Opening of the mitochondrial permeability transition pore links mitochondrial dysfunction to insulin resistance in skeletal muscle - PubMed (original) (raw)

. 2013 Nov 26;3(2):124-34.

doi: 10.1016/j.molmet.2013.11.003. eCollection 2014 Apr.

R C Laker 2, D S Breen 1, Y N Akhtar 2, B M Kenwood 1, J A Liao 1, M Zhang 2, D J Fazakerley 3, J L Tomsig 1, T E Harris 1, S R Keller 1, J D Chow 1, K R Lynch 4, M Chokki 5, J D Molkentin 6, N Turner 7, D E James 8, Z Yan 9, K L Hoehn 10

Affiliations

E P Taddeo et al. Mol Metab. 2013.

Abstract

Insulin resistance is associated with mitochondrial dysfunction, but the mechanism by which mitochondria inhibit insulin-stimulated glucose uptake into the cytoplasm is unclear. The mitochondrial permeability transition pore (mPTP) is a protein complex that facilitates the exchange of molecules between the mitochondrial matrix and cytoplasm, and opening of the mPTP occurs in response to physiological stressors that are associated with insulin resistance. In this study, we investigated whether mPTP opening provides a link between mitochondrial dysfunction and insulin resistance by inhibiting the mPTP gatekeeper protein cyclophilin D (CypD) in vivo and in vitro. Mice lacking CypD were protected from high fat diet-induced glucose intolerance due to increased glucose uptake in skeletal muscle. The mitochondria in CypD knockout muscle were resistant to diet-induced swelling and had improved calcium retention capacity compared to controls; however, no changes were observed in muscle oxidative damage, insulin signaling, lipotoxic lipid accumulation or mitochondrial bioenergetics. In vitro, we tested 4 models of insulin resistance that are linked to mitochondrial dysfunction in cultured skeletal muscle cells including antimycin A, C2-ceramide, ferutinin, and palmitate. In all models, we observed that pharmacological inhibition of mPTP opening with the CypD inhibitor cyclosporin A was sufficient to prevent insulin resistance at the level of insulin-stimulated GLUT4 translocation to the plasma membrane. The protective effects of mPTP inhibition on insulin sensitivity were associated with improved mitochondrial calcium retention capacity but did not involve changes in insulin signaling both in vitro and in vivo. In sum, these data place the mPTP at a critical intersection between alterations in mitochondrial function and insulin resistance in skeletal muscle.

Keywords: ANT, adenine nucleotide translocator; BKA, bongkrekic acid; CSA, cyclosporin A; CYPD, cyclophilin D; Cyclophilin D; DAG, diacylglycerol; ETC, electron transport chain; FFA, free fatty acid; Glucose; HFD, high fat diet; HK2, hexokinase 2; Insulin resistance; KO, knockout; LFD, low fat diet; MCAD, medium chain acyl-CoA dehydrogenase; MHC, myosin heavy chain; MIRKO, muscle insulin receptor knockout; MPTP, mitochondrial permeability transition pore; Mitochondrial dysfunction; Mitochondrial permeability transition pore; MnSOD, mitochondrial manganese superoxide dismutase; O2•, superoxide; OXPHOS, oxidative phosphorylation; PDH, pyruvate dehydrogenase; PDHa, active PDH; PDHt, total PDH; PM, plasma membrane; Rg′, rate of glucose transport; Skeletal muscle; TBARS, thiobarbituric acid reactive substances; TEM, transmission electron microscopy; VDAC, voltage-dependent anion channel; WT, wild type; [3H]-2-DOG, [3H]-2-deoxyglucose; β-HAD, β-hydroxyacyl-CoA dehydrogenase.

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Figures

Figure 1

Figure 1

CypD KO mice are protected from HFD-induced glucose intolerance. (A–D) CypD knockout (KO) mice and wild type (WT) control littermates were fed a LFD until 8 weeks of age when they were switched to a 45% high fat diet (HFD) for up to 11 weeks. Glucose tolerance was tested at weeks 0 (LFD), 1, 4, and 11 of HFD (2 g/kg glucose in (A) and 1.5 g/kg in (B–D)); ⁎ represents a significant difference in integrated AUC (p<0.05). (E and F) Gonadal, subcutaneous (inguinal), and retroperitoneal white adipose tissue (WAT) mass, liver mass, and total body mass were not statistically different between genotypes when fed a HFD diet for up to 11 weeks. Circulating insulin (G), free fatty acid (FFA) (H), and triglyceride (I) levels were similar between genotypes in either the fed or fasted states. Data are presented as means±standard error of the mean (SEM). _n_=8–16 for A–F and _n_=4–7 for G–I.

Figure 2

Figure 2

Improved skeletal muscle glucose clearance in CypD KO mice fed HFD for 11 weeks. (A) [3H]-2-DOG tracer appearance and disappearance curves for each genotype. (B) The calculated AUC for each genotype. (C and D) The estimated rate of glucose transport (Rg′) into quadriceps muscle and gonadal WAT as determined by phosphorylated-[3H]-2-DOG normalized to tracer availability over time and tissue mass. (E and F) [U-14C]-glucose storage as glycogen in quadriceps muscle and liver. Data are represented as means±SEM of 9 WT and 8 KO. ⁎p<0.05. ns, not significant.

Figure 3

Figure 3

Insulin signaling, GLUT4 protein expression, and lipid analysis of skeletal muscle from mice fed a HFD for 11 weeks. (A) Western blots for CypD, phospho-S473 Akt, total Akt, phospho-S9 GSK3β and total GSK3β in mixed quadriceps muscles. Expression of 14-3-3 was used as a loading control. (B) Quantification of phospho/total Akt and phospho/total GSK3β immunoblots. (C) Western blot of GLUT4 protein expression in quadriceps muscle. GAPDH served as a loading control. (D) Quantification of normalized GLUT4 expression. (E and F) Ceramide and diacylglycerol levels in quadriceps. Results are displayed as means±SEM, _n_≥3, ns, not significant.

Figure 4

Figure 4

Skeletal muscle morphology and mitochondrial calcium retention capacity in WT and CypD KO mice. (A–D) Representative TEM images (10,000× magnification) of intermyofibrillar and subsarcolemmal mitochondria in tibialis cranialis muscle from WT and CypD KO mice fed a HFD for 29 weeks. Scale bar equals 1 µm. (E) Quantification of mitochondrial length in tibialis cranialis muscles. Lengths from ≥170 mitochondria per genotype (intermyofibrillar) or ≥90 mitochondria per genotype (subsarcolemmal) were quantified in TEM images using ImageJ. (F) Representative trace from three calcium retention capacity assays of mitochondria isolated from gastrocnemius muscles of WT mice fed a LFD or HFD for 10 weeks. (G) Representative trace from three calcium retention capacity assays comparing WT and CypD KO mice fed HFD for 10 weeks. Arrows indicate calcium-induced mPTP opening.

Figure 5

Figure 5

mPTP opening drives insulin resistance in vitro. (A) Insulin-stimulated GLUT4 translocation was analyzed in L6 myotubes treated for 30 min with increasing concentrations of ferutinin (0.1 µM, 1 µM, and 5 µM) in the absence or presence of a 10 min pretreatment with mPTP inhibitors CsA (1 µg/mL) or BKA (10 µM). (B) Akt phosphorylation in basal or insulin-stimulated L6 myotubes treated with or without 5 µM ferutinin in the absence or presence of 1 µg/mL CsA or 10 µM BKA. Representative Western blots from three individual experiments are shown. (C) GLUT4 translocation assay after a 30 min treatment with 100 nM C2-ceramide or 50 nM antimycin A, with or without 10 min pretreatment with 1 µg/mL CsA, n_=3. Results are displayed as means±SEM, ⁎_p<0.05.

Figure 6

Figure 6

Palmitate-induced insulin resistance requires mPTP opening. (A) Calcium retention capacity was measured in mitochondria isolated from L6 myotubes treated for 16 h with 150 µM palmitate or control (BSA) in the absence or presence of CsA treatment (1 µg/mL). CsA improves mitochondrial calcium retention capacity in both palmitate- and BSA-treated cells. Representative traces are shown from one of three experiments that showed similar results. (B) Insulin-stimulated GLUT4 translocation to the PM was measured in control and palmitate-treated L6 myotubes in the absence or presence of CsA. Results are displayed as means±SEM. n_=3, ⁎_p<0.05. (C) Opening of the mPTP promotes insulin resistance in skeletal muscle. Insults that promote excessive mitochondrial O2• and/or mitochondrial calcium overload converge on the mPTP to induce skeletal muscle insulin resistance, which is rescued by pharmacological or genetic inhibition of the mPTP.

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