Temperature changes in brown adipocytes detected with a bimaterial microcantilever

Abstract

Mammalian cells must produce heat to maintain body temperature and support other biological activities. Methods to measure a cell's thermogenic ability by inserting a thermometer into the cell or measuring the rate of oxygen consumption in a closed vessel can disturb its natural state. Here, we developed a noninvasive system for measuring a cell's heat production with a bimaterial microcantilever. This method is suitable for investigating the heat-generating properties of cells in their native state, because changes in cell temperature can be measured from the bending of the microcantilever, without damaging the cell and restricting its supply of dissolved oxygen. Thus, we were able to measure increases in cell temperature of <1 K in a small number of murine brown adipocytes (n = 4-7 cells) stimulated with norepinephrine, and observed a slow increase in temperature over several hours. This long-term heat production suggests that, in addition to converting fatty acids into heat energy, brown adipocytes may also adjust protein expression to raise their own temperature, to generate more heat. We expect this bimaterial microcantilever system to prove useful for determining a cell's state by measuring thermal characteristics.

Figure 1

Decomposition of lipid droplets in a norepinephrine-stimulated brown adipocyte. (A) Heat production in brown adipocytes: (NE) norepinephrine; (cAMP) cyclic adenosine monophosphate; (PKA) protein kinase A; (HSL) hormone-sensitive lipase. (B–E) A brown adipocyte, observed with a confocal microscope. (B) A differential interference contrast image. (C) Fluorescence images of BODIPY 493/503-labeled lipid droplets; (D) mitochondria, labeled with MitoTracker Red CMXRos (Life Technologies); and (E) the merged images of panels C and D. Bar = 5 μm. (F) Changes in the cell’s lipid volume over time. Bars indicate ± SD (n = 5–8). The data were fitted to single-exponential decay; the rate constant was 9.4 × 10⁻³ min⁻¹. (Insets) Differential interference contrast images of the cell at each time point. Scale bar (inset, 0 min) = 10 μm. To see this figure in color, go online.

Figure 2

Bimaterial microcantilever bending associated with temperature changes in the environment. (A) A scanning electron microscope image of a 750 × 40 μm² bimaterial microcantilever. Scale bar = 100 μm. (Inset) An overview of the bimaterial microcantilever and its base. Bar = 1 mm. (B) Merged images of the side view of the bimaterial microcantilever at 40°C (red), 35°C (yellow), 30°C (blue), and 25°C (white). Bar = 50 μm. (C) The relationship between ambient temperature change and displacement of the tip of a 750 × 40 μm² bimaterial microcantilever in water (orange line). The slope (dotted line) was 9.15 ± 0.01 μm/K (mean ± SE). (Insets) The bimaterial microcantilever at each temperature point. (Inset) Scale bar at 40°C is 50 μm. To see this figure in color, go online.

Figure 3

Bimaterial microcantilever displacement over time. (A) A diagram of the experimental setup for detecting temperature changes in brown adipocytes. (B) Displacement traces of a 750 × 20 μm² bimaterial microcantilever, just before adding norepinephrine (0 min). Over a period of 120 s, seven cells on the end of a fine needle were moved from (i) a position ∼400 μm from the microcantilever (distant), (ii) toward (iii) and to within 1.8–2.4 μm of the microcantilever (close), (iv) and then away (v) to a distance of ∼400 μm (distant). The difference between the microcantilever’s average position when the cells were close or distant was considered to be the displacement. Bar = 10 μm. This was repeated (C) 30 min, (D) 45 min, (E) 60 min, (F) 75 min, (G) 90 min, (H) 105 min, and (I) 120 min after stimulation with norepinephrine. The experimental temperature was 25 ± 1°C. The color of vertical divisions in panels C–I corresponds to the states shown in panel B. (Red lines) Moving average for a set of 50 data points (calculated from the gray trace). (J) Plots of microcantilever displacement in panels B–I when cells were close to the tip (within 1.8–2.4 μm). To see this figure in color, go online.

Figure 4

Changes in cell temperature during norepinephrine stimulation. Data were plotted using Eq. 3 (described in the Discussion). (A) Tip displacement, with the following bimaterial microcantilever sizes and cell numbers: (red) 750 × 20 μm² bimaterial microcantilever, with seven cells; (orange) 750 × 40 μm² with six cells; (green) 500 × 20 μm² with four cells; (blue) 500 × 20 μm² with five cells; (purple) 750 × 40 μm² with seven cells; and (black) 750 × 40 μm² with four cells. (B) Changes in cell temperature over time at the experimental temperature of 25 ± 1°C; colors correspond to those in panel A. The data were converted from panel A with conversion factors: 750 × 20 μm² with seven cells, 0.368 nm/mK; 750 × 40 μm² with six cells, 0.354 nm/mK; 500 × 20 μm² with four cells, 0.238 nm/mK; 500 × 20 μm² with five cells, 0.253 nm/mK; 750 × 40 μm² with seven cells, 0.350 nm/mK; and 750 × 40 μm² with four cells, 0.328 nm/mK (see Fig. S1 in the Supporting Material). Values shown for time 0 were measured immediately before adding norepinephrine. To see this figure in color, go online.