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Endocrinology Vol. 148, No. 9 4185-4190
Copyright © 2007 by The Endocrine Society

Neuropeptidomics to Study Peptide Processing in Animal Models of Obesity

Lloyd D. Fricker

Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461

Address all correspondence and requests for reprints to: Lloyd Fricker, Ph.D., Professor, Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461. E-mail: fricker{at}aecom.yu.edu.


   Abstract
Top
 Abstract
Introduction
 Peptide Processing and fat/fat...
 Peptidomics and Quantitative...
 Altered Peptide Processing in...
 Conclusions
 References
 
Neuropeptidomics is the analysis of the neuropeptides present in a tissue extract. Most neuropeptidomic studies use mass spectrometry to detect and identify the peptides, which provides information on the precise posttranslationally modified form of each peptide. Quantitative peptidomics uses isotopic labels to compare the levels of peptides in extracts from two different samples. This technique is ideal for examining neuropeptide levels in a variety of systems and is especially suited for studies of mice lacking peptide-processing enzymes. This review is focused on the neuropeptidomics technique and its application to the analysis of mice with a mutation that inactivates carboxypeptidase E, a critical enzyme in the biosynthesis of many neuroendocrine peptides. Mice without carboxypeptidase E activity are overweight, and a key question is the identification of the peptide or peptides responsible. The quantitative peptidomics approach has provided some insights toward the answer to this question.


   Introduction
Top
 Abstract
 Introduction
Peptide Processing and fat/fat...
 Peptidomics and Quantitative...
 Altered Peptide Processing in...
 Conclusions
 References
 
A LARGE NUMBER of peptides function as intercellular messengers in the endocrine system (peptide hormones) and in brain (peptide neurotransmitters). Physiological functions of neuropeptides include feeding, energy balance, thermogenesis, glucose homeostasis, water retention, pain, memory, reproduction, arousal and sleep/wake cycles, and many others. For each of these functions, multiple peptides are thought to be involved. In the case of feeding/body weight regulation, a large number of peptides have been implicated (Table 1Go). Peptides that decrease body weight by suppressing food consumption (an anorexigenic effect) and/or by affecting energy balance include {alpha}-MSH, leptin, insulin, corticotrophic releasing factor (CRF), cocaine- and amphetamine-regulated transcript (CART), and many others (1, 2, 3, 4, 5). Some of these peptides have been well studied and their role in body weight regulation is widely accepted, whereas much less is known about other peptides (such as neuropeptide S and bombesin-like peptides). The list of peptides known or proposed to increase body weight, either by increasing food consumption (an orexigenic effect) and/or by affecting energy balance, is also long (Table 1Go). Some of these peptides, such as neuropeptide Y, agouti-related peptide (AgRP), galanin, melanin-concentrating hormone (MCH), and ghrelin have well-accepted roles in body weight regulation (1, 2, 3, 4, 5). Some peptides, such as those derived from the proteins named VGF and proSAAS, may also be involved in body weight regulation based on the results of transgenic or knockout mouse studies, but more work needs to be done to clearly establish the role of these peptides (6, 7). In addition to the peptides listed in Table 1Go, it is possible that other peptides contribute to body weight regulation.


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  		TABLE 1. Peptides implicated in food intake/body weight regulation

 

   Peptide Processing and fat/fat Mice
Top
 Abstract
 Introduction
 Peptide Processing and fat/fat...
Peptidomics and Quantitative...
 Altered Peptide Processing in...
 Conclusions
 References
 
Studies on mice lacking the various neuropeptide precursors or neuropeptide receptors have contributed to a better understanding of the role of neuropeptides in body weight regulation. In addition to targeted mutation of specific genes, there are also naturally occurring mutations that cause obesity. A well-studied example is the ob/ob mouse, which led to the discovery of leptin (8), and the db/db mouse, which has a mutation in the leptin receptor (9). A third naturally occurring mutation is the Cpefat/fat mouse, which spontaneously arose in the early 1970s (10). Unlike the early-onset obesity of the ob/ob and db/db mouse, the Cpefat/fat mouse develops obesity starting around 8–10 wk of age (10). By wk 14–20, Cpefat/fat mice are typically twice the weight of wild-type or heterozygous littermates (11). The fat mutation is a missense point mutation within the gene encoding carboxypeptidase E (CPE), a peptide-processing enzyme (12). CPE normally removes the C-terminal basic residues that are present on the processing intermediates after the action of prohormone convertases on the peptide precursor (13). In many cases, CPE is the final step in the biosynthesis of neuropeptides, although for some peptides additional steps occur; these include C-terminal amidation, N-terminal acetylation, Ser or Thr phosphorylation, and other modifications (14, 15).

The finding that the fat mutation caused a defect in CPE was unexpected because it was originally thought that such a mutation would be lethal. This single-nucleotide substitution replaces Ser202 with a Pro (12). Although Ser202 is not a catalytically important residue, structural modeling predicts that a Pro in this position would affect enzyme structure and eliminate activity. This is consistent with the observed results; mutant CPE has a short half-life in cell lines, presumably due to the misfolding, and has no detectable enzyme activity (16, 17). Because CPE was the only carboxypeptidase known to be present in the secretory pathway, it was expected that mice lacking CPE activity would not survive. However, further analyses found that Cpefat/fat mice had low but detectable levels of the mature forms of all neuropeptides examined (12, 18, 19, 20). The simplest explanation for this observation was that another carboxypeptidase contributed to the processing of neuropeptides. A search for novel enzymes led to the discovery of carboxypeptidase D (CPD), an enzyme that is broadly expressed in endocrine as well as nonendocrine tissues (21, 22). CPD only partially compensates for the deficient CPE in the Cpefat/fat mice because the two enzymes have different intracellular distributions. CPD is primarily localized to the trans-Golgi network and is not present in mature secretory vesicles, the site of much of the peptide processing (23, 24, 25).

A key question is why the Cpefat/fat mice are overweight. Specifically, what peptide or peptides contribute to the elevated body weight? Because peptides are involved in both sides of the weight balance, why is the net effect an increase rather than a decrease in mass? One study by Leiter and colleagues (11) reported that 8 wk-old Cpefat/fat mice are not hyperphagic. However, another study found that Cpefat/fat mice of 11–14 wk are slightly hyperphagic, eating 25–50% more than wild-type littermates (26). Interestingly, when given access to exercise wheels, the Cpefat/fat mice initially lose weight, but then after 1–2 wk, they further increase their food consumption and gain weight at the same rate as sedentary Cpefat/fat mice (26). The Cpefat/fat mice are defective in thermogenesis; when placed in a 4–7 C environment, their core temperature drops approximately 2 C within 30 min and continues to decline upon further cold exposure (27). When Cpefat/fat mice are limited to the amount of food consumed by wild-type littermates, the Cpefat/fat mice still gain significantly more weight than wild-type mice, although this gain is less than Cpefat/fat mice given free access to food (Fricker, L. D., and R. Biswas, unpublished observation). Taken together, these results suggest that the Cpefat/fat mice are overweight due to a combination of moderate overeating and also changes to the partitioning of energy into fat. The peptide or peptides that may contribute to these effects are discussed below, after a description of the peptidomics approaches recently used to analyze peptides in the Cpefat/fat mice.


   Peptidomics and Quantitative Peptidomics
Top
 Abstract
 Introduction
 Peptide Processing and fat/fat...
 Peptidomics and Quantitative...
Altered Peptide Processing in...
 Conclusions
 References
 
The standard method for analysis of peptides has been RIAs. Although RIAs are often sensitive, there are many limitations. First, it takes months to raise antisera to peptides, and characterization of the antisera requires additional time. Antisera are rarely specific for the exact form of the peptides to which they were raised, usually cross-reacting with N- and/or C-terminally extended or truncated forms of the peptides or with posttranslationally modified peptides. In some cases, related peptides can cross-react with the antisera, further complicating the analysis. Even if highly specific antisera have already been raised and characterized, it is time consuming to set up and analyze dozens of RIAs. Finally, any RIA analysis is limited to known peptides. Because it is possible that there are additional neuropeptides in brain and other tissues that have not yet been discovered, it would be ideal to have a technique for peptide analysis that would not be limited to known peptides but that could detect all major peptides in a single experiment.

The overall objective of peptidomics is to identify all peptides present in any given tissue (28, 29, 30). The general approach is to extract peptides from the tissue, size fractionate to exclude proteins, and then analyze by reverse-phase HPLC followed by mass spectrometry. If the mass spectrometry is performed on an instrument that is capable of fragmentation of the peptide (using collision-induced dissociation or a related technique), then it is often possible to determine the partial amino acid sequence of the peptide from the fragmentation pattern (28, 29, 30, 31). In addition to identifying the peptides present in the extract, this method can usually identify posttranslational modifications such as phosphorylation, acetylation, and C-terminal amidation (28, 29, 30, 31).

Quantitative peptidomics techniques are generally similar to the standard peptidomics approach, except that the samples are first labeled with one of two related chemicals that differ isotopically (Fig. 1Go). A number of isotopic labels have been tested for proteomics applications (32, 33, 34, 35). For peptidomics, the best isotopic labels are the active esters of trimethylammoniumbutyrate (TMAB) containing either nine deuteriums (heavy) or nine hydrogens (light) (30, 36). The N-hydroxysuccinimide ester of the TMAB reagent reacts with amines that are present on the N terminus of the peptide and/or on the side chain of lysine residues. Once the peptides in the samples are labeled, the two reactions are pooled and processed using the standard peptidomics approach shown in Fig. 1Go. The relative level of each peptide can be determined from the relative intensity of the signal for the heavy and light forms (Fig. 2Go). Then, as with the standard peptidomics approach, the sequence of each peptide is determined from the fragmentation pattern, i.e. tandem mass spectrometry (MS/MS) analysis, as shown in Fig. 2Go.


Figure 1
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  		FIG. 1. Outline of quantitative peptidomics approach using isotopic labels and mass spectrometry. Peptides are extracted from two different samples and labeled with the active N-hydroxysuccinimide (NHS) ester of TMAB containing either nine hydrogens or nine deuteriums on the methyl groups adjacent to the quaternary amine group. In a typical experiment, extracts from one group of wild-type (WT) mice are labeled with H9-TMAB and extracts from one group of Cpefat/fat mice (fat/fat) are labeled with the D9-TMAB. Additional groups of WT and fat/fat mice are labeled in the reverse order (WT with D9-TMAB and fat/fat with H9-TMAB) and similarly analyzed (not shown). After quenching of unreacted labels, the two samples are combined, the peptides isolated by microfiltration through a membrane with a 10-kDa cutoff, and then analyzed by liquid chromatography on a reverse-phase column followed by mass spectrometry (LC/MS) on an instrument capable of MS/MS. The relative levels of peptide in the two samples are calculated from the difference between peak height (or area) between the light and heavy form of each peptide. The peptide is identified based on partial sequence information provided by the MS/MS analysis after collision-induced dissociation.

 

Figure 2
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  		FIG. 2. Selected liquid chromatography/mass spectrometry (LC/MS) and MS/MS data from an experiment in which the wild-type (WT) hypothalamic extract was labeled with H9-TMAB and the Cpefat/fat hypothalamic extract was labeled with D9-TMAB. A, MS spectrum representing the relative intensity of ions that eluted between 62.5 and 63.5 min, over the mass/charge (m/z) range of 600-1300. In a typical experiment, data are collected for a 2-h period over the m/z range 300-1800, and so the spectrum in A represents a small fraction of the total data. The major ions in this spectrum are expanded in the subsequent panels. B, The charge state of the ions with m/z of 628.348 and 631.373 are both 3+ (this can be determined by analysis of the difference between the m/z of the monoisotopic peak and those containing one or more 13C atoms; because this difference is 0.33, the z is therefore 3). The mass difference between the ions with m/z of 628.348 and 631.373 is therefore 9 Da, which is equal to one isotopic tag. Quantification is performed by comparing the heights of the H-TMAB peaks and those of the D-TMAB peaks. In this example, the peptide is present in the fat/fat mouse hypothalamic extracts at about twice the level of the WT extracts. This peptide was subsequently identified as big LEN by MS/MS sequencing (not shown). C, The charge state of the ions with m/z of 820.923 and 829.983 are both 2+, and so the mass difference between them is 18 Da, corresponding to two isotopic tags. In this example, the peptide is present in the fat/fat mouse hypothalamus at a level about 10% that of the WT mouse hypothalamus. This peptide was subsequently identified as proenkephalin 197–208 by MS/MS sequencing shown in the bottom panel. D, MS/MS analysis of the ion with m/z 820.923. The indicated sequence was derived from the mass difference between fragment ions; most amino acids have distinct masses. In this example, the presence of Leu (and not Ile, which has the same mass) was based on the match of the sequence to proenkephalin 197–208 (SPQLEDEAKELQ). In addition, the number of tags incorporated, the charge state, and the mass of this proenkephalin-derived peptide match the expected values.

 
Using this quantitative peptidomics technique, it is possible to detect, quantify, and identify approximately 100 peptides in extracts of a single mouse hypothalamus (37, 38). A large number of peptides are also detected in other brain regions such as prefrontal cortex, striatum, hippocampus, and amygdala (39) (unpublished observation). Many of the hypothalamic peptides identified by this method are neuropeptides or other fragments of secretory pathway proteins, some of which may represent novel neuropeptides. In addition, degradation fragments of cytosolic fragments are also detected, although most of these can be greatly reduced by microwave irradiation of the brain before dissection and by extraction procedures that avoid hot acid (37, 40, 41). Although not every neuropeptide known or predicted to be present in brain is detected by the quantitative peptidomics approach, those peptides identified by this technique generally represent the most abundant peptides in each of the brain regions. However, abundant peptides cannot be quantified if they lack a free amine to react with the TMAB label (i.e. peptides that are acetylated or pyroglutamylated on the N terminus and that also lack lysine residues), although there are not many known peptides in this category. Examples include the N terminus of procholecystokinin (pyroE-PVVPAEATDPVEQRAQEAPRRQL) and a fragment of VGF (pyroE-QETAAAETETRTHTLTRVNLESPGPERVW). Also, some peptides just don’t give strong signals on mass spectrometry or are otherwise difficult to sequence for various reasons. There are many known peptides that are below or above the mass range that can readily be sequenced by MS/MS; examples include TRH (362 Da) and neuropeptide Y (4269 Da).

A number of analyses have been performed with the quantitative peptidomics technique, some of which are relevant to the area of food intake/body weight regulation and/or which involve mice lacking peptide-processing enzymes. An ongoing study is investigating the relative levels of peptides in various brain regions of Cpefat/fat mice vs. wild-type littermates. The key results of these analyses are described in the following section, with the goal being the identification of candidate peptides that contribute to the elevated body weight in these mice. Another study examined the effect of a 2-d fast on levels of hypothalamic peptides in Cpefat/fat mice and found a large number of peptides to be affected by this treatment (42). In addition to these studies, the Cpefat/fat mice were also used to study the effect of cocaine or morphine treatment on peptide levels (43, 44). The quantitative peptidomics approach was also used to examine the relative levels of peptides in mice lacking either prohormone convertase 1 or 2 (38, 45). Collectively, these studies have further defined the biological role of the various peptide-processing enzymes.


   Altered Peptide Processing in Cpefat/fat Mice
Top
 Abstract
 Introduction
 Peptide Processing and fat/fat...
 Peptidomics and Quantitative...
 Altered Peptide Processing in...
Conclusions
 References
 
As described in the preceding sections, a key question has been identifying the peptide (or peptides) that cause Cpefat/fat mice to be overweight. Based on the results of the neuropeptidomic analyses together with previous studies using RIAs, it appears that several peptides known to decrease body weight are greatly reduced in the Cpefat/fat mouse brain. For example, levels of {alpha}-MSH, CART, TRH, neurotensin, and cholecystokinin (CCK) are much lower in Cpefat/fat mouse brain compared with wild-type mouse brain (Table 1Go). Plasma levels of the mature form of insulin are also lower in Cpefat/fat mice. Other hypothalamic peptides implicated in decreasing body weight are also likely to be affected by the fat mutation (Table 1Go); although these other peptides were not yet measured by either peptidomics or RIA, they are predicted to be affected by the absence of CPE based on the cleavage sites required to generate the biologically active peptide and on the requirement for CPE to perform similar cleavages of other peptides in the same tissue. It is not possible to predict whether gut peptides such as peptide YY and neuromedin B are reduced in the Cpefat/fat mice; the fat mutation has a large effect on CCK produced in the brain but little effect on CCK produced in the duodenum (46). The level of leptin mRNA in Cpefat/fat mouse adipose depots is 2.4-fold the level in wild-type mice (47). Leptin does not require CPE for its biosynthesis, and so the increase in leptin mRNA in adipose tissue presumably means that plasma leptin levels are elevated in Cpefat/fat mice.

In contrast to the hypothalamic peptides that decrease body weight, some of the hypothalamic peptides that increase body weight are present at higher levels in Cpefat/fat mouse brain (Table 1Go). For example, MCH was found to be higher in the Cpefat/fat mouse brain than in wild-type brain (19). Similarly, two opioid peptides, enkephalin heptapeptide and ß-endorphin 1–31, are both elevated in the Cpefat/fat mouse. All three of these peptides are present on the C terminus of their precursors, and therefore the peptides do not require a carboxypeptidase for their production. Similarly, the proSAAS-derived peptide big LEN is present on the C terminus of its precursor, although in this example it is not clear which fragment of proSAAS is biologically active (if any). The increase in the levels of these C-terminal peptides presumably reflects an increase in the biosynthesis of their precursors in the Cpefat/fat mouse, although this has not been directly tested. Because AgRP is also located on the C terminus of its precursor, it is predicted that this peptide would be present at either normal or slightly elevated levels in the Cpefat/fat mice. However, not all peptides that increase body weight are normal or elevated in the Cpefat/fat mouse brain; some of these peptides require a carboxypeptidase step in their biosynthesis and would therefore be present in the Cpefat/fat mouse brain at lower than normal levels. Examples include neuropeptide Y, galanin, and several other peptides that have been suggested to play a role in body weight regulation (Table 1Go).


   Conclusions
Top
 Abstract
 Introduction
 Peptide Processing and fat/fat...
 Peptidomics and Quantitative...
 Altered Peptide Processing in...
 Conclusions
References
 
In summary, the use of a neuropeptidomics approach has greatly expanded our understanding of the peptide defects in the Cpefat/fat mice. Based on these analyses, together with previous studies that used RIA, it is likely that the elevated body weight of the Cpefat/fat mice is due to the reduced levels of peptides that decrease body weight such as {alpha}-MSH. Although some of the peptides that increase body weight are also decreased in the Cpefat/fat mice, several of the body weight-elevating peptides are present at levels equal to or even greater than levels in wild-type mice. Thus, the net balance in the Cpefat/fat mouse is tipped toward increasing body weight.


   Footnotes
 
This work was supported by National Institutes of Health Grants DA-04494 and DK-51271.

Disclosure Statement: The author has nothing to disclose.

First Published Online June 21, 2007

Abbreviations: AgRP, Agouti-related peptide; CART, cocaine- and amphetamine-regulated transcript; CCK, cholecystokinin; CPD, carboxypeptidase D; CPE, carboxypeptidase E; MCH, melanin-concentrating hormone; MS/MS, tandem mass spectrometry; TMAB, trimethylammoniumbutyrate.

Received January 29, 2007.

Accepted for publication May 3, 2007.


   References
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 Abstract
 Introduction
 Peptide Processing and fat/fat...
 Peptidomics and Quantitative...
 Altered Peptide Processing in...
 Conclusions
 References
 

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