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. 2011;6(6):e20832.
doi: 10.1371/journal.pone.0020832. Epub 2011 Jun 16.

Mice with a targeted deletion of the type 2 deiodinase are insulin resistant and susceptible to diet induced obesity

Alessandro Marsili  1 , Cristina Aguayo-Mazzucato, Ting Chen, Aditi Kumar, Mirra Chung, Elaine P Lunsford, John W Harney, Thuy Van-Tran, Elena Gianetti, Waile Ramadan, Cyril Chou, Susan Bonner-Weir, Philip Reed Larsen, Jorge Enrique Silva, Ann Marie Zavacki
Affiliations

Mice with a targeted deletion of the type 2 deiodinase are insulin resistant and susceptible to diet induced obesity

Alessandro Marsili et al. PLoS One. 2011.

Abstract

Background: The type 2 iodothyronine deiodinase (D2) converts the pro-hormone thyroxine into T3 within target tissues. D2 is essential for a full thermogenic response of brown adipose tissue (BAT), and mice with a disrupted Dio2 gene (D2KO) have an impaired response to cold. BAT is also activated by overfeeding.

Methodology/principal findings: After 6-weeks of HFD feeding D2KO mice gained 5.6% more body weight and had 28% more adipose tissue. Oxygen consumption (V0(2)) was not different between genotypes, but D2KO mice had an increased respiratory exchange ratio (RER), suggesting preferential use of carbohydrates. Consistent with this, serum free fatty acids and β-hydroxybutyrate were lower in D2KO mice on a HFD, while hepatic triglycerides were increased and glycogen content decreased. Neither genotype showed glucose intolerance, but D2KO mice had significantly higher insulin levels during GTT independent of diet. Accordingly, during ITT testing D2KO mice had a significantly reduced glucose uptake, consistent with insulin resistance. Gene expression levels in liver, muscle, and brown and white adipose tissue showed no differences that could account for the increased weight gain in D2KO mice. However, D2KO mice have higher PEPCK mRNA in liver suggesting increased gluconeogenesis, which could also contribute to their apparent insulin resistance.

Conclusions/significance: We conclude that the loss of the Dio2 gene has significant metabolic consequences. D2KO mice gain more weight on a HFD, suggesting a role for D2 in protection from diet-induced obesity. Further, D2KO mice appear to have a greater reliance on carbohydrates as a fuel source, and limited ability to mobilize and to burn fat. This results in increased fat storage in adipose tissue, hepatic steatosis, and depletion of liver glycogen in spite of increased gluconeogenesis. D2KO mice are also less responsive to insulin, independent of diet-induced obesity.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Male D2KO mice weight gain and body fat on a HFD.
(A) Body weight of wild type and D2KO mice on chow or a HFD for 6-weeks, n = 22–24 mice/genotype for HFD group, n = 5 mice/genotype for chow group. A significant interaction between genotype and diet were determined by two-way ANOVA for repeated measures (p<0.001; F = 33.55; Df = 312). After Bonferroni correction, there was a significant difference in weight gain of D2KO vs. WT after 4, 5 and 6 weeks of diet (p<0.05, 0.01 and 0.001). (B) Weight gain expressed as % of initial weight after 6 weeks on either chow or a HFD of mice shown in (A). Two-way ANOVA indicated a significant interaction between genotype and diet (2WA g×d) (p<0.05; F = 7.11; Df = 52). (C) % body fat as determined by microCT, n = 4–5 mice/group. Two-way ANOVA indicated a significant interaction between genotype and diet (p<0.05; F = 4.87; Df = 15). (D) Density of voxels falling in the Hounsfield Unit range defined for adipose tissue of a representative wild type and D2KO mouse. Red is more dense while purple is less in the colorbar range. (E) Average food intake of wild type and D2KO mice on either a chow or HFD was monitored for 7 days at 3 weeks of diet and is shown expressed as kcal/day/mouse. Two-way ANOVA indicated that diet significantly effected the overall caloric consumption independent of genotype (2WA d) (p<0.01; F = 10.25; Df = 16). When a significant interaction between genotype and diet was found individual means were compared within groups by unpaired Student's t-test. Data presented are the mean ± SEM; * = p<0.05, ** = p<0.01, *** = p<0.001, ns = not significant.
Figure 2
Figure 2. VO2 consumption and RER of wild type and D2KO mice on a HFD.
(A) 24-hour VO2 profile of wild type and D2KO mice on a HFD. Each point represents the mean of 5 mice measured every 35 minutes. The black bar below the X-axis represents the dark period during which mice would be feeding. (B) Histograms representative of the areas under the curve of (A). (C) 24-hour RER of wild type and D2KO mice on a HFD. Each point represents the mean of 5 mice measured every 35 minutes. (D) Histograms representative of the areas under the curve of (C) Data presented are the mean ± SEM; * = p<0.05, ns = not significant.
Figure 3
Figure 3. Glucose tolerance testing and corresponding insulin levels of wild type and D2KO mice on a chow and HFD.
(A) Wild type and D2KO mice maintained on a chow diet were fasted for 14 h, and then injected IP with 2 g/kg D-glucose at time 0 indicated by the arrow. Blood glucose levels were measured at the indicated time, and are not significantly different by two-way ANOVA for repeated measures. (B) Histograms representative of the areas under the curve of (A). (C) Corresponding serum insulin levels of mice in (A). A significant difference was found by two-way ANOVA for repeated measures after Bonferroni correction at 120′ (p<0.05). D) Histograms representative of the areas under the curve of (C). (E) Same as (A) except mice were maintained for 6-weeks on a HFD prior to testing, values are not significantly different by two-way ANOVA for repeated measures. (F) Histograms representative of the area under the curve of (E). (G) Corresponding serum insulin levels of mice in (E). A significant interaction was found by two-way ANOVA for repeated measures (p<0.05; F = 4.26, Df = 16). A significant difference was found after Bonferroni correction at 120′ (p<0.01). (H) Histograms representative of the area under the curve of (G). N = 5–6 mice/group, male mice were 15 weeks old time of at testing. Data shown are the mean ± SEM with * = p<0.05, ** = p<0.01, ns = not significant.
Figure 4
Figure 4. Insulin tolerance testing of wild type and D2KO mice on chow and HFD.
(A) wild type and D2KO mice maintained on a chow diet were fasted for 14 h, and then injected IP with 0.5 mU/g body weight insulin at time 0 indicated by the arrow. A significant difference was found by two-way ANOVA for repeated measures after Bonferroni correction at 90′ (p<0.05). (B) Histograms representative of the area under the curve of (A). (C) Same as (A) except mice were maintained for 6-weeks on a HFD prior to testing. Two-way ANOVA for repeated measures showed a significant interaction (p<0.05; F = 3.09; Df = 55). Significant differences were found after Bonferroni correction at 90′ and 120′ (p<0.001 and p<0.01) (D) Histograms representative of the area under the curve of (C). N = 5–7 mice/group, male mice were 15 weeks old time of at testing, data shown are the mean ± SEM with * = p<0.05, ** = p<0.01, *** = p<0.001.
Figure 5
Figure 5. Serum and liver biochemistry of wild type and D2KO mice on chow and HFD.
Levels of T3 (A) T4 (B), (C) triglycerides, (D) free fatty acids, or (E) β-hydroxybutyrate in serum are shown. Hepatic levels of (F) triglycerides or (G) glycogen are also indicated. Data are analyzed by two-way ANOVA. There was a significant effect of diet (2WA d) on T3 (p<0.001; F = 16.45; Df = 19) and T4 (p<0.05; F = 4.73; Df = 20) independent of genotype. Two-way ANOVA indicated a significant interaction between diet and genotype (2WA g×d) for serum FFA (p<0.001; F = 4.91; Df = 20); β-hydroxybutyrate (p<0.01; F = 8.55; Df = 19), hepatic triglycerides (p<0.05; F = 6.31; Df = 19) and hepatic glycogen (p<0.05; F = 5.37; Df = 20). When a significant interaction between genotype and diet was found individual means were compared within groups by unpaired Student's t-test and showed in the figure. Data shown are the mean ± SEM of 4–5 mice/group on chow diet or 7–10 mice/group on HFD with * = p<0.05, ** = p<0.01, *** = p<0.001, ns = not significant.
Figure 6
Figure 6. Levels of gene expression in wild type and D2KO mice on chow and HFD.
mRNA levels of the indicated genes were measured using qRT PCR, and then corrected by Cyclophillin A expression as a house-keeping gene. Data is normalized to expression of wild type mice on a chow diet, and expression levels in (A) BAT, (B) WAT, (C) liver, (D) soleus, and (E) vastus lateralis are shown. Significant effects were determined by two-way ANOVA. A significant interaction between genotype and diet (2WA g×d) was found in vastus lateralis for GLUT4 (p<0.01; F = 8.79; Df = 16). A significant effect of diet (2WA d) independent of genotype was found in BAT for ACC1 In BAT (p<0.05; F = 6.02; Df = 16), in WAT for ACC1 (p<0.05; F = 5.41; Df = 16), in liver for PPARα(p<0.05; F = 6.0; Df = 16), PEPCK (p<0.001; F = 30.19; Df = 16), αGPD (p<0.01; F = 12.21; Df = 16), CPT1α (p<0.001; F = 21.87; Df = 16), and in soleus for MCD (p<0.01; F = 6.95; Df = 8). A significant effect of genotype (2WA g) independent of diet was found in liver for PEPCK (p<0.01; F = 8.91; Df = 16) and in vastus lateralis for GYS1 (p<0.05; F = 8.02; Df = 16) and HK (p<0.01; F = 5.9; Df = 16). When a significant interaction between genotype and diet was found individual means were compared within groups by unpaired Student's t-test. N = 5 mice/group except for soleus were n = 3–4 with each sample being solei pooled from 3 mice. Data shown are the mean ± SEM; * = p<0.05, ** = p<0.01, *** = p<0.001, ns =  not significant.
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