Proc. Natl. Acad. Sci. USAVol. 87, pp. 8642-8646, November 1990Genetics

Molecular mapping of the mouse db mutation(obesity/genetic mapping/diabetes)

N. BAHARY*, R. L. LEIBELt, L. JOSEPH*t, AND J. M. FRIEDMAN*t§tHoward Hughes Medical Institute, *Laboratory of Molecular Cell Biology, tLaboratory of Human Behavior and Metabolism, Rockefeller University,New York, NY 10021

Communicated by Alexander G. Bearn, May 16, 1990

ABSTRACT Diabetes (db) is an autosomal recessive mu-tation located in the midportion of mouse chromosome 4 thatresults in profound obesity with hyperphagia, increased met-abolic efficiency, and insulin resistance. To clone this gene andgenerate a molecular map of the region around this mutation,two genetic crosses were established: an intraspecific backcrossbetween C57BL/6J db/db females and C57BL/6J db/db xDBA/2J +/+ F1 (B6D2 db/+ FI) male mice and an inter-specific intercross between B6D2 db/+ F1 males andC57BL/6J db/db x Mus spretus F1 (B6spretus db/+ F1)females. The progeny of both crosses were characterized forgenotype at the db locus to map a series of restriction fragmentlength polymorphisms relative to the db locus. Measurementsof body weight, body length, and plasma concentrations ofglucose and insulin in the animals allowed the assignment ofgenotype (db/db vs. db/+ or +/+). A total of 132 progeny ofthe intraspecific cross and 48 db/db progeny of the interspecificcross were typed for individual restriction fragment lengthpolymorphisms to generate a gene order of: centromere-brown(Mt4)-PAMm3_--Ifa (Inta)-Cjun-db-D4Rpl-Glutl-Mtv-13-Lck. Several of the genes that are linked to db [Cjun, glucosetransporter (Glut)) and Lck] map to human chromosome 1p,suggesting that db may be part of a syntenic group betweenhuman ip and the distal portion of mouse chromosome 4. Inaddition, phenotyping of the progeny of these crosses revealeda wide range in plasma concentrations of glucose and insulinamong the obese progeny, with some animals developing overtdiabetes and others remaining euglycemic. Distributions ofage-controlled plasma [glucose] and [insulin] among the in-traspeciffic-cross obese progeny were not bimodal, suggesting arole for polygenic differences between the progenitor strains(C57BL/6J and DBA/2J) in the development of overt diabe-tes.

Diabetes (db) is an autosomal recessive mutation located onmouse chromosome 4 that arose spontaneously in theC57BL/KsJ (BL/Ks) strain (1). At least five separate spon-taneous mutations allelic with db-db2J, dbad, db3A, db4A, anddb pasteur-have been detected since the initial report (2, 3).Animals homozygous for the db mutation are notably obeseas early as 4 weeks of age and often develop glucoseintolerance and diabetes, the severity of which depends uponthe inbred strain on which the mutant allele has been bred(4-8).Numerous attempts have been made to identify the pri-

mary molecular defect in db/db animals. However, becauseof the multiple metabolic and physiologic abnormalities seenin db/db animals, the separation of primary gene effects fromsecondary metabolic alterations has proven difficult. For thisreason we have taken the approach of "reverse genetics" toisolate the db gene product (9). This approach requires noknowledge or assumptions concerning the biological nature

of the defect and provides an approach for cloning genessolely on the basis of chromosomal location (10, 11).The initial phase of the reverse genetic approach involves

mapping of a mutant gene relative to chromosomal markers,usually restriction fragment length polymorphisms (RFLPs).Toward this end, both an intraspecific and an interspecificbackcross segregating db were established to enable thegenetic mapping of the db mutation relative to a series ofRFLPs on mouse chromosome 4. To unambiguously assigngenotype at the db locus and also to examine the effects ofgenetic (strain) background on the development of diabetes,plasma concentrations ofglucose and insulin were assayed inthe backcross and intercross progeny at the time of sacrifice.Variance of these parameters among these progeny can alsobe used to assess the effect of the differing genetic back-grounds of the progenitor strains on the severity of glucoseintolerance resulting from homozygosity for db.

MATERIALS AND METHODSMouse Crosses. The intraspecific and interspecific crosses

segregating the db mutation were performed as shown in Fig.1 a and b. Agouti females carrying the transplanted ovariesof C57BL/6J db/db mice (generously provided by DouglasColeman ofThe Jackson Laboratory, who has maintained dbcongenic stocks on C57BL/6J for seven generations) weremated to both DBA/2J +/+ and Mus spretus males (TheJackson Laboratory) (4). F1 black (indicating their originfrom transplanted ovarian tissue) male progeny of theC57BL/6J x DBA/2J cross were backcrossed to the femaletransplants. In addition, F1 males of a C57BL/6J x M.spretus cross were subsequently mated to F1 female offspringof the C57BL/6J x DBA/2J progenitors. All animals werefed an ad libitum diet ofPico laboratory mouse breeding chow5058 (Purina), containing 9% (wt/wt) fat, and water.Progeny Analysis. Animals were sacrificed by CO2 asphyx-

iation between 87 and 186 days of age. After a 15-hr fast eachmouse had -1.0 ml of whole blood withdrawn by cardiacpuncture into an Eppendorf tube containing 50 ml of 82 ttMEDTA as an anticoagulant. The plasma was decanted andfrozen at -80'C for subsequent assay of [insulin] (12) and[glucose] (13). All progeny were scored for sex, weight, andlength (nose to anus).DNA Preparation and Analysis. High molecular weight

DNA was prepared from kidney, spleen, or liver (14) andSouthern blotted to GeneScreenPlus (NEN Research Prod-ucts, Boston) by following the manufacturer's protocol.

Probes. The following probes for detecting the loci indi-cated were used in these experiments. Lck (lymphocyte-specific protein-tyrosine kinase, Lck) was the gift of R.Perlmutter (15), Inta (interferon a, Ifa) was from P. Pitha-Rowe (16), PAMm32 [variable number of tandem repeats(VNTR) probe] was from A. Jeffreys (17), MMTV (Mtv-13)

Abbreviations: RFLP, restriction fragment length polymorphism;BMI, body mass index.§To whom reprint requests should be addressed.

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

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aB -I|B D- D

db db +Id '- +

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S D~ eSlean obese obese leanParentals-J LRecombinantsi Parental

C

V:RFLP haplotypes, and phenotypes, of N2 animals

resulting from: C57BL/6J db/db X B6D2 F1 db'+

HecomoonantsRFLP haplotypes for obese progeny (F2) resulting

from: B6D2 F1 db/+ X B6spretus F1 db/+

FIG. 1. Description of the crosses performed. (a) The intraspecific backcross was accomplished by mating C57BL/6J db/db ovariantransplants to DBA/2J males. The F1 male progeny were then backcrossed to the ovarian transplants. (b) In an analogous fashion, C57BL/6Jdb/db ovarian transplants were mated to M. spretus males. Female F1 offspring were mated to F1 males of the C57BL/6J db/db x DBA/2J+/+ cross, and the obese progeny (F2) were analyzed. In this figure, B, D, and S represent the respective C57BL/6J, DBA/2J, and M. spretusalleles for a particular RFLP. For the C57BL/6J x DBA/2J backcross, recombinants are scored when (i) db/db animals carry the DBA/2J alleleat a particular locus or (ii) db/+ animals are homozygous for the C57BL/6J allele. For the C57BL/6J x M. spretus intercross, recombinantsare scored when obese (db/db) animals carry any combination of the DBA/2J (if one exists) or M. spretus alleles for a particular RFLP. (c)Photograph of db/db and db/+ animals; 5-month-old male littermates derived from the backcross. The animal on the left is db/+ and weighed26.7 g. The animal on the right is db/db and weighed 83.7 g.

from B. Callahan (18), Mt4 (brown locus, b) was from I.Jackson (19), Glutl (glucose transporter 1) was from M.Birnbaum (20), D4Rp1 (D4Rpl) was from R. Elliot (21), andCjun (avian sarcoma virus 17 oncogene homologue, Jun) wasfrom P. Vogt (22).

Inserts for each of theDNA probes were radiolabeled using[a-32P]dCTP and random hexamers (23) and purified over aSephadex G-50 spin column. Hybridizations were performedfollowing the manufacturer's protocol for GeneScreenPlus.

RESULTSPhenotypic Characterization of the N2 Animals. The prog-

eny of the intraspecific backcross between C57BL/6J db/dbfemale ovarian transplants and DBA/2J males (Fig. la) arereferred to as N2 animals. The offspring of the interspecificintercross (Fig. lb) between B6D2 F1 db/+ males x B6spretus F1 db/+ females are referred to as F2 animals.Mapping of loci relative to db requires both the assignment ofgenotype at the db locus and typing the animal for theinheritance of a particular RFLP. Generally, the phenotypicdifferentiation between db/db and db/+ in the N2 cross andbetween db/db and db (or +)/+ in the F2 cross was obviouson physical inspection of the animal (Fig. 1c). Nevertheless,to ensure the unambiguous assignment of db genotype, bodyweight, body mass index [BMI = weight/(nose-to-anuslength)2], plasma [glucose] and plasma [insulin] were mea-sured in each animal. Of these four parameters, only BMIproduced a clear separation of the animals into two distinctgroups with values that generally were either >0.450 or<0.400 g/cm2 (Fig. 2). The lean N2 animals had a mean BMIof 0.320 g/cm2 with a SD of0.037 g/cm2 (n = 76). N2 animalswith a BMI > 0.450 g/cm2 were tentatively designated asdb/db, whereas those N2 animals with a BMI < 0.400 g/cm2were considered to be db/ +. Plasma from the N2 animals wasassayed for [glucose] and [insulin]. The mean (± SD) [glu-cose] of the lean animals was 241 ± 78 mg/dl (n = 76) and themean (± SD) [insulin] was 102.1 ± 65.1 microunits/ml (n =76). All N2 animals with a BMI > 0.450 g/cm2 had either a

plasma [glucose] or [insulin] that was >3 SDs above the meanof their lean littermates, thus confirming their classificationas genotypically db/db. For the 58 db/db N2 animals, themean BMI was 0.582 g/cm2, the mean [glucose] was 488mg/dl, and the mean [insulin] was 717.4 microunits/ml. Thedb genotype of three animals with BMIs between 0.400 and0.450 g/cm2 was assigned on the basis of their plasma[glucose] and [insulin].

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FIG. 2. Analysis of individual N2 animals. Plasma [insulin],[glucose], weight, and BMI (g/cm2) were measured in all N2 animals.The BMI is comparable to the body mass index in man and is usedas an indirect measurement of adiposity. ,uu, Microunits; animal ID,animal identification number.

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Of concern in these experiments was the possibility thatsome ofthe db/db N2 animals might become overtly diabetic,lose weight, and thus be mistakenly classified by BMI(<0.400 g/cm2) as db/+. To identify such examples ofincomplete phenotypic penetrance of body weight (hence,BMI) among the N2 db/db animals, the concentrations ofglucose and insulin among all the low BMI (<0.400 g/cm2) N2animals were examined to identify N2 db/db animals whoseovert diabetes might have resulted in weight loss and misas-signment as db/+. In fact, two "low BMI" N2 animals hadplasma [glucose] >3 SDs above the mean of the low BMIgroup (i.e., plasma [glucose] 2 475 mg/dl). These two lowBMI animals had high concentrations of plasma glucose andrelatively low concentrations of plasma insulin: animal 123,whose [glucose] was 613 mg/dl and whose [insulin] was 119.8microunits/ml, and animal 191, whose [glucose] was 623mg/dl and whose [insulin] was 213.4 microunits/ml. Of note,the haplotype at all loci linked to db was homozygousC57BL/6J in both these animals (see below). Thus, these twoanimals probably represent examples of incomplete pene-trance of the db mutation with regard to body weight. Theseanimals were not included in the mapping panel. Of all theanimals characterized using this protocol, only one animalwas doubly recombinant for db and other linked loci onchromosome 4, supporting the proposal that the N2 animalswere correctly genotyped as db/db or db/+ (see below).As a result of these analyses of the N2 progeny, 74 animals

were designated db/+ (36 females and 38 males) and 58 weredesignated db/db (41 females and 17 males). The number ofdb/db N2 males is clearly less than expected (X2 p < 0.001),even though there was no statistical difference between thenumber of males and females in the interspecific intercrossdescribed below (x2 p > 0.5). The number of N2 males,according to our weekly census, did not change substantiallyafter weaning, suggesting that the less than expected numberof db/db N2 males was likely the result of death before 4weeks of age.RFLP Mapping in the Intraspecific Cross. Initially, five

genomic probes, known to map to the region of chromosome4 near db, were used to type the backcross (N2) progeny forthe inheritance of RFLPs between the C57BL/6J andDBA/2J inbred lines. For probes, Inta, PAMm32, and Lck, apolymorphism was detected with the restriction enzyme BglII. An RFLP for the Mtv-13 locus detected by the retroviralMMTV probe was scored with EcoRI. The brown locus wasmapped using the Mt4 probe for which an RFLP betweenC57BL/6J and DBA/2J animals is detectable using Taq I(19). The sizes of informative DBA/2J alleles for each probeare as follows: Lck [3.5 kilobases (kb), 1.7 kb], MMTV (9.0kb), PAMm32 (6.0 kb), Inta (3.2 kb), and Mt4 (4.9 kb). Theprobes were separately hybridized to restriction digests ofDNA obtained from both the db/db and db/+ progeny of thedb backcross and stored for the presence of the DBA/2Jallele. All N2 animals were scored for Mt4, PAMm32, Inta,and Lck.

Phenotypic Characterization of the F2 Animals. To facilitatemapping of probes for which B x D (C57BL/6J x DBA/2J)RFLPs were not readily identifiable, an interspecific inter-cross between C57BL/6J db/db and M. spretus was devel-oped (24) (Fig. lb). In this cross, DBA/2J +/+ x C57BL/6Jdb/db F1 males were mated to C57BL/6J db/db x M. spretus+/+ F1 females, and only obese F2 animals were scored.Genotype at the db locus was tentatively assigned on thebasis of BMI and of plasma concentrations of glucose andinsulin as described for the N2 cross. For the 85 lean controlanimals, the mean BMI was 0.319 ± 0.049 g/cm2 (+ SD); theplasma [glucose] was 237 + 93 mg/dl; and the plasma[insulin] was 28.2 + 33.4 microunits/ml. Forty-eight obese-appearing progeny from this cross had a BMI > 0.450 g/cm2,and either a plasma [insulin] or [glucose] that was elevated 3

SDs above the mean of lean F2 littermates (as defined byBMI, [glucose], and [insulin]). For these 48 animals, the meanBMI was 0.577 g/cm2, the mean [glucose] was 557 mg/dl, andthe mean [insulin] was 252.9 microunits/ml. These 48 prog-eny were tentatively designated as db/db F2 animals.One obese F2 animal rates special attention in this regard.

Animal 337, which was originally designated db/db, had aBMI of 0.459 g/cm2 and an [insulin] of 199.4 microunits/ml.This concentration of insulin is elevated >3 SDs above thelean F2 mean of 8.4 ± 10.8 microunits/ml. Despite these datathat suggest that this animal is db/db, RFLP analysis ofanimal 337 revealed M. spretus alleles for all the probeslinked to db (described above). Thus, animal 337 is either awild-type animal with an unusually high BMI or is doublyrecombinant between the flanking markers Cjun and D4Rp1.Since the animal carries the M. spretus allele at all loci tested,inclusion of this animal in the mapping panel does not affectthe order of the loci shown in the Fig. 4 but does slightly alterthe relative distances between the loci. Of note, progeny ofinterspecific crosses do occasionally attain weights of >50 g(unpublished observation).The F2 obese mice were typed for the presence of the M.

spretus allele with the same probes used in the intraspecificcross (with the exception of MMTV) as well as three addi-tional probes: Cjun, Glutl, and D4Rp1. Cjun was scoredusing Pst I; D4RpJ and Glut] were followed using Taq I. Inaddition, F2 obese mice were typed for the presence of theDBA/2J alleles for the probes Inta and Lck, by using theRFLPs described earlier for the typing of the N2 animals.By counting recombinants and minimizing double cross-

overs among the progeny of these crosses (Fig. 3), the geneticmap of the region adjacent to the db locus was refined (Fig.4). The gene order of these loci was: centromere-brown(Mt4)-PAMm32-Ifa (Inta)-Cjun-db-D4RpJ-Glutl-Mtv-13-Lck. In Fig. 4, the location ofthe loci mapped among progenyof the intraspecific cross are shown above the line, and thosefor the interspecific cross are shown below the line. Orderwas determined among the probes D4Rp1, Inta, and Glutl.However, definitive order could not be determined betweenprobes MMTV and either Glutl or D4Rp1, because theseprobes were not mapped in the same cross. There was onlyone double crossover across this interval (Fig. 3a), and thisorder is 10,000-fold more likely than any other (allowing theorder of probe MMTV relative to probes Glutl and D4Rp1 toremain undetermined) when the recombination frequenciesbetween all loci are analyzed with the MAPMAKER program(26). Of note, three of the genes that flank db, Cjun, Glutl,and Lck, all map to human chromosome 1p31-36 (27),suggesting that the human homologue ofdb may reside in thisinterval.

Glucose and Insulin Level in N2 db/db Animals. Values ofplasma [glucose] and [insulin] among the db/db N2 animalswere examined for possible effects of genetic background-including sex on the diabetogenicity of the db mutation. Theplasma [glucose] and [insulin] of the db/db N2 animals wereanalyzed in three nonoverlapping age groups (3-4, 4-5, and5-6 months). Analysis of variance of age and sex (28)demonstrated statistically significant effects ofage and sex on[insulin] but not on [glucose] (data not shown). Despite thelower [insulin] in males than females and the decline of[insulin] with age in both sexes, no age or sex effect on[glucose] was seen. Of note in all three age groups, a widerange of plasma [glucose] and [insulin] was evident; animalswith [glucose] varying from nondiabetic to frankly diabeticwere apparent with a relatively continuous distribution.Among the db/db N2 animals the plasma [glucose] rangedbetween 218 and 698 mg/dl and the plasma [insulin] rangedbetween 106.2 and 2160.8 microunits/ml. There was no

evidence of a bimodal distribution of plasma [glucose] or

[insulin] among the db/db backcross progeny (Fig. 2); i.e.,

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Mt4 MlMl*EJOPX Mm3 .0

Intadb * z EME

MMTV * ]*Lck *5E 5***

146 3 4 3 0 2

db/db N2 animals

2/1211.7 (1.2)

7/1285.5 (2.0)

7/1245.6 (2.1)

6/129 12/2324.7(1.9) 9.1 (2.5)

11/1328.3 (2.4)

81/1 328.3 (2.4)

000e00000z000000000005000

155 6 4 1 2 5

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MM3

Inta b

db

MMTV

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0

0

1/48.2.1 (2.1)

3/486.2 (3.4)

4/488.3 (4.0)

0/480.0 (0)

3/486.2 (3.4)

4/488.3 (4.0)

1/482.0 (2.0)4/96

4.1 (2.8)

4/488.3 (3.9)

5/4810.4 (4.4)

4/48 5/48 11/96 8/48 12/968.3 (4.0) 10.4 (4.4) 11.4 (3.4) 16.36(5.4) 12.5 (3.9)

Gt D4RP1 db

Inta flllOOcjun MElfM[]

D4RP1 MHO *GtI

Lck ESOOHE139 4 3 1 1 01

inheritance of themus spretus allele

Cjun Inta

END

MEN

143 2 3 iinheritance of

the DBA/2J allele

FIG. 3. Summary of the results from the intraspecific (a) and interspecific (b) crosses. The approximate map distances in centimorgans [mapdistance in cM = (no. recombinants/total no. of meioses)*100 between probes was calculated using the program "Spretus Madness" (25); valuesin parentheses are SD]. Each of the loci mapped in the crosses is listed on the left of the grid; each column represents a possible configurationof inherited DBA/2J and/or M. spretus alleles. The number underneath each column is the number of animals that have inherited that particularpattern of alleles. Solid boxes represent the C57BL/6J alleles and open boxes represent the DBA/2J or M. spretus allele. Gt, (Glutl); Inta (Ifa).

numerous animals with intermediate levels of [glucose] and[insulin] were notable.

DISCUSSIONWe have positioned db relative a series of RFLPs by char-acterizing 132 N2 offspring of an intraspecific backcrossbetween C57BL/6J db/db and B6D2 F1 db/+ mice, and 48obese progeny of a B6D2 F1 db/+ x B6spretus F1 db/+intercross. A protocol using BMI, plasma [glucose], andplasma [insulin] was used to assign genotype at the db locus.Inclusion of measurements of [glucose] and [insulin] in thisanalysis ensured correct assignment of genotype among theprogeny of the intraspecific cross. Nevertheless, similaranalysis of the progeny of the interspecific cross suggestedeither a phenotypic misassignment or a double crossover inone apparently obese (db/db) animal (animal 337). Mappingof additional RFLPs between the flanking markers Cjun andD4Rpl will be necessary to distinguish these possibilities.

Analysis of recombination frequencies among the variousprobes positions db: -10 centimorgans (cM) distal to Mt4 (thebrown locus), 4.5 cM distal to PAMm32, 3 cM distal to Ifa and

centromere

* 11w 11

,I E cc2 2 r db 61 5.5 1151 3

Ill 6 1 2]1-oc L

0

Cjun, 5cM proximal to D4RpJ and Mtv-13 (MMTV), 7 cMproximal to Gluti, and 12 cM proximal to Lck (see Fig. 4).This gene order is consistent with that reported (25, 27, 29).Specifically, our data position Mtv-13 distal, and Ifa proxi-mal, to db, as suggested by Huppi et al. (29). Because someprobes could not be mapped on both crosses, the order ofMtv-13 relative to Glutl and D4Rpl remains ambiguous.These data demonstrate that db is flanked by a series of

RFLPs that have been mapped to human chromosomelp31-36 (27) and suggest that, if there is a human homologueof db, it would probably map to chromosome ip. Thisinformation may be useful in the analysis of the heritability ofhuman obesity and type II diabetes. Humans with a defect inthe human homologue of the db gene (if one exists) might beexpected to cosegregate RFLPs from this chromosome withan obese and/or diabetic phenotype in human pedigrees.Experiments to test this possibility are under way.The characterization of the progeny of the crosses de-

scribed will permit fine genetic mapping of this region,ultimately narrowing the 95% confidence interval around db

to <1 cM between db and nonrecombinant markers. Expan-sion of this cross according to the protocols suggested should

C57BL/6J db/db X B6D2 F1 db/+ N2

-J

7

8iI

B6D2 Fl db/+ X B6spretus Fl db/+

FIG. 4. Genetic maps of the region surrounding db on mouse chromosome 4. The genetic map shown above the line was derived from theintraspecific N2 backcross animals (see Fig. 3a), the genetic map shown below the line was derived from the interspecific M. spretus cross (seeFig. 3b). For each N2 animal, the genotype at each of the loci typed was analyzed by pedigree analysis to generate a genetic map in the regionaround db. For the F2 animals, the genotype at the loci corresponding to the following probes Inta, Cjun, D4Rp1, Glutl, and Lck was determined,and the resulting map was aligned to the N2 map at the loci for Ifa (Inta) and Lck. The actual order of the probes MMTV and D4Rpl-Glutlrelative to one another cannot be determined since MMTV was analyzed in the interspecific (F2) cross and Glutl and D4Rp1 were not analyzedon the intraspecific (N2) cross. The loci are shown in their most likely order based upon the pedigree analysis, and this order is 10,000-fold morelikely than any other (allowing for the order of MMTV relative to Glutl and D4Rp1 to remain undetermined) by use of the MAPMAKER program(26). The N2 map is based on 132 meiotic events, the F2 map is based on the 48 M. spretus meioses scored with the probes Cjun, Glutl, andD4Rp1 and an additional 48 DBA/2J meiotic events (for a total of %) with the probes Inta and Lck.

a4/125

3.2 (1.6)

10/1327.6 (2.3)

8/1326.0 (2.1)

9/132 17/132 19/125 20/129 29/1326.8 (2.2) 12.8 (2.9) 15.2 (3.2) 15.5 (3.2) 121.9 (3.6)

MMTV db Inta MM3 Mt4

Cjun

db

D4RP1

Gt

Lck

000U00I

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allow even finer genetic mapping of this mutation to thesubcentimorgan level. In fact, the two crosses have beenexpanded to include a total of 760 informative meioses,generating a 95% confidence interval of <0.4 cM for anonrecombinant RFLP (30). To find additional RFLPs thatare linked to db, chromosomal microdissection (31) as well asflow sorting of a 4:6 Robertsonian (translocation) chromo-some have been used to generate region-specific genomiclibraries (unpublished data and refs. 14 and 31).As part of the phenotypic characterization of the N2

animals, we have measured plasma [glucose] and [insulin].Analysis of these data reveals that there is a wide range ofplasma [glucose] among the db/db progeny of the C57BL/6Jdb/db x B6D2 F1 db/+ backcross, demonstrating a contin-uous rather than a bimodal distribution. In fact, frank dia-betes is frequent in these animals, and in two cases in theintraspecific backcross would have resulted in a misassign-ment of genotype if only body weight or BMI had beenscored.

Previous investigators have demonstrated a potent effectof genetic background on the diabetes phenotype of ob anddb animals (5, 6, 8, 32). The diabetes of db/db animals isreported to be severe in congenic DBA/2J db/db strains andmilder in db/db congenic C57BL/6J animals (6). The geneticbasis for this phenotypic difference between C57BL/6J andDBA/2J mice is unknown.

If the difference in the propensity for diabetes between theC57BL/6J and DBA/2J strains were inherited as a singleallele, segregation of the N2 animals into two distinct groupswith regard to [glucose] and/or [insulin] would be expected.The wide range of plasma [glucose] and [insulin] in the db/dbN2 and the failure to detect a bimodal distribution in plasma[glucose] or [insulin] in these animals, (representing either theC57BL/6J or DBA/2J allele), indicate that more than onelocus in combination with environmental differences are

likely to be responsible for the differential strain susceptibil-ity to the diabetogenic influence of db. The conclusion thatpolygenes can contribute to differences in the diabetogenicityof the db/db genotype among inbred strains is consistent withthat reached in the context of an intercross between C3H/HeJ x C57BL/6J +/+ animals (33) and between db/+C57BL/KsJ and 129/J mice (34).One other interesting finding concerned the life span of the

male db/db N2 animals. Previous studies have shown thatcongenic C57BL/6J db/db animals of either sex have a mildlyreduced lifespan compared to the average of 22 months inwild-type (+/+) mice (35). Studies of the congenic strainDBA/2J-db/db have reported respective mortalities of 73%and 25% by 5 months of age in males and females (6). In thepresent study, male progeny of the intraspecific cross ap-peared to show a markedly increased rate of death thatapparently occurred prior to their weaning at 4 weeks of age.Premature death of males prior to weaning is surprising givenresults from earlier studies of DBA/2J and C57BL/KsJdb/db animals that report an increased mortality of malesbetween 3 and 5 months of age. In addition, the F2 offspringdid not demonstrate a premature loss of males. Althoughdifferences in diabetes susceptibility and viability betweenmale and female animals have been demonstrated in othercircumstances, the reason for premature death among our

male N2 animals remains unclear (36-38).In summary, we have established intraspecific and inter-

specific mouse crosses segregating the db mutation and havetyped the progeny for a series of RFLPs. Analysis of thesedata has generated a molecular map ofthe db mutation, whichwill aid in the cloning of the gene. In addition, we have noteda wide range and the lack of a bimodal distribution of plasma

[glucose] and [insulin] among db/db N2 animals. We con-

clude that variability in the development of diabetes among

the N2 db/db animals is consistent with a polygenic mode ofinheritance for the background strain's effect on the devel-opment of diabetes accompanying the db mutation.

We thank D. Coleman for helpful discussions and for the generousgift of the C57BL/6J db/db animals, D. Corow for performing theovarian transplants, and S. Cancellieri for assistance in animalhandling. We are grateful to X. F. Pi-Sunyer and Y. Dam forperformance of the assays for plasma glucose and insulin and to L.Cousseau for help in the preparation of this manuscript. Twoanonymous reviewers provided helpful suggestions. This work wassupported by National Institutes of Health Grants DK26687,DK41096, and 5P30DK26687. N.B. is the recipient of an Arnold andMabel Beckman Graduate Fellowship. R.L.L. is an EstablishedInvestigator of the American Heart Association. J.M.F. is an As-sistant Investigator of the Howard Hughes Medical Institute.

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