Is Ghrelin Synthesized in the Central Nervous System?
Abstract
:1. Introduction
2. Ghrelin Synthesis
3. Ghrelin Synthesis in the Central Nervous System
3.1. Presence of Pre-Proghrelin mRNA
3.2. Presence of Ghrelin Peptide in Tissue Extracts
3.3. Ghrelin Immunohistochemical Staining
3.4. Ghrelin Knock out and Ghrelin-Gene Reporter Mice
4. Discussion
5. Conclusions
Acknowledgments
Conflicts of Interest
References
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First Author-Date | Experimental Strategy | Main Observations | Reference |
---|---|---|---|
Kojima-1999 | Northern blot using a 620 bp probe against pre-proghrelin mRNA in rat brain. | No signal. | [51] |
RT-PCR using primers that amplify exon 1 to 4 sequence of pre-proghrelin mRNA in rat brain. | Positive gene expression. | ||
IHC using a non-commercial rabbit polyclonal antibody against C-terminal ghrelin (13–28) in colchicine-treated rat hypothalamus. | Ghrelin-IR cell bodies in the ARC. | ||
Hosoda-2000 | RP-HPLC separation of peptide extracts from rat hypothalamus followed by two RIAs, named N-RIA and C-RIA, developed using non-commercial rabbit polyclonal antisera against either N-terminal ghrelin (1–11) (#G606 antiserum) or C-terminal ghrelin (13–28) (#G107 antiserum), respectively. | Ghrelin-IR signal in hypothalamus, which was ~1000-times smaller than in the stomach, using C-RIA. In contrast to the stomach, no signal was detected in the hypothalamus using the N-RIA. | [52] |
Lu-2002 | IHC using #G606 antiserum against ghrelin (1–11) in colchicine-treated rat hypothalamus. | Ghrelin-IR cell bodies and their terminals in the ARC. Ghrelin-IR signal was detected in the Golgi apparatus and dense granular vesicles by electron microscopy. | [53] |
Cowley-2003 | RT-PCR in mouse and rat hypothalamus using primers against pre-proghrelin mRNA, as used by Kojima et al. 1999. | Positive gene expression. | [54] |
Western blot using a non-commercial affinity-purified polyclonal antisera against an unspecified part of ghrelin in protein extracts from rat hypothalamus. | A band of 13 kDa corresponding to the estimated proghrelin mass. | ||
IHC using same antibodies used for western blot in naïve and colchicine-treated rat as well as mouse hypothalamus. | Ghrelin-IR cell bodies located in the hypothalamic space between the LH, ARC, DMN, VMN, PVN and ependymal layer of the third ventricle. No labeling was detected in sections of the ghrelin-KO mice. Ghrelin-IR signal was also detected by electron microscopy in neuronal terminals, and located in dense-cored vesicles. | ||
Mozid-2003 | RT-PCR using primers that amplify exons 1 to 3 sequence of pre-proghrelin mRNA in rat hypothalamus.. | Positive gene expression. | [55] |
Competitive EIA using a rabbit polyclonal antibody against ghrelin (13–28) in rat hypothalamic homogenates and secretion media. | Ghrelin-IR signal. | ||
Guan-2003 | IHC using #G606 antiserum against ghrelin (1–11) in rat hypothalamus. | Ghrelin-IR cell bodies and their terminals in the ARC. Ghrelin-IR signal was detected in dense granular vesicles by electron microscopy. | [56] |
Wortley-2004 | Northern blot using a full length ghrelin probe in mouse hypothalamus. | No signal. | [57] |
Q-PCR (TaqMan) using an unspecified set of primers against pre-proghrelin mRNA in mouse hypothalamus. | Positive gene expression. | ||
IHC using a rabbit serum against rat octanoylated ghrelin (Phoenix Pharmaceuticals, Cat# H-191-031-31) in mouse brain. | No signal. | ||
Mondal-2005 | RT-PCR in rat ARC using primers against pre-proghrelin mRNA, as used by Kojima et al. 1999. | Positive gene expression. | [58] |
IHC using #G606 antiserum against ghrelin (1–11) in colchicine-treated rat ARC. | Ghrelin-IR cell bodies. | ||
RP-HPLC separation of peptide extracts from rat ARC followed by a RIA using the same antibody than for IHC. | A peak that elutes at the position of ghrelin. | ||
Turek-2005 | Q-PCR using an unspecified set of primers against pre-proghrelin mRNA in mouse hypothalamus. | Positive gene expression. | [59] |
Hu-2005 | RT-PCR using an unspecified set of primers against pre-proghrelin mRNA in hypothalamic mouse explants. | Positive gene expression. | [60] |
RP-HPLC separation of peptide extracts from mouse hypothalamus followed by commercial RIAs (Phoenix Pharmaceuticals) to measure ghrelin or ghrelin plus des-acyl-ghrelin. Some HPLC fractions were subjected to SDS/PAGE and immunoblotting using an unspecified anti-ghrelin antibody from Phoenix Pharmaceuticals. | Immunoblotting shows no ghrelin-IR signal, while RIAs of HPLC fractions detect ghrelin-IR signal that elutes at the position of ghrelin and accounts the majority of the total ghrelin-IR signal. | ||
RIA analysis of ghrelin content in and secreted from mouse hypothalamic explants using commercial kits form Phoenix Pharmaceuticals and Linco Research. | Ghrelin-IR signal in secretion medium. | ||
Sato-2005 | RT-PCR using primers that amplify exons 2 to 3 sequence of pre-proghrelin mRNA in rat hypothalamus. | Positive gene expression. | [61] |
IHC #G606 antiserum against ghrelin (1–11) in porcine hypothalamus. | Ghrelin-IR in the ARC, the PVN and periventricular regions. | ||
RP-HPLC separation of peptide extracts from rat hypothalamus followed by N-RIA and C-RIA, as described in Hosoda et al. 2000. | A peak that elutes at the position of ghrelin was detected by both N-RIA and C-RIA. | ||
Hou-2006 | RT-PCR in rat hypothalamus and frontal cortex using primers against pre-proghrelin mRNA, as used by Kojima et al. 1999. | Positive gene expression. | [62] |
IHC using a goat antiserum against ghrelin (Santa Cruz Biotechnology, Cat# sc-10368) in rat hypothalamus and frontal cortex. | Ghrelin-IR cells in the LH, the PVN, the ARC and the ependymal layer of the third ventricle. In cerebral cortex, Ghrelin-IR cells mainly distributed in sensorimotor cortex and cingulate gyrus. | ||
Canpolat-2006 | IHC using an unspecified rabbit polyclonal antibody in rat hypothalamus. | Ghrelin-IR cell bodies in the ARC. | [63] |
Menyhert-2006 | IHC in human hypothalamus using the non-commercial antibody, as used by Cowley et al. 2003. | Ghrelin-IR fibers, but not cell bodies in the SON, Pe, SQN, PVN, perifornical, infundibular nucleus, DMN and VMN. | [64] |
Luque-2007 | Q-PCR using primers that amplify exons 2 to 3 sequence of pre-proghrelin mRNA in mouse hypothalamus. | Positive gene expression. | [65] |
Yang-2008 | RT-PCR using sets of primers that amplify sequences from exons 1 to 4 of pre-proghrelin or GOAT mRNAs in mouse brain. | Negative gene expression of GOAT and very little expression of ghrelin. | [66] |
Gutierrez-2008 | TissueScan Real-Time with human cDNA panels (Origene Technologies) for pre-proghrelin and GOAT mRNAs. Primer sequences are not provided. | Negative gene expression for both genes in the brain. | [67] |
Guan-2008 | IHC using the #G606 antiserum and a goat antiserum both against anti-ghrelin (1–11) in rat hypothalamus. | Ghrelin-IR cell bodies and their terminals in the ARC. Ghrelin-IR signal was detected in dense granular vesicles by electron microscopy. | [68] |
Hori-2008 | IHC using #G606 and #G4-2 non-commercial rabbit polyclonal antisera against ghrelin (1–11) in rat hypothalamus. | Ghrelin-IR cell bodies and their terminals in the ARC. Ghrelin-IR signal was detected in dense granular vesicles by electron microscopy. | [69] |
Kageyama-2008 | RT-PCR using primers that amplify exons 2 to 4 of pre-proghrelin mRNA in mouse hypothalamus. | Positive gene expression. | [70] |
Ghrelin-EGFP transgenic mouse model, in which EGFP gene expression is driven by ghrelin promoter. | EGFP-IR cells in the ARC only detected by wide-band spectral confocal laser microscopy but not observed by conventional fluorescence microscopy. | ||
Grouselle-2008 | Competitive EIA using a rabbit polyclonal antibody against ghrelin (1–11) in sheep hypothalamic homogenates. | Ghrelin-IR signal in hypothalamus, which was ~30,000-fold smaller than in abomasums and ~1000-fold smaller than in small intestine. | [40] |
Ueberberg-2009 | Q-PCR using primers that amplify exon 1 sequence of pre-proghrelin mRNA in human brain. | Positive gene expression. | [71] |
Sakata-2009 | -RT-PCR using sets of primers that amplify sequences from exons 1 to 2 of pre-proghrelin or GOAT mRNAs in mouse brain. | Positive gene expression for both genes. | [72] |
Sakata-2009 | Ghrelin-hrGFP transgenic mouse models in which hrGFP gene expression is driven by ghrelin promoter. | No signal of hrGFP fluorescence or hrGFP-IR in the brain. | [73] |
IHC using two different commercial rabbit anti-ghrelin antisera (Phoenix Pharmaceuticals, Cat# H-191-031-31 or Santa Cruz Biotechnology, SC-10368) in ghrelin-hrGFP transgenic or wild-type mice brains. | No signal. | ||
ISHH targeting exon 4 sequence of pre-proghrelin mRNA in brains of ghrelin-hrGFP transgenic or wild-type mice. | No signal. | ||
Gahete-2010 | Q-PCR using sets of primers that amplify sequences from exon 2 to 3 of pre-proghrelin or GOAT mRNAs in mouse hypothalamus. | Positive gene expression for both genes. | [74] |
Yakabi-2010 | Q-PCR (TaqMan) using primers that amplify sequences from exon 2 to 3 of pre-proghrelin mRNA in rat hypothalamus. | Positive gene expression. | [75] |
ELISA (Mitsubishi Chemical Medience Corp.) against ghrelin in rat hypothalamic homogenates and secretion media. | Positive ghrelin-IR signal in content and secretion. | ||
Gahete-2010 | Q-PCR using sets of primers that amplify sequences from exon 1 to 2 of pre-proghrelin or GOAT mRNAs in human temporal lobe. | Positive gene expression for both genes. | [76] |
Furness-2011 | IHC in rat and mouse brains using different non-commercial rabbit polyclonal antisera: (1) RY1601, against rat ghrelin (1–15)-Cys coupled to keyhole limpet hemocyanin; (2) RY1595, against rat des-acyl-ghrelin (1–15)-Cys coupled to keyhole limpet hemocyanin; (3) GO-1, against human ghrelin coupled through glutaraldehyde to bovine serum albumin; (4) GM-2, against human ghrelin coupled through carbodiimide to bovine serum albumin. | No specific signal. | [77] |
ELISA (Chemicon) against ghrelin in rat and mouse hypothalamus. | Very low levels of ghrelin. | ||
Rucinski-2012 | RT-PCR and Q-PCR for GOAT mRNA in rat hypothalamus. | Positive gene expression. | [78] |
Francois-2015 | ISHH targeting exons 1 to 3 sequences of pre-proghrelin mRNA in mouse hypothalamus. | No signal. | [79] |
Wellman-2015 | Q-PCR using primers that amplify exons 1 to 4 sequence of pre-proghrelin and GOAT mRNAs in rat hypothalamus. | Positive gene expression for both genes. | [80] |
Level | Experimental Findings | Main Conclusions |
---|---|---|
pre-proghrelin mRNA | No signal by Northern blot or ISHH. Positive signal by RT-PCR or Q-PCR. | Pre-proghrelin mRNA measurable in low amounts |
Ghrelin peptide | Presence of ghrelin in amounts less than 100- to 5000-fold as compared to stomach levels | Ghrelin measurable in minute amounts but no conclusive evidence for local synthesis. |
Cellular localization. | Presence of ghrelin-IR positive neurons in many nuclei and regions | No reproducibility amongst studies. Lack of adequate specificity controls, namely absence in ghrelin KO mice. Cellular location of the positive signal unexpected, based on the epitope recognized by the antibodies. |
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Cabral, A.; López Soto, E.J.; Epelbaum, J.; Perelló, M. Is Ghrelin Synthesized in the Central Nervous System? Int. J. Mol. Sci. 2017, 18, 638. https://doi.org/10.3390/ijms18030638
Cabral A, López Soto EJ, Epelbaum J, Perelló M. Is Ghrelin Synthesized in the Central Nervous System? International Journal of Molecular Sciences. 2017; 18(3):638. https://doi.org/10.3390/ijms18030638
Chicago/Turabian StyleCabral, Agustina, Eduardo J. López Soto, Jacques Epelbaum, and Mario Perelló. 2017. "Is Ghrelin Synthesized in the Central Nervous System?" International Journal of Molecular Sciences 18, no. 3: 638. https://doi.org/10.3390/ijms18030638