Stimulation of Mitochondrial Fatty Acid Oxidation by Growth Hormone

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0021-972X/97/$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1997 by The Endocrine Society

Vol. 82, No. 12 Printed in U.S.A.

Stimulation of Mitochondrial Fatty Acid Oxidation by Growth Hormone in Human Fibroblasts* KIN-CHUEN LEUNG

AND

KEN K. Y. HO

Garvan Institute of Medical Research, St. Vincent’s Hospital, Sydney, New South Wales 2010, Australia ABSTRACT In vivo administration of GH induces lipolysis and lipid oxidation. However, it is not clear whether the stimulation of lipid oxidation is a direct effect of GH or is driven by increased substrate supply secondary to lipolysis. An in vitro bioassay has been established for assessing b-oxidation of fatty acids in mitochondria, based on the measurement of conversion of tritiated palmitic acid to 3H2O by fibroblasts in culture. We have modified this assay to investigate whether GH stimulates fatty acid oxidation. GH stimulated oxidation of palmitic acid maximally by 26.7 6 2.5% (mean 6 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 dif-

ferent 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. (J Clin Endocrinol Metab 82: 4208 – 4213, 1997)

G

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 b-oxidation (13).

H HAS COMPLEX effects on lipid metabolism (1). It stimulates lipolysis in adipocytes by increasing the activity of hormone-sensitive lipase (2, 3). It also increases the activity of hepatic lipase and the numbers of low-density lipoprotein receptors (4 –7), which facilitate hepatic degradation and uptake of lipoproteins. In vivo administration of GH induces lipid mobilization, elevates circulating free fatty acids and glycerol, and increases lipid oxidation and ketogenesis (8 –11). It is generally accepted that GH stimulates lipid oxidation by increasing the availability of substrates. However, the question of whether GH has a direct effect on oxidation of fatty acids has not been addressed. Fatty acids are mainly catabolized in mitochondria via the b-oxidation pathway (12). A number of in vitro bioassays, using radiolabeled fatty acids as substrates, have been established for measuring the rate of mitochondrial b-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 b-oxidation are converted to carbon dioxide (13, 14). Moon and Rhead (15) have previously reported a bioassay with an alternative approach for assessing mitochondrial b-oxidation, in which the rate of conversion of tritiated palmitic acid to 3H2O by cultured fibroblasts is measured. Received March 25, 1997. Revision received July 17, 1997. Accepted September 15, 1997. 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: [email protected]. * This work was supported by the National Health and Medical Research Council of Australia.

Materials and Methods Materials 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 PurvisSmith (Prince of Wales Hospital, Sydney, Australia). The cells were

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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 mCi of [9,10(n)-3H]-palmitic acid to 300 mL of 2.2 mmol/L unlabeled palmitic acid in absolute ethanol. After complete evaporation of the solvent, the fatty acid was resuspended in 300 mL 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 mmol/L palmitic acid and specific radioactivity of 5–7 3 104 cpm/nmol. Fibroblasts were plated at a density of 4 3 104 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 mL of the reaction mixture. Blanks were set by inactivating cells in triplicate wells with 200 mL 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 mL 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/hzmg protein. The intra- and interassay CVs were 6.3% (n 5 12) and 10.9% (n 5 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 3 104 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 3 105 cpm/ mL), with and without 10 mg/mL unlabeled GH, was added. Specific GH binding was determined after 2 h incubation at 22 C.

Statistical analyses Results are expressed as mean 6 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.

Results Assay standardization

To assess the effect of cell density, fatty acid oxidation rates of cultures at densities of 2, 4, 5, and 6 3 104 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 3 104 cells/cm2, and decreased significantly at 5 and 6 3 104 cells/cm2. The effects of substrate at concentrations of 22, 55, 110, 220, and 330 mmol/L were examined next (Fig. 1b). The oxidation rate increased with increasing substrate concentration and reached a plateau level at 110 mmol/L. The

FIG. 1. Assay standardization. a, Cell density. The rates of palmitic acid oxidation in cultures at the indicated cell densities were measured. Each point is the mean of triplicate measurements in a representative experiment. Similar results were obtained in three separate experiments. *, P 5 0.02; †, P 5 0.007 (both vs. culture of 2 3 104 cells/cm2). ‡, P 5 0.016; §, P 5 0.005 (both vs. culture of 4 3 104 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; ‡, P # 0.001 (both vs. 110, 220, and 330 mmol/L). †, P 5 0.03 vs. 55 mmol/L. c, Time course of 3H2O production. Cultures were incubated with 110 mmol/L of palmitic acid for 1, 2, 3, and 4 h. Each point is the mean of triplicate measurements in one experiment, which was repeated twice.

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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The time course of 3H2O production was investigated by measuring concentration of 3H2O in cultures after incubation for 1, 2, 3, and 4 h. As shown in Fig. 1c, 3H2O concentration increased linearly over the 4-h incubation period (r2 5 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 6 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 6 1.9%; P , 0.01) than in the last 2 h (7.2 6 5.1%; P 5 not significant). Therefore, all further studies were performed with 2 h incubation. Preliminary studies of GH effect on fatty acid oxidation revealed that the response to GH varied with the culture age. As shown in Fig. 2, the basal oxidation rates of young (passage number 12–16) and old cultures (passage number 26 – 31) were not significantly different (6.85 6 0.09 and 6.36 6 0.36 nmol/hzmg, respectively). However, the maximal GH stimulation in young cultures (20.7 6 5.3%) was about 5-fold higher than that of old cultures (4.4 6 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 6 74 cpm/106 cells) was significantly higher than that of old cultures (490 6 69 cpm/106 cells; n 5 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. GH

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 6 2.5% of control (n 5 10, P , 0.0001). Because IGF-I is a mediator for some of the biological actions of GH, its effect on fatty acid oxidation was investigated. As shown in Fig. 3b, IGF-I, over a concentration range of 0.08 –250 nmol/L, did not significantly stimulate fatty acid oxidation. TABLE 1. Time course of GH treatment Time

0– 4h 0–2 h 2– 4h

Fatty acid oxidation (nmol/hzmg) Control

GH-treated

4.61 6 0.30 5.00 6 0.21 4.65 6 0.19

5.49 6 0.18a 6.82 6 0.21b 4.98 6 0.13

The time course of fatty acid oxidation by cultures treated with and without 4.5 nM GH was investigated by measuring 3H2O concentration at 1, 2, 3, and 4 h of incubation. Rates of fatty acid oxidation were determined using a linear regression analysis program (Statview 4.02). Each value is the mean of triplicate measurements in a representative experiment. Similar results were obtained in three separate experiments. vs. control. a P , 0.05. b P , 0.01.

FIG. 2. Effect of culture age. The effects of GH at 0.045–15 nmol/L on young (passage number 12–16, F) and old cultures (passage number 26 –31, E) 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 6 0.09 and 6.36 6 0.36 nmol/hzmg, respectively. *, P , 0.01; †, P , 0.02 (both vs. control).

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 6 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. The effect of MAb5 on GH-stimulated fatty acid oxidation was examined next. As previously described, GH significantly increased the oxidation rate by 21.4 6 3.5% (P 5 0.0003), whereas MAb5 had no significant effect (Fig. 4b). However, the antibody significantly inhibited the increase in fatty acid oxidation induced by GH (P 5 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 6 0.22 nmol/hzmg; Fig. 5), compared with that of control (4.33 6 0.10 nmol/hzmg; P , 0.05). GHBP alone (3.96 6 0.21 nmol/hzmg) had no significant effect on fatty acid oxidation but did suppress GHstimulated oxidation (4.58 6 0.15 nmol/hzmg). Discussion

Using an in vitro bioassay, we demonstrate, for the first time, that GH directly stimulates fatty acid oxidation. The stimulation was acute and significant within 2 h treatment. The effect was attenuated by GHBP and MAb5 (an antagonistic GHR antibody) and mimicked by MAb263 (an agonistic GHR antibody). IGF-I had no detectable effect on fatty acid oxidation.

STIMULATION OF FATTY ACID OXIDATION BY GH

FIG. 3. Concentration effects of GH and IGF-I. a, GH. Each point represents the mean of triplicate measurements in one experiment, which was repeated three times. *, P , 0.05 vs. control. b, IGF-I. Each point is the mean of three separate experiments. Basal oxidation rate was 9.44 6 0.45 nmol/hzmg.

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 5 0.9 nmol/L) (16). The extent of stimulation (26.7 6 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

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FIG. 4. GHR antibodies. a, Concentration effects of antibodies. The effects of MAb263 (F) and MAb5 (E) at the indicated concentrations were investigated. Each point is the mean of three separate experiments. The basal oxidation rates for the two antibodies were 10.39 6 0.49 and 9.91 6 0.17 nmol/hzmg, respectively. *, P , 0.02 vs. control. b, GH and MAb5. To investigate the effect of MAb5 on GH-stimulated fatty acid oxidation, the oxidation rates of cultures with GH (1.5 nmol/L), the antibody (56 nmol/L), or both were measured. Each bar is the mean of triplicate measurements in one experiment, which was repeated three times. *, P 5 0.0003 vs. control. ‡, P 5 0.03; †, P 5 0.003 (both vs. GH).

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

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FIG. 5. GH and GHBP. To investigate the effect of GHBP on GH action, the oxidation rates of cultures with addition of 1.5 nmol/L GH, 1 nmol/L GHBP, or both were measured. Each bar is the mean of triplicate measurements in one experiment, which was repeated twice. *, P 5 0.047 vs. control.

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 b-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 NAD1. There is evidence that some of the key enzymes in b-oxidation are inhibited by acetyl-CoA and elevated NADH/NAD1 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 b-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 b-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 b-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 b-oxidation, and provide the first evidence that the hormone directly stimulates b-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. Acknowledgments We thank Erefili Peters, Irit Markus, and Nathan Doyle for excellent technical assistance. We also thank Dr. Michael Waters for generously providing the GHR antibodies, Dr. William Wood for GHBP, and Dr. Stewart Purvis-Smith for the fibroblast.

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