Quantitative Mass Spectrometry Reveals Food Intake-Induced Neuropeptide Level Changes in Rat Brain: Functional Assessment of Selected Neuropeptides as Feeding Regulators.
ABSTRACT: Endogenous neuropeptides are important signaling molecules that function as regulators of food intake and body weight. Previous work has shown that neuropeptide gene expression levels in a forebrain reward site, the nucleus accumbens (NAc), were changed by feeding. To directly monitor feeding-induced changes in neuropeptide expression levels within the NAc, we employed a combination of cryostat dissection, heat stabilization, neuropeptide extraction and label-free quantitative neuropeptidomics via a liquid chromatography-high resolution mass spectrometry platform. Using this methodology, we described the first neuropeptidome in NAc and discovered that feeding caused the expression level changes of multiple neuropeptides derived from different precursors, especially proSAAS-derived peptides such as Big LEN, PEN and little SAAS. We further investigated the regulatory functions of these neuropeptides derived from the ProSAAS family by performing an intra-NAc microinjection experiment using the identified ProSAAS neuropeptides, 'Big-LEN' and 'PEN'. Big LEN significantly increased rats' food and water intake, whereas both big LEN and PEN affected other behaviors including locomotion, drinking and grooming. In addition, we quantified the feeding-induced changes of peptides from hippocampus, hypothalamus and striatum to reveal the neuropeptide interplay among different anatomical regions. In summary, our study demonstrated neuropeptidomic changes in response to food intake in the rat NAc and other key brain regions. Importantly, the microinfusion of ProSAAS peptides into NAc revealed that they are behaviorally active in this brain site, suggesting the potential use of these peptides as therapeutics for eating disorders.
Project description:ProSAAS is the precursor of a number of peptides that have been proposed to function as neuropeptides. Because proSAAS mRNA is highly expressed in the arcuate nucleus of the hypothalamus, we examined the cellular localization of several proSAAS-derived peptides in the mouse hypothalamus and found that they generally colocalized with neuropeptide Y (NPY), but not ?-melanocyte stimulating hormone. However, unlike proNPY mRNA, which is upregulated by food deprivation in the mediobasal hypothalamus, neither proSAAS mRNA nor proSAAS-derived peptides were significantly altered by 1-2 days of food deprivation in wild-type mice. Furthermore, while proSAAS mRNA levels in the mediobasal hypothalamus were significantly lower in Cpe(fat/fat) mice as compared to wild-type littermates, proNPY mRNA levels in the mediobasal hypothalamus and in other subregions of the hypothalamus were not significantly different between wild-type and Cpe(fat/fat) mice. Intracerebroventricular injections of antibodies to two proSAAS-derived peptides (big LEN and PEN) significantly reduced food intake in fasted mice, while injections of antibodies to two other proSAAS-derived peptides (little LEN and little SAAS) did not. Whole-cell patch clamp recordings of parvocellular neurons in the hypothalamic paraventricular nucleus, a target of arcuate NPY projections, showed that big LEN produced a rapid and reversible inhibition of synaptic glutamate release that was spike independent and abolished by blocking postsynaptic G protein activity, suggesting the involvement of a postsynaptic G protein-coupled receptor and the release of a retrograde synaptic messenger. Taken together with previous studies, these findings support a role for proSAAS-derived peptides such as big LEN as neuropeptides regulating food intake.
Project description:ProSAAS, a recently discovered granin-like protein, potently inhibits prohormone convertase (PC)1, and might also perform additional functions. In the present study, the processing of proSAAS was compared in two neuroendocrine cell lines overexpressing this protein: the AtT-20 mouse pituitary corticotrophic line and the PC12 rat adrenal phaeochromocytoma line. The processing of proSAAS was examined by pulse-chase analysis using [(3)H]leucine, by MS, and by chromatography and radioimmunoassay. Various smaller forms of proSAAS were detected, including peptides designated as little SAAS, PEN and big LEN. Because the PC-12 cells used in the present study do not express either PC1 or PC2, the finding that these cells efficiently cleave proSAAS indicates that these cleavages do not require either enzyme. Two of the peptides identified in AtT-20 media represent novel C-terminally truncated forms of PEN. In both cell lines, the secretion of the small proSAAS-derived peptides is stimulated by secretagogues. However, long-term treatment of wild-type AtT-20 cells with two different secretagogues (8-bromo-cAMP and a phorbol ester) does not affect levels of proSAAS mRNA; this treatment significantly increases PC1 mRNA by approx. 60-80%. The lack of co-regulation of proSAAS and PC1 mRNA implies that enzyme activity can be induced without an accompanying increase in the inhibitor. In addition, the finding that the peptides are secreted via the regulated pathway is consistent with the proposal that they may function as neuropeptides.
Project description:Several neuropeptide systems in the hypothalamus, including neuropeptide Y and agouti-related protein (AgRP), control food intake. Peptides derived from proSAAS, a precursor implicated in the regulation of body weight, also control food intake. GPR171 is a heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor (GPCR) for BigLEN (b-LEN), a peptide derived from proSAAS. To facilitate studies exploring the physiological role of GPR171, we sought to identify small-molecule ligands for this receptor by performing a virtual screen of a compound library for interaction with a homology model of GPR171. We identified MS0015203 as an agonist of GPR171 and demonstrated the selectivity of MS0015203 for GPR171 by testing the binding of this compound to 80 other membrane proteins, including family A GPCRs. Reducing the expression of GPR171 by shRNA (short hairpin RNA)-mediated knockdown blunted the cellular and tissue response to MS0015203. Peripheral injection of MS0015203 into mice increased food intake and body weight, and these responses were significantly attenuated in mice with decreased expression of GPR171 in the hypothalamus. Together, these results suggest that MS0015203 is a useful tool to probe the pharmacological and functional properties of GPR171 and that ligands targeting GPR171 may eventually lead to therapeutics for food-related disorders.
Project description:To identify neuropeptides that are regulated by cocaine, we used a quantitative peptidomic technique to examine the relative levels of neuropeptides in several regions of mouse brain following daily intraperitoneal administration of 10 mg/kg cocaine or saline for 7 days. A total of 102 distinct peptides were identified in one or more of the following brain regions: nucleus accumbens, caudate putamen, frontal cortex, and ventral tegmental area. None of the peptides detected in the caudate putamen or frontal cortex were altered by cocaine administration. Three peptides in the nucleus accumbens and seven peptides in the ventral tegmental area were significantly decreased in cocaine-treated mice. Five of these ten peptides are derived from proSAAS, a secretory pathway protein and neuropeptide precursor. To investigate whether proSAAS peptides contribute to the physiological effects of psychostimulants, we examined acute responses to cocaine and amphetamine in the open field with wild-type (WT) and proSAAS knockout (KO) mice. Locomotion was stimulated more robustly in the WT compared to mutant mice for both psychostimulants. Behavioral sensitization to amphetamine was not maintained in proSAAS KO mice and these mutants failed to sensitize to cocaine. To determine whether the rewarding effects of cocaine were altered, mice were tested in conditioned place preference (CPP). Both WT and proSAAS KO mice showed dose-dependent CPP to cocaine that was not distinguished by genotype. Taken together, these results suggest that proSAAS-derived peptides contribute differentially to the behavioral sensitization to psychostimulants, while the rewarding effects of cocaine appear intact in mice lacking proSAAS.
Project description:Mice with a targeted mutation in proSAAS have been generated to investigate whether peptides derived from this precursor could function as an inhibitor of prohormone convertase 1/3 (PC1/3) in vivo as well as to determine any alternate roles for proSAAS in nervous and endocrine tissues. Fetal mice lacking proSAAS exhibit complete, adult-like processing of prodynorphin in the prenatal brain instead of the incomplete processing seen in the brains of wild-type fetal mice where inhibitory proSAAS intermediates are transiently accumulated. This study provides evidence that proSAAS is directly involved in the prenatal regulation of neuropeptide processing in vivo. However, adult mice lacking proSAAS have normal levels of all peptides detected using a peptidomics approach, suggesting that PC1/3 activity is not affected by the absence of proSAAS in adult mice. ProSAAS knockout mice exhibit decreased locomotion and a male-specific 10-15% decrease in body weight, but maintain normal fasting blood glucose levels and are able to efficiently clear glucose from the blood in response to a glucose challenge. This work suggests that proSAAS-derived peptides can inhibit PC1/3 in embryonic brain, but in the adult brain proSAAS peptides may function as neuropeptides that regulate body weight and potentially other behaviors.
Project description:OBJECTIVE:The hypothalamic control of energy balance is regulated by a complex network of neuropeptide-releasing neurons. Although the effect of these neuropeptides on individual aspects of energy homoeostasis has been studied, the coordinated response of these effects has not been comprehensively investigated. We have simultaneously monitored a number of metabolic parameters following intracerebroventricular (ICV) administration of 1 and 3 nmol of neuropeptides with established roles in the regulation of feeding, activity and metabolism. Ad libitum- fed rats received the orexigenic neuropeptides neuropeptide Y (NPY), agouti-related protein (AgRP), melanin-concentrating hormone (MCH) or orexin-A. Overnight-food-deprived rats received an ICV injection of the anorectic peptides alpha-melanocyte-stimulating hormone (MSH), corticotrophin-releasing factor (CRF) or neuromedin U (NMU). RESULTS:Our results reveal the temporal sequence of the effects of these neuropeptides on both energy intake and expenditure, highlighting key differences in their function as mediators of energy balance. NPY and AgRP increased feeding and decreased oxygen consumption, with the effects of AgRP being more prolonged. In contrast, orexin-A increased both feeding and oxygen consumption, consistent with an observed increase in activity. The potent anorexigenic effects of CRF were accompanied by a prolonged increase in activity, whereas NMU injection resulted in significant but short-lasting inhibition of food intake, ambulatory activity and oxygen consumption. alpha-MSH injection resulted in significant increases in both ambulatory activity and oxygen consumption, and reduced food intake following administration of 3 nmol of the peptide. CONCLUSION:We have for the first time, simultaneously measured several metabolic parameters following hypothalamic administration of a number of neuropeptides within the same experimental system. This work has shown the interrelated effects of these neuropeotides on activity, energy expenditure and food intake, thus facilitating comparison between the different hypothalamic systems.
Project description:Food intake is regulated by various neuromodulators, including numerous neuropeptides. However, it remains elusive at the molecular and cellular level as to how these important chemicals regulate internal processes and which regions of the neuronal organs are responsible for regulating the behavior. Here we report a comparative neuropeptidomic analysis of the brain and pericardial organ (PO) in response to feeding in two well-studied crustacean physiology model organisms, Callinectes sapidus and Carcinus maenas, using mass spectrometry (MS) techniques. A multifaceted MS-based approach has been developed to obtain complementary information on the expression changes of a large array of neuropeptides in the brain and PO. The method employs stable isotope labeling of brain and PO extracts for relative MS quantitation, capillary electrophoresis (CE)-MS for fractionation and high-specificity analysis, and mass spectrometric imaging (MSI) for in-situ molecular mapping of peptides. A number of neuropeptides, including RFamides, B-type allatostatins (AST-B), RYamides, and orcokinins exhibit significant changes in abundance after feeding in this investigation. Peptides from the AST-B family found in PO tissue were shown to have both altered expression and localization changes after feeding, indicating that they may be a class of vital neuropeptide regulators involved in feeding behavior. Graphical Abstract ?.
Project description:Five novel peptides were identified in the brains of mice lacking active carboxypeptidase E, a neuropeptide-processing enzyme. These peptides are produced from a single precursor, termed proSAAS, which is present in human, mouse, and rat. ProSAAS mRNA is expressed primarily in brain and other neuroendocrine tissues (pituitary, adrenal, pancreas); within brain, the mRNA is broadly distributed among neurons. When expressed in AtT-20 cells, proSAAS is secreted via the regulated pathway and is also processed at paired-basic cleavage sites into smaller peptides. Overexpression of proSAAS in the AtT-20 cells substantially reduces the rate of processing of the endogenous prohormone proopiomelanocortin. Purified proSAAS inhibits prohormone convertase 1 activity with an IC(50) of 590 nM but does not inhibit prohormone convertase 2. Taken together, proSAAS may represent an endogenous inhibitor of prohormone convertase 1.
Project description:Gut microbiota can regulate host physiological and pathological status through gut-brain communications or pathways. However, the impact of the gut microbiome on neuropeptides and proteins involved in regulating brain functions and behaviors is still not clearly understood. To address the problem, integrated label-free and 10-plex DiLeu isobaric tag-based quantitative methods were implemented to compare the profiling of neuropeptides and proteins in the hypothalamus of germ-free (GF)- vs conventionally raised (ConvR)-mice. A total of 2943 endogenous peptides from 63 neuropeptide precursors and 3971 proteins in the mouse hypothalamus were identified. Among these 368 significantly changed peptides (fold changes over 1.5 and a p-value of <0.05), 73.6% of the peptides showed higher levels in GF-mice than in ConvR-mice, and 26.4% of the peptides had higher levels in ConvR-mice than in GF-mice. These peptides were mainly from secretogranin-2, phosphatidylethanolamine-binding protein-1, ProSAAS, and proenkephalin-A. A quantitative proteomic analysis employing DiLeu isobaric tags revealed that 282 proteins were significantly up- or down-regulated (fold changes over 1.2 and a p-value of <0.05) among the 3277 quantified proteins. These neuropeptides and proteins were mainly involved in regulating behaviors, transmitter release, signaling pathways, and synapses. Interestingly, pathways including long-term potentiation, long-term depression, and circadian entrainment were involved. In the present study, a combined label-free and 10-plex DiLeu-based quantitative method enabled a comprehensive profiling of gut microbiome-induced dynamic changes of neuropeptides and proteins in the hypothalamus, suggesting that the gut microbiome might mediate a range of behavioral changes, brain development, and learning and memory through these neuropeptides and proteins.