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. 2024 Mar;300(3):105760.
doi: 10.1016/j.jbc.2024.105760. Epub 2024 Feb 16.

Differential responses to UCP1 ablation in classical brown versus beige fat, despite a parallel increase in sympathetic innervation

Qimuge Naren  1 , Erik Lindsund  2 , Muhammad Hamza Bokhari  2 , Weijun Pang  3 , Natasa Petrovic  4
Affiliations

Differential responses to UCP1 ablation in classical brown versus beige fat, despite a parallel increase in sympathetic innervation

Qimuge Naren et al. J Biol Chem. 2024 Mar.

Abstract

In the cold, the absence of the mitochondrial uncoupling protein 1 (UCP1) results in hyper-recruitment of beige fat, but classical brown fat becomes atrophied. Here we examine possible mechanisms underlying this phenomenon. We confirm that in brown fat from UCP1-knockout (UCP1-KO) mice acclimated to the cold, the levels of mitochondrial respiratory chain proteins were diminished; however, in beige fat, the mitochondria seemed to be unaffected. The macrophages that accumulated massively not only in brown fat but also in beige fat of the UCP1-KO mice acclimated to cold did not express tyrosine hydroxylase, the norepinephrine transporter (NET) and monoamine oxidase-A (MAO-A). Consequently, they could not influence the tissues through the synthesis or degradation of norepinephrine. Unexpectedly, in the cold, both brown and beige adipocytes from UCP1-KO mice acquired an ability to express MAO-A. Adipose tissue norepinephrine was exclusively of sympathetic origin, and sympathetic innervation significantly increased in both tissues of UCP1-KO mice. Importantly, the magnitude of sympathetic innervation and the expression levels of genes induced by adrenergic stimulation were much higher in brown fat. Therefore, we conclude that no qualitative differences in innervation or macrophage character could explain the contrasting reactions of brown versus beige adipose tissues to UCP1-ablation. Instead, these contrasting responses may be explained by quantitative differences in sympathetic innervation: the beige adipose depot from the UCP1-KO mice responded to cold acclimation in a canonical manner and displayed enhanced recruitment, while the atrophy of brown fat lacking UCP1 may be seen as a consequence of supraphysiological adrenergic stimulation in this tissue.

Keywords: MAO-A; UCP1; Western blot; adipocyte; beige adipose tissue; brown adipose tissue; gene knockout; immunohistochemistry; macrophage; sympathetic nerves.

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Conflict of interest statement

Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1
The effect of UCP1 ablation on biochemical and morphological parameters of IBAT and ingWAT. Wild-type (filled symbols) (n = 4–6) and UCP1-KO mice (open symbols) (n = 5–6) were acclimated to the indicated temperatures (as detailed in Experimental procedures). A, IBAT wet weight, (B) ingWAT wet weight, (C) IBAT protein content, (D) ingWAT protein content, (E) IBAT protein density and (F) ingWAT protein density. Each symbol represents one mouse sample. Where not visible, the error bars are smaller than the symbols. Values are means ± SEM. Data were analyzed using two-way ANOVA. (Full ANOVA statistics are given in Table S1). ∗Significant difference between wild-type and UCP1-KO mice for each tissue and temperature using the two-way ANOVA followed by Tukey’s multiple comparison test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. G–R, Histological appearance of IBAT (G, H, K, L, O and P) and ingWAT (I, J, M, N, Q and R) from the animals in (A–F). The tissues were analyzed using hematoxylin and eosin staining, and representative images are presented. Scale bar 100 μm. UCP1-KO, UCP1-knockout.
Figure 2
Figure 2
A notable accumulation of macrophages in IBAT and ingWAT of UCP1-KO mice housed below thermoneutrality.A–L, representative confocal images of IBAT (A, B, E, F, I and J) and ingWAT (C, D, G, H, K and L) from wild-type and UCP1-KO mice acclimated to 30 °C, 18 °C and 4 °C. The tissues were stained for MAC-2 (green), perilipin (red) and nuclei (blue). Scale bar 50 μm. M and N, representative western blots of MAC-2 in IBAT (M) and ingWAT (N) from animals acclimated to the indicated temperatures. O and P, MAC-2 content in IBAT (O) and ingWAT (P) in the indicated samples. The mean value in IBAT of UCP1-KO mice acclimated to 4 °C was set to 1.0, and the levels in all other samples were expressed relative to this value. Each symbol represents a sample from one mouse. Values are means ± SEM. Where not visible, the error bars are smaller than the symbols. Full ANOVA statistics are given in Table S1. ∗Significant difference between wild-type and UCP1-KO mice for each tissue using two-way ANOVA followed by Tukey’s multiple comparison test. ∗∗∗p < 0.001. To facilitate comparisons of MAC-2 levels between IBAT and ingWAT, the respective graphs were drawn with equal y-axis range. In the right panel of P, the graph was redrawn with optimal y-axis range. UCP1-KO, UCP1-knockout.
Figure 3
Figure 3
Macrophages in IBAT and ingWAT of UCP1-KO mice housed below thermoneutrality do not express tyrosine hydroxylase.A–F, representative confocal images of IBAT (A, C and E) and ingWAT (B, D and F) from UCP1-KO mice acclimated to 4 °C. The tissues were stained for tyrosine hydroxylase (TH) (green), MAC-2 (red) (C and D), perilipin (red pseudocolor) (E and F) and nuclei (blue). Scale bar 50 μm. UCP1-KO, UCP1-knockout.
Figure 4
Figure 4
Macrophages in IBAT and ingWAT of UCP1-KO mice housed below thermoneutrality are not equipped with the enzymes necessary for norepinephrine catabolism.A–D, representative confocal images of IBAT (A and C) and ingWAT (B and D) from UCP1-KO mice acclimated to 4 °C. In (A and B), the tissues were stained for monoamine oxidase-A (MAO-A) (red), MAC-2 (green) and perilipin (blue pseudocolor). In (C and D), the tissues were stained for norepinephrine transporter (NET) (red), MAC-2 (green) and TH (blue pseudocolor). Scale bar 50 μm. Individual antibody staining is shown in the top-inset panels (scale bar 20 μm). Images were acquired with settings in each case allowing the maximum signal detection below the saturation limits of the detectors and are therefore not directly quantitatively comparable. Note that the MAO-A staining (red in A and B) overlapped only with perilipin staining (blue). Similarly, the NET staining (red in C and D) overlapped only with TH staining (blue). UCP1-KO, UCP1-knockout.
Figure 5
Figure 5
A strong induction and unexpected adipocyte localization of MAO-A in IBAT and ingWAT of UCP1-KO mice housed below thermoneutrality.A–L, Representative confocal images of IBAT (A, B, E, F, I and J) and ingWAT (C, D, G, H, K and L) from wild-type and UCP1-KO mice acclimated to 4 °C. The tissues were stained for MAO-A (red), TH (green) and perilipin (blue pseudocolor). Scale bar 50 μm. Images were acquired with settings in each case allowing the maximum signal detection below the saturation limits of the detectors and are therefore not directly quantitatively comparable. M and N, representative western blots of MAO-A and β-tubulin (cannot be used as a loading control due to the plasticity of the tissues) in IBAT (M) and ingWAT (N) from animals acclimated to the indicated temperatures. O, IBAT MAO-A content and (P) ingWAT MAO-A content in the indicated samples. The mean value in IBAT of wild-type mice acclimated to 4 °C was set to 1.0, and the levels in all other samples were expressed relative to this value. Each symbol represents a sample from one mouse. Values are means ± SEM. Where not visible, the error bars are smaller than the symbols. ∗Significant difference between wild-type and UCP1-KO mice for each tissue using two-way ANOVA followed by Tukey’s multiple comparison test. ∗p < 0.05, ∗∗∗p < 0.001. To facilitate comparisons of MAO-A levels between IBAT and ingWAT, the respective graphs were drawn with equal y-axis range. In the right panel of P, the graph was redrawn with optimal y-axis range. UCP1-KO, UCP1-knockout.
Figure 6
Figure 6
Notably enhanced sympathetic innervation in IBAT and ingWAT of UCP1-KO mice housed below thermoneutrality.A–H, representative confocal images of IBAT (A, B, E and F) and ingWAT (C, D, G and H) from wild-type and UCP1-KO mice acclimated to 4 °C. The tissues were stained for TH (green), perilipin (red) and nuclei (blue). Scale bar 20 μm. Images were acquired with settings in each case allowing the maximum signal detection below the saturation limits of the detectors and are therefore not directly quantitatively comparable. I and J, representative western blots of tyrosine hydroxylase (TH) and FABP4 (was not used as a loading control) in IBAT (I) and ingWAT (J) from animals acclimated to the indicated temperatures. K, IBAT TH content, (L) ingWAT TH content, (M) IBAT TH density, and (N) ingWAT TH density in the indicated samples. The value in IBAT of wild-type mice acclimated to 4 °C was set to 1.0, and the levels in all other samples were expressed relative to this value. Each symbol represents a sample from one mouse. Values are means ± SEM. Where not visible, the error bars are smaller than the symbols. ∗Significant difference between wild-type and UCP1-KO mice for each tissue using two-way ANOVA followed by Tukey’s multiple comparison test. ∗∗p < 0.01, ∗∗∗p < 0.001. To facilitate comparisons of TH levels between IBAT and ingWAT, the respective graphs were drawn with equal y-axis range. In the right panels of L and N, the graphs were redrawn with optimal y-axis range. UCP1-KO, UCP1-knockout.
Figure 7
Figure 7
Significant upregulation of adrenergically regulated genes in both IBAT and ingWAT of UCP1-KO mice housed below thermoneutrality.A and B, expression levels of thermogenesis-related genes in IBAT (A) and ingWAT (B) of wild-type and UCP1-KO mice acclimated to the indicated temperatures. The expression levels were normalized using reference genes: 18 S rRNA for IBAT and TFIIB for ingWAT. Each symbol represents a sample from one mouse. Values are means ± SEM. For each gene, the reported value is the mean Ct value in the condition with the highest expression. Note that Ct values were consistently higher in ingWAT than in IBAT for every examined gene, indicating lower expression levels in ingWAT. ∗Significant difference between wild-type and UCP1-KO mice for each tissue using Student’s unpaired t test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. C and D, representative western blots of UCP1, glycerol kinase (GK) and CIDEA in IBAT (A) and ingWAT (B) from animals acclimated to the indicated temperatures. E, IBAT GK content, (F) ingWAT GK content, (G), IBAT CIDEA content, and (H) ingWAT CIDEA content in the indicated samples. The value in IBAT of wild-type mice acclimated to 4 °C was set to 1.0, and the levels in all other samples were expressed relative to this value. Each symbol represents a sample from one mouse. Values are means ± SEM. ∗Significant difference between wild-type and UCP1-KO mice for each tissue using two-way ANOVA followed by Tukey’s multiple comparison test. ∗∗p < 0.01, ∗∗∗p < 0.001. To facilitate comparisons of examined proteins between IBAT and ingWAT, the respective graphs were drawn with equal y-axis range. In the right panels of D and F, the graphs were redrawn with optimal y-axis range. UCP1-KO, UCP1-knockout.
Figure 8
Figure 8
Strongly reduced content of mitochondrial respiratory chain proteins in IBAT, but not in ingWAT, of UCP1-KO mice housed below thermoneutrality.A and B, representative western blots of mitochondrial respiratory chain proteins (ATP5A, subunit of complex V; MTCO1, subunit of complex IV; UQCRC2, subunit of complex III; SDHB, subunit of complex II; NDUFB8, subunit of complex I) and outer mitochondrial membrane protein VDAC in IBAT (A) and ingWAT (B) from animals acclimated to indicated temperatures. Note that the blots for each protein were acquired with optimal exposure. C–H, the content of the indicated mitochondrial proteins in IBAT and ingWAT from animals acclimated to 30 °C (C and D), 18 °C (E and F) and 4 °C (G and H). The mean value for each protein in IBAT of wild-type mice acclimated to 4 °C was set to 1.0, and the levels in all other samples were expressed relative to this value. Each symbol represents a sample from one mouse. Values are means ± SEM. Where not visible, the error bars are smaller than the symbols. ∗Significant difference between wild-type and UCP1-KO mice for each tissue using two-way ANOVA followed by Tukey’s multiple comparison test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. To facilitate comparisons of examined proteins between IBAT and ingWAT and also between the three temperatures, the respective graphs were drawn with equal y-axis range. I, representative western blots of mitochondrial respiratory chain proteins (ATP5A, subunit of complex V; MTCO1, subunit of complex IV; UQCRC2, subunit of complex III; SDHB, subunit of complex II; NDUFB8, subunit of complex I) in IBAT, ingWAT, liver and heart tissue lysates. IBAT and ingWAT are obtained from wild-type mice acclimated to 4 °C; liver and heart are obtained from wild-type mice acclimated to 21 °C. Note that whereas the subunits of respiratory complexes I – IV were expressed at similar levels in the tissues with high oxidative capacity - IBAT and heart, ATP5A was an exception by being expressed at much lower levels in IBAT (as well as in ingWAT). UCP1-KO, UCP1-knockout.
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