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School of Biosciences, Cardiff University, Cardiff, United Kingdom CF10 3US; Medical Research Council Bone Research Group, University of Cambridge, Department of Medicine, Addenbrookes Hospital (N.L.), Cambridge, United Kingdom CB2 2QQ; Department of Neuroendocrinology, Division of Neuroscience and Psychological Medicine, Imperial College London, Hammersmith Hospital (P.A.H.), London, United Kingdom W12 0NN; and Division of Molecular Neuroendocrinology, National Institute for Medical Research (I.C.A.F.R.), London, United Kingdom NW7 1AA
Address all correspondence and requests for reprints to: Dr. Timothy Wells, School of Biosciences, Cardiff University, P.O. Box 911, Museum Avenue, Cardiff, United Kingdom CF10 3US. E-mail: wellst{at}cardiff.ac.uk.
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Introduction |
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Despite the lipolytic action of increased GH release (11, 12), the overall in vivo effect of ghrelin is adipogenic (2). Earlier studies with GHSs, a group of synthetic ghrelin mimetics, demonstrated that short-term, continuous GHS-R activation markedly augments the secretion of GH (13, 14). However, the magnitude of this effect wanes rapidly, and in some circumstances, prolonged continuous GHS infusion can suppress GH secretion (15). In contrast, prolonged intermittent GHS-R activation continues to augment GH secretion (15) and accelerate skeletal growth (16). Thus, it is possible that the net effect of ghrelin on adipogenesis may depend upon the pattern of exposure.
We have now investigated the mechanism of ghrelin-induced adipogenesis in vivo in the following series of experiments. To determine whether the adipogenic actions of ghrelin are pattern dependent, equivalent doses of ghrelin were infused iv in either continuous or intermittent patterns. This experiment was performed in transgenic growth-retarded (Tgr) rats (17), a model of moderate GH-deficient dwarfism, whose skeletal growth can be accelerated by GHS-R activation (15, 16, 18) and in which we have previously demonstrated the pattern-dependent effects of ghrelin on the GH axis (15). In a second study, hypophysectomized (HX) rats were infused with the GHS, GH-releasing peptide-6 (GHRP-6), to investigate whether GHS-induced adipogenesis is mediated by the pituitary hormones. In this study an intermittent infusion was used to examine whether the adipogenic effect is pattern dependent per se. Finally, we sought evidence for a direct adipogenic action of ghrelin, des-octanoyl ghrelin, or L-163,255 [a potent spiropiperidine GHS-R1a agonist (19, 20)], by direct unilateral infusions into the tibial bone marrow cavity. This represents a novel treatment strategy, exploiting an isolated adipose depot in which adipocyte size and number can be readily quantified, the contralateral tibia serving as an excellent control for potential systemic overflow effects. In addition, to circumvent the potential interference of GH on marrow fat (21) that might result from systemic overflow, these experiments were performed in the profoundly GH-deficient dwarf (dw/dw) rat (22) in which the GH response to GHS-R activation is low (23), but the adipogenic response to ghrelin is maintained (2). A preliminary report of part of this work has previously been communicated (24).
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Materials and Methods |
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Study 1: patterned iv infusion of ghrelin in Tgr rats
Male Tgr rats (14 wk old; weighing 216–242 g) were placed in metabolic cages for 4 d before the implantation of a single-bore iv cannula into the right jugular vein under halothane anesthesia. Animals were permitted at least 48 h of recovery, during which time body weight and food intake were monitored daily. After this period, an automated infusion system was used to deliver an iv infusion of either vehicle [sterile saline containing BSA (1 mg/ml) and heparin (10 U/ml)], given either continuously (at 100 µl/h) or intermittently [300 µl (2 min) pulses every 3 h], or vehicle containing rat ghrelin, given either continuously (80 µg/d) or in 10-µg (300-µl) pulses every 3 h, for 7 d. Body weight and food intake were monitored daily throughout the 7-d infusion period; one animal showing erratic food intake/weight loss was excluded from further analysis.
At the end of the infusion period the rats were reanesthetized and killed by cervical dislocation. A terminal blood sample was removed by cardiac puncture, and separated plasma was stored at -20 C before determination of the plasma leptin concentration. The following tissues were dissected and weighed: liver, kidney, spleen, left visceral (retroperitoneal and perirenal), and epididymal fat pads. In addition, right tibiae were excised for subsequent determination of bone marrow adiposity, and samples of abdominal skin were collected for measurement of the thickness of the sc fat depot. Brains were dissected and snap-frozen in isopentane at -20 C for subsequent determination of hypothalamic neuropeptide Y (NPY) mRNA expression.
Study 2: pulsatile iv infusion of GHRP-6 in HX rats
Male CD rats were HX at 7 wk of age [frontal route under ketamine (90 mg/kg)/xylazine (5 mg/kg)/atimpamazole (1 mg/kg) anesthesia and carprofen analgesia (5 mg/kg); Charles River, Margate, UK], and body weights were monitored for 2 wk. Rats showing a rapid acceleration in body weight during this period were excluded from further study. Together with a group of age-matched intact CD males, HX rats were housed singly in metabolic cages for 4 d before the implantation of iv jugular cannulae (as described above). Body weight and food intake were monitored daily throughout the postoperative period. After at least 48 h recovery, HX rats received a pulsatile iv infusion of vehicle (as described above; n = 4) or vehicle containing GHRP-6 (10-µg pulses; n = 5) for 7 d. We have previously shown that equimolar doses of GHRP-6 and rat ghrelin have similar growth-promoting effects (15, 16). A cohort of intact CD rats also received a pulsatile iv infusion of vehicle containing GHRP-6 (10-µg pulses; n = 6). At the end of infusion rats were reanesthetized and killed by decapitation, and a sample of trunk blood was collected for subsequent determination of the plasma leptin concentration. Left visceral (retroperitoneal and perirenal) and epididymal fat pads were dissected and weighed. Right tibiae were excised for subsequent determination of epiphyseal plate width (EPW) and bone marrow adiposity. The pituitary fossae were examined to ensure the absence of pituitary remnants.
Study 3: infusion of rat ghrelin into tibial bone marrow in dw/dw rats
Male dw/dw rats (13 wk old; weighing 147–175 g) were anesthetized with halothane and prepared with stainless steel cannulas (0.8-mm diameter) introduced into the marrow cavity of the right tibia through the anterior middiaphyseal wall. The cannulas were secured to the tibial surface with cyanoacrylate cement and connected via a polythene cannula to an osmotic minipump (Alzet model 2001, Alza Corp., Palo Alto, CA) implanted sc in a dorsal location. Minipumps delivered vehicle alone [sterile saline containing BSA (1 mg/ml) and heparin (5 U/ml) at 1 µl/h] or vehicle containing rat ghrelin (720 ng/d) for 7 d. Rats were checked daily, and body weight was monitored. At the end of the infusion period, animals were killed by cervical dislocation and decapitation. Right and left tibiae were dissected for subsequent determination of bone marrow adiposity and measurement of EPW.
Study 4: infusion of des-octanoyl-ghrelin and L-163,255 into tibial bone marrow in dw/dw rats
Male dw/dw rats (12–13 wk old; weighing 145–164 g) were prepared with right tibial marrow cavity cannulas and osmotic minipumps as described above. Minipumps delivered vehicle alone (as described above) or vehicle containing rat ghrelin (catalog no. 1465, Tocris Cookson Ltd., Avonmouth, UK; 720 ng/d), des-octanoyl rat ghrelin (catalog no. 031-33, Phoenix Pharmaceuticals, Belmont, CA; 720 ng/d), or L-163,255 (1440 ng/d) for 7 d. Rats were checked daily, and body weight was monitored. At the end of the infusion period, animals were killed by cervical dislocation and decapitation. Right tibiae were dissected for subsequent determination of bone marrow adiposity and measurement of EPW.
Peptides used
The ghrelin used in studies 1 and 3 was donated by Pharmacia & Upjohn (Uppsala, Sweden), and the GHRP-6 used in study 2 was donated by Novo Nordisk A/S (Bagsvaerd, Denmark). The GHS-R 1a-specific agonist, L-163,255, was supplied by Merck & Co. (Rahway, NJ).
Tissue analysis
The plasma leptin concentration was determined by RIA (Linco Research, Inc., St. Charles, MO; intraassay variability, 2.7%; sensitivity, 1 pg/ml). Dissected tibiae were fixed in 10% buffered formal saline for 2 d and decalcified in 10% EDTA (in 0.3 M NaOH) for 2–3 wk. After embedding in paraffin wax, 8-µm longitudinal/anterior-posterior sections were taken, and alternate sections were stained with Masson’s Trichrome (25) or toluidine blue. EPW was measured on Masson’s Trichrome-stained sections under light microscopy with an ocular graticule. Bone marrow adiposity was measured on toluidine blue-stained sections following the method described by Gevers et al. (21). Briefly, digital images (1 x 186,304 µm2 field/section; 3 sections/tibia) were taken of middiaphyseal marrow. In infused tibia, these images represent marrow midway between the infusion site and the primary spongiosa. Images were analyzed using the public domain NIH Image program (version 1.62 for Macintosh, developed at NIH and available on the internet at http://rsb.info.nih.gov/nih-image/) to quantify adipocyte density (cells per field), adipocyte size, and degree of adiposity (total adipocyte area as percentage of field).
Measurement of hypothalamic gene expression
Coronal brain sections (12 µm) cut through the periventricular and arcuate nuclei of the hypothalamus were taken at -16 C, thaw-mounted onto gelatin and chrome alum-coated slides, and stored at -70 C for subsequent in situ hybridization. [35S]UTP (NEN Life Science Products, Stevenage, UK) was used to synthesize radiolabeled probes using an SP6/T7 transcription kit (Roche, Lewes, UK) and was purified on Sephadex G-50 columns (Amersham Pharmacia Biotech, Uppsala, Sweden). The NPY cDNA was a 370-bp fragment corresponding to nucleotides 79–449. No detectable signals were obtained from a corresponding sense RNA probe that was used as a negative control. Frozen sections were thawed at room temperature, fixed in 4% paraformaldehyde, acetylated, dehydrated through graded ethanol solutions, and then delipidated in chloroform before hybridization, as previously described (26). Sections were hybridized overnight at 45 C. The following day, posthybridization washes were performed, including a ribonuclease digestion. Slides were then air-dried and apposed to autoradiographic film (BioMax MR, Eastman Kodak Co., Rochester, NY) for up to 1 wk. X-ray images were analyzed densitometrically using NIH Image software (http://rsb.info.nih.gov/nih-image/). For each rat, two to four anatomically matched sections (identified by counterstaining with cresyl violet) were analyzed, with data generated in the form of integrated OD. Due to the arbitrary nature of OD units, results are displayed as the percent change compared with the control. All statistical analysis relates to the raw data.
Statistical analysis
All data are expressed as the mean ± SEM, with statistical comparisons performed using t test or one-way ANOVA, with Bonferroni or Dunnett’s post hoc test as appropriate.
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Results |
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), with continuous infusion being less effective than intermittent treatment. Both patterns of ghelin infusion elicited an increase in liver (P < 0.01) and kidney (P < 0.05) weights (data not shown). Although cumulative food intake was elevated by both patterns of ghrelin infusion (Fig. 1B), arcuate NPY mRNA expression was only significantly elevated after intermittent ghrelin infusion (Fig. 1C). Continuous infusion of ghrelin increased visceral adiposity (Fig. 1D), but had no effect on either epididymal fat pad weight or the thickness of sc fat (Fig. 1, E and F). Tibial bone marrow adiposity was more than doubled by continuous ghrelin infusion (Fig. 1G); both cell number and size appeared to contribute to this effect, although neither parameter was significantly different. Intermittent ghrelin infusion had no significant effect on any of the adiposity parameters measured (Fig. 1, D–I). Circulating leptin levels followed the same pattern as visceral and bone marrow adiposity, with the highest levels seen after continuous infusion (Fig. 1J).
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) accompanied by a small increase in EPW (Fig. 2B). Both parameters were significantly less than those in GHRP-6-treated, intact CD rats (Fig. 2, A and B). Cumulative food intake was unaffected by GHRP-6 treatment in HX rats (Fig. 2C). Food intake was significantly higher in GHRP-6-treated CD control rats even before the commencement of infusion (Fig. 2C). Visceral fat pad weight was highest in GHRP-6-treated HX rats and lowest in GHRP-6-treated intact rats. This pattern was also observed in bone marrow; the GHRP-6-induced elevation in tibial marrow adiposity (Fig. 2G) was due to an increase in adipocyte number (Fig. 2H), and adipocyte size was unchanged (Fig. 2I). GHRP-6 treatment did not affect epididymal fat pad weight or plasma leptin concentration in HX rats (Fig. 2, E and F).
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and Fig. 4, A and B). This was entirely due to an increase in the number of adipocytes (Fig. 3D and Fig. 4, A and B); mean adipocyte size was unaltered (Fig. 3E and Fig. 4, A and B). The absence of any change in either body weight gain (Fig. 3A) or EPW (Fig. 3B) indicates that residual GH secretion was not elevated, and the absence of any change in marrow adipocytes in the contralateral (untreated) tibiae (Fig. 3, C–E, and Fig. 4C) suggests that there was no significant leakage of ghrelin into the systemic circulation.
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), as described above (Fig. 4
), without affecting either body weight gain or EPW (Fig. 5, A and B). This effect was also observed after treatment with des-octanoyl ghrelin, but was not seen after infusion of L-163,255 (Fig. 5C). Adipocyte number showed the same pattern as percent adiposity (Fig. 5D), but neither adipocyte number nor size were significantly affected (Fig. 5, D and E).
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Discussion |
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Ghrelin is thought to act as a starvation signal (2, 3), promoting the resumption of energy supply (7, 8) and stimulating GH secretion (1). However, we have recently demonstrated that activation of the hypothamalo-pituitary-GH axis by ghrelin and the GHSs is dependent upon the pattern of treatment (15, 16). Intermittent ghrelin infusion, which may mimic the release of ghrelin from hypothalamic neurons (1, 27, 28), augments GH secretion and accelerates skeletal growth (15). In contrast, prolonged continuous ghrelin infusion, which is most likely to mimic gastric secretion in response to fasting (2), suppresses spontaneous GH secretion (15) and is ineffective in accelerating skeletal growth (15, 16). The growth-promoting activity of both GH and GRF is similarly pattern dependent (29, 30).
Ghrelin is also adipogenic (2), and we now show that although ghrelin-induced adipogenesis is pattern dependent, the nature of this pattern dependency is exactly the opposite of that for ghrelin-induced GH secretion.
Intermittent ghrelin infusion did not promote fat deposition in Tgr rats. As GH has potent lipolytic properties (11, 12), it is possible that in the context of a responsive hypothalamo-pituitary-GH axis, the adipogenic action of intermittent ghrelin exposure is masked by the lipolytic effect of enhanced GH secretion. This is supported by our observation that intermittent infusion of the ghrelin mimetic, GHRP-6, is clearly adipogenic in HX rats; similar findings were reported after twice daily GHS injections in lit/lit mice (31). This indicates that the adipogenic action of ghrelin is not pattern dependent per se, but that the pattern dependency is itself dependent upon GH status.
In contrast to intermittent treatment, continuous infusion of ghrelin is clearly adipogenic in the Tgr rat. This could account for the small, but significant, elevation in body weight gain that occurs with this treatment. Given that prolonged continuous GHS-R activation suppresses GH secretion (15), this raises the possibility that the adipogenic action of continuous ghrelin exposure is a consequence of the reduced lipolytic influence of GH. However, this cannot be the complete explanation, as ghrelin and the GHSs clearly promote adipogenesis in the absence of GH (2, 31). We are thus required to consider other, non-GH-dependent mechanisms by which ghrelin may promote adipogenesis.
Firstly, activation of hypothalamic feeding centers by ghrelin (7, 8) may increase substrate availability, thereby giving rise to adipogenesis. However, unlike the pattern-dependent adipogenesis observed in Tgr rats, both patterns of ghrelin treatment induced a positive energy balance, and arcuate NPY mRNA expression was only significantly elevated by the nonadipogenic intermittent infusion. This evidence in conjunction with the differential sensitivity of the fat pads and the more consistent changes in adipocyte number (rather than size) suggests that increased substrate availability does not contribute significantly to the observed adipogenesis. This is also supported by the observation that adipogenesis is stimulated by GHRP-6 in HX rats without the induction of a positive energy balance, possibly indicating an interaction between ghrelin-induced orexigenesis and pituitary status. This aside, although the adipogenic effects of ghrelin do not appear to be mediated by increased substrate availability over the time course of the current study, the orexigenic effects of ghrelin may become significant in the longer term. Suppression of GHS-R expression in transgenic rats reduces food intake and produces a relatively uniform reduction in fat pad weights (32).
Secondly, ghrelin may regulate adiposity via adenohypophysial hormones other than GH. For example, both PRL and ACTH secretion can be elevated by GHS-R activation (23, 33, 34, 35), and both hormones potentially contribute to the regulation of adipocyte differentiation (36, 37, 38, 39). However, the pituitary hormones do not appear to mediate ghrelin-induced fat accumulation, as GHRP-6 promoted adipogenesis in HX rats. This does not exclude the possibility that hormones from peripheral endocrine glands may contribute to the observed ghrelin-induced adipogenesis. Binding sites for the GHSs are present in a range of peripheral tissues, including thyroid (40, 41) and adrenal (41) glands, and both thyroid hormones and glucocorticoids appear to play a role in preadipocyte differentiation (42).
Our data indicate that ghrelin induces adipogenesis by a direct peripheral action. Previous in vitro evidence indicated that ghrelin activates the p42/p44 MAPK pathway in cultured 3T3-L1 preadipocytes (43) and promotes differentiation of preadipocytes in primary culture (44). To investigate the potential direct adipogenic action of ghrelin in vivo, we devised a method to infuse ghrelin into the tibial bone marrow cavity. The fact that body weight gain, epiphyseal plate width, and marrow adiposity in contralateral (untreated) tibiae were unchanged indicates that there was negligible systemic overflow of ghrelin using this approach. Thus, the combination of this novel technique with quantification of adipocyte number and size may have utility as a bioassay for the adipogenic activity of ghrelin and its isoforms.
The doubling of marrow adiposity after the infusion of ghrelin into tibial bone marrow is the first clear demonstration of a direct peripheral adipogenic action of this hormone in vivo. The effectiveness of ghrelin treatment in the dw/dw rat is remarkable, given the 5-fold increase in marrow adipocytes in this strain (21). This model of profound GH deficiency was used to exclude the potential confounding influence of increased GH secretion on adiposity.
The combination of our data with those previously reported in vitro (43, 44) represents a strong argument for a direct action of ghrelin on the adipocyte or preadipocyte, but an action via other cell types in bone marrow cannot be excluded. In the marrow, adipocytes and osteoblasts differentiate from a common mesenchymal precursor (42), but it is unclear at what stage in this lineage the cells are sensitive to ghrelin. Proliferation of mature adipocytes seems unlikely, and therefore, differentiation and maturation of preadipocytes, or even their precursors, represent the most plausible mechanism. Elucidation of the mechanisms underlying the observed adipogenic action of ghrelin may thus predicate a novel approach to manipulating stem cell differentiation, adipocyte proliferation, and the cellular components of bone marrow.
Given this evidence for a direct adipogenic action of ghrelin, we wanted to test the nature of the receptors mediating this effect. Binding sites for the GHS, hexarelin, in adipose tissue have previously been reported (41), but receptor identity remains unclear. A number of receptor mechanisms are possible, the most obvious of which is the GHS-R1a, through which ghrelin itself was first identified (1).
Although initial studies could not detect GHS-R1a mRNA in bone marrow (45), a recent (RT-PCR) study indicates that GHS-R1a mRNA is expressed in rat adipocytes, and that its expression increases with age (44). However, the adipogenic effect seen with acylated ghrelin was not reproduced with the GHS-R1a-specific agonist, L-163,255, suggesting that, at least in bone marrow, the adipogenic action of ghrelin is not mediated by this receptor. This result needs to be interpreted with some caution, as it is possible that the dose of L-163,255 employed was insufficient to generate an observable adipogenesis. We believe this to be unlikely, as the marrow adiposity parameters after L-163,255 treatment were identical to those after vehicle infusion. The possibility that the adipogenic action of ghrelin is not mediated by GHS-R1a was further corroborated by the robust adipogenesis seen with des-octanoyl ghrelin, which does not activate GHS-R1a (9, 10).
Our observation of the adipogenic action of des-octanoyl ghrelin is important in its own right. Although des-acyl ghrelin has been shown to possess a cytoprotective effect in cultured cardiomyocytes (46) and to inhibit cell proliferation in breast carcinoma cell lines (47), we believe that our data represent the first conclusive demonstration of a physiological effect of this form of ghrelin in vivo. As des-octanoyl ghrelin appears to constitute the majority of circulating ghrelin (9), it is possible that during periods of prolonged starvation, acylated ghrelin acts centrally to suppress the lipolytic action of GH (15), whereas both forms of ghrelin can promote adipogenesis. Thus, the ratio of ghrelin to des-octanoyl ghrelin could help to regulate the balance between adipogenesis and lipolysis in response to nutritional status.
In bone marrow, the adipogenic action of ghrelin appears to be mediated by a receptor mechanism other than GHS-R1a. Both ghrelin and the GHS-R are structurally related to motilin (48) and its receptor (49, 50). However, the motilin receptor is also unlikely to mediate ghrelin-induced adipogenesis, because binding of ghrelin to the motilin receptor requires the presence of the octanoyl modification, and ghrelin is unable to mimic the contractile responses of motilin in a smooth muscle bioassay (51).
Ghrelin and the GHSs also interact with elements of the lipid transport system, with ghrelin binding to a species of high density lipoprotein in plasma (52), and hexarelin binding to CD36 (53), a class B monocyte/macrophage scavenger receptor. CD36 appears to be a strong candidate to mediate the observed adipogenesis, because it plays an important role in fatty acid and lipoprotein metabolism (54) and is believed to mediate the increase in coronary perfusion pressure elicited by the GHS, hexarelin (53).
It is unclear whether bone marrow adipocytes are representative of adipocytes in extramedullary sites. In the context of GH treatment, marrow adipocytes appear to behave most like sc adipocytes (21), but this does not appear to be the case after changes in circulating ghrelin. The effect of systemic ghrelin infusion on bone marrow adiposity most closely resembles that seen in visceral fat, with epidydimal and sc depots being unresponsive. These differences in sensitivity may reflect expression levels of appropriate receptors. Indeed, our data do not exclude the possibility that ghrelin-induced adipogenesis in extramedullary fat depots may be mediated by GHS-R1a.
The sensitivity of marrow adipocytes to ghrelin exposure may be significant in the context of starvation, because, unlike other fat depots, marrow fat is not utilized (55), potentially conserving a local energy supply for the maintenance of hematopoiesis. The presence of ghrelin may favor differentiation into the adipocytic, rather than osteoblastic, pathway. As leptin, which is highly expressed in bone marrow adipocytes (56), inhibits osteoblast function and bone formation (57), ghrelin may also act to reduce the energy requirements of bone remodeling during starvation.
In conclusion, we present evidence that the adipogenic action of ghrelin is pattern dependent in the context of significant GH secretion, but is not dependent upon pituitary hormones. The adipogenic properties of acylated ghrelin in bone marrow are shared by des-octanoyl ghrelin, but not by a synthetic GHS-R1a agonist, and thus are likely to be mediated by a receptor other than GHS-R1a. We believe that this may represent the first conclusive demonstration of a physiological role for des-octanoyl ghrelin in vivo.
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Acknowledgments |
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rd, Denmark) for the gift of GHRP-6 for infusion, Phill Blanning (Cardiff University) for PCR identification of Tgr rats, and Derek Scarborough (Cardiff University) for bone histology. |
Footnotes |
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Abbreviations: EPW, Epiphyseal plate width; GHRP-6, GH-releasing peptide-6; GHS-R1a, type 1a GH secretagogue receptor; GRF, GH-releasing factor; HX, hypophysectomized; NPY, neuropeptide Y; Tgr, transgenic growth retarded.
Received July 18, 2003.
Accepted for publication October 2, 2003.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) or rat ghrelin given either continuously (c-ghrelin; 80 µg/d; 
) or in pulses (p-ghrelin; 10 µg pulse every 3 h; 
) on body weight gain (A), daily food intake (B), arcuate NPY mRNA expression (C), visceral (retroperitoneal and perirenal) fat pad weight (D), epididymal fat pad weight (E), thickness of abdominal sc fat (F), tibial marrow adiposity (G), marrow adipocyte number (H), marrow adipocyte size (I), and plasma leptin concentration (J) in male Tgr rats. Values shown are the mean ± SEM (n = 5 for all groups; *, P < 0.05; **, P < 0.01; ***, P < 0.001 (vs. vehicle-treated rats); 
, P < 0.05; 
