Differential effects of leptin receptor mutation on male and female BBDR Gimap5-/Gimap5- spontaneously diabetic rats

Abstract

Rodents homozygous for autosomal leptin receptor gene mutations not only become obese, insulin resistant, and hyperleptinemic but also develop a dysregulated immune system. Using marker-assisted breeding to introgress the Koletsky rat leptin receptor mutant (lepr-/lepr-), we developed a novel congenic BBDR.(lepr-/lepr-) rat line to study the development of obesity and type 2 diabetes (T2D) in the BioBreeding (BB) diabetes-resistant (DR) rat. While heterozygous lepr (-/+) or homozygous (+/+) BBDR rats remained lean and metabolically normal, at 3 wk of age all BBDR.(lepr-/lepr-) rats were obese without hyperglycemia. Between 45 and 70 days of age, male but not female obese rats developed T2D. We had previously developed congenic BBDR.(Gimap5-/Gimap5-) rats, which carry an autosomal frameshift mutation in the Gimap5 gene linked to lymphopenia and spontaneous development of type 1 diabetes (T1D) without sex differences. Because the autoimmune-mediated destruction of pancreatic islet beta-cells may be affected not only by obesity but also by the absence of leptin receptor signaling, we next generated BBDR.(lepr-/lepr-,Gimap5-/Gimap5-) double congenic rats carrying the mutation for Gimap5 and T1D as well as the Lepr mutation for obesity and T2D. The hyperleptinemia rescued end-stage islets in BBDR.(lepr-/lepr-,Gimap5-/Gimap5-) congenic rats and induced an increase in islet size in both sexes, while T1D development was delayed and reduced only in females. These results demonstrate that obesity and T2D induced by introgression of the Koletsky leptin receptor mutation in the BBDR rat result in islet expansion associated with protection from T1D in female but not male BBDR.(lepr-/lepr-,Gimap5-/Gimap5-) congenic rats. BBDR.(lepr-/lepr-,Gimap5-/Gimap5-) congenic rats should prove valuable to study interactions between lack of leptin receptor signaling, obesity, and sex-specific T2D and T1D.

Fig. 1.

Breeding strategy (left) and genotyping of chromosome 5 (right). Left: the breeding approach of total N15 backcrosses that led to the generation of the new congenic line. The first intercross and from N2 to N8 backcrosses were generated in the laboratory of Dr. J. T. Hansen at the National Institutes of Health (NIH). From N9 to N15 backcrosses were produced in the Robert H. Williams Laboratory, University of Washington, of Å. Lernmark. At each backcross the DNA was sent to Seattle for genotyping assessment. Numbers and names in and below each box represent rat ID. Right: simple sequence length polymorphism (SSLP) marker map of chromosome 5. Marker annotations are shown together with the University of California-Santa Cruz (UCSC) map marker location and the size of the marker in the 3 parental strain BioBreeding diabetes-resistant (BBDR) and Koletsky rats. Purple, localization of the Lepr gene expanded from 122.320.075 bp to 122.503.437 bp with the marker D5Wox39 inside the DNA fragment that comprised the gene; blue, markers polymorphic between the parental strains. Animals in lanes 1–3 represent the parental and new Lepr−/− congenic line. The animal in lane 1 is diabetes resistant (DR), lane 2 Koletsky (Kol), lane 3 BBDR.^Lepr− (D5Rat98-D5Rat233)Rhw. The numerical color code is green (2) DR/DR and yellow (1) Kol/Kol. Colors in left panel correspond with colors in lanes 1 and 3 in right panel.

Fig. 2.

Time course of changes in body weight and blood glucose of male and female congenic rats. Rat body weights (A and C) and blood glucose (B and D) of DR.^{lepr−/lepr−} [n = 3 (○) and n = 4 (□)] and DR.^+/+ [n = 4 (● and ■)]. All rats were monitored daily from 30 to 180 days of age. Data are means ± SE. Most bars are not visible because of small SE. P < 0.05 vs. age-matched wild-type control group, which persisted up to 180 days of age (ANOVA). Data for DR.^{lepr−/lepr+} were the same as for DR.^+/+. E: representative image of the 3 genotypes at 45 days of age.

Fig. 3.

Values for cholesterol, triglycerides, glucagon, insulin, and leptin in serum of female and male DR.^+/+, DR.^{lepr−/lepr−}, and DR.^{lepr−/lepr−,Gimap5−/Gimap5−} rats. Serum samples were evaluated at 180 days of age for the +/+ (open bars) and lepr−/lepr− (hatched bars) rats and at 150 days of age for lepr−/lepr−,Gimap5−/Gimap5− (filled bars) double congenic animals. Bars that share common symbols in the same chart are significantly different (P < 0.05) (Student's t-test). Leptin levels in DR.^{lepr−/lepr−,Gimap5−/Gimap5−}: female > male (P = 0.051). Data are means ± SE (n = 3–5/group).

Fig. 4.

Kaplan-Meier survival estimates in male (A) and female (B) rats. A: days of age of diabetes onset in DR.^+/+ [n = 9 (■)], DR.^{lepr−/lepr−} [n = 16 (○)], DR.^{Gimap5−/Gimap5−} [n = 11 (□)], and DR.^{lepr−/lepr−,Gimap5−/Gimap5−} [n = 5 (●)] male rats. B: time of diabetes onset in DR.^+/+ and DR.^{lepr−/lepr−} [n = 6 for each genotype (■)], DR.^{Gimap5−/Gimap5−} [n = 12 (□)], and DR.^{lepr−/lepr−,Gimap5−/Gimap5−} [n = 10 (●)] female rats.

Fig. 5.

Representative hematoxylin and eosin (H & E)-stained histological and insulin and glucagon immunofluorescent sections from female (A–F) and male (G–L) rats. Wild-type DR.^+/+ (left); DR.^{lepr−/lepr−} (center), and DR.^{lepr−/lepr−,Gimap5−/Gimap5−} (right) rats are shown. Age and no. of days with diabetes: DR.^+/+ control rat at 180 days of age (A, D, G, and J); nondiabetic female DR.^{lepr−/lepr−} rat at 180 days of age (B and E); 113-day diabetic DR.^{lepr−/lepr−} male rat at 180 days of age (H and K); 27-day diabetic DR.^{lepr−/lepr−,Gimap5−/Gimap5−} rat at 150 days of age (C and F), and 36-day diabetic DR.^{lepr−/lepr−,Gimap5−/Gimap5−} rats at 81 days of age (I and L). Wild-type islets are clearly demarcated from exocrine acinar cells with central insulin-producing β-cells (green) surrounded by a ring of glucagon-producing α-cells (red) in females (A and D) and males (G and J). In contrast, DR.^{lepr−/lepr−} islets in females (B and E) and males (H and K) are enlarged with an irregular shape, encroachment, and occasional entrapment of acinar exocrine cells (black arrowheads). Immunofluorescent sections stained for insulin and glucagon confirm that the enlarged islets noted on H & E-stained sections are composed of abundant β-cells and disrupted α-cells (E and K, open arrowheads) with multifocal black regions within the islets likely representing entrapped acinar cells (B and H, black arrowheads), lipocytes (B, #), ducts (B, *) or regions of inflammatory (H, black arrows) or degenerate islet cells characterized by enlarged cells with condensed to pyknotic nuclei, occasional karyorrhexis, and vacuolated cytoplasm (H, white arrow). Note the relative increase in degenerate cells within the male DR.^{lepr−/lepr−} islet compared with the female DR.^{lepr−/lepr−} islet. Enlarged islets were also noted in DR.^{lepr−/lepr−,Gimap5−/Gimap5−} double congenic rats (C and F, female; I and L, male), immunostaining again confirming that enlarged islets contained abundant β-cells and disrupted α-cells (F and L, open arrowheads). Note the perivascular, periductular, and intraislet mixed inflammation (C, inset and I, black arrow) composed of lymphocytes and macrophages, occasionally hemosiderin laden (I, arrow), eosinophils (C, inset and black arrows), and neutrophils.

Fig. 6.

Score assessment of pancreatic islet size (top), degeneration (middle), and inflammation (bottom). Values for female (1–4) and male (5–8) rats. Open bars, DR.^+/+ (1 and 5); hatched bars, DR.^{lepr−/lepr−} (2 and 6); checkerboard bars, DR.^{Gimap5−/Gimap5−} (3 and 7); filled bars, DR.^{lepr−/lepr−,Gimap5−/Gimap5−} (4 and 8). Ages for scores are as follows: 1, 2, 5, and 6, 180 days of age; 3 and 7, a day after diabetes onset; 4, 150 days of age; 8, between 15 and 27 days after diabetes onset. Groups marked by common symbols in the same chart are significantly different (P < 0.05). Data are means ± SE (n = 3–5/group).