Patch-Clamp Analysis of the Mitochondrial H+ Leak in Brown and Beige Fat

Figures

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  FIGURE 1. Mitochondrial ATP and heat production in brown/beige fat. Mitochondria have two membranes, the inner mitochondrial membrane (IMM) and the outer mitochondrial membrane (OMM). The IMM contains the energy conversion machinery. The Krebs cycle and electron transport chain (ETC) convert the chemical energy of nutrients into the electrical potential (ΔΨ) across the IMM, which is then converted into ATP or heat. ΔΨ (−160 mV, negative in the mitochondrial matrix) is directly generated by the ETC that pumps H+ across the IMM. ΔΨ is converted into ATP or heat by ATP synthase (AS) or UCP1, respectively.

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  FIGURE 2. Brown and beige fat. Representative sections of the inguinal fat depot (in two states – with and without beige adipocytes) and interscapular fat depot are shown along with diagrams demonstrating morphological features of brown, white, and beige adipocytes. In the adipocyte diagrams, mitochondria are shown in green, cytoplasmic lipid droplets in yellow, and cell nucleus in gray. Brown adipocytes are characterized by a large number of mitochondria (that give this tissue its brown color) and numerous small cytoplasmic lipid droplets. In contrast, white adipocytes have a very low number of mitochondria and contain a single large lipid droplet that occupies almost all of the cytosol. In thermoneutrality, white adipocytes are the predominant type of adipocyte in the inguinal depot. However, under certain conditions such as cold exposure or injection with β3 adrenergic agonists, the inguinal depot develops beige adipocytes that are morphologically similar to brown adipocytes.

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  FIGURE 3. FA-dependent H+ currents via UCP1. (A) Left panel: the H+ current via UCP1 (control, black) is inhibited by 1 mM guanosine 5′-diphosphate (GDP), a classical UCP1 inhibitor (red); Right panel: the same experiment performed in UCP1–/– mice; (B) H+ current via UCP1 (control, black) is de-activated by 10 mM MβCD (blue) and re-activated by 1.5 μM arachidonic acid (AA, red). The voltage protocols are indicated above the corresponding traces. Reproduced with permission from Bertholet et al. (2017).

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  FIGURE 4. Transport of FA anions by UCP1. (A) Transport of short-chain alkylsulfonates by UCP1. Left panel: application of 10 mM C6-sulfonate (C6) on the cytosolic side of the IMM results in a steady FA anion current via UCP1 (red). This FA anion current is inhibited by the classical UCP1 inhibitor GDP (blue). The control trace (before C6 addition) is shown in black. Voltage protocol is shown above. Right panel: the mechanism of FA anion translocation by UCP1. UCP1 has two conformation states, with the SBS exposed either to the cytosolic (c) or matrix (m) side of the IMM. UCP1 binds a short-chain FA anion in the c-state and upon a c-m conformation change, releases the short-chain FA anion on the matrix site of the IMM. To reflect the fact that long-chain FA anions cannot bind to UCP1 on the matrix side, the access to the SBS in the m-state is shown as narrower than that in the c-state. (B) Limited translocation of the long-chain FA within UCP1. Left panel: application of 3 μM C20-sulfonate (C20) on the cytosolic side of the IMM results in a transient FA anion current via UCP1 [red, compare to (A)]. The transient FA anion current is inhibited by the classical UCP1 inhibitor GDP (blue). The…

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  FIGURE 5. The proposed mechanism by which long-chain FA activate H+ current via UCP1. In this model, UCP1 cotransports one FA– and one H+ per a transport cycle. The H+ and the FA– bind to UCP1 on the cytosolic side of the IMM and, upon a conformational change, are translocated by UCP1 to the matrix side of the IMM. H+ is then released into the mitochondrial matrix, while the FA anion stays associated with UCP1 due to the hydrophobic interactions established by its carbon tail. Next, upon the second conformation change, UCP1 returns FA anion back to the cytosolic face of the IMM, where FA can initiate a new H+ translocation cycle. The translocation of FA anion to the cytosolic face of the IMM is assisted by the negative charge within the matrix. Also note that the charge translocation required for uncoupling occurs only in this step, while the previous step of FA–/H+ cotransport is electroneutral.

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  FIGURE 6. FA anions and purine nucleotides compete for binding to UCP1. (A) Left panel: representative H+ current via UCP1 induced by 0.2 mM OA/10mM MβCD in control (red), and in the presence of 100 μM (blue) and 1 mM (black) ATP. Right panel: representative H+ current via UCP1 induced by 2 mM OA/10 mM MβCD in control (red) and in the presence of 100 mM (blue) and 1 mM (black) ATP. (B) The dose-dependence of UCP1 inhibition by ATP at two different concentrations of activating OA. The H+ leak via UCP1 was activated either with 0.2 mM (red curve) or 2 mM OA (black curve) mixed with 10 mM MβCD. Error bars represent standard error of the mean (SEM). Reproduced with permission from Fedorenko et al. (2012).

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  FIGURE 7. Preparation of mitoplasts (Leech and Holz, 1994). Mitochondria isolated from tissue lysate are subjected to low-pressure French press to rupture the OMM and release the IMM (mitoplasts are formed) (Suchyna et al., 2009); when mitoplasts are incubated in KCl solution, the IMM is further released from the OMM and mitoplasts assume an 8-shaped form. Remnants of the OMM are attached to the IMM. The IMM is shown in green, while the OMM is shown in blue.

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  FIGURE 8. Formation of a gigaohm seal and breaking-in into a mitoplast. (A) Formation of the gigaohm seal between the glass patch pipette and the IMM (mitoplast-attached configuration). Upper panel: a photograph of a mitoplast attached to the glass patch-pipette in the mitoplast-attached configuration. Middle panel: a diagram illustrating seal formation between the patch pipette and the 8-shaped mitoplast. The pipette is attached to the IMM lobe (green). When observed with a DIC optics, the IMM lobe is less optically dense as compared to the lobe that contains both, IMM and OMM (blue). Lower panel: the current response to square 10-mV voltage pulses in the mitoplast-attached configuration after compensation of stray capacitance transients. (B) Formation of the whole-mitoplast (whole-IMM) configuration. Establishing the mitoplast-attached configuration is followed by break-in into the mitoplast to achieve a whole-mitoplast configuration for recording currents across the whole IMM. The IMM patch under the pipette is destroyed by high-amplitude voltage pulses (200–500 mV). Upper panel: a photograph of a mitoplast attached to the glass patch-pipette after breaking-in and formation of the…