The Leu34Phe ProCART Mutation Leads to Cocaine- and Amphetamine-Regulated Transcript (CART) Deficiency: A Possible Cause for Obesity in Humans
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《内分泌学杂志》
Section on Cellular Neurobiology (T.Y., Y.P.L.), National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892
Yerkes National Primate Center of Emory University (G.D., M.J.K.), Atlanta, Georgia 30329
Dipartimento di Pediatria (E.M.D.G.), Seconda Universita di Napoli, 80138 Napoli, Italy
Abstract
Cocaine- and amphetamine-regulated transcript (CART) is an anorexigenic neuropeptide synthesized in the hypothalamus. A Leu34Phe missense mutation in proCART has been found in an obese family in humans. Here we show that humans bearing the Leu34Phe mutation in proCART have severely diminished levels of bioactive CART, but elevated amounts of partially processed proCART in their serum. Expression of wild-type proCART in AtT-20 cells showed that it was sorted to the regulated secretory pathway, a necessity for proper processing to bioactive CART. However, expressed Leu34Phe proCART was missorted, poorly processed, and secreted constitutively. The defective intracellular sorting of Leu34Phe proCART would account for the reduced levels of bioactive CART in affected humans. These results suggest that the obesity observed in humans bearing the Leu34Phe mutation could be due to a putative deficiency in hypothalamic bioactive CART.
Introduction
SEVERAL HYPOTHALAMIC peptides have been shown to control feeding and appetite. These include neuropeptide Y, MSH, ACTH hormone, melanin-concentrating hormone, and recently, cocaine- and amphetamine-regulated transcript (CART) is one more peptide shown to be involved in control of feeding (1, 2, 3). Such a role for CART was demonstrated when recombinant CART injected intracerebroventricularly into rats inhibited feeding (4). Also, injection of antisera against CART increased feeding, indicating that CART may be an endogenous inhibitor of food intake (4, 5). Furthermore, a knockout (CART–/–) mouse showed increased body weight after being fed standard laboratory food (6), and another knockout mouse model, on a high-fat diet, showed an obese phenotype (7).
Mutations in human genes that encode precursors of such appetite-controlling peptides, e.g. proopiomelanocortin (8), can lead to early-onset obesity. Recently, a missense mutation in the CART gene that changes Leu at codon 34 of proCART to Phe (Fig. 1A) has been discovered in a 10-yr-old Italian boy who has been obese since age 2. His birth weight was normal. At 10 yr of age, his body mass index was 32 kg/m2 and his serum leptin concentration (23 ng/ml) was normal for his body mass index, sex, and pubertal development (9). The mutation also cosegregated for three generations in his maternal relatives who are obese. These patients show hyperphagic behavior (Del Giudice, E. M., unpublished data) and resting metabolic rates were lower than expected in the boy and his mother (9).
CART is expressed abundantly in the arcuate and paraventricular nuclei of the hypothalamus, as well as in pituitary and adrenal glands (10). Human proCART consists of 89 amino acids (Fig. 1A) preceded by a 27-residue signal peptide at the N terminus. It also contains several dibasic amino acids, which are processing sites that are cleaved by prohormone convertases, PC1/3 and PC2, to form intermediate 8-kDa CART (10–89), and two bioactive peptides, 5.2-kDa CART I (42–89), and 4.4-kDa CART II (49–89) (11) (Fig. 1A). We propose that the Leu34Phe mutation might cause conformational changes that could lead to processing and/or trafficking defects of proCART, and the lack of formation of bioactive CART would lead to obesity. Preliminary studies in AtT-20 cells, an endocrine cell line have suggested a possible defect in the processing of Leu34Phe proCART (12).
In this study, we examined the processing of Leu34Phe proCART in humans by analyzing the forms of CART in serum. We also investigated the trafficking and secretion of Leu34Phe proCART in AtT-20 cells. Our results indicate that poor processing of Leu34Phe proCART due to intracellular missorting leads to diminished bioactive CART, and increased levels of pro- and intermediate CART in the serum of obese humans bearing the Leu34Phe mutation.
Materials and Methods
Surface-enhanced laser desorption ionization-time-of-flight mass spectrometry
The ProteinChip antibody capture kit was used (Ciphergen Biosystems, Fremont, CA) for the detection of CART immunoreactive proteins directly from the serum, according to manufacturer’s instructions. Blood samples were drawn with the informed consent of the patients and the control participants. Serum was prepared by centrifugation (500 x g, 10 min), collected and stored at –80 C until analyzed. Anti-CART (55–102) (Phoenix Pharmaceuticals Inc., Belmont, CA), which recognizes the active form of CART (C terminus of proCART), was coupled to the antibody capture chips. The chips were washed in PBS with 0.5% Triton-X and incubated with serum (2 μl of 10 x diluted). To facilitate desorption and ionization of proteins on the ProteinChip array, 0.5 μl of matrix, cyno-4-hydroxy cinnamic acid (Ciphergen Biosystems) in 50% acetonitrile containing 1% trifluoroacetic acid was added to each spot on the chips. Data acquisition was performed by a ProteinChip reader (Ciphergen Biosystems). The individual proteins were displayed as unique peaks based on their molecular mass. Peaks were base-line subtracted, calibrated for mass accuracy, detected and clustered automatically by the analysis software. Spectral analysis was performed using software version 3.2.1 from Ciphergen Biosystems, which integrates the area under each peak.
Transfection of wild-type (WT) and mutant CART constructs into AtT-20 cells
AtT-20 cells from American Type Culture Collection (Manassas, VA) were grown in six-well plates with complete DMEM and 10% fetal bovine serum. The cells were then transfected with 5 μg of the plasmid carrying WT, or mutant (M) CART cDNA. In all experiments, cells were transiently transfected for 18 h using LipofectAMINE 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.
Western blot of cell extracts
Transfected cells were extracted in lysis buffer (Pierce, Rockford, IL) plus protease inhibitors (1x complete mini protease inhibitor cocktail; Roche Molecular Biochemicals, Indianapolis, IN). The soluble extracts, obtained after centrifugation of the total cell lysates at 15,000 x g for 10 min, were assayed for protein content. Forty micrograms of protein from each extract were analyzed by Western blot. The blots were probed with anti-CART (55–102) (1:5000 dilution; Phoenix Pharmaceuticals Inc.) overnight at 4 C. Visualization of the Western blot signal was by enhanced chemiluminescence (Pierce Chemical Co., Rockford, IL) using goat antirabbit IgG coupled to horseradish peroxidase (1:10,000; Sigma, St. Louis, MO) as the secondary antibody.
Secretion assay
For secretion assay, transfected cells were rinsed with the medium containing high-glucose DMEM supplemented with 0.01% BSA and then incubated in 1 ml of the same medium at 37 C for two 30-min periods (basal release). This was followed by a third 30-min incubation period (stimulated release) with 50 mM KCl/DMEM supplemented with 0.01% BSA. The medium was collected after each incubation period and analyzed. Equal aliquots (40 μl) of the basal and stimulated media were analyzed by Western blot for CART immunoreactivity using anti-CART (55–102) (1:5000 dilution; Phoenix Pharmaceuticals Inc.). Immunoreactive CART bands on the Western blots were quantified using ImageQuant software, version 5.2 (Molecular Dynamics, Sunnyvale, CA).
Pulse-chase secretion assay
AtT-20 cells were transfected with WT or M CART constructs as described above. Cells were then starved in DMEM without cysteine and methionine for 30 min and then pulsed labeled with 140 μCi 35S cys/met/1 ml for 30 min, followed by two 30-min chase periods in DMEM, and a third 30-min chase period in DMEM containing 50 mM KCl. Media from the incubation periods and cells were collected and CART-related proteins were by immunoprecipitated with anti-CART (Phoenix Pharmaceuticals, Inc.) The immunoprecipitates were analyzed by SDS-PAGE using 4–12% polyacrylamide Nu-PAGE gels (Invitrogen, San Diego, CA) and the radiolabeled signal on the gel was recorded and read by a phosphorimage system (Molecular Dynamics). The level of radiolabeled signal in the band corresponding to CART species was quantified using ImageQuant, version 5.2 (Molecular Dynamics). After background subtraction, radioactivity in each band was expressed as the percentage of total labeling, which included labeled CART from all the media plus cell lysate extracted at the end of the secretion assay.
Immunofluorescence microscopy
Immunofluorescence microscopy was performed as described by Arnaoutova et al. in 2003 (17). Primary antibodies used were polyclonal anti-CART (55–102) (Phoenix Pharmaceuticals, Inc.), and monoclonal anti-ACTH (Abcam, Cambridge, MA); and secondary antibodies used were Alexa Fluor 568 goat antirabbit and Alexa Fluor 488 goat antimouse (Molecular Probes, Eugene, OR).
Results
Figure 1B shows the analysis of circulating forms of CART in the obese boy, and his obese maternal relatives bearing the Leu34Phe mutation, his unaffected sister, and an unrelated, nonobese human (male). ProteinChip-antibody capture assays of serum from affected humans (Fig. 1B) revealed incomplete processing of mutant (M) Leu34Phe proCART to primarily an intermediate form of CART (7.7 kDa). Very low levels of bioactive CART II (4.4 kDa) and no CART I (5.2 kDa) were detected compared with normal humans. In the seven control nonobese humans tested in this study, CART II (4.4 kDa) was the predominant form observed in serum although a small amount of CART I (5.2 kDa) was present (Fig. 1B and data not shown). To analyze the amount of intermediate vs. mature CART present in serum of the obese patients and controls, we quantified the area under the 4.4-kDa and 7.7-kDa peaks (Table 1). Ninety-three to 97% of circulating CART was the intermediate form, and only 3–7% was CART II in affected individuals. In contrast, sera from the unaffected sister and six nonobese normal humans showed that 30–76% of the circulating CART was in the form of CART II. The average percentage of 4.4-kDa bioactive CART in nonobese normal humans including the unaffected sister was 59% ± 6 SEM (n = 7) compared with 5% ± 1 SEM (n = 3) in affected individuals. These results indicate that Leu34Phe proCART is poorly processed to active CART in humans.
To investigate the cellular basis for this lack of processing of Leu34Phe proCART, cDNAs were transiently transfected into AtT-20 cells, an endocrine cell line that is derived from pituitary, and synthesizes only very low levels of CART (12). Western blotting of the cell extracts using human anti-CART (55–102) revealed that WT proCART was largely processed to intermediate (8 kDa) and active CART I (5.2 kDa) (Fig. 2A). Because AtT-20 cells do not express the convertase, PC2, needed to generate CART II, this form was not detected. In contrast, Leu34Phe proCART was partially processed to intermediate forms (8 kDa), and only minimally processed to yield the (5.2 kDa) active CART form (Fig. 2A).
To determine whether the poor processing was due to a defect in intracellular trafficking, stimulated secretion of WT and M proCART from transfected AtT-20 cells was studied by Western blotting. Figure 2B shows that during two 30-min periods of basal secretion (B1 and B2), a small amount of bioactive CART I (5.2 kDa) was detected in the medium of cells expressing WT CART. Upon depolarization of these cells for 30 min, a 3.3 ± 0.4 SEM-fold increase (n = 5) in stimulated secretion of active CART I (5.2 kDa) was observed (Fig. 2B). In cells expressing M proCART, during the two 30-min periods of basal release, only the proform and an intermediate form were detected in the medium, and no stimulated secretion of CART (0.8 ± 0.1 SEM-fold; n = 4) was observed (Fig. 2C).
To assess the level of secretion relative to synthesis, release of newly synthesized proCART/CART was examined using a pulse-chase paradigm. AtT-20 cells were transfected with WT or M proCART cDNA, pulse labeled with 35S cys/met for 30 min followed by two 30-min chase periods, and then depolarized for 30 min. WT proCART/CART secreted during the second chase period (basal secretion), and after depolarization with 50 mM KCl, expressed as % total in cell lysate plus media were 13.7% ± 1.5 SEM (n = 3) and 27.7% ± 6 SEM (n = 3), respectively, showing stimulated secretion. In contrast, cells transfected with M proCART showed release of 13.2% ± 1.6 SEM (n = 3) during the second chase period and 12.7% ± 3 SEM (n = 3) with 50 mM KCl treatment, indicating constitutive secretion (13), and no stimulated secretion. The significant amount (13.2%) of newly synthesized mutant proCART secreted into the medium indicates that it was not trapped at the trans-Golgi network (TGN).
To investigate the subcellular localization of WT and M proCART, immunocytochemical studies were performed. After transfection of AtT-20 cells with WT or M proCART constructs, cells were fixed, permeabilized, and probed with antibodies against CART and ACTH, an endogenous secretory granule marker. Cells expressing WT proCART/CART showed punctate anti-CART immunostaining that colocalized with ACTH in secretory granules at the tips of the processes where the secretory granules reside (Fig. 3, A–C). In contrast, many of the labeled cells expressing M proCART showed no colocalization of immunoreactive CART with ACTH at the tips of the cell processes (Fig. 3, D–F). The number of cells showing colocalization of CART and ACTH immunoreactivity in the processes was quantified by counting 120 cells with processes in each of six separate experiments for both genotypes. Ninety percent ± 1 SEM (n = 6) of cells expressing WT proCART showed colocalization of immunoreactive CART with immunoreactive ACTH. In contrast, only 53% ± 5 SEM (n = 6) of cells expressing M proCART showed colocalization of immunoreactive CART with immunoreactive ACTH, indicating that a significant proportion of M proCART was not directed into the granules of the regulated secretory pathway.
The secretion and immunocytochemical studies taken together indicate that M proCART was not efficiently sorted into the regulated secretory pathway, but was secreted constitutively, primarily as the precursor and an intermediate. This is consistent with the presence of mutant pro- and intermediate CART in the circulation of affected humans.
Discussion
A Leu34Phe proCART mutation was uncovered in members of an Italian family that have early onset obesity (9). However, the relationship between the mutation and the obese phenotype was not understood. In this study, we show that affected members of the family have severely diminished circulating levels of bioactive CART, compared with an unaffected member of the same family, as well as six nonobese humans used as controls (Table 1). Instead, the obese members had high levels of circulating intermediate CART, indicating impaired processing of Leu34Phe proCART.
Further understanding of the inefficient processing of Leu34Phe proCART came from our cell biological studies in AtT-20 cells. We demonstrated that Leu34Phe proCART was missorted at the TGN to the constitutive secretory pathway, and secreted as the proform and a partially processed intermediate (Figs. 2 and 3). This finding suggests that Leu34 may be part of a regulated secretory pathway sorting signal because sorting motifs have been reported to contain leucine residues (14). Neuropeptide precursors are generally processed after packaging into granules of the regulated secretory pathway at the TGN. These granules have an acidic environment allowing the prohormone convertases (PCs) with a low pH optimum to work more efficiently (15). However, when Leu34Phe proCART was missorted to the constitutive pathway, which lacks the necessary PCs, it was poorly processed. The partial processing of the mutant proform to intermediate CART that did occur was likely due to the action of some active PC1/3 present in the Golgi apparatus (16).
Circulating CART forms, although only reflecting secretion from peripheral organs (pituitary and adrenals) (2), nevertheless provided an insight into the poor processing of Leu34Phe proCART in humans. We propose that, in hypothalamic neurons, missorting of Leu34Phe proCART to the constitutive pathway would result in impaired processing and lack of activity-dependent secretion of active CART at synapses. Because CART is an anorexigenic peptide, its deficiency in the brain of humans bearing the Leu34Phe missense mutation in the proCART gene would lead to poor appetite control and the obese phenotype seen in these individuals.
In summary, this study has 1) identified a deficiency of bioactive CART in humans bearing the Leu34Phe mutation, 2) elucidated the cellular basis for the deficiency by demonstrating an intracellular trafficking defect of Leu34Phe proCART that accounts for incomplete processing of the mutant precursor, and 3) suggested a link between the absence of bioactive CART and the onset of obesity in humans.
Footnotes
This research was supported in part by the Intramural Research Program of the National Institutes of Health (NIH), National Institute of Child Health and Human Development. G.D. and M.J.K. were supported by NIH Grant DA15162.
T.Y., G.D., E.M.D.G., and Y.P.L. have nothing to declare. M.J.K. has consulting fees from a law firm, Frommer, Lawrence, and Huag. M.K.J. also has stock options from AptoTec, Inc.
First Published Online October 6, 2005
Abbreviations: CART, Cocaine- and amphetamine-regulated transcript; M, mutant; TGN, trans-Golgi network; WT, wild type.
Accepted for publication September 28, 2005.
References
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Yerkes National Primate Center of Emory University (G.D., M.J.K.), Atlanta, Georgia 30329
Dipartimento di Pediatria (E.M.D.G.), Seconda Universita di Napoli, 80138 Napoli, Italy
Abstract
Cocaine- and amphetamine-regulated transcript (CART) is an anorexigenic neuropeptide synthesized in the hypothalamus. A Leu34Phe missense mutation in proCART has been found in an obese family in humans. Here we show that humans bearing the Leu34Phe mutation in proCART have severely diminished levels of bioactive CART, but elevated amounts of partially processed proCART in their serum. Expression of wild-type proCART in AtT-20 cells showed that it was sorted to the regulated secretory pathway, a necessity for proper processing to bioactive CART. However, expressed Leu34Phe proCART was missorted, poorly processed, and secreted constitutively. The defective intracellular sorting of Leu34Phe proCART would account for the reduced levels of bioactive CART in affected humans. These results suggest that the obesity observed in humans bearing the Leu34Phe mutation could be due to a putative deficiency in hypothalamic bioactive CART.
Introduction
SEVERAL HYPOTHALAMIC peptides have been shown to control feeding and appetite. These include neuropeptide Y, MSH, ACTH hormone, melanin-concentrating hormone, and recently, cocaine- and amphetamine-regulated transcript (CART) is one more peptide shown to be involved in control of feeding (1, 2, 3). Such a role for CART was demonstrated when recombinant CART injected intracerebroventricularly into rats inhibited feeding (4). Also, injection of antisera against CART increased feeding, indicating that CART may be an endogenous inhibitor of food intake (4, 5). Furthermore, a knockout (CART–/–) mouse showed increased body weight after being fed standard laboratory food (6), and another knockout mouse model, on a high-fat diet, showed an obese phenotype (7).
Mutations in human genes that encode precursors of such appetite-controlling peptides, e.g. proopiomelanocortin (8), can lead to early-onset obesity. Recently, a missense mutation in the CART gene that changes Leu at codon 34 of proCART to Phe (Fig. 1A) has been discovered in a 10-yr-old Italian boy who has been obese since age 2. His birth weight was normal. At 10 yr of age, his body mass index was 32 kg/m2 and his serum leptin concentration (23 ng/ml) was normal for his body mass index, sex, and pubertal development (9). The mutation also cosegregated for three generations in his maternal relatives who are obese. These patients show hyperphagic behavior (Del Giudice, E. M., unpublished data) and resting metabolic rates were lower than expected in the boy and his mother (9).
CART is expressed abundantly in the arcuate and paraventricular nuclei of the hypothalamus, as well as in pituitary and adrenal glands (10). Human proCART consists of 89 amino acids (Fig. 1A) preceded by a 27-residue signal peptide at the N terminus. It also contains several dibasic amino acids, which are processing sites that are cleaved by prohormone convertases, PC1/3 and PC2, to form intermediate 8-kDa CART (10–89), and two bioactive peptides, 5.2-kDa CART I (42–89), and 4.4-kDa CART II (49–89) (11) (Fig. 1A). We propose that the Leu34Phe mutation might cause conformational changes that could lead to processing and/or trafficking defects of proCART, and the lack of formation of bioactive CART would lead to obesity. Preliminary studies in AtT-20 cells, an endocrine cell line have suggested a possible defect in the processing of Leu34Phe proCART (12).
In this study, we examined the processing of Leu34Phe proCART in humans by analyzing the forms of CART in serum. We also investigated the trafficking and secretion of Leu34Phe proCART in AtT-20 cells. Our results indicate that poor processing of Leu34Phe proCART due to intracellular missorting leads to diminished bioactive CART, and increased levels of pro- and intermediate CART in the serum of obese humans bearing the Leu34Phe mutation.
Materials and Methods
Surface-enhanced laser desorption ionization-time-of-flight mass spectrometry
The ProteinChip antibody capture kit was used (Ciphergen Biosystems, Fremont, CA) for the detection of CART immunoreactive proteins directly from the serum, according to manufacturer’s instructions. Blood samples were drawn with the informed consent of the patients and the control participants. Serum was prepared by centrifugation (500 x g, 10 min), collected and stored at –80 C until analyzed. Anti-CART (55–102) (Phoenix Pharmaceuticals Inc., Belmont, CA), which recognizes the active form of CART (C terminus of proCART), was coupled to the antibody capture chips. The chips were washed in PBS with 0.5% Triton-X and incubated with serum (2 μl of 10 x diluted). To facilitate desorption and ionization of proteins on the ProteinChip array, 0.5 μl of matrix, cyno-4-hydroxy cinnamic acid (Ciphergen Biosystems) in 50% acetonitrile containing 1% trifluoroacetic acid was added to each spot on the chips. Data acquisition was performed by a ProteinChip reader (Ciphergen Biosystems). The individual proteins were displayed as unique peaks based on their molecular mass. Peaks were base-line subtracted, calibrated for mass accuracy, detected and clustered automatically by the analysis software. Spectral analysis was performed using software version 3.2.1 from Ciphergen Biosystems, which integrates the area under each peak.
Transfection of wild-type (WT) and mutant CART constructs into AtT-20 cells
AtT-20 cells from American Type Culture Collection (Manassas, VA) were grown in six-well plates with complete DMEM and 10% fetal bovine serum. The cells were then transfected with 5 μg of the plasmid carrying WT, or mutant (M) CART cDNA. In all experiments, cells were transiently transfected for 18 h using LipofectAMINE 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.
Western blot of cell extracts
Transfected cells were extracted in lysis buffer (Pierce, Rockford, IL) plus protease inhibitors (1x complete mini protease inhibitor cocktail; Roche Molecular Biochemicals, Indianapolis, IN). The soluble extracts, obtained after centrifugation of the total cell lysates at 15,000 x g for 10 min, were assayed for protein content. Forty micrograms of protein from each extract were analyzed by Western blot. The blots were probed with anti-CART (55–102) (1:5000 dilution; Phoenix Pharmaceuticals Inc.) overnight at 4 C. Visualization of the Western blot signal was by enhanced chemiluminescence (Pierce Chemical Co., Rockford, IL) using goat antirabbit IgG coupled to horseradish peroxidase (1:10,000; Sigma, St. Louis, MO) as the secondary antibody.
Secretion assay
For secretion assay, transfected cells were rinsed with the medium containing high-glucose DMEM supplemented with 0.01% BSA and then incubated in 1 ml of the same medium at 37 C for two 30-min periods (basal release). This was followed by a third 30-min incubation period (stimulated release) with 50 mM KCl/DMEM supplemented with 0.01% BSA. The medium was collected after each incubation period and analyzed. Equal aliquots (40 μl) of the basal and stimulated media were analyzed by Western blot for CART immunoreactivity using anti-CART (55–102) (1:5000 dilution; Phoenix Pharmaceuticals Inc.). Immunoreactive CART bands on the Western blots were quantified using ImageQuant software, version 5.2 (Molecular Dynamics, Sunnyvale, CA).
Pulse-chase secretion assay
AtT-20 cells were transfected with WT or M CART constructs as described above. Cells were then starved in DMEM without cysteine and methionine for 30 min and then pulsed labeled with 140 μCi 35S cys/met/1 ml for 30 min, followed by two 30-min chase periods in DMEM, and a third 30-min chase period in DMEM containing 50 mM KCl. Media from the incubation periods and cells were collected and CART-related proteins were by immunoprecipitated with anti-CART (Phoenix Pharmaceuticals, Inc.) The immunoprecipitates were analyzed by SDS-PAGE using 4–12% polyacrylamide Nu-PAGE gels (Invitrogen, San Diego, CA) and the radiolabeled signal on the gel was recorded and read by a phosphorimage system (Molecular Dynamics). The level of radiolabeled signal in the band corresponding to CART species was quantified using ImageQuant, version 5.2 (Molecular Dynamics). After background subtraction, radioactivity in each band was expressed as the percentage of total labeling, which included labeled CART from all the media plus cell lysate extracted at the end of the secretion assay.
Immunofluorescence microscopy
Immunofluorescence microscopy was performed as described by Arnaoutova et al. in 2003 (17). Primary antibodies used were polyclonal anti-CART (55–102) (Phoenix Pharmaceuticals, Inc.), and monoclonal anti-ACTH (Abcam, Cambridge, MA); and secondary antibodies used were Alexa Fluor 568 goat antirabbit and Alexa Fluor 488 goat antimouse (Molecular Probes, Eugene, OR).
Results
Figure 1B shows the analysis of circulating forms of CART in the obese boy, and his obese maternal relatives bearing the Leu34Phe mutation, his unaffected sister, and an unrelated, nonobese human (male). ProteinChip-antibody capture assays of serum from affected humans (Fig. 1B) revealed incomplete processing of mutant (M) Leu34Phe proCART to primarily an intermediate form of CART (7.7 kDa). Very low levels of bioactive CART II (4.4 kDa) and no CART I (5.2 kDa) were detected compared with normal humans. In the seven control nonobese humans tested in this study, CART II (4.4 kDa) was the predominant form observed in serum although a small amount of CART I (5.2 kDa) was present (Fig. 1B and data not shown). To analyze the amount of intermediate vs. mature CART present in serum of the obese patients and controls, we quantified the area under the 4.4-kDa and 7.7-kDa peaks (Table 1). Ninety-three to 97% of circulating CART was the intermediate form, and only 3–7% was CART II in affected individuals. In contrast, sera from the unaffected sister and six nonobese normal humans showed that 30–76% of the circulating CART was in the form of CART II. The average percentage of 4.4-kDa bioactive CART in nonobese normal humans including the unaffected sister was 59% ± 6 SEM (n = 7) compared with 5% ± 1 SEM (n = 3) in affected individuals. These results indicate that Leu34Phe proCART is poorly processed to active CART in humans.
To investigate the cellular basis for this lack of processing of Leu34Phe proCART, cDNAs were transiently transfected into AtT-20 cells, an endocrine cell line that is derived from pituitary, and synthesizes only very low levels of CART (12). Western blotting of the cell extracts using human anti-CART (55–102) revealed that WT proCART was largely processed to intermediate (8 kDa) and active CART I (5.2 kDa) (Fig. 2A). Because AtT-20 cells do not express the convertase, PC2, needed to generate CART II, this form was not detected. In contrast, Leu34Phe proCART was partially processed to intermediate forms (8 kDa), and only minimally processed to yield the (5.2 kDa) active CART form (Fig. 2A).
To determine whether the poor processing was due to a defect in intracellular trafficking, stimulated secretion of WT and M proCART from transfected AtT-20 cells was studied by Western blotting. Figure 2B shows that during two 30-min periods of basal secretion (B1 and B2), a small amount of bioactive CART I (5.2 kDa) was detected in the medium of cells expressing WT CART. Upon depolarization of these cells for 30 min, a 3.3 ± 0.4 SEM-fold increase (n = 5) in stimulated secretion of active CART I (5.2 kDa) was observed (Fig. 2B). In cells expressing M proCART, during the two 30-min periods of basal release, only the proform and an intermediate form were detected in the medium, and no stimulated secretion of CART (0.8 ± 0.1 SEM-fold; n = 4) was observed (Fig. 2C).
To assess the level of secretion relative to synthesis, release of newly synthesized proCART/CART was examined using a pulse-chase paradigm. AtT-20 cells were transfected with WT or M proCART cDNA, pulse labeled with 35S cys/met for 30 min followed by two 30-min chase periods, and then depolarized for 30 min. WT proCART/CART secreted during the second chase period (basal secretion), and after depolarization with 50 mM KCl, expressed as % total in cell lysate plus media were 13.7% ± 1.5 SEM (n = 3) and 27.7% ± 6 SEM (n = 3), respectively, showing stimulated secretion. In contrast, cells transfected with M proCART showed release of 13.2% ± 1.6 SEM (n = 3) during the second chase period and 12.7% ± 3 SEM (n = 3) with 50 mM KCl treatment, indicating constitutive secretion (13), and no stimulated secretion. The significant amount (13.2%) of newly synthesized mutant proCART secreted into the medium indicates that it was not trapped at the trans-Golgi network (TGN).
To investigate the subcellular localization of WT and M proCART, immunocytochemical studies were performed. After transfection of AtT-20 cells with WT or M proCART constructs, cells were fixed, permeabilized, and probed with antibodies against CART and ACTH, an endogenous secretory granule marker. Cells expressing WT proCART/CART showed punctate anti-CART immunostaining that colocalized with ACTH in secretory granules at the tips of the processes where the secretory granules reside (Fig. 3, A–C). In contrast, many of the labeled cells expressing M proCART showed no colocalization of immunoreactive CART with ACTH at the tips of the cell processes (Fig. 3, D–F). The number of cells showing colocalization of CART and ACTH immunoreactivity in the processes was quantified by counting 120 cells with processes in each of six separate experiments for both genotypes. Ninety percent ± 1 SEM (n = 6) of cells expressing WT proCART showed colocalization of immunoreactive CART with immunoreactive ACTH. In contrast, only 53% ± 5 SEM (n = 6) of cells expressing M proCART showed colocalization of immunoreactive CART with immunoreactive ACTH, indicating that a significant proportion of M proCART was not directed into the granules of the regulated secretory pathway.
The secretion and immunocytochemical studies taken together indicate that M proCART was not efficiently sorted into the regulated secretory pathway, but was secreted constitutively, primarily as the precursor and an intermediate. This is consistent with the presence of mutant pro- and intermediate CART in the circulation of affected humans.
Discussion
A Leu34Phe proCART mutation was uncovered in members of an Italian family that have early onset obesity (9). However, the relationship between the mutation and the obese phenotype was not understood. In this study, we show that affected members of the family have severely diminished circulating levels of bioactive CART, compared with an unaffected member of the same family, as well as six nonobese humans used as controls (Table 1). Instead, the obese members had high levels of circulating intermediate CART, indicating impaired processing of Leu34Phe proCART.
Further understanding of the inefficient processing of Leu34Phe proCART came from our cell biological studies in AtT-20 cells. We demonstrated that Leu34Phe proCART was missorted at the TGN to the constitutive secretory pathway, and secreted as the proform and a partially processed intermediate (Figs. 2 and 3). This finding suggests that Leu34 may be part of a regulated secretory pathway sorting signal because sorting motifs have been reported to contain leucine residues (14). Neuropeptide precursors are generally processed after packaging into granules of the regulated secretory pathway at the TGN. These granules have an acidic environment allowing the prohormone convertases (PCs) with a low pH optimum to work more efficiently (15). However, when Leu34Phe proCART was missorted to the constitutive pathway, which lacks the necessary PCs, it was poorly processed. The partial processing of the mutant proform to intermediate CART that did occur was likely due to the action of some active PC1/3 present in the Golgi apparatus (16).
Circulating CART forms, although only reflecting secretion from peripheral organs (pituitary and adrenals) (2), nevertheless provided an insight into the poor processing of Leu34Phe proCART in humans. We propose that, in hypothalamic neurons, missorting of Leu34Phe proCART to the constitutive pathway would result in impaired processing and lack of activity-dependent secretion of active CART at synapses. Because CART is an anorexigenic peptide, its deficiency in the brain of humans bearing the Leu34Phe missense mutation in the proCART gene would lead to poor appetite control and the obese phenotype seen in these individuals.
In summary, this study has 1) identified a deficiency of bioactive CART in humans bearing the Leu34Phe mutation, 2) elucidated the cellular basis for the deficiency by demonstrating an intracellular trafficking defect of Leu34Phe proCART that accounts for incomplete processing of the mutant precursor, and 3) suggested a link between the absence of bioactive CART and the onset of obesity in humans.
Footnotes
This research was supported in part by the Intramural Research Program of the National Institutes of Health (NIH), National Institute of Child Health and Human Development. G.D. and M.J.K. were supported by NIH Grant DA15162.
T.Y., G.D., E.M.D.G., and Y.P.L. have nothing to declare. M.J.K. has consulting fees from a law firm, Frommer, Lawrence, and Huag. M.K.J. also has stock options from AptoTec, Inc.
First Published Online October 6, 2005
Abbreviations: CART, Cocaine- and amphetamine-regulated transcript; M, mutant; TGN, trans-Golgi network; WT, wild type.
Accepted for publication September 28, 2005.
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