Purpose

To evaluate the oxidative fat metabolism in activated brown adipose tissue

Introduction

White adipose tissue (WAT) and Brown adipose tissue (BAT) play unique role in energy storage and energy expenditure respectively. Brown adipocytes are characterized by abundant iron-rich mitochondria with multiple smaller lipid droplets1. There is a need for understanding of mechanistic pathways of BAT activation considering it is a potential endocrine organ for treatment of metabolic diseases. In this context we have studied the dynamic oxidative fat metabolism in interscapular brown adipose tissue by activation of β3-adrenergic receptors.

Methods

All the experimental procedures carried out in this study were approved by institutional animal care and use committee. Male Wistar rats of twelve weeks old were randomized into two groups including β3-adrenergic agonist (n=5) treated and saline (n=5) treated groups. The β3-adrenergic agonist, CL-316243 was administered at 0.2 mg/Kg body weight of the animal. Animals were anesthetized with 1.5-2% Isoflurane in combination with air and catheterized for β3-adrenergic agonist or saline injections before imaging. The MR measurements were performed by using 7T MRI scanner (Clinscan). A 72 mm volume coil was used for radio frequency pulse transmission in combination with 2 x 2 phased array coil for signal reception. Fat-Water Dixon imaging was performed in interscapular BAT (iBAT) region with FOV 54x54 mm2, matrix size 256×256, in-plane resolution of 211μmx211μm, slice thickness of 1mm,TR=8 ms, averages=1, flip angle=8°, echo bandwidths of 1090 and 1500Hz/pixel, with out-of-phase (1.0ms) and in-phase (2.5ms) echo times. Localized PRESS sequence was employed in interscapular BAT and WAT with TR=4s, TE=13ms, NA 16, voxel volume-8mm3, SW= 3500 Hz, data points= 2048. Fat content from in vivo spectra were estimated using LC Model software2. Dynamic MRI/MRS was performed at 10 minutes (min), 30 min, 60 min, 90 min and 120 min after administration of β3-adrenergic agonist including baseline (T0 min) scans before intervention. After terminal experiments BAT and WAT samples were fixed in neutral buffered solution (10 %) for 24 hours. A slice of 5µm tissue section was stained for hematoxylin (H) and eosin (E) staining. The mRNA analysis of CPTI and UCP1 genes were performed in both WAT and BAT.

Results and Discussion

Figure 1 shows the in vivo dynamic spectra obtained from iBAT at various time points. The amplitude of (CH2)n at 1.3 ppm decreased progressively after the injection of β3 agonist. Figure 2 shows the kinetics of lipids oxidation at different time interval. In response to activation of sympathetic nervous system by β3-adrenergic agonist, series of metabolic events occur (Figure 3) leading to lipolysis induced thermogenesis. The β3-adrenergic agonist is taken up by the adrenergic receptors and activates the adenylate cyclase (AC) by Gα G-protein coupling with increased cellular concentration of cAMP. This in turn activates protein kinase A (PKA) and results in phosphorylation of lipid droplets3-5. The net effect is release of free fatty acids (FFA) by the hydrolysis of triglyceride (TG) lipid droplets. The FFAs activated to long chain fatty acyl CoAs by acyl-CoA synthetase and converted to long chain acylcarnitine by the up-regulation of CPT1 in the outer membrane of mitochondria. Long chain acylcarnitines are then transported into mitochondrial matrix by carnitine transporter which is a rate limiting step for beta oxidation. In the mitochondrial matrix, long chain acylcarnitines are cleaved into free carnitine and acyl CoA by the up-regulation of CPTII involved in beta oxidation leading to UCP1 uncoupled thermogenesis. In the current study, the CPTI expression was upregulated in iBAT indicating the elevated lipolysis. UCP1 expression was also upregulated in iBAT evidencing the activated BAT followed by thermogenesis. Upregulation of both CPTI and UCP1 confirms that lipolysis is inevitable for thermogenesis. The progressive depletion of the lipids from iBAT region is indicative of oxidative metabolism by utilizing the lipids as fuel substrate. To support the stimulated energy requirements, utilization of intracellular energy stores by iBAT has been reported by Cannon B et.al6. The iBAT might use intracellular lipid droplets within the brown adipocytes initially and later may recruit from neighboring white adipocytes. This was confirmed in histology of iBAT (Figure 4A, B). We noticed the significant depletion of lipid droplets in H & E sections from iBAT of β3-adrenergic agonist treated animal compared to saline treated.

Conclusion

We have investigated the lipid oxidation by dynamic imaging and spectroscopy during BAT activation. The β3-adrenergic mediated lipid oxidation followed by thermogenesis is supported by the up-regulation of CPTI and UCP1. Evaluation of lipid mobilization in real time is important to assess the altered metabolic rate and mitochondrial biogenesis involving lipid oxidative metabolism. Our results suggest that this approach can be extended for designing targeted lipolysis and oxidation.

Acknowledgements

No acknowledgement found.