From the Departments of $Medicine and llPathology,. Toronto General Hospital and St. Michael's Hospital,. University of Toronto, Toronto, Ontario M5G2C4, ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 263,No. 27,Issue of September 25, pp. 13475-13478,1988 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.
Communication
Printed in U.S.A.
Glucagon Gene Expressionin Vertebrate Brain*
for proglucagon-derived peptides in the nervous system stems from the observations that intracerebral injections of glucagon produces dosage-dependent hyperglycemia, and both glucagon and GLP-I' receptors have recently been identified in (Received for publication, March 9, 1988, and in revised form, June 14, 1988) rat brain (12-14). Recent studies have demonstrated that the glucagon-like peptides activate adenylate cyclase in brain Daniel J. DruckerSP and Sylvia Asa7 tissue and membrane preparations (14-15), providing addiFrom the Departments of $Medicine and llPathology, tional evidence for the importance of a brain-derived glucagon Toronto General Hospital and St. Michael's Hospital, system. University of Toronto, Toronto, Ontario M5G2C4, Canada Taken together, these data suggest that glucagon and the glucagon-like peptides may function as neuropeptides in seAn increasing number of regulatory peptide genes lected regions of the nervous system. However, whether these are known to be transcribed in neuroendocrine cells of peptides are actually synthesized in the brain or simply taken the intestine and neurons of the central and peripheral up from the circulation remains uncertain. To determine if nervous system. The discovery of the expression of peptide hormone genes in the nervous system has led the biosynthesis of glucagon and the glucagon-like peptides to the suggestion that these peptides may function as occurs in the central nervous system, we sought evidence for neurotransmitters, neuromodulators, and releasing orthe expression of the glucagon or a glucagon-related gene in different regions of the brain. We find that theglucagon gene inhibiting factors in different regions ofthebrain. Glucagon and the glucagon-like peptides are derived is expressed in neurons in both the hypothalamus and brainfrom proglucagon in the pancreatic islets and intestine. stem andthat glucagon geneexpression in the brain gives rise A role for these peptides in the central nervous system to an mRNA transcript that is identical in sequence to that has been proposed, but evidence for the biosynthesis offound in pancreas and intestine. proglucagon in brain has been lacking. We now report that the glucagon gene is expressed in the brainstem EXPERIMENTAL PROCEDURES and hypothalamus and that a glucagon mRNA tranMaterials-Restriction enzymes, T4 DNA ligase, DNA polymerase, script identical to that produced in pancreas intesand tine gives rise to proglucagon-related peptides in the and alkaline phosphatase were from Pharmacia LKB Biotechnology Inc. [cP~'P]ATP (>800 Ci/mmol) was from ICN Radiochemicals. [abrain. %]ATP (600 Ci/mmol) was from Amersham Corp. Nitrocellulose
The gene encoding preproglucagon is expressed in the A cells of the pancreatic islets and the neuroendocrine L cells of the intestine. Several studies have reported glucagon immunoreactivity in other tissues, including thymus, thyroid, and adrenal gland (1).Reports of glucagon biosynthesis in tissues other than pancreas and intestine, however, have not been widely accepted. For example, initial reports of a glucagon-like peptide with hyperglycemic activity in salivary gland extracts (2, 3) were subsequently discounted due to the presence of tracer degrading activity in thesalivary gland extracts (4).A growing body of evidence suggests, however, that the brain may be a potential site of glucagon biosynthesis. Neurons in the retina, hypothalamus, and medulla oblongata have been identified which stain positive for glucagon and glucagon-like peptide I immunoreactivity (5-8). Immunoreactive glucagon and glucagon-like peptide I of various molecular sizes have also been detected in different regions of canine and rat brain (9-12). Functional evidence of a biological role * This work was supported by grants from the Medical Research Council of Canada and the National Cancer Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisemnt" in accordance with 18U.S.C. Section 1734 solelyto indicate this fact. The nucleotide sequencefs)reported in this paper has been submitted to the GenBankTM/EMBL Data Bank withaccessionnumberfs) 504040. 5 Research Scholar of the Medical Research Council of Canada. TO whom correspondence should be addressedToronto General Hospital, 200 Elizabeth St.,CCRW3-838, Toronto, OntarioM5G2C4, Canada.
membranes were from Schleicher and Schuell. All chemicals were from Sigma or Fisher. Sprague-Dawley rats were obtained from Charles River, Canada. The human neonatal brainstemcDNA library (16) was a kind gift from M. Jaye, Meloy Laboratories, Springfield, VA. The rat glucagon and somatostatin cDNA probes were a gift of Dr. J. Habener, Massachusetts General Hospital. The rat cholecystokinin cDNA was obtained from Dr. J. Dixon, Purdue University. RNA Analysis-RNA was isolated from tissues as described previously (17). Polyadenylated RNA was prepared by two cycles of oligo(dT)-cellulose chromatography (18).RNA was size-fractionated through a 1.3% agarose-formaldehyde gel, ethidium-stained to assess the integrity and migration of the RNA, and transferred to a nylon membrane. The RNA was fixed on the membrane by UV irradiation, following which prehybridization was performed overnight in 1 X Denhardt's, 4 X SSC, 200 pg/ml salmon sperm DNA, 40% deionized formamide, 0.014 M Tris, pH 7.4. cDNA probes for rat glucagon, somatostatin, and cholecystokinin were labeled by the random priming technique (19) to a specific activity of 5 X 10' cpmlpg. Hybridization was performed in the same solution with 1 X IO6 cpm/ml of 32P-labeledcDNA probes for 24 h at 42 "C. Final washing conditions were 0.1 X SSC, 0.1% sodium dodecyl sulfate a t 65 "C. 1 X SSC is 0.15 M NaCl, 0.3 M sodium citrate. Autoradiography was carried out using Kodak X-Omat film a t -70 "C. Isolation of cDNAClones-A human neonatalbrainstem Xgtll cDNA library was plated at a density of 5 X lo' plaques per plate, and duplicate filters were denatured, neutralized, and baked at 80 "C for 2 h. Filters were prehybridized and hybridized in 1 M NaCl, 1% sodium dodecyl sulfate at 65 "C. Positive clones were purified by replating a t lower dilutions, and cDNA inserts were excised from phage DNA by cutting with the restriction enzyme EcoRI. The cDNA inserts were subcloned into the Bluescript plasmid (Stratagene) and sequenced as described previously (20). All ligations, transformations, and plasmid and phage preparations were carried out by standard techniques under Medical Research Council and National Cancer Institute of Canada guidelines. The abbreviations used are: GLP-I, glucagon-like peptide I; bp, base pair.
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Glucagon GeneExpression in Vertebrate Brain
Immunocytochemistry-immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissue using the avidin-biotin peroxidase complex technique (21).Aprimary antiserumdirected against synthetic GLP-I (22) and a primary antiserum directed against glucagon (Dako, Santa Barbara, CA) were used at dilutions of 1:200. The durationof exposure to primary antiserumwas 24 h a t 4 "C. The reaction product was visualized by detection of peroxidase activity usingasolution of 3,3'-diaminobenzidinetetrahydrochloride and hydrogen peroxide. Preabsorption of primary antiserum against GLPIwith synthetic GLP-I (1-37) (a kindgift of Dr. J. F. Habener, Massachusetts General Hospital, Boston) eliminated staining at concentrations of 6 pglml. RESULTS AND DISCUSSION
To determinewhether expression of the glucagon or a related gene could be detected in brain tissues, total cellular RNA was prepared from rat cortex, cerebellum, striatum, hippocampus, pons, pituitary, hypothalamus, and brainstem. Northern blot analysis of 30 pg of RNA from each region of the brain showed no hybridizable glucagon mRNA transcripts except for the brainstem, which contained a glucagon mRNA species of -1300 bp after a 7-day exposure. We next prepared poly(A+)RNA from the above tissues and repeated the Northern blot analysis (Fig. 1). Glucagon mRNA transcripts were easily detectable in 5 pg of poly(A+) RNA from adult brainstem. A considerably weaker signal was seen in fetal brainstem, despite loading double the amount of poly(A+) RNA in this lane. No glucagon mRNA transcripts could be convincingly detected in RNA prepared from adult orfetal hypothalamus, due to considerable background in the expected region of the glucagon mRNA transcript. Theglucagon mRNA transcript in rat brainstem (-1300 bp) was just slightly larger
FIG.2. Northern blot analysis of RNA from rat brainstem and hypothalamus. 2 pg of poly(A+) RNA isolated from adult rat brainstem and hypothalamus was analyzed by Northern blot analysis and hybridized with cDNA probes for glucagon (panels a and b ) and somatostatin (panel c). The blot in panel a was exposed for 20 h, in panel b for 11 days, and in panelc for 24 h.
than theglucagon mRNA transcripts detected in ratintestine (1250 bp). T o verify the integrity of the hypothalamic and brainstem RNAs and tocompare the relative abundance of neuropeptide mRNA transcripts in differentregions of the brain, the Northern blot shown in Fig. 1was rehybridized with cDNA probes for somatostatin andcholecystokinin, two neuropeptide genes expressed at relatively high levels in vertebrate brain (23). AB FB AH FH I NE These experimentsdemonstrated abundant yet differing 18s * amounts of somatostatin andCCK mRNAs in both adult and fetal hypothalamus, despite the apparent lack of detectable glucagon mRNA transcripts in the same RNA preparations (Fig. 1,B and C). Although the mRNA signals obtained with the somatostatin andCCK probes appeared similar, the relative levels of abundance of these mRNAs in poly(A+) RNA A from brainstem and hypothalamus were clearly different. In view of the suggestive, albeit inconclusive, presence of a faint glucagon mRNA transcript in RNA from rat hypothalamus (Fig. l ) , we attempted to more conclusively demonstrate the presence of glucagon mRNA transcripts in rat hypothalamus. A second Northern blot was run with 2 pg of poly(A') RNA prepared from rat brainstem ( B S )or hypothalamus (H) and is shown in Fig. 2. A 20-h exposure was sufficient to visualize the relatively abundant glucagon mRNA transcripts in the brainstem preparation, but no definiteglucagon mRNA transcript was detected in theRNA prepared from hypothalamus. However, prolonged autoradiographic exposure of the same blot for 12 days resulted in the appearance of a single band in the hypothalamiclane, the same size asthe glucagon mRNA transcript detected in the brainstem. Thus, the glucagon gene is also expressed in the hypothalamus, albeit a t C levels approximately 100-fold lower than inbrainstem. In FIG.1. Northern blot analysis of rat brain RNA with probes contrast to the marked regional variation in abundance of for glucagon ( A ) ,somatostatin ( B ) ,and cholecystokinin (C). glucagon mRNA transcripts, the same blot was rehybridized Poly(A+) RNA from adult ( A B ) and fetal ( F B ) brainstem, and adult with a somatostatin cDNA probe, as shown in Fig. 2C. Nearly ( A H ) and fetal ( F H ) hypothalamus and total cellularRNAfrom identical amounts of somatostatin mRNA transcripts were neonatal (day1) brainstem ( N B )and rat intestine(I).The migration position of 18 S ribosomal RNA is shown at the left of the panel. 5 detected in the brainstem and hypothalamic RNA preparapg ( A B and A H ) or 10 pg ( F B and F H ) of poly(A+) RNA and 30 pg tions, consistent with the results obtained in Fig. 1B. of total cellular RNA from neonatal rat brainstem ( N B ) and 10 pg To define in greater detail the structure of the preproglutotal cellular RNA from rat intestine ( I ) were loaded, respectively. cagon mRNA produced in cells of neural origin, we screened cDNA probes for glucagon, somatostatin, and cholecystokinin were labeled to the same specific activities. Blots were exposed to Kodak cDNA libraries prepared from both hypothalamus and brainX-Omat film for 24 h(glucagon), 3.5 h (somatostatin), or 22 h stem RNA with a full-length rat pancreatic preproglucagon (cholecystokinin). cDNA. No positive clones were obtained after screening 1 X
Glucagon Gene Vertebrate Expression in
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