Mitofusin 2 (Mfn2) links mitochondrial and endoplasmic reticulum function with insulin signaling and is essential for normal glucose homeostasis David Sebastiána,b,c,1, María Isabel Hernández-Alvareza,b,c,1, Jessica Segalésa,b,c,1, Eleonora Sorianelloa,b,c, Juan Pablo Muñoza,b,c, David Salaa,b,c, Aurélie Wagetd, Marc Liesaa,b,c, José C. Paza,b,c, Peddinti Gopalacharyulue, Matej Oresice, Sara Picha,b, Rémy Burcelind, Manuel Palacína,b, and Antonio Zorzanoa,b,c,2 aInstitute for Research in Biomedicine (IRB Barcelona), 08028 Barcelona, Spain; bDepartament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, 08028 Barcelona, Spain; cInstituto de Salud Carlos III, Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), 08017 Barcelona, Spain; dInstitut National de la Santé et de la Recherche Médicale Unité 1048, Institut de Recherche sur les Maladies Métaboliques et Cardiovasculaires de l’Hôpital Rangueil, 31432 Toulouse, France; and eQuantitative Biology and Bioinformatics, VTT Technical Research Centre of Finland, 02044 Espoo, Finland Edited* by Bruce M. Spiegelman, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, and approved February 7, 2012 (received for review June 21, 2011) Mitochondria are dynamic organelles that play a key role in en- 22). Alternatively, insulin resistance may be a consequence of ergy conversion. Optimal mitochondrial function is ensured by specific alterations in mitochondrial metabolism. In this regard, a quality-control system tightly coupled to fusion and fission. In a high-fat diet (HFD) causes increased hydrogen peroxide pro- this connection, mitofusin 2 (Mfn2) participates in mitochondrial duction and oxidative stress in skeletal muscle, which might ex- fusion and undergoes repression in muscle from obese or type 2 plain, at least in part, the development of insulin resistance (23, diabetic patients. Here, we provide in vivo evidence that Mfn2 24). On the basis of these studies, we sought to determine the plays an essential role in metabolic homeostasis. Liver-specific ab- effects of Mfn2 in mitochondrial function and glucose homeo- fi lation of Mfn2 in mice led to numerous metabolic abnormalities, stasis in vivo. We demonstrate that Mfn2 de ciency produces characterized by glucose intolerance and enhanced hepatic gluco- mitochondrial dysfunction, increases H2O2 concentration, and PHYSIOLOGY fi activates JNK, leading to insulin resistance in skeletal muscle neogenesis. Mfn2 de ciency impaired insulin signaling in liver and fi muscle. Furthermore, Mfn2 deficiency was associated with endo- and liver. Importantly, Mfn2 de ciency also leads to ER stress, contributing as well to the loss of insulin sensitivity. In this paper, plasmic reticulum stress, enhanced hydrogen peroxide concentra- we describe a unique mechanism by which mitochondrial and ER tion, altered reactive oxygen species handling, and active JNK. N function, both under the control of Mfn2, converge in the reg- Chemical chaperones or the antioxidant -acetylcysteine amelio- ulation of insulin signaling and glucose homeostasis in vivo. rated glucose tolerance and insulin signaling in liver-specific Mfn2 KO mice. This study provides an important description of a unique Results unexpected role of Mfn2 coordinating mitochondria and endoplas- Liver-Specific Mfn2 KO Mice Show Glucose Intolerance, Enhanced mic reticulum function, leading to modulation of insulin signaling Hepatic Glucose Production, and Impaired Response to Insulin. Liver- and glucose homeostasis in vivo. specific Mfn2 KO mice were generated, and expression of Mfn2 − − was analyzed in tissues from control (Alb-Cre / Mfn2loxP/loxP) − mitochondrial dynamics | insulin resistance | metabolism | oxidative stress and KO (Alb-Cre+/ Mfn2loxP/loxP; hereafter referred to as L-KO) mice. Mfn2 expression was absent in liver, whereas normal Mfn2 itochondrial morphology, response to apoptotic stimuli, expression was detected in other tissues (Fig. 1A). Isolation of Mmitochondrial metabolism, and quality control are in part mouse hepatocytes was performed to analyze the architecture controlled through the balance between mitochondrial fusion of the mitochondrial network. Mitochondria formed tubules in and fission (1, 2). Mitochondrial fusion in mammalian cells is control hepatocytes, whereas mitochondrial clusters were de- controlled by mitofusin 1 and 2 (Mfn1 and Mfn2) proteins and by tected in L-KO mice (Fig. 1B). L-KO mice at 8 wk of age showed optic atrophy 1 (OPA1). Mfn1 and Mfn2 explain outer mito- similar values of body weight or epididymal adipose depots chondrial membrane fusion. Mfn2 has pleiotropic cellular roles compared with control mice (Fig. S1 A and B). Plasma glucose and regulates cell proliferation, oxidative metabolism, autoph- was increased in Mfn2 KO mice under a normal chow diet (Fig. agy, and mitochondrial antiviral signaling protein (3–7). In ad- 1C), which occurred in the presence of normal plasma insulin or dition, Mfn2 is also localized in the endoplasmic reticulum glucagon levels (Fig. 1 D and E). L-KO mice at 8 wk of age showed (ER)–mitochondrial contact sites, and it regulates the tethering impaired glucose tolerance (Fig. 1F) without changes in plasma of the ER to mitochondria as well as calcium homeostasis in insulin levels (Fig. S1C). Glucose intolerance was also detected mouse embryonic fibroblasts (8). Double ablation of Mfn2 and in L-KO mice subjected to a HFD for 20 wk (Fig. 1G), despite no Mfn1 causes mitochondrial DNA depletion, indicating that these changes in body weight (Fig. S1 D and E). In mice subjected to proteins are required for mitochondrial DNA stability (9). Mfn2 is relevant in human disease, and mutations in Mfn2 have been – – reported in patients affected by Charcot Marie Tooth neurop- Author contributions: D. Sebastián, M.I.H.-A., J.S., E.S., J.P.M., D. Sala, A.W., J.C.P., S.P., athy type 2A (10–12). In addition, we have reported that Mfn2 R.B., and A.Z. designed research; D. Sebastián, M.I.H.-A., J.S., E.S., J.P.M., D. Sala, J.C.P., expression is reduced in skeletal muscle of obese subjects and in P.G., and S.P. performed research; M.L. contributed new reagents/analytic tools; D. Sebastián, type 2 diabetic patients (13, 14). M.I.H.-A., J.S., E.S., J.P.M., D. Sala, J.C.P., M.O., S.P., R.B., M.P., and A.Z. analyzed data; and A.Z. Insulin resistance in skeletal muscle and liver plays a primary wrote the paper. role in the pathogenesis of type 2 diabetes (15). There is sub- The authors declare no conflict of interest. stantial evidence in humans indicating that insulin-resistant *This Direct Submission article had a prearranged editor. conditions are characterized by alterations in mitochondrial ac- 1D. Sebastián, M.I.H.-A., and J.S. contributed equally to this work. tivity in skeletal muscle caused by either reduced mitochondrial 2To whom correspondence should be addressed. E-mail: antonio.zorzano@irbbarcelona. mass or functional impairment of mitochondria (16–19). How- org. ever, several studies have challenged the concept that insulin This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. resistance is caused by a deficient mitochondrial function (20– 1073/pnas.1108220109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1108220109 PNAS | April 3, 2012 | vol. 109 | no. 14 | 5523–5528 Downloaded by guest on September 28, 2021 AB CD ◄ Mfn2 C L-KO Liver 250 10 ◄ Porin ◄ * SM Mfn2 200 8 ◄ Porin ◄ Mfn2 150 * 6 WAT ◄ Porin 100 4 ◄ Insulin (ng/ml) Heart Mfn2 Glucose (mg/dl) ◄ Porin 50 2 ◄ Mfn2 Pancreas ◄ Porin 0 0 CL-KO CL-KO CL-KO CL-KO L-KO C ND HFD ND HFD EG400 F 400 C 600 C HFD H 250 C HFD L-KO L-KO HFD L-KO HFD 500 225 300 300 400 200 200 200 300 * 175 * Glucose (mg/dl) Glucagon (pg/ml) 150 Glucose (mg/dl) * Glucose (mg/dl) 200 100 100 100 125 0 0 0 100 0 25 50 75 100 C L-KO C L-KO 0 25 50 75 100 125 0 25 50 75 100 125 Time (min) Time (min) Time (min) ND HFD C IJKL-KO 160 C 2.0 L-KO * * Fed 4h Fast 140 ◄ 1.5 * ◄ pCRTC2 ◄ CRTC2 ◄ 120 * 1.0 ◄ β-actin ◄ 100 * mRNA levels mRNA Glucose (mg/dl) (Relative units) 0.5 80 C L-KO C L-KO 60 0.0 0 25 50 75 100 125 PC PEPCK G6Pase PGC-1α Time (min) Fig. 1. Liver-specific Mfn2 ablation causes glucose intolerance and a reduced response to insulin. (A) Mfn2 protein levels from various tissues in control and L-KO mice. (B) Representative images of mitochondria in isolated hepatocytes. Mitochondria were visualized by transfecting hepatocytes with DsRed2-mito vector. (C–E) Plasma glucose insulin and glucagon levels in mice fed a normal diet (ND) or a HFD (n =8–12). (F) Glucose tolerance test (GTT) on mice fed a normal diet (n =15–20). (G) GTT on mice fed a HFD (n =8–12). (H) Insulin tolerance tests on mice fed a HFD (n =8–12). (I) Pyruvate challenge (n =15–20). (J) Hepatic expression of gluconeogenic genes (n =8–12). (K) CRTC2 phosphorylation in livers from fed or 4-h fasting mice. Data represent mean ± SEM. *P < 0.05. C, control; G6Pase, glucose 6-phosphatase; PC, pyruvate carboxylase. a HFD, plasma glucose was similar in control and L-KO mice FoxO1 was detected in the L-KO group after insulin treatment (Fig. 1C). In contrast, plasma insulin levels were markedly (Fig. S2C). These results suggest that Mfn2 deficiency causes greater in L-KO mice (Fig. 1D). The L-KO mouse subjected to a greater activation of CRTC2 and FoxO1 as well as the induction a HFD showed impaired glucose tolerance and high basal plasma of target genes and gluconeogenesis. insulin levels, suggesting susceptibility to insulin resistance.