Comparison of Pulsatile Versus Continuous Administration of Human Placental Growth Hormone

Comparison of pulsatile versus continuous administration of human placental growth hormone in female C57BL/6J mice

Shutan Liao1,2,3, Mark H Vickers1,2, Angharad Evans1, Joanna L Stanley1,2, Philip N Baker1,2, Jo K Perry1,2

1Liggins Institute, University of Auckland, Auckland, New Zealand;

2Gravida: National Centre for Growth and Development, New Zealand;

3The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China

Abbreviated Title: Efficacy of different administration methods of human placental growth hormone in mice

Key words: placental growth hormone, insulin sensitivity, mice

Word count: 3460 (including the abstract)

Number of figures and tables: 7

Correspondence and reprint requests to be addresses to:

Dr Jo. K. Perry, PhD

The Liggins Institute, University of Auckland

85 Park Rd, Private Bag 92019 Auckland, New Zealand

Tel: +64(9) 3737599 Extn. 87873; Fax: +64(9) 3737497

Email:

This work was funded by Gravida: National Centre for Growth and Development.

Declaration of interest:

The authors have nothing to declare.

Abstract

Pituitary growth hormone (GH-N) has different actions depending on the method of administration. However, the effect of different modes of placental growth hormone variant (GH-V) administration on growth, body composition and glucose metabolism has not been investigated. In this study, we examined the dose-response effect of pulsatile versus continuous administration of recombinant GH-V in a normal mouse model. Female C57BL/6J mice were randomized to receive vehicle or GH-V (2 or 5 mg/kg per day) by daily injection or osmotic pump for 6 days. Pulsatile treatment with 2 and 5 mg/kg per day significantly increased body weight. There was also an increase in liver, kidneys and spleen weight via pulsatile treatment, whereas continuous treatment did not affect body weight or organ size. Pulsatile treatment with 5 mg/kg per day significantly increased fasting plasma insulin concentration, whereas with continuous treatment, fasting insulin levels were not significantly different from the vehicle treated control. However, a dose-dependent increase in fasting insulin concentration and decrease in insulin sensitivity assessed by HOMA was observed with both modes of treatment. At 5 mg/kg per day, hepatic GH receptor (Ghr) expression was increased compared to vehicle treated animals but not affected by the mode of administration. Pulsatile or infused GH-V did not alter plasma IGF-1 concentration or hepatic Igf-1 mRNA expression. Our findings suggest that pulsatile GH-V treatment was more effective in stimulating growth but caused marked hyperinsulinemia in mice.

Introduction

The growth hormone (GH) and insulin-like growth factor-1 (IGF-1) axis is a major regulator of mammalian growth, reproduction and cell regeneration. In humans, two GH genes encode two 22 kDa GH proteins: pituitary GH (GH-N; GH1) and placental GH variant (GH-V; GH2) (1). The protein sequences of GH-N and GH-V are highly conserved, differing by 13 out of 191 amino acids (2), but GH-V has an N-glycosylation site and is more basic in structure than GH-N (3). Apart from that, they have distinct expression profiles; GH-N is mainly secreted in a pulsatile fashion from the pituitary, while GH-V is secreted from the placenta in a non-pulsatile manner during human pregnancy. The continuous secretion of GH-V into the maternal compartment is thought to contribute to maternal metabolic alterations during pregnancy (4). Both proteins bind the GH receptor (GHR) with similar affinity and share similar physiological somatotrophic, lactogenic and lipolytic properties (5,6). However, compared with GH-N, GH-V binds the prolactin (PRL) receptor poorly and its lactogenic affects are greatly reduced (7,8). GH-V replaces GH-N as the dominant circulating GH at approximately 20 weeks of gestation (4). The increase in maternal circulating GH-V is positively associated with fetal growth and circulating IGF-1 concentrations during pregnancy (9-13).

The effects of exogenous GH-N on growth, body composition and carbohydrate metabolism have been well documented (9). A difference in the dose-response effect to modes of delivery was noted. Pulsatile infusions of human or bovine GH, compared to continues infusion, was more effective in stimulating growth in hypophysectomized rats in a dose-dependent manner (14,15). Differences on growth rate and body composition were also observed in intact rats (16,17). In humans, different administration methods of GH-N had differed impacts on glucose homeostasis and lipid profiles (18).

Although GH-V has been demonstrated to stimulate growth, alter body composition and induce insulin resistance in hypophysectomized rats and transgenic mice (8,19,20), there is relatively little information on the effects of GH-V utilising different modes of administration. In the present study, we examined the dose-response effect of pulsatile versus continuous administration of recombinant GH-V in a normal mouse model.

Materials and Methods

Materials

Mouse BA/F3 cells stably expressing the human GH receptor (BA/F3-GHR) were a kind gift from Professor Mike Waters (University of Queensland, Australia). Cells were cultured at 37°C, 5% CO2 in RPMI (Gibco) supplemented with 10% heat-inactivated FBS, 100U/ml penicillin, 100µg/ml streptomycin and Glutamax (Gibco). BA/F3 cells were grown in the presence of 100 ng/ml GH and 10 ng/ml interleukin-3.

Recombinant human GH-V (22 kDa) was purchased from Protein Laboratories Rehovot (Rehovot, Israel) and was reconstituted in 0.4% NaHCO3 pH 9 (21). Recombinant human GH-N (22 kDa) was obtained from the National Hormone and Peptide Program (Harbor-UCLA Medical Center, Torrance, CA, US).

Animals

All protocols were approved by the Animal Ethics Committee of the University of Auckland. Female C57BL/6J (B6) mice aged 6-8 weeks (Jackson Laboratories) were housed under standard conditions and maintained at 22°C with a 12h light/dark cycle and with ad-libitum access to food and water. Mice were housed in pairs. A total of 36 mice, averaging 19.5 ± 0.1g initial body weight, were assigned to treatment groups (n=6 per group as detailed below). Mean body weight and weight range within each group was the same at the start of treatment.

Treatments

Mice were randomized to receive GH-V (2 or 5 mg/kg per day; calculated on the basis of pretreatment body weight) or vehicle for six days by either subcutaneous injection (SC) or osmotic pump (OP) (Alzet model 1007D, Durect Corporation, Cupertino, CA).

For SC groups, GH-V or vehicle (100 µl) was administered subcutaneously in the skinfold at the nape of the neck using tuberculin syringes twice a day (8am and 5pm) from day 1 to day 6. Lyophilized powder was solubilized to the target concentration prior to use on each treatment day.

For OP groups, mice were implanted with Alzet osmotic pumps. Pump selection was based on the size, duration and flow rate. Model 1007D (100 µl) was designed to release its contents at a rate of 0.5 µl/h over one week duration. Pumps were filled with reconstituted recombinant human GH-V or vehicle and placed in sterile 0.9% saline at 37º C for priming overnight. At day 1, pumps were inserted on the back of animal, slightly posterior to the scapulae. At day 4, a blood sample was obtained via tail tip from OP groups. Delivery was verified by measurement of the residual volume in the pump reservoir after explanation at day 7.

Body weights and food intake were monitored daily from day 0 to day 7. On day 7, mice were fasted for 4h, and euthanized by cervical dislocation. Blood was collected by cardiac puncture. Glucose measurements were performed with a Freestyle Optium glucometer (Abbott, UK). The weights of the liver, kidneys, spleen, heart, perirenal fat, retroperitoneal fat and gonadal fat were recorded and tissues stored at -80°C.

AlphaScreen assay

To ensure continuous delivery in the OP treatment groups, the stability of recombinant GH-V protein at 37º C was verified using an AlphaScreen assay. 500 ng/ml GH-V solubilised in 0.4% NaHCO3 pH 9 was incubated at 37 °C for 7 days with samples taken each day and stored at -80°C for later analysis. Dose-response assays were also carried out with unfrozen GH-V on day 0, 3 and 6 and the half maximal effective concentration (EC50) calculated. BA/F3-GHR cells were serum starved for 16h and treated with GH-V for 10 min. An AlphaScreen SureFire p-STAT5 (PerkinElmer, US) was used to measure the phosphorylation level of STAT5A and STAT5B in cellular lysates and was performed as per the manufacturer’s instructions. F was read on an EnVision Multilabel plate reader (PerkinElmer).

Plasma analysis

Plasma IGF-1 (Mediagnost, Germany) and insulin (CrystalChem, USA) was assayed with a mouse-specific enzyme-linked immunosorbent assay (ELISA) as per the manufacturer’s instructions. The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated as: Fasting glucose (mmol/l)×fasting insulin (mU/l)/22.5 (22).

Statistical analysis

All normally distributed data are expressed as means ± S.E.M and were compared using Student’s t test, one way ANOVA with post-hoc analysis (Tukey's procedure), or regression analysis as appropriate. Maternal body weight and food intake data were analysed by repeated measures ANOVA. ANOVA analysis and regression analysis were conducted using SigmaPlot 12.0 and IBM SPSS Statistics 21, respectively. Linear comparisons were made among doses. A p-value of <0.05 was accepted as statistically significant.

Results

Recombinant GH-V protein stability

The bioactivity of GH-V in osmotic pumps over the treatment period was estimated by incubating GH-V at 37oC for 6 days and measuring the phosphorylation of STAT5 in GH-V-treated BA/F3-GHR cells using an AlphaScreen assay. A 26% reduction in GH-V bioactivity (p<0.01) was observed after 6 days incubation at 37oC (Figure 1) No statistically significant difference in bioactivity was observed from day 0-3. Dose response curves were carried out on day 0, 3 and 6 and the EC50 measured. A small increase in the EC50 concentration was observed at day 3 (152.2) and day 6 (122.8), compared to day 0 (90.9). However, this was not significantly different from the time-points.

Body weight and food intake

Mice were treated with GH-V (2 or 5 mg/kg per day; calculated on the basis of pre-treatment body weight) or vehicle for six days via either SC or OP. GH-V treatment with 2 and 5 mg/kg per day via SC significantly increased body weight without affecting food intake, compared with vehicle treated animals (Figure 2A and B). However, there was no statistically significant difference in maternal body weight or food intake among groups following OP treatment (Figure 2C and D). A transient reduction in maternal food intake was seen following osmotic pump implantation (Figure 2D).

Tissue weights

GH-V treatment with 5 mg/kg per day via SC significantly increased liver, kidney and spleen weights, but only liver weight was increased under 2 mg/kg SC treatment (Figure 3). GH-V treatment via OP did not affect the weight of the liver, kidneys, or spleen (Figure 3). Heart weight was not affected by different administration methods or treatment doses. There were no significant differences in adipose tissue weights across treatment groups (Figure 4).

IGF-1, fasting glucose and insulin levels

In the SC groups, there was no effect of GH-V treatment on IGF-1 plasma concentration after 6 days of treatment (Figure 5A). In the OP treatment group, GH-V treatment did not affect IGF-1 plasma concentration at day 4 (Figure 5B). However, we observed a significant dose effect of GH-V on IGF-1 concentration (linear trend, p<0.05), with a decrease in IGF-1 level associated with increasing GH-V dose at day 7 in OP groups (Figure 5C).

As no effect on circulating IGF-1 was observed, we tested whether GH-V was capable of stimulating IGF-1 mRNA expression in the mouse myoblast cell line C2C12, which is known to up-regulate IGF-1 mRNA expression in response to GH-N treatment (23,24). Both GH-N and GH-V increased expression of IGF-1 in this cell line (data not shown).

Fasting insulin concentrations were significantly increased in the 5 mg/kg SC treatment group, compared to vehicle (0.35 ± 0.03 vs. 0.51 ± 0.04, p<0.05) (Figure 6B). Although fasting insulin levels were not significantly different from the vehicle treated control in the OP groups (Figure 6D); interestingly, a dose-dependent increase in fasting insulin concentration and decrease in insulin sensitivity was observed in both SC and OP administration treatment groups, as assessed by HOMA (linear trend, p<0.05) (Figure 6A-D). No affect was seen on fasting glucose level (data not shown).

Hepatic mRNA expression

The effect of GH-V on hepatic mRNA expression was analysed by comparing gene expression in the vehicle-treated and 5 mg/kg GH-V treatment group (Supplementary Table 1). Mouse growth hormone binding protein (GHBP) is generated through alternative splicing of RNA transcripts from the Ghr/Ghbp gene. The primers used in our study do not distinguish between these two transcripts. GH-V treatment via SC and OP significantly increased hepatic Ghr/Ghbp expression (Supplementary Table 1). However, GH-V treatment did not alter the expression of hepatic insulin receptor substrate-1 (Irs-1), insulin receptor (Insr), v-akt murine thymoma viral oncogene homolog 3 (Akt3), Igf-1, Solute carrier family 2, member 4 (Slc2a4, Glut4), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3-kinase) catalytic subunit alpha (Pik3ca), or PI3-kinase regulatory subunit alpha (Pik3r1).

Discussion

Exogenous GH-N therapy has long been used as an effective treatment for growth disorders due to its somatotrophic properties. However, GH-N treatment can induce insulin resistance and alterations in carbohydrate and lipid metabolism (25,26). Previous studies have found that the induction of the growth and metabolic response to GH varies according to the dose administered and the delivery method used (14,27,28).

In the present study, we compared the effect of pulsatile versus continuous administration of recombinant GH-V, in terms of growth and metabolic outcomes in female C57BL/6J mice. GH-N secretion is highly episodic in mammals. This intermittent secretion is of importance for the biological effects in peripheral tissues. A sexually dimorphic GH-N secretory pattern has been observed in rodents and humans and regulates the expression of several sex-specific phenotypes (29-32). In females, the pulses of GH-N secretion are lower and plasma GH-N baseline level is higher than in males (30).

The effects of different modes of GH-N delivery on regulation of growth have been studied previously. Jansson et al. (33) and Thorngren et al. (34) observed that the frequency of GH administration influenced body growth in hypophysectomized rats. However, the growth response did not increase proportionally to an increased administration frequency. The "stress" associated with injections may contribute to this. To avoid frequent injections, an osmotic pump was designed to mimic the continuous fashion accumulated by multiple injections. In terms of growth stimulation, pulsatile GH administration has been inferred to be superior to continuous delivery (35). Clark et al. treated hypophysectomised rats with recombinant human growth hormone (0.04, 0.2, 1, or 5 mg/kg per day) for 7 days and found that growth responses depended on the pattern of GH administration (twice daily injections > continuous infusions > daily injections) (36). Consistent with previous studies, we found that GH-V treatment via twice daily SC is more effective in stimulating growth than OP and significantly increased body weight and organ size. However, despite in comparable doses, GH-V treatment via OP did not affect animal growth.