Introduction

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Most neuroendocrine peptides are produced from precursors by limited proteolysis. A number of enzymes involved in the processing of neuropeptides have been identified and characterized (for review, see Steiner, 1998). In a majority of cases, endoproteolysis by prohormone convertases is followed by the removal of C-terminal basic residue extensions by CPE (Fricker, 1991). A point mutation in the coding region of the CPE gene results in a loss of enzyme activity that correlates with the development of late onset obesity in Cpe^fat/Cpe^fat mice (Naggert et al., 1995). These mice exhibit a deficiency in the C-terminal trimming of basic residues (Che et al., 2001) and an impaired processing of a large number of neuropeptides and hormone precursors (Fricker et al., 1996). Previously we showed that, in adult Cpe^fat/Cpe^fat (obese) mice, impaired processing of prodynorphin (ProDyn) is characterized by a marked increase in the level of ir-Dyn A-17 and a decrease in the levels of ir-Dyn B-13 and ir-Dyn A-8 (Berman et al., 2001). Furthermore, the levels of high-molecular-weight enkephalin containing peptides are increased 2- to 3-fold in these mice (Fricker et al., 1996).

Opioid receptors can regulate a number of biological functions including feeding, analgesia, miosis, bradycardia, general sedation, and hypothermia (Herz, 1993). ProDyn- and ProEnk-derived peptides produce their biological effects by interacting with three types of opioid receptors: µ, , and . Dynorphin peptides bind with high affinity to  receptors (Kieffer, 1995 and references herein). Leu- and Met-enkephalins bind with high affinity to µ and  receptors and with low affinity to  receptors (Kieffer, 1995). Opioid receptors are coupled to G-proteins, and the efficacy of agonist activation can be determined using the hydrolysis-resistant GTP analog [³⁵S]GTPS. This assay has been used to explore the regional specificity of opioid activation in the brain (Sim et al., 1995, 1996; Sim and Childers, 1997). In general, µ-stimulated [³⁵S]GTPS binding predominates in the hypothalamus, amygdala, and brainstem, whereas -stimulated [³⁵S]GTPS binding is particularly high in the substantia nigra and cortex and is moderate in the cerebellum (Sim and Childers, 1997).

Previous studies have shown that opioid receptor activity can be regulated by long term treatment with opiates (Sim et al., 1996). Since the level of peptides derived from ProDyn is altered in adult Cpe^fat/Cpe^fat mice (Berman et al., 2001), we examined whether it is also altered in young mice at an age preceding obesity (6-8 weeks of age) and whether this correlates with the relative level of  receptor activity. We also examined whether the ir level of peptides derived from ProEnk is altered before and after the onset of obesity and whether this correlates with µ receptor activity. We find that impaired ProDyn and ProEnk processing does not correlate with the development of obesity since there is impaired processing in Cpe^fat/Cpe^fat mice at 6 to 8 weeks of age, whereas obesity starts to develop at 10 to 12 weeks of age (Naggert et al., 1995). We also find a decrease in µ opioid receptor activity in the striatum and midbrain of Cpe^fat/Cpe^fat mice with no significant changes in the  opioid receptor activity in any of the brain regions examined.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Materials. [³⁵S]GTPS (1000-1100 Ci/mmol) was from Amersham Biosciences, Inc. (Piscataway, NJ). [D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-Enkephalin (DAMGO) and ICI 199, 441 hydrochloride were obtained from Tocris Cookson, Inc. (Ballwin, MO). Dyn A and Dyn B peptides, Met-Enk peptide, antisera, and iodinated tracer were obtained from Peninsula Laboratories, Inc. (San Carlos, CA). Guanosine-5'-O-(-thio)-triphosphate, guanosine-5'-diphosphate, E-64, pepstatin, leupeptin, phenylmethylsulfonyl fluoride, aprotinin, and all other chemicals were obtained from Sigma-Aldrich (St. Louis, MO) or Fisher Scientific Co. (Fair Lawn, NJ).

Animals. Mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The identity of Cpe^fat/Cpe^fat (/) animals was confirmed by genotyping using primers (D8MIT69 F and R; D8MIT131 F and R) from Research Genetics (Huntsville, AL) according to the protocol supplied by The Jackson Laboratory. Nonobese littermates (+/) or (+/+) were used as controls. The age of the animals ranged from 6 to 17 weeks. In all studies, age- and sex-matched animals were used.

Tissue and Membrane Preparation. Cpe^fat/Cpe^fat or control mice were decapitated between 10:00 AM to 12:00 PM. Brains were collected and dissected into seven regions, as described by Glowinski and Iversen (1966). Frozen tissues were stored at 70°C until use. Brain structures from Cpe^fat/Cpe^fat and control mice (one midbrain, two hypothalami, or two striata) were homogenized with ice-cold 50 mM Tris-Cl, pH 7.4, containing 1 mM EDTA and 10% sucrose using a Teflon tissue grinder (15-20 strokes). The homogenates were centrifuged at 17,000g for 20 min. The supernatants were discarded, and pellets were resuspended in the buffer used for initial homogenization without sucrose and kept on ice for 30 min. The suspensions were centrifuged at 35,000g for 20 min, resulting pellets were resuspended in 50 mM Tris-Cl, pH 7.4, and the volume was adjusted to yield a concentration of 1 mg/ml. This homogenate was divided into small aliquots, which were then frozen quickly and stored at 70°C.

Peptide Extraction and Radioimmunoassay. For peptide analysis, brain structures were homogenized with 50 mM Tris-Cl, pH 7.5, containing 0.2% Triton X-100, 1 µM E-64, 1 µM pepstatin, 10 µM leupeptin, 300 µM phenylmethylsulfonyl fluoride, and 5 µg/ml aprotinin followed by the addition of an equal volume of 2 M CH₃COOH. After centrifugation, the supernatants were concentrated on a Speed Vac (Thermo Savant, Holbrook, NY) and stored at 20°C. Before RIA, samples were resuspended in methanol/0.1 N HCl or 0.15 M sodium phosphate buffer, pH 7.4. RIAs for ProDyn- or ProEnk-derived peptides were performed as described previously (Berman et al., 2001 and references herein). Dyn A-8 antiserum does not recognize C-terminal extensions, whereas Dyn B antiserum recognizes both the N- and C-terminally extended Dyn B-13 (Cone and Goldstein, 1982; Cone et al., 1983; Xie and Goldstein, 1987).

[³⁵S]GTPS Binding Assay. Hypothalamus, striatum, or midbrain membranes (10 µg) from Cpe^fat/Cpe^fat and control mice were incubated in 20 mM HEPES, pH 7.5, containing 5 mM MgCl₂, 100 mM NaCl, and 100 µM GDP, 0.1 nM [³⁵S]GTPS, and the agonist (0.01 to 10 µM DAMGO or ICI 199,441 for the stimulation of µ and  receptors, respectively) in a final volume of 500 µl. Basal binding was assessed in the presence of GDP and absence of the drug. Nonspecific binding was determined in the presence of 10 µM GTPS. After 1 h at 30°C, membranes were filtered and washed 3 times with 20 mM ice-cold HEPES, pH 7.5, using a Brandell cell harvester (Montreal, PQ, Canada). Bound radioactivity was determined following an overnight incubation in scintillation fluid. Data analysis was carried out using Prism software (GraphPad, San Diego, CA). Agonist stimulation of GTPS binding was expressed as a percentage of basal values.

Receptor Binding Experiments. Membranes (50 µg) were diluted in 50 mM Tris-Cl, pH 7.4, and incubated with [³H]DAMGO (0.3 to 10.0 nM) in the absence (total binding) or presence (nonspecific binding) of 1 µM diprenorphine in a total volume of 0.3 ml for 1 h at 37°C. Incubation mixture was rapidly filtered on GF/B filters (Whatman, Clifton, NJ) presoaked for at least 1 h in 0.5% polyethylenimine and washed with cold 50 mM Tris-Cl buffer, pH 7.4, using a Brandell cell harvester. For the calculation of B_max and K_d values Prism software was used.

Statistical Analysis. Statistical analysis of the data on ir peptide content (Dyn A and Dyn B) in Cpe^fat/Cpe^fat and control mice was performed by one-way analysis of variance (ANOVA) using Systat 8.0 program. Statistical analysis of the data on ir-Met-Enk content in Cpe^fat/Cpe^fat and control mice (at 8 and 17 weeks of age) was performed by two-way ANOVA with follow-up contrast. Stimulation as a function of animal type (control or Cpe^fat/Cpe^fat) and DAMGO or ICI 199,441 concentration was analyzed by two-way mixed ANOVA with ligand concentration as a within-subject factor and animal type as a between-subject factor. Receptor binding as a function of animal type (control or Cpe^fat/Cpe^fat) and DAMGO concentration was analyzed by two-way mixed ANOVA with DAMGO concentration as a within-subject factor and animal type as a between-subject factor. Results are presented as the mean ± S.E.M., and a value of p < 0.05 was considered to be statistically significant.

	Results

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ir-Dyn A-8, ir-Dyn B, and ir-Met-Enk Levels in Brain Regions of Developing Cpe^fat/Cpe^fat Mice. Previous studies have shown changes in the level of a number of neuropeptides in adult Cpe^fat/Cpe^fat mice (Fricker et al., 1996; Fricker and Leiter, 1999; Berman et al., 2001 and references herein). In this study, we focused on some of the peptides derived from ProDyn and ProEnk and examined their levels in discrete brain regions of control and Cpe^fat/Cpe^fat mice at ages preceding obesity. At 6 weeks of age, the levels of ir-Dyn A-8 in control mice are highest in the striatum and hypothalamus, lower in midbrain and cortex, and lowest in cerebellum (Table 1). Compared with control mice, the levels of ir-Dyn A-8 are significantly reduced in Cpe^fat/Cpe^fat mice. Since the latter lack CPE activity, they are expected to have increased levels of C-terminally extended basic residues. Consistent with this, CPB produced a greater increase in C-terminally extended Dyn A-8 peptides in Cpe^fat/Cpe^fat mice (as a percentage of their pre-CPB treatment levels) compared with control animals (Table 1). Nevertheless, the total level of ir-Dyn A-8 (completely processed peptides as well as its C-terminally extended forms) are lower in most brain regions of 6-week-old Cpe^fat/Cpe^fat mice compared with control animals (Table 1).

µ and  Receptor Activity in Hypothalamus, Striatum, and Midbrain Membranes from Control and Cpe^fat/Cpe^fat Mice 8 and 17 Weeks of Age. Next, we examined whether the changes in opioid peptide levels affected the activity of µ and  opioid receptors in hypothalamus, striatum, and midbrain membranes of young and adult Cpe^fat/Cpe^fat mice and their littermate controls using [³⁵S]GTPS binding assays. The assay was optimized by using a range of membrane and GDP concentrations. Low basal activity and best response to agonists were obtained when 10 µg of membranes and 100 µM GDP were incubated for 1 h at 30°C in 20 mM HEPES, pH 7.5, containing 5 mM MgCl₂ and 100 mM NaCl. The agonists used in this assay were ICI 199,441 for  receptors and DAMGO for µ opioid receptor. There were no differences in basal binding measured in the presence of GDP and absence of the agonist in Cpe^fat/Cpe^fat mice compared with controls.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   A comparison of the  receptor-selective agonist ICI 199,441 stimulated [35S]GTPS binding to membranes from hypothalamus, striatum, and midbrain of control and Cpefat/Cpefat mice of 8 and 17 weeks of age. Membranes were incubated with 100 pM [35S]GTPS, 10 µg of membrane, 100 µM GDP, and various concentrations of the agonist for 1 h at 30°C. The data are expressed as the percentage of basal binding measured in the presence of GDP and absence of the agonist and represent the mean ± S.E.M. of at least three independent experiments.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   A comparison of the µ receptor-selective agonist DAMGO stimulated [35S]GTPS binding to membranes from hypothalamus, striatum, and midbrain of control and Cpefat/Cpefat mice of 8 and 17 weeks of age. Membranes were incubated with 100 pM [35S]GTPS, 10 µg of membrane, 100 µM GDP, and various concentrations of the agonist for 1 h at 30°C. The data are expressed as the percentage of basal binding measured in the presence of GDP and absence of the agonist and represent the mean ± S.E.M. of at least three independent experiments.

View this table: [in this window] [in a new window]   TABLE 5 Pharmacological parameters from [35S]GTP S binding to membranes of control and Cpefat/Cpefat mice upon DAMGO stimulation [35S]GTP S binding assay was performed as described under Materials and Methods. Agonist stimulation of GTP S binding was expressed as a percentage of basal values. "Basal" is defined as the activity measured in the presence of GDP and absence of DAMGO. Stimulation as a function of animal type (control or Cpefat/Cpefat mice) and DAMGO concentration was analyzed by 2-way ANOVA.

µ Opioid Receptor Binding Assay in Midbrain of Cpe^fat/Cpe^fat and Control Mice at 8 and 17 Weeks of Age. Since a decrease in µ receptor activity is seen in the midbrain of 8- and 17-week-old Cpe^fat/Cpe^fat mice, we examined µ receptor binding in this region. The midbrain membranes were incubated with a range of [³H]DAMGO concentrations (0.3-10 nM) for 1 h at 37°C. Figure 3 illustrates saturation binding plots for two groups of mice at 8 and 17 weeks of age. The membranes from Cpe^fat/Cpe^fat mice displayed a 22 and 32% reduction in the number of [³H]DAMGO binding sites at 8 and 17 weeks of age, respectively, compared with control mice. Binding affinity of [³H]DAMGO for µ receptor was not significantly different in young and adult Cpe^fat/Cpe^fat mice compared with control mice (Fig. 3). These results are consistent with the reduced µ receptor activity observed in the midbrain of Cpe^fat/Cpe^fat mice.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   [3H]DAMGO binding to membranes from midbrain of control and Cpefat/Cpefat mice of 8 and 17 weeks of age. Membranes were incubated with different concentration of [3H]DAMGO in the absence or presence of 1 µM diprenorphine. The data represent the mean ± S.E.M. of at least three independent experiments. Receptor binding as a function of animal type (control or Cpefat/Cpefat mice) and DAMGO concentration was analyzed by 2-way ANOVA. **, a value of p < 0.01 was considered to be significantly different from the control group.

	Discussion

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Dynorphin peptide levels are decreased in all brain regions of Cpe^fat/Cpe^fat mice tested. Changes in peptide levels are detectable as early as 6 weeks of age, the earliest age tested. A similar decrease in peptide level was also seen in adult (17 weeks of age) Cpe^fat/Cpe^fat mice accompanied by a decrease in C-terminal trimming of basic residues. Nonetheless, the total level of Dyn peptides are not substantially altered in Cpe^fat/Cpe^fat mice (Fricker et al., 1996; Berman et al., 2001 and references herein). Thus, the  receptor activity is not likely to be affected in Cpe^fat/Cpe^fat mice. Furthermore, the C-terminally basic residue extended Dyn peptides do not significantly alter  receptor activity. For example, the binding affinities of C-terminally extended forms of Dyn A-8 for the  receptor do not substantially differ from those of Dyn A-8 (Mansour et al., 1995). The same is true for Dyn B-13 and higher molecular weight intermediates; Dyn B-13 and leumorphin (Dyn B-29) have essentially the same binding affinity for the  receptor. Thus, it is likely that in Cpe^fat/Cpe^fat mice  receptors were exposed to a pool of peptides with binding affinities not substantially different from those in control mice. This could account for the lack of significant differences in the functional activity of  opioid receptors in Cpe^fat/Cpe^fat mice.

In the present study, we find that ir-Met-Enk levels of control adult mice were highest in hypothalamus followed by pons, medulla, striatum, midbrain, hippocampus, and cortex. The lowest amount of ir-Met-Enk was found in the cerebellum. The relative levels of ir-Met-Enk in adult mice are in good agreement with previously reported findings (Nabeshima et al., 1992; Tejwani and Rattan, 1997). In Cpe^fat/Cpe^fat mice, ir-Met-Enk levels are differentially altered. Altered peptide levels affect µ receptor activity in discrete brain areas of Cpe^fat/Cpe^fat mice. It is difficult, however, to establish a correlation between ir-Met-Enk level and µ opioid receptor activity. Although there is an increase in µ receptor activity in the hypothalamus of Cpe^fat/Cpe^fat mice the level of ir-Met-Enk is not altered. In contrast, in the striatum of young Cpe^fat/Cpe^fat mice, there is no change in µ receptor activity, but there is an increase in peptide level, whereas in the midbrain of adult mice, there is a decrease in the peptide level and µ receptor activity. Met-enkephalin is an endogenous ligand for µ and  opioid receptors (Mansour et al., 1995). C-Terminally extended peptides containing Met-Enk core, such as Met-Enk-Arg-Gly-Leu and Met-Enk-Arg-Phe, are also ligands for µ receptor (Mansour et al., 1995). Interestingly, they exhibit higher affinity for µ receptor compared with Met-Enk. It is possible that the levels of these peptides are increased in Cpe^fat/Cpe^fat mice due the absence of CPE. To establish a correlation between peptide level and receptor activity, the levels of opioid peptides such as -endorphin 1-31, Met-Enk-Arg-Gly, Met-Enk-Arg-Phe, Met-Enk, and others need to be considered. It is possible that sustained exposure of the µ receptor to altered pool of peptides derived from opioid peptide precursors may result in a decrease in receptor density at the cell surface, as seen in developing and adult Cpe^fat/Cpe^fat mice. In midbrain, a decrease in receptor function is detectable in both developing and adult mice. This decrease in receptor function is accompanied by a reduction in the steady-state level of µ opioid receptor binding sites. It has been previously shown that the efficiency of µ receptor coupling to G-proteins varies across brain regions (Sim and Childers, 1997; Maher et al., 2000). Therefore, the decrease in µ receptor function in striatum and midbrain of Cpe^fat/Cpe^fat mice may be due to a decrease in receptor number and/or coupling to G-proteins. Similarly, the increase in µ receptor function in the hypothalamus of Cpe^fat/Cpe^fat mice compared with control mice may reflect an increase in coupling to G-proteins and/or increase in receptor numbers.

One of the major findings of the present study is that ProDyn and ProEnk processing is impaired in 6- to 8-week-old mice, an age that precedes obesity development in Cpe^fat/Cpe^fat mice. It is particularly striking that in Cpe^fat/Cpe^fat mice there is a high increase in ir-Met-Enk in the striatum of both young (2-fold) and adult (2.6-fold) Cpe^fat/Cpe^fat mice. A previous study with cholecystokinin injections (CCK) showed that the levels of Met-Enk were decreased in the brain of obese Zucker rats (McLaughlin et al., 1986). CCK levels are greatly reduced in the brain of Cpe^fat/Cpe^fat mice (Cain et al., 1997). Therefore, it is possible that a decrease in CCK levels in Cpe^fat/Cpe^fat mice may regulate the local levels of Met-Enk. Furthermore, the striatal neuropeptide levels are regulated by dopaminergic, glutamatergic, and serotoninergic systems (Liste et al., 2000 and references herein). Therefore, opioid peptide levels in the brain of Cpe^fat/Cpe^fat mice could be subject to multiple regulations leading to an increase in the ir-Met-Enk level in some regions and a decrease in others.

The role of the opioid peptides and their receptors in modulating ingestive behavior has been a source of intense study (for review see Gosnell and Levine, 1996; Glass et al., 1999). Opioids acting at µ receptors (Sanger and McCarthy, 1980) as well as direct microinjections of enkephalin analogs (Jackson and Sewell, 1985; Stanley et al., 1989) stimulate feeding behavior. Met-Enk levels are increased in the brain of genetically obese mice (ob/ob) (Khawaja et al., 1989) in the pituitary of obese diabetic mice (db/db) (Timmers et al., 1986) and in the striatum of Cpe^fat/Cpe^fat mice. Therefore, the increased Met-Enk levels detected in this study may contribute to the development of obesity in Cpe^fat/Cpe^fat mice by directly targeting the µ opioid receptor system and/or indirectly triggering other related pathways.