Three-Dimensional Adipose Tissue Imaging Reveals Regional Variation in Beige Fat Biogenesis and PRDM16-Dependent Sympathetic Neurite Density

Abstract

While the cell-intrinsic pathways governing beige adipocyte development and phenotype have been increasingly delineated, comparatively little is known about how beige adipocytes interact with other cell types in fat. Here, we introduce a whole-tissue clearing method for adipose that permits immunolabeling and three-dimensional profiling of structures including thermogenic adipocytes and sympathetic innervation. We found that tissue architecture and sympathetic innervation differ significantly between subcutaneous and visceral depots. Subcutaneous fat demonstrates prominent regional variation in beige fat biogenesis with localization of UCP1 beige adipocytes to areas with dense sympathetic neurites. We present evidence that the density of sympathetic projections is dependent on PRDM16 in adipocytes, providing another potential mechanism underlying the metabolic benefits mediated by PRDM16. This powerful imaging tool highlights the interaction of tissue components during beige fat biogenesis and reveals a previously undescribed mode of regulation of the sympathetic nervous system by adipocytes.

Leptin and brain–adipose crosstalks

“…Using volume fluorescence-imaging techniques to visualize the neural arborizations in mouse inguinal WAT, it was recently shown that sympathetic fibres are in close apposition to over 90% of adipocytes 34 . Supporting these observations, investigators recently developed an Adipo-Clear model in which fat pads were delipidated to enable 3D whole-tissue imaging and found dense staining of sympathetic fibres in close contact with the majority of adipocytes in inguinal WAT 99 . Interestingly, blocking the outgrowth of neural fibres to inguinal WAT using genetic or pharmacologic tools prevented the sympathetic arborization of the tissue and the ability of cold to promote the development of brite adipocytes 34 .…”

“…4). Moreover, the pattern of SNS innervation of fat pads differs in mice, with minimal fibres projecting to gonadal WAT compared with inguinal WAT 99 . The regional specificity of SNS innervation is further emphasized by functional changes (reflected by differences in noradrenaline turnover) observed following treatment with various stimuli.…”

An optimized tissue clearing protocol for rat brain labeling, imaging, and high throughput analysis

“…Toward this goal, we adapted the delipidation methods of the AdipoClear protocol (Chi, Wu et al 2018), which was developed to clear and immunolabel lipid dense adipose tissue without damaging tissue structure. A schematic of this modified protocol with relevant additions and substitutions is shown in Fig.1A, alongside rat brain hemisphere cleared with this new protocol (Fig1C), as compared to results of the previous version of the protocol (Fig1B).…”

A new mouse model for noninvasive fluorescence‐based monitoring of mitochondrial UCP1 expression

“…First, we can detect fluorescence without any injection, and this characteristic allows us to easily detect fluorescence in neonatal mice, as shown in Fig. This method revealed that UCP1 expression strongly correlates with sympathetic innervation [62,63]. UCP1-mRFP1 BAC Tg mice are also suitable for evaluation of pharmacological UCP1 stimulation.…”

“…Recently, a tissue clearing method is used as a new method for histological analysis of adipose tissue. This method revealed that UCP1 expression strongly correlates with sympathetic innervation [62,63]. However, three-dimensional distribution of the UCP1 is still unknown.…”