help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2008-0630
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Brøns, C.
Right arrow Articles by Vaag, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Brøns, C.
Right arrow Articles by Vaag, A.
Related Collections
Right arrow Diabetes and Insulin
Right arrow Male Endocrinology
Right arrow Metabolism
The Journal of Clinical Endocrinology & Metabolism Vol. 93, No. 10 3885-3892
Copyright © 2008 by The Endocrine Society

Mitochondrial Function in Skeletal Muscle Is Normal and Unrelated to Insulin Action in Young Men Born with Low Birth Weight

Charlotte Brøns, Christine B. Jensen, Heidi Storgaard, Amra Alibegovic, Stine Jacobsen, Emma Nilsson, Arne Astrup, Bjørn Quistorff and Allan Vaag

Steno Diabetes Center (C.B., C.B.J., H.S., A.Al., S.J., E.N., A.V.), 2820 Gentofte, Denmark; Department of Human Nutrition (C.B., S.J., A.As.), Faculty of Life Sciences, University of Copenhagen, 1870 Frederiksberg C, Denmark; and Department of Medical Biochemistry and Genetics (B.Q.), Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark

Address all correspondence and requests for reprints to: Charlotte Brøns, Steno Diabetes Center, Niels Steensens Vej 1, 2820 Gentofte, Denmark. E-mail: chbe{at}steno.dk.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Objective: Low birth weight (LBW) is an independent risk factor of insulin resistance and type 2 diabetes. Recent studies suggest that mitochondrial dysfunction and impaired expression of genes involved in oxidative phosphorylation (OXPHOS) may play a key role in the pathogenesis of insulin resistance in aging and type 2 diabetes. The aim of this study was to determine whether LBW in humans is associated with mitochondrial dysfunction in skeletal muscle.

Methods: Mitochondrial capacity for ATP synthesis was assessed by 31phosphorus magnetic resonance spectroscopy in forearm and leg muscles in 20 young, lean men with LBW and 26 matched controls. On a separate day, a hyperinsulinemic euglycemic clamp with excision of muscle biopsies and dual-energy x-ray absorptiometry scanning was performed. Muscle gene expression of selected OXPHOS genes was determined by quantitative real-time PCR.

Results: The LBW subjects displayed a variety of metabolic and prediabetic abnormalities, including elevated fasting blood glucose and plasma insulin levels, reduced insulin-stimulated glycolytic flux, and hepatic insulin resistance. Nevertheless, in vivo mitochondrial function was normal in LBW subjects, as was the expression of OXPHOS genes.

Conclusions: These data support and expand previous findings of abnormal glucose metabolism in young men with LBW. In addition, we found that the young, healthy men with LBW exhibited hepatic insulin resistance. However, the study does not support the hypothesis that muscle mitochondrial dysfunction per se is the underlying key metabolic defect that explains or precedes whole body insulin resistance in LBW subjects at risk for developing type 2 diabetes.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
Individuals born with low birth weight (LBW), a surrogate marker of impaired intrauterine growth, are at increased risk for developing insulin resistance and type 2 diabetes later in life (1, 2, 3, 4). In previous studies we have shown that LBW is associated with a redistribution of body fat to the abdominal region (5), impaired insulin-stimulated glucose uptake by the forearm tissue (6), and decreased whole body insulin-stimulated glycolytic flux (GF), even before the development of whole body peripheral insulin resistance (7) in 19-yr-old healthy men. Moreover, we recently showed that LBW is associated with altered muscle fiber distribution (8) and reduced basal expression in muscle and adipose tissue of the glucose transporter 4 (GLUT-4), as well as several key insulin-signaling proteins (9, 10). These findings suggest the presence of multiple abnormalities in insulin-sensitive tissues before the onset of whole body insulin resistance and type 2 diabetes, which could represent key metabolic defects linking LBW with the later development of insulin resistance and the metabolic syndrome, including type 2 diabetes.

Evidence has emerged over recent years that impaired mitochondrial function may represent a primary pathogenic defect involved in the development of insulin resistance in type 2 diabetes (11, 12, 13) as well as in aging (14, 15, 16). Impaired ATP synthesis due to reduced oxidative phosphorylation (OXPHOS) capacity, leading to accumulation of intramyocellular fat in skeletal muscle and subsequently muscle insulin resistance, appears to be particularly important in this respect. Obese individuals and subjects with type 2 diabetes display alterations in mitochondrial morphology (17), and patients with type 2 diabetes and their prediabetic offspring exhibit decreased expression of OXPHOS genes in skeletal muscle (18). In addition, mitochondrial dysfunction in liver and pancreatic β-cells has been linked to impaired insulin secretion in growth-retarded rats (19, 20).

Although challenged by other recent studies (21, 22, 23), the hypothesis that mitochondrial dysfunction is a general mechanism contributing to a variety of known key metabolic defects in type 2 diabetes remains attractive. Following this line of thinking, we hypothesized that programming in utero might be a potentially important etiological factor determining mitochondrial function.

To test our hypothesis, that LBW in humans is associated with impaired muscle mitochondrial function, we assessed in vivo mitochondrial function by 31phosphorus magnetic resonance spectroscopy (31P-MRS) after energy depleting exercise in young, prediabetic men born with LBW, and in matched controls. To clarify further the role of specific key OXPHOS genes involved in the electron transport chain and previously associated with insulin resistance and type 2 diabetes, quantitative real-time PCR (rt-PCR) was performed on muscle biopsies for selected genes.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Ethical approval

The protocol conformed to the Declaration of Helsinki and was approved by the ethics committee for Copenhagen County. All subjects signed an informed consent before participation.

Subjects

A total of 46 healthy males was recruited from the Danish National Birth Registry according to birth weight (BW). There were 20 men who had LBW (BW ≤10th percentile), and 26 were age-matched controls with normal BW (NBW) (50th ≤BW≤90th percentile). Of the 46 subjects, 14 had participated in a previous study by our group (7). All men were singletons, born in 1979–1980 at term in Copenhagen. Because a family history of type 2 diabetes is a risk factor for developing the disease in itself, subjects with a family history of diabetes in two generations were excluded in this study to exclude major genetic confounding. Subjects with a body mass index greater than 30 kg/m2 and a high physical activity level were also excluded from participation.

Experimental protocol

Study activities were performed over 3 d. Subjects were requested to refrain from strenuous physical activity and consuming alcohol 3 d before examination. To ensure standardized conditions, all meals the day before and throughout the study period were provided to the subjects.

Dual-energy x-ray absorptiometry scanning

On d1, fat-free mass (FFM) and fat mass (FM) were assessed by dual-energy x-ray absorptiometry (Lunar Radiation, Madison, WI).

31P-MRS

An Otsuka Electronics VivoSpec spectrometer was used, interfaced with a 2.9-tesla magnet (Magnex Scientific, Oxford, UK) with a 26-cm bore. 31P-MRS was performed on the 2nd day on two different muscle groups in separate experiments, and data were acquired as previously described (24).

Initially, maximal voluntary contraction was determined as the best of three 1-sec maximal contractions. Thereafter, 31P-MRS recording at a time resolution of 10 sec was performed for 3 min rest, 3 min exercise, and 6 min recovery. The protocol involved 18 successive, intermittent isometric contractions at 50% maximal voluntary contraction, each lasting 7 sec, interspersed by 3 sec rest. The 31P-MRS spectra during rest and the changes in response to work are depicted in Fig. 1Go. The contraction force was monitored throughout the experiment. The protocol was selected to obtain steady-state aerobic exercise, in which pH changes are minimal, and all measurements were performed on the right arm and leg.


Figure 1
View larger version (9K):
[in this window]
[in a new window]

  		FIG. 1. 31P-MRS spectra of human skeletal muscle at rest and during exercise. Only part of the spectra, including from the left Pi, phosphodiester (PDE), PCr, and the three phosphate groups ({alpha}, β, and {gamma}) of ATP, is included. ppm, Parts per million.

 
Hyperinsulinemic euglycemic clamp

On d 3 the clamp procedure was initiated at 0700 h after an overnight fast. A polyethylene catheter was placed in the antecubital vein for blood sampling. The hand was placed in a heated Plexiglas box to ensure arterialization of the venous blood. A second catheter was placed in the antecubital vein of the contralateral arm for test infusions. A primed-continuous infusion of [3-3H] tritiated glucose (bolus 10.9 µCi, 0.109 µCi/min) was initiated at 0 h and continued throughout the examination.

After a 120-min basal period, a 30-min iv glucose tolerance test (IVGTT) was initiated to determine β-cell function. A glucose bolus of 0.3 g/kg body weight was infused over 1 min. Blood samples for glucose, insulin, and C-peptide were collected at 0, 2, 4, 6, 8, 10, 15, 20, and 30 min.

After the IVGTT a primed-continuous insulin infusion was initiated and fixed at 80 mU/m2·min throughout the 180-min clamp. Steady-state was defined as the last 30 min of the basal and insulin clamp period, when tracer equilibrium was anticipated. Variable infusion of "cold" glucose (180 g/liter) enriched with tritiated glucose (110 µCi/500 ml) was used to maintain euglycemia during insulin infusion. Blood glucose concentration was monitored every 5 min during steady-state using a blood glucose meter (OneTouch; LifeScan Inc., Milpitas, CA). The target blood glucose concentration was 5 mmol/liter, and the infusion rate was adjusted if necessary immediately after each blood glucose assessment. During the clamp period, blood samples for measuring tritiated glucose and water were drawn every 10–15 min and determined as previously described (25).

Samples for measuring insulin and C-peptide were drawn every 30 min.

At the end of each steady-state period, a vastus lateralis muscle biopsy was taken using a Bergström needle under local anesthesia. The tissue was immediately frozen in liquid nitrogen and stored at –80 C.

Quantitative rt-PCR

Extraction of total RNA from the muscle biopsies was performed with TRI reagent (Sigma-Aldrich, St. Louis, MO). cDNA was synthesized using the QuantiTect Reverse Transcription kit (QIAGEN, Inc., Valencia, CA). rt-PCR was performed using the ABI 7900 Sequence Detection System (Applied Biosystems, Foster City, CA). Assays from Applied Biosystems were used: NDUFB6 (Hs00159583_m1), UQCRB (Hs00559884_m1), COX7A1 (Hs00156989_m1), ATP5O (Hs00426889_m1), and peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1{alpha}) (Hs00173304_m1). All samples were run in duplicate; values were calculated using the standard curve method and normalized to the mRNA level of Cyclophilin A (4326316E; Applied Biosystems).

Calculations

31P-MRS The spectra were subjected to a 5-Hz line broadening and a baseline correction, involving a semi-manual, cubic spline procedure. Subsequently, the phosphocreatine (PCr), inorganic phosphor (Pi), and the three ATP peaks were fitted by a least-squares routine, assuming a Lorentzian or Gaussian curve (26). The peak areas were corrected for partial saturation measured at rest, i.e. 1.22, 1.14, and 1.11 for PCr, Pi, and ATP, respectively. Metabolite concentrations were calculated, assuming a resting ATP concentration of 5.8 mM (27). Intracellular pH was calculated from the difference in the chemical shift between Pi and PCr (28).

The concentration of free ADP was estimated from the creatine kinase reaction, assuming equilibrium (29, 30). The aerobic capacity for ATP production was estimated from the PCr recovery kinetics, assuming a monoexponential model, and the maximum velocity (Vmax) was calculated from these values as described by Ratkevicius and Quistorff (31), assuming Michaelis-Menten kinetics and a Michaelis-Menten constant for ADP for OXPHOS of 30 µM (32). Similarly, the recovery kinetics of Pi after exercise was modeled by monoexponential kinetics and the recovery half-times presented.

Hyperinsulinemic euglycemic clamp and IVGTT Glucose turnover rates
During the predefined insulin stimulated steady-state period (150–180 min), rates of unlabeled glucose appearance (Ra), unlabeled glucose disappearance, and hepatic glucose production (HGP) were calculated using Steele’s nonsteady-state equation (33). During the steady-state period, the rate of unlabeled glucose disappearance, and HGP were calculated at 10-min intervals, and the distribution volume of glucose was set at 200 ml/kg body weight and the pool fraction as 0.65 (34). The GF was calculated from the appearance rate of tritiated water, and the total plasma water was assumed to be 93% of the total plasma volume (35, 36). HGP during the insulin-stimulated steady-state period was calculated as the difference between Ra and the glucose infusion rate. In cases in which Ra was lower than the exogenous glucose infusion (HGP negative), the M-value calculated from the glucose infusion rate was used in the calculations.

The area under the curve was calculated using a trapezoidal method for glucose and insulin during the first-phase insulin response (0–10 min) of the IVGTT. The hepatic insulin-resistance index was calculated as the product of mean fasting plasma insulin concentration and basal HGP (37).

Statistics

Statistical analysis was performed using SAS software (version 9.1; SAS Institute Inc., Cary, NC). An unpaired Student’s t test was used to identify statistically significant differences between NBW and LBW subjects. Correlations were calculated using Spearman’s or Pearson’s (normally distributed data) correlation coefficient. A P value of less than 0.05 was considered significant. Data are presented as mean values ± SD, except gene expression data, which are presented as means ± SEM. In a post hoc power calculation with recovery rates of PCr and Pi as endpoints, we had an 80% chance of detecting differences between NBW and LBW subjects of 23–35% in arm and leg muscles.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Clinical characteristics (Table 1Go)

At birth, the LBW subjects were approximately 1200 g lighter than the control group (3893 ± 207 vs. 2688 ± 269 g; P < 0.0001), current height was less (1.83 ± 0.07 vs. 1.77 ± 0.05 m; P = 0.004), as were the plasma high-density lipoprotein levels (1.38 ± 0.22 vs. 1.17 ± 0.23 mmol/liter; P = 0.003). LBW subjects had a higher trunk FM (g) to total FM (g) ratio (0.50 ± 0.05 vs. 0.53 ± 0.04; P = 0.02), a lower leg FM (g) to total FM (g) ratio (0.37 ± 0.04 vs. 0.34 ± 0.03; P = 0.02), and a higher percent trunk FM to leg FM ratio (1.10 ± 0.19 vs. 1.24 ± 0.16; P = 0.01) compared with controls.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Clinical characteristics of the study participants

 
Hyperinsulinemic euglycemic clamp (Table 2Go)

Fasting blood glucose (4.59 ± 0.46 vs. 4.96 ± 0.47 mmol/liter; P = 0.01) and plasma insulin levels (30.9 ± 14.1 vs. 40.7 ± 14.4 pmol/liter; P = 0.03) were higher in the LBW group. As demonstrated previously during lower clamp insulin levels in other individuals (7), the LBW subjects had a lower insulin-stimulated GF (4.62 ± 2.17 vs. 3.22 ± 1.98 mg/kg FFM/min; P = 0.03). The hepatic insulin resistance index was higher in the LBW group (68.7 ± 34.1 vs. 99.3 ± 49.1; P = 0.02). There were no differences in basal Ra or in insulin-stimulated glucose disposal rates (M-value) between the two groups.


View this table:
[in this window]
[in a new window]

  		TABLE 2. Hyperinsulinemic euglycemic clamp results

 
31P-MRS (Table 3Go)

No differences were observed between the two groups with regard to the calculated aerobic ATP turnover rate (Vmax) or the recovery kinetics of PCr and Pi after exercise in either arm or leg muscle.


View this table:
[in this window]
[in a new window]

  		TABLE 3. 31P-MRS findings at rest and after exercise (recovery) in the forearm flexor muscles (NBW n = 22, LBW n = 19) and in the tibialis anterior of the leg (NBW n = 20, LBW n = 16)

 
One subject almost completely used his entire amount of PCr during the contraction, causing a PCr/Pi ratio above 100. Despite the fact that all other measurements on this subject were in the normal range, the subject was excluded from the group comparison. Exclusion of the subject did not affect the level of significance.

Correlations (Table 4Go)

No significant correlations between any measurements of in vivo mitochondrial function and peripheral whole body insulin action were observed.


View this table:
[in this window]
[in a new window]

  		TABLE 4. Correlations between the M-value and MRS-generated measures of mitochondrial function

 
rt-PCR (Fig. 2Go)

No significant differences in OXPHOS gene expression levels between NBW and LBW subjects were found, either in the basal (Fig. 2AGo) or the insulin-stimulated states (Fig. 2BGo).


Figure 2
View larger version (11K):
[in this window]
[in a new window]

  		FIG. 2. A and B, Gene expression of OXPHOS genes in skeletal muscle obtained from NBW (white) vs. LBW (black) subjects for basal (A) or insulin-stimulated (B) states. mRNA levels were quantified by rt-PCR and normalized to the level of endogenous cyclophilin A (Cyc A). Values are means ± SEMs.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
The young LBW subjects exhibited a number of prediabetic features in support of a true prediabetic phenotype. A novel finding of this study is that the LBW subjects had a significant increased hepatic insulin resistance index. This was not observed in a previous study of LBW individuals from our group (7) and could potentially be explained by the higher age of the current subjects. The hepatic insulin resistance, which is present before the development of peripheral insulin resistance, is likely to contribute significantly to the mild elevations of fasting hyperglycemia in the LBW subjects. Finally, the LBW subjects had a higher area under the glucose curve than NBW subjects, in response to the IVGTT, but similar insulin secretion.

The most obvious metabolic defect of impaired mitochondrial function is reduced capacity for aerobic ATP synthesis, either due to a lowered number of otherwise normal mitochondria or a decreased capacity of the individual mitochondria. Although such changes are unlikely to cause complete loss of ATP synthesis, it may be expected that significant changes will become unmasked under demanding conditions, such as in response to energy depleting exercise (38). Recently, a number of studies have suggested that mitochondrial dysfunction measured in resting skeletal muscle by 31P-MRS is involved in the development of muscle insulin resistance in aging and type 2 diabetes (12, 14, 39). To determine the putative role of mitochondrial dysfunction and reduced ATP synthesis in LBW subjects, we estimated in vivo ATP synthesis based on the recovery of phosphagenic compounds and pH. Because the recovery kinetics of Pi and PCr after exercise is dependent on mitochondrial capacity for ATP synthesis, and thereby on oxidative metabolism, both of these parameters, together with the calculated capacity for ATP synthesis, were used as the primary measures of mitochondrial function. Somewhat surprisingly, the data showed that there were no differences in determinants of in vivo mitochondrial function between LBW and control subjects.

In this study we examined in vivo mitochondrial function in muscles that are normally different in fiber composition: tibialis anterior high in oxidative type I fibers; and the forearm flexor muscle, normally high in glycolytic type II fibers (24). As expected, recovery rates of Pi and PCr were twice as fast in oxidative compared with glycolytic muscles (Table 3Go). However, no differences were observed between the groups for either of the muscles, providing substantial evidence of normal mitochondrial function in LBW subjects. Thus, our data indicate that mitochondrial dysfunction in skeletal muscle of patients with type 2 diabetes associated with LBW (if existing), is a late-occurring phenomenon secondary to or unmasked by aging, high-fat diet, or low physical activity. We have previously shown reduced insulin-stimulated uptake of the forearm despite a normal whole body insulin-stimulated glucose uptake (similar to that found in this study) (6). This could indicate that LBW subjects maintain normal mitochondrial function, even in the presence of local impaired insulin-stimulated muscle glucose uptake. However, we cannot exclude that LBW subjects have decreased mitochondrial function if measured during a euglycemic clamp experiment. Because insulin stimulates mitochondrial function, the possibility exists that mitochondrial dysfunction, in some insulin-resistant states, may be secondary to insulin resistance, an issue particularly relevant when measuring mitochondrial function during insulin infusion (11). We believe that the present data obtained after energy depleting exercise are more accurate in determining the true function of the mitochondria, as opposed to resting measurements performed during clamp insulin infusions.

It was recently reported that young insulin-resistant subjects with a family history of type 2 diabetes exhibit dysregulation of intramyocellular fatty acid metabolism linked to mitochondrial dysfunction, with a 40% reduction of the number of mitochondria (12, 13). If LBW subjects, representing another significant prediabetic and (with age) insulin-resistant phenotype, exhibit a similar magnitude of mitochondrial dysfunction, it would beyond any doubt have been detected with the present 31P-MRS experiment and with the cycling test. Unlike previous studies of first-degree relatives and elderly, we examined our subjects during and after energy depleting exercise, and not only during resting conditions, allowing us to determine the true functional potential of the mitochondria. However, it is possible that an underestimation of the Vmax could have occurred, e.g. if ATP turnover measured during recovery was limited by oxygen supply (state 5 respiration), but an "overestimation" would seem to be out of the question. Thus, the 40% decreased mitochondrial capacity that has previously been reported in elderly patients (14) cannot be confirmed in this study comparing LBW and controls.

In addition, we measured the maximal rate of oxygen consumption (VO2 max) of the participants, and in accordance with a previous study (7), we found no difference between the groups. We thereby ensured an equal rate of maximal oxygen consumption, which could otherwise represent a confounding factor. The number of subjects in each group was approximately twice that of subjects in previous studies of first-degree relatives of patients with type 2 diabetes (11, 12, 39). Furthermore, mitochondrial function was measured in two different muscle groups in each subject, altogether ensuring sufficient statistical power to detect a physiologically relevant difference between the groups. In this study we did not select for known insulin resistance and/or low VO2 max in an a priori screened prediabetic population, thereby avoiding selection for common causes of insulin resistance acquired from dietary habits or a sedentary lifestyle. In that respect our aim was to determine whether LBW, a known and significant risk factor of insulin resistance and type 2 diabetes, was associated with mitochondrial dysfunction before, and not as a result of, these states of disease. Our previous findings of multiple molecular defects associated with, and/or explaining, muscle insulin resistance in similar groups of (whole body insulin sensitive) LBW subjects, including altered fiber type composition (8) and impaired insulin signaling (9), support our approach in this study of selecting for the criteria of LBW per se, and not for insulin resistance or reduced VO2 max.

Recent studies have demonstrated that mitochondrial OXPHOS genes are reduced in the skeletal muscle of subjects with a family history of diabetes, in patients with type 2 diabetes, and in insulin-resistant patients with polycystic ovary syndrome, and that these changes may be coordinated by decreased expression of PGC-1{alpha} (40, 41, 42). ATP5O, COX7A1, NDUFB6, and UQCRB are all genes involved in the tightly PGC-1{alpha}-regulated OXPHOS-coregulated cluster. Despite the documented prediabetic phenotype of the LBW subjects, no significant differences in the basal or insulin-stimulated expression of prominent type 2 diabetes related OXPHOS genes were observed between NBW and LBW subjects, as assessed in vastus lateralis muscle biopsies, indeed, supporting the normal in vivo measurements of mitochondrial function.

There is growing evidence that type 2 diabetes is a more heterogeneous state than previously thought, in which multiple minor susceptibility genes and a range of nongenetic factors together contribute to a complicated metabolic state involving many different organ dysfunctions. It should be mentioned that we cannot exclude the possibility that primary genetic defects of mitochondrial function contribute to some unknown extent to the metabolic disturbances in genetically predisposed prediabetic subjects, and in some groups of patients with overt type 2 diabetes (21). In this study we excluded subjects with a family history of type 2 diabetes to study the isolated component of an adverse intrauterine environment.

Using different techniques, other recent studies have challenged the entire concept of mitochondrial dysfunction and impaired OXPHOS in type 2 diabetes (23). Boushel et al. (23) measured ex vivo electron transport capacity in muscle biopsies using high-resolution respirometry and reported that mitochondrial function was normal in patients with type 2 diabetes. Another interesting finding of the same study was a reduced muscle mitochondrial content (but not function), which in turn was thought to reflect reduced VO2 max (or physical activity) in type 2 diabetic patients. Regardless, it is likely that the divergent results of in vivo mitochondrial function in type 2 diabetes patients reflect the heterogeneity of the disease.

A key feature of the hypothesis linking insulin resistance to mitochondrial dysfunction is that the latter limits the capacity to oxidize fat, which subsequently accumulates in muscle, where it causes impaired insulin signal transduction and thereby insulin resistance (43). Although this work involved the greatest number of subjects yet studied with simultaneous state-of-the-art measurements, we did not find any correlations between measurements of mitochondrial function in skeletal muscles and whole body insulin resistance. This suggests that mitochondrial function does not play any significant role in the day-to-day control of insulin action in young, lean men, including both LBW subjects at risk for developing diabetes as well as NBW controls, and that any impact of mitochondrial function on insulin action may not occur until a certain amount of ectopic fat has accumulated with time or age in skeletal muscle. This remains to be tested in a prospective study setting. During the chosen study conditions, skeletal muscle is supposed to account for around 80% or more of insulin-stimulated glucose uptake (44). Our previous finding of reduced insulin-stimulated muscle glucose uptake in LBW subjects, in the face of a normal whole body glucose uptake (6), indicates that this association may be disrupted in LBW subjects. This may in turn, to some unknown extent, contribute to the lack of association between muscle mitochondrial function and whole body insulin action, at least in this study.

Conclusions

This study provides further confirmation of an abnormal glucose metabolism, including the novel finding of hepatic insulin resistance in young, healthy men with LBW. However, given that in vivo mitochondrial capacity for ATP synthesis was normal in two different muscle groups, the study does not support the hypothesis of mitochondrial dysfunction per se being the underlying key defect of metabolism that explains (or precedes) peripheral insulin resistance in family history negative LBW subjects. This conclusion is further supported by the finding of normal OXPHOS gene expression, and the surprising absence of any correlations between in vivo insulin action and mitochondrial function.


   Acknowledgments
 
We thank Ib Terkelsen, Marianne Modest, Julie S. Appel, and Lars Sander Koch for providing excellent technical support and assistance with the experiments. We are especially appreciative of all the young men who participated in this study.


   Footnotes
 
This work was supported by The Danish Diabetes Association, The European Foundation for the Study of Diabetes, The European Union 6th Framework EXGENESIS Grant, The Strategic Danish Research Council, and The Aase and Ejnar Danielsen Foundation. C.B. was granted a Ph.D. scholarship from the Faculty of Life Sciences at the University of Copenhagen.

Disclosure Summary: C.B., C.B.J., and A.V. have stock options in Novo Nordisk. A.V. has consulting and lecture fees of less than $ 10,000. H.S., A.Al., S.J., E.N., A.As., and B.Q. have nothing to declare.

First Published Online July 15, 2008

Abbreviations: BW, Birth weight; FFM, fat-free mass; FM, fat mass; GF, glycolytic flux; HGP, hepatic glucose production; IVGTT, iv glucose tolerance test; LBW, low birth weight; M-value, insulin stimulated glucose uptake; NBW, normal birth weight; OXPHOS, oxidative phosphorylation; PCr, phosphocreatine; PGC, prostaglandin; Pi, inorganic phosphor; 31P-MRS, 31phosphorus magnetic resonance spectroscopy; Ra, rate of unlabeled glucose appearance; rt-PCR, real-time PCR; Vmax, maximum velocity; VO2 max, maximal rate of oxygen consumption.

Received March 18, 2008.

Accepted July 7, 2008.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Ravelli G, Stein ZA, Susser MW 1976 Obesity in young men after famine exposure in utero and early infancy. N Engl J Med 295:349–353[Abstract]
  2. Hales CN, Barker DJ, Clark PM, Cox LJ, Fall C, Osmond C, Winter PD 1991 Fetal and infant growth and impaired glucose tolerance at age 64. BMJ 303:1019–1022[Abstract/Free Full Text]
  3. Ravelli AC, van der Meulen JH, Michels RP, Osmond C, Barker DJ, Hales CN, Bleker OP 1998 Glucose tolerance in adults after prenatal exposure to famine. Lancet 351:173–177[CrossRef][Medline]
  4. Vaag A, Jensen CB, Poulsen P, Brøns C, Pilgaard K, Grunnet L, Vielwerth S, Alibegovic A 2006 Metabolic aspects of insulin resistance in individuals born small for gestational age. Horm Res 65(Suppl 3):137–143
  5. Rasmussen EL, Malis C, Jensen CB, Jensen JE, Storgaard H, Poulsen P, Pilgaard K, Schou JH, Madsbad S, Astrup A, Vaag A 2005 Altered fat tissue distribution in young adult men who had low birth weight. Diabetes Care 28:151–153[Free Full Text]
  6. Hermann TS, Rask-Madsen C, Ihlemann N, Dominguez H, Jensen CB, Storgaard H, Vaag AA, Kober L, Torp-Pedersen C 2003 Normal insulin-stimulated endothelial function and impaired insulin-stimulated muscle glucose uptake in young adults with low birth weight. J Clin Endocrinol Metab 88:1252–1257[Abstract/Free Full Text]
  7. Jensen CB, Storgaard H, Dela F, Holst JJ, Madsbad S, Vaag AA 2002 Early differential defects of insulin secretion and action in 19-year-old Caucasian men who had low birth weight. Diabetes 51:1271–1280[Abstract/Free Full Text]
  8. Jensen CB, Storgaard H, Madsbad S, Richter EA, Vaag AA 2007 Altered skeletal muscle fiber composition and size precede whole-body insulin resistance in young men with low birth weight. J Clin Endocrinol Metab 92:1530–1534[Abstract/Free Full Text]
  9. Ozanne SE, Jensen CB, Tingey KJ, Storgaard H, Madsbad S, Vaag AA 2005 Low birthweight is associated with specific changes in muscle insulin-signalling protein expression. Diabetologia 48:547–552[CrossRef][Medline]
  10. Ozanne SE, Jensen CB, Tingey KJ, Martin-Gronert MS, Grunnet L, Brons C, Storgaard H, Vaag AA 2006 Decreased protein levels of key insulin signalling molecules in adipose tissue from young men with a low birthweight: potential link to increased risk of diabetes? Diabetologia 49:2993–2999[CrossRef][Medline]
  11. Petersen KF, Dufour S, Shulman GI 2005 Decreased insulin-stimulated ATP synthesis and phosphate transport in muscle of insulin-resistant offspring of type 2 diabetic parents. PLoS Med 2:e233
  12. Befroy DE, Petersen KF, Dufour S, Mason GF, de Graaf RA, Rothman DL, Shulman GI 2007 Impaired mitochondrial substrate oxidation in muscle of insulin-resistant offspring of type 2 diabetic patients. Diabetes 56:1376–1381[CrossRef][Medline]
  13. Morino K, Petersen KF, Dufour S, Befroy D, Frattini J, Shatzkes N, Neschen S, White MF, Bilz S, Sono S, Pypaert M, Shulman GI 2005 Reduced mitochondrial density and increased IRS-1 serine phosphorylation in muscle of insulin-resistant offspring of type 2 diabetic parents. J Clin Invest 115:3587–3593[CrossRef][Medline]
  14. Petersen KF, Befroy D, Dufour S, Dziura J, Ariyan C, Rothman DL, DiPietro L, Cline GW, Shulman GI 2003 Mitochondrial dysfunction in the elderly: possible role in insulin resistance. Science 300:1140–1142[Abstract/Free Full Text]
  15. Short KR, Bigelow ML, Kahl J, Singh R, Coenen-Schimke J, Raghavakaimal S, Nair KS 2005 Decline in skeletal muscle mitochondrial function with aging in humans. Proc Natl Acad Sci USA 102:5618–5623[Abstract/Free Full Text]
  16. Rooyackers OE, Adey DB, Ades PA, Nair KS 1996 Effect of age on in vivo rates of mitochondrial protein synthesis in human skeletal muscle. Proc Natl Acad Sci USA 93:15364–15369[Abstract/Free Full Text]
  17. Ritov VB, Menshikova EV, He J, Ferrell RE, Goodpaster BH, Kelley DE 2005 Deficiency of subsarcolemmal mitochondria in obesity and type 2 diabetes. Diabetes 54:8–14[Abstract/Free Full Text]
  18. Ling C, Poulsen P, Simonsson S, Ronn T, Holmkvist J, Almgren P, Hagert P, Nilsson E, Mabey AG, Nilsson P, Vaag A, Groop L 2007 Genetic and epigenetic factors are associated with expression of respiratory chain component NDUFB6 in human skeletal muscle. J Clin Invest 117:3427–3435[CrossRef][Medline]
  19. Peterside IE, Selak MA, Simmons RA 2003 Impaired oxidative phosphorylation in hepatic mitochondria in growth-retarded rats. Am J Physiol Endocrinol Metab 285:E1258–E1266
  20. Simmons RA, Suponitsky-Kroyter I, Selak MA 2005 Progressive accumulation of mitochondrial DNA mutations and decline in mitochondrial function lead to β-cell failure. J Biol Chem 280:28785–28791[Abstract/Free Full Text]
  21. De Feyter HM, van den Broek NM, Praet SF, Nicolay K, van Loon LJ, Prompers JJ 2008 Early or advanced stage type 2 diabetes is not accompanied by in vivo skeletal muscle mitochondrial dysfunction. Eur J Endocrinol 158:643–653[Abstract/Free Full Text]
  22. Trenell M, Hollingsworth K, Lim E, Gerrard J, Taylor R 2007 Changes in resting and maximal mitochondrial ATP production in type 2 diabetes; non-invasive investigation by 31P-magnetic resonance spectroscopy. Diabetologia 50:S1–S538
  23. Boushel R, Gnaiger E, Schjerling P, Skovbro M, Kraunsoe R, Dela F 2007 Patients with type 2 diabetes have normal mitochondrial function in skeletal muscle. Diabetologia 50:790–796[CrossRef][Medline]
  24. Jeppesen TD, Quistorff B, Wibrand F, Vissing J 2007 31P-MRS of skeletal muscle is not a sensitive diagnostic test for mitochondrial myopathy. J Neurol 254:29–37[CrossRef][Medline]
  25. Hother-Nielsen O, Beck-Nielsen H 1990 On the determination of basal glucose production rate in patients with type 2 (non-insulin-dependent) diabetes mellitus using primed-continuous 3-3H-glucose infusion. Diabetologia 33:603–610[CrossRef][Medline]
  26. Hoch JC, Stern AS 1996 In: NMR data processing. New York: Wiley-Liss; 34–77
  27. Bangsbo J, Graham T, Johansen L, Strange S, Christensen C, Saltin B 1992 Elevated muscle acidity and energy production during exhaustive exercise in humans. Am J Physiol 263(4 Pt 2):R891–R899
  28. Arnold D, Matthews PM, Radda GK 1984 Metabolic recovery after exercise and the assessment of mitochondrial function in vivo in human skeletal muscle by means of 31P NMR. Magn Reson Med 1:307–315[Medline]
  29. Kushmerick MJ 1997 Multiple equilibria of cations with metabolites in muscle bioenergetics. Am J Physiol 272(5 Pt 1):C1739–C1747
  30. Conley KE, Esselman PC, Jubrias SA, Cress ME, Inglin B, Mogadam C, Schoene RB 2000 Ageing, muscle properties and maximal O(2) uptake rate in humans. J Physiol 526(Pt 1):211–217
  31. Ratkevicius A, Quistorff B 2002 Metabolic costs of force generation for constant-frequency and catchlike-inducing electrical stimulation in human tibialis anterior muscle. Muscle Nerve 25:419–426[CrossRef][Medline]
  32. Chance B, Leigh Jr JS, Kent J, McCully K, Nioka S, Clark BJ, Maris JM, Graham T 1986 Multiple controls of oxidative metabolism in living tissues as studied by phosphorus magnetic resonance. Proc Natl Acad Sci USA 83:9458–9462[Abstract/Free Full Text]
  33. Steele R 1959 Influences of glucose loading and of injected insulin on hepatic glucose output. Ann NY Acad Sci 82:420–430[Medline]
  34. Cowan JS, Hetenyi Jr G 1971 Glucoregulatory responses in normal and diabetic dogs recorded by a new tracer method. Metabolism 20:360–372[CrossRef][Medline]
  35. Rossetti L, Giaccari A 1990 Relative contribution of glycogen synthesis and glycolysis to insulin-mediated glucose uptake. A dose-response euglycemic clamp study in normal and diabetic rats. J Clin Invest 85:1785–1792[Medline]
  36. Del Prato S, Bonadonna RC, Bonora E, Gulli G, Solini A, Shank M, Defronzo RA 1993 Characterization of cellular defects of insulin action in type 2 (non-insulin-dependent) diabetes mellitus. J Clin Invest 91:484–494[Medline]
  37. Bajaj M, Suraamornkul S, Romanelli A, Cline GW, Mandarino LJ, Shulman GI, DeFronzo RA 2005 Effect of a sustained reduction in plasma free fatty acid concentration on intramuscular long-chain fatty Acyl-CoAs and insulin action in type 2 diabetic patients. Diabetes 54:3148–3153[Abstract/Free Full Text]
  38. James AM, Murphy MP 2002 How mitochondrial damage affects cell function. J Biomed Sci 9:475–487[Medline]
  39. Petersen KF, Dufour S, Befroy D, Garcia R, Shulman GI 2004 Impaired mitochondrial activity in the insulin-resistant offspring of patients with type 2 diabetes. N Engl J Med 350:664–671[Abstract/Free Full Text]
  40. Mootha VK, Lindgren CM, Eriksson KF, Subramanian A, Sihag S, Lehar J, Puigserver P, Carlsson E, Ridderstrale M, Laurila E, Houstis N, Daly MJ, Patterson N, Mesirov JP, Golub TR, Tamayo P, Spiegelman B, Lander ES, Hirschhorn JN, Altshuler D, Groop LC 2003 PGC-1{alpha}-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet 34:267–273[CrossRef][Medline]
  41. Patti ME, Butte AJ, Crunkhorn S, Cusi K, Berria R, Kashyap S, Miyazaki Y, Kohane I, Costello M, Saccone R, Landaker EJ, Goldfine AB, Mun E, DeFronzo R, Finlayson J, Kahn CR, Mandarino LJ 2003 Coordinated reduction of genes of oxidative metabolism in humans with insulin resistance and diabetes: potential role of PGC1 and NRF1. Proc Natl Acad Sci USA 100:8466–8471[Abstract/Free Full Text]
  42. Skov V, Glintborg D, Knudsen S, Jensen T, Kruse TA, Tan Q, Brusgaard K, Beck-Nielsen H, Hojlund K 2007 Reduced expression of nuclear-encoded genes involved in mitochondrial oxidative metabolism in skeletal muscle of insulin-resistant women with polycystic ovary syndrome. Diabetes 56:2349–2355[CrossRef][Medline]
  43. Krssak M, Falk Petersen K, Dresner A, DiPietro L, Vogel SM, Rothman DL, Roden M, Shulman GI 1999 Intramyocellular lipid concentrations are correlated with insulin sensitivity in humans: a 1H NMR spectroscopy study. Diabetologia [Errata (1999) 42:386, (1999) 42:1269] 42:113–116[CrossRef]
  44. Defronzo RA, Jacot E, Jequier E, Maeder E, Wahren J, Felber JP 1981 The effect of insulin on the disposal of intravenous glucose. Results from indirect calorimetry and hepatic and femoral venous catheterization. Diabetes 30:1000–1007[Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
W. Jorgensen, C. Gam, J. L. Andersen, P. Schjerling, M. Scheibye-Knudsen, O. H. Mortensen, N. Grunnet, M. O. Nielsen, and B. Quistorff
Changed mitochondrial function by pre- and/or postpartum diet alterations in sheep
Am J Physiol Endocrinol Metab, December 1, 2009; 297(6): E1349 - E1357.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
C. Brøns, C. B. Jensen, H. Storgaard, N. J. Hiscock, A. White, J. S. Appel, S. Jacobsen, E. Nilsson, C. M. Larsen, A. Astrup, et al.
Impact of short-term high-fat feeding on glucose and insulin metabolism in young healthy men
J. Physiol., May 15, 2009; 587(10): 2387 - 2397.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
L. G. Grunnet, C. Brons, S. Jacobsen, E. Nilsson, A. Astrup, T. Hansen, O. Pedersen, P. Poulsen, B. Quistorff, and A. Vaag
Increased Recovery Rates of Phosphocreatine and Inorganic Phosphate after Isometric Contraction in Oxidative Muscle Fibers and Elevated Hepatic Insulin Resistance in Homozygous Carriers of the A-allele of FTO rs9939609
J. Clin. Endocrinol. Metab., February 1, 2009; 94(2): 596 - 602.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Brøns, C.
Right arrow Articles by Vaag, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Brøns, C.
Right arrow Articles by Vaag, A.
Related Collections
Right arrow Diabetes and Insulin
Right arrow Male Endocrinology
Right arrow Metabolism

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals