Metabolic characterization of a mouse deficient in all known leptin receptor isoforms

Abstract

We have characterized a newly generated mouse model of obesity, a mouse strain deficient in all five previously described leptin receptor isoforms. These transgenic mice, named the db (333)/db (333) mice, were identified from an ENU mutagenesis screen and carry a point mutation in the seventh exon of the db gene encoding the leptin receptor, resulting in a premature stop codon (Y(333)Stop) and gene product that lacks STAT signaling domains. db (333)/db (333) mice have a morbidly obese phenotype, with body weights diverging from wild type as early as 4 weeks of age (P < 0.05). To determine the contribution of the short isoforms of the leptin receptor in this metabolic phenotype, we performed an extensive metabolic characterization of the db (333)/db (333) mouse in relation to the well-characterized db/db mouse lacking only the long form of the leptin receptor. db (333)/db (333) mice have similar endocrine and metabolic parameters as previously described in other leptin receptor transgenic mice including db/db mice that lack only the long isoform of the leptin receptor. However, db (333)/db (333) mice show a subtle trend toward higher body weight and insulin levels, lower oxygen, carbon dioxide production, respiratory exchange ratio (RER), and temperature than db/db mice suggesting the short isoforms may play an additional role in energy homeostasis.

Fig. 1

a The db₃₃₃/db₃₃₃ mutation. DNA sequence chromatograms from homozygous mutant db₃₃₃/db₃₃₃ mice (TAA/TAA), heterozygous db₃₃₃/db₃₃₃ mice (TAA/TAT), and C57Bl/6 WT mice (TAT/TAT). db₃₃₃/db₃₃₃ mice have a point mutation in the leptin receptor gene causing a premature stop codon. The db₃₃₃/db₃₃₃ mouse has a T to A transversion (TAT → TAA) in the seventh coding exon of the leptin receptor gene, resulting in a premature stop codon at Tyr₃₃₃. b Schematic presentation of leptin receptor mutations in mouse models of obesity with leptin signaling deficiencies. The predicted protein length is shown with the numbers at the end of each receptor representing the amino acid residue at the carboxy terminus. The STAT-3 binding site is located at amino acid 1138 and has an important role in the regulation of energy balance through the JAK/STAT signaling pathway

Fig. 2

Phenotypic characterization of the db ₃₃₃/db ₃₃₃ mice. a Photograph of db ₃₃₃/db ₃₃₃ and WT littermate. b Body weights of db ₃₃₃/db ₃₃₃ and WT mice at 6 months of age. c db ₃₃₃/db ₃₃₃ mice have significantly increased omental, mesenteric and subcutaneous fat, and increased liver and brown adipose tissue (BAT) weight compared with WT littermates. d Food intake in db ₃₃₃/db ₃₃₃ and WT mice during a 24 h monitoring period. e db ₃₃₃/db ₃₃₃ mice are hyperphagic and consume significantly more food than WT littermates in the dark cycle (db ₃₃₃/db ₃₃₃ 3.95 ± 0.05 g, WT 0.78 ± 0.05 g). Significant (P < 0.05) differences between group means were determined by a two-tailed independent T test and denoted by asterisk. Data are presented as means ± SE

Fig. 3

Growth curve of male db ₃₃₃/db ₃₃₃, db/db and WT mice. Both db ₃₃₃/db ₃₃₃ and db/db mice have significantly greater body weights than WT littermates from as early as 4 weeks of age and throughout this 24 weeks monitoring period. db ₃₃₃/db ₃₃₃ mice display consistently higher body weights than db/db mice from 8 to 24 weeks of age, but this trend only reached statistical significance at one time point (“†” P < 0.05 at 12 weeks). At 24 weeks of age db ₃₃₃/db _333, mice display a higher body weight than db/db mice, but this trend is not statistically significant at this time point. At 24 weeks of age db ₃₃₃/db ₃₃₃ and db/db, mice have significantly greater body weights than WT littermates (db ₃₃₃/db ₃₃₃ 65.0 ± 5.9 g; db/db 60.25 ± 1.38 g, WT 36.25 ± 1.70 g, P < 0.05). Data represent the mean of 5 male mice per genotype weighed monthly from 4 to 24 weeks of age. Significant (P < 0.05) differences between db ₃₃₃/db ₃₃₃ and WT group means are denoted by asterisk, differences between db/db and db ₃₃₃/db ₃₃₃ mice are denoted by “†”. All significant differences were determined by one way ANOVA and post hoc Tukey test. Data are presented as means ± SE

Fig. 4

a Insulin sensitivity test in db₃₃₃/db₃₃₃, db/db, and WT mice. Intraperitoneal insulin administration (1 unit per kg of body weight) revealed that both db₃₃₃/db₃₃₃ and db/db mice have an impaired glucose disposal ability relative to the hypoglycemic response to insulin observed in the WT littermates. Data are presented as percentage change in blood glucose from baseline before and after insulin injection for up to 120 min. b Intraperitoneal glucose tolerance test in db₃₃₃/db₃₃₃, db/db, and WT mice. After glucose challenge db₃₃₃/db₃₃₃ and db/db mice display impaired glucose disposal compared with WT mice. Data are plotted as mean blood glucose ± SE for n = 5 animals of each genotype before and after intraperitoneal glucose injection for up to 120 min. Significant (P < 0.05) differences between db₃₃₃ and WT group means are denoted by asterisk and were determined by one way ANOVA and post hoc Tukey test

Fig. 5

Core body temperature and spontaneous locomotor activity in db ₃₃₃/db ₃₃₃, db/db, and WT mice during a 24 h monitoring period. a db ₃₃₃/db ₃₃₃ and db/db mice display a significantly lower CBT relative to WT mice. b Average CBT over the 12 h light or dark cycle in db ₃₃₃/db ₃₃₃ and db/db mice is significantly lower when compared with WT littermates. c Average spontaneous physical activity of db ₃₃₃/db ₃₃₃, db/db, and WT mice during a 24 h monitoring period. db ₃₃₃/db ₃₃₃ and db/db mice display decreased locomotor activity relative to WT mice. d Average activity for db ₃₃₃/db ₃₃₃, db/db, and WT mice during the light and dark cycle. Both db ₃₃₃/db ₃₃₃ and db/db mice display lower average activity than WT mice. Data are plotted as mean ± SE for n = 4 animals of each genotype. Significant (P < 0.05) differences between group means are denoted by asterisk and were determined by one way ANOVA and post hoc Tukey test

Fig. 6

Effects of icv leptin administration on food intake in db ₃₃₃/db ₃₃₃, db/db, and WT mice. Injection of leptin (icv 4 μg/μl) caused a significant reduction in food intake in WT mice (P = 0.0038) while no significant appetite suppression was observed in db ₃₃₃/db ₃₃₃ or db/db mice. Data are plotted as mean ± SE for n = 4 animals of each genotype. Significant differences between group means are denoted by asterisk and were determined by one way ANOVA and post hoc Tukey test

Fig. 7

Metabolic comparison of db ₃₃₃/db ₃₃₃, db/db, and WT mice during a 24 h monitoring period. a Oxygen consumption in db ₃₃₃/db ₃₃₃, db/db, and WT mice monitored for 24 h. Both db/db and db ₃₃₃/db ₃₃₃ mice displayed similar oxygen consumption at all time points, and these obese mutants showed significantly lower oxygen consumption than WT mice throughout both the light and dark cycle. b CO₂ production in db ₃₃₃/db ₃₃₃, db/db, and WT mice monitored for 24 h. Both db/db and db ₃₃₃/db ₃₃₃ mice displayed similar levels of CO₂ production at all time points, and these obese mutants showed significantly lower CO₂ production than WT mice throughout both the light and dark cycle. c RER (VCO₂/VO₂) in db ₃₃₃/db ₃₃₃, db/db, and WT mice over a 24 h period. Both db ₃₃₃/db ₃₃₃ and db/db show a trend of lower RER relative to WT mice that is most pronounced in the dark cycle. db ₃₃₃/db ₃₃₃ mice show a consistently lower RER relative to db/db and WT mice. Data are plotted as mean ± SE for n = 4 animals of each genotype. Significant (P < 0.05) differences between group means are denoted by asterisk and were determined by one way ANOVA and post hoc Tukey test