Investigation of biophysical properties of white and brown adipose tissues by diffusion spectroscopy.

There are two types of fat tissues, white adipose tissue (WAT) and brown adipose tissue (BAT), which essentially perform opposite functions in whole body energy metabolism.¹ WAT consists of adipocytes containing large uni-locular lipid droplets, is used mainly for energy storage in the form of triglycerides. On other hand, BAT composed by small multi-locular droplet which is abundant in iron content because of mitochondria and highly vascularized. BAT dissipates energy directly by heat due to the presence of uncoupling protein 1 (UCP1) in the mitochondria. Diffusion properties of tissues provide information on microstructure, anisotropy, and pathology. In the presence of barriers, the diffusion is restricted to the characteristic length established by the barriers.² The goal of this study was to investigate the diffusion properties of white and brown adipose tissues obtained from rats at different temperature.All experiments were conducted after approval by the local institutional animal care and use committee. Tissue samples were obtained by postmortem dissection of ~12 week old male Wistar rats (n = 6, mean body weight 300 ± 20 g). A BAT depot was excised from the interscapular region, whereas WAT was removed from the gonadal fat pad. Approximately 50 mg of WAT or BAT tissue was weighed and set between Ultem susceptibility-matching plugs (Wilmad-LabGlass/Doty Scientific) in a thin-walled NMR tube (S-5-600-7; Norell) using a positioning rod. The translational diffusion was measured with a bipolar-stimulated echo sequence using a Bruker Avance III 400 NMR spectrometer and an inverse-detection broadband liquid NMR probe at 25 °C and 36 °C.³ Diffusion coefficient, D [m²/s], that corresponds to the slope of a diffusion signal intensity decay plot against q²Δ_r, where q = (γ/2π)Gδ and Δ_r = Δ − δ/3 − τ/2. During measurements, timing parameters were set as diffusion time (Δ = 0.25 ~ 2 s), gradient pulse width (δ/2 = 3 or 5 ms), and gradient polarization switching interval (τ = 0.5 ms). The gradient strength was calibrated with a trace amount of HDO in D₂O at 25 °C.⁴ The deuterium field lock was turned off when the measurements were being conducted. A mono-exponential function was used to fit the strongest fat CH₂ signal intensity integrated between 1.04 and 1.32 ppm, while a bi-exponential function was used to fit the water resonance intensity integrated between 4.45 to 4.94 ppm. Real time qPCR measurements were performed to estimate UCP1.Figure 1 shows the apparent diffusion coefficient (ADC) of WAT and BAT at two different temperature 25 °C and 36 °C. Both water and fat diffused significantly faster in WAT than in BAT at both temperatures. There was an increase in the ADC values for both water and fat with temperature. Water diffused faster than the fat in both WAT and BAT at 25 °C and 36 °C. Figure 2 shows the plot of signal attenuation curves as a function of q²Δ_r for different diffusion times. BAT showed an upward drift of the decay curve when ∆ was increased. Also, as a function of ∆, we observed WAT did not show significant changes in the diffusion coefficients whereas BAT shows significant difference. These findings suggest the restricted behavior of fat molecules in BAT and not in WAT. Figure 3 shows the real-time PCR mRNA expression analysis of UCP1 gene in BAT, WAT and muscle tissues. UCP1 was expressed significantly higher in BAT than WAT. BAT exhibits smaller sized adipocytes with considerable volume of cytoplasm. The nuclei are round and almost centrally located. Adipocytes within the WAT have a scant ring of cytoplasm surrounding a single large lipid droplet. These microscopic geometrical differences between BAT and WAT results in differences in their diffusion properties.⁵ We have demonstrated that diffusion of fat molecules in WAT is relatively free, whereas it is restricted in BAT, reflecting the fat droplet sizes in each. BAT is also rich in vasculature with high density of mitochondria contributing to the restriction lengths. The change in temperature also resulted in changes for the diffusion properties of both water and fat due to increased thermal energy. In conclusion, we have demonstrated the differences in biophysical properties of BAT and WAT by diffusion NMR techniques.