Genetic Modifiers ofLeprfaAssociated with Variability in Insulin Production and Susceptibility to NIDDM

GENOMICS

41, 332– 344 (1997) GE974672

ARTICLE NO.

Genetic Modifiers of Lepr fa Associated with Variability in Insulin Production and Susceptibility to NIDDM WENDY K. CHUNG,* MIN ZHENG,* MELVIN CHUA,* ERIN KERSHAW,* LORAINE POWER-KEHOE,* MICHAEL TSUJI,* X. SHARON WU-PENG,* JULIE WILLIAMS,† STREAMSON C. CHUA, JR.,* AND RUDOLPH L. LEIBEL*,1 *Laboratory of Human Behavior and Metabolism, The Rockefeller University, 1230 York Avenue, New York, New York 10021; and †Department of Biology, Animal Model Core Laboratory of the National Institutes of Health, Vassar College, Poughkeepsie, New York 12601 Received November 6, 1996; accepted February 11, 1997

In an attempt to identify the genetic basis for susceptibility to non-insulin-dependent diabetes mellitus within the context of obesity, we generated 401 genetically obese Lepr fa/Lepr fa F2 WKY13M intercross rats that demonstrated wide variation in multiple phenotypic measures related to diabetes, including plasma glucose concentration, percentage of glycosylated hemoglobin, plasma insulin concentration, and pancreatic islet morphology. Using selective genotyping genome scanning approaches, we have identified three quantitative trait loci (QTLs) on Chr. 1 (LOD 7.1 for pancreatic morpholology), Chr. 12 (LOD 5.1 for body mass index and LOD 3.4 for plasma glucose concentration), and Chr. 16 (P õ 0.001 for genotype effect on plasma glucose concentration). The obese F2 progeny demonstrated sexual dimorphism for these traits, with increased diabetes susceptibility in the males appearing at approximately 6 weeks of age, as sexual maturation occurred. For each of the QTLs, the linked phenotypes demonstrated sexual dimorphism (more severe affection in males). The QTL on Chr. 1 maps to a region vicinal to that previously linked to adiposity in studies of diabetes susceptibility in the nonobese Goto– Kakizaki rat, which is genetically closely related to the Wistar counterstrain we employed. Several candidate genes, including tubby (tub), multigenic obesity 1 (Mob1), adult obesity and diabetes (Ad ), and insulinlike growth factor-2 (Igf2), map to murine regions homologous to the QTL region identified on rat Chr. 1. q 1997 Academic Press

INTRODUCTION

Non-insulin-dependent diabetes mellitus (NIDDM) affects 15% of all Americans over the age of 60 and 100 million individuals worldwide (WHO study group, 1994) and accounts for 10% of all health care expendi1

To whom correspondence should be addressed. Telephone: (212) 327-8435. Fax: (212) 327-7150. E-mail: [email protected]. 0888-7543/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

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tures in the United States (Huse et al., 1989). Obesity is associated with approximately 70% of all instances of NIDDM (West and Kalbfleisch, 1971; Campbell and Carlson, 1993), and the integral relationship between body fatness and NIDDM is exemplified by the effects of modest perturbations in body weight to either ameliorate NIDDM with weight reduction (Henry et al., 1986) or to induce insulin resistance in normal-weight subjects overfed to increase body weight by 15– 20% (Sims, 1973; Olefsky et al., 1974). However, the mechanisms by which increased body fat predisposes to NIDDM are not understood. Evidence for genetic susceptibility to NIDDM in humans is based upon nearly 100% concordance in monozygotic twins followed for sufficient periods of time (Barnett et al., 1981; Newman et al., 1987), familial clustering of the disease, and varying disease prevalence among ethnic groups (Zimmet et al., 1982) and within populations with varying degrees of racial admixture (Chakraborty et al., 1986). However, with the exception of rare forms of NIDDM such as MODY1 (hepatocyte nuclear factor 4 a) (Yamagata et al., 1996a), MODY2 (glucokinase) (Froguel et al., 1992), MODY3 (hepatocyte nuclear factor 1 a) (Yamagata et al., 1996b), and mitochondrial mutations in tRNALeu (Vanden-Ouweland et al., 1992), the genes underlying genetic susceptibility to NIDDM in humans are unknown. Obese (Lepob/Lep ob) and diabetes (Lepr db/Lepr db) mice have mutations in the genes for leptin (Lep) (Zhang et al., 1994) and the leptin receptor (Lepr) (Chen et al., 1996; Chua et al., 1996a; Lee et al., 1996), respectively, and develop early onset, profound obesity. The fatty (Lepr fa/Lepr fa) rat has a Gln269Pro mutation in the leptin receptor that apparently restricts trafficking of the receptor to the cell surface (Chua et al., 1996b; Phillips et al., 1996; Takaya et al., 1996a). The Koletsky rat (Lepr fak/Lepr fak) has a Tyr763Stop (nonsense) mutation in the leptin receptor that affects all splice variants of the Lepr mRNA (Wu-Peng, 1997; Takaya et al., 1996b). With the availability of rat microsatellite ge-

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GENETIC MODIFIERS OF Lepr fa

netic markers at increasing density, it is now possible to begin to dissect the genetics of modifiers of singlegene obesity mutations such as Lepr fa in the rat. Strain-related differences in NIDDM susceptibility have been demonstrated in mice segregating for the obesity mutations obese (Lepob) and diabetes (Lepr db) (Leiter, 1989; Leiter et al., 1989). Similarly, the Zucker fatty rat (Lepr fa/Lepr fa on the 13M strain) is phenotypically obese, hyperinsulinemic, and glucose intolerant, yet euglycemic (Zucker and Antoniades, 1972), while the same mutation backcrossed onto the carbohydrateintolerant Wistar (WKY) stock produces males that are obese, hyperinsulinemic, and hyperglycemic (Ikeda et al., 1981; Kava et al., 1992). Expression of Lepr fa on the WKY strain is sexually dimorphic because only males become hyperglycemic and overtly diabetic. The basis for the sexual dimorphism is not known, but ovariectomy does not induce diabetes in the obese WKY females (Kava et al., 1992). Little sexual dimorphism with regard to insulin and carbohydrate homeostasis is observed in the Zucker (13M strain) rats (Zucker and Antoniades, 1972). To identify the genomic intervals (and eventually the genes) conferring diabetes susceptibility and resistance within the setting of obesity induced by a mutation in the leptin receptor (Lepr fa), we established interspecific intercrosses segregating for Lepr fa between the 13M (diabetes resistant) and the WKY (diabetes susceptible) strains of rats. Following characterization of the F2 progeny for body weight, body mass index, glycosylated hemoglobin, fasting plasma glucose and insulin concentrations, and pancreatic morphology, selective genotyping was conducted on the 5% of the obese males demonstrating the most extreme diabetic and nondiabetic phenotypes by scanning the genome with 122 microsatellite markers. Genomic intervals tentatively linked to diabetes susceptibility were then tested on all of the obese progeny (both sexes) with increased marker density, localizing a total of three discrete intervals on chromosomes 1, 12, and 16 that were linked to various NIDDM-related phenotypes. Interestingly, the interval on chromosome 1 is approximately the same interval linked to relative adiposity in two separate genetic analyses of NIDDM in the Goto–Kakizaki (GK) rat (Galli et al., 1996; Gauguier et al., 1996). Striking phenotypic differences were observed between the sexes as have been previously observed in genetically obese mice (Leiter, 1989; Leiter et al., 1989); these may be related to changes in circulating androgen concentrations associated with sexual maturation. MATERIALS AND METHODS Animal resources. Rats were bred and raised as previously described with ad libitum access to PicoLab Rodent Chow 20 (Purina Mills, Richmond, IN) (Kershaw et al., 1995). A total of 401 Lepr fa / Lepr fa F2 progeny (193 males and 208 females) and 206 lean (Lepr fa / / and ///) progeny (94 males and 112 females) were monitored until a mean (//0 standard deviation) age of 143 //0 18 days, when they were sacrificed. Rats were fasted for 2 h prior to sacrifice by

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CO2 asphyxiation. Following sacrifice, urine was tested for glucose and ketones by dipstick (Chemstrip uGK) (Boehringer Mannheim, Indianapolis, IN). Approximately 8 ml whole blood was withdrawn by cardiac puncture into a heparinized syringe and transferred to a tube containing 300 ml anticoagulant (82 mM EDTA and 10,000 U/ml heparin) and protease inhibitor (1000 U/ml aprotinin). The spleen, kidneys, liver, and heart were removed and immediately frozen at 0807C for subsequent isolation of genomic DNA. The pancreas was excised en bloc and sectioned longitudinally, half was immediately frozen at 0807C for subsequent analysis of pancreatic hormone content, and half was fixed in modified Bouin’s solution (750 ml saturated aqueous picric acid, 250 ml 40% formalin, and 50 ml glacial acetic acid) for 6 h for subsequent morphological analyses. Phenotypic characterization. Weight and nasoanal body length were determined weekly on rats beginning at age 15 days until the time of sacrifice. Plasma insulin concentration was determined by radioimmunoassay (Campfield and Smith, 1983), plasma glucose concentration by glucose oxidase (Kadish et al., 1968), and percentage of glycosylated hemoglobin by an affinity-based chromatography assay (Glyc-Affin GHb; Isolab Inc., OH). Pancreatic hormones were extracted with acid ethanol and precipitated with alcohol ether (Karam and Grodosky, 1962). Protein content of the pancreatic extracts was determined by the Lowry method using a modified Biuret reagent (Ohnishi and Barr, 1978) and was used to normalize pancreatic insulin concentration and glucagon concentration, which were determined by radioimmunoassay (Herbert et al., 1965). Pancreatic morphology. After fixation in Bouin’s solution, pancreata were washed with 70% ethanol until further leaching of picric acid from the tissues was minimal. Tissues were processed by dehydration through increasing percentages of ethanol, cleared with xylene, and saturated and embedded in paraffin. Five-micrometer longitudinal sections the length of the pancreas were cut and immunohistochemically stained using the avidin –biotin– immunoperoxidase technique (Hsu et al., 1981a,b). Endogenous peroxidases were inhibited by treating the sections with 0.3% hydrogen peroxide in methanol for 30 min. To minimize nonspecific staining, sections were incubated for 20 min with diluted normal serum from the species from which the secondary antibody was derived. Sections were incubated overnight with a 1:3000 dilution of primary polyclonal guinea pig anti-insulin antibody (DAKO, Carpinteria, CA) or a 1:1500 dilution of primary polyclonal rabbit anti-glucagon antibody followed by incubation with an anti-guinea pig or anti-rabbit secondary antibody bound to biotin. Diaminobenzidine tetrahydrochloride was used to visualize the sites of bound antibody with hematoxylin as a counterstain. Pancreatic sections from males were independently graded by two observers according to the criteria outlined below (Fig. 6): Grade 1: Islet hypertrophy (ú12 islets/section) and b cell hyperplasia within islets with no evidence of islet disorganization or insulinnegative b cells. Grade 2: Islet hypertrophy (6 –12 islets/section), b cell hyperplasia within islets, and at least 15 islets/section in which õ50% of the islets demonstrated islet disorganization or insulin-negative b cells. Grade 3: Evidence of islet hypertrophy (õ6 islets/section), b cell hyperplasia within islets, and at least 15 islets in which 50– 90% of the islets demonstrated islet disorganization or insulin-negative b cells. Grade 4: Variable islet hypertrophy and organization but at least one islet with normal organization. Islets may demonstrate fibrosis and neovascularization. Grade 5: No evidence of islet hypertrophy and minimal number of b cells still producing insulin. No organizationally intact islets. Variable degrees of fibrosis and neovascularization in remaining islets. Similar criteria were used to grade the pancreata from the females, although in general the numbers of islets used to define each stage was approximately twice those in the males. Pancreatic histology was also quantitatively analyzed using an image analysis system with Image Pro Plus (Version 1.3) by capturing images of pancreatic sections and quantifying the number of

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Note. Summary data of 10 male Lepr fa /Lepr fa animals at each extreme of phenotype (diabetic and insulin-resistant nondiabetic) used in a 122-marker genome scan for selective genotyping to identify genomic regions for genotyping all F2 progeny are also indicated. The 10 most extremely affected diabetic and 10 most extremely insulin-resistant nondiabetic males were selected on the basis of percentage HbA1c and plasma insulin concentration. Data are shown as the mean { standard devation (range). Pancreatic morphology for the lean animals was not formally measured because there was little variation among animals. Lean pancreata were distinct from those of any of the obese by virtue of fewer numbers of smaller islets that were all organizationally intact (see Fig. 5). ND, not determined.

9 58.2 0.080 1.8 156.4 38 0.5 25.0 2.3 31.0 162 { 714.6 { 1.073 { 6.8 { 1178.1 { 305 { 2.4 { 49.4 { 5.8 { 28.3 { 10 13 (110– 145) 40.0 (377.7– 505.5) 0.050 (0.720– 0.866) 1.6 (15.2– 18.9) 34.8 (27.5– 132.5) 76 (658– 896) 0.4 (3.8 –5.0) 15.7 (23– 69) 0.5 (0 –1) 14.8 (5 –54) 128 { 447.1 { 0.790 { 16.6 { 70.3 { 745 { 4.5 { 50.9 { 0.4 { 30.3 { 94 { 18 (106– 178) { 42.4 (345.9– 599.7) { 0.062 (0.607– 0.845) { 0.6 (2.5 – 5.9) { 55.3 (15.5 –288.4) { 95 (121– 582) ND ND ND ND 142 438.5 0.722 4.5 126.2 313 112 { 19 (106– 187) { 42.1 (194.0– 480.0) { 0.061 (0.360– 0.793) { 0.6 (2.7– 5.9) { 42.1 (5.0– 203.4) { 76 (52– 426) ND ND ND ND 142 252.2 0.571 4.1 72.4 255 193 17 (106– 178) 94.1 (347.0– 826.5) 0.112 (0.599– 1.278) 4.1 (3.8 – 21.4) 422.1 (28.6– 2036.1) 170 (210– 896) 1.1 (1 – 5) 18.2 (12– 112) 1.3 (0 – 22) 18.4 (0 – 103)

Extremely diabetic male Lepr fa/Lepr fa Male Lepr fa// and /// Female Lepr fa// and /// Male Lepr fa/Lepr fa

143 { 585.7 { 0.933 { 11.1 { 584.3 { 514 { 3.2 { 51.6 { 2.9 { 21.9 { 208 { 19 (106 –187) { 56.1 (340.5 –673.4) { 0.092 (0.691 –1.405) { 1.8 (3.6 – 16.9) { 383.1 (64.2 – 2123.1) { 101 (114 –724) { 1.9 (1 – 5) { 19.6 (11 – 114) { 3.2 (0 – 14) { 18.4 (0 – 80) 143 484.5 0.961 5.5 733.2 317 2.7 49.6 3.8 27.7 N Age (days) Body weight (g) BMI (g/cm2) HbA1c (%) Plasma insulin concentration (mU/ml) Plasma glucose concentration (mg/dl) Pancreatic grade Number of islets Number of hypertrophic islets Number of organized islets

Statistical analysis. Results of the selective genotyping were analyzed by x 2 analysis assuming that the expected distribution of genotypes would be 25% 13M/13M, 50% 13M/WKY, and 25% WKY/WKY for any autosome and 50% hemizygous 13M and 50% hemizygous WKY for the male X chromosome. Markers demonstrating significant deviation from the predicted allele frequency at P õ 0.05 in either the diabetic or the insulin-

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Female Lepr fa/Lepr fa Phenotype

Genotypic characterization. High-molecular-weight genomic DNA was purified from solid tissues using proteinase K digestion followed by extraction with phenol and chloroform and precipitation with ethanol. Polymerase chain reactions (PCRs) for amplification of small fragments of simple sequence repeats were performed with both unlabeled and radiolabeled primers as dictated by the allele sizes. End labeling of primers and PCR amplification and electrophoresis of PCR products were performed as previously described (Chung et al., 1996). Genotype at the Lepr fa locus was determined as previously described (Chua et al., 1996b) to ensure that phenotypically obese rats were homozygous for the mutation. All rat microsatellite markers currently genetically mapped at the Whitehead Institute and distributed through Research Genetics were tested for polymorphism between 13M and WKY. All polymorphic markers (a total of 122) were used for selective genotyping. Markers were on average 11.8 cM apart and were within 25 cM of other markers with the exception of the following four intervals: D1Mgh2 –D1Mgh18 (32.4 cM), D2Mgh11 –telomere (52.3 cM), D4Mgh1 –D4Mit5 (36.4 cM), and D13Mgh5 –D13Mgh6 (39.5 cM). After the data from the original genome scan were analyzed, additional chromosome 1 markers used by Gauguier et al. (1996) and Galli et al. (1996) were added to increase the density of markers on chromosome 1 and to allow direct comparison of the relative locations of the QTLs identified in the GK rat with the QTLs we had identified.

Phenotypic Characteristics of F2 WKY13M Progeny Displayed by Sex and Genotype at Lepr fa

TABLE 1

Selection of animals for selective genotyping. Because the males demonstrated greater phenotypic variation in NIDDM-related phenotypes, selective genotyping was limited to the 5% of the males at each extreme of the phenotypic distribution. The mean //0 SD plasma insulin concentration and percentage of HbA1c for all Lepr fa / Lepr fa males (N Å 193) were 584 //0 422 mU/ml and 11.1 //0 4.1%, respectively. The extremes of phenotype were selected by consideration of both plasma insulin concentration following a 2-h fast at the time of sacrifice and HbA1c, a stable measure of hyperglycemia over approximately 60 –90 days that is not affected by increases in stress hormones associated with sacrificing the animals. The diabetic class was selected from among the younger animals, while the insulinresistant relatively nondiabetic class was selected from the older animals to prevent bias of increasing disease severity with advancing age. Using measures of insulin production and sustained hyperglycemia (HbA1c) to define the extremes of phenotype, we sought to identify quantitative trait loci (QTLs) involved in: (1) the ability of the pancreatic b cells to meet the need for increased insulin production due to the insulin resistance induced by genetic obesity and (2) either glucose disposal or glucose production that might independently lead to hyperglycemia. The extremely affected diabetic males were defined as the 10 males with fasting plasma insulin concentration 1 SD or more below the mean of all Lepr fa/Lepr fa males (£162 mU/ml) and HbA1c 1 SD or more above the mean (§15.2%). These animals also had to have ages at sacrifice below that of the group’s mean to ensure that any increased severity of disease was not due to advanced age. The insulin-resistant nondiabetic males were defined as the 10 males with fasting plasma insulin concentration 1 SD or more above the mean (§1006 mU/ml) and with ages at sacrifice above the group’s mean to ensure that the rats had had sufficient time to develop NIDDM. All of the insulin-resistant nondiabetic males also had HbA1c’s less than 9.1%. The complete phenotypic data on each of the extreme classes are summarized in Table 1.

Extremely insulin-resistant male Lepc fa/Lepr fa

islets, the areas of the islets and average islet area, and the area of the entire pancreatic section. The number of hypertrophic islets (area ú0.05 mm 2) and the number of organizationally intact islets (homogeneous insulin staining throughout the islet without neovascularization or ductal proliferation) were quantified.

10 (145– 170) (645.2– 826.5) (0.992– 1.263) (3.8 – 9.1) (1015.0 – 1520.0) (258– 378) (1.5 – 3.0) (24– 108) (2 – 8) (4 – 103)

CHUNG ET AL.

GENETIC MODIFIERS OF Lepr fa resistant nondiabetic class were then used to type all 401 F2 obese progeny (both sexes). The data generated from genotyping the entire F2 progeny were analyzed by analysis of variance (ANOVA) between groups defined by genotypic class and sex for the following dependent variables: body weight, body mass index (BMI), fasting plasma glucose concentration, fasting plasma insulin concentration, HbA1c, pancreatic grade, pancreatic insulin concentration/mg protein, pancreatic glucagon concentration/mg protein, number of islets, number of hypertrophic islets, number of organizationally intact islets, and average islet area—using the BMDP statistical package with post hoc group comparisons using the Bonferroni test (Dixon et al., 1988). Interactions among selected genetic loci were also tested by ANOVA. The effect of genotype over time was tested by ANOVA with repeated measures. MapMaker QTL (Version 1.1, 1993) was used to analyze chromosomal segments for which three or more markers had been typed on a majority of the obese progeny. All phenotypic parameters were tested under all modes of inheritance (free, additive, dominant, and recessive). Marker order and interlocus distances were calculated using the ‘‘ripple’’ and ‘‘try’’ functions of MapMaker/Exp (Version 3.0b) with the Haldane correction for genetic distances.

RESULTS

A total of 607 F2 progeny comprised 208 Lepr fa/Lepr fa obese females, 193 Lepr fa/Lepr fa obese males, 112 Leprfa// and /// lean females, and 94 Lepr fa// and // / lean males. The Lepr fa/Lepr fa females weighed almost twice as much as the Lepr fa// and /// females, and the Lepr fa/Lepr fa males weighed 34% more than the Lepr fa// and /// males (Table 1). The lean (Lepr fa/ / and ///) and obese (Lepr fa/Lepr fa), by sex, were readily distinguishable when the BMI, defined as the weight (g)/nasoanal length (cm)2, and glycosylated hemoglobin (HbA1c), a measure of long-term glycemic status, were considered simultaneously (Fig. 1). None of the Lepr fa// or /// progeny were chronically hyperglycemic (defined as HbA1c ú7.5%). The Lepr fa/Lepr fa rats demonstrated sexual dimorphism with the males being much more susceptible to diabetes than the females. Such dimorphism was similar to related phenotypes observed in GK rat intercrosses (Gauguier et al., 1996). Less than 5% of the Lepr fa/Lepr fa females were overtly diabetic (HbA1c ú7.5%); however, the Lepr fa/ Lepr fa males demonstrated large variation in all diabetes-related phenotypes (plasma glucose and insulin concentrations, HbA1c, and pancreatic morphology). In general, adiposity in the Lepr fa/Lepr fa males was inversely related to severity of diabetes (Fig. 1). Percentage HbA1c and fasting plasma insulin concentration were used as measures of relative hyperglycemia, insulin resistance, and the capacity for b cell insulin secretion, to select Lepr fa/Lepr fa males at the extremes of diabetes-related phenotypes for selective genotyping (genome scan). In general, fasting plasma insulin concentration was inversely related to HbA1c

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in the males (Fig. 2). The phenotypic characteristics of each of the 10 males constituting the respective diabetic and insulin-resistant nondiabetic classes are summarized in Table 1. Selection criteria for these animals are summarized under Materials and Methods. The two groups were phenotypically extremely different: the insulin-resistant nondiabetic class weighed 60% more, demonstrated fasting plasma insulin concentration almost 17 times higher, and maintained fasting glucose concentration and HbA1c half that of the diabetic class. Selective genotyping identified seven possible QTLs on chromosomes 1, 7, 9, 12, 14, 16, and 20 (Table 2). Two or more adjacent markers demonstrated significant x2 results on chromosomes 1, 12, and 16. Genotypic analysis of all of the obese F2 progeny (male and female) for each of the markers in Table 2 indicated linkage with either obesity and/or diabetes phenotypes at P õ 0.01 only for the markers on chromosomes 1, 12, and 16. Additional markers were then used to genotype all of the Lepr fa/Lepr fa male and female progeny (a total of 17 markers on Chr. 1 spanning 97.7 cM, 5 markers on Chr. 12 spanning 34.8 cM, and 2 markers on Chr. 16 spanning 7.8 cM) to further localize the QTLs. Interval mapping of the genomic region on Chr. 1 using MapMaker QTL indicated significant relationships of genotype at the Chr. 1 interval with BMI (LOD Å 3.91), pancreatic grade (LOD Å 4.95), number of islets (LOD Å 5.59), and number of organizationally intact islets (LOD Å 7.05) when combining the results from both sexes (Fig. 3 and Table 3). Twelve percent of the variance in the number of intact islets in the females was attributable to genotype at the interval between Lsn and D1Wox8. The precise location of the quantitative trait locus or loci on chromosome 1 is difficult to define over the 19.6-cM distance between Lsn and D1Wox10 due to the large number of phenotypes associated with genotype in this interval and the sexual dimorphism in phenotypes most significantly associated with genotype in the interval. Genotype at the interval on Chr. 12 was significantly related to BMI (LOD Å 5.07) and plasma glucose concentration (LOD Å 3.37), accounting for 9% of the variance in plasma glucose concentration in the males. In a detection paradigm such as that employed here, use of a threshold of LOD ú 4.3 ensures that only 5% of the loci reported will achieve statistical significance by chance alone (Lander and Schork, 1994). Both the loci on Chr. 1 and on Chr. 12 exceeded this threshold for at least a single subphenotype and are most likely to be biologically significant because multiple NIDDM-related phenotypes were linked to the QTLs.

FIG. 1. Relationship between BMI and HbA1c at the mean age of 143 days for F2 WKY13M progeny arrayed by sex and genotype at Lepr fa (obese is Lepr fa /Lepr fa; lean is Lepr fa// and ///). Lepr fa /Lepr fa males at the extremes of phenotype (diabetic and insulin-resistant nondiabetic) used for genome-scanning experiments are indicated. Severe diabetes (high HbA1c) results in weight loss (lower BMI). FIG. 2. Relationship between 2-h fasting plasma insulin concentration and HbA1c at the mean age of 143 days for F2 WKY13M progeny arrayed by sex and genotype at Lepr fa . Lepr fa/Lepr fa males at the extremes of phenotype (diabetic and insulin-resistant nondiabetic) used for genome-scanning experiments are indicated.

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FIG. 4. Body weights over time (by sex and genotype at D1Wox8) in Lepr fa/Lepr fa males and females are illustrated. The period between 15 and 37 days of age is highlighted in the inset. FIG. 6. Qualitative pancreatic grading. Examples of one lean and five Lepr fa/Lepr fa male rat pancreata immunohistochemically stained with antibodies to insulin. Grading was based upon relative degrees of hypertrophy of the islets, islet number, insulin production by the b cells within the islets, and structural changes within the islets including fibrosis and neovascularization. Grade 1 represents the pancreata with the most islets and the greatest extent of hypertrophy and hyperplasia while grade 5 represents the opposite extreme with few insulinpositive b cells and a small number of islets.

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CHUNG ET AL.

TABLE 2 Selected Results from Genome Scans of 20 F2 WKY13M Lepr fa/Lepr fa Male Progeny at Extremes of Affection Status for NIDDM

Locus Chr. 1 Mtipa Lsn D1Mit5 D1Mgh22 D1Mgh10 D1Mgh11 Pbpc2 D1Mgh13 Chr. 7 D7Mit13 Chr. 9 D9Mgh1 Chr. 12 D12Mit5 D12Mgh1 Chr. 14 D14Csna Chr. 16 D16Mgh2 D16Mgh4 D16Mit2 Chr. 20 D20Mgh2

Distance from centromere (cM)

Diabetic genotype frequency 13M/13M

13M/WKY

WKY/WKY

98.2 98.2 105 105 106.1 109.6 Ç120 139.2

6 7 7 7 7 7 7 6

3 3 3 3 3 3 3 3

1 0 0 0 0 0 0 1

11.2

3

3

4

8

1

5

19 19

1 1

7

Insulin-resistant genotype frequency

x2

13M/13M

13M/WKY

WKY/WKY

x2

4 4 4 4 4 4 4 4

4 4 4 4 4 3 3 4

2 2 2 2 2 3 3 2

NS NS NS NS NS NS NS NS

NS

2

2

6

P õ 0.05

4

NS

1

3

6

P õ 0.05

7 7

2 2

NS NS

6 6

2 2

2 2

P õ 0.05 P õ 0.05

4

4

2

NS

5

1

4

P õ 0.05

9 9 16.8

1 1 2

3 3 2

6 6 6

P õ 0.05 P õ 0.05 P õ 0.05

1 1 1

6 6 7

3 3 2

NS NS NS

22.9

1

3

6

P õ 0.05

2

6

2

NS

P P P P P P P P

õ õ õ õ õ õ õ õ

0.05 0.01 0.01 0.01 0.01 0.01 0.01 0.05

Note. Markers demonstrating deviation from the expected Mendelian ratios are summarized with the genotypic distribution in the two classes of rats. For none of the markers did a randomly selected group of 96 Lepr fa // and /// F2 progeny deviate from the expected Mendelian ratios of genotypes, eliminating the possibility of segregation distortion as a possible explanation for non-Mendelian genotype ratios in Lepr fa/Lepr fa F2 progeny.

Results of the ANOVA for the markers demonstrating the greatest phenotypic effects are summarized in Table 4. The phenotypic effect of each of the three QTLs demonstrated sexual dimorphism, and there was only modest overlap of phenotypes correlated with each of the genomic intervals between the two sexes. For example, the Chr. 1 interval (D1Wox8 r D1Mgh11) demonstrated significantly decreased body weight and BMI, increased HbA1c, reduced fasting plasma insulin concentration, and increased (worse) pancreatic grade in the 13M/13M males relative to the 13M/WKY and WKY/WKY, acting in a recessive manner with regard to measures of adiposity while acting in a codominant manner for measures of diabetes affection. In the females, 13M alleles acted dominantly to increase diabetes susceptibility. However, only phenotypes related to pancreatic morphology, such as pancreatic grade, number of organizationally intact islets, and total number of islets, were significantly related to genotype at the Chr. 1 interval in females. Association of 13M alleles with increased diabetes susceptibility was not predicted on the basis of the inbred strain parental phenotypes and is an example of a ‘‘transgression’’ (see Discussion). WKY alleles in the region on Chr. 12 represented by D12Mit6 were dominantly associated with measures

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of hyperglycemia in the males (fasting plasma glucose concentration and HbA1c) and with decreased body weight, decreased BMI, and increased HbA1c in the females, even though few of the females were overtly diabetic. This QTL demonstrated a powerful influence on degree of hyperglycemia, because measures such as HbA1c were correlated with the genotype in both sexes even though the females were largely resistant to overt NIDDM. The decreased adiposity in the WKY/WKY females is likely secondary to the perturbations in glucose homeostasis. In the Chr. 16 interval, WKY alleles were recessively associated with fasting hyperglycemia in males alone. Because only a single NIDDM-related subphenotype in a single sex was correlated with the chromosome 16 interval, this QTL should be considered provisional until replicated. Genotypic analysis of the lean males and females for D1Mgh10, D1Wox23, and D12Mit6 demonstrated no association with phenotypic measures of obesity or diabetes (results not shown). Analysis of the relationship between genotype at any of the QTLs and the multiplicity of diabetes-related phenotypes at the age of approximately 140 days necessarily suffers from the confound of secondary and tertiary effects of the QTLs on the range of phenotypes

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FIG. 3. Results of MapMaker QTL interval mapping for the genomic regions on rat chromosomes 1 and 12 under a free genetic model. Genetic distances between markers are indicated in cM above the x axis. Genetically unresolvable markers are indicated on the same line. For the region on chromosome 1, the phenotypes of qualitative pancreatic grade and BMI are illustrated for the males and total number of islets and number of organizationally intact islets for the females. Only fasting plasma glucose concentration for the males is illustrated for chromosome 12.

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TABLE 3 Interval Mapping Results for the Genomic Regions on Rat Chromosomes 1 and 12 in Lepr fa /Lepr fa Animals Shown by Sex

Trait

Location

LOD

% variance attributable to QTL

Weight BMI Plasma insulin concentration Pancreatic grade Number of islets Number of intact islets BMI Number of islets Number of intact islets Plasma glucose concentration Weight BMI

D1Wox23/Pbpc2/D1Wox10 D1Wox23/Pbpc2/D1Wox10 D1Mgh22/D1Mgh10/D1Mgh11 D1Mgh11/D1Wox23/Pbpc2 D1Mit8 –D1Mgh14 D1Mgh11 –D1Wox23 D1Mgh5/D1Wox6/D1Mit2 Lsn– D1Wox8 Lsn– D1Wox8 D12Mit6 –D12Mgh3 D12Mit6/D12Mgh3/D12Mgh6 D12Mit5/D12Mit6/D12Mgh3/D12Mgh6

2.3 2.5 2.3 3.5 2.1 2.1 2.2 3.5 4.9 3.2 2.7 3.0

5.4 5.8 5.5 8 5.1 5 6.9 8.5 11.9 9 7.8 8.3

Sex Males

Females

Males Females

Note. Intervals demonstrating the highest LODs are indicated in addition to the percentage of the total variance attributable to the QTL. The total LOD for both sexes for a given phenotype over a defined interval was calculated by adding the LODs obtained for each sex.

analyzed. In an attempt to determine the ontogeny and the primary phenotype associated with each of the QTLs, the longitudinal weights and BMIs obtained approximately every 7 days, beginning at Day 15, were analyzed by ANOVA with repeated measures for effects of genotype at the three QTL intervals and for interactions of genotype with sex and age. In Lepr fa/Lepr fa animals, genotype at the marker D1Wox8 demonstrated a small effect of genotype on weight (P Å 0.03), but also demonstrated interactions of genotype with

sex (P Å 0.01), genotype with age (P Å 0.01), and genotype with sex and age (P Å 0.002) (Fig. 4). Similar results were not observed for BMI or for either of the QTLs on chromosome 12 or 16. Significant differences in body weight by genotypic class at D1Wox8 were evident as early as 22 days in the females (13M/13M Å 53.6 g, 13M/WKY Å 49.0 g, and WKY/WKY Å 45.1 g), and sexual dimorphism of weight began at approximately 45 days (Fig. 4). Interestingly, the 13M homozygotes consistently remained

TABLE 4 ANOVA— Grouping by Sex and Genotype at Candidate QTLs— for the Markers Demonstrating Greatest Association with Obesity and/or Diabetes-Related Phenotypes for the Intervals on Chromosomes 1, 12, and 16 Genotype at locus Marker

Phenotype

D1Wox8

13M/13M 13M/WKY WKY/WKY Males

Weight (g) BMI (g/cm 2) HbA1c (%) Plasma insulin concentration (mu/ml) Pancreatic grade Number of intact islets Number of islets D1Mgh11 Weight (g) BMI (g/cm 2) Plasma insulin concentration (mu/ml) Pancreatic grade Number of intact islets Number of islets D12Mit6 Weight (g) BMI (g/cm 2) HbA1c (%) Plasma glucose concentration (mg/dl) D16Mit2 Plasma glucose concentration (mg/dl)

550.8 0.891 12.4 434.1 3.7 27.7 54.3 550.8 0.892 436.7 3.7 26.7 55.3 611.3 0.958 9.8 445 484

595.9 0.948 11.5 599.1 3.1 25.1 49.1 600.8 0.953 628.6 3.0 24.9 49.0 577.3 0.932 11.8 553 473

596.5 0.493 10.0 704.9 2.8 33.4 53.3 592.4 0.94 665.8 2.9 33.8 52.9 566.9 0.908 12.0 521 587

Genotype at locus Pa

0.016 0.012 0.013 0.007 0.0006 0.007 0.008 0.007 0.01 0.0008 0.008

0.005 0.001 0.001

13M/13M 13M/WKY WKY/WKY Females 488.4 0.972 6.1 675.9 2.9 26.3 47.9 484.7 0.971 731.3 2.9 26.7 48.2 503.3 0.995 5.04 317 312

487.8 0.965 5.4 746.7 2.7 23.8 46.3 489.7 0.965 764.2 2.5 23.8 46 481.0 0.95 5.85 324 326

470.0 0.938 5.3 755.1 2.2 36.1 57.4 469.1 0.936 695.1 2.2 34.8 56.6 468.6 0.941 5.65 304 333

Pa

0.0004 0.003

0.003 0.002 0.003 0.002 0.001 0.001

Note. The mean for each genotypic class (by sex) is listed. P value indicated only for phenotypes demonstrating significance at P õ 0.01 (Bonferroni test). a Bonferroni test.

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the female genotypic class with the highest body weight over time (with increasing differences over time). However, this same genotypic class was associated with the lowest body weight in the males by the age of 45 days. Therefore, this genetic cross demonstrated both sex effects and interactions of sex with genotype on weight for the Chr. 1 QTL. The Chr. 1 QTL may have as one of its primary effects a rapid gain in body weight between the ages of 15 and 36 days, the precise early developmental stage that is critical in the remodeling of the pancreatic islets (Finegood et al., 1995). High velocities of weight gain may stress the pancreatic islets and such effects may be exacerbated by the hormonal milieu, which begins to differ between the sexes as the animals reach sexual maturity at approximately 6 weeks. The relevant gonadal hormone in this distinction is likely to be testosterone, since the sexual dimorphism in the WKY strain is not eliminated by ovariectomy of females (Kava et al., 1992). DISCUSSION

In males, rapid weight gain associated with the 13M/ 13M genotype at the QTL on Chr. 1, in combination with increasing testosterone production, could deleteriously affect developmental reorganization of the islets, leading to worse pancreatic grade and decreased number of total and intact islets. Such an effect would promote early diabetes and prevent the increased weight gain associated with the 13M/13M genotype seen in the females that have not had their threshold to diabetes susceptibility increased by ambient testosterone. Tests of this hypothesis will be possible using gonadectomized animals to which exogenous testosterone and estrogen are administered at various ages. In Otsuka–Long–Evans–Tokushima–Fatty rats, orchiectomy decreases the incidence of NIDDM from 100 to 20% in males; ovariectomy increases the incidence of NIDDM from 0 to 30% in females. These results suggest both a detrimental effect of testosterone and a protective effect of estrogen on NIDDM susceptibility (Shi et al., 1994). The balance of sex hormones has been hypothesized to contribute to NIDDM susceptibility in the Lepr db/ Lepr db mouse for which strain-specific differences of sulfotransferase enzymes alter the relative balance of active (to inactive, sulfated), intrahepatic testosterone and estrogen and correlate with NIDDM susceptibility (Leiter, 1989; Leiter et al., 1989). Androgens may also play a role in NIDDM susceptibility in humans. Women with elevated concentrations of blood androgens such as are seen in the polycystic ovary syndrome are at increased risk of developing NIDDM, and of normal women selected at large from the population, those in the lowest quintile of sex hormone-binding globulin (which is inversely related to bioavailability androgens) were at highest risk of developing NIDDM (Bjorntorp, 1996). Additionally, humans also demonstrate decreased insulin sensitivity at the age of puberty in asso-

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ciation with increased gonadal and/or adrenal androgen output (Bloch et al., 1987). Based upon the phenotypes of the 13M and WKY parental strains, it might have been assumed that WKY alleles would be associated with increased susceptibility to NIDDM for each of the QTLs identified. However, of the three NIDDM susceptibility QTLs identified, only those on Chrs. 12 and 16 demonstrated increased diabetes susceptibility with WKY alleles; 13M alleles were associated with increased diabetes susceptibility for the QTL on Chr. 1. Although the Zucker fatty rat (Lepr fa on 13M strain background) is NIDDM-resistant, portions of its genome contain diabetes-susceptibility alleles as evidenced by the diabetes phenotypes of the special subline, Zucker diabetic fatty (Peterson et al., 1990). In the Zucker fatty rat, these 13M NIDDM-susceptibility alleles are ‘‘masked’’ by more potent diabetes-resistance genes. Such susceptibility alleles are probably revealed when 13M is outcrossed to another strain containing relatively less potent diabetes-resistance alleles. Thus, there are evidently additional loci with sufficiently powerful effects on phenotype to counterbalance the ‘‘transgressed’’ 13M susceptibility locus. Therefore, the QTLs on Chrs. 12 and 16, and possibly additional undetected loci, may offset the effects of Chr. 1 in the Zucker fatty rat. The NIDDM susceptibility QTLs we have identified were detected in the context of profound, early onset obesity induced by a point mutation in Lepr. Mutations in Lepr are pleiotropic, having multiple phenotypic effects on glucose and lipid metabolism as well as early effects on b cell function (Zucker and Antoniades, 1972; Polonsky, 1995); however, it has been difficult to distinguish between the primary effects of mutations in Lepr and the secondary effects of increased adiposity. To distinguish effects due to the direct interactions between the NIDDM susceptibility QTLs and Lepr from those due to obesity per se will require additional studies using other rodent models of genetic and diet-induced obesity. If the QTLs for increased adiposity in the GK rat intercrosses (Galli et al., 1996; Gauguier et al., 1996) coincide with our NIDDM susceptibility QTL on Chr. 1 (see below), then it is more likely that this QTL is generally applicable to obesity per se rather than having relevance primarily in the context of mutations in Lepr. The homologous regions of the mouse and human genomes for each of the three rat NIDDM-associated genomic intervals are (Yamada et al., 1994) rat Chr. 1: mouse 7 and 19, human 16q13, 16p11, 11p15, and 11q13; rat Chr. 12: mouse 5, human 7q22; and rat Chr. 16: mouse 14, human 10q22–q23. Several physiologically relevant, positional candidate genes are suggested by these regions of the rat, mouse, or human genomes. For rat Chr. 1 Niddm1 and Nidd/gk1-NIDDM-susceptibility loci identified in the GK rat (Galli et al., 1996; Gauguier et al., 1996); the insulin gene (Ins2); tubby —the phosphodiesterase-like gene defective in the mouse mutant tub that results in

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adult onset, moderate obesity (Kleyn et al., 1996; Noben-Trauth et al., 1996); multigenic obesity 1 (Mob1), which is associated with increased carcass lipid in a mouse intersubspecific backcross (Warden et al., 1993); adult obesity and diabetes (Ad ), an extinct semidominant murine obesity mutation producing moderate obesity and hyperinsulinemia (Trayhurn et al., 1979); insulin-like growth factor 2 (Igf2), which stimulates growth, metabolism, and differentiation (Harvey and Kaye, 1992); and the inwardly rectifying potassium channel (Bir) and the sulfonylurea receptor (Sur) which comprise the b cell ATP– potassium channel complexes that are necessary for regulation of glucose-induced insulin secretion (Inagaki et al., 1995) are all candidate genes based upon their respective roles in regulating growth and adiposity or in mediating b cell function. Insulin-degrading enzyme (Ide), a cytosolic proteinase with high affinity for insulin, which is thought to be involved in the degradation of insulin in insulin-responsive tissues (Affholter et al., 1990), is a candidate gene for the QTL on rat Chr. 16. Of the candidate genes suggested above, two deserve further examination. The Niddm1 and Nidd/gk1 loci were identified with genome-scanning strategies similar to ours using the GK rat, a model of nonobese NIDDM. The GK rat was originally derived by selective breeding from an outbred Wistar stock (Goto et al., 1975; Suzuki et al., 1992) and should be genetically similar to the Wistar strain that we used to convey diabetes susceptibility. It is, therefore, of great interest that in WKY13M Lepr fa/Lepr fa obese rats there is also a major QTL for NIDDM susceptibility on Chr. 1. However, the interval we detected on Chr. 1 is centromeric of the intervals associated in the GK progeny with postprandial hyperglycemia (Galli et al., 1996) or glucose tolerance (Gauguier et al., 1996) and may coincide with the second, centromeric QTL hypothesized by Gauguier et al. to be more directly related to adiposity (Fig. 5). In the GK rat cross, GK homozygosity in this region was associated with increased body weight. This QTL is probably analogous to that for increased weight associated with the WKY allele observed in our Lepr fa/ Lepr fa females. The inherent difficulty in localizing these QTLs, which results from their association with multiple phenotypes and the limited number of molecular markers that have been typed across the relevant intervals in all studies, makes direct comparison of QTL locations among studies problematic. Although our NIDDM-associated QTL on Chr. 1 may recapitulate results in the GK rat, which is genetically similar to the WKY strain, it is also important to note that none of the other QTLs reported by Galli et al. or Gauguier et al. were replicated in our cross of obese rats. The disparity in the results could be due to differences in phenotypic assessment, the counterstrain used (13M versus Brown Norway or Fischer-344), the difference in metabolism associated with extreme obesity, or specific QTL interactions with Lepr. Such differences are important and may provide an opportunity for major

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FIG. 5. Locations of NIDDM-associated QTLs on rat chromosome 1 identified either in the GK rat [GKF or GKBN intercrosses between GK and either Fischer 344 (F) (Galli et al., 1996) or Brown Norway (BN) (Gauguier et al., 1996)] or in the Lepr fa/Lepr fa . The locations of the QTLs are indicated with brackets, and the phenotypes most strongly associated with each QTL are indicated in parentheses. 1 Based on data by Gauguier et al., 1996. 2Based on data by Galli et al., 1996.

mechanistic insights. In any event, these results emphasize the complexity of the NIDDM phenotype and the difficulties that have been and will be encountered in human genetic studies of this disorder (Hanis et al., 1996; Mahtani et al., 1996). In summary, we have identified three rat QTLs for NIDDM susceptibility within the setting of obesity on Chrs. 1, 12, and 16. The Chr. 1 QTL may be the same QTL previously reported as related to body weight and adiposity in GK rat intercrosses and may predispose rats to diabetes by virtue of the dynamic interaction between velocity of early weight gain, insulin resistance, change in sex steroid milieu, and restructuring of the pancreatic islets. ACKNOWLEDGMENTS We gratefully acknowledge the assistance provided by Renata Tenenbaum and L. Arthur Campfield in the radioimmunoassay for plasma insulin concentration and the glucose oxidase assay for plasma glucose concentration, by Yim Dam and Xavier Pi-Sunyer in the radioimmunoassay for pancreatic insulin and glucagon concentrations, by Sarita Whitehead in manuscript preparation, and by Michele Blum, Florence Chu, Johan Helmer, Charles LeDuc, Alexis Leibel, Mark Lindrud, David Markel, Ellen Murphy, Ephraim Tsalik, and Michael Wajnrajch in collection of relevant tissues. This work was supported by NIH Grants DK52431, DK47473, DK26687, and DK07569.

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