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GRH-SOMATOSTATIN-GH |
Department of Biochemistry and Molecular Biology (M.H., E.O., R.G., W.J.J., F.M.N.) and Department of Pharmacology (R.J.S.), Monash University, Clayton, Australia 3800; and Department of Medicine (A.T.), University of Melbourne, Parkville, Australia 3052
Address all correspondence and requests for reprints to: A/Prof. Frank Ng, Department of Biochemistry and Molecular Biology (13d), Monash University, Clayton, Victoria 3800, Australia. E-mail: frank.ng{at}med.monash.edu.au
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The nature of the response to both hGH and AOD9604 is not clearly understood. We hypothesized that both molecules may influence the expression of the ß3-adrenergic receptors (ß3-ARs), the major lipolytic receptor in fat tissue. Both AOD9604 and hGH can increase ß3-AR mRNA expression, as well as protein levels and function, in mouse and human cell lines in vitro (11). This response was investigated at the level of RNA and protein expression and function. The results for each mode of analysis were consistent in that both hGH and AOD9604 acted in a dose- and time-dependent manner to modulate the ß3-AR response.
In this paper, we investigated whether the changes observed in ß3-AR RNA expression in vitro also occur in an in vivo model. The in vivo model used was the obese (ob/ob) mouse model of obesity that has repressed levels of ß3-ARs, which in part contributes to reduced lipolytic sensitivity (12). Lean C57BL/6J mice were used as a control. Following a 14-d chronic administration with AOD9604 or hGH, adipose tissue weights were measured, and ß3-AR mRNA expression was determined. The decrease in weight of adipose tissue depots in the ob/ob mice was associated with increased ß3-AR expression. Further studies in ß3-AR knock-out (ß3-KO) mice showed that the presence of the ß3-AR is necessary to mediate the chronic effectiveness of hGH and AOD9604 with regards to weight loss and fat mass reduction. However, an acute dose of AOD9604 was capable of increasing energy expenditure in ß3-KO mice, although the response was less than that seen in the wild-type control mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals and treatment
Lean C57BL/6J and obese (ob/ob) mice aged 12 wk were
used in this study. There were 18 mice in each group, and they were
divided into three treatment groups [saline (n = 6); AOD (250
µg/kg·d; n = 6); hGH (1 mg/k·d; n = 6)]. The animals
were housed in the Departmental Animal Facility at a constant
temperature and humidity in a 12-h light, 12-h dark cycle. Animals were
injected with a single intraperitoneal dose of saline, AOD9604, or hGH
at 0800 h each day for 14 d using a 1-ml syringe and 23-gauge
needle. The body weights of the animals were recorded every second day
along with food intake.
After 14 d, the animals were killed with 35 mg/kg sodium pentobarbitone (Rhone Merieux, Pinkenba, Queensland, Australia) injected into the heart. Their adipose tissues (white epididymal and brown interscapular) were removed and weighed. The tissues were frozen in liquid nitrogen and stored at -80 C until RNA analysis was performed.
The male ß3-KO mice and wild-type (WT) that were used in this study were offspring of animals provided by Dr. Bradford Howell (Beth Israel Hospital, Harvard Medical School, Boston, MA). The animals were bred and housed in the central animal house facility (Monash University). For chronic studies, animals were housed in the Departmental Animal Facility (Biochemistry, Monash University). For acute energy expenditure studies, animals were transported to the Department of Medicine (University of Melbourne), acclimatized, and killed following experimentation. The WT and ß3-KO mice genotype were verified by breeding records and RT-PCR analysis performed in the laboratory of Professor Roger Summers.
Twelve WT and 11 ß3-KO male mice aged between 12 and 14 wk were used in the chronic administration study. The animals were housed individually in cages under the conditions described above. The animals were divided into three groups: WT [control (saline; n = 3); AOD (250 µg/kg·d; n = 4) and hGH (1 mg/kg·d; n = 5)] and ß3-KO [control (saline; n = 3); AOD (250 µg/kg·d; n = 4); and hGH (1 mg/kg·d; n = 4)]. On d 0, all animals were anesthetized with sodium pentobarbitone and a collection of blood (200 µl) was taken in heparinized tubes (Terumo, Somerset, NJ) for glycerol determination. The plasma was isolated by centrifugation and stored at -20 C until required for analysis. For the following 28 d, the animals were given a single ip dose of compound at 0800 h each morning. Their food intake and body weight were recorded every second day and results expressed as a change from d 0. On d 28, the animals were anesthetized with sodium pentobarbitone, blood was collected for plasma glycerol determination, and they were then killed by a lethal injection of sodium pentobarbitone to the heart (35 mg/kg). Their white epididymal and brown subscapular adipose tissues were collected and weighed.
Glycerol determination
The levels of plasma glycerol were determined according to the
method previously described (8) and expressed as a change
from d 0 values. The amount of glycerol present in the plasma was
enzymatically assayed using glycerol phosphate oxidase reactions
(catalog no. GPO-337, Sigma Diagnostics, St. Louis, MO).
Plasma glycerol was determined using a spectrophotometer and converted
to micromoles per deciliter.
RNA extraction and RT-PCR
Tissues from ob/ob and lean mice were cut into 100-mg
pieces and homogenized with 1 ml TRIzol (Life Technologies, Inc., Grand Island, NY) using a PolyTron homogenizer
(Kinematica, Lausanne, Switzerland). The samples were
incubated for 5 min at room temperature to permit the complete
extraction of the RNA from the tissues. An aliquot of 0.2 ml chloroform
was added to each milliliter of TRIzol. The method of RNA extraction
was as described by the manufacturer (Life Technologies, Inc.). The final RNA pellet was air dried for 5–10 min and
then resuspended in 20 µl ddH20. RNA was
quantitated spectrophotometrically using the
A260/280 ratio.
RT-PCR
cDNAs were synthesized by reverse transcription (RT) of 1.0 µg
of each total RNA using oligo (dT)15 as a primer.
The RNA in a volume of 7.7 µl H2O was heated to
70 C for 5 min then placed on ice for 2 min before the addition of a
reaction mix containing 1x RT buffer (Promega Corp.,
Madison, WI), 1 mM deoxynucleotide triphosphates
(dNTPs), 5 mM MgCl2, 18 U
Rnasin (Promega Corp.), 20 U avian myeloblastosis
virus RT (Promega Corp.), and 50 µg
ml-1 oligo(dT)15 in a
volume of 12.5 µl. Following a brief centrifugation, the reactions
were incubated at 42 C for 45 min and then 95 C for 5 min. The
completed RT reactions were stored at -20 C and used for PCR without
further treatment.
PCR amplification was carried out on cDNA equivalent to 100 ng of starting mRNA using the following murine oligonucleotide primers (expected and observed PCR product size): ß3-AR forward, 5'-TCTAGTTCCCAGCGGAGTTTTCATCG-3'; (234 bp) reverse, 5'-CGCGCACCTTCATAGCCATCAAACC-3'; ß-actin forward, 5'-ATCCTGCGTCTGGACCTGGCTG-3'; (559 bp) reverse, 5'-CCTGCTTGCTGATCCACATCTGCTG-3'.
The ß3-AR and actin primers were intron
spanning to potentially reveal contaminant genomic DNA (none observed).
Reverse primers were labeled before the PCR in a reaction mixture
containing 120 pmol oligonucleotide, 70 µCi
[
-33P]ATP (Bresagen, Adelaide,
Australia), 1x One-Phor-All Plus buffer (Pharmacia Biotech, Uppsala, Sweden) and 20 U T4 polynucleotide kinase
(Pharmacia Biotech) in a volume of 40 µl. Following
incubation at 37 C for 30 min, reactions were diluted to 100 µl with
H2O and heated at 90 C for 2 min.
The PCR reaction mixture contained 1 U Taq polymerase (Life Technologies, Inc.), the supplied buffer [20 mM Tris-HCl (pH 8.4) and 50 mM KCl], 200 µM dNTPs, 2 mM Mg-acetate, 2.5 pmol of forward primer, 2.5 pmol labeled reverse primer, and cDNA in a vol of 10 µl. The PCR reactions were carried out in a Hybaid PCR Sprint machine (Hybaid, Ltd., Middlesex, UK). Following the initial heating of the samples at 95 C for 2 min, each cycle of amplification consisted of 30 sec at 95 C, 30 sec at 64 C, and 30 sec at 72 C. It was found that 24 cycles were optimum for the amplification process.
Southern blot transfer
Following amplification, PCR products were electrophoresed on
1.3% agarose gels and transferred onto Hybond N+
membranes (RPN 303B, Amersham Pharmacia Biotech) by
Southern blotting in 0.4 M NaOH/1 M NaCl. The
membranes were rinsed for 5 min in 0.5 M Tris-HCl (pH
7.5)/1 M NaCl and then in 0.3 M NaCl/30
mM sodium citrate, and air dried. Membranes were apposed
directly to a phosphor imager screen for 18 h, and scanned using a
Storm PhosphorImager and data quantitated using MCID software
(Imaging Research, Inc., St. Catherines, Ontario, Canada).
The ß3-AR product bands were normalized against
the ß-actin control, averaged, and RNA isolated from treated animals
was expressed against control animals.
Acute effects of AOD9604 and BRL37344 in WT and
ß3-KO mice
WT (n = 9) and ß3-KO (n = 9)
mice were used in this study. Animals were fasted 2 h before being
individually placed in an indirect calorimeter. Calorimetry was
performed as in previous studies (8). After baseline
readings were taken, mice were injected with one of the following
compounds: saline (control; n = 3); AOD9604 (2 mg/kg body weight;
n = 3); or BRL37344 (250 µg/kg body weight; n = 3). Rates
of energy expenditure, fat oxidation, and glucose oxidation were
measured for an additional 30 min. The concentrations of AOD9604 and
BRL37344 were determined as lowest concentration needed to give a
maximal response in these mice (data not shown). Rates of energy
expenditure and fat and glucose oxidation were plotted as a change from
the average baseline values.
Statistical analysis
Results are expressed as the mean ± SE in each
experiment. The levels of significance were determined using a
t test, and P values <0.05 were considered
statistically significant when compared with control.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows the chronic effect
of saline, AOD9604, and hGH in lean C57BL/6J mice on body weight and
food intake. The hGH potently increased the body weight gain of these
mice, reaching significance by d 8. There was an increase in body
weight after AOD9604 only on the last day of treatment. In contrast,
ob/ob mice (Fig. 1B) showed a profound decrease in body
weight after both AOD9604 and hGH. Importantly, these effects were not
attributed to changes in food intake in either the lean or the
ob/ob mice (Fig. 1, C and D, respectively).
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shows that neither hGH nor
AOD9604 had any significant effect on the weight of epididymal adipose
tissue in the lean mice. However, in the ob/ob mice, AOD9604
induced a 28% decrease in epididymal adipose tissue mass, and hGH
induced a 40% reduction in epididymal adipose tissue mass
(P < 0.05). Brown adipose tissue weights were also
measured (Fig. 2B). In this tissue, both AOD9604 and hGH induced a
reduction in the weight of brown adipose tissue in both the lean and
ob/ob mice.
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) and brown (Fig. 3B) adipose tissues,
compared with lean C57BL/6J mice, in agreement with others
(14). Figure 3A shows the effect of saline, AOD9604, and
hGH on ß3-AR RNA expression in epididymal
adipose tissue from both lean and obese mice, respectively. The level
of ß3-AR expression increased significantly in
response to AOD9604 and hGH only in the obese mice, correlating with
the significant decrease in epididymal adipose tissue weights seen in
these mice. The same correlation was observed in brown adipose tissue,
where increased expression of ß3-AR was
accompanied by a decrease in brown adipose tissue mass in both lean and
obese mice (Fig. 3B).
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, both
AOD9604 and hGH increased body weight after 28 d in lean WT mice
similar to the effect seen after 14 d in lean C57BL/6J mice (Fig. 1A
). This effect was not observed in the ß3- KO
mice (Fig. 4B) in whom AOD9604 or hGH had no significant effect on body
weight.
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demonstrates that chronic
administration of AOD9604 or hGH has no significant effect on the
weight of white adipose tissue in either WT or
ß3-KO mice. However, in brown adipose tissue,
both AOD9604 and hGH significantly reduced the size of the brown
adipose tissue mass in the WT mice (Fig. 5B), by 20% and 31%
(P < 0.05), respectively, as was found previously in
the C57BL/6J ob/ob mice (Fig. 2B). Importantly, this effect
was not observed in ß3-KO mice.
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, both AOD9604 and hGH increased
glycerol levels following treatment in the WT mice, indicative of
enhanced lipolysis. In the ß3-KO mouse,
however, no effect was observed with AOD9604. A significant increase in
plasma glycerol from controls was observed following hGH treatment, but
this was markedly less than that observed in the WT mouse. These data
indicate the importance of ß3-AR in the
lipolytic response to AOD9604 in these animals and the necessity of the
receptor for the chronic effectiveness of AOD9604 and hGH on fat
reduction.
The final study involved an assessment of the acute effect of
AOD9604 and a ß3-AR agonist (BRL37344) on
energy expenditure, fat oxidation, and glucose oxidation in WT and
ß3-KO mice. When AOD9604 or BRL37344 were
administered to WT mice, an acute increase in fat oxidation and energy
expenditure occurred, with an associated reduction in glucose oxidation
(Fig. 6A). The effect plateaued 18 min
following injection and remained stable for the duration of the
experiment. The response to the two compounds was very similar, despite
the fact we have previously shown that AOD9604 does not directly
interact with the ß3-AR as demonstrated by
ligand binding studies (11). This clear separation of
pathways was further confirmed in Fig. 6B in which AOD9604 clearly
increases fat oxidation and energy expenditure in
ß3-KO mice, whereas BRL37344 does not. The KO
mice neither decrease their glucose oxidation in response to AOD9604
nor show a prolonged increase in fat oxidation and energy expenditure
in response to AOD9604.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Both AOD9604 and, to a greater extent, hGH increase body weight in lean mice, compared with saline-treated animals. This is in the absence of an increase in fat mass, which suggests an increase in lean body mass occurs with these compounds. This supports previous work with hGH in rodents and humans (17). Both compounds have also been previously shown to reduce body weight and adiposity in obese mice (11). The effects of hGH and AOD9604 occur without significant changes to caloric intake. It has been reported that hGH increases, reduces, or does not change food intake in which the differences are attributed to variations in hGH preparations, concentrations, and animals used between different laboratories.
The effects of hGH and AOD9604 on fat metabolism may be mediated by an alteration in the expression of a lipolytic/antilipogenic gene. The ß3-AR is a major lipolytic receptor identified in rodent fat cells (18) that mediates its effects through G protein coupling to adenylate cyclase, generation of cAMP, and stimulation of PKA (19). This enzyme then phosphorylates proteins in the lipolytic cascade, including hormone-sensitive lipase (20). In BAT, the ß3-AR stimulates uncoupling of the electron transport chain, enhancing the ability of mitochondria to generate heat in preference to ATP through the dissipation of the electron gradient (21). Mice that lack this receptor have lower rates of resting energy expenditure (0.0041 vs. 0.0047 kcal/min, P < 0.02) and lower rates of fat oxidation (0.00019 vs. 0.00030 g/min, P < 0.02) than control mice (data not shown).
AOD9604 and hGH appear to act in a similar manner to induce their effects on body weight regulation and adipose tissue mass in vivo. However, in vitro studies have demonstrated a number of differences suggesting that the two compounds operate via unique signaling pathways to control the regulation of the ß3-AR. These studies suggested that AOD9604 had no interaction with the ß3-AR or hGH receptors (11).
In lean animals, neither AOD9604 nor hGH had any effect on epididymal white adipose tissue mass or expression of ß3-AR RNA, indicating that in lean animals, this fat tissue is not a major target for these drugs in this study. In contrast, the mass of BAT in lean animals was reduced by both hGH and AOD9604, and ß3-AR RNA expression was increased by both these compounds. This could possibly suggest that the increased expression of ß3-ARs in brown adipocytes sensitizes catecholamines to dissipate heat.
In ob/ob mice, both AOD9604 and hGH reduced both white and brown adipose tissue mass and increased ß3-AR RNA expression. This suggested that an elevation in ß3-AR RNA expression is associated with increased fat metabolism and a reduction in the fat tissue mass in the ob/ob mouse model. Obese mice have lower levels of ß3-AR expression in their adipose tissues than lean mice, shown in this study and others (14). The ability of AOD9604 and hGH to increase the level of ß3-AR RNA expression in obese mice to a level that is comparable to those in lean mice is an exciting finding. However, it must also be considered that both hGH and AOD9604 may influence the expression of other members of the adrenergic pathway, such as the ß1-ARs, hormonesensitive lipase, and signaling proteins, which are all expressed in adipose tissue and associated with lipolysis. The importance of the change in ß3-AR expression with AOD9604 and hGH in humans is not established and will depend on the use of potent and selective ß3-AR agonists that are active at the human receptor.
From this study it appears that the ß3-AR is an important contributor to the effects observed on body weight in obese mice treated with AOD9604 and hGH. To determine whether the ß3-AR is partly responsible for this effect, we examined the effects of AOD9604 and hGH in the ß3-KO mouse. The ß3-KO mouse is not grossly obese, but female mice have increased fat depots (21) and the mice do develop late-onset obesity (Summers, R. J., personal communication). AOD9604 and hGH increased body mass and decreased BAT mass in the WT strain but had no effect in the KO animals. In WT mice, plasma glycerol was increased in response to AOD9604 and hGH treatment (4 wk). However, in the KO mice, only hGH resulted in increased levels of glycerol in the KO mice, and this effect was significantly less than that observed in the WT mice. This suggests that the regulation of the ß3-AR is essential in the ability of AOD9604 and hGH to mediate chronic effects on lipolysis and fat mass reduction.
The effect of AOD9604 and hGH on ß3-ARs in adipose tissue is believed to be a direct action of these compounds and not an effect secondary to the fat metabolism, given that both AOD9604 and hGH can influence ß3-AR expression and function in a nonadipocyte human cell line (11). Hence, the ß3-AR appears to be necessary for the chronic effectiveness of AOD9604 on lipolysis in BAT.
The acute effect of AOD9604 and BRL37344 (a ß3-AR agonist) on energy expenditure and substrate oxidation rates in WT and KO mice was also assessed. KO animals had lower energy expenditure, lower fat oxidation, and increased glucose oxidation, compared with the WT controls (data not shown). Injection of WT mice with a single dose of BRL37344 or AOD9604 increased energy expenditure and fat oxidation and decreased glucose oxidation. In the KO animals, BRL37344 failed to elicit any response in these metabolic parameters, clearly demonstrating that its effects are mediated exclusively through the ß3-AR. AOD9604 did elicit a response in the KO mice, increasing fat oxidation and energy expenditure, although the response was not as great as in WT mice, suggesting that ß3-ARs are not responsible for the acute biological response of AOD9604 on lipid metabolism. This is consistent with our previous findings in which AOD9604 was shown not to bind to the ß3-AR (11). The size and duration of the metabolic responses to AOD9604 in the ß3-AR KO animals was different from that observed in the control wild-type mice. The response was more rapid, shorter in duration, and greater in peak response. This may be because the KO animals are more acutely sensitive to lipolytic agents, a compensation for the ablation of the major lipolytic receptor.
These findings suggest that the acute effects of AOD9604 are quite different from the chronic effects. Enhanced ß3-AR expression appears to play a major role in the chronic effectiveness of the compound in terms of fat metabolism and weight loss. The acute effects observed in this study confirm that the ß3-AR is not the sole mediator of this action. The increase in ß3-AR expression in response to hGH and AOD9604 would permit enhanced lipolytic sensitivity. Identification of the components of the intracellular pathway(s) and effector(s) activated by AOD9604 are currently being investigated. The results presented in this paper suggest that the effectiveness of AOD9604 and hGH may partly rely on their ability to increase levels of ß3-AR RNA expression in models of obesity in which the numbers of the lipolytic receptor are low. These unique properties may give AOD9604 an advantage over other lipolytic agonists such as adrenergic agents and hGH, which exhibit undesirable side effects when administered chronically (22).
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Footnotes |
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Abbreviations: ß3-AR, ß3-Adrenergic receptor; ß3-KO, ß3-AR knock-out; BAT, brown adipose tissue; hGH, human GH; ob/ob, obese; RT, reverse transcription/transcriptase; WT, wild-type.
Received February 2, 2001.
Accepted for publication August 6, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-adrenergic systems of sheep
adipose tissue by growth hormone and insulin. Biochem J 273(Pt
1):39–42
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