Mitochondrial dynamics controlled by mitofusins regulate Agrp neuronal activity and diet-induced obesity

Abstract

Mitochondria are key organelles in the maintenance of cellular energy metabolism and integrity. Here, we show that mitochondria number decrease but their size increase in orexigenic agouti-related protein (Agrp) neurons during the transition from fasted to fed to overfed state. These fusion-like dynamic changes were cell-type specific, as they occurred in the opposite direction in anorexigenic pro-opiomelanocortin (POMC) neurons. Interfering with mitochondrial fusion mechanisms in Agrp neurons by cell-selectively knocking down mitofusin 1 (Mfn1) or mitofusin 2 (Mfn2) resulted in altered mitochondria size and density in these cells. Deficiency in mitofusins impaired the electric activity of Agrp neurons during high-fat diet (HFD), an event reversed by cell-selective administration of ATP. Agrp-specific Mfn1 or Mfn2 knockout mice gained less weight when fed a HFD due to decreased fat mass. Overall, our data unmask an important role for mitochondrial dynamics governed by Mfn1 and Mfn2 in Agrp neurons in central regulation of whole-body energy metabolism.

Figure 1. Mitochondria dynamics in the orexigenic Agrp neurons

(A) Mitochondria fusion and fission are multistep mechanisms that participate in mitochondria dynamics. (B) Agrp neuron labeled using immunohistochemistry and prepared for electron microscopy. (C) Labeled mitochondria profiles in a stained Agrp neuron. (D) Tethering mitochondria and (E) outer mitochondria membrane fusion in Agrp neurons. (F) NPY-GFP cells in the arcuate nucleus were captured using glass pipettes and utilized for RT-PCR of genes involved in mitochondrial dynamics (mean ± SEM). N = 32 cells/mouse, 3–4 mice/gene.

Figure 2. Mitochondria fusion-fission in Agrp neurons varies with metabolic status

(A) Mitochondria density, and (B) mitochondria coverage in Agrp neurons from fed and 24h food deprived (FD) mice. (C) Cumulative probability distribution of cross-sectional mitochondria area in Agrp neurons of fed and FD mice; insert, relative change in mean mitochondria area between fed and FD mice. (D) Cumulative probability distribution of the aspect ratio (AR) of mitochondria in Agrp neurons; insert, relative changes in mean AR between fed and FD mice. N (fed) = 527 mitochondria/19 cells/4 mice; N (FD) = 729 mitochondria/21 cells/4 mice. (E–H) Differences in mitochondria parameters between mice fed normal chow diet (ND) and a HFD similarly to (A–D). N (ND) = 629 mitochondria/18 cells/3 mice; N (HFD) = 417 mitochondria/18 cells/3 mice. (I) Representative electron micrographs of mitochondria profiles in Agrp neurons from fed and FD mice. (J) Similar to (I), mitochondria labeled in Agrp neurons of mice fed ND or a HFD. (K) Schematic showing the transition from negative energy balance to positive energy balance, and the concomitant changes in mitochondria fusion-fission. All mice were male. Bars represent mean ± SEM. Symbols represent individual values. * P < 0.05, *** P < 0.001. See also Figures S1 and S2.

Figure 3. Role of Mfn1 and Mfn2 in mitochondria morphology in Agrp neurons from normal chow fed mice

(A) Mitochondria density, and (B) mitochondria coverage in Agrp neurons from littermate control and Agrp-Mfn1^−/− mice. (C) Cumulative probability distribution of cross-sectional mitochondria area in Agrp neurons of control and Agrp-Mfn1^−/− mice; insert, relative change in mean mitochondria area. (D) Cumulative probability distribution of the aspect ratio (AR) of mitochondria in Agrp neurons; insert, relative changes in mean AR. N (Control) = 1329 mitochondria/42 cells/3 mice; N (Mfn1^−/−) = 1228 mitochondria/35 cells/4 mice. (E–H) Similarly to (A–D), the data describe mitochondria density and morphology in Agrp-Mfn2^−/− mice. N (Control) = 1565 mitochondria/49 cells/9 mice; N (Mfn2^−/−) = 728 mitochondria/21 cells/4 mice. (I–J) Representative electron micrographs of mitochondria profiles in Agrp neurons from (I) Agrp-Mfn1^−/− mice and (J) Agrp-Mfn2^−/− mice. Data are pooled from both male and female. Bars represent mean ± SEM. Symbols represent individual values. * P < 0.05, ** P < 0.01, *** P < 0.001. See also Figure S3.

Figure 4. Mfn1 and Mfn2 in Agrp neurons critically regulate mitochondria fusion in response to high-fat diet

(A) Mitochondria density, and (B) mitochondria coverage in Agrp neurons from littermate control and Agrp-Mfn1^−/− mice fed a HFD. (C) Cumulative probability distribution of cross-sectional mitochondria area in Agrp neurons of control and Agrp-Mfn1^−/− mice; insert, relative change in mean mitochondria area. (D) Cumulative probability distribution of the aspect ratio (AR) of mitochondria in Agrp neurons; insert, relative changes in mean AR. N (Control) = 333 mitochondria/15 cells/3 mice; N (Mfn1^−/−) = 1363 mitochondria/44 cells/3 mice. (E–H) Similarly to (A–D), the data describe mitochondria density and morphology in Agrp-Mfn2^−/− mice. N (Control) = 601 mitochondria/27 cells/4 mice; N (Mfn2^−/−) = 579 mitochondria/20 cells/5 mice. Data are pooled from both male and female mice. Bars represent mean ± SEM. Symbols represent individual values. * P < 0.05, ** P < 0.01, *** P < 0.001. See also Figure S3.

Figure 5. Mitochondria fusion regulates the electrical activity of Agrp neurons in response to high-fat feeding

(A, E) In normal chow conditions, both control and Agrp-Mfn1^−/− or Mfn2^−/− neurons have similar frequency of action potential (AP) as recorded using slice whole-cell recording. (B, F) When mice were fed a HFD, Agrp-Mfn1^−/− or Mfn2^−/− neurons have decreased AP frequency compared to control cells. (C, G) Percentage of silent Agrp neurons in control and Agrp-Mfn1^−/− or Mfn2^−/− mice fed a normal chow diet. (D, H) In HFD, increased percentage of silent Agrp neurons in both Agrp-Mfn1^−/− and Agrp-Mfn2^−/− mice compared to control mice (P < 0.05, Fisher’s test). Data are pooled from both male and female mice. In A, B, E and F bars represent mean ± SEM. In C, D, G and H bars represent absolute values. Representative traces are plotted in panels A–B, E–F. * P < 0.05. See also Figure S4.

Figure 6. Intracellular ATP levels determine electrical differences in Agrp-Mfn1^−/− and Agrp-Mfn2^−/− neurons

(A) Schematic illustration showing patch clamp recording utilizing perforated clamp with high ATP (5 mM) in the pipette solution or (B) traditional whole-cell recording after patch membrane rupture. (C) Membrane potential in control and Agrp-Mfn1^−/− neurons during perforated and whole-cell recordings. (D) Similar to (C), data represent recordings from control and Agrp-Mfn2^−/− neurons. (E) Firing rate of control and Agrp-Mfn1^−/− cells during perforated clamp and after patch rupture. Number of silent neurons is represented in parenthesis. (F) Similar to (E), data represent recordings from control and Agrp-Mfn2^−/− neurons. (G) Two representative traces to illustrate the electrical responses of Agrp neurons during perforated and whole-cell recordings. The cell on the top is silent during perforated clamp, and becomes highly active after successful membrane rupture and whole-cell recording. Bars represent mean ± SEM. * P < 0.05, ** P < 0.01.

Figure 7. Metabolic response of Agrp-Mfn1^−/− and Agrp-Mfn2^−/− mice to diet-induced obesity

(A) Body weight curve of control and Agrp-Mfn1^−/− male mice. (B) Fat mass and (C) lean mass in the same animals as measured by MRI. (D–F) Similar to (A–C), but data correspond to female mice. A–C, n = 25 (control) and n = 10 (Mfn1^−/−); D–F, n = 14 (control) and n = 10 (Mfn1^−/−). (G–L) Similar to (A–F), data correspond to control and Agrp-Mfn2^−/− mice fed a HFD. G–I, n = 19 (control) and n = 8 (Mfn2^−/−); J–L, n = 16 (control) and n = 13 (Mfn2^−/−). In A, D, G and J symbols represent mean ± SEM; shadow lines represent individual mouse body weight curve. Bars represent mean ± SEM. * P < 0.05, ** P < 0.01. See also Figures S5–S7.