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, 290 (13), 8243-55

Mitochondrial Turnover: A Phenotype Distinguishing Brown Adipocytes From Interscapular Brown Adipose Tissue and White Adipose Tissue

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Mitochondrial Turnover: A Phenotype Distinguishing Brown Adipocytes From Interscapular Brown Adipose Tissue and White Adipose Tissue

Emilia Gospodarska et al. J Biol Chem.

Abstract

To determine the differences between brown adipocytes from interscapular brown tissue (iBAT) and those induced in white adipose tissue (WAT) with respect to their thermogenic capacity, we examined two essential characteristics: the dynamics of mitochondrial turnover during reversible transitions from 29 °C to 4 °C and the quantitative relationship between UCP1 and selected subunits of mitochondrial respiratory complex in the fully recruited state. To follow the kinetics of induction and involution of mitochondria, we determined the expression pattern of UCP1 and other mitochondrial proteins as well as analyzed mtDNA content after cold stimulation and reacclimation to thermoneutrality. We showed that UCP1 turnover is very different in iBAT and inguinal WAT (ingWAT); the former showed minimal changes in protein content, whereas the latter showed major changes. Similarly, in iBAT both mtDNA content and the expression of mitochondrial proteins were stable and expressed at similar levels during reversible transitions from 29 °C to 4 °C, whereas ingWAT revealed dynamic changes. Further analysis showed that in iBAT, the expression patterns for UCP1 and other mitochondrial proteins resembled each other, whereas in ingWAT, UCP1 varied ∼100-fold during the transition from cold to warmth, and no other mitochondrial proteins matched UCP1. In turn, quantitative analysis of thermogenic capacity determined by estimating the proportion of UCP1 to respiratory complex components showed no significant differences between brown and brite adipocytes, suggesting similar thermogenic potentiality. Our results indicate that dynamics of brown adipocytes turnover during reversible transition from warm to cold may determine the thermogenic capacity of an individual in a changing temperature environment.

Keywords: Brown Adipocytes; Cold Adaptation; Energy Metabolism; Mitochondrial Respiratory Chain Complex; Mitophagy; Mouse; Obesity; Thermogenesis; UCP1.

Figures

FIGURE 1.
FIGURE 1.
The induction of brite adipocytes in ingWAT during cold stimulation and their involution in thermoneutral conditions. A, scheme of the experimental design to assess brite adipocytes turnover. Young adult (9 weeks) female AXB8 mice were singly housed for 10 days at 4 °C, after 7 days of initial acclimation at 29 °C (control group), and subsequently reacclimated to 29 °C. iBAT and ingWAT were collected from 4 animals per group at the following time points: day 7 of initial acclimation (0d4 °C); days 1, 2, 3, 5, 7, and 10 at 4 °C; days 1, 3, 5, 7, 14, and 21 of reacclimation to 29 °C. ad lib, ad libitum. B, hematoxylin and eosin-stained paraffin sections of ingWAT of cold-stressed and rewarmed mice.
FIGURE 2.
FIGURE 2.
Effect of ambient temperature on UCP1 expression in iBAT and ingWAT. Western blot analysis of UCP1 protein expression performed by using the whole tissue lysate (30 μg) from iBAT (A) and ingWAT (B) of mice maintained at 4 °C or 29 °C for different time points. The number of mice was four for each time point; β-actin was used as a protein loading control. Representative images are shown. Quantitative analysis of Western blot results (panel C and D). Signals corresponding to UCP1 protein in iBAT (C) and ingWAT (D) were normalized to that of β-actin and are shown as -fold change relative to the control group. Each time point is the average value from four animals for Ucp1 mRNA expression in iBAT (E) and ingWAT (F) based on the results of quantitative PCR analysis and expressed relative to the levels of cyclophilin. Results are shown as -fold change relative to the levels of the control group (0 days). G, Ucp1 gene expression in iBAT and ingWAT during cold and reacclimation conditions. Each time point is the average value from four individual animals analyzed in duplicate by quantitative PCR and related to cyclophilin. The asterisks in C–F indicate statistically significant differences in protein or mRNA expression between mice exposed to cold for 10 days and mice from selected time points of the experiment. Student's unpaired t test: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. a.u., absorbance units.
FIGURE 3.
FIGURE 3.
The kinetics of induction and regression of mitochondrial components in iBAT and ingWAT during reversible transitions from 29 to 4 °C. Above: protein expression analysis of selected mitochondrial proteins VDAC1, COX IV, and cytochrome c (CYT C) performed by using the whole tissue lysate (30 μg) of mice maintained at 4 or 29 °C for the different lengths of time. Proteins were detected on different membranes that were prepared in parallel from the same sample dilution (for iBAT and ingWAT separately). The protein loading control sample from only one of the membranes is shown. The number of mice was four for the time point. Representative images are shown. Below: quantitative analysis of Western blot results. Signals of the protein expression were normalized to that of β-actin and are shown as -fold change relative to the control group. Each time point is the average value from four animals. The asterisks indicate statistically significant differences in protein expression between mice exposed to cold for 10 days and mice from selected time points in the experiment. Student's unpaired t test: *, p < 0.05; **, p < 0.01.
FIGURE 4.
FIGURE 4.
Expression of selected subunits of mitochondrial respiratory chain complexes in iBAT and ingWAT in cold and thermoneutral conditions. Above: protein levels of representative subunits of the mitochondrial respiratory complexes (RC) I-V, NDUFB8 (RC I), SDHB (RC II), UQCRC2 (RC III), MTCO1 (RC IV), and ATP5A (RC V) were determined by using the whole tissue lysate (30 μg) of mice maintained at 4 or 29 °C for the different time lengths. Representative images are shown. Below: graphs present quantitative analysis of expression of subunits of the mitochondrial respiratory complexes and UCP1 protein in iBAT (filled bars) and ingWAT (open bars) as the average value from four individual animals. Signals of the protein expression are shown as -fold change relative to the control group (0 days). The asterisks indicate statistically significant differences in protein expression between mice exposed to cold for 10 days and mice from selected time point of the experiment; Student's unpaired t test: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).
FIGURE 5.
FIGURE 5.
Effect of ambient temperature on UCP1, respiratory chain components and mitochondrial DNA turnover in iBAT and ingWAT. A, line graphs of WB results (see Figs. 2A and 4) comparing the change in expression of UCP1 protein and selected subunits of mitochondrial respiratory chain during cold and warm acclimation. Signals of the protein expression are shown as a multiple of the levels in control group (-fold change). B, expression of Atp5a1 and Atp5g1 mRNA. Data are the means ± S.E. from 4–6 mice in each group. C, changes in mitochondrial DNA content. Mitochondrial DNA-encoded cytochrome b measurement relative to the nuclear gene cyclophilin was analyzed for four mice in duplicate. Student's unpaired t test: *, p < 0.05; ***, p < 0.001.
FIGURE 6.
FIGURE 6.
Estimation the optimal time of cold exposure needed for maximum expression of UCP1. A, scheme of the experiment for estimating the optimal time of cold exposure to reach the maximum induction of brite adipocytes in ingWAT. Young adult (9 weeks) female AXB8 mice were caged individually for 0, 5 10, 15, 20, and 25 days at 4 °C after 7 days of initial acclimation at 29 °C. Four mice from selected time points were sacrificed, iBAT and ingWAT were collected, and UCP1 gene and protein expression were analyzed. ad lib, ad libitum. Shown is Western blot analysis of UCP1 protein expression (B) and quantitative analysis (C) carried out by using the whole tissue lysate (30 μg) of mice maintained at 4 °C for selected time points. Signals corresponding to UCP1 protein were normalized to that of β-actin. D, Ucp1 mRNA expression based on the results of quantitative PCR analysis. Each time point is the average value from four mice analyzed in duplicate and expressed relative to the levels of cyclophilin. a.u., absorbance units.
FIGURE 7.
FIGURE 7.
Thermogenic capacity of mitochondria from brown adipocytes from iBAT and ingWAT from cold-acclimated mice. A, levels of UCP1 and mitochondrial respiratory chain subunits in purified mitochondria isolated from iBAT and ingWAT from a pool of 4 mice acclimated to 29 °C for 7 days (0d4°C) or to 4 °C for 15 days. Levels of UCP1 (B) and mitochondrial respiratory chain proteins (C) in iBAT and ingWAT mitochondria isolated from mice acclimated to 29 °C for 7 days (0d4°C) or to 4 °C for 15 days. a.u., absorbance units. D, the ratio of UCP1 protein to respiratory complex subunits in mice acclimated to 4 °C. In ingWAT, respiratory complex protein levels from initial acclimation conditions were subtracted from values obtained after 15 days of cold exposure (asterisk). It was assumed that at 29 °C brite adipocytes are not induced within ingWAT and have no signal corresponding to UCP1 (A). Thus, expression levels of RC components at 29 °C were treated as background originating from other non-brown adipocytes.

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