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Original Studies |
Garvan Institute of Medical Research, St. Vincent’s Hospital, Sydney, New South Wales 2010, Australia
Address all correspondence and requests for reprints to: Dr. Ken K. Y. Ho, Associate Professor of Medicine, Garvan Institute of Medical Research, St. Vincent’s Hospital, 384 Victoria Street, Sydney, New South Wales 2010, Australia. E-mail: k.ho{at}garvan.unsw.edu.au
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Abstract |
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GH stimulated oxidation of palmitic acid maximally by 26.7 ± 2.5% (mean ± SEM; P < 0.0001). The stimulation was biphasic, with the oxidation rate increasing with increasing GH concentration to a peak response at 1.5 nmol/L and declining to a level not significantly different from control thereafter. Insulin-like growth factor-I at concentrations of up to 250 nmol/L had no significant effect on fatty acid oxidation. GH-binding protein attenuated the effect of GH. An anti-GH receptor (GHR) antibody (MAb263), which dimerizes the receptor and induces GH-like biological actions, significantly stimulated fatty acid oxidation. Another anti-GHR antibody (MAb5), which prevents receptor dimerization, suppressed GH action. In summary, GH directly stimulated fatty acid oxidation, an action not mediated by insulin-like growth factor-I. Dimerization of GHRs was necessary for this effect. This bioassay is a practical tool for studying the regulatory effects of GH on lipid oxidation.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fatty acids are mainly catabolized in mitochondria via the ß-oxidation pathway (12). A number of in vitro bioassays, using radiolabeled fatty acids as substrates, have been established for measuring the rate of mitochondrial ß-oxidation (13). The most commonly used methods are based on quantifying 14CO2 released from 14C-labeled fatty acids. However, these assays are labor intensive and usually have a low sensitivity, because less than 25% of the 14C-labeled intermediates generated by ß-oxidation are converted to carbon dioxide (13, 14).
Moon and Rhead (15) have previously reported a bioassay with an alternative approach for assessing mitochondrial ß-oxidation, in which the rate of conversion of tritiated palmitic acid to 3H2O by cultured fibroblasts is measured. Unlike the 14C-labeled substrates, about 75% of the radiolabel released from the tritiated substrate is readily incorporated to H2O, which can be separated from the unreacted substrate by a simple chromatographic procedure. This bioassay has been shown to be sensitive, accurate, and reproducible (13, 15).
We have modified the in vitro bioassay of Moon and Rhead to investigate whether GH stimulates fatty acid oxidation. Human fibroblasts were chosen for this study, based on the observations that these cells express GH receptors (GHRs) (16, 17), produce insulin-like growth factor-I (IGF-I) in response to GH (18), and have the metabolic machinery for ß-oxidation (13).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Reagents for cell culture, including Eagle’s minimum essential medium (EMEM), FBS, HEPES, L-glutamine, penicillin/streptomycin, and trypsin-EDTA (1:250), were obtained from Cytosystems (Sydney, Australia). Recombinant human GH was produced as previously described (19). Recombinant human IGF-I was purchased from GroPep (Adelaide, Australia). Anti-GHR monoclonal antibodies, MAb5 and MAb263 (20, 21), were generous gifts from Dr. Michael Waters (University of Queensland, Brisbane, Australia). Recombinant human GH-binding protein (GHBP) (22) was obtained from Genentech (CA). Palmitic acid, fatty acid-free BSA, and Krebs-Ringer bicarbonate buffer were purchased from Sigma (St. Louis, MO), and [9,10(n)-3H]-palmitic acid (36 Ci/mmol) was from Du Pont (Sydney, Australia). AG1-X8 resin (100–200 mesh, chloride form) was obtained from Bio-Rad (Hercules, CA) and converted to the hydroxide form by treatment with 1 mol/L NaOH before use. Ready Safe scintillant was purchased from Beckman (Fullerton, CA) and the bicinchoninic acid protein assay kit (23) from Pierce (Rockford, IL).
Cell culture
Human skin fibroblasts were kindly provided by Dr. Stewart Purvis-Smith (Prince of Wales Hospital, Sydney, Australia). The cells were routinely grown in monolayer cultures at 37 C in 5% CO2/95% air in EMEM supplemented with 10% FBS, 25 mmol/L HEPES, 200 mmol/L L-glutamine, and antibiotics.
Fatty acid oxidation assay
The procedure of Moon and Rhead (15) was followed, with modifications of substrate concentration, cell density, and incubation time optimized for our studies. Briefly, the reaction mixture was prepared by adding 50 µCi of [9,10(n)-3H]-palmitic acid to 300 µL of 2.2 mmol/L unlabeled palmitic acid in absolute ethanol. After complete evaporation of the solvent, the fatty acid was resuspended in 300 µL Krebs-Ringer buffer containing 10 mg/mL BSA and incubated at 37 C for 30 min. The reaction mixture was further diluted with Krebs-Ringer buffer to a final concentration of 110 µmol/L palmitic acid and specific radioactivity of 5–7 x 104 cpm/nmol.
Fibroblasts were plated at a density of 4 x 104 cells/cm2 in a 24-well multidish (Corning, NY). After overnight incubation, the monolayer cultures, at 70–80% confluency, were washed with phosphate-buffered saline before addition of 200 µL of the reaction mixture. Blanks were set by inactivating cells in triplicate wells with 200 µL absolute methanol for 1 min before the addition of the reaction mixture. The effects of GH, IGF-I, anti-GHR antibodies, and GHBP were investigated by adding these factors at predetermined concentrations in triplicate at the beginning of the assay. The cultures were then incubated at 37 C for 2 h. At the end of the incubation, the reaction mixture was removed, and the cultures were washed twice with 150 µL of phosphate-buffered saline. Both the reaction mixture and the wash were transferred to AG1–8X columns set in Pasteur pipettes for separating 3H2O from the unreacted substrate. The eluates, containing 3H2O, were collected into scintillation counting vials. After the columns were rinsed twice with 1 mL deionized water, 10 mL scintillant was added to each vial, and the samples were counted with a Beckman LS6500 scintillation counter. To determine the protein content, the cells were lysed with 1 mol/L NaOH, neutralized with an equal vol of 1 mol/L HCl, and assayed by the bicinchoninic acid method (23). The reaction rate was expressed as nmol 3H2O/h·mg protein. The intra- and interassay CVs were 6.3% (n = 12) and 10.9% (n = 6), respectively.
GH-binding assay
Specific GH binding of fibroblast monolayers was determined as described previously (24). Briefly, the cells were plated at a density of 5 x 104 cells/cm2 in a 6-well multidish (Corning) and cultured overnight. To set up the binding assay, the culture medium was replaced with 1 mL EMEM with 0.2% BSA, and 125I-labeled GH (2 x 105 cpm/mL), with and without 10 µg/mL unlabeled GH, was added. Specific GH binding was determined after 2 h incubation at 22 C.
Statistical analyses
Results are expressed as mean ± SEM from triplicate determinations. All experiments were performed at least three times, unless otherwise stated. Differences between groups were analyzed by Student’s t test and ANOVA (Statview 4.02, Abacus Concepts, Berkeley, CA) wherever appropriate, and the degree of significance was set at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To assess the effect of cell density, fatty acid oxidation rates
of cultures at densities of 2, 4, 5, and 6 x 104
cells/cm2, corresponding to confluency of 30–40%,
70–80%, 100%, and over 100%, respectively, were investigated. As
shown in Fig. 1a
, the oxidation rates
were not different at cell densities of 2 and 4 x 104
cells/cm2, and decreased significantly at 5 and 6 x
104 cells/cm2. The effects of substrate at
concentrations of 22, 55, 110, 220, and 330 µmol/L were examined next
(Fig. 1b). The oxidation rate increased with increasing substrate
concentration and reached a plateau level at 110 µmol/L. The use of
2- to 4-fold higher mass of tritiated substrate, to alter the ratio of
labeled to unlabeled substrates, did not change the oxidation rate
significantly (data not shown).
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, 3H2O concentration increased linearly
over the 4-h incubation period (r2 = 0.975;
P < 0.0001). The effect of GH was investigated by
incubating cultures, with and without 4.5 nmol/L GH, for up to 4
h. GH significantly increased fatty acid oxidation by 19.0 ±
8.8% (P < 0.05; Table 1) at the end of 4 h. Examination of
the time course of GH effect revealed that the stimulating effect was
greater in the first 2 h (36.4 ± 1.9%; P <
0.01) than in the last 2 h (7.2 ± 5.1%; P =
not significant). Therefore, all further studies were performed with
2 h incubation.
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, the basal oxidation rates of young
(passage number 12–16) and old cultures (passage number 26–31) were
not significantly different (6.85 ± 0.09 and 6.36 ± 0.36
nmol/h·mg, respectively). However, the maximal GH stimulation in
young cultures (20.7 ± 5.3%) was about 5-fold higher than that
of old cultures (4.4 ± 1.2%). We next investigated whether the
difference in GH response was attributed to difference in GH binding of
the two cultures. Specific GH binding of young cultures (837 ± 74
cpm/106 cells) was significantly higher than that of old
cultures (490 ± 69 cpm/106 cells; n = 3,
P < 0.05). These results suggest that the response to
GH was related, in part, to the GHR status of the culture. All further
studies were performed in cultures having been passaged for less than
20 times.
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The effects of GH were investigated over the range of 0.015–150
nmol/L (Fig. 3a). GH stimulated fatty
acid oxidation in a biphasic manner; the oxidation rate increased with
increasing GH concentration to a peak response at 1.5 nmol/L and
declined to a level not significantly different from control. The
ED50 was 
0.2 nmol/L, and the mean maximal stimulation
was 26.7 ± 2.5% of control (n = 10, P <
0.0001).
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, IGF-I, over a concentration range of 0.08–250 nmol/L, did not
significantly stimulate fatty acid oxidation. GHR antibodies
The specificity of GH action was investigated by using two
antibodies to GHR, MAb263 and MAb5. These two antibodies have different
binding properties, in that MAb263 dimerizes GHRs and induces GH-like
biological actions (25), whereas MAb5 blocks receptor dimerization
(26). MAb263 caused a concentration-dependent, biphasic stimulation of
fatty acid oxidation, an effect similar to that of GH (Fig. 4a). The maximal response induced by the
antibody was 18.5 ± 5.6% (P < 0.02). In
contrast, MAb5 did not have a significant effect on fatty acid
oxidation over the range of 0.2–22 nmol/L.
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). However, the antibody
significantly inhibited the increase in fatty acid oxidation induced by
GH (P = 0.003). GHBP
The effect of GHBP on GH-stimulated fatty acid oxidation was
investigated. As previously described, GH significantly increased the
oxidation rate (5.02 ± 0.22 nmol/h·mg; Fig. 5), compared with that of control
(4.33 ± 0.10 nmol/h·mg; P < 0.05). GHBP alone
(3.96 ± 0.21 nmol/h·mg) had no significant effect on fatty acid
oxidation but did suppress GH-stimulated oxidation (4.58 ± 0.15
nmol/h·mg).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The stimulation of fatty acid oxidation by GH was concentration
dependent and biphasic, with a peak response at 1.5 nmol/L. These
concentrations are within the physiologic range of circulating GH of
0.04–1.5 nmol/L (27). The ED50 for stimulation was 0.2
nmol/L, which is comparable with that reported for lipolysis (0.1–0.5
nmol/L) (28) and corresponds well with the binding affinity for the GHR
(Kd = 0.9 nmol/L) (16). The extent of stimulation
(26.7 ± 2.5%) is comparable with those for lipolysis (35–50%)
(2, 29, 30), leucine oxidation (35%), and glucose oxidation
(50%) induced by GH (28). Although the present studies were
undertaken in the fibroblast, which is not generally appreciated as a
tissue type which oxidizes fat, these findings would provide a basis
for further research into the effects of GH on classical fat-oxidizing
tissues such as the liver and muscle. These data provide valid evidence
for a direct stimulatory effect of GH, but they do not allow us to
speculate on the magnitude of the contribution by this mechanism to
whole-body fat oxidation.
Studies with MAb5 suggest that the stimulatory effect of GH on fatty acid oxidation is specific and is mediated by GHRs. Moreover, the response to GH is significantly affected by the GHR status, as decreased GH binding was accompanied by a reduction in stimulation. We also showed that GHBP potently inhibited the effect of GH, a finding in accord with a previous report (31) that GHBP reduces the in vitro biopotency of GH by complexing the hormone and inhibiting its binding to GHRs (32).
The findings that the stimulation by GH and MAb263 were biphasic are consistent with the hypothesis that dimerization of GHR is necessary for initiating GH action (25, 26). A similar biphasic pattern of GH effect on cell proliferation has previously been described (25). According to this hypothesis, GH forms a dimeric complex through sequential binding at two distinct but adjacent sites on GH to GHRs, an event which is critical for triggering the postreceptor signaling cascade (33). Excess GH will antagonize signaling by preventing receptor dimerization, thus accounting for the loss of stimulatory effect.
GH stimulated fatty acid oxidation acutely, but no significant effect was demonstrable beyond 2 h of treatment. As GH down-regulates its own receptors (34, 35), the temporary nature of GH stimulation could be explained by this mechanism. However, we consider this possibility unlikely because cultures pretreated with GH for 3 h before the fatty acid oxidation assay had responses to GH in later incubation similar to those of cultures without GH pretreatment (unpublished observations). Alternatively, the refractoriness may be caused by end-product inhibition. In the mitochondrial ß-oxidation pathway, fatty acids are oxidized and degraded to acetyl-CoA through four consecutive reactions of acyl-CoA dehydrogenation, hydration, hydroxyacyl-CoA dehydrogenation, and thiolysis (12). During these processes, NADH is generated from its precursor NAD+. There is evidence that some of the key enzymes in ß-oxidation are inhibited by acetyl-CoA and elevated NADH/NAD+ ratio (12, 36, 37). It is possible that GH increased the production and accumulation of these end products, which prevented further increase in the oxidation rate.
It has been shown that IGF-I infusion enhanced lipid oxidation in humans (38, 39). However, it is not clear from these in vivo studies whether the effect is direct or indirect. In the present study, we demonstrate that IGF-I does not directly stimulate fatty acid oxidation. Thus, it is likely that the in vivo stimulation of lipid oxidation by IGF-I occurs indirectly through suppression of insulin secretion (38, 39) and reducing the inhibitory effect of insulin on whole-body lipid oxidation.
The mechanism(s) by which GH stimulates fatty acid oxidation is unknown. Cellular oxidation of fatty acids is a multistep process including cellular uptake of fatty acids, intracellular delivery to mitochondria, transport across the inner mitochondrial membrane, and ß-oxidation in the mitochondrial matrix (12). The uptake and intracellular trafficking of fatty acids are mediated by fatty acid-binding proteins (40). Fatty acids are transferred across the inner mitochondrial membrane by a carnitine-dependent transporting system composed of carnitine palmitoyltransferase-I, carnitine-acylcarnitine translocase, and carnitine palmitoyltransferase-II (12). Within the mitochondrial matrix, fatty acids are successively oxidized and degraded via the four-step process of ß-oxidation. GH has been shown to stimulate the transcription of fatty acid-binding proteins in the liver (41) and to increase the transcription and activity of medium-chain acyl-CoA dehydrogenase (42), one of the several acyl-CoA dehydrogenases catalyzing the first step of ß-oxidation. These findings suggest that GH may stimulate fatty acid oxidation by transcriptional regulation. However, because GH stimulation in the present study occurred acutely, it is unlikely that the stimulation occurred through induction of gene transcription, de novo protein synthesis, or cell proliferation. There is evidence that fatty acid oxidation can be regulated by allosteric modulation of key enzymes, such as carnitine palmitoyltransferase-I, in the pathway (12). Whether GH stimulates fatty acid oxidation through this mechanism is not known but worthy of further investigation.
We have developed an in vitro bioassay as a practical tool for studying the effects of GH on fatty acid ß-oxidation, and provide the first evidence that the hormone directly stimulates ß-oxidation. This bioassay also may be useful for evaluating the actions of other metabolic regulators of lipid oxidation, for investigating the interaction of GH with these regulators, and for assessing the metabolic actions of GH analogues.
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Acknowledgments |
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Footnotes |
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Received March 25, 1997.
Revised July 17, 1997.
Accepted September 15, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, P = 0.007 (both vs.
culture of 2 x 104 cells/cm2). 
,
P = 0.016; §, P = 0.005 (both
vs. culture of 4 x 104
cells/cm2). b, Substrate concentration. The fatty acid
oxidation assay was performed with palmitic acid at concentrations as
indicated. Each point is the mean of triplicate
measurements in one experiment, which was repeated three times. *,
P 
0.0003; 
) were investigated. Each
point is the mean of triplicate measurements in a
representative experiment, and similar results were obtained in a
repeated study. The basal oxidation rates for the two cultures were
6.85 ± 0.09 and 6.36 ± 0.36 nmol/h·mg, respectively. *,
P < 0.01; 
