The influence of age and aerobic fitness: effects on mitochondrial respiration in skeletal muscle.

AIM: Mitochondrial function has previously been studied in ageing, but never in humans matched for maximal oxygen uptake ((V)·O2max). Furthermore, the influence of ageing on mitochondrial substrate sensitivity is not known. METHODS: Skeletal muscle mitochondrial respiratory capacity and mitochondrial substrate sensitivity were measured by respirometry in young (23 ± 3 years) and middle-aged (53 ± 3 years) male subjects with similar (V)·O2max. Protocols for respirometry included titration of substrates for complex I (glutamate), complex II (succinate) and both (octanoyl carnitine) for calculation of substrate sensitivity (C(50) ). Myosin heavy chain (MHC) isoforms, citrate synthase (CS) and ß-hydroxy-acyl-CoA-dehydrogenase (HAD) activity, mitochondrial DNA (mtDNA) content, protein levels of complexes I-V and antioxidant defence system [manganese superoxide dismutase (MnSOD)] were measured. RESULTS: No differences were found in maximal mitochondrial respiration or C(50) with glutamate (2.0 ± 0.3 and 1.8 ± 0.3 mm), succinate (3.7 ± 0.2 and 3.8 ± 0.4 mm) or octanoyl carnitine (47 ± 8 and 56 ± 7 µm) in young and middle-aged subjects respectively. Normalizing mitochondrial respiration to mtDNA young subjects had a higher (P < 0.05) respiratory capacity per mitochondrion compared to middle-aged subjects. HAD activity and mtDNA per mg tissue were higher in middle-aged compared to young subjects. Middle-aged had a higher MHC I isoform and a lower MHC IIX isoform content compared to young subjects. CONCLUSION: Mitochondrial substrate sensitivity is not affected by ageing. When young and middle-aged men are carefully matched for (V)·O2max, mitochondrial respiratory capacity is also similar. However, per mitochondrion respiratory capacity was lower in middle-aged compared to young subjects. Thus, when matched for (V)·O2max, middle-aged seem to require a higher mitochondrial content than young subjects.

Obesity augments the age-induced increase in mitochondrial capacity for H2O2 release in Zucker fatty rats.

AIM: Mitochondrial dysfunction has been suggested to play a significant role in obesity and insulin resistance. The aim of the present study was to investigate if changes in obesity and insulin resistance were related to similar changes in mitochondrial capacity for hydrogen peroxide release in Zucker diabetic fatty rats and their lean littermates. METHODS: Thirty-four rats were used in this study. Rats were either lean or obese Zucker rats killed at 5-6 (young) or 12-14 (adults) weeks of age. Mitochondria were isolated from soleus muscles; respiration and release of hydrogen peroxide were determined and related to citrate synthase activity to determine intrinsic mitochondrial function. Mitochondrial-specific super-oxide dismuthase (MnSOD) protein content was determined in isolated mitochondria and muscle homogenate. Catalase protein content was determined in muscle homogenate. RESULTS: Young lean and obese rats had a higher mitochondrial respiration when using palmitoyl-l-carnitine as substrate compared with adult lean and obese rats. The obese strain had higher mitochondrial hydrogen peroxide release but only in the adult animals. In both lean and obese animals, increased age was associated with increased mitochondrial hydrogen peroxide release. MnSOD tended to be higher in the obese strain in the isolated mitochondria. Regardless of age, catalase protein content was significantly lower in the obese rats. CONCLUSIONS: This study shows that the augmented increase in obesity and insulin resistance seen in Zucker diabetic fatty rats is associated with increased capacity for mitochondrial hydrogen peroxide release.

Increased mitochondrial substrate sensitivity in skeletal muscle of patients with type 2 diabetes.

AIMS/HYPOTHESIS: Mitochondrial respiration has been linked to insulin resistance. We studied mitochondrial respiratory capacity and substrate sensitivity in patients with type 2 diabetes (patients), and obese and lean control participants. METHODS: Mitochondrial respiration was measured in permeabilised muscle fibres by respirometry. Protocols for respirometry included titration of substrates for complex I (glutamate), complex II (succinate) and both (octanoyl-carnitine). Myosin heavy chain (MHC) composition, antioxidant capacity (manganese superoxide dismutase [MnSOD]), citrate synthase activity and maximal oxygen uptake (VO2) were also determined. Insulin sensitivity was determined with the isoglycaemic-hyperinsulinaemic clamp technique. RESULTS: Insulin sensitivity was different (p < 0.05) between the groups (patients

Impaired insulin-induced site-specific phosphorylation of TBC1 domain family, member 4 (TBC1D4) in skeletal muscle of type 2 diabetes patients is restored by endurance exercise-training.

AIMS/HYPOTHESIS: Insulin-mediated glucose disposal rates (R(d)) are reduced in type 2 diabetic patients, a process in which intrinsic signalling defects are thought to be involved. Phosphorylation of TBC1 domain family, member 4 (TBC1D4) is at present the most distal insulin receptor signalling event linked to glucose transport. In this study, we examined insulin action on site-specific phosphorylation of TBC1D4 and the effect of exercise training on insulin action and signalling to TBC1D4 in skeletal muscle from type 2 diabetic patients. METHODS: During a 3 h euglycaemic-hyperinsulinaemic (80 mU min⁻¹ m⁻²) clamp, we obtained M. vastus lateralis biopsies from 13 obese type 2 diabetic and 13 obese, non-diabetic control individuals before and after 10 weeks of endurance exercise-training. RESULTS: Before training, reductions in insulin-stimulated R (d), together with impaired insulin-stimulated glycogen synthase fractional velocity, Akt Thr³°8 phosphorylation and phosphorylation of TBC1D4 at Ser³¹8, Ser588 and Ser75¹ were observed in skeletal muscle from diabetic patients. Interestingly, exercise-training normalised insulin-induced TBC1D4 phosphorylation in diabetic patients. This happened independently of increased TBC1D4 protein content, but exercise-training did not normalise Akt phosphorylation in diabetic patients. In both groups, training-induced improvements in insulin-stimulated R(d) (~20%) were associated with increased muscle protein content of Akt, TBC1D4, α2-AMP-activated kinase (AMPK), glycogen synthase, hexokinase II and GLUT4 (20-75%). CONCLUSIONS/INTERPRETATION: Impaired insulin-induced site-specific TBC1D4 phosphorylation may contribute to skeletal muscle insulin resistance in type 2 diabetes. The mechanisms by which exercise-training improves insulin sensitivity in type 2 diabetes may involve augmented signalling of TBC1D4 and increased skeletal muscle content of key insulin signalling and effector proteins, e.g., Akt, TBC1D4, AMPK, glycogen synthase, GLUT4 and hexokinase II.

Effect of physical training on mitochondrial respiration and reactive oxygen species release in skeletal muscle in patients with obesity and type 2 diabetes.

AIM/HYPOTHESIS: Studies have suggested a link between insulin resistance and mitochondrial dysfunction in skeletal muscles. Our primary aim was to investigate the effect of aerobic training on mitochondrial respiration and mitochondrial reactive oxygen species (ROS) release in skeletal muscle of obese participants with and without type 2 diabetes. METHODS: Type 2 diabetic men (n = 13) and control (n = 14) participants matched for age, BMI and physical activity completed 10 weeks of aerobic training. Pre- and post-training muscle biopsies were obtained before a euglycaemic-hyperinsulinaemic clamp and used for measurement of respiratory function and ROS release in isolated mitochondria. RESULTS: Training significantly increased insulin sensitivity, maximal oxygen consumption and muscle mitochondrial respiration with no difference between groups. When expressed in relation to a marker of mitochondrial density (intrinsic mitochondrial respiration), training resulted in increased mitochondrial ADP-stimulated respiration (with NADH-generating substrates) and decreased respiration without ADP. Intrinsic mitochondrial respiration was not different between groups despite lower insulin sensitivity in type 2 diabetic participants. Mitochondrial ROS release tended to be higher in participants with type 2 diabetes. CONCLUSIONS/INTERPRETATION: Aerobic training improves muscle respiration and intrinsic mitochondrial respiration in untrained obese participants with and without type 2 diabetes. These adaptations demonstrate an increased metabolic fitness, but do not seem to be directly related to training-induced changes in insulin sensitivity.