Mitochondrial fission is required for thermogenesis in brown adipose tissue

Abstract

Brown adipose tissue (BAT) thermogenesis is pivotal for maintaining body temperature and energy balance. Mitochondrial morphology is dynamically controlled by a balance between fusion and fission, which is crucial for cell differentiation, response to metabolic insults, and heat production. Dynamin-related protein 1 (Drp1) is a key regulator of mitochondrial fission. This study investigates the role of Drp1 in BAT development and thermogenesis by generating Drp1-deficient mice. These mice were created by crossing Drp1 floxed mice with fatty acid-binding protein 4-Cre (aP2-Cre) transgenic mice, resulting in aP2-Cre+/-Drp1flox/flox (aP2-Drp1f/f) mice. The aP2-Drp1f/f mice exhibited severe BAT and brain hypoplasia, with the majority dying within 48 hours postnatally, highlighting Drp1's crucial role in neonatal survival. Impaired thermogenic responses were observed in aP2-Drp1f/f mice, characterized by significantly decreased expression of thermogenic and lipogenic genes in BAT. Ultrastructural analysis revealed disrupted mitochondrial morphology and reduced lipid droplet content in BAT. The few surviving adult aP2-Drp1f/f mice also showed impaired BAT and brain development, along with BAT thermogenesis dysfunction during cold exposure. Our findings underscore the essential role of Drp1-mediated mitochondrial fission in BAT thermogenesis and neonatal survival, providing insights into potential therapeutic approaches for metabolic disorders.

Fig 1. Postnatal death of aP2-Drp1^f/f neonatal mice.

A. aP2-Drp1^f/f mice immediately after birth (0 h) compared with wild-type littermate controls. B. Body weight from control and aP2-Drp1^f/f mice at 0 hours after birth was measured and presented as means ± SEM. *P < 0.05 determined by an unpaired t-test. (n = 28–30). C. Body length from the nose to tail base from control and aP2-Drp1^f/f mice at 0 hours after birth were measured and presented as means ± SEM. (n = 3–4). D-E, Signs of life were closely observed to assess survival and were presented as Kaplan-Meier survival curves. Between-group difference was tested by the log-rank test. ***P < 0.001. F. Tissues were harvested from control and aP2-Drp1^f/f mice within 12 hours after birth and subjected to SDS-PAGE and western blot analysis. G. Gene expression profiles are shown for the heart, lung, kidney, iBAT, liver, muscle, skin, brain, spleen, adrenal gland, and thyroid gland from control and aP2-Drp1^f/f mice within 12 hours after birth. Data were shown as means ± SEM. * P < 0.05 determined by an unpaired t-test. (n = 3–6). H. Western blot analysis was performed to evaluate the expression of Drp1 in the iBAT and whole brain from control and aP2-Drp1^f/f mice within 12 hours after birth. Gapdh was used as a loading control. The biological duplicate samples in each condition numbered as #1 and #2. Results are expressed as means ± SEM. The results of quantitative analysis are shown in the right panel. *P < 0.05 determined by an unpaired t-test. (n = 3). Abbreviations: Drp1; dynamin-related protein 1, K; knock-out, W; wild-type, Gapdh; glyceraldehyde 3-phosphate dehydrogenase, iBAT; interscapular brown adipose tissue.

Fig 2. iBAT characterization of the aP2-Drp1^f/f neonatal mice.

A-B. Morphology (A) and relative mass (B) of iBAT from control and aP2-Drp1^f/f mice at 0 h after birth. Yellow dashed lines track iBAT boundaries. Scale bar = 1.0 mm. n = 5–6, *p < 0.05 C. Immunohistological staining for Fabp4 and Drp1 in control and aP2-Drp1^f/f mice at 0 h after birth. Scale bar = 50 μm. D. H&E staining of control and aP2-Drp1^f/f mice iBAT sections. The mice were treated at room temperature (26°C) or cold temperature (19°C) at 12 h after birth. Areas indicated with white dashed squares are enlarged and shown in the middle. Traces of adipocytes (purple) and lipid droplets (yellow) are shown on the right. Scale bar = 600 μm. E. Quantification of the average area of the adipocytes. n = 80–109, ***p < 0.001 F. Transmission electron microscopy (TEM) of mitochondrial morphology in iBAT from control and aP2- Drp1^f/f mice at room temperature. False color TEM micrograph showing mitochondria (red), lipid droplets (yellow), and nucleus (blue). Areas indicated with white dashed squares are enlarged and shown on the lower panel. Scale bar = 1μm. G. Quantification of the total area and the average area of the mitochondria and lipid droplets. n = 5, **p < 0.01, and ***p < 0.001 determined by an unpaired t-test. Abbreviations: Drp1; dynamin-related protein 1, Fabp4; Fatty acid binding protein 4, HE; Hematoxylin and Eosin.

Fig 3. Reduced expression of thermogenic and lipogenic genes of aP2-Drp1^f/f neonatal mice.

A. Rescuing effect of a hot plate (35°C) on the survival of aP2- Drp1^f/f mice relative to room temperature (26°C) at indicated time after birth. Signs of life were presented as Kaplan-Meier survival curves. Between-group difference was tested by the log-rank test. (n = 12–18). B-C. Thermogenic (B) and lipogenic (C) gene expression in iBAT from neonatal control and aP2-Drp1^f/f mice. Mice were chronically exposed at room temperature (26°C) or cold temperature (19°C), respectively, at 12 h after birth. Data are shown as means ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001 determined by two-way ANOVA with Bonferroni’s post hoc test. (n = 3–6). Abbreviations: Ucp1; uncoupling protein 1, Cpt1; carnitine palmitoyl transferase 1, Cox8b; cytochrome c oxidase subunit 8B, Prdm16; PR domain containing 16, Cidea; cell death-inducing DNA fragmentation factor alpha-like effector A, Fasn; fatty acid synthase, Acc; acetyl CoA carboxylase, and Srebp1c; sterol regulatory element binding protein 1.

Fig 4. Brain disintegration in aP2- Drp1^f/f neonatal mice.

A. The macroscopic view of the brains of the control and aP2-Drp1^f/f mice at 0 h after birth. Scale bar = 3.0 mm. B. Histological H&E staining (upper panel) and IHC staining for Drp1 (lower panel) of sagittal sections from control and aP2-Drp1^f/f brains at 0 h after birth. Scale bar = 3.0 mm. C. Western blotting was performed to evaluate the expression of Drp1 and Fabp4 in the following portions of the brain: cerebral cortex, cerebellum, midbrain, and hypothalamus. Gapdh was used as a loading control. D. Representative images of Fabp4 and Drp1 in the cerebellum of control and aP2-Drp1^f/f mice at 0 h after birth. Fabp4-positive cells using Alexa Fluor 488-conjugated anti-rabbit IgG (green) and Drp1-positive cells were visualized using Alexa Fluor 594-conjugated anti-mouse IgG (red) and the nuclei were stained with DAPI (blue). Areas indicated with white dashed squares are enlarged and shown on the right side of the picture. Scale bar = 10 μm. E. Brain sections from newborn mice were subjected to TUNEL staining and visualized with HRP-DAB (dark brown dots). Nuclei were stained with methyl green as a nuclear counterstain. Scale bar = 10 μm. F. The quantitative analysis of apoptotic cells in the whole brain of control and aP2-Drp1^f/f mice at 0 h after birth. n = 4, **p < 0.01 determined by an unpaired t-test. Abbreviations: Drp1; dynamin-related protein 1, V; lateral ventricles, Fabp4; Fatty acid binding protein 4, DAPI; 4′,6-diamidino-2-phenylindole, HE; Hematoxylin and Eosin, IHC; immunohistochemistry, TUNEL; terminal deoxynucleotidyl transferase dUTP nick end labeling.

Fig 5. Impaired development of the brain and iBAT of rarely alive adult aP2-Drp1^f/f mice.

A. The body weight of control and aP2-Drp1^f/f mice on a normal chow diet was measured and is presented as means ± SEM. ***P < 0.001, determined by two-way ANOVA with sidak’s multiple comparisons test. n = 4–10. B. Morphology of the brain and iBAT of control and aP2-Drp1^f/f mice at 24 weeks after birth. Photographs were taken under the stereoscope. C. Thermographic imaging showing single-cage control and aP2-Drp1^f/f mouse surface temperature. Mice that were 52 weeks of age were exposed to 4°C temperature for 4 h. Thermografic system FLIR E50 bx shows cooler color according to the temperature at the indicated time. D. Changes in rectal temperature during a cold challenge. *P < 0.05 and **P < 0.01 determined by two-way ANOVA with sidak’s multiple comparisons test (n = 4–5). E. Morphology of iBAT from control and aP2-Drp1^f/f mice after a cold test. Yellow dashed lines track iBAT boundaries. Abbreviations: Drp1; dynamin-related protein 1, iBAT; interscapular brown adipose tissue.