Specific knockout of p85α in brown adipose tissue induces resistance to high-fat diet–induced obesity and its metabolic complications in male mice

Figures

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  Figure 1. Characterization of BATp85αKO mice. (A) Representative gels of PCR for p85αloxP/loxP (upper panel) and UCP-1 Cre recombinase. In the upper gel, a 460-bp band was obtained for the floxed allele and a 270-bp band for the WT allele. As positive control, p85αloxP/+ mice were used to obtain two bands. In the lower gel, a 600-bp band and a 285-bp band were obtained for BATp85αKO mice and only a 285-bp band for control mice. (B) The Cre-mediated deletion of exon 7 of the p85α gene was verified by PCR. We observed a 225-bp fragment from the undeleted p85 loxP allele and a 103-bp fragment from the deleted allele. (C) Representative microphotographs of immunohistochemistry against p85α and its quantification to assess the presence or absence of this regulatory subunit in BAT from BATp85αKO or control mice, respectively. Image magnification: 20 ×. Negative controls without the primary antibody were included to check for nonspecific staining. Results are expressed as mean ± SEM. Statistical significance was assessed by two-tailed unpaired t-test by comparison with control mice (*p < 0.05).

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  Figure 2. Characterization of BATp85αKO mice at 6 and 12 months of age. (A) Representative microphotographs of BAT, gWAT, or iWAT, stained with hematoxylin and eosin, in control or BATp85αKO mice at 12 months of age. Image magnification: 20 × . (B) Size quantification of brown or white adipocytes from control and BATp85αKO mice at 12 months of age. (C) Graph of body temperature at different times to analyze the thermogenic response to cold exposure in control and BATp85αKO mice at 12 months of age. Results are expressed as mean ± SEM. Statistical significance was assessed by two-tailed unpaired t-test by comparison with control 12-month-old mice (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001). Twelve-month-old control (n = 10); 12-month-old BATp85αKO (n = 6). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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  Figure 3. Brown fat functionality in BATp85αKO mice under HFD. (A) Representative microphotographs and quantification of UCP-1 protein in BAT from control and BATp85αKO mice by immunohistochemistry. (B) qRT-PCR analysis of Ucp1 and Prdm16 mRNA levels in BAT from the different groups studied. Thus, the amount of target, normalized to endogenous gene and relative to the control, is given by qRT-PCR. The value represented on the y axis is the RQ, being: [(RQ) = 2−ΔΔCt; ΔCt (cycle threshold) = Ct (target gene) – Ct (Gapdh); ΔΔCt = ΔCt for any sample – ΔCt for the control]. Amplification of Gapdh was used in the same reaction of all samples as an internal control. We have used ΔΔCt referred to control STD. (C) Graphs of body temperature at different times (0–8 h, each hour) to analyze the thermogenic response to cold exposure from control and BATp85αKO mice under an HFD. Results are expressed as mean ± SEM. Statistical significance was carried out by one-way ANOVA with Bonferroni and Tukey post tests in B and C and two-tailed unpaired t-test in D by comparison with the control STD group (*p < 0.05, **p < 0.01, and ***p < 0.001) and with the control HFD group (##p < 0.01 and ###p < 0.001).…

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  Figure 4. The lack of p85α in BAT reduces obesity and adipocyte size. (A) Body weight (BW) (g), (B) BAT (mg)/BW (g) ratio, and (C) WAT (mg)/BW (g) ratio from control STD, BATp85αKO STD, control HFD, and BATp85αKO HFD groups. (D) Representative microphotographs and quantification of BAT, gWAT, or iWAT stained with hematoxylin and eosin from control and BATp85αKO HFD groups. Image magnification: 20×. Control STD (n = 5); control HFD (n = 8); BATp85αKO STD (n = 5); BATp85αKO HFD (n = 7). Results are expressed as mean ± SEM. Statistical significance was carried out by one-way ANOVA with Bonferroni and Tukey posttests by comparison with control STD mice (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001), with BATp85αKO STD mice (†p < 0.05, ††p < 0.01, †††p < 0.001, and ††††p < 0.0001), and with control HFD mice (#p < 0.05 and ###p < 0.001).

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  Figure 5. The lack of p85α in BAT reduces obesity, fatty liver, and insulin resistance. (A) Representative nuclear magnetic resonance images from the four groups studied to assess the percentage of fat volume versus total volume. (B) Plasma cholesterol and triglyceride levels from the four groups studied. (C) Representative microphotographs and quantification of livers stained with Oil Red O contrasted with hematoxylin to identify lipid depots. Image magnification: 10 × . (D) Glucose and insulin plasma levels of fasted mice by automatic monitor or by ELISA, respectively. (E) Glucose tolerance test (GTT) (upper) and insulin tolerance test (ITT) (lower) after 10 weeks on an HFD were performed in control HFD and BATp85αKO HFD nice. Control STD (n = 5); control HFD (n = 8); BATp85αKO STD (n = 5); BATp85αKO HFD (n = 7). Results are expressed as mean ± SEM. Statistical significance was carried out by one-way ANOVA with Bonferroni and Tukey posttests in A, B, and C; two-way ANOVA with Sidak posttest in D; and two-tailed unpaired t-test in E by comparison with the control STD group (*p < 0.05, **p < 0.01, and ***p < 0.001), with the BATp85αKO STD group (††p < 0.01 and ††††p < 0.0001), and with the…

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  Figure 6. The lack of p85α in BAT improves insulin signaling in adipose depots through a lower JNK activation and a higher IRB expression. (A) Western blot analysis of Akt phosphorylation in response to insulin in control versus BATp85αKO mice fed an HFD. Representative gels and quantification of in vivo insulin signaling studies performed in both groups after 10 weeks on an HFD. In BAT: control HFD (n = 7, with saline [n = 2] and with insulin [n = 5]), BATp85αKO HFD (n = 6, with saline [n = 2] and with insulin [n = 4]); in gWAT: control HFD (n = 7, with saline [n = 3] and with insulin [n = 4]), BATp85αKO HFD (n = 7, with saline [n = 3] and with insulin [n = 4]); and in iWAT: control HFD (n = 7, with saline [n = 2] and with insulin [n = 5]), BATp85αKO HFD (n = 8, with saline [n = 2] and with insulin [n = 6]). (B) Representative gels and quantification of JNK phosphorylation (T183/Y185) by Western blot of BAT from control HFD and BATp85αKO HFD mice. β-actin was used as a loading control. Control HFD (n = 6); BATp85αKO HFD (n = 7). (C) Representative gels and quantifications of IRA (left panels) and IRB (right panels) protein levels and their association with IRS-1 in BAT from control and…

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  Figure 7. The lack of p85α in BAT modified the expression profile of adipokines in the adipose organ.Tnfa, Leptin, or Adipoq mRNA levels were analyzed by qRT-PCR in (A) BAT, (B) gWAT, and (C) iWAT from the four groups studied. Thus, the amount of target, normalized to endogenous gene and relative to the control, is given by qRT-PCR. The value represented on the y axis is the RQ, being: [(RQ) = 2−ΔΔCt; ΔCt (cycle threshold) = Ct (target gene) – Ct (Gapdh); ΔΔCt = ΔCt for any sample – ΔCt for the control]. Amplification of Gapdh was used in the same reaction of all samples as an internal control. We have used ΔΔCt referred to control STD. Results are expressed as mean ± SEM. Statistical significance was assessed by one-way ANOVA with Bonferroni and Tukey posttests by comparison with control STD mice (*p < 0.05, **p < 0.01, and ***p < 0.001), with BATp85αKO STD mice (†p < 0.05, ††p < 0.01, and †††p < 0.001), and with control HFD mice (#p < 0.05, ##p < 0.01, and ###p < 0.001). Control STD (n = 5); control HFD (n = 8); BATp85αKO STD (n = 5); BATp85αKO HFD (n = 7).

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  Figure 8. The lack of p85α in BAT might induce iWAT browning. (A) Analysis of Ucp1 mRNA levels in gWAT or iWAT from the four groups studied by qRT-PCR. Gapdh was used as an endogenous target. The value represented on the y axis is the RQ and is referred to control STD. Western blot analysis of (B) UCP-1 and (C) p-AMPK protein levels in iWAT from the four groups studied. (D) Representative microphotographs and their quantification of UCP-1 protein by immunohistochemistry in iWAT from control HFD and BATp85αKO HFD mice. Image magnification: 10× and 20×. (E) Analysis of Prdm16 mRNA levels in iWAT from control HFD and BATp85αKO HFD mice by qRT-PCR. Gapdh was used as an endogenous target. The value represented on the y axis is the RQ and is referred to control HFD. Results are expressed as mean ± SEM. Statistical significance was carried out by one-way ANOVA with Bonferroni and Tukey post tests in A, B, and C and two-tailed unpaired t-test in D and E by comparison with control STD mice (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001), with BATp85αKO STD mice (††p < 0.01, †††p < 0.001, and ††††p < 0.0001) and with control HFD mice (#p < 0.05, ##p < 0.01, ###p < 0.001, and ####p <…

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