Mitochondrial energy deficiency leads to hyperproliferation of skeletal muscle mitochondria and enhanced insulin sensitivity

Abstract

Diabetes is associated with impaired glucose metabolism in the presence of excess insulin. Glucose and fatty acids provide reducing equivalents to mitochondria to generate energy, and studies have reported mitochondrial dysfunction in type II diabetes patients. If mitochondrial dysfunction can cause diabetes, then we hypothesized that increased mitochondrial metabolism should render animals resistant to diabetes. This was confirmed in mice in which the heart-muscle-brain adenine nucleotide translocator isoform 1 (ANT1) was inactivated. ANT1-deficient animals are insulin-hypersensitive, glucose-tolerant, and resistant to high fat diet (HFD)-induced toxicity. In ANT1-deficient skeletal muscle, mitochondrial gene expression is induced in association with the hyperproliferation of mitochondria. The ANT1-deficient muscle mitochondria produce excess reactive oxygen species (ROS) and are partially uncoupled. Hence, the muscle respiration under nonphosphorylating conditions is increased. Muscle transcriptome analysis revealed the induction of mitochondrial biogenesis, down-regulation of diabetes-related genes, and increased expression of the genes encoding the myokines FGF21 and GDF15. However, FGF21 was not elevated in serum, and FGF21 and UCP1 mRNAs were not induced in liver or brown adipose tissue (BAT). Hence, increased oxidation of dietary-reducing equivalents by elevated muscle mitochondrial respiration appears to be the mechanism by which ANT1-deficient mice prevent diabetes, demonstrating that the rate of mitochondrial oxidation of calories is important in the etiology of metabolic disease.

Keywords: ANT1; insulin sensitivity; mitochondria; skeletal muscle.

Fig. 1.

ANT1 deficiency provides resistance to HFD and modulates metabolism. (A) Kaplan–Meier survival curve of Ant1^+/+ and Ant1^−/− mice fed a HFD. (B) Body weight measured in mice fed a HFD. (C) Body composition measured by MRI in 8-mo-old mice fed a HFD. (D) RER measured over 24 h in 8–9-mo-old mice fed a HFD. (E) Activity levels measured over 24 h in 8–9-mo-old mice fed a HFD. Data are represented as mean ± SEM; n = 10–12 per group (A), 16–28 per group (B), and 6–9 per group (C–E). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. S1.

ANT1 deficiency modulates metabolism according to diet. (A) Kaplan–Meier survival curve of Ant1^+/+ and Ant1^−/− mice fed a LFD. (B) Body weight measured in mice fed a LFD. (C) Body composition measured by MRI in 8-mo-old mice fed a LFD. (D) RER measured over 24 h in 8–9-mo-old mice fed a LFD. (E) Activity levels measured over 24 h in 8–9-mo-old mice fed a LFD. VO₂ rate during a 24-h period divided into four 6-h phases measured in 8–9-mo-old mice fed (F) a LFD or (G) a HFD. Data are represented as mean ± SEM; n = 12–30 per group (A and B) and 6–9 per group (C–G). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 2.

ANT1 deficiency enhances insulin sensitivity. (A) Glucose tolerance test performed in 9-mo-old mice fed a HFD for 6 mo. (B) Plasma insulin levels during the glucose tolerance test. (C) Hyperinsulinemic–euglycemic clamp performed on 8–9-mo-old mice fed a HFD. GIR, glucose infusion rate; Rd, rate of glucose disposal. (D) ¹⁴C-2-deoxyglucose uptake in white fat and skeletal muscle. Data are represented as mean ± SEM; n = 16–28 per group (A and B) and 7–9 per group (C and D). **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. S2.

ANT1 deficiency enhances insulin sensitivity on LFD. (A) Glucose tolerance test performed in 24-mo-old mice fed a LFD. (B) Plasma insulin levels during the glucose tolerance test on LFD. (C) Hyperinsulinemic–euglycemic clamp performed on 8–9-mo-old mice fed a LFD. GIR, glucose infusion rate; Rd, rate of glucose disposal. (D) ¹⁴C-2-deoxyglucose uptake in white fat and skeletal muscle on LFD. Data are represented as mean ± SEM; n = 11–13 per group (A and B) and 7–9 per group (C and D). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 3.

Skeletal muscle morphology in Ant1^−/− mice. Ant1^+/+ and Ant1^−/− mice fed a HFD were killed at 12 mon. Ant1^−/− mice show increased reddening in (A) whole-body skeletal muscle and isolated gastrocnemius. (B) Representative electron micrographs of white gastrocnemius in the intermyofibrillar (IMF) and subsarcolemmal (SS) compartments. (C) Representative triple MHC labeling (MHCI, blue; MHCIIa, red; MHCIIb, green; MHCIIx, black) performed in Ant1^+/+ and Ant1^−/− mice. (Scale bar, 2,000 µm.). 1, white gastrocnemius; 2, red gastrocnemius; 3, plantaris; 4, soleus. (D) Fiber type proportion in white gastrocnemius from Ant1^+/+ and Ant1^−/− mice. Data are represented as mean ± SEM; n = 2 per group. *P < 0.01; **P < 0.01.

Fig. 4.

ANT1 deficiency causes alterations in mitochondrial enzymes and OXPHOS activity. Ant1^+/+ and Ant1^−/− mice were fed a HFD and killed at 12 mo. Enzyme activity of (A) CS, (B) SDH, and (C) COX in isolated white gastrocnemius muscle expressed as units of activity per wet weight of muscle tissue. (D) Relative mtDNA content is represented by fold change of ND1 normalized to B2M in white gastrocnemius and graphed as geometric mean with 95% confidence interval (CI). (E) Oxygen consumption in permeabilized muscle fibers from white gastrocnemius per unit of CS enzyme activity. ADP, G/M + ADP; G/M, glutamate + malate; Succ, G/M + ADP + succinate. (F) Oxygen consumption in permeabilized white muscle fibers per mg wet weight of muscle tissue. (G) H₂O₂ production in permeabilized white muscle fibers per unit of CS enzyme activity. AA = G/M + Succ + ADP + antimycin A; ADP = G/M + Succ + ADP; Basal = no substrates; G/M = glutamate + malate; Succ = G/M + succinate. (H) H₂O₂ production in permeabilized white muscle fibers per mg wet weight of muscle tissue. (I) Free radical leak in permeabilized white muscle fibers per unit of CS activity expressed as fold change of Ant1^+/+. (J) CRC in permeabilized white muscle fibers per unit of CS activity. Data are represented as mean ± SEM; n = 8 per group. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. S3.

ANT1 deficiency causes alterations in mitochondrial enzymes and OXPHOS activity. Ant1^+/+ and Ant1^−/− mice were fed a HFD and killed at 12 mo. Enzyme activity of (A) CS, (B) SDH, and (C) COX in isolated red gastrocnemius muscle. Data are expressed as units of activity per wet weight of muscle tissue. (D) Relative mtDNA content is represented by fold change of ND1 normalized to B2M in red gastrocnemius and graphed as geometric mean with 95% CI. (E) Oxygen consumption in permeabilized red muscle fibers per unit of CS enzyme activity. ADP = G/M + ADP; G/M = glutamate + malate; Succ = G/M + ADP + succinate. (F) Oxygen consumption in permeabilized red muscle fibers per mg wet weight of muscle tissue. RCR in permeabilized muscle fibers from (G) white and (H) red gastrocnemius, calculated as the ratio of state III respiration/state II respiration. (I) H₂O₂ production in permeabilized red muscle fibers per unit of CS enzyme activity. AA = G/M + Succ + ADP + antimycin A; ADP = G/M + Succ + ADP; Basal = no substrates; G/M = glutamate + malate; Succ = G/M + succinate. (J) H₂O₂ production in permeabilized red muscle fibers per mg wet weight of muscle tissue. (K) Free radical leak in permeabilized red muscle fibers per unit of CS activity expressed as fold change of Ant1^+/+. (L) CRC in permeabilized red muscle fibers per unit of CS activity. All data are means ± SEM; n = 8 per group. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 5.

Alterations in transcript levels of ANT1-deficient gastrocnemius. (A) Volcano plot showing distribution of the up- and down-regulated genes in Ant1^−/− versus Ant1^+/+ white muscle analyzed by RNA sequencing. (B) Mean nuclear DNA (nDNA) transcript levels of the electron transport chain complexes. Solid fill, Ant1^+/+; dashed fill, Ant1^−/−. (C) mtDNA transcript levels averaged by electron transport chain complexes, transfer RNAs (tRNA), and ribosomal RNAs (rRNA). Solid fill, Ant1^+/+; dashed fill, Ant1^−/−. (D) mtDNA transcripts shown individually in their sequential order on the mtDNA genome. Data are represented as mean ± SEM; n = 4 per group. *P < 0.01; **P < 0.001; ***P < 0.0001. (E) Fold change relative levels of FGF21 mRNA in gastrocnemius (gastroc) and liver and UCP1 mRNA in BAT by qPCR. H, HFD; L, LFD. Data are represented as geometric mean with 95% CIs. n = 4 per group. **P < 0.01; ****P < 0.0001. (F) Serum FGF21 levels in Ant1^+/+ and Ant1^−/− mice. n = 4 per group.

Fig. S4.

Altered transcriptome in ANT1-deficient mice up-regulates mitochondrial processes in white muscle. Twenty-five most significant biological process categories for genes enriched in Ant1^−/− skeletal muscle showing (A) up-regulated (>1.5-fold, n = 227) and (B) down-regulated categories (<0.3-fold, n = 453). Queried categories include Gene Ontology (GO), Protein Information Resource (PIR), Sequence (Seq) Features, Kyoto Encyclopedia of Genes and Genomes (KEGG), and InterPro protein sequence and analysis classification. Analysis was performed using DAVID (v6.7). (C–F) Fold change of relative mRNA levels of FGF21, GDF15, UCP1, and BDNF in Ant1^−/− versus Ant1^+/+ gastrocnemius (gastroc), liver, BAT, and WAT by qPCR. H, HFD; L, LFD. Data are represented as geometric mean with 95% CIs. n = 4 per group. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.