ATRAP Expression in Brown Adipose Tissue Does Not Influence the Development of Diet-Induced Metabolic Disorders in Mice

Figures

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  Figure 1. Generation of adipose tissue-specific angiotensin II type-1 receptor-associated protein (ATRAP) downregulated mice. (A) Schematic representation of Adipoq-Cre mediated recombination in adipocytes: (a) wild-type allele; (b) targeting vector; (c) targeted allele; (d) Flp recombinase-mediated allele; and (e) Cre-mediated allele. (B) Agarose-gel electrophoresis of PCR products amplified with following primer sets, (a) F23005 and R23744, (b) F24587 and R24917, and (c) Cre-fw and Cre-rw for Cre transgene, mAgtrapChIPF and mAgtrapChIPR for Agtrap gene as an internal control. These primer sequences are described in Table 1. (C) The relative ATRAP mRNA levels in each tissue (WAT, BAT, liver, muscle, kidney, heart, and brain) of ATRAPadipoq and ATRAPfl/fl mice aged 19–24 weeks on an LFD (n = 3–10). (D) Representative Western blots of ATRAP protein expression in WAT and BAT of ATRAPfl/fl mice on an LFD. Values are the means ± standard error of the mean (SEM). † p < 0.05, †† p < 0.01 vs. ATRAPfl/fl mice. Data were analyzed by unpaired Student’s t-test. EX: Exon; PGK-Neo: Phosphoglycerate kinase 1 promoter driven neomycin resistant gene; PGK-TK: Phosphoglycerate kinase 1 promoter driven SV40…

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  Figure 2. The relative expression of renin–angiotensin system (RAS) components in adipose tissues. Relative angiotensin II type-1 receptor (AT1R) mRNA (A) and angiotensinogen mRNA (B) expression in WAT and BAT of ATRAPfl/fl and ATRAPadipoq mice fed an LFD (n = 5–6). Values are the means ± standard error of the mean (SEM). AT1R: Angiotensin II type1 receptor; WAT: White adipose tissue; BAT: Brown adipose tissue; LFD: Low-fat diet.

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  Figure 3. Change in body weight in ATRAPfl/fl and ATRAPadipoq mice fed an LFD or HFD (n = 9). ∆, ATRAPfl/fl mice fed an LFD; ▲, ATRAPadipoq mice fed an LFD; ○, ATRAPfl/fl mice fed an HFD; ●, ATRAPadipoq mice fed an HFD. Values are the means ± SEM. *** p < 0.001 vs. LFD within the same group. Data were analyzed by two-way repeated measures analysis of variance (ANOVA). LFD: Low-fat diet; HFD: High-fat diet.

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  Figure 4. Glucose tolerance test (GTT) and insulin tolerance test (ITT). (A,B) The GTT and ITT in ATRAPfl/fl and ATRAPadipoq mice after 16-week LFD or HFD feeding (n = 5–15). ∆, ATRAPfl/fl mice fed an LFD; ▲, ATRAPadipoq mice fed an LFD; ○, ATRAPfl/fl mice fed an HFD; ●, ATRAPadipoq mice fed an HFD; (C,D) The area under the curve (AUC) of GTT and ITT in ATRAPfl/fl and ATRAPadipoq mice after 16-week LFD or HFD feeding (n = 5–15). Values are the means ± SEM. (A,B) *** p < 0.001 vs. LFD within the same group. Data were analyzed by two-way repeated measures ANOVA. (C,D) *** p < 0.001 vs. LFD within the same group. Data were analyzed by two-way ANOVA. LFD: Low-fat diet; HFD: High-fat diet.

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  Figure 5. Adipocyte morphology and inflammation of WAT. (A) Representative images of adipocyte cells in WAT of ATRAPfl/fl and ATRAPadipoq mice fed an LFD and HFD. (B) Relative monocyte chemotactic protein-1 (MCP-1) and F4/80 mRNA expression in WAT of ATRAPfl/fl and ATRAPadipoq mice fed an LFD and HFD (n = 5–6). Values are the means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. LFD within the same group. Data were analyzed by two-way ANOVA. LFD: Low-fat diet; HFD: High-fat diet.

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  Figure 6. The changes of ATRAP expression in adipose tissues and liver with HFD. (A) Relative WAT ATRAP mRNA expression in ATRAPfl/fl and ATRAPadipoq mice fed an LFD and HFD (n = 6). (B) Relative BAT ATRAP mRNA expression in ATRAPfl/fl and ATRAPadipoq mice fed an LFD and HFD (n = 6). Relative liver ATRAP mRNA expression in ATRAPfl/fl mice fed an LFD and HFD (n = 6). Values are the means ± SEM. (A, B) † p < 0.05, ††† p < 0.001 vs. ATRAPfl/fl mice on the same diet. * p < 0.05, *** p < 0.001 vs. LFD within the same group. Data were analyzed by two-way ANOVA. (C) * p < 0.05 vs. LFD group. Data were analyzed by an unpaired Student’s t-test. LFD: Low-fat diet; HFD: High-fat diet.

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  Figure 7. Agarose-gel electrophoresis of PCR products from embryonic stem (ES) cell clones’ DNA (#a2925–a2930, #a3006–a3020), amplified for homologous recombination (A) and for detecting second loxP (B). (A) 5′ PCR using primer set: 5AF4/neo100 (upper panel) and 3′ PCR using primer set: neo marker sense/3AR2 (lower panel); red-labeled clones were positive for amplification. (B) PCR using primer set: F24587/R2491; red-labeled clones were positive for amplification of mutant loxP within positive for homologous recombination.

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  Figure 8. Southern blot analysis using neo probe. 5′ EcoRI-digested (target mutant band is 8.3 kb in upper panel) and 3′ NdeI-digested (target mutant band is 10.3 kb in lower panel) DNA were analyzed. #a3006 and #a3008 clones (in red) were verified to have one inserted copy of the targeting vector.

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  Figure 9. Agarose-gel electrophoresis of PCR products amplified to detect first loxP using the primer set F23005 and R23744. Analyzed DNAs (F3-1–F3-15) were from F3 mice with heterozygous Agtrap floxed allele from which the PGK-Neo cassette was removed. The red-labeled sample were positive for loxP site amplification.