Mitochondrial dysfunction has recently been implicated in the etiology of insulin resistance and type 2 diabetes. This suggestion is mainly based on studies that determined in vivo mitochondrial function (6
). However, it is not yet elucidated whether intrinsic mitochondrial defects cause this lower in vivo mitochondrial function in type 2 diabetic patients and first-degree relatives. Ex vivo studies on mitochondrial function in type 2 diabetic patients appear less conclusive (13
); however, in these studies, in vivo mitochondrial function was not assessed. To identify the true nature of reduced mitochondrial function in type 2 diabetic patients, the combination of in vivo and ex vivo measurements are of importance. Thus, impaired in vivo mitochondrial function might be caused by an impaired intrinsic capacity per mitochondrion or lower mitochondrial content. In this study, we performed both in vivo and ex vivo measurement of mitochondrial function in male diabetic patients, first-degree relatives, and control subjects, all matched for age and BMI.
We confirmed our previous finding (8
) of compromised in vivo mitochondrial function, determined as the rate of PCr recovery after exercise in type 2 diabetic patients compared with age- and BMI-matched control subjects, and we extend these findings to a tendency to reduced in vivo mitochondrial function in normoglycemic first-degree relatives. In fact, this reduction in in vivo mitochondrial function was more pronounced in the seven subjects with multiple type 2 diabetic relatives (data not shown). Lower basal ATP synthesis rate in insulin-resistant first-degree relatives has been reported previously (6
) when compared with insulin-sensitive healthy control subjects. Here, we report tendency to compromised mitochondrial function in first-degree relatives who possessed (nonsignificant) intermediate values of insulin sensitivity (the Rd
of glucose, EGP, and nonoxidative glucose disposal) compared with age- and BMI-matched control subjects. This may suggest that a compromised mitochondrial function is an early defect in the pathogenesis of type 2 diabetes.
Interestingly, using high-resolution respirometry in permeabilized skinned muscle fibers, we identified lower ADP-driven state 3 respiration as an intrinsic mitochondrial defect (ex vivo) that underlies the mitochondrial dysfunction observed in vivo. However, mitochondrial dysfunction was not related to insulin sensitivity or IMCL content, and the latter was not different between groups. This is in line with earlier findings, in which no differences in IMCL were found between BMI-matched overweight diabetic patients and control subjects (8
). Most likely, the combination of low oxidative capacity and fat content in muscle will lead to the accumulation of fatty acid intermediates, leading to muscular insulin resistance. Interestingly, the compromised ex vivo mitochondrial function could not be attributed to the mitochondrial density because the differences across groups remained on normalization to mtDNA copy number or citrate synthase. Moreover, even if in vivo mitochondrial function was adjusted for mtDNA copy number, differences between groups remained similar, as could be deduced from the similarity in mtDNA copy number between groups (data not shown). Thus, here, we show intrinsic mitochondrial defects in type 2 diabetic patients and similar tendencies in diabetes-prone first-degree relatives, which may underlie the mitochondrial dysfunction found in vivo.
The interesting, novel outcome of our study is the finding that intrinsic mitochondrial function, if also normalized to mitochondrial content, is lower in type 2 diabetic patients, and tended to be lower in first-degree relatives. Thus, both ADP-stimulated state 3 respiration and FCCP-driven maximal state u respiration were significantly lower (~30%) in diabetic patients compared with control subjects. ADP-stimulated state 3 respiration reflects the capacity of the mitochondria to reduce the tricarboxylic acid cycle and β-oxidation–derived reducing equivalents NADH and FADH2 in the electron transport chain (ETC), resulting in oxidative phosphorylation of ADP to synthesize ATP at the level of ATP synthase. Under physiological conditions, the rate of state 3 respiration is limited by, or under control of, the activity of the phosphorylation system (including ATP synthase and ANT). Thus, bypassing the phosphorylation system ex vivo by the use of the chemical uncoupler FCCP results in a stimulation of respiration, called state u respiration. State u respiration hence reflects the maximal capacity of the ETC and the upstream dehydrogenases (malate, glutamate, and succinate dehydrogenase) involved in hydrolysis of the substrates supplied. This can therefore be referred to as maximal, uncontrolled mitochondrial oxidative capacity. The reduction of ~30% in state u respiration as observed in type 2 diabetic patients therefore indicates a reduced maximal mitochondrial oxidative capacity in these patients that may reside at the level of the ETC and the upstream dehydrogenases. However, we found that state 3 respiration was also affected to a similar extent. Although, this could theoretically be explained by the reduced maximal mitochondrial capacity mentioned above (and thus at the level of the ETC and/or dehydrogenases), it should be noted that respiration was still stimulated on FCCP addition, in fact to a similar extent in type 2 diabetic patients and control subjects. Thus, state u respiration (ETC activity and upstream dehydrogenases) exceeds the level of state 3 respiration in all groups, indicating that also in type 2 diabetic patients, state 3 respiration is being limited by the phosphorylation system. Therefore, the 30% reduction in state 3 in diabetic patients indicates that the capacity of the phosphorylation systems is also reduced in these patients. Therefore, taken together, these data point toward a reduced intrinsic mitochondrial defect that resides both at the level of the ETC activity and/or upstream dehydrogenases and at the level of the phosphorylation system.
In first-degree relatives, the tendency to a lower in vivo mitochondrial function was accompanied by a tendency to a lower maximal respiratory capacity (state u), but no defect in state 3 respiration. A lower state u respiration, with unaffected state 3 respiration, points toward a reduced maximal ETC activity, without a reduction in the phosphorylation system. Thus, a tendency toward lower state u respiration in first-degree relatives suggests that mitochondria of first-degree relatives are capable of compensating the reduced maximal mitochondrial capacity by respiring on a higher percentage of the ETC capacity, i.e., with less tight control by the phosphorylation system.
Mitochondrial entrance of fatty acids via the carnitine shuttle system is classically believed to be rate limiting for β-oxidation. Using palmitoyl-carnitine, this putative limitation in fat oxidation is circumvented, thereby maximizing the flow through the β-oxidation. Through the addition of palmitoyl-carnitine to the substrate cocktail, additional reducing equivalents not requiring malate, glutamate, or succinate dehydrogenases are fed into the ETC. The addition of palmitoyl-carnitine, however, did not rescue the defects detected when using malate, glutamate, or succinate as substrates. This is an important observation suggesting that the defects observed in type 2 diabetic patients are unlikely to be localized in the malate, glutamate, and succinate dehydrogenases but are more likely to be located at the level of the ETC and the phosphorylation system. This indicates a lower overall intrinsic mitochondrial oxidative capacity. The reason for the latter is unknown but may involve ultrastructural mitochondrial changes. In that respect, smaller mitochondria have been reported for type 2 diabetic patients (1
), and mitochondrial size was related to insulin sensitivity (1
). Whether mitochondrial size determines overall mitochondrial capacity in these patients needs further investigation.
A putatively confounding factor when studying mitochondrial function is the level of physical activity of the subjects. In our study, we included sedentary subjects and type 2 diabetic patients without comorbidities. All subjects filled out a physical activity questionnaire indicating low levels of physical activity without differences between groups. Also maximal aerobic performance, which is a reflection of physical fitness, was similar between groups. This suggests that in the present study, the lower mitochondrial function in type 2 diabetes and tendency in first-degree relatives was not due to differences in level of habitual physical activity. Because physical activity is the major driver of mitochondrial biogenesis (27
), the low and comparable level of physical activity may also explain why we did not observe any differences in mitochondrial content (determined by citrate synthase activity and mtDNA copy number) among diabetic patients, first-degree relatives, and control subjects.
Mitochondrial dysfunction implicated in type 2 diabetes, as shown in the present study, may alter substrate oxidation and might underlie the blunted increase in fat oxidation under conditions such as fasting, despite the presence of high circulating plasma levels of NEFA (28
). This defect has been termed “metabolic inflexibility” and can also be observed as a reduced response to insulin, i.e., a reduced enhancement of glucose oxidation during insulin stimulation (29
). A previous study indicated a link between defects in substrate switching and mitochondrial defects as shown in young, sedentary, healthy first-degree relatives (30
). In their study, first-degree relatives were metabolic inflexible after consuming a high-fat diet, suggesting a decreased ability to enhance fat oxidation. In the present study, we observed metabolic inflexibility in type 2 diabetic patients, but not in first-degree relatives, together with a (tendency to) reduced in vivo mitochondrial function in both type 2 diabetic patients and first-degree relatives. Therefore, our data do not entirely match with the suggestion that mitochondrial dysfunction underlies this metabolic inflexibility. However, the change in respiratory quotient on insulin stimulation is more likely to be a reflection of reduced skeletal muscle insulin sensitivity.
In conclusion, the present study supports the hypothesis that intrinsic mitochondrial dysfunction is implicated in the etiology of type 2 diabetes. In vivo mitochondrial function was accompanied by lower ADP-driven mitochondrial respiration, if corrected for mitochondrial density. These mitochondrial aberrations are located at the level of both electron-transport chain capacity and oxidative phosphorylation system. Our data suggest that a reduced intrinsic ADP-stimulated mitochondrial respiration is an important factor in the pathogenesis of type 2 diabetes.