Brown adipose tissue whitening leads to brown adipocyte death and adipose tissue inflammation

Abstract

In mammals, white adipose tissue (WAT) stores and releases lipids, whereas brown adipose tissue (BAT) oxidizes lipids to fuel thermogenesis. In obese individuals, WAT undergoes profound changes; it expands, becomes dysfunctional, and develops a low-grade inflammatory state. Importantly, BAT content and activity decline in obese subjects, mainly as a result of the conversion of brown adipocytes to white-like unilocular cells. Here, we show that BAT "whitening" is induced by multiple factors, including high ambient temperature, leptin receptor deficiency, β-adrenergic signaling impairment, and lipase deficiency, each of which is capable of inducing macrophage infiltration, brown adipocyte death, and crown-like structure (CLS) formation. Brown-to-white conversion and increased CLS formation were most marked in BAT from adipose triglyceride lipase (Atgl)-deficient mice, where, according to transmission electron microscopy, whitened brown adipocytes contained enlarged endoplasmic reticulum, cholesterol crystals, and some degenerating mitochondria, and were surrounded by an increased number of collagen fibrils. Gene expression analysis showed that BAT whitening in Atgl-deficient mice was associated to a strong inflammatory response and NLRP3 inflammasome activation. Altogether, the present findings suggest that converted enlarged brown adipocytes are highly prone to death, which, by promoting inflammation in whitened BAT, may contribute to the typical inflammatory state seen in obesity.

Keywords: adipocyte size; adipose triglyceride lipase; brown fat; macrophage; nucleotide-binding oligomerization domain-like receptor-3 inflammasome; white adipocyte; white adipose tissue.

Fig. 1.

Warm acclimation induces brown adipocyte whitening and CLS formation. A–F: Representative microscopy pictures of MAC-2-immunostained sections of iBAT, mBAT, and iWAT fat depots from mice kept at 6°C (A, C, E) or 28°C (B, D, F) showing BAT whitening (B, D) and increased adipocyte size and CLS formation in the warm-acclimated animals. G: Comparison of brown and white adipocyte area in iBAT, mBAT, and iWAT from cold-acclimated (6°C) and warm-acclimated (28°C) mice documenting a similar brown adipocyte size in both groups and a significant enlargement of subcutaneous inguinal white adipocytes from warm-acclimated mice (n = 5). H: Comparison of CLS density in cold- and warm-acclimated mice showing that warm acclimation significantly increases CLS density in iBAT, to some extent in mBAT, but not in iWAT (n = 5). Data are expressed as mean ± SEM. Statistical significance was determined using unpaired two-tailed Student’s t-test for iBAT and the Mann-Whitney test for mBAT and iWAT *P < 0.05; n.d., not detectable.

Fig. 2.

Increased CLS formation in whitened BAT from db/db and β-less mice. A–D: Representative microscopy pictures of MAC-2-immunostained sections of iBAT from genetically diabetic (db/db) and β-adrenergic signaling (β-less)-deficient mice and their respective controls showing whitened, enlarged brown adipocytes, and increased CLS formation in both models. E, F: Increased interscapular brown adipocyte area and iBAT CLS density in both db/db (E) and β-less (F) mice compared with the respective controls (n = 5). Data are expressed as mean ± SEM. Statistical significance was determined by using unpaired two-tailed Student’s t-test; ***P < 0.001.

Fig. 3.

Atgl deficiency promotes adipocyte hypertrophy and CLS formation in WAT and BAT. A–F: MAC-2-immunostained sections from iBAT, mBAT, and iWAT fat depots from wild-type (A, C, E) and Atgl-ko (B, D, F) mice, showing BAT whitening (B, D), hypertrophic adipocytes, and increased CLS formation in Atgl-ko mice. G: Comparison of brown and white adipocyte area showing enlarged adipocytes in all adipose depots from Atgl-deficient animals (n = 3). H: Comparison of CLS density in Atgl-ko and wild-type mice showing that Atgl deletion involves a significant increase in CLS density in all depots from the former mice (n = 3). Data are expressed as mean ± SEM. Statistical significance was determined using unpaired two-tailed Student’s t-test; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4.

Perilipin immunostaining in Atgl-ko mouse iBAT. A: In wild-type animals, brown adipocytes are strongly positive for perilipin, which is localized around the small lipid droplets. B–D: In Atgl-ko mice, the vast majority of whitened brown adipocytes exhibit reduced perilipin immunostaining, while some multilocular adipocytes (B, arrowheads) retain a fairly strong perilipin immunopositivity. At higher magnification (C), perilipin staining is absent in a CLS and in an adjacent whitened brown adipocyte (“W”), where even its small lipid droplets are completely negative (arrows). D: Two capillaries (cap) contain endothelial cells filled with some perilipin-positive small lipid droplets. A, artery; V, vein.

Fig. 5.

The CLS index. Comparison of the CLS indices (CLS density/mean adipocyte area) calculated in iBAT from the different mouse models examined in the study shows the highest value in Atgl-ko mice. Data are expressed as mean ± SEM. Statistical significance was determined by one-way ANOVA and Tukey’s multiple comparison post hoc test; **P < 0.01; ***P < 0.001.

Fig. 6.

- TEM of whitened brown adipocytes from Atgl-ko mice. A: In wild-type animals, brown adipocytes show their typical ultrastructural appearance and contain several small lipid droplets (L) and numerous large mitochondria filled with parallel cristae (m); one typical brown mitochondrion is shown in the inset. B: An enlarged brown adipocyte from an Atgl-ko mouse contains large and coalescing lipid droplets (L) and mitochondria (m) that, in addition to the typical packed and parallel cristae, show degenerating areas (compare the inset of B with the inset of A). C: High magnification of the cytoplasmic rim of a whitened brown adipocyte (“W”) from an Atgl-ko mouse containing dilated ER and a cholesterol crystal (Chol). Note the abundance of collagen fibrils (coll) in the extracellular space. D: Small lipid droplets (L) are seen in the endothelial cells of a capillary (cap) found in close proximity to an Atgl-ko mouse whitened brown adipocyte (“W”), where mitochondria (m) with degenerating areas are also visible. E: In the iBAT of an Atgl-ko mouse, several macrophages (M) are detected among brown adipocytes (“W”) showing different degrees of whitening; some macrophages are arranged into a CLS. Insets are enlargements of the corresponding framed areas. N, nucleus. Scale bars: 1.5 μm (A), 0.4 μm (inset of A); 0.9 μm (B), 0.3 (inset of B); 0.4 μm (C, D); 3 μm (E).

Fig. 7.

iBAT of Atgl-ko mice exhibits increased expression of inflammatory cytokines and NLRP3 inflammasome activation. A: Determination of the relative mRNA abundance of F4/80, Tnfα, IL-1β, Mcp-1, IL-10, Mgl1, Mgl2, and Arg in iBAT from wild-type and Atgl-ko mice showing higher expression levels in the latter mice (n = 5–6 per genotype, refed mice). B: The expression of inflammasome markers, Casp-1 and IL-18, is upregulated in Atgl-ko mouse iBAT (n = 6, refed mice). The mRNA levels of the inflammation and inflammasome markers were measured by quantitative real-time PCR. Target gene abundance was normalized to 36b4 and expressed relative to wild-type levels of each marker. Data are expressed as mean ± SEM. Statistical significance between groups was calculated using unpaired two-tailed Student’s t-test or Mann-Whitney test for Mgl1 mRNA abundance and is expressed as: *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 8.

iBAT from Atgl-ko mice does not show signs of ER stress or mitochondrial destruction. Assessment of ER stress markers, such as Xbp-1 splicing (A), Grp78 and Chop mRNA levels (B), and eIF2a phosphorylation (C), shows no difference between wild-type and Atgl-ko mice, thus excluding ER stress in iBAT from Atgl-ko mice (n = 4, refed mice). Cytochrome c release in iBAT lysates (D) and qPCR analysis of iBAT mitochondrial (mt) DNA content (E): the relative abundance of the mtDNA-encoded gene Mt-Co1 was measured by qPCR, normalized to the nuclear single copy gene, Ndufv1, and expressed relative to wild-type levels (n = 4–6, refed mice), showing that mitochondrial integrity is not affected in Atgl-ko mouse iBAT. The mRNA abundance of catalase (F) and Gpx1 (G) in Atgl-ko mouse iBAT, which reflect oxidative stress, remained unchanged (n = 5–6, refed mice). Relative mRNA levels were measured by quantitative real-time PCR. Target gene abundance was normalized to 36b4 and expressed relative to wild-type levels of each marker. Data are expressed as mean ± SEM. Statistical significance between groups was calculated with unpaired two-tailed Student’s t-test or Mann-Whitney test for Gpx1 mRNA abundance.