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. 2017 Jun 1;36(11):1543-1558.
doi: 10.15252/embj.201694914. Epub 2017 Mar 27.

Mfn2 is critical for brown adipose tissue thermogenic function

Marie Boutant  1 , Sameer S Kulkarni  1 , Magali Joffraud  1 , Joanna Ratajczak  1   2 , Miriam Valera-Alberni  1   2 , Roy Combe  3 , Antonio Zorzano  4   5   6 , Carles Cantó  7   2
Affiliations

Mfn2 is critical for brown adipose tissue thermogenic function

Marie Boutant et al. EMBO J. .

Abstract

Mitochondrial fusion and fission events, collectively known as mitochondrial dynamics, act as quality control mechanisms to ensure mitochondrial function and fine-tune cellular bioenergetics. Defective mitofusin 2 (Mfn2) expression and enhanced mitochondrial fission in skeletal muscle are hallmarks of insulin-resistant states. Interestingly, Mfn2 is highly expressed in brown adipose tissue (BAT), yet its role remains unexplored. Using adipose-specific Mfn2 knockout (Mfn2-adKO) mice, we demonstrate that Mfn2, but not Mfn1, deficiency in BAT leads to a profound BAT dysfunction, associated with impaired respiratory capacity and a blunted response to adrenergic stimuli. Importantly, Mfn2 directly interacts with perilipin 1, facilitating the interaction between the mitochondria and the lipid droplet in response to adrenergic stimulation. Surprisingly, Mfn2-adKO mice were protected from high-fat diet-induced insulin resistance and hepatic steatosis. Altogether, these results demonstrate that Mfn2 is a mediator of mitochondria to lipid droplet interactions, influencing lipolytic processes and whole-body energy homeostasis.

Keywords: brown adipose tissue; insulin resistance; lipid droplet; mitochondrial dynamics; mitofusin 2.

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Figures

Figure 1
Figure 1. Energy homeostasis in Mfn2‐adKO mice
  1. A–C

    Mfn2 gene expression (A) and protein levels (B), as well as Mfn1 gene expression (C), were measured in tissues from 20‐week‐old male control and Mfn2‐adKO mice.

  2. D, E

    Three‐month‐old male control and Mfn2‐adKO mice were used to analyze body weight (D) and composition (E) through EchoMRI.

  3. F–H

    Daily activity (F), food intake (G), and energy expenditure (EE) (H) were measured during indirect calorimetry tests using a comprehensive laboratory animal monitoring system (CLAMS).

  4. I

    Thermogenic capacity was evaluated by placing 16‐week‐old male control and Mfn2‐adKO mice at 6°C for 5 h.

  5. J

    Non‐shivering thermogenesis in mice kept at regular housing temperature (˜22°C) was evaluated by measuring baseline and CL (1 mg/kg)‐induced O2 consumption in anesthetized mice at 30°C (n = 8 mice per genotype).

  6. K, L

    Immediately after the experiment in (J), the mice were housed at thermoneutrality (˜30°C) for 4 weeks. Then, non‐shivering thermogenesis was measured as in (J). Total O2 consumption (K) and the difference between CL‐induced increases in O2 consumption at 22°C and 30°C (L) are shown.

  7. M

    Baseline and CL (1 μM)‐induced O2 consumption rates were evaluated in isolated mature adipocytes using an O2K Oxygraph. Then, the relative effect of CL was calculated for each preparation (n = 11 per genotype).

Data information: All values are presented as mean ± SEM of, unless otherwise stated, n = 10 mice for each genotype. *, **, and *** indicate statistically significant difference between control (white bars and circles) and Mfn2‐adKO mice (black bars and circles) at P < 0.05, P < 0.03, and P < 0.01, respectively (two‐tailed Student's t‐test).
Figure EV1
Figure EV1. Brown adipose tissue function is not affected in Mfn1‐adKO mice

  1. Mfn1 protein levels were measured in tissues from 20‐week‐old male control and Mfn2‐adKO mice.

  2. Three‐month‐old control and Mfn1‐adKO mice were fed ad libitum with a low‐fat diet, and energy expenditure (EE) was measured during indirect calorimetry tests using a comprehensive laboratory animal monitoring system (CLAMS).

  3. A cold resistance test was used to evaluate thermogenic activity in control and Mfn1‐adKO mice.

Data information: All values are presented as mean ± SEM of n = 9 mice for each genotype.
Figure 2
Figure 2. Impaired brown adipose tissue function in Mfn2‐adKO mice

  1. Brown adipose tissue (BAT) pictures from male control and Mfn2‐adKO mice.

  2. Hematoxylin/eosin staining of BAT (scale bar, 100 μm).

  3. Total mRNA was extracted from BAT and used for qPCR analysis.

  4. Western blots were performed to evaluate the ACC phosphorylation in BAT.

  5. Lipolysis was evaluated by measuring glycerol release in isolated BAT from mice after 5‐h treatment with vehicle or isoproterenol (1 μM).

  6. Fatty acid oxidation and respiratory chain‐related gene expression in BAT was analyzed by qPCR.

  7. Respirometry analyses of uncoupled respiration (leak), Complex I respiration (CI), Complex I + Complex II respiration (CI + CII), maximal electron transfer system (ETS) capacity, and maximal Complex II driven respiration (ETS CII) in BAT.

  8. Respirometry analyses were performed in isolated mitochondria from BAT of male (n = 3 per genotype) and female (n = 3 per genotype) control and Mfn2‐adKO mice. On the left, malate (2 mM) and glutamate (10 mM) were used to stimulate Complex I and State 2, State 3 and maximal respiration were evaluated. On the right, succinate (10 mM) and rotenone (0.5 μM) were used to evaluate Complex II State 2, State 3, and maximal respiration.

  9. Mitochondrial proteins levels in total homogenates from BAT. Quantifications are shown below the images.

Data information: Unless otherwise stated, all values are presented as mean ± SEM of n = 10 mice for each genotype. *, **, and *** indicate statistically significant difference between control (white bars) and Mfn2‐adKO mice (black bars) at P < 0.05, P < 0.03, and P < 0.01, respectively (two‐tailed Student's t‐test).
Figure EV2
Figure EV2. eWAT function is not affected in Mfn2‐adKO mice and ER stress is increased in Mfn2‐adKO BAT on low‐fat diet
  1. A

    H&E staining of eWAT (scale bar, 100 μm).

  2. B

    Lipolysis was evaluated by measuring glycerol release in isolated eWAT stimulated or not with 1 μM isoproterenol for 5 h.

  3. C

    Oxidative phosphorylation and electron transfer capacity in eWAT.

  4. D

    Mitochondrial proteins levels in eWAT.

  5. E, F

    mRNA (E) and proteins levels (F) for ER stress markers in BAT.

Data information: All values are presented as mean ± SEM of n = 9–10 mice for each genotype. *, **, and *** indicate statistically significant difference between control (white bars) and Mfn2‐adKO mice (black bars) at P < 0.05, P < 0.03, and P < 0.01, respectively (two‐tailed Student's t‐test).
Figure 3
Figure 3. Mfn2 enhances mitochondria/lipid droplet interactions

  1. Electron microscopy (EM) images from BAT (scale bar, 2 μm) of 20‐week‐old male mice.

  2. Mitochondrial length quantifications from EM images, corresponding to 12 independent images per BAT sample (n = 3 per genotype).

  3. Mitochondria–lipid droplet interaction was evaluated by measuring the ratio between mitochondria displaying direct contact with lipid droplet membranes and the total number of mitochondria (12 independent images per BAT sample; n = 3 per genotype).

  4. Mitochondrial fractions and crude protein extract material from BAT were used to evaluate the presence of lipid droplet (PLIN1, PLIN3), mitochondrial (Mfn2, porin), cytosolic (LDH), or membrane (GLUT4) proteins.

  5. The interaction between Mfn2 and PLIN1 was evaluated by immunoprecipitating PLIN1 from BAT of control and Mfn2‐adKO mice.

  6. Differentiated brown adipocytes were stimulated with vehicle or 1 μM CL316,243 (CL) for 5 h. Then, total proteins were extracted and immunoprecipitated against PLIN1. Quantifications for the increase in Mfn2‐PLIN1 interaction after CL treatment are shown on the right.

  7. Brown adipocytes were transfected with either FLAG‐tagged wild‐type Mfn2 or with a FLAG‐tagged GTPase dead Mfn2 mutant (K109A). Then, adipocytes were differentiated for 3 days, and total protein homogenates were obtained to test the interaction between Mfn2 and PLIN1 via FLAG immunoprecipitation. The graph on the right displays quantification for the decrease in Mfn2‐PLIN1 interaction observed when the K109A mutant is present.

Data information: All values are presented as mean ± SEM. * and *** indicate statistically significant difference between control (white bars) and Mfn2‐adKO mice (black bars) at P < 0.05 and P < 0.01, respectively (two‐tailed Student's t‐test).
Figure EV3
Figure EV3. Mfn2 influences mitochondria–lipid droplet connectivity and specifically interacts with PLIN1

  1. Low‐magnification EM images of BAT from control and Mfn2‐adKO mice.

  2. Mfn2–PLIN1 interaction was evaluated by immunoprecipitating PLIN1 from BAT of control and Mfn2‐adKO mice using an antibody recognizing the N‐terminal region of PLIN1.

  3. Mfn1–PLIN1 interaction was evaluated by immunoprecipitating PLIN1 from BAT of control mice. The results illustrate the lack of co‐IP between these two proteins.

  4. The interaction between Mfn2 and PLIN3 was evaluated by immunoprecipitating PLIN3 from BAT of control and Mfn2‐adKO mice. The results illustrate the lack of co‐IP between these two proteins.

Figure EV4
Figure EV4. PLIN1 and Mfn2 are both required for forskolin‐induced fat oxidation in MEF cells

  1. MEF cells were transfected with PLIN1, wild‐type Mfn2, or Mfn2K109A as indicated. Then, 36 h later, cells were loaded with oleic acid (0.2 mM). After 12 h, protein extracts were obtained to test the markers indicated by Western blot.

  2. MEFs were treated as in (A). After 12 h of oleic acid loading, cells were trypsinized and counted. Then, two million cells were placed in respirometry chambers with air‐equilibrated serum‐free minimum essential medium. After evaluating basal respiration (basal; white bars), cells were treated with forskolin (Fsk; 1 μM; gray bars) and respiration rates were further evaluated. In the left graph, absolute respiration values are shown. In the right graph, the relative increase in respiration induced by Fsk is depicted. Values are presented as mean ± SEM of n = 6 independent experiments. * indicates statistically significant difference vs. the respective basal state at P < 0.05 (two‐tailed Student's t‐test). In the right panel, the ANOVA with Bonferroni post‐hoc test was used to evaluate the differences in Fsk‐induced respiration.

  3. MEFs were transfected with MitoDsRed to visualize mitochondria (in red) and with PLIN1 (in green) to visualize lipid droplets. Then, 36 h later, cells were loaded with oleic acid. After 12 h, cells were fixed with 4% paraformaldehyde and PLIN1 and mitochondria were visualized using fluorescence microscopy.

  4. Cells were treated as in (B). At the end of the treatment protein extracts were obtained and used for Western blot analysis of the indicated markers.

Figure 4
Figure 4. Improved insulin sensitivity in Mfn2‐adKO mice fed high‐fat diet (HFD)
Three‐month‐old male control and Mfn2‐adKO mice were fed ad libitum with a HFD for 8 weeks.
  1. A, B

    Body weight (A) and composition (B) were measured through EchoMRI.

  2. C

    After a 12‐h fast, mice were euthanized and tissue weights were measured.

  3. D

    H&E staining from BAT (scale bar, 100 μm).

  4. E

    Thermogenic capacity was evaluated by placing WT and Mfn2‐adKO mice at 6°C for 5 h.

  5. F, G

    An intraperitoneal glucose tolerance test (F) and an intraperitoneal insulin tolerance test (G) were performed on HFD‐fed control and Mfn2‐adKO mice.

  6. H–J

    Hyperinsulinemic–euglycemic clamps were performed on HFD‐fed control and Mfn2‐adKO mice. Glucose infusion rate (GIR) (H), tissues glucose utilization (I), and glycolysis rates (J) are represented.

  7. K

    Protein levels from total BAT homogenates from HFD‐fed mice.

Data information: All values are presented as mean ± SEM of n = 9–10 mice for each genotype. *,**, and *** indicate statistically significant difference between control (white bars and circles) and Mfn2‐adKO mice (black bars and circles) at P < 0.05, P < 0.03, and P < 0.01, respectively (two‐tailed Student's t‐test).
Figure EV5
Figure EV5. Mfn2‐adKO mice display brown adipose tissue dysfunction on high‐fat diet
  1. A

    H&E staining of eWAT on HFD (scale bar, 100 μm).

  2. B

    Non‐shivering thermogenesis was evaluated by measuring baseline and CL (1 mg/kg)‐induced O2 consumption in anesthetized mice at 30°C (n = 8 mice per genotype).

  3. C, D

    Oxidative phosphorylation and electron transfer system (ETS) capacity in eWAT (C) and BAT (D) from HFD‐fed control and Mfn2‐adKO mice.

  4. E

    An intraperitoneal glucose tolerance test was performed on low‐fat diet‐fed control and Mfn2‐adKO mice.

  5. F

    Glycolytic and ER stress markers gene expression in BAT of HFD‐fed control and Mfn2‐adKO mice.

  6. G

    Protein levels of glycolytic markers in mitochondrial fractions from control and Mfn2‐adKO BAT.

  7. H

    Insulin signaling in ex vivo incubated BAT with or without insulin (50 nM) from control and Mfn2‐adKO mice fed on HFD.

  8. I

    Adiponectin plasma level from HFD‐fed control and Mfn2‐adKO mice.

Data information: All values are presented as mean ± SEM of, unless otherwise stated, n = 9 mice per genotype. *, **, and *** indicate statistically significant difference between control (white bars and circles) and Mfn2‐adKO mice (black bars and circles) at P < 0.05, P < 0.03, and P < 0.01, respectively (two‐tailed Student's t‐test).
Figure 5
Figure 5. Thermoneutrality blunts the glycolytic rewiring in the BAT of HFD‐fed Mfn2‐adKO mice and sensitizes them to cold

  1. High‐fat diet (HFD)‐fed control and Mfn2‐adKO littermates were housed for 8 weeks at thermoneutrality. Then, mice were sacrificed and protein homogenates were obtained from their BAT in order to test the indicated markers.

  2. Mice were housed as in (A). After 6 weeks at thermoneutrality, mice were fasted for 6 h and injected with insulin (1 U/kg). Then, blood glucose levels were measured at the indicated times.

  3. Mice were housed as in (A). After 7 weeks at thermoneutrality, mice were placed on a cold chamber at 6°C and body temperature was evaluated using a rectal thermometer at the indicated times. Mice were excluded from the test if their body temperature was ≤ 30°C.

Data information: All results are expressed as mean ± SEM of n = 10 (control; white circles; five males and five females) and n = 6 (Mfn2‐adKO; black circles; three males and three females) mice. * indicates statistically significant difference at P < 0.05 vs. respective control group (two‐tailed Student's t‐test).
Figure 6
Figure 6. Hepatic steatosis is reduced in Mfn2‐adKO mice on high‐fat diet (HFD)

  1. FGF21 plasma levels in HFD‐fed male control and Mfn2‐adKO mice.

  2. FGF21 mRNA level in BAT from HFD‐fed control and Mfn2‐adKO mice.

  3. H&E and Oil Red O staining of liver from HFD‐fed control and Mfn2‐adKO mice (scale bar, 100 μm).

  4. Liver triglycerides (TG) content from HFD‐fed control and Mfn2‐adKO mice.

  5. mRNA levels in liver from HFD‐fed control and Mfn2‐adKO mice.

Data information: All values are shown as mean ± SEM of n = 9 mice per genotype. *, **, and *** indicate statistically significant difference between control (white bars) and Mfn2‐adKO mice (black bars) at P < 0.05; P < 0.03; P < 0.01, respectively (two‐tailed Student's t‐test).
Figure 7
Figure 7. Representation of the effects of Mfn2 deficiency in BAT

  1. Schematic representation of Mfn2 deficiency in BAT on mitochondria–lipid droplet (LD) interaction. In the left graph, Mfn2 allows the docking of mitochondria to LDs, ensuring an efficient transfer of fatty acids to the mitochondria for beta‐oxidation. On the right, Mfn2‐deficient BAT displays defects in mitochondrial oxidative capacity and LD docking, prompting the accumulation of fat within the LD.

  2. Representation of the metabolic adaptation of the HFD‐BAT from Mfn2‐adKO mice to compensate for the impaired ability to use fat as energy source. In control mice, fat is used as main energy source through oxidative paths. In the right, the Mfn2‐deficient BAT fails to oxidize fat, forcing the adaptation to massively use glycolytic paths for energy production.

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