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1School of Biosciences, Cardiff University, Cardiff; 2Division of Molecular Neuroendocrinology, National Institute of Medical Research, London, United Kingdom; 3Department of Biomedical Sciences, College of Osteopathic Medicine, Ohio University, Athens, Ohio; 4Department of Child Health, Wales College of Medicine, Cardiff University, Cardiff, United Kingdom; and 5Edison Biotechnology Institute, Ohio University, Athens, Ohio
Submitted 14 July 2006 ; accepted in final form 15 January 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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adipose tissue; bone marrow fat; leptin; dwarfism
The dwarf (dw/dw) rat is a widely used model of profound GH deficiency (8) in which an unknown genetic defect leads to a 95% reduction in pituitary GH and a 90% reduction in the amplitude of the spontaneous episodes of GH secretion (7, 27). Although we have previously reported that tibial bone marrow adiposity is increased three- to fivefold in adult dw/dw females (21), we had observed that circulating leptin was profoundly reduced (unpublished data), indicating that truncal adiposity may not be elevated parallel to that in marrow. Thus, in the present study, we have tested the hypothesis that the profound GH deficiency in dw/dw rats is not accompanied by truncal obesity.
We have investigated the development of visceral and bone marrow adiposity in dw/dw rats and compared the developing adiposity profile with that in the moderately GH-deficient transgenic growth retarded (Tgr) rat (17). In the Tgr model, the expression of human GH (hGH) in the GH-releasing hormone neurons leads to a 70–90% reduction in pituitary GH (13, 17) and a 70% reduction in the amplitude of spontaneous GH pulses (17, 36). To determine whether the differences in adiposity between these two models reflect the degree of GH insufficiency, we have also quantified visceral and bone marrow adiposity in two murine models in which moderate or complete loss of GH signaling arises from the expression of the GH receptor antagonist G119KbGH [in GH antagonist (GHA)-transgenic mice] (9, 25) or disruption in the GHR/BP gene (GHR/BP–/–) (39), respectively.
We have also extended this study to investigate whether the reduction in proportionate fat mass is due to a reduction in energy intake and whether the degree of adiposity in dw/dw rats can be restored by GH treatment. In addition, we have investigated whether the adipose depots in dw/dw rats respond appropriately to two developmental manipulations known to cause truncal obesity, neonatal monosodium glutamate (MSG) treatment and maintenance on a high-fat diet. Preliminary reports of elements of this work have previously been communicated (20, 33).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The animal procedures described conformed to the respective institutional and national ethical guidelines for animal experimentation, including those involving genetically modified animals, and were specifically approved by local ethical review. Homozygous dw/dw rats bred on an Albino Swiss (AS) background, used in studies 3, 5, 6, and 8, were housed in the Division of Biological Services, National Institute of Medical Research (NIMR; London, UK), under conditions of 12:12-h light-dark (lights on at 0600), with food and water available ad libitum. The hemizygous Tgr rats, wild-type (WT; AS) littermates, and homozygous dw/dw rats used in studies 1, 2, and 7 were derived from the original colonies at NIMR and were bred in the Transgenic Unit (School of Biosciences, Cardiff University) under conditions of 14:10-h light-dark (lights on at 0500), with food and water available ad libitum. Tgr rats were identified by PCR analysis of a tail biopsy. Standard chow diet (Cardiff) consisted of 4.0% fat, 14.2% protein, 4.5% fiber, 63.9% carbohydrate, and 4.7% ash (metabolizable energy: 12.99 MJ/kg, Rodent Maintenance Diet 2014; Harlan Teklad, Harlan, UK). Standard chow diet (NIMR) consisted of 3.4% fat, 18.8% protein, 3.7% fiber, 60.3% carbohydrate, and 3.8% ash (metabolizable energy: 15.6 MJ/kg). The high-fat diet used in study 5 was made by mixing normal rat chow (NIMR) with 60% fat containing chow (Special Diet Services, Witham, UK) to give a diet consisting of 41.1% fat, 19.6% protein, 29% carbohydrate, and 2.3% ash (metabolizable energy: 24.0 MJ/kg).
The GHR/BP–/– and WT control littermates, as well as the GHA and nontransgenic (WT) control littermates, have been described previously (11). The genetic backgrounds of the GHR/BP–/– and of the GHA mice were Ola/BalbC and C57BL/6J, respectively. Genotypes were determined by PCR analysis of tail biopsies. Mice were weaned onto a standard rodent chow (14% of calories from fat, 16% from protein, and 60% from carbohydrates, Prolab RMH 3000; PMI Nutrition International, Brentwood, NJ) at 28 days of age and housed in a temperature-controlled room at 22°C with a light-dark cycle of 14:10-h in the mouse facility of Edison Biotechnology Institute at Ohio University (Athens, OH). Food and water were supplied ad libitum.
Study 1: Transpubertal Growth and Adiposity Profiles in GH-Deficient Rats
To consider the effects of GH deficiency on adiposity, 3-, 6-, and 9 wk-old male dw/dw, Tgr, and AS littermates were weighed and anesthetized with halothane prior to decapitation. Trunk blood was collected and centrifuged, and separated plasma was stored at –20°C for subsequent determination of plasma leptin and IGF-I concentrations by radioimmunoassay (RIA) and immunoenzymometric assay (IEA), respectively. Visceral (perirenal and retroperitoneal) and epididymal fat pads were excised and weighed. In addition, the right tibiae were removed for subsequent determination of epiphyseal plate width (EPW) and quantification of bone marrow adiposity.
Study 2: Adiposity Profiles in 1-yr-Old GH-Deficient Rats
To determine whether the lean phenotype observed in dw/dw rats was maintained with age, cohorts of 1-yr-old male AS and Tgr littermates and male dw/dw rats were weighed, concussed, and decapitated. Trunk blood was collected and treated as above for subsequent determination of plasma leptin concentration by RIA, and visceral fat pads were excised and weighed.
Study 3: Adiposity Profiles in Female dw/dw Rats
To determine whether the lean phenotype observed in the male dw/dw rat was sex specific, 6- to 7-wk-old dw/dw female rats along with their respective WT controls were weighed, concussed, and killed by decapitation. Visceral (perirenal and retroperitoneal) and ovarian fat pads were excised postmortem and weighed. Right tibiae were removed for subsequent quantification of bone marrow adiposity.
Study 4: Adiposity Profiles in Murine Models of Profound and Moderate GH Resistance
The role of reduced GH signaling was analyzed in GHR/BP–/– and GHA mice. Ten-week-old male GHR/BP–/– and GHA mice, with their respective WT controls, were weighed and killed by cervical dislocation. Following immediate decapitation, trunk blood was collected from GHR/BP–/– and GHR/BP+/+ mice and centrifuged, and separated plasma was stored at –20°C for subsequent determination of plasma leptin concentration by RIA. Right tibiae were removed for subsequent quantification of bone marrow adiposity. Visceral (perirenal and retroperitoneal) and epididymal fat pads were excised postmortem and weighed.
Study 5: Measurement of Food Intake in dw/dw Rats
To determine whether the lean phenotype observed in peripubertal dw/dw rats was due to a reduction in food intake, 14-wk-old male AS and dw/dw rats were acclimatized to metabolic cages for 1 wk, during which body weight continued to increase. Subsequently, food intake was measured for an additional 6 days.
Study 6: Effect of GH Treatment on Adiposity Profiles in Male dw/dw Rats
To determine whether the lean phenotype observed in the dw/dw rat is reversible by GH treatment, two groups of 12-wk-old dw/dw males [average weight: 149.8 ± 2.7 (control) and 150.4 ± 1.8 g (hGH)] were either sham operated (control) or prepared with subcutaneous osmotic minipumps primed to deliver hGH (200 µg/day for 14 days) under halothane anaesthesia. At the end of the 14-day treatment period these rats, together with a cohort of sham-operated, age-matched AS males (average initial weight: 265.9 ± 8.1 g), were weighed, concussed, and decapitated. Trunk blood was collected and treated as above for subsequent determination of plasma leptin concentration by RIA, and perirenal and epididymal fat pads were excised and weighed.
Study 7: Effect of Neonatal MSG Treatment on Adiposity Profiles in dw/dw Rats
To determine whether the reduced abdominal adiposity in dw/dw rats can be corrected by a stimulus known to induce obesity, we analyzed adiposity in dw/dw rats following neonatal MSG treatment (6). Three litters of dw/dw rats (8–10 pups/litter) received intraperitoneal injections of either vehicle (50 µl of 0.9% sterile saline) or MSG (4 mg/g body wt in 50 µl of vehicle) on postnatal days 2, 4, 6, 8, and 10 (5) and were carefully monitored for potential adverse effects. At 8 wk of age these rats were weighed, concussed, and decapitated. Trunk blood was collected and treated as above for subsequent determination of leptin and IGF-I concentrations by RIA and IEA. In addition, the visceral (retroperitoneal and perirenal) fat pads and liver were dissected and weighed. The left tibiae were excised, with the length measured using a hand-held micrometer, and then processed for subsequent determination of EPW and bone marrow adiposity.
Study 8: Effect of a High-Fat Diet on Adiposity Profiles in dw/dw Rats
To determine whether fat accretion in dw/dw rats may be augmented by pubertal exposure to elevated dietary fat, 5- to 7-wk-old female dw/dw and AS rats (n = 5/group for dw/dw rats, n = 6/group for AS rats) were fed normal chow or a 40% high-fat diet for 4 wk. Rats were weighed weekly. At the end of the treatment period rats were concussed and decapitated. Trunk blood was collected and treated as above for subsequent determination of leptin and IGF-I concentrations by RIA and IEA. Perirenal and ovarian fat pads were excised and weighed, and tibiae were dissected for subsequent analysis of bone marrow adiposity and epiphyseal plate width.
Tissue Analysis
Dissected tibiae were fixed in 10% buffered formal saline for 2 days and decalcified in 10% EDTA (in 0.3 M NaOH) for 3 wk (studies 1, 3, and 5) or fixed in 4% paraformaldehyde for 5 days and decalcified in 20% EDTA for 3 wk (study 4). After being embedded in paraffin wax, 7-µm-longitudinal/anterior-posterior sections were taken and alternate sections stained with Masson's Trichrome (28) or toluidine blue. EPW was measured on Masson's Trichrome-stained sections under light microscopy with an ocular graticule, with three measurements taken per section and three sections measured per bone. Bone marrow analysis was measured on toluidine blue-stained sections following the method previously described by Gevers et al. (21). Briefly, digital images of mid-diaphyseal marrow (1 x 343,313 µm2 field/section, 3 sections/tibia) were taken (with a Leica DFC300FX digital camera mounted on a Leica DMLB microscope) and analyzed with National Institutes of Health (NIH) Image (version 1.62 for Macintosh, available at http://rsb.info.nih.gov/nih-image/) to quantify adipocyte density (cells/field), adipocyte size, and degree of adiposity (total adipocyte area as percentage of field). In study 5, frontal sections of the middle of the proximal half of the tibia were used, and the complete section was reconstructed by scanning and computer montage. Images were then imported into NIH Image to analyze adipocyte size, number, and total adipocyte area.
Plasma Hormone Analysis
The plasma leptin concentrations were determined by RIA (catalog no. RL-83K, intra-assay variability, 4.4% sensitivity, 1 pg/ml; Linco Research, St. Charles, MO), and plasma IGF-I (total) concentrations were determined by IEA (reference no. AC-18F1, intra-assay variability, 9.2% sensitivity, 82 ng/ml; Immunodiagnostic Systems, Tyne and Wear, UK).
Statistical Analysis
All data are expressed as means ± SE (n values shown in the table and figure legends), with statistical comparisons performed using either Student's t-test or one-way ANOVA with either Bonferroni's selected comparison or Student-Newman-Keuls post hoc tests, as indicated in the table and figure legends.
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RESULTS |
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Growth parameters. Between 3 and 9 wk of age, the body weight of male AS rats increased sixfold (Table 1). Although similar at 3 wk of age, body weights of dw/dw and Tgr rats increased at a lower rate than in AS males (P < 0.001; Table 1) and in parallel with each other. Over the same period, plasma IGF-I concentration increased threefold in AS males but was profoundly suppressed at all ages in both models of GH deficiency (P < 0.001; Table 1), being reduced by 60% in Tgr males and 70% in dw/dw males at 9 wk. Tibial EPW declined with age in all strains, with dw/dw rats having significantly narrower plate widths at 3 and 9 wk (P < 0.001 vs. AS; Table 1).
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Study 2: Adiposity Profiles in 1-Yr-Old GH-Deficient Rats
To determine whether this lean phenotype is maintained in dw/dw rats, measurements of body weight and adiposity were made in 1-yr-old males. In Tgr males, a 16% reduction in body weight (P < 0.001; Table 2) was accompanied by a 30% increase in proportional visceral fat pad weight (P < 0.001) and a twofold increase in circulating leptin (P < 0.01). In contrast, at 1 yr of age, dw/dw males were neither lean nor obese and were normoleptinemic despite a 35% lower body weight compared with AS rats (P < 0.001; Table 2).
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Analysis of adiposity profiles in female dw/dw rats in the NIMR colony confirmed that the observed reduction in truncal adiposity in dw/dw rats was neither sex nor institution specific. A 39% reduction in body weight in dw/dw females (P < 0.001; Table 3) was accompanied by 54 and 42% reductions in proportionate visceral (P < 0.001) and ovarian (P < 0.001) fat pad weights, respectively (Table 3). In contrast, tibial marrow adiposity was doubled in dw/dw females (P < 0.01), which was attributable to the increase in adipocyte number (P < 0.01), with adipocyte size being unaltered (Table 3).
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GHR/BP–/– mice. Deletion of the GHR/BP gene resulted in a 47% reduction in body weight (P < 0.001; Table 4), a severe reduction in circulating IGF-1 (39), and a 20% reduction in tibial epiphyseal plate width (P < 0.01). Although there was no significant alteration in the proportion of visceral or epididymal fat (Fig. 2, A and B), there was a slight increase in the proportion of retroperitoneal fat in male GHR/BP–/– mice (separate fat pad data not shown, P < 0.05). In contrast, deletion of the GHR/BP gene resulted in a fivefold elevation in tibial marrow adiposity (P < 0.001; Fig. 2D), which resulted from the combination of a threefold increase in adipocyte number (P < 0.05; Fig. 2E) and a smaller elevation in adipocyte size (P < 0.05; Fig. 2F). Plasma leptin concentration was almost doubled in GHR/BP–/– mice (P < 0.05; Fig. 2C).
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To determine whether the lean phenotype observed in the dw/dw rat was due to a reduction in energy intake, we measured daily food intake in male AS and dw/dw rats for 6 days. When expressed in proportion to body weight, food intake was elevated in dw/dw rats (P < 0.01; Table 5), but when corrected for body surface area, food intake in dw/dw and AS rats was identical.
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To determine whether the lean phenotype observed in the dw/dw rat was reversible by GH treatment, a group of dw/dw males was infused with hGH for 14 days. GH treatment accelerated body weight gain to that seen in untreated AS males (P < 0.001 vs. vehicle-treated dw/dw males; Fig. 4A). Although proportionate perirenal fat pad weight appeared to change in parallel with body weight (Fig. 4B), none of the means were significantly different. In contrast, neither proportionate epididymal fat pad weight (Fig. 4C) nor plasma leptin concentration (Fig. 4D) were increased by hGH treatment, with both remaining significantly lower than that seen in untreated AS males (P < 0.01).
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Growth parameters. Neonatal MSG treatment had no significant effect on body weight (Table 6) or liver weight (data not shown) in either male or female dw/dw rats. Residual circulating IGF-I concentrations were halved in both sexes (P < 0.01 vs. vehicle treated; Table 6), and both measures of skeletal growth, EPW (P < 0.05 vs. vehicle-treated; Table 6) and tibial length (P < 0.05 vs. vehicle-treated, males only; Table 6), were significantly reduced.
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Study 8: Effect of a High-Fat Diet on Adiposity Profiles in dw/dw Rats
Growth parameters. Maintenance on the high-fat diet for 4 wk did not produce a significant elevation in final body weight, or average daily weight gain in AS rats, but elicited a significant decrease in plasma IGF-I concentration and tibial EPW (P < 0.05; Table 7). On the standard chow diet, dw/dw females continued to gain less weight than their AS counterparts (P < 0.001; Table 7). Average daily weight gain in dw/dw females was not significantly affected by maintenance on the high-fat diet. When the additional weight gain of fat-fed rats was expressed as a proportion of the weight gain of female rats on standard chow, fat-fed AS rats gained 5.8 ± 4.5%, whereas fat-fed dw/dw females gained 12.5 ± 7.6% (not significantly different, P > 0.05). Neither plasma IGF-I concentration nor tibial epiphyseal plate width in dw/dw rats was affected by the increase in dietary fat content (Table 7).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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This study of the adiposity profile in dw/dw rats not only confirms a previous assertion (10) that these animals are not obese but demonstrates for the first time that, over the peripubertal period, dw/dw males actually developed proportionately lower abdominal fat reserves, accompanied by a marked hypoleptinemia. This feature was not unique to the Cardiff colony and was not affected by sex, with similar reductions in visceral and gonadal fat and plasma leptin concentration being observed in male and female dw/dw rats from the Cardiff and Mill Hill colonies. However, the severity of truncal leanness in the dw/dw rat declined with age, so that in 1-yr-old males both proportionate abdominal fat reserves and circulating leptin were normal.
Given the severity of GH deficiency in the dw/dw model (7, 27), this abdominal leanness is surprising, contrasting with the obese/hyperleptinemic phenotype of both the profoundly GH-deficient spontaneous dwarf rat (24) and the moderately GH-deficient Tgr rat. It should be noted here that, although hyperleptinemia was not observed in 9-wk-old Tgr males in the current study, circulating leptin was highly elevated at 3 and 6 wk of age and becomes progressively elevated in Tgr rats after 9 wk of age (13), with plasma leptin concentrations being increased more than twofold in 1-yr-old Tgr males [study 2 (16)]. Our direct comparison of the Tgr and dw/dw models suggests that either the regulation of adipose tissue is specifically disrupted in the dw/dw rat or the relationship between the degree of adiposity and the degree of GH deficiency (or GH resistance) may be nonlinear.
To test the latter hypothesis, we examined the adiposity profiles in two mouse models of reduced GH signaling, the GHA-transgenic mouse and the GHR/BP-null mouse. Our observation that visceral adiposity is doubled in the GHA model of partially reduced GH receptor signaling, but not significantly increased in GHR/BP-null mice, appears to lend support to our hypothesis. However, this may describe only the relationship between GH deficiency and adiposity in pubertal animals, since retroperitoneal fat in 3- to 6-mo-old GHR/BP–/– mice is increased more than in similarly aged GHA-transgenic mice (3). It is also important to note that, although the proportions of visceral, epididymal, and marrow fat were comparable in the two wild-type mouse strains (Fig. 2, A, B, and D, and Fig. 3, A–C), the number and size of the marrow adipocytes were considerably different (Fig. 2, E and F, and Fig. 3, D and E). Such differences may arise from an underlying differential sensitivity of these strains to the genetic manipulations influencing adiposity (3). However, strain-dependent differences cannot explain the leanness in the dw/dw model, since both this and the Tgr rat are maintained on the same genetic background.
In the context of this hypothesis, it is interesting to note that, whereas GH inhibits the differentiation of primary preadipocytes (22), it promotes the differentiation of 3T3-F442A fibroblasts into adipocytes (36). This latter process, which is dependent upon activation of the STAT5 signaling pathway (32), suggests that initial exposure to GH (or possibly prolactin) may be a prerequisite for early adipogenic events that enable the subsequent accumulation of adipose reserves during the peripubertal period. This is supported by our observation that GH treatment in postpubertal dw/dw rats was unable to restore proportionate epididymal fat weight (data for perirenal fat being less unequivocal) or circulating leptin levels in this model (study 6). Although such a mechanism might account for the absence of obesity in dw/dw rats and GHR/BP-null mice, it does not account for our demonstration that similarly aged dw/dw rats and GHR/BP-null mice do not share a lean/hypoleptinemic phenotype. Taken together, our data do not support the hypothesis that the unusual adiposity pofile in the dw/dw rat is a direct consequence of its profound GH deficiency.
The degree of GH-deficiency is considered to be important in the regulation of intra-abdominal fat, but there is now growing evidence for differential regulation of specific adipose depots (17, 18, 34). As reported previously (21), the adiposity in the tibial marrow compartment was not regulated parallel to that in abdominal fat (studies 1 and 3), but the advantage of analyzing marrow fat is that it enables ready discrimination between adipocyte hypertrophy (lipid accumulation) and adipocyte hyperplasia. Our data indicate that, over the age range studied, the population of marrow adipocytes in male dw/dw rats appears to change in parallel with that in AS males (Fig. 1). In contrast, the occurrence of elevated marrow fat in female dwarves is almost entirely due to the expansion in adipocyte number (Table 3), which we (21) have previously shown to be completely reversible by hGH (but not IGF-I) treatment. These findings, coupled with our observation of an increase in the marrow adipocyte population in GHR/BP-null mice (Fig. 2), suggest that the absence of the expected elevation in marrow adiposity in pubertal dw/dw males may be due to retarded preadipocyte differentiation or adipocyte proliferation. Whether a similar mechanism is responsible for the truncal leanness in dw/dw rats remains unclear.
In contrast with the control of differentiation and proliferation, the regulation of adipocyte size in dw/dw rats appears normal, or even exaggerated, with lipid being accumulated in response to both neonatal MSG exposure (Fig. 5) and high-fat feeding (Fig. 6). These manipulations induce similar responses in the weight of abdominal fat reserves. Our data, which corroborate a previous report that high-fat feeding induces obesity in dw/dw females (10), indicates that dw/dw adipocytes not only remain responsive to lipogenic stimulation but appear more responsive than adipocytes in GH-replete controls (Fig. 6), possibly as a result of altered insulin sensitivity (29). A similar increase in the sensitivity of adipose tissue has recently been reported in GHR/BP-null mice (4). Thus, abdominal fat in the dw/dw rat may be particularly sensitive to the lipolytic influence of residual circulating GH. Although we have not made any direct measurements of GH receptor expression in fat depots from dw/dw rats, the increase in adiposity following MSG treatment in dw/dw rats lends support to this proposal. Although neonatal MSG treatment does not reduce the somatotroph population in the dw/dw pituitary (23), the reductions in circulating IGF-I and tibial length are consistent with a suppression of residual GH secretion (29) that may in turn contribute to the elevated adiposity. However, some caution is necessary in interpreting the data obtained following MSG treatment. In particular, it should be noted that, although neonatal MSG treatment destroys the majority of the arcuate GH-releasing hormone neurons, this ablation is not selective, with 70–90% of all neuronal perikarya in the arcuate nuclei being destroyed (1, 5). Thus, MSG treatment may also influence adiposity via the disruption of additional hypothalamic pathways, including those regulating orexigenesis and energy homeostasis.
However, this raises the possibility that the lean phenotype in dw/dw rats may be the consequence of reduced energy intake. The converse scenario has been proposed to occur in the GHA-antagonist mice, viz., that elevated adiposity and circulating leptin may be due in part to a proportionate hyperphagia (3), which does not occur in GHR/BP-null mice (3). Our measurement of food intake, however, does not support this hypothesis, since proportionate food intake was not reduced in dw/dw males but may in fact have been increased (study 5).
In summary, this study of the adiposity profile in dw/dw rats has produced evidence of developmental abdominal leanness and hypoleptinemia in the context of profound GH deficiency. The mechanisms underlying this paradoxical leanness are yet to be established but do not appear to be the direct result of a reduction in either GH signaling or energy intake. Identification of the genetic defect responsible for the dw/dw phenotype may uncover a novel mechanism contributing to the regulation of adiposity. In addition, given the divergent status of adipose reserves in the dw/dw and Tgr rats, further comparative characterization of these models may also reveal the relative contributions of elevated leptin and reduced circulating GH to the development of the pleiotropic abnormalities associated with GH-deficient dwarfism.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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P < 0.05; 
