Dependence of brown adipose tissue function on CD36-mediated coenzyme Q uptake

Abstract

Brown adipose tissue (BAT) possesses the inherent ability to dissipate metabolic energy as heat through uncoupled mitochondrial respiration. An essential component of the mitochondrial electron transport chain is coenzyme Q (CoQ). While cells synthesize CoQ mostly endogenously, exogenous supplementation with CoQ has been successful as a therapy for patients with CoQ deficiency. However, which tissues depend on exogenous CoQ uptake as well as the mechanism by which CoQ is taken up by cells and the role of this process in BAT function are not well understood. Here, we report that the scavenger receptor CD36 drives the uptake of CoQ by BAT and is required for normal BAT function. BAT from mice lacking CD36 displays CoQ deficiency, impaired CoQ uptake, hypertrophy, altered lipid metabolism, mitochondrial dysfunction, and defective nonshivering thermogenesis. Together, these data reveal an important new role for the systemic transport of CoQ to BAT and its function in thermogenesis.

Figure 1. CD36 is required for CoQ transport

(A) HEK293 cells stably expressing CD36 or parent vector (control) were treated with CoQ₉ for 24 h (n=5–6 per group). CoQ levels were measured by HPLC and normalized to µg of protein. (B) Primary mature brown adipocytes were isolated from WT and Cd36^−/− BAT and treated with either vehicle or CoQ₁₀ for 3 h (n=5). (Ci–iv) WT and Cd36^−/− mice were injected intraperitoneally with either vehicle (Intralipid) or CoQ₁₀. After 24 h tissues were isolated and CoQ₁₀ levels were measured by HPLC and normalized to µg of protein or µl of serum (n=8–9). (D) BAT, liver, heart, soleus, gastrocnemius, quadriceps, adrenal gland, brain, and serum CoQ₉ and CoQ₁₀ levels in Cd36^−/− relative to WT (100%) were measured by HPLC and normalized to µg of protein (n=3–6). A complete table with the absolute CoQ values is included in Supplementary Table 1. (E) CoQ₁₀ levels were measured in lipoprotein fractions from human serum by HPLC and normalized to ml of fraction (n=6). *p<0.05; **p<0.005; ***p<0.0005. Error bars, SEM. See also Figure S1 and Table S1.

Figure 2. Pathologic TAG storage in Cd36^−/− BAT

(A) Western blot for CD36 and β-tubulin was performed on lysates from WT and Cd36^−/− BAT, WAT, heart, lung, quadriceps muscle, spleen, and liver (n=5). Data is presented in arbitrary units as a ratio of CD36 normalized to tubulin. (B) Co-localization of CD36 (green), UCP1 (red), and DAPI (blue) in a WT BAT cryosection. Scale bar represents 10µm. (C) Images of BAT from WT and Cd36^−/− mice and their weights before and after cold exposure (n=3–6). (D) Representative images of 3-D reconstructions from cold-exposed mouse BAT sections stained with the fluorescently-labeled TAG probe BODIPY 493/503 (green) and DAPI (blue). (E) Genome-wide expression analysis was performed on RNA from WT and Cd36^−/− BAT (n=3 per group). A summary table of misregulated genes identified using a p≤0.05 threshold is presented. A complete list of misregulated genes is included in Supplementary Tables 2 and 3. (F) Lipase activity (expressed as nmol of FFA released/hour/mg protein of tissue lysate) from WT and Cd36^−/− BAT chunks (n=4). (G) Maximal BODIPY 510/512-labeled FFA uptake capacity of untreated and CL-316,243-treated primary mature brown adipocytes from WT and Cd36^−/− mice (n=3). (H) WT and Cd36^−/− fasting plasma FFA levels at 0, 2.5 and 5 h of cold exposure (n=4). (I) Cholesterol was extracted from BAT, liver, quadriceps, and serum and normalized to either mg of tissue or µl of serum (n=3). *p<0.05; **p<0.005; ***p<0.0005. Error bars, SEM. See also Figures S2 and S3 and Tables S2 and S3.

Figure 3. Impaired substrate utilization and mitochondrial dysfunction in Cd36^−/− BAT

(A) Production of ¹⁴CO₂ from ¹⁴C-[Palmitic acid] in Cd36^−/− primary mature brown adipocytes relative to WT (100%) and normalized for mg of protein (n=3). (B) WT and Cd36^−/− BAT mitochondrial respiration rates using glycerol-3-phosphate (G3P) with and without CoQ2, succinate, and pyruvate/malate (Pyr/Mal). Oxygen consumption rates (OCR) were measured and presented as µg O₂/min/50 µg protein (n=6–9). (C) Representative electron microscopy (EM) images from WT and Cd36^−/− BAT. 43,000× magnification. Scale bar represents 0.2µm. (D) Quantification of the mitochondrial area from WT and Cd36^−/− BAT EM images (n=4). (E) Quantification of the mitochondrial volume density from WT and Cd36^−/− BAT EM images (n=4). (F) Cytochrome content was measured in isolated mitochondria from WT and Cd36^−/− BAT and normalized to mg of mitochondrial protein (n=3). (G) The rate of H₂O₂ production was measured in WT and Cd36^−/− BAT mitochondria and presented as pmol/min/mg protein (n=4). *p<0.05; ***p<0.0005. Error bars, SEM.

Figure 4. Defective non-shivering thermogenesis in Cd36^−/− BAT

(A) Mouse core body temperature measured rectally before and after 5 h of cold exposure (n=6). (B) The wireless accelerometer used in this study was built at UC Berkeley and is approximately the size of a coin (a US quarter). Shivering and movement were monitored by attaching the device shown to the mouse back. (C) Accelerograph showing millisecond-resolution monitoring of movement and shivering in a WT mouse. The large bursts of acceleration indicate animal movement. A zoomed-in accelerograph displays regular shivering spikes in a cold-exposed WT mouse. Motor activity (D), time spent shivering (E), and shivering amplitude (F) of fasted mice exposed to 4°C for 2 h (n=5). (G) Oxygen consumption and carbon dioxide and heat production measurements 3 h after intraperitoneal administration of the β3-adrenergic receptor agonist CL-316,243 (n=3). Data is presented as fold change compared to before CL injection. *p<0.05. Error bars, SEM.