1. Mitochondrial Dynamics Controlled by Mitofusins Regulate Agrp Neuronal Activity and Diet-Induced Obesity 
Marcelo O. Dietrich, Zhong-Wu Liu, Tamas L. Horvath 
Cell 
Volume 155, Issue 1, Pages (September 2013)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. 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 and E) Tethering mitochondria (D) and outer mitochondria membrane fusion (E) 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.
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4. Figure 2
Mitochondria Fusion-Fission in Agrp Neurons Varies with Metabolic Status
(A–D) Mitochondria density (A), and mitochondria coverage (B) in Agrp neurons from fed and 24 hr 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 those fed a HFD are similar 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.
Scale bars, mean ± SEM. Symbols represent individual values. ∗p < 0.05, ∗∗∗p < See also Figures S1 and S2.
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5. Figure 3
Role of Mfn1 and Mfn2 in Mitochondria Morphology in Agrp Neurons from Normal Chow-Fed Mice
(A and B) Mitochondria density (A), and mitochondria coverage (B) 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) = 1,329 mitochondria/42 cells/3 mice; n (Mfn1−/−) = 1,228 mitochondria/35 cells/4 mice.
(E–H) Similar to (A–D), the data describe mitochondria density and morphology in Agrp-Mfn2−/− mice. n (Control) = 1,565 mitochondria/49 cells/9 mice; n (Mfn2−/−) = 728 mitochondria/21 cells/4 mice.
(I and 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.
Scale bars, mean ± SEM. Symbols represent individual values. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < See also Figure S3.
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6. Figure 4
Mfn1 and Mfn2 in Agrp Neurons Critically Regulate Mitochondria Fusion in Response to HFD
(A and B) Mitochondria density (A), and mitochondria coverage (B) 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) Similar 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.
Scale bars, mean ± SEM. Symbols represent individual values. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < See also Figure S3.
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7. Figure 5
Mitochondria Fusion Regulates the Electrical Activity of Agrp Neurons in Response to High-Fat Feeding
(A and 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 and F) When mice were fed a HFD, Agrp-Mfn1−/− or Mfn2−/− neurons have decreased AP frequency compared to control cells.
(C and G) Percentage of silent Agrp neurons in control and Agrp-Mfn1−/− or Mfn2−/− mice fed a normal chow diet.
(D and 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.
Scale bars: mean ± SEM in (A), (B), (E), and (F); absolute values in (C), (D), (G), and (H). Representative traces are plotted in (A)–(B), (E)–(F). ∗p < See also Figure S4.
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8. Figure 6
Intracellular ATP Levels Determine Electrical Differences in Agrp-Mfn1−/− and Agrp-Mfn2−/− Neurons
(A and B) Schematic illustration(A) 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.
Scale bars, mean ± SEM. ∗p < 0.05, ∗∗p < 0.01.
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9. 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. Scale bars, mean ± SEM. ∗p < 0.05, ∗∗p < See also Figures S5, S6, and S7.
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10. Figure S1
Mitochondria Changes in POMC Neurons in Response to Food Deprivation, Related to Figure 2
(A) Mitochondria density decreases in POMC neurons in response to 24 hr food deprivation (FD).
(B) Mitochondria coverage of cell or cytosol also decreased after FD.
(C) Probability distribution of mitochondria profiles cross-sectional area in POMC cells showing no effect of FD on mitochondria size.
(D) Similar to C, but showing a parameter utilized to describe changes in mitochondria shape (aspect ratio – AR). No effects of FD were found. Overall, the data indicate decrease in mitochondria density in POMC neurons in response to FD, without changes in size and/or shape. n (Fed) = 852 mitochondria/15 cells/3 mice. n (FD) = 616 mitochondria/15 cells/3 mice.
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11. Figure S2
Levels of mfn1 and mfn2 in Agrp Neurons during Different Metabolic States, Related to Figure 2
We used ribose profiling to isolated RNA bound to the ribosomes selectively from Agrp neurons.
(A) Data represent enrichment for agrp, npy, pomc, and s100b (marker for astrocyte contamination).
(B) Data from fasted, fed and HFD-fed mice related to fed mice. n = 3–4/group. Five animals were pooled for each n. Both male and female mice were used. ∗p < 0.05, ∗∗p < 0.01.
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12. Figure S3
Hypothalamic Transcripts and Agrp Neuronal Projection in Mitofusin-Deficient Mice, Related to Figures 3 and 4
(A) Amount of agrp, npy and pomc transcripts in the arcuate nucleus of the hypothalamus of littermate control and Agrp-Mfn1−/− mice.
(B) Similar to (A), data correspond to Agrp-Mfn2−/− mice. Female mice were used in these studies.
(C) Agrp neuronal projection was measured in the paraventricular nucleus of the hypothalamus (PVN). Quantification of fluorescent fibers in control and Agrp-Mfn1−/− mice.
(D) Similar to (A), but comparing control and Agrp-Mfn2−/− mice. Both males and females were used for these studies.
(E) Levels of bip, chop, atf3, atf4, atf6, and xbp1s in control and Agrp-Mfn1−/− mice.
(F) Similar to (E), data correspond to Agrp-Mfn2−/− mice. Female mice were used in these studies.
(G and H) Amount of agrp, npy and pomc transcripts in the arcuate nucleus of the hypothalamus of (G) Agrp-Mfn1−/− and (H) Agrp-Mfn2−/− mice related to littermate control mice.
(I) Levels of bip, chop, atf3, atf4, atf6, and xbp1s in control and Agrp-Mfn1−/− mice.
(J) Similar to (I), data correspond to Agrp-Mfn2−/− mice. Female mice were used in these studies. Data are expressed in relative quantities related to control mice. Bars represent mean ± SEM. ∗p < 0.05.
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13. Figure S4
Mitochondria Fusion Regulates the Electrical Activity of Agrp Neurons in Response to High-Fat Feeding, Related to Figure 5
(A) In normal chow conditions, both control and Agrp-Mfn2−/− neurons have similar frequency of action potential (AP) as recorded using slice whole-cell recording.
(B) When mice were fed a HFD, Agrp-Mfn2−/− neurons have decreased AP frequency compared to control cells.
(C) Percentage of silent Agrp neurons in control and Agrp-Mfn2−/− mice fed a normal chow diet.
(D) In HFD, increased percentage of silent Agrp neurons in Agrp-Mfn2−/− mice compared to control mice (p < 0.05, Fisher’s test).
In (A) and (B), bars represent mean ± SEM. In (C) and (D), bars represent absolute values. All cells were recorded using perforated clamp with amphotericin B in the pipette solution.
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14. Figure S5
Metabolic Adaptations of Agrp-Mfn1−/− and Agrp-Mfn2−/− Mice Fed a Normal Chow Diet, Related to Figure 7
(A) Body weight curve of female control (gray) and Agrp-Mfn2−/− mice (pink).
(B and C) Fat mass (B) and lean mass (C) in the same animals as measured by MRI.
(D) Leptin levels in two different cohorts of mice.
(E–I) Water intake (E), food intake (F), energy expenditure (G), ambulatory and vertical activities (H), and RER (I) in female control (gray) and Agrp-Mfn2−/− mice (pink).
(J–L) Similar to (A)–(C), but data correspond to male mice.
(M) Body weight curve of female control (gray) and Agrp-Mfn1−/− mice (blue).
(N) Fat mass and (O) lean mass in the same animals as measured by MRI.
(P) Leptin levels in two different cohorts of mice.
(Q–U) Water intake (Q), food intake (R), energy expenditure (S), ambulatory and vertical activities (T), and RER (U) in female control (gray) and Agrp-Mfn1−/− mice (blue).
(V–X) Similar to (M)–(O), but data correspond to male mice.
(Y) Leptin levels in male mice.
In (A), (J), (M), and (V) symbols represent mean ± SEM; shadow lines represent individual mouse body weight curve. Scale bars, mean ± SEM. ∗p < 0.05.
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15. Figure S6
Glucose Profile of Agrp-Mfn1−/− and Agrp-Mfn2−/− Mice Fed a Normal Chow Diet, Related to Figure 7
(A) GTT (A) and ITT (B) in female control and Agrp-Mfn1−/− mice.
(C) Insulin level in two different cohorts of mice.
(D–F) Similar to (A)–(C), data correspond to male mice.
(G–H) GTTs in two different cohorts of female control and Agrp-Mfn2−/− mice.
(I) ITT in the same groups of mice.
(J) Insulin level in two different cohorts of mice.
(K–N) Similar to (G)–(J), data correspond to male Agrp-Mfn2−/− mice.
Symbols represent mean ± SEM. Scale bars, mean ± SEM. p value is stated in the graphics when a trend is observed or when statistical difference was found. Differences in GTT and ITT were tested using two-way ANOVA with time as a repeated-measure. Statistical differences in insulin levels were tested using t test.
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16. Figure S7
Metabolic Adaptations of Agrp-Mfn1−/− and Agrp-Mfn2−/− Mice Fed a HFD, Related to Figure 7
(A–H) Food intake (A), water intake (B), energy expenditure (C), activity (D), RER (E), GTT (F), ITT (G), insulin levels (H) in female control and Agrp-Mfn1−/− mice.
(I–J) GTT (I) and ITT (J) male mice fed a HFD.
(K) Leptin levels in female mice.
(L–S) Food intake, water intake (M), energy expenditure (N), activity (O), RER (P), GTT (Q), ITT (R), insulin levels (S) in female control and Agrp-Mfn2−/− mice.
(T–V) GTT (T), insulin (U), and leptin levels (V) in male mice fed a HFD.
(W) Leptin levels in female mice.
Symbols represent mean ± SEM. Scale bars, mean ± SEM. ∗p < 0.05.
Cell  , DOI: ( /j.cell )
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