Fig. 4: mAC3-AT inhibits oxidative metabolism in vitro and in vivo.
From: Cold-induced expression of a truncated adenylyl cyclase 3 acts as rheostat to brown fat function
a,b, Expression of Adcy3 (a) and Adcy3-at (b) in BAT of chow diet-fed male mice housed at 22 °C (n = 4) and for 24 h at 5 °C (n = 3) as determined by RNA-seq. c, Quantification of intracellular cAMP levels in 1°BAs. 1°BAs, differentiated from the SVF of LoxP (n = 5) and Adcy3-atΔKO (n = 5) female and male mice, were stimulated with 10 μM CL for 6 or 24 h. Bar graphs represent the mean + s.e.m. with all data points plotted (n numbers indicated in parentheses). Statistical significance was determined using an unpaired, non-parametric and two-tailed Mann–Whitney test; P value is indicated. d,e, Oxygen consumption rate (OCR) in 1°BAs derived from the SVF of LoxP or Adcy3∆AT mice and stimulated with oligomycin (O), FCCP (F) and antimycin A plus rotenone (A/R) (d) or 1°BAs stimulated with 10 µM CL at the indicated time point (e); n = 5. Unpaired, two-tailed Student’s t-tests were performed to assess statistical significance. P values are indicated. f, BWs in HFD-fed, male LoxP (n = 14) and Adcy3∆AT (n = 15) mice. g, Blood glucose levels during intraperitoneal glucose tolerance tests in HFD-fed, male LoxP (n = 14) and Adcy3∆AT (n = 16) mice. h, Blood glucose levels during intraperitoneal insulin tolerance tests in HFD-fed, male LoxP (n = 14) and Adcy3∆AT (n = 7) mice. f–h, Graphs represent the mean + s.e.m. Statistical significance was determined by performing two-way ANOVA with repeated measurements for x values (mixed models). Post hoc P-value correction to account for multiple testing was performed using Bonferroni adjustment. The source of variation, percentage of the variation and exact P values are given in the table insets. Genotype P = 0.85 (d), P = 0.422 (e), P = 0.0003 (f), P = 0.0001 (g) and P = 0.0001 (h). i, Daily mean food intake in HFD-fed, male LoxP (n = 3) and Adcy3∆AT (n = 3) mice as determined using custom-made food hoppers averaged over 4 days. Unpaired, two-tailed and non-parametric Mann–Whitney tests were performed to assess statistical significance. j, iBAT proximal temperature measurements in HFD-fed, male LoxP (n = 13) and Adcy3∆AT (n = 15) mice exposed to 5 °C. Temperatures were recorded using implanted subdermal probes and telemetry devices. Genotype P = 0.147. k, Indirect calorimetry measurement of oxygen consumption in HFD-fed, male LoxP (n = 6) and Adcy3∆AT (n = 6) mice. l, Relative (BW adjusted) tissue weights from indicated adipose tissue depots and liver of LoxP (n = 14) and Adcy3∆AT (n = 12) mice fed a HFD and housed at 22 °C. m, Fractional body composition of HFD-fed LoxP (n = 10) and Adcy3∆AT (n = 10) mice, determined by NMR. l,m, Bar graphs represent the mean + s.e.m. with all data points plotted. Unpaired, two-tailed, and non-parametric Mann–Whitney tests were performed to assess statistical significance between genotypes within each tissue. n,o, Western blot analyses of BAT from HFD-fed male LoxP and Adcy3 mice (n = 4) after 5 °C for 6 days. Anti-phospho-PKA substrate antibodies were used for detection of PKA phosphorylation substrates and anti-phospho-HS levels were normalized to total HSL protein. anti-HSC70 served as the loading control. Densitometric quantification was performed, and relative values are indicated above the blots.
