Supplemental Information Exercise and metformin counteract altered mitochondrial function in the insulin resistant brain 1 1 2 1 Gregory N. Ruegsegger , Patrick M. Vanderboom , Surendra Dasari , Katherine A. Klaus , Parijat Kabiraj3, Christina B McCarthy3. Claudia F. Lucchinetti3, 4, K. Sreekumaran Nair1* Affiliations 1 Division of Endocrinology, Diabetes, Metabolism, and Nutrition, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA 2 Division of Biomedical Statistics and Informatics, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA 3 Department of Neurology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA 4 Center for Multiple Sclerosis and Autoimmune Neurology, Mayo Clinic, Rochester, MN, USA. Figure S1. Aerobic exercise attenuates weight gain, inflammation, and increased ceramide content accompanying HFD. (A-B) 4 weeks of HFD led to greater body weight (A) and body fat (B), which was corrected in HFD-EX (n = 16-17) [for panel A: *p < 0.05: HFD-SED vs. Chow-SED; # p < 0.05: HFD-EX vs. HFD-SED; ‡ p < 0.05: Chow-EX vs. Chow-SED]. (C) Food intake was greater in HFD-fed mice independent of exercise status, while exercise increased food intake in Chow-fed mice (n = 10). (D) HFD did not influence running distance (n = 17). (E) Tnfa, Il1b, and Il6 mRNA expression was higher in the hypothalamus (hypo), hippocampus (hippo), and cortex of HFD-SED mice, which was partially attenuated in HFD-EX (n = 6). (F) C16:0, C18:0, C20:0, and C22:0 ceramides were greater in the hippocampus of HFD-SED, which was corrected in HFD-EX (n = 6). (G) Cer1, Cer2, Cer4 mRNA expression in the hippocampus did not differ between groups (n = 6) Data represent means ± SEM and were analyzed with two-way ANOVA followed by Tukey’s multiple comparisons post hoc test (A-G); *p < 0.05, **p < 0.01, ***p <0.001. Figure S2. Aerobic exercise corrects altered mitochondrial respiration accompanying HFD. (A) State 3 mitochondrial respiration in isolated cerebral mitochondria was lower in HFD- SED, which was corrected in HFD-EX (n = 10-11). (B) The respiratory control ratio (RCR), which is indicative of mitochondrial coupling, was lower in HFD-SED, and normalized in HFD- EX (n = 10-11). Data represent means ± SEM and were analyzed with two-way ANOVA followed by Tukey’s multiple comparisons post hoc test; *p < 0.05. Figure S3. Intranasal insulin receptor antagonism influences mitochondrial respiration. (A-B) Daily intranasal injection of the insulin receptor antagonist S961 for 7 days did not influence plasma glucose (A) and insulin (B) concentrations (n = 8). (C) S961 increased state 4 mitochondrial respiration in isolated cerebral mitochondria (n = 8). (D) S961 decreased the respiratory control ratio (RCR), indicative of uncoupled mitochondrial respiration (n = 8). Data represent means ± SEM and were analyzed with two-tailed Student’s t-test; **p < 0.01. Figure S4. Intranasal S961 blocKs exercised-induced in glucose and insulin tolerance. (A- B) Exercise attenuated gains in body weight (A) and body fat (B) independent of S961 treatment (n = 8). (C) HFD and exercise independently increased food intake independent of S961 treatment (n = 8). (D) S961 did not alter voluntary running distance (n = 8). (E-F) S961 prevented exercise-induced improvements in fasting plasma glucose (E) and insulin (F) in HFD- fed mice (n = 7). (G-H) S961 prevented exercise-induced improvements in oral glucose tolerance (G) and intraperitoneal insulin tolerance (H) in HFD-fed mice (n = 7). Data represent means ± SEM and were analyzed with Mixed Model Analysis and Student’s t-test post hoc comparisons; *p < 0.05: HFD-SED vs. Chow-SED; # p < 0.05: HFD-EX vs. HFD-SED; † p < 0.05: HFD-EX vs. HFD-EX + S961; ‡ p < 0.05: HFD-EX vs. Chow-SED. Figure S5. Metformin crosses the blood-brain barrier. (A-B) Metformin administration in drinking water dose-dependently increased metformin concertation in plasma (A) and hippocampal tissue (B) (n = 3). (C) Metformin administration in drinking water dose-dependently increased AMPK phosphorylation at Thr-172 in the hippocampus (n = 3). Data represent means ± SEM and were analyzed with two-tailed Student’s t-test; *p < 0.05. Figure S6. Metformin improves whole-body glucose and insulin tolerance. (A) Following 4-weeks of HFD, mice were provided ad libitum (HFD-AL), HFD provided ad libitum while receiving metformin (2mg/mL; ~300mg/kg/day) in the drinking water (HFD-MFN), or HFD restricted to 85% (HFD-PF) for 8 additional weeks. HFD-PF and HFD-MFN prevented further gains in body weight compared to HFD-AL (n = 9-10). (B) HFD-MFN and HFD-PF attenuated gains in body fat compared to HFD-AL (n = 9-10). (C) HFD-AL lead to greater food intake, which was partially attenuated in HFD-MFN (n = 9-10). (D-E) HFD-AL increased fasting plasma glucose (D) and insulin (E) which were partially attenuated in HFD-AL and further reduced in HFD-MFN (n = 9-10). (F-G) HFD-AL increased oral glucose tolerance (F) and intraperitoneal insulin tolerance (G) which were partially attenuated in HFD-AL and further reduced in HFD- MFN (n = 9-10) (H) AMPK phosphorylation at Thr-172 in the hippocampus was increased by HFD independent of treatment (n = 6). Data represent means ± SEM and were analyzed with Mixed Model Analysis and Student’s t-test post hoc comparisons. Symbols in panels A-E, H: *p < 0.05 compared to Chow; # p < 0.05 compared to HFD-MFN; † p < 0.05 compared to HFD-AL. Symbols in panels F, G: *p < 0.05: Chow vs. HFD-AL; # p < 0.05: HFD-AL vs. HFD-MFN; † p < 0.05: HFD-AL vs. HFD-PF; ‡ p < 0.05: HFD-PF vs. HFD-MFN; ¶ p < 0.05: Chow vs. HFD-MFN. Figure S7. Metformin corrects altered mitochondrial respiration accompanying HFD. (A) State 3 mitochondrial respiration in isolated cerebral mitochondria was lower in HFD-SED, which was corrected in HFD-MFN (n = 9-10). (B) The respiratory control ratio (RCR) was lower in HFD-SED and HFD-PF, and normalized in HFD-MFN (n = 9-10). Data represent means ± SEM and were analyzed with Mixed Model Analysis and Student’s t-test post hoc comparisons; *p < 0.05, ** p < 0.01. Table S1. Individual protein differential expression data from the 159 significantly affected proteins shown in the Figure 3A volcano plot (Chow-SED vs. HFD-SED comparison). Table lists protein symbols, and names as given by UniProt database, together with their expression ratio (in log2 scale), FDR (in log2 scale) and protein affiliate group as shown in Figure 3A (Group). Protein group was determined by cross-reference against the MitoCarta database from protein affiliation. Proteins are listed by log2 fold-change from most up-regulated to most-downregulated in HFD-SED compared to Chow-SED. Gene Symbol Log2 (HFD-SED/Chow-SED) Log2FDR Group H2-M10.3 29.81 7.32 Ptpn12 28.68 7.38 Ccdc175 27.29 7.07 Mrpl2 27.08 7.32 Mitochondrial Cwf19l2 26.92 4.60 Thg1l 25.97 8.76 Mitochondrial Mrpl53 25.89 8.14 Mitochondrial Rundc3b 25.89 7.38 Ccdc170 25.56 7.38 Tifa 25.13 18.12 Tor2a 24.48 5.71 S100a1 24.39 7.37 Ranbp17 23.44 5.77 Cacna1h 21.85 7.28 Ppig 20.82 4.94 Unc5a 3.49 5.82 Apoa4 3.15 8.55 Ndufs6 2.97 4.76 Mitochondrial Dnal1 2.73 5.86 Vimp 2.61 5.70 Lrrc20 2.21 19.55 Ccdc115 2.13 5.87 Fhl2 2.06 11.59 Commd8 2.02 7.38 Prrt1 1.97 11.39 Fkbp1a 1.88 4.60 H1fx 1.71 12.56 Ercc1 1.69 26.76 Nrp2 1.66 7.20 Pcsk2 1.62 7.47 Ociad1 1.58 6.11 Mitochondrial Macrod2 1.58 7.47 Fmnl2 1.46 9.94 Ddn 1.45 5.10 Tceal6 1.44 6.68 Lpin2 1.44 7.26 Pdzd2 1.41 5.55 Rwdd4 1.40 10.88 Txn 1.38 7.87 Mitochondrial Pycard 1.38 8.35 Jph3 1.37 5.96 Rpl18a 1.30 9.29 Dbi 1.30 6.33 Nek9 1.28 5.42 Mif 1.26 10.40 Ppp1cb 1.25 4.65 Kiaa1671 1.23 6.24 Cox5a 1.22 16.76 Mitochondrial Tceal5 1.20 6.46 Tmem263 1.19 4.39 Fibp 1.16 4.52 Pnck 1.12 4.94 Crym 1.05 4.60 Naip6 1.05 5.18 Hpca 1.04 5.49 Gm10020 1.02 10.15 Nebl 1.02 17.19 Synpr 1.02 7.79 Trappc1 0.98 4.60 Ptprs 0.96 18.21 Arpc2 0.91 18.12 Mtmr12 0.91 5.10 Ccdc43 0.90 7.55 Kbtbd11 0.89 5.42 Asphd2 0.88 9.75 Dgkb 0.88 6.33 Ube2g2 0.87 7.38 Capzb 0.87 4.68 Hebp1 0.86 7.26 Ncs1 0.82 6.33 Ccsap 0.80 5.07 Eif3d 0.80 6.56 Frrs1l 0.80 6.74 Kctd12 0.78 7.29 Fdx1l 0.77 4.52 Mitochondrial Begain 0.75 6.20 Cpeb4 0.74 5.12 Letmd1 0.74 7.38 Mitochondrial Rogdi 0.73 7.22 Anks1b 0.72 11.39 Coq3 0.71 6.93 Mitochondrial Srr 0.71 5.09 Nfu1 0.71 5.77 Mitochondrial Mcu 0.71 33.49 Mitochondrial Faah 0.69 6.94 Stxbp6 0.68 9.29 Dcakd 0.66 4.52 Mitochondrial Grb2 0.65 12.30 Csnk2b 0.63 5.16 Adprh 0.62 6.96 Cttnbp2 0.60 12.80 Cenpv 0.60 7.37 Chchd6 0.59 5.96 Mitochondrial Nptxr 0.59 5.30 Mta1 0.58 5.42 Map6d1 0.57 6.68 Pebp1 0.55 10.01 Mgll 0.54 7.26 Sdcbp 0.53 6.25 Ak4 0.53 7.59 Mitochondrial Src 0.52 8.09 Acy1 -0.51 19.63 Gap43 -0.51 6.95 Mettl7a1 -0.52 6.24 Atg2b -0.56 7.29 Vat1 -0.60 5.73 Acaa2 -0.61 4.42 Mitochondrial Osbpl1a -0.64 5.78 Suclg2 -0.65 4.65 Mitochondrial Trim23 -0.65 5.98 Aplp2 -0.67 7.78 Enpp6 -0.67 5.86 Mag -0.69 8.74 Syt2 -0.70 4.52 Tpp1 -0.76 6.09 9030624J02Rik -0.78 4.76 Btbd17 -0.79 12.30 Eml1 -0.80 9.29 Atpaf1 -0.89 4.46 Mitochondrial Arf2 -0.93 6.71 Pygm -0.97 7.80 Xdh -1.04 6.89 Sccpdh -1.05 12.30 Mitochondrial Fntb -1.06 4.73 Astn2 -1.08 7.38 Esyt1 -1.12 15.34 Ptgfrn -1.19 9.46 Trdn -1.21 20.27 Limk1 -1.24 7.60 Ctnna3 -1.36 5.46 Serpinh1 -1.40 7.75 Itih5 -1.47 4.69 Cbln1 -1.72 5.47 Elovl5 -1.75 8.57 Hbs1l -1.78 8.26 C3 -1.85 12.30 Kcnj10 -1.97 4.82 Rplp2 -2.32 5.27 Krt79 -2.69 5.71 Krt13 -2.74 5.86 Zmat4 -2.76 7.85 Pcdha9 -2.88 11.56 Fxyd1 -3.28 6.10 Faxc -3.29 5.96 Lsm3 -3.88 5.42 Rbx1 -3.98 8.44 mt-Nd3 -23.72 7.38 P2rx5 -24.08 7.38 Gm5814 -24.67 7.38 Nexn -25.05 7.38 Uaca -25.15 6.47 Ccdc12 -25.16 7.09 Fbxo45 -25.53 7.29 Spag1 -25.77 7.38 Trpv3 -27.74 6.93 Wdr93 -28.12 7.38 Timm10b -28.68 10.06 Ddx18 -29.67 7.20 Efcab5 -30.77 7.06 Table S2.