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APPETITE, OBESITY, AND DIGESTION

Role of adrenomedullin system in lipid metabolism and its signaling mechanism in cultured adipocytes

Departments of ¹Hypertension and Cardiorenal Medicine, and ²Laboratory Medicine, Dokkyo Medical University, Mibu, Tochigi, Japan; and ³Department of Medicine and ⁴Research Institute, National Cardiovascular Center, Fujishirodai, Suita, Osaka, Japan

Submitted 2 June 2008 ; accepted in final form 4 August 2008

	ABSTRACT

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We investigated the levels of adrenomedullin (AM) system during the process of preadipocyte differentiation and its role in lipid metabolism and cellular signaling mechanism in differentiated adipocytes. We cultured rat preadipocytes and measured the following during the process of differentiation: two molecular forms of AM in the culture medium using a specific immunoradiometric assay and gene expression of AM and its receptor component using RT-PCR analysis. In differentiated adipocytes, we measured the effects of AM on the intracellular cAMP level, lipolysis, glucose incorporation, and the protein levels. Two molecular forms of AM were secreted into the medium, and the AM-mature/AM-total ratio was increased after 6 days of differentiation. Cultured rat preadipocytes highly expressed the genes of AM and its receptor components at day 1, and they increased at day 10. Administration of AM to preadipocytes increased the number of Oil Red O-positive adipocytes and spectrophotometric absorbance of Oil Red O. AM dose dependently increased cAMP level and lipolysis, and its effect was blocked by CGRP(8-37). Isoproterenol increased lipolysis, and AM had additive effects on isoproterenol-induced lipolysis. KT5720 and U0126 significantly inhibited the AM-induced lipolysis, whereas KT5720, but not U0126, significantly inhibited the isoproterenol-induced lipolysis. AM increased glucose incorporation and its effect was blocked by wortmannin. Western blot analysis revealed that AM increased phospho PKA, ERK, and Akt. These results indicate that AM and its receptor component are highly expressed in cultured adipocytes and may play a role in lipid metabolism via a different signaling pathway.

adipocyte; calcitonin receptor like receptor; receptor activity-modifying proteins; lipolysis

Recent studies demonstrated that rat adipose tissue contains AM mRNA (14, 23), and adipose tissue obtained from rats fed a high-fat diet expresses greater amounts of AM than that obtained from rats fed a normal diet (2). In addition, studies in humans showed that plasma AM correlates with body mass index, and circulating AM is elevated in overweight patients with essential hypertension (34) and is decreased with a reduction of body weight by a hypocaloric diet (18), suggesting a role of AM in human adipose tissue.

However, the molecular form of AM and gene expression pattern of AM and its receptor system (CLR and RAMPs) in differentiating adipocytes are scarcely investigated. Furthermore, the role of AM in lipid metabolism in differentiated adipocytes is not fully understood. We, therefore, investigated the levels of different molecular forms of AM and the mRNA expression of AM and its receptor system during the process of preadipocyte differentiation. We also examined the role of AM in lipolysis and glucose incorporation in differentiated adipocytes and its cellular signaling mechanism.

	MATERIALS AND METHODS

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All procedures were in accordance with our institutional guidelines for animal research, and our experiments were approved by the Dokkyo Medical University Animal Care Committee. The investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health.

Cell culture. Rat preadipose cells (MK427) were purchased from the Takara (Osaka, Japan) and were grown in DMEM supplemented with 10% FBS at 37°C in an atmosphere of 5% CO₂. Confluent cells were allowed to differentiate into adipocytes in DMEM containing 10% FBS supplemented with 2.5 µM dexamethasone, 10 µg/ml insulin, and 0.5 mM 3-isobutyl-1-methylxanthine (differentiation medium) for 48 h and then with postdifferentiation medium containing 10% FBS and insulin, which was changed every other day.

Oil red O staining. Adipogenesis was monitored by morphological examination of the cellular accumulation of lipid droplets and Oil Red O staining. Confluent rat preadipocytes were treated for 2 days with DMEM supplemented with 10% FBS and then treated with differentiation medium (day 0). On days 0, 3, 7, and 10, adipocytes were fixed with 10% formaldehyde, washed with PBS, and stained with Oil Red O (0.3% in 60% isopropanol), followed by extensive washes, and the stained triglyceride droplets were visualized and photographed.

Assay for two molecular forms of AM. One milliliter of the conditioned medium of the adipocytes incubated in a 10-cm dish was collected at 2, 4, 6, 8, and 10 days after the cells were transferred to differentiation medium (32). Each sample of conditioned medium was boiled for 10 min after the addition of one-tenth volume of 1 mol/l acetic acid to inactivate intrinsic proteases. After cooling, the boiled medium was evaporated to dryness in a vacuum. AM-mature and AM-total (AM-mature+AM-glycine) in the culture medium were measured by immunoradiometric assays using specific kits (AM mature RIA Shionogi, AM RIA Shionogi, Shionogi, Osaka, Japan) with some modifications as previously reported (30). The amount of AM-glycine, the inactive form of AM, was calculated using the following equation: (AM-glycine) = (AM-total) – (AM-mature). The assay's minimal detectable concentration of AM-m or AM-T was 0.5 pmol/l for both kits (30).

Quantification of messenger RNA (mRNA). Total RNA from adipocytes was extracted using the acid guanidinium thiocyanate-phenol-chloroform method, and first-strand complementary DNA was synthesized, as previously reported (29). The gene expression levels of AM, CLR, RAMP2, RAMP3, PPAR leptin, adiponectin, FAS, IR, ISR-1, AP2, GLUT4, peptidyl-glycine alpha-amidating monooxygenase (PAM), and GAPDH in the adipocytes were determined by real-time quantitative RT-PCR (qRT-PCR) with the use of ABI 7700 and specific primers, as reported previously (25). Quantification of each mRNA was performed using the following formula: amount of original template of each molecule/amount of original template of GAPDH.

Quantification of Oil Red O. To investigate the effect of AM on adipogenesis, confluent rat preadipocytes were treated for 2 days with differentiation medium with and without AM (10^–6 M) and then with postdifferentiation medium containing 10% FBS and insulin with and without AM (10^–6 M), which was changed every other day. On days 5 and 7, adipocytes were fixed with 10% formaldehyde, washed with PBS, and stained with Oil Red O (0.3% in 60% isopropanol), followed by extensive washes. The stained triglyceride droplets were quantified using an adipogenesis assay kit (Chemicon, Temecula, CA).

Assay for cAMP and cGMP. Differentiated adipocytes incubated in 24-well plates were washed two times with prewarmed PBS and were switched to 0.5 ml of Dulbecco's balanced salt solution containing 2% BSA and 4.5 mg/ml glucose. Following each treatment of adipocytes with various concentrations of AM with or without CGRP[8-37] or AM[22-52] in the presence of 0.5 nM 3-isobutyl-1 methyl-xanthine, the medium was removed, and the cellular extract was obtained with the use of cold 70% ethanol (27). The incubation time was 10 min. Each ethanol sample was evaporated in a vacuum until dry. The eluate was dissolved in RIA buffer. The RIA for cAMP and cGMP was performed with a RIA kit (cyclic AMP and GMP assay kit; Yamasa Shoyu, Chiba, Japan), as previously reported (29).

Lipolysis. Differentiated adipocytes incubated in 24-well plates were washed two times with prewarmed PBS and were switched to 0.5 ml of Dulbecco's balanced salt solution containing 2% BSA and 4.5 mg/ml glucose (lipolysis buffer) (20). After incubation for 1.5 h, the buffer was changed to fresh lipolysis buffer with or without inhibitor, and the cells were further incubated for 1 h. Then, in the presence or absence of AM and/or isoproterenol, the cells were further incubated for 1 h, 180 µl of buffer was collected, and the glycerol concentration was determined using the tryglyceride E-test Wako (Wako Chemical, Tokyo, Japan).

Glucose incorporation measurement using 2-deoxy-D-[1-³H]glucose. The differentiated adipocytes incubated in 24-well plates were washed two times with prewarmed PBS and were changed to 0.5 ml of DMEM containing 0.1% FBS and further incubated overnight h at 37°C. Next, the cells were washed with PBS and changed to Krebs-Ringer phosphate buffer with or without inhibitor. After incubation for 1 h, the buffer was changed to Krebs-Ringer phosphate buffer with or without inhibitor in the presence or absence of AM or insulin, and the incubation was continued for 1 h. Then, 2-deoxy-D-[1-³H]glucose was added, and the cells were subsequently incubated for 30 min. The cells were washed twice with PBS and then solubilized with 0.1% SDS in 1 N NaOH. The radioactivity incorporated into the cells was measured with a liquid scintillation counter (1).

Western blot analysis. The differentiated adipocytes incubated in 60-mm plates were washed two times with prewarmed PBS and were changed to 0.5 ml of DMEM containing 0.1% FBS and further incubated for 24 h at 37°C. Next, the cells were washed with PBS and changed to Krebs-Ringer phosphate buffer with or without inhibitor. After incubation for 1 h, the buffer was changed to the Krebs-Ringer phosphate buffer with or without inhibitor in the presence or absence of AM, isoproterenol, or insulin, and the cells were further incubated for 10 min. The cells were then lysed, and immunoblot analysis was performed by using antibodies against PKA, phosphoPKA, Akt, and phosphoAkt, as described previously (33). Peroxidase-labeled proteins were detected with the enhanced chemiluminescence detection system (Amersham International, Little Chalfont, UK) on X-ray film, and the results were quantified by densitometry.

Statistical analysis. All data are expressed as means ± SD. Multiple comparisons were performed by one-way ANOVA followed by by Bonferroni's test. P values of less than 0.05 were considered to indicate statistical significance.

	RESULTS

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Time-course of adipocyte differentiation. Oil Red O staining of the adipocytes at days 1, 3, 7, and 10 is shown in Fig. 1. There were no Oil Red O-positive adipocytes on day 1. On day 3, Oil Red O-positive cells appeared; however, the size of the droplets was small. On day 7, Oil Red O-positive adipocytes increased, and the size of the droplets was also enlarged. On day 10, Oil Red O-positive cells with large droplets further increased. At this stage, the expression of adipocyte-specific genes, including PPAR, FAS, IR, ISR-1, leptin, adiponectin, AP2, and GLUT4, was detected by real-time PCR analysis.

View larger version (114K): [in this window] [in a new window]   Fig. 1. Oil Red O staining of each stage of rat preadipocytes during differentiation. Cells were fixed and stained with Oil Red O at each indicated time (days 1, 3, 7, and 10) during differentiation.

View larger version (23K): [in this window] [in a new window]   Fig. 2. Line graph shows the time course of the secretion of two molecular forms of adrenomedullin (AM) into the medium (A) and the AM-mature/AM-total ratio (B) during preadipocyte differentiation. The medium was replaced every other day (days 2, 4, 6, 8, and 10), and the medium was collected just before medium replacement. AM-mature and AM-glycine were measured by immunoradiometric assays. Solid circles show AM-mature, and open squares show AM-glycine. Values are expressed as means ± SD; n = 10. **P < 0. 01 vs. 2 days, ***P < 0.001 vs. 2 days.

View larger version (18K): [in this window] [in a new window]   Fig. 3. Bar graph shows the time course of mRNA expression of AM (A), calcitonin receptor-like receptor (CLR) (B), receptor activity-modifying proteins 2 (RAMP2) (C), RAMP3 (D), and peptidyl-glycine alpha-amidating monooxygenase (PAM) (E) in preadipocytes during differentiation (days 1, 5, and 10). Quantitative analysis of AM, CLR, RAMP2, RAMP3, and PAM mRNA levels normalized by the GAPDH mRNA level. Data are expressed as means ± SD. *P < 0.05 vs. 1 day, ***P < 0.001 vs. 1 day, P < 0.001 vs. 5 days.

View larger version (56K): [in this window] [in a new window]   Fig. 4. Oil Red O staining and Oil Red O absorbance of rat preadipocytes during differentiation. Cells were fixed and stained with Oil Red O at day 7. A and B: magnification is x10. C and D: magnification is x200. Oil Red O absorbance was higher in AM-treated preadipocytes than in the control. E: bar graph shows the quantitative analysis of leptin (F), PPAR (G), and adiponectin (H) mRNA levels normalized by the GAPDH mRNA level. *P < 0.05 vs. control.

View larger version (21K): [in this window] [in a new window]   Fig. 5. Values are expressed as means ± SD; n = 4. A: bar graph shows the dose-dependent effect of AM (10–10 to 10–6 M) on the intracellular cAMP level in adipocytes. *P < 0.05 vs. control. **P < 0.01 vs. control. B: bar graph shows the dose-dependent effect of AM (10–10 to 10–6 M) on the intracellular cGMP level in adipocytes. C: bar graph shows the effect of AM (10–7 M) on the intracellular cAMP level and antagonistic effects of CGRP[8-37] (10–7 to 10–6 M) or AM[22-52] (10–7 to 10–6 M) on AM (10–7 M) -induced intracellular cAMP level in adipocytes. *P < 0.05 vs. Cont; **P < 0.01 vs. Cont; P < 0.05 vs. AM 10–7 M; P < 0.01 vs. AM 10–7 M. D: bar graph shows the dose-dependent effect of AM (10–9 to 10–6 M) on the glycerol level in adipocytes. *P < 0.05 vs. control; **P < 0.01 vs. control. E: line graph shows the dose-dependent effect of isoproterenol (10–10 to 10–6 M) and combined effects of isoproterenol (10–10 to 10–6 M) and AM (10–7 M) on the glycerol levels in adipocytes. Open circles show isoproterenol, and solid circles show AM + isoproterenol. *P < 0.01 vs. isoproterenol, P < 0.01 vs. control. F: bar graph shows the dose-dependent effect of AM (10–7 M) on the glycerol level and antagonistic effects of CGRP[8–37] (10–7 to 10–6 M) or AM[22–52] (10–7 to 10–6 M) on AM (10–7 M)-induced glycerol level in adipocytes. *P < 0.05 vs. Cont, **P < 0.01 vs. Cont, P < 0.05 vs. AM10–7 M, P < 0.01 vs. AM10–7 M.

To investigate the intracellular signaling mechanisms responsible for the effects of AM on lipolysis, we evaluated the effects of various inhibitors on AM-induced lipolysis. KT5720, a PKA inhibitor, significantly decreased the AM-induced lipolysis (Fig. 6A). Isoproterenol-induced lipolysis was also inhibited by KT5720 (Fig. 6D). U0126, an ERK inhibitor, significantly reduced basal lipolysis, indicating that the MEK/ERK pathway regulated basal lipolysis in these cells (Fig. 6B). Interestingly, the AM-induced lipolytic effect was completely inhibited by U0126 (Fig. 6B), whereas U0126 attenuated the isoproterenol-induced lipolysis slightly and not significantly (Fig. 6E). The lipolytic effects of AM or isoproterenol were not inhibited by wortmannin, a PI-3kinase inhibitor (Fig. 6C, F).

View larger version (32K): [in this window] [in a new window]   Fig. 6. A–F: Values are expressed as means ± SD; n = 4. A: bar graph shows the dose-dependent effect of AM (10–7 to 10–6 M) on the glycerol level and the effects of KT5720 (2 x 10–5) on AM (10–7 to 10–6 M)-induced glycerol level. *P < 0.01 vs. Cont, P < 0.01 vs. AM 10–7 M, P < 0.01 vs. AM 10–6 M. B: bar graph shows the dose-dependent effect of AM (10–7 to 10–6 M) on the glycerol level and the effects of U0126 (10–4) on AM (10–7 to 10–6 M)-induced glycerol level. *P < 0.01 vs. Cont. P < 0.01 vs. AM 10–7 M. P < 0.01 vs. AM 10–6 M. C: bar graph shows the dose-dependent effect of AM (10–7 to 10–6 M) on the glycerol level and the effects of wortmannin (10–7 M) on AM (10–7 to 10–6 M)-induced glycerol level. *P < 0.01 vs. Cont. D: bar graph shows the dose-dependent effect of isoproterenol (10–8 to 10–7 M) on the glycerol level and the effects of KT5720 (2 x 10–5 M) on isoproterenol (10–8 to 10–7 M)-induced glycerol level. *P < 0.01 vs. Cont; P < 0.01 vs. ISP10–8 M; P < 0.01 vs. ISP10–7 M. E: bar graph shows the dose-dependent effect of isoproterenol (10–8 to 10–7 M) on the glycerol level and the effects of U0126 (10–4 M) on isoproterenol (10–8 to 10–7 M)-induced glycerol level. *P < 0.01 vs. Cont. F: bar graph shows the dose-dependent effect of isoproterenol (10–8 to 10–7 M) on the glycerol level and the effects of wortmannin (10–7 M) on isoproterenol (10–8 to 10–7 M)-induced glycerol level. *P < 0.01 vs. Cont. G: cell lysates were subjected to immunoblot analysis using the anti-total PKA and anti-phospho-PKA antibodies. Adipocytes were treated with or without AM (10–7 M) or isoproterenol (10–8 M) in the presence or absence of KT5720 (2 x 10–5 M), U0126 (10–4 M), or wortmannin (10–7 M). H: cell lysates were subjected to immunoblot analysis using the anti-ERK1/2 and anti-phospho-ERK1/2 antibodies. Adipocytes were treated with or without AM (10–7 M) or isoproterenol (10–8 M) in the presence or absence of KT5720 (2 x 10–5 M), U0126 (10–4 M), or wortmannin (10–7 M).

Effect of AM on glucose incorporation. Fig. 7A shows the effect of AM on glucose incorporation. Glucose incorporation was evaluated by measuring 2-deoxy-D-[1-³H]glucose levels in the adipocytes. AM at 10^–7 M significantly increased glucose incorporation and AM dose dependently increased the glucose incorporation at concentrations of 10^–7 to 10^–6 M (Fig. 7A). Insulin treatment significantly increased glucose incorporation at 10^–8 M, and insulin dose dependently increased the glycerol level at concentrations of 10^–8 to 10^–6 M (Fig. 7B).

View larger version (23K): [in this window] [in a new window]   Fig. 7. A–D: values are expressed as means ± SD; n = 4. A: bar graph shows the dose-dependent effect of AM (10–9 to 10–6 M) on 2-deoxy-D-[1-3H]glucose incorporation level in adipocytes. *P < 0.05 vs. control, **P < 0.01 vs. Cont. B: bar graph shows the dose-dependent effect of insulin (10–9 to 10–6 M) on 2-deoxy-D-[1-3H]glucose incorporation level in adipocytes. *P < 0.05 vs. control, **P < 0.01 vs. Cont. C: Bar graph shows the dose-dependent effect of AM (10–7 to 10–6 M) on 2-deoxy-D-[1-3H]glucose incorporation level and the effects of wortmannin (10–7 M) on AM-(10–7 to 10–6 M) induced 2-deoxy-D-[1-3H]glucose incorporation level. *P < 0.05 vs. Cont, **P < 0.01 vs. Cont, P < 0.01 vs. AM 10–7, P < 0.01 vs. AM 10–6 M. D: bar graph shows the dose-dependent effect of insulin (10–7 to 10–6 M) on 2-deoxy-D-[1-3H]glucose incorporation level and the effects of wortmannin (10–7) on insulin - (10–7 to 10–6 M) induced 2-deoxy-D-[1-3H]glucose incorporation level. **P < 0.01 vs. Cont,P < 0.01 vs. Insulin10–7 M, P < 0.01 vs. Insulin 10–6 M. E: cell lysates were subjected to immunoblot analysis using the anti-total Akt and anti-phospho-Akt antibodies. Adipocytes were treated with or without AM (10–7 M) or insulin (10–8 M) in the presence or absence of wortmannin (10–7 M).

AM also caused a significant increase in Akt phosphorylation in adipocytes, as insulin did, and this effect was abolished by wortmannin (Fig. 7E), but not KT5720 or U0126 (data not shown). Insulin-induced Akt phosphorylation was inhibited by genistein (data not shown).

	DISCUSSION

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In the present study, we examined the role of AM and its receptor system (CLR, RAMP2, and RAMP3) in the differentiation of rat cultured preadipocytes, and showed that the mRNAs of AM, CLR, RAMP2, and RAMP3 were highly expressed in cultured rat preadipocytes. Preadipocytes secreted a considerable amount of AM, most of which was AM-glycine. With the differentiation of preadipocytes, the AM-mature/AM-total ratio increased with an increase of mRNA levels of PAM, and differentiated adipocytes mainly secreted AM-mature. In differentiated adipocytes, AM had effects on lypolysis and glucose incorporation, and these effects were mediated by different cellular signaling mechanisms. Our data suggest that endogenous local AM secreted by adipocytes may modulate the adipogenesis and lipid metabolism.

AM is strongly expressed in the cardiovascular system (12), and previous studies have demonstrated that AM exerts cardiorenoprotective effects via pleiotropic effects, including antioxidative stress, angiogenic, and antiproliferative effects (8, 22, 28). However, reported studies of the expression of AM in preadipocytes during differentiation and in adipocytes are limited and inconsistent. Li et al. (14) studied the expression of AM and resistin in 3T3-L1 preadipocytes and adipocytes by Northern blot analysis and showed that AM mRNA was expressed in 3T3-L1 preadipocytes but was undetectable in adipocytes, whereas resistin mRNA was not detected in 3T3-L1 preadipocytes but was expressed in adipocytes. It was also reported that preadipocytes at early differentiation stages secreted AM, but mature adipocytes did not secrete AM (34). In contrast, a recent study showed that the expression of AM mRNA increased in human mesenchymal stem cell-derived adipocytes at days 4, 8, 12, and 18 (16). Fukai et al. (2) showed that AM mRNA expression in a mouse preadipocyte cell line (3T3-L1) transiently decreased by day 3, returned to the basal level by day 6, and then increased by day 9 during preadipocyte differentiation. Our observations that rat preadipocytes expressed the AM gene at day 1, and AM mRNA levels increased at day 5, and this increase was maintained through day 10 seems to be consistent with the latter reports. This increased expression of AM and receptor component in undifferentiated preadipocytes suggests that AM may play a role in adipogenensis. Indeed, administration of AM increased the number of Oil Red O-positive cells and the spectrophotometric absorbance of intracellular Oil Red O, suggesting the role of AM in adipogenesis. Because the role of AM in adipogenesis appears complicated (5), further studies will be required to elucidate the exact role of AM in adipogenesis. The reasons for the discrepancies of the reported results regarding AM mRNA expression during adipocyte differentiation remain unknown at present; however, these inconsistent data may reflect differences in cell types, species, and culture conditions. Further studies will be necessary to resolve these discrepancies.

In the present study, we analyzed the molecular forms of AM that were secreted by preadipocytes and differentiated adipocytes. In the biosynthesis of AM, AM precursor is converted to COOH-terminal glycine-extended AM (AM-glycine), which is an inactive intermediate form of AM. Subsequently, inactive AM-glycine is converted to the active form of AM-mature by PAM, an amidation enzyme (13). Recent studies showed that two molecular forms of plasma AM circulate in human plasma (13), the major circulating form of AM is AM-glycine, and both plasma AM-mature and AM-glycine levels are increased in parallel in patients with hypertension and heart failure (6, 24, 26). In the present study, we first showed that the major molecular form of AM in cultured preadipocytes was AM-glycine and that the AM-mature/AM-glycine ratio increased during differentiation, and differentiated adipocytes mainly secreted AM-mature rather than AM-glycine. This higher AM-mature/AM-total ratio is associated with an increase of mRNA levels of PAM. Interestingly, the dissociation between mRNA levels of AM and AM total levels in the medium was observed. The dissociation between the mRNA levels and peptide levels have been often observed. Indeed, we previously reported the dissociation between the mRNA levels and peptide levels of AM in pressure-overloaded cardiac hypertrophy (21, 39). Although the exact mechanism of dissociation between the mRNA levels and protein levels still remains unknown, the process of translation and/or processing of AM precursor may be involved. Increased AM-mature/AM-total ratio and receptor mRNA levels in differentiated adipocytes may lead to enhancement of AM signaling. In addition, AM-mature levels in the medium might be underestimated because AM bound to the receptor in the autocrine fashion could not be secreted into the medium.

The AM level per 10⁵ cells was 250 fmol/12 h in adipocytes, which was almost consistent with the previous report by Li et al. (15), who stimulated 3T3-L1cells adipocytes by TNF- (15). However, these values are considerably higher compared with those in cultured rat endothelial cells (20 fmol/12 h), vascular smooth muscle cells (5 fmol/12 h), cardiac fibroblasts (35 fmol/12 h), myocytes (20 fmol/12 h), human skin fibroblasts (100 fmol/12 h), and human lung fibroblasts (100 fmol/12 h) (9, 10, 15, 32, 36). Thus, the higher levels of the active form of AM in the medium of differentiated adipocytes suggest the role of endogenous AM in lipid metabolism in adipocytes.

Harmancey et al. (4) recently examined the effect of AM on lipolysis in the 3T3-F442A cell line. They showed that AM inhibited isoproterenol-stimulated lipolysis by a nitric oxide (NO)-dependent mechanism. However, the effect was cGMP independent, as it occurred in the presence of an NO-sensitive guanylate cyclase inhibitor. Liquid chromatography-tandem mass spectrometry showed that AM-produced NO-oxidized isoproterenol to generate its aminochrome, namely isoprenochrome, suggesting that AM downregulates lipolysis in adipocytes through the chemical modification of a β-agonist by producing NO. In contrast to the finding of Harmancey and colleagues, we found that even AM monotreatment dose dependently increased lipolysis, and cotreatment with isoproterenol showed an additive effect on lipolysis. An additive effect of AM and isoproterenol on lipolysis was observed at their lowest concentrations and the maximal lipolytic effects of isoproterenol were amplified by the addition of 10^–7 M AM, suggesting that the cellular signaling pathways of the two compounds may be different, although both AM and isoproterenol are known to elevate intracellular cAMP levels. Indeed, the isoproterenol-induced lipolytic effect was PKA dependent, because the PKA inhibitor abolished the isoproterenol-induced lipolytic effect. The AM-induced lipolytic effect was also inhibited by KT5720. However, U0126, which directly prevents MEK from phosphorylating ERK, completely inhibited the AM-induced lipolytic effect, but not the isoproterenol-induced lipolytic effect. U0126 inhibited the AM-induced phosphorylation of ERK in adipocytes without affecting the AM-induced phosphorylation of PKA. Phosphorylation of PKA is known to moderately increase hormone-sensitive lipase (HSL) hydrolytic activity and to translocate HSL onto lipid droplets (3), thereby enhancing the lipolytic activity. ERK activation also increases the phosphorylation of HSL at Ser⁶⁰⁰ and increases HSL hydrolytic activity (35). Therefore, we propose that isoproterenol-induced lipolytic effects might be mainly due to PKA-mediated translocation and activation of HSL, and AM-induced lipolytic effects might be due to an ERK-mediated increase of HSL phosphorylation and hydrolytic activity. Further studies will be required to investigate these possibilities. The question of why AM-induced lipolysis is directly linked to ERK activation, but not PKA activation, awaits further study. The discrepancy between the findings of the two studies may be due to the different effects of AM in the 3T3-F442A cell line and rat differentiated adipocytes.

The effect of insulin on glucose transport is known to be dependent upon translocation of the muscle and adipocyte glucose transporter GLUT4 from microsomes to the plasma membrane via the PI 3-kinase pathway (11, 38). This effect of insulin is known to be abolished by wortmannin, a PI 3-kinase inhibitor. In the current study, we examined the effect of AM on glucose incorporation and its signaling mechanism compared with that of insulin. AM significantly increased 2-deoxy-D-[1-³H]glucose incorporation, and this effect of AM was abolished by wortmannin, but not KT5720 or U0126. Furthermore, AM increased the phosphorylation of Akt, and this effect was also inhibited by wortmannin. Thus, we have demonstrated for the first time that AM enhances glucose transport in adipocytes through the PI-3 kinase/Akt pathway, as shown for insulin. Recently, Tsuchiya et al. (37) showed that glucose disposal was significantly increased in chronic AM-infused mice compared with control mice after a glucose tolerance test. Taken together, these results suggest that AM may improve glucose intolerance via improving glucose incorporation in adipose tissue. Further studies will be required to elucidate the exact role of AM in the metabolism of adipose tissue.

The present study demonstrated the high expression of AM and its receptor system in differentiated adipocytes. AM enhanced lipolysis and glucose incorporation via different intracellular signaling mechanisms in differentiated adipocytes. A schematic diagram depicting the signaling supported by the present study is shown in Fig. 8. Such roles of the local AM system in adipocytes might be physiologically important in the function of adult adipose tissue. Thus, our results may provide insight into the pathophysiology of obesity-related conditions such as metabolic syndrome.

View larger version (33K): [in this window] [in a new window]   Fig. 8. Our working hypothesis of autocrine role of AM on lipid metabolism and adipogenesis in adipocytes. AR, adrenergic receptor; GLUT4, glucose transporter type 4; HSL, hormone sensitive lipase; NEFA, nonesterified fatty acids; TG, triglyceride.

We and others have previously reported that plasma AM levels are increased in obese patients. We also have found that adipocytes secrete a considerable amount of AM, and AM has an effect on lipolysis and glucose incorporation. Given the potent lipolysis and glucose incorporation of AM, it may be interesting to investigate whether AM administration is beneficial in patients with severe obesity and obesity-related metabolic syndrome.

	GRANTS

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This work was supported in part by Scientific Research Grant-in-Aid 14570692 and 18590787 from the Ministry of Education, Culture, Sports, Science and Technology, by the Science Research Promotion Fund from the Promotion and Mutual Aid for Private Schools of Japan, by the Research Grant for Cardiovascular Diseases 17A-1 from the Ministry of Health, Labour and Welfare, and by the Seki Minato Prize.

	ACKNOWLEDGMENTS

 
We thank Keiko Ishikawa, Masako Minato, Noriko Suzuki, Kyoko Tabei, Fumie Yokotsuka, and Machiko Sakata for technical assistance.

	FOOTNOTES

 

Address for reprint requests and other correspondence: T. Nishikimi, Dept. of Hypertension and Cardiorenal Medicine, Dokkyo Univ. School of Medicine, Mibu, Tochigi 321-0293, Japan (e-mail: nishikim{at}dokkyomed.ac.jp)

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.

	REFERENCES

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1. Filippis A, Clark S, Proietto J. Possible role for gp160 in constitutive but not insulin-stimulated GLUT4 trafficking: dissociation of gp160 and GLUT4 localization. Biochem J 330: 405–411, 1998.[Web of Science][Medline]
2. Fukai N, Yoshimoto T, Sugiyama T, Ozawa N, Sato R, Shichiri M, Hirata Y. Concomitant expression of adrenomedullin and its receptor components in rat adipose tissues. Am J Physiol Endocrinol Metab 288: E56–E62, 2005.[Abstract/Free Full Text]
3. Greenberg AS, Shen WJ, Muliro K, Patel S, Souza SC, Roth RA, Kraemer FB. Stimulation of lipolysis and hormone-sensitive lipase via the extracellular signal-regulated kinase pathway. J Biol Chem 276: 45456–45461, 2001.[Abstract/Free Full Text]
4. Harmancey R, Senard JM, Pathak A, Desmoulin F, Claparols C, Rouet P, Smih F. The vasoactive peptide adrenomedullin is secreted by adipocytes and inhibits lipolysis through NO-mediated beta-adrenergic agonist oxidation. FASEB J 19: 1045–1057, 2005.[Abstract/Free Full Text]
5. Harmancey R, Senard JM, Rouet P, Pathak A, Smih F. Adrenomedullin inhibits adipogenesis under transcriptional control of insulin. Diabetes 56: 553–563, 2007.[Abstract/Free Full Text]
6. Hirayama N, Kitamura K, Imamura T, Kato J, Koiwaya Y, Tsuji T, Kangawa K, Eto T. Molecular forms of circulating adrenomedullin in patients with congestive heart failure. J Endocrinol 160: 297–303, 1999.[Abstract]
7. Ishimitsu T, Nishikimi T, Saito Y, Kitamura K, Eto T, Kangawa K, Matsuo H, Omae T, Matsuoka H. Plasma levels of adrenomedullin, a newly identified hypotensive peptide, in patients with hypertension and renal failure. J Clin Invest 94: 2158–2161, 1994.[Web of Science][Medline]
8. Ishimitsu T, Ono H, Minami J, Matsuoka H. Pathophysiologic and therapeutic implications of adrenomedullin in cardiovascular disorders. Pharmacol Ther 111: 909–927, 2006.[CrossRef][Web of Science][Medline]
9. Isumi Y, Minamino N, Katafuchi T, Yoshioka M, Tsuji T, Kangawa K, Matsuo H. Adrenomedullin production in fibroblasts: its possible function as a growth regulator of Swiss 3T3 cells. Endocrinology 139: 2552–2563, 1998.[Abstract/Free Full Text]
10. Isumi Y, Shoji H, Sugo S, Tochimoto T, Yoshioka M, Kangawa K, Matsuo H, Minamino N. Regulation of adrenomedullin production in rat endothelial cells. Endocrinology 139: 838–846, 1998.[Abstract/Free Full Text]
11. Kanzaki M. Insulin receptor signals regulating GLUT4 translocation and actin dynamics. Endocr J 53: 267–293, 2006.[CrossRef][Web of Science][Medline]
12. Kitamura K, Kangawa K, Kawamoto M, Ichiki Y, Nakamura S, Matsuo H, Eto T. Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem Biophys Res Commun 192: 553–560, 1993.[CrossRef][Web of Science][Medline]
13. Kitamura K, Kato J, Kawamoto M, Tanaka M, Chino N, Kangawa K, Eto T. The intermediate form of glycine-extended adrenomedullin is the major circulating molecular form in human plasma. Biochem Biophys Res Commun 244: 551–555, 1998.[CrossRef][Web of Science][Medline]
14. Li Y, Totsune K, Takeda K, Furuyama K, Shibahara S, Takahashi K.Decreased expression of adrenomedullin during adipocyte-differentiation of 3T3–L1 cells. Hypertens Res 26 Suppl: S41–S44, 2003.[CrossRef][Web of Science][Medline]
15. Li Y, Totsune K, Takeda K, Furuyama K, Shibahara S, Takahashi K. Differential expression of adrenomedullin and resistin in 3T3–L1 adipocytes treated with tumor necrosis factor-alpha. Eur J Endocrinol 149: 231–238, 2003.[Abstract]
16. Li Y, Zhang Y, Furuyama K, Yokoyama S, Takeda K, Shibahara S, Takahashi K. Identification of adipocyte differentiation-related regulatory element for adrenomedullin gene repression (ADRE-AR) in 3T3–L1 cells. Peptides 27: 1405–1414, 2006.[CrossRef][Web of Science][Medline]
17. McLatchie LM, Fraser NJ, Main MJ, Wise A, Brown J, Thompson N, Solari R, Lee MG, Foord SM. RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature 393: 333–339, 1998.[CrossRef][Web of Science][Medline]
18. Minami J, Nishikimi T, Ishimitsu T, Makino Y, Kawano Y, Takishita S, Kangawa K, Matsuoka H. Effect of a hypocaloric diet on adrenomedullin and natriuretic peptides in obese patients with essential hypertension. J Cardiovasc Pharmacol 36 Suppl 2: S83–S86, 2000.[CrossRef][Web of Science][Medline]
19. Miyao Y, Nishikimi T, Goto Y, Miyazaki S, Daikoku S, Morii I, Matsumoto T, Takishita S, Miyata A, Matsuo H, Kangawa K, Nonogi H. Increased plasma adrenomedullin levels in patients with acute myocardial infarction in proportion to the clinical severity. Heart 79: 39–44, 1998.[Abstract/Free Full Text]
20. Morito S, Yaguchi K, Imada M, Tachikawa C, Nomura M, Moritani S, Igarashi M, Yokogawa K, Miyamoto K. Insulin signaling in adipocytes differentiated from mouse stromal MC3T3–G2/PA6 cells. Biol Pharm Bull 28: 2040–2045, 2005.[CrossRef][Web of Science][Medline]
21. Morimoto A, Nishikimi T, Yoshihara F, Horio T, Nagaya N, Matsuo H, Dohi K, Kangawa K. Ventricular adrenomedullin levels correlate with the extent of cardiac hypertrophy in rats. Hypertension 33: 1146–1152, 1999.[Abstract/Free Full Text]
22. Nagaya N, Mori H, Murakami S, Kangawa K, Kitamura S. Adrenomedullin: angiogenesis and gene therapy. Am J Physiol Regul Integr Comp Physiol 288: R1432–R1437, 2005.[Abstract/Free Full Text]
23. Nambu T, Arai H, Komatsu Y, Yasoda A, Moriyama K, Kanamoto N, Itoh H, Nakao K. Expression of the adrenomedullin gene in adipose tissue. Regul Pept 132: 17–22, 2005.[CrossRef][Web of Science][Medline]
24. Nishikimi T, Asakawa H, Iida H, Matsushita Y, Shibasaki I, Tadokoro K, Mori Y, Mori H, Mochizuki Y, Okamura Y, Miyoshi S, Kangawa K, Matsuoka H. Different secretion patterns of two molecular forms of cardiac adrenomedullin in pressure- and volume-overloaded human heart failure. J Card Fail 10: 321–327, 2004.[CrossRef][Web of Science][Medline]
25. Nishikimi T, Hagaman JR, Takahashi N, Kim HS, Matsuoka H, Smithies O, Maeda N. Increased susceptibility to heart failure in response to volume overload in mice lacking natriuretic peptide receptor-A gene. Cardiovasc Res 66: 94–103, 2005.[Abstract/Free Full Text]
26. Nishikimi T, Horio T, Kohmoto Y, Yoshihara F, Nagaya N, Inenaga T, Saito M, Teranishi M, Nakamura M, Ohrui M, Kawano Y, Matsuo H, Ishimitsu T, Takishita S, Matsuoka H, Kangawa K. Molecular forms of plasma and urinary adrenomedullin in normal, essential hypertension and chronic renal failure. J Hypertens 19: 765–773, 2001.[CrossRef][Web of Science][Medline]
27. Nishikimi T, Horio T, Yoshihara F, Nagaya N, Matsuo H, Kangawa K. Effect of adrenomedullin on cAMP and cGMP levels in rat cardiac myocytes and nonmyocytes. Eur J Pharmacol 353: 337–344, 1998.[CrossRef][Web of Science][Medline]
28. Nishikimi T, Matsuoka H. Cardiac adrenomedullin: its role in cardiac hypertrophy and heart failure. Curr Med Chem Cardiovasc Hematol Agents 3: 231–242, 2005.[CrossRef][Medline]
29. Nishikimi T, Miyata A, Horio T, Yoshihara F, Nagaya N, Takishita S, Yutani C, Matsuo H, Matsuoka H, Kangawa K. Urocortin, a member of the corticotropin-releasing factor family, in normal and diseased heart. Am J Physiol Heart Circ Physiol 279: H3031–H3039, 2000.[Abstract/Free Full Text]
30. Nishikimi T, Mori Y, Kobayashi N, Tadokoro K, Wang X, Akimoto K, Yoshihara F, Kangawa K, Matsuoka H. Renoprotective effect of chronic adrenomedullin infusion in Dahl salt-sensitive rats. Hypertension 39: 1077–1082, 2002.[Abstract/Free Full Text]
31. Nishikimi T, Saito Y, Kitamura K, Ishimitsu T, Eto T, Kangawa K, Matsuo H, Omae T, Matsuoka H. Increased plasma levels of adrenomedullin in patients with heart failure. J Am Coll Cardiol 26: 1424–1431, 1995.[Abstract]
32. Nishikimi T, Tadokoro K, Akimoto K, Mori Y, Ishikawa Y, Ishimura K, Horio T, Kangawa K, Matsuoka H. Response of adrenomedullin system to cytokine in cardiac fibroblasts-role of adrenomedullin as an antifibrotic factor. Cardiovasc Res 66: 104–113, 2005.[Abstract/Free Full Text]
33. Nishikimi T, Tadokoro K, Wang X, Mori Y, Asakawa H, Akimoto K, Yoshihara F, Horio T, Minamino N, Matsuoka H. Cerivastatin, a hydroxymethylglutaryl coenzyme A reductase inhibitor, inhibits cardiac myocyte hypertrophy induced by endothelin. Eur J Pharmacol 453: 175–181, 2002.[CrossRef][Web of Science][Medline]
34. Paulmyer-Lacroix O, Desbriere R, Poggi M, Achard V, Alessi MC, Boudouresque F, Ouafik L, Vuaroqueaux V, Labuhn M, Dutourand A, Grino M. Expression of adrenomedullin in adipose tissue of lean and obese women. Eur J Endocrinol 155: 177–185, 2006.[Abstract/Free Full Text]
35. Su CL, Sztalryd C, Contreras JA, Holm C, Kimmel AR, Londos C. Mutational analysis of the hormone-sensitive lipase translocation reaction in adipocytes. J Biol Chem 278: 43615–43619, 2003.[Abstract/Free Full Text]
36. Sugo S, Minamino N, Shoji H, Kangawa K, Matsuo H. Effects of vasoactive substances and cAMP related compounds on adrenomedullin production in cultured vascular smooth muscle cells. FEBS Lett 369: 311–314, 1995.[CrossRef][Web of Science][Medline]
37. Tsuchiya K, Yoshimoto T, Suzuki N, Iwashima F, Sakai H, Hirata Y. Adrenomedullin ameliorates high-fat diet-induced obesity and glucose intolerance in mice (Abstract). J Hypertens 24: S96, 2006.
38. Watson RT, Kanzaki M, Pessin JE. Regulated membrane trafficking of the insulin-responsive glucose transporter 4 in adipocytes. Endocr Rev 25: 177–204, 2004.[Abstract/Free Full Text]
39. Yoshihara F, Nishikimi T, Horio T, Yutani C, Nagaya N, Matsuo H, Ohe T, Kangawa K. Ventricular adrenomedullin concentration is a sensitive biochemical marker for volume and pressure overload in rats. Am J Physiol Heart Circ Physiol 278: H633–H642, 2000.[Abstract/Free Full Text]

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