Inhibition of Pancreatic Glucagon Gene Expression in Mice Bearing Subcutaneous Glucagon-Producing GLUTag Transplantable Tumor

a

Daniel J. Drucker*, Ying C. Lee, Sylvia L. Asa, and Patricia

L. Brubakert

Departments of Medicine, Clinical Biochemistry, Genetics, Pathology and Physiology University of Toronto Toronto, Ontario, Canada

Molecular

induced fibrosarcoma. The suppression of pancreatic A-cell function and islet size in mice with elevated plasma levels of the proglucagon-derived peptides raises the possibility that a proglucagonderived peptide may participate in a negative feedback loop, inhibiting expression of the glucagon gene in the A-cells of the endocrine pancreas. (Molecular Endocrinology 6: 2175-2184, 1992)

Transgenic mice that express a glucagon genesimian virus-40 large T-antigen (GLUTag) fusion gene develop neuroendocrine carcinoma of the large bowel. This glucagon-producing tumor was implanted SC and reproducibly formed tumors in nude mice. The transplanted GLUTag tumor expressed large amounts of proglucagon mRNA transcripts, and the levels of proglucagon mRNA transcripts remained constant during 2-8 weeks of tumor growth. The posttranslational processing of proglucagon in the transplantable tumor resembled that detected in the original transgenic tumor, with the liberation of glicentin, oxyntomodulin, glucagon, glucagon-like peptide (l-37) [GLP-l-( l-37)] and GLP-l-(7-37). Tumor-bearing mice demonstrated progressive elevations in the plasma levels of proglucagon-derived peptides. Elevated plasma levels of glucagon-like immunoreactive peptides and immunoreactive glucagon were associated with a marked reduction in the levels of pancreatic glucagon mRNA transcripts by 4 weeks, and after 8 weeks of tumor growth, the levels of glucagon mRNA transcripts in the pancreas were not detectable by Northern blot analysis. Synthesis of the proglucagonderived peptides was also significantly suppressed at 4-8 weeks in the pancreas of tumor-bearing animals. Histological examination of the endocrine pancreas in mice carrying the GLUTag tumor for 6-8 weeks demonstrated a marked reduction in the number and size of the islets of Langerhans and a disproportionately greater decrease in the number of cells exhibiting glucagon immunoreactivity. By electron microscopy, the residual A-cells were small, compressed at the periphery of the islets, and had poorly developed cytoplasmic organelles. In contrast, no changes in mouse glucagon gene expression or islet morphology were detected in control animals without tumors or mice carrying a SC v-jun0888.8809/92/2175-2184$03.00/O Molecular Endocrinology Copynght 0 1992 by The Endocrine

and Medical

INTRODUCTION

The glucagon gene is expressed in the A-cells of the endocrine pancreas, the L-cells of the intestine, and brainstem and hypothalamic neurons (l-3). A number of different peptides encoded within proglucagon are liberated by tissue-specific posttranslational processing and exhibit important bioactive properties. Glucagon released from the pancreatic A-ceil controls the regulation of gluconeogenesis and glycogenolysis in the hepatocyte. More recent studies have revealed that a truncated form of glucagon-like peptide 1 (GLP-I), secreted by the intestinal L-cell, is a potent stimulator of glucose-dependent insulin secretion (4, 5). Studies directed at elucidating the molecular control of glucagon gene expression have shown that a single proglucagon mRNA species is transcribed from an identical promoter in pancreas, intestine, and brain (6). The results of gene transfer experiments in vitro have lead to the identification of specific &-acting glucagon gene sequences that direct islet cell-specific glucagon gene expression in pancreatic islet cell lines (7). DNA-binding proteins have been identified in nuclear extracts prepared from islet cell lines that bind to specific sequences in the 5’-flanking region of the rat glucagon gene (8, 9). In contrast, much less is known about the factors important for the control of glucagon gene expression in the intestine, in part due to a lack of suitable experimental models. Primary cultures of fetal rat intestinal cells have provided useful data about the control of

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intestinal proglucagon biosynthesis (2, lo), but glucagon-producing intestinal cell lines suitable for cell transfection studies have not yet been established. Transgenie mice [containing 1.3 kilobases (kb) of rat glucagon gene 5’-flanking sequences up-stream of the simian virus-40 (SV40) large T-antigen] were found to express the GLUTag transgene in the brain and pancreas, but not in the gastrointestinal tract (11). These results suggested that the cis-acting sequences that specify intestine-specific expression of the glucagon gene were different from the sequences that targeted expression to the brain and endocrine pancreas. We recently generated mice with a transgene containing 2.0 kb of the rat glucagon gene 5’-flanking region up-stream of SV40 large T-antigen-coding sequences. Analysis of four different transgenic lines demonstrated expression of this larger GLUTag transgene in brain, pancreas, stomach, and small and large intestine (12). Expression of the transgene in the large intestine produced hyperplasia of the glucagon-producing cells, which invariably progressed to invasive neuroendocrine carcinoma, detectable by 4-8 weeks of age. In contrast, expression of the transgene in the pancreas of mice 4-8 weeks of age produced focal islet cell hyperplasia, which progressed in older mice to neoplastic transformation of the endocrine pancreas (12). Analysis of the expression of the endogenous glucagon gene in different tissues of the GLUTag transgenie mice demonstrated a reduction of glucagon mRNA transcripts in transgenic compared to control pancreas (13). The levels of pancreatic glucagon-like immunoreactive peptides (GLI) and immunoreactive glucagon peptides (IRG) were also substantially reduced in transgenic animals, and islets from the transgenic mice exhibited a marked reduction in the number of glucagon-immunoreactive cells. To distinguish the effects of SV40 T-antigen expression in the transgenic pancreas from the possible effects of high concentrations of circulating proglucagon-derived peptides on the suppression of pancreatic glucagon gene expression, we established a transplantable glucagon-producing (GLLtTag) tumor from the original transgenic large bowel glucagon-producing neuroendocrine carcinoma. This tumor has been propagated for multiple passages SC in nude mice. We now report that the tumor-bearing mice contain substantially reduced pancreatic levels of the proglucagon-derived peptides in association with a marked inhibition of pancreatic proglucagon gene expression. These observations suggest that elevated levels of circulating proglucagon-derived peptides may inhibit pancreatic A-cell function, defining a novel mechanism for the control of pancreatic glucagon gene expression in vivo.

RESULTS

Nude mice were injected SC with equal volumes of a GLUTag tumor suspension prepared from a single 0.8-

g GLUTag tumor, and mice were killed 2-8 weeks after tumor innoculation. Control mice were injected with medium alone or with v-jun fibrosarcoma cells. After 8 weeks, the SC GLUTag tumors weighed 1.4 f 0.3 g; however, the body weights of the animals carrying the tumors were not significantly different from those of the control animals (21.2 + 1.6 vs. 21.8 + 0.4, respectively; P 2 0.60). To analyze for variability in the expression of the glucagon gene at different time points during the growth of the SC GLLfTag tumor in vivo, animals were killed at the intervals described above, and tumor fragments were homogenized for RNA isolation and analysis. The results of these experiments are shown in Fig. 1. Glucagon mRNA transcripts were readily detectable

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Fig. 1. Northern Blot Analysis of RNA Prepared from RIN 56 Islet Cells, the Large Bowel of a g-Week-Old GLUTag-Y Transgenie Mouse, or the GLUTag SC Transplantable Tumor Passaged for 2, 4,6, or 8 Weeks The blots that contained 10 pg total cellular RNA were hybridized sequentially with cDNA probes for glucagon (G), insulin (I), somatostatin (S), PYY, or tubulin (T). The blots were exposed for 12-48 h.

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Inhibition

of Pancreatic

Glucagon

Gene

Expression

in RNA isolated from tumors propagated SC for 2-8 weeks. The relative levels (per gg tumor RNA) of glucagon mRNA transcripts remained fairly constant, although they were somewhat lower (40% by densitometry) than the levels detected in the original transgenic mouse large bowel tumor (GLUTag LB) or in RIN 56 islet cells, a glucagon-producing rat islet cell line (14) analyzed for comparative purposes. Insulin mRNA transcripts were detected in RNA from RIN 56 cells, but not in the transgenic mouse large bowel tumor (12) or the GLUTag SC tumors, in keeping with the intestinal origin of this neoplasm (Fig. 1). In contrast to the relatively constant levels of glucagon mRNA transcripts in the GLUTag tumors, the levels of somatostatin and peptide-YY (PYY) mRNA transcripts were considerably more variable. Both somatostatin and PYY mRNA transcripts were less abundant in RNA prepared from the SC tumors than in RNA extracted from the transgenic large bowel tumor (23% and 10% of GLUTag LB values for somatostatin and PYY mRNAs, respectively, by densitometry), as depicted in Fig. 1. The levels of tubulin mRNA transcripts did not differ significantly in the SC tumors from 2-8 weeks. No mRNA transcripts for glucagon, somatostatin, insulin, or PYY were detectable in RNA prepared from the v-jun fibrosarcomas (data not shown). Plasma levels of GLI and IRG rose significantly in parallel with tumor growth during the experimental period, from 526 + 28 (n = 8) and 142 + 23 (n = 7) pg/ ml in controls to 14,290 + 2,078 (n = 5) and 2,514 f 720 (n = 3), respectively (P < 0.001) by 8 weeks (Fig. 2A). Plasma levels of immunoreactive insulin (IRI) did not change significantly during the experimental period, and blood glucose levels rose only transiently at 2 and 4 weeks of tumor growth. Consistent with the constant amounts of proglucagon mRNA transcripts detected in GLUTag tumors from 2-8 weeks, the concentrations of the proglucagon-derived peptides in the tumor, expressed per pg protein, did not change over the course of the experiment (Fig. 28). The v-jun-transformed fibrosarcomas reached a mean weight of 4.9 + 0.9 g by 2.5 weeks of tumor growth (vs. 1.4 + 0.3 g at 8 weeks for the GLUTag tumors), at which time the animals were killed. No GLI, IRG, or GLP-1 was detected in any of the fibrosarcoma extracts (data not shown). In keeping with these findings, the plasma levels of GLI, IRG, IRI, and glucose were normal in these animals (Fig. 2A, FS). The levels of the proglucagon-derived peptides were significantly suppressed in the pancreases of nude mice carrying the GLUTag tumor for 4-8 weeks (Fig. 3). Strikingly, pancreatic concentrations of GLI and IRG decreased to 8% of control levels (P 5 0.001) and GLP-1 levels decreased to 17% of control levels (P 5 0.001) after 8 weeks of tumor growth. Pancreatic levels of IRI rose transiently during tumor growth (P 5 O.Ol0.05 at 2-4 weeks; Fig. 3), but then returned to normal control levels for the remainder of the experimental period. Mice carrying the v-jun fibrosarcomas exhibited a small generalized increase in the levels of all of the pancreatic hormones examined (P 5 0.05 to P 5 0.01).

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Fig. 2. A, Concentrations of GLI (A), IRG (B), IRI (C), and Glucose (D) in Plasma from Age-Matched Control Mice (c), Mice Carrying the Fibrosarcoma Tumor (FS), and Nude Mice Harboring GLUTag Tumors for 2, 4, 6, or 8 Weeks Due to limitations in plasma volume, some groups contain data for only two animals; no SE was calculated for these values. Insufficient sample remained for insulin determinations at 4 weeks; *, P < 0.05; ++, P < 0.01; l ‘*, P < 0.001. B, Concentrations of GLI (A), IRG (B), and GLP-1 (C) in GLUTag tumors passaged in nude mice for 2, 4, 6, or 8 weeks. *, P < 0.05; If, P < 0.01; ***, P < 0.001.

To characterize the molecular forms of the proglucagon-derived peptides synthesized by the GLUTag tumors, tumor extracts were examined by HPLC and RIA after 8 weeks of growth SC. Normal nude mouse intes-

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3. Concentrations of GLI (A), IRG (B), GLP-1 (C), and RI (D) in the Pancreas of Age-Matched Control Mice (c), Mice Carrying the v-jun Fibrosarcoma (FS), and Nude Mice with the GLUTag Tumor after 2,4,6, or 8 Weeks *, P < 0.05; l *, P < 0.01; ***, P < 0.001, Fig.

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site of the GLUTag tumor in GLUTag-Y transgenic mice (12). The profiles of GLI and IRG in normal mouse large intestine were identical to those in normal rat and mouse intestine (13, 15) and included two main peaks of GLI, identified as glicentin (56% of the total GLI) and oxyntomodulin (37%) and a minor peak of both GLI and IRG, identified as glucagon (7%; Fig. 4). HPLC analysis of the GLUTag tumor also demonstrated peaks of glicentin, oxyntomodulin, and glucagon; however, the proportion of glicentin was diminished to 25% of the total peak GLI, and that of oxyntomodulin remained at 33%, whereas glucagon now constituted 36% of the total GLI (Fig. 4). An additional peak of GLI and IRG was detected at fraction 81 (6% of the total peak GLI). This peak may represent either the 9K peptide (16) an N-terminally extended pancreatic form of glucagon, or glucagon (19-29), a peptide known to be cleaved from glucagon in the liver (17). These data are consistent with our observation that the ratio of GLI/IRG in the large intestine of the normal nude mouse was 73 f 8, but was only 2.2 f 0.2 (n = 17) in the GLUTag tumor (Fig. 2B). HPLC analysis of the GLP-1 immunoreactive peptides in the normal mouse large intestine and in 8-week propagated GLUTag tumors demonstrated two principle peaks of immunoreactivity corresponding in elution position to synthetic GLP-1 -(l-37) and GLP-i-(7-37) (Fig. 5). No marked differences were detected between the profiles obtained from the two different tissues. To ascertain whether the increased levels of circulating proglucagon-derived peptides and the decreased pancreatic content of proglucagon-derived peptides were associated with any change in the expression of the endogenous mouse glucagon gene, RNA was prepared from pancreas and small and large intestine, after

0 60

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Fraction # Fig. 4. HPLC Separation of GLI (m) and IRG (0) Contained in Control Mouse Large Intestine (A) and a GLUTag Tumor after 8 Weeks of Growth in a Nude Mouse (B) The peaks are identified based on their elution positions as glicentin (a), oxyntomodulin (b), and glucagon (c), based upon immunoreactivity and similar analyses of rat pancreatic and rat and mouse intestinal extracts (3, 10, 13). The elution position of synthetic glucagon is indicated by the arrowhead. IR, Immunoreactive.

which the levels of mRNA transcripts were quantitated by Northern blot analysis. There was no difference in the pancreatic weight of age-matched control mice or mice carrying the GLUTag tumor (mean f SEM, 0.121 + 0.005 vs. 0.139 f 0.016 g; control vs. experimental, respectively). The difference was not statistically significant by unpaired Student’s r test. Pancreatic glucagon mRNA transcripts were slightly reduced after 2 weeks (50% of control values by densitometry) in the tumorbearing animals, after which a progressive and marked reduction (13% of control values at 4 weeks) in the levels of pancreatic glucagon mRNA transcripts was observed (Fig. 6). No glucagon mRNA transcripts were detectable in the pancreatic RNA preparations from mice carrying the tumor for 8 weeks even after a lweek exposure of the blot. The extremely low levels of glucagon mRNA transcripts in the tumor-bearing animals at 6 and 8 weeks precluded accurate densitometric assessment of the quantitative reduction in the levels of glucagon mRNA transcripts at these time points.

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lnhlbltlon

of Pancreatic

Glucagon

Gene

Expression

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Blot Analysis of Total Cellular RNA (10 pg/ Lane) from Pancreas of Experimental (E) Nude Mice Carrying the GLUTag Tumor for 2, 4, 6, or 8 Weeks, from Age- and Sex-Matched Control Mice without Tumors (C), or from Nude Mice Carrying the v-jun Fibrosarcoma (v-jun) G, Glucagon; I, insulin; S, somatostatin; T, tubulin mRNA transcripts. The blots were exposed for 24-72 h.

Fig. 5. HPLC Separation

of GLP-1 lmmunoreactive Peptides Contained in Control Mouse Large Intestine (A) and a GLUTag Tumor after 8 Weeks of Growth in a Nude Mouse (B) The peaks are identified as GLP-1 -(l-37) (a) and GLP-1-(737) (b) based upon the elution profile of the synthetic standards, as designated by the arrowheads.

mRNA, despite the much larger size of the tumors in these animals. A progressive, although less marked, reduction in the levels of insulin mRNA transcripts was also observed in RNA from mice carrying the GLUTag tumor (3.2- and 4.3-fold reduction compared to controls at 6 and 8 weeks, respectively), as shown in Fig. 6. The marked changes in islet mRNA transcripts, but lack of changes in tubulin RNA transcripts (Fig. 6), in total pancreatic RNA suggests that the inhibition of gene expression is largely confined to the islets and not generalized to the exocrine pancreas. Somatostatin mRNA transcripts were also reduced in the tumorbearing mice, although this reduction was not progressive and was also seen in the control mice carrying the v-jun fibrosarcomas. The demonstration that animals carrying the GLUTag tumor, but not the fibrosarcoma, exhibited marked reductions in the levels of pancreatic glucagon mRNA transcripts and proglucagon-derived peptides prompted us to examine the histology of the endocrine pancreas and the morphology of the glucagon-producing pancreatic A-cells. Morphometric analysis confirmed a marked reduction in pancreatic area occupied by islets. Control mice (bearing no tumors or fibrosarco-

mas) had islet areas representing 0.83 +. 0.14% of the total pancreatic area in multiple random sections; 6 weeks after implantation of GLUTag tumors, mean islet areas were markedly reduced to 0.14 f 0.03% of the total pancreatic area, and after 8 weeks, mean islet areas were 0.09 f 0.04% of the total pancreatic area (P < 0.01). Analysis of the pancreatic islets in tumorbearing animals also demonstrated a consistent reduction in the size of the islets in mice carrying the GLUTag tumors for 6-8 weeks. The control animals had islets measuring 109 f 27 pm*; 6 and 8 weeks after GLUTag tumor implantation, islets measured 33 f 9 and 19 -I- 7 pm*, respectively (P c 0.05). The majority of the islet cells from tumor-bearing mice contained cytoplasmic insulin immunopositivity (Fig. 7C). In contrast to the islets from control mice (Fig. 7) or mice carrying the v-jun fibrosarcoma, the islets from mice carrying the GLUTag tumor for 6 or 8 weeks contained few or no detectable cells with glucagon and GLP-1 immunopositivity (Fig. 7D). Examination of the islets by electron microscopy demonstrated that the Bcells exhibited well developed cytoplasmic organelles and contained numerous secretory granules of normal morphology. In contrast, A-cells were difficult to identify (Fig. 7, E and F). They were few in number and small, and were found compressed at the periphery of the islets. Their scanty cytoplasm contained poorly developed organelles and a reduced number of secretory granules.

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Fig. 7. A, The pancreatic islets of a control nude mouse are normal in size and contain numerous insulin-immunoreactive cells (avidin-biotin-peroxidase technique; magnification, x135). B, The islets of a control mouse also contain numerous glucagonimmunoreactive cells, distributed along the periphery of the islet (avidin-biotin-peroxidase technique; magnification, x135). C, The pancreas of a nude mouse carrying a GLUTag tumor SC for 8 weeks demonstrates only small sparse islets. The immunoreactivity for insulin is intense in the majority of islet cells (avidin-biotin-peroxrdase technique; magnification, xl 35). D, The small islets in the mice carrying the GLUTag tumors contain few cells with glucagon immunopositiwty. Most islets contained only zero to two glucagon-immunopositive cells (arrow), with faint cytoplasmic staining (avidin-biotin-peroxidase technique; magnification, x135). E, The islet from an age-matched control nude mouse contains A-cells (A) and B-cells (B) that are easily recognized by electron microscopy. The glucagon-producing A-cells have well developed cytoplasmic organelles, including a Golgi complex (arrow) and numerous secretory granules of homogenous electron density (electron micrograph; magnification, X31 00). F, The islet of a nude mouse harboring the GLUTag tumor SC for 8 weeks contains B-cells (B) of normal morphology, but the single small A-cell (A) identified in this islet is compressed at the periphery of the islet; it has scant cytoplasm, with poorly developed organelles and a moderate number of secretory granules of typical morphology (electron micrograph; magnification, x31 00).

DISCUSSION A number of different glucagon-producing transplantable tumors derived from pancreatic endocrine neoplasms have been established and propagated in vivo. These tumors have been used for the derivation of glucagon-producing islet cell lines in vitro, including rat RIN 1056A cells, hamster InRl -G9 cells, and mouse aTC cell lines (14, 18, 19). Analysis of the patterns of hormone gene expression in these pancreatic glucagon-producing tumor cell lines has demonstrated that these tumors are largely plurihormonal in phenotype, expressing multiple peptide hormone mRNA transcripts (including glucagon, somatostatin, insulin, and cholecystokinin mRNAs) both in vitro and in viva (20-22). The results of our experiments indicate that the intestine-derived GLUTag tumor carried SC is also plurihormonal, as indicated by the detection of glucagon, PYY, and somatostatin mRNA transcripts in tumor RNA at different times during tumor propagation. In contrast to the pancreatic tumors studied previously (14, 18, 19) insulin mFiNA transcripts could not be detected in either the original transgenic mouse GLLJTag tumor (12) or

the GLUTag tumors propagated SC in nude mice. The pattern of hormone gene expression (presence of glucagon, somatostatin, and PYY mRNA transcripts) detected in GLUTag tumors is consistent with an intestinal endocrine phenotype (2). However, the results of the HPLC and RIA analyses of proglucagon-derived peptides demonstrated that the pattern of posttranslational processing of proglucagon in the GLUTag tumor is intermediate between that of intestine and pancreas (3, 15) with glicentin, oxyntomodulin, GLP-1, as well as 29-amino acid glucagon present in tumor extracts. This intermediate phenotype of proglucagon posttranslational processing has been previously described in studies of proglucagon cleavage in RIN 1056A and InRlG9 islet cell lines (14, 18). The observation that progressive GLUTag tumor growth was associated with marked suppression of pancreatic glucagon gene expression and a reduction in islet size raises the possibility that a tumor-derived factor(s) circulates and directly suppresses proglucagon biosynthesis and islet cell growth in the endocrine pancreas. Candidates for this factor include the proglucagon-derived peptides, which may circulate and bind

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Inhibition

of Pancreatic

Glucagon

Gene Expression

to a receptor on the pancreatic A-ceil, leading to suppression of proglucagon gene expression. Alternatively, the elevated levels of plasma GLI may only reflect the critical tumor mass necessary to achieve levels of one or more nonproglucagon-derived factors that may contribute, either directly or indirectly, to suppression of A cell glucagon gene expression. The levels of plasma GLI measured in these mice are clearly higher than those that might be achieved under different pathophysiological conditions in human subjects in vivo, with the exception of patients with glucagon-producing tumors (23-26). A single case report described a reduction in the number of A-cells from nontumorous pancreas in a patient with a glucagon-producing pancreatic endocrine carcinoma (27). Histological analysis of multiple islets demonstrated a marked reduction in the number of glucagon-immunoreactive cells. In contrast to the ultrastructural findings described in mice carrying the GLUTag tumor, however, A-cells from this patient’s pancreas did not demonstrate decreased numbers of cellular organelles or depletion of secretory granules (27). Paradoxically, the B-cells, but not the A-cells, were noted to have decreased numbers of secretory granules. The pancreatic content of proglucagon-derived peptides and the relative levels of proglucagon mRNA transcripts in this patient were not reported. Different studies have documented the complexity of intraislet interactions and the reciprocal control of islet hormone biosynthesis (28). The structural organization of the endocrine pancreas and the direction of intraislet perfusion suggest that insulin may regulate the control of glucagon biosynthesis and secretion (29, 30). Experiments using immortalized cell lines have demonstrated insulin suppression of glucagon gene transcription (31); however, no insulin-binding sites were detected on purified populations of rat islet A-cells (32). In contrast, the observations that GLP-1 released from the intestine is a potent glucose-dependent stimulator of insulin biosynthesis and secretion (5, 33-35) are consistent with the finding of GLP-1 receptors on rat insulinoma cell lines (36) and B-cells in the rat endocrine pancreas (37). Despite the increased plasma concentrations of proglucagon-derived peptides, the levels of insulin mRNA transcripts in total pancreatic RNA were relatively decreased in the GLUTag tumor-bearing animals, and insulin secretion was not elevated. This may be explained at least in part by the smaller size of the islets in mice carrying the GLUTag tumors, but could also be due to GLP-1 receptor desensitization (38) or the lack of concomitant hyperglycemia in the mice. GLP-1 has also been shown to bind to rat pancreatic A-cells in studies using a combination of immunocytochemistry and autoradiography (37). However, variable results have been obtained after the exposure of pancreatic A-cells to synthetic forms of GLP-1. Infusion of GLP-l-(7-36) amide into normal and diabetic humans to achieve concentrations of GLP-1 in the physiological range (39) or into perfused preparations of porcine pancreas (40) inhibited glucagon secretion, whereas

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the full-length GLP-1 and GLP-2 peptides were without effect (40). Furthermore, GLP-l-(7-36) amide inhibited the arginine-stimulated increase in glucagon secretion and intracellular glucagon content, but had no effect on glucagon mRNA levels in isolated rat islets (41). These observations suggest that a GLP-1 receptor on the pancreatic A-cell may mediate the feedback inhibition of glucagon secretion. In contrast, GLP-1(7-36) amide stimulated insulin release, but had no effect on the secretion of glucagon by rat pancreatic islet monolayer cultures (42), and GLP-1(7-37) stimulated insulin, but not glucagon, secretion in the perfused rat pancreas (43). The reasons for the conflicting results obtained in these studies remain to be determined. In summary, we have described the establishment and SC propagation in nude mice of a glucagon-producing tumor originally derived from a neuroendocrine intestinal carcinoma in GLUTag-Y transgenic mice. This endocrine neoplasm is plurihormonal and secretes multiple immunoreactive proglucagon-derived peptides in vivo. The observation that elevated plasma levels of these peptides are associated with marked suppression of glucagon gene expression in the pancreatic A-cell raises the possibility that one or more peptides encoded by the glucagon gene mediates a novel feedback loop, resulting in the inhibition of pancreatic glucagon biosynthesis. This hypothesis should be directly testable in future experiments.

MATERIALS

AND METHODS

Reagents All chemicals and molecular biology grade reagents were from Sigma (St. Louis, MO) or Baxter Travenol Canada (Toronto, Ontario, Canada). Restriction enzymes and DNA-modifying enzymes were obtained from Boeringher Mannheim Canada (Toronto, Ontario, Canada). [cY-32P]Deoxy-ATP was purchased from Amersham Canada (Toronto, Ontario, Canada).

Nude mice were obtained from Charles River Laboratory (Charles River Canada, Toronto, Ontario, Canada). The original transplantable GLUTag tumor was established by mincing a 2-cm segment of large bowel (containing a glucagon-producing neuroendocrine carcinoma) from an 8-week-old GLUTag-Y transgenic mouse (12). The tissue fragments were washed in PBS, resuspended in Dulbecco’s Modified Eagle’s Medium (DMEM), and injected SC into three mice. All three mice developed visible SC tumors, first detectable 2-3 weeks after injection. These tumors grew SC for 8-12 weeks, after which they exhibited signs of ulceration, central necrosis, and hemorrhage, and the mice were killed. The minced tumor fragments were frozen in DMEM supplemented with 10% (vol/vol) fetal calf serum plus 10% (vol/vol) dimethylsulfoxide and stored in liquid nitrogen for 3-12 months. Once thawed and injected SC, the previously frozen tumor cells consistently produced SC tumor nodules, visible after 2-3 weeks. For the experiments described here, a tumor cell suspension (from passage 4) was resuspended in DMEM supplemented with 10% (vol/vol) fetal calf serum and injected SC into mice that carried the tumor for 2, 4, 6, or 8 weeks (n = 4, 4, 5, and 5, respectively). Controls included age- and sex-matched sham-injected an-

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1992

tmals (n = 8) that did not carry tumors SC. As a control for the nonspecific effects of SC tumors, we used v-@-transformed fibroblasts derived from fibrosarcomas isolated from v-jun transgenic mice (44) (n = 5). Injection of 5 x 1 O6 fibrosarcoma ceils produced visible tumors within 3-4 days. These tumors grew faster than the GLUTag tumors, and the animals harboring the fibrosarcomas were killed after 2.5 weeks. Mice were anesthetized with CO1, and blood was collected into 50 ~1 Trasylol-EDTA (5000 kallikrein inhibitor units/ml: 1.2 mg/ml) by cardiac puncture. Plasma was prepared immediately by centrifugation at 4 C and stored at -70 C before analysis. For peptide extraction, the pancreas was homogenized twice in 5 ml extraction medium [l N HGI containing 5% (vol/vol) formic acid, 1% (vol/vol) trifluoroacetic acid (TFA), and 1% (wt/ vol) NaCI] at 4 C, as described previously (10, 15). The combined supernatants were passed twice through a cartridge of Cl 8 silica (Cl 8 SepPak, Waters Associates, Milford, MA), and adsorbed peptides and small proteins were eluted with 4 ml 80% (vol/vol) isopropanol containing 0.1% (vol/vol) TFA. Extracts were stored at -70 C before analysis. These extraction methods have previously been shown to permit greater than 88% recovery of intact proglucagon-derived peptides from tissues (10, 45). Assays RlAs for GLI and IRG were carried out using two different antisera, respectively: 1) antiserum K4023 (Novo Alle, Bagsvaerd, Denmark), which cross-reacts with the midsequence of glucagon and recognizes glucagon and both N- and C-terminally extended forms of glucagon (glicentin and oxyntomodulin); and 2) antiserum 04A (Dr. R. H. Unger, Dallas, TX), which cross-reacts with the free C-terminal end of glucagon and recognizes glucagon and N-, but not C-, terminally extended forms of glucagon (i.e. 9K glucagon or glucagon) (19-29). The range of the assays for GLI and IRG was 4-400 pg/tube. The ratio of GLI to IRG was used as an index of glucagon plus glicentin plus oxyntomodulin levels compared to those of glucagon. A ratio of 1 .O indicates the presence of only glucagon, whereas a ratio greater than 1 indicates increased amounts of glicentin and oxyntomodulin. Antiserum b5 (a gift from Dr. S. Mojsov, New York, NY) was used to determine the levels of immunoreactive GLP-1 in tissues; it was not found suitable for use with plasma samples. This antiserum crossreacts with the C-terminal end of GLP-1 and recognizes GLP1 -(l-37) and GLP-l-(7-37), but not C-terminally modified forms of GLP-1 (i.e. GLP-1 -(7-36)NHz or the major proglucagon fragment, which contains GLP-1 and GLPi2] (46i The ranae of the assav for GLP-1 was 1 O-500 pa/tube. IRI levels were determined by RIA using Wright’s ant;erum, as previously described (22). Protein assays were carried out using the method of Lowry et al. (47) and blood glucose levels were determined using the glucose oxidase method (Glucose Autoanalyzer 2, Beckman Instruments, Fullerton, CA). HPLC HPLC separation of immunoreactive peptides was performed using a Waters Associates Liquid Chromatography System with a Cl8 KBondpak column. The solvent system used for analysis of GLI and IRG was a 45-min linear qradient of 25625% (vol/vol) solvent B [solvent A = 1% (vol/vol) TFA buffered to DH 2.5 with diethvlamine; solvent B = 80% (vol/ vol) acetonitrile], as described previously (15). The flow rate was 1.5 ml/mm, and 0.3-min fractions were collected. HPLC of GLP-l-containing peptides was carried out using a modification of the method of Mojsov et al. (3). In brief, peptides were separated on a Cl 8 PBondapak column using a 30-min linear gradient of 45-68% (vol/vol) solvent B [solvent A = 0.085% (vol/vol) phosphoric acid and 0.3% (vol/vol) triethylamine, pH 7.0; solvent B = 40% (vol/vol) solvent A in 60% acetonitrile]. The flow rate was 1.5 ml/min, and 1-min fractions

Vol6No.12

were collected. before RIA. RNA Isolation

All HPLC

fractions

were

stored

at -20

C

and Analysis

For the preparation of total cellular RNA, tissues were homogenized in guanidine isothiocyanate, using a Polytron (Brinkmann Instruments, Westbury, NY), and RNA was extracted using the phenol-acid precipitation procedure (48). For Northern blot analysis, 10 pg total cellular RNA were size-fractionated on a 1.2% (wt/vol) agarose formaldehyde gel, and the gel was stained with ethidium bromide before transfer to ensure the integrity and equal loading of the RNA. The RNA was fixed to a nylon membrane with UV light, after which prehybridization and hybridization were carried out in 1 x Denhardt’s, 4 X SSC (1 x SSC = 0.15 M NaCI, 0.015 M Na citrate), 200 pg/ml salmon sperm DNA, 40% (vol/vol) deionized formamide, and 5% (wt/vol) dextran sulfate in 0.014 M Tris, pH 7.4. Hybridization was performed in the same solution with 1 X lo6 cpm/ml 3ZP-labeled cDNA probe for 24 h at 42 C. Complementary DNA probes were labeled by the random priming technique, and final washing conditions were 0.1 x SSC-0.1% (wt/vol) sodium dodecyl sulfate at 60 C. The cDNA probes for glucagon, insulin, somatostatin, PYY, and tubulin have been previously described (6, 49). Autoradiography was performed using Kodak X-Omat film (Eastman Kodak, Rochester, NY) at -70 C. Autoradiograms were quantitated by scanning with a laser densitometer. All RNA samples (four or five per group) were characterized for each tissue (pancreas and GLUTag or v-jun fibrosarcoma tumor), and each time point (2-8 weeks) was characterized by Northern blot analysis to ensure that the results/changes depicted were representative. lmmunocytochemistry

and Electron

Microscopy

For light microscopy, freshly collected tissues were fixed in 10% (vol/vol) buffered formalin, dehydrated, and embedded in paraffin. Sections 4-6 pm thick were cut and stained with hematoxylin and eosin for tissue identification. For immunocytochemistry, the avidin-biotin-peroxidase complex technique was applied, as previously described (50). The dilutions of primary antisera used were 1:400 for neuron-specific enolase, 1:lOOO for insulin (Dakopatts, Glostrup, Denmark), 1:lOOO for glucagon (Diagnostic Products, Los Angeles, CA), and 1 :lOOO for GLP-1 (49); incubations in primary antisera were performed for 24 h at 4 C. The specificity of immunostaining was verified by replacing primary antisera with nonimmune rabbit sera and absorbing the primary antisera with synthetic insulin or GLP1, as appropriate. Positivity was accepted only in sections in which no immunopositivity was detected with nonimmune sera and where staining was completely abolished by the addition of synthetic peptide (10 fig/ml) to primary antisera. For electron microscopy, tissue fragments were fixed in 25% glutaraldehyde, postfixed in osmium tetroxide, and embedded in an Epon-Araldite mixture. Ultrathin sections were stained with uranyl acetate and visualized with a Philips 410-LS electron microscope (Philips Electronics, Mahway, NJ). Morphometry Islet areas were measured by morphometric analysis, using a Bioquant System IV computer (R&M Biometrics, Inc., Nashville, TN). Measurements were performed by an independent observer blinded to the experimental conditions. Sections stained for neuron-specific enolase were measured as follows. The total pancreatic area was measured, and the total area of all islets in each section was expressed as a percentage of the total pancreatic area. The area of islets, measured in square microns, was also expressed as the mean f SEM for all islets in each experimental group.

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lnhlbltlon

Data

of Pancreatic

Glucagon

Gene Expression

Analysis

All data are expressed as the mean i SEM. Statistical differences between samples of three or more were assessed by analysis of variance, using a StatIstical Analysis System package for personal computers (SAS Institute, Inc., Cary, NC). Significance was assumed at the P 5 0.05 level.

Acknowledgments

Received August 18, 1992. Revision received September 28, 1992 Accepted September 28, 1992. Address requests for reprints to: Dr. Daniel J. Drucker, Toronto General Hospital, 200 Elizabeth Street CCRW3-838, Toronto, Ontario, Canada M5G 2C4. This work was supported In part by operating grants from the Medical Research Council of Canada and the Canadian Diabetes Assoclatlon. ’ Career Scientist of the Ontario Ministry of Health t Diabetes Canada Scholar.

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14.

15.

16.

17.

18.

19.

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