Proc. Natl. Acad. Sci. USA Vol. 89, pp. 10036-10040, November 1992 Medical Sciences

Insulitis in transgenic mice expressing tumor necrosis factor 13 (lymphotoxin) in the pancreas DoMINIc E. PICARELLA*, ALEXANDER KRATZt, CHANG-BEN Lit, NANCY H. RUDDLEt, AND RICHARD A. FLAVELL*t *Section of Immunobiology, tHoward Hughes Medical Institute, and tDepartment of Epidemiology and Public Health, Yale University School of Medicine, New Haven, CT 06510

Communicated by Joan Steitz, June 23, 1992 (received for review April 29, 1992)

Tumor necrosis factor 13 (TNF-P) (lymphoABSTRACT toxin) may play an important role in the immune response and pathologic inflammatory diseases. Insulitis is an important early step in the development of insulin-dependent diabetes mellitus. To understand better the role of TNF-fl in the regulation of infamation and type 1 diabetes, we produced transgenic mice in which the murine TNF-fi gene was regulated by the rat insulin H promoter. The transgene was expressed in the pancreas, kidney, and skin of transgenic mice. The expression of TNF-f8 in the pancreas of transgenic mice resulted in a leukocytic inflammatory infiltrate consisting primarily of B220+ IgM+ B cells and CD4+ and CD8+ T cells. The insulitis is reminiscent of the early stages of diabetes, though the mice did not progress to diabetes.

In the present study, we addressed two issues with respect to the biological activity of TNF-,B in vivo. We demonstrate that local production of TNF-,f is sufficient to initiate and maintain an islet-specific inflammatory response consisting primarily of B and T lymphocytes but that this is not sufficient to cause IDDM.

MATERIALS AND METHODS

The relationship between inflammation and autoimmunity is of considerable interest. Our goal was to determine whether the inappropriate expression of inflammatory cytokines within the islets of Langerhans would be sufficient to induce an inflammatory infiltrate and, if so, whether that inflammatory infiltrate was a sufficient condition to lead to insulindependent diabetes mellitus (IDDM). Such a model could prove useful in defining basic mechanisms of autoimmune pathogenesis and provide insight into prophylaxis and treatment. Although little is known about the contribution of tumor necrosis factor 1 (TNF-(3) to the inflammatory response in vivo, several in vitro studies implicate TNF-13 in processes that may contribute to inflammation and pathogenesis in general and in IDDM in particular. TNF-,B activates human endothelial cells in vitro to express a number of leukocyte adhesion molecules (1), and major histocompatibility complex class I and class II molecules (2) and also synergizes with interferon 'y (IFN-y) in inducing HLA class II gene expression in human islets (3). As such, TNF-,8 might be expected to enhance lymphocyte traffic and antigen presentation to T cells in those tissues in which the cytokine is present. IDDM is an autoimmune inflammatory disease that results in destruction of 3 cells in the pancreatic islets of Langerhans. The trigger for this inflammation, presumably due to reactivity with an as yet unidentified autoantigen, has not been defined. Susceptibility to diabetes in the human population, and in rodent models of the disease, is linked to the major histocompatibility complex. Recently, it was shown that diabetic individuals heterozygous for DR3.4 have a higher frequency of a particular TNF-,( polymorphism (a 10.5-kilobase Nco I restriction fragment length polymorphism) than nondiabetic DR3.4 individuals (4). This implicates additional genes that are linked to the major histocompatibility complex on chromosome 6, such as TNF, in diabetes.

Production of Transgenic Mice. A 600-base-pair HindIIIXho I rat insulin II promoter (RIP) fragment was blunted with Klenow fragment of DNA polymerase I and subcloned into the Sma I site in pBluescript SK (Stratagene) (pSK-RIP). The murine TNF-,f gene from EMBL 7 clone 13 subcloned into pUC-12 (5) was the generous gift of V. Jongeneel (Ludwig Institute for Cancer Research, Epilanges, Switzerland) and S. Nedospasov (Engelhardt Institute for Molecular Biology, Moscow). The TNF-,B gene was isolated as a 2.0-kilobase BamHI fragment and subcloned into the BamHI site in pSK-RIP (6). The RIP-TNF-(3 plasmid was extracted from bacterial suspensions and purified through two sequential CsCl gradients (7). The purified DNA was digested with Sst II, EcoRV, and Pvu I to generate the RIP-TNF-P3 fragment, separated by electrophoresis through a 1% agarose gel (SeaKem GTG; FMC), and isolated by electroelution into dialysis tubing (7). The DNA fragment was purified through Elutip-D columns by following the manufacturer's instructions (Schleicher & Schuell) and dialyzed on filters against injection buffer (0.5 mM Tris.HCl/25 mM EDTA, pH 7.5). Transgenic mice were made in (CBA x C57BL/6)F2 animals as described (8), and positive founder animals were identified by Southern blot analysis of tail DNA by using a 32P-labeled TNF-(3 cDNA probe (pM7A) (9). Analysis by Northern Blot Hybridization of Total RNA Extracted from Tissue. Total RNAs from mouse tissues were extracted by guanidinium thiocyanate and purified by ultracentrifugation (7). Total RNAs were electrophoresed through 1% agarose/formaldehyde gels, transferred to Nytran membranes (Schleicher & Schuell), and hybridized to 32P-labeled mouse TNF-P cDNA probe prepared by random-primer labeling (Boehringer Mannheim). The 22-mer oligonucleotide probe, homologous to the 3' end of the rat insulin promoter, was labeled by phosphorylation with T4 kinase and hybridized to Nytran membranes by following the manufacturer's instructions. TNF-13 Activity in Supernatants from in Vitro Islet Cultures. Islets were isolated by collagenase digestion of whole pancreata as described (10) and cultured in 0.5 ml of Bruffs medium containing 20 mM glucose and 10% (vol/vol) fetal calf serum in 4-well tissue culture multidishes (Nunc) at 370C

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Abbreviations: TNF-,3, tumor necrosis factor P; IDDM, insulindependent diabetes mellitus; IFN-'y, interferon y; FACS, fluorescence-activated cell sorter. 10036

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FIG. 1. TNF-(3 mRNA is transcribed from the RIP-TNF-,3 transgene. Whole RNA, extracted from tissues of transgene-positive and negative littermates, was hybridized to a murine TNF-,B cDNA probe (A) or an oligonucleotide probe from the 3' untranslated region of the rat insulin II promoter (B). (A and B) Lanes: 1, PD-31 a subclone of the Abelson murine leukemia virus-transformed pre-B cell line PD (obtained from J. Hesse, National Institutes of Health); 2 and 10, kidney; 3 and 9, pancreas; 4 and 8, skin; 5, lung; 6, liver; 7, heart; 2-7, RIP-TNF-P; 8-10, negative control. Locations of 28S and 18S rRNAs are indicated.

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in 5% C02/95% air. After 48 hr, supernatants were analyzed for TNF-3 cytotoxic activity (11). Preparation ofTissue Samples for Histology. Tissue samples were placed in Tissue-Tek III unicassettes (Miles), fixed for a minimum of 18 hr in phosphate-buffered 10%6 (vol/vol) formalin (Sigma), paraffin-embedded, and stained with hematoxylin and eosin. Immunocytochemistry to Detect Insulin and Glucagon. Pancreatic tissue sections were deparaffinized in xylene and rehydrated with a graded alcohol series. Endogenous peroxidase activity was blocked with 0.6% H202 in methanol. Glucagon was detected with a polyclonal antibody to synthetic human glucagon (BioGenex, San Ramon, CA), using biotin-streptavidin-peroxidase and diaminobenzidine (Polysciences). Insulin was detected with a monoclonal antibody against human insulin (BioGenex) using biotin-streptavidinalkaline phosphatase. The slides were counterstained with 0.03% methyl green (Sigma), and mounted in an aqueous medium. Immunocytochemistry on Frozen Sections. Tissue was minced in periodate/lysine/paraformaldehyde fixative (12), kept on ice for a minimum of 4 hr, processed through three consecutive sucrose solutions [10%, 20%6, and 30%o (wt/vol)], and snap-frozen in Tissue-Tek OCT compound (Miles) by submersion into 2-methylbutane (Aldrich). Tissue sections were cut, transferred onto silane-treated glass slides, and stained with various antibody reagents as described (12). Sections were blocked with Triton X-100 and bovine serum albumin or normal goat serum prior to reaction with biotinylated primary antibodies. The slides were then washed three times in 0.1 M Tris buffer (pH 7.5) and the tissue sections were incubated with a prediluted streptavidin-alkaline phosphatase solution (KPL; Gaithersburg, MD) for 1 hr. The sections were washed and developed using HistoMark-Red staining system (Kirkegaard and Perry Laboratories) in accordance with the manufacturer's instructions. The slides were counterstained in Meyer's hematoxylin and then mounted with Permount histological mounting medium (Fisher). In Situ Hybridization. A modified protocol of Mueller et al. (13) was followed. The prehybridization and hybridization solutions were 2x standard saline citrate (SSC)/45% (vol/ vol) formamide/0.5 x Denhardt's solution/10%o (wt/vol) dextran sulfate/10 mM dithiothreitol/tRNA (0.1 mg/ml). Probes were prepared from a pBS vector (Stratagene) carrying a 900-base-pair TNF-,B cDNA fragment using T3 (sense) or T7

(antisense) polymerase (Boehringer Mannheim). Unhybridized RNA was digested with RNase A (Boehringer Mannheim; 20 ,ug/ml). The slides were washed in 50%6 formamide/2x SSC/0.2% 2-mercaptoethanol and in 0.1x SSC/

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0.2% 2-mercaptoethanol. The tissue was dehydrated and dipped in NTB2 nuclear track emulsion (International Biotechnologies), developed after a 2-week exposure, and stained with hematoxylin/eosin. Determination of Insulin and Glucose Levels. Glucose levels were determined with a One-Touch II meter (Lifescan, Milpitas, CA); insulin levels were determined by RIA using rat insulin standards (Eli Lilly). For insulin determinations, mice were fasted for 6 hr, injected i.p. with 100 mg of glucose plus 50 mg of Travasol, and sacrificed 20 min after stimulation. Blood was collected by cardiac puncture, and the pancreas was extracted in acid ethanol. Insulin levels in the pancreas are expressed per gram of wet weight. For glucose tolerance, fasting glucose levels were determined at 6 hr after removal of food, mice were injected with 100 mg of glucose, and glucose levels were again measured 2 hr after stimulation. Fluorescence-Activated Cell Sorter (FACS) Analysis of Mononuclear Ceils. Islets were isolated, cultured overnight in 0.5 ml of Bruffs medium with 10% fetal calf serum at 37°C in 5% C02/95% air, and then disrupted by trituration through a 27-gauge needle to form a single-cell suspension. Fc receptor sites were blocked with 1% purified mouse immunoglobulin and the cells were incubated on ice for 30-60 min with 10080

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FIG. 2. Production of biologically active TNF-,3 by islets from transgene-positive (solid lines) and transgene-negative (dashed lines) littermates. One hundred islets per well were cultured for 48 hr in 0.5 ml of culture medium containing 20 mM glucose. Cytotoxic activity was evaluated by measurement of viability of WEHI-164 cells in a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Supernatant from the F1-28 cell line was used as a positive control.

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biotinylated, fluorescein isothiocyanate, or RED-613 directconjugated primary antibody. Biotinylated antibodies were developed by incubation for 30 min on ice with an avidinphycoerythrin conjugate. The cells were analyzed on a FACStar Plus (Becton Dickinson) for three-color fluorescence. RESULTS Generation of Transgenic Mice. We constructed several lines of transgenic mice in which the expression of the murine TNF-f3 gene was regulated by the rat insulin II promoter. Of 70 progeny screened by Southern blot analysis of tail DNA, 12 were positive for the RIP-TNF-f3 transgene with copy numbers ranging from 2 to 100 copies per genome (data not

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shown). Founders 5 (410 copies), 16 (-100 copies), and 42 (-100 copies) were bred with C57BL/6 mice. The RIP-TNF-P Transgene Is Transcribed and mRNA Is Translated into Biologically Active TNF-f3 in the Pancreas. To determine whether the RIP-TNF-P transgene was appropriately expressed, RNA was extracted from tissues and analyzed on a Northern blot (Fig. 1) using a combination of probes to distinguish endogenous and transgene TNF-f3. The TNF-3 cDNA probe hybridizes to transcripts encoded by both the transgene and the endogenous gene, and the oligonucleotide probe from the 3' end of the rat insulin DNA segment hybridizes only to transgene-encoded RNA. TNF-/3 RNA was detected in pancreas, kidney, and skin of transgene-positive mice, using both probes (Fig. 1), but not in

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FIG. 3. Mononuclear cell infiltration and RIP-TNF-(3 transgene expression in islets of transgenic mice. Paraffin-embedded tissue sections stained with hematoxylin/eosin reveal the presence of infiltrating leukocytes in the pancreas of RIP-TNF-.3 transgenic mice: normal islets from C57BL/6 F1 transgene-negative mice (A), predominant perivascular accumulation ofmononuclear cells with mild insulitis (B), and massive insulitis resulting in disruption of normal islet architecture (C). (D-F) In situ hybridization offrozen sections. (D) Antisense RNA probe, transgene-negative littermate. (E) Antisense RNA probe, RIP-TNF-,3 transgenic mouse. (F) Sense RNA probe, sequential section of the same transgenic islet.

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transgene-negative littermates. The transgene expression in the kidney was not unprecedented, as previous work with this promoter had demonstrated such expression in the proximal tubules (14). The expression of the TNF-(3 transgene in the kidney was associated with an extensive mononuclear infiltration in the region of the proximal tubules (data not shown). Expression of the rat insulin II promoter in the skin had not been reported, to our knowledge. In this case the TNF-/3 expression in the skin correlated with a ruffled-fur phenotype (data not shown). TNF-j3 was produced and secreted from P cells, since TNF-,/ activity was detected in supernatants from islets of three RIP-TNF-f3 transgenic lines but not in negative littermate controls (Fig. 2). Production of TNF-(3 in the Pancreas Results in Insulitis. TNF-P expression in the pancreas was sufficient to induce an inflammatory response, since periinsulitis and insulitis were apparent in hematoxylin/eosin-stained sections ofislets from transgenic progeny but not in transgene-negative littermates of C57BL/6 matings (Fig. 3). Infiltration ofmononuclear cells was restricted to islets and was not observed in surrounding acinar tissue. The histological pattern ofindividual transgenic mice was heterogeneous. Many islets appeared uninfiltrated; some islets had interstitial accumulation of mononuclear cells around the islet capsule without obvious infiltration (Fig. 3B); and some islets were partially or completely infiltrated by mononuclear cells (Fig. 3C). The percentage of islets without apparent infiltration ranged from 30 to 45% in transgenic mice from 1 month to 5 months of age. The percentage ofislets with periinsulitis and insulitis ranged from 1o in 1-month-old transgenic mice to 35-40o in 5-month-old transgenic mice. Most TNF-p in the Pancreas of Transgenic Mice Is Produced by the Islets and Not by Infiltrating Mononuclear Cells. In situ hybridization shows that the RIP-TNF-f3 transgene is expressed in cells in the islets of Langerhans and not by infiltrating cells (Fig. 3). Furthermore, TNF-(3 is produced in

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islets with and without infiltration (data not shown), suggesting that the lack of infiltration of some islets is not due to the absence of transgene expression. Insulitis Does Not Prevent Insulin Production in the Islets of RIP-TNF-fi Transgenlc Mice. To determine whether the infiltrating leukocytes affected (8-cell physiology, paraffinembedded pancreatic sections were analyzed by immunocytochemistry for insulin and glucagon. In normal islets, the glucagon-producing a cells (stained brown) are aligned along the perimeter of the islet capsule, and the insulin-producing P cells (stained red) are predominantly in the center portion of the islet (Fig. 4A). Although the organization of a and ( cells appears to be abnormal in islets that have leukocytic involvement, the level of reactivity with the insulin-specific antibody in transgene-positive islets (Fig. 4B) suggests that a significant level of insulin production remains in some of the islets. By 8 months to 1 year of age, these transgenic mice continue to produce levels of insulin, in response to stimulation, that are not diminished relative to control mice (Table 1). The Inlating Leukotc Population Consists of CD4+ and CD8+ T Cells and B220+, IgM+ B Cells. Infiltrating cells were examined by immunocytochemistry of frozen pancreatic sections and by FACS analysis of leukocytes recovered from infiltrated islets in vitro. Both CD4+ (Fig. 4C) and CD8+ (Fig. 4D) cells were present in the infiltrates, as were significant numbers of B220+ cells (Fig. 4E). FACS analysis suggested that 90-95% of the B220+ cells were also IgM+ (data not shown). None of the antibodies reacted with islet sections from the pancreas of negative littermates (data not shown). This result was confirmed by FACS analysis of mononuclear cells isolated after 24 hr of in vitro culture of individual islets from 4-monthold RIP-TNF-,B transgenic mice (data not shown).

DISCUSSION Local expression of TNF-(3 in the islets and kidney is sufficient to cause a tissue-specific inflammatory response. The RIP-

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FIG. 4. Immunocytochemical staining of pancreatic tissue sections. (A and B) Islet-cell staining for the presence of insulin and glucagon in paraffin-embedded sections. (C-E) Mononuclear cell staining for CD4, CD8, and B220 in frozen sections. (A) Typical cellular organization with production of insulin (stained red) and glucagon (stained brown) in a normal islet from a transgene-negative littermate. (B) Disrupted cellular architecture in islets from RIP-TNF-, transgene-positive mice. The infiltrating leukocytes stain green. (C) Biotinylated monoclonal antibody specific for CD4 (cloie YTS191.1.2; Immunoselect, GIBCO/BRL). (D) CD8 (clone 53-6.7; Immunoselect, GIBCO/BRL). (E) B220 (RM2615; Caltag, South San Francisco, CA).

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Table 1. RIP-TNF-,8 transgenic mice produce normal levels of insulin in response to stimulation with glucose and amino acids Glucose tolerance, mg/dl Insulin, ,ug/g 2 hr 0 hr Serum Pancreas RIP-TNF-,B + 369.0 ± 90.9 161.1 ± 6.0 143.0 ± 168.0 425.1 ± 90.0 242.7 ± 152.9 122.0 ± 17.3 84.5 ± 55.6 294.7 ± 60.0 Mice used were C57BL/6 F1 (8-12 months), three transgene-positive, and four negative controls.

TNF-P mice exhibit perivascular accumulation of mononuclear cells that eventually progresses to insulitis in the case of many, but not all, islets. Despite the extent of insulitis in these mice, fasting serum glucose levels remained within normal limits. Analysis of insulin levels in the pancreas and serum of the transgenic mice suggests that insulin production is not diminished and may be slightly elevated above the levels observed in littermate controls, suggesting that production of TNF-,8 in the pancreas does not induce diabetes. In contrast to our results, transgenic mice that produce murine IFN-y controlled by the human insulin promoter developed a generalized pancreatitis that progressed to diabetes (15). The predominantly lymphocytic infiltrate was not restricted to the islets but was demonstrated to be specific for self pancreatic antigens. The difference in the outcome between the IFN-y and TNF-P transgenic mice could be due to the activation of antigen-specific lymphocytes in the former case by the induction of costimulatory activity (16) and the failure of this to occur in the TNF-13 transgenic mice. Alternatively, antigen-specific lymphocytes could be present in both transgenic models and the diabetes produced in the IFN-y transgenic mice could be caused by direct effects of IFN-,y on P cells, coupled with lymphocytic infiltration. A third possibility is that antigen-specific cells are present in both cases, but the effector function of those cells differs between the two systems. Indeed, recent data show that culture of CD4+ T cells in the presence of IFN-y results in the expansion of inflammatory type Thl T helper cells (17). Finally, we cannot rule out that differences in the relative levels of the two cytokines contribute to the different outcomes. The gene products responsible for insulitis and the relationship between insulitis and diabetes in rodent and human IDDM remain poorly defined. Although three genetic loci (idd-3, idd4, and idd-S) that appear to influence the development of insulitis and diabetes in mice have been mapped to chromosomes 3, 11, and 1, respectively (18-20), evidence that these loci by themselves may not be sufficient to induce diabetes comes from analysis of the NOR/Lt strain (21). NOR/Lt mice are homozygous for the nonobese diabetes (NOD) allele of the currently defined diabetogenic loci (idd-1 to idd-6) but do not develop insulitis or diabetes, suggesting that additional gene products are necessary for insulitis (21). By contrast, in the model described here, we can unambiguously implicate the product of a single well-defined gene (TNF-P) in periinsulitis and insulitis. Thus TNF-3 may be substituting for idd gene products in the development of insulitis. Although other mouse strains in which insulitis has been uncoupled from diabetes, such as NOD/WEHI, have been reported, a low frequency of these mice will spontaneously develop diabetes, and this frequency can be dramatically increased by various treatments such as cyclophosphamide (22, 23). In our model, we have completely uncoupled insulitis from diabetes. These mice may prove valuable to elucidate additional factors to continue progression from inflammation to autoimmunity. We thank Cindy Hughes and Debbie Butkus for producing the transgenic mice used in these experiments, Robert Sherwin for helpful discussions and review of this manuscript, and Jennifer

Morgen, Alesia Barrett, Andrea Belous, and Aida Groszmann for performing the insulin analysis. This work is supported by National Institutes of Health Grants RO1 CA 47878 (N.H.R.) and P01 DK 43078 (R.A.F.). R.A.F. is an Investigator of the Howard Hughes Medical Institute. D.E.P. was supported by training Grants Al 07019 and Al 07174. 1. Pober, J. S., Gimbrone, M. A., Lapierre, L. A., Mendrick, D. L., Fiers, W., Rothlein, R. & Springer, T. A. (1986) J. Immunol. 137, 1893-18%. 2. Lapierre, L. A., Fiers, W. & Pober, J. S. (1988) J. Exp. Med. 167, 794-804. 3. Pujol-Borrell, R., Todd, I., Doshi, M., Bottazzo, G. F., Sutton, R., Gray, D., Adolf, G. R. & Feldmann, M. (1987) Nature (London) 326, 304-306. 4. Pociot, F., Molvig, J., Wogensen, L., Worsaae, H., Dalboge, H., Baek, L. & Nerup, J. (1991) Scand. J. Immunol. 32, 297-311. 5. Semon, D., Kawashima, E., Jongeneel, C. V., Shakov, A. N. & Nedospasov, S. A. (1987) Nucleic Acids Res. 15, 9083-9084. 6. Gray, P. W., Chen, E., Li, C.-b., Tang, W.-L. & Ruddle, N. (1987) Nucleic Acids Res. 15, 3937. 7. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold Spring Harbor, NY). 8. Hogan, B., Costantini, F. & Lacy, E. (1986) Manipulating the Mouse Embryo: A Laboratory Manual (Cold Spring Harbor

Lab., Cold Spring Harbor, NY). 9. Li, C.-b., Gray, P. W., Lin, P.-F., McGrath, K. M., Ruddle, F. H. & Ruddle, N. H. (1987) J. Immunol. 138, 4496-4501. 10. McInerney, M. F., Rath, S. & Janeway, C. A. (1991) Diabetes 40, 648-651. 11. Ruddle, N. H., Bergman, C., McGrath, K. M., Lingenheld, E. G., Grunnet, M. L., Padula, S. J. & Clark, R. B. (1990) J. Exp. Med. 172, 1193-1200. 12. DeCamilli, P., Cameron, R. & Greengard, P. (1983) J. Cell Biol. 96, 1337-1354. 13. Mueller, C., Gershenfeld, H., Lobe, C. G., Okada, C. Y., Bleakley, R. C. & Weissman, I. L. (1988) J. Exp. Med. 167, 1124-1136. 14. Lo, D., Burkly, L. C., Widera, G., Cowing, C., Flavell, R. A., Palmiter, R. D. & Brinster, R. L. (1988) Cell 53, 159-168. 15. Sarvetnick, N., Liggitt, D., Pitts, S. L., Hansen, S. E. & Stewart, T. A. (1988) Cell 52, 773-782. 16. Sarvetnick, N., Shizuru, J., Liggitt, D., Martin, L., McIntyre, B., Gregory, A., Parslow, T. & Stewart, T. (1990) Nature (London) 346, 844-847. 17. Swain, S. L., Bradley, L. M., Croft, M., Tonkonogy, S., Atkins, G., Weinberg, A. D., Duncan, D. D., Hedrick, S. M., Dutton, R. W. & Huston, G. (1991) Immunol. Rev. 123, 115144. 18. Todd, J. A., Aitman, T. J., Cornall, R. J., Ghosh, S., Hall, J. R. S., Hearne, C. M., Knight, A. M., Love, J. M., McAleer, M. A., Prins, J.-B., Rodrigues, N., Lathrop, M., Pressey, A., DeLarato, N. H., Peterson, L. B. & Wicker, L. S. (1991) Nature (London) 351, 542-547. 19. Garchon, H.-J., Bedossa, P., Eloy, L. & Bach, J.-F. (1991) Nature (London) 353, 260-262. 20. Cornall, R. J., Prins, J.-B., Todd, J. A., Pressey, A., DeLarato, N. H., Wicker, L. S. & Peterson, L. B. (1991) Nature (London) 353, 262-265. 21. Prochazka, M., Serreze, D. V., Frankel, W. N. & Leiter, E. H. (1992) Diabetes 41, 98-106. 22. Charlton, B., BaceU, A., Slattery, R. M. & Mandel, T. E. (1989) Diabetes 38, 441-447. 23. Baxter, A. G., Adams, M. A. & Mandel, T. E. (1989) Diabetes 38, 1296-1300.