Isolation and Characterization of a Novel Proopiomelanocortin-Derived Peptide from Hemofiltrate of Chronic Renal Failure Patients
http://www.100md.com
内分泌学杂志 2005年第4期
IPF PharmaCeuticals GmbH, D-30625 Hannover, Germany
Address all correspondence and requests for reprints to: Dr. Erik Maronde, IPF PharmaCeuticals GmbH, Feodor Lynen Strasse 31, D-30625 Hannover, Germany. E-mail: erik.maronde@ipf-pharmaceuticals.de.
Abstract
We report the isolation of a novel human circulating proopiomelanocortin-derived peptide, VA-?-MSH, from hemofiltrate and its pharmacological characterization. Screening for lipolytic activity in differentiated 3T3-L1 adipocytes led to the isolation from a hemofiltrate peptide library by alternating reverse phase and cation exchange chromatography. In the course of this isolation, we also identified human ?-MSH-(1–22). We synthesized VA-?-MSH by the N-(9-fluorenyl)-methoxycarbonyl (F-moc) solid phase method and used synthetic ?-MSH-(1–22) to confirm that both isolated peptides are lipolytically active in a dose-dependent manner in differentiated 3T3-L1 adipocytes in the nanomolar range. Using cAMP ELISA, we demonstrate that stimulation with both peptides caused a strong cAMP elevation in this cell system. Furthermore, we show that the selective inhibitors of cAMP-dependent protein kinase, 8-(4-Chlorophenylthio)adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-CPT-cAMPS); N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89), significantly reduce VA-?-MSH- and ?-MSH-(1–22)-mediated lipolysis. Although isolated after its lipolytic activity on 3T3-L1 cells, this newly identified circulating human melanocortin may serve other functions in human physiology. Moreover, the fact that these peptides have been identified after a functional assay, but have been overseen in large proteomic approaches, underscores the importance of such approaches in identifying previously undescribed circulating bioactive molecules.
Introduction
THE IMPORTANCE OF peptides in the diagnosis and therapy of human disease is well accepted. Regulatory peptide hormones have been isolated from various sources, including pituitary gland (1), hypothalamus (2), and placenta (3).
Human hemofiltrate from patients with chronic renal failure, due to its comparably high concentrations of peptides and proteins, is a comprehensive source of regulatory peptides (4). The specific concentration of a given peptide compared with plasma is increased by a factor of 1000 by hemofiltration, thus increasing the probability of subsequent isolation of regulatory peptides (4).
The importance of lipolysis in the body fat and weight control system is generally understood. A large number of pharmacological approaches to the treatment of diet-induced obesity have been made recently, and one target is the control of fat stores through modulation of lipolysis and lipogenesis in white adipose tissue. One such approach is the development of ?3-adrenoceptor agonists as lipid-mobilizing drugs for the treatment of obesity (5, 6, 7). Often, cancer-induced weight loss (cachexia) is associated with increased turnover of both glycerol and nonesterified fatty acids. This differs from the situation in cancer patients without weight loss (8). These observations led Todorov et al. (9) to isolate and identify a tumor-derived, lipid-mobilizing factor and suggest a role for lipid-mobilizing substances in the control of body weight. A variety of hormones stimulate the rate of fat-mobilizing lipolysis of adipose tissue triglycerides. In humans, these include catecholamines (10), ACTH (11), TSH (12), and GH (13).
The aim of this study was to detect human circulating lipolytic peptides. For this purpose, human hemofiltrate was used as a source of the isolation and identification of such peptides. As a screening system, differentiated 3T3-L1 adipocytes were used. This cell line has been shown to exhibit many of the characteristics of normal white adipocytes, including responsiveness to insulin and lipolytic hormones (14). The bioactivity in our assay was based on lipolytic activity, measured as glycerol release from differentiated 3T3-L1 adipocytes. This assay system allowed fast and sensitive detection of lipolytic substances.
We describe the isolation and identification of a novel circulating melanocyte-stimulating peptide hormone, VA-?-MSH, from human hemofiltrate, based on a bioactivity-oriented isolation strategy. Moreover, using this isolation strategy, we found ?-MSH-(1–22) as a lipolytic circulating human peptide hormone whose existence in the circulation has long been doubted (15). We characterize the signal transduction mechanism of the lipolytic pathway of VA-?-MSH and ?-MSH-(1–22) in 3T3-L1 adipocytes and show that activation of adenylyl cyclase and cAMP-dependent protein kinase activation is the main pathway mediating the strong lipolytic effect of these circulating peptide hormones. Immunoblot analysis displayed protein expression of melanocortin receptor isoforms 2 and 5, and a possible mediation of the pharmacological effect of the isolated peptides via melanocortin receptor-5 (MCR5) is discussed.
Materials and Methods
Cell culture and differentiation
Murine 3T3-L1 fibroblasts (American Type Culture Collection, Manassas, VA) were grown in DMEM containing 10% fetal bovine serum. Cells were differentiated into adipocytes after reaching 90–100% confluence, as described previously (16). Adipocytes were used for experiments 7–9 d after the differentiation protocol was initiated.
Lipolysis
3T3-L1 adipocytes cultured in 96-well plates were transferred into fresh DMEM and treated for 5 h as described in Results. Glycerol content in the supernatants was measured using a colorimetric assay (GPO-Trinder, Sigma-Aldrich Corp., Deisenhofen, Germany). Results are expressed either as nanomoles of glycerol per well or as a percentage of the lipolytic effect of 1 μM isoproterenol, which was used as a positive control.
cAMP ELISA
Intracellular cAMP was measured using the cAMP ELISA kit (IHF GmbH, Hamburg, Germany) (17). 3T3-L1 adipocytes were treated with various concentrations of the indicated stimulants, followed by an incubation at 37 C. At the indicated time points, supernatants were removed, 70% ethanol was added to each well, and the cells were scraped into Eppendorf tubes. The tubes were dried under vacuum, and 100 μl cAMP working buffer were added to each tube. cAMP was assayed using the supplier’s protocol.
Preparation of a peptide library from human hemofiltrate
A peptide library was prepared from 20,000 liters human hemofiltrate from patients suffering from chronic renal failure, as described previously (4). Each batch of hemofiltrate is pooled from the filtration sessions of approximately 30 patients over 8 wk, given that a patient undergoes filtration three times a week, providing a total of 60–90 liters/wk. The patients were of different ages and genders and using various medications, but most were older than 50 yr of age. All subjects gave consent for the use of their hemofiltrates for research purposes. Briefly, the crude hemofiltrate was immediately cooled to 4 C, acidified to pH 3, and loaded onto a strong cation exchange column (25 x 10 cm; Fractogel TSK SP 650 M, Merck & Co., Darmstadt, Germany), followed by stepwise elution using seven buffers with increasing pH from 3.6–9. Each of the seven pH pools was subjected to a second purification step using a reverse phase column (20 x 15.5 cm; Fineline Source RPC Polymer, Amersham Biosciences, Braunschweig, Germany). Fractionation was performed by collecting 46–63 fractions/pH pool, and aliquots of each fraction were tested in the lipolysis bioassay.
Isolation of circulating human VA-?-MSH and ?-MSH- (1–22) from hemofiltrate
All gradients used for chromatographic separation were linear. HPLC fractions 12 and 13 of pH pool 3 (which was eluted with maleic acid, pH 5.0) were diluted with 10 mM HCl and loaded onto a Fineline RPC Polymer (10 x 15.5-cm) column (Amersham Biosciences). Elution was performed by an increase in acetonitrile from 0–24% for 53 min at a flow rate of 150 ml/min (see Fig. 2A). The active fraction 12 was diluted with 50 mM Na2PO4 (pH 4.5) and loaded onto a TSK SP 650(S) cation exchange column (Merck & Co.), and peptides were eluted using a gradient from 0.15–1.5 M NaCl within 45 min at a flow rate of 50 ml/min (Fig. 2B). Fractions 26–34 containing the active material were diluted with 10 mM HCl and further purified using a 47 x 300-mm Bakerbond Prep Pak C18 cartridge (Waters Corp., Milford, MA) with an increase in methanol from 20–80% in 63 min at a flow rate of 30 ml/min (Fig. 2C). Lipolytically active fractions 22 and 23 were diluted with 50 mM Na2PO4 (pH 4.5) and loaded onto a PepKat cation exchange column (20 x 150 mm; Biotek, ?stringen, Germany). A gradient with an increase from 0.075 to 0.75 M NaCl in Na2PO4 (pH 4.5) within 45 min at a flow rate of 6 ml/min was used. Active material eluted in fractions 25–30 (Fig. 2D) and was subjected to the next purification step using a Source Rp C15 (20 x 250-mm) column (Pharmacia Biotech, Freiburg, Germany). An increase in methanol in the eluent from 16–64% in 70 min at a flow rate of 4 ml/min was performed (Fig. 2E). Active fractions 41–44 were diluted with 50 mM KH2PO4 (pH 4.5) and loaded onto a PepKat (10 x 125-mm) column (Biotek). Elution was performed by a gradient increase in the KCl concentration from 0 to 1.05 M in 70 min at a flow rate of 1.8 ml/min (Fig. 2F). The active material of the sixth purification step was found in fractions 21–25 and divided into peaks I and II. Fractions 21–22, referred to as peak I, and fractions 24–25 (peak II) were individually purified using the same strategy thereafter.
FIG. 2. Purification of human VA-?-MSH from hemofiltrate. Elution profiles of a preparative Fineline RPC Polymer column (A), a TSK SP 650 (S) cation exchange column (B), a Bakerbond Prep Pak C18 column (C), a Biotek PepKat cation exchange HPLC column (20 x 125 mm; D), a Pharmacia Source C15 HPLC column (20 x 250 mm; E), a Biotek PepKat cation exchange HPLC column (10 x 125 mm; F), a YMC ODS AQ HPLC column (10 x 250 mm; G), a Biotek PepKat cation exchange HPLC column (4 x 125 mm; H), and a Vydac Rp HPLC column (4.6 x 250 mm; I). After each cation exchange chromatography step, salt was removed from aliquots using a 96-well, high performance extraction disk plate C18 SD (3M, St. Paul, MN) with 80% acetonitrile in 0.1% TFA as eluent. 3T3-L1 adipocytes were incubated for 5 h with aliquots of each fraction, and glycerol content in the supernatants was measured. , Lipolytic activity of the collected fractions. The gradient profiles are indicated by the dotted lines. Results are expressed as percentages of the lipolytic effect of 1 μM isoproterenol and are representative for two separate experiments.
The corresponding fractions were loaded onto a YMC ODS AQ (10 x 250-mm) column (YMC Europe, Scherbeck, Germany). Peptides were eluted with an increase of 8–32% acetonitrile in 0.1% trifluoroacetic acid (TFA) in 60 min at a flow rate of 1.2 ml/min (Fig. 2G). Active material found in fractions 15 and 16 was diluted with 50 mM KH2PO4 (pH 4.5), loaded onto a PepKat column (4 x 125 mm; Biotek), and eluted using a linear gradient from 0–0.45 M KCl in 30 min at a flow rate of 1 ml/min (Fig. 2H). Fractions 19–20 were diluted with 0.1% TFA, injected onto a protein and peptides C18 (4.6 x 250-mm) column (Vydac, Hesperia, CA), and eluted with a gradient increase in acetonitrile from 8–32% in 60 min at a flow rate of 0.7 ml/min (Fig. 2I). The active fractions (50, 51) were pooled as purified peptide.
Peptide analysis and synthesis
The purity of the isolated peptides was determined with a P/ACE MDQ 2000 capillary zone electrophoresis system (Beckman, Munich, Germany), using a fused silica capillary (TSP 075375, Composite Metal Services, West Yorkshire, UK) and a buffer containing 0.1 M H3PO4 in 0.02% hydroxypropylmethylcellulose at a voltage of 20 kV. Molecular weight determination was carried out by matrix-assisted-laser-desorption-ionization-time of flight (MALDI-TOF)-mass spectrometric analysis performed on a Voyager DE Pro mass spectrometer (Applied Biosystems, Darmstadt, Germany). Positive ions were accelerated at 25 kV. Time of flight data were externally calibrated for each sample plate and sample preparation. The amino acid sequences of the purified peptides were analyzed with a protein sequencer (494, Applied Biosystems) using Edman N terminal sequencing. VA-?-MSH was prepared by the N-(9-fluorenyl)methoxycarbonyl (F-moc) solid- phase method using a peptide synthesizer (433A, Applied Biosystems).
Immunoblot analysis
3T3-L1 adipocytes were incubated with the indicated stimulants for the indicated time periods, supernatants were removed, and whole cell extracts were prepared by adding 50 μl lysis buffer (NuPAGE, Invitrogen Life Technologies, Inc., Karlsruhe, Germany) containing 0.05 M dithiothreitol. Lysates were transferred to microcentrifuge tubes and sonicated three times for 3 sec each time. After heat denaturation at 70 C for 10 min and centrifugation at 13,000 rpm for 5 min at 4 C, equal amounts of samples were separated by SDS-PAGE under denaturing conditions. Proteins were transferred to polyvinylidene difluoride membranes, which were then blocked in Rotiblock (Carl Roth, Karlsruhe, Germany) for 1 h at room temperature. Blots were probed overnight at 4 C with the primary antibody. Immunoblot analysis was performed by enhanced chemiluminescence (Super Signal West Dura, Pierce Chemical Co., Rockford, IL) after 1-h incubation with the secondary antibody conjugated to horseradish peroxidase at room temperature. To confirm equal loading of the lanes, immunoblots were stained with India ink (Pelikan, Hannover, Germany) after chemiluminescence detection.
Chemicals and reagents
All reagents and the organic solvents were of the highest analytical grade. Acetonitrile, methanol, and TFA were purchased from Merck & Co. Water was purified in a Milli-QUF Plus System (Millipore Corp., Bedford, MA). Fluorenylmethoxycarbonyl-protected amino acids were purchased from Merck Biosciences (Schwalbach, Germany). DMEM was purchased from Invitrogen Life Technologies, Inc., and fetal bovine serum was obtained from Biochrom (Berlin, Germany). ?-MSH-(1–22) was obtained from Bachem (Merseyside, UK), Rp-8-CPT-cAMPS was purchased from BIOLOG Life Science Institute (Bremen, Germany), H89 was obtained from Biomol (Hamburg, Germany), and isoproterenol and forskolin were purchased from Sigma-Aldrich Corp. (Taufkirchen, Germany). Antibodies against cAMP response element-binding protein (CREB) and phosphorylated CREB were purchased from New England Biolabs (Frankfurt, Germany), and the polyclonal antibody against perilipin was obtained from Progen (Heidelberg, Germany). Antiserum directed against MCR2 and affinity-purified antibody against MCR5 were obtained from Biotrend (Cologne, Germany), and mouse ascites-derived antibody against ?-actin was purchased from Sigma-Aldrich Corp. (Deisenhofen, Germany). A peroxidase-conjugated antibody against rabbit was purchased from New England Biolabs (Beverley, MA), and the antiguinea pig antibody conjugated to peroxidase was obtained from Sigma-Aldrich Corp.
Statistical analysis
Results are expressed as the mean ± SD. One-way ANOVA and Bonferroni post hoc test were used for statistical analyses. P 0.05 was considered statistically significant. All calculations were performed with PRISM software 3.02 (GraphPad, Inc., San Diego, CA).
Results
Isolation of human circulating VA-?-MSH and ?-MSH- (1–22) from hemofiltrate
We isolated human circulating VA-?-MSH and ?-MSH-(1–22) from hemofiltrate in nine purification steps by alternating reverse phase HPLC and cation exchange chromatography, using a lipolysis assay that allowed simple and sensitive screening of fractions for the presence of lipolytic substances. As shown in Fig. 1, the primary screening of the human hemofiltrate peptide library for lipolytic factors detected two fractions with lipolytic activity in pool 3, which were further purified as described in Materials and Methods (Fig. 2, A–E). Within the sixth purification step (Fig. 2F), the active material was separated into two peaks (peaks I and II) that were isolated separately by the same strategy (Fig. 2, G–I) for further characterization. Peaks I and II could only be separated by cation exchange chromatography in the sixth and eighth purification steps, whereas the bioactive material of both peaks eluted at the same percentage of organic solvent in the mobile phase when using reversed phase chromatography in isolation steps 7 and 9. Both of the two resulting purified active peaks were subjected to capillary electrophoresis to confirm the purity of the selected fractions. Figure 3a shows that peaks I and II contained a single peptide each, which were further analyzed.
FIG. 1. Primary screening of pool 3 of the human hemofiltrate peptide library for lipolytic factors. 3T3-L1 adipocytes were incubated with aliquots of each fraction for 5 h, and glycerol content in the supernatants was measured as described in Materials and Methods. , Lipolytic activity of the collected fractions. The gradient profile is indicated by the dotted line. Fractions 12 and 13 showed lipolytic activity and underwent additional purification. The results shown are expressed as percentages of the lipolytic effect of 1 μM isoproterenol and are representative for two independent experiments.
FIG. 3. Confirmation of purity and determination of molecular masses of the isolated peptides. A, Capillary zone electropherograms of the pooled fractions of each last purification step of the two isolated peptides. The purities of peaks I and II were 91% and 94%, respectively. B, MALDI-TOF mass spectra of the purified peptides. Molecular masses of peaks I and II were 2831 and 2661 Da, respectively, calculated by the single-charged ions. The MALDI-TOF mass spectrum of peak I also shows the double-charged ion at m/z 1415.5.
Structural analysis of human VA-?-MSH and human ?-MSH-(1–22)
The two purified peptides were subjected to protein sequencing, showing that both peaks I and II contained the amino acid sequence AEKKDEGPYRMEHFRWGSPPKD, identical except for the extended N terminus of peak I prolonged by the amino acids valine and alanine, yielding the amino acid sequence VAAEKKDEGPYRMEHFRWGSPPKD. The molecular masses of the two isolated peptides determined by MALDI-TOF-mass spectrometry were 2831 and 2661 for peaks I and II, respectively, and confirmed these results (Fig. 3B). The C termini were not amidated. Using MS-Edman 2.2.1 (ProteinProspector 3.2.1, Prof. Alma Burlingame, University of California-San Francisco, San Francisco, CA), the amino acid sequence of peak II was shown to be the peptide hormone ?-MSH-(1–22), whereas the prolonged amino acid sequence of peak I was lipotropin--(33–57), which has not previously been described as an isolated peptide hormone entity and was named VA-?-MSH.
Pharmacological characterization of human VA-?-MSH and ?-MSH-(1–22)
Biological activity of human VA-?-MSH and ?-MSH-(1–22).
To further characterize the biological activity of the isolated peptides, we synthesized VA-?-MSH and tested this and synthetic human ?-MSH-(1–22) purchased from Bachem (Merseyside, UK). As shown in Fig. 4, both of the synthetic peptides exerted a lipolytic effect in 3T3-L1 adipocytes in a concentration-dependent manner. VA-?-MSH and ?-MSH-(1–22) had statistically indistinguishable 50% effective concentration values (Table 1), and displayed similar potency.
FIG. 4. Lipolytic effect of synthetic VA-?-MSH and ?-MSH-(1–22). Dose-response relationships of increasing concentrations of both peptides. After incubation of 3T3-L1 adipocytes with either peptide for 5 h, the glycerol content in the supernatants was measured. Results are expressed as percentages of the lipolytic response of cells to 1 μM isoproterenol and are the mean ± SD (n = 3). One of three independent experiments is presented.
TABLE 1. Lipolytic potency and intracellular cAMP formation of VA-?-MSH, ?-MSH-(1–22), human (h) ACTH-(1–39), and forskolin
Involvement of cAMP-dependent protein kinase A (PKA).
To confirm that VA-?-MSH- and ?-MSH-(1–22)-stimulated lipolysis is mediated by activation of PKA, 3T3-L1 adipocytes were incubated in the presence or absence of the two peptides and the PKA inhibitors Rp-8-CPT-cAMPS and H89 for 5 h after preincubation with the inhibitor for 40 min. Exposure of 3T3-L1 adipocytes to 0.3 and 1 mM Rp-8-CPT-cAMPS resulted in an inhibition of VA-?-MSH-stimulated glycerol release of 50% and 72%, respectively. Treatment with H89 inhibited lipolysis induced by both peptides by approximately 50%, whereas basal lipolysis (control) was not significantly changed by either inhibitor (Fig. 5).
FIG. 5. Effects of specific PKA inhibitors on VA-?-MSH- and ?-MSH-(1–22)-induced lipolysis. 3T3-L1 adipocytes were preincubated with or without inhibitor for 40 min, followed by the incubation with 20 nM VA-?-MSH or ?-MSH-(1–22) for 5 h. A, Preincubation of 3T3-L1 adipocytes with 0.3 and 1 mM Rp-8-CPT-cAMPS significantly decreased VA-?-MSH-induced lipolysis. B, VA-?-MSH- and ?-MSH-(1–22)-induced glycerol release was inhibited in the presence of H89. Basal lipolysis was not significantly changed by either inhibitor. Data points are the mean ± SD (n = 3). Results shown are representative for three separate experiments. *, P 0.05 vs. no inhibitor.
Stimulation of adenylyl cyclase activity.
Next, the ability of the isolated ligands to stimulate adenylyl cyclase activity was investigated. Functional coupling of the receptors involved in signal transduction of the lipolytic effect in response to VA-?-MSH and ?-MSH-(1–22) was demonstrated by cAMP ELISA. Intracellular cAMP was elevated in a dose- and time-dependent manner, as shown in Fig. 6 and Table 1. It is noteworthy that the intracellular cAMP level remains high even after 30-min incubation. These results strongly suggest that the formation of cAMP is involved in the lipolytic effect of these peptide hormones.
FIG. 6. Characterization of VA-?-MSH- and ?-MSH-(1–22)-stimulated cAMP production in 3T3-L1 adipocytes. The differentiated adipocytes were transferred into fresh DMEM on the day of the experiment, and various concentrations of ?-MSH-(1–22) and VA-?-MSH were added and incubated at 37 C for the indicated time periods. The supernatants were removed, and cells were lysed with 70% ethanol. Cell lysates were assayed for cAMP by ELISA. A, Time dependency of VA-?-MSH- and ?-MSH-(1–22)-stimulated cAMP production in 3T3-L1 cells. Isoproterenol (1 μM) was used as a positive control. B, Dose-response relationship of cAMP production in differentiated 3T3-L1 cells by VA-?-MSH and ?-MSH-(1–22). Each point represents the mean ± SD of triplicate values. Results are representative for three (A) and two (B) independent experiments.
Analysis of PKA substrate proteins.
To address whether increased cAMP activated PKA and subsequently increased CREB phosphorylation, we performed immunoblot analysis using cell extracts of 3T3-L1 adipocytes treated with or without 300 nM VA-?-MSH for 15, 30, 60, or 120 min or 5 h. CREB phosphorylation was increased by VA-?-MSH, compared with nonstimulated control values, whereas the level of total CREB remained unchanged (Fig. 7A). This indicates that treatment with VA-?-MSH results in PKA activation, followed by CREB phosphorylation.
FIG. 7. Effects of VA-?-MSH on CREB and perilipin phosphorylation. A, 3T3-L1 adipocytes were serum-deprived overnight and subsequently stimulated with 300 nM VA-?-MSH for the times indicated. At each time point, cells were lysed, and equal amounts of lysate protein were subjected to immunoblot analysis. Blots were probed with antibodies against serine 133-phosphorylated CREB (pCREB) and total CREB. One of three independent experiments, each with two replicates, is shown. The positions of the pCREB and CREB bands are indicated. B, After 15-min stimulation with VA-?-MSH, cell lysates were subjected to immunoblot analysis, and blots were probed with an antibody against perilipin. The characteristic upward shift observed with VA-?-MSH reveals the phosphorylation of perilipin A in stimulated cells compared with cells treated with control medium (representative blot of three independent experiments).
To determine whether VA-?-MSH induces phosphorylation of perilipin A, immunoblot analysis was performed using an antibody against perilipin A on cell extracts from 3T3-L1 adipocytes, which were treated for 15 min with either 300 nM VA-?-MSH or control medium. As depicted in Fig. 7B, VA-?-MSH treatment induced a marked shift in the migration of perilipin A, indicating that VA-?-MSH induces PKA-dependent phosphorylation of this protein as well.
Analysis of MCR protein expression
Melanocortins exhibit their biological effect via MCRs, of which five isoforms (MCR1 to MCR5) exist. Gene expression of MCR2 and MCR5 has previously been shown in 3T3-L1 adipocytes (18). To address whether these receptor proteins are also expressed in differentiated 3T3-L1 adipocytes, extracts of untreated adipocytes were subjected to SDS-PAGE. The subsequent immunoblot analysis showed MC2R as well as MC5R protein expression in differentiated 3T3-L1 cells, whereas neither receptor isoform protein was detected in undifferentiated 3T3-L1 preadipocytes. ?-Actin served as a loading control (Fig. 8).
FIG. 8. Protein expression of melanocortin receptor isoforms 2 and 5 in 3T3-L1 adipocytes. Untreated 3T3-L1 adipocytes were lysed and subjected to SDS-PAGE, followed by immunoblot analysis. Blots were probed using a specific antiserum directed against MC2R (left) or an affinity-purified antibody against MC5R (right). ?-Actin served as a loading control.
Discussion
We have identified the novel peptide hormone VA-?-MSH and ?-MSH-(1–22) as lipolytically active peptides present in human hemofiltrate in the lower picomolar range (roughly estimated to 16 pM for both). The isolation strategy was based on measuring the increase in lipolytic activity in 3T3-L1 adipocytes after application of potentially bioactive fractions. In addition, we characterized the pharmacology of these peptides and the signal transduction pathway involved in their lipolytic effect using protein kinase inhibitors, by measuring intracellular cAMP levels, and by immunoblot analysis to detect phosphorylation of the PKA substrate proteins, perilipin A and CREB.
It is well known that peptide hormones, such as the enkephalins (19), endorphins, dynorphins (20), and ?-lipotropins (21), are cleaved to multiple forms by processing proteases. Many cleavage events occur at pairs of basic amino acid residues (Lys or Arg), which are usually removed from the resultant products by carboxypeptidase E (22). The enzymes involved are the prohormone convertases (PCs). The family members, PC1 (also named PC3) and PC2, are of particular importance in the proopiomelanocortin (POMC) processing cascade (23, 24). However, other processing enzymes, such as proprotein convertase 4, may also play a role in POMC cleavage (25).
It is likely that both the 22- and 24-amino acid MSH molecules isolated in this study are produced through alternative processing of the same precursor peptide, POMC. In the hypothalamus, this 32-kDa POMC precursor peptide is cleaved to generate ACTH and ?-lipotropin (?-LPH) and -MSH. ?-LPH is the precursor of ?-endorphin and -LPH; the latter is further processed to ?-MSH-(5–22) in humans (26). The existence of human ?-MSH-(1–22), however, has been controversially discussed. Its structure was determined by Harris (27) and was shown to be similar to that of ?-MSH isolated from the pituitary glands of other species, except for the presence of an extra four amino acids at the N terminus. Abe et al. (28) detected ?-MSH in similar amounts as ACTH in human plasma and pituitaries. In contrast, Bertagna et al. (26) were unable to identify this peptide in human pituitary glands. However, we have now isolated circulating VA-?-MSH as well as ?-MSH-(1–22), the latter containing the same sequence as ?-MSH from human pituitary by Dixon (29) and characterized by Harris (27). Presumably, the PCs play only a minor role in the formation of the N termini of VA-?-MSH and ?-MSH-(1–22) from -LPH, because the cleavage here occurs between the nonpolar amino acid pairs Leu and Val and between Ala and Ala, respectively, raising the question of whether such peptides form within the isolation process. Bloomfield et al. (30) suggested that human ?-MSH-(1–22) from pituitary glands occurs during the extraction procedure using mild acetic conditions. Barat et al. (31) have shown that ?-MSH-(1–22) can be formed from ?-LPH by hydrolysis with cathepsin D, which selectively splits the Ala34-Ala35-peptide bond of human ?-LPH at pH 4–5 and 37 C. In contrast, Bertagna et al. (26) compared two methods for the extraction of MSHs from human pituitary tumor tissues using stronger acidic conditions and a working temperature of 4 C, but did not observe formation of ?-MSH-(1–22) from added [125I]human -LPH by either extraction procedure. Upon hemofiltration, blood cells containing lysosomal enzymes such as cathepsin D are separated from plasma. Therefore, the presence of cathepsin D-like enzymes in the hemofiltrate, in contrast to tissue extraction leading to the liberation of lysosomal enzymes, can be ruled out. We also acidified the hemofiltrate to pH 3 and cooled it to 4 C to prevent proteolysis immediately after collection. We conclude that the isolated peptides are existing forms in the circulation of chronic renal failure patients. However, it remains to be determined whether these peptides are present in the circulation of healthy subjects. It is also unclear which proteases besides PCs are responsible for the formation of these peptides.
The lipolytic action of POMC-derived peptides has been shown previously. Richter and Schwandt (32) investigated the lipolytic activity of ACTH-(1–39) as well as ?-LPH and truncated forms, including human ?-MSH-(1–22) and human ?-MSH-(5–22), in primary rabbit adipocytes. The lipolytic effect of VA-?-MSH and ?-MSH-(1–22) that we observed in this study has not previously been demonstrated in 3T3-L1 adipocytes.
It is generally known that a major pathway of lipolysis in adipocytes is the activation of adenylyl cyclase. The resulting increase in intracellular cAMP levels leads to the dissociation of cAMP-dependent PKA, whose free catalytic subunits phosphorylate, among other substrates, perilipin and hormone-sensitive lipase. This results in enhanced hydrolytic activity, translocation of hormone-sensitive lipase from cytosol to the lipid droplet surface, and subsequent release of glycerol and free fatty acids (reviewed in Ref. 33). However, stimulation of lipolysis in adipocytes via other signal transduction mechanisms involving the extracellular signal-regulated kinase pathway (16, 34) or the Janus kinase-signal transducer and activator of transcription pathway (35) have been described. In the present study, experiments using specific PKA inhibitors demonstrate significant inhibition of VA-?-MSH-stimulated lipolysis, showing PKA involvement, but because no full inhibition was achieved, additional mechanisms may be involved.
The isolated peptides strongly stimulate cAMP production in 3T3-L1 adipocytes, supporting PKA participation in the lipolytic action of these peptides. However, cAMP elevation was 10 times less sensitive to treatment than lipolysis. Exactly the same discrepancy between cAMP formation and the pharmacological effect of ACTH was described for adrenal glomerulosa cells, where both cAMP and Ca2+ influx act synergistically on aldosterone secretion (36). Therefore, an additional signaling pathway may be involved here as well. This is also supported by our findings that forskolin showed similar 50% effective concentration values in the lipolysis assay and in intracellular cAMP formation, leading to the conclusion that for forskolin, only adenylyl cyclase stimulation is the signaling pathway responsible for lipolysis. However, to clarify the relative impact of other signaling pathways, additional experiments will be necessary.
The perilipin proteins are specifically expressed in adipocytes and are located on the surface of the lipid droplet, presenting the major cAMP-dependent PKA substrate in fat cells (33). In 3T3-L1 adipocytes, VA-?-MSH leads to a shift in perilipin migration similar to the effect of isoproterenol in human preadipocytes (37) and cAMP analogs in 3T3-L1 adipocytes (16). Furthermore, CREB was initially characterized as a PKA substrate in 3T3-L1 cells. PKA activation results in the elevation of CREB transcriptional transactivation activity through the phosphorylation of CREB at Ser133 (38). However, CREB phosphorylation was used only as a marker for activation of PKA. In contrast to perilipin phosphorylation, which is involved in lipolysis, whether CREB phosphorylation plays a role in glycerol release was not examined. Together, our results strongly indicate that PKA activation occurs after treatment of 3T3-L1 adipocytes with the peptides isolated in this study.
Of all five melanocortin receptor isoforms described to date, 3T3-L1 adipocytes only express MCR2 and MCR5 (18) (our unpublished observations), which functionally couple to adenylyl cyclase (18). In this study immunoblotting shows the presence of both MC2R and MC5R protein in 3T3-L1 adipocytes. The sizes of the immunoreactive signals are similar to those described previously (39). Notably, MCR2 is activated only by ACTH and does not bind -, ?-, or -MSH (40). Thus, MCR5 or an as yet undescribed MCR mediates lipolysis induced by VA-?-MSH and ?-MSH-(1–22), which will be investigated in future studies.
Activation of hypothalamic MCR4 strongly inhibits food consumption and causes weight loss by regulating energy balance (41, 42). Also, peripheral daily injection of -MSH over the course of 4 wk led to a reduction in fat mass in relation to total body mass (43). Melanocortins have therefore been discussed as candidate molecules for the treatment of obesity, which may also apply for the peptides described in this study, because Harrold et al. (44) suggested that centrally administered ?-MSH is one of the endogenous ligands of the hypothalamic MCR4 that acts to inhibit food intake.
A new aspect of VA-?-MSH and ?-MSH-(1–22) representing circulating peptide hormones is raised in this study, although the lipolytic potential of ?-MSH was previously known. Whether lipolysis or one of the other known actions of melanocortins is their true physiological role needs to be determined. Although isolated in this study after its lipolytic activity on 3T3-L1 adipocytes, this newly identified circulating human melanocortin may also serve other functions in human physiology. Moreover, the fact that these peptides have been identified after a functional assay, but have been overseen in large proteomic approaches (45, 46), underscores the importance of such approaches in identifying previously undescribed circulating bioactive molecules.
Acknowledgments
We thank Ilka Herberz and Kathleen Listemann for their excellent technical assistance.
References
Kwa HG, van der Bent EM, Feltkamp CA, Rumke P, Bloemendal H 1965 Studies on hormones from the anterior pituitary gland. I. Identification and isolation of growth hormone and prolactin from the "granular" fraction of bovine pituitary. Biochem Biophys Acta 111:447–465
Schally AV, Bowers CY, Redding TW, Barrett JF 1966 Isolation of thyrotropin releasing factor (TRF) from porcine hypothalamus. Biochem Biophys Res Commun 25:165–169
Lee CY, Wong S, Lee AS, Ma L 1977 Purification and properties of choriogonadotropin from human term placenta. Hoppe Seylers Z Physiol Chem 358:909–914
Schulz-Knappe P, Schrader M, St?ndker L, Richter R, Hess R, Jürgens M, Forssmann WG 1997 Peptide bank generated by large-scale preparation of circulating human peptides. J Chromatogr A 776:125–132
Louis SN, Jackman GP, Nero TL, Iakovidis D, Louis WJ 2000 Role of ?-adrenergic receptor subtypes in lipolysis. Cardiovasc Drugs Ther 14:565–577
Candelore MR, Deng L, Tota L, Guan XM, Amend A, Liu Y, Newbold R, Cascieri MA, Weber AE 1999 Potent and selective human ?3-adrenergic receptor antagonists. J Pharmacol Exp Ther 290:649–655
Sasaki N, Uchida E, Niiyama M, Yoshida T, Saito M 1998 Anti-obesity effects of selective agonists to the ?3-adrenergic receptor in dogs. I. The presence of canine ?3-adrenergic receptor and in vivo lipomobilization by its agonists. J Vet Med Sci 60:459–463
Shaw JH, Wolfe RR 1987 Fatty acid and glycerol kinetics in septic patients and in patients with gastrointestinal cancer. Ann Surg 205:368–375
Todorov PT, McDevitt TM, Meyer DJ, Ueyama H, Ohkubo I, Tisdale MJ 1998 Purification and characterization of a tumor lipid-mobilizing factor. Cancer Res 58:2353–2358
Carlson LA 1963 Studies on the effect of nicotinic acid on catecholamine stimulated lipolysis in adipose tissue in vitro. Acta Med Scand 173:719–722
Kiwaki K, Levine JA 2003 Differential effects of adrenocorticotropic hormone on human and mouse adipose tissue. J Comp Physiol [B] 173:675–678
Goodman HM, Bray GA 1966 Role of thyroid hormones in lipolysis. Am J Physiol 210:1053–1058
Lucidi P, Parlanti N, Piccioni F, Santeusanio F, De Feo P 2002 Short-term treatment with low doses of recombinant human GH stimulates lipolysis in visceral obese men. J Clin Endocrinol Metab 87:3105–3109
Rosen OM, Smith CJ, Hirsch A, Lai E, Rubin CS 1979 Recent studies of the 3T3–L1 adipocyte-like cell line. Recent Prog Horm Res 35:477–499
Bertagna X, Lenne F, Comar D, Massias JF, Wajcman H, Baudin V, Luton JP, Girard F 1986 Human ?-melanocyte-stimulating hormone revisited. Proc Natl Acad Sci USA 83:9719–9723
Fricke K, Heitland A, Maronde E 2004 Cooperative activation of lipolysis by protein kinase A and protein kinase C pathways in 3T3–L1 adipocytes. Endocrinology 145:4940–4947
Maronde E, Pfeffer M, Olcese J, Molina CA, Schlotter F, Dehghani F, Korf HW, Stehle JH 1999 Transcription factors in neuroendocrine regulation: rhythmic changes in pCREB and ICER levels frame melatonin synthesis. J Neurosci 19:3326–3336
Boston BA, Cone RD 1996 Characterization of melanocortin receptor subtype expression in murine adipose tissues and in the 3T3–L1 cell line. Endocrinology 137:2043–2050
Noda M, Furutani Y, Takahashi H, Toyosato M, Hirose T, Inayama S, Nakanishi S, Numa S 1982 Cloning and sequence analysis of cDNA for bovine adrenal preproenkephalin. Nature 295:202–206
Kakidani H, Furutani Y, Takahashi H, Noda M, Morimoto Y, Hirose T, Asai M, Inayama S, Nakanishi S, Numa S 1982 Cloning and sequence analysis of cDNA for porcine ?-neo-endorphin/dynorphin precursor. Nature 298:245–249
Nakanishi S, Inoue A, Kita T, Nakamura M, Chang AC, Cohen SN, Numa S 1979 Nucleotide sequence of cloned cDNA for bovine corticotropin-?-lipotropin precursor. Nature 278:423–427
Fricker DL 1988 Carboxypeptidase E. Annu Rev Physiol 50:309–321
Benjannet S, Rondeau N, Day R, Chretien M, Seidah MG 1991 PC1 and PC2 are proprotein convertases capable of cleaving proopiomelanocortin at distinct pairs of basic residues. Proc Natl Acad Sci USA 88:3564–3570
Thomas L, Leduc R, Thorne BA, Smeekens S, Steiner DF, Thomas G 1991 Kex2-like endoproteases PC2 and PC3 accurately cleave a model prohormone in mammalian cells: evidence for a common core of neuroendocrine processing enzymes. Proc Natl Acad Sci USA 88:5297–5301
Dong W, Marcinkiewicz MN, Vieau D, Chretien M, Seidah NG, Day R 1995 Distinct mRNA expression of the highly homologous convertases PC5 and PACE4 in the rat brain and pituitary. J Neurosci 15:1778–1796
Bertagna X, Seidah N, Massias JF, Lenne F, Luton JP, Girard F, Chretien M 1988 Microsequencing evidence for the maturation of human proopiomelanocortin into an 18 amino acid hormone [h?MSH(5–22)] in nonpituitary tissue. Peptides 10:83–87
Harris JI 1959 Structure of a melanocyte-stimulating hormone from the human pituitary gland. Nature 184:167–169
Abe K, Nicholson WE, Liddle GW, Orth DN, Island DP 1969 Normal and abnormal regulation of ?-MSH in man. J Clin Invest 48:1580–1585
Dixon HBF 1960 Chromatographic isolations of pig and human melanocyte-stimulating hormones. Biochim Biophys Acta 37:38–42
Bloomfield GA, Scott AP, Lowry PJ, Gilkes JJ, Rees LH 1974 A reappraisal of human ? MSH. Nature 252:492–493
Barat E, Patthy A, Graf L 1979 Action of cathepsin D on human ?-lipotropin: a possible source of human "?-melanotropin." Proc Natl Acad Sci USA 76:6120–6123
Richter WO, Schwandt P 1987 Lipolytic potency of proopiomelanocorticotropin peptides in vitro. Neuropeptides 9:59–74
Holm C 2003 Molecular mechanisms regulating hormone-sensitive lipase and lipolysis. Biochem Soc Transact 31:1120–1124
Greenberg AS, Shen WS, Muliro K, Patel S, Souza SC, Roth RA, Kraemer FB 2001 Stimulation of lipolysis and hormone-sensitive lipase via the extracellular signal-regulated kinase pathway. J Biol Chem 276:45456–45461
Asada N, Takahashi Y, Wada M, Naito N, Uchida H, Ikeda M, Honjo M 2000 GH induced lipolysis stimulation in 3T3–L1 adipocytes stably expressing hGHR: analysis on signaling pathway and activity of 20 K hGH. Mol Cell Endocrinol 162:121–129
Kojima I, Kojima K, Rasmussen H 1985 Role of calcium and cAMP in the action of adrenocorticotropin on aldosterone secretion. J Biol Chem 260:4248–4256
Sengenès C, Bouloumié A, Hauner H, Berlan M, Busse R, Lafontan M, Galitzky J 2003 Involvement of a cGMP-dependent pathway in the natriuretic peptide-mediated hormone-sensitive lipase phosphorylation in human adipocytes. J Biol Chem 278:48617–48626
Gonzalez GA, Montminy MR 1989 Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell 59:675–680
Akbulut S, Byersdorfer CA, Larsen CP, Zimmer SL, Humphreys TD, Clarke BL 2001 Expression of the melanocortin 5 receptor on rat lymphocytes. Biochem Biophys Res Commun 281:1086–1092
Schioth HB, Chhajlani V, Muceniece R, Klusa V, Wikberg JE 1996 Major pharmacological distinction of the ACTH receptor from other melanocortin receptors. Life Sci 59:797–801
Fan W, Boston BA, Kesterson RA, Hruby WJ, Cone RD 1997 Role of melanocortinergic neurons in feeding and the agouti obesity syndrome. Nature 385:165–168
Huszar D, Lynch CA, Fairchild-Huntress V, Dunmore JH, Fang Q, Berkemeyer LR 1997 Targeted disruption of the melanocortin-4-receptor results in obesity in mice. Cell 88:131–141
Cornish J, Callon KE, Mountjoy KG, Bava U, Lin JM, Myers DE, Naot D, Reid IR 2003 -Melanocyte-stimulating hormone is a novel regulator of bone. Am J Physiol 284:E1181–E1190
Harrold JA, Widdowson PS, Williams G 2003 ?-MSH: a functional ligand that regulated energy homeostasis via hypothalamic MC4-R? Peptides 24:397–405
Rose K, Bougueleret L, Baussant T, Bohm G, Botti P, Colinge J, Cusin I, Gaertner H, Gleizes A, Heller M, Jimenez S, Johnson A, Kussmann M, Menin L, Menzel C, Ranno F, Rodriguez-Tome P, Rogers J, Saudrais C, Villain M, Wetmore D, Bairoch A, Hochstrasser D 2004 Industrial-scale proteomics: from liters of plasma to chemically synthesized proteins. Proteomics 4:2125–2150
Chan KC, Lucas DA, Hise D, Schaefer CF, Xiao Z, Janini GM, Buetow KH, Issaq HJ, Veenstra TD, Conrads TP 2004 Analysis of the human serum proteome. Clin Proteomics 1:101–226(Katrin Fricke, Axel Schul)
Address all correspondence and requests for reprints to: Dr. Erik Maronde, IPF PharmaCeuticals GmbH, Feodor Lynen Strasse 31, D-30625 Hannover, Germany. E-mail: erik.maronde@ipf-pharmaceuticals.de.
Abstract
We report the isolation of a novel human circulating proopiomelanocortin-derived peptide, VA-?-MSH, from hemofiltrate and its pharmacological characterization. Screening for lipolytic activity in differentiated 3T3-L1 adipocytes led to the isolation from a hemofiltrate peptide library by alternating reverse phase and cation exchange chromatography. In the course of this isolation, we also identified human ?-MSH-(1–22). We synthesized VA-?-MSH by the N-(9-fluorenyl)-methoxycarbonyl (F-moc) solid phase method and used synthetic ?-MSH-(1–22) to confirm that both isolated peptides are lipolytically active in a dose-dependent manner in differentiated 3T3-L1 adipocytes in the nanomolar range. Using cAMP ELISA, we demonstrate that stimulation with both peptides caused a strong cAMP elevation in this cell system. Furthermore, we show that the selective inhibitors of cAMP-dependent protein kinase, 8-(4-Chlorophenylthio)adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-CPT-cAMPS); N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89), significantly reduce VA-?-MSH- and ?-MSH-(1–22)-mediated lipolysis. Although isolated after its lipolytic activity on 3T3-L1 cells, this newly identified circulating human melanocortin may serve other functions in human physiology. Moreover, the fact that these peptides have been identified after a functional assay, but have been overseen in large proteomic approaches, underscores the importance of such approaches in identifying previously undescribed circulating bioactive molecules.
Introduction
THE IMPORTANCE OF peptides in the diagnosis and therapy of human disease is well accepted. Regulatory peptide hormones have been isolated from various sources, including pituitary gland (1), hypothalamus (2), and placenta (3).
Human hemofiltrate from patients with chronic renal failure, due to its comparably high concentrations of peptides and proteins, is a comprehensive source of regulatory peptides (4). The specific concentration of a given peptide compared with plasma is increased by a factor of 1000 by hemofiltration, thus increasing the probability of subsequent isolation of regulatory peptides (4).
The importance of lipolysis in the body fat and weight control system is generally understood. A large number of pharmacological approaches to the treatment of diet-induced obesity have been made recently, and one target is the control of fat stores through modulation of lipolysis and lipogenesis in white adipose tissue. One such approach is the development of ?3-adrenoceptor agonists as lipid-mobilizing drugs for the treatment of obesity (5, 6, 7). Often, cancer-induced weight loss (cachexia) is associated with increased turnover of both glycerol and nonesterified fatty acids. This differs from the situation in cancer patients without weight loss (8). These observations led Todorov et al. (9) to isolate and identify a tumor-derived, lipid-mobilizing factor and suggest a role for lipid-mobilizing substances in the control of body weight. A variety of hormones stimulate the rate of fat-mobilizing lipolysis of adipose tissue triglycerides. In humans, these include catecholamines (10), ACTH (11), TSH (12), and GH (13).
The aim of this study was to detect human circulating lipolytic peptides. For this purpose, human hemofiltrate was used as a source of the isolation and identification of such peptides. As a screening system, differentiated 3T3-L1 adipocytes were used. This cell line has been shown to exhibit many of the characteristics of normal white adipocytes, including responsiveness to insulin and lipolytic hormones (14). The bioactivity in our assay was based on lipolytic activity, measured as glycerol release from differentiated 3T3-L1 adipocytes. This assay system allowed fast and sensitive detection of lipolytic substances.
We describe the isolation and identification of a novel circulating melanocyte-stimulating peptide hormone, VA-?-MSH, from human hemofiltrate, based on a bioactivity-oriented isolation strategy. Moreover, using this isolation strategy, we found ?-MSH-(1–22) as a lipolytic circulating human peptide hormone whose existence in the circulation has long been doubted (15). We characterize the signal transduction mechanism of the lipolytic pathway of VA-?-MSH and ?-MSH-(1–22) in 3T3-L1 adipocytes and show that activation of adenylyl cyclase and cAMP-dependent protein kinase activation is the main pathway mediating the strong lipolytic effect of these circulating peptide hormones. Immunoblot analysis displayed protein expression of melanocortin receptor isoforms 2 and 5, and a possible mediation of the pharmacological effect of the isolated peptides via melanocortin receptor-5 (MCR5) is discussed.
Materials and Methods
Cell culture and differentiation
Murine 3T3-L1 fibroblasts (American Type Culture Collection, Manassas, VA) were grown in DMEM containing 10% fetal bovine serum. Cells were differentiated into adipocytes after reaching 90–100% confluence, as described previously (16). Adipocytes were used for experiments 7–9 d after the differentiation protocol was initiated.
Lipolysis
3T3-L1 adipocytes cultured in 96-well plates were transferred into fresh DMEM and treated for 5 h as described in Results. Glycerol content in the supernatants was measured using a colorimetric assay (GPO-Trinder, Sigma-Aldrich Corp., Deisenhofen, Germany). Results are expressed either as nanomoles of glycerol per well or as a percentage of the lipolytic effect of 1 μM isoproterenol, which was used as a positive control.
cAMP ELISA
Intracellular cAMP was measured using the cAMP ELISA kit (IHF GmbH, Hamburg, Germany) (17). 3T3-L1 adipocytes were treated with various concentrations of the indicated stimulants, followed by an incubation at 37 C. At the indicated time points, supernatants were removed, 70% ethanol was added to each well, and the cells were scraped into Eppendorf tubes. The tubes were dried under vacuum, and 100 μl cAMP working buffer were added to each tube. cAMP was assayed using the supplier’s protocol.
Preparation of a peptide library from human hemofiltrate
A peptide library was prepared from 20,000 liters human hemofiltrate from patients suffering from chronic renal failure, as described previously (4). Each batch of hemofiltrate is pooled from the filtration sessions of approximately 30 patients over 8 wk, given that a patient undergoes filtration three times a week, providing a total of 60–90 liters/wk. The patients were of different ages and genders and using various medications, but most were older than 50 yr of age. All subjects gave consent for the use of their hemofiltrates for research purposes. Briefly, the crude hemofiltrate was immediately cooled to 4 C, acidified to pH 3, and loaded onto a strong cation exchange column (25 x 10 cm; Fractogel TSK SP 650 M, Merck & Co., Darmstadt, Germany), followed by stepwise elution using seven buffers with increasing pH from 3.6–9. Each of the seven pH pools was subjected to a second purification step using a reverse phase column (20 x 15.5 cm; Fineline Source RPC Polymer, Amersham Biosciences, Braunschweig, Germany). Fractionation was performed by collecting 46–63 fractions/pH pool, and aliquots of each fraction were tested in the lipolysis bioassay.
Isolation of circulating human VA-?-MSH and ?-MSH- (1–22) from hemofiltrate
All gradients used for chromatographic separation were linear. HPLC fractions 12 and 13 of pH pool 3 (which was eluted with maleic acid, pH 5.0) were diluted with 10 mM HCl and loaded onto a Fineline RPC Polymer (10 x 15.5-cm) column (Amersham Biosciences). Elution was performed by an increase in acetonitrile from 0–24% for 53 min at a flow rate of 150 ml/min (see Fig. 2A). The active fraction 12 was diluted with 50 mM Na2PO4 (pH 4.5) and loaded onto a TSK SP 650(S) cation exchange column (Merck & Co.), and peptides were eluted using a gradient from 0.15–1.5 M NaCl within 45 min at a flow rate of 50 ml/min (Fig. 2B). Fractions 26–34 containing the active material were diluted with 10 mM HCl and further purified using a 47 x 300-mm Bakerbond Prep Pak C18 cartridge (Waters Corp., Milford, MA) with an increase in methanol from 20–80% in 63 min at a flow rate of 30 ml/min (Fig. 2C). Lipolytically active fractions 22 and 23 were diluted with 50 mM Na2PO4 (pH 4.5) and loaded onto a PepKat cation exchange column (20 x 150 mm; Biotek, ?stringen, Germany). A gradient with an increase from 0.075 to 0.75 M NaCl in Na2PO4 (pH 4.5) within 45 min at a flow rate of 6 ml/min was used. Active material eluted in fractions 25–30 (Fig. 2D) and was subjected to the next purification step using a Source Rp C15 (20 x 250-mm) column (Pharmacia Biotech, Freiburg, Germany). An increase in methanol in the eluent from 16–64% in 70 min at a flow rate of 4 ml/min was performed (Fig. 2E). Active fractions 41–44 were diluted with 50 mM KH2PO4 (pH 4.5) and loaded onto a PepKat (10 x 125-mm) column (Biotek). Elution was performed by a gradient increase in the KCl concentration from 0 to 1.05 M in 70 min at a flow rate of 1.8 ml/min (Fig. 2F). The active material of the sixth purification step was found in fractions 21–25 and divided into peaks I and II. Fractions 21–22, referred to as peak I, and fractions 24–25 (peak II) were individually purified using the same strategy thereafter.
FIG. 2. Purification of human VA-?-MSH from hemofiltrate. Elution profiles of a preparative Fineline RPC Polymer column (A), a TSK SP 650 (S) cation exchange column (B), a Bakerbond Prep Pak C18 column (C), a Biotek PepKat cation exchange HPLC column (20 x 125 mm; D), a Pharmacia Source C15 HPLC column (20 x 250 mm; E), a Biotek PepKat cation exchange HPLC column (10 x 125 mm; F), a YMC ODS AQ HPLC column (10 x 250 mm; G), a Biotek PepKat cation exchange HPLC column (4 x 125 mm; H), and a Vydac Rp HPLC column (4.6 x 250 mm; I). After each cation exchange chromatography step, salt was removed from aliquots using a 96-well, high performance extraction disk plate C18 SD (3M, St. Paul, MN) with 80% acetonitrile in 0.1% TFA as eluent. 3T3-L1 adipocytes were incubated for 5 h with aliquots of each fraction, and glycerol content in the supernatants was measured. , Lipolytic activity of the collected fractions. The gradient profiles are indicated by the dotted lines. Results are expressed as percentages of the lipolytic effect of 1 μM isoproterenol and are representative for two separate experiments.
The corresponding fractions were loaded onto a YMC ODS AQ (10 x 250-mm) column (YMC Europe, Scherbeck, Germany). Peptides were eluted with an increase of 8–32% acetonitrile in 0.1% trifluoroacetic acid (TFA) in 60 min at a flow rate of 1.2 ml/min (Fig. 2G). Active material found in fractions 15 and 16 was diluted with 50 mM KH2PO4 (pH 4.5), loaded onto a PepKat column (4 x 125 mm; Biotek), and eluted using a linear gradient from 0–0.45 M KCl in 30 min at a flow rate of 1 ml/min (Fig. 2H). Fractions 19–20 were diluted with 0.1% TFA, injected onto a protein and peptides C18 (4.6 x 250-mm) column (Vydac, Hesperia, CA), and eluted with a gradient increase in acetonitrile from 8–32% in 60 min at a flow rate of 0.7 ml/min (Fig. 2I). The active fractions (50, 51) were pooled as purified peptide.
Peptide analysis and synthesis
The purity of the isolated peptides was determined with a P/ACE MDQ 2000 capillary zone electrophoresis system (Beckman, Munich, Germany), using a fused silica capillary (TSP 075375, Composite Metal Services, West Yorkshire, UK) and a buffer containing 0.1 M H3PO4 in 0.02% hydroxypropylmethylcellulose at a voltage of 20 kV. Molecular weight determination was carried out by matrix-assisted-laser-desorption-ionization-time of flight (MALDI-TOF)-mass spectrometric analysis performed on a Voyager DE Pro mass spectrometer (Applied Biosystems, Darmstadt, Germany). Positive ions were accelerated at 25 kV. Time of flight data were externally calibrated for each sample plate and sample preparation. The amino acid sequences of the purified peptides were analyzed with a protein sequencer (494, Applied Biosystems) using Edman N terminal sequencing. VA-?-MSH was prepared by the N-(9-fluorenyl)methoxycarbonyl (F-moc) solid- phase method using a peptide synthesizer (433A, Applied Biosystems).
Immunoblot analysis
3T3-L1 adipocytes were incubated with the indicated stimulants for the indicated time periods, supernatants were removed, and whole cell extracts were prepared by adding 50 μl lysis buffer (NuPAGE, Invitrogen Life Technologies, Inc., Karlsruhe, Germany) containing 0.05 M dithiothreitol. Lysates were transferred to microcentrifuge tubes and sonicated three times for 3 sec each time. After heat denaturation at 70 C for 10 min and centrifugation at 13,000 rpm for 5 min at 4 C, equal amounts of samples were separated by SDS-PAGE under denaturing conditions. Proteins were transferred to polyvinylidene difluoride membranes, which were then blocked in Rotiblock (Carl Roth, Karlsruhe, Germany) for 1 h at room temperature. Blots were probed overnight at 4 C with the primary antibody. Immunoblot analysis was performed by enhanced chemiluminescence (Super Signal West Dura, Pierce Chemical Co., Rockford, IL) after 1-h incubation with the secondary antibody conjugated to horseradish peroxidase at room temperature. To confirm equal loading of the lanes, immunoblots were stained with India ink (Pelikan, Hannover, Germany) after chemiluminescence detection.
Chemicals and reagents
All reagents and the organic solvents were of the highest analytical grade. Acetonitrile, methanol, and TFA were purchased from Merck & Co. Water was purified in a Milli-QUF Plus System (Millipore Corp., Bedford, MA). Fluorenylmethoxycarbonyl-protected amino acids were purchased from Merck Biosciences (Schwalbach, Germany). DMEM was purchased from Invitrogen Life Technologies, Inc., and fetal bovine serum was obtained from Biochrom (Berlin, Germany). ?-MSH-(1–22) was obtained from Bachem (Merseyside, UK), Rp-8-CPT-cAMPS was purchased from BIOLOG Life Science Institute (Bremen, Germany), H89 was obtained from Biomol (Hamburg, Germany), and isoproterenol and forskolin were purchased from Sigma-Aldrich Corp. (Taufkirchen, Germany). Antibodies against cAMP response element-binding protein (CREB) and phosphorylated CREB were purchased from New England Biolabs (Frankfurt, Germany), and the polyclonal antibody against perilipin was obtained from Progen (Heidelberg, Germany). Antiserum directed against MCR2 and affinity-purified antibody against MCR5 were obtained from Biotrend (Cologne, Germany), and mouse ascites-derived antibody against ?-actin was purchased from Sigma-Aldrich Corp. (Deisenhofen, Germany). A peroxidase-conjugated antibody against rabbit was purchased from New England Biolabs (Beverley, MA), and the antiguinea pig antibody conjugated to peroxidase was obtained from Sigma-Aldrich Corp.
Statistical analysis
Results are expressed as the mean ± SD. One-way ANOVA and Bonferroni post hoc test were used for statistical analyses. P 0.05 was considered statistically significant. All calculations were performed with PRISM software 3.02 (GraphPad, Inc., San Diego, CA).
Results
Isolation of human circulating VA-?-MSH and ?-MSH- (1–22) from hemofiltrate
We isolated human circulating VA-?-MSH and ?-MSH-(1–22) from hemofiltrate in nine purification steps by alternating reverse phase HPLC and cation exchange chromatography, using a lipolysis assay that allowed simple and sensitive screening of fractions for the presence of lipolytic substances. As shown in Fig. 1, the primary screening of the human hemofiltrate peptide library for lipolytic factors detected two fractions with lipolytic activity in pool 3, which were further purified as described in Materials and Methods (Fig. 2, A–E). Within the sixth purification step (Fig. 2F), the active material was separated into two peaks (peaks I and II) that were isolated separately by the same strategy (Fig. 2, G–I) for further characterization. Peaks I and II could only be separated by cation exchange chromatography in the sixth and eighth purification steps, whereas the bioactive material of both peaks eluted at the same percentage of organic solvent in the mobile phase when using reversed phase chromatography in isolation steps 7 and 9. Both of the two resulting purified active peaks were subjected to capillary electrophoresis to confirm the purity of the selected fractions. Figure 3a shows that peaks I and II contained a single peptide each, which were further analyzed.
FIG. 1. Primary screening of pool 3 of the human hemofiltrate peptide library for lipolytic factors. 3T3-L1 adipocytes were incubated with aliquots of each fraction for 5 h, and glycerol content in the supernatants was measured as described in Materials and Methods. , Lipolytic activity of the collected fractions. The gradient profile is indicated by the dotted line. Fractions 12 and 13 showed lipolytic activity and underwent additional purification. The results shown are expressed as percentages of the lipolytic effect of 1 μM isoproterenol and are representative for two independent experiments.
FIG. 3. Confirmation of purity and determination of molecular masses of the isolated peptides. A, Capillary zone electropherograms of the pooled fractions of each last purification step of the two isolated peptides. The purities of peaks I and II were 91% and 94%, respectively. B, MALDI-TOF mass spectra of the purified peptides. Molecular masses of peaks I and II were 2831 and 2661 Da, respectively, calculated by the single-charged ions. The MALDI-TOF mass spectrum of peak I also shows the double-charged ion at m/z 1415.5.
Structural analysis of human VA-?-MSH and human ?-MSH-(1–22)
The two purified peptides were subjected to protein sequencing, showing that both peaks I and II contained the amino acid sequence AEKKDEGPYRMEHFRWGSPPKD, identical except for the extended N terminus of peak I prolonged by the amino acids valine and alanine, yielding the amino acid sequence VAAEKKDEGPYRMEHFRWGSPPKD. The molecular masses of the two isolated peptides determined by MALDI-TOF-mass spectrometry were 2831 and 2661 for peaks I and II, respectively, and confirmed these results (Fig. 3B). The C termini were not amidated. Using MS-Edman 2.2.1 (ProteinProspector 3.2.1, Prof. Alma Burlingame, University of California-San Francisco, San Francisco, CA), the amino acid sequence of peak II was shown to be the peptide hormone ?-MSH-(1–22), whereas the prolonged amino acid sequence of peak I was lipotropin--(33–57), which has not previously been described as an isolated peptide hormone entity and was named VA-?-MSH.
Pharmacological characterization of human VA-?-MSH and ?-MSH-(1–22)
Biological activity of human VA-?-MSH and ?-MSH-(1–22).
To further characterize the biological activity of the isolated peptides, we synthesized VA-?-MSH and tested this and synthetic human ?-MSH-(1–22) purchased from Bachem (Merseyside, UK). As shown in Fig. 4, both of the synthetic peptides exerted a lipolytic effect in 3T3-L1 adipocytes in a concentration-dependent manner. VA-?-MSH and ?-MSH-(1–22) had statistically indistinguishable 50% effective concentration values (Table 1), and displayed similar potency.
FIG. 4. Lipolytic effect of synthetic VA-?-MSH and ?-MSH-(1–22). Dose-response relationships of increasing concentrations of both peptides. After incubation of 3T3-L1 adipocytes with either peptide for 5 h, the glycerol content in the supernatants was measured. Results are expressed as percentages of the lipolytic response of cells to 1 μM isoproterenol and are the mean ± SD (n = 3). One of three independent experiments is presented.
TABLE 1. Lipolytic potency and intracellular cAMP formation of VA-?-MSH, ?-MSH-(1–22), human (h) ACTH-(1–39), and forskolin
Involvement of cAMP-dependent protein kinase A (PKA).
To confirm that VA-?-MSH- and ?-MSH-(1–22)-stimulated lipolysis is mediated by activation of PKA, 3T3-L1 adipocytes were incubated in the presence or absence of the two peptides and the PKA inhibitors Rp-8-CPT-cAMPS and H89 for 5 h after preincubation with the inhibitor for 40 min. Exposure of 3T3-L1 adipocytes to 0.3 and 1 mM Rp-8-CPT-cAMPS resulted in an inhibition of VA-?-MSH-stimulated glycerol release of 50% and 72%, respectively. Treatment with H89 inhibited lipolysis induced by both peptides by approximately 50%, whereas basal lipolysis (control) was not significantly changed by either inhibitor (Fig. 5).
FIG. 5. Effects of specific PKA inhibitors on VA-?-MSH- and ?-MSH-(1–22)-induced lipolysis. 3T3-L1 adipocytes were preincubated with or without inhibitor for 40 min, followed by the incubation with 20 nM VA-?-MSH or ?-MSH-(1–22) for 5 h. A, Preincubation of 3T3-L1 adipocytes with 0.3 and 1 mM Rp-8-CPT-cAMPS significantly decreased VA-?-MSH-induced lipolysis. B, VA-?-MSH- and ?-MSH-(1–22)-induced glycerol release was inhibited in the presence of H89. Basal lipolysis was not significantly changed by either inhibitor. Data points are the mean ± SD (n = 3). Results shown are representative for three separate experiments. *, P 0.05 vs. no inhibitor.
Stimulation of adenylyl cyclase activity.
Next, the ability of the isolated ligands to stimulate adenylyl cyclase activity was investigated. Functional coupling of the receptors involved in signal transduction of the lipolytic effect in response to VA-?-MSH and ?-MSH-(1–22) was demonstrated by cAMP ELISA. Intracellular cAMP was elevated in a dose- and time-dependent manner, as shown in Fig. 6 and Table 1. It is noteworthy that the intracellular cAMP level remains high even after 30-min incubation. These results strongly suggest that the formation of cAMP is involved in the lipolytic effect of these peptide hormones.
FIG. 6. Characterization of VA-?-MSH- and ?-MSH-(1–22)-stimulated cAMP production in 3T3-L1 adipocytes. The differentiated adipocytes were transferred into fresh DMEM on the day of the experiment, and various concentrations of ?-MSH-(1–22) and VA-?-MSH were added and incubated at 37 C for the indicated time periods. The supernatants were removed, and cells were lysed with 70% ethanol. Cell lysates were assayed for cAMP by ELISA. A, Time dependency of VA-?-MSH- and ?-MSH-(1–22)-stimulated cAMP production in 3T3-L1 cells. Isoproterenol (1 μM) was used as a positive control. B, Dose-response relationship of cAMP production in differentiated 3T3-L1 cells by VA-?-MSH and ?-MSH-(1–22). Each point represents the mean ± SD of triplicate values. Results are representative for three (A) and two (B) independent experiments.
Analysis of PKA substrate proteins.
To address whether increased cAMP activated PKA and subsequently increased CREB phosphorylation, we performed immunoblot analysis using cell extracts of 3T3-L1 adipocytes treated with or without 300 nM VA-?-MSH for 15, 30, 60, or 120 min or 5 h. CREB phosphorylation was increased by VA-?-MSH, compared with nonstimulated control values, whereas the level of total CREB remained unchanged (Fig. 7A). This indicates that treatment with VA-?-MSH results in PKA activation, followed by CREB phosphorylation.
FIG. 7. Effects of VA-?-MSH on CREB and perilipin phosphorylation. A, 3T3-L1 adipocytes were serum-deprived overnight and subsequently stimulated with 300 nM VA-?-MSH for the times indicated. At each time point, cells were lysed, and equal amounts of lysate protein were subjected to immunoblot analysis. Blots were probed with antibodies against serine 133-phosphorylated CREB (pCREB) and total CREB. One of three independent experiments, each with two replicates, is shown. The positions of the pCREB and CREB bands are indicated. B, After 15-min stimulation with VA-?-MSH, cell lysates were subjected to immunoblot analysis, and blots were probed with an antibody against perilipin. The characteristic upward shift observed with VA-?-MSH reveals the phosphorylation of perilipin A in stimulated cells compared with cells treated with control medium (representative blot of three independent experiments).
To determine whether VA-?-MSH induces phosphorylation of perilipin A, immunoblot analysis was performed using an antibody against perilipin A on cell extracts from 3T3-L1 adipocytes, which were treated for 15 min with either 300 nM VA-?-MSH or control medium. As depicted in Fig. 7B, VA-?-MSH treatment induced a marked shift in the migration of perilipin A, indicating that VA-?-MSH induces PKA-dependent phosphorylation of this protein as well.
Analysis of MCR protein expression
Melanocortins exhibit their biological effect via MCRs, of which five isoforms (MCR1 to MCR5) exist. Gene expression of MCR2 and MCR5 has previously been shown in 3T3-L1 adipocytes (18). To address whether these receptor proteins are also expressed in differentiated 3T3-L1 adipocytes, extracts of untreated adipocytes were subjected to SDS-PAGE. The subsequent immunoblot analysis showed MC2R as well as MC5R protein expression in differentiated 3T3-L1 cells, whereas neither receptor isoform protein was detected in undifferentiated 3T3-L1 preadipocytes. ?-Actin served as a loading control (Fig. 8).
FIG. 8. Protein expression of melanocortin receptor isoforms 2 and 5 in 3T3-L1 adipocytes. Untreated 3T3-L1 adipocytes were lysed and subjected to SDS-PAGE, followed by immunoblot analysis. Blots were probed using a specific antiserum directed against MC2R (left) or an affinity-purified antibody against MC5R (right). ?-Actin served as a loading control.
Discussion
We have identified the novel peptide hormone VA-?-MSH and ?-MSH-(1–22) as lipolytically active peptides present in human hemofiltrate in the lower picomolar range (roughly estimated to 16 pM for both). The isolation strategy was based on measuring the increase in lipolytic activity in 3T3-L1 adipocytes after application of potentially bioactive fractions. In addition, we characterized the pharmacology of these peptides and the signal transduction pathway involved in their lipolytic effect using protein kinase inhibitors, by measuring intracellular cAMP levels, and by immunoblot analysis to detect phosphorylation of the PKA substrate proteins, perilipin A and CREB.
It is well known that peptide hormones, such as the enkephalins (19), endorphins, dynorphins (20), and ?-lipotropins (21), are cleaved to multiple forms by processing proteases. Many cleavage events occur at pairs of basic amino acid residues (Lys or Arg), which are usually removed from the resultant products by carboxypeptidase E (22). The enzymes involved are the prohormone convertases (PCs). The family members, PC1 (also named PC3) and PC2, are of particular importance in the proopiomelanocortin (POMC) processing cascade (23, 24). However, other processing enzymes, such as proprotein convertase 4, may also play a role in POMC cleavage (25).
It is likely that both the 22- and 24-amino acid MSH molecules isolated in this study are produced through alternative processing of the same precursor peptide, POMC. In the hypothalamus, this 32-kDa POMC precursor peptide is cleaved to generate ACTH and ?-lipotropin (?-LPH) and -MSH. ?-LPH is the precursor of ?-endorphin and -LPH; the latter is further processed to ?-MSH-(5–22) in humans (26). The existence of human ?-MSH-(1–22), however, has been controversially discussed. Its structure was determined by Harris (27) and was shown to be similar to that of ?-MSH isolated from the pituitary glands of other species, except for the presence of an extra four amino acids at the N terminus. Abe et al. (28) detected ?-MSH in similar amounts as ACTH in human plasma and pituitaries. In contrast, Bertagna et al. (26) were unable to identify this peptide in human pituitary glands. However, we have now isolated circulating VA-?-MSH as well as ?-MSH-(1–22), the latter containing the same sequence as ?-MSH from human pituitary by Dixon (29) and characterized by Harris (27). Presumably, the PCs play only a minor role in the formation of the N termini of VA-?-MSH and ?-MSH-(1–22) from -LPH, because the cleavage here occurs between the nonpolar amino acid pairs Leu and Val and between Ala and Ala, respectively, raising the question of whether such peptides form within the isolation process. Bloomfield et al. (30) suggested that human ?-MSH-(1–22) from pituitary glands occurs during the extraction procedure using mild acetic conditions. Barat et al. (31) have shown that ?-MSH-(1–22) can be formed from ?-LPH by hydrolysis with cathepsin D, which selectively splits the Ala34-Ala35-peptide bond of human ?-LPH at pH 4–5 and 37 C. In contrast, Bertagna et al. (26) compared two methods for the extraction of MSHs from human pituitary tumor tissues using stronger acidic conditions and a working temperature of 4 C, but did not observe formation of ?-MSH-(1–22) from added [125I]human -LPH by either extraction procedure. Upon hemofiltration, blood cells containing lysosomal enzymes such as cathepsin D are separated from plasma. Therefore, the presence of cathepsin D-like enzymes in the hemofiltrate, in contrast to tissue extraction leading to the liberation of lysosomal enzymes, can be ruled out. We also acidified the hemofiltrate to pH 3 and cooled it to 4 C to prevent proteolysis immediately after collection. We conclude that the isolated peptides are existing forms in the circulation of chronic renal failure patients. However, it remains to be determined whether these peptides are present in the circulation of healthy subjects. It is also unclear which proteases besides PCs are responsible for the formation of these peptides.
The lipolytic action of POMC-derived peptides has been shown previously. Richter and Schwandt (32) investigated the lipolytic activity of ACTH-(1–39) as well as ?-LPH and truncated forms, including human ?-MSH-(1–22) and human ?-MSH-(5–22), in primary rabbit adipocytes. The lipolytic effect of VA-?-MSH and ?-MSH-(1–22) that we observed in this study has not previously been demonstrated in 3T3-L1 adipocytes.
It is generally known that a major pathway of lipolysis in adipocytes is the activation of adenylyl cyclase. The resulting increase in intracellular cAMP levels leads to the dissociation of cAMP-dependent PKA, whose free catalytic subunits phosphorylate, among other substrates, perilipin and hormone-sensitive lipase. This results in enhanced hydrolytic activity, translocation of hormone-sensitive lipase from cytosol to the lipid droplet surface, and subsequent release of glycerol and free fatty acids (reviewed in Ref. 33). However, stimulation of lipolysis in adipocytes via other signal transduction mechanisms involving the extracellular signal-regulated kinase pathway (16, 34) or the Janus kinase-signal transducer and activator of transcription pathway (35) have been described. In the present study, experiments using specific PKA inhibitors demonstrate significant inhibition of VA-?-MSH-stimulated lipolysis, showing PKA involvement, but because no full inhibition was achieved, additional mechanisms may be involved.
The isolated peptides strongly stimulate cAMP production in 3T3-L1 adipocytes, supporting PKA participation in the lipolytic action of these peptides. However, cAMP elevation was 10 times less sensitive to treatment than lipolysis. Exactly the same discrepancy between cAMP formation and the pharmacological effect of ACTH was described for adrenal glomerulosa cells, where both cAMP and Ca2+ influx act synergistically on aldosterone secretion (36). Therefore, an additional signaling pathway may be involved here as well. This is also supported by our findings that forskolin showed similar 50% effective concentration values in the lipolysis assay and in intracellular cAMP formation, leading to the conclusion that for forskolin, only adenylyl cyclase stimulation is the signaling pathway responsible for lipolysis. However, to clarify the relative impact of other signaling pathways, additional experiments will be necessary.
The perilipin proteins are specifically expressed in adipocytes and are located on the surface of the lipid droplet, presenting the major cAMP-dependent PKA substrate in fat cells (33). In 3T3-L1 adipocytes, VA-?-MSH leads to a shift in perilipin migration similar to the effect of isoproterenol in human preadipocytes (37) and cAMP analogs in 3T3-L1 adipocytes (16). Furthermore, CREB was initially characterized as a PKA substrate in 3T3-L1 cells. PKA activation results in the elevation of CREB transcriptional transactivation activity through the phosphorylation of CREB at Ser133 (38). However, CREB phosphorylation was used only as a marker for activation of PKA. In contrast to perilipin phosphorylation, which is involved in lipolysis, whether CREB phosphorylation plays a role in glycerol release was not examined. Together, our results strongly indicate that PKA activation occurs after treatment of 3T3-L1 adipocytes with the peptides isolated in this study.
Of all five melanocortin receptor isoforms described to date, 3T3-L1 adipocytes only express MCR2 and MCR5 (18) (our unpublished observations), which functionally couple to adenylyl cyclase (18). In this study immunoblotting shows the presence of both MC2R and MC5R protein in 3T3-L1 adipocytes. The sizes of the immunoreactive signals are similar to those described previously (39). Notably, MCR2 is activated only by ACTH and does not bind -, ?-, or -MSH (40). Thus, MCR5 or an as yet undescribed MCR mediates lipolysis induced by VA-?-MSH and ?-MSH-(1–22), which will be investigated in future studies.
Activation of hypothalamic MCR4 strongly inhibits food consumption and causes weight loss by regulating energy balance (41, 42). Also, peripheral daily injection of -MSH over the course of 4 wk led to a reduction in fat mass in relation to total body mass (43). Melanocortins have therefore been discussed as candidate molecules for the treatment of obesity, which may also apply for the peptides described in this study, because Harrold et al. (44) suggested that centrally administered ?-MSH is one of the endogenous ligands of the hypothalamic MCR4 that acts to inhibit food intake.
A new aspect of VA-?-MSH and ?-MSH-(1–22) representing circulating peptide hormones is raised in this study, although the lipolytic potential of ?-MSH was previously known. Whether lipolysis or one of the other known actions of melanocortins is their true physiological role needs to be determined. Although isolated in this study after its lipolytic activity on 3T3-L1 adipocytes, this newly identified circulating human melanocortin may also serve other functions in human physiology. Moreover, the fact that these peptides have been identified after a functional assay, but have been overseen in large proteomic approaches (45, 46), underscores the importance of such approaches in identifying previously undescribed circulating bioactive molecules.
Acknowledgments
We thank Ilka Herberz and Kathleen Listemann for their excellent technical assistance.
References
Kwa HG, van der Bent EM, Feltkamp CA, Rumke P, Bloemendal H 1965 Studies on hormones from the anterior pituitary gland. I. Identification and isolation of growth hormone and prolactin from the "granular" fraction of bovine pituitary. Biochem Biophys Acta 111:447–465
Schally AV, Bowers CY, Redding TW, Barrett JF 1966 Isolation of thyrotropin releasing factor (TRF) from porcine hypothalamus. Biochem Biophys Res Commun 25:165–169
Lee CY, Wong S, Lee AS, Ma L 1977 Purification and properties of choriogonadotropin from human term placenta. Hoppe Seylers Z Physiol Chem 358:909–914
Schulz-Knappe P, Schrader M, St?ndker L, Richter R, Hess R, Jürgens M, Forssmann WG 1997 Peptide bank generated by large-scale preparation of circulating human peptides. J Chromatogr A 776:125–132
Louis SN, Jackman GP, Nero TL, Iakovidis D, Louis WJ 2000 Role of ?-adrenergic receptor subtypes in lipolysis. Cardiovasc Drugs Ther 14:565–577
Candelore MR, Deng L, Tota L, Guan XM, Amend A, Liu Y, Newbold R, Cascieri MA, Weber AE 1999 Potent and selective human ?3-adrenergic receptor antagonists. J Pharmacol Exp Ther 290:649–655
Sasaki N, Uchida E, Niiyama M, Yoshida T, Saito M 1998 Anti-obesity effects of selective agonists to the ?3-adrenergic receptor in dogs. I. The presence of canine ?3-adrenergic receptor and in vivo lipomobilization by its agonists. J Vet Med Sci 60:459–463
Shaw JH, Wolfe RR 1987 Fatty acid and glycerol kinetics in septic patients and in patients with gastrointestinal cancer. Ann Surg 205:368–375
Todorov PT, McDevitt TM, Meyer DJ, Ueyama H, Ohkubo I, Tisdale MJ 1998 Purification and characterization of a tumor lipid-mobilizing factor. Cancer Res 58:2353–2358
Carlson LA 1963 Studies on the effect of nicotinic acid on catecholamine stimulated lipolysis in adipose tissue in vitro. Acta Med Scand 173:719–722
Kiwaki K, Levine JA 2003 Differential effects of adrenocorticotropic hormone on human and mouse adipose tissue. J Comp Physiol [B] 173:675–678
Goodman HM, Bray GA 1966 Role of thyroid hormones in lipolysis. Am J Physiol 210:1053–1058
Lucidi P, Parlanti N, Piccioni F, Santeusanio F, De Feo P 2002 Short-term treatment with low doses of recombinant human GH stimulates lipolysis in visceral obese men. J Clin Endocrinol Metab 87:3105–3109
Rosen OM, Smith CJ, Hirsch A, Lai E, Rubin CS 1979 Recent studies of the 3T3–L1 adipocyte-like cell line. Recent Prog Horm Res 35:477–499
Bertagna X, Lenne F, Comar D, Massias JF, Wajcman H, Baudin V, Luton JP, Girard F 1986 Human ?-melanocyte-stimulating hormone revisited. Proc Natl Acad Sci USA 83:9719–9723
Fricke K, Heitland A, Maronde E 2004 Cooperative activation of lipolysis by protein kinase A and protein kinase C pathways in 3T3–L1 adipocytes. Endocrinology 145:4940–4947
Maronde E, Pfeffer M, Olcese J, Molina CA, Schlotter F, Dehghani F, Korf HW, Stehle JH 1999 Transcription factors in neuroendocrine regulation: rhythmic changes in pCREB and ICER levels frame melatonin synthesis. J Neurosci 19:3326–3336
Boston BA, Cone RD 1996 Characterization of melanocortin receptor subtype expression in murine adipose tissues and in the 3T3–L1 cell line. Endocrinology 137:2043–2050
Noda M, Furutani Y, Takahashi H, Toyosato M, Hirose T, Inayama S, Nakanishi S, Numa S 1982 Cloning and sequence analysis of cDNA for bovine adrenal preproenkephalin. Nature 295:202–206
Kakidani H, Furutani Y, Takahashi H, Noda M, Morimoto Y, Hirose T, Asai M, Inayama S, Nakanishi S, Numa S 1982 Cloning and sequence analysis of cDNA for porcine ?-neo-endorphin/dynorphin precursor. Nature 298:245–249
Nakanishi S, Inoue A, Kita T, Nakamura M, Chang AC, Cohen SN, Numa S 1979 Nucleotide sequence of cloned cDNA for bovine corticotropin-?-lipotropin precursor. Nature 278:423–427
Fricker DL 1988 Carboxypeptidase E. Annu Rev Physiol 50:309–321
Benjannet S, Rondeau N, Day R, Chretien M, Seidah MG 1991 PC1 and PC2 are proprotein convertases capable of cleaving proopiomelanocortin at distinct pairs of basic residues. Proc Natl Acad Sci USA 88:3564–3570
Thomas L, Leduc R, Thorne BA, Smeekens S, Steiner DF, Thomas G 1991 Kex2-like endoproteases PC2 and PC3 accurately cleave a model prohormone in mammalian cells: evidence for a common core of neuroendocrine processing enzymes. Proc Natl Acad Sci USA 88:5297–5301
Dong W, Marcinkiewicz MN, Vieau D, Chretien M, Seidah NG, Day R 1995 Distinct mRNA expression of the highly homologous convertases PC5 and PACE4 in the rat brain and pituitary. J Neurosci 15:1778–1796
Bertagna X, Seidah N, Massias JF, Lenne F, Luton JP, Girard F, Chretien M 1988 Microsequencing evidence for the maturation of human proopiomelanocortin into an 18 amino acid hormone [h?MSH(5–22)] in nonpituitary tissue. Peptides 10:83–87
Harris JI 1959 Structure of a melanocyte-stimulating hormone from the human pituitary gland. Nature 184:167–169
Abe K, Nicholson WE, Liddle GW, Orth DN, Island DP 1969 Normal and abnormal regulation of ?-MSH in man. J Clin Invest 48:1580–1585
Dixon HBF 1960 Chromatographic isolations of pig and human melanocyte-stimulating hormones. Biochim Biophys Acta 37:38–42
Bloomfield GA, Scott AP, Lowry PJ, Gilkes JJ, Rees LH 1974 A reappraisal of human ? MSH. Nature 252:492–493
Barat E, Patthy A, Graf L 1979 Action of cathepsin D on human ?-lipotropin: a possible source of human "?-melanotropin." Proc Natl Acad Sci USA 76:6120–6123
Richter WO, Schwandt P 1987 Lipolytic potency of proopiomelanocorticotropin peptides in vitro. Neuropeptides 9:59–74
Holm C 2003 Molecular mechanisms regulating hormone-sensitive lipase and lipolysis. Biochem Soc Transact 31:1120–1124
Greenberg AS, Shen WS, Muliro K, Patel S, Souza SC, Roth RA, Kraemer FB 2001 Stimulation of lipolysis and hormone-sensitive lipase via the extracellular signal-regulated kinase pathway. J Biol Chem 276:45456–45461
Asada N, Takahashi Y, Wada M, Naito N, Uchida H, Ikeda M, Honjo M 2000 GH induced lipolysis stimulation in 3T3–L1 adipocytes stably expressing hGHR: analysis on signaling pathway and activity of 20 K hGH. Mol Cell Endocrinol 162:121–129
Kojima I, Kojima K, Rasmussen H 1985 Role of calcium and cAMP in the action of adrenocorticotropin on aldosterone secretion. J Biol Chem 260:4248–4256
Sengenès C, Bouloumié A, Hauner H, Berlan M, Busse R, Lafontan M, Galitzky J 2003 Involvement of a cGMP-dependent pathway in the natriuretic peptide-mediated hormone-sensitive lipase phosphorylation in human adipocytes. J Biol Chem 278:48617–48626
Gonzalez GA, Montminy MR 1989 Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell 59:675–680
Akbulut S, Byersdorfer CA, Larsen CP, Zimmer SL, Humphreys TD, Clarke BL 2001 Expression of the melanocortin 5 receptor on rat lymphocytes. Biochem Biophys Res Commun 281:1086–1092
Schioth HB, Chhajlani V, Muceniece R, Klusa V, Wikberg JE 1996 Major pharmacological distinction of the ACTH receptor from other melanocortin receptors. Life Sci 59:797–801
Fan W, Boston BA, Kesterson RA, Hruby WJ, Cone RD 1997 Role of melanocortinergic neurons in feeding and the agouti obesity syndrome. Nature 385:165–168
Huszar D, Lynch CA, Fairchild-Huntress V, Dunmore JH, Fang Q, Berkemeyer LR 1997 Targeted disruption of the melanocortin-4-receptor results in obesity in mice. Cell 88:131–141
Cornish J, Callon KE, Mountjoy KG, Bava U, Lin JM, Myers DE, Naot D, Reid IR 2003 -Melanocyte-stimulating hormone is a novel regulator of bone. Am J Physiol 284:E1181–E1190
Harrold JA, Widdowson PS, Williams G 2003 ?-MSH: a functional ligand that regulated energy homeostasis via hypothalamic MC4-R? Peptides 24:397–405
Rose K, Bougueleret L, Baussant T, Bohm G, Botti P, Colinge J, Cusin I, Gaertner H, Gleizes A, Heller M, Jimenez S, Johnson A, Kussmann M, Menin L, Menzel C, Ranno F, Rodriguez-Tome P, Rogers J, Saudrais C, Villain M, Wetmore D, Bairoch A, Hochstrasser D 2004 Industrial-scale proteomics: from liters of plasma to chemically synthesized proteins. Proteomics 4:2125–2150
Chan KC, Lucas DA, Hise D, Schaefer CF, Xiao Z, Janini GM, Buetow KH, Issaq HJ, Veenstra TD, Conrads TP 2004 Analysis of the human serum proteome. Clin Proteomics 1:101–226(Katrin Fricke, Axel Schul)