRT-PCR, quantitative real-time RT-PCR; SRIF, somatostatin; TBS-T, Tris-buffered saline containing 0.1% Tween 20.

Received December 5, 2005.

Accepted for publication February 21, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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H. Iwakura, T. Akamizu, H. Ariyasu, T. Irako, K. Hosoda, K. Nakao, and K. Kangawa
Effects of ghrelin administration on decreased growth hormone status in obese animals
Am J Physiol Endocrinol Metab, September 1, 2007; 293(3): E819 - E825.
[Abstract] [Full Text] [PDF]

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