Insulin and IGF-1 receptors regulate complex I–dependent mitochondrial bioenergetics and supercomplexes via FoxOs in muscle
Gourav Bhardwaj, Christie M. Penniman, Jayashree Jena, Pablo A. Suarez Beltran, Collin Foster, Kennedy Poro, Taylor L. Junck, Antentor O. Hinton Jr., Rhonda Souvenir, Jordan D. Fuqua, Pablo E. Morales, Roberto Bravo-Sagua, William I. Sivitz, Vitor A. Lira, E. Dale Abel, Brian T. O’Neill
Gourav Bhardwaj, Christie M. Penniman, Jayashree Jena, Pablo A. Suarez Beltran, Collin Foster, Kennedy Poro, Taylor L. Junck, Antentor O. Hinton Jr., Rhonda Souvenir, Jordan D. Fuqua, Pablo E. Morales, Roberto Bravo-Sagua, William I. Sivitz, Vitor A. Lira, E. Dale Abel, Brian T. O’Neill
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Research Article Endocrinology

Insulin and IGF-1 receptors regulate complex I–dependent mitochondrial bioenergetics and supercomplexes via FoxOs in muscle

Abstract

Decreased skeletal muscle strength and mitochondrial dysfunction are characteristic of diabetes. The actions of insulin and IGF-1 through the insulin receptor (IR) and IGF-1 receptor (IGF1R) maintain muscle mass via suppression of forkhead box O (FoxO) transcription factors, but whether FoxO activation coordinates atrophy in concert with mitochondrial dysfunction is unknown. We show that mitochondrial respiration and complex I activity were decreased in streptozotocin (STZ) diabetic muscle, but these defects were reversed in muscle-specific FoxO1, -3, and -4 triple-KO (M-FoxO TKO) mice rendered diabetic with STZ. In the absence of systemic glucose or lipid abnormalities, muscle-specific IR KO (M-IR–/–) or combined IR/IGF1R KO (MIGIRKO) impaired mitochondrial respiration, decreased ATP production, and increased ROS. These mitochondrial abnormalities were not present in muscle-specific IR, IGF1R, and FoxO1, -3, and -4 quintuple-KO mice (M-QKO). Acute tamoxifen-inducible deletion of IR and IGF1R also decreased muscle pyruvate respiration, complex I activity, and supercomplex assembly. Although autophagy was increased when IR and IGF1R were deleted in muscle, mitophagy was not increased. Mechanistically, RNA-Seq revealed that complex I core subunits were decreased in STZ-diabetic and MIGIRKO muscle, and these changes were not present with FoxO KO in STZ-FoxO TKO and M-QKO mice. Thus, insulin-deficient diabetes or loss of insulin/IGF-1 action in muscle decreases complex I–driven mitochondrial respiration and supercomplex assembly in part by FoxO-mediated repression of complex I subunit expression.

Authors

Gourav Bhardwaj, Christie M. Penniman, Jayashree Jena, Pablo A. Suarez Beltran, Collin Foster, Kennedy Poro, Taylor L. Junck, Antentor O. Hinton Jr., Rhonda Souvenir, Jordan D. Fuqua, Pablo E. Morales, Roberto Bravo-Sagua, William I. Sivitz, Vitor A. Lira, E. Dale Abel, Brian T. O’Neill

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Figure 6

Mitophagy flux is not increased by loss of IR/IGF1R in vivo and in vitro.

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Mitophagy flux is not increased by loss of IR/IGF1R in vivo and in vitro...
(A and B) Western blot images and densitometry of Bnip3 in quad tissue homogenate from MIGIRKO (A) and M-QKO (B) with respective controls (n = 3–4). (C and D) Western blot images of Bnip3 (C) and densitometry (D) in mitochondrial isolates in quad/gast muscle from IND-IGIRKO with respective controls (n = 5–6). (E and F) Western blot image from IND-MIGIRKO (E) with densitometry (F) of Bnip3 in quad tissue homogenate after 2-day colchicine or saline treatment (n = 3–4). (G) Western blot of IR and IGF1R from IR/IGF1Rfl/fl primary myotubes on day 6 of differentiation after Ad-Cre or Ad-Luc. (H) Measurement of mitophagy in primary myotubes at day 7 of differentiation, previously infected with Adeno–mito-Keima, Ad-Cre, and Ad-Luc. Myotubes were treated with or without insulin and CCCP for 4 hours prior to imaging for mitophagy index (n = 3 experiments). Mitophagy index is displayed as the ratio of mito-Keima fluorescence using 550 nm excitation versus 450 nm excitation (550/450 nm). (I) Confocal microscopy analysis of TA muscle from day-21 IND-IGIRKO and control mice after coelectroporation with the mitochondrial oxidation probe, MitoTimer, and lysosomal LAMP1-YFP. Merged panels are also presented in Supplemental Figure 6E. Original magnification, ×40. (J and K) Quantification of mitochondrial oxidation measured as MitoTimer red/green ratio (I) and colocalization of oxidized mitochondria (MitoTimer red/green ratio >2) and LAMP1-YFP measured as Manders’ coefficient (J) (n = 9–10). Results are represented as mean ± SEM. *P < 0.05; **P < 0.01 vs. littermate control, t test for 2 groups. ##P < 0.01; ###P < 0.001, 3-way ANOVA.