The thermogenic actions of natriuretic peptide in brown adipocytes: The direct measurement of the intracellular temperature using a fluorescent thermoprobe

Figure 1. The experimental protocol showing the duration and the time course of the intracellular temperature measurement. Brown and white adipocytes treated with the cellular thermoprobe were incubated at 37 °C, and were placed in the microscopic chamber at 35 °C or 37 °C. The fluorescence intensity and ratio were measured every 6 minutes. After stabilization for 40 minutes, atrial natriuretic peptide (ANP), isoproterenol, or CL316,243 was added to the indicated sample dish.

Figure 2. Representative microscopic images of rat brown adipocytes treated with the fluorescent polymeric thermometer. A differential interference contrast image (a), a fluorescence image (490 nm excitation, 525 nm emission), (b) and a fluorescence image (490 nm excitation, 605 nm emission) (c) of the cellular thermoprobe in rat brown adipocytes on day 8. A merged image of (b) and (c) with the sampling square of the measurement are also shown in (d). Scale bar: 40 µm.

Figure 3. The calibration curve of the fluorescent polymeric thermometer in rat brown adipocytes. The responses of the fluorescence ratio (605 nm/525 nm) were analyzed (n = 4). The data indicate the mean ± SEM.

Figure 4. The profile of the intracellular temperature change in rat brown adipocytes incubated with ANP. The intracellular temperature was indicated by the fluorescence ratio (605 nm/525 nm). The changes of the fluorescence ratio in rat brown adipocytes (day 8) after treatment with ANP (10−9 M, n = 5; 10−7 M, n = 6), isoproterenol (10−7 M, n = 6), or CL316,243 (0.5 µM, n = 4) were recorded every 6 minutes (Control, n = 6) at 35 °C (a) or 37 °C (b). The data represent the mean ± SEM. *P < 0.03 and **P < 0.01 versus the controls at each time point (one-way ANOVA followed by Bonferroni’s multiple comparisons test).

Figure 5. ANP increases the UCP1 levels in rat brown adipocytes. The quantification of the Ucp1 gene expression levels in rat brown adipocytes (day 7 or 8) after one hour of incubation at 35 °C with ANP (10−7 M), isoproterenol (10−7 M), or CL316,243 (0.5 µM) (n = 3 each) (a) and those after 30 minutes of incubation at 35 °C with ANP (10−7 M) (n = 3) (b) were shown. The Ucp1 gene expression levels after one hour of incubation at 37 °C with ANP (10−9 M or 10−7 M, n = 4), isoproterenol (10−7 M, n = 4), or CL316,243 (0.5 µM) (n = 3) (c) and those after 30 minutes of incubation at 37 °C with ANP (10−7 M) (n = 3) (d) were also shown. The qPCR data were normalized to GAPDH. The data are shown as the fold change normalized to the levels found in untreated cells (control) *P < 0.05, **P < 0.03, ***P < 0.01 versus control (unpaired two-tailed Student’s t-test). (e) The ATP content in the adipocytes was measured after one hour of incubation with either ANP or isoproterenol (n = 3). Iso, isoproterenol, CL, CL316,243.

Figure 6. Thermogenic actions of ANP in rat brown adipocytes through p38MAPK-UCP1 pathway. (a) Phosphorylation of p38MAPK was evaluated in rat brown adipocytes treated with or without ANP (10−7 M) for one hour. Representative immunoblots obtained using the indicated antibodies are shown and full-length blots are presented in Supplementary Fig. S6. Averaged densitometry data normalized to the control at the same time points are shown in the bar graphs (n = 3). *P < 0.05 versus control. (b) The quantification of the Ucp1 gene expression levels in rat brown adipocytes (day 7 or 8) after one hour of incubation at 35 °C with or without ANP (10−7 M) stimulated with either SB203580 or DMSO (n = 3) are shown. The qPCR data were normalized to GAPDH. The data are shown as the fold change normalized to the levels found in untreated cells (control). **P < 0.01 versus control; ††P < 0.01 versus ANP + SB. (c) The changes of the fluorescence ratio in rat brown adipocytes (day 8) after treatment with or without ANP (10−7 M) stimulated with either SB203580 or DMSO were recorded every 6 minutes at 35 °C (n = 3). The data represent the mean ± SEM. **P < 0.01 versus control; †P < 0.03 and ††P < 0.01 versus…