Hyperphagia in male melanocortin 4 receptor ... - Wiley Online Library

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J Physiol 594.24 (2016) pp 7309–7326

Hyperphagia in male melanocortin 4 receptor deficient mice promotes growth independently of growth hormone H. Y. Tan1 , F. J. Steyn1,2

, L. Huang1 , M. Cowley3 , J. D. Veldhuis4 and C. Chen1

1

School of Biomedical Sciences, University of Queensland, Brisbane, Queensland, Australia The University of Queensland Centre for Clinical Research, The University of Queensland, Brisbane, Queensland, Australia 3 Department of Physiology Monash University, Melbourne, Victoria, Australia 4 Department of Medicine, Endocrine Research Unit, Mayo School of Graduate Medical Education, Clinical Translational Science Center, Mayo Clinic, Rochester, MN, USA 2

Key points

The Journal of Physiology

r Loss of function of the melanocortin 4 receptor (MC4R) results in hyperphagia, obesity and increased growth.

r Despite knowing that MC4Rs control food intake, we are yet to understand why defects in the function of the MC4R receptor contribute to rapid linear growth.

r We show that hyperphagia following germline loss of MC4R in male mice promotes growth while suppressing the growth hormone–insulin-like growth factor-1 (GH–IGF-1) axis.

r We propose that hyperinsulinaemia promotes growth while suppressing the GH–IGF-1 axis. r It is argued that physiological responses essential to maintain energy flux override conventional mechanisms of pubertal growth to promote the storage of excess energy while ensuring growth.

Abstract Defects in melanocortin-4-receptor (MC4R) signalling result in hyperphagia, obesity and increased growth. Clinical observations suggest that loss of MC4R function may enhance growth hormone (GH)-mediated growth, although this remains untested. Using male mice with germline loss of the MC4R, we assessed pulsatile GH release and insulin-like growth factor-1 (IGF-1) production and/or release relative to pubertal growth. We demonstrate early-onset suppression of GH release in rapidly growing MC4R deficient (MC4RKO) mice, confirming that increased linear growth in MC4RKO mice does not occur in response to enhanced activation of the GH–IGF-1 axis. The progressive suppression of GH release in MC4RKO mice occurred alongside increased adiposity and the progressive worsening of hyperphagia-associated hyperinsulinaemia. We next prevented hyperphagia in MC4RKO mice through restricting calorie intake in these mice to match that of wild-type (WT) littermates. Pair feeding of MC4RKO mice did not prevent increased adiposity, but attenuated hyperinsulinaemia, recovered GH release, and normalized linear growth rate to that seen in pair-fed WT littermate controls. We conclude that the suppression of GH release in MC4RKO mice occurs independently of increased adipose mass, and is a consequence of hyperphagia-associated hyperinsulinaemia. It is proposed that physiological responses essential to maintain energy flux (hyperinsulinaemia and the suppression of GH release) override conventional mechanisms of pubertal growth to promote the storage of excess energy while ensuring growth. Implications of these findings are likely to extend beyond individuals with defects in MC4R signalling, encompassing physiological changes central to mechanisms of growth and energy homeostasis universal to hyperphagia-associated childhood-onset obesity. (Received 18 May 2016; accepted after revision 22 August 2016; first published online 25 August 2016) Corresponding author F. J Steyn: The University of Queensland, University of Queensland Centre for Clinical Research, Building 71/918, Royal Brisbane & Women’s Hospital Campus, Herston, QLD, 4029. Email: [email protected]

C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society

DOI: 10.1113/JP272770

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Abbreviations α-MSH, alpha melanocortin stimulating hormone; AgRP, agouti-related releasing peptide; ARC, arcuate nucleus; BMI, body mass index; DAPI, 4 ,6-diamidino-2-phenylindole; DMH, dorsomedial hypothalamus; ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence-activated cell sorting; FFA, free fatty acids; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; GH, growth hormone; GHRH, growth hormone releasing hormone; IGF-1, insulin-like growth factor; KO, knock out; LM, littermates; MC4R, melanocortin 4 receptor; NEFA, non-esterified free fatty acid; PeVN, periventricular nucleus; PF, pair-fed; PVN, paraventricular nucleus; SRIF, somatotroph release inhibiting factor (somatostatin); Thal, thalamus; TG, triglycerides; WT, wild type.

Introduction The hypothalamic melanocortin system regulates body weight, appetite and energy expenditure (Cone, 1999). Within this system, the melanocortin 4 receptor (MC4R) is the main regulator of energy homeostasis, conveying signals from alpha-melanocortin stimulating hormone (α-MSH) and agouti-related peptide (AgRP) expressing neurons (Tao, 2010). Dysfunction of MC4Rs promotes hyperphagia, rapid weight gain, and adult obesity, accounting for 5.8% of reported cases of early-onset obesity in humans (Farooqi et al. 2003). Loss of MC4R function increases body length in rodents (Huszar et al. 1997; Mul et al. 2012), and height in adults (Farooqi et al. 2003). While the cause for obesity in this instance is mostly known, it remains unclear how defects in MC4R signalling alter linear growth. Growth hormone (GH) is a key regulator of pubertal linear growth (Rose et al. 1991; Martha et al. 1992). GH deficiency in humans (Laron et al. 1966; Kaufman et al. 1992; de Boer et al. 1995; Dattani & Preece, 2004) and rodents (Zhou et al. 1997; Sims et al. 2000; Sjogren et al. 2000) results in severe postnatal growth retardation, and GH treatment in GH deficient patients restores growth rate to improve final adult height (Wit et al. 1996; Eiholzer et al. 1998). As GH release is increased in MC4R deficient adults when compared with adults matched for body mass index (BMI), it is thought that increased adult growth in this population may occur as a consequence of excess GH release in puberty and/or early-adulthood (Martinelli et al. 2011). Given that GH excess worsens insulin resistance (Cutfield et al. 2000) and advances the pathophysiological implications of adult obesity (Cornford et al. 2012), pubertal GH excess in children with defects in MC4R signalling may imprint additional and significant adult health consequences. To determine whether loss of MC4R signalling results in pubertal GH excess, we assessed GH release relative to linear growth in a MC4R deficient (MC4RKO) mouse model. Contrasting our predictions, we demonstrate that MC4RKO mice present with suppressed GH release secondary to the loss of MC4R signalling, suggesting that GH does not contribute to accelerated growth in this mouse model. Our extended observations provide new insights that define the impact of pubertal

hyperphagia and calorie mechanisms of growth.

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Methods Ethical approval

All experiments and procedures were approved by the University of Queensland Animal Ethics Committee, and were conducted in accordance with the National Health and Medical Research (Australia) guidelines for the ethical use of animals in research. Male homozygous MC4RKO mice on a C57Bl6/J background with a targeted deletion of functional neuronal MC4R were provided by Professor Michael Cowley (Monash University, Melbourne, Australia) (Huszar et al. 1997). MC4RKO mice carry a loxp-flanked transcriptional blocking (loxTB) sequence that prevents endogenous gene transcription of MC4R and thus its translation from the endogenous locus. As such, homozygous MC4RKO mice are devoid of functional Mc4r mRNA in all regions of the brain. Male homozygous MC4RKO and age-matched wild-type (WT) litter mate (LM) mice were obtained from heterozygote breeding pairs and identified using established MC4R primer sequences (Shaw et al. 2005). Transgenic adult C57/BL6 mice expressing GFP in GH-secreting cells (GH-GFP mice) and hypothalamic GHRH neurons (GHRH-GFP) were provided by Professor Iain Robinson (MRC National Institute for Medical Research, London) (Magoulas et al. 2000; Balthasar et al. 2003). WT C57/BL6 male mice were obtained from institutional breeding colonies from The University of Queensland Biological Resources (UQBR). All mice were group housed (n = 2–3) in a temperature-controlled room (22 ± 2°C), maintained on a 12:12-hour light–dark cycle (lights on at 06.30 h and off at 18.30 h) unless otherwise specified. Groups were littermate-matched for genotype, age and body weight (pair-fed group; n = 5, ad libitum fed n = 6–8). Animals were handled daily and had free access to food and water unless otherwise specified. For the collection of terminal tissue and blood samples, mice were anaesthetized with a single I.P. injection of sodium pentobarbitone (32.5 mg ml−1 , 1PO643-1; Virbac Animal Health, Milperra, NSW, Australia). We acknowledge and C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society

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Hyperphagia promotes growth without growth hormone

confirm compliance to the ethical principles under which The Journal of Physiology operates.

Phenotypic characteristics of MC4RKO mice

Starting at 4 weeks of age, body weight and body length (nasal–anal distance) of WT LM and MC4RKO mice were assessed at weekly intervals. To assess food intake in ad libitum fed mice, food was measured daily from 5 weeks of age. A pre-weighed amount of food was placed into the food hopper and the remaining food in the hopper weighed at the same time on the following day. Food pellets were replaced daily, and cages were checked on a daily basis for collection and inclusion of spilled fragments of food pellets. Pair feeding (PF) intervention

Starting at 4 weeks of age, WT LM and MC4RKO mice were individually housed and food intake was controlled to limit the amount of food consumed by MC4RKO mice. Pair-fed MC4RKO (PF MC4RKO) mice were given the equivalent amount of food consumed by age-matched WT LM. The pair feeding strategy carefully considered food anticipatory (Mendoza et al. 2010) and diurnal feeding habits of mice. This feeding strategy further prevented hyperphagic MC4RKO mice from consuming all food within a single meal. Food pellets were weighed and placed inside the cage of individually housed mice prior to the onset of the dark cycle (2.5 g per mouse per meal; between 17.30 and 18.00 h in anticipation of the dominant period of night time feeding) and following the onset of the light cycle (1 g per mouse per meal; between 07.30 and 08.00 h, to allow daytime feeding). The total amount of food provided was based on the average daily food consumption of WT mice as determined above, and detailed in Fig. 1A. Separate groups of WT LM and MC4RKO mice were allowed ad libitum access to food, and included for comparison. All mice were given free access to water at all times. Characterizing pulsatile growth hormone (GH) secretion in MC4RKO mice

For measures of pulsatile GH secretion, blood samples were collected and processed as previously described (Steyn et al. 2011). For assessment of changes of pulsatile GH secretion from puberty into early adulthood, weaned WT LM and MC4RKO mice at 3 weeks of age were individually housed and allowed 1 week to acclimate before the commencement of all experiments. Changes in pulsatile GH secretion throughout pubertal growth were assessed in WT LM, MC4RKO mice, PF WT LM and PF MC4RKO mice at 4 and/or 8 and 16 weeks of age. C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society

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Assessment of in vivo metabolic profile of MC4RKO mice

WT LM, MC4RKO mice, PF WT LM and PF MC4RKO mice were killed at 8 and 16 weeks of age. At the time of killing (between 09.00 h and 10.00 h), terminal blood samples were collected into ethylenediamine tetra-acetate (EDTA) tubes (1.6 mg (ml blood)−1 ). Plasma was collected, placed on dry ice, and stored at −80°C for future analysis. To assess circulating metabolites (glucose, insulin and NEFAs) from freely moving mice at respective ages, blood samples were collected from the tail tip as previously described (Steyn et al. 2011). A 20-μl tail-tip blood sample was collected using a heparinized pipette tip (100 IU ml−1 ), and plasma was separated via centrifugation (6000 rpm for 3 min at room temperature). Aliquoted plasma was placed on dry ice, and stored at −80°C for further analysis. To determine the adiposity of mice, fat pads (gonadal and inguinal) were isolated by dissection and immediately weighed. Hormone and metabolite analysis

Plasma levels of leptin, insulin, IGF-I and muscle IGF-1 were determined by commercial ELISA kits (EZML-82 K mouse leptin and EZRMI-13 K rat/mouse insulin (Millipore, Billerica, MA, USA) and SMG100 mouse/rat IGF-I (R&D Systems, Minneapolis, MN, USA)). For fasting insulin levels, a single 20-μl tail-tip blood sample was collected using a heparinized pipette tip following a 6-h fast. Circulating levels of FFAs and glucose were determined by colorimetric assay (FFA kit, 279–75401, NEFA C assay (Wako, Osaka, JPN); 10009582 glucose assay kit). To determine hepatic triglyceride content, liver samples were digested by saponification in ethanolic KOH and neutralized with MgCl2 as described previously (Salmon & Flatt, 1985). Glycerol content was determined using a glycerol standard (G7793; Sigma, St Louis, MO, USA) and free glycerol reagent (F6428; Sigma). Hepatic and muscle glycogen content were determined by anthrone reaction (319899; Sigma) as described previously (Illingworth & Russell, 1951). Assessment of GH profiles was performed using an in-house GH ELISA (Steyn et al. 2011). Within- and between-assay coefficients of variation for all ELISAs were below 2.8 and 3.9%, respectively. Isolation of mouse somatotrophs

Adult GH-GFP transgenic male mice (8 weeks old) were decapitated and whole pituitary glands were collected aseptically into a tissue culture dish containing ice-cold Ca2+ and Mg2+ free-Hank’s buffered solution (HBSS, 137 mM NaCl, 5.4 mM KCl, 0.3 mM Na2 HPO4 , 0.4 mM KH2 PO4 , 4.2 mM NaHCO3 , 1.0 mM D-glucose, pH 7.2). Tissues were digested in pre-warmed filtered protease


solution (10 μl ml−1 , Aspergillus oryzae, Sigma) and incubated at 37°C for 30 min. Dissociated tissues were incubated in HBSS-supplemented bovine serum albumin (BSA) (Bovogen, Victoria, Australia) at room temperature for 5 min and filtered using sterile 50 μm nylon gauze, and centrifuged at 1300 rpm for 5 min. The cell pellet A Food intake (g/mouse/day)

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was resuspended in 1 ml warmed-Dulbecco’s Modified Eagle Medium (DMEM, Sigma). Fluorescence-activated cell sorting (FACS, BD FACSAria Influx Cell Sorter, BD Biosciences) was used to specifically isolate somatotrophs amongst other pituitary cells. Clusters of cells/cell doublets were omitted to ensure only GFP-expressing cells were B

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Figure 1. Hyperphagic MC4RKO mice grow rapidly and become obese A, MC4RKO mice are hyperphagic by 5 weeks of age. B, hyperphagia results in increased weight gain, adult obesity and an overall increase in linear length. C and D, growth curves illustrating cumulative weekly body weight gain (C) and total body weight change (D) in MC4RKO mice compared to WT LM controls. E–G, hyperphagia results in the progressive rise in fat mass. H shows a corresponding rise in circulating levels of leptin. I and J, growth curves illustrating cumulative weekly body length gain (I; black arrowheads indicate age of assessment of pulsatile GH secretion – 4, 8 and 16 weeks of age) and total body length change (J) in MC4RKO mice compared to WT LM. Data are presented as means ± SEM. A P value < 0.05 was accepted as significant. WT, n = 8; KO, n = 6. [Colour figure can be viewed at wileyonlinelibrary.com] C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society

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isolated. Cells were collected in TRIzol (Invitrogen, CA, USA) and processed immediately for RNA analysis. Total RNA isolation and reverse transcriptase-polymerase chain reaction

Total RNA was extracted from freshly dissected hypothalamus, anterior pituitary glands, sorted GH-GFP somatotrophs and liver tissues. Tissues were homogenized in TRIzol and RNA was extracted using a PureLinkTM RNA Mini Kit (Invitrogen). Expression of MC4Rs in the hypothalamus, pituitary gland and somatotrophs was determined by conventional RT-PCR using established MC4R primers (Shaw et al. 2005), with GAPDH as an internal control. Mouse hypothalamus and liver tissues were used as positive and negative controls respectively. To generate first-strand cDNA, cDNA was transcribed with 1 μg total RNA using an iScript cDNA synthesis kit (Biorad Laboratories Inc., CA, USA). The intensity of the PCR bands was visualized on a Molecular Imaging GelDoc XR System (Biorad Laboratories). Colocalization of MC4R with somatotrophs and GHRH neurons in mice

Brain tissues and pituitary glands of WT C57Bl6/J mice, transgenic GH-GFP and GHRH-GFP, and MC4RKO mice (8–9 weeks old) were collected for immunohistochemistry and immunofluorescence, respectively. Following established methodology (Huang et al. 2014), mice were anaesthetized with a single I.P. injection of sodium pentobarbitone and transcardially perfused using a 20-ml syringe containing ice-cold 0.1 M phosphate buffer (5 ml), followed by freshly prepared ice-cold paraformaldehyde (4% PFA; 10 ml). The brain and pituitary glands were excised and post-fixed in 4% PFA overnight at 4°C and then replaced in 30% sucrose. For immunohistochemistry, coronal brain sections were cut at a thickness of 30 μm on a cryostat (LEICA CM 1850) and every fourth section through the hypothalamus was collected and stored in cryoprotectant (30% sucrose, 1% polyvinyl pyrolidone and 30% ethylene glycol in 0.1 M phosphate buffer) at −20°C. To unmask antigenic sites in PFA-fixed brain tissues, heat-induced antigen retrieval was performed by incubating slides in retrieval buffer (10 mM sodium citrate, pH 6.0) at 80°C for 30 min. Following antigen retrieval, sections were incubated with rabbit polyclonal primary MC4R antibody (Abcam No. 24233, Cambridge, UK; diluted in blocking buffer) for 72 h at 4°C on an orbital shaker. The anti-MC4R antibody (diluted in the blocking buffer) was first preincubated with MC4RKO hypothalamus (frozen-powdered hypothalamic tissue (0.9 g)) at 4°C overnight. Supernatant containing unbound MC4R antibodies were collected following centrifugation (13,000 rpm for 30 min at 4°C) C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society

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and diluted (at a final working concentration of 1:250) prior to use. For immunofluorescence, coronal sections of pituitary glands were cut at a thickness of 8 μm. Mounted sections were incubated in blocking buffer before incubating in rabbit polyclonal primary MC4R antibody (1:250, Abcam) overnight at 4°C on an orbital shaker. Following incubation in primary antibodies, sections were washed and incubated with secondary antibodies (Alexa Fluoro 555-conjugated donkey anti rabbit 555 1:500, Invitrogen). To test the specificity of MC4R antibody (Chitravanshi et al. 2009), brain tissues and pituitary glands harvested from MC4RKO mice were processed under similar conditions. Additional negative controls were included by omitting the primary antibody. To visualize immunoreactivity, sections were air-dried, mounted with Prolong gold anti-fade reagent with 4·,6-diamidino-2-phenylindole (DAPI; Invitrogen), and examined under a ×60 oil immersion objective (numerical aperture 1.35) unless otherwise specified. Images were collected on an Olympus FluoView FV1000 Confocal Microscope (Olympus, USA). Data and statistical analysis

Data are presented as means ± SEM unless otherwise stated. The rate of progressive weight gain and linear growth were assessed by linear regression, comparing the slope of each regression between WT and MC4RKO mice at respective ages and following intervention. For clarity, statistical results for regression analysis are presented alongside each comparison (Figs 1, 3 and 6). Comparisons between genotypes (WT LM and MC4RKO mice, or PF WT LM and PF MC4RKO mice) were analysed by Student’s unpaired t test and restricted to age. All measures (excluding deconvolution analysis) were performed using GraphPad Prism 6.0c (GraphPad, Inc., San Diego, CA, USA). The threshold level for statistical significance was set at P < 0.05. The quantitative features underlying GH secretion and clearance associated with the observed GH concentration profiles were determined by deconvolution analysis (Tan et al. 2013). Measurements of output parameters are indicative of GH secretion from the anterior pituitary gland, and thus observed circulating GH measures in this study reflect representative measures of hypothalamic-pituitary function. Results Loss of MC4R results in early onset hyperphagia, weight gain and rapid linear growth

We assessed growth rate (body weight and body length via nasal-anal distance) from 4 weeks to 20 weeks of age in ad libitum fed MC4RKO and WT LM mice. Loss

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of MC4R signalling resulted in early onset hyperphagia (starting by 5 weeks of age; Fig. 1A), weight gain and increased linear growth, resulting in a larger and fatter phenotype (Fig. 1B). Hyperphagia occurred in parallel with rapid weight gain (Fig. 1C) and increased total body weight (Fig. 1D). Weight gain occurred alongside the progressive accumulation of excess adipose mass (Fig. 1E–G), and a corresponding increase in circulating levels of the adipose-derived hormone leptin (Fig. 1H) (Considine et al. 1996; Beckers et al. 2009). Loss of MC4R signalling in mice resulted in increased linear growth rate (Fig. 1I), contributing to in an overall increased adult body length (Fig. 1J). The rate of linear growth between 5 and 8 weeks, and 8 and 16 weeks of age in MC4RKO mice was significantly greater than that of WT LM mice (P = 0.03 and P = 0.01, respectively), and slowed to that seen in WT LM by 16 weeks of age (Fig. 1I; P = 0.07). Thus increased linear growth in MC4RKO mice occurred during pubertal maturation and in early adulthood. GH release is suppressed in MC4RKO mice

We assessed spontaneous GH release in MC4RKO mice as increased GH secretion is thought to promote rapid pubertal linear growth in MC4R deficient individuals (Martinelli et al. 2011). Assessed ages corresponded to the initial onset of rapid linear growth rate (4 weeks), sustained rapid linear growth rate (8 weeks), and the eventual slowing of linear growth rate to that seen in adult WT LM mice (16 weeks). The amount of GH release (Fig. 2A, D–G) and the GH secretory pattern (Fig. 2A, H and I) from MC4RKO mice and WT LM mice were similar at 4 weeks of age. Total, pulsatile and the mass of GH release per secretory burst in MC4RKO mice was markedly reduced by 8 and 16 weeks of age (Fig. 2D–G), whereas basal GH release (Fig. 2G) and the patterning of GH release (pulse number and approximate entropy; Fig. 2H and I) remained unchanged. Suppressed GH release in MC4RKO mice corresponds with a reduction in tissue-specific IGF-1 levels

GH promotes linear growth indirectly via autocrine and paracrine effects of IGF-1 (Attanasio & Shalet, 2007), and IGF-1 may promote linear growth independently of GH (Laron, 2001). We therefore determined whether increased IGF-1 levels may have contributed to the observed increase in linear growth in MC4RKO mice. IGF-1 levels were assessed in plasma and tissues of mice at 4, 10 and 20 weeks of age. Circulating levels of total IGF-1 did not increase in rapidly growing MC4RKO mice (Fig. 2J). Rather, congruent with the progressive suppression of GH release, we observed a progressive loss of muscle- (Fig. 2K) and liver-specific IGF-1 (Fig. 2L) expression in MC4RKO mice.

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Prevention of hyperphagia normalized linear growth in MC4KO mice

Rapid linear growth in hyperphagic obese children occurs alongside the development of GH deficiency (Kleber et al. 2011), and thus factors other than GH may promote rapid growth in these children. Given the progressive suppression of GH release in MC4RKO mice, we anticipated that hyperphagia in MC4RKO mice may have contributed to an acceleration of growth, and not GH. Indeed, prevention of hyperphagia in MC4RKO mice by pair feeding slowed linear growth rate (Fig. 3A, B, D and E; weeks 4–8, P = 0.36 and weeks 8–16, P = 0.22) and restored final adult body length relative to starting length to that seen in pair-fed (PF) WT LM mice (Fig. 3C and F). Hyperphagia contributes to a reduction in GH and IGF-1 release in ad libitum fed MC4RKO mice

Hyperphagia is associated with decreased GH secretion and this precedes dietary-induced weight gain (Cornford et al. 2011), suggesting that excess calorie intake may play a critical role in the development of GH deficiency. Prevention of hyperphagia through pair feeding attenuated the suppression of GH secretion in MC4RKO mice at 8 weeks of age (Fig. 3G and I–K). While pair feeding restored GH release by 16 weeks of age (Fig. 3H and I), pulsatile GH release was modestly reduced (Fig. 3J), whereas the mass of GH secreted per burst was suppressed (Fig. 3K). Circulating total IGF-1 levels remained unchanged (Fig. 3M), and pair feeding in MC4RKO mice completely restored muscle-specific IGF-1 content to that seen in PF WT LM mice (Fig. 3N), presumably due to the recovery in peak GH release. Importantly, GH patterning (number of pulses and approximate entropy) remained unchanged regardless of dietary intervention, suggesting that interactions between hypothalamic components thought to modulate GH patterning remained intact. Loss of MC4R does not directly contribute to altered GH release in MC4RKO mice

Recovery of GH release in PF MC4RKO mice suggest that loss of MC4R contributes to suppressed GH release via an indirect mechanism, and therefore MC4Rs may not directly modulate GH release. To address this, we next assessed the expression of MC4Rs in the hypothalamus, anterior pituitary gland, and in somatotrophs isolated from the pituitary glands of transgenic mice expressing green fluorescence protein (GFP) in GH-secreting somatotrophs. While Mc4r mRNA was expressed in the hypothalamus and in the anterior pituitary gland of WT mice, Mc4r mRNA was not detected in somatotrophs (Fig. 4A and B; GH-GFP). This was confirmed by C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society


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Figure 2. Loss of MC4R results in the progressive suppression of GH–IGF-1 A–C, representative examples of pulsatile GH secretion profiles in WT LM and MC4RKO mice at 4 (A), 8 (B) and 16 weeks (C) of age. Deconvolution analysis of GH secretion profiles reveals the progressive suppression in total (D), pulsatile (E) and the mass of (F) GH secreted per burst. G–I, basal GH secretion (G), and pulse number (H) and entropy (I) remained unchanged, suggesting that loss of MC4R does not alter GH secretory patterning. J–L, while circulating levels of total IGF-1 remain unchanged (J), the progressive suppression of GH release was seen alongside a reduction in both muscle-specific (K) and liver-specific (L) IGF-1 levels in MC4RKO mice at 4, 10 and 20 weeks of age. Data are presented as means ± SEM. A P value < 0.05 was accepted as significant. Genotype symbols: WT, +/+; MC4RKO, −/−. WT, n = 8; KO, n = 6.


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Figure 3. Pair feeding reverses aberrant linear growth and restores normal GH–IGF-1 secretion in MC4RKO mice A, pair feeding restored normal body length in MC4RKO mice by 16 weeks of age. B and C, growth curves illustrating cumulative weekly body length (B), and the percentage change in body length (C) in PF WT LM and PF MC4RKO mice relative to body length at 4 weeks of age. D, increased body length in ad libitum fed MC4RKO mice by 16 weeks of age. E and F, growth curves illustrating cumulative weekly body length gain (E) and total body length change (F) in ad libitum fed WT LM and MC4RKO mice relative to body length at 4 weeks of age. G and H, representative examples of pulsatile GH secretion profiles in PF WT LM and PF MC4RKO mice at 8 (G) and 16 weeks (H) of age. I–K, pair feeding recovered total (I), pulsatile (J) and the mass of (K) GH secreted per burst in 8 week old mice. L, pair feeding did not alter the number of GH secretory bursts. M, circulating IGF-1 remained unchanged regardless of dietary intervention. N, muscle IGF-1 was restored in PF MC4RKO mice at 16 weeks of age. Data are presented as means ± SEM. A P value < 0.05 was accepted as significant. Genotype symbols: WT, +/+; MC4RKO, −/−. WT, n = 8; KO, n = 6. [Colour figure can be viewed at wileyonlinelibrary.com]


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Figure 4. MC4R is not expressed in somatotrophs, and loss of MC4R does not alter hypothalamic expression of Srif or Ghrh mRNA A, fluorescence-activated cell sorting (FACS) of somatotrophs isolated from pituitary glands of transgenic mice expressing GH-GFP. B, expression of MC4Rs in WT hypothalamus, anterior pituitary gland and somatotrophs isolated using FACS in GH-GFP mice at 8 weeks of age. WT liver tissue was used as a negative expression control. C–E, immunofluorescence of MC4R expression in the pituitary gland of transgenic mice expressing GFP in somatotrophs. MC4Rs (red, D) are not expressed on somatotrophs (green, C); E, merge. F–H, negative controls show no/little MC4R immunoreactivity following the omission of primary MC4R antibody in pituitary tissue of WT LM mice (F and G), and the absence of MC4R immunoreactivity in the pituitary glands of MC4KO mice (H). Nuclei (blue) indicated by 4 ,6-diamidino-2-phenylindole (DAPI) labelling. I, hypothalamic tissues containing the PeVN, PVN and ARC were collected from 300 µm thick frozen brain sections. Micropunch biopsies were obtained from brain sections located between bregma −0.34 and −1.82 (tissue collected at representative levels corresponding to the PeVN, PVN and ARC nucleus complex outlined and shaded in red). J, no differences in Srif or Ghrh mRNA expression were observed between WT LM and MC4RKO mice (WT, n = 8; KO, n = 6). For PCR, data are presented as means ± SEM. C–E and G–H, scale bars = 20 µm. F, scale bar = 200 µm. Images representative of observations verified across 4 mice. For J, a P value of < 0.05 was accepted as significant.


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immunohistochemical observations demonstrating that MC4Rs are not expressed on somatotrophs (Fig. 4C–E; negative staining illustrated in Fig. 4F–H). To determine whether the loss of MC4R may suppress GH secretion through inhibition of hypothalamic GHRH neurons (the dominant neuronal population known to stimulate pituitary GH release; Steyn et al. 2016), we next assessed the abundance of hypothalamic gene expression for growth hormone releasing hormone (Ghrh) and somatostatin (Srif) neurons – key hypothalamic regulators of the pulsatile release of GH (Steyn et al. 2016). Hypothalamic tissues of WT LM and MC4RKO mice containing the periventricular nucleus (PeVN) and arcuate nucleus (ARC) (Fig. 4I) were isolated for mRNA analysis. Compared to WT LM mice, no discernible change in Srif or Ghrh (Fig. 4J) mRNA expression was observed in the PeVN/ARC complex of MC4RKO mice. While suggesting that the loss of MC4R does not alter GHRH–SRIF interactions, these measures do not exclude possible direct effects of MC4R on the activity of GHRH neurons. Thus, to determine whether MC4Rs may regulate GH secretion via direct interactions with GHRH neurons, we next examined co-expression of MC4R with GHRH neurons. We observed widespread distribution of MC4Rs in the brain (Fig. 5A and B). Aggregated MC4R immunoreactivity (identified by neuronal cell bodies) was evident in the cortex (Fig. 5C; Cx), hippocampus (Fig. 5D; HippoC), and thalamus (Fig. 5E; Thal) (in comparison to the absence of MC4R immunoreactivity in MC4RKO mice, Fig. 5M). MC4R immunoreactivity (demonstrated by punctuate staining) was observed in the paraventricular nucleus (Fig. 5F; PVN), the dorsal medial hypothalamus (Fig. 5G; DMH), and the ARC (Fig. 5H). Observations are consistent with previous studies characterizing MC4R expression in rats (Mountjoy et al. 1994). GHRH neurons lie along the ventral border of the ARC (Fig. 5I). We found little or no co-expression of MC4Rs (Fig. 5J) with GHRH neurons (Fig. 5K, higher magnification).

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mice (Fig. 3A–F), PF MC4RKO mice continued to gain weight relative to PF WT LM mice (Fig. 6A and B) (as is observed in ad libitum fed MC4RKO mice (Fig. 6C and D)), and continue to accumulate excess fat mass (Fig. 6E and F). Given the recovery of GH release and IGF-1 in PF MC4RKO mice (Fig. 3G–L), it appears unlikely that increased adipose mass directly contributes to the suppression of GH release in ad libitum fed MC4RKO mice. Hyperphagic MC4RKO mice develop hyperinsulinaemia while maintaining plasma fatty acid and glucose homeostasis

Hyperinsulinaemia is thought to suppress GH release during dietary-induced weight gain in mice (Steyn et al. 2013), and thus we considered hyperphagia-associated hyperinsulinaemia in MC4RKO mice as a potential cause for the early-onset suppression of GH release. Assessment of circulating insulin in ad libitum fed MC4RKO mice revealed a striking elevation in fed (Fig. 7A) and fasting (Fig. 7B) insulin levels at 6 and 12 weeks of age. In contrast, pair feeding mostly prevented hyperinsulinaemia in fed (Fig. 7A) and fasting (Fig. 7B) MC4RKO mice. The suppression of GH release that occurs alongside an elevation in circulating levels of insulin promotes the uptake and storage of NEFAs and glucose (Cornford et al. 2011, 2012). Accordingly, circulating glucose (Fig. 7C) and NEFAs (Fig. 7D) in ad libitum or PF MC4RKO mice were maintained within a similar range as that seen in WT mice. Circulating glucose and NEFA homeostasis was maintained in MC4RKO mice, regardless of weight gain and increase in adiposity (Figs 1 and 6). Maintained glucose and NEFA homeostasis was likely sustained through the progressive accumulation of hepatic TG (Fig. 7E) and glycogen stores (Fig. 7F), as was observed in ad libitum fed MC4R KO mice. Discussion

Prevention of hyperphagia in MC4RKO mice does not completely prevent weight gain or increased adiposity

GH deficiency occurs in obesity (de Boer et al. 1995), and is characterized by a progressive reduction in GH release in humans (Iranmanesh et al. 1991; Veldhuis et al. 1995) and mice (Steyn et al. 2013) relative to increased adiposity. Restoration of GH release following weight loss (Williams et al. 1984; Rasmussen et al. 1995) suggests that factors secreted in proportion to adipose mass may directly contribute to the development of GH deficiency in obesity. We thus considered that increased adipose mass might have directly contributed to the suppression of GH release in ad libitum fed MC4RKO mice. While pair feeding normalized linear growth rate in MC4RKO

Loss of function of the MC4R is thought to contribute to a recovery in GH release (Martinelli et al. 2011), normally suppressed as a consequence of dietary weight gain and obesity (Scacchi et al. 1999). As such, it is suggested that the MC4R may promote the suppression of GH release in obesity, and thus that loss of MC4R signalling contributes to increased linear growth as a consequence of increased pubertal GH release. Using the mouse as a model, we assessed GH–IGF-1 levels in MC4RKO mice relative to pubertal and early-adult growth. We provide evidence to show that hyperphagia in MC4RKO mice contributes to the progressive suppression of GH–IGF-1, and thus that rapid linear growth in MC4RKO mice does not occur as a consequence of the hypersecretion of GH–IGF-1. Our observations further suggest that hyperinsulinaemia C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society


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Figure 5. MC4R is expressed throughout the brain, but not in GHRH neurons A and B, schematic coronal diagrams of mouse brain mapping melanocortin 4 receptor (MC4R) immunoreactivity at bregma between −0.94 and −1.82. C–H, representative ×20 images of MC4R immunoreactivity in the cerebral cortex (Cx, panel C), hippocampus (HippoC, panel D), thalamus (Thal, panel E), paraventricular nucleus (PVN, panel F), dorsal medial hypothalamus (DMH, panel G) and the arcuate nucleus (ARC, panel H) of the hypothalamus. I–K, immunofluorescence assessment of growth hormone releasing hormone (GHRH) expressing green fluorescent protein (GFP; green) neurons (I) and MC4R neurons (red; J); K, merge. C–K, scale bars = 50 µm. L and M, representative examples showing minimal/no MC4R staining in the absence of the primary antibody (−1°AB; inset


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illustrates higher magnification (×60) within the thalamus of WT tissue; panel L) and the absence of comparable MC4R immunoreactivity in the thalamus between MC4KO mice and wild-type (WT) mice (inset) (panel M). Nuclei (blue) indicated by 4 ,6-diamidino-2-phenylindole (DAPI) labelling. L, scale bar = 500 µm. M, scale bar = 20 µm. Insets illustrate higher magnification (×60) of representative areas. Images representative of observations verified across 4 mice.

as a consequence of hyperphagia may contribute to reduced GH–IGF-1 levels while also promoting increased linear growth in these mice. This is in keeping with the documented effects of insulin in regulating GH release (Steyn et al. 2016) and growth (Geffner, 1996), furthering our understanding of mechanisms that may contribute to reduced GH release and increased growth following sustained childhood and pubertal hyperphagia. Increased adiposity is negatively associated with decreased GH secretion (Iranmanesh et al. 1991; Veldhuis et al. 1991), with the degree of GH attenuation correlating with the amount of total and visceral adipose mass (Clasey et al. 2001; Johannsson, 2008). As a consequence, GH deficiency is commonly observed in obese individuals (Scacchi et al. 1999). A similar relationship is observed in 40

B

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10 0

mice, wherein a progressive reduction in peak GH release occurs relative to increased adipose mass (Steyn et al. 2013), and where dietary-induced weight gain accelerates the naturally occurring reduction in peak GH release that occurs with age (Huang et al. 2012). Given that a loss of adipose mass following calorie restriction in obese adults restores spontaneous and stimulated GH release (Williams et al. 1984; Rasmussen et al. 1995), it is thought that factors specific to adipose mass selectively inhibit GH release. Our observations challenge this notion, as we observed the recovery of GH release in PF MC4RKO mice, regardless of the continued accumulation of excess adipose mass. This accumulation of excess adipose mass likely occurred as a consequence of metabolic defects seen in these mice (Ste Marie et al. 2000), wherein activation of the MC4R

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Figure 6. Prevention of hyperphagia results in continued weight gain and increased fat mass in MC4RKO mice Growth curves illustrating cumulative weekly body weight gain (A and C) and total body weight change (B and D) in PF WT LM and MC4RKO mice, and ad libitum fed WT LM and MC4RKO mice. E and F, epididymal (E) and inguinal fat mass (F) of PF WT LM and MC4RKO mice, and ad libitum fed WT LM and MC4RKO mice at 8 and 16 weeks of age. Data are presented as means ± SEM. A P value < 0.05 was accepted as significant. PF WT, n = 5; KO, n = 5. Ad libitum fed WT, n = 6; KO, n = 8. [Colour figure can be viewed at wileyonlinelibrary.com]



J Physiol 594.24

In the hypothalamus, insulin may stimulate the release of catecholamines (Sauter et al. 1983) to activate SRIF neurons, thereby inhibiting GHRH-induced GH secretion (Ishibashi & Yamaji, 1984). Promotion of weight loss in obese adults improves dietary-induced hyperinsulinaemia and restores impaired GH secretion (Rasmussen et al. 1995). While the accumulation of fat mass remained unchanged, pair feeding significantly reduced circulating insulin levels in MC4RKO mice. Given that we observed a coincident recovery in GH release at this time, it is likely that the prevention of hyperphagia-associated hyperinsulinaemia may have prevented the insulin-mediated suppression of GH release. Observations suggest that insulin may regulate GH release independently of fat mass. Further justification is needed before we can confirm insulin as the key inhibitor of GH release in this instance,

would normally promote increased energy expenditure (Haynes et al. 1999). Observations suggest that the progressive suppression of GH release observed in ad libitum fed MC4RKO mice occurred independently of fat mass, and therefore that factors other than adipose mass may promote the suppression of GH release in response to dietary-induced weight gain. Prior assessment of adipose mass as a possible regulator of GH release during weight gain in the mouse showed an inverse relationship between circulating levels of insulin and GH (Steyn et al. 2013). Specifically, peak and total GH secretion in mice was found to decrease relative to a rise in circulating levels of insulin. Insulin is thought to directly suppress GH release, acting at the level of the anterior pituitary gland (Luque & Kineman, 2006) or indirectly within the hypothalamus.

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Figure 7. Prevention of hyperphagia prevents the development of hyperinsulinaemia, while glucose and NEFAs balance is maintained in ad libitum fed and pair-fed MC4RKO mice A and B, assessment of fed (A) and fasting (B) insulin levels in ad libitum fed and PF WT LM and MC4RKO mice at 6 and 12 weeks of age. C and D, fed glucose (C) and NEFA levels (D) in ad libitum fed and PF WT LM and MC4RKO mice at 6 and 12 weeks of age. E and F, accumulation of hepatic triglycerides in hyperphagic MC4RKO mice at 4, 10 and 20 weeks of age (E), and corresponding levels of hepatic glycogen content (F). Data are presented as means ± SEM. A P value < 0.05 was accepted as significant. PF WT, n = 5; KO, n = 5. Ad libitum fed WT, n = 6; KO, n = 8. C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society

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and it remains unclear how rapid growth in MC4RKO mice is sustained alongside suppressed GH–IGF-1. Rapid and early pubertal linear growth is common in hyperphagic obese children, regardless of the development of GH deficiency (Kleber et al. 2011). Thus, while GH is a major regulator of postnatal somatic growth in childhood (Rose et al. 1991; Martha et al. 1992), linear growth in pubertal obese individuals generally occurs in the absence of GH–IGF-1 actions (Geffner, 1996). It has been proposed that hyperphagia-associated hyperinsulinaemia promotes linear growth in obese children (Kleber et al. 2011). In line with this, we show that prevention of hyperphagia-associated hyperinsulinaemia in PF MC4RKO mice is able to normalize GH release and linear growth rate. Thus, as may occur in other cases of childhood obesity (Kleber et al. 2011), hyperinsulinaemia appears to suppress GH release while also promoting growth in young MC4RKO mice. While the mechanism by which a reduction of calorie intake can normalize linear growth in MC4RKO mice remains unknown, it is plausible that the restriction of substrate supply in PF MC4RKO mice in this study might emulate effects seen in these animals following exercise intervention (Borghouts & Keizer, 2000). Indeed, the prevention of hyperinsulinaemia through exercise slows growth rate and normalizes body length in MC4RKO mice (Haskell-Luevano et al. 2009), presumably by enhancing lipid metabolism and hepatic glucose output to improve insulin sensitivity, thereby reducing insulin secretion (Borghouts & Keizer, 2000). Considering that insulin may promote growth in the absence of GH (Geffner, 1996), prevention of hyperinsulinaemia through exercise (Haskell-Luevano et al. 2009) or following normalization of calorie intake may have largely normalized linear growth in MC4RKO mice. Sustained hyperphagia contributes to decreased GH release and increased insulin secretion (Cornford et al. 2011). Pharmacological reversal of this suppression in GH release in hyperphagic adults results in impaired insulin action. Subsequent disruptions in fatty acid and glucose flux culminate in hyperlipidaemia (Cornford et al. 2012). Similarly, excess GH release severely impairs insulin-mediated lipid uptake (Olsson et al. 2005; Boparai et al. 2010). Therefore, the suppression of GH release that occurs alongside prolonged hyperphagia appears to be a physiological phenomenon that serves to facilitate insulin action to maintain energy uptake. This prevents adverse pathophysiological sequelae associated with the development of insulin resistance, including increased risk for cardiovascular disease and the eventual development of type 2 diabetes (Reaven, 1995). Accordingly, the suppression of GH release in ad libitum fed MC4RKO mice most likely promoted continued clearance and storage of endogenous dietary fuels by aiding the endogenous actions of insulin. It is anticipated that, in ad libitum fed

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MC4RKO mice, the progressive suppression of GH release promoted insulin-driven lipid uptake in adipose tissue and in the liver (accounting for the observed accumulation of fat mass and hepatic triglycerides and glycogen stores). Accordingly, it was not surprising that the prevention of an oversupply of energy substrates in PF MC4RKO mice resulted in the slowing of the development of hyperinsulinaemia, and a delay in the suppression of GH release. Based on our observations, we propose that metabolic requirements to maintain energy flux during periods of sustained hyperphagia in MC4RKO mice are likely to override the anticipated actions of GH in regulating growth at this time. In this instance, hyperinsulinaemia promotes the continued uptake and storage of energy, suppressing GH release and potentially promoting growth. This physiological adaptation to sustained hyperphagia is schematically illustrated in Fig. 8. Our observations provide novel insights to aid in our understanding of altered mechanisms of growth in children with monogenetic defects in MC4R signalling. Observations are currently limited to MC4RKO mice, and the proposed mechanisms of GH-independent growth can only be adopted once verified across other instances of increased growth that occurs alongside hyperphagia-associated hyperinsulinaemia. This includes verification of GH release in children with defects in MC4R signalling. Accurate assessment of GH release requires repeat blood sampling, an arduous and expensive process. Prior to this study, evidence to justify comprehensive GH assessment in children with defects in MC4R function did not exist. In light of our findings, it may now be imperative that closer clinical assessment of the release of GH relative to insulin release be conducted in this population. Measures may provide additional mechanistic insights to aid in our understanding of the cause and physiological significance of suppressed GH release. This is important, as proper care of GH deficient obese children may prevent the eventual development of metabolic complications that arise in adulthood, including a worsening of a predisposition to the development of insulin resistance and thus the development of type 2 diabetes (Kahn et al. 2001). While offering new data to show that loss of MC4R function may not directly contribute to increased linear growth, additional studies are needed to comprehensively exclude MC4R as a regulator of GH release in obesity. The assessment of GH release in individuals with defects in MC4R was done alongside a BMI-matched control population (Martinelli et al. 2011). Current measures are limited by inclusion of lean littermate controls; with the assessment of GH release in MC4RKO mice not matched to WT mice with a similar degree of adiposity. Therefore, we are unable to draw direct comparisons with existing measures that document GH release relative to dysfunction of MC4R (Martinelli et al. 2011). To discount possible direct effects of MC4R on central C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society


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regulation of GH release we show that the MC4R is not widely expressed on GHRH neurons or somatotrophs. While demonstrating negligible immunoreactivity in MC4RKO tissue, documentation of MC4R expression throughout the brain has been hampered by the lack of reliable antibodies for MC4R (Cowley et al. 1999). Therefore, while our observations demonstrate the lack of MC4R expression in key components of central GH regulatory mechanisms, ancillary studies are needed to verify this data. Such studies should focus on central mechanisms of GH release, assessing the direct (MC4R) and indirect (hyperinsulinaemia) consequences of loss of MC4R function on GHRH and SRIF neuron activity. We documented normal expression of Srif and Ghrh mRNA in the hypothalamus of MC4RKO mice, but these measures cannot define the functional capacity of these neurons, and in particular the response of SRIF and GHRH neurons to feedback mechanisms that may be activated during prolonged periods of hyperphagia. Indeed, while measures from 4-week-old MC4RKO mice demonstrate normal GH secretion, confirming that loss of MC4R may not directly impact on GH release, it

A

Linear growth

remains unknown if sustained hyperphagia-associated hyperinsulinaemia might affect interactions between these central regulators of GH secretion. Such studies may further advance our understanding of possible feedback effects of key peripheral factors (including insulin and IGF-1) in the regulation of GH release. For example, we found reduced liver and muscle IGF-1 levels in ad libitum fed MC4RKO, whereas circulating levels of IGF-1 remained unchanged. This suggests derangement of IGF-1 production and or clearance. Given the complexities of IGF-1 production and action (including interactions of IGF-1 with IGF-1 binding proteins), follow-up studies may uncover changes in the function and feedback of IGF-1 as a regulator of somatic growth following loss of MC4R function, and the extent of the effect of hyperinsulinaemia within this system. In summary, confirmation of the impairment of GH–IGF-1 release in hyperphagic MC4R KO mice suggests a role for insulin in regulating both the release of GH, but also in mediating growth during periods of physiologically suppressed GH–IGF-1 levels. These observations build on prior evidence to suggest that insulin may be a

Balanced energy intake and maintenance of GH/insulin balance: 1.Movement of dietary fat into and out of liver/adipose stores relative to metabolic demand.

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1. Hyperphagia-associated hyperinsulinemia promotes increased storage of fat in liver/adipose. 2. Insulin drives the progressive decrease in GH secretion to reduce FFA use and to anhance FFA storage. 3. Insulin suppresses canonical mechanisms of growth (GH/IGF-1 axis). 4. Linear growth maintained via anabolic actions of insulin.

Figure 8. Proposed schematic illustrating the effects of hyperphagia on insulin-mediated growth and GH release Proposed feedback and regulation of fatty acid uptake and storage, GH release and growth in WT (A) and hyperphagic MC4RKO (B) mice. Hyperphagia-associated hyperinsulinaemia may contribute to the progressive suppression of GH release. Linear growth is sustained during periods of suppressed GH release, potentially in response to hyperinsulinaemia. [Colour figure can be viewed at wileyonlinelibrary.com] C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society

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Author contributions

Additional information

Acknowledgements

Competing interests

The authors gratefully acknowledge the assistance and support from staff and animal technicians from the University of Queensland Biological Resources (UQBR).

The authors have no competing interests to disclose.

H.Y.T. designed and conducted research, analysed and interpreted data, prepared manuscript. F.J.S. developed research concepts, analysed and interpreted data, prepared manuscript. L.H. assisted with collection of data, assisted with preparation of manuscript. M.C. provided essential research support, assisted with preparation of manuscript. J.D.V. assisted with essential data analysis and interpretation. C.C. provided research support, designed research, assisted with preparation of final manuscript. All authors have approved the final version of the manuscript and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. Funding This work was supported by The University of Queensland. H.Y.T. was a recipient of an International Postgraduate Research Scholarship (IPRS) and an Australian Postgraduate Scholarship (APS) at The University of Queensland.
