Clinical and in vitro data suggest a link between the elevation of the melanocortin peptide, ACTH, and longitudinal growth. Overproduction of ACTH in familial glucocorticoid deficiency (FGD) is associated with increased growth and ACTH increases the differentiation of chondrocytes along the endochondral pathway in vitro. Using the leptin-deficient obese (ob/ob) mouse along with lean control littermates (n = 9–10), we investigated the effects of adrenalectomy (ADX)-induced elevated ACTH with and without peripheral administration of the MC3-R-specific agonist, γ2-melanocyte stimulating hormone (γ2-MSH), on longitudinal growth. Naso-anal and tibial growth were measured together with growth plate parameters; both total and zonal heights together with the proliferative index. Data were analyzed using two-way ANOVA with post hoc comparisons made using the Bonferroni correction. ADX significantly increased naso-anal length in lean mice and ADX plus γ2-MSH administration significantly increased naso-anal length above ADX alone in ob/ob mice. γ2-MSH administration to ADX lean and ob/ob mice significantly increased tibial length. In ob/ob mice, these changes occurred in the context of reduced food intake. Analysis of total and zonal growth plate heights suggest an increase in hypertrophic differentiation and an overall increase in growth plate turnover in ADX lean and ob/ob mice. These in vivo data show that ADX enhances linear growth and the results of γ2-MSH treatment suggest that the melanocortin system plays a role in linear growth.
Longitudinal growth is accomplished through the process of endochondral ossification, which occurs in the cartilaginous growth plate located at both ends of the vertebrae and long bones. It follows a sequential pattern of biological events, beginning with chondrocyte maturation and ending in the development of mature hypertrophic chondrocytes. The growth plate is comprised of three distinct groups of chondrocytes: round periarticular chondrocytes, proliferating columnar chondrocytes, and hypertrophic chondrocytes. In developing growth plates, the periarticular chondrocytes near the articular surface proliferate, differentiate into flat proliferating columnar chondrocytes, eventually stop dividing, and differentiate into hypertrophic cells (Kobayashi et al. 2002). We have hypothesized that ACTH positively influences this process of differentiation along the endochondral pathway.
A direct correlation between elevated ACTH production and increased longitudinal growth is observed with the clinical syndrome, familial glucocorticoid deficiency (FGD). FGD is an ACTH-insensitivity disorder characterized by an overproduction of ACTH, enhanced linear growth, and advanced bone age (Clark & Weber 1998, Elias et al. 2000). In FGD, systemic levels of ACTH are extremely elevated due to a mutation in the ACTH-receptor/melanocortin-2 receptor (MC2-R). When the FGD patients are treated with glucocorticoid, their ACTH levels are reduced and their growth patterns normalize somewhat (Elias et al. 2000).
In vitro, ACTH directly affects chondrocyte development; it increases the differentiation of resting chondrocytes and mesenchymal chondroprogenitors (Evans et al. 2004). ACTH also evokes transient elevations in intracellular free calcium [Ca2+]i and increases basal [Ca 2+]i (Evans et al. 2005) and differentiation of chondrocytes along the endochondral pathway is associated with an incremental increase in basal [Ca2+]i (Zuscik et al. 2002). The MC3-R/MC4-R antagonist, SHU9119, attenuates ACTH evoked increases in [Ca2+]i (Evans et al. 2004) and the MC3-R is highly expressed by chondrocytes and chondrocyte progenitors (Clark & Weber 1998). To investigate the effects of elevated ACTH in vivo, both leptin-deficient obese mice (ob/ob) and their lean littermates were adrenalectomized (ADX) or adrenalectomized and given the MC3-R-specific agonist, γ2-melanocyte stimulating hormone (γ2-MSH) (50 μg/day, s.c.), for 21 days. The MC3-R-specific agonist, γ2-melanocyte stimulating hormone (γ2-MSH), was used because we expected it to augment the effects of elevated ACTH either through further activation of MC3-R or another MC-R. Activation of the MC3-R results in transient elevations in [Ca2+]i in chondrocytes and other cell types (Mountjoy et al. 2001, Evans et al. 2004), and in pituitary cells the γ-MSH ligand produces transient elevations in [Ca2+]i through an undefined MC-R (Langouche et al. 2001). Therefore, γ2-MSH activation of the MC3-R or another MC-R on growth plate chondrocytes could translate to increased hypertrophy and lead to increases in linear growth. After treatment, naso-anal and tibial length were measured along with growth plate parameters, i.e. total and zonal heights and the proliferative index.
The ob/ob mouse model of obesity was used in these studies because these mice have been described as 5–10% shorter than their lean littermates (Cone 2000). It was thought that this would allow room for significant changes in growth with ADX and ADX plus γ2-MSH treatment. Additionally, with the use of this model, we were able to determine if ADX and ADX+γ2-MSH treatment enhance linear growth parameters in the absence of hypothalamic and peripheral leptin signaling.
Materials and Methods
Forty obese (ob/ob) and 40 lean (+/?) female 5-week-old mice were obtained from The Jackson Laboratory (Bar Harbor, Maine, USA). The exact genotype of the lean littermate control mice was not determined, therefore this group was likely a mixture of the +/+ and +/− leptin genotypes.
Lean and ob/ob animals were divided into four groups of ten animals each. Five-week-old basal control (basal), age-matched sham-operated control (Sham), adrenalectomized (ADX), and adrenalectomized with 50 μg/day γ2-MSH administration (ADX+γ2-MSH). Lean controls and ob/ob mice were adrenalectomized by the vendor. Sham operation was also performed by the vendor. ADX served to remove endogenous corticosterone and significantly elevate ACTH levels. ADX groups were maintained on drinking water supplemented with 0.9% NaCl and received an s.c. 50 μg corticosterone pellet (Innovative Research of America, Sarasota, FL, USA) designed to deliver 2.4 μg corticosterone per day over 21 days implanted to maintain basal metabolism. The γ2-MSH peptide (acetyl-(lys0,Nle3)-γ2-MSH amide, BACHEM, King of Prussia, PA, USA) was used in these experiments because it is a specific MC3-R agonist and the MC3-R has been detected in cultured chondrocytes. The ADX+γ2-MSH treatment groups received approximately 50 μg/day acetyl-(lys0,Nle3)-γ2-MSH amide over the 21-day experimental period delivered through an s.c. pellet (Innovative Research of America) containing a total of 1.05 mg peptide. The pellets were implanted at the time of surgery. The γ2-MSH dose used in this study can be calculated as 12.5 pmol/ml blood volume per min in lean control mice and 8.42 pmol/ml blood volume per min in ob/ob mice, or 1.07 nmol/kg per min and 0.672 nmol/kg per min respectively. These values are ~500 and 337 times greater respectively than known circulating levels of immunoreactive γ-MSH in mice (Ni et al. 2003).
Animals arrived at our institution’s animal facility 8 days after surgery and were kept under local vivarium conditions (12 h light:12 h darkness cycle, 23 °C) for the remainder of the experiment. Body weight and food intake were monitored every 3–4 days for the duration of the experiment. All animals were injected with 50 μg/kg BrdU to label chondrocytes in S-phase, 24 h prior to euthanasia. At the end of the 3-week experimental period, animals were euthanized by exsanguination under 100 mg/kg ketamine per 20 mg/kg xylazine anesthesia. The method of euthanasia is consistent with the recommendations of the 2000 Report of the American Veterinary Medical Association Panel on Euthanasia. The Institution’s Animal Care and Use Committee approved all animal protocols. Naso-anal length was measured prior to euthanasia and length of the left tibia measured after dissection using a dial type 6932 Bel Art caliper (Switzerland).
The leptin-deficient ob/ob mice did not tolerate the adrenalectomy surgery as well as the wild-type mice. One sham operated and one ADX animal receiving low-dose corticosterone treatment died shortly after arrival in the animal facility.
ACTH, corticosterone, and leptin measurements
Blood was collected in EDTA containing tubes via intra-cardiac puncture and stored at −70 °C until assay. To confirm completeness of adrenalectomy, ACTH and corticosterone were measured via RIA using kits from MP Biomedicals, Inc (Irvine, CA, USA). ACTH levels in plasma from ADX and ADX+γ2-MSH groups were significantly elevated and thus were outside the range of standards. Therefore, these samples were run a second time at a 10× dilution along with samples from the SHAM groups. Since the volume of plasma collected from these mice was limited, data were available for only seven animals per ADX and ADX+γ2-MSH groups. To confirm animal genotype, leptin was measured via RIA with a kit from Linco Research (St Charles, MO, USA).
After being thoroughly cleaned of soft tissues, distal portions of each right femur were cut using an Isomet saw (Buehler, Lake Bluff, IL, USA) and stained at 4 °C using Villanueva Osteochrome Bone Stain (Polysciences, Warrington, PA, USA) for 5 days. The bones were then destained and dehydrated in sequential changes (70, 95, and 100%) of ethanol solutions and xylene and then cold embedded in methylmethacrylate (Erben 1997). Distal femora were frontal cut longitudinally to 8 μm with a microtome (Leica RM2155, Germany). Sections were placed on gelatin-coated slides and cover slipped using Eukitt (Calibrated Instruments, Hawthorne, NY, USA). Growth plate width was measured using the ‘Osteomeasure’ (Osteometrics) digitizing morphometry system and the standard nomenclature (Parfitt et al. 1987).
Eight micrometers methylmethacrylate embedded sections were de-plastified using 2-methyloxyethyl acetate overnight followed by 5-min incubation in acetone. Sections were re-hydrated through decreasing graded concentrations of EtOH ending with distilled water. De-calcification was carried out in 10% EDTA in PBS for 2 h followed by washing in 1×PBS. Sections were exposed to proteinase K for 15 min at room temperature prior to a 3 h blocking with 2.5% horse serum. Avidin D (Vector Laboratories) was included in the blocking serum according to the manufacturer’s instructions. After washing thrice with 0.5% Tween in PBS (PBST), sections were incubated overnight in a 1:100 dilution of mouse monoclonal anti-BrdU antibody (Sigma) in blocking serum. Biotin (Vector Laboratories, Burlingame, CA, USA) was included with the primary antibody solution according to the manufacturer’s instructions. The appropriate normal IgG at the same concentration as the primary antibody was used as a negative control. Secondary antibody from the RTU Universal Vectastain kit (Vector Laboratories) was diluted in the ratio of 1:1 in blocking serum and incubated for 30 min followed by 30-min incubation with ABC reagent from the same kit. Three 10-min washes with PBST followed all steps including and subsequent to blocking. Dab was used as the chromogenic substrate. Nuclear-associated BrdU labeling was analyzed in four random growth plate fields of one femoral section from each animal. As an additional negative control, sections from mice not injected with BrdU were processed according to the described protocol. No staining was observed in these sections. Data were expressed as number of cells positive for BrdU divided by the total number of cells×100 = % BrdU positive cells (proliferative index).
Data were analyzed using one-way or two-way ANOVA as indicated. With two-way ANOVA, treatment and genotype were used as the main factors. Post hoc test P values were adjusted using the Bonferroni correction and a nominal significance level of 0.05 was used. When analyzing growth parameters, one significant outlier was identified using Grubbs’ test (Iglewicz & Hoaglin 1993). The outlier was detected in the SHAM-operated lean control group. Data from this animal were removed from all analyses.
Obese (ob/ob) mice demonstrate reduced longitudinal growth compared with lean littermate controls
Body weight and longitudinal growth parameters were measured in both ob/ob and lean mice at 5 weeks of age (basal; Table 1). As expected, ob/ob mice showed significantly greater body weight than their lean littermates. Conversely, ob/ob mice demonstrate significantly decreased naso-anal length and tibial length compared with lean controls. These data are consistent with previous reports on somatic and longitudinal growth in ob/ob mice (Cone 2000). Examination of total growth plate height and height of individual growth zones indicate that total growth plate height is reduced in ob/ob animals due to a significant reduction in hypertrophic zone height (Table 1, Fig. 1). This reduction appears to be due to significantly less hypertrophic cells in number but not in size in growth plate of the ob/ob mice (Table 1).
Adrenalectomy (ADX) alone and ADX plus the administration of γ2-MSH reduces body weight and food intake in obese (ob/ob) mice
Pro-opio melano cortin (POMC) peptides have known anorexigenic effects (Millington et al. 2001). This may, in turn, impact linear growth, as calorie, restriction is known to limit linear growth (Brochman et al. 2003). Therefore, body weight and food intake were monitored in sham-operated and adrenalectomized (ADX) groups upon arrival in our animal care facility and until the end of the experimental period of 3 weeks. No significant changes in body weight were observed among the lean controls with the exception that the ADX group had a slightly lower body weight upon arrival in our animal care facility (Fig. 2A). This was likely due to the combined stresses of surgery and shipping as body weight in this group was comparable with sham-operated controls after 3 days. On the other hand, significant differences in body weight were observed among ob/ob groups (Fig. 2B). ADX significantly reduced body weight among ob/ob mice and ADX+γ2-MSH supplementation decreased body weight significantly below ADX alone.
As with body weight measurements, neither ADX nor ADX+γ2-MSH affected food intake among lean controls (Fig. 3). Conversely, ADX significantly reduced food intake in obese animals. The administration of γ2-MSH to ADX ob/ob mice also reduced food intake to a level significantly below ADX alone. In fact, food intake among ADX+γ2-MSH-treated ob/ob mice was not significantly different from food intake of lean control mice (Fig. 3).
ADX and ADX+γ2-MSH increase longitudinal growth in both lean and obese (ob/ob) mice
As an index of longitudinal growth, the changes in naso-anal length and tibial length from basal measurements were analyzed (Fig. 4). ADX increased Δtibial length in both ob/ob and lean animals compared with sham-operated controls, yet among lean mice this effect was not quite significant (Fig. 4). However, the administration of γ2-MSH to ADX lean mice significantly increased tibial length compared with SHAM-operated controls. ΔNaso-anal length in the ADX and ADX+γ2-MSH lean groups were also significantly greater than the SHAM-operated controls. Administration of γ2-MSH to the ADX lean animals did not significantly increase Δtibial length or Δnaso-anal length above ADX alone. In the ob/ob groups, however, γ2-MSH administration to the ADX animals significantly increased Δnaso-anal length above ADX alone. Two-way ANOVA with treatment and genotype as the main factors show no interaction effect on either Δnaso-anal length or Δfinal tibial length. However, both treatment (F(2,51) = 13.92, P<0.001) and genotype (F(1,51) = 56.17, P = 0.0001) significantly affected Δnaso-anal length. Treatment (F(2,51) = 16.89, P<0.0001) and genotype (F(1,51) = 27.34, P<0.0001) were also significant factors in Δtibial length.
Total and individual zone measurements of the growth plate reveal differential responses to treatment among ob/ob and lean control mice
To gain insight into how melanocortin peptides may increase linear growth, the effects of ADX and ADX+γ2-MSH on the zones of the growth plate were examined (Table 2, Fig. 5). These data illustrate differential effects of ADX and ADX+γ2-MSH administration on growth plate chondrocytes of lean and obese mice. A significant interaction effect of treatment and genotype was found for total growth plate height (F(2108) = 10.93, P<0.0001), proliferating zone height (F(2108) = 6.67, P = 0.0019), and hypertrophic zone height (F(2108) = 19.23, P<0.0001). Post hoc analyses point to differences in the response to treatment between genotypes as one source of the interaction effect. Significant increases in total growth plate height and proliferating and hypertrophic zone heights were observed between γ2-MSH-treated ADX ob/ob mice and ADX ob/ob mice. These significant increases were not observed among ADX+γ2-MSH-treated compared with ADX lean mice. Interestingly, no significant interaction effect was found for stem cell zone height (F(2108) = 1.01, P = 0.3672), and treatment (F(2108) = 26.38, P<0.0001) but not genotype had a significant main effect on the height of this zone. Therefore, the stem cell zone of the tibial growth plate experienced a similar increase in height in lean and ob/ob mice.
ADX and ADX +γ2-MSH increase the proliferative index of the growth plate in both lean and obese (ob/ob) mice
In vitro ACTH increases the proliferation of chondrocyte precursors and increases their differentiation toward the chondrogenic phenotype (Evans et al. 2004). To confirm an in vivo effect of elevated melanocortin on proliferation of growth plate cells, both treated and untreated lean and obese mice were injected with 50 mg/kg BrdU 24 h prior to euthanasia and DNA-incorporated BrdU was detected using immunohistochemistry. In the stem cell and proliferating cell zones, the percentage of BrdU positive cells (proliferative index) was significantly increased by ADX in lean mice (Fig. 6A and B). Treatment with γ2-MSH significantly increased the proliferative index above ADX alone in the proliferating zone (Fig. 6B) but not in the stem cell zone of lean animals (Fig. 6A). In obese mice, ADX showed a trend toward increasing the proliferative index of the stem and proliferating cell zones but this was not significant. However, the proliferative index of both stem and proliferating zones of γ2-MSH-treated ADX ob/ob mice was significantly increased above SHAM (Fig. 6A and B).
Plasma measurements confirmed leptin deficiency and completeness of ADX
Plasma was collected from each animal at the time of euthanasia and circulating levels of leptin, ACTH, and corticosterone were measured via RIA. Leptin levels of ob/ob animals were at undetectable levels (data not shown) confirming the ob/ob genotype. Corticosterone and ACTH levels in the ADX groups reflect the absence of adrenal tissue and function (Fig. 7). However, plasma levels of ACTH and corticosterone in sham-operated groups were elevated compared with published basal values (Maor et al. 2002), which indicate that sample collection occurred post stress induction. Interestingly, a significant interaction effect of treatment and genotype on plasma ACTH level was found (F(2,40) = 7.86, P = 0.0013). This effect can be attributed to a significantly lower ACTH level found in the lean ADX+γ2-MSH group. No interaction effect between treatment× genotype was found with plasma corticosterone and treatment was responsible for the main effect on this variable (F(2,51) = 65.56, P<0.0001) with no effect of genotype (F(1,51) = 0.74, P = 0.3928).
Buffett & Wyman (1954) were the first to report adrenalectomy-induced increases in the width of the rat epiphyseal growth plate. Since then others have documented increases in longitudinal growth after ADX and have attributed these changes to the removal of glucocorticoid (Li et al. 1996a,b). Here, we introduce the possibility that melanocortin signaling, i.e. ACTH, at the level of the growth plate contributes to ADX-induced increases in linear growth. In this study, ADX increased both the proliferative index and the hypertrophic zone height of the femoral growth plate demonstrating that the removal of glucocorticoid along with elevated ACTH exposure increases the overall turnover of the growth plate leading to an overall increase in longitudinal growth. In vitro, ACTH increases the proliferation of chondrocyte precursors and increases their differentiation toward the chondrogenic phenotype (Evans et al. 2004) suggesting that ACTH in vivo acts on the tibial stem cell zone in a similar manner. Consistent with this is the fact that stem cell zone height is increased in ADX ob/ob and lean mice, and in lean mice the proliferative index of both the stem and the proliferating growth plate zones are significantly increased by ADX. In vitro findings also show that elevated exposure to ACTH increases hypertrophic differentiation among chondrocytes likely through an increase in intra-cellular calcium (Evans et al. 2004, 2005). These data are consistent with in vivo findings where the hypertrophic zone of both the ADX ob/ob and the lean mice are significantly greater than the sham-operated controls pointing toward a direct action of ACTH at the growth plate. Further studies are necessary to definitively determine if ACTH does indeed contribute to ADX-induced increases in growth plate height.
However, several factors suggest that melanocortin stimulation at the level of the growth plate can lead to changes in longitudinal growth. First, γ2-MSH administration to ADX obese mice increased naso-anal length above ADX alone; secondly, γ2-MSH administration to both lean and obese mice enhanced tibial growth above ADX alone, and lastly, γ2-MSH administration increased growth plate proliferative indices in ADX lean and ob/ob mice. All the five melanocortin receptors are expressed in the mouse growth plate (Zhong et al. 2005); however because γ2-MSH is an MC3-R-specific agonist, our data implicate the MC3-R as a receptor involved in the regulation of endochondral growth. This is supported by both in vivo and in vitro findings. MC3-R-deficient mice experience decreased linear growth. Chen et al.(2000) have reported that the average femoral length in female MC3-R-deficient mice was significantly shorter than wild-type littermates suggesting growth is stunted in the absence of MC3-R. Additionally, the MC3-R is highly expressed by both chondrocytes and chondrocyte precursors (Evans et al. 2004). Moreover, the MC3-R-/MC4-R-specific inhibitor, SHU9119, attenuates ACTH-evoked transient elevations in intracellular calcium in resting chondrocytes and the RCJ3.1C5.18 chondrogenic cell line (Evans et al. 2005).
However, studies of the cardiovascular system and of brain indicate the possible existence of an undefined γ-MSH-responsive MC-R. γ2-MSH, when centrally administered, evokes cardiovascular responses even in the presence of the SHU9119 antagonist (Li et al. 1996a,b). SHU9119 is also an antagonist of the other two melanocortin receptors expressed in brain. This suggests that γ2-MSH can act through a melanocortin receptor other than the five receptors that have been described. In vitro data support the existence of another yet to be described γ-MSH-responsive melanocortin receptor. The pituitary cell line, GH3, responds to γ-MSH with transient elevations in calcium and a high dose of SHU9119 does not block this response (Langouche et al. 2001). These data introduce the possibility that this undefined MC-R is expressed in the growth plate and that γ2-MSH exerts its effects through it.
Regardless of the receptor through which γ2-MSH exerts its effects, it likely augments adrenalectomy effects on growth through a mechanism involving an elevation in intracellular calcium. All the MC-Rs, including the putative new γ-MSH-responsive MC-R, are known to evoke transient elevations in intracellular calcium or calcium influx upon activation (Langouche et al. 2001, Mountjoy et al. 2001, Al-Majed et al. 2004). In the case of chondrocytes, ACTH stimulation of the MC3-R also leads to increases in basal intracellular calcium (Evans et al. 2005). Chondrocyte hypertrophic differentiation is accompanied by an incremental increase in basal intracellular calcium and increased hypertrophy due to the activation of MC-R among growth plates cells would translate to increases in linear growth. The possibility that γ2-MSH augments the response to adrenalectomy through increasing hypertrophic differentiation is supported by the fact that hypertrophic zone height is increased in γ2-MSH-treated ADX ob/ob mice when compared with ADX ob/ob mice.
The changes in linear growth parameters of ADX and ADX+γ2-MSH-treated ob/ob mice occurred in the context of significantly decreased food intake and body weight. The effect of ADX on food intake and body weight gain in ob/ob mice has been previously documented (Makimura et al. 2000); however, the significant reduction in these parameters with ADX and concomitant γ2-MSH treatment has not been reported. It is not clear why ADX and γ2-MSH treatment did not also produce body weight changes in lean mice. Our results however, are consistent with previous findings where ADX of lean mice did not reduce body weight or food intake (Saito & Bray 1984).
As mentioned, the ob/ob mouse model has been reported to be 5–10% shorter than its lean littermates (Langouche et al. 2001) and our data confirm this finding (Table 1). A role for leptin in longitudinal growth has recently been defined (Gat-Yablonski et al. 2004). Therefore, the possibility exists that the absence of leptin is responsible for the growth deficiency of the ob/ob mouse. In fact, researchers have shown that administration of peripheral leptin ameliorates decreased tibial length in ob/ob mice (Steppan et al. 2000). On the other hand, glucocorticoid levels are elevated in ob/ob mice (Sainsbury et al. 2002) and adrenal steroids are known to inhibit endochondral growth (Chrysis et al. 2003, Smink et al. 2003). These data create the possibility that linear growth in ob/ob mice is inhibited through mechanisms related to elevated glucocorticoid. Our present data support this latter possibility. Removal of glucocorticoid by ADX enhanced tibial growth in ob/ob mice independently of leptin. Previous findings, where endogenous leptin treatment improved tibial growth in ob/ob mice may have been due to the fact that leptin treatment normalizes circulating levels of glucocorticoid (Halaas et al. 1995, Pelleymounter et al. 1995).
Contrary to what was expected, at the end of the study period the SHAM-operated lean mice and the SHAM ob/ob mice had similar naso-anal lengths. Stress among lean controls could have contributed to this discrepancy. This is supported by the lack of weight gain by this group during the last 10 days of the study. Another possibility is that hyperphagia among the ob/ob mice led to a more rapid increase in naso-anal length between 5 and 8 weeks. The precise role of the melanocortin system is yet to be determined but it is tempting to speculate that it is involved in relaying signals of nutritional status at the level of the growth plate. Expression of the precursor to the melanocortin peptides, POMC (Solomon 1999), in the hypothalamus is highly regulated by food intake, i.e. expression increases with increased food intake and decreases in response to caloric restriction (Ziotopoulou et al. 2000, Bi et al. 2003). An increase in POMC expression in the ob/ob mice as a result of increased food intake could lead to increased circulating melanocortin peptide and signal an increased growth rate at the level of the growth plate. This along with increased stress or poor health among the controls could explain the discrepancy.
Greater changes in naso-anal length were also observed among γ2-MSH-treated ADX ob/ob mice and greater changes in tibial length were observed in ADX and γ2-MSH-treated ADX ob/ob mice compared with lean controls. In vitro data have shown that ACTH-evoked [Ca2+]i transients and increases in basal calcium are greater among populations of chondrocytes that have been primed with the glucocorticoid dexamethasone (Evans et al. 2005). As stated above, ob/ob mice have increased circulating levels of glucocorticoid (Sainsbury et al. 2002). Therefore, the possibility exists that because growth plate chondrocytes of ob/ob mice have been exposed to increased levels of glucocorticoid prior to exposure to elevated ACTH they are more responsive to its effects. More data are necessary to confirm this hypothesis.
Both ACTH and corticosterone levels in sham-operated lean mice were elevated compared with published basal values (Millington et al. 2001), which indicates that sample collection occurred post stress induction. The restraint required for injection of the ketamine/xylazine anesthesia combined with the stress of the injection itself is the likely cause of the elevated ACTH and corticosterone values. ADX will induce hypersecretion of ACTH and stress to ADX animals will increase ACTH levels further (Mukherjee et al. 2004). In this study, ADX mice experienced significant increases in ACTH; however, in the γ2-MSH-treated lean control mice, these values were significantly less than ADX alone. This may be explained by melanocortin effects on the hypothalamic-pituitary-adrenal axis (HPA). Central γ-MSH administration can inhibit IL-1β activation of the HPA (Cragnolini et al. 2004). In addition, the MC3-R, the hypothalamic target of γ2-MSH, may act as an autoinhibitory receptor on POMC neurons (Cone et al. 2001). Therefore, it is possible that γ2-MSH administration reduced the stress response to restraint. Although γ2-MSH was administered peripherally, it likely reached its target receptor among POMC hypothalamic neurons. POMC neurons are located in the arcuate nucleus of the hypothalamus, which functions as a circumventricular organ, existing functionally outside the blood-brain barrier (Cone et al. 2001). This is supported by the fact that γ2-MSH treatment reduced food intake in ob/ob mice. Central administration of γ2-MSH is known to reduce food intake in fasted rats (Millington et al. 2001). The reduction in plasma ACTH level in response to stress by γ2-MSH was not observed among ob/ob mice likely reflecting a functional dependence on leptin signaling.
While plasma ACTH levels among sham-operated ob/ob mice reflect a response to stress, corticosterone levels in these mice are not significantly elevated above published basal values for these mice (Sainsbury et al. 2002). In ob/ob mice, a smaller fractional increase in restraint-induced cortisol increase is observed (Rowland & Dunn 1995) along with an increased clearance of 3H-corticosterone from the plasma (Naeser 1975). These two factors may have contributed to the reduced cortisol response to stress observed in the ob/ob mice of this study. These findings are similar to those in obese humans where elevated plasma ACTH is associated with a paradoxically low plasma cortisol in response to hydrocortisone injection (Jessop et al. 2001) and where an increased metabolic clearance of cortisol is observed (Dunklemann et al. 1964).
In summary, clinical data suggest a link between the melanocortin system and linear growth. This and other studies performed in rodents show that ADX, which removes glucocorticoid and elevates ACTH, results in increased longitudinal growth. Our in vitro data taken together with the present in vivo data support a role for the melanocortin, ACTH, in ADX-induced changes in growth plate height (Evans et al. 2004, 2005). Additionally, γ2-MSH treatment of ADX ob/ob and lean mice resulted in increases in tibial length above ADX alone, suggesting that the melanocortin system plays a role in endochondral ossification. However, the precise role of the melanocortin system in longitudinal growth is yet to be determined.
This work was supported by research grant R03-HD044692 from the National Institutes of Health. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
Somatic and linear growth parameters in 5-week-old (basal) lean and obese (ob/ob) mice. Data are presented as means ± s.d. with n = 10 animals.
|For growth plate measurements, two sections were measured per animal. For hypertrophic cell number, four random fields were analyzed per section. Statistical significance between groups was determined using Student’s unpaired t-test, *P < 0.001.|
|Body weight (g)||17.42 ± 0.62||31.13 ± 2.4*|
|Naso-anal length (mm)||85.62 ± 1.29||82.07 ± 0.80*|
|Tibial length (mm)||16.34 ± 0.283||15.32 ± 0.175*|
|Total growth plate height (μm)||165.72 ± 16.61||148.04 ± 11.62*|
|Stem zone height (μm)||24.76 ± 6.19||21.63 ± 4.68|
|Proliferating zone height (μm)||71.01 ± 10.60||73.43 ± 9.36|
|Hypertrophic zone height (μm)||69.19 ± 10.72||53.21 ± 7.59*|
|Hypertrophic cells (no. per field)||55.14 ± 8.39||42.08 ± 7.58*|
Growth plate measurements in adrenalectomized (ADX) and ADX + γ2-MSH-treated lean and obese (ob/ob) mice. Data presented in μm and as means ± s.d., two sections were analyzed per group with and n = 9–10 animals.
|Results of two-way ANOVA are presented in Results. Post hoc analysis with the Bonferroni correction was used to determine differences between individual groups. aSignificantly different from Sham of same genotype (P < 0.05); bsignificantly different from ADX of same genotype (P < 0.05); csignificantly different from lean counterpart (P < 0.05).|
|Total growth plate height||96.2 ± 14.1||129.1 ± 13.6a||118.1 ± 14.0a||99.8 ± 11.8||134.9 ± 17.5a||150.7 ± 18.1a,b,c|
|Stem zone height||12.9 ± 4.6||21.0 ± 5.2a||19.6 ± 5.9a||13.6 ± 4.2||22.3 ± 6.3a||23.9 ± 7.8a|
|Proliferating zone height||58.8 ± 8.3||70.4 ± 6.6a||64.4 ± 6.8||69.1 ± 7.5c||75.2 ± 7.2||82.4 ± 10.3a,b,c|
|Hypertrophic zone height||24.7 ± 6.9||37.9 ± 5.4a||34.1 ± 6.1a||17.0 ± 4.4c||37.4 ± 7.9a||44.4 ± 7.0a,b,c|
Al-MajedHT Jones PM Persaud SJ Sugden D Huang GC Amiel S & Whitehouse BJ 2004 ACTH stimulates insulin secretion from MIN6 cells and primary mouse and human islets of Langerhans. Journal of Endocrinology180155–166.
BiS Robinson BM & Moran TH 2003 Peptides that regulate food intake: acute food deprivation and chronic food restriction differentially affect hypothalamic NPY mRNA expression. American Journal of Physiology. Regulatory Integrative and Comparative Physiology285R1030–R1036.
BrochmanEJ Duarte ME Zaidi HA & Murray SS 2003 Effects of dietary restriction on total body femoral and vertebral bone in SENCAR C57BL/6 and DBA/2 mice. Metabolism521265–1273.
BuffettRF & Wyman L 1954 Changes in width of uncalcified tibial epiphyseal cartilage of the rat following adrenalectomy and gonadectomy. American Journal of Physiology18016–20.
ChenAS Marsh DJ Trumbauer ME Frazier EG Guan X-M Yu H Rosenblum CI Vongs A Feng Y Cao L et al.2000 Inactivation of the mouse melanocortin-3 receptor results in increased fat mass and reduced lean body mass. Nature Genetics2697–102.
ChrysisD Ritzen EM & Savendahl L 2003 Growth retardation induced by dexamethasone is associated with increased apoptosis of the growth plate chondrocytes. Journal of Endocrinology176331–337.
ConeRD2000 The melanocortin-4 receptor. In The Melanocortin Receptors pp 405–445. Ed. RD Cone. Humana Press: Totowa New Jersey USA.
ConeRD Cowley MA Butler AA Fan W Marks DL & Low MJ 2001 The arcuate nucleus as a diverse conduit for diverse signals relevant to energy homeostasis. International Journal of Obesity and Related Metabolic Disorders25563–567.
CragnoliniAB Perello M Schioth HB & Scimonelli TN 2004 Alpha-MSH and gamma-MSH inhibit IL-1beta induced activation of the hypothalamic–pituitary–adrenal axis through central melanocortin receptors. Regulatory Peptides122185–190.
EliasL Huebner A Metherell L Canas A Warne G Manca Bittis M Cianferani S Clayton P Savage M & Clark A 2000 Tall stature in familial glucocorticoid deficiency. Clinical Endocrinology53423–430.
ErbenRG1997 Embedding of bone samples in methylmethacrylate: an improved method suitable for bone histomorphometry histochemistry and immunohistochemistry. Journal of Histochemistry and Cytochemistry45307–313.
EvansJF Niu Q-T Canas JA Shen C-L Aloia JF & Yeh JK 2004 ACTH enhances chondrogenesis in multipotential progenitor cells and matrix production in chondrocytes. Bone3596–107.
EvansJF Shen C-L Pollack S Aloia JF & Yeh JK 2005 Adrenocorticotropin evokes transient elevations in intracellular free calcium ([Ca2+]i) and increases basal [Ca2+]i through a phospholipase C-dependent mechanism. Endocrinology1463123–3132.
Gat-YablonskiG Ben-Ari T Shtaif B Poteivsky O Moran O Eshet R & Maor G 2004 Leptin reverses the inhibitory effect of caloric restriction on longitudinal growth. Endocrinology145343–350.
HalaasJL Gajiwala KS Mafei M Cohen SL Chait BT Rabinowitz D Lallone RL Burley SK & Friedman JM 1995 Weight-reducing effects of the plasma protein encoded by the obese gene. Science269543–546.
IglewiczB & Hoaglin DC 1993 How to detect and handle outliers ASQ Quality Press: Milwaukee WI USA.
JessopDS Dallman MF Fleming D & Lightman SL 2001 Resistance to glucocorticoid feedback in obesity. Journal of Clinical Endocrinology and Metabolism864109–4114.
KobayashiT Chung U-I Schipani E Starbuck M Karsenty G Katagiri T Goad DL Lanske B & Kronenberg HM 2002 PTHrP and Indian hedgehog control differentiation of growth plate chondrocytes at multiple steps. Development1292977–2986.
LangoucheL Roudbaraki M Pals K & Denef C 2001 Stimulation of intracellular free calcium in GH3 cells by γ3-melanocyte-stimulating hormone. Involvement of a novel melanocortin receptor. Endocrinology142257–266.
LiM Shen Y Halloran BP Baumann BD Miller K & Wronski TJ 1996a Skeletal response to corticosteroid deficiency and excess in growing male rats. Bone1981–88.
LiS-J Varga K Archer P Hruby V Sharma SD Kesterson RA Cone RD & Kunos G 1996b Melanocortin antagonists define two distinct pathways of cardiovascular control by α- and γ-melanocyte-stimulating hormones. Journal of Neuroscience165182–5188.
MakimuraH Mizuno TM Roberts J Silverstein J Beasley J & Mobbs CV 2000 Adrenalectomy reverses obese phenotype and restores melanocortin tone in leptin-deficient ob/ob mice. Diabetes491917–1923.
MaorG Rochwerger M Segev Y & Phillip M 2002 Leptin acts as a growth factor on the chondrocytes of skeletal growth centers. Journal of Bone and Mineral Research171034–1043.
MillingtonGW Tung YC Hewson AK O’Rahilly S & Dickson SL 2001 Differential effects of alpha- beta- and gamma(2)-melanocyte-stimulating hormones on hypothalamic neuronal activation and feeding in the fasted rat. Neuroscience108437–445.
MountjoyKG Kong PL Taylor JA Willard DH & Wilkison WO 2001 Melanocortin receptor-mediated mobilization of intracellular free calcium in HEK293 cells. Physiological Genomics711–19.
MukherjeeK Knisely A & Jacobson L 2004 Partial glucocorticoid agonist-like effects of imipramine on hypothalamic–pituitary–adrenocortical activity thymus weight and hippocampal glucocorticoid receptors in male C57BL/6 mice. Endocrinology1454185–4191.
NaeserP1975 Disappearance of 3h-corticosterone from the serum of obese-hyperglycemic mice (gene symbol ob). Acta Physiologica Scandinavica9310–14.
NiX-P Pearce D Butler AA Cone RD & Humphreys MH 2003 Genetic disruption of γ-melanocyte-stimulating hormone signaling leads to salt-sensitive hypertension in the mouse. Journal of Clinical Investigation1111251–1258.
ParfittA Drezner M Glorieux F Kanis J Malluche H Meunier P Ott S & Recker R 1987 Bone histomorphometry: standardization of the nomenclature symbols and units. Journal of Bone and Mineral Research2595–610.
PelleymounterMA Cullen MJ Baker MB Hecht R Winters D Boone T & Collins F 1995 Effects of the obese gene product on body weight regulation in ob/ob mice. Science269540–543.
RowlandNE & Dunn AJ 1995 Effect of dexfenfluramine on metabolic and neurochemical measures in restraint-stressed ob/ob mice. Physiology and Behavior58749–754.
SainsburyA Schwarzer C Couzens M Jenkins A Oakes SR Ormandy CJ & Herzog H 2002 Y4 receptor knockout rescues fertility in ob/ob mice. Genes and Development161077–1088.
SminkJJ Gresnigt MG Hamers N Koedam JA Berger R & Van Buul-Offers SC 2003 Short-term glucocorticoid treatment of prepubertal mice decreases growth and IGF-I expression in the growth plate. Journal of Endocrinology177381–388.
SteppanCM Crawfordd DT Chidsey-Frink KL Ke H & Swick AG 2000 Leptin is a potent stimulator of bone growth in ob/ob mice. Regulatory Peptides9273–78.
ZhongQ Sridhar S Ruan L Ding KH Xie D Insogna K Kang B Xu J Bollag RJ & Isales CM 2005 Multiple melanocortin receptors are expressed in bone cells. Bone36820–831.
ZiotopoulouM Mantzoros CS Hileman SM & Flier JS 2000 Differential expression of hypothalamic neuropeptides in the early phase of diet-induced obesity in mice. American Journal of Physiology. Endocrinology and Metabolism279E838–E845.
ZuscikMJ D’Souza M Ionescu AM Gunter KK Gunter TE O’Keefe RJ Schwarz EM Puzas JE & Rosier RN 2002 Growth plate chondrocyte maturation is regulated by basal intracellular calcium. Experimental Cell Research276310–319.