## Main

Cystic fibrosis (CF) is a fatal autosomal recessive disease. The absence or reduction of functional CFTR (cystic fibrosis transmembrane conductance regulator) affects multiple organs; the impact on the lungs is the cause of most morbidity and mortality in humans, and the majority of CF patients eventually succumb to lung failure. CF patients are also growth retarded (13), which is clinically relevant not only because it affects the quality of life for patients, but also because the severity of growth retardation correlates with the severity of lung disease (2,3). Furthermore, studies in which CF patients have benefited from chronically administered growth hormone suggest that generation of a growth-promoting physiologic state may improve lung function and clinical status (4,5).

Malnutrition is a major cause of growth retardation in CF patients and results from loss of exocrine pancreatic function, elevated energy requirements (likely due to increased work of breathing), anorexia during pulmonary exacerbations, and other factors (6). Other proposed contributors to the growth deficit include a down-regulation of growth by inflammatory cytokines (7), a primary cellular energy wasting defect (8,9), and a direct effect on growth or metabolism from loss of hypothalamic CFTR (10,11). Modern therapies including aggressive nutritional intervention have led to improved growth in CF patients; nonetheless, studies show that average height and weight are still significantly below normal (12,13). The goal of achieving normal growth for all CF patients would benefit from better implementation of existing therapies as well as development of novel treatments. Further study of the mechanisms of CF growth failure may benefit from utilization of an animal model.

We propose that study of CF mice, which also have reduced growth (14,15), will yield insight into the mechanisms of growth retardation. CF mice containing a null mutation in Cftr have severe intestinal disease, evidence of pancreatic changes that are mild compared with humans, and normal lung function (16,17). CF mouse growth including length measurements and major regulatory hormones has not been described in detail. To establish a mouse model of CF growth retardation, this paper describes growth and aspects of growth regulation in mice with a null mutation in Cftr.

## METHODS

### Mice.

CF mice used in this study were homozygous for a null mutation in Cftr (Cftrtm1Unc/tm1Unc) that produces no functional protein (15,18) and were bred to congenicity on C57BL/6J for at least 12 generations. Homozygous wildtype littermates (Cftr +/+) served as controls. All mice were offspring of Cftr heterozygotes (Cftrtm1Unc/+). Hormones and mRNA were assayed at 31, 45, and 84 d to achieve a temporal profile extending from juvenile to adult ages. Mice were housed at constant temperature (22°C) on a 12 h light/dark cycle alternating at 0600h and 1800h. To aid survival of CF mice, all mice were weaned at 28 d of age, fed an enriched diet (9F Sterilizable Rodent Diet 7960, Harlan Teklad, Madison, WI) and CF mice were provided a Colyte solution (peg-3350 & electrolytes for oral solution - Schwarz Pharma, Milwaukee, WI) in place of water (19). Known deaths accounted for 40% of CF mice assigned to this study. Mice were anesthetized with avertin (0.5 mg tribromoethanol/g body weight ip) and euthanized by exsanguination via heart puncture between 1000h and 1200h. Whole pituitaries and small portions (20 mg) of the left lateral lobe of the liver were dissected, frozen in liquid nitrogen, and stored at −80°C for use in quantitative PCR. Blood was stored on ice during dissection and centrifuged at 3500g to separate serum which was stored at −80°C until use in hormone measurements. This study was approved by the Institutional Animal Care and Use Committee.

#### Growth analysis.

All mice were weighed weekly. Only mice surviving until 84 d were included in growth curve analysis, to avoid artefactual shifts from deaths of mice. Mice were measured from the tip of the nose to the base of the tail when they were anesthetized before euthanization to avoid innacuracy from movement of mice.

#### Food consumption study.

WT and CF mice were separately caged beginning at day 35 and continuously provided with 50 to 100g of food. Weights of food and mice were recorded daily between 900h and 1100h. Food intake was measured from day 37 through day 46 (provided food was initially measured on day 36) and only mice that survived through day 46 were included in analysis.

#### RNA extraction and cDNA synthesis.

RNA was extracted from pituitaries and livers using 1 mL Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions and reconstituted in The RNA Storage Solution (Ambion). RNA was quantified spectrophotometrically. cDNA was synthesized in duplicate for each RNA sample with 1 μg liver RNA or 0.25 μg pituitary RNA using M-MLV-RT (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. RT-PCR samples excluding M-MLV-RT served as negative controls in quantitative PCR.

#### Quantitative PCR.

Oligonucleotide primers for quantitative PCR were based on mRNA sequences obtained from NCBI; Accession numbers were as follows: 18S subunit ribosomal RNA (18S): ×00686, Gh: ×02891, Igf-1: NM_184052. Primers designed using MacVector 7.2 (Accelrys, San Diego, CA) and product sizes were as follows: 18S, 5′-ATTGACGGAAGGGCACCACCAG-3′, 5′-CAAATCGCTCCACCAACTAAGAACG-3′, 161 bp; Gh, 5′-TGAGAAACTGAAGGACCTGGAAGAG-3′, 5′-GTTGGCGTCAAACTTGTCATAGG-3′, 115bp; Igf-1, 5′-CGCTCTGCTTGCTCACCTTCAC-3′, 5′-CACTCATCCACAATGCCTGTCTG-3′, 178 bp.

Real-time quantitative PCR was performed on a Rotor-Gene 3000 (Corbett Research, Sydney, Australia) using Sybr Green I (Sigma Chemical Co.) as a fluorescent indicator. Sybr Green I, provided as a 10,000× concentrate, was diluted to a 10× solution in TAE. This solution was found to be stable at 4°C for at least one month. PCR was performed with Platinum Taq (Invitrogen) according to the manufacturer's instructions except for the addition of 0.2 μL of 10× Sybr Green I to each 20 μL reaction. Five percent of synthesized cDNA was used in each PCR reaction, except for 18S amplification for which 0.05% was used.

Relative values were calculated such that all samples were compared with the WT 31 d mean, which was set to 1. The relative value of each sample was based on a threshold fluorescence set in the exponential phase of amplification. The following equation determined the relative value of each sample: ${\text{E}}^{{\text{(Ct}}_{{\text{wt}}^{\text{-}}}{\text{Ct}}_{\text{s}}\text{)}}$, where E is the efficiency of the reaction, Ctwt is the average threshold-crossing cycle number for the 31 d WT samples, and Cts is the threshold-crossing cycle number of the sample. The efficiency was determined empirically for each gene by amplifying 4 serial 4-fold dilutions of cDNA with each primer set. The slope of the line created by plotting log10(concentration) by Ct was used to find the efficiency with the equation: E = 10(−1/slope) (20). The efficiencies found for each gene were as follows:18S: 2.00; Gh: 1.97, Igf-1: 1.98. The relative values for Gh and Igf-1 were normalized to the relative value of the housekeeping gene 18S, to correct for variation in RNA concentration or cDNA synthesis efficiency.

To ensure the quality of reported data, each RNA sample was used to make cDNA in duplicate. Each duplicate was quantified separately and then averaged to yield a final result. If duplicates differed by more than a factor of two, the result was discarded. All analyses compare samples within the same PCR reaction, to avoid interassay variation.

#### Hormone measurements.

Serum Gh was measured by RIA (National Hormone & Peptide Program, Harbor-UCLA Medical Center, Torrance, CA). Serum Igf-1 was measured using Rat Igf-1 EIA (Diagnostic Systems Laboratories, Webster, TX) which is designed to measure total hormone concentration; all positive controls were within acceptable ranges. Hormone measurements were performed in duplicate and averaged for a final result.

#### Statistics.

Logistic growth curves were estimated and compared using nonlinear mixed effects regression techniques (SAS Proc NLMIXED) where the final length of the mouse was a random effect. The van Elteren test was used to analyze serum Gh data separately by gender with undetectable values set to the lower limit of the assay at 0.2 ng/mL. To examine the association between Igf-1 and growth, z-scores were assigned to Igf-1, weight, and length values: Z-scores were calculated from the mean and SD for each genotype/sex/age group separately. Linear regression was then used to regress weight and length z-scores on Igf-1 z-scores. Differences of mean food intake between WT and CF mice were compared using a t test separately by gender. All other data were analyzed separately by gender using two-way ANOVA with age and genotype as independent variables followed by Bonferroni posttests to compare CF and WT means within single age groups. A P-value of <0.05 was considered significant.

## RESULTS

### Growth analysis.

CF mice of both genders were consistently smaller than WT mice (Fig. 1A). Mean weight (in grams) ± SE for mice at time of euthanization was as follows: 31 d males, WT = 18.6 ± 0.7 (n = 15), CF = 10.3 ± 0.9 (n = 10); 45 d males, WT = 22.2 ± 0.7 (n = 13), CF = 14.2 ± 0.8 (n = 10); 84 d males, WT = 27.2 ± 0.6 (n = 14), CF = 18.3 ± 0.7 (n = 11); 31 d females, WT = 16.6 ± 0.4 (n = 11), CF = 9.5 ± 0.4 (n = 12); 45 d females, WT = 18.5 ± 0.4 (n = 15), CF = 14.0 ± 0.6 (n = 7); 84 d females, WT = 20.8 ± 0.4 (n = 11), CF = 15.4 ± 0.5 (n = 7); p < 0.0001 for all pairwise comparisons (t tests). Length measurements indicate that CF mice of both genders were significantly shorter than WT mice for all ages (Fig. 1B).

#### IGF-1.

Insulin like growth factor-1 (Igf-1) is an important regulator of growth in mice (21). There was a significant deficit of serum Igf-1 in both genders of CF mice for all ages ranging from 31-55% (Fig. 2A). As most circulating Igf-1 is derived from the liver (22,23) hepatic Igf-1 mRNA was measured to determine whether message levels corresponded with depressed serum levels (Fig. 2B). CF males and females both had significantly less hepatic Igf-1 mRNA than WT mice at 31 d, but this difference was not significant at 45 d.

To better determine the relationship between serum Igf-1 and growth in our mouse population, we compared serum Igf-1 with both weight and length using linear regression. To isolate the relationship between serum Igf-1 and size, the analysis was designed to control for the effects of sex, genotype, and age, which would have confounded the results. Serum Igf-1 positively varied with weight and length in both WT and CF mice (Fig. 3).

#### Growth hormone.

Growth hormone (Gh) positively regulates serum Igf-1 and also independently stimulates growth (21). There was no significant difference of serum Gh between CF and WT mice in males (Fig. 4A; p = 0.90; van Elteren test), but there was a nominally significant deficit in CF females (p = 0.048; van Elteren test). Pituitary Gh mRNA was also measured (Fig. 4B). In males, CF mice had less Gh mRNA at all ages (p < 0.05 for day 31 and day 45 mice, p < 0.001 for 84 d mice; Bonferroni posttests). In females, Gh mRNA was not different between CF and WT mice.

#### Food consumption.

The observations of low Igf-1 and reduced growth are consistent with models of malnutrition in rodents (2426). Since anorexia, or reduced food intake, is one simple explanation for malnutrition, food consumption was measured from age 37 through 46 d. CF males ate more than WT males per unit body weight (p = 0.0074; t test), whereas CF females ate the same as WT females (Fig. 5). Specifically, food consumption in units of “daily food intake (g)/mouse weight (g)” ± SE was as follows: WT males, 0.164 ± 0.0057 (n = 6); CF males, 0.202 ± 0.0096 (n = 6); WT females, 0.195 ± 0.0044 (n = 7); CF females, 0.204 ± 0.0093 (n = 7).

## DISCUSSION

Growth in CF patients is clinically relevant because it correlates with lung function (2,3). For example, Konstan et al. (2003) showed that growth parameters at age 3 positively predict lung function at age 6 in CF patients (3). Further evidence that growth stimulation is beneficial to CF patients is that treatment with GH not only stimulates growth and elevates serum Igf-1 levels, but may also improve clinical status (5). For example, a randomized, controlled trial found that administration of GH to CF patients improves clinical status as measured by lung capacity and reduced hospital visits (4). It is suggested that the beneficial effects of growth hormone may be attributed to increased respiratory muscle strength and larger absolute lung capacity (5).

To assess growth regulation in CF patients, several studies have measured serum concentrations of GH and IGF-1. Several studies have shown significant deficits of Igf-1 in CF patients (2729). The behavior of GH in CF patients is more equivocal, with studies showing higher, normal, or lower levels of the hormone [reviewed in (28)].

Many mouse models of CF have been created which contain various levels of functional Cftr. (30). Several of these strains are smaller than their wild-type counterparts (14,15), including the one used in this study which contains a null mutation in Cftr (15). The absence or reduction of functional Cftr in mice affects several organs. CF mice with null or severe mutations in Cftr have intestinal disease characterized by mucous accumulation and goblet cell hyperplasia, and a large portion of them die of bowel obstruction before reaching maturity (17,30). Exocrine pancreatic disease in CF mice is mild compared with humans (who experience fibrosis and complete loss of function) and includes progressive histologic changes without fibrosis (17,31), as well as evidence of decreased functional enzymes (32). CF mice do not have overt lung disease, although they do develop more pronounced disease than healthy mice if inoculated with bacteria (16).

This paper presents a study of growth and major growth regulating hormones in CF mice. CF mice were small in mass and linear size (Fig. 1) and also had consistently low levels of serum Igf-1 (Fig. 2A). This is significant in that Igf-1 is the main effector of Gh and likely accounts for the majority of the growth stimulating effects of Gh (21). Indeed, the present study was consistent with a growth-stimulating role of serum Igf-1 in that levels of the hormone correlated significantly with the size of mice after controlling for genotype, sex, and age (Fig. 3). Recent evidence has suggested, however, that paracrine-acting Igf-1 from connective tissues such as muscle and bone may be more important for stimulating growth (22,23). It is possible, therefore, that the altered levels of serum Igf-1 in CF mice are reflective of locally produced levels of the hormone, but not directly functionally relevant. The data presented here demonstrate that circulating levels of Igf-1 can serve as a gauge of growth regulation in the context of the CF mouse growth deficiency.

In mice, Gh is a potent regulator of serum Igf-1 as demonstrated by the fact that mice lacking the Gh receptor have an 80% reduction of (33) or undetectable levels of serum Igf-1 (21). We hypothesized that low serum Gh may be responsible for the observed change in Igf-1. There was a marginally significant reduction of serum Gh in female CF mice but no difference in males (Fig. 4A). Conversely, male CF mice had reduced pituitary Gh mRNA but females did not (Fig. 4B). It is difficult to detect changes in Gh levels by measuring single time points due to the pulsatile nature of the hormone (34). Unfortunately, temporal profiles of Gh designed to detect such changes are impractical in the mouse due to the need for multiple blood samples over a short period of time. Our findings, therefore, may not have adequately resolved differences in pulse amplitude or frequency and thus our measurements may underestimate GH differences. Despite the inherent difficulty in detecting changes in levels of serum Gh, our data show some evidence of Gh down-regulation in both sexes of CF mice.

Our findings in CF mice are consistent with a model of malnutrition causing growth retardation. Various types of induced malnutrition in rodents including starvation and caloric restriction cause reduced serum Gh (24,25) and Igf-1 (24,26). [Note that this differs from the human response to malnutrition which includes elevated GH and reduced IGF-1 (35)]. This study found that CF mice had low serum Igf-1, as well as evidence of a Gh deficit (although only females had low serum Gh). One simple explanation for this finding is that CF mice consume less food leading to malnutrition and growth retardation. We found, however, that CF mice ate at least as much as age-matched WT mice (Fig. 5). It is important to note that although CF mice ate adequately for the studied ages, it is possible that younger CF mice did not. The ages examined in this study were limited by poor survival in younger, separately caged CF mice. Despite this caveat, the food intake data suggest that CF mouse growth retardation is caused by consumption-independent mechanisms.

One likely consumption-independent mechanism for growth retardation is malabsorption leading to an energy deficit. CF mice have severe gastrointestinal disease (16,17) and CF mice with a null Cftr mutation have lipid malabsorption (36). We speculate that loss of Cftr function results in intestinal epithelial cell dysfunction, including altered fluid and electrolyte transport, but also malabsorption in this tissue. Malabsorption would be predicted to cause an energy deficit and suppress growth, including reduced production and secretion of Igf-1. To quantify this phenomenon, an important area of future research would be to measure total fecal energy losses in CF mice. Since the CF mouse has minor pancreatic disease (16,36), this experiment would help elucidate the role of primary intestinal disease on malabsorption and growth.

The CF mouse may also be used to study other proposed contributors to the growth defect. First, it has been suggested that hypothalamic Cftr regulates neuropeptide secretion or energy balance regulation, ultimately affecting the growth and reproductive axes (10,11). Study of mice with a neuron-specific deletion of Cftr would help elucidate the role of neuronal Cftr. Second, chronic inflammation may also contribute to growth retardation in CF (7). There is evidence that CF patients have elevated systemic cytokines (37,38) and CF mice have elevated expression of inflammatory mediators in the intestine (39). Chronic systemic cytokines have been shown to blunt growth and growth hormones (40). To determine whether chronic inflammation is affecting growth in CF mice, systemic inflammation would have to be demonstrated and subsequently manipulated pharmacologically or genetically to elicit a change in growth. Third, several studies have shown that CF patients have elevated resting energy expenditure which is likely due to increased work of breathing (6). Studies have also suggested, however, that loss of Cftr causes primary cellular energy wasting (8,9). Measurements of energy expenditure in the CF mouse, which has no lung disease but may share a primary energy-wasting defect with humans, would help define the etiology of elevated resting energy expenditure.

In summary, growth has been identified as an important clinical target in cystic fibrosis, but normal growth in CF patients has not been universally achieved. We propose that a mouse model of CF, which is here shown to have severely inhibited growth and low serum Igf-1 despite adequate food intake, will serve to model several potential mechanisms of CF growth retardation in humans. Specifically, the CF mouse may elucidate the effects on growth of intestinal disease, inflammatory cytokines, hypothalamic Cftr, and a proposed energy wasting defect.